*Article* **Use of Chemical Nano-Selenium as an Antibacterial and Antifungal Agent in Quail Diets and Its Effect on Growth, Carcasses, Antioxidant, Immunity and Caecal Microbes**

**Mahmoud Alagawany 1,\* , Shaza Y. A. Qattan <sup>2</sup> , Youssef A. Attia 3,\* , Mohamed T. El-Saadony <sup>4</sup> , Shaaban S. Elnesr <sup>5</sup> , Mohamed A. Mahmoud <sup>6</sup> , Mahmoud Madkour <sup>7</sup> , Mohamed E. Abd El-Hack <sup>1</sup> and Fayiz M. Reda <sup>1</sup>**


**Simple Summary:** The chemical Nano-Selenium (Che-SeNPs) is a good example of applied nanotechnology used in the area of nutritional supplements due to its advantages and properties. From our results, dietary supplementation with Che-SeNPs could improve the performance of growing quails; the best level was 0.4 g Che-SeNPs/kg feed. Thus, this study supports the application of Che-SeNPs in quail diets in an effort to improve the productive and physiological performance. The results revealed that Che-SeNPs boosts the growth, blood biochemistry, antioxidant indices, immunity, and bacterial environment of the intestine of quail.

**Abstract:** Nano-minerals are used to enhance mineral bioavailability, which helps improve animal growth and health. The use of chemical nano-selenium (Che-SeNPs) has lately attracted great scientific interest, mainly due to its potential benefits for poultry. The current study was conducted to investigate the impact of the dietary supplementation of Che-SeNPs on the growth performance, carcass traits, blood constituents, antioxidant status, immunity, and gut microbiota of Japanese quails. A total of one week-old 180 Japanese quails were randomly distributed into four equal groups, and each group consisted of 45 unsexed birds with five replications (nine birds each). The first group was fed a basal diet without supplementation (0 g/kg Che-SeNPs), and the second, third, and fourth groups were fed diets containing 0.2, 0.4, and 0.6 g/kg Che-SeNPs, respectively. The results showed that the dietary supplementation of Che-SeNPs significantly (*p* < 0.0001) increased body weight, body weight gain, and feed conversion ratio, but decreased feed intake (*p* < 0.0001) compared to the control group. The highest values of growth performance were recorded in the group fed 0.4 g Che-SeNPs g/kg feed. Che-SeNPs levels did not affect the carcass traits, relative organs (except liver), or blood hematology (except platelet count and hemoglobin level) of quails. Plasma total protein, albumin, aspartate amino transferase (AST), and urea values were not affected by dietary Che-SeNPs, but alanine aminotransferase and lactate dehydrogenase values declined. Globulin and creatinine values were linearly increased with the inclusion of Che-SeNPs (0.4 and 0.6 g/kg) in quail diets compared to the control. The supplementation of Che-SeNPs in quail diets significantly improved (*p* < 0.05) the plasma lipid profile and activities of antioxidant enzymes compared to the control group. Immunoglobulin G values of Che-SeNPs (0.4 and 0.6 g/kg) were higher (*p* < 0.05)

**Citation:** Alagawany, M.; Qattan, S.Y.A.; Attia, Y.A.; El-Saadony, M.T.; Elnesr, S.S.; Mahmoud, M.A.; Madkour, M.; Abd El-Hack, M.E.; Reda, F.M. Use of Chemical Nano-Selenium as an Antibacterial and Antifungal Agent in Quail Diets and Its Effect on Growth, Carcasses, Antioxidant, Immunity and Caecal Microbes. *Animals* **2021**, *11*, 3027. https://doi.org/10.3390/ani11113027

Academic Editor: Valentina Virginia Ebani

Received: 24 August 2021 Accepted: 19 October 2021 Published: 21 October 2021

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**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/).

than those in the control group. The groups fed diets supplemented with Che-SeNPs showed lower (*p* < 0.0001) total bacterial count, total yeast and molds count, *Coliform*, *Escherichia coli*, *Enterococcus* spp., and *Salmonella* spp. colonization, and higher (*p* = 0.0003 and 0.0048) lactic acid bacteria counts than those in the control group. In conclusion, Che-SeNPs supplemented up to 0.4 g/kg can improve the performance, lipid profile, antioxidant indices, and immunity, as well as decrease intestinal pathogens in quails during the fattening period (1–5 weeks of age).

**Keywords:** nano particles; selenium; performance; blood; pathogens; quails

#### **1. Introduction**

Selenium (Se) is one of the elements that can be used in diets as the chemical nanoselenium (Che-SeNPs). Se is required for the maintenance of physiological functions, growth, and health of birds. It also plays a crucial role in nutritional value and feed metabolism, leading to considerable growth [1]. Che-SeNPs has attracted more attention because of its strong adsorbing ability, high catalytic efficiency, high surface activity, and low toxicity compared to that of other chemical Se forms [2]. The high absorption of Che-SeNPs from the intestinal lumen into the body was observed. Shirsat et al. [3] highlighted that Che-SeNPs has antioxidant, anticancer, antibacterial, and antiprotozoal properties. El-Deep et al. [4] stated that dietary Che-SeNPs supplementation enhanced growth performance by improving immune or antioxidative properties in broiler chicks. Additionally, Ahmadi et al. [5] revealed that the dietary supplementation of Che-SeNPs improved growth performance and immune function without the deleterious effects on the internal organs of broiler chickens.

Previous investigations exhibited that Che-SeNPs augmented body weight gain and improved antioxidant functions of Arbor Acres broilers [3,6]. Se nanoparticles have also been utilized in food preservation methods such as packing food items and antiseptic coating over food materials. Studies have been conducted to highlight the disinfectant properties of Se nanoparticles against *Pseudomonas aeruginosa*, and *Proteus mirabilis* [7]. On the other hand, natural agents and trace elements including nanoparticles as feed additives may affect the diversity of gut microbiota and health [8]. Se is one of the important elements that can help microbiota to complete its action within the gut [9]. In this concern, the caecal counts of *Salmonella* and *E. coli* of quails were decreased in birds fed diets containing nano-curcumin when compared to the control diets [8].

Selenium can be considered an essential trace element and micronutrient for living creatures at low concentrations, but it becomes toxic and harmful at higher dose [2]. The extensive use of nano Se in nanotechnologies and medicine has increased the risk of their contamination in the environment, which could harm living species; however, it is useful to understand the assessment of Se-NPs toxicity to the biological ecosystem [10]. Nano-Se has lower toxicity than selenomethionine and is now the least toxic of all supplments of Se. Nano-Se has a threefold lower toxicity than organic Se and a sevenfold lower toxicity than inorganic Se [10].

The positive impacts of nanotechnology involving Se are well-known in many pathological conditions [11]. However, the inclusion of Che-SeNPs in quail diet during the growth period remains limited. It is hypothesized that the dietary addition of Che-SeNPs is expected to exert beneficial effects on growing quails. Therefore, the purpose of this study was to evaluate the antibacterial and antifungal activities of Che-SeNPs, and its beneficial effects on the growth, feed utilization, carcass traits, hematology, blood constituents, and cecal microbiota of growing quails.

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

#### *2.1. Source of Selenium Nanoparticles*

The study was carried out at Zagazig University, Zagazig, Egypt in conjunction with King Abdulaziz University, Jeddah, Saudi Arabia under protocol no: (FP-73-43). In this study, Che-SeNPs were prepared using wet chemicals. Sodium selenite (Na2SeO3) was used for producing Se nanoparticles with ascorbic acid (C6H8O6) as a reducing agent. A stock of aqueous solution of 100 mM Na2SeO<sup>3</sup> and 50 mM C6H8O<sup>6</sup> was prepared in a 1: 4 ratio. The solution was kept under a magnetic stirring condition at different rpm and ambient temperature for 30 min. The mixtures were allowed to react with each other in the concentrated form until the mixture changed from colorless to red. Next, the solution was centrifuged at 3000 rpm, pellets were collected, and Che-SeNPs was obtained [12,13]. Chemically synthesized nano-selenium was determined via UV–Vis spectroscopy using an automated spectrometer (Spectro UV–Vis double beam UVD 3500). The morphology and element percentage of selenium nanoparticles were measured using transmission electron microscopy and an energy dispersive X-ray analytical instrument. Fourier transform infrared spectroscopy (JASCO) was used to determine the properties of produced selenium nanoparticles including size, shape, charge, and stability. Characterization of Che-SeNPs; maximum UV absorbance at 300 nm, spherical shape by TEM, size (75.68 nm) and charge (−23.26 mV) by zeta seizer, and zeta potential, respectively.

#### *2.2. Antibacterial Activity of Che-SeNPs*

*Listeria monocytogenes* ATCC 15313, *Staphylococcus aureus* MTCC 1809, *Bacillus cereus* ATCC 11778, *E. coli* ATCC 25922, *P. aeruginosa* ATCC 27853, and *Salmonella enterica* MTCC 1253 were used in this study. The antibacterial activity of Che-SeNPs against animal and human pathogenic Gram-negative bacteria, *P. aeruginosa* ATCC 27853, *E. coli* ATCC 25922, and *S. enterica* MTCC 1253, and Gram-positive bacteria, *L. monocytogenes* ATCC 15313, *B. cereus* ATCC 11778, and *S. aureus* MTTC 1809, were estimated using the disc diffusion assay method. Mueller–Hinton agar medium consisting of peptone, beef extract, yeast extract, NaCl, and agar with 5, 3, 5, 5, and 20 g, respectively in 1 L of distilled water was prepared in slant to preserve all bacterial strains. Mueller–Hinton broth was used to activate bacterial cells; one hundred microliters of each bacterium (1 <sup>×</sup> <sup>10</sup><sup>9</sup> CFU/mL) were spread with sterile swabs in Mueller–Hinton agar plates. Freshly prepared selenium nanoparticles with different concentrations (50, 100, 200, 400, and 800 µg/mL) were loaded on paper discs (disc diameter was about 6 mm) and then were placed on the Muller–Hinton agar plates. Sodium selenite (50 µg/mL) and sterilized deionized water were loaded on paper discs and used as a positive and negative control, respectively. Mueller-Hinton agar plates were incubated for 24 h at 37 ◦C. After incubation, the obtained zones of inhibition surrounded the Che-SeNPs discs were measured and recorded as the mean ± standard deviation if they were greater than 6 mm. The minimum inhibitory concentration (MIC) of the Che-SeNPs was calculated based on a broth micro dilution method. Briefly, six pathogenic bacteria were cultured overnight at 37 ◦C in Mueller–Hinton broth and were adjusted to a final density of 10<sup>9</sup> CFU/mL by 0.5 McFarland standards. The Che-SeNPs (1 mg/mL) were homogenized with sterilized deionized water and dilutions of 50, 100, 200, 400, and 800 µg/mL were made. Next, 10 µL of different concentrations of Che-SeNPs was mixed in sterile test tubes contain 10 µL of bacterial inoculum and 90 µL of Mueller– Hinton broth. The test tubes were incubated for a day at 37 ◦C. The lower concentration of Che-SeNPs which inhibited bacterial strains growth or turbidity was considered the MIC. The lower concentration of Che-SeNPs which totally killed bacterial strains was defined as the minimum bactericidal concentration (MBC). The experiments were carried out in triplicate [8].

### *2.3. Antifungal Activity of Che-SeNPs*

The antifungal activity of the Che-SeNPs was tested against animals and human pathogenic *Candida* strains. *Candida albicans* ATCC 4862, *C. glabrata* ATCC64677, *C. parapsilosis* ATCC 22019, and *C. guilliermondii* ATCC 6260 were used in this study. The antifungal activity of Che-SeNPs against these four strains was evaluated via the disc diffusion method [14] using sterile cotton swab lawn cultures of selected fungi that were prepared on Sabouraud Dextrose agar (SDA) plates. Che-SeNPs was loaded on paper discs (disc diameter was about 6 mm) and then was placed in SDA surface. Selenium selenite and sterilized deionized water were used as the positive and negative controls, respectively. The plates were then incubated for 36h at 30 ◦C. The Che-SeNPs were tested for MIC using the broth dilution method [14]. Sabouraud broth was used as diluents for fungal species. About 10<sup>6</sup> CFU/mL cells could be inoculated. The Che-SeNPs levels (50 to 800 µg/mL) were prepared in sterilized deionized water and homogenized. Next, 10 µL of different concentrations of Che-SeNPs were mixed in sterile test tubes containing 10 µL of *Candida* inoculum and 90 µL of Sabouraud broth. The test tubes were incubated at 30 ◦C for 36 h. The obtained turbidity was estimated at 600 nm to determine the MIC values. The minimum concentration of Che-SeNPs that reduced fungi growth by 90% was considered the minimum inhibitory concentration (MIC). The concentration of Che-SeNPs at which complete fungal growth was not observed was defined as the minimum fungicidal concentration (MFC). The experiments were replicated in triplicate.

#### *2.4. Animals, Design, and Diets*

A total of 180 one-week-old Japanese quails with an average weight of 27.17 ± 0.075 g were used. Quail chicks were randomly allocated into four groups, and each group consisted of 45 unsexed birds with five replications (nine birds each). Quails were kept in conventional cages (90 × 40× 40 cm), and feed and water were open during the study (4 weeks). The treatments were as follows: the first group was fed the basal ration which containing 150 mg of Se as Se selenite, whereas the second, third, and fourth groups were fed diets supplemented with 0.2, 0.4, and 0.6 g/kg of Che-SeNP, respectively. The Che-SeNP was added at the top of the basal diet at the highest level and then diluted with the unsupplemented basal diet to achieve the desire concentration. The basal diet (Table 1) was formulated to meet the birds' requirements according to NRC [15]. The Ethics statement for Animal care and maintenance were in accordance with the guidelines of the Egyptian Research Ethics Committee and the guidelines for the Care and Use of Laboratory Animals by Zagazig University (ZU-IACUC/2/F/56/2021).


**Table 1.** Ingredients and nutrient contents of basal diet for growing Japanese quail.

**Table 1.** *Cont.*


<sup>1</sup> Provides per kg of diet: Vitamin A, 12,000 I.U.; Vitamin D3, 5000 I.U.; Vitamin E, 130.0 mg; Vitamin K3, 3.605 mg; Vitamin B1 (thiamin), 3.0 mg; Vitamin B2 (riboflavin), 8.0 mg; Vitamin B6, 4.950 mg; Vitamin B12, 17.0 mg; Niacin, 60.0 mg; D-Biotin, 200.0 mg; Calcium D-pantothenate, 18.333 mg; Folic acid, 2.083 mg; manganese, 100 mg; iron, 80 mg; zinc, 80 mg; copper, 8 mg; iodine, 2 mg; cobalt, 500 mg; and selenium, 150 mg.

#### *2.5. Growth Performance and Carcass Measurements*

All growth parameters and feed utilization were measured at 1, 3, and 5 weeks of age. For carcass examinations, at 5weeks old, 20 birds (5 per treatment) were randomly selected, weighed, and euthanized. All edible parts were weighed and expressed as a percent of the live body weight before slaughter.

#### *2.6. Microbiological Analysis*

Ten grams of quail cecum samples (five samples per each treatment) were homogenized and transferred to a 250 mL conical flask containing 90 mL of sterile physiological saline solution consisting of 0.1% peptone and 0.85%NaCl; the mixture was well-mixed to obtain a 10−<sup>1</sup> dilution. Serial dilutions from the previous dilution (10−<sup>1</sup> ) were prepared to obtain up to 10−<sup>6</sup> dilution. The total bacterial count was counted using plate count agar medium at 30 ◦C for 24 h, and the total count of *Enterococcus* spp., was counted using Chromocult enterococci agar medium [16,17]. Total coliforms were enumerated by using MacConkey agar medium. Biochemical methods such as indole test, citrate reactions, methyl red, and Voges–Proskauer were used to identify *Escherichia coli*. DeManRogosa Sharpe agar was used to enumerate the lactic acid bacteria. *Salmonella Shigella* agar (SSA) media (Oxide CM 99) was used to count the *Salmonella* spp. The appearance of black colonies on SSA indicated the presence of *Salmonella* spp. SSA plates were incubated at 37 ◦C for 1 day. Sabouraud Dextrose agar (SDA) was used to count the molds and yeasts. SDA plates were incubated at 25 ◦C for 3–7 days. All the obtained microbiological results were then converted to logarithmic colony-forming units per gram (CFU/g) [8,18,19].

#### *2.7. Blood Chemistry*

After euthanization, blood samples were randomly collected from five quails per treatment into heparinized tubes. Hematological parameters were measured. Regarding biochemical parameters, we used a centrifuge (Janetzki, T32c, 5000 rpm, Germany) at 2146.56× *g* for 15 min to separate the plasma. The biochemical blood parameters were determined using commercial kits from Biodiagnostic Company (Giza, Egypt).

#### *2.8. Statistics*

The statistical analyses were carried out using SAS. The data of growth rate, feed efficiency, carcass parameters, hematology, blood chemistry, and microbiology were analyzed with a one-way analysis of variance using a normal distribution and the replicate as the experimental unit. Orthogonal polynomial contrasts were used to test the significance (linear and quadratic) of the gradual levels of dietary Che-SeNPs using the post-hoc Tukey's test (*p* < 0.05).

#### **3. Results**

#### *3.1. Antibacterial Activity of Che-SeNPs*

Three animal and human pathogenic Gram-negative bacteria (*E. coli, P. aeruginosa*, and *S. enterica*) and three Gram-positive bacteria (*L. monocytogenes*, *B. cereus*, and *S. aureus*) were selected to test Che-SeNPs antibacterial activity (Table 2). The maximum zones of inhibitions were observed in the three Gram-positive bacterial strains *L. monocytogenes* ATCC 15313, *B. cereus* ATCC 11778, and *S. aureus* MTTC 1809. The antibacterial activity of Che-SeNPs increased with increasing concentrations of Che-SeNPs. The effect of Che-

SeNPs was superior to that of sodium selenite as an antimicrobial agent against tested pathogenic microorganisms; moreover, the deionized water did not show any antimicrobial activity. The highest MIC of Che-SeNPs against *E. coli* ATCC 25922, *P. aeruginosa* ATCC 27853, and *S. enterica* MTCC 1253 was 45, 40, and 50µg/mL, respectively, whereas, the lowest MIC was 30, 35, and 25 µg/mL against *L. monocytogenes* ATCC 15313, *S. aureus* MTTC 1809, and *B. cereus* ATCC 11778, respectively (Table 2).

