A genomic biomarker for the rapid identification of the rob(1;29) translocation in beef cattle breeds.

Robertsonian translocations, specifically rob(1;29) translocation, have reportedly been the most prevalent chromosomal abnormalities in cattle, affecting various breeds and leading to a decrease in fertility and reproductive value. Currently, the identification of rob(1;29) carriers relies on cytogenetic analysis that has limitations in terms of accessibility, cost, and sample requirements. To address these limitations, a novel genomic biomarker was developed in this study for the rapid and precise identification of rob(1;29) carriers. Using q-PCR, a specific copy number variation associated with translocation was targeted, which effectively distinguished between wild-type, homozygous and heterozygous carriers. Crucially, the biomarker can be applied to DNA extracted from various biological matrices, such as semen, embryos, oocytes, milk, saliva, coat, and muscle, and it is compatible with fresh, refrigerated, or frozen samples. Furthermore, this approach offers significant reductions in cost compared to those associated with traditional cytogenetic analysis and provides results within a short turnaround time. The successful development of this genomic biomarker has considerable potential for widespread adoption in screening programs. It facilitates timely identification and management of rob(1;29) carriers while mitigating economic losses and preserving genetic integrity in bovine populations.

Reproductive traits in Pura Raza Española mares manifest inbreeding depression from low levels of homozygosity.

Inbreeding depression is a genetic phenomenon associated with the loss of fitness and mean phenotypic performance due to mating between relatives. Historically, inbreeding coefficients have been estimated from pedigree information. However, the onset of genomic selection programs provides large datasets of individuals genotyped using SNP arrays, enabling more precise assessment of an individual's genomic-level inbreeding using genomic data. One of the traits most sensitive to issues stemming from increased inbreeding is reproduction. This is particularly important in equine, in which fertility is only moderate compared to other livestock species. To explore this further, we evaluated the effect of inbreeding on five reproductive traits (age at first foaling (AFF), average interval between foalings (AIF), total number of foalings (NF), productive life (PL) and reproductive efficiency (RE)) in Pura Raza Español mares using genomic data. Residual predicted phenotypes were obtained by purging these traits through the REML (wgResidual ) and ssGREML (gResidual ) approaches in reproductive data of 29,847 PRE mares using the BLUPF90+ program. Next, we used pedigree-based (Fped ) and ROH-based genomic (FROH ) inbreeding coefficients derived from 1018 animals genotyped with 61,271 SNPs to estimate the inbreeding depression (linear regression). Our results indicated significant levels of inbreeding depression for all reproductive traits, with the exception of the AIF trait when Fped was used. However, all traits were negatively affected by the increase in genomic inbreeding, and FROH was found to capture more inbreeding depression than Fped . Likewise, REML models (ssGREML) using genomic data for estimated predicted residual phenotypes resulted in higher variance explained by the model compared with the models not using genomics (REML). Finally, a segmented regression analysis was conducted to evaluate the effect of inbreeding depression, revealing that the levels of genealogical and genomic homozygosity do not manifest uniformly in reproductive traits. In contrast, the levels of inbreeding depression ranged from low to high as homozygosity increased. This analysis also showed that reproductive traits are very sensitive to inbreeding depression, even with relatively low levels of homozygosity.

Genomic screening of allelic and genotypic transmission ratio distortion in horse.

The phenomenon in which the expected Mendelian inheritance is altered is known as transmission ratio distortion (TRD). The TRD analysis relies on the study of the transmission of one of the two alleles from a heterozygous parent to the offspring. These distortions are due to biological mechanisms affecting gametogenesis, embryo development and/or postnatal viability, among others. In this study, TRD phenomenon was characterized in horses using SNP-by-SNP model by TRDscan v.2.0 software. A total of 1,041 Pura Raza Español breed horses were genotyped with 554,634 SNPs. Among them, 277 horses genotyped in trios (stallion-mare-offspring) were used to perform the TRD analysis. Our results revealed 140 and 42 SNPs with allelic and genotypic patterns, respectively. Among them, 63 displayed stallion-TRD and 41 exhibited mare-TRD, while 36 SNPs showed overall TRD. In addition, 42 SNPs exhibited heterosis pattern. Functional analyses revealed that the annotated genes located within the TRD regions identified were associated with biological processes and molecular functions related to spermatogenesis, oocyte division, embryonic development, and hormonal activity. A total of 10 functional candidate genes related to fertility were found. To our knowledge, this is the most extensive study performed to evaluate the presence of alleles and functional candidate genes with transmission ratio distortion affecting reproductive performance in the domestic horse.

