Effect of protein acetylation on capacitation of stallion sperm.

Sperm capacitation is considered the main factor limiting conventional in vitro fertilization (IVF) in horses. A recent scientific breakthrough in sperm processing for IVF in horses has resulted in embryos and foals being produced; however, various aspects of the IVF process remain to be fully elucidated. Lysine acetylation has been shown to play a role in sperm capacitation in several species and the objective of this study was to detect and evaluate this process in the horse. Ejaculates of two stallions were collected and incubated in different conditions with deacetylase inhibitors to induce a hyperacetylation state. Although lysine acetylation was successfully detected in all experimental groups, sperm hyperacetylation could not be induced following incubation with deacetylase inhibitors. In addition, no hyperactivation was detected by kinematic sperm evaluation and tyrosine phosphorylation increased only in the positive control group. Treatments with high doses of deacetylase inhibitors increased acrosome reaction indicating a possible connection between induction of acrosome reaction and protein acetylation. Future studies investigating the effect of longer incubation periods with different doses of deacetylase inhibitors are warranted to elucidate the ability of protein acetylation to induce capacitation of stallion sperm.

Calcium affects stallion spermatozoa parameters in different incubation temperatures.

This study was aimed to determine the effect of CaCl2 on the motility and viability of stallion spermatozoa during different incubation temperatures. Experimental samples were prepared by diluting the ejaculates (n = 10) from three uniformly housed and fed breeding stallions with six different concentrations of CaCl2 (A: 0.1125, B: 0.225, C: 0.45, D: 0.938, E: 1.25, and F: 1.875 mg/mL). The control samples (CON) were prepared by diluting ejaculate only with physiological solution. Samples were divided into two aliquots for analyses at different storage temperatures (5 °C and 37 °C). The motility parameters were analysed by Computer Assisted Semen Analysis system at several time intervals (0, 1, 2 and 3 h) and the viability was assessed using a mitochondrial toxicity test (MTT) realized at the end of incubation at both temperatures. Addition of CaCl2 to stallion semen showed significant effect on motility parameters, especially in the highest concentrations at 5 °C. Significant objectionable effect of CaCl2 on both total and progressive motility was observed at temperature 37 °C compared to control sample. However, results of velocity curved line in samples C, D and F at time 1 h and also at time 2 h in sample F showed significant positive effect of CaCl2. Sperm viability in experimental samples did not show a significant difference compared to the control at either 5 °C or 37 °C. The results of this study did not confirm essential effect of calcium on reproductive parameters of stallion. To conclude, our study demonstrated that the effect of CaCl2 on stallion sperm motility differs in a dose-dependent manner; however, the overall impact on motility parameters does not seem to be beneficial.

Localization of ß-Nerve Growth Factor in the Stallion Reproductive Tract.

ß-Nerve growth factor (ß-NGF) is a protein produced in the reproductive tract of camelids (camels, llamas, and alpacas) that has been identified as the ovulation inducing factor in seminal plasma. ß-NGF from seminal plasma deposited into the reproductive tract of the female camelid acts systemically to stimulate the secretion of luteinizing hormone (LH) from the anterior pituitary, which in turn induces follicle maturation and ovulation. The objectives of the present study were to determine if ß-NGF is present in the reproductive tract of the stallion and identify the specific site(s) of production. The hypotheses were that ß-NGF would be present in the stallion reproductive tract and would primarily be localized in Sertoli cells of the testes and the prostate gland. Immunohistochemistry on paraffin-embedded paraformaldehyde-fixed tissues was performed using a rabbit polyclonal anti-ß-NGF antibody on a total of six male equine reproductive tracts, including a one-day old colt, a one-year-old colt, and four adult stallion tracts. Strong immunostaining was observed in the efferent ducts of the testes and the epithelial cells of the prostate, seminal vesicles, bulbourethral glands, and ampullae. Weaker ß-NGF staining was noted in Leydig cells, Sertoli cells, and spermatogonia within the testes and in epithelial cells of the epididymis. In conclusion, immunohistochemistry revealed that ß-NGF is present in the stallion reproductive tract, and the protein is primarily present in the efferent ducts of the testes and in all accessory sex glands.

