Fertility Assessment in Sorraia Stallions by Sperm‐Fish and Fkbp6 Genotyping

The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm‐FISH and molecular analysis of FKBP6 – a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 64,XY karyotype. The majority of sperm (89%) had normal haploid sex chromosome content, although 11% of sperm carried various sex chromosome aneuploidies. No correlation was found between the percentage of sperm sex chromosome abnormalities and inbreeding, sperm morphology or stallion age. Direct sequencing of FKBP6 exon 4 for SNPs g.11040315G>A and g.11040379C>A revealed that none of the stallions had the susceptibility genotype (A/A‐A/A) for IAR. Instead, all animals had a G/G‐A/A genotype – a testimony of low genetic variability. The findings ruled out chromosomal abnormalities and genetic predisposition for IAR as contributing factors for subfertility. However, low fertility of the Sorraia stallions could be partly attributed to relatively higher rate of sex chromosome aneuploidies in the sperm.     

Evaluation of Prolactin Receptor (PRLR) as Candidate Gene for Male Fertility in Hanoverian Warmblood Horses

Stallion fertility has increasing importance as the artificial insemination is employed in horses more intensely. Molecular genetic markers may be useful tools to evaluate the stallion fertility before breeding. The prolactin receptor gene (PRLR) was chosen as a candidate for stallion fertility because of its influence on testicular and accessory sex gland function. Screening the equine PRLR gene for polymorphisms in Hanoverian stallions revealed two single nucleotide polymorphisms (SNPs). Association and haplotype analyses were performed in 162 Hanoverian warmblood stallions for these intragenic SNPs using the least square means (LSM) of the pregnancy rate per oestrus for stallions and the paternal component and embryonic component of the breeding values (BV) of the pregnancy rate per oestrus. The two SNPs (BIEC2‐589441, BIEC2‐560860) showed significant associations using single marker and haplotype analysis with the embryonic and paternal component of BV and one SNP (BIEC2‐560860) was also significantly associated with the LSM of the pregnancy rate per oestrus. This is the first report on an association of PRLR‐associated genetic markers with fertility traits in stallions.     

INHBA‐Associated Markers as Candidates for Stallion Fertility

The inhibin beta A (INHBA) gene was chosen as candidate for stallion fertility and analysed for intragenic markers to find associations with pregnancy rate per oestrus. Intragenic single nucleotide polymorphisms (SNPs) were developed in order to perform an association and haplotype analysis using the least square means (LSM) of the pregnancy rate per oestrus for stallions as well as breeding values (BVs) for the embryonic and paternal component of the pregnancy rate per oestrus. The polymorphisms were genotyped in 161 Hanoverian warmblood stallions. Insemination records from approximately 20 000 Hanoverian warmblood mares were used to calculate LSM for stallions and to predict the paternal and embryonic component of BVs for the pregnancy rate per oestrus. We demonstrated significant associations of single markers and haplotypes with the LSM and the embryonic and paternal component of BVs for the pregnancy rate per oestrus. This is the first report on INHBA as an associated candidate gene with the LSM of stallions and the paternal and embryonic component of BVs for the pregnancy rate per oestrus.     

Genetic Parameters and Breeding Values for Semen Characteristics in Hanoverian Stallions

The objectives of this study were to show whether semen traits of 30 Hanoverian stallions regularly used in AI may be useful for breeding purposes. Semen characteristics were studied using 15 149 ejaculates from 30 Hanoverian stallions of the State Stud Celle of Lower Saxony. Semen samples were collected between 2005 and 2009. Traits analysed were gel‐free volume, sperm concentration, total and motile sperm number and progressive motility. A linear multivariate animal model was employed to estimate heritabilities and permanent environmental variances for stallions. The same model was used to predict breeding values for all traits simultaneously. Heritabilities were high for gel‐free volume (h² = 0.43) and moderate for total number of sperm (h² = 0.29) and progressive motility (h² = 0.20). Gel‐free volume, sperm concentration and total number of sperm were genetically negatively correlated with progressive motility. The effect of the permanent environment for stallions accounted for 9–55% of the trait variance. The total variance among stallions explained 37–69% of the trait variance. The average reliabilities of the breeding values were 0.43–0.76 for the 30 Hanoverian stallions. In conclusion, the study could demonstrate large effects of stallions, routinely employed in a breeding programme, on semen characteristics analysed here. We could demonstrate that estimated breeding values (EBV) with sufficient high reliabilities can be predicted using data from these stallions and these EBV are useful in horse breeding programmes to achieve genetic improvement in semen quality.     