The Bovine Pangenome Consortium: democratizing production and accessibility of genome assemblies for global cattle breeds and other bovine species.

The Bovine Pangenome Consortium (BPC) is an international collaboration dedicated to the assembly of cattle genomes to develop a more complete representation of cattle genomic diversity. The goal of the BPC is to provide genome assemblies and a community-agreed pangenome representation to replace breed-specific reference assemblies for cattle genomics. The BPC invites partners sharing our vision to participate in the production of these assemblies and the development of a common, community-approved, pangenome reference as a public resource for the research community ( https://bovinepangenome.github.io/ ). This community-driven resource will provide the context for comparison between studies and the future foundation for cattle genomic selection.

Estimation of the Genetic Components of (Co)variance and Preliminary Genome-Wide Association Study for Reproductive Efficiency in Retinta Beef Cattle.

In this study, we analyzed the variation of reproductive efficiency, estimated as the deviation between the optimal and real parity number of females at each stage of the cow's life, in 12,554 cows belonging to the Retinta Spanish cattle breed, using classical repeatability and random regression models. The results of the analyses using repeatability model and the random regression model suggest that reproductive efficiency is not homogeneous throughout the cow's life. The h2 estimate for this model was 0.30, while for the random regression model it increased across the parities, from 0.24 at the first calving to 0.51 at calving number 9. Additionally, we performed a preliminary genome-wide association study for this trait in a population of 252 Retinta cows genotyped using the Axiom Bovine Genotyping v3 Array. The results showed 5 SNPs significantly associated with reproductive efficiency, located in two genomic regions (BTA4 and BTA28). The functional analysis revealed the presence of 5 candidate genes located within these regions, which were previously involved in different aspects related to fertility in cattle and mice models. This new information could give us a better understanding of the genetic architecture of reproductive traits in this species, as well as allow us to accurately select more fertile cows.

Prevalence of Sex-Related Chromosomal Abnormalities in a Large Cohort of Spanish Purebred Horses.

Chromosomal abnormalities are largely associated with fertility impairments in the domestic horse. To date, over 600 cases of individuals carrying abnormal chromosome complements have been reported, making the domestic horse the species with the highest prevalence. However, studies analyzing the prevalence of chromosomal diseases in whole populations are scarce. We, therefore, employed a two-step molecular tool to screen and diagnose chromosomal abnormalities in a large population of 25,237 Pura Raza Español horses. Individuals were first screened using short tandem repeats parentage testing results and phenotypic evaluations. Those animals showing results suggesting chromosomal abnormalities were re-tested using a single nucleotide polymorphism (SNP)-based diagnostic methodology to accurately determine the chromosomal complements. Thirteen individuals showed a positive screening, all of which were diagnosed as chromosomally abnormal, including five 64,XY mares with sex development disorders (DSD) and four cases of blood chimerism (two male/female and two female/female cases). In addition, we detected one Turner and one Klinefelter syndrome and two individuals carrying complex karyotypes. The overall prevalence in the entire population was ~0.05%, with the prevalence of 64,XY DSD and blood chimerism ~0.02% and ~0.016%, respectively. However, the overall results should be taken with caution since the individuals carrying Turner syndrome (in full (63,X) or mosaic (mos 63,X/64,XX) forms) cannot be detected due to limitations in the methodology employed. Finally, the lack of agreement between populational studies performed using karyotyping or molecular methods is discussed. To our knowledge, this is the largest populational study performed evaluating the prevalence of the most common chromosomal abnormalities in the domestic horse.

