**Preface to "The Era of Assisted Reproductive Technologies Tailored to the Specific Necessities of Species, Industry and Case Reports"**

This book covers the manuscripts of the Special Issue of Animals titled: "*The Era of Assisted Reproductive Technologies Tailored to the Specific Necessities of Species, Industry and Case Reports*" with a total of 12 original manuscripts and three reviews. All of them are focused on different strategies to enhance the reproductive outcome of our main domestic farm animals (pig, cattle, horse, rabbit and sheep) by the use of novel assisted reproductive technologies (ARTs).

Fertilization is a complex event to orchestrate that can be affected by countless factors, for instance, toxicants, diseases or genetic issues. Thus, for example, it has been demonstrated that the ovine gene BMP15 presents a mutation named FecX. Interestingly, in heterozygosis, ewes exhibit an increase in the ovulation rate, producing more lambs per birth. Unfortunately, homozygosis induces ewe sterility. It has been described that rams carrying the FecX mutation exhibited a better sperm motility parameters and a higher pregnancy rate than non-carrier rams after performing artificial inseminations (AIs) (Abecia et al., 2020). Therefore, when looking to increase profit associated with a better farrowing rate, the selection of males carrying FecX is highly recommended. Nevertheless, before performing AI with a male carrying FecX, the ewe population should test as FecX wild-type or non-carrier. In the same line of thought, in the improvement of farm industry profits, it is a common strategy to select parents that transmit a desired phenotype. For instance, in rabbits, selection can be based on post-weaning daily weight gain. Nevertheless, this selected trait seems to be exhausted after 37 generations of paternal selection (Juarez et al., 2020). At this point, other inherited phenotype selection criteria must be considered (Juarez et al., 2020), for example, feed efficiency, body fat content, litter size, etc.

One of the first steps of the fertilization process begins with ejaculation into the vagina and the female tract undergoes dynamic modifications after it interacts with the ejaculate. Thus, using the rabbit as an animal model, a species where ovulation is induced after mating, it was shown that seminal plasma triggers the differential expression of the glucocorticoid receptor (NR3C1/GR) in the female rabbit reproductive tract (Ruiz-Conca et al., 2020). This exhaustive work analyzed the expression levels of the glucocorticoid receptor changes along seven different subsections of the female rabbit reproductive tract. In addition, differential NR3C1/GR expression throughout early embryo development suggested a relevant role of GR action, mediated by NR3C1 in oviductal and uterine embryo transport (Ruiz-Conca et al., 2020). Further investigation should confirm this observation and be kept in mind when seeking protocols with the final aim of enhancing ART outcome in rabbits.

Sperm, oocyte and embryo cryopreservation is one of the most common ARTs used worldwide that allows long-term preservation of genetic diversity. Nevertheless, it is not a harmless process and cells show declines after the freezing/thawing process. Although a great improvement has been achieved in the cryopreservation medium, cryobiology extenders are still under constant development. The bovine industry is by far one of the sectors where sperm cryopreservation has been increasingly developed in recent decades and where cryopreserved sperm are used routinely to perform AI. Recently, it has been shown that bovine extenders can be successfully used to cryopreserve stallion sperm (Nikitkina et al., 2020). The comparison of commercial versus homemade cryopreservation extenders showed that both can be used interchangeably, with the only advantage being that commercial ones already contain egg yolk within their components, saving time in the process of extender elaboration (Nikitkina et al., 2020).

In their travel along the reproductive tract, sperm must undergo several events, named sperm capacitation, that allow them to have the ability to fertilize an oocyte. Hypermotility and an increase in phospho-tyrosine (PY) levels have been historically used as hallmarks of sperm capacitation status. Recently, it has been shown that two pollutants, perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS), impair boar sperm capacitation (Oseguera-Lopez et al., 2020). ´ The harmful effects were associated with the induction of higher sperm mortality associated with a higher level of reactive oxygen species (ROS) production that led to an increase in the percentage of damaged DNA and lower sperm PY levels (Oseguera-Lopez et al., 2020). ´

