Expression pattern of germ cell markers in cryptorchid stallion testes.

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.

Efficacy, safety, and pharmacokinetics of teduglutide in adult Japanese patients with short bowel syndrome and intestinal failure: two phase III studies with an extension.

PURPOSE: The short- and long-term efficacy, safety, and pharmacokinetics of teduglutide were analyzed in adult Japanese patients with short bowel syndrome and intestinal failure (SBS-IF). METHODS: Patients received teduglutide 0.05 mg/kg/day in clinical trials (TED-C14-004, SHP633-306, and extension SHP633-307). Data were analyzed at 24 weeks and an interim data cut-off of 4.5 years. RESULTS: The parenteral support (PS) volume decreased by ≥ 20% for 9/18 patients at 24 weeks and in all 11 patients by data cut-off in SHP633-307. The mean (standard deviation) PS volume decreased from baseline at 24 weeks in TED-C14-004 (-30.1 ± 25.9%) and SHP633-306 (-25.6 ± 25.5%), and at data cut-off in SHP633-307 (-57.08 ± 28.49%). Teduglutide was absorbed quickly. The adverse events were consistent with the underlying disease and known adverse drug reactions. Anti-teduglutide antibody titers declined with long-term treatment. CONCLUSIONS: In Japanese adults with SBS-IF, teduglutide treatment was associated with clinically meaningful reductions in PS requirements, similar to findings in prior international studies. No new safety concerns specific to the Japanese SBS-IF patient population were identified with short- or long-term teduglutide treatment. Anti-teduglutide antibody titers disappeared in most Japanese adults with long-term treatment. These results constitute the longest evaluation of teduglutide treatment within clinical trials reported to date.

Effects of Stocking Density on Stress, Hematological Responses, and Growth of Black Rockfish Sebastes schlegelii.

The objective of the current study was to evaluate the effect of stocking density on juvenile Black Rockfish Sebastes schlegelii (average weight = 12 g) in terms of stress, hematological responses, and growth performance during a 4-month growth trial in a flow-through system. The initial stocking densities were 1.3 kg/m3 (low), 1.8 kg/m3 (medium), and 2.3 kg/m3 (high), and the final densities were 4.9 kg/m3 (low), 5.6 kg/m3 (medium), and 6.3 kg/m3 (high). At the end of the trial, the high stocking density significantly affected growth characteristics, levels of growth hormone and insulin-like growth factor 1, and hematological indices (hematocrit, red blood cell count, and hemoglobin level) compared to the medium and low stocking densities. The plasma cortisol and immunoglobulin-M levels were significantly higher at the high density than at the other two densities. Taken together, while the low and medium stocking densities (final densities of up to 5.6 kg/m3 ) did not affect stress and hematological indices or growth, the high stocking density (final density of 6.3 kg/m3 ) significantly impacted those variables, which suggests an allostatic load at that density. Thus, the use of a final stocking density less than 6.3 kg/m3 should be considered to avoid compromising the stress and health condition and growth of Black Rockfish at this size and temperature range.

A retrospective study of outcomes of device-associated osteomyelitis treated with daptomycin.

BACKGROUND: Daptomycin appears well tolerated and effective for osteomyelitis treatment. However, limited data exist regarding daptomycin use for treatment of device-associated osteomyelitis (DAO). METHODS: We used a retrospective, observational database (Cubicin® Outcomes Registry and Experience [CORE® 2007-2009]) that assessed patients treated with daptomycin to evaluate the characteristics of patients with DAO, outcomes after daptomycin treatment, and safety of daptomycin in this setting. Information from 54 institutions for patients with prosthetic joint infection (PJI) and other hardware-associated osteomyelitis (OHAO) who received daptomycin from January 2007 to December 2008 with follow-up data in 2009 was collected using a standardized data collection form. RESULTS: Eighty-two patients receiving daptomycin were identified in CORE 2007-2009; 48 patients (59 %) had follow-up data. Sixty-seven percent of patients had received a previous antibiotic. Surgical intervention was similar between the 2 groups: PJI, 22 of 27 (82 %) and OHAO, 17 of 21 (81 %). However, device removal or replacement was more frequent in the PJI patients (17 of 27, 63 %) than in the OHAO patients (8 of 21, 38 %). Clinical success was reported in 22 of 27 (82 %; 95 % confidence interval [CI], 62-94 %) patients with PJI and 18 of 21 (86 %; 95 % CI, 64-97 %) patients with OHAO at follow-up (13-402 days). Adverse events occurred in 8 of 50 (16 %) patients in the safety population and did not differ by daptomycin dose. CONCLUSION: Daptomycin appeared effective and well tolerated in patients with DAO, including PJI or OHAO.

