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Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro , the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse. • Different treatments for equine sperm capacitation were evaluated in two media. • Combination of capacitating inducers boosts phosphorylation and acrosomal exocytosis. • Capacitating conditions improve sperm's zona pellucida binding capacity. [ABSTRACT FROM AUTHOR]

Simple Summary: Freezing stallion semen yields variable results, heavily dependent on individual differences, categorizing stallions as good or poor freezers. This variability was investigated by analyzing sperm kinetics, mitochondrial membrane potential, and hydrogen peroxide content in sperm samples treated with different extenders and procedures. Additionally, antioxidant activities based on ABTS and FRAP assays, along with nitric oxide stable metabolites (NOx), were assessed in seminal plasma, blank extenders, and extenders conditioned by spermatozoa either before or after freezing. Strong individual variability was observed in most sperm functions both before and after freezing. Several physical and chemical differences were noted between the shipping and freezing extenders. However, no significant differences in sperm function, antioxidant activities, and NOx values were found among the shipping extenders. In contrast, significant differences in antioxidant activities and NOx values were found between the freezing extenders. The significant interaction between stallion and extender suggests that customizing freezing procedures can improve sperm freezing outcomes in stallions. Sensitivity to freezing remains a critical issue in stallion semen cryopreservation procedures. To explore this topic in-depth, semen was collected from ten stallions, diluted with three different extenders, transported to the laboratory, and then centrifuged and frozen with four different extenders. We conducted analyses of sperm kinetics, mitochondrial membrane potential (MMP), and hydrogen peroxide content both before and after freezing. Additionally, we assessed antioxidant activity using the ABTS and FRAP methods and measured nitric oxide stable metabolites (NOx) in the blank extenders, seminal plasma, and extenders conditioned by spermatozoa before and after freezing. We found significant variability in the antioxidant activity and NOx content of the blank extenders and the seminal plasma. In the seminal plasma, ABTS-based antioxidant activity and NOx values were correlated with some sperm kinematic parameters and MMP in refrigerated semen, while no correlation was observed in frozen sperm parameters. Sperm function varied significantly between stallions but not between extenders, either before or after freezing. However, significant differences in antioxidant activities and NOx values were found among extenders conditioned following freezing. These results provide new insights into the factors contributing to the variability in individual stallions' tolerance to sperm freezing. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs) are essential for the maintenance of male fertility and survival of species. Environmental conditions, notably heat stress, have been identified as important causes of male infertility and have a negative impact on SSCs. Animals with cryptorchid testes (CT) are optimal models for the study of long-term heat stress-related changes in germ cells. The effect of heat stress on germ cells differs depending on the spermatogenesis stage. Thus, verifying whether the specific phase of spermatogenesis is dependent or independent of heat stress in stallions is important. We evaluated the heat stress-related response of SSCs by comparing the relative abundance of mRNA transcripts and expression patterns of the undifferentiated embryonic cell transcription factor 1 (UTF-1) and deleted in azoospermia-like (DAZL) in the seminiferous tubules of CT and normal testes (NT) of stallions using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blotting. We also analyzed the relative abundance of mRNA of different proliferative markers, including minichromosome maintenance 2 (MCM2), marker of proliferation Ki-67 (MKI-67), and proliferating cell nuclear antigen (PCNA). Testicular tissues from four Thoroughbred unilateral cryptorchid postpubertal stallions were used in this study during the breeding season. The relative abundance of the mRNA transcripts of UTF-1 and MCM2 was significantly upregulated in the CT group than that of those in the NT group. In contrast, the relative abundance of the mRNA transcripts of DAZL was significantly downregulated in the CT group than that of those in the NT group. Western blot quantification showed that the relative intensity of UTF-1 protein bands was significantly higher, while that of DAZL protein bands was significantly lower in the CT group than in the NT group. Immunofluorescence studies showed that the number of germ cells immunostained with UTF-1 was significantly higher while immunostained with DAZL was significantly lower in the CT group than that in the NT group. The higher expression level of UTF-1 in the CT group shows that undifferentiated SSCs are not affected by long-term exposure to heat stress. These results also indicate that germ cells after differentiation phase are directly affected by heat-stress conditions, such as cryptorchidism, in stallions. • UTF-1 was found significantly higher and DAZL significantly lower in cryptorchid testes. • The undifferentiated SSCs are not affected by long-term exposure to heat stress. • The germ cells after differentiation phase are directly affected by heat-stress conditions. • Cryptorchid testes are optimal models for the study of long-term heat stress-related changes in germ cells. [ABSTRACT FROM AUTHOR]

Summary: This case report describes the diagnosis and surgical treatment of a 12‐year‐old Friesian stallion that presented with colic and was diagnosed with an unilateral abdominal seminoma. The diagnostic work‐up included haematology, blood chemistry, rectal examination and ultrasonography before the tentative diagnosis of a neoplastic intra‐abdominal testicle was made. The mass was removed surgically and diagnosed as a seminoma via histopathology. Regular follow‐up was performed since suspected metastasis was seen on transrectal ultrasonography and laparoscopy. Unfortunately, the patient was subjected to euthanasia 18 months post‐surgery due to clinical deterioration. Post‐mortem examination was performed and confirmed the cause of clinical deterioration was due to metastasis of the previously removed seminoma. Colic is, to the author's knowledge, rarely seen in stallions with an abdominal testicular neoplasm, in contrast to humans and dogs. [ABSTRACT FROM AUTHOR]

Although the experimental found footage film recycles Hollywood films so that the outcome may radically differ from the original story, there are no accounts on how it adapts images from mainstream cinema to represent human–animal relations, linking to gender and masculinity. To fill this gap, I discuss how experimental film Horsey recycles footage from three Hollywood productions— Reflections in a Golden Eye , The Swimmer , and The Black Stallion —to construct a new narrative, which displays a very close conformity to the prior text, shifting the focus to human–horse interactions. Raising questions about the traditional understanding, scope, and limits of adaptation in avant-garde film studies, Horsey fits in with the broader tradition of cinematic recycling of mainstream cinema as it exemplifies intertextuality as a direct form of quotation, taking quotation as appropriation through cuts, detournement, compilation and free association. Particularly, following Guy Barefoot's understanding of adaptation as an intertextual form of recycling, Horsey is distinctive in its sole use of found footage from the three Hollywood films as it fully acknowledges the recycled material, strongly alluding to the original stories, and simultaneously re-processes them through a collage of pre-used footage, slow motion, washed-out colours, and an altered soundtrack. Despite appearing to merely extract images and sounds from Reflections , The Swimmer , and The Black Stallion , Horsey emerges as a productive site for recycling Hollywood cinema, placing it into new contexts and audio-visual configurations and offering more complex, engaging ways of looking at how humans connect to horses. [ABSTRACT FROM AUTHOR]

Virological evaluation was performed on equine semen to detect the presence of papillomaviruses (PVs) using droplet digital polymerase chain reaction (ddPCR) as the aim of this study was to investigate whether the sperm from asymptomatic stallions harbors ovine papillomaviruses (OaPVs). Twenty-seven semen samples were analyzed, 18 of which were commercially acquired. The remaining nine samples comprising semen and peripheral blood, were collected from nine stallions with no apparent signs of PV-related diseases during clinical examination at the Didactic Veterinary University Hospital (DVUH) of Naples. OaPV was detected in 26 semen samples. OaPV1 was the most prevalent virus infecting equine semen. OaPV1 infected 21 semen samples (~80.8%) and showed a high number of DNA and RNA copies per microliter. qPCR was used to detect OaPV1 DNA in the 18 semen samples. ddPCR was used to detect and quantify the expression of OaPV2, OaPV3, and OaPV4. qPCR failed to detect DNA for these genotypes. Additionally, ddPCR was used to detect the transcriptionally active OaPV1 in six blood and semen samples from the same stallion. ddPCR failed to detect any nucleic acids in OaPVs in peripheral blood samples from the three stallions. In one semen sample, ddPCR detected OaPV1 DNA but failed to detect any nucleic acid in the remaining two semen samples, and peripheral blood from the same animals of the remaining 18 semen samples was not available, OaPV1 and OaPV4 were responsible for nine and five single infections, respectively. No single infections with either OaPV3 or OaPV4 were seen. [ABSTRACT FROM AUTHOR]

