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.     

Implication of FKBP6 for Male Fertility in Horses

In stallions, impaired acrosome reaction (IAR) may often cause subfertility. Single nucleotide polymorphisms (SNPs) within FK506‐binding protein (FKBP6) seem to be associated with IAR in stallions. However, their effect on stallion fertility has not yet been quantified. Using whole‐genome sequence data of seven stallions, we searched FKBP6 for mutations to perform an association study in Hanoverian stallions with estimated breeding values for the paternal component of the pregnancy rate per oestrus cycle (EBV‐PAT) as target trait. Genotyping five exonic mutations within FKBP6 revealed a significant association of the SNP g.11040379C>A (p.167H>N) with EBV‐PAT in 216 Hanoverian stallions. The difference among the two homozygous genotypes was 7.62% in EBV‐PAT, corresponding to one standard deviation of EBV‐PAT. In conclusion, in Hanoverian stallions, the FKBP6‐associated SNP g.11040379C>A confers higher conception rates in A/A homozygous and lower conception rates in C/C homozygous Hanoverian stallions. Thus, an FKBP6‐associated missense mutation is significantly associated with stallion 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.     

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.     

Management Options for the Aged Breeding Stallion with Declining Testicular Function

In our experience, the testicular dysfunction that develops in aged stallions is typically progressive, contributing to a gradual deterioration in sperm output and quality over 2-4 years. As the ability to produce sufficient numbers of normal sperm in ejaculates declines, so do pregnancy rates until the stallion eventually becomes so subfertile that it is no longer commercially feasible to continue breeding. However, more intensive breeding management can sometimes result in pregnancy rates (per cycle and per season) that are sufficient to justify breeding of the aged stallion to a diminishing number of mares during the period of declining fertility.     

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.     

Reproductive efficiency of Flatrace and National Hunt Thoroughbred mares and stallions in England

REASONS FOR PERFORMING STUDY: Previous surveys of reproductive efficiency in British Thoroughbreds included only mares and stallions standing on studfarms in and around Newmarket. The present study was widened to compare Flatrace (FR) (Group A) and National Hunt (NH) (Group B) mares and stallions on studfarms throughout England. OBJECTIVES: To assess the influences of mare type, status and age, and veterinary manipulations on reproductive efficiency parameters. To compare the inherent fertility of stallions, based on singleton and twin pregnancy rates and pregnancy loss rates, in Groups A and B Thoroughbred breeding stock. METHODS: Managers of 24 FR and 9 NH public studfarms were asked to complete a questionnaire for each mated oestrous cycle shown by 2321 Group A and 1052 Group B mares throughout the 2002 mating season. Parameters such as per cycle singleton and twin pregnancy rates, and pregnancy loss rates were noted, and the success of hormone treatments to induce oestrus and ovulation assessed. The number of matings per oestrus and per pregnancy were recorded, together with the incidence and effectiveness of uterine and other veterinary treatments. The inherent fertility of 84 Group A and 43 Group B stallions in the study, as measured by the singleton and twin early pregnancy rates and the pregnancy loss rates recorded in the mares they mated, was also estimated. RESULTS: Per cycle early pregnancy (Days 13-16) was 63.2% for Group A and 65.3% for Group B mares; and 10.3% and 13.1%, respectively, of those pregnancies were twins or triplets. Early, middle and late pregnancy loss rates were 7.2% vs. 8.0% (Days 15-42), 3.6% vs. 6.1% (Days 42-1st October) and 2.7% vs. 2.1% (October-foaling), respectively. Matings per oestrus and per early pregnancy were significantly higher in Group B vs. Group A mares. For stallions that mated > or = 30 mares, overall early pregnancy rates per cycle in mares mated ranged from 30-89% across the 2 groups. CONCLUSIONS: No major differences in reproductive efficiency were identified between FR and NH mares and stallions. Increasing mare age was the single biggest limiting factor to an otherwise high rate of fertility in well-managed English Thoroughbreds. POTENTIAL RELEVANCE: This study identified factors that influence reproductive efficiency in the Thoroughbred.     

The influence of mare numbers, ejaculation frequency and month on the fertility of Thoroughbred stallions

Reason for performing the study: Although considerable variation in per cycle pregnancy rates exists between Thoroughbred (TB) stallions, there is little information on factors that may influence this figure. Objective: To assess the influence of month, mare numbers and mating frequency on the fertility of TB stallions standing on studfarms in East Anglia, England. Methods: The daily breeding records of 31 TB stallions mating 3034 mares on 4851 occasions during the 2010 season were surveyed and related to first scan pregnancy rates. The influences of mare book size, month, number of matings per day and mating frequency or abstinence on per mating pregnancy rates were analysed. Results: The overall per mating pregnancy rate for all the stallions was 59.6%, but for individual stallions the figures ranged from 19.0% to 80.1%. The first mating occurred on 9 February and the last on 24 July and the per mating pregnancy rate per month was significantly reduced in June and July. The number of mares mated by individual stallions ranged from 15 to 161, giving a mean overall workload of 160 matings per 100 mares. The per mating pregnancy rate was not related to book size, the number of matings in the season or the mating frequency per day. However, some stallions showed differences in per mating pregnancy rate related to month or the number of ejaculations in the preceding 3 days. Conclusions: The majority of TB stallions are able to maintain good fertility despite large books of mares. However, 5 of the 31 stallions surveyed showed a per mating pregnancy rate of ≤50%. Potential relevance: This survey has identified wide differences between the per mating pregnancy rate in TB stallions. Identification of the factors involved through more comprehensive surveys would provide useful information for mare and stallion owners.     

