Journal of Equine Veterinary Science最新文献

{"title":"Association of seminal plasma and alternative cryoprotectants with the antioxidant and the fertilizing capacity of cryopreserved sperm in donkeys","authors":"J.D. Montoya ,&nbsp;A. Usuga ,&nbsp;B. Rojano ,&nbsp;G. Restrepo","doi":"10.1016/j.jevs.2024.105306","DOIUrl":"10.1016/j.jevs.2024.105306","url":null,"abstract":"<div><div>Seminal plasma (SP) in Equidae is involved in several events that precede sperm fertilization, such as motility activation, antimicrobial action, metabolite neutralization, and a mediating effect on sperm capacitation and postcoital uterine inflammatory response. In the donkey species, it has been shown that SP can provide to sperm subjected to cryopreservation, a greater antioxidant capacity, since SP contains various molecules that can protect spermatozoa from oxidative stress. The aim of this study was to evaluate the association between seminal plasma components (proteins and enzymes) and alternative cryoprotectants, with the antioxidant and the fertilizing capacity of cryopreserved sperm of Colombian Creole donkeys. Ten Colombian Creole donkeys (Equus asinus) were collected using the artificial vagina method to obtain three ejaculates per animal, for a total of 30 ejaculates. A fraction of each ejaculate was centrifuged to obtain the SP. Evaluation of some components of seminal plasma was performed. The enzymes catalase, superoxide dismutase and glutathione peroxidase were evaluated by spectrophotometry and the concentration of the cysteine-rich secretory 2 (CRISP-2) protein 2 by the ELISA assay. For semen cryopreservation, treatments with different combinations of dimethylformamide (DMF), sucrose (SAC) and homologous seminal plasma (SP) were used. In thawed semen, total antioxidant capacity was evaluated by the ABTS and ORAC assays; additionally, the reactive oxygen species (ROS) production was evaluated. Fertilizing capacity was evaluated using a sperm binding assay to the zona pellucida of oocytes. The semen quality index (SQi) was calculated for total motility, progressive motility, vitality and plasma membrane integrity. Correlation and regression analysis were performed between the SP components, TAC, ROS and fertilizing capacity. For the comparisons of means, a Tukey Test was carried out. For CRISP-2 a positive correlation coefficient of 0.17 with the seminal quality index (SQi) of thawed semen, was obtained (P&lt; 0.05). The sperm binding to the zona pellucida was 265.34±11.03, 135.2±7.66, 268.37±9.01 and 299.14±10.22 for treatments of 5% DMF; 0.2M SAC and 1% BSA; 5%DMF, 0.2M SAC and 10% SP; and 5% DMF, 0.2M SAC and 10% SP, respectively. The last treatment being the one with a higher value (P&lt; 0.05). For the combination 5% DMF and 10% SP; 0.2 SAC,1%BSA and 10% SP, it could be found that the antioxidant capacity measured by the (ABTS) method had a negative regression coefficient with total ROS (P&lt;0.05). In conclusion, SP components, TAC and ROS are associated with semen quality and the fertilizing capacity of donkey sperm. Additionally, freezing of semen under a medium composed of dimethylformamide (DMF), bovine serum albumin (BSA) and seminal plasma (PS), improves the adherence of sperm to the zona pellucida, being a favorable indicator of the fertilizing capacity of donkey sperm.</div></div>","PeriodicalId":15798,"journal":{"name":"Journal of Equine Veterinary Science","volume":"145 ","pages":"Article 105306"},"PeriodicalIF":1.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143386974","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ice application without water drainage supports optimal hoof cooling in adult horses","authors":"K.M. Folk,&nbsp;R.R. White,&nbsp;C.B. Gleason","doi":"10.1016/j.jevs.2024.105255","DOIUrl":"10.1016/j.jevs.2024.105255","url":null,"abstract":"<div><div>Cryotherapy is often used to reduce inflammation in acute equine laminitis cases. Certain hoof temperatures have been suggested as effective in minimizing the inflammatory process; however, there is limited evidence on which methods are best at achieving these temperatures. Our objective was to determine how different methods of cryotherapy influence the rate and extent of cooling for the equine hoof wall. Four horses received three hoof cooling treatments and a control (CON; no treatment application) in a 4 × 4 Latin square design. Treatments included (1) ice surrounding the hoof in a 5 L fluid bag with water