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.     

Detection of Sex Chromosome Aneuploidy in Equine Spermatozoa Using Fluorescence In Situ Hybridization

The aim of our study was to diagnose aneuploidy in equine spermatozoa by multicolour fluorescence in situ hybridization (FISH) technique using specific molecular probes for equine sex chromosomes and autosome pair four (EGFR probe) labeled by different fluorochromes. These were applied on decondensed spermatozoa of four stallions. In total, more than 8800 sperm cells were examined. The total frequency of aberrant cells was 0.496%: aneuploidy of XX (0.135%), YY (0.023%), XY (0.102%), diploidy (0.057%), lack of sex chromosome (0.18%). In one stallion the ratio of normal X‐ and Y‐bearing cells was different from the expected 1 : 1 ratio (p = 0.0002), in all three other stallions this ratio was close to 1 : 1. The present study demonstrated that the FISH technique is a powerful method to identify sex chromosome aberrations in equine spermatozoa and allows for the determination of the ratio between X–Y‐spermatozoa.     

Alkaline and Acid Phosphatase, β‐Glucuronidase and Electrolyte Levels in Fractionated Stallion Ejaculates

Seminal plasma (SP) is a mixture of contents from the testes, epididymides and accessory sex glands. The sperm concentration is highest in the first few jets, or fractions, of the ejaculate, and the composition of SP varies between these fractions because accessory gland secretions are released in a specific order. The aim of this study was to compare the levels of Na, Cl, K, Mg, Ca, inorganic phosphate (Pi) and the enzymes alkaline phosphatase (AP), acid phosphatase (ACP) and β‐glucuronidase (BG) in the different fractions of the ejaculate and in different stallions. All semen collections were done using a computer‐controlled phantom that collects the ejaculatory jets separately in five cups. The cups with the highest (HIGH) and the lowest (LOW) sperm concentration were analysed. In Trial I, semen was collected from three reproductively normal stallions. In Trial II, ejaculates of two reproductively normal stallions were compared to those of two subfertile stallions. In Trial III, semen was collected from seven stallions with varying reproductive history. The sperm‐rich fractions contained the highest levels of AP, ACP, BG and inorganic phosphate, and the values were positively correlated to the sperm concentration. Significant differences between the subfertile and the fertile stallions pairs in HIGH : LOW ratios were found in Pi and Cl concentrations. The highest concentrations of Ca and Mg were found in the last fractions with low sperm concentrations, with no significant differences between the fertile and the subfertile stallion pairs. The concentrations of K, Na and Cl were similar in HIGH and LOW fractions and in whole ejaculate samples. Pre‐sperm fluid contained the highest concentrations of Na and Cl. Some of the possible variation in storage tolerance between ejaculates and ejaculatory fractions could perhaps be explained by differences in the composition of SP.     

Investigation of genetic diversity and inbreeding in a Japanese native horse breed for suggestions on its conservation

Because native breeds can serve as genetic resources for adapting to environment changes, their conservation is important for future agroecosystems. Using pedigree analysis, we investigated genetic diversity and inbreeding in Japanese Hokkaido native horses, which have adapted to a cold climate and roughage diet. Genetic diversity was measured as the number of founders and the effective number of founders, ancestors and genomes. All metrics imply a decrease in genetic diversity. A comparison of these metrics suggested that pedigree bottlenecks contributed more than did random gene losses to the reduction of genetic diversity. Estimates of marginal contributions of ancestors suggest that the bottlenecks arose mainly because related stallions had been used for breeding. A tendency for an increase in inbreeding coefficients was observed. F‐statistics revealed that a small effective population size majorly contributed to this increase, although non‐random mating in particular regions also contributed. Because the bottlenecks are thought to have reduced the effective population size, our results imply that mitigation of bottlenecks is important for conservation. To this end, breeding should involve genetically diverse stallions. In addition, to prevent non‐random mating observed in particular regions, efforts should be made to plan mating with consideration of kinships.     

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.     

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.     

