**The Presence of D-Penicillamine during the In Vitro Capacitation of Stallion Spermatozoa Prolongs Hyperactive-Like Motility and Allows for Sperm Selection by Thermotaxis**

**Sara Ruiz-Díaz 1,2,**†**, Ivan Oseguera-López 3,**†**, David De La Cuesta-Díaz 1, Belén García-López 1, Consuelo Serres 4, Maria José Sanchez-Calabuig 4, Alfonso Gutiérrez-Adán 1,\* and Serafin Perez-Cerezales <sup>1</sup>**


Received: 9 July 2020; Accepted: 18 August 2020; Published: 21 August 2020

**Simple Summary:** Capacitation of stallion semen in vitro is still a suboptimal procedure. The main objective of this study was the use of thermotaxis as a novel method for sperm selection and determining the most adequate media for maintaining frozen/thawed horse sperm longevity in vitro. Our results show that the most common media (Whitten's) used in this species is not the best for capacitating the semen in terms of hyperactive-like motility, and tyrosine phosphorylation being synthetic human tubal fluid supplemented with D-penicillamine is the most adequate in preserving these parameters during 180 min of incubation. Therefore, this media (with and without D-penicillamine) was chosen for performing thermotaxis. The selection conditions were a gradient of 3 ◦C of difference (35–38 ◦C) for 1 h. The results revealed that the selected fraction showed higher levels of tyrosine phosphorylation in the whole flagellum and lower levels of DNA fragmentation when compared to the unselected fraction (kept at 37 ◦C) when human tubal fluid with D-penicillamine was used. These results are promising for improving the in vitro embryo production rates in these species by improving the sperm selection methodology.

**Abstract:** Assisted reproductive technologies (ARTs) in the horse still yield suboptimal results in terms of pregnancy rates. One of the reasons for this is the lack of optimal conditions for the sperm capacitation in vitro. This study assesses the use of synthetic human tubal fluid (HTF) supplemented with D-penicillamine (HTF + PEN) for the in vitro capacitation of frozen/thawed stallion spermatozoa by examining capacitation-related events over 180 min of incubation. Besides these events, we explored the in vitro capacity of the spermatozoa to migrate by thermotaxis and give rise to a population of high-quality spermatozoa. We found that HTF induced higher levels of hyperactive-like motility and protein tyrosine phosphorylation (PTP) compared to the use of a medium commonly used in this species (Whitten's). Also, HTF + PEN was able to maintain this hyperactive-like motility, otherwise lost in the absence of PEN, for 180 min, and also allowed for sperm selection by thermotaxis in vitro. Remarkably, the selected fraction was enriched in spermatozoa showing PTP along the whole flagellum and lower levels of DNA fragmentation when compared to the unselected fraction (38% ± 11% vs 4.4% ± 1.1% and 4.2% ± 0.4% vs 11% ± 2% respectively,

*t*-test *p* < 0.003, *n* = 6). This procedure of in vitro capacitation of frozen/thawed stallion spermatozoa in HTF + PEN followed by in vitro sperm selection by thermotaxis represents a promising sperm preparation strategy for in vitro fertilization and intracytoplasmic sperm injection in this species.

**Keywords:** stallion sperm; capacitation; penicillamine; thermotaxis; selection

#### **1. Introduction**

Despite nearly two decades of efforts, in vitro fertilization (IVF) in the horse remains unavailable, and intracytoplasmic sperm injection (ICSI) in this species still yields suboptimal results. One of the main causes of these limitations are suboptimal in vitro conditions for sperm capacitation, preventing successful IVF [1] and low ICSI outcomes [2].

Synthetic media successfully used for the in vitro capacitation of sperm in other mammalian species are also being tested in the stallion. Thus, reports exist of capacitation-related events that occur in equine spermatozoa under different incubation conditions employing the media Biggers–Whitten–Whittingham (BWW), Tyrode's, Whitten's, or human tubal fluid [3–8]. However, the available data indicate that using these media, spermatozoa are not fully capacitated, with the consequence that in vitro fertilization remains elusive in this species [1]. One of the main events occurring during sperm capacitation in mammals is the phosphorylation of multiple proteins at their tyrosine residues [9]. Specifically, protein tyrosine phosphorylation (PTP) in the sperm flagellum has been related to the hyperactive motility of sperm, and both these factors are considered hallmarks of mammalian sperm capacitation [10,11]. However, while PTP in the equine spermatozoon flagellum is elevated under different capacitating conditions in vitro, so far all attempts to establish its relationship with hyperactive motility have had limited success [12]. Romero-Aguirregomezcorta [13] showed that the type of hyperactivated motion induced in vitro by species-specific hyperactivation agonists significantly differed in stallion in relation to human or ram spermatozoa, and led to the absence of a rheotactic response in stallion sperm. As an explanation, the authors suggested a different role of sperm hyperactivation in the horse. However, we propose here that it could be the suboptimal in vitro conditions for stallion spermatozoa that prevent a true or complete hyperactivation response.

The proportions of mammalian spermatozoa that acquire a capacitated status at a given time point can be low (roughly around 10%) [14–16]. Further, in humans, this capacitated state of spermatozoa is transient (>50 min to <4 h) and occurs only once in a sperm's lifespan [14]. Harrison [17] described capacitation as a series of positive destabilizing events that eventually lead to sperm death. Later on, Aitken et al. [18] related the physiological production of reactive oxygen species (ROS) to capacitation, followed by apoptosis and sperm senescence. The concept of sperm death as a consequence of capacitation implies that too-high or too-rapid induction of capacitation can shorten the lifespan to the extent that fertilization is prevented [19]. Aitken et al. [20] suggested that the reduced lifespan of spermatozoa incubated in vitro is the outcome of an excessive production of free radicals that eventually provokes lipid peroxidation and the generation of electrophilic cytotoxic aldehydes. To counteract this effect, these authors showed that the addition of penicillamine (PEN), a molecule that neutralizes these aldehydes and slows down their production, improved the maintenance of motility of fresh equine spermatozoa using a non-capacitating medium. In earlier work, Pavlok [21] established that the addition of PEN to the capacitating medium significantly prolonged the lifespan of frozen/thawed bovine spermatozoa, maintaining their fertilization ability for at least 8 h. Accordingly, we hypothesized that capacitated stallion spermatozoa incubated under in vitro conditions for capacitation acquire PTP in their flagella, rapidly losing their viability. This means that physiological hyperactivation is prevented or defective, at least for a short time, and as a consequence, fertilizing ability is lost. This effect could be even more pronounced in some horses because of the initial low quality of their

semen [2]. To test this hypothesis, herein we examined the effect of supplementing the capacitating medium with PEN to prolong the lifespan of capacitated equine spermatozoa.

Recently, we proposed that capacitated spermatozoa could be selected from the whole pool of spermatozoa by an in vitro thermotaxis assay [22]. Sperm thermotaxis, defined as the ability of spermatozoa to navigate within a temperature gradient towards the warmer temperature, seems to be exclusive to capacitated spermatozoa [23,24]. In recent work, we found that the DNA of human and mouse spermatozoa selected by thermotaxis in vitro is of very high integrity when compared to the DNA of unselected spermatozoa [22]. Further, the use of these selected spermatozoa significantly increased successful ICSI outcomes in mice. For selection by thermotaxis, capacitated spermatozoa need to preserve their motility during migration within the temperature gradient. This study aimed to evaluate if PEN could prolong the lifespan of frozen/thawed sperm in different capacitating media, thus allowing spermatozoa migration by thermotaxis. Also, this selection could serve to obtain a sperm fraction of high genetic quality for its use in both IVF and ICSI.

The objectives of this study were: (i) to assess the effect of penicillamine supplementation on capacitation-related events during incubation under capacitating conditions in frozen/thawed stallion spermatozoa, (ii) to examine the capacity of these spermatozoa to migrate by thermotaxis, and (iii) to determine DNA integrity and tyrosine phosphorylation in the spermatozoa selected by thermotaxis.

#### **2. Materials and Methods**

#### *2.1. Reagents*

All reagents were purchased from Sigma–Aldrich (Saint Louis, MO, USA) unless specified otherwise.

#### *2.2. Experimental Design*

In an initial experiment, we examined the effect of incubating frozen/thawed stallion spermatozoa processed by density gradient centrifugation (DGC) in Whitten's medium (WHI) and synthetic human tubal fluid (HTF), two media commonly used for the capacitation of mammalian spermatozoa. Because in our preliminary experiments HTF induced more signs of capacitation (confirmed in the experiments shown here), we supplemented it with 750 μM of penicillamine (HTF + PEN), as this concentration has been shown to prolong sperm motility in the horse [20]. Over an incubation period of 180 min, we evaluated the sperm integrity and capacitation, analyzing the plasma membrane integrity, acrosomal exocytosis, protein tyrosine phosphorylation, and motility (total motility and motion kinetics). The percentage of motile spermatozoa was determined after DGC (time 0) and at 30 and 180 min of incubation. Plasma membrane integrity, acrosomal exocytosis, and protein tyrosine phosphorylation were analyzed at time 0 and after 180 min of incubation.

After 30 min of incubation, HTF and HTF + PEN induced higher capacitation levels than WHI. Therefore, in the second experiment, spermatozoa were selected by thermotaxis after 30 min of incubation employing an in vitro system previously used in mouse and human sperm [22]. This selection was conducted for 60 min using a gradient from 35 to 38 ◦C (see the section below for details). Next, we determined the percentage of migration by thermotaxis. As migration by thermotaxis was only achieved using HTF + PEN, we analyzed in these samples the PTP of the migrating spermatozoa, non-migrating spermatozoa (those that did not migrate in the in vitro system), and unselected spermatozoa (aliquot incubated in parallel for 90 min at 37 ◦C in 5% CO2). In addition, DNA fragmentation was examined in the migrating and unselected spermatozoa.

#### *2.3. Semen Collection and Cryopreservation*

Semen was collected from six fertile purebred Lusitano stallions aged 3 to 13 years housed at the Centro de Selección y Reproducción Animal (CENSYRA) using an artificial vagina (Hannover model, Minitüb, Landshut, Germany). All experimental procedures were performed according to institutional and European regulations. A nylon in-line filter (Animal Reproduction Systems, Chino, CA, USA) was

used to eliminate the gel fraction. The sperm-rich fraction was diluted 1:2 (*v:v*) in INRA96 medium (IMV, L'Aigle, France) and subsequently processed for cryopreservation. Diluted ejaculates were centrifuged for 10 min at 900× *g* and the supernatant discarded. The sperm pellet was re-suspended in an egg yolk-based freezing extender (Gent, Minitube Ibérica, Tarragona, Spain) to obtain a final concentration of 200 <sup>×</sup> 106 sperm/mL, loaded into straws (0.5 mL) and sealed using sealing balls. Subsequently, the straws were equilibrated for 20 min at 4 ◦C and frozen by exposure to liquid nitrogen vapor at 4 cm above the liquid nitrogen level for 20 min. At the end of the cryopreservation process, the straws were submerged into liquid nitrogen at −196 ◦C where they were stored until analysis.

#### *2.4. Sperm Sample Preparation and Incubation*

Cryopreserved samples were thawed at 37 ◦C for 45 s in a water bath and processed by DGC. The contents of two straws were recovered into a microtube and transferred to a 15 mL centrifuge tube on top of 500 <sup>μ</sup>L of equipure TM (Nidacon, Mölndal, Sweden) and centrifuged for 20 min at 400<sup>×</sup> *g*. Next, the supernatant was discarded and each pellet was resuspended in one of the following media: (i) WHI (100 mM NaCl, 4.7 mM KCl, 4.8 mM L-lactic acid hemicalcium salt, 1.2 mM MgCl2 × 6H2O, 5.5 mM glucose, 22 mM HEPES, and 1.0 mM pyruvic acid), (ii) HTF (2.04 mM CaCl2 × 2H2O, 101.6 mM NaCl, 4.69 mM KCl, 0.37 mM KH2PO4, 0.2 mM MgSO4 × 7H2O, 21.4 mM sodium lactate, 0.33 mM sodium pyruvate, and 2.78 mM glucose), or (iii) HTF supplemented with 750 μM of penicillamine (HTF + PEN). All media were supplemented with 25 mM NaHCO3, 4 mg/mL of bovine serum albumin (BSA), 100 U/mL penicillin, 50 μg/mL streptomycin SO4, and 0.001% (*w*/*v*) phenol red (pH = 7.4 and 280–300 mOsm/kg). Before their use, the media were preincubated overnight at 37 ◦C in a 5% CO2 humidified atmosphere. The sperm concentration was adjusted to 20 <sup>×</sup> 106 spermatozoa/mL and samples were incubated for 3 h at 37 ◦C in a 5% CO2 humidifying atmosphere for capacitation. During the 3 h of incubation, pH was monitored and confirmed stable at 7.4.

#### *2.5. Sperm Thermotaxis*

Sperm thermotaxis was conducted as described elsewhere [22]. Briefly, our thermotaxis selection assay is based on recovering the spermatozoa who have migrated through a capillary between two drops of the same medium (in this experiment, HTF or HTF + PEN). For selection under thermotactic conditions, a 3 ◦C temperature gradient was set up between both drops from 35 to 38 ◦C. Between 5 and 6 <sup>×</sup> 10<sup>6</sup> spermatozoa were loaded into the 35 ◦C drop and allowed to migrate for 1 h. After this time, migrated spermatozoa were recovered from the 38 ◦C drops and processed for tyrosine phosphorylation or DNA fragmentation analysis. As controls for random migration, two drops were placed in parallel to the thermotactic assay at the same temperature (35 to 35 ◦C and 38 to 38 ◦C, non-gradient controls). The percentage of net thermotaxis was calculated as follows: 100 × (number of spermatozoa migrating within the temperature gradient (35 to 38 ◦C) minus number of spermatozoa migrating within the temperature non-gradient (35 or 38 ◦C, selecting the temperature which resulted in higher random migration)/number of spermatozoa loaded].

#### *2.6. Plasma Membrane Integrity*

We employed propidium iodide (PI) to stain spermatozoa with damaged membranes. Sperm plasma membrane integrity was assessed using propidium iodide (PI) to stain spermatozoa with damaged membrane [25] and the fluorochrome Hoechst 33342 to stain the nuclei. Semen samples were diluted into PBS at a concentration of 2 <sup>×</sup> 106 spermatozoa/mL, then PI and Hoechst 33342 were added to a final concentration of 10 and 15 μM, respectively. After 5 min, the stained samples were analyzed by flow cytometry in a FASCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). Spermatozoa were gathered in the forward scatter and side scatter (FSC/SSC) dot plot to exclude debris and confirmed with the violet laser (405 nm) and the blue filter (450/50 nm) to detect nuclear staining with Hoechst 33342. A total of 1 <sup>×</sup> 104 spermatozoa were acquired per determination. For PI, the blue laser (488 nm) and the orange filter (585/42 nm) were used. Acquired data were analyzed

using FlowJo software (Becton–Dickinson, Franklin Lakes, NJ, USA) to determine the percentage of PI-stained spermatozoa per each sample.

#### *2.7. Acrosomal Exocytosis*

The method employed was based on acrosome staining using *Arachis hypogaea* (peanut) lectin conjugated with fluorescein isothiocyanate (PNA–FITC) following a standard protocol described previously [26], with minor modifications. Briefly, the spermatozoa were washed twice in fhosphate-buffered saline (PBS) by centrifugation (1 min at 500× *g*) and subsequently smeared on a microscope glass slide and air-dried on a heat plate at 37 ◦C. Next, the slides were immersed in absolute methanol for 30 s, air-dried, and rinsed in PBS twice for 5 min before incubation with PNA–FITC and Hoechst 33342 (15 μg/mL and 0.0065 mg/mL respectively, in H2O) in a wet-mount box/humidified box for 30 min at room temperature. Finally, the slides were washed with distilled water for 15 min and mounted with Fluoromount TM aqueous mounting medium. Slides were examined in a fluorescence microscope (Nikon Eclipse i50, Nikon, Tokyo, Japan) and numbers of acrosome-reacted and non-acrosome-reacted spermatozoa were counted by randomly moving across different fields of the slide (counting 200 cells per slide, 2 slides per sample).

#### *2.8. Motility and Kinetics*

Ten microliters of sperm suspension was placed in a Mackler chamber on the stage heated to 37 ◦C of a Nikon Eclipse E400 (Nikon, Tokyo, Japan) fitted with a digital camera, Basler acA1300-200uc (Basler AG, Ahrensburg, Germany). Three to five movies of 1.5 s were recorded at 60 frames/s using the software Pylon Viewer provided by Basler, capturing at least 100 moving spermatozoa. The motility and sperm kinetics were analyzed using the free software ImageJ 1.x [27] with the plugin CASA\_bmg following instructions for analyzing stallion spermatozoa [28]. The parameters analyzed were as described by Mortimer et al. [29]: straight-line velocity (VSL; μm/s), curvilinear velocity (VCL; μm/s), average path velocity (VAP; μm/s), linearity (LIN) (defined as (VSL/VCL) × 100), straightness (STR) (defined as (VSL/ VAP) × 100), wobble (WOB) (defined as (VAP/VCL) × 100), amplitude of lateral head (ALH) displacement (μm), and beat-cross frequency (BCF; Hz). Also, we examined the percentage of spermatozoa showing more signs of hyperactivation (HYP) by determining out of all the analyzed spermatozoa (9340) the lower VCL and ALH values of the 10% of spermatozoa with the highest VCL and ALH. These values were: VCL = 150 μm/s and ALH = 5.5 μm. Thus, we defined spermatozoa showing hyperactive-like motility as those showing VCL > 150 μm/s and ALH > 5.5 μm (following the definition used in Su et al. [30]).

#### *2.9. DNA Fragmentation*

DNA fragmentation was analyzed employing the neutral version of the single cell gel electrophoresis assay (SCGE or Comet assay), as described previously [22]. Briefly, the samples were pelleted by centrifugation (600<sup>×</sup> *g*) and diluted to a maximum of 20 <sup>×</sup> 104 spermatozoa/mL in 0.5% low melting point agarose in PBS. Because of the low numbers obtained in the thermotaxis assay, the samples of migrated spermatozoa were used entirely. Immediately after dilution, 85 μL were placed on a slide previously coated with 1% agarose and covered with a 22 × 22 mm coverslip. The slides were then left in a wet-mount box/humidified box at 4 ◦C for 1 h for agarose polymerization. After removing the coverslips, slides were incubated at 37 ◦C for 1 h in lysis solution (2 M NaCl, 55 mM EDTA-Na2, 8 mM Tris, 4% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM ditiotreitol (DTT), and 0.5 mg/mL of proteinase K, pH 8). Next, the slides were washed twice in neutral electrophoresis solution (90 mM Tris, 90 mM boric acid, and 2 mM EDTA, pH 8.5) and subjected to electrophoresis (25 V for 10 min). The slides were then washed with distilled water, fixed in methanol for 3 min, air-dried, and stored upon microscope examination. The samples were stained with 50 μL of 0.02 mg/mL ethidium bromide, covered with a 22 × 22 mm coverslip, and immediately observed in a fluorescence microscope Nikon Optiphot-2 (Nikon, Tokyo, Japan). Comets were digitalized with a Nikon 5100 digital

camera (Nikon, Tokyo, Japan) coupled to the microscope. At least 150 comets were analyzed using the free software Casplab 1.2.3beta2 (CaspLab.com) [31].

#### *2.10. Protein Tyrosine Phosphorylation*

Protein tyrosine phosphorylation was analyzed by immunofluorescence. Spermatozoa were diluted in 500 <sup>μ</sup>L of PBS to a concentration of 4 <sup>×</sup> 106 spermatozoa/mL. Due to the low numbers obtained in the thermotaxis assay, the samples of migrated spermatozoa were used entirely and undiluted. Samples were centrifuged (600× *g* for 5 min) and the resultant pellet was fixed in 2% paraformaldehyde in PBS for 10 min and stored at –20 ◦C for, at most, one week, until continuing with immunodetection. After defrosting at room temperature, the fixed samples were washed 3 times in PBS by centrifugation (600× *g* for 5 min) and the pellet was resuspended in 50 μL of PBS. Two drops of 25 μL were each smeared on a glass microscope slide and left to dry. Subsequently, slides were washed three times with PBS and 100 μL of PBS with 0.2% of Triton-X were placed on each slide and covered with a 20 × 60 mm coverslip, placed in a wet box, and incubated at 37 ◦C for 15 min. Then, slides were washed once in PBS and incubated in a wet box with 100 μL of PBS and 1% BSA (again using a coverslip) for 1 h at 37 ◦C. Subsequently, slides were drained, and 100 μL of the primary antibody (phosphor-tyrosine monoclonal antibody (pY20), reference 14-5001-82, ThermoFisher Scientific, Waltham, MA, USA) diluted 1:100 in PBS was added to each slide, covered with a coverslip, and incubated overnight at 4 ◦C. On the next day, slides were washed three times in PBS and the secondary antibody (goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488, reference A-11029, ThermoFisher Scientific, Waltham, MA, USA) was added (100 μL of a 1:100 dilution in PBS and covered with a coverslip) and incubated for 1 h in a wet box at 37 ◦C. Slides were then washed three times in PBS and nuclei-counterstained with 100 μL of 15 μM Hoechst 33342 by incubating for 5 min in a wet box. After an additional wash in PBS, the slides were mounted with Fluoromount TM aqueous mounting medium and examined in a fluorescence microscope (Nikon Eclipse i50, Nikon, Tokyo, Japan). Numbers of tyrosine-phosphorylated spermatozoa were counted by randomly moving across different fields of the slide (counting 200 cells per slide, 2 slides per sample).

#### *2.11. Statistical Analysis*

Statistical analysis was carried out using the software package GraphPad Prism 8.0.2 for Windows (GraphPad Software, San Diego, CA, USA). Results are expressed as means ± standard error of the mean (SEM). Means were compared and analyzed using a one-tailed paired-sample Student's *t*-test or repeated measures one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. Significance was set at *p* < 0.05.

#### **3. Results**

We employed frozen/thawed spermatozoa from 6 stallions that were prepared by density gradient centrifugation and incubated for 180 min in WHI, HTF, or HTF + PEN. To assess sample integrity, we determined percentage motility, plasma membrane integrity, and the occurrence of acrosomal exocytosis before and during incubation. The percentage of motile spermatozoa was similar between the three studied media at every time point during incubation, with a significant decrease detected in the initial 30 min (Figure 1A) (*p* < 0.002). From 30 to 180 min of incubation, motility slightly diminished, though not significantly (*p* > 0.38). Incubation with the three media for 180 min also provoked a significant reduction in the percentage of spermatozoa showing an intact plasma membrane (Figure 1B) and a significant increase in spermatozoa undergoing acrosomal exocytosis (Figure 1C) (*p* < 0.0001 and *p* < 0.0019, respectively).

**Figure 1.** Sperm integrity over a 180 min period of capacitation. (**A**) Percentage of motile spermatozoa. (**B**) Percentage of spermatozoa with an intact plasma membrane as confirmed by propidium iodide staining. (**C**) Percentage of spermatozoa showing acrosomal exocytosis as determined by *Arachis hypogaea* (peanut) lectin conjugated with fluorescein isothiocyanate (PNA-FITC) staining. Spermatozoa after density gradient centrifugation (T0) and incubation for 30 or 180 min under capacitating conditions in three media: Whitten's medium (WHI), synthetic human tubal fluid (HTF), and HTF supplemented with 750 μM of penicillamine (HTF + PEN). \*\*\* *p* < 0.0001, \*\* *p* < 0.0019; a,b different letters indicate significant differences (*p* < 0.05), (*n* = 6, 12 determinations).

#### *3.1. E*ff*ects of the Incubation Medium on Sperm Kinetics and Protein Tyrosine Phosphorylation*

To determine which of the media, WHI or HTF, could potentially induce more spermatozoa to acquire the capacitation state, we conducted a comparative analysis of sperm kinetics and protein tyrosine phosphorylation over the 180 min of incubation. At the onset of incubation, VCL and ALH of swimming spermatozoa were higher in the HTF medium than WHI (*p* = 0.045 and *p* = 0.04, respectively) (Table 1). Thus, in HTF, we detected a higher fraction of spermatozoa showing a motility classified as indicating hyperactivation (relatively high VCL (>150 μm/s) and ALH (>5.5 μm), hereafter referred to as hyperactive-like motility) (*p* = 0.02) (Figure 2). Moreover, sperm kinetics in WHI did not significantly vary during the 180 min of incubation, while in HTF, sperm gradually acquired a less progressive motility type (LIN and STR reduced after 30 min (*p* = 0.04 and *p* = 0.01, respectively), as well as a decrease in WOB and beat cross frequency detected after 180 min (*p* = 0.04 and *p* = 0.014). These motion changes produced during incubation in HTF were recorded as a significant increase in the percentage of spermatozoa showing hyperactive-like motility after 30 min of incubation (*p* = 0.045) (Figure 2). However, after 180 min, hyperactive-like motility returned to the levels observed at the start of incubation.


**Table 1.** Kinetics of stallion spermatozoa measured at time 0 (T0), and after 30 and 180 min of incubation at 37◦C in a 5% CO2 atmosphere in Whitten's medium

(ALH). a, b Different letters indicate significant differences between time points for each medium. Asterisks indicate significant differences between media for each time point (*<sup>p</sup>* < 0.05,repeated measures one-way analysis of variance (ANOVA)).

**Figure 2.** Percentage of spermatozoa showing hyperactive-like motility. Kinetics were examined at the time points 0, 30, and 180 min of incubation in Whitten's medium (WHI), synthetic human tubal fluid (HTF), or HTF supplemented with 750 μM of penicillamine (HTF + PEN). Spermatozoa showing hyperactive-like motility were defined as those showing VCL > 150 and ALH > 5.5. a, b, c, d Different letters indicate significant differences (*p* < 0.05, *n* = 6, 12 determinations).

Immunofluorescence PTP analyses revealed two staining patterns of the flagella: (i) staining showing PTP only in the midpiece (pattern I) and (ii) staining showing PTP along the whole flagellum (pattern II) (Figure 3A). The percentage of spermatozoa showing either staining pattern increased during incubation in the two media (Figure 3B,C). This increase was significantly higher for pattern I when the incubation medium was HTF rather than WHI (*p* = 0.027) (Figure 3B). No significant differences in pattern II emerged between HTF and WHI (*p* = 0.1) (Figure 3C).

**Figure 3.** Protein tyrosine phosphorylation in stallion spermatozoa after 180 min of incubation for capacitation. (**A**) Micrograph of fluorescence microscopy of stallion spermatozoa after 180 min of capacitation and immune-labelled for protein tyrosine phosphorylation (PTP) (green). Nuclei were labelled with Hoechst 33342 (blue), bar = 10 μm. Pattern I: spermatozoa showing PTP at the midpiece. Pattern II: Spermatozoa showing PTP along the whole flagellum. (**B**,**C**) Percentage of spermatozoa showing pattern I (B) and pattern II (**C**) staining at time 0 and after 180 min of incubation in Whitten's medium (WHI), synthetic human tubal fluid (HTF), or HTF supplemented with 750 μM of penicillamine (HTF + PEN). a, b, c Different letters indicate significant differences (*p* < 0.05, *n* = 6, 12 determinations).

#### *3.2. Effects of HTF Supplementation with Penicillamine on Sperm Kinetics and Protein Tyrosine Phosphorylation*

To examine the effect of PEN on capacitated spermatozoa, it was added to HTF at a final concentration of 750 μM [20] and sperm kinetics and PTP were analyzed over the 180 min of incubation. At the start of incubation, we detected no significant differences in kinetics for HTF versus HTF + PEN (Table 1). However, at this early stage, the percentage of spermatozoa showing hyperactive-like motility was higher for HTF (*p* = 0.01). Interestingly, after 30 min of incubation, kinetics and hyperactive-like

motility were similar in both media, but after 180 min, the spermatozoa incubated in HTF + PEN swam with significantly higher VCL (*p* = 0.03), VAP (*p* = 0.033), and ALH (*p* = 0.04), and lower STR (*p* = 0.02). Accordingly, the percentage of spermatozoa showing hyperactive-like motility after 180 min of incubation was higher in HTF + PEN (*p* = 0.004) (Figure 2). Our immunofluorescence analyses, nevertheless, revealed no differences in PTP for both staining patterns (Figure 3B,C). Compared to incubation in HTF alone, supplementation with PEN gave rise to a significantly higher percentage of spermatozoa showing PTP staining pattern II compared to WHI (*p* = 0.04) which, in turn, could indicate a slightly higher incidence of PTP related to incubation in HTF + PEN compared to HTF alone.