Ceftolozane/Tazobactam Plus Metronidazole for Complicated Intra-abdominal Infections in an Era of Multidrug Resistance: Results From a Randomized, Double-Blind, Phase 3 Trial (ASPECT-cIAI).

BACKGROUND: Increasing antimicrobial resistance among pathogens causing complicated intra-abdominal infections (cIAIs) supports the development of new antimicrobials. Ceftolozane/tazobactam, a novel antimicrobial therapy, is active against multidrug-resistant Pseudomonas aeruginosa and most extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae. METHODS: ASPECT-cIAI (Assessment of the Safety Profile and Efficacy of Ceftolozane/Tazobactam in Complicated Intra-abdominal Infections) was a prospective, randomized, double-blind trial. Hospitalized patients with cIAI received either ceftolozane/tazobactam (1.5 g) plus metronidazole (500 mg) every 8 hours or meropenem (1 g) every 8 hours intravenously for 4-14 days. The prospectively defined objectives were to demonstrate statistical noninferiority in clinical cure rates at the test-of-cure visit (24-32 days from start of therapy) in the microbiological intent-to-treat (primary) and microbiologically evaluable (secondary) populations using a noninferiority margin of 10%. Microbiological outcomes and safety were also evaluated. RESULTS: Ceftolozane/tazobactam plus metronidazole was noninferior to meropenem in the primary (83.0% [323/389] vs 87.3% [364/417]; weighted difference, -4.2%; 95% confidence interval [CI], -8.91 to .54) and secondary (94.2% [259/275] vs 94.7% [304/321]; weighted difference, -1.0%; 95% CI, -4.52 to 2.59) endpoints, meeting the prespecified noninferiority margin. In patients with ESBL-producing Enterobacteriaceae, clinical cure rates were 95.8% (23/24) and 88.5% (23/26) in the ceftolozane/tazobactam plus metronidazole and meropenem groups, respectively, and 100% (13/13) and 72.7% (8/11) in patients with CTX-M-14/15 ESBLs. The frequency of adverse events (AEs) was similar in both treatment groups (44.0% vs 42.7%); the most common AEs in either group were nausea and diarrhea. CONCLUSIONS: Treatment with ceftolozane/tazobactam plus metronidazole was noninferior to meropenem in adult patients with cIAI, including infections caused by multidrug-resistant pathogens. CLINICAL TRIALS REGISTRATION: NCT01445665 and NCT01445678.

Immunolocalization of insulin-like growth factor-I (IGF-I) and its receptors (IGF-IR) in the equine epididymis.

Insulin-like growth factor plays a paracrine/autocrine role in regulating testicular function in the stallion, but its presence in the equine epididymis remains unknown. The aim of this study was to test the hypothesis that insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) are localized in the caput, corpus, and cauda of the epididymis in an age-dependent manner. Immediately after castration, epididymal tissue was fixed, paraffin-embedded, and processed for immunohistochemistry (IHC). Western blot was also performed using equine epididymal extracts to verify the specificity of the antibodies against IGF-I and IGF-IR. Immunolabeling of IGF-I was observed in the cytoplasm of principal and basal cells in the caput, corpus, and cauda at the pre-pubertal (3-7 months), pubertal (12-18 months), post-pubertal (2-4 years), and adult stages (4.5-8 years). Immunolabeling of IGF-IR was observed in the cytoplasm of principal cells in all regions of the epididymis in each age group. Immunolabeling of IGF-IR was also detected in the cytoplasm of basal cells from animals of all ages. Bands observed by Western blot corresponded to the molecular weights of IGF-I and IGF-IR, ~23 kDa and 95 kDa, respectively. These results suggest that IGF-I might function as an autocrine and/or paracrine factor during the development, maintenance and/or secretions of the stallion epididymis.

