Plasma concentration of dopamine varies depending on breed, sex, and the genotype of DRD4 in horses.

Dopamine (DA) is known to be a key modulator of animal behaviors. Thus, the plasma concentration of DA might be used as a biomarker for the behavioral characteristics of horses. The behavioral characteristics of horses vary depending on the breed, age, and sex. Moreover, the DA receptor genotypes are also related to horse behaviors. Thus, the aim of this study was to investigate the DA concentration variations of horse plasma by breed, age, sex, or genotype of its receptor. The horses were divided by breed into Thoroughbred (n = 13), Pony (n = 9), Warmblood (n = 4), and Haflinger (n = 5). The age variable was divided into three different groups: post-pubertal (2-5 years, n = 6), adult (6-13 years, n = 19), and aged horses (15-24 years, n = 6). The sex variable was divided into geldings (n = 8) and mares (n = 23). Approximately 10 mL of blood was collected, and an ELISA kit was used to measure the plasma concentration of DA. Polymerase chain reaction analysis was performed to identify the genetic variation in the DA D4 receptor gene (DRD4). SPSS statistical software was used for statistical analysis. The DA concentrations in geldings were significantly lower than those in mares. There was no significant difference in DA concentrations among breed and age groups. Horses with the GG and GA genotypes had significantly higher plasma concentrations of DA compared to horses with the AA genotype for the G292A gene. Briefly, the plasma concentration of DA varied depending on the sex and genotype of G292A. These factors should be considered when the concentration of DA is used as a biomarker for the behavioral characteristics of horses. In conclusion, the DA concentration or DRD4 genotype of horse plasma has the potential to be used as a biomarker that can predict the behavioral characteristics of horses.

Seasonal changes in the expression of molecular markers of stallion germ cells.

The economic impacts of infertility and subfertility of stallions greatly influence the horse breeding industry. Self-renewal and differentiation of spermatogonial stem cells are the initial processes to maintain an adequate sperm population. Thus, understanding these processes may provide useful information to reveal the causes and remedies of subfertile and infertile stallions. Stallions are seasonal breeders. About 50% of the sperm population is reduced during the non-breeding season (NBS) in stallions. The seasonal regulation of spermatogenesis renders stallions as ideal models to understand the process of sperm production. Furthermore, comparing internal and external factors related to spermatogenesis during the breeding season (BS) and NBS may provide a solution for subfertile/infertile stallions. It is especially pertinent to study the expression pattern of different protein markers during undifferentiated, differentiating, and differentiated spermatogonia. Deleted in azoospermia-like (DAZL), undifferentiated cell transcription factor 1 (UTF-1), and protein gene product 9.5 (PGP9.5) are the molecular markers expressed at different stages of spermatogenesis. However, whether the expression pattern of these molecular markers is similar throughout the year in stallion remains undetermined. The objectives of this study were to (1) investigate the expression pattern and localization of DAZL, UTF-1, and PGP9.5 within seminiferous tubules and (2) evaluate the relative mRNA levels of these three germ cell markers in stallion testes during BS and NBS. Immunohistochemistry was performed to check and compare the expression pattern and localization of DAZL, UTF-1, and PGP9.5 antibodies. Reverse transcription-quantitative PCR analysis was performed to calculate the relative mRNA expression levels in the testes. Testicular tissues from thoroughbred stallions were collected during routine castration that was carried out in field conditions. Immunostaining of germ cells with DAZL and UTF-1 in BS and NBS were not significantly different. However, the relative mRNA expression levels of DAZL and UTF-1 were significantly different in both groups. Interestingly, the immunolabeling and the relative mRNA expression of PGP9.5 were significantly different between BS and NBS. From these results, it is hypothesized that the expression level of these putative molecular markers might be gonadotropin-dependent in stallion testes.

Efficiency of Equilume light mask on the resumption of early estrous cyclicity and ovulation in Thoroughbred mares.