#### *3.3. Sperm Thermotaxis*

As thermotaxis is a capacitation-dependent process that requires spermatozoa to maintain their swimming capacity to migrate within a temperature gradient, we employed our in vitro thermotaxis assay to analyze the effect of PEN in prolonging the migration ability of capacitated spermatozoa. When incubated in HTF + PEN and not HTF alone, the number of migrated spermatozoa within the temperature gradient was significantly higher compared to those migrating in the absence of a gradient (constant temperature of 35 or 38 ◦C) (*p* < 0.002) (Figure 4A). These results confirm the occurrence of thermotaxis, the percentage of net thermotaxis being 1.1% ± 0.5% for HTF + PEN (percentage of spermatozoa migrated in vitro by thermotaxis referred to the loaded spermatozoa) and confirmed the protective effect of PEN supplementation. Further, as thermotaxis allows for the selection of a sperm subpopulation of high genetic integrity in humans and mice [22], we analyzed DNA fragmentation of the selected spermatozoa. Our results indicate significantly lower DNA fragmentation in selected spermatozoa compared to the unselected sample (aliquot incubated in parallel at 37 ◦C) (4.4% ± 0.4% and 11% ± 2% respectively, *p* = 0.009). These selected spermatozoa fractions also showed enrichment in populations with low DNA fragmentation (Figure 4B). Thus, the percentage of spermatozoa showing 0–5% DNA fragmentation (high DNA integrity) was significantly higher in the selected fraction (69% ± 4% vs 23% ± 4% respectively, *p* = 0.0002).

**Figure 4.** Sperm selection by thermotaxis. (**A**) The number of spermatozoa in vitro migrating from 35 to 38 ◦C or across a constant temperature (35 or 38 ◦C) for 60 min after 30 min of incubation in synthetic human tubal fluid (HTF) or HTF supplemented with 750 μM of penicillamine (HTF + PEN). The initial number of spermatozoa loaded in the thermotaxis system was between 5 and 6 <sup>×</sup> 106 per separation. (**B**) Histograms show the distributions of % fragmented DNA in individual spermatozoa unselected or selected by thermotaxis after incubation for 30 min in HTF + PEN. \*\*\* *p* = 0.009, *n* = 6.

To examine the relationship between PTP and the ability of the spermatozoa to migrate by thermotaxis, we also conducted PTP immunofluorescence analyses on selected and unselected spermatozoa. Our results revealed lower percentages of spermatozoa showing the PTP staining pattern I in the migrated spermatozoa (in all the thermotaxis and both non-gradient controls) compared to the unselected sample (*p* < 0.0002) (Figure 5A). In contrast, the percentage of spermatozoa showing PTP

staining pattern II was significantly higher in the spermatozoa migrating in the non-gradient control at 38 ◦C and in those spermatozoa selected by thermotaxis when compared to the unselected sample (*p* < 0.02) (Figure 5B,C). In the non-migrating spermatozoa (those remaining in the drop where they were first loaded in the thermotaxis system), percentages of PTP staining patterns I and II were similar for all the conditions analyzed and in the unselected sample (Figure 5D).

**Figure 5.** Protein tyrosine phosphorylation in the thermotaxis assay. (**A**,**B**) Percentage of spermatozoa showing staining patterns I or II for tyrosine phosphorylation in the unselected sample, in the spermatozoa migrating across constant temperatures (35 or 38 ◦C), or across a temperature gradient (from 35 to 38 ◦C, thermotaxis). (**C**) Representative micrograph of immunofluorescence for tyrosine phosphorylation (green) in stallion spermatozoa migrating by thermotaxis. Nuclei were labelled with Hoechst 33342 (blue). Bar = 10 μm. (**D**) Percentage of spermatozoa showing staining patterns I and II for protein tyrosine phosphorylation in the unselected sample or in those spermatozoa that did not migrate during the thermotaxis assay in the conditions described above. \*\*\* *p* = 0.001, a, b, c different letters indicate significant differences (*p* < 0.05, *n* = 6).

#### **4. Discussion**

Several hypotheses have been put forward to explain the unsuccessful in vitro capacitation of stallion spermatozoa [1]. The results of our study along with the literature findings detailed below suggest that the media employed should be rethought by optimizing concentrations of energy sources and adding supplements to modulate and prolong the lifespan of spermatozoa once capacitated. In our study, the HTF medium induced the greater occurrence of capacitation-related events during incubation when compared to WHI, presumably because of its higher lactate content. We also observed that supplementation with PEN prolonged the duration of hyperactive-like motility and this allowed the sperm migration by thermotaxis, suggesting a pro-survival effect on the capacitated sperm population. Interestingly, we also found that spermatozoa selected by thermotaxis showed relatively good DNA integrity, corroborating our previous results in the mouse and human [22] and opening the possibility of employing this method to improve ARTs. Another significant result that we also discuss here was the

lack of a relationship found between PTP in the whole flagellum and sperm migration by thermotaxis. This might indicate the existence of a physiological PTP-independent hyperactivation response.

#### *4.1. Capacitation-Related Events During Incubation in HTF*

Incubation in HTF led to a time-dependent effect on sperm kinetics whereby hyperactive-type motility was acquired by a fraction of the spermatozoa. This effect was not observed when spermatozoa were incubated in WHI, which is a medium commonly used for stallion sperm capacitation. Further, although both media significantly increased PTP levels after 180 min of incubation, higher levels were attained with HTF. This difference was not detected by Arroyo-salvo et al. [8], who conducted a similar comparative study. In contrast, they found no time-dependent changes in sperm kinetics compatible with hyperactivation over 120 min, and PTP induction levels were similar to both media after 120 and 240 min of incubation. However, Arroyo-salvo et al. [8] employed a fresh sample washed by centrifugation, while we used frozen/thawed samples washed by DGC, which could explain the differences between both studies. Cryopreservation provokes significant changes in the sperm plasma membrane, increasing membrane peroxidation and permeability that could trigger capacitation-related events [32,33]. Further, even if capacitation is not immediately triggered, freezing/thawing may leave the spermatozoa in a poised status, making them more susceptible to capacitation than fresh sperm, as confirmed elsewhere [34]. We also detected the significant occurrence of spontaneous acrosomal exocytosis after 180 min of incubation, not detected by others employing fresh stallion semen [3,8]. This also supports the higher susceptibility of frozen/thawed spermatozoa to the destabilizing changes occurring during capacitation, as has been also shown for bull spermatozoa [35].

Differences in composition between WHI and HTF could explain the observed differences in both sperm kinetics and PTP. Both media differ in the amount of glucose and pyruvate they contain, and these are ~2 and 3 times less concentrated in HTF, respectively. However, HTF contains ~4.5 times more lactate than WHI (21.4 and 4.8 mM, respectively), which can be directly transformed to pyruvate in the sperm mitochondria by the Krebs cycle [36]. In effect, lactate and pyruvate are the main sources of energy utilized by stallion spermatozoa, and glucose may even reduce mitochondrial function [37]. Thus, the higher concentration of an energy source that can be rapidly and effectively utilized by the mitochondria could increase their activity [37], enhancing ROS production which will subsequently trigger PTP and its associated hyperactive-like motility [9]. Hence, a greater mitochondrial activity could explain the higher VCL and hyperactive-like motility observed from the onset of incubation and the higher PTP levels reported here after 180 min when using HTF rather than WHI. This could also explain the effect observed in enhancing kinetics when incubating stallion spermatozoa with follicular fluid from pre-ovulatory follicles [38]. As examined in buffalo, bull, sheep, rat, and mouse, this fluid also contains higher concentrations of lactate than glucose + pyruvate, ranging from ~7 to 27 mM depending on the species [39–41]. In humans, similar levels of glucose and lactate are reported, of around 3 mM [42]. Recently González-Fernández et al. [43] reported that in the mare's pre- and post-ovulatory oviductal fluids, concentrations of lactate were 54.66 ± 10.7 and 69.25 ± 7.3 mM, while concentrations of glucose were 0.18 ± 0.04 and 0.57 ± 0.2 mM, respectively. It is also important to point out that lactate is the most abundant source of energy within the oviduct [43,44] where capacitation, and thus hyperactivation, is triggered in vivo [45].

#### *4.2. E*ff*ect of Penicillamine on Capacitation-Related Events During Incubation with HTF*

Under our capacitating conditions, PEN was not able to rescue the time-dependent loss of motility and plasma membrane integrity. This contrasts with the results reported by Aitken et al. [20], where PEN prolonged the motility of fresh sperm incubated in BWW medium without BSA. The pro-survival effect of PEN on spermatozoa has been attributed to its ability to inactivate and slow down the production of cytotoxic aldehydes by lipid peroxidation provoked by ROS produced by the mitochondria [20]. As capacitation enhances mitochondrial function, ROS production is increased and modulates intracellular signaling for sperm capacitation, stimulating adenylyl cyclase and inhibiting tyrosine

phosphatases, causing a downstream increase in PTP [9,46]. However, when oxidative stress exceeds a certain limit, the spermatozoa undergo an apoptotic-like process [18]. Thus, in our experiment, the sperm fraction that abruptly lost motility within the first 30 min of incubation and lost membrane integrity after 180 min of incubation, could have exceeded ROS production, overwhelming the protective effect of PEN. Another option is that a different deleterious process associated with sperm capacitation may have compromised sperm lifespan in a fraction of our samples. The significant changes observed in the architecture of the plasma membrane produced during capacitation, such as cholesterol removal, glycoprotein redistribution, and loss of phospholipid asymmetry [47], affects the lifespan of the spermatozoa, making them more vulnerable to damage. Thus, the plasma membrane of capacitated spermatozoa becomes more permeable to vital stains such as propidium iodide [48] and/or, as occurs with the acrosomal membrane, becomes more prone to destabilization [49]. This was likely more pronounced in our samples as we employed frozen/thawed spermatozoa, which are known to be more sensitive to the destabilizing conditions of capacitation, as the abundance of spermatozoa sustaining sublethal damage could be high. In agreement, Pommer et al. [34] showed that, in contrast to fresh sperm, frozen/thawed stallion spermatozoa incubated under capacitating conditions lost motility and membrane integrity within an hour of incubation.

We found that, unlike the case of HTF alone, supplementation with PEN led to a sustained fraction of motile spermatozoa (between 16% ± 5% and 22% ± 3%) with relatively high VCL and ALH, indicating hyperactive-like motility from 30 to 180 min of incubation. Using this medium, after 180 min, PTP along the whole flagellum reached 6% ± 1% of the total spermatozoa, from close to 0 at the onset of incubation. Assuming that only motile spermatozoa (20% ± 2% after 30 min of incubation with HTF + PEN) will acquire the capacity for PTP during incubation, then the percentage of PTP on the whole flagellum within the motile population may represent some 30%. Thus, we hypothesize that the spermatozoa that showed hyperactive-like motility could be those undergoing PTP in the whole flagellum [10,11], as each indicator was present in similar percentages of spermatozoa. No differences in PTP were observed when we compared the use of HTF + PEN to HTF alone, indicating that PEN did not induce more spermatozoa to enter a capacitated-like state, but protected those that did and also lengthened the duration of this acquired hyperactive-like motility. As commented above, physiological ROS are needed to induce hyperactivation in human sperm [50,51], but as a consequence, oxidative stress generates cytotoxic aldehydes, damaging the cell [20]. The first structure injured by this oxidative stress is the mitochondrial membrane, thus motility is the first function affected [52,53]. Accordingly, the protective effect of PEN in this setting has been shown to enhance the velocity of the motile sperm fraction in horse, rat, and human [20]. In our experiment, we also found that after 180 min of incubation in HTF + PEN, spermatozoa showed significantly higher VCL than in HTF alone. Thus, our results suggest that PEN was able to maintain the observed hyperactive-like motility, possibly prolonging the lifespan of the capacitated spermatozoa fraction. Our theory is in line with the results reported by Pavlok [21], in which PEN prolonged the fertilizing ability of frozen/thawed bovine spermatozoa.

#### *4.3. Penicillamine Enables Sperm Selection by Thermotaxis*

To carry out thermotaxis, spermatozoa must be motile and capacitated so that they can move across the temperature gradient [23,24]. Thus, for the thermotaxis experiments, spermatozoa were capacitated for 30 min in HTF or HTF + PEN, as at this time point, we had observed hyperactive-like motility with both media. However, only when incubated in HTF + PEN were spermatozoa able to migrate by thermotaxis. This observation supports the protective effect of PEN on the fraction of capacitated spermatozoa. Thus, in addition to maintaining the specific sperm kinetics needed for migration, PEN could be protecting intracellular signaling involved in the thermotactic response itself. This signaling is mediated by the phosphodiesterase and phospholipase C pathways whose thermosensors are thought to be opsins [54,55] as well as transient receptor potential cation channel subfamily V member 1 (TRPV1) [56].

In the HTF + PEN medium, thermotaxis selection yielded a net thermotaxis of 1.1% ± 0.5%, similar to the response reported in humans and mice using the same protocol [22] or employing other devices [54]. Our DNA damage assessment revealed that the fraction migrating by thermotaxis was significantly enriched in spermatozoa bearing high DNA integrity, as reported for human and mouse sperm [22]. As suggested for these two species and now also for horses, thermotaxis might be a bi-functional mechanism for the navigation and selection of high-quality capacitated spermatozoa in mammals.

Both the fraction of spermatozoa selected by thermotaxis and the fraction of spermatozoa showing random movement at a constant temperature of 38 ◦C showed similar strong enrichment in spermatozoa with PTP along the whole flagellum (37% ± 8% and 24% ± 8%, respectively). These percentages are similar to the percentage of PTP in the whole flagellum estimated above for the motile fraction (~30%), assuming that only motile spermatozoa can trigger PTP in the whole flagellum. This suggests that thermotaxis selects a fraction within the motile spermatozoa independently of the PTP status of the flagellum and argues against the involvement of PTP-dependent hyperactivation in the behavioral thermotaxis response of spermatozoa. Further, the lower levels of PTP detected in the sample of spermatozoa migrating in the non-gradient control at 35 ◦C indicates a direct PTP-inducing effect of temperature within the motile and migrating sperm fraction. This direct relationship between absolute temperature and PTP is a well-known phenomenon [57] and could indicate that PTP in the thermotactic fraction occurs during spermatozoa migration or once they have migrated. Boryshpolets et al. [57] reported that changes in the direction of swimming during the thermotactic response of human spermatozoa occur as turns that may be subtle or generated by episodes of hyperactivation. Thus, the model proposed by Boryshpolets et al. [58] for the thermotactic behavior response implies that hyperactive-like motility is transient and more frequent at lower temperatures. This contrasts with the longstanding nature of hyperactivation related to flagellum PTP and explains why in our experiment there was no significant PTP enrichment in the spermatozoa migrated by thermotaxis compared to those moving across the non-gradient control at 38 ◦C. We, therefore, propose the hyperactive-like motility involved in thermotaxis is PTP-independent and possibly directly linked to opsin and TRPV1 signaling for a rapid transient response. As support for this theory of PTP-independent hyperactive motility, procaine and caffeine have been shown to induce hyperactive-like motility in stallion and ram spermatozoa independently of PTP, respectively [59,60]. Further work is needed to elucidate the full transduction signaling pathway coupled to the sperm temperature sensing machinery along with the behavior changes involving the transient acquisition of hyperactive-like motility.

#### **5. Conclusions**

In this study, we observed a protective effect of penicillamine used for the in vitro capacitation of stallion spermatozoa in prolonging the duration of hyperactive-like motility of a fraction of the sperm sample and in allowing sperm migration by thermotaxis, a process that is capacitation-dependent. In addition, we report here that thermotaxis selects a sperm fraction enriched in PTP also showing high DNA integrity, thus supporting its potential use for sperm preparation before assisted reproductive techniques in the horse. The results reported here also point to a relevant role of lactate in the capacitation of stallion spermatozoa and also identify no relationship between protein tyrosine phosphorylation in the sperm flagellum and migration by thermotaxis.

**Author Contributions:** S.P.-C., I.O.-L., and S.R.-D. conceived the study, conducted the experiments, and wrote the paper; D.D.L.C.-D., C.S., and B.G.-L. conducted the experiments; M.J.S.-C. and A.G.-A. conceived the study and wrote the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is supported by Grants RTI2018-096736-A-I00 and RTI2018-093548-B-I00 from the Spanish Ministry of Science, Innovation, and Universities. I.O.L. and S.R.D. are supported by CONACYT fellowship of the Mexican government (283833) and "Doctorados Industriales 2018" fellowship of Comunidad de Madrid (IND2018/BIO-9610), respectively. S.P.C. is supported by a Ramón y Cajal contract from the Spanish Ministry of Science, Innovation, and Universities (RYC-2016-20147).

**Acknowledgments:** We thank L. González-Fernández and B. Macías-García from the University of Extremadura for providing the cryopreserved semen samples. This research is especially dedicated to the memory of Serafín Pérez-Cerezales, an invaluable person, mentor, and friend.

**Conflicts of Interest:** The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen–Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status**

**Brenda Florencia Gimeno 1,†, María Victoria Bariani 1,†, Lucía Laiz-Quiroga 1, Eduardo Martínez-León 2, Micaela Von-Meyeren 1, Osvaldo Rey 2, Adrián Ángel Mutto 1,\* and Claudia Elena Osycka-Salut 1,\***


**Simple Summary:** The use of assisted reproductive techniques, which involve the manipulation of sperm and oocytes in the laboratory, support owner production of valuable animals' offspring. However, several limitations remain underlining the need to further optimize existing protocols as well as to develop new strategies. For example, the required conditions to make equine spermatozoa competent to fertilize an oocyte in vitro (IVF) have not been established. Therefore, our initial goal was to optimize different conditions associated with frozen equine sperm manipulations in order to improve their quality. We observed that simple factors such as sample concentration, incubation period and centrifugation time affect the sperm motility. Since in vivo fertilization involves the interaction between spermatozoa and epithelial cells in the mare's oviductal tract, our next goal was to mimic this environment by establishing primary cultures of oviductal cells. Using this in vitro system, we were able to select a sperm population capable of fertilization. In short, this study provides a novel protocol that improves the yield of fertilization-capable sperm obtained from equine frozen spermatozoa.

**Abstract:** Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 <sup>×</sup> 106/mL compared to lower concentrations (*<sup>p</sup>* < 0.05) and when a short centrifugation at 200× *g* was performed (*p* < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we established an in vitro OEC culture model to select a sperm population with potential fertilizing capacity in this species. We demonstrated E-cadherin and cytokeratin expression in cultures of OECs obtained. When sperm–OEC cocultures were performed, the attached spermatozoa were motile and presented an intact acrosome, suggesting a selection by the oviductal model. When co-cultures were incubated in capacitating conditions a greater number of alive (*p* < 0.05), capacitated (*p* < 0.05), with progressive motility (*p* < 0.05) and with the intact acrosome sperm population was observed (*p* < 0.05) suggesting that the sperm population released from OECs in vitro presents potential fertilizing capacity. Improvements in handling and selection of cryopreserved sperm would improve efficiencies in ARTs allowing the use of a population of higherquality sperm.

**Citation:** Gimeno, B.F.; Bariani, M.V.; Laiz-Quiroga, L.; Martínez-León, E.; Von-Meyeren, M.; Rey, O.; Mutto, A.Á.; Osycka-Salut, C.E. Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen–Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status. *Animals* **2021**, *11*, 74. https://doi.org/ 10.3390/ani11010074


Received: 25 November 2020 Accepted: 28 December 2020 Published: 3 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** cryopreserved sperm; sperm–oviduct interaction; sperm selection; ARTs; equines

#### **1. Introduction**

The equine industry is a very strong, economically diverse and productive business worth US\$300 billion worldwide [1]. Moreover, horses represent an enormous value as sports and companion animals as well as a valuable model for the study of several human pathologies [2–5]. Nevertheless, the low success rate of assisted reproduction techniques (ARTs) available for in vitro equine embryo production is insufficient to satisfy the current needs.

The development of protocols for gametes and embryo cryopreservation has facilitated the implementation of ARTs. For example, cryopreserved sperm are widely employed in domestic animal production since cryopreservation facilitates the transport and storage of the samples for later use in different reproductive biotechnologies [6]. Nevertheless, cryopreservation procedures can negatively affect spermatozoa quality, causing changes at the structural and molecular levels, compromising sperm function [7]. Membranes are thought to be the primary site of cryopreservation injury, an injury associated with an increase in the intracellular concentration of reactive oxygen species (ROS) [8,9]. Generally, the key for cryopreservation of stallion semen is the individual stallion itself [10,11]. However, success in cryopreserving has been variable [10,12,13] as revealed by limited pregnancy rates [10] very likely due to differences in sperm freezing efficiencies [11]. For example, 20% of stallions produce semen that freezes well (high cryosurvival rates and post-thaw sperm progressive motility and total motility figures of 40–60% and >70%), 60% freezes acceptably (post-thaw sperm progressive motilities figures of 25–35%) and 20% freezes poorly (low tolerance to cryopreservation and post-thaw sperm progressive motilities as low as 10–15%) [11,14]. Therefore, the optimization of semen cryopreservation is critical for a successful ART.

The conditions to induce in vitro capacitation of stallion spermatozoa have not yet been described, a limitation that specially affects the efforts to perform equine in vitro fertilization (IVF), a widely used ART [15,16]. Thus, intracytoplasmic sperm injection (ICSI) is a technique widely employed within the equine breeding industry despite its modest yield [17,18]. This technique employs mature oocytes and fresh, cryopreserved, and/or low-quality stallion semen selected by sperm motility and morphology [19]. On the contrary, high fertilization rates in equines are obtained via artificial insemination (AI) [14,20], very likely due to the promoting effects of the oviductal environment upon sperm capacitation [21]. Therefore, the use of an in vitro system that mimics the in vivo conditions should significantly improve spermatozoa selection, fertilization and embryonic development employing ARTs protocols [22].

The oviductal epithelial cells (OECs), which form the oviductal sperm reservoir [23], are specialized cells associated with the selection of sperm suitable for oocyte fertilization [24]. The sperm–OEC interaction is highly specific [25–27] and takes place between the sperm plasma membrane at the acrosome region and the ciliated cells of the oviductal epithelium [28]. Several studies showed that spermatozoa attached to the oviductal cells have an intact acrosome and chromatin [29,30] and are morphologically normal [31] and motile [31,32]. Furthermore, OECs preferentially bind non-capacitated sperm [33–35] indicating that in vivo sperm capacitation is associated with its release from OECs [23,26,36,37] by a mechanism that involves sperm plasma membrane remodeling and molecular processes including an increase in cyclic adenosine monophosphate (cAMP) levels, increase in calcium influx, changes in protein phosphorylation and protein kinases activity, and an increment in the production of reactive oxygen species, such as nitric oxide [38–40]. These modifications lead to hyperactivated motility and prepare the spermatozoa to undergo the acrosome reaction to fertilize the oocyte [41,42].

Considering the high demand for in vitro developed equine embryos, first, we aimed to evaluate the effect of different conditions associated with the manipulation of stallion cryopreserved sperm. Furthermore, the second aim of this work was to establish an in vitro OEC primary culture to select a sperm population from cryopreserved samples with potential fertilizing capacity.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Fetal bovine serum (FBS), gentamicin and fungizone were purchased from GIBCO (Thermo Fisher Scientific, Waltham, MA, USA). Dulbecco's Modified Eagle Mediumhigh glucose (DMEM-HG) medium, *Pisum sativum* agglutinin-FITC staining (PSA-FITC), Hoechst 33258 (viability studies), Hoechst, L-glutamine, penicillin–streptomycin and bovine serum albumin (BSA; V fraction) were obtained from Sigma Chemicals (St. Louis, MI, USA). Glass wool columns for sperm selection were acquired from MicroFiber Manville. Salts used to prepare sperm, Whitten's medium (WM), were purchased from MERK (Darmstadt, Germany). All PCR reagents were obtained from Biodynamics and Genbiotech (Buenos Aires, Argentina). Cytokeratin-7 antibody (catalogue # ab9021; lot # Gr3225265-2) and E-cadherin antibody (catalogue # ab76055; lot # Gr317373-14) were purchased from Abcam Inc. (Cambridge, MA, USA). Antibodies against protein kinase A (PKA) phosphorylated Ser/Thr containing-substrates (clone 100G7E catalogue #9624S, lot # 21) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-phosphorylated tyrosine antibody (clone 4G10 catalogue #05-321; lot # 3272262) was purchased from EMD Millipore (Burlington, MA, USA). Secondaries antibodies, Alexa 488-conjugated chicken anti-mouse IgGs and Alexa 568-conjugated goat anti-rabbit IgGs, were obtained from Invitrogen (Carlsbad, CA, USA). All the other chemicals were of analytical grade and obtained from standard sources.

#### *2.2. Culture Media*

DMEM-HG medium supplemented with 10% FBS, 0.25 mg/mL gentamicin, 1 μg/mL fungizone, 2 mM L-glutamine and 50 U/mL penicillin–streptomycin (complete DMEM-HG) was employed during oviduct handling and monolayer cultures establishment. Sperm handling and co-culture experiments were performed with non-capacitating modified Whitten's base medium (NCWM) (BSA and bicarbonate free: 100 mM NaCl; 4.7 mM KCl; 1.2 mM MgCl2 \* 6H2O, 22 mM HEPES acid-free; 4.8 mM L-lactic acid hemicalcium; 5.5 mM D-glucose; 1 mM pyruvate; pH = 7.4; Osm = 300 mOsm). Capacitating medium (CWM) was prepared by adding 25 mM NaHCO3 and 7 mg/mL BSA to the NCWM (pH = 7.4; Osm = 300 mOsm) [43,44].

#### *2.3. Cryopreserved Stallion Sperm Handling and Processing*

For each set of experiments, four cryopreserved semen straws (ejaculates) from four different stallions (200 × <sup>10</sup><sup>6</sup> spermatozoa/0.5 mL straw) were used. These samples were obtained from GeneTec by Ativet (Pilar, Buenos Aires, Argentina; website: www. genetec.com.ar) and Los Pingos del Taita (Rio Cuarto, Córdoba, Argentina; website: www. lospingosdeltaita.com). Straws were thawed in a water bath at 37 ◦C for 30 s. Spermatozoa were selected using glass wool columns and washed by centrifugation at 600× *g* for 5 min at room temperature (RT). Pellets were resuspended in 100 μL of NCWM and sperm concentration and motility were examined using a hemocytometer mounted on a brightfield microscope stage heated at 38.5 ◦C at 100× magnification (Nikon Instruments Inc., Tokyo, Japan).

2.3.1. Effect of Incubation Time, Sample Concentration and Centrifugation Time on Sperm Motility

After glass wool column selection, sperm were incubated in NCWM at two different concentrations, 12 × <sup>10</sup><sup>6</sup> sperm/mL and 30 × 106 sperm/mL, at different times (0, 30, 60 and 120 min) and sperm motility was measured by Computer-Assisted Sperm Analysis (CASA system: IVOS II™ Animal—Hamilton Thorne). These concentrations were chosen based on the CASA system optimal-concentration working range (10–50 × <sup>10</sup><sup>6</sup> sperm/mL). To study the effect of centrifugation time on sperm motility, sperm were incubated at <sup>30</sup> × <sup>10</sup><sup>6</sup> sperm/mL and centrifugated for 0 (control), 1 min or 2 min at 200× *<sup>g</sup>* at RT. We did centrifugations studies with 30 × <sup>10</sup><sup>6</sup> sperm/mL because we observed that sperm motility was not affected for short times. Motility was studied using the CASA system. Results are expressed as % of total motile sperm.

#### 2.3.2. Sperm Motility Assay

Sperm motility was analyzed with CASA-system (IVOS II™ Animal—Hamilton Thorne). Fourteen randomly selected microscopic fields were scanned at 60 Fr/s with ~45 sperm per field (*n* = at least 3 independent replicates). Moreover, sperm total motility was subjectively evaluated using a field microscope stage heated at 38.5 ◦C at 100× magnification.

#### *2.4. Oviducts Collection*

Mare oviducts were obtained from the Lamar S.A. slaughterhouse (Mercedes, Buenos Aires, Argentina). Oviducts were collected at the time of slaughter and transported to the laboratory at 4 ◦C in saline solution with 50 μg/mL of gentamycin.

#### 2.4.1. Oviductal Cell Collection and Cultures

The oviducts were cleaned of surrounding tissues, and the oviductal content was collected by flushing the oviducts with PBS and squeezing (applying pressure) the entire oviduct with tweezers within a laminar flow hood. The ampulla and isthmus OECs from 6 different animals were collected, pooled, resuspended in 10 mL of PBS and pelleted by centrifugation at 1500× *g* for 5 min at RT. The pelleted cells were resuspended in complete DMEM-HG, plated in 24-well tissue culture plates, and maintained at 38.5 ◦C in a 5% CO2 atmosphere. After 24 h, the explants were collected by micropipette-aspiration, subjected to the same clarification procedure, plated again and further incubated with complete DMEM-HG at 38.5 ◦C in a 5% CO2 atmosphere until the cultures reached confluence (10–13 days). The medium was changed every 48 h. The epithelial phenotype of the cultured cells was confirmed by immunocytochemical analysis and RT-PCR. To perform co-cultures with sperm, confluent cell monolayers were washed three times with NCWM medium and maintained in the same medium for 1 h before sperm addition.