Review on Effects of Fescue Grass Ergot Alkaloids in the Horse and Preliminary Study on Effect of Fescue Grass Ergot Alkaloid in the Stallion

Feed with ergot alkaloids ingested by horses has a deleterious clinical and economic impact on the industry. The clinical manifestation of the effects in mares is early embryonic mortality, abortions, prolonged gestation, dystocia, thickened (edematous) or retained placental membranes, agalactia, and increased rates of newborn mortality. For the stallion, very little is known, although ergot alkaloids decrease the ejaculate volume. However, a large number of breeding stallions graze endophyte infected (E+) pastures. This is especially true in the southeastern United States, and clinically we do not perceive that any stallions have any defined problems that could be attributed to ergot alkaloids. However, the number of spermatozoa produced by any stallion might mask the effects. The effects on fertility may be more subtle and only evident with sperm cell manipulation such as chilling or freezing. A preliminary study was performed on six breeding stallions fed feed containing infected fescue seed. We were not able to determine any significant (P < .05) detrimental effects on sperm motility, number morphology, and sperm morphology when compared with controls.     

Key Factors Affecting Reproductive Success of Thoroughbred Mares and Stallions on a Commercial Stud Farm

To evaluate factors contributing to fertility of thoroughbred mares, data from 3743 oestrous periods of 2385 mares were collected on a large thoroughbred farm in Ireland. Fourteen stallions (mean age 8.3 years; range 4–15 years) had bred 2385 mares (mean age 9.4 years; range 3–24 years). Maiden mares accounted for 12%, mares with a foal at foot for 64%, and barren, slipped or rested mares for 24% of the total. The mean pregnancy rate per cycle was 67.8% (68.6% in year 1 and 66.9% in year 2). Backward stepwise multivariable logistic regression analysis was utilized to develop two models to evaluate mare factors, including mare age, reproductive status, month of foaling, dystocia, month of cover, foal heat, cycle number, treatments, walk‐in status and stallion factors including stallion identity, stallion age, shuttle status, time elapsed between covers and high stallion usage on the per cycle pregnancy rate and pregnancy loss. Old age (p < 0.001) and cover within 20 days post‐partum (p < 0.003) were associated with lowered pregnancy rates. High mare age (p < 0.05) and barren, slipped or rested reproductive status (p = 0.05) increased the likelihood of pregnancy loss. Uterine inflammation or infection, if appropriately treated, did not affect fertility. Only high usage of stallions (used more than 21 times in previous week) was associated with lowered (p = 0.009) pregnancy rates. However, shuttle stallions were more likely to have increased (p = 0.035) pregnancy survival, perhaps reflecting a bias in stallion selection. In conclusion, mare age exerted the greatest influence on fertility; nonetheless, thoroughbreds can be effectively managed to achieve high reproductive performance in a commercial setting.     

Artificial insemination and preservation of semen.

Artificial insemination is an effective technique for improving utilization of stallions in breeding programs. When proper semen handling and insemination procedures are used, optimal pregnancy rates are attainable. When AI techniques are employed for mares and stallions with marginal fertility, pregnancy rates may be improved in comparison with natural mating. Preservation of stallion semen in the liquid or frozen state reduces the costs and potential health hazards incurred by transporting mares and provides easier access to genetic material that may otherwise be unavailable. Acceptable pregnancy rates are consistently obtained with cooled semen. Conversely, techniques for cryopreservation of stallion semen will require more refinement before the procedure can be considered commercially viable on a wide scale.     

Trypan Blue/Giemsa Staining to Assess Sperm Membrane Integrity in Salernitano Stallions and its Relationship to Pregnancy Rates

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty‐one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty‐five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June–July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June–July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut‐off value of 48% LSIA. Trypan blue‐Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor‐quality ejaculates.     

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.     

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.     

Management Strategies Aiming to Improve Horse Welfare Reduce Embryonic Death Rates in Mares

The objective of this retrospective study was to evaluate the effect of management strategies aiming to improve animal well‐being on pregnancy and embryonic death (ED) rates. Breeding records of a cohort of 1206 Thoroughbred mares brought to a stallion station facility, to be bred with the stallions housed there, were evaluated during ten breeding seasons. Mares were blocked according to management strategies in two groups: Stress and Relax. Strategies used to improve animal well‐being (Relax group) were as follows: stopping the teasing routine, reducing or eliminating stall confinement, reducing the number of mares per group and maintaining herd stability during the breeding season. In barren mares, the pregnancy rate was higher in the Relax group (91.8%) when compared to the observed in Stress group (84.7%). However, no difference in pregnancy rates were observed (Stress = 85.2% vs. Relax = 86.2) in foaling mares. ED rate was higher in barren and foaling mares of the Stress group mares (25.5% and 26.8%, respectively) compared with the Relax group (16.1% and 14.7%, respectively). No significant differences were observed on foal heat pregnancy rate between groups; yet, the embryo loss on foal heat was significant reduced in Relax mares (Relax = 8.7% vs Stress = 24.5%). In conclusion, management strategies aimed to reduce social stress can reduce early pregnancy losses and the average cycles per pregnancy, improving reproductive performance in mares.     