Birth of puppies of predetermined sex after artificial insemination with a low number of sex‐sorted,frozen–thawed spermatozoa in field conditions

The aim of this study was to evaluate fertility and sex ratios after artificial insemination in dogs under field conditions. Semen was cryopreserved as unsorted (control) or was separated into X‐ and Y‐chromosome‐bearing sperm using a cell sorter. Sixty female dogs were inseminated with frozen–thawed spermatozoa of 100 × 10⁶ unsorted (a dose in practice) and 4 × 10⁶ sorted (X and Y group, respectively). A total of 20 dogs became pregnant and 126 puppies were born from the three groups. The percentage of parturition was similar for the X (5/20; 25.0%) and Y (4/20; 20.0%) group (P > 0.05), but lower than controls (11/20; 55.0%) (P < 0.05). Ultimately 28 out of the 32 puppies produced from X group were female (87.5%) and 19/22 (86.4%) puppies of Y group were male. In contrast, sex ratio (51.4% to 48.6%) in the control was significantly different from the X, Y group (P < 0.05). However, male and female puppies in the control had similar birth weights and weaning weights to those from the X and Y groups. This preliminary information indicated that normal puppies of predicted sex can be produced with low numbers of sorted cryopreserved dog spermatozoa at a farm level, making sperm‐sexing technology potentially applicable for elite breeding units.     

High or Low Body Fat Deposition in the Presence of a Normal Oral Sugar Test is Not Associated With Postthaw Semen Parameters in Stallions

This study compared the postthaw semen parameters of stallions with high and low body condition score (BCS) and evaluated associations between body morphometric parameters and postthaw semen parameters. Twenty stallions were split into Low BCS (BCS<7, n = 11) and High BCS (BCS ≥7, n = 9) groups, and underwent a complete morphometric analysis (e.g., neck scores and circumference, crest neck height, body weight, and height), and subcutaneous body fat thickness (SFT) at the tail head, withers, shoulders, and retroperitoneal space. A fasted oral sugar test (OST) was conducted on all stallions. One ejaculate from each stallion was frozen with a commercial egg yolk-based extender. Postthaw sperm motility parameters, plasma membrane integrity, mitochondrial membrane potential, hydrogen peroxide and intracellular superoxide production, and lipid peroxidation were analyzed for all stallions. The circumference at 25% and 50% of the neck’s length were larger for High-BCS stallions (P < .05). There were no differences between groups for the neck crest height (P > .05). Stallions with High BCS had greater SFT at the tail head than stallions with Low BCS (P < .05); however, there were no differences between groups in the SFT at the shoulders and withers (P > .05). All stallions had resting blood glucose below the cutoff for equine metabolic syndrome. There were no differences between groups for resting glucose concentrations or for a peak at 30 or 60 minutes after initiation of the OST (P > .05). There were no differences in sperm parameters between groups (P > .05). Collectively, the findings of the present study suggest that High BCS or Low BCS in the presence of normal OST do not explain post-thaw semen parameters.     

Genetic variability in Hanoverian warmblood horses using pedigree analysis

A data set constituting a total of 310,109 Hanoverian warmblood horses was analyzed to ascertain the genetic variability, coefficients of inbreeding, and gene contributions of foreign populations. The reference population contained all Hanoverian horses born from 1980 to 2000. In addition, Hanoverian stallions born from 1980 to 1995 and Hanoverian breeding mares from the birth years 1980 to 1995 with registered foals were analyzed for the same genetic parameters. The average complete generation equivalent was approximately 8.43 for the reference population. The mean coefficient of inbreeding was 1.33, 1.19, and 1.29% for the reference population, stallions, and breeding mares, respectively. The effective number of founders was largest in stallions (364.3) and smallest in the reference population (244.9). The ratio between the effective number of founders and the effective number of ancestors was 3.15 for the reference population, 3.25 for the stallions, and 3.06 for the breeding mares. The effective population size in the Hanoverian warmblood reference population was 372.34. English Thoroughbreds contributed nearly 35% of the genes to the Hanoverian reference population and even slightly greater contributions (39%) to the stallions. Trakehner and Arab horses contributed approximately 8 and 2.7%, respectively, to the Hanoverian gene pool. The most important male ancestors were Aldermann I from the A/E line, Fling from the F/W line, and Absatz from the Trakehner line, whereas the breeding mare Costane had the greatest contribution to the reference population, stallions, and breeding mares. From 1996 onward, the stallions Weltmeyer and Donnerhall had the largest genetic impact on the Hanoverian horse population.     