An Overview of Low Dose Insemination in the Mare

The need for relatively high numbers of spermatozoa for artificial insemination limits our application of recently available technologies such as sex‐sorted semen. The fertility of two different methods of low dose insemination using fresh, frozen and sex‐sorted semen are compared in this overview. Satisfactory conception rates are described using very low doses of spermatozoa inseminated by either hysteroscopic or deep uterine insemination methods, proving the stallion is fully fertile. The hysteroscopic method appears to give higher conception rates when inseminating fewer than 5 × 10⁶ spermatozoa and is therefore, the preferred method of insemination for sex‐sorted spermatozoa. However, hysteroscopic deposition of low numbers of spermatozoa from infertile stallions does not appear to improve their fertility.     

The Predictive Value of Semen Analysis in the Evaluation of Stallion Fertility

Pregnancy rates in managed horse populations depend on the innate fertility of the mares and stallions involved and on the quality of breeding management. Of course, because a single stallion usually mates many mares, stallion fertility is a critical factor in the overall success of a breeding program. Unfortunately, accurate evaluation of stallion fertility per se requires a large number of normal mares to be mated and is necessarily retrospective. Rather, the ideal is to predict fertility in advance of the stallion's breeding career, and this is currently attempted by way of a thorough physical examination and a routine analysis of semen quality. However, while such a ‘breeding soundness examination’ identifies stallions that clearly lack the capacity for adequate fertility, it is of limited use for predicting the level of fertility and fails to identify some seriously sub‐fertile animals. Similarly, while various sperm function tests (e.g., sperm head morphometry, the hypoosmotic swelling test, glass wool‐sephadex filtration, progesterone receptor exposure) have been shown to correlate fairly well with fertility in the field, most examine only a single or a narrow range of the attributes that a sperm must possess if it is to fertilize an oocyte in vivo, and are thus more useful for identifying specific causes of sub‐fertility than for predicting the level of fertility. On the other hand, combining the results of the various sperm function tests does improve the reliability of fertility estimation and current research is therefore concentrated on identifying a range of tests that covers as many important sperm attributes as possible but that can be performed rapidly and cheaply. In this respect, flow‐cytometry has proven to be an ideal tool because it allows the objective, rapid and simultaneous analysis of a number of properties in a large number of sperm. Moreover, stains are available for an increasing range of sperm characteristics including viability, capacitation and acrosome status, mitochondrial activity and chromatin integrity. Flow‐cytometric analysis of sperm with appropriate probes thus offers considerable promise for the prediction of stallion fertility.     

Relationship of cysteine‐rich secretory protein‐3 gene and protein with semen quality in stallions

Cysteine‐rich secretory protein‐3 (CRISP‐3) and some of its nonsynonymous polymorphism have been related to the fertility and freezability of stallion semen; however, the role of the CRISP‐3 gene and its seminal plasma protein in the raw semen quality is still unknown. The aim of this study was to evaluate the relationship of CRISP‐3 with semen quality in stallions. DNA was obtained from blood samples of 100 stallions, from which 30 stallions were randomly selected to obtain 60 ejaculates. Through PCR amplification and sequencing, the variation of four nonsynonymous SNPs from CRISP‐3 was identified and haplotypes were derived. Semen quality was assessed through the total motility (MOT), sperm vitality (SV), normal morphology (NM), functional integrity of membrane (MI) and a seminal quality index (SQi). CRISP‐3 protein content of seminal plasma (SP) was determined by ELISA. The effect of the genotype, the haplotype and the concentration of the CRISP‐3 protein on the seminal quality were evaluated through generalized linear models and linear regression analyses. Homozygous genotypes for SNP1, SNP2 and SNP3 and the heterozygous genotype for SNP4 showed a positive effect on seminal quality. Different haplotypes with positive effect on MOT, SV, NM, MI and SQi were identified. The allelic substitution analysis resulted in positive regression coefficients for MOT (SNP2) and MI (SNP2 and SNP3). A high level of CRISP‐3 resulted in a higher MOT and SQi. It is concluded that the quality of stallion semen is influenced by the genotype of CRISP‐3 and the concentration of CRISP‐3 protein in SP.     