The importance of capacitation has been highlighted in equines, where it has been suggested that in vitro fertilization (IVF) fails due to sperm capacitation defects. As a consequence, a lot of effort have been made lately to improve stallion sperm capacitation medium. For instance, the addition of D-penicillamine (PEN) to the capacitation medium improved stallion sperm hypermotility and PY levels (Ruiz-D´ıaz et al., 2020). In addition, PEN allowed for the selection of sperm in vitro by thermotaxis. Thus, those sperm selected by a temperature gradient had better levels of PY and lower levels of DNA fragmentation when compared to the unselected fraction (Ruiz-D´ıaz et al., 2020). The authors proposed to improve equine IVF and intracytoplasmic sperm injection (ICSI) outcomes by selecting sperm by thermotaxis after capacitating them in a medium containing PEN (Ruiz-D´ıaz et al., 2020). Others proposed a protocol to select cryopreserved stallion spermatozoa with higher fertilizing capacity by incubating them in an in vitro model of oviduct epithelial cells (OECs) (Gimeno et al., 2021). The sperm that detached from OECs after capacitation presented higher potential fertilizing capacity, described by an increase in living sperm with progressive motility and with the acrosome intact (Gimeno et al., 2021).

Following from stallion sperm cryopreserved as a model of study, the irradiation of these cells with red light has been proposed as a tool to enhance sperm quality after the process of cryopreservation. Red light induces changes in sperm mitochondrial membrane potential, and intracellular ROS, without affecting the integrity of the plasma membrane and acrosome (Catalan et al., 2021). Interestingly, because different results can be obtained, the turbidity of the ´ cryopreservation extender and the color of the straw chosen (Catalan et al., 2021) must be taken into ´ consideration. Recently, extracellular vesicles have emerged in the field of reproduction due to their roles in reproductive processes. Moreover, extracellular vesicles have been used as a novel tool to ameliorate the harmful effects of sperm cryopreservation (Saadeldin et al., 2020) as well as to improve the outcome of other ARTs (Gervasi et al., 2020).

Female reproductive tract peristaltic movement helps sperm to advance in their trip seeking an oocyte. Once sperm are close to the fertilization place (ampulla), they are guided to the oocyte by chemotaxis (mainly by substances secreted by the oocyte, cumulus cells and others poured into the oviduct). The development of chemotactic chambers specially designed for sperm selection has allowed the discovery that boar sperm selected by chemotaxis using follicular fluid provide better IVF outcomes in comparison to other chemoattractants tested, such as periovulatory oviductal fluid (pOF), conditioned medium (CM) from the in vitro maturation of oocytes and progesterone (P4) (Vieira et al., 2020).

The success of IVF is determined, among other factors, by the quality of both sperm and oocytes. To increase the efficiency of embryo production by in vitro procedures, different non-invasive selection strategies have been summarized by Aguila et al. (2020) to help to predict bovine oocyte competence. In addition, before performing IVF, oocytes need to be maturated in vitro. Nevertheless, ovaries of farm animals are usually obtained in distant locations from the assisted reproductive laboratory. Consequently, the development of appropriate media is needed to transport ovaries and to keep oocyte developmental competence in the best condition. In this regard, it has been shown that transport of up to 5 h in a saline solution protected ovine oocytes better than TCM199 (Mart´ın-Maestro et al., 2020). In addition, IVF outcomes were improved when using an oocyte obtained from ovaries transported in saline solution in comparison to TCM199 (Mart´ın-Maestro et al., 2020). In brief, when ovary transport is needed, a saline solution is the recommended medium to preserve oocyte developmental competence and to produce more and better quality embryos (Mart´ın-Maestro et al., 2020).

The final goal of IVF is the obtention of in vitro embryo production (IVP) that eventually will transfer back to a receptor female. As happens with IVF medium, IVP medium production is under constant optimization to enhance its final yield. For instance, the successful equine pregnancy and foaling rates obtained after ICSI are only 10% of the oocytes matured in vitro. These poor results underline that conditions used for oocyte in vitro maturation are not optimized for equine oocytes. Following the rationale that before ovulation, oocytes are surrounded by follicular fluid (FF), Fernandez-Hern ´ andez et al. (2020) studied the metabolome of periovulatory FF. Their final aim was ´ to improve our insight into the in vivo conditions to eventually translate this knowledge to optimize equine in vitro maturation protocols. The authors found nine metabolites in the FF not included in traditional IVP medium, confirming that bigger efforts have to be made to adapt current ART media to every species' specific needs (Fernandez-Hern ´ andez et al., 2020). Finally, also intrigued by ´ a better IVP medium composition, no differences were found when a comparison of bovine embryo transference results of classic IVP medium containing BSA versus IVP medium containing female reproductive fluids was made (Lopes et al., 2020). Interestingly, some hormone levels (anti-Mullerian ¨ hormone and estrogen:progesterone ratio) were different in the receptor of embryos depending on the IVP medium chosen in comparison with control (pregnant by AI) (Lopes et al., 2020).