#### 2.4.2. E-Cadherin and Cytokeratin-7 MRNA Expression in OEC Primary Cultures

The expression of mRNAs encoding for the epithelial markers, cytokeratin-7 (e-KRT7) and E-cadherin (e-CDH1), was examined by RT-PCR. Specifically, total RNA was isolated from OECs using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. Only samples with a 260 nm/280 nm ratio greater than 1.7 were used for further analysis. cDNA was synthesized from 1 μg of total mRNA by using SuperScriptIII enzyme (Invitrogen TM, Carlsbad, CA, USA) and random primers (Invitrogen TM, Carlsbad, CA, USA), according to the manufacturer's instructions in the presence of recombinant RNAase inhibitor (Invitrogen TM, Carlsbad, CA, USA). After first-strand synthesis, PCR was performed with the following oligonucleotide primers: e-KRT7 (cytokeratin-7): 5 -GTGGTGAATTCTTCTGGCGG-3 (sense), 5 - AATAGGCTTTGAGGACCCCC-3 (antisense); e-CDH1 (E-cadherin): 5 -TCACCACAGAC CCAGTAACC-3 (sense), 5 -CGTTCACATCCATCACGTCC-3 (antisense); equine GAPDH: 5 -CATCATCCCTGCTTCTACTGG-3 (sense), 5 -TCCACGACTGACACGTTAGG-3 (antisense). Amplifications were performed using Taq DNA polymerase enzyme (Invitrogen, Carlsbad, California, USA). PCR was performed as follows: 95 ◦C for 5 min (initial denaturation) and 35 cycles at 95 ◦C for 30 s, 56 ◦C for 30 s, 72 ◦C for 1 min and finally 72 ◦C for 10 min. Negative controls were performed without cDNA template. PCR products were

separated on a 2% (*w*/*v*) agarose gel, stained with ethidium bromide, and recorded under UV light with an Olympus C5060 digital camera (Olympus Corp., Japan).

#### 2.4.3. Cytokeratin and E-Cadherin Distribution in OEC Primary Cultures

OECs were fixed for 10 min at RT in 4% *w*/*v* paraformaldehyde and permeabilized with PBS-Tritón X-100 0.4%. Non-specific binding sites were blocked (60 min, PBS-1% gelatin IRA grade, Bio-Rad Laboratories, Hercules, CA, USA) and samples incubated with anti-cytokeratin antibody (1:50) or anti E-cadherin antibody (1:50) diluted in PBS-Tween 0.05% for 18 h at 4 ◦C. After three washes in PBS-Tween 0.05% at RT, the samples were incubated with anti-mouse antibody (1:1000) diluted in PBS-Tween 0.05%. After three washes with PBS-Tween 0.05% at RT, DNA was stained for 7 min with Hoechst 33352 (1 μg/mL). The specificity of the immunodetection was assessed by a) omitting the primary antibody and b) replacing the primary antibody with serum from non-immunized rabbits at the same concentration as the corresponding primary antibody (IgG control). Samples were examined with a Nikon Eclipse Ti-E microscope (Nikon Instruments Inc., Tokyo, Japan) and fluorescence images were captured with an Andor Neo 5.5 sCMOS camera (Oxford Instruments, Abingdon, United Kingdom) driven by NIS-Elements AR v 4.30.01 software (Nikon Instruments Inc., Tokyo, Japan). Not less than 20 fields per experiment were analyzed, and both markers were studied at least on three different pools of OECs (*n* = 3). Results are shown with one representative image.

#### *2.5. Co-Cultures of OECs and Spermatozoa*

To perform co-cultures with sperm, confluent OEC monolayers were washed three times with NCWM medium and maintained in the same medium for 1 h before sperm addition. OEC monolayers were co-cultured with sperm suspensions (7 × 106 sperm/mL of NCWM/well) for 60 min at 38.5 ◦C in a 5% CO2 atmosphere. Sperm concentration used was selected to recover enough numbers of sperm released from OECs to perform the different assays, where 7 × 106 sperm/mL was the optimal sperm concentration. When lower concentrations were used (<7 × <sup>10</sup><sup>6</sup> sperm/mL), we did not recover enough sperm to perform the assays, whereas higher concentrations (>7 × <sup>10</sup><sup>6</sup> sperm/mL) showed a similar number of sperm released and attached to the OECs to that obtained with <sup>7</sup> × <sup>10</sup><sup>6</sup> sperm/mL, likely due to the saturation of the sperm-binding sites on the OECs. The sperm's viability and motility were not affected by sperm concentration because the gametes were selected and immediately co-incubated with oviductal cells. Unbound spermatozoa were removed by washing the monolayers three times with NCWM. At this point, we evaluated the acrosome status and motility of sperm bound to OECs (Figure 1a).

#### Evaluation of Acrosome Status of Sperm Bound to OEC Primary Cultures

To evaluate the acrosome status of spermatozoa bound to OECs (Figure 1a), cocultures were washed and fixed in 0.4% *w*/*v* paraformaldehyde in PBS for 1 h at RT and permeabilized with methanol for 5 min at 4 ◦C. The fixed co-cultures were incubated with PSA-FITC (10 μg/mL) for 1 h at RT. After three washes with PBS, DNA was stained with Hoechst 33352 (1 μg/mL) for 10 min at RT. Samples were mounted with Fluor Save (Merck Millipore, Burlington, MA, USA) and examined with a fluorescence Nikon 80i microscope (Nikon Instruments Inc., Tokyo, Japan) coupled to a digital camera. Not less than 20 fields per experiment were analyzed and acrosome status was studied at least on three different sperm–OEC co-cultures (*n* = 3). Results are shown with one representative image.

#### *2.6. Retrieval of the Released and Bound Sperm Population after the Incubation of the Co-Cultures under Capacitating Conditions*

The OEC/sperm co-cultures were washed three times with NCWM to remove unbound sperm. Then, were incubated with CWM or NCWM (control) for 15 min and washed again three times with NCWM to recover released sperm (Figure 1b) and the population that remained bound to the OECs (Figure 1c). The following analyses were performed in the released sperm population retrieved: the number of sperm, viability, acrosome integrity, motility and capacitation status. Additionally, we evaluated the number of sperm bound to the OECs. A replicate (*n*) in these experiments was defined as the co-culture of an OEC monolayer co-cultured with sperm from one stallion (total 4 stallions). All the treatments (including the control) were performed for each replicate.

**Figure 1.** Oviductal epithelial cells and sperm co-culture experimental design. Confluent oviductal epithelial cell (OEC) monolayers were stabilized with non-capacitating modified Whitten's base medium (NCWM) 1 h before sperm addition. Scheme 7 <sup>×</sup> <sup>10</sup><sup>6</sup> sperm/mL of NCWM) were added to each well and were co-cultured with OECs for 60 min at 38.5 ◦C in a 5% CO2 atmosphere. Unbound spermatozoa were removed by washing the monolayers three times with NCWM. Acrosome status and motility were evaluated on (**a**) sperm that remained bound to OECs. Next, the sperm–OEC co-cultures were incubated with NCWM or capacitating medium (CWM) for 15 min. After that time, monolayers were washed three times with NCWM to analyze (**b**) the number of released sperm and (**c**) the number, viability, motility, acrosome integrity and capacitation status of the sperm that remained bound to the OECs after treatments. Created with BioRender.com.

2.6.1. Evaluation of the Number of Sperm Bound to the OECs after Capacitating Treatment

Co-cultures were fixed in glutaraldehyde 2.5% *v*/*v* for 60 min at RT and washed three times with PBS. The number of sperm that remained attached (Figure 1c) was determined by examining 20 fields under a phase-contrast microscope (Olympus, Tokyo, Japan). Results were expressed as the mean of the average number of bound spermatozoa in a 0.11 mm2 area per replicate.

2.6.2. Evaluation of the Number of Sperm Released from the OECs under Capacitating Conditions

The released sperm population recovered (Figure 1b) was fixed (0.2% *w*/*v* paraformaldehyde) for 30 min at RT and the number of sperm was determined using a hemocytometer (results are shown as the total number of released spermatozoa).

2.6.3. Evaluation of Viability of the Released Sperm Population from the OECs under Capacitating Conditions

To assess viability, the released sperm population recovered was incubated with Hoechst 33258 (2 μg/mL) for 5 min, fixed (1% *w*/*v* paraformaldehyde) for 8 min at RT, washed with PBS and aliquots were air-dried onto glass slides. Hoechst 33258 is a fluorescent DNA-binding supravital stain with limited membrane permeability [45–47]. At least 200 stained cells/treatment were scored in an epifluorescence Nikon 80i microscope (Nikon Instruments Inc., Tokyo, Japan). Results are shown as the percentage of live spermatozoa.

2.6.4. Evaluation of Acrosome Status of the Released Sperm Population from the OECs under Capacitating Conditions

To assess acrosome status, spermatozoa were incubated with Hoechst 33258 as described before (see Section 2.6.3.). Aliquots were air-dried onto glass slides and permeabilized in methanol for 10 min at 4 ◦C. Slides were incubated with *Pisum sativum* agglutinin-FITC (PSA-FITC, 50 mg/mL) for 1 h at RT. At least 200 stained cells per treatment were evaluated using an epifluorescence Nikon 80i microscope (Nikon Instruments Inc., Tokyo, Japan). Results are shown as the percentage of live spermatozoa with intact acrosomes.

2.6.5. Evaluation of Total Motility of the Released Sperm Population from the OECs under Capacitating Conditions

Sperm released were briefly concentrated bya3s centrifugation and total sperm motility was analyzed using CASA-system (IVOS II™ Animal—Hamilton Thorne) as described before (see Section 2.3.2). Moreover, sperm total motility was subjectively evaluated using a field microscope stage heated at 38.5 ◦C at 100× magnification with similar results. Results are shown as the % of total motile sperm.

2.6.6. Evaluation of Capacitation Status of the Released Sperm Population from the OECs under Capacitating Conditions

The capacitation status of released sperm was analyzed by examining the phosphorylation of protein kinase A substrates (pPKAs) and tyrosine-phosphorylation of proteins (pY) using immunofluorescence ([48] with some modifications). Specifically, we first determined sperm viability with Hoechst 33258 as described before (see Section 2.6.3.). Then, spermatozoa were fixed (20 min, at RT with 0.2% *w*/*v* paraformaldehyde), immobilized on slides and permeabilized with TPBS-Triton X100 0.5% for 20 min at RT. Non-specific binding sites were blocked (60 min, at RT with 3% *w*/*v* BSA TPBS) and incubated with pPKA (1:500) and pY (1:500) antibodies diluted in PBS for 18 h at 4 ◦C. Samples were washed and further incubated with Alexa 555-conjugated goat anti-rabbit IgG (red, 1:500) and Alexa 488 chicken anti-mouse IgG (green, 1:500) diluted in PBS for 1 h at RT. The specificity of the immunodetection was assessed by omitting the first antibody. Sperm cells were mounted and examined under a fluorescence Nikon 80i microscope (Nikon Instruments Inc., Tokyo, Japan). The proportion of spermatozoa with green and red fluorescent tails among the live sperm population (without Hoechst 33258 fluorescent heads) was determined by randomly scoring 200 spermatozoa. Results are shown as the percentage of live spermatozoa with both stains: positive pPKA and positive pY.

#### *2.7. Statistical Analysis*

The effect of the incubation time and sample concentration on % total motility was analyzed by two-way ANOVA in a completely randomized design with repeated measures. Comparisons were made with Tukey's post hoc test (*p* < 0.05). The statistical analysis of

% total motility between the times of centrifugation was analyzed by one-way ANOVA (time) in a completely randomized design. Comparisons between the mean of each time of centrifugation and control (0 min) were made with Dunnett's post hoc test (*p* < 0.05). For the evaluation of the number of sperm that remained attached to OECs after treatments (Section 2.6.1., number of bound sperm), comparison between groups was performed with a one-way ANOVA in blocks, where each pool of OECs with spermatozoa was considered a block, and all treatments were applied to it (randomized blocks). When the ANOVA tests were significant (*p* < 0.05), multiple comparisons were performed by Tukey´s post hoc test (*p* < 0.05). The effect of dependent variables measured in the released sperm population from OEC primary cultures (i.e., number of released sperm, % viability, % acrosome-intact sperm, % motile sperm and % capacitated sperm) were analyzed by *t*-test (*p* < 0.05). The assumptions of normality (ANOVA and *t*-test) and homogeneity of variances (ANOVA) were assessed prior to performing the statistical analysis by Shapiro–Wilks test and Levene test, respectively. In the case of the two-way ANOVA, as we chose not to accept the assumption of the sphericity, we used the method of Geisser and Greenhouse to correct for violations of the assumption. All values represent the mean ± S.E.M. Statistical analyses were performed using the Prism 7 software package (GraphPad, La Jolla, CA, USA).

#### **3. Results**

#### *3.1. Effect of Different Processing Conditions on Sperm Motility*

To determine whether different processing conditions of cryopreserved samples affect sperm motility, we evaluated the effect of incubation time, centrifugation time and sample concentration on this parameter. A repeated-measures two-way ANOVA was applied to study the interaction between time of incubation and sample concentration on the % of sperm total motility. The results showed a statistically significant interaction between the two independent variables on the dependent variable (*p* < 0.05), i.e., that the changes in the motility during the incubation depended on the sample concentration. Specifically, we observed that samples incubated at 12 × 106 sperm/mL did not show changes in motility at different times of incubation, while samples incubated at <sup>30</sup> × 106 sperm/mL showed a decrease in motility after 30 and 120 min of incubation (Figure 2A). It is important to highlight that these equine cryopreserved sperm samples present poor motility after the thawing process (0 min; total motility <35%) (Figure 2A). However, the motility of the lower-concentrated samples was always lower than 10% and we found that at time 0 samples incubated at 30 × 106 sperm/mL presented higher motility compared to <sup>12</sup> × 106 sperm/mL samples, suggesting that it is convenient to use more concentrated samples (Figure 2A). After more than 60 min of incubation, samples at <sup>30</sup> × <sup>10</sup><sup>6</sup> sperm/mL did not show differences in motility compared to lower-concentrated samples (Figure 2A). Centrifugation steps could be necessary for some ART protocols and in the study of sperm physiology. In this sense, we evaluated how two different centrifugation (at 200× *g*) times may affect sperm motility when sperm were incubated at concentrations of <sup>30</sup> × 106 sperm/mL. This sperm concentration was used because sperm motility was not affected for short times (Figure 2A). We did not find differences in this parameter after centrifugations for 1 min in comparison to samples without centrifugation (control) (Figure 2B). However, we observed a significant decrease in sperm motility when the samples were centrifuged for 2 min compared with control (Figure 2B).

**Figure 2.** Parameters that affect cryopreserved equine sperm motility. (**A**) Time of incubation and sample concentration. Spermatozoa were incubated in NCWM at 12 <sup>×</sup> <sup>10</sup>6/mL or at 30 <sup>×</sup> <sup>10</sup>6/mL and motility was determined at different times (0, 30, 60 and 120 min). Two-way ANOVA with repeated measures (*p* < 0.05). *n* = 4. Means with different letters are significantly different (Tuckey post hoc test). (**B**) Time of centrifugation. Spermatozoa were incubated at 30 <sup>×</sup> <sup>10</sup>6/mL centrifuged for 0 min (control), 1 min or 2 min at 200× *g*. One-way ANOVA (*p* < 0.05). ns: not statistically different. \* Indicates statistically significant differences (Dunnett's post hoc test between the mean of each time of centrifugation and control). *n* = 4. Bars represent the mean ± SEM of total motile sperm. Percentage of total motility was assessed by Computer Assisted Sperm Analysis (CASA).

#### *3.2. OEC Primary Culture Characterization*

To characterize the obtained OEC primary cultures, we examined the expression of the epithelial markers E-cadherin and cytokeratin-7 using RT-PCR and immunofluorescence. As Figure 3A shows, OECs cultured in vitro expressed mRNAs for both epithelial cell markers whereas equine fibroblasts did not (data not shown). In agreement with these observations, E-cadherin and cytokeratin-7 protein expression was detected in OECs, with E-cadherin mainly present on cell membranes and cytokeratin-7 in the cytoplasm (Figure 3B).

#### *3.3. Characterization of Cryopreserved Semen Bound to OEC Primary Cultures*

As shown in Figure 4A, sperm not only adhered to the OECs immediately after insemination (0 min), but it remained bound even after 1 h of co-culture and several washes with NCWM. Moreover, we observed that the majority of the OEC attached sperm were motile (Supplementary Materials, Video S1) and with an intact acrosome (Figure 4B).

**Figure 3.** Characterization of OEC primary cultures derived from equine oviduct explants. (**A**) Expression of epithelial markers in equine OECs was determined by RT-PCR employing equine specific primers for eCDH1 (E-cadherin), eKRT7 (cytokeratin-7) and GAPDH. Representative results of three different pools of OECs are shown (P1, P2, P3; *n* = 3); (**B**) Intracellular distribution of E-cadherin (green) and cytokeratin (red) were examined using immunofluorescence (see Section 2.4.3., Materials and Methods). DNA was labeled with Hoechst 33352 (blue), E-cadherin: 200× magnification, cytokeratin: 400× magnification (*n* = 3). Bar = 10 μm.

#### *3.4. Characterization of Cryopreserved Sperm Released after Co-Culture with OECs under Capacitating Conditions*

Previous studies indicated that spermatozoa release from oviductal cells in vivo and in vitro is associated with sperm capacitation. Based on that, our results showed that cryopreserved stallion sperm's attachment to OECs in vitro did not impact their motility or acrosomes integrity, we examined whether released spermatozoa from OECs under capacitating condition (CWM) affected sperm viability, acrosome status and progressive motility. We also assessed PKA phosphorylated substrates (pPKA) and protein tyrosine phosphorylation (pY), two widely employed sperm capacitation indicators.

**Figure 4.** Characterization of cryopreserved semen bound to OEC primary cultures (**A**) Phase-contrast representative images of cryopreserved equine sperm co-cultured with OECs, 400× magnification, *n* = 6. Bar = 50 μm. (**B**) Representative images of intact acrosomes (white arrows) examined by immunofluorescence 1 h after co-culture with OECs stained with PSA-FITC (green, acrosome) and Hoechst 33352 (blue, DNA). 1000× magnification, *n* = 3. Bar = 10 μm.

As Figure 5A,B shows, the treatment with CWM reduced the number of OEC-bound spermatozoa (*p* < 0.05) concurrently increasing the number of released ones (*p* < 0.05). The released sperm in response to CWM treatment also showed a significantly higher percentage of alive spermatozoa (*p* < 0.05) with intact acrosomes (*p* < 0.05) and total motility (*p* < 0.05) (Figure 5C–E). In contrast, this effect on motility was not observed during the incubation of cryopreserved spermatozoa at the same conditions (7 × 106 sperm/mL, 60 min) in OECs' absence where total motility was around 0% (subjectively evaluated using a field microscope, data not shown). Moreover, spermatozoa released in response to CWM showed an increase in PKA activity and protein tyrosine phosphorylation (pY) (*p* < 0.05) (Figure 5F).

**Figure 5.** Characterization of sperm released from OECs primary cultures. OEC and sperm cocultures were incubated for 15 min at 37 ◦C in the presence of NCWM (no capacitating condition) or CWM (capacitating condition) and the spermatozoa bound (**A**) to OECs were quantified using bright field microscopy while (**B**) the released sperm were recovered and quantified with a hemocytometer. Bars represent the mean <sup>±</sup> SEM of bound spermatozoa/0.11 mm2 monolayer (**A**) and (**B**) the mean ± SEM of the number of released sperm. \* *p* < 0.05, *n* = 6. (**C**) Sperm released from the cocultures were incubated with Hoechst 33258, fixed and the percentage of live sperm was determined using fluorescence microscopy. Bars represent the mean ± SEM of live spermatozoa. \* *p* < 0.05, *n* = 6. (**D**) Sperm viability and acrosome status were evaluated as described in Section 2. Bars represent the mean ± SEM of live spermatozoa with intact acrosomes. \* *p* < 0.05, *n* = 6. (**E**) The total motility of released sperm was determined by CASA. Bars represent the mean ± SEM of total motile spermatozoa. \* *p* < 0.05, *n* = 6. (**F**) OEC-released sperm positive for pPKA and pY were evaluated using immunofluorescence. Bars represent the mean ± SEM of live sperm. \* *p* < 0.05, *n* = 6.

#### **4. Discussion**

The use of stallion frozen semen minimizes the spread of diseases, eliminates geographic barriers, and preserves the genetic material of the animal for an unlimited time [12]. The efficiency of equine semen cryopreservation depends mainly on the animal's characteristics such as genetics and age [49]. Additionally, stallions that are satisfactorily fertile under normal field conditions can produce semen that after freezing and thawing results in

very low pregnancy rates [10]. Several factors influence the cryo-survival of stallion sperm including freezing regimes [12,50,51], oxidative and osmotic stress, ice crystal formation, toxicity of the cryoprotectants [8,52,53], sample processing [54] and variability among stallions [11]. Consequently, the attachment of cryopreserved equine spermatozoa to equine OECs or zona pellucida in vitro is reduced compared to that of fresh spermatozoa [55]. This limitation is very likely due to a reduction in post-thaw motility associated with changes in the integrity of the sperm membrane, suggesting a possible mechanism to explain the reduced fertility achieved with cryopreserved samples versus fresh spermatozoa in horses. These negatives effects of cryopreservation on sperm function result in low ART success rates. Given the characteristics of cryopreserved semen samples, it is important to know how to manipulate them, not only for their application in ART but also for their use in research studies.

Previously, Hayden et al. showed that raw stallion semen dilution with commercial extenders decreased the total motility [56]. In agreement with these observations, our results indicate that sperm total motility—measured shortly after thawing—was concentrationdependent, i.e., more concentrated samples displayed higher motility. Moreover, after more than 60 min of incubation, samples at 30 × 106 sperm/mL did not show differences in motility compared to lower-concentrated samples. We studied these sperm concentration and sperm total motility from 0 to 120 min because there are different protocols described for post-thawed sperm incubation times for research studies, such as sperm capacitation a process with high relevance in ARTs applied in equines. Those concentrations range between 10 × <sup>10</sup><sup>6</sup> sperm/mL to 200 × 106 sperm/mL [57–62] and times ranged between 10 and 120 min [58,63,64]. Sperm concentrations used were chosen based on the CASA system optimal-concentration working range (10–50 × <sup>10</sup><sup>6</sup> sperm/mL). Moreover, several sperm selection protocols used during ARTs require multiple centrifugations. It was previously described that different conditions of centrifugation alter motility and oxidative status, and consequently increase the DNA damage of cryopreserved stallion sperm [65,66]. We tested a 200× *g* force, not previously described for cryopreserved stallion sperm and its effect on motility. As previously reported, we found that total sperm motility was negatively affected by centrifugation times longer than 2 min, even at lower *g*-forces [65]. In this work, we have used samples that were frozen poorly (<35% motility post-thawing) [67], suggesting that a better outcome could be possible using samples with better post-thaw motility.

The mammalian oviduct plays an essential role during the selection of competent sperm subpopulations, and it is involved in the maintenance of fertilization capacity during sperm storage (sperm reservoir) [23,68]. Accordingly, we developed an in vitro OEC culture model to select sperm populations from cryopreserved samples with fertilizing potential.

In view of the role of OECs in sperm selection under physiological conditions, we speculated whether the co-culture of equine cryopreserved semen and equine-derived OEC primary cultures would replicate parameters observed in the intact reproductive tract including OEC–sperm binding, motility and acrosome integrity [23,69,70].

Thomas et al. have shown that a subpopulation of morphologically normal and motile spermatozoa attach to equine OEC monolayers using fresh stallion semen [31]. In agreement with these results, we observed that cryopreserved equine sperm, processed under our optimized conditions, not only attached to the OEC culture model but also maintained their motility and presented an intact acrosome. These spermatozoa maintained their motility after 1 h of in vitro co-culture with OECs. In contrast, this effect on motility was not observed during the incubation of cryopreserved spermatozoa at the same conditions in OECs' absence. Thus, the in vitro equine OEC monolayer culture established in this work would be a useful tool for the selection of a sperm population with fertilization potential from cryopreserved samples.

Previous studies indicated that spermatozoa release from oviductal cells in vivo and in vitro is associated with sperm capacitation and hyperactivation [23,34,36,37,69,71,72]. These processes stimulate sperm release from the oviductal epithelium, to come into contact and fertilize an oocyte [23,73]. Based on that, our results showed that cryopreserved stallion

sperm's attachment to OECs in vitro did not impact the motility or the acrosome's integrity, we examined whether spermatozoa released from OECs under capacitating conditions (CWM) presented affected sperm viability, acrosome status and progressive motility. We also assessed PKA phosphorylated substrates (pPKA) and protein tyrosine phosphorylation (pY), two widely utilized sperm capacitation indicators [74].

In previous works, it has been described that the in vitro incubation of equine sperm in CWM medium increases protein phosphorylation in tyrosine residues, progressive motility and the induction of the acrosomal reaction, all events associated with sperm capacitation [44]. In agreement with these observations, our results showed that the incubation of sperm–OEC cultures in the presence of CWM promoted the release of a greater number of sperm. The released sperm were alive, motile and presented an intact acrosome and an increase in molecular markers, i.e., PKA activity and Tyr phosphorylation, associated with sperm capacitation. Thus, these results indicate that the frozen equine sperm–OEC co-culture model provides a useful system to enrich spermatozoa populations with fertilization potential.

ICSI is a low-embryo-yield technique within the equine breeding industry that can achieve similar embryo development using frozen or fresh equine spermatozoa [17,75]. Different methods are employed to select stallion sperm prior to ICSI (swim-up procedure, density gradient centrifugation or microfluidics) in order to increase the probability of selecting sperm that when used will result in optimal fertility [76–78]. Within this context, the probability that sperm-injected oocytes develop into an embryo (morula or blastocyst) improves when frozen–thawed stallion sperm show high membrane integrity [79]. In this regard, we speculated that sperm population released from OEC co-culture under capacitating conditions could be used to enhance ICSI efficiency and embryo quality in equines due to their potential fertilizing capacity.

Further studies to achieve a better understanding of the molecular mechanisms that regulate the acquisition of spermatozoa fertilization capacity during their transit through the female reproductive tract will favor the development of new sperm selection methods to be incorporated into ARTs for equines and other animal species.

#### **5. Conclusions**

Our results show that the total motility of previously frozen equine sperm samples is dependent on its concentration, the incubation time and the centrifugation duration applied during processing. We also found that cryopreserved spermatozoa interacted with OEC cultures in vitro and that this equine sperm–OEC co-culture model could be a useful tool to select a sperm population with potential fertilizing capacity under capacitating conditions.

In conclusion, this work contributed to the existing knowledge on the effect of different conditions associated with the manipulation of stallion cryopreserved sperm. Although further analyses are needed, we speculated that the selection of higher-quality male gametes using the in vitro OEC primary culture established in this study would improve, in future, the efficiency of ARTs as well as the quality of the obtained embryos in equines.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-2 615/11/1/74/s1, Video S1: Cryopreserved equine sperm attached to OECs in vitro after 1 h of co-culture.

**Author Contributions:** Conceptualization, C.E.O.-S.; methodology, B.F.G., M.V.B., L.L.-Q., E.M.-L., M.V.-M. and C.E.O.-S.; formal analysis, B.F.G., L.L.-Q. and M.V.B.; investigation, B.F.G., L.L.-Q., M.V.B. and C.E.O.-S.; resources, O.R. and A.Á.M.; writing—original draft preparation, C.E.O.-S.; writing review and editing, M.V.B., E.M.-L., O.R., A.Á.M. and C.E.O.-S.; supervision, A.Á.M. and C.E.O.-S.; funding acquisition, A.Á.M. and C.E.O.-S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by grants from Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación of Argentina PICT 2016-3138 to C.E.O.-S and PICT 2015-1548 to A.A.M.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available within the article or supplementary material.

**Acknowledgments:** We thank to the Equine Breeding Centers (GeneTec by Ativet and Los Pingos del Taita) and Lamar S.A. slaughterhouse for providing semen and oviducts respectively. Authors thank to Francisco Guaimas for his technical support.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**


### *Article* **The Effects of Red Light on Mammalian Sperm Rely upon the Color of the Straw and the Medium Used**

**Jaime Catalán 1,2,3, Iván Yánez-Ortiz 1, Sabrina Gacem 1, Marion Papas 1, Sergi Bonet 2,3, Joan E. Rodríguez-Gil 1, Marc Yeste 2,3,\*,† and Jordi Miró 1,\*,†**

	- +34-93-5814293 (J.M.)

**Simple Summary:** Several studies have shown that the exposure of semen to red light improves sperm quality and fertilizing ability, which could improve the efficiency of assisted reproductive techniques with irradiated semen. However, despite being considered as possible sources of variation, the effects of the color of the container (straws) or the medium have not yet been evaluated. In this study, 13 ejaculates from different stallions were split into equal fractions, diluted either with Kenney or Equiplus extender, and subsequently packed into straws of five different colors. After storage at 4 ◦C for 24 h, the sperm were irradiated and different variables, including sperm motility, plasma membrane integrity, and mitochondrial membrane potential, were evaluated. Our results confirm that irradiation increases some motion characteristics and mitochondrial membrane potential without affecting sperm viability and demonstrate that the effects depend on the color of the straw and the extender used.