Equilume light masks had no impact on hastening the resumption of estrous cyclicity in mares maintained in outdoor pastures on the mainland of Korea due to the cold weather conditions. Jeju Island is a major horse-breeding site in Korea and is warmer than the mainland during the winter season. Therefore, the primary objective of this study was to explore the efficiency of the Equilume light mask on the resumption of seasonal estrous cycles in Thoroughbred mares on Jeju Island. A total of 20 nonpregnant mares were randomly divided into the Equilume light mask (n = 9) and stable lighting (n =11) groups. The experiment was performed at seven different horse-breeding farms located on Jeju Island from November 15, 2020, to February 15, 2021. The mares were exposed to the respective lights from 16:00 to 23:00. Follicle size and uterine edema were measured by ultrasound scanning. Body condition scores (BCS) were also monitored during the experiment. Statistical analysis was conducted using the SAS and SPSS software, and p-values of < 0.05 were considered statistically significant. Two of the nine (22.2%) mares in the Equilume light mask group and three of the 11 (27.28%) mares in the stable lighting group were still cycling in December and January, which were considered as all-year-round cycling mares. On February 15, there was no difference between groups in the resumption of early seasonal estrus cycle, which was determined by follicles > 25 mm in addition to uterine edema. All mares in the Equilume light mask group and five of the eight mares (62.5%) in the stable lighting group had resumed cycling. Interestingly, six of the seven mares (87.5%) in the Equilume light mask and four of eight mares (50%) in the stable lighting group had already ovulated on February 15 (p > 0.05), as determined by the presence of a recent corpus luteum. No difference was observed in BCS and uterine edema between groups (p > 0.05). In conclusion, the Equilume light mask can be an effective approach to induce early seasonal estrus cycles of mares in Jeju Island, and it also enhances the efficiency of farm management by reducing labor.

Effects of intravenous multiple busulfan injection on suppression of endogenous spermatogenesis in recipient stallion testes.

Preparation of recipient stallions is critical step to produce donor spermatogonial stem cell (SSC) derived sperm using transplantation technique. This study was conducted to evaluate the effects of intravenous busulfan infusion on germ cell depletion, semen production, and libido in stallions. Six Thoroughbred stallions were separated into two treatment groups: 1) a multiple low-dose (2.5 mg/kg bw for the first 4 weeks and 5 mg/kg bw for the 5th week); and 2) control group treated with PBS. Testicular samples were obtained at 11 weeks and classified into three different patterns of spermatogenesis, such as normal, Sertoli cell only, and destroyed. Semen collection and libido experiments were performed 1 week before treatment, and 4 and 8 weeks after treatment. For the sperm analysis, total spermatozoa and motility were measured using a light microscope with a motility analyzing system. In the multiple low-dose group, the numbers of tubules categorized as Sertoli cell only were significantly higher than those in the control as well as the total population and total/progressive motility of sperm were significantly decreased 8 weeks after the start of the treatment. The sperm production and motility in the multiple low-dose group appears to be reduced, while libido was maintained. In conclusion, multiple administration of 2.5 mg/kg bw busulfan depletes endogenous germ cells in the stallion recipients for SSC transplantation.

Germ Cell Transplantation in Stallion Testes.

The production of donor-derived sperm using spermatogonial stem cell transplantation has been studied in various animals including mice, rats, goats, boar, dogs, sheep, and monkeys. However, germ cell transplantation has not been applied in stallions. The objective of this study was to produce donor germ cell-derived sperm using germ cell transplantation in stallions. Donor germ cells were transplanted into the parenchyma of 3 recipient stallions that had been treated with busulfan IV injections of 15 mg/kg body weight. For the preparation of donor single germ cells, tissue (20 g) from each testis was subjected to a 2-enzyme digestion procedure. Donor testicular germ cells in minimum essential medium α supplemented with 10% fetal bovine serum were transplanted in the testis of recipient stallions at a rate of 2 ml/min. The semen of each recipient stallion was collected using an artificial vagina at 8 weeks after germ cell transplantation. General sperm evaluation and libido tests were performed. Microsatellite fingerprinting with 17 markers was performed to identify the presence of donor-derived sperm in the semen of the recipient stallions. Sperm were observed to have total and progressive motility exceeding 50% throughout the experimental period. The libido of the recipient stallions was unchanged. No donor-derived sperm could be detected in the semen of the recipient stallions by genotyping. In conclusion, the transplantation of donor germ cells into the testicular parenchyma of stallions was not an optimal transplantation technique for producing donor-derived sperm.

Relationship between oxytocin and serotonin and the fearfulness, dominance, and trainability of horses.

Oxytocin (OXT) and serotonin (5-HT) are essential neurotransmitters associated with the behavior of animals. Recently, we found that the plasma concentration of OXT is positively correlated with horse docility and friendliness toward humans. However, the relationships between the neurotransmitters and other temperaments such as fearfulness, dominance, and trainability are unknown. This study aimed to identify whether the plasma concentration of OXT or 5-HT is correlated with fearfulness, dominance, and trainability of horses. Blood samples of 34 horses were collected at the Horse Industry Complex Center of Jeonju Kijeon College. The concentration of OXT and 5-HT was measured in the plasma samples using enzyme-linked immunosorbent assays. The fearfulness, dominance, and trainability of horses were scored by three professors who were very familiar with the horses. One-way analysis of variance with the least significant difference post-hoc analysis was used to compare the scores for fearfulness and dominance among groups. The trainability of horses was compared using the student t-test. The 5-HT was negatively correlated with dominance, but it had no relation with fearfulness. The OXT appeared to be negatively correlated with fearfulness and dominance in horses. Furthermore, OXT was positively correlated with the trainability of horses. Additionally, 5-HT appeared to enhance trainability. In conclusion, the concentration of OXT or 5-HT in horse blood plasma can be used as a biomarker to monitor the fearfulness, dominance, or trainability of horses.