**Item Sod. Selenite (50** µ**g/mL) Selenium Nanoparticles (**µ**g/mL) DI Water 50 100 200 400 800** Bacteria Inhibition zones (mm) *Listeria monocytogenes* ATCC 15313 <sup>14</sup> <sup>±</sup> 0.2 <sup>f</sup> <sup>15</sup> <sup>±</sup> 0.3 <sup>e</sup> <sup>19</sup> <sup>±</sup> 0.1 <sup>d</sup> <sup>23</sup> <sup>±</sup> 0.2 <sup>c</sup> <sup>26</sup> <sup>±</sup> 0.2 <sup>b</sup> <sup>32</sup> <sup>±</sup> 0.1 <sup>a</sup> - *Staphylococcus aureus* MTTC 1809 <sup>11</sup> <sup>±</sup> 0.4 <sup>f</sup> <sup>13</sup> <sup>±</sup> 0.2 <sup>e</sup> <sup>18</sup> <sup>±</sup> 0.2 <sup>d</sup> <sup>20</sup> <sup>±</sup> 0.1 <sup>c</sup> <sup>23</sup> <sup>±</sup> 0.3 <sup>b</sup> <sup>28</sup> <sup>±</sup> 0.35 <sup>a</sup> - *Bacillus cereus* ATCC 11778 <sup>13</sup> <sup>±</sup> 0.2 <sup>f</sup> <sup>16</sup> <sup>±</sup> 0.15 <sup>e</sup> <sup>20</sup> <sup>±</sup> 0.1 <sup>d</sup> <sup>23</sup> <sup>±</sup> 0.2 <sup>c</sup> <sup>27</sup> <sup>±</sup> 0.15 <sup>b</sup> <sup>33</sup> <sup>±</sup> 0.14 <sup>a</sup> - *Escherichia coli* ATCC 25922 <sup>9</sup> <sup>±</sup> 0.5 <sup>f</sup> <sup>11</sup> <sup>±</sup> 0.45 <sup>e</sup> <sup>15</sup> <sup>±</sup> 0.3 <sup>d</sup> <sup>17</sup> <sup>±</sup> 0.4 <sup>c</sup> <sup>21</sup> <sup>±</sup> 0.2 <sup>b</sup> <sup>25</sup> <sup>±</sup> 0.2 <sup>a</sup> - *Pseudomonas aeruginosa* ATCC 27853 <sup>10</sup> <sup>±</sup> 0.5 <sup>f</sup> <sup>11</sup> <sup>±</sup> 0.45 <sup>e</sup> <sup>16</sup> <sup>±</sup> 0.4 <sup>d</sup> <sup>20</sup> <sup>±</sup> 0.1 <sup>c</sup> <sup>22</sup> <sup>±</sup> 0.3 <sup>b</sup> <sup>27</sup> <sup>±</sup> 0.19 <sup>a</sup> - *Salmonella enterica* MTCC 1253 <sup>8</sup> <sup>±</sup> 0.5 <sup>f</sup> <sup>11</sup> <sup>±</sup> 0.45 <sup>f</sup> <sup>14</sup> <sup>±</sup> 0.5 <sup>d</sup> <sup>17</sup> <sup>±</sup> 0.4 <sup>c</sup> <sup>20</sup> <sup>±</sup> 0.5 <sup>b</sup> <sup>24</sup> <sup>±</sup> 0.3 <sup>a</sup> - Fungi *Candida albicans* ATCC 4862 <sup>11</sup> <sup>±</sup> 0.4 <sup>e</sup> <sup>12</sup> <sup>±</sup> 0.3 <sup>d</sup> <sup>14</sup> <sup>±</sup> 0.2 <sup>c</sup> <sup>15</sup> <sup>±</sup> 0.2 <sup>b</sup> <sup>16</sup> <sup>±</sup> 0.15 ab <sup>17</sup> <sup>±</sup> 0.1 <sup>a</sup> - *Candida glabrata* ATCC 64677 <sup>8</sup> <sup>±</sup> 0.5 <sup>e</sup> <sup>9</sup> <sup>±</sup> 0.5 <sup>d</sup> <sup>9</sup> <sup>±</sup> 0.5 <sup>d</sup> <sup>10</sup> <sup>±</sup> 0.4 <sup>c</sup> <sup>11</sup> <sup>±</sup> 0.4 <sup>b</sup> <sup>13</sup> <sup>±</sup> 0.2 <sup>a</sup> - *Candida parapsilosis* ATCC 22019 <sup>10</sup> <sup>±</sup> 0.35 <sup>d</sup> <sup>11</sup> <sup>±</sup> 0.4 <sup>c</sup> <sup>12</sup> <sup>±</sup> 0.3 <sup>b</sup> <sup>13</sup> <sup>±</sup> 0.3 <sup>b</sup> <sup>14</sup> <sup>±</sup> 0.2 <sup>a</sup> <sup>14</sup> <sup>±</sup> 0.3 <sup>a</sup> - *Candida guilliermondii* ATCC 6260 <sup>8</sup> <sup>±</sup> 0.5 <sup>d</sup> <sup>8</sup> <sup>±</sup> 0.5 <sup>d</sup> <sup>9</sup> <sup>±</sup> 0.5 <sup>c</sup> <sup>9</sup> <sup>±</sup> 0.5 <sup>c</sup> <sup>10</sup> <sup>±</sup> 0.3 <sup>b</sup> <sup>11</sup> <sup>±</sup> 0.5 <sup>a</sup> -

**Table 2.** Zone of inhibition produced by Sodium Selenite and selenium nanoparticles.

Mean ± SE, Means in the same row with a similar superscript letter following them are not significantly different (*p* < 0.05).

#### *3.2. Antifungal Activity of Che-SeNPs*

Che-SeNPs showed acceptable antifungal activity, ranging from 50 µg/mL to 800 µg/mL, against all the tested fungal strains. *C. albicans* ATCC 4862 was the most sensitive strain to Che-SeNP when compared to other tested strains. The MICs for *C. albicans* ATCC 4862, *C. glabrata* ATCC64677, *C. parapsilosis* ATCC 22019, and *C. guilliermondii* ATCC 6260 were 70, 90, 80, and 100 µg/mL, respectively (Table 3).

**Table 3.** The MIC (Minimum Inhibitory Concentration), MBC (Minimum Bactericidal Concentration), and MFC (Minimum fungicidal concentration) of the selenium nanoparticles.


**Table 3.** *Cont.*


#### *3.3. Growth Performance*

The effects of dietary Che-SeNPs supplementation on the growth performance of Japanese quails are presented in Table 4. From the results, quails fed diets containing Che-SeNPs had significantly higher body weight (BW) (linear, *p* < 0.0001 and quadratic, *p* = 0.0004) and body weight gain (BWG) (linear, *p* < 0.0001 and quadratic, *p* = 0.0005) during the whole experimental period. The group fed diets containing Che-SeNPs (0.4 g/kg diet) had the highest BW and BWG. Feed intake was decreased (linear and quadratic, *p* < 0.001) in the Che-SeNPs groups compared with that in the control group. The feed conversion ratio was linearly and quadratically improved with the addition of Che-SeNPs in quail diets during all periods. Generally, the best growth performance parameters were recorded in the group fed 0.4 g Che-SeNPs/kg feed.



Means in the same row with no superscript letters after them or a similar superscript letter following them are not significantly different (*p* < 0.05).

#### *3.4. Carcass Traits*

As indicated in Table 5, dietary Che-SeNPs levels did not affect carcass traits and relative organs (except liver) of Japanese quails. Supplementation of Che-SeNPs significantly increased the relative weight of the liver (linear and quadratic, *p* < 0.05) compared to the control group.

#### *3.5. Blood Hematology*

The effects of the addition of Che-SeNPs on the blood hematology of growing quails are presented in Table 6. Dietary Che-SeNPs levels did not affect (*p* > 0.05) white blood cells (WBCs), lymphocytes, mid-range, granulocytes, red blood cells (RBCs), hematocrit, and mean corpuscular volume of the growing quails. Platelet count and hemoglobin (Hb) levels were increased (linear, *p* < 0.05) by the addition of Che-SeNPs at 0.4 and 0.6 g/kg. The mean corpuscular volume value was augmented (linear, *p* < 0.05) by the addition of Che-SeNPs level compared to the control group (without Che-SeNPs). The supplementation of dietary Che-SeNPs at levels of 0.6 g/kg decreased values of red blood cell distribution width linearly (*p* = 0.0091). In contrast, the dietary levels of 0.4 and 0.6 g/kg declined the values of red blood cell distribution volume linearly (*p* = 0.0019) compared to the control group.

**Table 5.** Carcass traits and relative organs of growing Japanese quail as affected by dietary treatments.


Means in the same row with no superscript letters after them or a similar superscript letter following them are not significantly different (*p* < 0.05).

**Table 6.** Hematological parameters of growing Japanese quail as affected by dietary treatments.


Means in the same row with no superscript letters after them or with a common superscript letter following them are not significantly different (*p* < 0.05). <sup>1</sup> WBCs: white blood cells; LYM: lymphocytes; MID: mid-range; GRA: granulocytes; RBCs: red blood cells; HGB: hemoglobin; HCT: hematocrit; MCV: Mean corpuscular volume; MCH: Mean corpuscular hemoglobin; RDWSD: Red blood cell distribution width; RDWCV: Red blood cell distribution volume; PLT: Platelet count.

#### *3.6. Blood Constituents*

Liver and kidney function data are presented in Table 7. The total protein and albumin were not affected (*p* > 0.05) by dietary Che-SeNPs. The globulin value was lowest (linear, *p* < 0.05) in the Che-SeNPs levels of 0.4 and 0.6 g/kg compared with the Che-SeNPs levels of 0.2 g/kg and the control group. The quails fed diets containing Che-SeNPs had higher alanine aminotransferase (ALT) and lactate dehydrogenase (*p* < 0.05) than those in the control group. Dietary Che-SeNPs had no significant effect on AST and urea values. The inclusion of Che-SeNPs (0.4 and 0.6 g/kg) in quail diets increased the creatinine value (linear, *p* < 0.05) compared with that in the control and 0.2 g/kg Che-SeNPs groups.

**Table 7.** Liver and kidney function of growing Japanese quail as affected by dietary treatments.



**Table 7.** *Cont.*

Means in the same row with no superscript letters after them or a similar superscript letter following them are not significantly different (*p* < 0.05). <sup>1</sup> TP: total protein; ALB: albumin; GLOB: globulin; A/G: albumin/ globulin ratio; LDH: Lactate dehydrogenase, AST: aspartate aminotransferase and ALT: alanine aminotransferase.

> The effects of Che-SeNPs inclusion in diets on the lipid profile of quails are presented in Table 8. Total cholesterol, triglyceride, and very-low-density lipoprotein were significantly decreased (*p* < 0.05) in Che-SeNPs-treated groups compared to those in control. The dietary supplementation of Che-SeNPs (0.2 and 0.4 g/kg) quadratically increased high-density lipoprotein (HDL) (*p* = 0.0019).

**Table 8.** Lipid profile of growing Japanese quail as affected by dietary treatments.


Means in the same row with no superscript letters after them or a similar superscript letter following them are not significantly different (*p* < 0.05). <sup>1</sup> TC: total cholesterol; TG: triglycerides; HDL: high-density lipoprotein; LDL: low-density lipoprotein. SOD: superoxide dismutase; MDA: malondialdehyde; GSH: reduced glutathione; GPX: glutathione peroxidase; IgG and M: immunoglobulin G.

### *3.7. Antioxidant Indices*

The results of the antioxidant indices in the serum are given in Table 8. The activities of superoxide dismutase (SOD) and glutathione peroxidase, and the levels of reduced glutathione (GSH) were significantly increased (linear and quadratic, *p* < 0.05) by the dietary supplementation of Che-SeNPs compared with those in control. Dietary Che-SeNPs levels decreased malondialdehyde (MDA) levels linearly (*p* < 0.0001) compared to the control group. The values of immunoglobulin G (IgG) of Che-SeNPs-treated groups were higher (linear and quadratic, *p* < 0.05) than those in the control group. IgM and IgA values of Che-SeNPs (0.4 and 0.6 g/kg) were higher (*p* < 0.05) than those in the control group. The quails fed a diet supplemented with Che-SeNPs showed higher plasma selenium concentrations when compared to those fed the control diet (linear, *p* = 0.0001).

#### *3.8. Microbiological Analysis*

The different Che-SeNPs levels significantly affected the cecal microbiota of growing Japanese quails (Table 9). The groups fed a diet supplemented with Che-SeNPs showed lower total bacterial count, total yeast and molds count, *Coliform*, *E. coli*, *Enterococcus* ssp., and *Salmonella* spp. colonization than those in the control group (linear and quadratic, *p* < 0.0001). However, the dietary supplementation of Che-SeNPs levels increased the lactic acid bacteria count (linear and quadratic, *p* < 0.05) compared to the control group.


**Table 9.** Caecal microbiota of growing Japanese quail as affected by dietary treatments.

Means in the same row with no superscript letters after them or a similar superscript letter following them are not significantly different (*p* < 0.05). TBC: Total bacterial count. TYMC: total yeasts and molds count.

#### **4. Discussion**

Antimicrobial agents are critical in the pharmaceutical and textile industries, water purification, and food packaging. One notable disadvantage of organically synthesized compounds is toxicity in the body; therefore, the trend is to use inorganic nanoparticles such as Che-SeNPs with antimicrobial activity [20]. These Che-SeNPs have an inhibitory effect on many microorganisms. Currently, antimicrobial drugs are becoming less effective for many diseases globally because of the drug resistance capability of microbes. Microorganisms use their biofilm to resist antimicrobial drugs, and the membranes are the primary source of food contamination. Che-SeNPs have been used to control the growth and formation of biofilms of food spoilage bacteria, including *B. cereus*, *Enterococcus faecalis*, *S. aureus*, *E. coli* O157:H7, *S. typhimurium*, and *S. enterica* [21]. The development more effective antibacterial agents is vital for a wide range of applications in various diseases for better public health. However, the emergence of multiple antibiotic-resistant bacteria presents a public health threat. Many developed antimicrobial drugs have limited effective applications due to chemical imbalances, low biocompatibility, and poor long-term antibacterial efficiency. Che-SeNPs conjugated with quercetin and acetylcholine have shown a tremendous antimicrobial effect on the pathogen [22]. Probiotics are microorganisms that can improve intestinal microbial balance and benefit poultry health after consumption in adequate amounts. *Lactobacillus plantarum* and *L. johnsonii* cells are resistant against selenium dioxide, and their cell-free extracts were tested against *C*. *albicans* ATCC 14053 [7]. Selenium particles extracted from cultures of *S. carnosus* stabilized by their natural protein coating, for instance, show considerable activity against the nematode *Steinernema feltiae*, *Saccharomyces cerevisiae,* and *E. coli*. Natural SeNPs were found to be more active than mechanically generated selenium particles and can be applied as antimicrobial materials in medicine and agriculture [23]. Antimicrobial tests show SeNPs activity against *S. epidermidis*, but not against *E*. *coli* in a low Se concentration of 2 ppm. *S. aureus* is an important bacterium commonly found in numerous infections. *S. aureus* infections were difficult to treat due to their biofilm formation and defined antibiotic resistance. SeNPs were used effectively in the prevention and treatment of disease caused by *S. aureus* [8].

The antifungal activity of SeNPs was evaluated against *C. albicans* ATCC 4862, *C. glabrata* ATCC64677, *C. parapsilosis* ATCC 22019, and *C. guilliermondii* ATCC 6260 using the disk diffusion method [14] (Table 2). The common antifungal agents are enormously irritant and lethal, and it is necessary to formulate newer types of safe and cost-effective fungicidals. Accordingly, the present study illustrates that selenium nanoparticles have good antifungal activity against all pathogenic animals and human *Candida* species. Selenium nanoparticles showed better activity against *C. albicans* ATCC 4862 compared to other *Candida* species used in this study. In addition, it was proved that SeNPs ranging in size from 100 to 550

nm, with an average size of 245 nm, have low toxicity and high biological activities [24]. A similar observation was reported by Shakibaie et al. [7], who studied the antifungal activity of selenium nanoparticles against *Aspergillus fumigatus* and *C. albicans,* and found that the MICs for *A. fumigatus* and *C. albicans* were 100 and 70 µg/mL, respectively. However, the high surface-to-volume ratios and their nanoscale sizes provide better activity against biological materials. In addition, Che-SeNPs have significantly lower toxicity than other inorganic and organic forms of supplemental selenium [7].

The current data demonstrated that dietary supplementation with Che-SeNPs substantially affected BW, BWG, feed intake, and feed conversion ratio (FCR). A similar observation was stated by Zhou and Wang [25], who clarified a significant improvement in the FCR and growth performance by supplementation with Che-SeNP up to a 0.5-mg/kg basal diet. Khazraie and Ghazanfarpoor [26] illustrated that weight gain was significantly increased in quail chicks fed the Che-SeNPs-supplemented diet compared to the control. Selim et al. [27], using the Che-SeNPs form (0.15 to 0.30 ppm), showed a marked improvement in BW, BWG, and FCR of broiler chicks. Ibrahim et al. [28] indicated that dietary Che-SeNPs supplementation significantly improved BW, BWG, and FCR of broiler chicks compared to the control group. The improved performance may be due to (1) higher utilization of Che-SeNPs associated with the unique properties of the nano form, such as excellent bioavailability, higher solubility, high cellular uptake, and greater surface activity [2]; (2) the involvement of Se in regulating several enzymatic systems, which interfere in energy metabolism and metabolism of the essential fatty acid apurinic and apyrimidinic base; and (3) Che-SeNPs having high biological activity, immune regulation, and oxidation resistance [22]. In addition, the improved FCR can be elucidated by the Che-SeNPs role in enhancing the activity of intestinal microbiota to digest and absorb the nutrients via the intestinal barriers [9].

The results of the present study in carcass traits and relative organ weight of growing Japanese quails were in line with the study of Khazraie and Ghazanfarpoor [26], who stated that the supplementation of Che-SeNPs to the diet did not affect carcass traits of chicks. Additionally, Cai et al. [6] reported no significant effect of Che-SeNPs on the weights of carcass parts in broilers. Selim et al. [27] indicated that giblets were not affected due to the inclusion of Che-SeNPs in the diet. Recently, Bakhshalinejad et al. [29] reported that neither carcass yield nor carcass yield parts such as thigh and breast muscles and liver, gizzard, and heart of broilers were affected by different Che-SeNPs levels at 42 d of age. In the present study, the relative liver weight was significantly increased with Che-SeNPs; this increase (21–28% relative to control) may be due to the increase in live body weight in Che-SeNPs groups. However, the widespread use of Nano Se in medication and nanoelectronics has increased the risk of their environmental contaminations, which might affect animal species and humans, although it is useful to understand the assessment of the toxicity of Se-NPs to the biological ecosystem. It should be mentioned that the increase in WBCs was insignificant in the Che-SeNPs supplemented-groups; these change along with the change in liver percentage, even if not significant, warrant further investigation to confirm the safety of Che-SeNPs in animal and human nutrition.