Analysis of the genomic landscape of inbreeding in two divergent groups of Spanish Florida goats.

The analysis of the genomic landscape of inbreeding using runs of homozygosity (ROH) patterns is becoming an interesting tool to partially understand phenotypic differences among individuals. In this study, we analysed genome-wide ROH patterns in two groups of Florida goats. We first determined the inbreeding levels of each individual by calculating ROH-based inbreeding coefficients (FROH ). Then, the individuals were divided into two groups based on FROH : high inbreeding (HI, FROH >0.1) and low inbreeding (LI, FROH <0.03). Finally, we performed an extensive in-depth analysis of ROH distribution in each group separately. We found a higher abundance of short ROH in LI, whereas long ROH was more frequent in HI. Furthermore, ROH abundance was not evenly distributed among chromosomes within groups, with some chromosomes showing larger numbers of ROH, like CHI6, CHI7 and CHI27. A different landscape was observed in recent inbreeding (ROH >8 Mb), with significant increases in CHI6, CHI11 and CHI28. Determination of genomic regions with significantly increased ROH (ROH islands-ROHi) showed 13 ROHi related to whole inbreeding and five ROHi associated with recent inbreeding analysis. Within these genomic regions, 123 and 101 genes were identified in HI and LI, respectively, including 10 and seven candidate genes previously related to production, fertility and heat resistance in goats and livestock species.

Fine-tuning genomic and pedigree inbreeding rates in equine population with a deep and reliable stud book: the case of the Pura Raza Española horse.

BACKGROUND: Estimating inbreeding, which is omnipresent and inevitable in livestock populations, is a primary goal for management and animal breeding especially for those interested in mitigating the negative consequences of inbreeding. Inbreeding coefficients have been historically estimated by using pedigree information; however, over the last decade, genome-base inbreeding coefficients have come to the forefront in this field. The Pura Raza Española (PRE) horse is an autochthonous Spanish horse breed which has been recognised since 1912. The total PRE population (344,718 horses) was used to estimate Classical (F), Ballou's ancestral, Kalinowski's ancestral, Kalinowski's new and the ancestral history coefficient values. In addition, genotypic data from a selected population of 805 PRE individuals was used to determine the individual inbreeding coefficient using SNP-by-SNP-based techniques (methods of moments -FHOM-, the diagonal elements of the genomic -FG-, and hybrid matrixes -FH-) and ROH measures (FRZ). The analyse of both pedigree and genomic based inbreeding coefficients in a large and robust population such as the PRE horse, with proven parenteral information for the last 40 years and a high degree of completeness (over 90% for the last 70 years) will allow us to understand PRE genetic variability better and the correlations between the estimations will give the data greater reliability. RESULTS: The mean values of the pedigree-based inbreeding coefficients ranged from 0.01 (F for the last 3 generations -F3-) to 0.44 (ancestral history coefficient) and the mean values of genomic-based inbreeding coefficients varied from 0.05 (FRZ for three generations, FH and FHOM) to 0.11 (FRZ for nine generations). Significant correlations were also found between pedigree and genomic inbreeding values, which ranged between 0.58 (F3 with FHOM) and 0.79 (F with FRZ). In addition, the correlations between FRZ estimated for the last 20 generations and the pedigree-based inbreeding highlight the fact that fewer generations of genomic data are required when comparing total inbreeding values, and the opposite when ancient values are calculated. CONCLUSIONS: Ultimately, our results show that it is still useful to work with a deep and reliable pedigree in pedigree-based genetic studies with very large effective population sizes. Obtaining a satisfactory parameter will always be desirable, but the approximation obtained with a robust pedigree will allow us to work more efficiently and economically than with massive genotyping.

Genetic analysis of the effects of heat stress before and after lambing on pre-weaning live weight in Spanish Merino lambs.