Changes in characteristics of spermatogonial stem cells in response to heat stress in stallions.

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.

Bifenthrin Diminishes Male Fertility Potential by Inducing Protein Defects in Mouse Sperm.

A synthetic pyrethroid pesticide, bifenthrin, has been commonly used as an effective exterminator, although the rise in its usage has raised concerns regarding its effects on the environment and public health, including reproduction, globally. The current study investigated the function-related molecular disparities and mechanisms in bifenthrin-exposed sperm cells and the underlying mechanism. Therefore, epididymal spermatozoa were released, and various concentrations of bifenthrin were treated (0.1, 1, 10, and 100 µM) to evaluate their effects on sperm. The findings showed that although bifenthrin had no effect on sperm viability, various other sperm functions (e.g., motility, spontaneous acrosome reaction, and capacitation) related to male fertility were decreased, commencing at a 1 µM treatment. Molecular studies revealed nine differentially expressed sperm proteins that were implicated in motile cilium assembly, sperm structure, and metabolic processes. Furthermore, bifenthrin affected sperm functions through abnormal diminution of the expression of specific sperm proteins. Collectively, these findings provide greater insights into how bifenthrin affects male fertility at the molecular level.

Effects of insulin-like growth factor-1 on the proliferation and apoptosis of stallion testicular cells under normal and heat stress culture conditions.

This study investigated the effect of heat stress on stallion testicular cells (TCs) and the effect of insulin-like growth factor (IGF)-1 on TC viability, proliferation, and apoptosis, including different stages of germ cells. TCs were divided into control or treatment groups with 0.01, 0.1, 1, 10, and 100 ng/mL of recombinant human IGF-1 (rhIGF-1) for 24 h at 34 °C and 37 °C. The population and viability were measured before and after treatment. The effects of rhIGF-1 on TC viability, proliferation, and apoptosis were determined using RT-qPCR. Proliferating cell nuclear antigen (PCNA) and marker of proliferation Ki-67 (MKI-67) were used as proliferation markers. Myeloid leukemia-1 (MCL-1) was used as an antiapoptotic marker. BCL2 antagonist/killer-1 (BAK-1) was used as a proapoptotic marker. The relative abundance of mRNA transcript of undifferentiated cell transcription factor 1 (UTF-1), protein gene product 9.5 (PGP9.5), and deleted in azoospermia-like (DAZL), was measured for spermatogenesis progression. TCs treated with 1 ng/mL rhIGF-1 at 34 °C exhibited the highest viability. Significant upregulation of the relative abundance of mRNA transcript of PCNA, MKI-67, and MCL-1 was observed in treated TCs compared with untreated TCs; however, BAK-1 was significantly downregulated in treated TCs. Germ cells treated with 1 ng/mL rhIGF-1 exhibited the highest relative abundance of mRNA transcript of UTF-1 and DAZL, whereas TCs exposed to 0.1 ng/mL showed the highest PGP9.5 level. These data confirm that heat stress in stallions decreases TC viability. These findings may help identify a basal IGF-1 level for TC proliferation and apoptosis during heat stress-induced testicular degeneration in stallions.

Heat stress and stallion fertility.

The threat posed by increased surface temperatures worldwide has attracted the attention of researchers to the reaction of animals to heat stress. Spermatogenesis in animals such as stallions is a temperature-dependent process, ideally occurring at temperatures slightly below the core body temperature. Thus, proper thermoregulation is essential, especially because stallion spermatogenesis and the resulting spermatozoa are negatively affected by increased testicular temperature. Consequently, the failure of thermoregulation resulting in heat stress may diminish sperm quality and increase the likelihood of stallion infertility. In this review, we emphasize upon the impact of heat stress on spermatogenesis and the somatic and germ cells and describe the subsequent testicular alterations. In addition, we explore the functions and molecular responses of heat shock proteins, including HSP60, HSP70, HSP90, and HSP105, in heat-induced stress conditions. Finally, we discuss the use of various therapies to alleviate heat stress-induced reproductive harm by modulating distinct signaling pathways.