drainage holes (DB), (2) ice surrounding the hoof in an undrained bag (UB), and (3) ice in a commercial wader boot (CW). Hoof wall temperatures were collected via thermal imaging for 12 h. Thermally imaged body temperatures and thermometer-based rectal temperatures, heat index, relative humidity, and ambient temperature were recorded. A treatment × time interaction (<em>P</em> &lt; 0.001) was observed for all hoof temperatures. All treatments differed from CON after 2 h post-application, with the UB treatment resulting in the greatest and most sustained reduction in hoof temperatures over the 12 h period (a change of −23.7 °C ± 1.6). The wader boot showed similar trends to the UB treatment but was poorly tolerated by the horses. Environmental effects differed between hoof and body surface. Our findings indicate that cryotherapy treatments that maintain an ice-water slurry around the hoof result in greater decreases in hoof temperatures 2 h post-treatment compared to drained ice application.</div></div>","PeriodicalId":15798,"journal":{"name":"Journal of Equine Veterinary Science","volume":"145 ","pages":"Article 105255"},"PeriodicalIF":1.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142791747","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ProAKAP4 correlation with flow cytometry parameters in semen of Asturcon ponies","authors":"M. Dordas Perpinyà ,&nbsp;I. Yanez-Ortiz ,&nbsp;N. Sergeant ,&nbsp;J. Catalan ,&nbsp;C. Tamargo ,&nbsp;C.O. Hidalgo ,&nbsp;A. Fernandez ,&nbsp;J. Miro ,&nbsp;L. Briand-Amirat Lamia","doi":"10.1016/j.jevs.2024.105279","DOIUrl":"10.1016/j.jevs.2024.105279","url":null,"abstract":"<div><div>ProAKAP4, AKAP4 precursor, is a specific protein from spermatozoa flagella, essential for maintaining motility efficiency. In all mammals studied proAKAP4 levels have been shown to correlate with total and progressive motility. In stallions and donkeys, proAKAP4 serves as an indicator of sperm quality and sustained motility over the time once thawed. This study aimed to compare proAKAP4 concentration with cytometry parameters: acrosome integrity, ROS production, mitochondrial potential and lipidic disorder. Animal cohort consisted of eight Asturcon ponies. Three ejaculates of each pony (24 ejaculates) were collected and frozen by commercial protocol. Post-thawed semen was analyzed by CASA and flow cytometry (PNA, JC1, HE, Fluo3 and M540 stains) and the proAKAP4 amount was quantified by ELISA commercial kit, Horse 4MID^Ⓡ Kit. Results show a positive correlation between proAKAP4 concentration and flow cytometry dyes: PNA stain (r= 0.34, p=0.0178), indicating membrane integrity; a negative correlation with membrane lipid disorder, M540 stain (r= -0.327, p=0.0234); a positive correlation with ROS production, H2 stain intensity (r= 0.48, p=0.005); and a negative correlation with mitochondrial potential measured by JC-1 (r=-0.41, p=0.0042). These results suggest that a spermatozoon with an intact membrane possesses more proAKAP4. Additionally, a spermatozoon with a high proAKAP4 concentration and an intact membrane also implies higher motility, resulting in increased ROS production. Conversely, a spermatozoon exhibiting significant lipid disorder shows a lower proAKAP4 concentration, indicating lower quality and reduced likelihood of reaching the fertilization site. As observed in previous publications, mitochondrial activity is not associated with proAKAP4 concentration, suggesting that the energy expenditure of the spermatozoon is not linked to the quality of movement defined by proAKAP4 concentration. A quality threshold of 42.10 ng of proAKAP4/10M of spermatozoa was calculated using a cluster between viability and motility parameters. This concentration is consistent with the 37.77 ng/10M of spermatozoa identified in a previous study, it may serve as a criterion for determining the sperm quality in post-thaw conditions. A relationship with cytometry parameters is observed showing that proAKAP4 is positively correlated with intact acrosome membrane and production of ROS which it suggests that an intact and rapid spermatozoon will show more proAKAP4 concentration. On the contrary, proAKAP4 is negatively correlate with a lipid disorder of the membrane which it means that a less quality spermatozoa will show less proAKAP4 concentration. Even if correlations are not high, their significance is strong, further analysis with a bigger cohort are needed to confirm these results.