BACKGROUND: Heat stress (HS) is a major environmental effect on sheep production. Hereby, we estimated the genetic (co)variance component of HS on the pre-weaning performance of 19,022 Merino lambs by analysing the climatological index of temperature and relative humidity (recorded 30 days before lambing and after lambing) using transversal and longitudinal mixed linear models. METHODS AND RESULTS: The global impact of HS during the last 30 days of pregnancy was -17% for birthweight and ranged between -4% and -8% for live weight at 15, 30 days of age (W30), and average daily gain from birth at 30 days. The results from both statistical approaches showed very similar heritabilities (h2 ), ranging from 0.192 to 0.237 for the direct genetic (D) effects and from 0.072 to 0.082 for the maternal genetic (M) effects, but the antagonism between (D) and (M) was higher when a longitudinal model was used. A significant genotype-environmental effect was also found regardless of whether the climatological covariables were considered in the model. In addition, we employed D and M breeding values for W30 as an example to create a new subjacent index by first using a principal component analysis and employing the leading eigenvalues as a weighted factor that provides the information needed to identify those genotypes that maximise the response for both genetic effects over a wide range of climate-environment levels. CONCLUSIONS: Our study revealed that the HS indexes of the mother during the gestation period have a significant effect on the growth of the lambs during the early stages of life.

Copy Number Variation (CNV): A New Genomic Insight in Horses.

Copy number variations (CNVs) are a new-fangled source of genetic variation that can explain changes in the phenotypes in complex traits and diseases. In recent years, their study has increased in many livestock populations. However, the study and characterization of CNVs in equines is still very limited. Our study aimed to investigate the distribution pattern of CNVs, characterize CNV regions (CNVRs), and identify the biological pathways affected by CNVRs in the Pura Raza Española (PRE) breed. To achieve this, we analyzed high-density SNP genotyping data (670,804 markers) from a large cohort of 654 PRE horses. In total, we identified 19,902 CNV segments and 1007 CNV regions in the whole population. The length of the CNVs ranged from 1.024 kb to 4.55 Mb, while the percentage of the genome covered by CNVs was 4.4%. Interestingly, duplications were more abundant than deletions and mixed CNVRs. In addition, the distribution of CNVs across the chromosomes was not uniform, with ECA12 being the chromosome with the largest percentage of its genome covered (19.2%), while the highest numbers of CNVs were found in ECA20, ECA12, and ECA1. Our results showed that 71.4% of CNVRs contained genes involved in olfactory transduction, olfactory receptor activity, and immune response. Finally, 39.1% of the CNVs detected in our study were unique when compared with CNVRs identified in previous studies. To the best of our knowledge, this is the first attempt to reveal and characterize the CNV landscape in PRE horses, and it contributes to our knowledge of CNVs in equines, thus facilitating the understanding of genetic and phenotypic variations in the species. However, further research is still needed to confirm if the CNVs observed in the PRE are also linked to variations in the specific phenotypical differences in the breed.

Genomic Population Structure of the Main Historical Genetic Lines of Spanish Merino Sheep.

According to historiographical documentation, the Romans first began to select Merino sheep in the Iberian Peninsula during the first century, with the aim of obtaining a breed appreciated for the quality of its wool. This process continued locally during the Middle Ages, when Spanish sheep were protected, and their export to foreign countries was banned. It was during the 16th century when individual Merino sheep were allowed to spread around the world to be used to improve the wool quality of local breeds. However, the wool crisis of the 1960s shifted the selection criteria of the Merino breed towards meat production at the expenses of wool. Consequently, individuals that display the genetic and phenotypic characteristics of those sheep originally bred in the kingdom of Spain in the Middle Ages are extremely difficult to find in commercial herds. In this study, we characterized the genetic basis of 403 individuals from the main historical Spanish Merino genetic lines (Granda, Hidalgo, Lopez-Montenegro, Maeso, Donoso and Egea), which were bred in isolation over the last 200 years, using a genomic approach based on genotyping data from the Axiom™ Ovine 50K SNP Genotyping Array. Our analysis included measuring population structure, genomic differentiation indexes, runs of homozygosity (ROH) patterns, and an analysis of molecular variance (AMOVA). The results showed large genetic differences between the historical lines, even though they belong to the same breed. In addition, ROH analysis showed differences due to increased inbreeding among the ancient generations compared with the modern Merino lines, confirming the breed's ancestral and closed origin. However, our results also showed a high variability and richness within the Spanish historical Merino lines from a genetic viewpoint. This fact, together with their great ability to produce high-quality wool, suggests that ancestral Merino lines from Spain should be considered a valuable genetic population to be maintained as a resource for the improvement of wool-producing sheep breeds all around the world.

