Function In Stallions Research Articles

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.

Effects of implants containing the GnRH agonist deslorelin on testosterone release and semen characteristics in Shetland stallions

The hypothesis in this study was continuous treatment of stallions with the GnRH agonist deslorelin inhibits reproductive functions. A 2-week pre-experimental period was followed by an 11-week deslorelin implant treatment. Stallions received 4.7 (D1, n = 7), or 18.8 mg deslorelin (D2, n = 5) or remained untreated (C, n = 5). Libido, sperm motility, membrane integrity, DNA fragmentation, estrogen receptors, basal plasma testosterone and Anti Muellerian hormone (AMH) concentrations were evaluated once weekly during the treatment period. The testosterone response to the GnRH agonist buserelin and hCG was evaluated twice. In Week 2, stallions in Group C but not Groups D1 and D2 responded to buserelin with testosterone release (P < 0.001), while in Week 9, stallions in Group C and D1 but not D2 released testosterone after buserelin administration (group P < 0.01, week P = 0.01). Stallions of all groups responded to hCG with testosterone release at both times of hCG administration (P < 0.001). The AMH concentration was similar in all groups. Deslorelin thus reduced pituitary responsiveness to GnRH but only with a large dose and this effect persisted for several weeks. Total sperm count increased transiently with the D2 treatment but not in stallions of the D1 and C groups after implant insertion (time P < 0.01, time x group P < 0.001). The percentage of ESR1-positive spermatozoa decreased transiently in Group D2 (time P < 0.01, time × group P < 0.01). There was no difference among groups at any time during the study in percentage of motile and membrane-intact spermatozoa and sperm with DNA fragmentation. In conclusion, deslorelin implants modulate pituitary function in stallions but not to an extent that affects testicular function.

Effect of stallion age on the expression of LH and FSH receptors and aromatase P450 in equine male reproductive tissues.

The aim of the present study was to characterise receptors for LH and FSH (LHR and FSHR, respectively) and aromatase in epididymal and testicular tissue from stallions of different ages (prepubertal, young, mature and old). Gene and protein expression were assessed by real-time quantitative polymerase chain reaction (real-time qPCR), immunohistochemistry and multiple immunofluorescence labelling. There were no differences in LHR mRNA expression in epididymal and testicular parenchyma in stallions of different age. In contrast, expression of FSHR and CYP19A1 in caput, corpus and cauda epididymis and in testicular parenchyma increased with age (P<0.001). Immunolabelling for LHR, FSHR and aromatase was influenced by puberty. In postpubertal stallions, positive staining for LHR and aromatase was detected in Leydig cells, whereas protein expression of FSHR was present in Sertoli cells and primary spermatocytes. In prepubertal colts, staining for LHR, FSHR and aromatase was detected in seminiferous tubules. In epididymal tissue, aromatase was present in the cauda epididymis only, regardless of age. In conclusion, the results highlight the significance of gonadotropin action and oestrogen production for the maturation of male reproductive tissue in the horse. The presence of FSHR in the seminiferous tubules suggests effects of FSH on spermatogenesis in this species. The importance of oestrogen production for maintenance of testicular function in stallions was confirmed.

Increased cortisol release and transport stress do not influence semen quality and testosterone release in pony stallions

The use of breeding stallions for equestrian competitions requires that fertility is not negatively affected by competition or transport to the competition site. In this study, effects of cortisol release induced by road transport (600 km), adrenocorticotropic hormone (ACTH) administration (3 × 0.5 mg synthetic ACTH) and placebo treatment on semen quality and testosterone release were investigated in Shetland stallions (N = 13) using a crossover design. Saliva for cortisol and blood for testosterone analysis were collected for 10 weeks after treatments. Semen was collected daily for 5 days directly after treatments and twice weekly for another 9 weeks. Total sperm count, sperm morphology, motility, and membrane integrity were analyzed. We hypothesized that elevated cortisol decreases testosterone concentration and semen quality. Cortisol concentrations increased in response to transport and ACTH (P < 0.001) but not control treatments (peak concentration, transport: 7.6 ± 2.4, ACTH: 13.7 ± 1.5, control: 3.8 ± 0.9 ng/mL). No treatment effects on testosterone existed. Total sperm count decreased with daily semen collections in week 1 (P < 0.01) but did not differ between the treatments. The percentage of motile, progressively motile, membrane-intact, and morphologically defective spermatozoa did not change over time from Days 2 to 6, and there existed no differences between the treatments. In conclusion, road transport evoked a stress response which was mimicked by ACTH treatment. Both treatments had no effect on testosterone release and semen quality. Testicular function in stallions is apparently well protected against transiently elevated cortisol concentrations, and stallions can be transported over longer distances without negatively affecting their fertility.