Effect of Feeding Fescue Seed Containing Ergot Alkaloid Toxins on Stallion Spermatogenesis and Sperm Cells

The cellular effects of tall fescue grass–associated toxic ergot alkaloids on stallion sperm and colt testicular tissue were evaluated. This was a continuation of an initial experiment where the effects of toxic ergot alkaloids on the stallion spermiogram were investigated. The only spermiogram parameter in exposed stallions that was affected by the toxic ergot alkaloids was a decreased gel‐free volume of the ejaculate. This study examined the effect of toxic ergot alkaloids on chilling and freezing of the stallion sperm cells. The effect of toxic ergot alkaloids on chilled extended sperm cells for 48 h at 5°C was to make the sperm cells less likely to undergo a calcium ionophore–induced acrosome reaction. The toxic ergot alkaloids had no effect on the freezability of sperm cells. However, if yearling colts were fed toxic ergot alkaloids, then the cytological analysis of meiotic chromosome synapsis revealed a significant increase in the proportion of pachytene spermatocytes showing unpaired sex chromosomes compared to control spermatocytes. There was little effect of ergot alkaloids on adult stallions, but there might be a significant effect on yearling colts.     

Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre‐sperm fluid and the first sperm‐rich jets (HIGH‐1), the main sperm‐rich portion (HIGH‐2), the jets with low sperm concentrations (LOW), and a combined whole‐ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH‐2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion’s own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse‐phase liquid chromatography (RP‐HPLC), N‐terminal sequencing and mass spectrometry. The area‐under‐the‐curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS‐PAGE and densitometry. No significant differences emerged between fractions in the AUC‐values of the Horse Seminal Protein‐1 (HSP‐1) and HSP‐2 peaks, or the peak containing HSP‐3 and HSP‐4 (HSP‐3/4). Levels of HSP‐1, HSP‐2 and HSP‐3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60–70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.     

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.     

Fertility in Single‐ovulating and Superovulated Dairy Heifers after Insemination with Low Dose Sex‐sorted Sperm

The present study was performed to test fertility in single‐ovulating and superovulated dairy heifers after insemination with low dose sex‐sorted sperm under field conditions. Some parameters, including the dosage, deposition site and timing, were assessed with the pregnancy rates after artificial insemination (AI). Moreover, the use of oestrus synchronization in combination with sorted sperm was evaluated. Besides that, we also improved the embryo production efficiency in superovulated dairy heifers by optimizing the timing of inseminations and repartitioning the sexed sperm dosage among multiple inseminations. The conception rate (52.8%) in heifers after low dose (2 × 10⁶) insemination with sorted sperm deep into the uterine horn did not differ (p > 0.05) from that (59.6%) of conventional AI (1 × 10⁷ non‐sorted sperm) and that of deep insemination with low dose non‐sorted sperm (57.7%). There was also no difference (p > 0.05) between conception rates after single (51.7%) and double (53.8%) deep insemination with sorted semen. Heifers inseminated with sorted sperm at synchronous oestrus had a lower pregnancy rate (48.1%) than heifers at spontaneous oestrus (53.6%), but this did not reach statistical difference (p > 0.05). The average number of transferable embryos collected in vivo from heifers inseminated with sorted sperm (4.81 ± 2.04) did not differ (p > 0.05) from that obtained from heifers after insemination with non‐sorted sperm (5.36 ± 2.74). Thus, we concluded that the pregnancy rate after deep intra‐uterine insemination with low dose sorted sperm was similar to that of non‐sorted sperm, which was either also deposited at a low dose deep intra‐uterine or into the uterine body. Sychronization of oestrus can be beneficial in combination with sorted sperm to optimize the organization and management of dairy herds. The results from superovulated heifers demonstrated that our insemination regime can be used to obtain a comparable embryo production efficiency with sorted sperm than with non‐sorted sperm.     

Semen parameters and hormone concentrations in stallions subjected to long-term estrogen administration

Eight pony stallions were paired by estimated daily sperm output (DSO) and randomly assigned to one of two treatments in a randomized block experiment. Stallions received 44 μg/kg BW estradiol cypionate (ECP) or an equivalent volume of physiological saline solution on alternate days during the breeding season. Blood samples collected immediately preceding each injection were assayed for luteinizing hormone (LH), estradiol-17β (E₂) and testosterone (T). Semen was collected twice weekly, 3.5 days apart, to evaluate sperm motility and total number of sperm per ejaculate. Prior to and after 4, 8 and 12 weeks on treatment, semen was collected once daily for 7 days to determine DSO. Data were separated into 9 periods (10 days each) for statistical analysis and subjected to analysis of variance for a randomized block design to determine treatment effects.There were no differences (p>.05) between groups for DSO or LH prior to initiation of treatment. Testosterone was higher (p<.05) in ECP stallions compared with C stallions prior to treatment and at all time points measured. As expected, E₂ was higher (p<.05) in the ECP stallions compared to C stallions after 20 days (period 2) of treatment and for the remainder of the experiment. However, E₂ was higher (p<.05) in the C group prior to treatment, but there was no difference between the groups after 10 d of treatment (period 1). ECP stallions had higher (p<.05) DSO than C stallions after 30 d on treatment. After 40 and 50 d (periods 4 and 5), ECP stallions demonstrated higher (p<.05) total sperm per period than C stallions. This was preceded by higher (p<.05) LH values for ECP stallions than for C stallions after 10 and 20 d (periods 1 and 2). No differences were found between the ECP and C groups for LH between 30 and 60 d. Although numerically higher, no significant differences (p>.05) were seen after 60 days for DSO or after 60, 70 or 80 days for total sperm per period. ECP stallions had higher (p<.05) DSO and total sperm per period after 90 d than C stallions. Additionally, LH remained significantly higher (p<.05) in the ECP group after 60 days (periods 7, 8 and 9). Elevated LH concentrations in ECP stallions demonstrated that estrogen treatment did not inhibit LH secretion in this study.     