In summary, this Special Issue has shown an overview of the new alternatives and protocols to enhance domestic animals' ART outcomes. The editor expects that this group of manuscripts will be used as an essential tool to help veterinarians to increase farm profits and offer them new options to bypass cases of subfertility. At the same time, it is expected to serve as a source of inspiration for researchers of this amazing branch of the biology of reproduction interested in the enhancement of fertility results by the use of novel ARTs, and new ideas may emerge after the reading this Special Issue.

> **David Mart´ın Hidalgo** *Editor*

### *Article* **Semen Quality of Rasa Aragonesa Rams Carrying the** *FecXR* **Allele of the** *BMP15* **Gene**

**José Alfonso Abecia 1,\*, Ángel Macías 2, Adriana Casao 1, Clara Burillo 2, Elena Martín 2, Rosaura Pérez-Pé <sup>1</sup> and Adolfo Laviña <sup>2</sup>**


Received: 16 August 2020; Accepted: 9 September 2020; Published: 11 September 2020

**Simple Summary:** It has been demonstrated that the ovine gene *BMP15* presents a mutation in the Rasa Aragonesa Spanish sheep breed, which has been called *FecXR*. In heterozygosis, ewes exhibit a variable increase in the ovulation rate, producing 0.35 additional lambs per birth and, in homozygosis, sterility. Since the importance of carrying this polymorphism in rams has not been studied, sperm quality and fertility of rams carrying the *FecXR* mutation of the ovine gene *BMP15* has been determined, comparing semen quality, testicle characteristics, and fertility rate of rams presenting or not the allele. FecXR rams exhibited a higher masal motility and a higher proportion of rapid sperm than did non-carrier rams; however, no differences in scrotal circumference or testicular length and diameter were found, although FecXR rams produced a higher proportion of pregnant ewes after artificial insemination. Thus, it seems that the *FecXR* allele creates high-quality semen and improves some sperm parameters in this breed, making these males especially valuable for artificial insemination to produce prolific ewes, when wild-type, non-carrier ewes, are inseminated.

**Abstract:** The *FecXR* mutation is a variant of the ovine gene *BMP15* in the Rasa Aragonesa breed. Information on the physiological importance of carrying the *FecX* polymorphism in rams is limited. The aim of this study was to compare semen quality, testicle characteristics, and fertility rate of rams that carry the *FecXR* allele. Rams (*n* = 15) were either FecXR allele carriers (*n* = 10) or non-carriers, wild type (++) (*n* = 5). FecXR rams exhibited higher mass motility (*p* < 0.05), proportion of rapid sperm (*p* < 0.05), and a lower proportion of slow sperm (*p* < 0.0001) than did ++ rams. The presence of the *FecXR* allele was not associated with mean scrotal circumference or testicular length and diameter, although season had a significant (*p* < 0.05) effect on these traits. Genotype (*p* < 0.05) and season (*p* < 0.01) had a significant effect on mean fertility rate, FecXR rams had a higher proportion of pregnant ewes than did ++ rams (*p* < 0.05). In conclusion, the *FecXR* allele produced high-quality semen throughout the year, and corresponded with an improvement in some sperm parameters, particularly, mass motility and the proportion of rapid sperm.