Background: The Franches-Montagnes (FM) is the last native horse breed of Switzerland, established at the end of the 19th century by cross-breeding local mares with Anglo-Norman stallions. We collected high-density SNP genotype data (Axiom™ 670 K Equine genotyping array) from 522 FM horses, including 44 old-type horses (OF), 514 European Warmblood horses (WB) from Sweden and Switzerland (including a stallion used for cross-breeding in 1990), 136 purebred Arabians (AR), 32 Shagya Arabians (SA), and 64 Thoroughbred (TB) horses, as introgressed WB stallions showed TB origin in their pedigrees. The aim of the study was to ascertain fine-scale population structures of the FM breed, including estimation of individual admixture levels and genomic inbreeding (FROH) by means of Runs of Homozygosity. Results: To assess fine-scale population structures within the FM breed, we applied a three-step approach, which combined admixture, genetic contribution, and FROH of individuals into a high-resolution network visualization. Based on this approach, we were able to demonstrate that population substructures, as detected by model-based clustering, can be either associated with a different genetic origin or with the progeny of most influential sires. Within the FM breed, admixed horses explained most of the genetic variance of the current breeding population, while OF horses only accounted for a small proportion of the variance. Furthermore, we illustrated that FM horses showed high TB admixture levels and we identified inconsistencies in the origin of FM horses descending from the Arabian stallion Doktryner. With the exception of WB, FM horses were less inbred compared to the other breeds. However, the relatively few but long ROH segments suggested diversity loss in both FM subpopulations. Genes located in FM- and OF-specific ROH islands had known functions involved in conformation and behaviour, two traits that are highly valued by breeders. Conclusions: The FM remains the last native Swiss breed, clearly distinguishable from other historically introgressed breeds, but it suffered bottlenecks due to intensive selection of stallions, restrictive mating choices based on arbitrary definitions of pure breeding, and selection of rare coat colours. To preserve the genetic diversity of FM horses, future conservation managements strategies should involve a well-balanced selection of stallions (e.g., by integrating OF stallions in the FM breeding population) and avoid selection for rare coat colours. [ABSTRACT FROM AUTHOR]

Background: Most commerce of equine seminal doses is carried out using commercial extenders under refrigeration at 5°C. Objectives: To determine if 10 mm pyruvate in a 67 mm glucose extender and storage at 22°C could be the basis of an alternative storage method to cooling to 5°C. Material and methods: Stallion ejaculates were extendedin: INRA96 (67 mm glucose, non‐pyruvate control), modified Tyrode's (67 mm glucose–10 mm pyruvate), supplemented with 0, 10, 50, and 100 μM itaconate. As itaconate was vehiculated in DMSO, a control vehicle was also included. Sperm motility, viability, mitochondrial membrane potential, and production of reactive oxygen species were measured after collection and again after 48 and 96 h of storage at 22°C. To disclose molecular metabolic changes, spermatozoa were incubated up to 3 h in modified Tyrode's 67 mm glucose–10 mm pyruvate and modified Tyrode's 67 mm glucose, and metabolic analysis conducted. Results: After 96 h of storage aliquots stored in the control, INRA96 had a very poor total motility of 5.6% ± 2.3%, while in the 67 mm glucose–10 mm pyruvate/10 μm itaconate extender, total motility was 34.7% ± 3.8% (p = 0.0066). After 96 h, viability was better in most pyruvate‐based media, and the mitochondrial membrane potential in spermatozoa extended in INRA96 was relatively lower (p < 0.0001). Metabolomics revealed that in the spermatozoa incubated in the high pyruvate media, there was an increase in the relative amounts of NAD+, pyruvate, lactate, and ATP. Discussion and conclusions: Aliquots stored in a 67 mm glucose–10 mm pyruvate‐based medium supplemented with 10 μM itaconate, maintained a 35% total motility after 96 h of storage at 22°C, which is considered the minimum acceptable motility for commercialization. Improvements may be related to the conversion of pyruvate to lactate and regeneration of NAD+. [ABSTRACT FROM AUTHOR]

Summary: The use of cryopreserved stallion semen is a significant aspect of equine reproduction, despite the tendency for lower per cycle pregnancy rates than cooled or fresh semen. This decreased fertility of thawed frozen semen is due in part to the innate subfertility of the chosen male breeding population, but also to the direct insults to the sperm cell during the freezing and subsequent thawing process. The mare aspect of frozen semen breeding is intensively managed to compensate for the aforementioned damages, and acceptable pregnancy rates can be achieved. Here we describe the stallion semen cryopreservation process including semen collection, expected causes of damage in processing, freezing and thawing, mare management and future directions. [ABSTRACT FROM AUTHOR]

Males of some species, from horses to humans, require medical help for subfertility problems. There is an urgent need for novel molecular assays that reflect spermatozoal function. In the last 25 years, studies examined RNAs in spermatozoa as a window into gene expression during their development and, more recently, for their functions in early embryo development. In clinics, more dense spermatozoa are isolated by density gradient centrifugation before use in artificial insemination to increase pregnancy rates. The objectives of the current study were to discover and quantify the microRNAs in stallion spermatozoa and identify those with differential expression levels in more dense versus less dense spermatozoa. First, spermatozoa from seven stallions were separated into more dense and less dense populations by density gradient centrifugation. Next, small RNAs were sequenced from each of the 14 RNA samples. We identified 287 different mature microRNAs within the 11,824,720 total mature miRNA reads from stallion spermatozoa. The most prevalent was miR‐10a/b‐5p. The less dense spermatozoa had fewer mature microRNAs and more microRNA precursor sequences than more dense spermatozoa, perhaps indicating that less dense spermatozoa are less mature. Two of the most prevalent microRNAs in more dense stallion spermatozoa were predicted to target mRNAs that encode proteins that accelerate mRNA decay. Nine microRNAs were more highly expressed in more dense spermatozoa. Three of those microRNAs were predicted to target mRNAs that encode proteins involved in protein decay. Both mRNA and protein decay are very active in late spermiogenesis but not in mature spermatozoa. The identified microRNAs may be part of the mechanism to shut down those processes. The microRNAs with greater expression in more dense spermatozoa may be useful biomarkers for spermatozoa with greater functional capabilities. [ABSTRACT FROM AUTHOR]

Equus caballus papillomavirus 2 (EcPV2) is known to cause genital neoplasms in horses. However, reports on EcPV2 in Japan and Asia are limited. Herein, we present the histopathological and virological findings of the first reported case of an EcPV2-associated penile mass in Japan. The patient was a 22-year-old stallion with a history of breeding in Japan and abroad. Histopathological examination contained RNA in situ hybridization targeting the E6/E7 region and an immunohistochemical approach, and whole-genome sequencing was conducted within the viral examination. Proliferating epidermal cells were observed, and EcPV2 E6/E7 mRNA was detected within the epidermis, which was interpreted as viral papilloma. The detected EcPV2 virus was genetically close to foreign strains and different from the strain previously reported from a Japanese mare. This suggests that various types of EcPV2 might already exist among horses in Japan. Although the mass reported herein was not malignant based on histopathological findings and the absence of recurrence, its presence on the penis would be an obstacle to breeding. These results provide a better understanding of the pathogenesis and diversity of EcPV2. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells originate from gonocytes and undergo self-renewal and differentiation to generate mature spermatozoa via spermatogenesis in the seminiferous tubules of the testis in male mammals. Owing to the unique capacity of these cells, the spermatogonial stem cell transplantation technique, which enables the restoration of male fertility by transfer of germlines between donor and recipient males, has been developed. Thus, spermatogonial stem cell transplantation can be used as an important next-generation reproductive and breeding tool in livestock production. However, in large animals, this approach is associated with many technical limitations and inefficiency. Furthermore, research regrading spermatogonial stem cell transplantation in stallions is limited. Therefore, this review article describes the history and current knowledge regarding spermatogonial stem cell transplantation in animals and challenges in establishing an experimental protocol for successful spermatogonial stem cell transplantation in stallions, which have been presented under the following heads: spermatogonial stem cell isolation, recipient preparation, and spermatogonial stem cell transplantation. Additionally, we suggest that further investigation based on previous unequivocal evidence regarding donor-derived spermatogenesis in large animals must be conducted. A detailed and better understanding of the physical and physiological aspects is required to discuss the current status of this technique field and develop future directions for the establishment of spermatogonial stem cell transplantation in stallions.