Stage-Dependent Expression of Protein Gene Product 9.5 in Donkey Testes.

Molecular markers can be used to identify and isolate specific developmental stages of germ cells and Leydig cells. Protein gene product (PGP)9.5 expression in spermatogonia and Leydig cells has been reported in several species. The stages of spermatogonia and Leydig cells expressing PGP9.5 vary depending on the species and reproductive stages. Thus, the objectives of this study were (1) to identify the localization of PGP9.5 in donkey testicular cells, and (2) to compare the expression patterns of PGP9.5 in donkey testicular cells between pre- and post-pubertal stages. Testes samples were collected following the routine field castration of six donkeys. Western blotting was performed to verify the cross-reactivity of the rabbit anti-human PGP9.5 antibody to donkey testes. Immunofluorescence was performed to investigate the expression pattern of PGP9.5 in testicular tissues at different reproductive stages. In Western blotting, the protein band of the PGP9.5 antibody appeared at approximately 27 kDa, whereas the band was not observed in the negative control treated with normal mouse IgG. In the pre-pubertal stage, the expression of deleted in azoospermia-like (DAZL) was found in some spermatogonia in pre-pubertal testicular tissues. However, the immunolabeling of PGP9.5 in testicular tissue was not observed in the seminiferous tubules. In stages 1 and 2, spermatogonia were immunolabeled with either PGP9.5 or DAZL. In contrast, PGP9.5 and DAZL were co-immunolabeled in some of the spermatogonia in stages 3 to 8. Interestingly, some Leydig cells were immunolabeled with PGP9.5 in both pre- and post-pubertal stages. In conclusion, the PGP9.5 antibody can be used as a tool to identify and isolate spermatogonia from seminiferous tubules.

Effects of Hemicastration on Testes and Testosterone Concentration in Stallions.

The endocrine system is critical to the maintenance of testicular function. The homeostasis of sex hormone levels is orchestrated by positive and negative feedback systems controlled by the hypothalamic-pituitary-gonadal axis. This study investigated the long-term effects of hemicastration on testicular size and function in stallions. Four Thoroughbred stallions, 4-6 years of age, were included in this study. Several parameters, including testicular weight and volume, plasma testosterone concentrations, VASA-positive germ cell populations and cross-sectional areas of the seminiferous tubules were compared in stallions that underwent two hemicastrations, approximately 11 months apart. The weights and volumes of testes harvested at the second hemicastration were significantly higher than those of testes collected at the first hemicastration. However, VASA-positive germ cell populations and the cross-sectional areas of seminiferous tubules were not significantly different between testes harvested at the first and second hemicastrations. Similarly, plasma testosterone concentrations measured weekly for 3 weeks before the first hemicastration, 3 weeks after the first hemicastration, and 3 weeks before the second hemicastration were not significantly different. Our results suggest that hemicastration results in compensatory enlargement of the remaining testis and compensatory steroidogenesis to maintain normal reproductive function in stallions.

The Lin28 Expression in Stallion Testes.

The molecular markers for specific germ cell stages can be utilized for identifying, monitoring, and separating a particular stage of germ cells. The RNA-binding protein Lin28 is expressed in gonocytes of human fetal testes. The Lin28 expression is restricted to a very small population of spermatogonial cells in human, mice, and monkey. The main objective of this study was to investigate the expression pattern of Lin28 in stallion testes at different reproductive stages. Based on the presence or absence of full spermatogenesis and lumina in seminiferous tubules, the testicular samples were categorized into two reproductive stages pre-pubertal and post-pubertal. We performed a reverse transcription polymerase chain reaction to confirm the presence of Lin28 mRNA in the testicular tissues and a western blot analysis to verify the cross-reactivity of rabbit Lin28 antibody with horse testicular tissue. For immunohistochemistry, Lin28 (rabbit anti-human), GATA4 (goat anti-human) or DAZL (goat anti-human) antibodies were used. The results of RT-PCR confirmed the expression of Lin28 mRNA in the stallion testes. The western blot analysis showed that the expression of 28 kDa Lin28 protein was localized in the cytoplasm of spermatogonia at both reproductive stages. The numbers of Lin28-positive germ cells per 1000 Sertoli cells in pre- and post-pubertal stages were 253 ± 8.66 and 29.67 ± 2.18, respectively. At both reproductive stages, all Lin28 positive cells showed no co-stained with GATA4 antibody, whereas only some of the Lin28-positive germ cells showed co-staining with DAZL antibody. The results from whole-mount staining showed that the Lin28 expression was limited to Asingle (As) and Apaired (Apr) spermatogonia. In conclusion, Lin28 might be utilized as a molecular marker for undifferentiated spermatogonial stem cells when used with DAZL antibody.