Boostani et al. [30] exhibited that packed cell volume, RBCs and WBCs were not different between the birds supplemented with Che-SeNPs and the control birds, which is in line with the current results. Likewise, Chen et al. [31] revealed no significant difference in WBCs, RBCs, and packed cell volume of broilers fed different Se sources. Additionally, Mohamed et al. [32] illustrated that using Che-SeNPs in the diet of Sinai chicks did not significantly affect WBCs, eosinophils, and monocytes. However, our study indicated that Hb level was increased by adding Che-SeNPs, in agreement with Khazraie and Ghazanfarpoor [26], who reported a significant increase in Hb concentration in quails fed a diet containing Che-SeNPs. These findings may be caused by Se enhancing the activity of hemopoietic organs [33]. Se protects the neutrophils, RBCs, WBCs, and other blood components against peroxidative damage [34]. Deficiency of Se can increase ROS in body

tissues, the significant adverse impacts on the consistency of immunity cells' performance and biological membranes [35].

The results of the current study on the blood biochemistry of quails were in agreement with previous studies. Serum total protein and albumin were not significantly affected due to Che-SeNPs supplementation to the broiler diet [27]. However, serum globulin levels were increased with the addition of Che-SeNPs in the diet [36]. Additionally, no significant difference in serum AST activity was observed of chicks fed a diet supplemented with Che-SeNPs [27]. However, our results are similar to the study of Elsaid [37], who reported increased serum ALT activity in birds fed a diet supplemented with Che-SeNPs. Selim et al. [27] found that increasing the Che-SeNPs level in broiler diets increased plasma creatinine levels compared to the control group. However, some studies showed that blood creatinine levels declined in birds fed a diet containing Che-SeNPs Elsaid [37]. The potential reason for these differences is possibly related to the dose and time of animal exposure. We conclude from the current study that the higher Che-SeNPs level is the cause of increased ALT and creatinine as indicators of liver and kidney oxidative damage, whereas lower levels showed less damage.

Selenium has a hypocholesterolemic activity. A significant reduction in plasma TC and an increase in HDL were detected in the Che-SeNPs-treated birds. The dietary addition of nano forms of selenium for hens caused substantial declines in serum levels of cholesterol as compared to that of the control [38]. Rizk [39] stated that Che-SeNPs addition in the chicken diet decreased cholesterol, triglycerides, and low-density lipoproteins and increased HDL compared with the control group. These results might be attributed to the lipolysis that increased with Se intake. Additionally, the reduction of cholesterol may be due to the role of Se in the activation of peroxisome proliferator-activated receptor-γ that can decrease sterol regulatory element-binding protein-2 level, resulting in decreased cholesterol synthesis [40].

The nutritional status of an animal greatly influences the antioxidant system. Se nanoparticles have vital roles in protecting the body cells from reactive oxygen species abundance by decreasing the production of free radicals and lipid peroxidation [41]. Se is well-known for its ability to boost the antioxidant capacity as it forms selenocysteine, a portion of the active center of GSH-peroxidase (Px) [42]. Therefore, a dietary supplementation of Se is essential to improve Se-dependent antioxidant enzymes. These enzymes can help in decreasing the concentration of lipid peroxides and hydrogen peroxide. Dietary Che-SeNPs enhanced oxidative stability and antioxidant ability in broilers [6]. Mohamed et al. [32] reported a positive effect on birds' plasma total antioxidant capacity when fed a diet containing Che-SeNPs. Aparna and Karunakaran [43] detected an increase in glutathione peroxidase and SOD cellular activity in birds fed Che-SeNPs compared to the control group. El-Deep et al. [4] displayed that Che-SeNPs enhanced the activities of SOD and GSH-Px and reduced MDA content in the liver of broilers. The improvement of antioxidant status in quails fed Che-SeNPs in the current study may be attributed to the fact that (1) Che-SeNPs had high antioxidant activity, because it has an augmented ability to trap free radicals with better antioxidant influence, (2) Che-SeNPs can act as a chemopreventive agent when administered at a smaller particle size, (3) Se plays a vital role as an antioxidant that could protect intestinal mucosa against pathogens and oxidative damage, and (4) Se has immunomodulation properties [44].

Nanominerals such as Che-SeNPs can increase immune parameters and disease resistance [4]. In the current study, we presented a potential approach to the application of Che-SeNPs to improve the immunity of quails. These findings can be due to the higher absorption of selenium nanoparticles. The present data are in harmony with the study of Cai et al. [6], who stated that dietary Che-SeNPs supplementation improved humoral immunity by increasing the levels of IgG and IgM of broiler chicks. Dietary Che-SeNPs supplementation showed immunostimulatory impacts in broiler chicks [45]. The improvement in serum immunoglobulins levels may be attributed to the essential

biological role of Che-SeNPs in increasing T helper cells and enhancing the secretion of cytokines [46].

Additionally, Se plays a crucial role in the production of GSH-Px. Selenium inhibits arachidonic acid peroxidation and protects cells and tissues of the immune system from damage caused by free radicals. Therefore, it can be stated that Che-SeNPs boosts birds' immunity and antioxidant metabolites [45]. Studies have shown that the use of nanominerals in poultry production and its effect on performance and immunity, and reproduction is promising [47,48]. It has been suggested that the application of Se can help to strengthen immunity and decrease inflammation [49,50]. Se, according to Rooke et al. [51], may be involved in a variety of immune functions at the cellular and molecular levels, including lowering immunosuppressive markers such as glucocorticoids; reducing the duration and rate of intramammary infections; and regulating the function of lymphocytes, neutrophils, and natural killer cells. Our results suggest that feeding a diet enriched with Che-SeNPs might have immunostimulatory impacts on quails.

The regulation of microbiota in the gut can be achieved through dietary supplements that can encourage the growth of beneficial bacteria or selectively suppress pathogenic bacteria. Trace elements and natural agents as feed additives may affect the diversity of gut microbiota [8]. The present study found that supplementation of Che-SeNPs in quail diets declined harmful bacteria and increased beneficial bacteria. Se is one of the critical elements that can help microbiota complete its action within the gut [9]. Furthermore, Se supplementation augmented the population of caecum such as *Bifidobacterium* spp. and *Lactobacillus* spp. compared to the basal diet [9]. Therefore, using Che-SeNPs is one of the recommendations for reducing the population of harmful gut bacteria due to its inhibitory effect against many pathogenic bacteria.

Nanotechnology has been found to have advantageous uses in the food chain of humans, mainly through enhancing the bioavailability and delivering enough levels of vital nutrients, vitamins, and minerals in animal products used by humans [10,52–56]. Moreover, the consumers' demand for foods and their knowledge has been enhanced as consumers want safe and high-quality foods with high sensory quality, favorable health qualities, and prolonged shelf life [57]. Several studies proved the possibility of supplementing nanomaterials to improve mineral contents in animal products; nevertheless, most of these studies were carried out on chicken, meat, and eggs [58,59]. Therefore, more research is needed to analyze the ability of nanomaterials to affect the quality and nutritional content of meat and egg. In addition, the influence of nanomaterials on the environment and health needs further examination [60,61]. Thus, the application of nanoparticles in the poultry industry must be further investigated before they can be applied.

#### **5. Conclusions**

The current study's findings demonstrated that dietary supplementation with Che-SeNPs could improve the performance of growing quails. The highest values of growth performance were recorded in the group fed 0.4 g Che-SeNPs g/kg feed during the fattening periods (1–5 wk of age). Moreover, the dietary addition of Che-SeNPs improved the lipid profile, antioxidant indices, and immunity and decreased the intestinal pathogens of growing quails. The groups fed diets supplemented with Che-SeNPs showed lower total yeast and mold count, *Coliform*, *Escherichia coli*, *Enterococcus* spp., and *Salmonella* spp. colonization, and higher lactic acid bacteria counts than those in the control group. However, further studies are warranted to understand the effect of nanominerals and their mechanisms of action, sites of absorption, and transcript expression analysis of distribution.

**Author Contributions:** Conceptualization, M.A., M.T.E.-S., S.S.E. and F.M.R.; methodology, M.A., M.T.E.-S., S.S.E. and F.M.R.; formal analysis, M.A., M.T.E.-S. and F.M.R.; investigation, M.A., M.T.E.-S. and F.M.R.; data curation, M.A. and F.M.R.; writing—original draft preparation, M.A., S.Y.A.Q., Y.A.A., M.T.E.-S., S.S.E., M.A.M., M.M., M.E.A.E.-H. and F.M.R.; writing—review and editing, M.A., S.Y.A.Q., Y.A.A., M.T.E.-S., S.S.E., M.A.M., M.M., M.E.A.E.-H. and F.M.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** The Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Saudi Arabia has funded this project, under grant No. (FP-073-43).

**Institutional Review Board Statement:** The Ethics statement for Animal care and maintenance were in accordance with the guidelines of the Egyptian Research Ethics Committee and the guidelines for the Care and Use of Laboratory Animals by Zagazig University (ZU-IACUC/2/F/56/2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are published in the cited literature and reported in the text of this manuscript.

**Acknowledgments:** The authors acknowledged the administrative, technical, and financial support by DSR, King Abdulaziz University, Jeddah, Saudi Arabia.

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

#### **References**


## *Article* **Antioxidant and Antiapoptotic Effects of a** *Turraea fischeri* **Leaf Extract on Cryopreserved Goat Sperm**

**Soha A. Hassan 1,† , Wael A. Khalil 2,\* ,† , Mahmoud A. E. Hassan <sup>3</sup> , Ahmed I. Yousif <sup>3</sup> , Omar M. Sabry <sup>4</sup> , Michael Wink <sup>5</sup> and Mansour Sobeh 6,\***

	- omar.sabry@pharma.cu.edu.eg

**Simple Summary:** The excessive production of reactive oxygen species (ROS) in cryopreservation and post-thawing affects sperm quality and subsequent fertilizing ability. Antioxidants of natural origin, such as plant extracts, rich in flavonoid and phenolic compounds, are of special interest in scavenging ROS. The supplementation of goat semen extender with 375 µg/mL *T. fischeri* leaf extract improved the functional and ultrastructural characteristics of cryopreserved sperm by maintaining antioxidant capacity, thus preventing membrane injury and reducing apoptosis.

**Abstract:** This study evaluated the efficacy of *Turraea fischeri* leaf extract for maintaining the viability of cryopreserved goat sperm. Ejaculated semen was collected from 5 mature Baladi bucks (50–60 kg, 2–4 years of age) and those samples with mass motility ≥ 70% and sperm concentration <sup>≥</sup> 2.5 <sup>×</sup> <sup>10</sup>9/mL were selected, pooled, and divided into 4 aliquots. Each aliquot was diluted in Tris-citric-soybean lecithin extender containing a different concentration of *T. fischeri* leaf extract (0, 125, 250, or 375 µg/mL). Treated semen samples were cooled to 5 ◦C, transferred to 0.25-mL French straws, and stored in liquid nitrogen (LN<sup>2</sup> ) at −196 ◦C. After thawing, membrane integrity was examined by transmission electron microscopy, apoptotic activity by Annexin/propidium iodide staining and flow cytometry, and both enzyme activities and antioxidant capacity by spectroscopic assays. The leaf extract at 375 µg/mL significantly improved semen quality as indicated by enhanced total antioxidant capacity, reduced H2O<sup>2</sup> concentration, a greater proportion of structurally intact motile sperm, and concomitant reductions in apoptosis and necrosis. The extract also significantly increased the proportion of sperm with a contiguous plasma membrane and intact acrosome (*p* < 0.05). Furthermore, LC-MS revealed numerous secondary metabolites in the extract that may contribute to sperm cryopreservation.

**Keywords:** *Turraea fischeri*; polyphenolics; semen cryopreservation; sperm ultrastructure; antioxidant biomarker; apoptosis

### **1. Introduction**

Artificial insemination (AI) is used widely in agriculture to optimize and spread commercially valuable genetic traits, including in goats. Effective cryopreservation of semen

**Citation:** Hassan, S.A.; Khalil, W.A.; Hassan, M.A.E.; Yousif, A.I.; Sabry, O.M.; Wink, M.; Sobeh, M. Antioxidant and Antiapoptotic Effects of a *Turraea fischeri* Leaf Extract on Cryopreserved Goat Sperm. *Animals* **2021**, *11*, 2840. https:// doi.org/10.3390/ani11102840

Academic Editors: Woo Kyun Kim, Youssef A. Attia, Maria de Olivera and Nesrein Hashem

Received: 26 July 2021 Accepted: 23 August 2021 Published: 29 September 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/).

samples is critical for efficient AI. However, cryopreservation can reduce goat sperm quality, motility, and viability, resulting in lower fertility rates [1]. The reduced semen quality results in part from cellular oxidative stress, which causes the peroxidation of unsaturated fatty acids in the biomembrane. In healthy mammalian sperm cells, total antioxidant capacity (TAC) and reactive oxygen species (ROS) production remain in balance [2], but an increase in ROS production, a decrease in TAC, or both during cryopreservation can result in reduced motility and cell death [3]. Excessive ROS production during cryopreservation and after thawing not only degrade cellular membranes but may also damage DNA [4], further enhancing dysfunction [5]. To reduce oxidative stress, exogenous antioxidants are frequently added to cryopreservative solutions to sustain semen quality prior to AI [6]. Several studies have confirmed ROS scavenging capacity of various medicinal plant extracts to improve sperm motility and increase fertility rates [7–10].

The genus *Turraea* L., a genus of the Meliaceae (mahogany) family of tropical flowering trees and shrubs, includes 70 species distributed throughout Africa and Asia [11,12]. *Turraea fischeri* is widely used in east Africa to treat stomachache and infertility [13]. Previous studies have also documented antioxidant and hepatoprotective properties [14]. Furthermore, biochemical analyses of *T. fischeri* extracts have identified numerous potentially bioactive secondary metabolites such as limonoids as well as various sterols and flavonoids with antioxidant and anti-inflammatory activities [15].

To date, there are no studies available on the effect of the leaf extract from *T. fischeri* in semen extenders on semen cryopreservation. In the current study, the chemical profile of a leaf methanol extract from *T. fischeri* was characterized using HPLC-MS/MS, and its antioxidant activity was measured by in vitro spectroscopic assays. The effects of *T. fischeri* leaf extract on goat sperm viability, motility, and morphology, and antioxidant capacity were then examined after thawing by vital staining under light microscopy, flow cytometry following Annexin/propidium iodide staining, various spectroscopic assays, and transmission electron microscopy.

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

#### *2.1. Plant Material, Extraction, LC-MS and Antioxidant Acctivities*

Leaves of *Turraea fischeri* were collected from the Lupaga Site in Shinyanga, Tanzania, and stored at the Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, under accession number P7336 [16].

The dried and ground leaves (100 g) were extracted three times in 100% methanol (3 × 500 mL) at ambient temperature and the three extracts combined. Pooled extracts were then filtered and dried under a vacuum at 40 ◦C. The obtained residue was lyophilized, yielding a fine dried powder (15 g). LC-MS analyses and in vitro antioxidant activities were performed according to Sobeh et al. [14]. Detailed methods are included in the Supplementary File.

#### *2.2. Animals*

Semen samples were collected from 5 mature, fertile Baladi bucks (50–60 kg LBW; 2–4 years of age) at the Animal Production Research Station, El-Karada, Kafrelsheikh, Animal Production Research Institute (APRI), Agricultural Research Center, Ministry of Agriculture, Egypt, using an artificial vagina. Sample collection was conducted in cooperation with the Physiology and Biotechnology Laboratory, Animal Production Department, Faculty of Agriculture, Mansoura University, Egypt, according to animal welfare guidelines of Mansoura University.

#### *2.3. Animal Management*

All 5 bucks were raised under the same environmental conditions. Feeding requirements were calculated according to the recommendations of Animal Production Research Institute, Ministry of Agriculture, Egypt. Each buck was fed 1.0 kg/day concentrate feed mixture containing 14% crude protein and 70% total digestible nutrients, plus 1.25 kg/day

berseem hay from August to November or 5 kg/day Egyptian fresh berseem clover (*Trifolium alexandrinum*) from December to February. Animals had free access to trace mineralized salt and drinking water at all times.

#### *2.4. Collection of Semen*

Semen was collected from each buck by artificial vagina once weekly before feeding at 7–8 a.m. for five consecutive weeks. In total, 25 samples were obtained. Samples were transferred immediately into a water bath at 37 ◦C and only those with mass motility <sup>≥</sup> 70% and sperm concentration of at least 2.5 <sup>×</sup> <sup>10</sup>9/mL were retained for experiments. Samples were then pooled and divided into 4 aliquots for each treatment group.

#### *2.5. Preparation of Extender*

The Tris-citric-soybean lecithin extender contained 3.025 g/dL Tris (Sigma Chemical Co., St. Louis, MO, USA), 1.66 g/dL citric acid monohydrate (Sigma, Darmstadt, Germany), 1.25 g/dL glucose (Sigma Aldrich, St. Louis, MO, USA), 5% glycerol (Honeywell, Regen, Germany), 1% soybean lecithin (L-a-phosphatidylcholine, LAB: product number MC041), 100 IU/mL penicillin, and 100 µg/mL streptomycin. The components were mixed in a water bath at 37 ◦C and adjusted to 300 mOsm/kg in H2O (pH 6.8) before addition of extract.

#### *2.6. Cryopreservation*

Pooled semen was diluted 1:10 in extender (*v*/*v*) containing the indicated extract concentration (0, 125, 250, and 375 µg/mL) and adjusted to a final sperm concentration of 2.5 <sup>×</sup> <sup>10</sup>8/mL. The mixture was then gradually cooled from 37 ◦C to 5 ◦C over 4 h (equilibration period) and transferred to 0.25-mL French straws (IMV Technologies, L'Aigle, France) for cryopreservation. Straws were first exposed to liquid nitrogen vapor for 10 min and then immersed in liquid nitrogen at −196 ◦C.

#### *2.7. Thawing*

After one month, the straws were thawed at 37 ◦C for 30 s in a water bath, and the various assessments conducted immediately.

#### *2.8. Semen Evaluation*

#### 2.8.1. Progressive Motility

The proportion of sperm cells showing progressive motility was examined under a phase-contrast microscope (DM 500, Leica, Switzerland) with heated stage set to 37 ◦C. Briefly, aliquots of diluted sperm (10 µL) were placed on pre-warmed glass slides and sealed with coverslips. A total of 200 spermatozoa/slide from 3 randomly chosen fields were counted by the same investigator and the mean proportion (%) recorded.

#### 2.8.2. Viability

Semen samples were double-stained with a mixture of 5% eosin (vital stain) and 10% nigrosin (background stain) to estimate the live: dead ratio [17]. The live: dead ratio was calculated by counting the unstained head area among 300 sperm at high magnification (400×) using a light microscope.

#### 2.8.3. Gross Structural Abnormalities

Abnormalities in gross structure were assessed in 300 sperm cells during viability measurements using a light microscope. The following criteria were considered: (i) tail defects (abnormal tails), (ii) abnormal heads, and (iii) cytoplasmic droplets [18].