Simple Summary: Stallion mules are infertile but still able to produce testosterone, which influences undesirable behavior. Surgical castration is required to eliminate testosterone synthesis, which results in pain and the risk of postoperative complications. To the best of our knowledge, there are no existing studies regarding immunocastration as an alternative method for surgical castration in mules. The objective of this study was to evaluate whether a gonadotropin-releasing hormone (GnRH) vaccine could be used as a method of immunocastration in stallion mules via the assessment of anti-GnRH antibody concentration, serum testosterone concentration, clinical adverse effects, and changes in the selected behaviors. Intact and unilateral cryptorchid mules received the GnRH vaccine at weeks 0, 4, and 8. The serum testosterone concentrations in GnRH-vaccinated mules were lower than before vaccination from weeks 6 to 14. Subcutaneous edema adjacent to the injection site was observed in intact mules after the second or third vaccination. The stallion mules responded to the GnRH vaccine, resulting in a temporary decrease in serum testosterone. Based on the results of this study, the GnRH vaccine may be administered as a temporary immunocastration method for 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Equine gastric ulcer syndrome is a common disease in horses worldwide, with different prevalences in various categories and breeds. It is divided into two different illnesses, based on the mucosa affected: Equine Squamous Gastric Disease, if it involves the squamous mucosa, and Equine Glandular Gastric Disease, if the lesions are located in the glandular portion of the stomach. The effect of sex on gastric ulcers, especially on the squamous mucosa, is not completely elucidated: some studies found a higher prevalence of lesions of the squamous mucosa in geldings and stallions, while others failed to identify a sex as a risk factor. The few studies on glandular lesions did not show any correlation between sex and ulcers. A paper investigating only Thoroughbred mares at pasture found that about 70% of them had gastric ulcers, especially on the squamous mucosa. The aim of this study was to determine the prevalence of the disease in a population of male horses, and determine if their breeding or sports career can influence the development of gastric lesions. Gastroscopies were performed in 101 males, after recording data about their breeding and exercise history, management, and presence of clinical signs. Breeding activity does not determine the presence or severity of gastric lesions in either mucosa, while exercise and a combination of both exercise and breeding is associated with ulcers in the glandular mucosa. Clinical signs are correlated only with the severity of lesions in the squamous one. Equine gastric ulcer syndrome (EGUS) is a worldwide disease, and includes two different syndromes, Equine Squamous Gastric Disease (ESGD), affecting the squamous mucosa, and Equine Glandular Gastric Disease (EGGD), affecting the glandular mucosa. These two diseases are present in different categories (different activities, ages, etc.) and breeds of horses. The effect of sex on gastric health is not clear: some studies found a higher prevalence of ESGD in geldings and stallions, while others found no influence of sex on the squamous mucosa. The few studies conducted on glandular diseases failed to identify sex as a risk factor. The only study on breeding horses, focused on Thoroughbred mares at pasture, found that 70% of them were affected by gastric ulcerations especially in the squamous mucosa. The aim of this study was to determine the prevalence of EGUS, ESGD, and EGGD in intact males while also investigating the potential influences of breeding and exercise activity on the occurrence and severity of the diseases. A total of 101 intact males were admitted for gastroscopic examination. Comprehensive data regarding their breeding and sports history, management, and presence of clinical signs were recorded. A statistical analysis was performed. Within this equine population, no discernible relationship was found between breeding activity and occurrence and severity of ESGD or EGGD. Exercise and a combination of exercise and breeding activities were found to be associated with the occurrence of EGGD. The presence of clinical signs was correlated only with the grade of ESGD in this cohort of horses. [ABSTRACT FROM AUTHOR]

The breeding history of the Einsiedler horse is closely connected with the Benedictine cloister Einsiedeln. In the mid‐nineteenth century, it was decided to use European Warmblood stallions for cross‐breeding and to abandon the selection of stallions. Since that time, it has only been possible to trace back the origin of Einsiedler horses using maternal ancestry information. Here, we collected high‐density genotype data for European Warmblood horses (Selle Français, Swiss Warmblood and Einsiedler) and Franches‐Montagnes horses, the last native Swiss horse breed, to unravel the current population structure of the Einsiedler horse. Using commonly applied methods to ascertain fine‐scale population structures, it was not possible to clearly differentiate the Einsiedler from other European Warmblood horses. However, by means of runs of homozygosity (ROH) we were able to detect breed‐specific ROH islands for the Einsiedler horse, including genes involved in domestication and adaptation to high altitude. Therefore, future breeding activities should involve the screening of these breed‐specific ROH segments, the revival of cryopreserved sperm and the selection of Einsiedler stallions. [ABSTRACT FROM AUTHOR]

The International Society for Animal Genetics (ISAG) currently advocates for a transition towards single nucleotide polymorphism (SNP) markers as a potential alternative for equine parentage verification. To ascertain the efficacy of this transition, it is imperative to evaluate the performance of parentage testing using SNPs in juxtaposition with short tandem repeats (STRs). As per ISAG's recommendation, we used an equine genotyping‐by‐sequencing panel with 144 SNPs for this purpose. Equine parentage is currently realized using 16 microsatellites (STRs) excluding the LEX3 marker. In this study, 1074 horses were genotyped using the 144 SNPs panel, including 432 foals, 414 mares, and 228 stallions, from five different breeds: 293 Arabians, 167 Barbs, 189 Thoroughbreds, 73 Anglo‐Arabians, and 352 Arabian‐Barbs. As a result, two SNPs markers were eliminated from the panel system due to inconsistent amplification across all examined individuals leaving 142 SNPs markers for analysis. A comparative analysis between SNPs and STRs markers revealed that the mean expected heterozygosity was 0.457 for SNPs and 0.76 for STRs, while the mean observed heterozygosity stood at 0.472 for SNPs and 0.72 for STRs. Furthermore, the probability of identity was calculated to be 5.722 × 10−57 for SNPs and 1.25 × 10−15 for STRs markers. In alignment with the Hardy–Weinberg equilibrium in polyploids test, 110 out of the total SNPs were consistent with the Hardy–Weinberg equilibrium in polyploids test (p > 0.05). Employing both SNPs and STRs markers, the mean polymorphic information content was discerned to be 0.351 for SNPs and 0.72 for STRs. The cumulative exclusion probabilities for SNP markers exceeded 99.99%, indicating that the 142 SNPs panel might be adequate for parentage testing. In contrast, when utilizing STRs markers, the combined average exclusion probabilities for one and both parents were determined to be 99.8% and 99.9%, respectively. Our comprehensive study underscores the potential of SNPs in equine parentage verification, especially when compared to STRs in terms of exclusion probabilities. As a corollary, the application of SNPs for parentage verification and identification can significantly contribute to the conservation initiative for the five Moroccan horse breeds. Nonetheless, further research is required to address and replace the deficient SNPs within the panel. [ABSTRACT FROM AUTHOR]

The article offers information on the health challenges faced by wild horse herds in New Zealand, particularly during times of scarcity such as summer and droughts. Topics include the management of wild horse populations through musters, a case study of a black stallion named Boomerang, and the veterinary care and treatment of Boomerang's fractured limb, including radiographs and follow-up examinations.