Association between the plasma concentration of melatonin and behavioral temperament in horses.

Aggression in horses may cause serious accidents during riding and non-riding activities. Hence, predicting the temperament of horses is essential for selecting suitable horses and ensuring safety during the activity. In certain animals, such as hamsters, plasma melatonin concentrations have been correlated with aggressive behavior. However, whether this relationship applies to horses remains unclear. To address this research gap, this study aimed to evaluate differences in the plasma melatonin concentrations among horses of different breeds, ages, and sexes and examine the correlation between plasma melatonin concentrations and the temperament of the horses, including docility, affinity, dominance, and trainability. Blood samples from 32 horses were collected from the Horse Industry Complex Center of Jeonju Kijeon College. The docility, affinity, dominance, and trainability of the horses were assessed by three professional trainers who were well-acquainted with the horses. Plasma melatonin concentrations were measured using an enzyme-linked immunosorbent assay. The consequent values were compared between the horses of different breeds, ages, and sexes using a three-way analysis of variance and least significant difference post hoc test. Linear regression analysis was employed to identify the relationship between plasma melatonin concentrations and docility, affinity, dominance, and trainability. The results showed that the plasma melatonin concentrations significantly differed with breeds in Thoroughbred and cold-blooded horses. However, there were no differences in the plasma melatonin concentrations between the horse ages and sexes. Furthermore, plasma melatonin concentrations did not exhibit a significant correlation with the ranking of docility, affinity, dominance, and trainability.

Daptomycin therapy for osteomyelitis: a retrospective study.

BACKGROUND: Daptomycin is a rapidly bactericidal agent with broad coverage against Gram-positive organisms, including Staphylococcus aureus, the most frequent cause of osteomyelitis. The objective of this study was to describe the clinical outcome of patients with non-hardware associated osteomyelitis, and the safety profile of daptomycin in the treatment of these infections. METHODS: All patients with osteomyelitis, excluding concurrent orthopedic foreign body infections, treated with daptomycin and identified between 2007-2008 in a retrospective, multicenter, observational registry, were included. Investigators assessed patient outcome (cured, improved, failed, non-evaluable) at the end of daptomycin therapy. Patients with a successful outcome at the end of daptomycin therapy were reassessed in 2009. All patients were included in the safety analysis; evaluable patients were included in the efficacy analysis. Data was assessed using descriptive statistics. A Kaplan Meier analysis was used to assess time to clinical failure. RESULTS: Two-hundred and nine osteomyelitis patients successfully completed daptomycin therapy in 2007-2008, 71 of which (34%) had a follow-up visit in 2009 and had an evaluable clinical outcome. The median (min, max) daptomycin dose and duration were 6 mg/kg (4, 10) and 42 days (1, 88), respectively. Of the 52 patients with a documented pathogen, S. aureus was the most common (42%); primarily methicillin-resistant S. aureus. All patients were included in the safety analysis; evaluable patients were included in the efficacy analysis. Clinical resolution was reported in 94% (CI - 86.2%, 98.44%) of patients. A Kaplan Meier analysis of time to clinical failure showed that approximately 85% (CI - 64%, 95%) of patients had a continued successful outcome at the time of re-evaluation. Eighteen patients (25%) in the safety population experienced an adverse event; 13 patients (18%) had an adverse event that was possibly-related to daptomycin treatment. CONCLUSIONS: Daptomycin appears to be an effective therapeutic choice with an acceptable safety profile in the management of osteomyelitis that does not involve hardware.

Functions of somatic cells for spermatogenesis in stallions.