</div></div>","PeriodicalId":15798,"journal":{"name":"Journal of Equine Veterinary Science","volume":"145 ","pages":"Article 105279"},"PeriodicalIF":1.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143386518","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Does the equilibration time prior to freezing affect post-thaw motion characteristics of stallion spermatozoa?","authors":"H. Farnia,&nbsp;P. Sautejeau-Rizzoni,&nbsp;J.-M. Galharret,&nbsp;M. Le-Guiriec,&nbsp;D. Bencharif","doi":"10.1016/j.jevs.2024.105280","DOIUrl":"10.1016/j.jevs.2024.105280","url":null,"abstract":"<div><div>In stallions, around 20% of ejaculates suffer damage during the freezing process, leading to significant declines in sperm quality. Consequently, numerous studies are dedicated to refining techniques to enhance fertility rates using frozen semen. One factor to consider is the equilibration time, whereby sperm cells are in contact with cooling/ freezing media components at a temperature of 4°C, providing a proper osmotic balance between the intra- and extra-cellular milieu. Our research aimed to assess the impact of preserving semen at 4°C for varying periods (2, 4, or 6 hours) following dilution before freezing, and its subsequent effect on sperm motility after thawing. To achieve this, we collected 16 ejaculates from five stallions. The spermatozoa were separated from the seminal plasma via centrifugation in INRA^Ⓡ 96 medium. After removal of the supernatant, the pellet was mixed with INRA FREEZE^Ⓡ extender at a concentration of 100 million sperm per milliliter. Subsequently, the semen was frozen in 0.5 ml straws after being kept at 4°C (Equilibration) for either 2, 4, or 6 hours. Post-thaw motility and progressive motility were assessed using computer-assisted sperm analysis (CASA). Our results indicate that an equilibration period of 6 hours led to higher percentages of motile spermatozoa after thawing (average of 46.3% P ≤ 0.05), compared to equilibration periods of 4 hours (average of 40%) and 2 hours (average of 33%). Additionally, samples frozen 2 hours after cooling exhibited lower percentages of progressively motile spermatozoa (average of 15.1% P≤ 0.05) compared to samples frozen 4 hours (average 18.8%) and 6 hours (average 20.6 %) after cooling, which showed no significant difference. Based on our initial findings, we suggest that semen should be maintained at 4°C for 6 hours before being frozen to yield higher sperm motility.</div></div>","PeriodicalId":15798,"journal":{"name":"Journal of Equine Veterinary Science","volume":"145 ","pages":"Article 105280"},"PeriodicalIF":1.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143386519","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative analysis of equine semen preservation techniques and DNA damage detection using Comet and SCSA assays","authors":"M. Yaseen,&nbsp;A. Swegen,&nbsp;Z. Gibb","doi":"10.1016/j.jevs.2024.105335","DOIUrl":"10.1016/j.jevs.2024.105335","url":null,"abstract":"&lt;div&gt;&lt;div&gt;Maintaining the DNA integrity of equine spermatozoa during preservation is crucial for the success of assisted reproductive techniques. Sperm preservation generally relies on low temperatures to reduce or halt sperm metabolism to preserve functionality, but more recently methods of storing sperm above 15°C have been developed to reduce the effects of cold shock and cryodamage. The major damage that spermatozoa experience during preservation at any temperature is oxidative damage, which may lead to DNA damage. There are numerous DNA damage assays available, but their relative prognostic value in horses has yet to be evaluated. Therefore, the objective of the present study was to compare the sensitivity of two popular sperm DNA damage assays, the sperm chromatin structure assay (SCSA) and the single cell gel electrophoresis (neutral comet) assay in identifying sperm DNA damage in the horse. A total of 3 split ejaculates (diluted 2:1 with EquiPlus) from individual stallions were utilised. High-quality spermatozoa were isolated via single-layer colloidal centrifugation (EquiPure). Sperm pellets were resuspended in: L-EDTA cryodiluent with 20% egg yolk and 3% dimethylformamide; EquiPlus; and SpermSafe, after which they were either cryopreserved, chilled (5°C), or stored at 18°C respectively. The SCSA (DNA fragmentation index; DFI) and comet (tail intensity) analyses were performed at various relevant timepoints. While the SCSA revealed no