#### 2.8.4. Plasma Membrane Integrity

The hypo-osmotic swelling test (HOS-t) was used to assess plasma membrane (PM) integrity according to a previously described protocol [19]. Briefly, semen (50 µL) was incu-

bated at 37 ◦C for 30 min in a hypo-osmotic solution (500 µL at 75 mOsm/kg) containing fructose (6.75 g/L) and sodium citrate (3.67 g/L) in H2O. A sample of the mixture was placed on a slide and covered with a coverslip. The number of spermatozoa with coiled or swollen tails (indicative of intact membranes under hypoosmotic conditions) among 300 sperm per slide was counted in each sample at 400× under phase-contrast microscopy.

#### 2.8.5. Antioxidant Capacity and Enzyme Activities

The following biochemical parameters were measured in post-thawed extender: total antioxidant capacity (TAC, linearity up to 2 mM/L) [20], hydrogen peroxide concentration (H2O2, linearity up to 1.5 mM/L) [21], lactic dehydrogenase (LDH, linearity up to 1700 units/L) activity [22], aspartate transaminase (AST, linearity up to 150 units/mL) activity, and alanine transaminase (ALT, linearity up to 120 units/mL) activity [23]. All measurements were performed using a spectrophotometer (Spectro UV-VIS Auto, UV-2602, Labomed, Los Angeles, CA, USA) and commercial kits (Biodiagnostic, Giza, Egypt) according to the manufacturer's instructions.

#### 2.8.6. Apoptosis and Necrosis

Semen samples were stained with Annexin-V (AV, calcium-dependent probe) for tracking phosphatidylserine (PS) externalization in the membrane and propidium iodide (PI) as an indicator of genomic DNA exposure using a commercial PS Detection Kit (IQP, Groningen, The Netherlands) according to the manufacturer's instructions. Briefly, semen samples were thawed and washed twice by centrifugation (300× *g* for 10 min at 4 ◦C) with phosphate-buffered saline. After the second centrifugation, the supernatant was removed, and the sperm pellet resuspended in binding buffer at 1 <sup>×</sup> <sup>10</sup><sup>6</sup> sperm cells/mL. Then, 100 µL of semen sample was transferred to culture tubes (5 mL) containing 5 µL AV (fluorescein isothiocyanate, FITC label, BD Biosciences, San Jose, CA, USA) and 5 µL PI (BD Biosciences). The mixed suspension was then incubated in the dark at room temperature (25 ◦C) for 15 min, followed by the addition of 400 µL binding buffer to each tube. Staining patterns were then evaluated by flow cytometry using an Accuri C6 Cytometer and Accuri C6 software (BD Biosciences) [24]. Cells negative for both AV and PI staining (A−/PI−) were classified as viable, those positive for AV and negative for PI (A+/PI−) as early apoptotic, those positive for both AV and PI (A+/PI+) as apoptotic, and those negative for AV and positive for PI (A−/PI+) as necrotic.

#### 2.8.7. Ultrastructure

Sperm ultrastructure was examined by transmission electron microscopy (TEM) as described [25] with some modifications. Briefly, 500 µL of each semen sample was centrifuged and resuspended in cold (4 ◦C) fixative solution (2.5% glutaraldehyde in phosphate buffer) for 2 h. Samples were then washed and post-fixed in osmium tetroxide (1%) for 90 min at room temperature, dehydrated, cleared in gradient ethanol and propylene oxide, and embedded in Epon 812 (Fluka Chemie, AG, Buchs, Switzerland). Ultrathin sections (60–70 nm) were prepared using glass knives and observed using a JEOL-JEM 2100 TEM at 80 kV. Changes in PM and acrosome ultrastructure were examined from 300 sperm per sample.

#### *2.9. Statistical Analysis*

Arcsine transformation was performed before statistical analyses because that helps in dealing with percentage values for semen characteristics including progressive motility, viability, membrane integrity, acrosome integrity, structural abnormality, sperm viability by Annexin-V, plasma membrane integrity, acrosomal ultrastructure. Treatment group means were compared by one-way analysis of variance (ANOVA) [26] and Duncan's multiple range tests [27]. A *p* < 0.05 was considered statistically significant for all tests.

### **3. Results**

#### *3.1. Chemical Composition and In Vitro Antioxidant Activities*

Analysis of a leaf methanol extract from *T. fischeri* by HPLC-MS/MS tentatively identified 17 compounds, including particularly high concentrations of phenolic acids (e.g., compounds 2, 3, and 4), flavonoids (compounds 10–17), and corresponding glycoside derivatives (Table 1). On the other hand, the bark extract from the same plant was rich in 20 secondary metabolites belonging to cinchonains and phenylpropanoid-substituted catechin [14]. The extract exhibited substantial antioxidant activity in two commonly used assays; FRAP and DPPH (Table 2). The observed results might be attributed to the existence of several phenolic acids (*p*-coumaroylquinic acid, feruloylquinic acid and caffeoylquinic acid) and flavonoids (Quercetin rutinoside, quercetin glucoside, kaempferol rutinoside, kaempferol glucoside and isorhamnetin glucoside). Comparable activities were reported from the bark extract [14].

**Table 1.** Characterization of secondary metabolites of *T. fischeri* by HPLC-MS/MS analyses. Precursor ions and corresponding fragment ions.


Rt: retention time, M-H: pseudo molecular ion in the negative ion mode, MS/MS: mass fragmentation pattern.

**Table 2.** Antioxidant activities of a leaf methanol extract from *T. fischeri* as measured by FRAP, DPPH, and TPC assays. GAE = gallic acid equivalents.


Ascorbic acid and quercetin are positive controls. DPPH, 2,2-diphenyl-1-picryl-hydrazyl-hydrate; FRAP, ferricreducing antioxidant power assay; TPC, total phenolic content.

### *3.2. Effects of T. fischeri Leaf Extract on the Viability, Morphology, Function, and Membrane Integrity of Thawed Cryopreserved Goat Sperm*

#### 3.2.1. Effects on Sperm Motility, Viability, and Membrane Integrity

Post-thaw motility, viability, and membrane integrity were all significantly (*p* < 0.05) improved by addition of *T. fischeri* leaf extract (TFLE) to semen extender during cryopreservation, and the degree of improvement increased with TFLE concentration compared to semen extender alone, Table 3. Alternatively, acrosome integrity and the proportion of abnormal sperm were not affected significantly by TFLE (*p* > 0.05, Table 3).


**Table 3.** Effects of *T. fischeri* leaf extract on cryopreserved goat sperm characteristics (means ± SE, *n* = 5).

<sup>a</sup> and <sup>b</sup> : Values in the same column with different superscripts are significantly different at *p* < 0.05.

3.2.2. Effects on Apoptosis and Necrosis Rates

The proportion of viable sperm was significantly higher in TFLE-treated cryopreserved semen than control cryopreserved semen (*p* < 0.05), and the viable fraction increased progressively with TFLE concentration (Table 4). Consistent with this dose-dependent improvement in viability, the proportions of early apoptotic, total apoptotic, and necrotic spermatozoa decreased progressively with TFLE dose compared to controls. Further, at 375 µg/mL TFLE, the proportion of necrotic sperm was significantly lower than at all other concentrations.

**Table 4.** Effects of *T. fischeri* leaf extract on goat sperm viability (means ± SE, *n* = 3).


a , b , <sup>c</sup> and <sup>d</sup> : Values in the same column with different superscripts are significantly different at *p* < 0.05.

3.2.3. Effects on Membrane Ultrastructure

Examination of sperm cell ultrastructure by TEM revealed that the plasma membrane (PM) was damaged by cryopreservation in semen extender alone, while supplementation with TFLE dose-dependently improved sperm ultrastructure after thawing. Table 5 summarizes the proportions of sperm cells in each treatment group demonstrating an intact, slightly swollen, swollen, or disrupted PM. Consistent with viability analyses, there was a TFLE dose-dependent increase in the portion of cells with an intact PM and decreases in the proportions with slightly swollen and swollen PM.



a , <sup>b</sup> and <sup>c</sup> : Values in the same column with different superscripts are significantly different at *p* < 0.05.

> Figure 1A–G illustrates the different ultrastructural abnormalities resulting from cryopreservation. Four injury patterns were defined according to the degree of PM damage [28]. (i) Sperm with intact PM exhibited a normal head region with an intact acrosome (IA) and contiguous PM tightly surrounding the acrosomal ground substance (Figure 1A–C). In addition, the mid-sectional region of healthy sperm contained a contiguous mitochondrial sheath (MS) completely enclosing morphologically typical mitochondria. The axoneme

3).

**Concentration** 

also exhibited the normal 9 + 2 arrangement of microtubules (Figure 1B,C). (ii) Sperm with slightly damaged PMs exhibited an acrosomal physiological reaction (AR) characterized by the initial formation of small vesicles under a dilated and slightly separated plasma membrane (SP) (Figure 1A,B). (iii) In the third category, sperm exhibited a swollen PM (S) with a wavy appearance, and mitochondrial sheathes were also dilated (Figure 1D,F). (iv) Finally, another fraction of sperm showed discontinuous or disintegrated (DS) PMs, damaged membranes (DMs) around mitochondria, and various mitochondrial ultrastructural abnormalities. The axoneme structure at this stage also showed an abnormal microtubule arrangement. The distribution of these categories differed among treatment groups, particularly at higher TFLE concentrations. In the 375 µg/mL group, there was a significantly greater proportion of cells with an IA and a smaller proportion with atypical acrosomes (*p* < 0.05). There was also a trend for lower frequencies of typical AR and lost acrosome in TFLE-treated groups compared to controls cryopreserved in extender only (Table 6). a , <sup>b</sup> and <sup>c</sup> : Values in the same column with different superscripts are significantly different at *p* < 0.05. Figure 1A–G illustrates the different ultrastructural abnormalities resulting from cryopreservation. Four injury patterns were defined according to the degree of PM damage [28]. (i) Sperm with intact PM exhibited a normal head region with an intact acrosome (IA) and contiguous PM tightly surrounding the acrosomal ground substance (Figure 1A–C). In addition, the mid-sectional region of healthy sperm contained a contiguous mitochondrial sheath (MS) completely enclosing morphologically typical mitochondria. The axoneme also exhibited the normal 9 + 2 arrangement of microtubules (Figure 1B,C). (ii) Sperm with slightly damaged PMs exhibited an acrosomal physiological reaction (AR) characterized by the initial formation of small vesicles under a dilated and slightly separated plasma membrane (SP) (Figure 1A,B). (iii) In the third category, sperm exhibited a swollen PM (S) with a wavy appearance, and mitochondrial sheathes were also dilated

**Table 5.** Effect of *T. fischeri* leaf extract on sperm plasma membrane (PM) integrity (means ± SE, n =

**(%)**

Control 39.3 ± 0.88 <sup>c</sup> 17.3 ± 0.88 <sup>a</sup> 33.7 ± 0.88 <sup>a</sup> 9.7 ± 0.88 Extract 125 µg/mL 43.0 ± 1.15 <sup>c</sup> 19.0 ± 1.00 <sup>a</sup> 30.0 ± 1.53 <sup>b</sup> 8.0 ± 1.15 Extract 250 µg/mL 54.3 ± 2.33 <sup>b</sup> 11.0 ± 1.53 <sup>b</sup> 24.7 ± 0.88 <sup>c</sup> 10.0 ± 1.53 Extract 375 µg/mL 64.4 ± 2.33 <sup>a</sup> 6.0 ± 2.31 <sup>b</sup> 21.3 ± 0.88 <sup>c</sup> 8.3 ± 1.20

**Swollen PM** 

**(%) Lost PM (%)**

**Table 6.** Effect of *T. fischeri* leaf extract on acrosomal ultrastructure (means ± SE, *n* = 3). (Figure 1D,F). (iv) Finally, another fraction of sperm showed discontinuous or disinte-

*Animals* **2021**, *11*, x FOR PEER REVIEW 7 of 13

**(mg/mL) Intact PM (%) Slightly Swollen PM** 


<sup>a</sup> and <sup>b</sup> : Values in the same column with different superscripts are significantly different at *p* < 0.05. in extender only (Table 6).

**Figure 1.** *Cont*.

**Figure 1.** Addition of *T. fischeri* leaf extract to semen extender during cryopreservation significantly improved post-thaw sperm ultrastructure. (**A**–**C**): Intact sperm with structurally intact acrosomes (IA) completely enclosed by contiguous plasma membranes (PM). The PM appears continuous alongside the nucleus in longitudinal sections and around the mitochondrial sheath (MS) in cross-sections. Few sperm cells exhibited a separated plasma membrane (SP) or diffusion of ground substance (GS) under a detached PM. (**D**): Damaged sperm showing a swollen 'wavy' PM leaving a large space around the nucleus (S). €: Cross-sections of the tail region showing swollen PM containing cytoplasmic residue (CR) and mitochondrial sheathes enveloping damaged mitochondria (DM). (**F**): Damaged acrosomal cap (DAC), dilated PM in the tail region (Arrows), and cytoplasmic residue (CR). (**G**): Discontinuous (DS) PM in longitudinal and cross-sections of different sperm regions. **Figure 1.** Addition of *T. fischeri* leaf extract to semen extender during cryopreservation significantly improved post-thaw sperm ultrastructure. (**A**–**C**) Intact sperm with structurally intact acrosomes (IA) completely enclosed by contiguous plasma membranes (PM). The PM appears continuous alongside the nucleus in longitudinal sections and around the mitochondrial sheath (MS) in cross-sections. Few sperm cells exhibited a separated plasma membrane (SP) or diffusion of ground substance (GS) under a detached PM. (**D**) Damaged sperm showing a swollen 'wavy' PM leaving a large space around the nucleus (S). (**E**) Cross-sections of the tail region showing swollen PM containing cytoplasmic residue (CR) and mitochondrial sheathes enveloping damaged mitochondria (DM). (**F**) Damaged acrosomal cap (DAC), dilated PM in the tail region (Arrows), and cytoplasmic residue (CR). (**G**) Discontinuous (DS) PM in longitudinal and cross-sections of different sperm regions.

#### **Table 6.** Effect of *T. fischeri* leaf extract on acrosomal ultrastructure (means ± SE, n = 3). 3.2.4. Effects on Oxidative Biomarkers and Enzyme Activity

**Concentration (mg/mL) Intact Acrosome (%) Atypical AR (%) Typical AR (%) Lost Acrosome (%)** Control 69.3 ± 2.40 <sup>b</sup> 19.3 ± 0.88 <sup>a</sup> 8.4 ± 1.45 3.0 ± 1.15 Addition of TFLE also dose-dependently enhanced insignificantly post-thaw TAC and reduced significantly H2O<sup>2</sup> (*p* < 0.05, Table 7). Alternatively, TFLE had no significant effects on LDH, AST, and ALT activities as shown in Table 7.

Extract 125 µg/mL 70.3 ± 1.20 <sup>b</sup> 19.0 ± 0.58 <sup>a</sup> 8.0 ± 0.58 2.7 ± 0.88 Extract 250 µg/mL 76.3 ± 1.45 <sup>a</sup> 14.7 ± 1.45 <sup>b</sup> 6.7 ± 0.33 2.3 ± 1.20 Extract 375 µg/mL 79.7 ± 0.88 <sup>a</sup> 11.6 ± 1.20 <sup>b</sup> 7.0 ± 0.58 1.7 ± 0.33

: Values in the same column with different superscripts are significantly different at *p* < 0.05.

<sup>a</sup> and <sup>b</sup>


**Table 7.** Effect of *T. fischeri* leaf extract supplementation on seminal antioxidant capacity and enzymatic activities (means ± SE, *n* = 3).

<sup>a</sup> and <sup>b</sup> : Values in the same column with different superscripts are significantly different at *p* < 0.05. TAC = total antioxidant capacity, H2O<sup>2</sup> = hydrogen peroxide, LDH = lactic dehydrogenase. AST = aspartate transaminase, ALT = alanine transaminase.

#### **4. Discussion**

Supplementation of goat semen extender with 375 µg/mL TFLE improved the functional and ultrastructural characteristics of cryopreserved sperm by maintaining antioxidant capacity, thereby preventing membrane injury and reducing apoptosis.

Semen cryopreserved in semen extender alone (control group) demonstrated the lowest proportion of viable sperm compared to samples preserved with extender containing varying concentrations of the leaf extract. Freezing and thawing can damage cellular membranes, reducing sperm number and quality as shown by flow cytometry and TEM. Our results are in accord with previous studies demonstrating the harmful effects of thawing on sperm structure and function. Oxidative stress during cryopreservation reduces the reproductive potential of semen by impairing sperm motility, reducing mitochondrial activity, damaging DNA, and activating apoptotic pathways [8,9,29]. Thus, supplementation of semen extender with antioxidants prior to cryopreservation is recommended to facilitate efficient goat breeding [30]. Here, we demonstrate improved cryopreservation using a natural plant extract containing multiple bioactive agents with known beneficial effects against cellular stress.

Freezing and thawing induced apoptosis as evidenced by Annexin staining, which reveals the translocation of phosphatidylserine from the inner to the outer PM layer. Further, some sperm cells became necrotic during cryopreservation as evidenced by PI staining [31]. Both apoptosis and necrosis are associated with loss of PM integrity, which is necessary to maintain sperm function within the female reproductive tract [32]. During cryopreservation, rearrangement of membrane lipids alters fluidity and increases susceptibility to disruption, which then induces further cellular damage and ultimately death [33]. These pathological effects are manifested by changes in sperm morphology during the freezingthawing process [34]. High concentrations of polyunsaturated fatty acids (PUSFAs) such as arachidonic and docosahexaenoic acids in the PM increase the vulnerability to ROS-induced peroxidative damage and membrane dysfunction [35]. Further, oxidative injury may be spread throughout the spermatozoa population by a subset of cells overproducing ROS [36], leading to generally reduced mitochondrial metabolic activity, motility, and viability [37]. Maintenance of cell membrane integrity and mitochondrial function under oxidative stress are thus essential for successful fertilization using cryopreserved semen [33].

Following cryopreservation, damage to the PM and acrosomal cap was predominantly observed in the head region, in accordance with previous observations of human sperm [28]. Membrane swelling is most probably caused by changes in the extracellular osmotic pressure during freezing and thawing, causing cells to accumulate or lose water. The sperm PM is known to mediate the exchange of sodium, potassium [38,39], and calcium [40], and these ion fluxes regulate motility and mitochondrial function as well as osmotic balance. An intact PM is also necessary for fusion with the outer acrosomal membrane and induction of the acrosome reaction [41]. Acrosomal integrity is also essential for fertilization as this organelle contains hydrolytic enzymes such as hyaluronidase, acrosin, and esterases required for lysis of the zona pellucida and penetration of the oocyte corona radiata [42]. Freezing and thawing significantly increased the number of sperm cells with

atypical acrosomal structure, which has previously been attributed to degeneration and apoptosis [43].