Simple Summary: Management of breeding stallions is crucial to equine reproduction. The long-life use of a stallion for a breeding career is the ultimate objective, whether it happens through natural mating or through semen collection and artificial insemination. Stud farm veterinarians should be aware of the techniques used to evaluate testicular function and the diagnostic approach to testicular disorders in cases of emergency. This paper presents the clinical methods used to assess testicular health, including palpation, ultrasonography, biopsy, and fine-needle aspiration. The discussion of testicular disorders is broken down into four categories: congenital (present at birth) disorders (cryptorchidism, monorchidism, and testicular hypoplasia), differential diagnosis of scrotal enlargement, differential diagnosis of causes of progressive testicular enlargement, and differential diagnosis of testicular asymmetry or reduction in size with an emphasis on testicular degeneration. Severe clinical signs often accompany a sudden increase in testicular size and are a major cause of stallions being referred for surgery. Testicular disorders are illustrated with clinical cases seen by the authors. Management of breeding stallions is crucial to equine reproduction. The longevity of the breeding career is the ultimate objective, whether the stallion is used for natural cover or for semen collection and artificial insemination. Stud farm veterinarians should be aware of the techniques used to evaluate testicular function and the diagnostic approach to testicular disorders in cases of emergency. This paper presents the clinical methods used to evaluate testicular health, including palpation, ultrasonography, biopsy, and fine-needle aspiration. The discussion of testicular disorders is broken down into four categories: congenital disorders (cryptorchidism, monorchidism, and testicular hypoplasia), differential diagnosis of scrotal enlargement, differential diagnosis of causes of progressive testicular enlargement, and differential diagnosis of testicular asymmetry or reduction in size with an emphasis on testicular degeneration. The sudden increase in testicular size is often accompanied by severe clinical signs and is a major cause for referral of stallion for surgery. Testicular disorders are illustrated with clinical cases seen by the authors. [ABSTRACT FROM AUTHOR]

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the "metabolism" and the "metabolism of lipids" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction. [ABSTRACT FROM AUTHOR]

Simple Summary: ProAKAP4, as a AKAP4 precursor, is the main protein of the spermatozoa flagellum in mammals. AKAP4, A-kinase anchor protein, is involved in sperm motility. The objective of the present study was to evaluate the evolution of proAKAP4 in horse semen post-thawing conditions during 3 h and its relationship with motility descriptors and mitochondrial membrane potential in the same conditions. The evolution in post-thawing conditions is the same either in proAKAP4 and motility parameters which gather that proAKAP4 can predict the evolution of the motility over the time. ProAKAP4, a precursor of AKAP4 (A-kinase anchor protein) found in the flagellum of mammalian and non-mammalian spermatozoa, serves as a structural protein with established correlations to motility parameters across diverse species. This study aimed to determine the proAKAP4 level evolution in thawed stallion semen over a 3 h period, examining its correlation with motility descriptors and mitochondrial membrane potential. Utilizing sixteen ejaculates from four French warmblood stallions, this study involved maintaining thawed samples at 37 °C for 3 h, conducting proAKAP4 enzyme-linked immunosorbent assays (ELISA), computer-assisted sperm analysis (CASA), and mitochondrial membrane potential by JC-1 probe and flow cytometry at 0, 1, and 3 h post-thawing. The findings indicate significant positive correlations (p ≤ 0.05) between proAKAP4 levels and sperm total or progressive motility at all time points analyzed. Spermatozoa velocity descriptors (VAP, VCL, VSL) and spermatozoa lateral head displacement (ALH) display positive correlations (p ≤ 0.05) with ProAKAP4 at the 0 h post-thawing. ProAKAP4 concentration exhibits no discernible difference between batches with or without a cryoprotectant. Notably, proAKAP4 consumption remains insignificant within the initial hour after thawing but becomes significant (p ≤ 0.05) between 1 and 3 h post-thawing. In summary, proAKAP4 demonstrates positive correlations with total and progressive motility in stallion semen for up to 3 h after thawing, albeit showing a noticeable decrease starting from the first hour post-thawing, indicating a progressive consumption as a result of spermatozoa motile activity. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the impact of including hydroxytyrosol (HT) antioxidant on the viability of sperm after the processes of cooling and freezing. HT antioxidants were used in the HF-20 extender at concentrations of 1.25, 2.5, 5, and 10 μg/ml. The HF-20 extender was a basic extender and was used for the control group. The post-thawed semen exhibited significantly higher total motility in the 2.5 HT and 10 HT treatment groups than the control group. The straight line velocity (VSL) of the 2.5 HT group exhibited a significantly high value compared with the control group. However, the average path velocity (VAP), linearity (LIN), straightness index (STR), and wobble (WOB) revealed identical findings across all groups. The findings of the analysis of HOST, normal morphology, major abnormalities, and minor abnormalities revealed that there were no significant differences between the HT groups and the control groups. Nevertheless, the use of HT antioxidant for freezing semen led to a notable enhancement (p < 0.05) in both acrosome integrity and vitality tests when compared to the control group. In this case, the lower quantities of HT (1.25 and 2.5 μg/ml; p < 0.05) preserve the DNA fragmentation compared with the 5 HT, 10 HT, and control groups. In conclusion, the HT antioxidant has shown the capacity to enhance the quality of frozen-thawed spermatozoa and positively influence the viability and integrity of DNA inside the frozen-thawed spermatozoa. Additional research should be conducted to assess the fertility potential of cryopreserved stallion semen. [ABSTRACT FROM AUTHOR]

In the current study, we examined: 1) the agreement (bias) between fluorescence-based methods (NucleoCounter-SP100 [NC] vs. flow cytometry [FC]) for determining the viability (VIAB) of frozen/thawed stallion sperm; 2) the agreement between post-thaw sperm total motility (TMOT) and VIAB; 3) whether a difference between TMOT and VIAB [VIAB – TMOT] in frozen/thawed stallion sperm could be explained by the level of lipid peroxidation in viable sperm (VLPP); 4) the repeatability of post-thaw analysis of sperm quality; and 5) the effect of final post-thaw semen dilution (10, 30, or 50 x 106 sperm/mL) on sperm motion characteristics. Post-thaw VIAB was similar between NC and FC (P > 0.05), and the agreement between these two methods was high (bias : 1 to −3). The agreement between post-thaw TMOT and VIAB decreased as the pre-freeze percentages of morphologically normal sperm and DNA quality decreased: bias – 4 to – 25. The bias between [VIAB – TMOT] and VLPP ranged from – 5 to 7. Differences in post-thaw sperm quality (TMOT, PMOT, VIAB, and sperm concentration) were not observed when analyzing one or three straws per ejaculate (P > 0.05). There was no effect of post-thaw sperm concentration (i.e., 10 vs. 30 vs. 50 x 106 sperm/mL) on sperm motion characteristics (P > 0.05). This study reports factors other than post-thaw sperm motility that warrant further consideration when analyzing frozen/thawed stallion sperm. • Post-thaw sperm viability was similar between NucleoCounter and Flow Cytometry across multiple levels of measurement. • The agreement between post-thaw motility and viability decreased as the intrinsic sperm quality decreased. • Lipid peroxidation was associated with reduced motility in the face of high viability in fresh but not frozen/thawed sperm. • Analysis of one straw per ejaculate yielded similar results than analyzing three straws per ejaculate. • Increasing the sperm concentration in thawed semen prior to motility analysis did not affect the CASA outcomes. [ABSTRACT FROM AUTHOR]