Effect of Sire-Associated Factors on Secondary Sex Ratio of Offspring in Equine

The objective of the present study was to evaluate the effect of breed of stallion and individual stallion on secondary sex ratio (SSR; the proportion of male foals at birth). Data associated with the sex of foals and the sire, as well as the breed and age of sire were retrieved from the database of the Equestrian Federation of the Islamic Republic of Iran. In total, data consisted of 4,491 birth records from 92 stallions. Stallions were from three breeds of Arabian, Thoroughbred, and Akhal-Teke. Data were analyzed using univariable and multivariable logistic regression. Proportion of colts was 63.0% (427/678), 46.1% (1,545/3,355), and 53.9% (247/458) in Arabian, Thoroughbred, and Akhal-Teke stallions, respectively. In Arabian stallions, SSR was skewed toward males (P < .0001; odds ratio, 1.701), whereas in Thoroughbred stallions, it was skewed toward females (P = .001; odds ratio, 0.853). Secondary sex ratio was not skewed in Akhal-Teke stallions (P > .05). Secondary sex ratio in Thoroughbred stallions was lower than that in Arabian (P < .0001; adjusted odds ratio, 1.983) and Akhal-Teke (P = .010; adjusted odds ratio, 1.527) stallions, but SSR did not differ between Arabian and Akhal-Teke stallions (P > .05). There was the effect of individual stallion on SSR in Arabian and Thoroughbred breeds (P < .0001) but not in Akhal-Teke breed (P > .05). In conclusion, the present study showed the effect of breed of stallion and stallion itself on sex ratio of foals.     

Quality and Fertilizing Ability In Vivo of Sex‐Sorted Stallion Spermatozoa

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex‐sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX^® flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14‐propidium iodide), mitochondrial function (JC‐1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 × 10⁶ X‐bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non‐sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post‐thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.     

Cryopreservation of Stallion Spermatozoa with INRA96 and Glycerol

The aim of the present study was to improve success of cryopreservation of stallion spermatozoa. Semen from eleven stallions was collected and frozen in INRA 96 with two different concentrations of glycerol (3.5% and 6.0%) and compared with a control freezing process. The mean post-thaw motility for the eleven stallions of 57.93% (3.5% glycerol) and 66.50% (6.0% glycerol), which was statistically higher (P < 0.05) when compared with the mean post-thaw motility (39.7%) for semen in a control egg-yolk extender (Equipro^® CryoGuard™ Complete, Minitube). The Equipro^® CryoGuard™ Complete is a commercial semen freezing protocol that has been one of the standard processes used in our laboratory for freezing equine spermatozoa. INRA 96 with 6% added glycerol was used in the fertility trial as it provided the highest spermatozoa survival. To evaluate fertility of the frozen semen, eight mares were bred over two cycles with both fresh and frozen semen. The pregnancy rate of mares bred with frozen semen (55.6%) was not statistically different (P > 0.05) from the pregnancy rate of mares bred with fresh semen (55.6%). INRA 96 with 6.0% glycerol improved the survivability of stallion spermatozoa through the cryopreservation process, and subsequent fertility was not different (P > 0.05) from fresh, extended semen.     

Influence of Seminal Plasma Antioxidants and Osteopontin on Fertility of the Arabian Horse

This study was designed to investigate enzymatic antioxidants’ activity and nonenzymatic antioxidants’ levels in seminal plasma of stallions and to relate them with season, age, and fertility of stallions. Fifty ejaculates were collected from six healthy Arabian stallions, 4-22 years old. Ejaculates were evaluated by conventional methods. Five milliliters of each semen sample was centrifuged, and the supernatant seminal plasma was stored at −20°C. Five antioxidants, in addition to osteopontin (OPN) and testosterone, were determined in stallion seminal plasma by using commercial enzyme-linked immunosorbent assay kits. Results revealed that uric acid, ascorbic acid, OPN, and testosterone concentrations and glutathione peroxidase (GPx) activity in stallions’ seminal plasma were high (P < .05) during spring. GPx activity was higher (P < .05) in age group B (11-18 years old) than in age group A (4-10 years old). The effect of stallions’ age on GPx activity in the fertility groups was highly significant (P < .01). OPN concentration was highest (P < .05) in age group A. Uric acid and OPN concentrations and GPx activity in stallions’ seminal plasma and percent of sperm motility were higher (P < .05) in fertility group III (>70%) than in fertility group I (<50%). However, ascorbic acid concentration, catalase activity and percentage of sperm abnormalities were lower (P < .05) in fertility group III than in fertility group I. It was concluded that season and stallion age may affect antioxidant defense systems in stallions’ seminal plasma. The impairment of seminal antioxidants and OPN could lead to low fertility.     