The addition of TFLE to sperm extender dose-dependently increased the TAC of goat semen and reduced the concentration of H2O2, a major ROS generator. On the other hand, TFLE had little effect on the activities of LDH, AST, and ALT. The dosedependent increase in TAC was strongly associated with the progressive rise in sperm cell viability and the decreases in apoptosis, necrosis, and structural abnormalities. Consistent with these findings, Salimi, et al. [44] reported positive correlations between TAC and both sperm motility and normal sperm morphology, while Pahune, et al. [45] observed positive correlations between TAC and multiple seminogram parameters including sperm concentration, sperm motility, and normal sperm morphology. Collectively, these findings suggest that an imbalance between TAC and ROS production is a major contributor to impaired sperm function following cryopreservation [46].

Mitochondrial enzymatic activities in human spermatozoa are strongly correlated with motility [47]. Aspartate transaminase (AST) and ALT are essential for metabolic processes that provide energy for sperm survival, motility, and fertility [48], and so are good indicators of sperm membrane stability and semen quality [49]. An increase in spermatozoa damage within the liquid storage medium results in an elevated concentration of transaminase enzymes [50]. Indeed, AST and ALT activities were slightly higher in control samples than samples containing 375 µg/mL TFLE, although the difference did not reach statistical significance.

TFLE contains secondary metabolites such as malic acid, quercetin, and kaempferol that may contribute to these improved functional and structural characteristics. Indeed, malic acid decreases the accumulation of ROS and enhances the glutathione cycle by regulating various endogenous antioxidant pathways [51,52], while the flavonoids can directly scavenge ROS, thereby resisting oxidative damage during cryopreservation [53]. Finally, kaempferol is a flavonoid compound with potent activity against inflammation caused by oxidative stress [54].

The effect of TFLE on semen cryopreservation was stronger compared with other plant extracts such as *Albizia harveyi* leaf extract in bull [8], *Entada abyssinica* bark extract in ram [9], and nanoformulations of mint, thyme, and curcumin in goat [10]. All these extracts enhanced semen preservability and sperm characteristics after freezing and thawing.

TFLE is rich in phenolic and flavonoid compounds, which have antioxidant properties. This is evident in the antioxidant activity of the extract via DPPH, FRAP, and TPC assays or by reducing the concentration of hydrogen peroxide in the semen extender after thawing. This can be explained by the ability of polyphenolic compounds to scavenge reactive oxygen species such as superoxide anion radicals and hydroxyl radicals, thus interrupting free radical chain reaction [55]. Therefore, we expect the same effect in preserving the semen in other species.

#### **5. Conclusions**

The addition of 375 µg/mL TFLE to Tris-soybean lecithin extender significantly improved the cryopreservation of goat semen as evidenced by a greater proportion of cells retaining robust motility, viability (low apoptosis rate), and normal ultrastructure after thawing. These benefits were associated with elevation of semen antioxidant capacity. The efficacy of the extract in artificial insemination needs to be studied in more detail.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/ani11102840/s1, Detailed methods of the LC-MS analysis and the in vitro assays (DPPH, FRAP and TPC) are included in the Supplementary File.

**Author Contributions:** Conceptualization, W.A.K., S.A.H. and M.S.; methodology, W.A.K., M.S., S.A.H., M.A.E.H. and A.I.Y.; software, W.A.K., M.S. and S.A.H.; formal analysis, W.A.K. and M.S.; investigation, W.A.K., M.S., S.A.H. and M.A.E.H.; data curation, W.A.K., M.S., S.A.H., O.M.S. and M.A.E.H.; writing—original draft preparation, W.A.K., M.S., S.A.H., M.A.E.H. and A.I.Y.; writing review and editing, W.A.K., M.S., O.M.S., M.W. and S.A.H.; visualization, W.A.K. and M.S.; supervision, W.A.K., M.S. and M.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding. APC was paid by UM6P.

**Institutional Review Board Statement:** The animal study was reviewed and approved by the Ethics Committee of Mansoura University.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author, W.A.K., upon reasonable request.

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

#### **References**


## *Article* **Correlations between Antioxidant and Biochemical Parameters of Blood Serum of Duroc Breed Pigs**

**Sergei Yu. Zaitsev \*, Anna A. Belous , Oksana A. Voronina, Roman A. Rykov and Nadezhda V. Bogolyubova**

Federal Research Center for Animal Husbandry Named after Academy Member L.K. Ernst, Dubrovitsy 60, 142132 Moscow, Russia; belousa663@gmail.com (A.A.B.); voroninaok-senia@inbox.ru (O.A.V.); brukw@bk.ru (R.A.R.); 652202@mail.ru (N.V.B.)

**\*** Correspondence: s.y.zaitsev@mail.ru; Tel.: +74-95-6765-1363

**Simple Summary:** Human nutrition is currently one of the most important factors that determine health, performance, duration and quality of life. The increasing demand for high-quality livestock products requires scientists and practitioners to develop an advanced complex approach to assessing the composition of animal meat and methods of its regulation. One of these products is pork, the value of which lies primarily in the lipid and protein content, which are necessary for human nutrition. The significance of this paper is also determined by the high popularity of pork in Russia and in a number of other countries worldwide.

**Abstract:** Correlations between the major biochemical (BC) and antioxidant (TAWSA) parameters of pigs' blood are necessary to study in order to assess physiological–biochemical status (PhBS), animal health, production, etc. Blood samples were obtained from Duroc breed boars (*n* = 77), divided into groups 1 (*n* = 25), 2 (*n* = 40) and 3 (*n* = 12), which were fattened for 65, 72 and 100 days, respectively. Significant positive and negative correlations were found between TAWSA and BC parameters of pigs' blood for group 3: very high in the case of total protein (TP) (−0.75) and aspartate aminotransferase (AST) (−0.79); high in the case of cholesterol (−0.72), glucose (0.66), alkaline phosphatase (0.66), calcium ions (−0.60) and globulins (0.53); moderate in the case of albumins (−0.36), triglycerides (−0.35), magnesium (−0.32) and phosphorus (−0.27). The same was found for group 2: high in the case of TP (0.51); moderate in the case of globulins (0.48), cholesterol (0.33) and phosphates (0.25). The only moderate correlation was found for group 1: magnesium (−0.48), glucose (0.36) and calcium (−0.25). This tendency indicated the stabilization of pig PhBS during growth and fattening, which can be useful for understanding the PhBS and antioxidant features of pigs, the factors of their nutrition, maintenance, etc.

**Keywords:** Duroc breed boars; antioxidants; biochemistry; blood parameters; feeding time; correlation coefficients

### **1. Introduction**

It is known that animal products are important sources of high-quality proteins, fats, vitamins, minerals and macro- and microelements in human nutrition [1]. In this regard, some approaches have been developed to improve the introduction of animal breeding [2–4] in order to ensure a higher production efficiency and meat quality (including pork), which is especially important for Russia [2,4–6]. Duroc is one of the most popular breeds of pig, possessing numerous positive characteristics in growth rate; total body weight; chemical composition of meat and fat, i.e., high intramuscular fat content in meat [6–8].

To assess the physiological–biochemical status (PhBS) and animal health, safety and quality of meat products [8–10], a complex of biochemical and hematological, antioxidant and "zootechnical" indicators must be studied.

**Citation:** Zaitsev, S.Y.; Belous, A.A.; Voronina, O.A.; Rykov, R.A.; Bogolyubova, N.V. Correlations between Antioxidant and Biochemical Parameters of Blood Serum of Duroc Breed Pigs. *Animals* **2021**, *11*, 2400. https://doi.org/ 10.3390/ani11082400

Academic Editors: Youssef A. Atti, Woo Kyun Kim, Nesrein Hashem and Maria de Olivera

Received: 27 July 2021 Accepted: 10 August 2021 Published: 13 August 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/).

To date, the PhBS of pigs, taking into account the changes in the content of antioxidants in the blood and other tissues of fattening animals, has not been sufficiently studied (both in the Russian Federation and in Asian countries) even for purebred animals. The assessment of PhBS of breeding pigs in Russia is mainly based on the study of changes in biochemical parameters in connection with feeding conditions [11–13], genotype [14,15], sex [16], age [17] and others (only some Russian works is cited as references in this part of the paper). For example, there was an interesting study on selective biochemical and hematological parameters of blood, as well as indicators of mineral metabolism in pigs of the Landrace and Kemerovo breeds, fed at large industrial enterprises of the "Chistogorsk" and "Altaymyasoprom" complexes in Russia [18]. These indicators are close to those obtained in our study and are within physiological norms [18].

On the other hand, some hematological and biochemical parameters of blood were studied in novel hybrids of boars (Large White Landrace) [19]. In industrial combinations (three breeds), where the meat breeds Pietrain and Duroc were used, the content of hemoglobin and erythrocytes was higher as compared to purebred animals [19]. From a practical point of view, data on the relationship of blood biochemical parameters with fattening and meat qualities [20–22] are especially important. There are a number of dissertations and articles on the assessment of PhBS in pigs of the Russian population based on the biochemical parameters of blood and meat of animals [4,9,17,23,24], which we will discuss in detail in the part 4 of our article. The least studied issue in this area is the study of the total amount of water-soluble antioxidants (TAWSA) in the blood serum of fattening pigs. In Russia in this area, only the groups from our Federal Research Center work in collaboration with physiologists and livestock specialists [24–26]. At the same time, individual indicators of antioxidant protection (for example, a concentration of TBA-active products) have been studied more fully [26,27] than TAWSA, but do not provide a complete knowledge of the antioxidant status of the pigs' organisms.

Some authors [8–10,28] believe that the assessment of PhBS through the specified set of indicators is very important, and "the average population values of biochemical parameters are the starting point for the subsequent monitoring of breeding populations" [28]. For example, using these data, it is possible to analyze "in which direction the biochemical status of the population changes when pigs are selected for various characteristics of productivity" and to carry out the ecological monitoring of populations of certain breeds of pigs "in zones with different anthropogenic pressures" [8–10,28]. In addition, the obtained data may be the "average population norm" for healthy animals [28].

The studies carried out to assess the PhBS and health of animals of the Duroc breed, which showed a comparison of hematological and biochemical parameters (more than 40 parameters) between purebred and crossbred offspring [29]. The data obtained are consistent with previous studies [30] on crossbred and purebred offspring, in which there were small differences in some biochemical parameters of pigs at 15 and 27 weeks [30], including creatine, alkaline phosphatase, phosphorus and calcium [29], and were "corrected" with the further growth of animals [29,30].

For a correct assessment of PhBS, it is necessary to study the possibilities of protecting the body from reactive oxygen species (ROS) [31–33]. ROS are formed during many metabolic processes in human and animal cells, capable of oxidizing biologically active compounds (BAC), and damaging the membranes and cells of the body [31–33]. The body's antioxidant defense system is designed to maintain the balance of bioactive substances (lipids, peptides, vitamins and other compounds) in organs and tissues of humans and animals, protecting them from ROS [34–36]. Antioxidant activity is a valuable source of information about the state of health and the level of stress resistance in humans and productive animals in industrial conditions [34–36], which led to a huge variety of methods for its study [33,36]. All these methods are based on a model reaction (most often oxidation) of an individual compound proceeded by a radical mechanism [33–36]. Among these, one of the relatively simple and reliable methods is the electrochemical method based on the amperometric detection of the oxidation reaction signal [33–36].

Duroc is one of the most popular breeds in Russia and Asian countries (including China), which is used not only for meat production, but also for reproduction in pig breeding. It is important to highlight that pork is one of the key directions in Russian livestock production.

In connection with the above-stated relevance, the aim of the work was to study the biochemical and antioxidant parameters of the blood of Duroc breed boars of the Russian population and to identify the most significant correlations between these parameters. These data will be used by the Ministry of Agriculture of the Russian Federation and are especially important in order to establish the range of particular biochemical references (standard norms) for the Duroc breed of pigs in the Russian population.

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

#### *2.1. The Samples of the Pig Blood Serum*

The studies were carried out on the basis of a selection-hybrid center on boars of the Duroc breed (*n* = 77), from which blood samples were taken from the ear vein during setting and withdrawing from fattening. These pigs were divided onto groups 1 (*n* = 25), 2 (*n* = 40) and 3 (*n* = 12) that were fattened for 65, 72 and 100 days, respectively. The age of boars' setting at the feeding stations was 75 ± 1 days, with an initial live weight of approximately 35.0 ± 0.5 kg. The times of boars' withdrawing were from 130 to 175 days, the final live weights were from 92 to 122 kg. Only clinically healthy animals that were periodically examined by veterinary specialists participated in the studies.

The experimental protocols concerning these animals were approved by the Bioethical Committee of the Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst. All experiments and conditions (animal care, feeding, biological material sampling, etc.) are fulfilled in accordance with the applicable regulations (internationally recognized guidelines and local acts).

#### *2.2. Measurements of the Biochemical Parameters of Pig Blood Serum Samples*

The biochemical parameters of animal blood serum samples were determined using a "ChemWell" automatic biochemical analyzer (Awareness Technology, Palm City, FL, USA). The open system of this analyzer allows the use of any method or reagent. Therefore, in this study, we used the reagents "Analyticon Biotechnologies AG" (Lichtenfels, Germany) and "Spinreact" (Carretera Santa Coloma, Spain). All reactions were performed in standard "microwell" plates. Pre-dilution, mixing, incubation, rinsing and measurement of the samples were controlled automatic. The following biochemical indicators were determined [37–39]: the concentration of total protein (TP)—by the biuret method; albumin (A)—by the colorimetric method with bromcresol green; urea—by enzymatic colorimetric analysis (Berthelot method); creatinine—by the kinetic Yaffe method; glucose—by the enzymatic glucose oxidase method; cholesterol (Chol) and triglycerides (TG) by the enzyme-colorimetric method; bilirubin (quantification by the Walters and Gerarde method); calcium (Ca)—by the O-cresolphthalein complexon method; phosphorus (P), magnesium (Mg) and iron (Fe) by the colorimetric method; alanine aminotransferase (ALT) activity—by the UV-kinetic method; aspartate aminotransferase (AST) activity by—the UV-kinetic method; alkaline phosphatase (ALP) activity—by the kinetic method. The following ratios and indicators were determined by calculation: A/G, Ca/P, ALT/AST and the concentration of globulins (G) [37–39]. The results of these measurements were statistically processed using the MS Excel program.

### *2.3. Measurements of the Total Amount of Water-Soluble Antioxidants of Pig Blood Serum Samples*

The amperometric method [33–36,39] was used to study the total amount of watersoluble antioxidants (TAWSA). The measurements were carried out on a "Tsvet-Yauza 01-AA" device [39]. The TAWSA values were determined by measuring the strength of the electric current arising during the oxidation of molecules on the surface of the working

electrode at a potential of ~500 mV. TAWSA was measured in equivalent to gallic acid as in reference [35]. For this, the "working solutions" were prepared from a gallic acid solution (100 mg/dm<sup>3</sup> ) for calibration with a mass concentration of 0.2, 0.5, 1.0 and 4.0 mg/dm<sup>3</sup> . An amount of 2.2 mmol/dm<sup>3</sup> phosphoric acid solution was used as an "eluent" [35,39]. The results of measuring the total antioxidant activity of the samples were statistically processed using the MS Excel program.

When analyzing the studied indicators, the methods of variation statistics were used to calculate the mean and standard error (M ± m) and the coefficient of variation (Cv,%), as well as performing correlation analysis using the STATISTICA 10 program (StatSoft, Moscow, Russia).

The obtained datasets of the antioxidant and biochemical parameters boars of the Duroc breed (*n* = 77) are available online on the website of the L.K. Ernst Federal Research Center for Animal Husbandry (https://www.vij.ru/institut/struktura-organizatsii/ nauchnye-podrazdeleniya/52-gruppa-analiticheskoj-biohimii accessed on 1 June 2021).

#### **3. Results**

The blood of animals has a complicated biochemical composition: proteins (including albumins, globulins and enzymes), lipids (triglycerides, cholesterol, etc.), minerals (calcium, magnesium, phosphate ions, etc.), etc. From the point of view of biological chemistry, blood serum is a multiphase colloidal system in which the main phase is aqueous moderate, and one of the important integral characteristics is the total amount of water-soluble antioxidants (TAWSA). The authors determined the main biochemical and antioxidant parameters of the blood serum of the Duroc breed of pigs, divided onto groups 1 (*n* = 25), 2 (*n* = 40) and 3 (*n* = 12), which were fattened for 65, 72 and 100 days, respectively (Tables 1–3).

**Table 1.** Biochemical and antioxidant parameters of the blood serum of the Duroc breed pigs (*n* = 25) in group 1 (65 days of fattening).


1 same unit as mentioned in the "Parameters" (i.e., g/L, mM/L, µM/L, etc.); <sup>2</sup> rel. units; <sup>3</sup> %.

**Table 2.** Biochemical and antioxidant parameters of the blood serum of the Duroc breed pigs (*n* = 40) in group 2 (72 days of fattening).



**Table 2.** *Cont.*

1 same unit as mentioned in the "Parameters" (i.e., g/L, mM/L, µM/L, etc.); <sup>2</sup> rel. units; <sup>3</sup> %.

**Table 3.** Biochemical and antioxidant parameters of the blood serum of the Duroc breed pigs (*n* = 12) in group 3 (100 days of fattening).


1 same unit as mentioned in the "Parameters" (i.e., g/L, mM/L, µM/L, etc.); <sup>2</sup> rel. units; <sup>3</sup> %.

> By measuring the biochemical and antioxidant (TAWSA) blood parameters of the Duroc breed pigs (*n* = 77), the most significant differences between these parameters in connection with the days of animal fattening were revealed. It was shown that all biochemical and antioxidant parameters of the blood of pigs of both groups were within the physiological norms for this animal species. The main indicators of protein metabolism in the blood were fairly constant for groups 1 and 2, but changed significantly for group 3. Thus, the parameters of total protein (TP) and albumins (A) changed by less than 2% for groups 1 and 2, but significantly decreased (−7.1% and −11.9% for TP and A, respectively) for group 3 as compared to group 1 (Table 1). The content of globulins (G) varied in the range of +3.3% for group 2 up to −1.2% for group 3 versus group 1, respectively (Table 1).

> It seemed logical that the A/G ratio changed by less than 1% for group 2 and more than 11.8% for group 3 (as compared to group 1); however, there was a significant decrease in the albumin values (−11.9%) for group 3 as compared with group 1, respectively, and insignificant changes in the globulin content for all the groups studied (Tables 1–3).

> Some of these biochemical parameters differed slightly in a number of values: urea by −5.3% and +4.7%, and creatinine—by −14.0% and −7.8%, for groups 2 and 3 compared to group 1, respectively (Tables 1–3). This is normal for a young growing organism and

indicates an improvement in protein metabolism in a number of animals [37–39]. Similar values of the total protein content in the blood serum of piglets were also noted by other authors [9,10,37–39], and it was indicated that this parameter "differed by variability" [28].