Artificial insemination (AI) with liquid-preserved stallion semen is a widely used reproductive technology. As the demand for AI doses of high-class stallions is transnational, they are frequently exposed to long-distance transport. Since recent studies in boars indicated that vibration emissions caused by transport negatively affected sperm quality in vitro , this study questioned whether sperm quality in stallions is similarly impaired. Furthermore, we investigated stallion and extender-related differences in the spermatozoa's resistance to transport-related quality loss. Stallion ejaculates (n = 30) were collected at a German AI center, split in half, and subsequently diluted to a final sperm concentration of 50 × 106 sperm/mL using the semen extenders EquiPlus or Gent (both Minitüb GmbH, Germany). Four 12 mL aliquots of each sample were filled in plastic syringes according to a split-sample design and exposed to vibration (Displacement index D i = 3.0 ± 0.1) at 5 °C for 0 h (control), 3 h, 6 h or 9 h. All samples were stored for four days at 5 °C after transport simulation and analyzed for total sperm motility, thermo-resistance, membrane integrity, and mitochondrial activity determined by flow cytometry as well as the pH. After calculating generalized linear mixed models for each sperm quality trait, a negative impact of the duration of transport simulation could be shown on total sperm motility (P = 0.001), thermo-resistance (P = 0.030), and the pH (P = 0.001). Simulated transport for 6 h and 9 h diminished sperm quality (P ≤ 0.01), with 9 h reducing thermo-resistance by 5 ± 2.2% points (PP) for EquiPlus and sperm motility by 2.2 ± 1.7 PP for Gent compared to the control group. In contrast, samples exposed to vibration for 3 h showed no decline in sperm quality (P > 0.05). The individual stallion influenced every semen trait (P < 0.05) and transport-related losses in sperm thermo-resistance of up to 15.9 PP were demonstrated. Furthermore, EquiPlus was superior to Gent in all semen assessments (P < 0.001). We conclude that in vitro sperm quality is impaired by vibration. As the quality loss depends on the transport time, we recommend keeping shipping time as short as possible especially for spermatozoa of stallions that are susceptible to vibration-induced sperm quality loss. • Transport-related vibrations affect the quality of AI doses in stallions. • Long transport times reduce sperm motility and thermo-resistance in stallions. • The ability of sperm to resist transport stress varies greatly between stallions. • There is an individual stallion effect influencing all sperm quality traits. • EquiPlus is superior to Gent over all investigated stallion sperm parameters. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs) are a group of molecular chaperones that are crucial for the proper folding and assembly of proteins and the protection of somatic and germ cells from damage caused by temperature changes. However, the localization and seasonal variation patterns of HSPs in stallions' testes remain unclear. Thus, we investigated the expression and seasonal variations in the localization of HSP60 and HSP90 in the testes of stallions. Testes from nine Thoroughbred stallions collected following castration during the non-breeding season (NBS) and two different environmental temperature conditions (normal temperature, NT; March-May and hot temperature, HT; June-August) during the breeding season (BS) were used. Testis tissues were subjected to Reverse transcription-quantitative polymerase chain reaction, Western Blot (WB) analysis and immunofluorescence (IF) assay. The results revealed that the relative abundance of HSP60 and HSP90 mRNA transcripts was significantly upregulated under NT and HT conditions during BS compared with that during NBS conditions. The WB analysis showed that the relative intensity of protein bands was significantly higher in NT and HT conditions during the BS than that of those during the NBS. Immunofluorescence assay showed localization of HSP60 in the cytoplasm of the Leydig cells during both seasons. HSP90 primarily showed immunostaining in undifferentiated spermatogonia and primary spermatocytes in NT and HT conditions and was additionally expressed in the spermatids during the NBS. The spermatogenesis was not monitored in the present study; however, findings of this study suggest that the localization of HSP60 and HSP90 varies in different seasons depending on the temperature, providing insight into the potential mechanisms influencing spermatogenesis during breeding and non-breeding seasons in stallions. [ABSTRACT FROM AUTHOR]

Cryptorchidism affects spermatogenesis and testis development, often resulting in stallion subfertility/infertility. This study aims to identify the specific germ cells impacted by cryptorchism in stallions. In a previous study, we found that PGP9.5 and VASA are molecular markers expressed in different germ cells within stallions. Herein, we assessed the heat stress-induced response of spermatogonial stem cells (SSCs) in the seminiferous tubules (ST) of cryptorchid stallion testes (CST) and normal stallion testes (NST). This goal was accomplished by comparing PGP9.5 and VASA expression patterns through reverse transcription quantitative PCR and immunofluorescence assays. We also compared the cross-sectional ST area between groups. Six post-pubertal Thoroughbred unilateral cryptorchid stallions were used. The relative abundance of the mRNA transcripts of PGP9.5 and VASA was significantly upregulated in the NST group than in the CST group. Additionally, the cross-sectional ST area and localization of PGP9.5 and VASA in germ cells were significantly higher in the NST group than in the CST group. Regarding Leydig cells, PGP9.5 staining was observed in both groups. Spermatogonia, primary spermatocytes and secondary spermatocytes were immunostained with VASA in the NST group, while immunostaining was only observed in spermatogonia in the CST group. These results indicate long-term exposure to heat stress conditions, such as cryptorchidism, directly impacts germ cell proliferation and differentiation, leading to impaired spermatogenesis and compromised fertility in stallions. [ABSTRACT FROM AUTHOR]

Scrotal surface thermography is a non-invasive method for assessing testicular thermoregulation in stallions; however, few studies have explored the application of this technique concerning the thermal physiology of equine reproductive systems. This study aimed to evaluate the consistency of testicular thermoregulation in stallions over a year using thermography to measure the scrotal surface temperature (SST). Moreover, we assessed the best region for measuring the surface body temperature compared with the SST. Ten light-breed stallions were used in the experiment. Thermographic images of the scrotal and body surfaces (neck and abdomen) were captured. Fresh, cooled and frozen-thawed semen samples were evaluated to verify the impact of thermoregulation on semen quality. Testicular thermoregulation was maintained throughout the year in stallions amidst changes in the external temperature, as evidenced by the weak correlation between the SST and ambient temperature. A lower correlation was observed between the environmental temperature and body surface temperature (BTS) obtained from the abdomen (BTS-A; R = .4772; p < .0001) than with that obtained from the neck (BTS-N; R = .7259; p < .0001). Moreover, both BTS-A and SST were simultaneously captured in a single image. The consistent quality of the fresh, cooled and frozen semen suggests efficient thermoregulation in stallions throughout the year. [ABSTRACT FROM AUTHOR]

Virological evaluation was performed on equine semen to detect the presence of papillomaviruses (PVs) using droplet digital polymerase chain reaction (ddPCR) as the aim of this study was to investigate whether the sperm from asymptomatic stallions harbors ovine papillomaviruses (OaPVs). Twenty-seven semen samples were analyzed, 18 of which were commercially acquired. The remaining nine samples comprising semen and peripheral blood, were collected from nine stallions with no apparent signs of PV-related diseases during clinical examination at the Didactic Veterinary University Hospital (DVUH) of Naples. OaPV was detected in 26 semen samples. OaPV1 was the most prevalent virus infecting equine semen. OaPV1 infected 21 semen samples (~80.8%) and showed a high number of DNA and RNA copies per microliter. qPCR was used to detect OaPV1 DNA in the 18 semen samples. ddPCR was used to detect and quantify the expression of OaPV2, OaPV3, and OaPV4. qPCR failed to detect DNA for these genotypes. Additionally, ddPCR was used to detect the transcriptionally active OaPV1 in six blood and semen samples from the same stallion. ddPCR failed to detect any nucleic acids in OaPVs in peripheral blood samples from the three stallions. In one semen sample, ddPCR detected OaPV1 DNA but failed to detect any nucleic acid in the remaining two semen samples, and peripheral blood from the same animals of the remaining 18 semen samples was not available, OaPV1 and OaPV4 were responsible for nine and five single infections, respectively. No single infections with either OaPV3 or OaPV4 were seen.

The presence of bacteria poses a significant challenge to the quality of stallion semen used in artificial insemination. The bacterial content of insemination doses arises from various sources, such as the healthy stallion, environment, and collection equipment, and is implicated in fertility problems as well as reduced sperm quality during storage. The conventional approach of adding antibiotics to semen extenders raises concerns about antimicrobial resistance and potential negative effects on sperm characteristics, and may not be effective in inhibiting all bacteria. The objective of this study was to determine whether an innovative alternative to antibiotic usage – centrifugation through a single layer of a low density colloid (SLC) – could reduce the bacterial load in stallion semen, and to compare sperm characteristics in samples arising from this procedure, or simple extension of the ejaculate in semen extender, or from sperm washing, i.e. adding extender and then centrifuging the sample to allow the removal of most of the seminal plasma and extender. Eighteen semen samples were collected from six stallions. The semen samples were split and extended prior to washing or SLC, or received no further treatment other than extension. After preparation aliquots from each type of sample were sent for bacteriological examination; the remaining samples were stored for up to 72 h, with daily checks on sperm quality. The low density colloid SLC outperformed sperm washing or extension for bacterial reduction, effectively removing several bacterial species. The bacterial load in the samples was as follows: extended semen, 16 ± 6.7 × 105; washed, 5.8 ± 2.0 × 105; SLC, 2.3 ± 0.88 × 105, p < 0.0001. In addition, SLC completely removed some bacterial species, such as Staphylococcus xylosus. Although there is no selection for robust spermatozoa with the low density colloid, sperm motility, membrane integrity, and DNA fragmentation were not different to washed sperm samples. These findings suggest that SLC with a low density colloid offers a promising method for reducing bacterial contamination in stallion semen without resorting to antibiotics. • Stallion ejaculates were prepared without antibiotics by extension, washing or SLC. • Bacterial load was highest in extended semen and lowest in SLC samples. • Sperm characteristics were not different between the three treatments. • Total motility was less in extended samples than in other treatments at 72 h. • SLC through a low density colloid could be an alternative to antibiotics. [ABSTRACT FROM AUTHOR]