Effect of Mitoquinone on sperm quality of cryopreserved stallion semen.

This study aimed to investigate the effect of mitochondria-targeted antioxidants (Mitoquinone, MitoQ) on the quality of frozen-thawed stallion semen. Semen samples collected from three fertile stallions aged 10 - 13 years, were filtered, centrifuged in a skimmed milk-based extender, and diluted to a final concentration of 50 × 106 sperm/mL in freezing medium. Diluted semen was divided into five experimental groups supplemented with MitoQ at concentrations of 0 (control), 25, 50, 100, and 200 nM and then subjected to freezing after cooling and equilibration. After thawing, semen was evaluated for motility and kinetics at different time points. Sperm viability, plasma membrane, acrosome, DNA integrity, mitochondrial membrane potential, apoptosis, and intracellular reactive oxygen species (ROS) concentrations were evaluated. The results revealed that MitoQ at concentrations of 25, 50, and 100 nM improved (P< 0.01) the total sperm motility after 30 minutes of incubation. In addition, 25 nM MitoQ improved the sperm amplitude of lateral head displacement values (P< 0.01) after 30 minutes of incubation. Conversely, negative effects on sperm motility, kinetics, and viability were observed with the highest tested concentration of MitoQ (200 nM). The various concentrations of MitoQ did not affect the plasma membrane, acrosome, and DNA integrity, or the mitochondrial membrane potential and intracellular ROS concentrations. In conclusion, supplementation of MitoQ during cryopreservation, had a mild positive effect on sperm motility and kinetics especially at a concentration of 25 nM, while the highest concentration (200nM) has a detrimental effect on motility and viability parameters of frozen-thawed stallion sperm.

Imputation of single nucleotide polymorphism genotypes in ungenotyped sport horses from the genotypes of their progeny.

Many sport horse studbooks worldwide use microsatellite markers for parentage verification. However, many have expressed a desire to introduce genomic selection using genome-wide dense single nucleotide polymorphism (SNP) genotypes to complement their current breeding programmes. Hence, it does not make sense to genotype the same animal for both microsatellite markers and SNP markers. Transitioning to SNP-based parentage verification is an obvious solution but one barrier to this transition is the lack of SNP data on parents from which to verify parentage against. Therefore, the objective of this study was to assess the ability to impute the SNP genotype of a stallion from the genotypes of its progeny, with or without the consideration of the genotype of the progeny's dam. Genotype information from 55 935 SNPs was available on 13 327 horses. A total of 98 stallions had genotype data on 10 progeny and the genotypes of these stallions were used as a test population. Genome-wide genotype imputation was undertaken by combining a family- and population-based imputation approach. Several different scenarios were assessed to quantify the ability to accurately impute the genotype of a stallion based on genotype data of incrementally more half-sibling progeny. Using genomic information from four progeny the average genotype concordance of the imputed sire genotype compared to the actual sire's genotype was 0.932. The average genotype concordance rate increased to 0.960 when the genotypes of 10 progeny were included in the imputation process. The inclusion of the genotypes of the dams of the progeny improved the concordance rate from 0.932 to 0.977 when based on the genotype of just four progeny and their dams and from 0.960 to 0.996 when based on the genotype of 10 progeny and their dams. These results suggest it is possible to impute the genotype of a non-genotyped horse from the genotypes of its progeny and that the inclusion of the genotypes of the dams of the progeny improves this imputation accuracy further.

Taylorella equigenitalis in Icelandic intact males compared with other horse breeds using natural cover.