significant increase in DNA damage at any timepoint, for any storage treatment, the comet assay was more sensitive, revealing a significant increase in DNA damage after 72h of storage in SpermSafe (0h: 21.1±11.4% vs 72h: 53.5±0.2%; p≤0.05), though this fell to non-significant levels by the 7-day timepoint (43.675±3.8%), and after cryopreservation (pre-freeze: 21.1±11.4% vs post-thaw: 67.2±3.5%; p≤0.05). There was no significant increase in DNA damage at any timepoint for the chilled, EquiPlus-stored samples. This study shows that the comet assay is more sensitive than the SCSA in detecting sperm DNA damage following various preservation methods. Notably, sperm stored in SpermSafe at 18°C exhibited unexpectedly high tail intensity, which contrasts with high pregnancy rates in commercial settings. We hypothesise that this may result from an artefact associated with capacitation-like changes and chromatin decondensation, due to the SpermSafe formulation mimicking oviduct fluid. Ongoing studies aim to investigate this further. Post-thaw, tail intensity (comet) more than tripled following cryopreservation, which could also be linked to capacitation-like changes or represent actual DNA damage that contributes to lower pregnancy rates seen with frozen-thawed sperm. A key limitation to this study is the small sample size, but additional replicates are being included in ongoing research. Despite this, our findings offer insights into sperm DNA integrity during preservation and suggest that the comet assay and SCSA m","PeriodicalId":15798,"journal":{"name":"Journal of Equine Veterinary Science","volume":"145 ","pages":"Article 105335"},"PeriodicalIF":1.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143386620","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessment of sperm longevity after diluting in different extenders","authors":"B. Vincze ,&nbsp;I. Arki ,&nbsp;S. Cseh","doi":"10.1016/j.jevs.2024.105334","DOIUrl":"10.1016/j.jevs.2024.105334","url":null,"abstract":"&lt;div&gt;&lt;div&gt;Differences in semen viability are still in the focus of clinicians working in artificial insemination (AI) centres. Clinical experience indicates that the quality of the produced fresh cooled and frozen semen is depending on individual stallion properties as well as the sensitivity to different extenders.&lt;/div&gt;&lt;div&gt;With the use of hormonal treatments (e.g. ovulation induction), pregnancy rates can be increased but the role of semen extenders is still a key factor in achieving good or excellent quality semen. Extenders provide the optimal environment for sperm cells during their journey and companies try to develop newer recipes; but over the last decades the base components of these liquids remained the same. When producing AI doses, the question arises: which extender will yield the best results for a particular sire with a specific sperm quality. In this study three different stallions with different sperm quality (excellent, good and poor) have been tested for viability with commercially available semen extenders offered for treating even lower quality semen. All stallions were kept at the same equine AI centre located in central Hungary under the same conditions. The animals have been clinically healthy and all of them were used as AI sires with good and acceptable pregnancy rates (65-100%). Fresh semen was collected, filtered and assessed at first for raw semen volume, total (TM%) and progressive motility (PM%), and concentration. Then the semen was diluted with 14 different commercially available extenders (INRA96, minitube EquiPlus, minitube Beyond, Hippex Prime, Hippex Plus, Hippex Equine Semen Extender, Spervital OVD white cap, EVD Plus red cap, EVD blue-old cap, EVD blue-new cap, EVD Special green cap, Nidacon BotuSemen, BotuSemen Gold, BotuTurbo, BotuSemen Special) according to the manufacturer's instructions. Diluted semen doses were cooled and stored at 4°Celsius and checked at 24 hours intervals for total and progressive motility at 12, 36, 60, 84, 108, 132 and 156 hours post collection. All data were inserted into Microsoft Excel and analysed for different parameters (sperm quality, stallion and extender as variables) relative to time (T) post collection. Significant differences were observed in TM% and PM% between stallions’ samples when applying different extenders. Best TM% and PM% could be seen at 12 hrs (37±9% mean±SD) and 36 hrs (23±8% mean±SD) motility assessment. Thereafter, the