Spermatogenesis and testis development are highly structured physiological processes responsible for post-pubertal fertility in stallions. Spermatogenesis comprises spermatocytogenesis, meiosis, and spermiogenesis. Although germ cell degeneration is a continuous process, its effects are more pronounced during spermatocytogenesis and meiosis. The productivity and efficiency of spermatogenesis are directly linked to pubertal development, degenerated germ cell populations, aging, nutrition, and season of the year in stallions. The multiplex interplay of germ cells with somatic cells, endocrine and paracrine factors, growth factors, and signaling molecules contributes to the regulation of spermatogenesis. A cell-to-cell communication within the testes of these factors is a fundamental requirement of normal spermatogenesis. A noteworthy development has been made recently on discovering the effects of different somatic cells including Leydig, Sertoli, and peritubular myoid cells on manipulation the fate of spermatogonial stem cells. In this review, we discuss the self-renewal, differentiation, and apoptotic roles of somatic cells and the relationship between somatic and germ cells during normal spermatogenesis. We also summarize the roles of different growth factors, their paracrine/endocrine/autocrine pathways, and the different cytokines associated with spermatogenesis. Furthermore, we highlight important matters for further studies on the regulation of spermatogenesis. This review presents an insight into the mechanism of spermatogenesis, and helpful in developing better understanding of the functions of somatic cells, particularly in stallions and would offer new research goals for developing curative techniques to address infertility/subfertility in stallions.

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.

Development of a foaling alarm system using an accelerometer.

Horse breeders suffer massive economic losses due to dystocia, abortion, and stillbirths. In Thoroughbred mares, breeders often miss the foaling process because approximately 86% of the foaling events occur from 19:00 to 7:00; consequently, breeders cannot assist mares experiencing dystocia. To solve this problem, various foaling alarm systems have been developed. However, there is a need to develop a new system to overcome the shortcomings of the existing devices and improve their accuracy. To this end, the present study aimed to (1) develop a novel foaling alarm system and (2) compare its accuracy with that of the existing Foalert™ system. Specifically, eighteen Thoroughbred mares (11.9 ± 4.0 years old) were included. An accelerometer was used to analyze specific foaling behaviors. Behavioral data were transmitted to a data server every second. Depending on the acceleration value, behaviors were automatically classified by the server as categorized behaviors 1 (behaviors without change in body rotation), 2 (behaviors with sudden change in body rotation, such as rolling over), and 3 (behaviors with long-term change in body rotation, such as lying down laterally). The system was designed to alarm when the duration of categorized behaviors 2 and 3 was 12.9% and that of categorized behavior 3 was 1% during 10 min. The system measured the duration of each categorized behavior every 10 min and transmitted an alarm to the breeders when foaling was detected. To confirm its accuracy, the foaling detection time of the novel system was compared with that of Foalert™. The novel foaling alarm system and Foalert™ alarmed foaling onset respectively 32.6 ± 17.9 and 8.6 ± 1.0 min prior to foal discharge, and the foaling detection rate of both systems was 94.4%. Therefore, the novel foaling alarm system equipped with an accelerometer can precisely detect and alert foaling onset.

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.

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.

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.

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.

The Effects of Androstenone on the Plasma Serotonin, ß-Endorphin, and Cortisol Concentrations in Thoroughbred Horses.

Androstenone influences the changing behaviors of animals. Previous studies discovered that an androstenone receptor was expressed in horses and treatment with androstenone induced horses to be more compliant. As changes in the level of neuroendocrine factors result in animal behavioral changes, the objective of the study was to monitor the changes in the concentrations of 5-HT, ß-endorphin, and cortisol in response to androstenone. Eight thoroughbred horses (five mares and three geldings) were treated with androstenone diluted in jojoba oil (10 µg/mL) and only oil for a control cross-overly. A handler applied the treatments to the horses' nostril and rubbed for 5 s. Blood samples were collected before, 15, 30, and 60 min after each treatment. The concentrations of each neurotransmitter were analyzed by enzyme-linked immunosorbent assay. The concentrations of each neurotransmitter after the treatment were compared to its baseline concentration. The concentration of 5-HT of the androstenone-treated horses remained consistent throughout the experiment, while the concentration of the control group significantly decreased over time. The plasma concentration of ß-endorphin in the androstenone-treated group also remained constant, whereas the concentration increased in the control group. Cortisol levels did not change in either the treated or untreated groups. An androstenone treatment triggers changes in the secretion of 5-HT and ß-endorphin in horses.