Reproductive efficiency of Thoroughbred and Standardbred horses in north-east Victoria

Objective To evaluate the reproductive efficiency of horse farms in north-east Victoria and identify aspects of management to be targeted for improving reproductive efficiency. Design Retrospective study. Procedure Records from seven Thoroughbred (TB) and four Standardbred (STB) studs in north-east Victoria from 1990 to 2001 were reviewed; 8813 cycles in 4455 mares were analysed. TB mares were inseminated by natural mating, whereas STB mares (89%) were artificially inseminated. Results The overall early pregnancy rate per cycle was 68.8% for TB mares and for STB mares, 68.3%. Multiple pregnancy per cycle was more frequent in TB (8.3%) than in STB (4.6%) mares (P < 0.001). Early embryonic death occurred in 7.1% of TB and 7.5% of STB pregnancies. TB mares had fewer inseminations per cycle (1.03) than STB mares (1.43) (P < 0.001). There was a significantly lower proportion of barren reproductive status within the TB than the STB mares. Pregnancy rate per cycle among stallions ranged from 48% to 79%. Conclusions On-farm pregnancy rates in both breeds were higher than previously reported and likely reflect improvements in reproductive management. The disparity between breeds in the inseminations per cycle and proportion of barren mares exposed the differing structures of the two industries, and presents a target for improving the reproductive efficiency in STBs. The difference between breeds in the multiple pregnancy rate per cycle likely reflects the higher ovulation rate of TB mares. The variability in pregnancy rate per cycle between the 22 stallions was associated with differences in individual inherent fertility and the quality of stallion management.     

Endocrine abnormalities and hormonal therapy.

Routine measurement of estrogens, testosterone, T4, insulin, FSH, and LH at least four times per year (e.g., during each of the four seasons) may improve the efficiency of stallion management. Benefits may not be realized in the short term but will provide valuable historical data on individual stallions that, when added to other data, will improve ability of management personnel to initiate early treatment and delay or slow declining fertility. This ability will be greatly improved as more data and products become available. There appears to be a relationship between low total estrogen concentration/high FSH concentration and subfertility. This condition is associated with high average breedings per pregnancy. A decrease in concentration of estrogen and an increase in FSH concentration often precede a decline in fertility associated with oligospermia. Hypogonadotropic stallions have not been reported. This condition is not likely to be a cause of declining fertility in stallions and greatly limits the potential efficacy of GnRH therapy in subfertile stallions. Much research must be done to elucidate the etiology of testicular degeneration associated with increased FSH concentrations and decreased estrogen concentrations in stallions. At present, no reliable hormonal therapeutic protocols exist that will improve fertility in subfertile stallions.     

Contribution of Reproduction Management and Technologies to Genetic Progress in Horse Breeding

Reproductive technologies aim at improving fertility with the ultimate result of improving genetic selection. In equidae, the respective contribution of different methods of horse management and breeding to genetic progress remain difficult to evaluate as breeding strategies affect the number of offspring per mare or stallion whereas different selection methods (based on pedigree, performance, genomics or progeny’s performance) will be applicable at different ages, leading to different accuracy in the estimation of the breeding value. Here, a mathematical model was applied to evaluate theoretical genetic progress depending on breeding conditions in horses. The model showed that for breeding systems ranging from 0.6 to 2 foals/year/mare and from 10 to 150 foals/year for stallions, when selection of the best animals is strictly made by a truncation, the genetic progress is accelerated by (1) increasing the number of offspring per year, (2) the start of reproduction as soon as the age of 2 in both sexes, and (3) reducing the number of years of use for stallions from 10 to 5 years. The calculation showed that using all ways of improvement could provide an increase in genetic progress of up to +270% and +226% in mares and stallions, respectively, above the basal reference situation of 100%. In the Selle Français breed, the observed reproductive management parameters (10 years generation interval, 10 foals/stallion and 0.55 foals/mare) are close to the worst conditions of the model. In addition, the best mares are not selected for breeding. In conclusion, new reproductive technologies, genomic selection, and breeding younger animals will increase genetic gain.     

Mesothelioma of the tunica vaginalis testes in a stallion

Scrotal enlargement is a problem in stallions that often leads to poor semen quality. Although there are many causes for scrotal enlargement, testicular neoplasia is often high on the differential list, especially in an aged stallion. This case report describes a stallion with scrotal enlargement caused by a rare neoplasia located in the tunica vaginalis, and not in the testicular parenchyma. Diagnosis of mesothelioma was made following hemicastration. Prognosis in other species is variable, but this stallion returned to his successful breeding career following treatment and has remained healthy one year following the diagnosis.