results slowly decreased until 60th hrs. Stallions with excellent or good initial motility had sperm with acceptable total and progressive motility for a longer time; four extenders-maintained sperm cells viable for more than 132 hours. Stallion-3 (low quality semen) produced poor results at the start, but interestingly, at 60 hrs, post-collection, both PM% and TM% increased for the next 24 hrs which may have clinical relevance. In conclusion, testing the extender before producing AI doses from a particular stallion is crucial for achievi","PeriodicalId":15798,"journal":{"name":"Journal of Equine Veterinary Science","volume":"145 ","pages":"Article 105334"},"PeriodicalIF":1.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143386708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Testicular volume measurements obtained with ultrasound images and its association with total sperm count in ejaculates from stallions","authors":"M. Leão Freitas ,&nbsp;M.A. De Oliveira Viu ,&nbsp;R. Arruda de Oliveira","doi":"10.1016/j.jevs.2024.105298","DOIUrl":"10.1016/j.jevs.2024.105298","url":null,"abstract":"<div><div>Testicular volume is usually related to sperm production capacity. A non-invasive method for assessing testicular measurements has been using ultrasound images, which allows a detailed analysis of the testicular parenchyma and isolate it from the surrounding structures. This study aimed to evaluate the most effective method for measuring testicular volume using ultrasound images and compare it to the total number of sperm per ejaculation, over a year. This was done by either measuring the width of each testicle to get the total testicular width (TTW; cm), or by determining the height, width, and length of both testes to calculate total testicular volume (TTV; cm³). Eight light-breed stallions were followed-up for one-year, testicular measurements using ultrasound were carried out every 15 days, prior to semen collection from each stallion. Semen, without the gel fraction, was evaluated immediately after collection to estimate the total sperm count in the ejaculate (x10⁹). The seasons of the year (summer, autumn, winter and spring) were considered to track the values throughout the year; differences between the means were compared using Tukey's test. Associations between variables were computed using the Pearson correlation method. Results are presented as mean ± standard error of the mean (SEM), and statistical significance was established at P&lt;0.05. Testicular volume varied throughout the year for both TTW and TTV (respectively presented; P&lt;0.0.05), with higher values observed in summer (9.39 ± 0.11; 179.30 ± 4.08), and spring (9.15 ± 0.07; 172.30 ± 2.38), lower values in autumn (8.92 ± 0.09; 166.18 ± 3.83), and winter (8.48 ± 0.09; 156.57 ± 2.79). A moderated correlation was observed between TTW and TTV (R=0.51; p&lt;0.05). For total sperm count in the ejaculate, both summer and spring showed higher values (6.03 ± 0.36 and 6.51 ± 0.34, respectively) compared to autumn and winter (5.00 ± 0.04 and 4.86 ± 0.36, respectively). A moderated correlation was observed between total sperm count in the ejaculate and either TTW (R = 0.6; P&gt;0.05) or TTV (R = 0.54; P&gt;0.05). Testicular dimensions are typically measured using calipers to determine total scrotal width or via ultrasound to calculate TTV. Ultrasound has the advantage of assessing only the testicular parenchyma, whereas caliper measurements also include surrounding structures. However, positioning the ultrasound probe to accurately measure testicular length requires more technical skill due to the positioning of the stallion's testicles within the scrotum. Therefore, obtaining TTW through ultrasound is a valuable tool for assessing testicular volume in stallions.</div></div>","PeriodicalId":15798,"journal":{"name":"Journal of Equine Veterinary Science","volume":"145 ","pages":"Article 105298"},"PeriodicalIF":1.