**Abstract:** Previous research has determined that irradiation of mammalian sperm with red light increases motility, mitochondrial activity, and fertilization capacity. In spite of this, no study has considered the potential influence of the color of the straw and the extender used. Therefore, this study tests the hypothesis that the response of mammalian sperm to red light is influenced by the color of the straw and the turbidity/composition of the extender. Using the horse as a model, 13 ejaculates from 13 stallions were split into two equal fractions, diluted with Kenney or Equiplus extender, and stored at 4 ◦C for 24 h. Thereafter, each diluted fraction was split into five equal aliquots and subsequently packed into 0.5-mL straws of red, blue, yellow, white, or transparent color. Straws were either nonirradiated (control) or irradiated with a light–dark–light pattern of 3–3–3 (i.e., light: 3 min, dark: 3 min; light: 3 min) prior to evaluating sperm motility, acrosome and plasma membrane integrity, mitochondrial membrane potential, and intracellular ROS and calcium levels. Our results showed that irradiation increased some motion variables, mitochondrial membrane potential, and intracellular ROS without affecting the integrities of the plasma membrane and acrosome. Remarkably, the extent of those changes varied with the color of the straw and the extender used; the effects of irradiation were more apparent when sperm were diluted with Equiplus extender and packed into red-colored straws or when samples were diluted with Kenney extender and packed into transparent straws. As the increase in sperm motility and intracellular ROS levels was parallel to that of mitochondrial activity, we suggest that the impact of red light on sperm function relies upon the specific rates of energy provided to the mitochondria, which, in turn, vary with the color of the straw and the turbidity/composition of the extender.

**Citation:** Catalán, J.; Yánez-Ortiz, I.; Gacem, S.; Papas, M.; Bonet, S.; Rodríguez-Gil, J.E.; Yeste, M.; Miró, J. The Effects of Red Light on Mammalian Sperm Rely upon the Color of the Straw and the Medium Used. *Animals* **2021**, *11*, 122. https://doi.org/10.3390/ani11010122

Received: 16 October 2020 Accepted: 5 January 2021 Published: 8 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** horse; sperm; red light irradiation; extender; straw

#### **1. Introduction**

Artificial insemination (AI) is a tool widely used today for horse breeding, especially when looking for genetic improvement [1]. The increasing use of this technology, both in the horse and other species, has augmented the interest for semen processing techniques and their optimization, aimed at maximizing their survival and fertilization capacity [2,3]. Unfortunately, semen quality often deviates from expectations and leads to unsatisfactory pregnancy rates [1]. In this context, any protocol or procedure that optimizes its use and helps increase reproductive performance should be considered; for this reason, several approaches have been undertaken in recent years [3,4]. One of these approaches is sperm irradiation; in effect, previous research has demonstrated that red light stimulation, either with low-level lasers or light-emitting diodes (LEDs), increases the motility, ability to elicit in vitro capacitation, fertilizing ability, and lifespan of fresh, liquid-stored, and frozenthawed sperm in fish [5], birds [6], humans [7–12], pigs [13–15], sheep [16], dogs [17,18], buffalos [19] donkeys [3], and horses [20,21]. In addition to this, recent studies have shown that the increase in sperm motility in response to LED-based red light is concomitant with that of mitochondrial activity in pigs, donkeys, and horses [3,13,20,22].

The mechanisms through which light exerts its effects are not entirely clear. Three potential mechanisms have been surmised to explain the response of mammalian sperm to red light (reviewed in Yeste et al. [23]). The first of these hypotheses is related to the possible influence of light on transient receptor proteins (TRPs) [24–26], which reside in sperm plasmalemma and have been purported to participate in the modulation of thermotaxis [27,28]. The second hypothesis is related to the presence of opsins in mammalian spermatozoa, which absorb light from different spectra [23]; despite being mainly related to the response to thermotaxis [28], they could also be involved in the sperm response to light. Finally, mounting evidence supports the third hypothesis, which confers a crucial role on endogenous cellular photosensitizers, especially those present in the mitochondria [22,23,29]. These photosensitizers absorb light from electromagnetic radiation and then ionize and transfer the absorbed energy into adjacent molecules [30]. This increased energy induces a rise in electrochemical mitochondria potential, which may result in an augmentation of ATP and Ca2+ levels [23]. In spite of this, it cannot be ruled out that more than one of the mechanisms proposed by these hypotheses are involved in the sperm response to red light [22,29].

Previous studies carried out with low-level laser therapy devices and light-emitting diodes (LEDs) have reported an increase in ATP production via the mitochondrial electron chain [31,32] without damaging the irradiated cells [32,33] or the integrity of their DNA [32,34]. Therefore, it has been suggested that light stimulation can have a safe and positive effect on sperm motility and fertilizing ability both in vivo and in vitro [32]. Nevertheless, the sperm response to irradiation has been reported to depend on different factors, including the type (i.e., fresh, cooled-stored or frozen-thawed) and state of the sample [3], the irradiation of the light beam used [32], the time or pattern of exposure [13], and the species [5]. Given the properties of light emission/absorption, other factors such as the color of the straw and choice of extender could also affect the sperm response to red light. However, to the best of our knowledge, no previous study has examined this possibility despite the wide variety of extenders and colors of commercial straws.

Taking the results obtained in the aforementioned studies (especially those conducted with fresh, cooled-stored, and frozen-thawed horse sperm) [20,21] into account, this study aims at determining whether the color of the straw and the extender used affect the response of cooled-stored sperm to LED-based red light (620–630 nm). Our hypothesis is that the effects of red light on horse sperm depend on the color of the straw and the extender.

#### **2. Materials and Methods**

#### *2.1. Suppliers*

All reagents used were of analytical grade and were purchased from Boehringer-Mannheim (Mannheim, Germany), Merck (Darmstadt, Germany), and Sigma-Aldrich (Saint Louis, MO, USA). As far as fluorochromes are concerned, unless otherwise stated, all were purchased from Molecular Probes (Thermo Fisher Scientific; Waltham, MA, USA) and were previously prepared with dimethyl sulfoxide (Sigma-Aldrich). Plastic materials were provided by Nunc (Roskilde, Denmark), and empty straws of different colors (transparent, red, white, blue, and yellow) were purchased from Minitüb GmbH (Tiefenbach, Germany).

#### *2.2. Animals and Ejaculates*

This study included 13 ejaculates from 13 different adult stallions (age: 5–8 years old) with proven fertility. Animals were housed at the Equine Reproduction Service, Autonomous University of Barcelona (Bellaterra, Cerdanyola del Vallès, Spain), which is an EU-approved semen collection center (Authorization code: ES09RS01E) that operates under strict protocols of animal welfare and health control. All animals were semen donors and were collected under CEE health conditions (free of equine arteritis, infectious anemia, and contagious metritis). As indicated in Catalán et al. [21], this Service runs under the rules of the Regional Government of Catalonia, Spain, and no manipulation of the animals other than semen collection was carried out. The study was approved by the Ethics Committee, Autonomous University of Barcelona (Code: CEEAH 1424).

Ejaculates were collected through a Hannover artificial vagina (Minitüb GmbH, Tiefenbach, Germany), and an in-line nylon mesh filter was used to remove the gel fraction. Upon collection, gel-free semen was split into two fractions of equal volume and immediately diluted 1:5 (*v:v*) either in Kenney [35] or Equiplus extender (Minitüb GmbH; Tiefenbach, Germany), which were selected for their different turbidity. The absorbance of these two extenders was evaluated at 625 nm with a spectrophotometer (Biochrom WPA, Lightwave II; Cambridge, UK) and sterilized; ultrafiltered Milli-Q water was used as blank. Absolute absorbance values of the Equiplus and Kenney extenders were 0.090 and >2.5, respectively. Both extenders were preheated to 37 ◦C, and sperm concentration was adjusted in all cases to 30 × 106 sperm/mL with a Neubauer chamber (Paul Marienfeld GmbH and Co. KG; Lauda-Königshofen, Germany). Following this, sperm motility (Computer Assisted Semen Analysis, CASA), morphology (eosin–nigrosin staining), and plasma membrane integrity (SYBR14/PI) of each sample were evaluated. All samples were confirmed to fulfill the standard thresholds: ≥60% SYBR14+/PI<sup>−</sup> spermatozoa and ≥70% morphologically normal spermatozoa. Thereafter, semen samples were stored in a refrigerator at 4 ◦C for 24 h.

#### *2.3. Experimental Design*

After 24 h of storage, samples extended in either Kenney or Equiplus were split and packed into 0.5-mL straws (Minitüb GmbH) of five different colors (blue, red, yellow, white, and transparent); the sperm concentration was maintained at 30 × 106 sperm/mL at all experimental points. Straws were placed within a programmable photoactivation system (MaxiCow; IUL, SA, Barcelona, Spain). In this device, each straw is in contact with a triple-LED configuration system that emits red light (wavelength window: 620 to 630 nm). The apparatus is equipped with software (IUL, SA) that allows the regulation of intensity and time of exposure. In all cases, the intensity was set at 100%.

Straws of different colors containing sperm diluted by both extenders were irradiated with a light–dark–light interval pattern of 3–3–3 min. Nonirradiated samples (control) were also packed into 0.5-mL straws and left for 9 min in the dark, which was the same time used to irradiate the samples. Upon light stimulation, irradiated and nonirradiated samples were transferred into 1.5-mL tubes. Sperm motility was evaluated with a computer-assisted sperm analysis (CASA) system, and plasma membrane and acrosome integrity, mitochondrial membrane potential, intracellular ROS (peroxides and superoxides), and calcium levels were determined through flow cytometry.

#### *2.4. Analysis of Sperm Motility*

Sperm motility was evaluated using a computer-assisted sperm analysis (CASA) system (Integrated Sperm Analysis System V1.0; Proiser S.L.; Valencia, Spain). In brief, samples were incubated at 38 ◦C in a water bath for 5 min, and 5 μL of each sperm sample was placed onto a Makler chamber (Sefi Medical Instruments; Haifa, Israel), previously warmed at 38 ◦C. Samples were then analyzed under a 10× negative phase-contrast objective (Olympus BX41 microscope; Olympus, Tokyo, Japan). A minimum of 1000 sperm cells was counted per analysis. In each evaluation, percentages of total motility (TMOT, %) and progressively motile spermatozoa (PMOT, %) were recorded together with the following kinetic measures: curvilinear velocity (VCL, μm/s), which is the mean path velocity of the sperm head along its actual trajectory; straight-line velocity (VSL, μm/s), which is the mean path velocity of the sperm head along a straight line from its first to its last position; average path velocity (VAP, μm/s), which is the mean velocity of the sperm head along its average trajectory; percentage of linearity (LIN, %), which is the quotient between VSL and VCL multiplied by 100; percentage of straightness (STR, %), which is the quotient between VSL and VAP multiplied by 100; percentage of oscillation (WOB, %), which is the quotient between VAP and VCL multiplied by 100; mean amplitude of lateral head displacement (ALH, μm), which is the mean value of the extreme sideto-side movement of the sperm head in each beat cycle; frequency of head displacement (BCF, Hz), which is the frequency at which the actual sperm trajectory crosses the average path trajectory.

CASA settings were those recommended by the manufacturer, i.e., frames: 25 images captured per second; particle area >4 and <75 μm2; connectivity = 6; minimum number of images to calculate ALH: 10. The cut-off value for motile spermatozoa was VAP ≥ 10 μm/s; for progressively motile spermatozoa, the cut-off value was STR ≥ 75%.

#### *2.5. Flow Cytometry*

The integrity of sperm plasma membrane (SYBR14/PI), acrosome integrity (PNA-FITC/PI), mitochondrial membrane potential (JC1), and intracellular levels of peroxides (H2DCFDA/PI), superoxides (HE/YO-PRO-1), and calcium (Fluo3/PI) were determined through flow cytometry. Samples were stained and evaluated following the protocol described by Prieto-Martínez et al. [36] and adjusted to horse spermatozoa.

Management of the flow cytometer and analysis of the samples were carried out in accordance with the recommendations of the International Society of Cytometry [37]. The flow cytometer used in this study was a Cell Lab Quanta SC™ (Beckman Coulter, Fullerton, CA, USA), and particles were excited with an argon laser (488 nm) at a power of 22 mW. Prior to staining, sperm concentration was adjusted to 1 × 106 sperm/mL. Every day, the electronic volume (EV) channel was calibrated with 10-μm diameter fluorescent beads (Beckman Coulter), following the manufacturer's instructions. The flow rate was set at 4.17 μL/min, and the analyzer threshold was established to exclude cell aggregates (particles with a diameter >12 μm) and debris (particles with a diameter < 7 μm). Sperm cells were gated on the basis of EV and side scatter (SS) distributions. Three different optical filters were used (FL1 for the analysis of SYBR14, PNA, H2DCFDA, Fluo3, and JC1 monomers, detection width: 505–545 nm; FL2 for the analysis of JC1 aggregates, detection width: 560–590 nm; FL3 for the analysis of PI and HE, detection width: 655–685 nm).

Dot plots were examined using Cell Lab Quanta SC™ MPL Analysis Software (version 1.0; Beckman Coulter) and data from PNA/PI, JC1, H2DCFDA/PI, HE/YO-PRO-1; Fluo3/PI were corrected using the percentage of nonstained debris particles found in SYBR14/PI staining, as recommended by Petrunkina et al. [38].

#### 2.5.1. Analysis of Plasma Membrane Integrity

Sperm viability (plasma membrane integrity) was assessed using the LIVE/DEAD® Sperm Viability Kit (SYBR14/PI; Molecular Probes, Thermo Fisher Scientific; Waltham, MA, USA), according to the protocol described by Garner and Johnson [39], and adapted to horse spermatozoa. In brief, samples were first incubated with SYBR14 (final concentration: 100 nM) at 38 ◦C for 10 min, and then with PI (final concentration: 12 μM) at 38 ◦C for 5 min. Three sperm populations were distinguished: (i) viable spermatozoa emitting green fluorescence (SYBR14+/PI<sup>−</sup>), which appeared on the right side of the lower half of the FL1/FL3 dot plots; (ii) nonviable spermatozoa emitting red fluorescence (SYBR14−/PI+), which appeared on the left side of the upper half of the FL1/FL3 dot plots; (iii) nonviable spermatozoa emitting both green and red fluorescence (SYBR14+/PI+), which appeared on the right side of the upper half of the FL1/FL3 dot plots. Nonstained particles (SYBR14−/PI−), which appeared on the left side of the lower half of the FL1/FL3 dot plots, showed EV/SS distributions similar to spermatozoa and were considered non-DNA debris particles. Percentages of nonstained particles were used to correct the percentages of double-negative sperm populations in the other assessments. Spill-over of FL1 into the FL3 channel was compensated (2.45%).

#### 2.5.2. Analysis of Acrosome Integrity

Plasma membrane integrity was evaluated through PNA/PI costaining, following the procedure described for horse spermatozoa by Rathi et al. [40]. With this purpose, spermatozoa were stained with PNA conjugated with FITC (final concentration: 5 μg/mL) and PI (final concentration: 12 μm) and incubated at 38 ◦C for 10 min in the dark. Green fluorescence from PNA was collected through FL1, whereas red fluorescence from PI was collected through FL3. As spermatozoa were not previously permeabilized, they were identified and placed in one of the four following populations: (i) spermatozoa with intact plasma membranes (PNA−/PI−); (ii) spermatozoa with damaged plasma membranes that presented an acrosome membrane that could not be fully intact (PNA+/PI+); (iii) spermatozoa with damaged plasma membranes and lost outer acrosome membranes (PNA−/PI+); (iv) spermatozoa with damaged plasma membranes (PNA+/PI−). Therefore, after PNA/PI staining, two main categories were detected: (i) spermatozoa with an intact plasma membrane (PNA−/PI−) and (ii) spermatozoa that had damaged their plasma membrane and/or their acrosome membrane (these were represented by the other three categories: PNA+/PI−, PNA+/PI+, PNA−/PI+). Unstained and single-stained samples were used for setting the EV gain, FL1 and FL3 PMT voltages, and for compensation of PNA spill over into the PI channel (2.45%).

#### 2.5.3. Analysis of Mitochondrial Membrane Potential

Mitochondrial membrane potential (MMP) was determined through incubation with JC1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 tetraethyl-benzimidazolylcarbocyanine iodide; final concentration: 0.3 μM) at 38 ◦C for 30 min in the dark. When MMP is low, JC1 forms monomers emitting green fluorescence (JC1mon), which are collected through FL1. When mitochondrial membrane potential is high, JC1 forms aggregates emitting orange fluorescence (JC1agg), which are detected through FL2. Three sperm populations were distinguished: (i) spermatozoa with green-stained mitochondria (low MMP), (ii) spermatozoa with orange-stained mitochondria (high MMP), and (iii) spermatozoa with heterogeneous mitochondria, stained both green and orange in the same cell (intermediate MMP). Ratios between FL2 (JC1agg) and FL1 fluorescence (JC1mon) for each of these sperm populations were also evaluated. Spill-over of FL1 into the FL2 channel was compensated (68.5%). Percentages of debris particles found in SYBR14/PI staining (SYBR14−/PI−) were subtracted from those of spermatozoa with low MMP, and the percentages of all sperm populations were recalculated.

#### 2.5.4. Analysis of Intracellular ROS Levels: H2O2 and O2 −

Intracellular ROS levels were determined through two oxidation sensitive fluorescent probes, 2 ,7 -dichlorodihydrofluorescein diacetate (H2DCFDA) and hydroethidine (HE), which detect hydrogen peroxides (H2O2) and superoxide anions (·O2 −), respectively [41]. Following a modified procedure from Guthrie and Welch [42], a simultaneous differentiation of viable and nonviable sperm was performed using PI (H2DCFDA) or YO-PRO-1 (HE).

In the case of peroxides, spermatozoa were incubated with H2DCFDA (final concentration: 200 μM) and PI (final concentration: 12 μM) at room temperature for 30 min in the dark. H2DCFDA is a stable, cell-permeable, nonfluorescent probe that is converted into 2 ,7 -dichlorofluorescein (DCF) in the presence of H2O2 [42]. Fluorescence of DCF+ was measured through FL1 and that of PI was detected through FL3. Four sperm populations were distinguished: (i) viable spermatozoa with low levels of peroxides (DCF−/PI−), (ii) viable spermatozoa with high levels of peroxides (DCF+/PI−), (iii) nonviable spermatozoa with low levels of peroxides (DCF−/PI+), and (iv) nonviable spermatozoa with high levels of peroxides (DCF+/PI+). Percentages of debris particles found in SYBR14/PI staining (SYBR14−/PI−) were subtracted from those of viable spermatozoa with low levels of peroxides (DCF−/PI−) and the percentages of all sperm populations were recalculated. Spill-over of FL1 into the FL3 channel was compensated (2.45%). Data are shown as corrected percentages of viable spermatozoa with high levels of peroxides (DCF+/PI−) and the geometric mean of DCF+-fluorescence intensity in the DCF+/PI<sup>−</sup> sperm population.

Regarding superoxide anions, samples were incubated with HE (final concentration: 4 μM) and YO-PRO-1 (final concentration: 25 nM) at room temperature for 30 min in the dark [42]. Hydroethidine diffuses freely through the plasma membrane and converts into ethidium (E+) in the presence of superoxide anions (O2 −) [43]. Fluorescence of ethidium (E+) was detected through FL3 and that of YO-PRO-1 was detected through FL1. Four sperm populations were distinguished: (i) viable spermatozoa with low levels of superoxides (E−/YO-PRO-1−), (ii) viable spermatozoa with high levels of superoxides (E+/YO-PRO-1−), (iii) nonviable spermatozoa with low levels of superoxides (E−/YO-PRO-1+), and (iv) nonviable spermatozoa with high levels of superoxides (E+/YO-PRO-1+). Percentages of debris particles found in the SYBR14/PI test (SYBR14−/PI−) were subtracted from those of viable spermatozoa with low levels of superoxides (E−/YO-PRO-1−) and the percentages of all sperm populations were recalculated. Spill-over of FL3 into the FL1 channel was compensated (5.06%). Data are shown as corrected percentages of viable spermatozoa with high levels of superoxides (E+/YO-PRO-1<sup>−</sup>) and the geometric mean of E+-fluorescence intensity in the E+/YO-PRO-1<sup>−</sup> sperm population.

#### 2.5.5. Intracellular Calcium Levels

Previous studies found that Fluo3 mainly stains mitochondrial calcium in mammalian sperm [44]. For this reason, we combined this fluorochrome with propidium iodide (Fluo3/PI), as described by Kadirvel et al. [45]; the following four populations were identified: (i) viable spermatozoa with low levels of intracellular calcium (Fluo3−/PI−), (ii) viable spermatozoa with high levels of intracellular calcium (Fluo3+/PI<sup>−</sup>), (iii) nonviable spermatozoa with low levels of intracellular calcium (Fluo3−/PI+), and (iv) nonviable spermatozoa with high levels of intracellular calcium (Fluo3+/PI+). FL1 spill-over into the FL3 channel (2.45%) and FL3 spill-over into the FL1 channel (28.72%) were compensated.

#### *2.6. Statistical Analyses*

Statistical analyses were conducted using a statistical package (SPSS® Ver. 25.0 for Windows; IBM Corp., Armonk, NY, USA). Data were first tested for normal distribution (Shapiro–Wilk test) and homogeneity of variances (Levene test), and, if required, they were transformed with arcsin √x. The effects of the color of the straw and the extender on the response of horse sperm to red light were tested with a two-way analysis of variance (ANOVA), followed by a post hoc Sidak test. Sperm motility measures; percentages of spermatozoa with an intact plasma membrane (SYBR14+/PI−), acrosome-intact spermato-

zoa (PNA-FITC−/PI−), spermatozoa with high and intermediate mitochondrial membrane potential, viable spermatozoa with high intracellular calcium levels (Fluo3+/PI−), viable spermatozoa with high superoxide levels (E+/YO-PRO-1<sup>−</sup>), and viable spermatozoa with high peroxide levels (DCF+/PI<sup>−</sup>); and geometric mean fluorescence intensities (GMFI) of JC1agg, Fluo3+, E+, and DCF+ were analyzed.

Motile sperm subpopulations were determined through the protocol described in Luna et al. [46]. In brief, individual kinematic variables (VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF) recorded for each spermatozoon were used as independent variables in a principal component analysis (PCA). Kinematic measures were sorted into PCA components, and the obtained matrix was subsequently rotated using the Varimax method with Kaiser normalization. As a result, each sperm cell was assigned a regression score for each of the new PCA components, and these values were subsequently used to run a two-step cluster analysis based on the log-likelihood distance and Schwarz's Bayesian criterion. Four sperm subpopulations were identified, and each individual spermatozoon was assigned to one of these subpopulations (SP1, SP2, SP3, or SP4). Following this, percentages of spermatozoa belonging to each subpopulation were calculated per sample and used to determine the effects of the color of the straw and the extender on the response of horse sperm to red light through two-way ANOVA and Sidak's post hoc test.

In all analyses, the level of significance was set at *p* ≤ 0.05. Data are shown as mean ± standard error of the mean (SEM).

#### **3. Results**

As expected, no differences in variables were observed between the straws of different colors in the nonirradiated group. For this reason, and in order to simplify the presentation of data, all these results have been grouped and identified as "control" (nonirradiated samples).

#### *3.1. Plasma Membrane Integrity*

Percentages of membrane-intact spermatozoa (Figure S1a) did not differ between nonirradiated and irradiated samples. In addition, neither the color of the straw nor the type of extender had any effect on the percentages of membrane-intact spermatozoa in irradiated and nonirradiated samples (e.g., nonirradiated sperm in Equiplus extender: 46.2% ± 3.9% vs. sperm diluted in Equiplus, packed into blue straws, and irradiated: 47.9% ± 3.6% vs. sperm diluted in Kenney, packed into blue straws, and irradiated: 38.6% ± 3.0%).

#### *3.2. Acrosomal Integrity*

In a similar fashion to that observed for plasma membrane integrity, percentages of acrosomal-intact spermatozoa (Figure S1b) did not differ between nonirradiated and irradiated samples, regardless of the color of the straw or the extender (e.g., nonirradiated sperm in Kenney extender: 39.2% ± 3.1% vs. sperm diluted in Kenney, packed into red straws, and irradiated: 39.7% ± 3.2% vs. sperm diluted in Equiplus, packed into red straws, and irradiated: 49.8% ± 3.6%).

#### *3.3. Sperm Motility*

No significant differences were observed in the percentages of sperm with total (Figure S2a) and progressive motility (Figure S2b) between nonirradiated and irradiated samples when compared within each extender. In addition, neither the color of the straw nor the type of extender had any effect on the percentages of sperm with total and progressive motility when the two extenders were compared within each of the colored straws (e.g., total motility: nonirradiated sperm in Equiplus: 56.0% ± 4.5% vs. sperm diluted in Equiplus, packed into yellow straws, and irradiated: 56.5% ± 3.8% vs. sperm diluted in Kenney, packed into yellow straws, and irradiated: 56.3% ± 3.4%; progressive motility: nonirradiated sperm diluted in Kenney: 27.1% ± 2.4% vs. sperm diluted in Kenney, packed into transparent straws, and irradiated: 31.9% ± 2.9% vs. sperm diluted in Equiplus, packed into transparent straws, and irradiated: 28.4% ± 2.6%).

Regarding sperm kinetic variables (Table 1), VCL, VSL, and VAP were significantly (*p* < 0.05) higher in samples diluted in Equiplus and packed into red straws than in their respective control (nonirradiated samples). In addition, VCL and VAP were significantly (*p* < 0.05) higher in sperm diluted in Kenney extender and packed into transparent straws than in the nonirradiated control. In addition to this, STR in samples packed into blue straws and irradiated was significantly higher (*p* < 0.05) in sperm diluted in Kenney than in those diluted in Equiplus extender.

As shown in Table 2, four different motile sperm subpopulations were identified (SP1, SP2, SP3, and SP4); SP1 was characterized as the fastest subpopulation since it exhibited the highest values in VCL, VSL, and VAP. SP2 was the slowest sperm subpopulation. SP3, although characterized by intermediate speed values (but lower than SP1 and SP4) and LIN and STR values similar to SP1, was the one that showed the highest BCF. Finally, SP4 was characterized by intermediate speed values, which were higher than in SP3, but it was the least linear.

Figure 1a shows the percentages of sperm belonging to SP1. Compared to their respective controls, these percentages were significantly (*p* < 0.05) higher in irradiated samples diluted in Equiplus extender and packed into blue, yellow, or red straws and in irradiated samples diluted in Kenney extender and packed into transparent straws. Percentages of sperm belonging to SP2 were significantly (*p* < 0.05) higher in the control than in irradiated samples diluted in Equiplus extender and packed into yellow, red, or transparent straws (Figure 1b). On the contrary, no significant differences (*p* > 0.05) between nonirradiated and irradiated samples were observed for SP3 and SP4 (Figure 1c,d).

#### *3.4. Mitochondrial Membrane Potential*

As shown in Figure 2a and Figure S3, percentages of sperm with high MMP were significantly (*p* < 0.05) higher in samples diluted in Equiplus and packed into yellow, red, and transparent straws and in those diluted in Kenney and packed into transparent straws than in their respective controls. In contrast, no significant differences between extenders were observed when nonirradiated and irradiated samples packed into straws of different color were compared. With regard to the percentages of sperm with intermediate MMP, no significant differences between nonirradiated and irradiated samples were observed, regardless of the color of the straw and the extender (Figure 2b).

No significant differences in the geometric mean of JC1agg intensity (orange, FL2) of sperm populations with high (Figure 2c) and intermediate MMP (Figure 2d) were observed between nonirradiated and irradiated samples, regardless of the color of the straw and the extender used. However, the geometric mean of JC1agg intensity (orange, FL2) of the sperm population with a high MMP (Figure 2c) was significantly (*p* < 0.05) higher in samples diluted in Kenney extender and nonirradiated (control) or packed into blue, yellow, red, white, or transparent straws than in their counterparts diluted in Equiplus extender. In addition, the geometric mean of JC1agg intensity (orange, FL2) of the sperm population, with an intermediate MMP in nonirradiated samples (control), was significantly (*p* < 0.05) higher when they were diluted in Kenney than when they were diluted in Equiplus extenders (Figure 2d).

Finally, we also evaluated JC1agg/JC1mon ratios of sperm populations with high (Figure 2e) and intermediate MMP (Figure 2f). No significant differences (*p* > 0.05) were observed when comparing nonirradiated and irradiated samples, regardless of the color of the straw and extender used, either within the same diluent or when comparing the two extenders.




(%): wobble; ALH (μm): amplitude of lateral head displacement;

 BCF (Hz): beat-cross frequency.

**Figure 1.** Effects of the color of the straw, extender, and light stimulation on the structure of motile sperm subpopulations in control (nonirradiated) and irradiated samples packed into straws of different color and extended either with Equiplus or Kenney extender. (**a**) Subpopulation 1 (SP1, which was the fastest subpopulation for VCL, VSL and VAP); (**b**) Subpopulation 2 (SP2, the slowest); (**c**) Subpopulation 3 (SP3); (**d**) Subpopulation 4 (SP4). The different numbers (1, 2) indicate significant differences (*p* < 0.05) between irradiated and nonirradiated samples packed into different colored straws within the same diluent. The absence of numbers indicates the lack of statistical differences between irradiated and nonirradiated samples packed into different colored straws within the same diluent. On the other hand, no significant differences between nonirradiated and irradiated samples were observed when the two extenders were compared within the same treatment. Data are shown as mean ±SEM of 13 separate experiments.