Acute and Chronic Effects of a Contraceptive Compound RTI‐4587‐073(l) on Testicular Histology and Endocrine Function in Miniature Horse Stallions

The objective of this study was to evaluate acute endocrine effects as well as histological changes in testicular parenchyma induced by the contraceptive compound RTI-4587-073(l). Six miniature stallions were used in this experiment. The treatment group (n=3) received one oral dose of 12.5mg/kg of RTI-4587-073(l), and the control group (n=3) received placebo only. The stallions' baseline parameters (semen, testicular dimensions, endocrine values) were collected and recorded for 5weeks before treatment and for 6weeks after treatment. Multiple blood samples were collected for endocrine analysis. Testicular biopsies were obtained before treatment, 1day after treatment and every other week after treatment. Ultrasound exams were performed to monitor the dimensions of the stallions' testes. All stallions were castrated 6weeks after treatment. Sperm numbers, motility and percentage of morphologically normal sperm decreased (p<0.05), while the number of immature germ cells increased in ejaculates from treated animals (p<0.05). Serum concentrations of inhibin and follicle-stimulating hormone did not change. Testosterone concentrations initially transiently decreased (p<0.05) after administration of RTI-4587-073(l), and increased several days later (p<0.05). Testicular content of testosterone and estradiol 17-β was lower in treated stallions than in control stallions on Day 1 after treatment (p<0.05). Severe disorganization of the seminiferous tubules, significant loss of immature germ cells and complete depletion of elongated spermatids were observed in testicular biopsies obtained from treated stallions 1day, 2 and 4weeks after treatment. These changes were still present in the testicular samples taken from treated stallions after castration. The results of this study confirmed that RTI-4587-073(l) has antispermatogenic effects in stallions. Furthermore, we concluded that this compound causes acute sloughing of immature germ cells from the seminiferous tubules. RTI-4587-073(l) has significant but transient effects on Leydig cell function in stallions.

Indenopyride derivative RTI-4587-073(l): A candidate for male contraception in stallions

The objective of this study was to determine whether an indenopyridine derivative RTI-4587-073(l) was a good candidate for male contraception in horses. We hypothesized that a single administration of RTI-4587-073(l) causes significant suppression of testicular function in stallions without affecting sexual behavior. Three Miniature horse stallions received a single dose of 12.5 mg/kg RTI-4587-073(l) orally (group “treated”), whereas three other Miniature horse stallions received placebo only (group “control”). Semen was collected and evaluated from all stallions twice a week for three baseline weeks and 13 post-treatment weeks. Sexual behavior was video-recorded and analyzed. Testicular dimensions were measured using ultrasonography, and blood samples were drawn for endocrine evaluation once before treatment and once a week during the post-treatment period. Single administration of RTI-4587-073(l) caused severe oligoasthenozoospermia (low sperm number and low motility), shedding large numbers of immature germ cells in semen, and increased FSH concentrations in treated stallions. These effects were fully reversible within ∼71 days. However, libido and copulatory behavior remained unchanged throughout the entire experiment. We concluded that RTI-4587-073(l) was a promising candidate for male contraceptive in domestic stallions. Further research should be performed to test this compound for fertility control in wildlife and humans.

Expression of anti‐Müllerian hormone, CDKN1B, connexin 43, androgen receptor and steroidogenic enzymes in the equine cryptorchid testis