Effects of porcine leptin receptor gene polymorphisms on backfat thickness,fat area ratios by image analysis,and serum leptin concentrations in a Duroc purebred population

The leptin receptor (LEPR) gene is considered a candidate gene for fatness traits. It is located on SSC 6 in a region in which quantitative trait loci (QTLs) for backfat thickness (BF), fat area ratios, and serum leptin concentration (LEPC) have previously been detected in a Duroc purebred population. The objectives of the present study were to identify porcine LEPR polymorphisms and examine the effects of LEPR polymorphisms on fatness traits in this same population. The Duroc pigs (226 to 953 pigs) were evaluated for BF, fat area ratios using image analysis, and LEPC. A total of seven single nucleotide polymorphisms (SNPs) in the full‐length LEPR coding region were identified in pigs from the base population. Four non‐synonymous SNPs of the LEPR gene and 15 microsatellite markers on SSC 6 were then genotyped in all pigs. During candidate gene analysis, we detected significant effects of the non‐synonymous SNP c.2002C>T in exon 14 on all traits. In fine mapping analysis, significant QTLs for BF, fat area ratios, and LEPC were detected near the LEPR gene in the same region. These results indicated that the c.2002C>T SNP of LEPR has a strong effect on BF, fat area ratios and LEPC.     

Some Factors Associated With Fertility of Thoroughbred Stallions

The results of 3 years (2005–2007) of observations and mating (5,646 estrous cycles of 3,788 mares bred to 1 of 15 stallions) at one Thoroughbred breeding farm in central Kentucky were analyzed by a multiple logistic regression model using Bayesian statistics to evaluate the relationship between data entries (factors) and pregnancy outcomes. Factors found to be significantly (P < .05) associated with pregnancy outcome included stallion (one stallion had lower OR for pregnancy higher odds ratio [OR] for pregnancy, and one had, than other stallions), date of mating (OR for pregnancy declined slightly in May – July), mare age (OR for pregnancy were higher for mares <13 years old, and lower for mares >18 years old), mare beginning status (foaling mares had a higher OR for pregnancy), mating on foal heat (lowered OR for pregnancy), mating of the day for the stallion (OR for pregnancy was 4.16 times lower for fifth compared with first mating of day), reinforcement breeding (increased OR for pregnancy), dismount semen neutrophil score (lowered OR for pregnancy when neutrophils were present in dismount semen samples), and tranquilization before breeding (lowered OR for pregnancy in foaling and barren mares). The influence of dismount sample sperm motility scores on OR for pregnancy was weak, so motility scores were not included in the final logistic regression model. The majority of variation in pregnancy outcome was because of mare factors, with only approximately one-third of the variation in fertility explained by stallion.     

Mitochondrial DNA Copy Number in Spermatozoa of Fertile Stallions

Predicting male fertility on non‐invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer‐assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four age groups (3–5, 6–10, 11–14 and >15 years) and were subjected to a complex investigation including conventional analysis, CASA, flow cytometry and mtDNA content (real‐time PCR). The mean (±SD) number of mtDNA copies equalled 14 ± 9 and varied from 3 to 64. Considering the great number of sperm parameters monitored in this study, only few of them were correlated with the mtDNA content: ejaculate volume (a positive correlation), the amplitude of lateral head displacement (ALH; a negative correlation) and the high mitochondrial activity index (a negative correlation). The stallion age was not correlated with the mtDNA copy number. This study provides the first set of data on mtDNA content in equine sperm and confirms phenomena previously described for humans and dog on associations between sperm mtDNA content and selected motility parameters monitored by the CASA. Basing our study on spermatozoa from fertile stallions could however limit the extent of detected associations.     

UTS2R gene polymorphisms are associated with fatty acid composition in Japanese beef cattle