Genetic Parameters of Somatic Cell Score in Florida Goats Using Single and Multiple Traits Models.

A total of 1,031,143 records of daily dairy control test of Spanish Florida goats were used for this study. The database was edited, and only the records of the first three lactations were kept. The final database contained 340,654 daily-test somatic cell counts from 27,749 daughters of 941 males and 16,243 goats. The evolution of this count in the last 14 years was analyzed following French and American international associations' criteria for the risk of mastitis in goats, and confirmed the slight increase in SCS in the last years and the importance of this problem (50% of dairy control tests show a risk of suffering mastitis). For the genetic analysis, the SCS records were log-transformed to normalize this variable. Two strategies were used for the genetic analysis: a univariate animal model for the SCS assuming that SCS does not vary throughout the parities, and a multi-character animal model, where SCS is not considered as the same character in the different parities. The heritabilities (h2) were higher in the multiple traits models, showings an upward trend from the first to the third parity (h2 between 0.245 to 0.365). The genetic correlations of the same trait, as well as between breeding values (GVs) between different parities, were different from unity. The breeding values (EBVs) obtained for both models were subjected to a PCA: the first eigenvector (λ1) explained most of the variations (between 74% to 90%), while the second λ2 accounted for between 9% to 20% of the variance, which shows that the selection will be proportionally favorable but not equivalent in all parities and that there are some variations in the type of response.

Fine-Scale Analysis of Runs of Homozygosity Islands Affecting Fertility in Mares.

The loss of genetic variability in livestock populations bred under strict selection processes is a growing concern, as it may lead to increased inbreeding values and lower fertility, as a consequence of the "inbreeding depression" effect. This is particularly important in horses, where inbreeding levels tend to rise as individuals become more and more closely related. In this study, we evaluated the effect of increased inbreeding levels on mare fertility by combining an SNP-based genomic approach using runs of homozygosity and the estimation of genetic breeding values for reproductive traits in a large population of Pura Raza Española mares. Our results showed a negative correlation between whole-genome homozygosity and fertility estimated breeding values (EBVs) at the genome level (ρ = -0.144). However, the analysis at chromosome level revealed a wide variability, with some chromosomes showing higher correlations than others. Interestingly, the correlation was stronger (-0.241) when we repeated the analysis in a reduced dataset including the 10% most and least fertile individuals, where the latter showed an increase in average inbreeding values (FROH) of around 30%. We also found 41 genomic regions (ROHi, runs of homozygosity islands) where homozygosity increased 100-fold, 13 of which were significantly associated with fertility after cross-validation. These regions encompassed 17 candidate genes previously related to oocyte and embryo development in several species. Overall, we demonstrated the relationship between increased homozygosis at the genomic level and fertility in mares. Our findings may help to deal with the occurrence of inbreeding depression, as well as further our understanding of the mechanisms underlying fertility in mares.

500 years of breeding in the Carthusian Strain of Pura Raza Español horse: An evolutional analysis using genealogical and genomic data.