**Figure 2.** *Cont*.

**Figure 2.** Effects of the color of the straw, extender, and light stimulation on mitochondrial membrane potential in control (nonirradiated) and irradiated samples packed into straws of different color and extended either with Equiplus or Kenney extender. The results are presented as percentages of sperm with high mitochondrial membrane potential (MMP; **a**) and with intermediate mitochondrial membrane potential (MMP; **b**), geometric mean of fluorescence intensity of JC1agg (GMFI, FL2) in sperm populations with high (**c**) and intermediate MMP (**d**), and JC1agg/JC1mon ratios (GMFI FL2/GMFI FL1) in sperm populations with high (**e**) and intermediate MMP (**f**) in nonirradiated (control) and irradiated samples. Different numbers (1, 2) indicate significant differences (*p* < 0.05) between nonirradiated and irradiated samples packed into straws of different color within the same diluent. Different letters (a, b) indicate significant differences (*p* < 0.05) between the two extenders within nonirradiated or samples irradiated and packed into straws of different color. The absence of numbers indicates the lack of statistical differences between irradiated and nonirradiated samples within the same diluent, and the absence of letters indicates the lack of differences when comparing a given treatment between both diluents. Data are shown as mean ± SEM of 13 separate experiments.

#### *3.5. Intracellular Peroxide and Superoxide Levels*

Figure 3a shows the percentage of viable sperm with high peroxide levels. No significant differences between nonirradiated and irradiated samples were observed, regardless of the color of the straw or the extender used. However, as Figure 3b shows, GMFI of DCF<sup>+</sup> in the population of viable sperm with high levels of peroxides (DCF+/PI−) was significantly higher (*p* < 0.05) in transparent, irradiated straws diluted in Equiplus extender than in their respective control (i.e., nonirradiated samples diluted in Equiplus) and transparent, irradiated straws diluted in Kenney extender.

**Figure 3.** Effects of the color of the straw, extender, and light stimulation on intracellular ROS levels in control (nonirradiated) and irradiated samples packed into straws of different color and extended either with Equiplus or Kenney extender. Data are shown as (**a**) percentages of viable spermatozoa with high peroxide levels (DCF+/PI<sup>−</sup>); (**b**) geometric mean of DCF+-intensity (GMFI, FL1 channel) in the population of viable spermatozoa with high peroxide levels (DCF+/PI−); (**c**) percentages of viable spermatozoa with high superoxide levels (E+/YO-PRO-1<sup>−</sup>); (**d**) geometric mean of E+-intensity (GMFI, FL3 channel) in the population of viable spermatozoa with high superoxide levels (E+/YO-PRO-1<sup>−</sup>). Different numbers (1, 2) indicate significant differences (*p* < 0.05) between nonirradiated and irradiated samples packed into straws of different color within the same diluent. Different letters (a, b) indicate significant differences (*p* < 0.05) between the two extenders within nonirradiated or samples irradiated packed into straws of different color. The absence of numbers or letters indicates the lack of statistical difference between irradiated and nonirradiated samples within the same diluent or when comparing a given treatment between Kenny and Equiplus extenders. Data are shown as mean ± SEM of 13 separate experiments.

As shown in Figure 3c, percentages of viable spermatozoa with high levels of superoxides (E+/YO-PRO-1−) and GMFI of E+ in the population of viable spermatozoa with high levels of superoxide (Figure 3d) did not differ (*p* > 0.05) between irradiated and nonirradiated samples, regardless of the color of the straw and the extender used.

#### *3.6. Intracellular Calcium Levels*

Percentages of viable sperm with high intracellular calcium levels (Fluo3+/PI−; Figure S4a) did not differ between nonirradiated and irradiated samples, regardless of the color of the straw and the extender used (e.g., nonirradiated samples diluted in Equiplus: 0.5% ± 0.1% vs. sperm diluted in Equiplus, packed into red straws, and irradiated: 0.8% ± 0.1% vs. samples diluted in Kenney, packed into red straws, and irradiated: 0.8% ± 0.2%). Similar results were observed for the GMFI of Fluo3<sup>+</sup> in the viable sperm population with high intracellular calcium levels (Figure S4b; e.g., nonirradiated sperm diluted in Kenney: 4.4 ± 0.2 vs. sperm diluted in Kenney, packed into white straws, and irradiated: 4.1 ± 0.2 vs. sperm diluted in Equiplus, packed into white straws, and irradiated: 4.1 ± 0.1).

#### **4. Discussion**

The results of this study agree with previous research, as irradiation with LED-based red light was found to modify some sperm motion variables and increase mitochondrial membrane potential and intracellular ROS of horse sperm without affecting the integrity of the plasma membrane and acrosome. The most remarkable and novel finding, however, was that these effects varied with the color of the straw used to pack sperm before irradiation and with the turbidity of the extender.

Regarding the effects on sperm motility, red light stimulation did not affect TMOT or PMOT, regardless of the color of the straw or the type of diluent used, which agrees with the data reported for dogs [17,18], bulls [47], and horses [20,21]. However, other studies found that irradiation of sperm with red light increases total and progressive motility in humans [7–11], buffaloes [19], sheep [4], pigs [13], and donkeys [3]. In evaluating the presence of motile subpopulations of sperm in horse ejaculates, we identified four separate subpopulations. These results are similar to those previously reported for this species [21,48]. In addition to this, we observed that the percentages of sperm belonging to SP1, which was the fastest subpopulation according to VCL, VSL, and VAP, were significantly higher in irradiated samples that were either diluted with Equiplus extender and packed into blue, yellow, and red straws or diluted with Kenney extender and packed into transparent straws. Furthermore, samples diluted in Equiplus extender and packed into red, yellow, and transparent straws showed significantly lower percentages of sperm belonging to SP2 (the slowest subpopulation) than the control. Therefore, our data confirm the results obtained in previous studies, where irradiation with red light was found to modify the structure of motile sperm subpopulations by decreasing the percentages of the slowest sperm subpopulation [21] and increasing those of the fastest one [18,21,22]. Moreover, we observed that light-stimulation increased some kinetic measures, which agrees with the data reported for other species such as humans [9,34], dogs [17,18], cattle [47], buffaloes [19], pigs [13], donkeys [3,22], and horses [20,21]. These observed differences reinforce the hypothesis that the effects of red light on spermatozoa depend on the specific irradiation pattern [3,13,22,29] and also differ between species [3,5,20–22]. At this point, the increase of VCL, VSL, and VAP observed in sperm diluted in Equiplus extender, packed into red straws, and irradiated and the increase of VCL and VAP found in semen diluted in Kenney extender, packed into transparent straws, and irradiated should be emphasized. Moreover, STR also increased in sperm diluted in Kenney extender, packed into blue straws, and irradiated. All these data suggest that the effects of red light on sperm depend on the color of the straw and the medium used. Based on these results, it is reasonable to surmise that the color of the straw and the turbidity of the extender modify the amount of light/energy that reaches the sperm cells.

At present, there is no clear explanation of how irradiation affects these sperm motion measures as the exact mechanism(s) through which red light stimulates sperm still remains unknown. However, one of the established hypotheses postulates that red light may boost mitochondrial activity, which could be relevant to explain the effects observed in sperm kinetics. Related to this, our data on the analysis of mitochondrial membrane potential (JC1) would agree with this possibility because there was an increase in the percentages of sperm with high mitochondrial membrane potential in samples packed into transparent and red straws and irradiated, regardless of the extender used (Equiplus or Kenney). This matches with Siqueira et al. [47], who found that irradiation of bovine sperm with a He-Ne laser at a wavelength of 633 nm increases the percentage of sperm cells with high mitochondrial membrane potential, and with Yeste et al. [13], who observed that irradiation with red LED light at a wavelength between 620–630 nm augments the percentages of pig sperm with high mitochondrial membrane potential. All these data suggest that red light stimulation could increase mitochondrial activity through photosensitizers present in the electronic chain, such as cytochrome C [13,22,29,49], which would underlie the increase observed in sperm motility.

In addition to the aforementioned, because ROS are mainly generated in the mitochondria as a byproduct of the electronic chain and following the previously established hypothesis, which points out that one of the first effects of light on sperm is the production of ROS [5,50], the generation of intracellular peroxide and superoxide levels was also evaluated. While sperm irradiation did not affect superoxide generation, we found an increase in the levels of peroxides in those irradiated after dilution in Equiplus and packing into transparent straws. This rise in intracellular ROS levels agrees with Zan-Bar et al. [5], Catalán et al. [20], and Cohen et al. [50], who suggested that ROS formation would be mediated through specific endogenous cellular photosensitizers such as mitochondrial cytochromes. In this sense, it has been reported that although an excess of ROS production produced by irradiation with light could be detrimental to sperm cells [5,50], low ROS levels are beneficial for sperm motility and fertilizing ability [5]. Whilst more studies are needed to set a relationship between fertilization ability and high mitochondrial membrane potential, intracellular ROS, and sperm motility, H2O2 has been suggested to be the active molecule involved in the light-mediated changes of sperm fertilizing capacity [50], which is consistent with Zan-Bar et al. [5] and de Lamirande et al. [51], who indicated that low concentrations of ROS participate in the signaling transduction pathways related to sperm capacitation and acrosomal reaction. Therefore, ROS can have both harmful and beneficial effects on sperm, and the delicate balance between the amounts of ROS produced and ROS scavengers at any time point determines whether a particular sperm function parameter is compromised or boosted [50]. In this sense, the extent of increase in intracellular ROS levels observed in this study was not enough to negatively affect sperm motility and viability, which is similar to that reported by Catalán et al. [3] in a study conducted with fresh and cooled-stored donkey semen. This increase in intracellular ROS (peroxides), observed herein after irradiation, together with the variation seen due to the color of the straw and the extender used, was concomitant with a rise in mitochondrial activity. These findings reinforce the conjecture that ROS formation caused by light would be mediated by specific endogenous cellular photosensitizers such as mitochondrial cytochromes. Furthermore, cytochrome complexes are also known to be implicated in the intrinsic apoptotic pathway [52], and both ROS generation and modulation of apoptotic-like changes are crucial to cause and control sperm capacitation [53]. Therefore, red light-induced changes in cytochrome C complex activity could ultimately affect sperm capacitation and survival. Surprisingly, however, our results did not show an increase in intracellular calcium levels, which is a crucial secondary messenger involved in the modulation of sperm motility and capacitation [54,55]. Related to this, it is worth noting that our data differ from those reported in previous studies, where light-stimulation was found to increase intracellular levels of calcium [30,50]. This could be explained by different conditions of time and intensity of radiation between the current study and the others, as previous research indicates that sperm irradiation can have stimulatory or inhibitory effects on calcium transport, depending on the intensity of the light used [56].

Regarding the effects of irradiation on the integrity of the plasma membrane, no significant differences were found between irradiated and nonirradiated samples. These results were similar to those reported by Yeste et al. [13] and Pezo et al. [14] in pigs and by Catalán et al. in horses and donkeys [3,20,22]. Similarly, no negative impact of irradiation on acrosomal integrity was observed, which concurs with previous studies in rabbits [16], pigs [13,57], and donkeys [22]. This supports the idea that under the conditions tested herein, stimulation of sperm with red light is safe and can have a positive effect on sperm motility and mitochondrial membrane potential, in agreement with Gabel et al. [32].

Finally, the differences observed in this study between straw colors and extenders with regard to mitochondrial activity, intracellular levels of peroxides, and motility suggest that these two factors also influence the sperm response to light. In fact, the impact of red light on mammalian sperm has been previously reported to rely on the precise rhythm and intensity of light [13] and the functional status of the cell [3]. In agreement with this and with the hypothesis that light acts on endogenous cellular photosensitizers of mitochondria, it is reasonable to suggest that the energy supplied to the mitochondrial electron chain by red light is proportional to the exposure time and the intensity of the light used. The final consequence of this phenomenon would be that the color of the straw and the opacity/turbidity of the medium influence the intensity of the light that feeds mitochondria, which would generate a different effect on sperm cells.

#### **5. Conclusions**

Our results confirm that LED-based red light irradiation increases some sperm motion variables, mitochondrial membrane potential, and intracellular ROS without affecting the integrity of the sperm membrane and acrosome. However, these effects vary with the color of the straw and the extender/medium used. Given that increased motility and intracellular ROS levels are concomitant with a rise in mitochondrial activity, we suggest that the impact of irradiation on sperm depends on the precise rates of energy provided by the light that feeds the mitochondria. Remarkably, such an energy rate, sensed by mitochondrial photosensitizers, varies with the color of the straw and the extender/medium used, so that these two aspects have to be taken into consideration when sperm are irradiated. In effect, as could be observed in this study, the greatest effects were obtained in samples diluted in Equiplus extender, packed into red straws, and irradiated and samples diluted in Kenney extender, packed into transparent straws, and irradiated.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-261 5/11/1/122/s1: Figures S1–S3.

**Author Contributions:** Conceptualization, S.B., J.E.R.-G., J.M., and M.Y.; methodology, J.C., I.Y.- O., S.G., and M.P.; validation, J.E.R.-G., J.M., and M.Y.; formal analysis, J.C.; investigation, J.C., S.G., M.P., J.E.R.-G., M.Y., and J.M.; resources, S.B., J.E.R.-G., J.M., and M.Y.; data curation, J.C.; writing—original draft preparation, J.C.; writing—review and editing, S.B., J.E.R.-G., J.M., and M.Y.; supervision, S.B., J.E.R.-G., J.M., and M.Y.; project administration, S.B., J.E.R.-G., J.M., and M.Y.; funding acquisition, S.B., J.E.R.-G., J.M., and M.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** J.C. was funded by the National Agency for Research and Development (ANID), Ministry of Education, Chile (Scheme: Becas Chile Doctorado en el Extranjero, PFCHA; Grant: 2017/72180128). I.Y.-O. was funded by the Secretary of Higher Education, Science, Technology and Innovation (SENESCYT), Ecuador (Scheme: Programa de Becas Internacionales de Posgrado 2019; Grant: CZ02-000507-2019). The authors also acknowledge support from the Ministry of Science and Innovation, Spain (Grants: RYC-2014-15581 and AGL2017-88329-R) and the Regional Government of Catalonia, Spain (2017- SGR-1229).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee, Autonomous University of Barcelona (Code: CEEAH 1424).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors would like to thank Sebastián Bonilla-Correal from Autonomous University of Barcelona, Spain, for his support and Marc Llavanera, Ariadna Delgado-Bermúdez, Sandra Recuero, Yentel Mateo-Otero, Beatriz Fernandez-Fuertes, Estela Garcia, and Isabel Barranco from University of Girona for their technical support.

**Conflicts of Interest:** J.E.R.-G. and M.Y. are inventors of a patent entitled "Method and apparatus for improving the quality of mammalian sperm" (European Patent Office, No. 16199093.2; EP-3-323-289- A1), which is owned by Instruments Útils de Laboratori Geniul, SL (Barcelona, Spain).

#### **References**


### *Review* **The Current Trends in Using Nanoparticles, Liposomes, and Exosomes for Semen Cryopreservation**

#### **Islam M. Saadeldin 1,2,\*, Wael A. Khalil 3, Mona G. Alharbi <sup>4</sup> and Seok Hee Lee 5,\***


Received: 19 November 2020; Accepted: 2 December 2020; Published: 3 December 2020

**Simple Summary:** Long-term preservation of semen is a pivotal step for artificial insemination in most farm animal species, but it is associated with cellular insults at the cell membrane and cytoskeleton level as well as the generation of reactive oxygen species (ROS). We highlight the recent strategies to combat these negative effects through defending against the ROS via antioxidant nanoparticles or through repairing/regenerating the damaged sperm through using liposomes and most recently exosomes derived from the reproductive tract or stem cells.

**Abstract:** Cryopreservation is an essential tool to preserve sperm cells for zootechnical management and artificial insemination purposes. Cryopreservation is associated with sperm damage via different levels of plasma membrane injury and oxidative stress. Nanoparticles are often used to defend against free radicals and oxidative stress generated through the entire process of cryopreservation. Recently, artificial or natural nanovesicles including liposomes and exosomes, respectively, have shown regenerative capabilities to repair damaged sperm during the freeze–thaw process. Exosomes possess a potential pleiotropic effect because they contain antioxidants, lipids, and other bioactive molecules regulating and repairing spermatozoa. In this review, we highlight the current strategies of using nanoparticles and nanovesicles (liposomes and exosomes) to combat the cryoinjuries associated with semen cryopreservation.

**Keywords:** nanoparticles; liposomes; exosomes; semen; cryopreservation; livestock production

#### **1. Introduction**

Semen cryopreservation contributes to genetic improvement through artificial insemination, eliminates geographical barriers in artificial insemination (AI) application, and supports the preservation of endangered breeds, thus the conservation of biodiversity. However, the sperm freezing process induces ultrastructural, biochemical, and functional changes of spermatozoa. Especially, spermatozoa membranes and chromatin can be damaged, sperm membrane permeability is increased, and hyper oxidation and formation of reactive oxygen species takes place, affecting fertilizing ability and subsequent early embryonic development [1].

Cryopreservation of mammalian sperm is a complex process affected by several factors for obtaining good quality semen for AI [2], such as type of cryoprotectants or extenders, rates of cooling and thawing, and method of packaging [3,4]. Cryopreservation is associated with damage on the level of the cell membrane, cytoskeleton, DNA, and mitochondria due to the generation of reactive oxygen species (ROS), which affect the entire cellular functions and genome instability [5]. Post-thawing trauma and cellular injury in gametes have been illustrated to affect the cell membrane, organelles, and biochemical perturbation [6]. Sperm cooling and freezing causes membrane phospholipids to accumulate due to van der Waals forces, and transition occurs from liquid crystal phase to gel phase. During thawing, irregular voids occur in the cell membrane that lead to damage to the membrane structure and irregular ion and water leakage both into and out of the cell [7].

In living organisms, generation of ROS, such as hydrogen peroxide (H2O2), superoxide anions (O2 −), and hydroxyl radicals (OH−), may be produced as a result of radiation [8], bio-activation of xenobiotics [9], inflammation [10], cell metabolism [11], decompartmentalization of transition metal ions [12], activities of redox enzymes [13], and deficit in the antioxidant defense [14,15]. Physiologically, free radicals level has a positive impact on sperm cells, including capacitation, hyper-activation, and sperm-oocyte fusion [14]. Therefore, ROS with a physiological limit are required for spermatozoa to attain the fertilizing ability [16], acrosome reaction/acrosomal exocytosis, and sperm motility [17]. However, during semen cryopreservation, the cold shock and the atmospheric oxygen [18,19] increase ROS production and cause an imbalance between free radicals and the antioxidant defense in the semen [20]. Increased ROS production can cause toxic effects in the sperm function [21], in terms of inactivating glycolytic enzymes through acrosomal damage [22], lipid peroxidation (LPO), and reducing sperm fertility [23–25]. Notably, LPO is a pathological outcome of several diseases and stress conditions [26]. The LPO process caused by ROS (H2O2) is detrimental to sperm survivability. As a result of high contents of polyunsaturated fatty acids in the plasma membrane and lack of antioxidant enzymes, mammalian spermatozoa are susceptible to LPO induced damage and loss of sperm functions [27,28]. Increasing ROS generation under oxidative stress (OS) leads to increased sperm plasma membrane failure, damaged spermatozoa [29], reduced sperm cell cytoplasm [30], and finally a marked reduction in viability, the integrity of the sperm membrane, and fertilizing ability and increased damage to sperm DNA [31].

Moreover, the process of freezing has resulted in a significant reduction in GSH content in frozen semen [32,33]. Baghshahi et al. [34] showed that cryopreservation of ram spermatozoa may cause damage to the function and structure of sperm cells, in terms of reduced semen quality and sperm characteristics. This is due to the reduction of the temperature that is associated with the OS, which has been defined as an imbalance between oxidants and cellular antioxidant mechanisms and is induced by the generation of ROS [35].

In the last few decades, most of the research work was focused on methods/approaches to improve the freezing efficiency of semen, considered to be a significant issue among reproductive biotechnologists. The approaches employed were mostly based on the protection of spermatozoa against the damaging effects of the freezing procedure, including the use of different extenders, cryoprotectant agents, antioxidants, and nutritional components. Moreover, some reports focused on the repair of the damaged spermatozoa during freezing and thawing.

There are many potential applications of nanomaterials in farm animal reproduction such as transgenesis and targeted delivery of substances to a sperm cell, antioxidants, antimicrobial properties, and special surface binding ligand functionalization as well as their application in sperm processing and cryopreservation. The antioxidant properties of some nanoparticles (NPs) are among the most promising characteristics for their application in protecting sperm cell functions during cryopreservation [36]. The use of NPs has markedly increased in various fields of animal reproduction including herd fertility issues [36]. Moreover, recent approaches showed the beneficial effects of using liposomes and extracellular vesicles (EVs) including exosomes of different origins to ameliorate the damaging effects of cryopreservation on spermatozoa. In this review, we highlight the recent strategies to defend against or repair the damage that occurs during cryopreservation of semen such as the use of nanoparticles as a defensive approach and nanovesicles including exosomes and liposomes as a repair and defense mechanism for improving the outcomes of semen cryopreservation in different animal species.

#### **2. Seminal Plasma, Antioxidants, and Their E**ff**ect on Sperm Function**

Antioxidants are compounds that scavenge or oppose the actions of ROS [37]. Antioxidants work as chelators or binding proteins, and their three main functions are to suppress the generation of ROS and eliminate ROS that are already present [38].

The antioxidant defense system includes an enzymatic mechanism in seminal plasma and sperm cells such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase. However, the nonenzymatic mechanism includes reduced glutathione (GSH), vitamins (A, C, and E), taurine, and hypotaurine. The rate of LPO in sperm cells is determined by the balance between antioxidative and pro-oxidative mechanisms in the semen [32].

Catalase and superoxide dismutase are antioxidant enzymes, which activate scavenging of ROS. Exposure of spermatozoa, primarily to anaerobic conditions during natural mating, may reduce the number of damaged spermatozoa by ROS. Female oviduct fluids contain substantial taurine levels, as it is an important protective factor of spermatozoa from ROS accumulation [39]. For instance, the catalase enzyme exists in ejaculate for the protection of the spermatozoa through the conversion of H2O2 into oxygen and water [26]. This prevents the generation of hydroxyl radicals (OH−), which are powerful oxidants, by the Fenton reaction [40]. However, bull spermatozoa contain little expression of catalase, which makes them prone to OH− toxicity [41]. Moreover, the concentration of catalase is reduced during semen processing [42]. The addition of antioxidants such as CAT in the buffalo [43], ram [33], boar [44], and bull [45–47] semen protected spermatozoa from the damaging effects of ROS and improved motility and membrane integrity during cooling storage. Elevation of the amount of H2O2 can occur as a result of abnormal sperm with residual cytoplasm or abnormal mid-piece [48]. Equine semen is rich in prostate-derived catalase, and therefore dilution or removing the seminal plasma decreases or adversely affects the scavenging capacity of the ROS [49].

Glutathione (GSH) is a tripeptide that comprises cysteine, glutamate, and glycine ubiquitously expressed in the cells. The cysteine subunit plays a pivotal role in scavenging free radicals. GSH acts as an intracellular defense against OS [50].

Exposure of semen to oxygen and visible light radiation during in vitro fertilization or AI resulted in ROS generation and damaged spermatozoa, reduced motility, and reduced membrane integrity in humans and bovines [51–53]. Under these conditions, exogenous addition of catalase, GSH, taurine, superoxide dismutase, and other antioxidants can lead to the maintenance of bovine sperm motility [52]. Supplementation of the whole milk semen extender with hypotaurine or taurine did not improve the motility of bovine spermatozoa in post-thawed semen [54]. In horses, the usage of catalase in extended semen was reported for cooled semen storage [55].

As antioxidants reduce the production of free radicals following the freeze–thaw process [56], the application of ROS scavengers is likely to improve sperm function and protect sperm from the deleterious effects of cryopreservation [57,58]. The detrimental effects of cryopreservation could be ameliorated by adding an exogenous source of antioxidants to the freezing medium to reverse OS [32]. This strategy together with other techniques for the removal of defective spermatozoa and cellular debris from semen could be used for gains in the viability of spermatozoa and reducing the necessary spermatozoa to a minimum number per AI dose [20].

#### **3. Nanoparticles (NPs)**

Several factors affect semen quality and fertilizing ability, including genetic, health, nutrition, season, stresses, and semen cryopreservation [59,60]. Multiple factors lead to poor quality semen [61]. The generation of ROS by nonviable sperm cells in the semen samples impairs sperm function [62]. To obtain good male reproduction, removing unviable or degenerated sperm cells and scavenger ROS from semen samples is important. Recent nanotechnologies reflect new prospects for developing novel and noninvasive techniques for sperm manipulation [63–65].

#### *3.1. Definition and Characterization of NPs*

NPs are molecules with <100 nm diameter and can be applied for different bioapplications including reproductive biology because they have unique physical and chemical properties [60,66].

Compared to molecules or bulk solids, there are several differences in the structural properties of the NPs [67]. The key factor of NP activity is the characteristics of their surface, such as size, charge density, and hydrophobicity [68,69]. Manipulation into a nanoform can increase the absorption and bioavailability of the functional ingredients [70]. Particle size can affect or change the properties of the original material [71]. The rapid progress in nanotechnology shows great potential for application in both medical and nutritional sciences because NPs possess unusual and advantageous properties that are different from ordinary or microscale materials in terms of their size and high surface reactivity [72]. NPs have been included in pharmaceuticals to increase the bioavailability of drugs and to target particular tissues/organs [73]. Moreover, NPs show increased cellular uptake, binding properties, and reactivity. Furthermore, the antioxidant properties of NPs recently contributed to optimizing the cryopreservation protocols [74].

Small sizes of nanoparticles have shown better integration possibilities in cellular processes and physiological pathways without interfering with the normal biological system. Nanomaterials used in drug delivery have great potential to carry large amounts and different types of biological cargo. The nanosystem abates the drug from rapid degradation and clearance through the reticuloendothelial system. The surface can be modified to react with environmental factors giving responsive drug release [75–77]. Different types of NPs are new forms of materials with promising biological properties and low toxicity and seem to have a high potential for passing through physiological barriers and accessing specific target tissues [78].

#### *3.2. Metal Nanoparticles and Sperm Cryopreservation*

Apoptosis, reduced cellular metabolism, and defective acrosome reaction are commonly caused by the increase of ROS levels [79]. Durfey et al. [80] used conjugated magnetic NPs for molecular-based selection of boar spermatozoa, and results showed that the nanoselected spermatozoa had improved motion characteristics with a higher proportion of progressive spermatozoa and straightness. Other reports [81,82] used NPs from FeO conjugated with annexin V to determine the early apoptosis of porcine and bovine sperm cells, respectively.

The use of antioxidants, such as nano-zinc oxide, can be important in reducing ROS generation and increasing sperm survival [75–77,83,84]. Using zinc nanoparticles (50 μg/mL) or selenium nanoparticles (1 μg/mL) in a SHOTOR extender enhanced morphological characteristics and ultrastructure of camel epididymal spermatozoa after cryopreservation via the reduction of apoptosis and lipid peroxidation [60].

In Holstein bulls, supplementing a semen extender with Se-NPs (1.0 μg/mL) improved post-thaw sperm quality and conception rate through reducing apoptosis, LPO, and sperm damage [85]. Moreover, in rams, Hozyen et al. [86] and Nateq et al. [87] used SeNPs (1 μg/mL) and showed improvement in motility, viability index, and membrane integrity, while acrosome defects, DNA fragmentation, and malondialdehyde (MDA) concentrations were reduced.

The addition of green synthesized gold nanoparticles (GSGNPs) (10 ppm) to a Tris-based extender improved buck semen freezing by maintaining the sperm membrane and acrosome integrity post-thawing. In addition, GSGNPs improved antioxidant capacity and consequently scavenged ROS in a buck semen extender [88]. GSGNPs are nontoxic and possess several medical applications [89]. While gold and silver NPs can penetrate the plasma membrane and can be detected inside the human sperm nucleus [90], no evidence regarding their spermatoxicity has been reported (Table 1).