Fatty acid composition of beef adipose tissue is one of its important traits because a high proportion of monounsaturated fatty acid is related to favorable beef flavor and tenderness. In this study, we searched polymorphisms in full length coding DNA sequence of urotensin 2 recepter and investigated the effects on fatty acid composition (C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, C18:2, monounsaturated fatty acid, saturated fatty acid). Eight single nucleotide polymorphisms (SNP) were identified by sequence comparison among eight animals, including five Japanese Black and three Holstein cattle. One of these SNP (c.866C>T) was predicted to cause amino acid substitutions (P289L) and the other seven synonymous SNP, including c.267C>T, were presumed to be in linkage disequilibrium. Therefore we selected two SNP (c.267C>T and c.866C>T) for further analysis. We investigated associations between these genotypes and fatty acid composition in three Japanese Black populations (n = 560, 245 and 287) and a Holstein population (n = 202). Tukey‐Kramer's honestly significant difference test revealed that CC genotype in c.267C>T indicated lower C14:0 and higher C18:1 than the other genotypes in Japanese Black cattle and CC genotype in c.866C>T showed lower C16:1 than CT genotype in Holstein cattle (P < 0.05). These results suggested that these genotypes would contribute to production of high‐grade meat as selection markers in beef cattle.     

Genetic risk factors for osteochondrosis in various horse breeds

Osteochondrosis (OC) is an injury to cartilage canals with a following necrosis in the growth cartilage, from there it can develop to osteochondrosis dissecans (OCD). Due to its high impact in the equine industry, new insights into predisposing factors and potential high‐risk genetic variants are warranted. This article reviews advancements in quantitative and molecular genetics in refining estimation of genetic parameters and identifying predisposing genetic loci. Heritabilities were highest for hock OC with estimates at 0.29–0.46 in Hanoverian warmblood and Norwegian trotters, whereas in Thoroughbreds only very low genetic variation seemed to be present in hock OC lesions. Whole genome scans using the Illumina Equine SNP50 or SNP70 Beadchip were performed in Thoroughbred, Standardbred, French and Norwegian trotter, Hanoverian and Dutch warmblood. Validation studies in Spanish Purebred and Hanoverian warmblood horses corroborated OC risk loci on ECA 3, 14, 27 and 29. Particularly, a strong association with hock‐OCD was found for a single nucleotide polymorphism (SNP) on horse chromosome (ECA) 3 upstream to the LCORL gene. Gene expression and microRNA analyses may be helpful to understand pathophysiological processes in equine OC and to connect OCD‐associated genomic regions with potential candidate genes. Furthermore progress in elucidating the underlying genetic variants and pathophysiological changes in OC may be expected from whole genome DNA and RNA next‐generation sequencing studies.     

Multiple marker effects of single nucleotide polymorphisms in three genes,AKIRIN2, EDG1 and RPL27A,for marbling development in Japanese Black cattle

Marbling in beef, measured by Beef Marbling Standard (BMS) number, is an economically important trait for beef cattle breeding and markets in Japan. We previously detected three single nucleotide polymorphisms (SNPs) associated with BMS number of Japanese Black in Oita prefecture: c.*188G>A in AKIRIN2, g.1471620G>T in EDG1 and g.3109537C>T in RPL27A. Here, we carried out single and multiple marker association analyses for the three SNPs in a different commercial Japanese Black population of 892 genotyped animals. The single marker analyses with the model including a single SNP showed significant associations of all SNPs with BMS number. The multiple marker analysis with the model including the main effects of the three SNPs and their interactions detected only significant main effects of g.1471620G>T and g.3109537C>T and a significant interaction between c.*188G>A and g.1471620G>T. These findings suggest the presence of inter‐allelic interactions among genes affecting the development of beef marbling. For effective marker‐assisted selection for BMS number, interactions among these markers need to be considered.     

Sodium Caseinate and Cholesterol Improve Bad Cooler Stallion Fertility

This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk–based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combination of the E2 added to cholesterol. At 24 hours after cooling, sperm motility parameters, plasma membrane stability (PMS), and mitochondrial membrane potential were assessed. In addition, cooled semen (1 billion sperm at 5°C/24 hours) from one “bad cooler” and one “good cooler” stallions, split into four extenders was used to inseminate 30 light breed mares (30 estrous cycles/extender). Milk-based extenders (E1 and E2) had superior sperm kinetics than E3 and E4 (P < .05). Plasma membrane stabilization was significantly higher (P < .05) in E4 than E1, whereas E2 and E3 presented intermediate values (P > .05). The mitochondrial potential intensity was lower (P < .05) in E2 and E4 groups compared with E1 and E3. The good cooler stallion had high fertility (∼80%) in all extenders. However, for bad cooler stallion, E1 40% (8/20) and E2 45% (9/20) had poor fertility (P < .05) compared with E4 85% (17/20), whereas E3 55% (11/20) had intermediate value (P > .05). In conclusion, the association of sodium caseinate and cholesterol improved fertility of bad cooler stallion semen cooled for 24 hours.