Cryptorchidism affects 2-8% of male horses and the affected testis undergoes a disruption of normal spermatogenesis. The underlying molecular changes are poorly understood in the cryptorchid equine testis. Compare the expression of anti-Müllerian hormone (AMH), anti-Müllerian hormone receptor (AMHR2), androgen receptor (AR), cyclin kinase inhibitor (CDKN1B), connexin 43 (Cx43), 3β hydroxysteroid dehydrogenase/Δ(5) -Δ(4) - isomerase (3βHSD), P450c17 hydroxylase/lyase (P450c17) and cytochrome P450 aromatase (P450arom) in the undescended testis of cryptorchid stallions with that of normal stallions. Undescended, abdominal testes from four cryptorchid stallions between 2 and 3 years of age were collected during routine castrations along with normally descended testes from normal stallions between 2 and 3 years of age (n = 7). Samples were analysed by immunohistochemistry and quantitative real-time PCR. Cryptorchid testes had increased AMH and AMHR2 immunolabelling when compared with normal testes, which indicates failure of maturation of Sertoli cells and/or lack of testosterone suppression. Failure of Sertoli cell maturation in the cryptorchid testis may also be attributed to AR abnormalities and/or a consequence of lack of testosterone suppression due to decreased 3βHSD. Cyclin-dependent kinase (CDKN1B) was not expressed in Sertoli cells of cryptorchid testes suggesting that Sertoli cells are still proliferating, which is also a characteristic of the immature testis. In addition, Cx43 expression is decreased in the cryptorchid testis, indicating a disruption in intercellular communication. Undescended testes of cryptorchid horses present characteristics of immaturity suggesting that the failure of Sertoli cell maturation may be a consequence of cryptorchidism. This study provides a better understanding of the effect of cryptorchidism on testicular function in stallions.

Regulation of testicular function in the stallion: An intricate network of endocrine, paracrine and autocrine systems

It is well established in many mammalian species, including the horse that normal testicular function is dependent upon a functional hypothalamic–pituitary–testicular (HPT) axis, which involves classic feedback mechanisms. The major HPT hormones involved in the stallion are gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), estrogens (Es) and inhibin (INH). Although prolactin (PRL) fluctuates with season in the stallion and both PRL and thyroid hormone (TH) affect reproduction in other male species, their effects on stallion reproduction have not been elucidated. Growth hormone (GH) in the stallion may be involved in sperm motility, production and secretion of insulin-like growth factor-1 (IGF-1) and LH-induced testosterone release. The action of these hormones and the products involved for normal spermatogenesis require cell to cell communication within the testis. The somatic cell types, Leydig, Sertoli and peritubular myoid cells, all support germ cell development, maturation and release into the seminiferous tubule lumen. The cell to cell crosstalk involves an intricate network of paracrine–autocrine systems that support the endocrine input to modulate cell function. In other male species, researchers have demonstrated the reproductive effects of such paracrine–autocrine factors as IGF-1, transferrin, androgens, estrogens, inhibin, insulin like peptide 3 (INSL3), beta-endorphin and oxytocin. The specific nature and relative contribution of these various factors on testicular function in fertile and subfertile stallions are under investigation. This review summarizes current information regarding the nature of the multiple endocrine–paracrine–autocrine systems that may be necessary for normal testicular function in the stallion.

Influence of long-term treatment with equine somato-tropin (EquiGen®) on gonadal function in stallions with poor semen quality

The aim of the present study was to investigate the spermatogenic and Leydig cell activity in stallions with impaired semen quality after treatment with equine somatotropin. Experiments were performed using 18 adult clinically healthy stallions with poor semen quality which did not pass breeding soundness evaluation. The animals were randomly divided into a treatment (n = 9) and a control (n = 9) group. Over a period of 90 days, nine stallions received a daily intramuscular injection of 10 mg recombinant equine somatotropin (EquiGen, BresaGen Limited, Adelaide, Australia) and 9 control animals were injected with the same amount of physiological saline solution. During and until 2 months after treatment, semen characteristics and daily sperm output as well as plasma testosterone concentrations were determined monthly in all stallions. In addition, testosterone concentration measurement after stimulation with hCG was performed in all animals immediately before and at the end of the treatment period as well as 2 months later. Our results demonstrate that equine somatotropin (EquiGen) given daily in a dose of 10 mg per animal during 90 days had no significant effect neither on plasma testosterone concentrations and hCG-induced testosterone release nor on semen quality parameters in adult stallions with poor semen characteristics.