BACKGROUND: Contagious equine metritis (CEM) is caused by Taylorella equigenitalis. It is a venereal disease that is detected in some breeds more than others and can cause temporary infertility with substantial costs for regular testing, sanitation and retesting. There was a perceived increase in T. equigenitalis-positive cases in Icelandic intact males where natural cover is common. OBJECTIVES: We aimed to investigate the prevalence of T. equigenitalis in Icelandic intact males and compare to draught horse and Haflinger intact males. We hypothesised that prevalence of T. equigenitalis is higher in Icelandic compared with draught and Haflinger intact males. STUDY DESIGN: Cross sectional. METHODS: Swabs from 76 Icelandic, 35 Haflinger, and 51 draught horse intact males were collected on 38 different farms and analysed by qPCR. Animals were further stratified into active breeding and non-breeding animals and age groups (1.5-7.0 and 8.0-26.0 years). Fisher's exact tests and mixed effect logistic regression with 'farm' as random effect were used to estimate differences in odds for T. equigenitalis-positive test results. RESULTS: The overall prevalence of T. equigenitalis in included intact males was 16.7% (27/162). The odds for T. equigenitalis-positive intact males were significantly higher in Icelandic compared with draught and Haflinger intact males (Odds ratio [OR] = 6.42, 95% confidence interval (CI) = 1.43-28.8, p = 0.02). Odds for T. equigenitalis-positive intact males were significantly lower in active breeding compared with non-breeding animals (OR = 0.09, 95% CI = 0.01-0.54, p = 0.009). Age had no significant influence on test results. MAIN LIMITATIONS: Convenience sampling with regional restrictions to Southern Germany and Austria, small sample size. CONCLUSIONS: Significantly higher odds for T. equigenitalis-positive intact males were found within Icelandic over draught and Haflinger and within non-breeding animals compared with active breeding animals. Findings suggest that non-breeding animals could be a reservoir for T. equigenitalis. Testing for CEM should therefore be routinely performed in Icelandic horses prior to breeding and investigations into epidemiology and reservoirs on affected farms should be initiated.

A scoping review on intraoperative and postoperative surgical castration complications in domesticated equids.

BACKGROUND: Castration is the most common surgical procedure in domesticated equids; surgical techniques used and perioperative management vary considerably. OBJECTIVES: To identify and chart the current evidence on perioperative complications associated with different methods of surgical castration in domesticated equids. STUDY DESIGN: Joanna Briggs Institute systematic scoping review. METHOD: CAB Abstracts, Medline and Embase databases were searched using terms related to equine castration complications. Two authors independently and blindly screened publications against eligibility criteria. Data on study methods, perioperative management, surgical techniques, and perioperative complications were extracted. Surgical techniques were grouped into categories depending on technique; open, closed or half-closed, and whether the parietal tunic was open or closed at the end of surgery. RESULTS: The search identified 1871 publications; 71 studies met the final inclusion criteria. The data reported 76 734 castrations, most of which were open or closed, with the vaginal tunic remaining open at the end of surgery. Twenty-five studies reported information regarding surgical techniques and perioperative management, allowing detailed charting and comparisons, of which analgesia and antimicrobial usage varied notably. Eighteen different complications were reported, with swelling or oedema being the most common. Evisceration was most commonly reported in draught breeds and Standardbreds, and the risk appeared low if the parietal tunic was closed at the end of surgery. MAIN LIMITATIONS: Grey literature and studies not available in English were not included. Existing studies varied greatly in perioperative management, surgical techniques and reporting of outcomes, making evidence consolidation problematic. CONCLUSION: A lack of consensus regarding complication definitions creates uncertainty and discrepancies between complication rates associated with different surgical techniques and perioperative management. The implementation of standardised systems for describing surgical techniques and complications is recommended for future studies. A number of studies did not follow current recommendations for perioperative analgesia and use of antimicrobials.

16s gene metagenomic characterization in healthy stallion semen.