Urospermia in stallions can occur intermittently, consistently, or as an isolated event, and may result in reduced sperm quality which is often assumed to reduce fertility. Although sperm quality declines in urospermic ejaculates, fertility has not been assessed in mares bred with urine contaminated semen. The aims of this study were to compare sperm quality after simple dilution (SD), cushioned centrifugation (CC) alone, or cushioned centrifugation combined with a 40 % silane-coated silica solution (SC) in semen contaminated with 0, 20, or 40 % (v/v) urine. Sperm quality values tended to decrease as the percent urine increased within all treatments (SD, CC, SC) after 24 h of cooled storage. However, SC treated groups had higher sperm quality compared to SD and CC when exposed to 20 or 40 % (v/v) urine. Differences in pregnancy rates among treatment groups (SD with 0 or 40 % (v/v) urine, or 40 % (v/v) urine followed by SC) were unable to be detected. • Urine contaminated semen can be improved with cushion/gradient centrifugation. • Negative effects in sperm quality are associated with the composition of urine. • Use of sperm viability to assess quality is valuable in low motility ejaculates. • Mares can become pregnant when bred with urospermic ejaculates. [ABSTRACT FROM AUTHOR]

The aim of this work was to evaluate selected influencing factors during the breeding of young Czech warmblood stallions in test farms on the results of the performance test. In six breeding farms for young horses (Měník, Písek, Skály, Suchá, Tlumačov and Železnice), 119 stallions were tested and 413 offspring were evaluated. During the performance test, the factors test farm, year of birth, father and parental descent were examined with regard to their influence on the characteristics of cannon bone circumference, conformation values, movement mechanics, jumping ability and overall evaluation. The data were collected from the Morgan ČT program and then analyzed using the GLM (Generalized Linear Model) analysis of variance method. The rearing operation, the year of the performance test, the test person's father and the ancestry of the parents had a significant influence. The influence of breeding on the overall evaluation, the exterior and the movement mechanics could be proven. The year of the test was proven to have an influence on chest circumference. The Tlumačov breeding farm was the best-rated testing station for young horses. The results confirmed the hypothesis that the farm influences the test results of young Czech Warmblood horses. Therefore, it is crucial to focus further on the evaluation system for the performance of young stallions. [ABSTRACT FROM AUTHOR]

Context. Little is known about the microbial composition ofstallion semen. Aims. To describe the microbiota detected in equine semen of healthy miniature pony stallions. Methods. Semen specimens were collected using a Missouri artificial vagina at a single time point. PacBio (Pacific Biosciences) genomic DNA sequencing of the 16S rRNA gene was performed on these specimens, following which next-generation microbiome bioinformatics platform QIIME2 was used to process fastq files and analyse the amplicon data. The data were categorised into genus, family, class, order and phylum. Key results. Firmicutes and Bacteroidetes phyla predominated (76%), followed by Proteobacteria (15%). Bacteroidales, Clostridiales and Cardiobacteriales predominated the microbial rank of order (86%). Class was mainly composed of Bacteroidia, Clostridia and Gammaproteobacteria (87%), while family was mainly composed of Porphyromonadaceae, Family_XI and Cardiobacteriaceae (62%). At the level of genus, 80% of the abundance was composed of seven genera, namely Porphyromonas, Suttonella, Peptoniphilus, Fastidiosipila, Ezakiella, Petrimonas and an unknown taxon. Conclusions. The findings indicate that specific microbiota may be characteristic of healthy miniature pony stallions’ semen with some inter-individual variations observed. Implications. Larger equine studies involving fertile and infertile subjects could be informed by this study and could explore the relationship of the semen microbiome to male fertility. [ABSTRACT FROM AUTHOR]

The members of the Chlamydiaceae family are important pathogens that infect a wide range of vertebrate hosts, including humans. Among them, Chlamydia psittaci, historically considered as an avian agent, has recently been identified in livestock, primarily sheep and cattle, but also in horses, with the infection being linked to reproductive disorders, such as abortion, absorption of embryos, stillbirth, and the birth of weak foals. Much less is known about chlamydial infections in the Sardinian equine population. This study aimed to identify the chlamydial diversity in genital samples from asymptomatic Sardinian horses. However, some horses had a previous history of reproductive disorders, i.e., abortion and infertility. A total of 60 horses (39 mares and 21 stallions) were opportunistically recruited from 17 equine farms in central-northern Sardinia. Vaginal and uterine swabs from mares and urethral swabs and seminal fluid from stallions were sampled for the presence of chlamydial DNA. Samples from environments where the horses lived were also tested for the detection of Chlamydia spp. Eight vaginal swabs (8/39; 20%), two uterine swabs (2/27; 7%), two seminal fluid samples (2/20; 10%), and one urethral swab (1/21; 4.7%) were found to be positive for Chlamydia spp. by PCR analysis. In addition, results from environmental samples showed the presence of Chlamydia spp. in three environmental swabs (3/8; 37.5%) and five water samples (5/16; 31.2%). Sequencing results revealed that strains here identified were 99–100% similar to members belonging to the Chlamydiaceae family, including C. abortus, C. psittaci, and uncultured Chlamydia genotypes. ompA species-specific PCR performed on samples was found to be positive after 16S rRNA amplification gave positive results for C. psittaci. These results reveal the first presence of C. psittaci in the genital tract of horses and in the environment in Sardinia and indicate that this pathogen could be the prevailing cause of infertility and abortion in the tested equines. However, these findings need further proof and highlight the importance of adopting a 'One Health' approach to control the presence of this zoonotic bacteria in domestic animals in order to understand its impact on people exposed to the infection risk. [ABSTRACT FROM AUTHOR]

Background: Equine spermatozoa appear to differ from spermatozoa of other species in using oxidative phosphorylation preferentially over glycolysis. However, there is little information regarding effects of different energy sources on measured parameters in equine spermatozoa. Objective: To determine the effect of three individual energy substrates, glucose, pyruvate, and lactate, on motion characteristics, membrane integrity, and acrosomal status of stallion spermatozoa. Materials and methods: Freshly ejaculated stallion spermatozoa were incubated with combinations of glucose (5 mm), pyruvate (10 mm), and lactate (10 mm) for 0.5 to 4 h. Response to calcium ionophore A23187 (5 μm) was used to evaluate capacitation status. Motility was evaluated using computer‐assisted sperm analysis, and plasma membrane and acrosomal integrity were evaluated by flow cytometry. Results: Incubation with lactate alone for 2 h increased acrosomal sensitivity to A23187. Notably, incubation with lactate alone for 4 h induced a significant spontaneous increase in acrosome‐reacted, membrane‐intact (viable) spermatozoa, to approximately 50% of the live population, whereas no increase was seen with incubation in glucose or pyruvate alone. This acrosomal effect was observed in spermatozoa incubated at physiological pH as well as under alkaline conditions (medium pH approximately 8.5). Sperm motility declined concomitantly with the increase in acrosome‐reacted spermatozoa. Sperm motility was significantly higher in pyruvate‐only medium than in glucose or lactate. The addition of pyruvate to lactate‐containing medium increased sperm motility but reduced the proportion of live acrosome‐reacted spermatozoa in a dose‐dependent fashion. Discussion: This is the first study to demonstrate that incubation with a specific energy substrate, lactate, is associated with spontaneous acrosome reaction in spermatozoa. The proportion of live, acrosome‐reacted spermatozoa obtained is among the highest reported for equine spermatozoa. Conclusion: These findings highlight the delicate control of key sperm functions, and may serve as a basis to increase our understanding of stallion sperm physiology. [ABSTRACT FROM AUTHOR]