The Carthusian horse is a Pura Raza Español (PRE) strain (CS), bred as a closed population since its creation more than 500 years ago. The aim of this study was to analyse for the first time its population structure and situation of variability combining both genealogical (GEL) and genomic (GEN) data. The GEL data comprised 348,429 pedigree records (56,105 CS horses), while the GEN analysis included the high-density genotypes (670,804 SNPs) of 287 horses. Pedigree completeness demonstrated its accuracy, showing a good correlation of GEL (F) and GEN (FROH ) inbreeding coefficient in the case of PRE subpopulations partially related and non-related to Carthusian strain (0.68) but a lower value in the 100% Carthusian horses (0.42), due to the high weight of founders not detected by GEL analysis. GEN (PCA, AMOVA, and Admixture) and GEL analysis showed a good differentiation of subpopulations, but also a high level of introgression of the CS in the breed during past decades. A recent change in this trend was noteworthy, with a considerable reduction in CS variability and a genetic bottleneck (effective population sizes of 31.57 and 30.20 in GEL and GEN analysis, respectively, in last generation). The PRE has maintained its variability, and a considerable difference in estimated Ne by GEL (60.77) and GEN (188.0) data was observed. Using two sources of complementary information, it was found the existence of an ancient PRE strain with a unique genetic landmark, practically free from the influence of other equine populations.

Effect of the rob(1;29) translocation on the fertility of beef cattle reared under extensive conditions: A 30-year retrospective study.

The Robertsonian translocation 1/29 (rob(1;29)) is the most worldwide widespread chromosomal abnormality in domestic animals. Previous studies have demonstrated its negative effect on fertility in dairy herds, but not in beef cattle extensively bred. In this study, we analysed the effect of rob(1;29) in a Retinta cattle breed data set gathered during the last 30 years. The data presented herein include rob(1;29) analysis of 11,505 cows from 251 herds, pedigree information of 24,790 animals and 67,457 calving records. Fertility was evaluated using estimated breeding values for the reproductive efficiency (Re), calculated as the percentage ratio between the number of calvings of an individual and the number expected in an optimal situation. Our results showed that cows carrying the heterozygote genotype showed a significant decrease in their Re (-5.10%, p < .001). No decrease was detected in free rob(1;29) animals and homozygous carriers. In addition, the incidence of rob(1;29) in the breed fertility was decreased to very low values after 30 years of avoiding selection of bulls' carrier as stallions. The effect of rob(1;29) on cattle fertility is only significant when the prevalence of carrier individuals is high. Selecting against the disease only by the paternal side reduced the incidence to negligible values.

Sexual Differentiation and Primordial Germ Cell Distribution in the Early Horse Fetus.

It was the aim of this study to characterize the development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localization of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size 23-fold from 45 to 60 days of gestation, and an even greater increase was observed in the metanephros volume. Signs of mesonephros atrophy were detected during this time. Tubular structures of the fetal testes were present from day 50 onwards, whereas cell clusters dominated in the fetal ovary. The genital ducts were well-differentiated and presented a lumen in all samples. No sign of mesonephric or paramesonephric duct degeneration was detected. Expression of AMH was strong in the fetal testes but absent in ovaries. Irrespective of sex, primordial germ cells selectively expressed LIN28. Migration of primordial germ cells from the mesonephros to the gonad was detected at 45 days, but not at 60 days of development. Their number and distribution within the gonad were influenced (p < 0.05) by fetal sex. Most primordial germ cells (86.8 ± 3.2% in females and 84.6 ± 4.7% in males) were characterized as pluripotent according to co-localization with CD117. However, only a very small percentage of primordial germ cells were proliferating (7.5 ± 1.7% in females and 3.2 ± 1.2% in males) based on co-localization with Ki67. It can be concluded that gonadal sexual differentiation in the horse occurs asynchronously with regard to sex but already before 45 days of gestation.

Sex Reversal Syndrome in an Egyptian Arabian Horse Detected Using Genomic Data - A case report.

A 4-year-old Straight Egyptian Arabian horse was evaluated in 2016 due to a malformation of external genitalia and male sexual behavior. On physical examination, small teats in the inguinal area and a rudimentary penis-like structure surrounded by a clitoral fossa could be seen. There was no evidence of vulva and vaginal canal. A stallion like behavior was observed, especially in the presence of mares in heat, when the animal was excited and aggressive and had erection of the penis-like structure. Blood samples were collected for two purposes: hormonal (testosterone and estradiol plasma concentration analyses) and genetic (cytogenetic and molecular analysis). The karyotype showed 32 pairs of chromosomes in all cells (2n = 64) including 14 and 18 pairs of metacentric and acrocentric chromosomes respectively, in agreement with a presumptive 64, XX complement. This result agree with STR and SNP molecular analysis, which also ruled out the possibility of hematopoietic chimerism. In addition, SNP genotyping showed no numerical chromosomal aberrations or large deletions or duplications, that can be linked to the phenotype in any autosome, nor numerical chromosomal abnormalities in the father and mother of the horse analyzed. In conclusion, we determined that the animal in the present study is a male pseudohermaphrodite.