**Keywords:** *BMP15*; ram; semen

#### **1. Introduction**

Several mutations in genes of the transforming growth factor-beta (TGF-β) superfamily have positive effects on ovulation rate and litter size; e.g., FecB or Bone Morphogenetic Protein (BMP) R1B, FecX or BMP15, and FecG or GDF9 (for a review [1]). Galloway et al. [2] identified a mutation in the *BMP15* gene, that introduced a stop codon on the X chromosome, which prevents the normal translation of the protein encoded by this gene and, subsequently, demonstrated its effect on the

ovulation rate in a population of the Inverdale (*FecXI*) sheep breed. The mutation is sex-linked because it is located in the non-recombinant region of the X chromosome and, therefore, males can have one copy of the gene, only, but females can be hetero or homozygous for the mutation (review [3]). A male carrier transmits the mutation to all of his daughters but to none of his sons, and heterozygous females pass on the mutation to, on average, half of their offspring. However, homozygous females are sterile because they do not develop ovarian follicles correctly.

Since the discovery of the mutation in the Inverdale breed, mutations in *BMP15* have been identified in other prolific breeds including Belclare and Cambridge (*FecXB*), Hanna (*FecXH*), Galway (*FecXG*), Lacaune (*FecXL*), Rasa Aragonesa (*FecXR*), Grivette (*FecXGr*), and Olkuska (*FecXO*) [2,4–9]. In all, the mechanism of action is similar (amino acid substitutions, deletions, or stop codons), and all have received the same name (*FecX*) and the first initial of the breed in which it was discovered because the phenotypic effects are similar; i.e., in heterozygosis, a variable increase in ovulation rate and, in homozygosis, sterility [10].

The *FecXR* mutation is a variant of the ovine gene *BMP15* in the Rasa Aragonesa breed. Rasa Aragonesa is one of the most important meat sheep breeds in Spain, where there are about 1.1 million head, and 360,000 are registered in the Stud Book of the National Association of Rasa Aragonesa Breeders (ANGRA) [11]. Mean litter size is 1.2–1.5 lambs/birth [12], and ANGRA is developing a genetic improvement program that includes prolificacy as one of the important objectives. *FecXR* has been included in the selection scheme under the commercial denomination "Gen ANGRA Santa Eulalia", and there are >5000 ewes that carry this mutation. The allele has been used to increase prolificacy in Rasa Aragonesa sheep through artificial insemination (AI) of wild type, non-carrier ewes, which are used to disseminate the allele across those farms interested in improving litter size. The positive effect of *FecXR* on prolificacy is well known; viz., 0.35 additional lambs per birth [11], which has increased cost effectiveness and profits.

Most of the studies on the expression of *FecXR* have involved female sheep and information on the physiological importance of the *FecXR* polymorphism in males, particularly, rams, is limited. Studies on *BMP15* in rams have investigated tissue expression pattern in rams that differ in fecundity [13], fertility rate [14], and the influence of the *FecB* genotype on semen attributes [15]. The aim of this study was to compare the semen quality, testicle characteristics, and fertility rate, through AI, of Rasa Aragonesa rams that carry the *FecXR* allele during different seasons, so that it is hypothesized that the efficiency of AI using these particular rams may be improved.

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

#### *2.1. Animals*

Rams were housed at CERSYRA (Regional Centre for Animal Selection and Reproduction) in Zaragoza, Spain (41◦N), and were breeding males for AI in the stud book of ANGRA. Inseminations were performed on commercial farms by veterinarians of ANGRA. Approval from the Ethics Committee of the University of Zaragoza was not a prerequisite for this study. The study met the Spanish Policy for Animal Protection RD1201/05, which meets the European Union Directive 2010/63 on the protection of animals used for experimental and other scientific purposes.

Fifteen adult Rasa Aragonesa rams (age: 5.7 ± 2.8 yr) used in the study were either FecXR allele carriers (*n* = 10) or non-carriers, wild type (++) (*n* = 5). The laboratory procedures (DNA extraction, polymerase chain reaction (PCR) amplification prior to sequencing, DNA sequencing and analysis) after they had been exposed the localization of the allele are described by Monteagudo et al. [8].