Subfertility is one of the main issues in horse breeding and the study of mRNAs in sperm might help in elucidating the reasons that lead to this diagnosis. The present study aims at assessing the differences in the expression of 10 potential candidate genes in stallions of different fertility. Frozen-thawed semen of 29 stallions was included. Each sample was classified into two groups according to pregnancy rates (PR) achieved with this semen: "good fertility" (GF; n = 17; PR ≥ 30 %) or "poor fertility" (PF; n = 12; PR <20 %). All stallions underwent a breeding soundness examination (BSE) before semen production and were only included into the semen cryopreservation program when raw semen characteristics at BSE met minimal requirements. Semen was cryopreserved following European Union regulations and all stallions met the respective health requirements. Each sample was assessed for concentration (NucleoCounter SP-100), motility (CASA), membrane functionality (SYBR-14/PI), mitochondrial membrane potential (JC-1), morphology (SpermacStain), acrosome integrity (SpermacStain), membrane integrity (HOS test) and chromatin integrity (Aniline blue). Sperm RNAs were extracted using the Direct-zol RNA Miniprep Kit (Zymo Research) and RT-qPCR was performed for each target gene. ACTB and RPL32 were included as reference genes (RGs) for normalization. For each variable of each group, mean, standard deviation and SEM were calculated. The difference in gene expression levels between the GF and PF group were analyzed using the Mann-Whitney U test and Spearman's rank correlation. Significant results were considered with p < 0.05. Sperm quality parameters did not differ significantly between the two groups except for concentration, that was significantly higher in GF (p = 0.043). In GF a positive correlation was identified for PRM1 / PRM2 with r = +0.6, while PRM1 / ACR (r = −0.495), PRM2 / ZPBP (r = −0.645) and CRISP3 / ACR (r = −0.551) were inversely correlated. In PF direct correlations were registered for PRM1 / PRM2 (r = +0.629), PRM1 / PRM3 (r = +0.657), PRM2 / SPA17 (r = +0.685), SPA17 / PLCZ1 (r = +0.786) and PRM3 / ACR (r = +0.627). In the total sample (GF + PF), positive correlations were detected for PRM1 / PRM2 (r = +0.625), PRM1 / PRM3 (r = +0.368); PRM2 / SPA17 (r = +0.465), SPA17 / PLCZ1 (r = +0.637) and PLCZ1 / ZAN (r = +0.587). Only two of the genes considered were differentially expressed in the 2 groups: PRM2 and PLCZ1 , that were significantly (p < 0.05) overexpressed in the GF group. Stallions frozen-thawed semen with higher expression levels of PRM2 and PLCZ1 are more likely to belong to animals with a good pregnancy rate. Further studies are needed to investigate the role of sperm transcripts in male subfertility in stallions. • Sperm concentration was increased in frozen-thawed stallion semen with good fertility (GF). • The mRNA abundance of protamine 2 (PRM2) was increased in GF semen. • The mRNA abundance of phospholipase C zeta 1 (PLCZ1) was increased in GF semen. • Analysis of PRM2 and PLCZ1 mRNA abundance helps to predict frozen semen fertility. [ABSTRACT FROM AUTHOR]

If a mechanism of more efficient glycolysis depending on pyruvate is present in stallion spermatozoa, detrimental effects of higher glucose concentrations that are common in current commercial extenders could be counteracted. To test this hypothesis, spermatozoa were incubated in a 67 mM Glucose modified Tyrode's media in the presence of 1- or 10-mM pyruvate and in the Tyrode's basal media which contains 5 mM glucose. Spermatozoa incubated for 3 h at 37 °C in 67 mM Tyrode's media with 10 mM pyruvate showed increased motility in comparison with aliquots incubated in Tyrode's 5 mM glucose and Tyrode's 67 mM glucose (57.1 ± 3.5 and 58.1 ± 1.9 to 73.0 ± 1.1 %; P < 0.01). Spermatozoa incubated in Tyrode's with 67 mM glucose 10 mM pyruvate maintained the viability along the incubation (64.03 ± 15.4 vs 61.3 ± 10.2), while spermatozoa incubated in 67 mM Glucose-Tyrode's showed a decrease in viability (38.01 ± 11.2, P < 0.01). 40 mM oxamate, an inhibitor of the lactate dehydrogenase LDH, reduced sperm viability (P < 0.05, from 76 ± 5 in 67 mM Glucose/10 mM pyruvate to 68.0 ± 4.3 %, P < 0.05). Apoptotic markers increased in the presence of oxamate. (P < 0.01). UHPLC/MS/MS showed that 10 mM pyruvate increased pyruvate, lactate, ATP and NAD+ while phosphoenolpyruvate decreased. The mechanisms that explain the improvement of in presence of 10 mM pyruvate involve the conversion of lactate to pyruvate and increased NAD+ enhancing the efficiency of the glycolysis. • 10 mM pyruvate in a 67 mM Glucose-Tyrode's media maintains sperm functionality in comparison with a 67 mM glucose- Tyrode's. • Motility, linear motility, velocities membrane integrity, mitochondrial membrane potential and glutathione content were higher in the 10 mM pyruvate, 67 mM glucose media. • LACTATE and NAD+ content were higher in the spermatozoa incubated in the 10 mM pyruvate in a 67 mM Glucose-Tyrode's media. • The mechanism explaining the improvements may involve conversion of pyruvate to lactate and regeneration of NAD+. • The stallion spermatozoa may produce significant amounts of lactate under high Oxygen (Warburg effect). [ABSTRACT FROM AUTHOR]

Context: Resumption of testicular function after gonadotrophin-releasing hormone (GnRH) immunisation varies among individual animals and some stallions regain fertility only after a prolonged time. Aims: This study evaluated endocrine effects of GnRH immunisation and early subsequent re-stimulation with a GnRH agonist. We hypothesised that GnRH agonist treatment advances resumption of normal endocrine function in GnRH-vaccinated stallions. Methods: Shetland stallions were assigned to an experimental and a control group (n = 6 each). Experimental stallions were GnRH-immunised twice, 4 weeks apart. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3 ng/mL. Three weeks later, daily treatment with the GnRH agonist buserelin was initiated (4 μg/day for 4 weeks followed by 8 μg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5 ng/mL in vaccinated stallions. Blood was collected for LH, FSH, oestradiol and anti-müllerian hormone (AMH) analyses, and testicular and epididymal tissue were conserved for real-time qPCR and histology. Key results: GnRH vaccination reduced blood concentrations of LH and FSH, with a structural deterioration of testicular tissue and disruption of spermatogenesis. Daily buserelin treatment for approximately 60 days partially restored gonadotropin secretion and induced a recovery of the functional organisation of the testicular tissue with effective spermatogenesis. Conclusions: Endocrine testicular function can be restored in GnRH-vaccinated stallions by daily low-dose buserelin treatment. The buserelin treatment protocol may potentially be improved regarding the dose, interval and duration. Implications: Daily buserelin treatment can be recommended for treatment of GnRH-vaccinated stallions with prolonged inhibition of testicular function. GnRH vaccination is used in male horses to reduce sexual behaviour and gonadal function, but the time to regain fertility varies considerably among individual animals. Daily GnRH agonist treatment for approximately 60 days in stallions vaccinated against GnRH successfully stimulated the production of sex steroid hormones and spermatogenesis. This treatment may be recommended for GnRH-vaccinated stallions with persistent inhibition of testicular function and fertility. Image by Camille Gautier. This article belongs to the Collection Dedication to Jim Cummins. [ABSTRACT FROM AUTHOR]

Simple Summary: Most artificial inseminations in horses currently use cooled semen. It has not been possible to develop a freezing protocol that is suitable for all stallions and all ejaculates. Furthermore, the thawed spermatozoa have a short life, which necessitates depositing the semen in the uterus close to the time of ovulation. Using frozen semen has many potential advantages: there would be greater choice of stallions, the supply of semen is guaranteed, and the amount of antibiotics used would be reduced compared to fresh semen. However, not all ejaculates can be frozen successfully. This review looks at some of the factors that might affect the successful cryopreservation of semen, including stallion nutrition, the frequency of semen collection, the medium that is added to protect the sperm during freezing, the speed of cooling, etc. It would be helpful to identify ejaculates that will freeze well, but as yet, there are no means of identifying them other than carrying out a test freeze. Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics used in semen extenders, since the volume of frozen semen is smaller than when cooled semen is inseminated. However, there is considerable variability in the cryosurvival of spermatozoa from different stallions, leading to the classification of stallions as good or bad freezers. Improvements could be made at the level of stallion nutrition, the semen collection regimen, the extender, the removal of seminal plasma, and the cooling protocol, among others. Stallion sperm membranes are highly susceptible to lipid peroxidation, but research on antioxidants has failed to identify an additive that would benefit all stallions. In the future, biomarkers for sperm freezability could be used as an aid in identifying suitable ejaculates for cryopreservation. [ABSTRACT FROM AUTHOR]