On the other hand, a significant (more than −27.2% and −25.7%) decrease in pigs' blood glucose values in groups 2 and 3 as compared to group 1 is surprising (Tables 1–3). Generally, with the increase in the age of pigs, there is a slight increase in the values of glucose in their blood. Therefore, in [28], it was found that the glucose content in piglets (at the age of 20–60 days) was at the level of 5.2–5.3 mmol/L, although the author pointed out the highest coefficient of variation (Cv = 42.2%) among all other biochemical parameters. In our studies, the coefficient of variation in the case of glucose ranged from 4% to 10%, which was typical for most other biochemical parameters.

It is interesting that for groups 1 and 2 (fed for 65 days and 72 days), such an important biochemical parameter of lipid metabolism as the content of triglycerides in the blood practically did not change, while the content of TG for group 3 animals (fed for 100 days) increased by 232%, i.e., almost 2.3 times (Tables 1–3).

The cholesterol content increased by 18.0% and 4.2% for groups 1 and 2 (Tables 1–3), respectively, which is indirect evidence of changes in lipid metabolism in a number of animals [39–41]. Some authors [9,10,39–41] note that at a young age, piglets of the Large White breed have the highest serum cholesterol content (for example, 4.21 ± 0.90 mmol/L at the age of 2–3 weeks [28], whereas by the age of two months, its content in piglets decreases to 2.70 ± 0.58 mmol/L and remains practically at the same level as in adult sows (2.67 ± 0.75 mmol/L) [28]. Of course, the coefficient of variation of this feature, according to the same authors [28], is a fairly large value of the order of 21–28%. These authors explain the tendency to a high cholesterol content in piglets by "more active metabolic processes in their body, including glycolysis" [28].

The following changes in enzyme activity were observed: ALT—by 21.0% and 17.1% (for groups 2 and 3) and AST—by −19% and approximately 0% (for groups 2 and 3), which did not go beyond the physiological norms. Since for such enzymes, only changes in values from 30% and higher are significant, only the change in the "de Ritis coefficient" should be considered, which decreased by more than −32% for the 2nd group of pigs as compared to the 1st and changed in the ALP activity by −37.0% for the 3rd group of pigs as compared to the 1st, respectively. Changes in ALP activity by −8.4% for the 2nd group of pigs as compared to the 1st are not significant (Tables 1–3).

The calcium content in the blood serum fell by −9.5% and −13.5%, whereas magnesium content sometimes increased by 10.2%, then fell by −24.2%, and the phosphorus content always increased by 17.6% and by 23.0% for the 2nd and 3rd groups of pigs as compared to the 1st one, respectively (Tables 1–3). These changes had a positive effect on the ratio of calcium to phosphorus in the blood serum of Duroc pigs.

Finally, the total amount of water-soluble antioxidants decreased by more than −23% in the 2nd group of pigs, and then increased by 96.1% in the 3rd group compared to the 1st group, respectively (Tables 1–3). This is directly related to the changes in a number of basic biochemical parameters of the blood of pigs of the Duroc breed, for example, albumin and TG listed above (Tables 1–3).

The experimental conclusions are summarized later. It should be noted that an increase in the duration of feeding (from 65 to 72 and 100 days) led to a tendency for a significant decrease in the coefficients of variation (Cv) for most biochemical parameters. This indicates the stabilization of the physiological and biochemical status of the growing organism in the 2nd and 3rd pig groups in comparison with the 1st one (Tables 1–3).

#### **4. Discussion**

#### *4.1. The Relationship between the Biochemical Parameters of the Blood Serum of Boars of the Duroc Breed*

In recent years, lipid peroxidation has become the subject of extensive research in terms of mechanisms, dynamics, product analysis, disease involvement, inhibition and biological signaling. Some types of antioxidants with different functions inhibit lipid peroxidation and the harmful effects caused by lipid peroxidation products. Much attention has recently been paid to the biological role of lipid peroxidation products, but it is topical to study the relationship between biochemical parameters and indicators of antioxidant protection [38,42].

The relationships of biochemical parameters in group 1 are presented in Table S1 (65 days of fattening, *n* = 25). The presence of 5 very strong, 5 strong (i.e., subtotal—10 significant) and 36 moderate correlations (i.e., subtotal—46 meaningful) from the 136 total independent correlations was found. In particular, there were only 2 very strong, 1 strong and 16 moderate correlations between the 4 protein indicators in group 1 (i.e., correlations— 8 for TP, 6 for albumins and 5 for globulins) correlations from the 58 total independent correlations. There was only 1 very strong correlation between enzymes and 2 strong correlations between enzymes and inorganic ions, as well as 2 strong correlations between cholesterol and inorganic ions. There were only 2 very strong correlations between inorganic ions, as well as numerous moderate correlations in the case of glucose, triglycerides, cholesterol, enzymes, magnesium ions, phosphates, inorganic ions and the Ca/P ratio.

The relationships of biochemical parameters in group 2 are presented in Table S2 (72 days of fattening, *n* = 40). The presence of 4 very strong, 11 strong (i.e., subtotal 15 significant) and 37 moderate (i.e., subtotal 52 meaningful) correlations from the 136 total independent correlations was found. In particular, there were only 3 very strong, 7 strong and 16 moderate correlations between the 4 protein indicators in group 2 (i.e., correlations—9 for TP, 7 for albumins, 5 for globulins and 3 for the A/G ratio) from the 58 total independent correlations. There was only 1 very strong correlation between enzymes and 3 strong correlations between inorganic ions, as well as 1 strong correlation between urea and Mg2+ ions. There were numerous moderate correlations in the case of glucose, triglycerides, cholesterol, enzymes and magnesium ions with Ca/P ratio.

The relationships of biochemical parameters in group 3 are presented in Table S3 (100 days of fattening, *n* = 12). The presence of 12 very strong, 28 strong (i.e., subtotal 40 significant) and 47 moderate correlations (i.e., subtotal 87 meaningful) from the 136 total independent correlations was found. In particular, there were 6 very strong, 9 strong and 20 moderate correlations between the 4 protein indicators in group 3 (i.e., correlations—11 for TP, 9 for albumins, 10 for globulins and 5 for the A/G ratio) correlations from the 58 total independent correlations. There were 6 very strong, 19 strong and numerous moderate correlations in the case of all other organic compounds and inorganic ions, as well as their ratios.

Thus, animal group 3 was preferential for blood biochemistry correlations as compared to groups 1 or 2. There were 2.5–3.0 times more very strong correlations, 2.5–5.6 times more strong correlations and approximately 1.3 times more moderate correlations in the case of animal group 3 (in total) as compared to the groups 1 or 2. Moreover, there were 2.0–3.0 times more very strong correlations, 1.3–9.0 times more strong correlations and approximately 1.25 times more moderate correlations between the 4 protein indicators in the case of animal group 3 (in total) as compared to the groups 1 or 2. It is important to highlight that there were no meaningful correlations of the total protein indicator and its fractions with the A/G ratio for group 3 as compared to groups 1 or 2.

It is well known [42–46] that the PhBS of animals is initially determined by the multilevel and complex interaction of the systems of the animal body. Therefore, a comparative analysis of the biochemical parameters of blood is especially important. For example, according to Molyanova G.V. [17], the total protein content in pigs' blood at the age of 120 days is 61.12 or 62.02 g/L and at the age of 180 days—63.02 or 72.05 g/L (for Duroc breed pigs in Samar region) in the cold or warm weather periods, respectively. According to Giro T.M. et al. [44], the total protein content in pigs' blood at the age of 120 days is approximately 74.6 g/L; albumins—38.9 g/L; globulins—35.5 g/L (for Duroc breed pigs in Saratov region). In the work of Nikolaev D.V. et al. [45], the level of total protein in pigs' blood at the age of 180 days is 78.5 g/L; albumins—33.7 g/L; globulins—44.8 g/L (for Duroc breed pigs in the Volgograd region). We compared the data of 11 biochemistry indicators (total protein,

albumin, globulins, A/G, glucose, triglycerides, ALT, AST, ALP, Ca and P) from all of these studies, which have different values between them, with our results (for Duroc breed pigs in the Voronezh region), but within the range of general data of blood biochemistry for healthy pigs [38]. This is why a detailed study on the major biochemical parameters of the blood serum of such pigs is especially important in order to establish the range of particular biochemical norms for the Duroc breed pigs in the Russian population.

The data of biochemical analysis, obtained in the work of Gu T. [46], did not show significant differences in more than 40 parameters in purebred Duroc pigs, their clones and offspring (including two-breed hybrids in various variations). According to Gu T. et al. [46], on the 112th day, the level of total protein in the blood of Duroc pigs was 76.38 g/L; albumin—40.08 g/L; globulins—36.30 g/L; glucose—3.35 mmol/L; calcium— 2.65 mmol/L. However, in the work of Gu T. [46], we did not find data characterizing the state of the antioxidant system in pigs. At the same time, the activity of enzymes such as ALT, AST and ALP is more variable and can vary significantly. Thus, in our study, ALT and AST activity was observed up to 30 U/L for all studied groups of animals (from 140 days of fattening to 175 days), while in [19], the ALT activity was 91.66 U/L and AST activity was 89.63 U/L, which is within the normal range (the norms for ALT 22–98 IU/L and AST 13– 95 IU/L according to Gusev I.V. [39]). The data of biochemical analysis, which we obtained in the study of animals of the Duroc breed, are in good agreement with the data obtained in the above works [44–46], and there are also relatively close values in the levels of total protein, glucose and calcium content in [19] for three-breed hybrids. As a literature search showed, most of the works are focused on biochemical parameters, while scarce attention is paid to the study of antioxidant systems in a comprehensive assessment of health status. Of course, the rate of metabolic processes in the body directly affects the formation of free radicals and their neutralization by both enzymatic and low molecular weight antioxidants. The assessment of these processes is extremely important, as confirmed by the work of Kotenkova E.A. [47], which studied the antioxidant potential of the pig spleen, heart and aorta extracts (by determining their total antioxidant capacity after slaughter). The highest total antioxidant capacity was observed in the spleen extract. However, no attention was paid to blood in the context of this work, which could be interesting for the development of a strategy to increase the antioxidant activity of food products.

#### *4.2. The Relationship of TAWSA with Biochemical Parameters of the Blood Serum of Boars of the Duro Breed*

For the first time, the calculation of phenotypic correlations of biochemical and antioxidant parameters of the blood of pigs of the Duroc breed was carried out (Table 4).



As shown in our previous works on the study of correlations between TAWSA and biochemical parameters of sheep blood [38], positive or negative values of correlations are not as important as their absolute values, i.e., whether these correlations are very strong (0.75–1.0), strong (0.50–0.74), moderate (0.25–0.49) or weak (0.01–0.24) [38]. In the latter case (weak correlations), it makes no sense to discuss their direction, i.e., whether they are positive or negative [38]. Here, we focused on describing very strong, strong and moderate correlations (in this sequence mainly) between the studied parameters.

Significant positive and negative correlations were found between TAWSA and the following biochemical parameters of pigs' blood for group 3: very high in the case of TP (−0.75) and AST (−0.79); high in the case of cholesterol (−0.72), glucose (0.66), alkaline phosphatase (0.66), calcium ions (−0.60) and globulins (0.53); and moderate in the case of albumin (−0.36), triglycerides (−0.35), magnesium ions (−0.32) and phosphorus (−0.27) (Table 4).

Significant positive correlations were found between the following biochemical and antioxidant parameters of pigs' blood for group 2 (Table 4): high in the case of TP (0.51) and moderate in the case of globulins (0.48), cholesterol (0.33) and phosphates (0.25).

The correlation between TAWSA and biochemical parameters was the most significant only in the case of magnesium ions (−0.48), glucose (0.36) and calcium ions (−0.25) for group 1 (Table 4), i.e., only moderate correlations were found.

Significantly higher values of the correlation coefficients between TAWSA and biochemical parameters (Table 4) and a large number of significant correlations were obtained for group 3 (11 in total, including 7 strong and very strong) as compared to groups 2 (4 in total, including 1 strong) and 1 (3 in total, only moderate). Thus, a clear tendency indicated the stabilization of the PhBS of animals during their growth and fattening. The same dependences concerning correlations of TAWSA and the biochemical parameters of pigs' blood were found in our ongoing research of hybrid animals (Duroc, Landras and Large White pigs of the Russian population).

It is important to highlight that the pronounced correlations between antioxidant and biochemical blood parameters were found earlier in our previous research in hybrid sheep breeds [38] and only in a short presentation concerning different sheep breeds [43]. For example, the author of [43] found the relationship of lipid peroxidation (LPO) parameters with some hematological and biochemical blood parameters of the following breeds: Texel x Manych Merino, Grozny Small-haired Merino and Soviet Merino. At a high level of lipid peroxidation (5.2 mmol/L), an inverse correlation was established among hemoglobin, total protein and the gamma-globulin fraction of protein for crossbred animals [43]. The high (r = −0.6–0.7) and very high (r = −0.9) negative correlations were revealed between LPO and total proteins (or albumins) for Soviet merino at the same antioxidant background (MDA = 5.0 mmol/L) [43]. Significant correlations were also established for the Grozny Yark breed (r = 0.8–0.7 for hemoglobin and albumin and r = −0.9 for globulins) [43]. All of these data indicated a particular balance between oxidative and reduction processes in the body of these sheep breeds, as considered by the authors of [34,35,38,43].

#### **5. Conclusions**

The presented data on the biochemical and antioxidant parameters of the blood serum of the Duroc breed pigs and their specific correlations were obtained for the first time. The tendency towards the optimization of blood biochemical parameters in adult animals can be useful for understanding the features of PhBS and the antioxidant status of pigs. The revealed tendencies can be explained only by a closer (than previously thought) relationship between the biochemical and antioxidant functionality of the pig body. In our opinion, the rate of ROS neutralization can be related to the rate of reaching a live weight of 100 kg, which is an important economic feature. This can be explained by the fact that the metabolic energy with an increase in the fattening period will be directed mainly to gaining muscle mass, but not "to fight free radicals" (ROS). The authors assume that the revealed tendency of changes in the biochemical and antioxidant parameters of

Duroc breed pigs (at a longer feeding duration) will be monitored in our further ongoing experiments with hybrid animals. These data will be used by the Ministry of Agriculture of the Russian Federation and are especially important in order to establish the range of particular biochemical references (standard norms) for the Duroc breed pigs of the Russian population.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ani11082400/s1, Table S1: Correlations of biochemical parameters\* of the blood serum of the Duroc breed pigs in group 1 (65 days of fattening), Table S2. Correlations of biochemical parameters of the blood serum of the Duroc breed pigs in group 2 (72 days of fattening), Table S3. Correlations of biochemical parameters of the blood serum of the Duroc breed pigs in group 3 (100 days of fattening).

**Author Contributions:** Conceptualization, S.Y.Z.; methodology, A.A.B., O.A.V. and R.A.R.; validation, N.V.B. and O.A.V.; formal analysis, A.A.B.; investigation, A.A.B., R.A.R. and O.A.V.; resources, S.Y.Z. and N.V.B.; data curation, A.A.B. and O.A.V.; writing—original draft preparation, S.Y.Z. and N.V.B.; writing—review and editing, S.Y.Z. and A.A.B.; supervision, S.Y.Z. and N.V.B.; project administration, S.Y.Z. and N.V.B.; funding acquisition, S.Y.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** The methodological topics of this research (parts 1 and 2) were supported by the Ministry of Science and Higher Education of the Russian Federation (registration number of the topic of the state task: 121052600314–1); parts 3, 4 and 5 were supported by a grant from the Russian Science Foundation (project no. 20–16-00032).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, internationally recognized guidelines (concerning experiments with animals), and approved by the Ethics Committee of L.K. Ernst Federal Research Center for Animal Husbandry (protocol code: 2021–2303; date of approval: 23 March 2021).

**Data Availability Statement:** Data supporting reported results can be found at: https://www.vij.ru/ goszadanie-i-proekty/proekty/proekty-rnf/749-proekt-20-16-00032-rnf (accessed on 1 June 2021).

**Acknowledgments:** The authors are grateful to T.V. Karpushkina and A.A. Savina for technical assistance.

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

#### **References**


## *Article* **Bayesian Analysis of the Effects of Olive Oil-Derived Antioxidants on Cryopreserved Buck Sperm Parameters**

**Ander Arando Arbulu 1,2, Francisco Javier Navas González 1,3,\* , Alejandra Bermúdez-Oria <sup>4</sup> , Juan Vicente Delgado Bermejo <sup>1</sup> , África Fernández-Prior <sup>4</sup> , Antonio González Ariza <sup>1</sup> , Jose Manuel León Jurado <sup>5</sup> and Carlos Carmelo Pérez-Marín 6**

	- 14014 Cordoba, Spain; pv2pemac@uco.es

**Simple Summary:** The use of olive oil by-products for caprine sperm cryopreservation offers an interesting opportunity to improve post-thawed sperm quality, as antioxidants such as hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG) could reduce lipid peroxidation. Therefore, this study provides evidence of the positive effect of the addition of HT, DHPG, or the mixture of both antioxidants in cryopreserved buck sperm. In addition, the application of Bayesian statistics for data analysis may enable quantifying the dimensionality of the real effect of antioxidants on sperm.

**Abstract:** The present study evaluates the effect of olive oil-derived antioxidants, hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG), on cryopreserved caprine sperm using Bayesian inference of ANOVA. For this proposal, sperm was collected, pooled and diluted in freezing media supplemented with different concentrations of HT, DHPG and the mixture (MIX) of both antioxidants. Sperm motility, viability, acrosome integrity, mitochondrial status, and lipid peroxidation (LPO) were assessed in fresh and frozen-thawed sperm samples. The results provided evidence that HT at low concentrations improves sperm motility and viability, and reduces the LPO. Contrastingly, DHPG and MIX exert a positive effect by reducing LPO values as concentration increases. Additionally, mitochondrial potential was reduced when samples were supplemented with HT at low concentrations and mixture of both antioxidants. Conclusively, the addition of olive oil-derived antioxidants (HT at 10 µg/mL and DHPG at 30 µg/mL) implements a protective effect in cryopreserved buck sperm. Bayesian analysis alternatives offer new possibilities to determine the repercussion of antioxidants on sperm, both quantitatively and qualitatively.