Reproductive stage-dependent effects of additional cryoprotectant agents for the cryopreservation of stallion germ cells.

The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1-1.5 yr, n=6) and post-pubertal (3-7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell population, and viable population were assessed after 1 and 3 months of cryopreservation. The viable UTF1-positive population of pre-pubertal stallion germ cells was also measured using immunocytochemistry after 1 and 3 months of cryopreservation. As expected, the viability, cell population, and viable cell population were significantly reduced after 1 and 3 months of cryopreservation. At the pre-pubertal stage, the addition of trehalose or PEG to 10% DMSO did not show any effect on the viability, cell population, viable cell population, or viable UTF1-positive germ cells at either 1 or 3 months after cryopreservation. However, at the post-pubertal stage, the viable population was significantly higher in germ cells that were cryopreserved with 5% or 10% PEG, than in the cells cryopreserved with 10% DMSO only. In conclusion, PEG at 5% or 10% added to 10% DMSO serves as an optimal cryoprotectant agent for the cryopreservation of germ cells from post-pubertal stallions.

The KIT is a putative marker for differentiating spermatogonia in stallions.

Putative markers have been discovered and are used to identify and separate certain lineage of spermatogonia. The KIT is a marker for differentiating spermatogonial stem cells in several species including mice and goats. The objectives of this study were (1) to investigate reproductive stage-dependent KIT expression patterns in stallions and (2) to identify spermatogonia subpopulations expressing KIT in stallion testes. To achieve these objectives, testicular samples were obtained during routine field castration of stallions. The reproductive stage of the stallions was classified as pre-pubertal (<1 year, n=3), pubertal (1-1.5 year, n=4), post-pubertal (2-3 year, n=6), or adult (4-8 year, n=6). For immunohistochemistry, KIT was used at a dilution of 1:200. In the pre-pubertal and pubertal stage, most germ cells were immunolabeled with KIT. In the post-pubertal and adult stages, immunolabeling of KIT was evident in the germ cells attached to the basement membrane of the seminiferous tubules with exception of some spermatogonia. Co-immunolabeling with KIT and deleted in azoospermia like (DAZL) showed different co-staining patterns, including KIT only, both KIT and DAZL), or DAZL positive germ cell populations alone. The KIT was not immunolabeled in Sertoli or Leydig cells at any reproductive stages. The result of Western blot analysis verified the cross-activity of the KIT antibody with horse testes tissue. In conclusion, KIT appears to be expressed in differentiating spermatogonia, and may be used to identify and isolate differentiating germ cells from stallions.

UTF1, a putative marker for spermatogonial stem cells in stallions.

Spermatogonial stem cells (SSCs) continuously undergo self-renewal and differentiation to sustain spermatogenesis throughout adulthood in males. In stallions, SSCs may be used for the production of progeny from geldings after cryopreservation and therapy for infertile and subfertile stallions. Undifferentiated cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans and rats. The main purposes of this study are to determine the following: 1) changes in the expression pattern of UTF1 at various reproductive stages of stallions, 2) subpopulations of spermatogonia that express UTF1. Testicular samples were collected and categorized based on the age of the horses as follows: pre-pubertal (<1 yr), pubertal (1-1.5 yr), post-pubertal (2-3 yr), and adult (4-8 yr). Western blot analysis was utilized to determine the cross-activity of the UTF1 antibody to horse testes tissues. Immunohistochemistry was conducted to investigate the UTF1 expression pattern in germ cells at different reproductive stages. Whole mount staining was applied to determine the subpopulation of UTF1-positive spermatogonia. Immunohistological analysis showed that most germ cells in the pre-pubertal and pubertal stages were immunolabeled with UTF1, whereas only a few germ cells in the basal compartment of the seminiferous tubule cross-sections of post-pubertal and adult tissues were UTF1-positive. No staining was observed in the Sertoli or Leydig cells at any reproductive stages. Whole mount staining showed that A(s), A(pr), and chains of 4, 8, 16 A(al) spermatogonia were immunolabeled with UTF1 in the post-pubertal stallion tubule. Isolated single germ cells were also immunolabeled with UTF1. In conclusion, UTF1 is expressed in undifferentiated spermatogonia, and its antibody can be used as a putative marker for SSCs in stallions.