One of the breeding objectives for Shagya Arabian breed in Rădăuți Stud Farm, Romania, focuses on increasing the height at withers for mares. The current research aimed to study the entire female progeny (175 foals) of 58 broodmares and 44 stallions included in the breeding activity for 40 years The objective was to determine the influence of breeders' height and the foaling number (rank) on the foals' biometrics. The average value of this measurement in female foals has consistently increased from first foaling (157.63 cm) to the fifth one (158.80 cm) throughout 40 years of breeding, indicating the fulfilment of the breeding goal over consecutive reproductive seasons. According to the simulation based on the linear trend equation, the progeny's height at withers could match the fathers' height by the 10th foaling if the same breeding practices were continued. In addition, stallions would have much more influence than broodmares in increasing the height of the foals. Continuing the forecast analysis based on the variations, the required average height at withers values in stallions and mares can be computed to attain a certain value for the descendants. [ABSTRACT FROM AUTHOR]

A body condition index for equines was proposed by Potter et al in 2015. This index showed, as the Henneke body condition score, a high correlation with the estimation of body fat content by the deuterium oxide dilution method. The purpose of this research was to validate the index by Potter as an objective method to estimate body fat content in Thoroughbred racing horses in competition, through its correlation with the Henneke score modified by Koehne (1992). Fifty Thoroughbred racing horses in training were used (2 stallions, 26 geldings and 22 mares) between 3 to 9 year old. Modified Henneke body condition score was estimated for each horse, along their height at withers, neck circumference, heart girth and belly girth were measured to calculate the body condition index. Measurements were compared between sexes by analysis of variance and Tukey test. Pearson correlation was calculated between the modified Henneke body condition score and the body condition index. The body condition index showed a high correlation with the modified Henneke body condition score in Thoroughbred racing horses (r=0.922, p<0.00001). This study suggests that the body condition index is a good method to estimate the modified Henneke body condition score in Thoroughbred racing horses in training. [ABSTRACT FROM AUTHOR]

Context. There has been a contraction within the New Zealand Thoroughbred racing industry and there are limited data demonstrating how this has affected, or been reflected, within the breeding industry that supplies horses for the racing industry. A reduction in the breeding industry, to a greater extent in the sector servicing domestic rather than export market has been speculated. Aim. To describe the demographics, breeding pattern, reproductive career and workload of New Zealand Thoroughbred stallions between 2005 and 2015. Methods. The breeding records of all Thoroughbred stallions covering >10 mares during 2005/2006-2015/2016 breeding seasons and the lifetime breeding records of all the mares covered by these stallions were extracted from the New Zealand Thoroughbred Racing website. The demographics, population numbers, career pattern, and 10-year trend in market share of stallions in different stud fee categories were examined using descriptive analysis. The stallion's reproductive workload was modelled deterministically. Results. The breeding population reduced over the 10 years examined (mares by 20%, sires by 32%). This industry consolidation was almost entirely due to the 50% reduction in the number of mares mated to the low-priced stallions, which generated horses for the domestic market. The relative proportion of mares sent to medium- and high-priced stallions (breed to sell and export sector) doubled. The commercial breeding career of low- and medium-priced stallions and shuttle stallions was short (4-5 years). The number of mares covered by a stallion increased with stud fee category. Stallions covered a greater number of mares in October and November than in September and December because of the restricted opportunity to cover foaled mares early in the season, caused by the long gestation length of mares and the need to cover mares before December. Conclusions. Findings in this study support that the contraction in the New Zealand Thoroughbred breeding industry was due to a reduction in the industry sector that focuses primarily on supplying horses for domestic racing. Commercial pressure and biological constraints heavily influenced the reproductive management of the breeding industry. The breeding management of stallions during the season is heavily influenced by stallion service fee category, which reflects his book size and the commercial appeal of the resultant progeny. Implications. The contraction within the domestic sector of the breeding industry was reflected in the concurrent contraction within New Zealands domestic racing population and fewer colts and geldings entering racing. Industry breeding trends demonstrate that economic viability and optimisation of revenue depend on the breeding industry focusing on the export rather than domestic market. [ABSTRACT FROM AUTHOR]

Background: Sperm protein at 22 kDa has been associated with fertility. Objectives: The objectives of this study were to determine (1) the localization pattern of SP22 on ejaculated and caudal epididymal equine spermatozoa and in epididymal fluid, and to (2) characterize SP22 protein and mRNA expression in testicular and epididymal tissues in response to heat‐induced testicular degeneration. Materials and methods: Semen was collected before and after hemi‐castration, as well as prior to and following insulation of the remaining testes, and tissue specimens were collected for analysis. Results: Histopathology confirmed degeneration in insulated testes. Ejaculated and epididymal spermatozoa from samples collected prior to insulation of the testicles had a predominant staining pattern of SP22 over the equatorial region. However, the equatorial pattern in the pre‐insulation epididymal semen samples was significantly lower than in the pre‐insulation ejaculated semen samples (68 ± 3, 81 ± 2.6, respectively). Ejaculated and epididymal samples collected after insulation of the testicles showed a complete loss of staining as the predominant pattern. Western blot analysis verified the presence of SP22 on fresh ejaculated spermatozoa prior to and following heat‐induced degeneration, on epididymal spermatozoa after testicular insulation, and in testicular and epididymal tissues. Heat insulation significantly reduced messenger RNA expression in the head of the epididymis and testicular tissues. Immunohistochemistry of the testicular and epididymal tissues pre‐heating showed considerably weaker staining than the same tissues post‐heating. Discussion and conclusion: It was concluded that heat‐induced testicular damage causes both loss and relocation of SP22 on the sperm membrane. Future studies are warranted to determine the diagnostic value of these findings. [ABSTRACT FROM AUTHOR]

Objective: To describe the successful restoration of superior eyelid function in a horse following traumatic avulsion using an advancement flap blepharoplasty and subdermal hyaluronic acid filler. Animal Studied: A 21‐year‐old American Paint Horse stallion who was attacked by a fellow stallion resulting in numerous traumatic injuries including avulsion of approximately 75% of the left superior eyelid. Procedures: With standing sedation and locoregional anesthesia, the superior eyelid wound was debrided and an advancement flap blepharoplasty (H‐plasty) and temporary tarsorrhaphy were performed. Routine healing of the surgical site occurred over the subsequent weeks, though lagophthalmos persisted. At 2 and 4 weeks post‐operatively, 2.4% cross‐linked hyaluronic acid was injected subdermally into the superior eyelid to attempt to improve corneal coverage. At 8 weeks post‐operatively, a complete blink was restored and the cosmetic outcome was good. Conclusions: Injection of subdermal hyaluronic acid filler following eyelid injuries or blepharoplastic procedures that result in lagophthalmos can improve corneal coverage by the eyelids and allow for maintenance of a comfortable and visual eye. [ABSTRACT FROM AUTHOR]

Summary: Simple dilution of semen is the oldest method to process semen for artificial insemination with either fresh or cooled semen. This was developed prior to the optimisation of centrifugation techniques for semen processing through a combination of trial and error and research. Simple dilution is a faster method to process semen, is easy to use and works in many circumstances. For on‐farm semen use a 1:1 (v/v) semen to extender ratio is used. A variety of other techniques have been developed, such as cushion centrifugation, density gradient centrifugation and sperm filtration. Cushion centrifugation is used to concentrate the sperm and allows higher centrifugation speeds than centrifugation alone. This allows for improved recovery rates of sperm. Density gradient centrifugation has been used to select for morphologically normal sperm but suffers from low recovery rates. Processing sperm with this technique can result in improved motility and improved DNA integrity of sperm. Sperm filtration is another method to concentrate sperm without the use of centrifugation; however, it has lower recovery rates compared to cushion centrifugation. This review will discuss these techniques in the application of processing equine semen for either fresh or cooled‐shipped use. [ABSTRACT FROM AUTHOR]