**Keywords:** phenolic antioxidant; olive oil; caprine; spermatozoa; Bayesian inference

### **1. Introduction**

For the goat industry, the combination of artificial insemination and sperm cryopreservation is an optimal manner in which to speed up genetic improvement while reducing the incidence of sexually transmitted diseases. However, sperm cryopreservation procedures are associated with irreversible damage in sperm cells, which compromises sperm fertility due to of cold shock, osmotic stress, and intracellular ice crystals formation, among others [1]. One of the reasons of the vulnerability of goat spermatozoa to freezing-thawing procedures is the composition of their plasma membrane, which contains large amounts of

**Citation:** Arando Arbulu, A.; Navas González, F.J.; Bermúdez-Oria, A.; Delgado Bermejo, J.V.; Fernández-Prior, Á.; González Ariza, A.; León Jurado, J.M.; Pérez-Marín, C.C. Bayesian Analysis of the Effects of Olive Oil-Derived Antioxidants on Cryopreserved Buck Sperm Parameters. *Animals* **2021**, *11*, 2032. https://doi.org/10.3390/ani11072032

Academic Editors: Woo Kyun Kim, Youssef A. Attia, Maria de Olivera, Nesrein Hashem and Manuel Álvarez-Rodríguez

Received: 21 May 2021 Accepted: 6 July 2021 Published: 7 July 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/).

polyunsaturated fatty acids [2]. As a consequence, such sperm turns highly susceptible to membrane peroxidation derived from the lipid oxidation of membrane by reactive oxygen species (ROS) [3]. The presence of prooxidant molecules, such as free radicals and ROS, is strongly related to metabolic stress and spermatozoa damage during cryopreservation [4].

Nevertheless, ROS have a variable effect on spermatozoa and their impact hinges on the nature and concentration of these substances. When in physiological concentrations, a promoting effect has been reported on capacitation, acrosomal reaction, and sperm zona pellucida interactions [5]. By contrast, high concentrations of ROS are related to spermatozoa normal function inhibition, thus the reduction of sperm viability due to the oxidative stress (OE) and the subsequent peroxidation of polyunsaturated fatty acids in their membranes [6].

Under normal conditions, spermatozoa have endogenous mechanisms to deal with OE. In the sperm plasma, enzymatic and non-enzymatic antioxidants are present, and they participate in the balance mechanism to prevent OE [7]. By contrast, when spermatozoa are exposed to stress conditions, endogenous antioxidants cannot counteract the excess of free radicals; thus, the addition of exogenous antioxidants may be essential to preserve the quality of these cells. In this sense, several studies have evaluated the use of exogenous antioxidants in goat sperm [3,8–11]. In this framework, a large number of natural compounds has been tested in cell cultures assessing the antioxidant, anti-inflammatory or chelating properties of antioxidants, in recent years, e.g., mentha [12], *Feijoa sellowiana* [13], grapes [14], or olives [15].

Olive fruits (*Olea europea*), olive oil, and its derivates present a large amount of phenolic components, for which remarkable antioxidant properties have been reported [16], with corroborated advantages for human health [17]. Two of the most important phenolic compounds present in olive fruit are hydroxytyrosol (3,4 dyhydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), which are isolated from the alperujo olive pulp (semi-solid waste generated in the two-phase system used in olive oil extraction). HT is a simple phenol with significant antioxidant properties [16], which reduces the oxidation of low-density lipoproteins, protect against H2O<sup>2</sup> cytotoxicity and minimize lactate dehydrogenase activity [18–20]. Regarding DHPG, powerful antioxidant and potential anti-inflammatory effects have been reported which even compare to those reported for vitamin E [21].

The use of HT and DHPH is widespread in human health studies. By contrast, there are few studies on the effects derived from the addition of these antioxidants to sperm dilution media in animals. Contextually, HT-supplemented sperm extender has previously been evaluated in studies conducted on rats [22], humans [23], and rams [15,24,25]. However, the effect of DHPG supplementation has only been reported in ram sperm [15,25]. Taking into account the properties of both antioxidants, the present study hypothesizes that extenders supplemented with these compounds might counteract the sperm damage inflicted by the cryopreservation process. Thus, the aim of the present study was to evaluate the effect of freezing extenders supplemented by different concentrations of HT, DHPG, and the mixture of both substances on the post-thawed sperm quality of goat semen. The effects of the increasing concentrations on each sperm parameter pair correlation were studied.

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

#### *2.1. Chemicals*

HT and DHPG, stock solution 76.9 mM and 14.7 mM, respectively (Figure 1), were extracted and purified from olive by-products (alperujo) following the processes described by Fernandez-Bolaños et al. [26] and Fernández-Bolaños Guzmán et al. [27]. A commercial TRIS-based extender (Biladyl, Minitube Iberica, Tarragona, Spain) was used to centrifuge and freeze sperm. LIVE/DEAD® sperm viability kit, composed by SYBR-14 and propidium iodide (PI), Mitotracker Red CMXRos and C11-BODIPY581/591 were purchased from Molecular Probes Europe (Leiden, The Netherlands). Peanut agglutinin conjugated

with fluorescein isothiocyanate (PNA-FITC) was obtained in Sigma-Aldrich (St. Louis, MO, USA). Louis, MO, USA).

to centrifuge and freeze sperm. LIVE/DEAD® sperm viability kit, composed by SYBR-14 and propidium iodide (PI), Mitotracker Red CMXRos and C11-BODIPY581/591 were purchased from Molecular Probes Europe (Leiden, The Netherlands). Peanut agglutinin conjugated with fluorescein isothiocyanate (PNA-FITC) was obtained in Sigma-Aldrich (St.

*Animals* **2021**, *11*, x 3 of 14

**Figure 1.** Chemical structure of olive oil-derived antioxidants used in the present study.

#### **Figure 1.** Chemical structure of olive oil-derived antioxidants used in the present study. *2.2. Animals and Semen Collection*

*2.2. Animals and Semen Collection*  Semen was collected from six Murciano-Granadina breed bucks (4–5 years old). The animals involved in the study were located at the Centro Agropecuario Provincial de Córdoba (Córdoba, Spain) and were managed following the prescriptions and regulations of the European Union (2010/63/EU) in its transposition to Spanish law (RD 53/2013). A total Semen was collected from six Murciano-Granadina breed bucks (4–5 years old). The animals involved in the study were located at the Centro Agropecuario Provincial de Córdoba (Córdoba, Spain) and were managed following the prescriptions and regulations of the European Union (2010/63/EU) in its transposition to Spanish law (RD 53/2013). A total of 12 ejaculates per animal (72 ejaculates in total) were collected with an artificial vagina twice a week during the non-breeding season. Previously, semen had been collected for one month in order to ensure the renewal of the epididymal reserves.

of 12 ejaculates per animal (72 ejaculates in total) were collected with an artificial vagina twice a week during the non-breeding season. Previously, semen had been collected for one month in order to ensure the renewal of the epididymal reserves. After collection, the ejaculates were placed in a water bath at 37 °C during evaluation and they were assessed to determine volume by graduated tubes, sperm concentration by photometer (Accurread, IMV technologies, France) and mass motility, by placing 5 µL of raw semen on a preheated slide (37 °C) and observed in the optical microscope (40× magnification; Olympus, Tokyo, Japan). Sperm mass motility was scored subjectively from 0 After collection, the ejaculates were placed in a water bath at 37 ◦C during evaluation and they were assessed to determine volume by graduated tubes, sperm concentration by photometer (Accurread, IMV technologies, France) and mass motility, by placing 5 µL of raw semen on a preheated slide (37 ◦C) and observed in the optical microscope (40× magnification; Olympus, Tokyo, Japan). Sperm mass motility was scored subjectively from 0 (no motile spermatozoa) to 5 (numerous rapid waves) as described by Evans and Maxwell [28] and Lopes et al. [29]. The inclusive criteria for ejaculates to be considered in the study were: volume <sup>≥</sup> 0.5 mL, concentration <sup>≥</sup> <sup>3000</sup> <sup>×</sup> <sup>10</sup><sup>6</sup> spz/mL and masal motility ≥ 4 (ejaculates with more than 70% total motility).

(no motile spermatozoa) to 5 (numerous rapid waves) as described by Evans and Maxwell

µg/mL); DHPG4 (70 µg/mL); MIX1 (5 µg/mL HT + 5 µg/mL DHPG); MIX2 (15 µg/mL HT + 15 µg/mL DHPG); MIX3 (25 µg/mL HT + 25 µg/mL DHPG); MIX4 (35 µg/mL HT + 35 µg/mL DHPG). In order to maintain the aforementioned final antioxidant concentrations, the fact that the extender used (Biladyl) requires a two-step process was considered. Therefore, the same antioxidant concentration was added to both fractions used (FAey

Sperm motility, viability, acrosome integrity, mitochondrial membrane potential

#### [28] and Lopes et al. [29]. The inclusive criteria for ejaculates to be considered in the study *2.3. Experimental Design*

were: volume ≥ 0.5 mL, concentration ≥ 3000 × 106 spz/mL and masal motility ≥ 4 (ejaculates with more than 70 % total motility). *2.3. Experimental Design*  As schematized in Figure 2, every sperm collection day, sperm samples were splitted into 13 different aliquots and diluted with extenders containing different (or null) concentrations of HT, DHPG, or a mixture of both antioxidants (MIX), to obtain the final concentrations as follows: Control (without antioxidant); HT1 (10 µg/mL); HT2 (30 µg/mL); HT3 (50 µg/mL) and HT4 (70 µg/mL); DHPG1 (10 µg/mL); DHPG2 (30 µg/mL); DHPG3 (50 As schematized in Figure 2, every sperm collection day, sperm samples were splitted into 13 different aliquots and diluted with extenders containing different (or null) concentrations of HT, DHPG, or a mixture of both antioxidants (MIX), to obtain the final concentrations as follows: Control (without antioxidant); HT1 (10 µg/mL); HT2 (30 µg/mL); HT3 (50 µg/mL) and HT4 (70 µg/mL); DHPG1 (10 µg/mL); DHPG2 (30 µg/mL); DHPG3 (50 µg/mL); DHPG4 (70 µg/mL); MIX1 (5 µg/mL HT + 5 µg/mL DHPG); MIX2 (15 µg/mL HT + 15 µg/mL DHPG); MIX3 (25 µg/mL HT + 25 µg/mL DHPG); MIX4 (35 µg/mL HT + 35 µg/mL DHPG). In order to maintain the aforementioned final antioxidant concentrations, the fact that the extender used (Biladyl) requires a two-step process was considered. Therefore, the same antioxidant concentration was added to both fractions used (FAey and FBey), as described below.

and FBey), as described below.

**Figure 2.** Schematic overview of the experimental design. **Figure 2.** Schematic overview of the experimental design.

*2.4. Semen Dilution and Freezing*  After collection, ejaculates were diluted at 1:2 with TRIS-based extender (FA of Bila-Sperm motility, viability, acrosome integrity, mitochondrial membrane potential (HMMP), and membrane lipid peroxidation (LPO) were assessed in frozen-thawed sperm samples. Twelve repetitions of the experiment were performed.

#### dyl without egg yolk) for individual evaluation and, if inclusion criteria were reached, *2.4. Semen Dilution and Freezing*

bath at 37 °C for 30 s.

2.5.1. Motility

these were pooled and diluted to reach a dilution of 1:10. To remove seminal plasma, pooled samples were centrifuged at 600 g for 15 min. The supernatant was removed and the pellet was resuspended, adding a volume of fraction A of Biladyl containing egg yolk (FAey). Then, the pooled sample was split into 13 different aliquots; 12 samples were prepared by adding FAey supplemented with the previously described HT, DHPG and MIX antioxidant concentrations. A control group (no antioxidant) was also prepared. The samples were then immediately placed in a programmable freezer (cell incubator SH-020S, Welson, Korea) to reach 5 °C and maintained for two hours at 5 °C. After collection, ejaculates were diluted at 1:2 with TRIS-based extender (FA of Biladyl without egg yolk) for individual evaluation and, if inclusion criteria were reached, these were pooled and diluted to reach a dilution of 1:10. To remove seminal plasma, pooled samples were centrifuged at 600× *g* for 15 min. The supernatant was removed and the pellet was resuspended, adding a volume of fraction A of Biladyl containing egg yolk (FAey). Then, the pooled sample was split into 13 different aliquots; 12 samples were prepared by adding FAey supplemented with the previously described HT, DHPG and MIX antioxidant concentrations. A control group (no antioxidant) was also prepared. The samples were then immediately placed in a programmable freezer (cell incubator SH-020S, Welson, Korea) to reach 5 ◦C and maintained for two hours at 5 ◦C.

Then, each aliquot was diluted with Tris-egg yolk-glycerol extender (FBey) supplemented with HT, DHPG and/or MIX, obtaining a final volume of 1000 µL per sample (with a concentration of 400 × 106 spz/mL). The samples were loaded into 0.25 mL straws (100 × 106 spz/straws) and maintained for two hours at 5 °C. Then, each aliquot was diluted with Tris-egg yolk-glycerol extender (FBey) supplemented with HT, DHPG and/or MIX, obtaining a final volume of 1000 µL per sample (with a concentration of 400 <sup>×</sup> <sup>10</sup><sup>6</sup> spz/mL). The samples were loaded into 0.25 mL straws (100 <sup>×</sup> <sup>10</sup><sup>6</sup> spz/straws) and maintained for two hours at 5 ◦C.

Three straws per sample were frozen using liquid nitrogen vapors. Straws were horizontally placed in racks 4 cm above the liquid nitrogen level for 10 min and then plunged Three straws per sample were frozen using liquid nitrogen vapors. Straws were horizontally placed in racks 4 cm above the liquid nitrogen level for 10 min and then

in liquid nitrogen pending analysis. For thawing, the samples were immersed in a water

ISAS software v.1.2 (Integrated Semen Analyser System, Proiser, Valencia, Spain)

equipped with an HS640C video camera was used to assess sperm motility. Sperm samples were diluted in FAey at a final concentration of 25 × 106 spz/mL and, after 10 min incubation, 5 µL of each diluted sample was evaluated using a slide and covered (22 × 22 plunged in liquid nitrogen pending analysis. For thawing, the samples were immersed in a water bath at 37 ◦C for 30 s.

#### *2.5. Sperm Quality Assessment*

#### 2.5.1. Motility

ISAS software v.1.2 (Integrated Semen Analyser System, Proiser, Valencia, Spain) equipped with an HS640C video camera was used to assess sperm motility. Sperm samples were diluted in FAey at a final concentration of 25 <sup>×</sup> <sup>10</sup><sup>6</sup> spz/mL and, after 10 min incubation, 5 µL of each diluted sample was evaluated using a slide and covered (22 × 22 mm). Four fields and a minimum of 500 spermatozoa were randomly captured at 10× magnification using a UB203i phase contrast microscope (Chongqing UOP Photoelectric Technology Co., Ltd, Beibei District, Chongqing, China). A total of 25 images per second were acquired, selecting particles with an area of between 10 and 70 µm<sup>2</sup> and categorized as motile when VAP >10 µm/s, and linearly motile when they deviated >75% from a straight line. The analyses provided information about total motility (TM, %) and progressive motility (PM, %), curvilinear velocity (VCL, µm/s), straight line velocity (VSL, µm/s), average path velocity (VAP, µm/s), straightness (STR, %), linearity (LIN, %), wobble (WOB, %), amplitude of lateral head displacement (ALH, µm), and beat/cross frequency (BCF, Hz).

#### 2.5.2. Flow Cytometer

The recommendations of the International Society for Advancement of Cytometry were followed to perform flow cytometric analyses [30] using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with a 488 nm argon blue laser. Sheath flow rate was set at 12.0 ± 3 µL/min in all analyses (LOW mode). Green fluorescence from SYBR-14, PNA-FITC and C11-BODIPY581/591 was read with an FL1 photodetector (530/30 band-pass filter). Red fluorescence PI and Mitotracker Red CMXRos was read with an FL2 photodetector (585/42 nm bandpass filter). Approximately 10,000 events of a gated population were counted per sample.

Forward scatter (FSC) and side scatter (SSC) were recorded in a linear mode (in FSC vs. SSC dot plots). Data were acquired as FSC files using BD Cell Quest Pro v. 6.0, (Becton Dickinson Immunochemistry, San Jose, CA, USA). FlowJo® Version 7.6.2 software (FlowJoTM, Ashland, OR, USA) was used to analyze the acquired data, using dot plots with the relative cell size (FSC), the internal complexity (SSC) and the specific fluorescence intensity for each probe.

#### 2.5.3. Viability

A LIVE/DEAD® sperm viability kit was used to evaluate sperm viability and the evaluation was conducted along the lines recommended by Arando et al. [31]. A total of 100 µL of sperm was diluted with 150 µL of cytometer buffer to reach a final concentration ~4 <sup>×</sup> <sup>10</sup><sup>6</sup> spz/mL. Then, 2.5 µL SYBR-14 (2 µM) and 5 µL PI (480 µM) were added and incubated in darkness conditions for 15 min. After incubation, the proportion of live/dead sperm cells was measured. Spermatozoa emitting in the green wavelength were deemed to be spermatozoa with intact plasma membranes and the results were reported as the percentage of spermatozoa with intact plasma membrane. Unstained and single-stained samples were used for calibrating the FSC gain, FL-1 and FL-2 PMT voltages and for compensation of SYBR-14 spill over into the PI channel (9.8%). Non-sperm particles (also called "alien events") were located in the SYBR-14−/PI− quadrant and they did not contain DNA. Spermatozoa with intact plasma membrane were located in the SYBR-14+/PI<sup>−</sup> quadrant.

#### 2.5.4. Acrosome Integrity

Acrosome integrity was assessed using the combination of PNA-FITC and PI. Onehundred microliters of sperm (containing around 40 <sup>×</sup> <sup>10</sup><sup>6</sup> spz) was incubated in darkness for 5 min with 5 µL of PNA-FITC stock solution (100 µg/mL in DMSO) and 5 µL PI

(480 µM). After incubation, 400 µL of cytometer buffer was added and samples were analyzed. PI− and PNA-FITC− cells were categorized as sperm with intact acrosome and plasma membrane. Unstained and single-stained samples were used for setting the FSC gain, FL-1 and FL-2 PMT voltages and for compensation of PNA-FITC spill over into the PI channel (9.8%). The percentages of alien particles (f) determined by SYBR-14/PI staining were used to correct the percentages of non-stained spermatozoa (q1) in each sample in order to obtain the corrected percentage of non-stained spermatozoa (q10 ), in accordance with Petrunkina & Harrison [32]:

$$\mathbf{q1'} = \left[ (\mathbf{q1} - \mathbf{f}) / (100 - \mathbf{f}) \right] \times 100. \tag{1}$$

#### 2.5.5. Mitochondrial Membrane Potential

The combination of Mitotracker Red CMXRos and SYBR-14 was used to estimate mitochondrial membrane potential and it was assessed using a modified protocol [33,34]. A volume of 50 <sup>µ</sup>L of sperm (containing 20 <sup>×</sup> <sup>10</sup><sup>6</sup> spz) was mixed with 350 µL of cytometer buffer and immediately loaded with 2 µL SYBR-14 (2 µM) and 2 µL Mitotracker Red CMXRos (20 µM) was added. Sperm doses were incubated for 10 min at 37 ◦C in the dark and only sperm with high mitochondrial potential (HMMP) were reported. Unstained and single-stained samples were used for setting the FSC gain, FL-1 and FL-2 PMT voltages. Data were not compensated.