#### *2.2. Semen Collection and Analyses*

Rams were housed together and were fed to meet their maintenance requirements. Throughout the year, semen samples (96 per ram) were collected twice a week [16], starting at 9:00 am, in an artificial vagina at 35–40 ◦C lubricated with petroleum jelly. Each collection day, a routine, simplified semen analysis was performed that included concentration measured by spectrophotometry (AccRead, IMV Technologies, L'Aigle, France) (1:400 dilution in saline solution plus 0.2% glutaraldehyde), volume (ml), measured in a graduated collection tube, and mass motility estimated by optical microscopy at 100× magnification and scored from 0 to 5. Once per month, the proportion of static sperm, total motile (TM) sperm, non-progressive (NPM) and progressive (PM), and motile sperm subpopulations (rapid, medium, or slow sperm) were measured in a computer-assisted sperm analysis (CASA) using ISAS software (Integrated Semen Analysis System, Proiser, Paterna, Valencia, Spain). Semen sample processing and motility and viability assessment followed the method of Palacín et al. [17]. Briefly, before motility or viability analysis, 200 <sup>×</sup> <sup>10</sup><sup>6</sup> sperm/mL semen samples were mixed and re-diluted to a final concentration of 50 <sup>×</sup> <sup>10</sup><sup>6</sup> sperm/mL using INRA 96 (IMV Technologies, L'Aigle, France) extender. An Olympus BX40 microscope under 100× magnifications, provided with heated stage set at 37 ◦C, was used to estimate sperm motility. The grade of the forward progression (fast progressive, slow progressive and motile but not progressive) determined on the TM sperm were recorded. Sperm with curvilinear velocity (VCL) ≥ 75 m/sg and straightness (STR) ≥ 80% were considered rapid progressive and with VCL < 5 m/sg and STR ≥ 80% slow progressive.

Thereafter, the semen was diluted (INRA 96) and put into French mini-straws for AI (0.25 mL, 300 × 106 spermatozoa/mL).

#### *2.3. Testicular Measurements*

Once every month, scrotal circumference (SC) (pulling the testes firmly down into the lower part of the scrotum and placing a measuring tape into a loop around the greatest diameter over the scrotum), length (placing the fixed arm of a caliper at the proximal end and the sliding arm at the distal end of the testes) and diameter (placing one arm of a caliper at the medial aspect and the other at the lateral aspect of the testes, at the point of maximum width) of each testicle, were determined. Testicular length (TL) and diameter (TD) were calculated as the mean of both testicles.

#### *2.4. Artificial Inseminations (AI)*

In the 12 months of the study, 1412 AI were performed on 29 farms. To synchronize estrus, vaginal sponges containing 30 mg of fluorogestone were applied for 12 d. At pessary withdrawal, ewes received 480 IU of eCG. Cervical AI [18] was performed 54 ± 1 h after sponge withdrawal (14:00 p.m.), using an ovine AI gun (IMV, Instruments de Medicine Veterinaire, L'Aigle, France) and 0.25 mL French mini-straws. All of the inseminated ewes were *FecXR* allele non-carriers.

Births from AI were recorded on the farms throughout the year of the study. Fertility rate was the proportion of ewes lambing after AI, prolificacy was the number of lambs born per lambing, and fecundity rate was the number of lambs born per inseminated ewe.

#### *2.5. Statistical Analyses*

Semen quality, testicular dimensions, and reproductive performance after AI were evaluated statistically based on a multifactorial model that included the presence/absence of the *FecXR* allele (FecXR or ++ wild rams) and season as fixed effects in the Least-Squares Method of the GLM procedure in SPSS v.26 (IBM Corp., Released 2019). The seasons were defined based on the Northern Hemisphere Meteorological Season Division [19]. An ANOVA identified significant differences between genotypes and between seasons. The general representation of the model is as follows: y = xb + e, where y is N × 1 vector of records, b denotes the fixed effect in the model within the association matrix x, and e is the vector of residual effects. To test for significant differences between effect combinations, a post-hoc Fisher's Least Significant Difference (LSD) test was used.

#### **3. Results**

#### *3.1. Semen Quality*

Mean (±S.E.M.) sperm count (3762 <sup>±</sup> 1060), ejaculate volume (0.93 <sup>±</sup> 0.04 cm3) and semen concentration (4055 <sup>±</sup> 100 <sup>×</sup> 106) did not differ between the two genotypes, but concentration was significantly (*p* < 0.05) higher in summer than it was in autumn and winter (*p* < 0.05). Mass motility (4.26 ± 0.19) was significantly (*p* < 0.05) affected by the presence of the allele and season, with a significant (*p* = 0.01) interaction between effects. FecXR rams exhibited a higher mass motility (*p* < 0.05), a higher proportion of rapid sperm (*p* < 0.05), and a lower proportion of slow sperm (*p* < 0.0001) than did ++ rams (Figure 1). Mean proportion of NPM, PM, TM, and medium-speed sperm did not differ significantly between genotypes or among seasons.