**Table 1.** Summary of the current reports using nanoparticles (NPs) for semen cryopreservation.

#### *3.3. Herbal Extract Nanoparticles and Sperm Cryopreservation*

Recently, several studies examined herbal extracts as natural antioxidants and suppressors of lipid peroxidation in semen preservation of farm animals. For instance, *Moringa oleifera* leaf extract improved the antioxidative defense for cryopreserved ram and buffalo spermatozoa [96,97]. *Arctiumlappa* root extract improved spermatozoa survivability and abnormality with appropriate progressive motility when used as a supplement with cryopreserved ram semen [98]. Curcumin extract exerted antioxidative effects and improved spermatozoa post-thaw quality when used as a supplement with cryopreserved bovine and rabbit semen [95,99,100]. Moreover, *Alnusincana* bark extract [101] and *Albiziaharveyi* leaf extract [102] showed protective antioxidative effects when used as a supplement with cryopreserved ram and bovine semen, respectively. Ginger and echinacea extracts improved the spermatozoa quality and fertilization ability when used as a supplement with cryopreserved ram semen [103]. To this end, Ismail et al. [94] reported that mint, thyme, and curcumin extract nanoformulations enhanced sperm functions and redox status of post-thawed buck semen and decreased sperm apoptosis and chromatin decondensation. Supplementing the extender with curcumin nanoparticles (1.5 μg/mL) also improved the quality of post-thawed rabbit sperm by reducing apoptosis and enhancing antioxidative defense [95] (Table 1).

#### *3.4. Vitamins Nanoparticles and Sperm Cryopreservation*

Vitamin E nanoemulsions (NEs) protected red deer epididymal sperm from oxidative damage, maintained mitochondrial activity, protected the acrosome integrity, prevented cell death, and reduced ROS and LPO after OS induction (with 100 μM Fe2+/ 500 μM ascorbate) and hence improved sperm velocity and progressive motility [104].

#### **4. Artificial Exosome-Like Vesicles (Liposomes) for Semen Cryopreservation**

A liposome is a spherical nanovesicle with a single lipid bilayer that is produced artificially through disrupting plasma membranes via sonication [105]. Liposomes can be used as a vehicle for delivering nutrients and drugs to target tissues [106,107]. Liposomes can be loaded with

antioxidants such as lycopene [108] and quercetin [109] and result in a significant increase in sperm total and progressive motility as well as increased viability, plasma membrane integrity, and mitochondria activity in rooster spermatozoa. Moreover, liposomes can be loaded with lipid-related content (such as lecithin [110]) to improve the plasma membrane regeneration efficacy during the freeze–thaw process of ram spermatozoa. Liposomes were used as a cryoprotectant additives in several animal species including equine [111,112], buffalo [113], ovine [107,114,115], porcine [116], and bovine [117] with reported improvement in fertility after AI [118]. It has been proposed that liposomes with their contents of phospholipids (phosphatidylserine, dioleoylphosphatidylcholine, phosphatidylcholine, dipalmitoylphosphatidylcholine, and dimyristoylphosphocholine) and saturated and unsaturated fatty acids can fuse with the sperm plasma membrane and abate the damage to spermatozoa caused by the freeze–thaw process [119,120] (Figure 1). For instance, in rams, liposomes comprising egg-phosphatidylcholine and dipalmitoylphosphatidylcholine used as a supplement with washed spermatozoa provided immediate protection against cold shock as indicated by motility preservation [121]. Similarly, in stallions, liposomes comprising a mixture of egg phosphatidylcholine and phosphatidylethanolamine (named E80-liposomes) were efficient in preserving post-thaw sperm motility [112]. In contrast, in bovines, liposomes composed of dioleoyl-glycero-phosphocholine and dioleoyl-glycero-phospho-glycerol resulted in higher post-thaw survival, progressive motility, and acrosome reaction when compared to dioleoyl-glycero-phosphocholine alone [117]. The transition of lipid to gel phase during cooling and freezing is highly dependent on the lipid composition of the membranes, and therefore the liposome fusion facilitates lipid and cholesterol transfer, which leads to rearrangement of cell membrane components and modifies the membrane physicochemical properties, thereby improving the cryostability of the spermatozoa [117,118]. OptiXcell® is one such commercial product that uses the liposome-based commercial extender and is currently used for several animal species [122–125].

**Figure 1.** The proposed mechanism of spermatozoa protection through exosomes and liposomes. Liposomes with their contents of fatty acid can replenish the damaged sperm plasma membrane caused by freezing/thawing. Liposomes when artificially loaded with certain chemicals and exosomes with their contents of miRNA, mRNA, proteins, and metabolites can fuse and transfer their cargo into the subacrosomal space and inside the spermatozoa.

#### **5. Potential Uses of Exosomes in Semen Cryopreservation**

Extracellular vesicles (EVs) including exosomes are membrane-bounded nanovesicles containing proteins, lipids, and nucleic acids (microRNAs and mRNAs) involved in cellular communication [126,127]. A wide variety of cells release EVs in physiological and pathological circumstances [128]. EVs play major roles in numerous biological communications, including reproduction, serving as potential theranostic candidates for normal and abnormal conditions [129].

Unlike other EVs, exosomes are secreted from cells by the exocytosis pathway. Exosomes are like a snapshot of the originating cells, and the variability of the secreting cell is reflected in the exosomal compositions [126]. Once these exosomes are taken by target cells, they transfer their cargo, which includes proteins [130,131], miRNA [132,133], and mRNA [134–136], to the target cells (Figure 1). This cargo may participate in energy pathways, protein metabolism, and maintenance of recipient cells.

Thus, exosomes confer different epigenetic and phenotypic modifications on recipient cells that affect the viability, tolerance to the external factors, and regenerative capabilities of their target cells [137]. Exosomes have also been found to play important bioactive functions such as sperm maturation, capacitation, acrosome reaction, and fertilization [138]. Recent findings regarding the regenerative potential of exosomes have guided the research towards the exploitation of exosomal potential for improving the outcomes of sperm freezing [137].

Different growth factors associated with exosomes have been reported to play an active role in the repair and accelerated healing of damaged tissue [139]. Additionally, the therapeutic potential of exosomes has also been reported to be effective in arthritis, diabetes [140], immunotherapy, nervous system-related issues [141], cellular aging, and tumors [142]. Similarly, the treatment of spermatozoa with exosomes during the freezing procedure was found effective in improving the post-thaw quality of canine [137], porcine [138], and rat semen [143].

#### *5.1. E*ff*ect of Exosomes on Sperm Motility and Viability*

Motility and viability of spermatozoa are very important quality-related parameters that have a direct influence on fertility. A strong correlation was found between increasing the concentration of seminal plasma exosomes and the sperm motility and viability of boar spermatozoa [138] when preserved at liquid stage (17 ◦C for 10 days). Moreover, mesenchymal stem cell (MSC)-derived microvesicles improved the frozen/thawed quality of rat spermatozoa [143]. It has been proposed that MSC-derived microvesicles shuttle surface adhesion molecules, such as CD54 (ICAM-I), CD106 (VCAM-I), CD29 (β1-Integrin), and CD44, and consequently increase the adhesive properties of sperm [143]. This improved motility was demonstrated in liquid storage (17 ◦C) [138] as well as frozen dog [137] and rat spermatozoa [143]. The amplitude of lateral head displacement also improved in exosome-treated dog spermatozoa [137]. Interestingly, stem-cell-derived conditioned medium and exosomes improved motility, viability, mitochondrial activity, and membrane integrity post-thawing in canine semen cryopreservation [144,145].

#### *5.2. E*ff*ect of Exosomes on Sperm Capacitation and Structural Integrity*

The structural integrity of spermatozoa is considered imperative for the proper functioning and fertilization of oocytes. The structures including the plasma membrane (physiological barrier [138]), acrosome (sperm penetration), and chromatin (embryo quality [146]) affect gamete interaction and embryonic development. Damage to these structures can lead to fertilization failure. Exosomes could transfer spermadhesins (AWN and porcine seminal protein, PSP-1) to the sperm membrane that could help to maintain sperm function through inhibiting premature capacitation (decapacitation) during long-term liquid storage [138]. Similarly, exosomes derived from mesenchymal stem cells increased the fraction of sperm with an intact acrosome and increased the expression of transcripts related to the repair of the plasma membrane (ANX 1, FN 1, and DYSF) and chromatin material (H3 and HMGB 1) in frozen/thawed dog spermatozoa [137]. In bovines, oviduct-derived EVs significantly stimulated the acrosome reaction by increasing the levels of protein tyrosine phosphorylation (PY) and increasing intracellular calcium levels in frozen/thawed spermatozoa [147]. In wildlife animals (red wolves and cheetahs), oviduct-derived EVs showed improvement in sperm motility and acrosome integrity and prevented the premature acrosome reaction post-thawing [148].

Capacitation is a physiological process that enables the spermatozoa to fertilize the oocytes. Naturally, capacitation occurs during spermatozoa transit through the uterus and oviduct. In vitro storage of spermatozoa requires inhibition of premature capacitation for maintaining sperm survival [138]. The higher concentration of seminal plasma isolated exosomes significantly decreased the percentage of capacitated spermatozoa upon artificially induced capacitation using 3 mg/mL BSA [138].

#### *5.3. E*ff*ect of Exosomes on Antioxidant Capacity*

Oxidative stress is one of the major causes of low fertility of post-thaw spermatozoa [149]. A. Mokarizadeh et al. [143] reported increased antioxidant activity in frozen/thawed rat spermatozoa treated with exosomes during freezing, i.e., decreased expression of mitochondrial ROS modulator (*ROMO1*) gene in exosome-treated spermatozoa [137]. Moreover, Du et al. [138] showed increased total antioxidant capacity activity and decreased malondialdehyde content when diluents were supplemented with seminal plasma exosomes. It was hypothesized that the enhanced antioxidant capacity of spermatozoa was either due to the horizontally transferred antioxidant and other factors including mRNA and proteins from exosomes or due to the modified hydrophobic character of the membrane. Table 2 describes the available literature that used exosomes for semen preservation either in cooling or in freezing.


**Table 2.** Main literature reporting the beneficial effects obtained following the supplementation of exosomes for semen preservation.

#### **6. Conclusions**

Current trends include using nanoparticles and natural or artificial nanovesicles such as exosomes and liposomes to improve the cryopreservation of semen. Nanoparticles mostly work as antioxidants (Figure 2) with significant effects when compared with corresponding metals or herb extracts. The functional molecules present inside the exosomes such as miRNA, mRNA, and proteins (Figure 2) are involved in the proper execution of a wide variety of physiological interactions that can help resolve issues related to the fertility of male gametes. Liposomes, with their contents of phospholipids and lipid chains, can replace the damaged lipid skeletons of the frozen/thawed spermatozoa. The treatment of spermatozoa with exosomes improved the efficiency of freezing procedures; however, further in vivo and fertility studies are essential to investigating the influence of exosome treatment on sperm functions. Since liposomes are currently available as a commercial product for semen cryopreservation, nanoparticles and nanoformulations as well as EVs and exosomes derived from the reproductive tract or stem cells should adhere to the appropriate manufacturing practices, quality control measurements, and safety and efficacy protocols for commercial purposes in AI.

**Figure 2.** The overall effects of nanoparticles, exosomes, and liposomes in improving semen cryopreservation and reducing cryoinjury. Nanoparticles either from metals or from natural herbs act mainly as antioxidants, while exosomes can deliver bioactive components such as antioxidant enzymes, proteins, lipids, mRNA, and miRNA to protect sperm against cryoinjury such as that caused by reactive oxygen species (ROS) and lipid peroxidation (LPO). Liposomes can fuse with the sperm plasma membrane and replenish the damaged phospholipids caused by freezing/thawing.

**Author Contributions:** Conceptualization, I.M.S., W.A.K., and S.H.L.; Writing and editing, I.M.S., W.A.K., M.G.A., and S.H.L. Project administration, I.M.S.; funding acquisition, I.M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work is supported by the Deputyship for Research & Innovation, Ministry of Education, Saudi Arabia through the project number IFKSURP-13.

**Acknowledgments:** The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education, in Saudi Arabia for funding this research work through the project number IFKSURP-13.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Review* **Extracellular Vesicles, the Road toward the Improvement of ART Outcomes**

**Maria G. Gervasi 1, Ana J. Soler 2, Lauro González-Fernández 3,4, Marco G. Alves 5, Pedro F. Oliveira <sup>6</sup> and David Martín-Hidalgo 3,7,\***


Received: 24 October 2020; Accepted: 19 November 2020; Published: 21 November 2020

**Simple Summary:** Nowadays, the farm and pet industries cannot be sustained without assisted reproductive technologies (ART). Nevertheless, ART outcomes still are far from ideal. Recently, the emerging role of bioactive molecules—known as "extracellular vesicles" (EVs)—in the reproductive processes has been reported. EVs originate in different sections of the reproductive tract, and they can be isolated from reproductive fluids. Here, we update recent advances in the use of EVs as additive to media used in ART to enhance their reproductive outcome, mainly in domestic mammal animals.

**Abstract:** Nowadays, farm animal industries use assisted reproductive technologies (ART) as a tool to manage herds' reproductive outcomes, for a fast dissemination of genetic improvement as well as to bypass subfertility issues. ART comprise at least one of the following procedures: collection and handling of oocytes, sperm, and embryos in in vitro conditions. Therefore, in these conditions, the interaction with the oviductal environment of gametes and early embryos during fertilization and the first stages of embryo development is lost. As a result, embryos obtained in in vitro fertilization (IVF) have less quality in comparison with those obtained in vivo, and have lower chances to implant and develop into viable offspring. In addition, media currently used for IVF are very similar to those empirically developed more than five decades ago. Recently, the importance of extracellular vesicles (EVs) in the fertility process has flourished. EVs are recognized as effective intercellular vehicles for communication as they deliver their cargo of proteins, lipids, and genetic material. Thus, during their transit through the female reproductive tract both gametes, oocyte and spermatozoa (that previously encountered EVs produced by male reproductive tract) interact with EVs produced by the female reproductive tract, passing them important information that contributes to a successful fertilization and embryo development. This fact highlights that the reproductive tract EVs cargo has an important role in reproductive events, which is missing in current ART media. This review aims to recapitulate recent advances in EVs functions on the fertilization process, highlighting the latest proposals with an applied approach to enhance ART outcome through EV utilization as an additive to the media of current ART procedures.

**Keywords:** spermatozoa; oocyte; in vitro fertilization; extracellular vesicles; assisted reproductive technologies; embryo

#### **1. Assisted Reproductive Technologies and Their Handicaps**

Countless advantages can be quoted for the use of assisted reproductive technologies (ART). ART have been used to preserve valuable genetic material (cryobiology), to perform offspring sex selection (by sperm sorting), to reduce the incidence of venereal diseases (by artificial insemination (AI)), to bypass sub-fertility issues (by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI)), to increase reproductive outcomes and maximize the number of offspring that can be obtained by a single female (by inducing superovulation, performing IVF and eventually transferring embryos to female recipients by embryo transference (ET)), and to enhance reproduction of males (by increasing the performance of a single ejaculate that can be cryopreserved and then used by AI). Consequently, ART have a critical role on the management of the herd; for example, a high proportion of pigs and bovines are produced by AI [1,2].

One of the most popular ART used is IVF. Current IVF protocols are based on the basic knowledge provided in 1951 by two investigators that independently discovered that ejaculated mammalian sperm require a period of incubation in the female reproductive tract to acquire the ability to fertilize [3,4]. This phenomenon is described as sperm capacitation [5], and it settled the cornerstone for the development of IVF [6]. Twenty years after the description of sperm capacitation, the first successful IVF in mouse using a defined medium in absence of female fluids was performed [7]. Before that, successful IVF were performed by including female fluids from the reproductive tract in the incubation media [8,9]. These results highlighted the importance of factors present in female reproductive fluids to accomplish fertilization. Nevertheless, current IVF media do not differ than those developed empirically more than 50 years ago [7].

It is well documented that the quantity and quality of embryos obtained by ART are lower in comparison to those obtained in vivo by mating [10,11]. In addition, ART-derived embryos present lower chances to fully develop and derive in live offspring [10,11], underlining the need to enhance the current media used during ART protocols. It is clear that current ART procedures lack the interaction of gametes with several components present in the reproductive tract during fertilization and the first stages of development. Recent advances point out that extracellular vesicles (EVs) present in the reproductive environment help to achieve in vitro-derived embryos with development levels similar to in vivo-derived embryos. In the following sections, novel manuscripts that emphasize the roles of EVs from male and female reproductive fluids on reproductive processes are summarized. These insights on gametes and embryo production and culture must be considered to enhance the poor success rates of some of the ART procedures. For instance, an effective method for horse IVF [12] or a method to improve the less than 45 % of IVF/ICSI blastocyst rate in the ovine model [13] have not been established yet, despite decades of research.

#### **2. Extracellular Vesicles (EVs) and Their Role on ART Outcome Improvement**

Nowadays, neither the farm animal industry nor pet reproductive management can be conceived without the use of ART. However, improvement of IVF and embryo culture media are required to overcome differences in quality and developmental potential between in vivo- and in vitro-derived embryos. EVs, present in both the female and male reproductive tracts, play important roles in gamete maturation and ultimately in the fertilization process. The omission of EVs in current media used for ART is one of the causes that might explain the lower embryo quality obtained in these in vitro conditions.

EVs are defined as spherical bilayers containing proteins, genetic material and lipids that transport their cellular content (cargo) to other cells acting as intercellular communicators [14]. EVs can be classified according to the organ where are originated, in particular, for the reproductive tracts EVs are called: epididymosomes if derived from the epididymis; prostasomes if derived from the prostate; vaginosomes if derived from the vagina; uterosomes if derived from the uterus; and oviductosomes if derived from the oviduct. In addition, EVs can be classified according to their diameter size in microvesicles or ectosomes (100–1000 nm) or exosomes (30–100 nm) [15]. To avoid confusion, in this manuscript we refer to EVs based on their origin without considering the diameter size classification (see Table 1 for more classification details). In this section we focus on the role of EVs on the gain of gametes function as well as embryo development competence with special emphasis in those manuscripts whose findings are applied to the improvement of ART outcome.

**Table 1.** Classification of extracellular vesicles (EVs) according to their localization in the reproductive tract or their diameter size.


#### *2.1. Relationship between Spermatozoa and EVs as a Tool to Enhance ART Results*

The spermatozoon is an extraordinary cell that is designed to survive in different microenvironments including a body where it was not created with the ultimate goal of fertilization. Due to the large amount of protamines that replace histones, sperm chromatin is highly compacted [16]. Thus, sperm are not able to transcribe gene information, do not synthesize proteins, and therefore, regulate their function by post-translational modifications [17]. Nevertheless, sperm are surrounded by a plasma membrane; and during their maturation in the epididymis, the ejaculation process, and along their journey through the female reproductive tract, sperm can acquire information from the surrounding milieu by exchanging information with EVs found in these fluctuating environments [18]. It has been shown that EVs cargos are up taken by sperm by a fusogenic mechanism of their membranes [19,20] or by lipid-rafts domain mediated-endocytosis [21].

Sperm are produced in the testis and transported to the epididymis. Sperm maturation occurs while the sperm transit from the caput region of the epididymis towards the cauda, where they are ultimately stored until ejaculation takes place [22]. Several works described the role of epididymosomes on the sperm maturation process through the transfer of cargo proteins and small RNAs to sperm (for review see [23]) while others focused on their applied roles on ART. Here we focus on the latter (Summarized in Table 2).


**Table 2.** Effects of co-incubation of extracellular vesicles and spermatozoa on ART outcome.

List of abbreviations: EECs: endometrial epithelial cells; ASCs: adipose-derived mesenchymal stem cells; SPAM1: sperm adhesion molecule 1; PMCA: plasma membrane calcium-transporting ATPase; ↑: increase; ↓: decrease.

It has been shown in cats that epididymosomes affect sperm motility in vitro. Co-incubation of immature sperm obtained from the caput epididymis with epididymosomes for a short period of time (up to 1 h), showed a modest enhancement of total motility. Interestingly, longer exposure to epididymosomes (1.5 to 3 h) increased the percentage of sperm displaying progressive motility [33]. This could help maximize the chances of obtaining sperm with fertilization potential from epididymis of animals with high genetic value that present ejaculation issues or sudden deaths of endangered species.

At the moment of ejaculation, sperm get exposed to EVs-containing seminal fluid originated in three accessory glands: the seminiferous vesicle, the bulbourethral glands, and the prostate. Nevertheless, prostasomes are the most widely studied EVs in the seminal fluid. Once ejaculated, sperm initiate their journey through the female reproductive tract. The acquisition of hyperactivated motility, the ability to undergo the acrosome reaction, and, at the molecular level, the increase of protein tyrosine phosphorylation have been historically used as hallmarks of capacitation status [37,38].

Contradictory results have been found on the role of prostasomes on sperm capacitation. On one hand, a protective function against premature capacitation and acrosome reaction has been described on human and stallion sperm [24,25,39,40]. These findings were associated to the membrane composition of EVs with high content of cholesterol and sphingomyelin that decrease fluidity of sperm plasma membrane once the EVs and sperm fusion occurs [40,41]. The inhibition of premature capacitation might have a functional application on ART. For instance, the prevention of premature sperm capacitation is desirable when seminal doses are stored before performing AI or to counteract capacitation-like events during the sperm cryopreservation procedure [42]. In addition, an exhaustive work described that prostasomes added to boar seminal doses preserved at 17 ◦C for a long-term period was able to: (1) prolong sperm motility; (2) increase the total sperm antioxidant capacity, and; (3) protect plasma membrane integrity [31]. In that work, they also showed that prostasomes protection of sperm against premature capacitation was associated to their seminal plasma protein 1 (PSP-1) and carbohydrate-binding protein AWN (AWN) cargo. Nevertheless, prostasomes did not affect sperm ability to undergo capacitation when they were stimulated [31]. On the other hand, other authors described that in vitro incubation of boar sperm with isolated prostasomes enhanced the acrosome reaction [30]. Interestingly, qualitative but not quantitative differences were found on prostasomes of normozoospermic and severe asthenozoospermic men [43]. Prostasomes from normozoospermic men transferred cysteine-rich secretory protein 1 (CRISP1) to sperm [43]. CRISP1 is a protein with the ability to regulate murine CatSper channel enhancing the acrosome reaction induced by Ca2<sup>+</sup> ionophore [27] and also to participate in the sperm-zona pellucida binding through the interaction with ZP3 [44]. Similarly, the plasma membrane calcium ATPase pump 4 (PMCA4), a vital machinery to regulate sperm calcium homeostasis was delivered in vitro into spermatozoa either by epididymosomes [45] or by prostasomes [46]. Future research on prostasomes must explore their applied function on ART considering that variation between species might be found that could be related to differences in their evolutive reproductive strategies.

In most species, sperm are deposited in the vagina during ejaculation and spend there a short period of time before continuing to travel through the female reproductive tract. This short time in the vagina is sufficient for sperm to be exposed to vaginosomes (VGS), which could have an effect on sperm functionality. Mice sperm incubated in non-capacitating conditions exposed for 30 min to VGS presented an enhanced progesterone-induced acrosome reaction [29]. In addition, co-incubation of sperm with vaginal luminal fluid (containing VGS) resulted in sperm incorporation of SPAM1, PMCA1/4, PMCA4, all proteins with roles on calcium homeostasis and the capacitation process as well as an overall increase in sperm protein tyrosine phosphorylation [29]. The transfer of tyrosine phosphorylated proteins by VGS could explain why sperm lacking the tyrosine kinase FER do not display tyrosine phosphorylation and do not fertilize in vitro although they are able to fertilize in vivo [47].

After leaving the vagina sperm enter the uterus. In vitro studies have shown that a short exposure (15 min) of sperm with uterosomes secreted by endometrial epithelial cells simulating the time that they spend in the uterus is enough to enhance sperm capacitation status in human spermatozoa [26]. Others authors have described the same results but with longer exposure times [21].

Finally, sperm pass through the uterotubal junction and reach the oviduct where the encounter with the oocyte and fertilization take place. Here, sperm interact with oviductosomes (OVS) produced by the oviductal epithelium. The impact of OVS on sperm function has been studied in several species. In mice, the OVS cargo is incorporated into sperm and is responsible for the rise of the protein PMCA1 and an increase in tyrosine-phosphorylated proteins levels in sperm [48]. Interestingly, it was observed that capacitated spermatozoa uptake higher quantity of OVS cargo that their non-capacitated counterparts [48]. These results were associated to a higher plasma membrane permeability found in capacitated spermatozoa since they lost sterol during the capacitation process [48].

In the bovine model, OVS have been used as a supplement of non-capacitating media for thawing cryopreserved sperm. The results obtained were dependent on the OVS origin: ampulla or isthmus. After incubation with isthmus-originated OVS, sperm displayed characteristic features associated to control capacitated sperm such as high levels of protein tyrosine phosphorylation, increased acrosome reaction and intracellular calcium responsiveness to progesterone [36]. The co-incubation of sperm with ampulla-originated OVS displayed an augmented capacitation response even when compared to capacitated control [36]. In summary, incubation of non-capacitated spermatozoa with OVS induced sperm capacitation and enhanced sperm survival with similar levels to those described in capacitated spermatozoa [36].

The role of OVS on sperm function was also studied in cats. In vitro experiments showed that cat's OVS bind to the acrosomal region of the sperm head and to the mid-piece of the sperm tail. In addition, OVS used as additive to regular capacitating media enhanced the percentage of motile sperm as well as increased the rates of cleavage and blastocyst formation (23% and 8%, respectively) in comparison with control (no OVS co-incubated) [34]. The authors then investigated the cargo proteins in the OVS by mass spectrometry. The analysis of protein content of OVS identified a total of 4879 proteins, and between other functions, proteins involved on the sperm-oocyte interaction and fertilization process as cluster differentiation 9 (CD9), CCTs (cytosolic chaperonin containing TCP-1;) and TCP1 were found [34].

It has been shown in mice that EVs derived from either the epididymis or the oviduct can transfer miRNAs into the sperm [49,50]. For instance, OVS miR-34c-5p is delivered to the sperm [50] and has an important role on the fertilization process by initiating the first cleavage division. Bypassing the interaction between sperm and OVS as occurs in in vitro conditions could lead to the failure of first cleavage division. Consequently, it might negatively impact ART results where for example a single semen donor can be used to inseminate hundreds of females.

Besides the studies focusing on EVs obtained from the reproductive tract, other study evaluated the effect of EVs obtained from adipose-derived mesenchymal stem cells (ASCs) cultivated in vitro and used as additive to dog sperm cryopreservation media [35]. Surprisingly, ASCs-EV lead to a significant improvement of sperm motility and mucus penetration ability [35]. In addition, ASCs-EV protected spermatozoa against damage of the plasma membrane, the acrosome membrane, and the chromatin. The authors associated EVs beneficial effects during the freezing/thawing process to their protein and mRNA cargo associated to plasma membrane and chromatin repair process [35].

In summary, sperm receive pivotal elements through their interaction with EVs produced in the different sections of the male and female reproductive tracts (Figure 1), and these evidences should not be underestimated for the development of future enhanced sperm culture media that mimic physiological environmental conditions.

**Figure 1.** Schematic representation of the sperm travel through the female reproductive tract. Sperm enter in contact with the different extracellular vesicles produced in the vagina, the uterus and the oviduct. Once fertilization takes place, the embryo will come into contact with the EVs produced by the oviduct and the uterus where the embryo and the future fetus will remain for the rest of the pregnancy until delivery. Note that the embryo also produces EVs that allow bidirectional communication with the mother tissue (oviduct).

#### *2.2. Relationship between Oocyte Maturation and EVs Used as a Tool to Enhance ART Results*

In most mammalian species, before ovulation, oocytes are in an immature stage (germinal vesicle (GV)), in order to be competent for fertilization, they need to undergo meiotic resumption and arrive to the meiotic competence stage (metaphase II (MII)). Collection of immature GV oocytes from the ovaries followed by incubation in specific conditions that allow for the resumption of meiosis is a common practice in ART such as IVF [51]. This process is named in vitro maturation (IVM) and current IVM media described are deficient to obtain a proper oocyte maturation in some species [52,53]. For instance, the canine industry also has to address the issue of low efficiency of oocytes IVM. Thus, OVS used as additive along the canine oocyte IVM renders better results in comparison with control 21.82% and 8.66%, respectively [52]. Recently, it has been elegantly demonstrated that the percentage of mature canine oocytes after IVM in presence of OVS is enhanced through OVS cargo [54,55]. Leet et al., demonstrated that EGFR/MAPK signaling is the responsible for this improvement by the use of an inhibitor of this pathway (gefitinib) and an inhibitor of exosomes generation (GW4869) [54]. In addition, canine OVS enhance antioxidant capacity, viability and proliferation of canine cumulus cells [54].

In the ovary, oocytes are contained in follicles that will gradually mature from primordial into pre-ovulatory. The latter are follicles larger in size, filled with follicular fluid, and composed by theca and granulosa cells that surround an oocyte. It has been shown in several species that the follicular fluid contains EVs and that these EVs may have a role in cellular communication within the follicle [56,57]. In bovines, EVs from follicular fluid induce granulose cells proliferation through Src, PI3K/Akt and MAPK signaling pathways [58]. In addition, granulose cells preferentially uptake EVs from small over EVs from larger follicles [58]. The follicular fluid of mares contains EVs, and their proteins and miRNAs cargo were analyzed and described as a pathway of communication between oocytes and ovaries [56]. It was elegantly shown—in both in vivo and in vitro conditions—that EVs from follicular fluid are uptaken by the granulosa cells that surround the oocyte [56]. Interestingly, the authors described that miRNAs' EVs cargo from follicular fluid changes along with the age of the mare and this fact might explain age-related decline of oocyte quality in this species [56]. Interestingly, EVs isolated from ovarian follicular fluid used as an additive during bovine oocyte maturation and embryo development in in vitro conditions enhanced blastocyst rate and decreased global DNA methylation and hydroxymethylation levels [59]. Nevertheless, caution needs to be taken when using follicular fluid EVs as additive to enhance oocyte competence, as it was shown that the EVs cargo vary along the estrus cycle [60–62], the size of the follicle [63], and with the age of the female [56].