Studies on the bacterial composition of seminal samples have primarily focused on species isolated from semen and their effects on fertility and reproductive health. Culture-independent techniques, such as 16S rRNA gene sequencing and shotgun metagenomics, have revolutionized our ability to identify unculturable bacteria, which comprise >90% of the microbiome. These techniques allow for comprehensive analysis of microbial communities in seminal samples, shedding light on their interactions and roles. In this study, we characterized the taxonomic diversity of seminal microbial communities in healthy stallions using 16S rRNA gene sequencing. Semen samples were collected from four stallions during the reproductive season, and DNA was extracted for sequencing. The results revealed a diverse array of bacterial taxa, with Firmicutes, Bacteroidota, and Proteobacteria being predominant phyla. At the family and genus levels, significant variations were observed among individuals, with individual variability in microbial richness and diversity standing out. Moreover, each stallion showed a distinct microbial fingerprint, indicating the presence of a characteristic microbial core for each stallion. These results underscore the importance of considering individual microbial profiles in understanding reproductive health and fertility outcomes.

Comparison of Nanoparticles and Single-Layer Centrifugation for Separation of Dead from Live Stallion Spermatozoa.

The goal of this study was to compare the efficacy of coated iron-core nanoparticles and single-layer centrifugation for separation of dead from live stallion spermatozoa. Our hypothesis was that nanoparticles would bind to dead sperm and allow for separation from live sperm using a magnet, resulting in a population of spermatozoa with a high percentage of total and progressive motility. Treatment Group 1 was an untreated control. Treatment Group 2 (nanoparticles, NP) utilized sperm incubated with nanoparticles followed by application of a magnet to remove dead sperm adhered to the coated nanoparticles. Treatment Group 3 (single-layer centrifugation, SLC) layered sperm above EquiPure™ followed by centrifugation. Semen samples were subsequently evaluated for sperm motility parameters, plasma membrane integrity, acrosome status, and morphology. The SLC technique yielded higher (p < 0.05) progressive motility (76 ± 9.2%) than the NP separation technique (59 ± 12.2%) or the untreated control (47.3 ± 5.1%). However, the total number of sperm recovered was higher (p < 0.05) in the NP technique (526.2 ± 96.6 × 106) than the SLC procedure (211.7 ± 70 × 106), yielding a higher total number of progressively motile sperm (317.6 ± 109 × 106) recovered using the NP technique than the SLC technique (157.8 ± 43.6 × 106). The percentage of live, acrosome intact sperm recovered was higher for SLC than NP. In summary, the SLC technique yielded a higher percentage of sperm motility, intact plasma membranes, and acrosome integrity, but yielded lower total sperm than with the nanoparticle separation technique.

GnRH Vaccine Could Suppress Serum Testosterone in Stallion Mules.

Stallion mules have been used as working equids in several countries. Aggressiveness under the influence of testosterone results in the necessity for surgical castration before work training. The gonadotropin-releasing hormone (GnRH) vaccine may be an alternative method for immunocastration in mules. The objective of this study was to evaluate the effect of the GnRH vaccine on anti-GnRH antibody concentration, serum testosterone concentration, clinical adverse effects, and behavioral changes in response to receiving selected physical manipulations from humans. Twenty-five mules were separated into three groups: Control-intact, Control-castrated, and Treatment. The Treatment group was further divided according to condition (intact or unilateral cryptorchid) and age. The Treatment group received 195 µg of the GnRH vaccine intramuscularly at weeks 0, 4, and 8. The anti-GnRH antibody concentrations increased at weeks 6 and 10, and then they gradually decreased to baseline at week 24. The Treatment-intact-young group had the highest concentration of anti-GnRH antibody. The serum testosterone concentrations in the Treatment group were lower than before vaccination from weeks 6 to 14. Subcutaneous edema adjacent to the injection site was detected in the Treatment-intact group after booster vaccination. In conclusion, the mules responded to the GnRH vaccine, which could temporarily suppress testosterone for up to 14 weeks.