#### 2.5.6. Lipid Peroxidation

Lipid peroxidation (LPO) was estimated using C11-BODIPY581/591 (Molecular Probes Europe, Leiden, The Netherlands) using a modified protocol [35]. A volume of 100 µL of diluted sperm (containing around 2 <sup>×</sup> <sup>10</sup><sup>6</sup> spz) was mixed with 1 µL C11-BODIPY581/591 (0.2 mM) and incubated at 37 ◦C for 30 min. After incubation, 1 mL of PBS was added for centrifugation at 600× *g* for 8 min. The pellet was resuspended with 100 µL of PBS prior to assessment. Spermatozoa with LPO emitted light in the green wavelength and were deemed to be BODIPY-positive cells. Unstained and single-stained samples were used for setting the FSC gain, FL-1 and FL-2 PMT voltages. Data were not compensated.

#### *2.6. Data Analysis*

Bayesian inference for ANOVA was run to test for statistical differences in the mean across antioxidants (HT, DHPG and MIX) at different concentrations (Control, HT1, HT2, HT3, HT4, DHPG1, DHPG2, DHPG3, DHPG4, MIX1, MIX2, MIX3 and MIX4) on buck sperm parameters (Table 1).

The Bayes factor (BF) quantifies the strength of the evidence of null and alternative hypotheses and is used instead of frequentist *p* values when Bayesian approaches are applied to issue conclusions. As BF increases, the degree at which evidence favors the alternative hypothesis compared to the null hypothesis increases as well. In this context, Cleophas and Zwinderman [36], suggested a method to extrapolate between the Bayes factor used in Bayesian approaches and *p* values from frequentist approaches to favor the interpretability of results.

Sample descriptive posterior statistics are modeled from the means and variances of the measured unpaired groups and are provided as sources of variation, while the prior element was modeled as an uninformative prior using the Jeffreys–Zellener–Siow (JZS) method or, equivalently, from the computation of a reference prior based on a gamma distribution with a standard error of 1. As suggested by Martins-Bessa et al. [37], the 95% credibility interval shows that there is a 95% probability that these regression coefficients (posterior distribution mean value for each covariate and factor) in the population lie within the corresponding credibility intervals. When 0 is not contained in the credibility interval, a significant effect for such factor is detected. Integral calculation of factors is required for BF accuracy purposes. Therefore, afterwards, it can be used as a precise statistical index to measure the amount of support in favor of either H<sup>1</sup> (the difference between the unpaired means is larger than zero) or H<sup>0</sup> (the difference between the unpaired means is not larger

than zero). Contextually, Bayesian approaches provide a better perspective of the structure model of the H<sup>1</sup> and H0. Therefore, the maximal likelihoods of likelihood distributions are not always identical to the mean effect of traditional tests, which specifically fits the context of biological inferences, given biological likelihoods may better respond biological questions than numerical means of non-representative subgroups do.

**Viability (%) Acrosome Integrity (%) HMMP (%) LPO (%)** FRESH 84.4 ± 4.5 72.0 ± 6.4 79.8 ± 7.1 1.9 ± 0.8 CONTROL 43.9 ± 7.2 43.0 ± 7.7 40.3 ± 6.6 2.1 ± 0.5 HT1 50.2 ± 6.5 40.2 ± 9.8 32.8 ± 4.4 1.7 ± 0.6 HT2 44.5 ± 5.9 37.7 ± 5.5 32.6 ± 7.0 2.2 ± 0.4 HT3 44.5 ± 8.8 42.2 ± 11.4 38.2 ± 5.6 2.5 ± 0.7 HT4 43.1 ± 9.0 42.3 ± 6.9 40.3 ± 7.1 2.2 ± 0.8 DHPG1 49.9 ± 14.2 47.5 ± 11.3 40.8 ± 9.0 2.2 ± 1.3 DHPG2 43.8 ± 6.7 41.7 ± 6.4 35.8 ± 8.2 1.6 ± 0.5 DHPG3 43.6 ± 7.0 38.8 ± 6.2 36.8 ± 9.1 1.8 ± 0.9 DHPG4 47.0 ± 8.5 35.0 ± 8.3 40.0 ± 6.0 1.3 ± 0.7 MIX1 43.4 ± 7.9 39.0 ± 5.2 35.8 ± 7.3 2.9 ± 1.5 MIX2 44.2 ± 8.6 39.9 ± 7.9 36.5 ± 7.3 1.6 ± 0.7 MIX3 42.6 ± 13.3 41.7 ± 12.7 38.0 ± 3.1 1.6 ± 0.6 MIX4 43.2 ± 8.5 38.8 ± 8.9 34.0 ± 8.1 1.6 ± 0.7

**Table 1.** Descriptive statistics of sperm viability, acrosome integrity, HMMP, and LPO parameters in fresh and frozen-thawed buck sperm. Data are expressed as Mean ± SD.

HMMP: mitochondrial potential; LPO: lipid peroxidation; HT: hydroxytyrosol; DHPG: 3,4-dihydroxyphenylglycol; CONTROL (without antioxidant); HT1 (10 µg/mL); HT2 (30 µg/mL); HT3 (50 µg/mL) and HT4 (70 µg/mL); DHPG1 (10 µg/mL); DHPG2 (30 µg/mL); DHPG3 (50 µg/mL); DHPG4 (70 µg/mL); MIX1 (5 µg/mL HT + 5 µg/mL DHPG); MIX2 (15 µg/mL HT + 15 µg/mL DHPG); MIX3 (25 µg/mL HT + 25 µg/mL DHPG); MIX4 (35 µg/mL HT + 35 µg/mL DHPG).

IBM SPSS Statistics Algorithms version 25.0 by IBM Corp. [38] suggests Bayesian inference of ANOVA is approached as a special case of the Bayesian general multiple linear regression model. The algorithms used by SPSS to perform Bayesian Inference on Analysis of Variance (ANOVA) in this study are described in IBM SPSS Statistics Algorithms version 25.0 by IBM Corp. [38]. The tolerance value for the numerical methods and the number of method iterations were set as a default by SPSS v25.0 (IBM Corp., Armonk, NY, USA) [39].

The around 0 symmetric JZS prior was used as it is appropriate for Bayesian inference of ANOVA, provided positive and negative values of the slope parameters a priori have the same probability of occurring [40]. Furthermore, its scale-invariant properties, permits comparing parameters measured in different units, as it occurs in the present study. Bayesian inference for ANOVA was performed using the Bayesian Package of SPSS v25.0 (IBM Corp., Armonk, NY, USA) [39].

#### **3. Results**

#### *3.1. Prior Descriptive Statistics*

The descriptive statistics of sperm viability, acrosome integrity, HMMP, and LPO parameters in fresh and frozen-thawed buck sperm are shown in the Tables 1 and 2.

#### *3.2. Bayesian Inference of Olive Oil Derived Antioxidant Effect*

Table 3 and Supplementary Table S1 report the outputs from Bayesian ANOVA analysis and present posterior distribution statistics for sperm parameters across antioxidants and concentrations. Regarding to motility parameters, an evident/significant (*p* < 0.05) increase in PM was observed when HT2 treatment when compared to control treatment. However, at higher HT concentrations (70 µg/mL) an evident/significant (*p* < 0.05) decrease in PM occurred.




When velocity parameters (VCL, VSL, and VAP) were evaluated, the mixture of both antioxidants produced a dose-dependent decrease in VSL and VAP values, with this being evidently/significantly (*p* < 0.05) lower when high concentrations were used. However, the rest of the studied kinetic parameters were not evidently/significantly affected (*p* > 0.05) by the addition of antioxidants, except for LIN when high concentrations of HT were added (70 µg/mL).

Samples supplemented with low concentrations of HT and DHPG (10 µg/mL) reported evident/significant (*p* < 0.05) sperm viability increases in comparison to Control. This positive evident/significant (*p* < 0.05) effect was also observed for acrosome integrity in samples supplemented with low DHPG concentrations (10 µg/mL). By contrast, higher DHPG concentrations offered evidently lower acrosome integrity. Similarly, a negative effect was observed when high concentrations (70 µg/mL) of the mixture of both antioxidants were used.

Mitochondrial potential evidently/significantly decreased (*p* < 0.05) in comparison to when the Control treatment was considered in samples supplemented with HT at low concentrations (10 and 30 µg/mL). Similarly, the addition of a mixture of both antioxidants evidently/significantly (*p* < 0.05) reduced the mitochondrial potential in frozen-thawed spermatozoa. Regarding to LPO values, the addition of 10 µg/mL of HT reported an evident/significant (*p* < 0.05) protective effect, reducing its value in comparison with Control treatments. However, when the dose increased, an opposite evident/significant (*p* < 0.05) effect was observed. By contrast, DHPG and MIX provided a dose-dependent better protection, diminishing LPO values as antioxidant concentration increases.

#### **4. Discussion**

Sperm cryopreservation offers goat breeders several benefits over fresh sperm storage. However, recent studies suggested that ROS concentration increases considerably during cryopreservation, disrupting sperm functions and subsequent fertilization [41]. Sometimes the endogenous antioxidant capacity of sperm cells is compromised due to the proliferation of ROS, producing an imbalance that promotes oxidative stress and consequently LPO, which affects membrane structure and distorts its functions such as membrane fluidity, membrane enzymes, ion gradients, receptor transduction, and transport processes [42]. In this context, the addition of antioxidants to the semen extender seems to have the potential to mitigate the negative impact of oxidative stress, as antioxidants capture free radicals and conclude the chain reaction, maintaining a redox state and offsetting their capacity to reduce molecular oxygen [43].

The addition of natural or synthetic antioxidants to the cryopreservation medium in goat sperm has attracted the attention of researchers as an alternative to diminish the negative effect of oxidative stress produced by ROS and to improve post-thawed sperm quality [11,44,45]. However, it is not easy to prove the exact nature of the action of antioxidants on sperm quality, and the degree to which interactions with other factors such as the species, the extender and the type or the concentration of antioxidant may be involved [46]. There is a knowledge gap on whether antioxidants are absorbed unchanged or metabolized into completely different compounds. Furthermore, the efficacy of the common antioxidants, such as vitamins C and E, selenium, and herbal supplements to reduce pathological ROS has not yet been determined [47].

In this context, HT is soluble in both lipid and water solutions, and therefore soluble in all phases of the heterogeneous system studied in the present research. The concentration of HT in biological systems is very similar in both aqueous and lipid areas [48]. As far as DHPG is concerned, this component has only recently been isolated and there is relatively little information regarding the way it behaves. On the basis of its chemical structure, which is very similar to HT, it should be broadly analogous to HT, and it is also soluble in both lipid and water solutions.

To the present authors' knowledge, this is the first study in which HT and DHPG were tested as antioxidants for caprine sperm cryopreservation. Biological activity and

risk/benefit of polyphenolic compounds are dependent on their diversity, dual-effects, biological activity, and source [49]. In this regard, the effect of olive oil and olive oil-derived antioxidants on sperm quality has previously been investigated in other species. In rabbits, olive oil administered at 7% *v*/*w* for 16 weeks succeeded in recovering the loss of volume, count, motility, and normal spermatozoa in males exposed to a hypercholesterolemic diet [50]. Similarly, the oral administration of olive oil in healthy rats at 0.4 mL daily for six weeks improved the sperm parameters [51]. Banihani [52] concluded that the addition of olive oil preserves semen quality by enhancing the gonadal function, reducing oxidative injury and lipid peroxidation, and promoting nitric oxide signaling.

Results derived from the present study reported the fact that in samples supplemented with 30 µg/mL of HT an increase in progressive motility of 11% was reported in comparison to Control treatment which agrees the results reported by Hamden et al. [22], who supplemented rat sperm with 50 µg/mL HT and those by Krishnappa et al. [24] who reported an improvement in total motility when 80 mM HT was added to ovine sperm. These findings suggest that the presence of HT could mitigate ROS concentration, preventing the negative impact of moderately elevated ROS concentrations on the sperm movement, mostly via depletion of intracellular ATP and the successive reduction in the phosphorylation of axonemal proteins [53]. However, in the present study, PM considerably decreased when high concentrations (70 µg/mL) of HT were added. One of the reasons for this negative effect associated to the increase of the dose would be the extender acidification, as previously described by Ibrahim et al. [54], who supplemented goat sperm with alpha lipoic acid. By contrast, no effect was observed in the present study for TM and PM when DHPG and MIX were added, in line with previous studies carried out on sheep using the same antioxidants and concentrations [15,25] and in incubated human sperm [23] after HT supplementation.

In regard to kinematic parameters, no effect was observed when HT and DHPG were independently added. However, the mixture of both antioxidants induced a decrease of VSL and VAP in dose-dependent manner, being ~13% when high concentrations were used. This similar trend was previously described by Arando, et al. [15] in liquid ram sperm stored at 5 ◦C, suggesting that high concentrations of these antioxidants could be deleterious for spermatozoa. Contextually, broad evidence suggests higher concentrations may not necessarily translate into better quality, but indeed may be detrimental [55]. In agreement with the present study, the use of other antioxidants such as arbutin, butylated hydroxyanisole, rosemary or lycopene, have reported a positive effect on goat sperm motility [11,44,45,56].

A high amount of PUFA in sperm membranes could interact with ROS, affecting membrane fluidity, facilitating Ca2+ influx, and provoking membrane protein reorganization and the destabilization of the plasma membrane [57]. Based on the current results, the addition of HT at low concentrations (10 µg/mL) may improve membrane integrity, showing an increase of 14% compared to Control treatment, as reported by Hamden et al. [22] when HT was added. Similarly, some authors reported an increase of membrane integrity when other antioxidants were added in extender medium [8,56,58,59].

Acrosome is a specialized sperm structure comprising membranes and proteins which makes it highly susceptible to ROS-derived damage [60]. In this context, a positive effect was observed in acrosome integrity when samples were supplemented with low DHPG concentrations (10 µg/mL). By contrast, as DHPG concentration increases or when high concentrations of mixture (70 µg/mL) were used, acrosome integrity decreased, as previously described by Arando et al. [25]. On the other hand, Hashem et al. [61] reported higher acrosome integrity values when oleic acid was added to ram sperm. Similarly, recent studies in buck sperm showed that the addition of different antioxidants, as vitamin C or lycopene, could mitigate the acrosome damage [11,62].

Mitochondria are involved in the generation of ROS in spermatozoa through the pathway of nicotinamide adenine dinucleotide-dependent oxide reductase reactions, which directly affects their normal functions [63]. Endogenous antioxidant supplementation has

bene hypothesized to reduce oxidative stress and, as a consequence, to maintain postthawed mitochondrial potential. In this sense, the present study reveals that mitochondrial membrane potential is not improved in freezing-thawing sperm samples supplemented with HT, DHPG, and/or MIX, in comparison to the Control treatment, as reported by Zanganeh et al. [45] and Arando et al. [25].

However, in a recent study using mitochondria-targeted antioxidants, the authors noted a slight significant mitochondrial potential increase [64]. The olive-oil antioxidants tested in the present study did not offer any advantage for the mitochondrial activity after cryopreservation. However, further studies should be conducted to elucidate why the intense impact of freezing-thawing process on the mitochondrial activity in buck sperm cannot be counteract by the mentioned antioxidants. By contrast, other authors observed a significant increase in mitochondrial potential when cysteine, coenzyme Q10, lycopene or alpha-lipoic acid were used [10,11,65].

As far as LPO is concerned, the present study showed that the addition of 10 µg/mL of HT may present a protective function against lipid peroxidation, reducing it by 19% in comparison to Control group. In reference to oil-derived antioxidants, a recent study also reported that the addition of HT and DHPG improved LPO values in ram sperm [25]. However, the opposite effect was observed as dose increased. DHPG and the mixture of both antioxidants provided better protection properties, diminishing LPO values around 20%, when sperm samples were supplemented with high doses of antioxidants.

#### **5. Conclusions**

HT could mitigate ROS concentration, preventing their negative impact on the sperm movement. Dose-dependent extender acidification produces a considerable reduction of PM. The mixture of both antioxidants induced a decrease of VSL and VAP in dose dependent manner, being approximately 13% when high concentrations were used. Higher concentrations may not necessarily lead to better results but may be detrimental for sperm quality. The addition of HT at low concentrations (10 µg/mL) may improve membrane integrity. Acrosome integrity improves when supplementing with low DHPG concentrations (10 µg/mL). By contrast, as DHPG concentration increases or when high concentrations of mixture (70 µg/mL) were used, acrosome integrity decreased. Mitochondrial membrane potential is not improved in freezing-thawing sperm supplemented with HT, DHPG and/or MIX. Olive-oil antioxidants did not benefit mitochondrial activity after cryopreservation. The addition of 10 µg/mL of HT may present a protective function against lipid peroxidation, reducing it by 19%. However, the opposite effect was observed as dose increased. DHPG and the mixture of both antioxidants provided better protection properties, diminishing LPO values around 20%, when sperm was supplemented with high doses of antioxidants. Therefore, in light of the obtained results the addition of HT at 10 µg/mL and DHPG at 30 µg/mL were the most suitable treatments since they improved post-thawing sperm quality.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ani11072032/s1, Table S1. Summary of the posterior distribution of Bayesian statistics and the percentage of improvement (+) or detriment (−) on the quality of buck sperm parameters ascribed to the addition of olive oil-derived antioxidants.

**Author Contributions:** Conceptualization, F.J.N.G., J.M.L.J. and C.P.-M.; Data curation, A.A.A., F.J.N.G., A.G.A., J.M.L.J. and C.P.-M.; Formal analysis, A.A.A., F.J.N.G., A.G.A. and J.M.L.J.; Funding acquisition, Á.F.-P. and J.M.L.J.; Investigation, A.A.A., A.G.A., J.M.L.J. and C.P.-M.; Methodology, A.A.A., F.J.N.G., Á.F.-P., A.G.A. and C.P.-M.; Project administration, Alejandra Bermúdez-Oria and C.P.-M.; Resources, F.J.N.G., A.B.-O., J.V.D.B. and Á.F.-P.; Software, A.A.A., F.J.N.G. and C.P.-M.; Supervision, F.J.N.G., A.B.-O., J.V.D.B., Á.F.-P. and C.P.-M.; Validation, F.J.N.G.; Visualization, F.J.N.G., J.V.D.B., Á.F.-P. and C.P.-M.; Writing—original draft, A.A.A., F.J.N.G., A.G.A. and J.M.L.J.; Writing review and editing, A.A.A., F.J.N.G., A.B.-O., J.V.D.B., Á.F.-P. and C.P.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Torres Quevedo Programme from the Ministry of Science and Innovation of Spain, grant number PTQ2019-010670, granted to Ander Arando Arbulu.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of the University of Córdoba (n. 2018PI/29).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data will be made accessible from corresponding authors upon reasonable request.

**Acknowledgments:** The authors would like to acknowledge the Agricultural and Livestock Center of the Córdoba Provincial Council (Diputación de Cordoba, Spain) for their help with the supply of semen samples.

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

#### **References**