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143386762","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Whole genome sequence-based analysis of Thoroughbred stallions with impaired acrosomal exocytosis and subfertility","authors":"T. Raudsepp,&nbsp;S. Stroupe,&nbsp;C. Hernández-Avilés,&nbsp;R. Juras,&nbsp;H.J. Kjöllerström,&nbsp;H. Anderson,&nbsp;B.W. Davis","doi":"10.1016/j.jevs.2024.105317","DOIUrl":"10.1016/j.jevs.2024.105317","url":null,"abstract":"<div><div>An idiopathic form of subfertility in Thoroughbred stallions with normal physical and semen parameters has been attributed to impaired acrosomal exocytosis (IAE) (see Hernández-Avilés et al 2023 PMID: <span><span>36413868</span><svg><path></path></svg></span>). This impairment is significantly associated with a double-homozygous A/A-A/A genotype in FKBP6 exon5 on horse chromosome 13 (Raudsepp et al 2012 PMCID: PMC3527208; Castaneda et al 2021 PMID: <span><span>34610162</span><svg><path></path></svg></span>). While this genotype is present in 4% of global horse population, it is associated with subfertility only in Thoroughbreds, suggesting FKBP6 tags Thoroughbred case-specific haplotypes containing causative variants. To find these variants, we designed a TaqMan genotyping assay for FKBP6 exon5 and identified 26 subfertile Thoroughbred stallions with the A/A-A/A susceptibility genotype. Whole genome sequencing short-read data (Illumina) was generated for 25 stallions and long-read data (PacBio Hi-Fi) for four stallions. Sequences of all Thoroughbred cases were aligned to EquCab3 reference and the recently available Telomere-to-Telomere (T2T) Thoroughbred genome in combination with over 200 other horse genomes. Comparison of sequence variant landscape between Thoroughbred cases and non-case horses, identified a 1,447,268 bp haplotype region around the FKBP6 gene that was specific to Thoroughbred cases. The region contains 372 unique or rare single nucleotide variants and several deletions of interest. For example, a 66 bp deletion in the BAZ1B gene was in complete linkage disequilibrium with the FKBP6 exon5 A/A-A/A genotype in case Thoroughbreds only. Functional relevance of this and other variants is under investigation. In addition, we are conducting sequence-based whole genome association study to identify additional genomic regions that could be associated with the subfertility phenotype in Thoroughbred cases. While molecular studies on Thoroughbred stallion subfertility due to impaired acrosome exocytosis are still ongoing, the generated short-read and long-read whole genome sequence data contribute to the Equine Genome Variant Database and the Equine Pangenome Project, respectively.</div></div>","PeriodicalId":15798,"journal":{"name":"Journal of Equine Veterinary Science","volume":"145 ","pages":"Article 105317"},"PeriodicalIF":1.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143386763","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Anti Mullerian Hormone as a potential biomarker of stallions fertility","authors":"O.Z. Sajecka,&nbsp;J. Morrell","doi":"10.1016/j.jevs.2024.105321","DOIUrl":"10.1016/j.jevs.2024.105321","url":null,"abstract":"&lt;div&gt;&lt;div&gt;Anti-Müllerian hormone (AMH) is an inhibiting hormone that is crucial for sexual differentiation during foetal development. It continues to influence reproductive health into adulthood. While its role in mares has been extensively studied, its function in stallion fertility is less well understood. Secreted by Sertoli cells, AMH concentrations are notably high before puberty, then decrease during adolescence as rising testosterone levels actively regulate its expression. The AMH concentration is valuable for diagnosing cryptorchidism and has been used to evaluate testicular activity in behavioural studies, although its direct relationship to fertility remains underexplored. Recent findings have shown a correlation between low serum AMH levels and poor semen quality in infertile men and dogs, suggesting its potential as a biomarker for reproductive health in stallions. Thus, the objective of this study was to evaluate whether AMH concentrations can be used as an indicator of potential fertility in stallions. Jugular blood samples were collected from 10 breeding stallions and 1 stallion with known testicular degeneration; breeding results from the same season were recorded. Based on anamnesis, the stallions were categorized into two groups: SG0 (good breeders) and SG1 (poor breeders) according to empirical observations of responsible veterinarians throughout the stallion's breeding career. The AMH concentrations were measured using an ELISA (Beckman-Coulter Gen II, California), followed by statistical analyses (t-test and linear regression) to evaluate the relationship between AMH concentrations and breeding outcomes. The AMH concentrations ranged from 7.71 ng/ml to 35.91 ng/ml for all stallions (median 14.60 ng/ml), 9.31-35.91 ng/ml for SG0 (median 21.91 