Genetic and Environmental Factors Influencing Gestation Length in Lusitano Horses

Breeding and parturition records collected over a period of 35 years in the Alter Real stud of Lusitano horses were used to calculate gestation length (GL). The 1027 gestations by 209 mares mated to 60 stallions had a mean GL of 338.1 ± 9.26 days. The mixed model analysis of variance indicated that the sex of the foal and inbreeding of the dam and foal had no significant effect on GL (P > .05). On the other hand, GL increased linearly with mare age, with an estimated regression coefficient of 0.155 ± 0.069 days/year (P < .05). Year and conception month affected GL (P < .05), with longer gestations observed when breeding occurred between January and April, followed by a decline of about 5 and 10 days in GL of mares bred in May and in June–July, respectively. Important differences were observed between stallions (P < .05), with most stallions resulting in a distribution of GL in an interval of ±5 days. The inclusion of the mare as a random effect in the mixed model resulted in an estimated repeatability of GL of 0.427, indicating that mares tend to be regular in having long or short gestations across their lifetime. Variance components estimated in an Animal Model resulted in heritability estimates of 0.39 for maternal genetic effects and 0.19 for direct genetic effects, with no association between the two components. Overall, the mare seems to have the major genetic influence on GL in Lusitano horses, but environmental factors, such as month of conception and also the age of the mare, have a noticeable effect.     

Determination of the correlation between stallion's age and number of sex chromosome aberrations in spermatozoa

The aim of this study was a cytogenetic analysis of stallions semen to find sex chromosome aberrations and to determine if there was an association between stallion's age and aberration frequency for the sex chromosomes. Sperm samples were collected from 22 stallions of various age from 3 to 23 years. Multicolour FISH was performed on each sample, using probes for the sex chromosomes and EGFR gene, localized on 4p12 in domestic horse. A total of 26199 sperm cells were analysed (from 1 070 to 1 532 per animal). Among the analysed cells, there were 50.318% with X chromosome, 48.543% with Y chromosome and 1.139% with aberrant chromosomes. The frequency of aberrations was: sex chromosomes nullisomy (0.466%), XY aneuploidy (0.454%), XX disomy (0.146%), YY disomy (0.041%), diploidy (0.024%) and trisomy XXY (0.008%). Additionally there was a correlation between the age of an animal and the frequency of sex chromosome aberration and a significant positive correlation between age and disomy of XY, XX, YY, trisomy of XXY, autosomal disomy was seen. A Correlation between the age of a stallion and the level of nullisomy was negative. The present study demonstrated that FISH technique is a powerful method to identify sex chromosome aberrations in equine spermatozoa and might be very helpful for a breeder during a selection for the best stallion.     

Single layer centrifugation of stallion spermatozoa consistently selects the most robust spermatozoa from the rest of the ejaculate in a large sample size

Reasons for performing study: An improvement in sperm quality after single layer centrifugation (SLC) has been seen in previous studies using small sample sizes (for example, n = 10 stallions). There is a need to investigate whether this improvement is repeatable over several breeding seasons with a larger number of stallions (n ≥ 30 stallions). Objective: To make a retrospective analysis of the results of SLC performed on more than 250 sperm samples (176 ejaculates) from 31 stallions in 3 consecutive breeding seasons. Methods: Sperm quality (motility, proportion of morphologically normal spermatozoa and the proportion of spermatozoa with undamaged chromatin) was assessed before and after SLC. Results: All parameters of sperm quality examined were significantly better in sperm samples after SLC than in their unselected counterparts (P<0.001 for each parameter). The yield of spermatozoa obtained after SLC was influenced by the type of extender used and also by the concentration of spermatozoa in the original ejaculate, with fewer spermatozoa being recovered when the loading dose contained a high concentration of spermatozoa. The optimal concentration was approximately 100 × 10⁶/ml. Sperm concentration in the samples loaded on to the colloid influenced the sperm yield while the type of semen extender affected sperm quality and survival. Furthermore, the scaled‐up SLC method was found to be suitable for use with a range of ejaculates, with similar sperm kinematics being observed for standard and scaled‐up preparations. Conclusions: SLC consistently improved the quality of stallion sperm samples from a large number of ejaculates. The method could be scaled‐up, allowing larger volumes of ejaculate to be processed easily from a wide range of stallions.     