**Figure 1.** Seminal traits (mean ± S.E.M.) of Rasa Aragonesa rams of the wild genotype (+ +; *n* = 5) or those carrying the *FecXR* allele of the BMP15 gene (*n* = 10) (a,b indicate *p* < 0.05) (spz: spermatozoon). Values calculated from semen samples collected twice a week for one year.

The proportion of slow sperm was significantly (*p* < 0.05) lower in summer (Figure S1). In winter, FecXR rams tended to present a higher mass motility and a lower proportion of static sperm than did ++ rams (*p* < 0.10) (Table 1). Furthermore, FecXR rams had higher proportions of rapid sperm in spring (*p* < 0.10) and winter (*p* < 0.001), and lower proportions of slow sperm in spring (*p* < 0.05), summer (*p* < 0.05), and winter (*p* < 0.01) than did ++ rams (Table 1).



#### *3.2. Testicular Measurements*

SC (FecXR: 32.9 ± 0.6; ++: 31.8 ± 1.7 cm), TD (FecXR: 6.5 ± 0.2; ++: 6.2 ± 0.5 cm), and TL (FecXR: 9.3 ± 0.2; ++: 8.6 ± 0.7 cm) did not differ significantly between carriers and non-carriers of the *FecXR* allele; however, SC was highest in summer and winter (*p* < 0.05), TD was highest in summer and autumn (*p* < 0.01), TL was lowest in spring and winter (*p* < 0.05) (Table S1).

#### *3.3. Reproductive Parameters*

FecXR rams impregnated a significantly higher proportion (*p* < 0.05) of ewes (62.5 ± 2.5%) than did ++ rams (56.7 ± 2.9%), and fertility rates were lowest in spring and winter inseminations (Table S1). FecXR rams had significantly (*p* < 0.05) higher fertility rates than did ++ rams in winter inseminations, only (0.66 ± 0.08 vs. 0.40 ± 0.06%).

Prolificacy (FecXR: 1.73 ± 0.06; ++: 1.71 ± 0.06 lambs/lambing) and fecundity (FecXR: 1.10 ± 0.06; ++: 0.98 ± 0.06 lambs/ewe) did not differ significantly between FecXR and ++ rams, but differed significantly (*p* < 0.001) among seasons (Table S1).

#### **4. Discussion**

To our knowledge, this is the first study of the semen quality of rams carrying the *FecXR* allele. Ejaculate volume and sperm concentration did not differ significantly between the FecXR and wild rams in any season, which suggests that the production of seminal plasma or spermatogenesis are not affected by the *BMP15* gene. These results are similar to the observations of Kumar et al. [20] in Garole x Malpura rams carrying the *FecB* allele, and parallel the absence of differences in testicular size. Mass motility and the proportion of rapid sperm were significantly higher, and the proportion of slow sperm lower in the FecXR than they were in the wild-type rams.

Furthermore, ewes that had been inseminated with semen collected from FecXR rams had the highest mean annual fertility rate. Sperm motility and velocity are two of the most important aspects of semen quality because they are correlated with fertility [21]. In a study of Rasa Aragonesa breed at the same latitude as in our study, it has been reported that high-fertility rams produced a higher proportion of fast and linear spermatozoa than did low-fertility rams [22]. In Iberian deer, mean and maximum straight-line velocity of sperm and fertility are significantly correlated, and it appears that sperm swimming velocity is a main determinant of fertility in mammals [23]. Thus, it is likely that high mass motility and high proportion of rapid sperm contributed to the high fertility rates in FecXR rams. On the other hand, Lahoz et al. [24] did not detect significant differences between genotypes in a program that involved cervical insemination. Given the number of external factors that can affect the proportion of ewes that become pregnant after AI (year, farm, technician) [25] including weather [26] and climate [27], differences in the conditions at the time of experiments involving AI might have contributed to the presence or absence of differences between genotypes. Further study is needed to determine how external factors might influence the effect of *FecX* on reproductive parameters.