ng/ml) and 7.71-14.60 ng/ml for SG1 group (median 13.27 ng/ml). The significant differences in AMH concentrations between the two groups, with the SG0 group having higher AMH concentration than the SG1 group (t-test: t = 3.53, p = 0.008). The stallion with testicular degeneration had the lowest AMH concentration in the sample (7.71 ng/ml). A linear regression model demonstrated a positive relationship between AMH levels and pregnancy success rates: a 1 ng/ml increase in AMH concentration was associated with a 2 percentage point increase in success rate (p = 0.033). These results suggest that AMH concentration could serve as a predictor for stallion fertility. However, the study has some limitations, including the small sample size and the need to include additional variables in a multiple regression model. The current model accounts for 41.41% of the variance in fertility outcomes. In conclusion, this study presents promising preliminary evidence supporting the use of AMH concentration as a biomarker for stallion fertility. It serves as a valuable pilot study for future research with larger samples and more comprehensive models that include other factors influencing fertility.&lt;/div&gt;&lt;div&gt;","PeriodicalId":15798,"journal":{"name":"Journal of Equine Veterinary Science","volume":"145 ","pages":"Article 105321"},"PeriodicalIF":1.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143386770","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Donkey sperm activating potential in heterologous ICSI","authors":"J. Otero ,&nbsp;D. Neild ,&nbsp;S.M. Gaviria ,&nbsp;A. Camporino ,&nbsp;S. Morado ,&nbsp;C.B. Castex ,&nbsp;P. Cetica ,&nbsp;C. Arraztoa","doi":"10.1016/j.jevs.2024.105311","DOIUrl":"10.1016/j.jevs.2024.105311","url":null,"abstract":"<div><div>There is a growing interest in the preservation and production of donkeys and mules, both species of significant importance in Argentina. The aim of this study was to evaluate the ability of cryopreserved donkey sperm to activate and induce pronuclear formation when injected into heterologous oocytes, as a potential predictor of future seminal capacity to produce embryos in vitro. Porcine cumulus-oocyte complexes (COCs) were obtained from ovaries of slaughtered gilts, selected, and then incubated, for 44 hours at 39°C, with 5% CO2 and 100% humidity, in 199 medium supplemented with 50 μg/ml gentamicin sulfate, 10% (v/v) porcine follicular fluid, 0.57 mM cysteine, 2 IU/ml equine chorionic gonadotropin, and 10 ng/ml epidermal growth factor. The matured COCs were denuded using serial passages through glass Pasteur pipettes. Donkey and equine sperm, cryopreserved with an egg yolk-free extender using a slow-freezing curve in an automatic freezer, were thawed in a water bath at 37°C for 1 minute and selected using Androcoll-ETM. A small aliquot was placed in a drop of 10% (v/v) polyvinylpyrrolidone in modified Whitten's (MW) injection medium. The oocytes were each placed in a drop of MW and intracytoplasmic sperm injection (ICSI) was performed. Porcine oocytes were injected with either equine sperm (control group) or donkey sperm (experimental group). As a control for the ICSI technique, and to assess parthenogenetic activation, mature oocytes were injected without depositing any sperm into the cytoplasm (Sham). Presumed zygotes were cultured for 16-18 hours in NCSU-23 medium at 39°C, with 5% CO2 and 100% humidity. They were then fixed for 15 minutes in 0.5% (v/v) glutaraldehyde in phosphate-buffered solution and stained for 10 minutes with Hoechst 33342 (5 µl/ml). Finally, they were classified as: 1) Activated: two pronuclei (2-PN) or one pronucleus with the presence of a decondensed sperm and 2) Non-activated: one pronucleus with the presence of a condensed sperm or no evidence of pronuclei. No significant difference was observed (p= 0.73) between activated porcine oocytes injected with donkey sperm: 66.67% (52/78) and those injected with equine sperm: 62.86% (44/70). Parthenogenetic development in the Sham group was 4.6% (3/65). Therefore, similar to equines, heterologous ICSI using donkey sperm could predict the future capacity of donkey semen for in vitro embryo production.</div></div>","PeriodicalId":15798,"journal":{"name":"Journal of Equine Veterinary Science","volume":"145 ","pages":"Article 105311"},"PeriodicalIF":1.3,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143386864","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}