Sperm morphometry is affected by increased inbreeding in the Retinta cattle breed: A molecular approach.

The effect of inbreeding depression on sperm motility is well documented, but its influence on sperm morphometry has been scarcely examined to date. Here, we combined the use of computer-assisted sperm morphometry analysis (CASMA) with a SNP-based genomic approach to determine and characterize the effect of inbreeding on the sperm shape of a highly inbred cattle population. We determined seven morphometric parameters on frozen-thawed sperm samples of 57 Retinta bulls: length (L, µm), width (W, µm), area (A, µm2 ), perimeter (P, µm), ellipticity (ELI; L/W), elongation (L-W)/(L + W) and perimeter-to-area shape factor (p2a; P2 /4 × π × A). The comparison of highly inbred (HI) and lowly inbreed (LI) individuals based on runs of homozygosity (ROH) inbreeding values (F ROH ) showed no differences between groups. An additional two-step unsupervised sperm subpopulation analysis based on morphometric parameters showed significant differences in the abundance of different sperm subpopulations between groups (p < 0.05). This analysis revealed that HI bulls harbored a higher percentage of narrow-head sperm as opposed to the higher percentage of large- and round-headed sperm detected in LI. A further genomic characterization revealed 23 regions differentially affected by inbreeding in both groups, detecting six genes (SPAG6, ARMC3, PARK7, VAMP3, DYNLRB2, and PHF7) previously related to different spermatogenesis-associated processes.

Impaired Reproductive Function in Equines: From Genetics to Genomics.

Fertility is one of the key factors in the economic and productive success of the equine industry. Despite this, studies on the genetic causes affecting reproductive performance are scarce, especially in mares, where the genetic architecture of the reproductive traits is extremely complex. Today, with the increasing availability of new genomic methodologies for this species, we are presented with an interesting opportunity to understand the genetic basis of equine reproductive disorders. These include, among others, novel techniques for detecting chromosomal abnormalities, whose association with infertility in horses was established over 50 years ago; new sequencing technologies permitting an accurate detection of point mutations influencing fertility, as well as the study of inbreeding and molecular homozygosity, which has been widely suggested as one of the main causes of low reproductive performance in horses. Finally, over the last few years, reproductive performance has also been associated with copy number variants and candidate genes detected by genome-wide association studies on fertility traits. However, such studies are still scarce, probably because they depend on the existence of large and accurate phenotypic datasets of reproductive and/or fertility traits, which are still difficult to obtain in equines.

The Effect of Different Vitrification and Staining Protocols on the Visibility of the Nuclear Maturation Stage of Equine Oocytes.

In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 µg/mL; P1 and 2.5 µg/mL; P2) combined with 50 µg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 oocytes were evaluated using the staining protocol P2, before (C, control) and after vitrification following a two-step conventional protocol with (15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose; V1) or without (1 M sucrose; V2) using permeable cryoprotectants. Our results showed that P2 provided a higher percentage of oocytes with outstanding visibility of the nuclear chromatin stage (52.17%; P < .05) in comparison with P1 (19.04%). In the second experiment, no cryoprotectant-free vitrified oocytes reached the metaphase II maturation stage. This result was significantly lower (P < .05) than conventional vitrification (15.38%) and both lower in comparison with the nonvitrified control group (42.11%). In conclusion, permeable cryoprotectant-free vitrification of equine oocytes obtained poor results and therefore cannot be considered an alternative to vitrification using permeable cryoprotectants. In addition, a staining protocol with a low concentration of HO is recommended to evaluate the nuclear chromatin stage of equine oocytes after in vitro maturation.