Another clear example of EVs used to enhance current ART was shown when EVs isolated from follicular fluid were added during the process of vitrification/thawing of immature cat oocytes, a procedure that compromises oocytes ability to undergo meiotic resumption [64]. In this case, the addition of EVs did not protect against the loss of oocyte viability during vitrification/thawing but enhanced the oocyte IVM rate after vitrification as higher numbers of oocytes arrived to MII stage (28.3%) in comparison with controls (8.6%) [64].

#### *2.3. Relationship between EVs Used as a Tool to Enhance Embryos and Conceptus Development Obtained by ART*

After ovulation, oocytes transit through the oviduct from the ampulla towards the isthmus. Fertilization occurs in the ampulla and is followed by the initiation of embryo development. Early embryo development and the transit of the embryo towards the uterus occur simultaneously. Valuable information was acquired in the 90s when it was described that embryos cultivated in groups displayed better cleavage and blastocyst formation rates than those incubated individually [65]. This fact has been associated to embryo secretion of autocrine/paracrine growth factors (secretome) that lead to a better embryo development [66,67]. In addition to the secretome, it was confirmed that EVs produced by the embryos contribute to the better embryo competence when they are cultured together [68]. Hence, information carried by embryo-derived EVs is not only important to communicate with the female tract, but also to communicate between them and achieve better embryo competence.

The oviduct has an important role in fertilization and embryo development. It is not surprising to find that the oviductal fluid of several species contains EVs [19,34,48,69,70], and as mentioned above these EVs were specifically named oviductosomes (OVS). The OVS cargo varies along the estrus cycle [48,71]. For example, OVS cargo of plasma membrane Ca2+-ATPase (PMCA), with Ca2<sup>+</sup> clearance-homeostasis role [72], changes along the estrus cycle where the PMCA levels in proestrus/estrus are higher than in metestrus/diestrus [48]. Interestingly, it was shown that the concentration and size of OVS is stable along the bovine estrus cycle; however the OVS content varies [71]. For example, higher mRNA composition was found in OVS recovered in the post-ovulatory stages when compared to OVS recovered during the rest of the cycle [71]. Differences were also found at the protein level, and the major differences were found between OVS recovered at the post-ovulatory and pre-ovulatory stages [71]. Similarly, differences were found along the estrus cycle when the protein cargo of porcine OVS was analyzed [62]. Due to the variations found during the estrous cycle, the authors hypothesized that OVS cargo changes are regulated by hormonal changes during the estrous cycle [62,71].

Primary cultures of bovine oviductal epithelial cells (BOECs) in monolayers are commonly used as an in vitro model for the study of gametes/embryo interaction with the oviduct in the bovine model [73,74]. It has been shown that BOECs secrete EVs [75]. The supplementation of the embryo culture media with BOEC-derived EVs did not affect embryo development outcome. However, these BOEC-derived EVs improved cryotolerance of embryos vitrified as they increased survival rate and number of cells, and upregulated genes related to implantation (PAG1) and metabolism (GADPH) [75]. In that work, the authors also described a negative effect of EVs present in fetal calf serum (FCS), a common component used on embryo culture media, over the bovine embryo vitrification/thawing process [75]. Oviductal region specificity was evidenced as isthmus-derived EVs were more effective than ampulla-derived EVs [76]. Interestingly, similar results were found when OVS were used to supplement in vitro bovine embryo cultures: the OVS did not have any effect on embryo development rate but improve embryo cryotolerance [76]. Almiñana and collaborators showed that the addition of oviduct-derived EVs to the culture media did not improve IVF fertilization rates; although, it enhanced the blastocyst embryo quality and the embryo hatching rate [70]. They also showed that frozen EVs had better reproductive outcome (as hatching rate) when used as additive to the embryo culture media in comparison to fresh EVs [70]. These results highlight that EVs can be frozen without any detriment in their cargo capacities simplifying the logistics of their application to different ART.

Despite similar results found by addition of BOECs-derived or oviduct-derived EVs to enhance ART outcome, it was revealed that there are qualitative and quantitative differences in the protein cargo between them [70]. A total of 319 proteins (47 only expressed in BOEC derived and 97 only expressed from oviduct derived) were identified by mass spectrometry, where only 175 of them were common to both populations [70]. Oviduct-specific glycoprotein 1 (OVGP1), a protein that enhances embryo development [77] and quality [78] was only present in in vivo EVs. One explanation for this discrepancy between in vitro and in vivo could be that static BOEC cultures induce cell dedifferentiation and therefore these cultured cells might lose some functions. Then, invaluable information could be obtained when using the novel dynamic culture system oviduct-in-a-chip developed by Ferraz an collaborators (2018). This culture system of oviductal epithelial cells allows the investigators to simulate physiological conditions by applying hormonal waves, and has shown to maintain epithelial cell differentiation and presence of cilia [79]. Interestingly, OVGP1 levels also increased when 3D-Chip were used to grow BOECs [79]. More cell culture studies using the chip strategy could help extrapolate results to in vivo conditions (see Figure 2).

**Figure 2.** Schematic representation of embryo production using a Chip-model or an in vitro model (in a dish). Hypothetically in a Chip-model, the system mimics estrus cycle hormones concentrations that allows oviductal cells to develop cilia and produce EVs that will vary their cargo along the estrus cycle, increasing the pregnancy and delivery rate. In contrast, in embryo production by classic ART in a dish there is no interaction with EVs oviduct, consequently the embryos produced have lower quality and less chance of implantation and ending in delivery.

The use of OVS in ART was proven beneficial also in other species. In rabbits, embryos co-incubated with OVS decreased reactive oxygen species (ROS) and DNA methylation levels that lead to an increase of the blastocyst development rate [80]. The authors showed that the antioxidant properties were associated to the melatonin OVS cargo by the use of luzindole, a selective melatonin receptor antagonist, that attenuated the positive effect of OVS on embryos [80]. In pigs, OVS have been used successfully to face the problem of polyspermy in porcine IVF, doubling the percentage of oocyte penetrated by a single spermatozoon [69].

Another study using a murine model described that OVS obtained from pregnant females used as additive for the IVF procedure enhanced embryo transference efficiency in comparison with the supplementation with OVS obtained from pseudo-pregnant females [81]. Pregnant females OVS increased the percentage of blastocysts, and the embryo quality determined by an increase of gene expression related to successful embryo development (Bcl-2 and Oct-4), an increased inner cell mass: trophectoderm ratio, and a decreased embryo apoptosis. In addition, birth rates after embryo transference were also enhanced [81].

An appealing application of EVs for human ART was found by the use of EVs obtained from human endometrial-derived mesenchymal stem cells (EV-endMSCs) isolated from menstrual blood [82–84]. IVF-embryos obtained from aged murine females and supplemented with human EV-endMSCs in the embryo culture media presented enhanced embryo development. Furthermore, blastocyst rate was doubled by the addition of 20 μg/mL of EV-endMSC in comparison to controls [83,84]. Positive effects of EV-endMSC in the embryo competence and quality of those embryos obtained in aged oocyte were associated to different levels of mRNA expression of genes associated to cellular response to oxidative stress (Sod1), metabolism (Gadph), placentation (Vegfa) and trophectoderm/ICM formation (Sox2) [83]. Moreover, human EV-endMSC used as additive to the culture media of mouse zygotes obtained in vivo increased the number of total cells by blastocyst and the hatching rate [82]. Recently, EVs derived from human umbilical cord mesenchymal stem cells (HUCMSCs) were successfully used to restore fertility on female mice with premature ovarian insufficiency (POI) [85]. Isolated EVs-HUCMSCs administered by only one intravenous injection rescued ovarian function, hormones levels (FSH and E2), as well as enhanced the mating behavior and the numbers of pups born after four weeks of treatment [85]. In addition, the POI group treated with EVs-HUCMSCs increased parameters associated to IVF procedures such as number of oocytes retrieved, percentage of fertilized oocytes, cleaved embryos, and blastocyst rates although did not reach values of the control group [85]. The effects of EVs on ART and embryo development are summarized in Table 3.

Once fertilization has occured, the embryo starts a series of developmental changes that orchestrate the transformation from a zygote into a blastocyst. During this period of early embryonic development, the embryo is still enclosed in the zona pellucida (ZP, a glycoproteic layer that surrounds the plasma membrane of the oocyte). Embryos in blastocyst stage will hatch from the ZP and implant by attaching and invading the uterus of the mother. Thus, embryo-uterus interaction plays vital roles in the recognition of pregnancy, maintenance of the corpus luteum, and consequently the allowing for the progression of pregnancy [86]. It has been shown that exist differences between non and pregnates mares' miRNA EVs cargo obtained from serum [87]. In adddition this EVs cargo might contribute to the pathway of maternal recognition of the pregnancy [87]. In other work it was shown that murine EVs produced by endometrium that contained miRNAs involved in the implantation process were internalized by embryo through the trophectoderm and increased embryo adhesion levels [88].

On the other hand, it was shown that conceptus-derived EVs also promote communication between the conceptus and the maternal tissue during the establishment of pregnancy in ovines [89]. In that study, the conceptus (14 days) originated in vivo were cultivated for a day in a dish and then the EVs were collected from the medium. Later on, the EVs originated by the conceptus in in vitro conditions were labelled (PKH67) and transferred to pregnant ewes. The authors showed that the embryos and the uterine epithelium of the pregnant female receptors internalized the conceptus-derived EVs [89]. In the same work, it was also shown that EVs obtained from the uterine luminal fluid are internalized by the conceptus trophectoderm and uterine epithelia [89]. This work ilustrates that embryo-derived EVs might have an impact on the receptivity of the uterus and that maternal-fetal communication could be key for a successful implantation and progression of pregnancy. Considering this, implantation failure of certain ART such as embryo transference might be related to the lack of embryo-mother communication during the first stages of development.


**Table 3.** Effects of co-incubation of extracellular vesicles and embryo on ART outcome.

List of abbreviation: Bax: BCL2-associated X protein; BOEC: bovine oviduct epithelial cells; E2: estradiol; ET: embryo transference; endMSCs: human endometrial-derived Mesenchymal Stem Cell; FSH: follicle stimulating hormone; Gadph: glyceraldehyde-3-phosphate dehydrogenase; HUCMSCs: human umbilical cord mesenchymal stem cells; ICM: inner cell mass; IVC: in vitro culture; IVM: in vitro maturation; Oct-4: transcription factor-1; POI: premature ovarian insufficiency; ROS: reactive oxygen species; Sod1: superoxide dismutase 1; Sox2: SRY-related HMG-box gene 2; Vegfa: vascular endothelial growth factor A. ↑: increase; ↓: decrease.

#### **3. Challenges for the New Era of ART Development**

Based on the broad-spectrum possibilities for EVs application, EVs have been postulated as potential non-invasive biomarkers. For example, EVs can be used as a biomarker for embryo quality [90–92] to select good embryos before transferring them to female recipients or to know the receptivity/health of a uterus before performing an embryo transference [93]. In addition, the increasing knowledge about the role of EVs on reproductive events and ART efficiency will lead to the next challenge for the biology of reproduction field: the large-scale production of EVs to use them as additives to gametes and embryo culture media. This might be addressed easily in livestock animals by the use of animal's remains from abattoirs; where moreover this strategy is in accordance with the three rules of animal welfare. Similarly, EVs can be obtained from the biological material originated in the daily practice of vet clinics, as it is the castration of domestic animals (i.e., cats and dogs). Nevertheless, both strategies are time consuming and it might present contaminations with other biological fluids. The future of EVs development as a real alternative tool to boost ART results involves the optimization of the oviduct-in-a-chip model prototype to culture in vitro embryos before transferring them to female recipients (See Figure 2).

Another possible therapeutic application of EVs for ART is to use it as cargo of information that the gametes or embryos are lacking in current ART protocols to succeed in fertilization and embryo development. It has been shown that EVs can be loaded with the desired information by electroporation [94]. Then, EVs could be preloaded with the biological deficient information found for instance in proteins of sperm of asthenozoospermic men or with those miRNAs transported by sperm that have shown to enhance embryo development.

The study of EVs for ART should also contemplate possible adverse effects of EVs on specific ART outcome. EVs overdose presented detrimental effects over embryo development due to elevated levels of ammonium that might be correlated to the EVs protein cargo in mice [81]. Negative effects of OVS overdose were also found when these EVs were used for canine oocyte IVM (less oocytes arrived to MII phase), and this effect was associated to higher levels of miR-375 [52], a microRNA that has been linked to poor oocyte quality in aged mares [56]. Consequently, EVs titration obtained from every subsection of the reproductive tract must be performed, and their effects on gametes and embryos must be determined before the universalization of the use of EVs protocols. In addition, these protocols must be adapted to the specific species under study.

#### **4. Concluding Remarks**

Although this review focused on EVs used to enhance ART outcome in mammals, it should be noted that EVs have also drawn the attention of the avian reproduction sector as it has been recently postulated that uterosomes might have a role on avian sperm survival [95]. All the information described above should be considered for the development of improved media such as gamete collection, IVF, and embryo culture media used in vitro for ART. Besides the supplementation of media with specific EVs, the possibility of re-use EVs should be explored. For instance, once an in vitro embryo culture is finalized, the EVs contained in the media could be recovered, isolated and used as additive for a new culture media in those females with a record of low oocytes and embryo quality.

We expect that researchers working in the reproductive field will increase their interest in the study and use of EVs. This is a fundamental step for the development of new tools that could improve ART outcomes. From our point of view, the broad applications of EVs will have a lasting impact on the field of reproductive biotechnology.

**Author Contributions:** Conceptualization, M.G.G., A.J.S. and D.M.-H.; writing—original draft preparation, M.G.G. and D.M.-H.; writing—review and editing, A.J.S., L.G.-F., P.F.O. and M.G.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** David Martín-Hidalgo was recipient of a post-doctoral grant from the Government of Extremadura (Spain) and "Fondo Social Europeo"; Reference: PO17020. L. González-Fernández was supported by the regional grant "Atracción y retorno de talento investigador a Centros de I+D+i pertenecientes al Sistema Extremeño de Ciencia, Tecnología e Innovación" from "Junta de Extremadura" (Spain); Reference: TA18008.

**Acknowledgments:** Figures were made using www.biorender.com software.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Article* **Selection of Boar Sperm by Reproductive Biofluids as Chemoattractants**

**Luis Alberto Vieira 1,2, Alessia Diana 1,2, Cristina Soriano-Úbeda 1,2,3 and Carmen Matás 1,2,\***


**Simple Summary:** Both in natural breeding and some assisted reproduction technologies, spermatozoa are deposited into the uterus. The journey the spermatozoa must take from the place of semen deposition to the fertilization site is long, hostile, and selective of the best spermatozoa. For the fertilization to succeed, spermatozoa are guided by chemical stimuli (chemoattractants) to the fertilization site, mainly secreted by the oocyte, cumulus cells, and other substances poured into the oviduct in the periovulatory period. This work studied some sources of chemotactic factors and their action on spermatozoa functionality in vitro, including the fertility. A special chemotactic chamber for spermatozoa selection was designed which consists of two wells communicated by a tube. The spermatozoa are deposited in well A, and the chemoattractants in well B. This study focuses on the use of follicular fluid (FF), periovulatory oviductal fluid (pOF), conditioned medium from the in vitro maturation of oocytes (CM), and progesterone (P4) as chemoattractants to sperm. The chemotactic potential of these substances is also investigated as related to their action on CatSper which is a calcium channel in the spermatozoa known to be sensitive to chemoattractants and essential for motility.

**Abstract:** Chemotaxis is a spermatozoa guidance mechanism demonstrated in vitro in several mammalian species including porcine. This work focused on follicular fluid (FF), periovulatory oviductal fluid (pOF), the medium surrounding oocytes during in vitro maturation (conditioned medium; CM), progesterone (P4), and the combination of those biofluids (Σ) as chemotactic agents and modulators of spermatozoa fertility in vitro. A chemotaxis chamber was designed consisting of two independent wells, A and B, connected by a tube. The spermatozoa are deposited in well A, and the chemoattractants in well B. The concentrations of biofluids that attracted a higher proportion of spermatozoa to well B were 0.25% FF, 0.25% OF, 0.06% CM, 10 pM P4 and 0.25% of a combination of biofluids (Σ2), which attracted between 3.3 and 12.3% of spermatozoa (*p* < 0.05). The motility of spermatozoa recovered in well B was determined and the chemotactic potential when the sperm calcium channel CatSper was inhibited, which significantly reduced the % of spermatozoa attracted (*p* < 0.05). Regarding the in vitro fertility, the spermatozoa attracted by FF produced higher rates of penetration of oocytes and development of expanded blastocysts. In conclusion, porcine reproductive biofluids show an in vitro chemotactic effect on spermatozoa and modulate their fertilizing potential.

**Keywords:** sperm selection; chemotaxis; reproductive fluids; IVF; IVC; porcine

#### **1. Introduction**

Assisted reproductive technologies (ART) have been widely used in humans and farm animals in the last decades. Even though the female plays a critical role in the success of an ART program, we cannot ignore that the spermatozoa preparation has been essential to selecting the best and most capable spermatozoa population in an ejaculated sample [1],

**Citation:** Vieira, L.A.; Diana, A.; Soriano-Úbeda, C.; Matás, C. Selection of Boar Sperm by Reproductive Biofluids as Chemoattractants. *Animals* **2021**, *11*, 53. https://doi.org/10.3390/ ani11010053


Received: 5 December 2020 Accepted: 21 December 2020 Published: 30 December 2020

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**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**<sup>\*</sup>** Correspondence: cmatas@um.es

thus contributing to the increasing success of ART. However, the efficiency of many ART is still low, in part due to spermatozoa quality which directly influences fertilization and embryo development.

In standard methodologies of in vitro fertilization (IVF) in mammals, both male and female gametes are co-cultured without barriers regulating their interaction which causes a high number of spermatozoa to be simultaneously at the place of fertilization. Consequently, increased rates of polyspermy (more than one spermatozoon penetrating the oocyte) are achieved, compromising the embryo development. This is of particular interest to the porcine species, representing one of the major detrimental factors for an efficient and successful porcine IVF (reviewed by Romar et al. [2]).

There are several mechanisms within the female reproductive tract that regulate the number and quality of spermatozoa reaching the fertilization site to reduce the risk of polyspermy [3]. The in vitro simulation of the physico-chemical conditions in the oviduct at the time of fertilization can reduce the high incidence of polyspermy in porcine animals [4].

Under in vivo conditions, only progressively motile spermatozoa will move toward the oocytes. Current sperm selection assays based on sperm chemotaxis towards progesterone (P4) provide a sperm subpopulation enriched with spermatozoa that are capacitated, with intact DNA and low levels of oxidative stress [1]. Since the selected subpopulation of spermatozoa are in an optimum physiological state, it would be reasonable to suggest that the application of spermatozoa selection methods may improve the efficiency of the current ART. Despite the fact that P4 has been one of the most studied chemoattractant agents, other substances have also been shown to have this effect such as atrial natriuretic peptide (ANP), heparin, adrenaline, oxytocin, calcitonin, acetylcholine, and nitric oxide [5,6]. On the other hand, it has been suggested that estradiol (E2), cyclic AMP (cAMP) and cyclic GMP (cGMP) could be essential for chemotaxis because they increase the levels of intracellular calcium (Ca2+) in the sperm [7–9].

Villanueva-Diaz et al. [10], showed for the first time that crude human follicular fluid (FF) produced a chemical attraction for spermatozoa. FF increases the in vitro motility of spermatozoa especially since the original follicle is mature and stimulates capacitation and the acrosomal reaction of human sperm [11]. The presence of chemotactic agents has been shown in the FF of several species [5]. Therefore, FF facilitates spermatozoa reaching the fertilization site. Likewise, oviductal fluid (OF) and secretions of the cumulus cells (conditioned medium, CM) have been demonstrated to have chemotactic components [12–15].

In an in vitro chemotactic system, it is important to consider the optimum concentration at which a chemoattractant has an effect. A typical chemotaxis response is represented by a bell-shaped curve [12–14,16,17]. The present work analyzed the chemotactic effect of different biofluids (FF, OF, CM) and P4 individually or in combination on spermatozoa as well as their potential as sperm fertility modulators.

#### **2. Material and Methods**

#### *2.1. Ethics Statement*

The study was carried out in accordance with the Spanish Policy for Animal Protection RD 53/2013, which meets the European Union Directive 2010/63/UE on animal protection. All experimental protocols were approved by the Ethical Committee of Animal Experimentation of the University of Murcia and by the Animal Production Service of the Agriculture Department of the Region of Murcia (Spain) (ref. no. A13160609).

#### *2.2. Reagents, Culture Media, and Solutions*

All the chemicals used in this study were purchased from Sigma-Aldrich Química S.A. (Madrid, Spain) unless otherwise indicated. Tyrode's albumin lactate pyruvate medium (TALP; [18]) was supplemented with 1.10 mmol/L sodium pyruvate and 3 mg/mL bovine serum albumin (BSA; A9647). Dulbecco's PBS (DPBS) was supplemented with 1 mg/mL PVA and 0.005 mg/mL red phenol. North Carolina State University 37 medium (NCSU37; [19]) was supplemented with 0.57 mmol/L cysteine, 1 mmol/L dibutyryl

cAMP, 5 μg/mL insulin, 50 μmol/L β-mercaptoethanol, 1 mmol/L glutamine, 10 IU/mL equine chorionic gonadotropin (eCG; Foligon; Intervet International BV, Boxmeer, Holland), 10 IU/mL human chorionic gonadotropin (hCG; Veterin Corion; Divasa Farmavic, Barcelona, Spain), and 10% (*v*/*v*) porcine FF [20]. A fixation solution of glutaraldehyde was prepared at 0.5% in phosphate-buffered saline (PBS). A staining solution of bisbenzimide (Hoechst 33342; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, H1399) was prepared at 1% (*w*/*v*) in PBS. The P4 (P8783) solution was prepared in DMSO (D2650) at 1mg/mL and frozen at −20 ◦C until use. The solution of the CatSper channel inhibitor NNC 55-0396 (NNC; Cayman 17216) was prepared in DMSO at 8.8 mM and frozen at −20 ◦C until use.

#### *2.3. Spermatozoa Collection and Preparation*

Fresh ejaculated spermatozoa were obtained by the manual method from mature and tested fertility boars. Once in the laboratory, the spermatozoa were separated from the seminal plasma (SP) by centrifugation on a discontinuous density gradient (Percoll®, Pharmacia, Uppsala, Sweden) 45/90% (*v*/*v*) at 700× *g* for 30 min [21]. The pellet was resuspended in TALP medium previously equilibrated in a humified atmosphere of 5% CO2 in air at 38.5◦C and centrifuged at 700× *g* for 5 min. Finally, the pellet of spermatozoa was resuspended in 5 mL of fresh TALP medium and the concentration of spermatozoa/mL was determined.

#### *2.4. In Vitro Maturation (IVM) of Oocytes*

Ovaries were obtained from prepuberal gilts at the local slaughterhouse and transported to the laboratory in saline solution at 38.5 ◦C within 1 h after the death of the animals. Cumulus oocyte complexes (COCs) were collected from antral follicles (3–6 mm diameter) and in vitro matured as described previously [4].

#### *2.5. Follicular Fluid (FF), Periovulatory Oviductal Fluid (pOF) and Conditioned Medium (CM) Collection and Preparation*

The FF was obtained making a pool by aspirating the liquid content of antral follicles (3–6 mm diameter) of prepuberal gilts ovaries, as described previously [22]. Periovulatory oviductal fluid (pOF) was obtained from a pool of porcine oviducts with ovaries close to ovulation according to the classification of Carrasco et al. [23]. The conditioned medium (CM), consisting of the pooled secretions of the cumulus cells, was obtained by pippeting NCSU37 medium from wells where groups of 50 COCs had completed the second phase of IVM (without dbAMPc, eCG and hCG) [4]. After collection, FF, pOF and CM were centrifugated at 7000× *g* and 4 ◦C for 10 min. After centrifugation, the supernatants were collected discarding the cellular debris and/or mucus at the bottom of the centrifuge tubes. All fluids, FF, pOF, and CM were aliquoted and frozen at −20 ◦C until use, avoiding repeated freezing-thawing cycles.

#### *2.6. HPLC-MS Analysis*

The separation and analysis of samples were performed with a HPLC-MS system consisting of an Agilent 1290 Infinity II Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an Automated Multisampler module and a High-Speed Binary Pump and connected to an Agilent 6550 Q-TOF Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA) using an Agilent Jet Stream Dual electrospray (AJS-Dual ESI) interface.

Standards or samples (20 uL) were injected onto an Agilent Zorbax Eclipse Plus C18 (2.1 × 100 mm, 1.8 um) HPLC column, at a flow rate of 0.4 mL/min. The column was equilibrated at 40 ◦C. Solvents A (MilliQ water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid) were used for the compound separation with the following elution program: 2 min at 3% B, linear gradient from 3 to 100% B in 9 min, and 1 min at 100% B.

The mass spectrometer was operated in the positive mode. Extracted ion chromatograms of the following compounds were analyzed: 273.1849 > 255.1749 m/z for

β-estradiol (E2), 315.2319 > 109.0660 for P4, 330.0566 > 136.0623 for AMPc and 346.0547 > 152.05686 for GMPc.

#### *2.7. Sperm Chemotaxis System*

For this study, a new chemotaxis chamber for spermatozoa was designed (Figure S1). It consisted of two 500 μL wells with stoppers connected by a tube of 250 μm in diameter and 850 μm in length. Spermatozoa suspension in TALP were added to well A in a final concentration of 20 × <sup>10</sup><sup>6</sup> spermatozoa/mL. The chemoattractants under study were added to well B in TALP. This system was kept for 20 min in a humified atmosphere of 5% CO2 in air at 38.5 ◦C. After that incubation, the percentage of spermatozoa recovered in well B were determined.

#### *2.8. Spermatozoa Motility*

Spermatozoa motility was evaluated by computer assisted semen analysis (CASA) using the ISAS® system (PROISER R + D S.L., Valencia, Spain), as protocolized in our laboratory [24]. The motion parameters determined into 3 different fields per sample were: the percentage of total motile spermatozoa (Mot, %), motile progressive spermatozoa (MotPro, %), curvilinear velocity (VCL, μm/s), straight line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity of the curvilinear trajectory (LIN, ratio of VSL/VCL, %), straightness (STR, ratio of VSL/VAP, %), wobble of the curvilinear trajectory (WOB, ratio of VAP/VCL, %), amplitude of lateral head displacement (ALH, μm), and beat crossfrequency (BCF, Hz).

#### *2.9. Spermatozoa Plasma Membrane Integrity*

The plasma membrane integrity was analyzed by eosin-nigrosin staining as a reflection of spermatozoa viability, as described by Soriano-Úbeda et al. [24]. The percentage of membrane-intact spermatozoa (non-stained spermatozoa) were determined and considered viable spermatozoa.

#### *2.10. In Vitro Fertilization (IVF) and Embryo Development (IVC)*

The matured oocytes from IVM were mechanically denuded, washed in fresh TALP, and transferred in groups of 50 oocytes to 4-well plates (Nunc, Roskilde, Denmark) with 500 μL/well TALP. The insemination of oocytes was carried out giving a final concentration of 25 × 103 spermatozoa/mL. The spermatozoa for insemination were those recovered in well B of the chemotactic system. Eighteen hours after insemination, putative zygotes were fixed in a 0.5% glutaraldehyde solution, stained in a bisbenzimide solution (Hoechst 33342) and examined under an epifluorescence microscopy (Leica® DMR, USA), as it has been previously described [25]. The IVF parameters analyzed were the percentage of penetrated oocytes (Pen, %), percentage of monospermy of penetrated oocytes (Mon, %), mean number of spermatozoa per penetrated oocyte (Spz/O) and mean number of spermatozoa bound to zona pellucida (Spz/ZP). For the embryo development assessment, 18 h after insemination, putative zygotes were transferred to a culture dish for 7 days (168 h), as previously described [20]. For the first 48 h, the media used for IVC was NCSU23 [19]. At 48 h after the addition of spermatozoa to the chemotactic system, the cleavage was assessed under the stereomicroscope. On day 7 (168 h after the addition of spermatozoa to the chemotactic system), the blastocyst stage of development was assessed under the stereomicroscope and classified as in Bó and Mapletoft [26]. Blastocysts and expanded blastocysts were fixed and stained as described for putative zygotes, and the mean number of blastomeres was determined under an epifluorescence microscope (Leica® DMR, Richmond, IL, USA).