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.     

Morphology and head morphometric characters of sperm in Thai native crossbred stallions

Background

One of the semen quality parameters use to determine fertility is the percentage of sperm that express normal morphology. Sperm head morphometry is also correlated with fertility. The objectives of this study were 1) to investigate the sperm morphology and normal sperm head morphometry of Thai native crossbred stallions, and 2) to compare our results with the characteristics of proven fertile sperm from purebred stallions.

Methods

Semen samples were collected monthly from nine stallions, of which five were Thai native crossbred (T) and four were purebred of proven fertility (F: F1 was a Standard-bred; F2 was a Warm-blood; F3 and F4 were Thoroughbreds). All the animals were aged between 5 and 12 years. Sperm morphological examination was performed using formaldehyde-fixed samples under phase-contrast microscopy (1000×). Normal sperm head morphometry characteristics were measured by Computer-Assisted Semen Analysis (Hamilton Thorne, USA.) after applying the Harris'' haematoxylin staining technique.

Results

The percentages of morphologically normal and abnormal sperm varied among individual stallions in both the T and F groups. The mean percentage of morphologically normal sperm was not significantly different (P > 0.05) between T and F stallions (mean ± SE, 49.7 ± 1.3 and 48.1 ± 2.8, respectively). A comparison between the T and F sperm heads revealed that all the dimensional parameters were significantly different (P < 0.05). The coefficients of within-animal variation (CVs) ranged from 2.6 (shape factor 1) to 7.5 (elongation) and 2.9 (shape factor 1) to 8.1 (elongation) in T and F, respectively. In the case of the T group, those sperm head parameters that featured a low within-animal CV and a high between-animal CV were perimeter (2.9, 19.1), shape factor 1 (2.6, 25.8) and shape factor 3 (3.8, 32.0). In the case of the F group, only shape factor 1 (2.9, 26.1) featured such characteristics.

Conclusion

We found variability in the percentage of morphologically normal and abnormal sperm, as well as in sperm head dimensions among Thai native crossbred stallions, and these results were similar to those of purebred stallions. Our findings demonstrate that the heads of the T sperm specimens were larger and rounder than that of the F sperm. Perimeter, shape factor 1 and shape factor 3 could be used as parameters for the identification of individual T stallions based on a sperm sample.     

Testicular Measurements and Daily Sperm Output of Tori and Estonian Breed Stallions

Evaluation of testicular measurements and daily sperm output (DSO) yields valuable information for predicting the reproductive capacity of stallions. The present study evaluated testicular measurements (height, length, width and circumference) and DSO of eight Tori and eight Estonian breed stallions. One ejaculate of semen was collected daily for 10 subsequent days from each stallion. The gel‐free volume of semen was measured with a graduated glass cylinder and the sperm concentration was assessed with a Chorjajev chamber. The volume of gel‐free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). The DSO was calculated as mean TSN of collection on days 8–10 in Tori breed stallions and on days 4–10 in Estonian breed stallions. The DSO of Tori breed stallions was 12.9 × 10⁹ spermatozoa and of Estonian breed stallions 4.5 × 10⁹ spermatozoa (p < 0.001). Testicular measurements (in cm) 1 day after the last semen collection were as follows: left testis– height 7.3, length 10.4 and width 7.3 in Tori breed stallions, and 5.9, 8.1 and 5.9, respectively, in Estonian breed stallions; right testis– height 7.4, length 10.6 and width 7.4 in Tori breed stallions, and 5.5, 7.4 and 5.3, respectively, in Estonian breed stallions. All these testicular measurements were significantly smaller in Estonian than in Tori breed stallions (p < 0.001). Testicular circumference was 45.4 and 35.4 cm in Tori and Estonian breed stallions, respectively (p < 0.001). The testicular circumference was correlated with DSO in both Estonian (p < 0.05) and Tori breed stallions (p = 0.071). The results give us valuable information on the reproductive capacity of Tori and Estonian breed stallions.