The finding that the highest fertility rate occurred in summer is similar to previous observations [28] in the same breed and at the same latitude as in our study, where the lowest AI fertility was between March and June, and the highest was in the first months of increasing daylength (July and August). Rasa Aragonesa is a reduced-seasonal anestrous breed [29], in which females exhibit an onset of the breeding season in July and a peak in ovulation rate in late August. Thus, our study confirms that summer is the peak breeding season for rams and ewes of this breed.

Differences in the pregnancy rates related to polymorphisms of the *BMP15* gene have been reported by Sun et al. [30], who found that Chinese Holstein bulls of the CT genotype had a significantly lower sperm motility than did bulls of the CC or TT genotypes. In sheep, Chen et al. [13] reported the expression of *BMP15* in the epididymis of rams, which was significantly higher in a less-fecund breed (Sunite) than it was in a high-fecundity breed (Small Tail Han). Possibly, the expression level of *BMP15* and fecundity in rams are negatively correlated. Garole × Malpura rams that carry the *FecB* genotype had a significantly higher proportion of rapid motile sperm and with higher linearity, and a higher FSH concentration than did the wild type [15].

In our study, testicular dimensions did not differ significantly between rams that carried the *FecXR* allele and those that did not. Rasa Aragonesa light lambs that did or did not carry the *FecXR* allele did not exhibit significant differences in birth weight, growth rate, or carcass quality [31]; moreover, it appears that *FecXR* allele may not influence testicular morphology or fleece weight at 13 months of age in carrier Romney rams [32]. The absence of differences in testicular measurements between genotypes parallels the lack of differences in sperm volume and concentration, which are highly correlated to testicle size [33].

Season had a significant effect on testicular measurements, which was similar to the effects reported by Avdi et al. [34] in Chios and Serres rams. Similarly, Chios and Friesian rams had semen characteristics that were generally better in summer and peaked in quality in autumn [35]. Although seasonal variations in reproductive traits in sheep are less marked in rams than they are in ewes, the consequences of the non-reproductive season are smaller testicular volume and diameter, lower semen quality, and hormone profiles that differ from those in the breeding season [36]. Photoperiod is the key environmental signal that dictates the timing of the reproductive cycle of the ram [37], which is synchronized through changes in daily melatonin secretion [38]. Rams exhibit a seasonal decrease in sexual behavior and spermatogenesis at about the time that ewes are in sexual rest, but with a 1- to 2-month advance in phase [39].

#### **5. Conclusions**

In conclusion, this study demonstrated that carriers of the *FecXR* allele produce good-quality semen throughout the year, and corresponded with an improvement in some sperm characteristics—particularly mass motility and the proportion of rapid sperm—along with an interaction effect with season. In addition, the ability to pass the allele to their female offspring, through the insemination of wild type, non-carriers ewes, makes these males especially valuable for AI to produce prolific ewes.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-2615/10/9/1628/s1, Figure S1: Annual seminal traits (mean ±S.E.M.) of Rasa Aragonesa rams (a,b indicate *p* < 0.05) (spz: spermatozoon). Values calculated from semen samples collected twice a week for one year, Table S1: Mean (± S.E.M.) testicular measurements and reproductive traits of Rasa Aragonesa rams (*n* = 15) (a,b,c indicate significant differences *p* < 0.05). Values calculated from semen samples collected twice a week for one year.

**Author Contributions:** Conceptualization, J.A.A., Á.M., R.P.-P. and A.L.; methodology, J.A.A., Á.M., C.B., E.M.; formal analysis, A.C., R.P.-P., Á.M.; investigation, Á.M., C.B. and E.M.; writing—original draft preparation, J.A.A.; writing—review and editing, Á.M., A.C. and R.P.-P.; supervision, J.A.A., Á.M. and A.L.; project administration, J.A.A.; funding acquisition, J.A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by Gobierno de Aragón, grant number A07\_20R.

**Acknowledgments:** The authors thank the farmers involved in the study and the CERSYRA staff for their collaboration in the collection of semen samples and the preparation of doses for AI. We thank Bruce MacWhirter for the English revision of the manuscript. Partially funded by Gobierno de Aragón, group A07\_20R.

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

#### **References**


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

*Article*