#### *2.11. Experimental Design*

The present study investigated the chemotactic ability of the female reproductive fluids (FF, pOF, CM) and P4 on the selection of porcine spermatozoa and the fertility of those selected spermatozoa (Figure 1). Firstly, the characterization in content of E2, P4, cAMP, and cGMP in each pool of biofluids used in the present work was carried out. The concentration of those components was measured twice by HPLC-MS in the pools of FF, pOF and CM, and the results are shown in Table 1. Once characterized, the same pools of biofluids were used in the two experiments of this work.

**Table 1.** Concentration of β-estradiol (E2), progesterone (P4), 3 ,5 -cyclic adenosine monophosphate (cAMP), and guanosine 3 -5 -cyclic monophosphate (cGMP) in reproductive biofluids used as chemoattractants. HPLC-MS was performed twice in each pool of biofluids.


FF: Follicular fluid; pOF: periovulatory oviductal fluid; CM: secretions of the cumulus cells. Results are expressed as mean ± standard deviation (SD).

#### Experiment 1. Spermatozoa selection by FF, pOF, CM, and P4

(1.1) Effective concentration of chemoattractants. Increasing concentrations of the chemoattractants (FF at 0.13, 0.25, 0.50, 1.00 and 1.50%; pOF at 0.13, 0.25, 0.50, 1.00 and 1.50%; CM at 0.03, 0.06, 0.13, 0.25 and 0.50%; P4 at 1.00, 2.50, 5.00, 7.50 and 10.00 pM) were added to well B of the chemotactic system. One experimental group in which there was no supplementation with any chemoattractant was included as control (C). Spermatozoa were added to the well A and incubated for 20 min. The percentage of spermatozoa recovered in well B at the end of the incubation period was determined, and the most effective concentration of each chemoattractant was established as the lowest concentration that attracted the highest proportion of spermatozoa. Four replicates were performed.

(1.2) Chemotactic potential of chemoattractants. Since all the chemoattractants coexist in combination in the female reproductive tract during the periovulatory stage, this work compared the effective concentration of the chemoattractants (FF, pOF, CM, and P4) and their possible synergy. For this purpose, a combination of the most effective concentrations of FF, pOF and CM were studied as the summation of the most effective concentrations of chemoattractants obtained in experiment 1.1 (Σ1 = 0.25% FF + 0.25% pOF + 0.06% CM). Due to the possible saturation of receptors [27], a second Σ group (Σ2) was included in which the total proportion of chemoattractants was limited to 0.25%. The proportion of each chemoattractant was maintained at 1/3 of the total 0.25% of biofluids in the chemotactic system (Σ2 = 1/3 of 0.25% FF + 1/3 of 0.25% OF + 1/3 of 0.06% CM). The chemotactic potential of chemoattractants and their combination was compared. A group in which no chemoattractants were added to well B was included as a control (C). Four replicates were performed.

(1.3) Motility of spermatozoa selected by chemoattractants. The possible influence of chemoattractants in motility of spermatozoa was studied in those spermatozoa selected in the chemotactic system by the chemoattractants FF, pOF, CM, P4 and Σ2. A group in which no chemoattractants were added to well B was included as a control (C). Four replicates were performed.

of

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 recovered in well B attracted by each

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 recovered in well B, it was also determined

 recovered, motility, in vitro fertility, and in vitro

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 the effective

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 selected by follicular fluid (FF)]. With the % of

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 through

(1.4) Mechanism of action of chemoattractants. The P4 triggering of Ca2+ uptake into the spermatozoa takes place through CatSper [28] and seems to play a key role in chemotaxis [14,29–32]. To elucidate P4 dependence on the chemotactic potential, spermatozoa selected by the most effective concentration of each chemoattractant (FF, pOF, CM, P4) were compared to the selected spermatozoa preincubated for 10 min with 2 μM of the CatSper inhibitor NNC 55-0396 [28] (FF , pOF , CM , P4 ). The most effective combination of chemoattractants (Σ2 and Σ2 ) were included in this study, and groups without chemoattractant supplementation in well B were included as controls (C and C ). Four replicates were performed. Additionally, the possible collateral effect of NNC on spermatozoa motility by CASA and the membrane integrity was evaluated in three replicates. For the membrane integrity study, 200 spermatozoa per experimental group and replicate were analyzed.

Experiment 2. In vitro fertility of spermatozoa selected by chemotaxis and in vitro development of embryos.

To study the functionality of the selected spermatozoa, IVF was performed with those spermatozoa recovered in well B of the chemotactic chamber. The experimental groups were established according to the chemoattractant used for sperm selection: 0.25% FF, 0.25% pOF, 0.06% CM, 10.00 pM P4, and Σ2. One group in which there was no supplementation with any chemoattractant in well B was included as control (C). A total of 702 oocytes in four replicates were inseminated, and they were fixed at 18 h post-insemination for IVF parameter evaluation. The experimental group with higher penetration in IVF, the FF, was tested also in IVC. For that purpose, a total of 1,520 oocytes in six replicates were inseminated and 18 h post-insemination they were transferred to NCSU23 medium for up to 7 days of culture.

#### *2.12. Statistical Analysis*

The statistical analyses were performed using SPSS v.20 (SPSS Inc. Chicago, IL, USA). The variables were analyzed by analysis of variance (ANOVA). When ANOVA revealed a significant effect, values were compared by the least significant difference pairwise multiple comparison Tukey post hoc test. The results were expressed as the mean ± standard error of the mean (SEM) and *p* < 0.05 was established to indicate statistical significance.

#### **3. Results**

#### *3.1. Concentration of E2, P4, cAMP and cGMP in FF, pOF and CM*

Despite chemotaxis being mainly attributed to P4, other components have been suggested as potential chemoattractants in the porcine species. This work characterized the pools of biofluids used in all experiments through the concentration of some molecules responsible for chemotaxis in some reproductive biofluids (Table 1). FF showed the highest amount of E2, P4, and cGMP, followed by pOF which showed the highest amount of cAMP. CM showed the lowest cAMP values but higher than pOF for E2 and P4. The concentration of cGMP was higher in FF than in E2, P4, and cAMP. cGMP represented the main component in pOF, also, followed by cAMP, P4, and E2. On the other hand, CM was richer in E2 than in the rest of the components analyzed. Interestingly, P4 was the third highest component in concentration of each biofluid analyzed.

#### *3.2. Effective Concentration of Chemoattractants Selecting Spermatozoa*

High concentrations of chemoattractants can saturate the cognate receptors and consequently the chemotactic response decreases. The results of the percentage of spermatozoa recovered in well B from those initially added to well A (called most effective concentration) for each chemoattractant are shown in Figure 2. The most effective concentration (*p* < 0.05) of each chemoattractant was: 0.25% FF (7.6 ± 1.6%), 0.25% pOF (8.4 ± 1.0%), 0.06% CM (3.3 ± 0.7%), 10.00 pM P4 (9.6 ± 1.6%).

**Figure 2.** Effective concentration of chemoattractants. Increasing concentrations of the chemoattractants were added to well B of the chemotactic system. A group in which there was not a supplementation of any chemoattractant in well B was included as control (C) for each chemoattractant. Spermatozoa were added to well A in a concentration of <sup>20</sup> <sup>×</sup> <sup>10</sup><sup>6</sup> spermatozoa/mL. The system was incubated for 20 min, 38 ◦C, 5% CO2 and 95% humidity. The results are expressed as the percentage of spermatozoa recovered in well B. The most effective concentration of each chemoattractant was established as the lowest concentration that attracted the highest proportion of spermatozoa. Four replicates were performed. Asterisks (\*, \*\*) indicate statistical differences between groups within the same chemoattractant (*p* < 0.05).

#### *3.3. Chemotactic Potential of Chemoattractants*

The possible synergies between chemoattractants on spermatozoa are shown in Figure S2. The combination of a lower concentration of each chemoattractant (FF, pOF, CM) to a final concentration of 0.25% (Σ2) attracted a higher percentage of spermatozoa (Σ2 = 12.3 ± 1.3%; *p* < 0.05) than Σ1 (Σ1 = 8.0 ± 2.1%) and C (C = 8.9 ± 1.8%).

The results of the chemotactic potential of chemoattractants are shown in Figure 3. The % of spermatozoa recovered when a chemoattractant (FF: 6.5 ± 1.0%; pOF: 6.0 ± 0.9%; CM: 6.7 ± 0.8%; P4: 6.6 ± 0.6%; Σ2: 6.1 ± 0.5%) was added to well B was higher (*p* < 0.05) than the control (C: 4.4 ± 0.4%).

**Figure 3.** Chemotactic potential of chemoattractants. The percentage of spermatozoa attracted by the most effective concentration of each chemoattractant (0.25% FF, 0.25% pOF, 0.06% CM, 10.00 pM P4) was determined. The combination of the most effective concentration of chemoattractants (Σ2) according to experiment 1.1 was included in this study. A group in which there was not a supplementation of any chemoattractant in well B was included as control (C). The results are expressed as man ± SEM. Four replicates were performed. The asterisk (\*) indicates significant statistical differences (*p* < 0.05).

#### *3.4. Motility of Spermatozoa Selected by Chemoattractants*

The motility of spermatozoa selected by the effective concentration of each chemoattractant is shown in Table 2. No statistical differences were found for any of the CASA parameters (*p* > 0.05).

#### *3.5. Mechanism of Action of Chemoattractants through CatSper*

Figure 4 shows the results of the concentrations of biofluids with higher chemotactic activity and it also shows that the chemotactic activity is related to CatSper. There was no difference in the % of spermatozoa attracted between chemoattractants or their combination (FF, pOF, CM, P4, Σ2; *p* > 0.05), or even when the spermatozoa were previously incubated with NNC (FF , pOF , CM , P4 , Σ2 ; *p* > 0.05). However, in both cases the % spermatozoa attracted were higher than their respective controls C and C . The chemotactic action of all chemoattractants through CatSper was demonstrated by the reduction of the % of spermatozoa attracted by all chemoattractants until the point of being statistically similar to the control C. In other words, all chemoattractants in which spermatozoa had CatSper inhibited by NNC attracted the same % of spermatozoa as in the absence of chemoattractants. When comparing the effect of NNC on the controls (without chemoattractants), in C a significantly lower proportion of spermatozoa were recovered in well B as compared to C (*p* < 0.05). Additionally, Table S1 and Figure S3 showed that the motility and membrane integrity of spermatozoa is not affected by NNC.

**Figure 4.** Chemotactic potential of chemoattractants and mechanism of action through CatSper. The percentage of spermatozoa attracted by the most effective concentration of each chemoattractant (0.25% FF, 0.25% pOF, 0.06% CM, 10.00 pM P4) was determined. That parameter was compared Table 10. min with 2 μM of the CatSper inhibitor NNC 55-0396 [28] (FF , pOF , CM , P4 ). The combination of the most effective concentration of chemoattractants according to experiment 1.1 (Figure S2; Σ2 and Σ2 ) was included in this study. The groups in which there was not a supplementation of chemoattractant in well B for selecting spermatozoa were included as control (C and C ). The results are expressed as man ± SEM. Four replicates were performed. Different letters (a–c) indicate statistical differences between groups (*p* < 0.05).



BCF (Hz): beat

cross-frequency.

 Two-way ANOVA and multiple pairwise Tukey test (*<sup>p</sup>* < 0.05) was carried out. Results are expressed as mean ± SEM.

#### *3.6. Spermatozoa Selected by Chemotaxis to the Follicular Fluid (FF) Increases the Penetration of Oocytes In Vitro and the Rate and Quality of Blastocysts Production*

During in vivo fertilization in the oviduct, the sperm must be guided towards the oocyte by different mechanisms, one of them is chemotaxis. Female reproductive biofluids can guide the spermatozoa and probably modulate their functionality, fertility, and potential in forming viable embryos. Table 3 shows the results of IVF with spermatozoa selected by chemotaxis. Spermatozoa selected by FF produced a higher % Pen (*p* < 0.05) but the same % Mon (*p* > 0.05) when compared to the rest of chemoattractants. In addition, spermatozoa selected by FF produced the highest proportion of spermatozoa bound to ZP (Spz/ZP), which was statistically the same to that obtained in C (*p* > 0.05). On the contrary, the spermatozoa selected by pOF and Σ2 showed the lowest % Pen, and Σ2 also produced the lowest Spz/ZP (*p* < 0.05).

**Table 3.** In vitro fertilization (IVF) with spermatozoa selected by chemotaxis to reproductive biofluids in the chemotaxis system. The effective concentration of chemoattractants were added to well B of the chemotaxis system: Follicular fluid (FF), periovulatory oviductal fluid (pOF), secretions of the cumulus cells (CM) progesterone (P4), and a combination of chemoattractants (Σ2 = 1/3 of 0.25% FF + 1/3 of 0.25% pOF + 1/3 of 0.06% CM). Spermatozoa (20 <sup>×</sup> 106 cells/mL) were added to well A, and the system was incubated for 20 min. A group in which spermatozoa were selected without chemoattractant was used as the control (C). Spermatozoa recovered in well B were used to perform the IVF at 25 <sup>×</sup> <sup>10</sup><sup>3</sup> spermatozoa/mL. Eighteen hours after insemination, putative zygotes were fixed with glutaraldehyde and stained with the bisbenzimide solution. Evaluation was carried out by fluorescence microscopy. Four replicates were performed.


Pen (%): percentage of oocytes penetrated; Mon (%): percentage of oocytes penetrate by one spermatozoon; Spz/O: mean number of spermatozoa penetrating one oocyte; Spz/ZP: mean number of spermatozoa bound to the zona pellucida. One-way ANOVA was performed and the Tukey test of multiple comparisons. Results are expressed as the mean ± SEM. Different superscript (a–d) within the same column indicate significant differences between experimental groups (*p* < 0.05).

Since the chemoattractant with the higher % Pen in IVF was FF, this group was also tested for IVC and the results are shown in Table 4. Although spermatozoa selected by FF did not modify the % of cleaved putative zygotes (2-cells; *p* > 0.05), they produced a significantly higher (*p* < 0.05) % of blastocysts (38.5 ± 3.7%) and expanded blastocyst (36.0 ± 2.5%) compared to the control group (27.6 ± 3.8% and 27.0 ± 2.2%, respectively). Furthermore, the number of cells of those blastocysts and expanded blastocyst were also significantly higher (*p* < 0.05) in the FF group (40.4 ± 2.3 and 54.5 ± 2.4, respectively) than in the control (35.9 ± 2.4 and 49.6 ± 2.1, respectively).

**Table 4.** In vitro embryo development (IVC) of putative zygotes produced by in vitro fertilization (IVF) with spermatozoa selected by follicular fluid (FF) in the chemotaxis system. IVF was performed with 25 <sup>×</sup> 103 selected spermatozoa/mL. After fertilization, putative zygotes continued the incubation in IVC medium (NCSU23) for up to 7 days (168 h). The percentage of embryos reaching 2-cells stage (48 h after insemination) and blastocyst and expanded blastocyst stages were analyzed (at 168 h after insemination). A group in which the spermatozoa were selected without chemoattractant was used as the control (C). Embryos after 7 days of culture were fixed with glutaraldehyde and stained with bisbenzimide solution. Evaluation was carried out by fluorescence microscopy. As an indicator of embryo quality, the number of cells forming each blastocyst was determined. Six replicates were performed.


2-cells (%): percentage of oocytes that cleaved to the 2-cell stage of embryo development were evaluated at 48 h after insemination. Blastocyst and expanded blastocysts: percentage of 2-cells embryos that developed to blastocyst or expanded blastocyst stage (according to the classification proposed by Bó and Mapleroft, [26]) after 7 days (168 h) of culture. The number of cells were determined by fluorescence microscopy in blastocyst and expanded blastocysts fixed in 0.5% glutaraldehyde and stained with bisbenzimide solution. One-way ANOVA was performed and the Tukey test of multiple comparisons. Results are expressed as the mean ± SEM. The different superscripts (a–b) within the same column indicate the significant differences between groups (*p* < 0.05).

#### **4. Discussion**

After ovulation, spermatozoa that remain attached to the oviductal epithelial cells in the sperm reservoir are released and continue their journey to meet the oocyte. Since the oviduct is a very narrow and full of crypts duct, the process of gametes encountering cannot be random. Not all sperm will reach the oocyte, and a strict and selective process occurs during their path. It has been suggested that spermatozoa could be attracted by chemical factors that would be released by the oocyte, cumulus cells, and/or the epithelial oviductal cells. Some possible chemoattractants that facilitate spermatozoon-oocyte interaction are substances present in FF, OF [12,33], and other secretions surrounding the oocyte [12,13,34]. Chemotaxis has been described as a concentration-dependent [27] and species-specific phenomenon [12–14,17]. However, the chemical nature of chemoattractants, their receptors and the underlying signaling pathways are still a subject up for debate. In the porcine species, the main sperm-chemotactic role was attributed to P4 [14,32,35,36], but other components were also suggested as potential chemoattractants [37,38].

A typical chemotaxis response is represented by a bell curve, where at lower or higher attractant dilutions no chemotactic response is observed. However at intermediate attractant dilutions, the proportion of chemotactic cells is the greatest [39]. The chemoattractant concentrations obtained in this study were higher than those described in the literature for other species [12,13,17,40]. These differences can be attributed, on the one hand, to the working conditions, since the devices and volumes used by those authors are different compared to ours. On the other hand, those differences could be attributed to some molecular species-specific events during sperm capacitation. However, this last assumption cannot be corroborated with other studies since, to the best of our knowledge, there are not any published studies developed in the porcine species. Regarding the concentration of P4, there is an in-depth study, not only concerning its chemotactic activity [1] but also its participation in the activation of calcium channels involved in the hyperactive movement of the spermatozoa. The concentration of P4 that attracted the largest number of porcine spermatozoa to well B was 10.00 pM, and above this amount there was no greater response. Similar results were observed in human [36], rabbit [36] and mouse sperm [41].

Regarding the chemotactic potential, we did not find statistical differences between experimental groups, but the phenomenon of spermatozoa attraction was clear since there was a significantly lower % of spermatozoa attracted in their absence. This result was unexpected since, despite all biofluids having common components, the concentrations of those components are different. Thus, it would be reasonable to think that the chemoattraction potential can vary between biofluids. It is possible that the effective concentrations of chemoattractants in the biofluids used in the present work were too high. In that case, the saturation of receptors could be a plausible explanation for the lack of effect of the biofluids. Thus, the reduction in the concentration of these biofluids to elucidate their chemotactic potential would make sense. To our knowledge, there are not any studies that show the physiological concentration of chemoattractants in porcine reproductive biofluids. And those may also vary according to the different phase of the estrous cycle. In mice, Oliveira et al. [12] observed that pOF induced a high proportion of spermatozoa travelling longer distances toward the chemotactic gradient, while FF caused an increase in spermatozoa velocity. However, the reasons why the values of spermatozoa attraction obtained in this work with pOF were lower than those obtained with FF remains to be clarified.

It seems logical to think that a chemoattractant whose composition is closest to that found by the spermatozoa in vivo should be the one that attracts more spermatozoa. That is represented in this work as a combination of biofluids (Σ). Nevertheless, the results in this study did not show that. Considering that the in vivo contact of the sperm with chemoattractants occurs sequentially, one hypothesis could be that some receptors may be activated at a given moment and consequently induce the activation of other receptors later [13,36]. The exposure of spermatozoa to chemoattractants in this study was not sequential and the chemotactic receptors could have been activated at the same time preventing a greater chemotactic response.

Although Armon and Eisenbach [42] provided evidence that hyperactivation is part of the chemotactic response of the spermatozoa, the motility results found here by CASA showed no differences between the experimental groups. The results in other studies carried until now about whether motility is affected by chemotaxis are quite controversial. Other authors such as Fabbri et al. [43] observed, in humans, an increase in motility and hyperactivation, but Isobe et al. [44] did not observe variations of motility derived from chemotaxis. The main differences of the present study with previous studies can be that the sperm motility was analyzed after sperm migration towards the chemoattractant. Armon and Eisenbach [42] analyzed the sperm trajectory of swimming in a spatial chemoattractant gradient and observed that hyperactivation was significantly reduced by chemoattractants compared to controls. These authors suggested that with the increase in the chemoattractant concentration the capacitated sperm represses the hyperactivation but keeps swimming in favor of the concentration gradient of chemoattractant.

The importance of increased intracellular Ca2+ in spermatozoa has been demonstrated during chemotaxis [45]. In the chemotactic process the hyperactive movement is produced (regulated by the activation of CatSper channels) and the directionality of the spermatozoa towards the chemoattractant changes [29]. It has been shown in porcine species that the CatSper inhibitor NNC blocked the spermatozoa release from oviduct isthmic epithelial cells [46]. This study used the same concentration of NNC and showed a decrease in the percentage of spermatozoa that migrated to the chemoattractant in all groups, including the control. However, the sperm accumulation in well B appeared not to be completely abolished by NNC, since that reduction was also observed when no chemoattractant was added to the system. Other substances with chemotactic activity could be present in the biofluids. Some candidates could be the 8.6 kDa protein similar to apolipoprotein B2 [38], or maybe the antithrombin III [37] which is responsible for this migration when CatSper are inhibited. However, the mechanism by which these substances stimulate motility has not yet been elucidated. The inhibitor NNC is quite unspecific for CatSper, which means that it could be unsuitable for chemotaxis assays. Rennhack et al. [47] showed that NNC exhibit serious adverse reactions in human sperm and evoke a sizeable and sustained increase of [Ca2+]i and pHi [48–50] in addition to stimulating the acrosomal exocytosis [50].

Although P4 has been identified as the main chemoattractant in pOF, FF, and CM, other components with chemotactic activity are due to be present in biofluids. Therefore, we decided to determine the concentration of E2, P4, cAMP, and cGMP in the biofluids studied. The FF showed the highest concentrations of all of them except for cAMP. Interestingly, the FF was not the biofluid that showed the highest chemoattraction power. Perhaps some components offset the effect of others found in lower concentrations. For instance, cells are less sensitive to cGMP than to cAMP, but cGMP produces a more intense response [51]. Biofluids exhibit complex activity and the spermatozoa are simultaneously exposed to multiple ligands. This can lead to multiple effects and/or separate interactions. In this sense, the estrogen pretreatment elevates Ca2+ in spermatozoa apparently by a CatSper independent mechanism [28,52]. The estrogen pretreatment also reduces the concentration of Ca2+ in response to the stimulation with P4. However, that inhibition could be not produced when the concentrations of P4 are high [53].

Other questions that still need to be solved include why second messengers, such as cAMP and cGMP, are present in biofluids and their roles in chemotaxis. Purinergic signaling has been found to be a key component in the physiology of several tissues. Through the self-production of nucleotides and nucleosides and their binding to specific receptors, a wide range of cellular responses are modulated such as cell growth, differentiation, and motility. A clear example of the importance of these nucleotides was shown by Osycka-Salut et al. [54]. These authors demonstrated that bicarbonate in the extracellular medium produced an early increase in cAMP dependent on the soluble adenylyl cyclase (sAC) in the intra- and extracellular space in spermatozoa. Thus, it suggests that if the existence of a cAMP flow from the spermatozoa to the extracellular space were blocked, that it could result in the inhibition of capacitation. Another possible function of these nucleotides could be as an indicator of the direction to which the spermatozoon must go to find the oocyte, as in the case of sea urchins [55].

The functionality of the spermatozoa selected by pOF, FF, CM, and P4 was analyzed through IVF. The highest oocyte penetration was achieved when FF was used as the chemoattractant but the number of spermatozoa per oocyte was not affected by the biofluids used. A possible explanation of this would be that the FF increases the penetration of oocytes by modulating capacitation and acrosome reaction (reviewed by Hong et al. [11]) which consequently increases the penetration of oocytes. It would be logical to think that the number of spermatozoa per oocyte should also increase [56]. In this sense, Funahashi and Day [57] observed that the pre-fertilization incubation of porcine spermatozoa in suitable concentrations of porcine FF effectively reduces the incidence of polyspermy. Their results indicated that polyspermy blocking is produced by an interaction between FF and spermatozoa and not with oocytes. Another plausible explanation would be that the FF was the attractant that selects the subpopulation of spermatozoa with the highest quality and, therefore, with the highest penetration rate. These hypotheses are supported by the results of Ralt et al. [40], who reported that the FF potential to attract human spermatozoa is strongly correlated with the potential of those spermatozoa to fertilize the oocyte. However, under in vivo conditions, the spermatozoon contacts with other biofluids and biomolecules while it is attracted to the oocyte.

The lowest penetration rate obtained when spermatozoa were selected by pOF or Σ could be attributed to the presence of some factors that prevent capacitation before the spermatozoa contact with the oocyte. Soriano-Úbeda et al. [4] showed that pOF and CM decreases spermatozoa protein kinase A substrates and that tyrosine residues phosphorylation. And later, Zapata-Carmona et al. [25] showed that this process is reversible. During capacitation, the decapacitation factors from the SP are lost and that the intracellular Ca2+ concentration rises [58]. After that, the spermatozoa acquire additional PMCA4 from the oviduct via exosomes [59] which provide an adequate Ca2+ efflux to promote spermatozoa viability and prevent a premature acrosome reaction. Therefore, it cannot be discarded that some factors of pOF decreased spermatozoa capacitation, and other factors produced by the oocyte attracted the spermatozoa and prepared it for fertilization [60].

Until a few years ago, the research on embryo quality focused mainly on evaluating the impact of maternal factors on the embryo. However, it has recently been determined that paternal factors also share responsibility for contributing to better embryo quality. In our work, we have analyzed the quality of embryos obtained with spermatozoa selected by a density gradient and then migrated towards the FF. The results showed that there were no differences in the percentage of embryos divided at 48 h. However, those differences appeared later at the blastocysts and expanded blastocyst stages as well as in the number of cells that form those blastocysts. In both cases, the best quality was higher for the group of spermatozoa that migrated towards the FF. Therefore, the FF somehow selected the best spermatozoon. Gatica et al. [1] obtained better quality embryos when using sperm selected by chemotaxis and argued that this was due to less DNA damage that usually is very low in AI doses. In a previous study (data not shown) we analyzed DNA damage using acridine orange and the Halomax® kit and observed that the % altered DNA was very close to 0%.

It is interesting to observe that the differences between the groups appear at 168 h (7 days) of embryo culture in the blastocyst stage. This fact leads us to think that not only selection by FF is important to obtain spermatozoa of higher quality, but this fluid can provide the spermatozoa with certain factors that improve subsequent embryonic development. Based on this data, we can hypothesize that the results obtained here could be regulated by the extracellular vesicles (EVs) present in the FF. It has been observed that a brief co-incubation is sufficient for transferring components from EVs to the spermatozoa. These EVs participate in this way in the sperm formation process while providing them different types of RNA [61]. All this could explain the improvement in the quality of the embryos obtained with spermatozoa selected by chemotaxis towards the FF [62]. In this work we have observed that the speed of the blastocysts to expand is greater when the spermatozoa are selected by FF. Supporting this theory, Fatehi et al. [63] reported that bovine embryos in the faster division stage are more likely to become blastocysts, and that embryos of rapid division are associated with higher gestation rates.

#### **5. Conclusions**

Under in vitro conditions, the FF selects the best spermatozoa for an optimum and more physiological interaction with the oocyte. The knowledge acquired with this work can be useful in improving the current ART performed in animal production and as model for human clinic assays. From this point on, more studies are necessary to deepen the knowledge of gamete interaction in the physiological environment.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-261 5/11/1/53/s1, Figure S1: Chemotaxis chamber diagram. Figure S2: Chemoattractant potential of chemoattractant combinations. Figure S3: Effect of CatSper inhibition on the membrane integrity of spermatozoa. Table S1: Effect of CatSper inhibition on the motility of spermatozoa.

**Author Contributions:** Conceptualization, L.A.V. and C.M.; Methodology, L.A.V., A.D., C.S.-Ú. and C.M.; Software, L.A.V.; Validation, L.A.V., C.S.-Ú. and C.M.; Formal Analysis, L.A.V., A.D., C.S.-Ú. and C.M.; Investigation, L.A.V. and A.D.; Resources, L.A.V. and C.M.; Data Curation, L.A.V., C.S.-Ú. and C.M.; Writing—Original Draft Preparation, L.A.V. and A.D.; Writing—Review & Editing, C.S.-Ú. and C.M.; Visualization, L.A.V., A.D., C.S.-Ú. and C.M.; Supervision, C.M.; Project Administration, L.A.V. and C.M.; Funding Acquisition, L.A.V. and C.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grants 20040/GERM/16 and 20020/SF/16 from Fundación Séneca (Agencia de Ciencia y Tecnología de la Región de Murcia, Spain) and PID2019-106380RB-I00/AEI/10.13039/501100011033 from the Spanish Ministry of Science and Innovation.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available within the article or supplementary material.

**Acknowledgments:** The authors would like to thank Alejandro Torrecillas for his technical assistance, Florentin Daniel Staicu for his valuable support, Joaquín Gadea for his scientific advice, Department of Research and Development of CEFUSA and El Pozo S.A. (Alhama, Murcia, Spain) for providing the biological samples, and Melinda Furse for editing the language.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Review*