Somatostatin treatment affects testicular function in stallions

This study investigated the regulation of growth hormone (GH) release in stallions and tested the hypothesis that the somatotrophic axis influences testicular function. Basal plasma GH concentrations, effects of an experimental decrease of GH release on testicular function and an opioidergic regulation of GH release were investigated in Shetland stallions ( n=6). No seasonal variations in plasma GH concentrations were found over a 12-month period. Treatment with the somatostatin analogue octreotid (100 mg twice daily over 10 days) caused a decrease in semen motility from 38.7±8.4% progressively motile spermatozoa before treatment to 18.3±5.4% on day 3 after end of treatment ( P<0.05). Values returned to 35.0±8.5% on day 5 after treatment. On the last day of octreotid treatment, a hCG stimulation test was performed (3000 IU hCG i.v.). The hCG-induced testosterone release was significantly higher in saline treated than in octreotid pretreated animals ( P<0.05). Neither plasma GH concentrations nor volume and density of ejaculates, total sperm count, or semen morphology were different between saline and octreotid treatments. Injection of the opioid antagonist naloxone (0.5 mg/kg) significantly increased GH release in June (from 1.1±0.3 ng/ml before to 3.7±2.2), while a minor and not significant increase occurred in January. In conclusion, our results indicate a non-seasonal basal GH release with a fine-modulation by season-dependent opioidergic mechanisms in the male horse. A transient decrease in semen motility and hCG-induced testosterone release following ocreotid treatment indicate a role of GH in the regulation of testicular function in stallions.

Measurements of reproductive function in stallions treated with trimethoprim-sulfamethoxazole and pyrimethamine

Objective To evaluate the effects of trimethoprim-sulfamethoxazole and pyrimethamine treatment on various measures of reproductive function in healthy pony stallions. Design Randomized complete block study. Animals 12 healthy, mature pony stallions. Procedure Stallions were assigned to treatment and control groups balanced for age and various characteristics of reproductive function. The treated group received trimethoprim-sulfamethoxazole and pyrimethamine for 90 days during summer and fall; the control group was not treated. Semen characteristics, sexual behavior, testicular volume, and sperm production efficiency were evaluated before treatment started and at 30-day intervals until 60 days after treatment ended. Results Effects of treatment were not detected for semen characteristics, testicular volume, sperm production efficiency, libido, erection, and quantitative measures of ejaculatory efficiency. At 30, 60, and 90 days, 4 of 6 treated stallions had unsteadiness upon mounting, clumsy or weak thrusting, failure to flex the back, and thready or inapparent ejaculatory pulses that resulted in dribbling of semen rather than forceful expulsion. Conclusions and Clinical Relevance Although treatment with trimethoprim-sulfamethoxazole and pyrimethamine may not affect semen quality, testicular volume, sperm production efficiency, erection, or libido of healthy stallions, treatment may induce changes in copulatory form and agility and alter the pattern and strength of ejaculation. Stallions that develop neurologic signs during treatment should be used with caution for breeding. (J Am Vet Med Assoc 1999;215:1317–1319)

Relationship between running speed, isoenzymes of serum creatine kinase and lactate dehydrogenase and left ventricular function in stallions.

The purpose of this study was to assess the possible relationship between maximal running speed, serum isoenzyme patterns of creatine kinase (CK) and lactate dehydrogenase (LDH) and echocardiographic indices of left ventricular function. A group of 15 healthy, 3-year-old Maremmano stallions were given a 100 day training programme. At the end of this the animals carried out a maximum speed test and were divided into 2 groups (A and B) according to whether or not they had attained a speed of 15 m/s. Venous blood samples were taken from each horse before exercise (T0), 2 min (T1) and 24 h (T2) after exercise. Total serum activity of CK and LDH was measured and their isoenzyme distribution pattern determined. The day before the speed test echocardiographic examination was carried out at rest to assess the left ventricular function by calculating telediastolic, telesystolic and stroke volume, ejection fraction and stroke index. Statistically significant differences were found for the CK isoenzyme pattern at T2, where Group A showed an increase in the MM fraction (P = 0.003) and a decrease in the MB fraction (P = 0.014). These changes were thought to be linked to an increased membrane leakage due to exercise and not to muscle fibre disruption because the CK and LDH total activities remained within the normal range. In Group A there was also greater left ventricular telediastolic volume (P = 0.044) and length (P = 0.033) at rest as well as a greater stroke index (P = 0.032). We concluded that the evaluation of CK pattern after exercise and of echocardiographic left ventricular function indices at rest made it possible to select for the fastest horses (Group A).

Endocrine basis for testicular function in the stallion

The specific nature and relative contribution of the various factors involved in the endocrine/paracrine/autocrine control of reproductive function in normal stallions are not well defined nor have they been elucidated in the idiopathic subfertile/infertile stallion. Over the last 9 years, work in our laboratory has been focused in characterizing the hypothalamic-pituitary-testicular axis in fertile, subfertile (idiopathic oligospermia) and infertile (idiopathic azoospermia) stallions. We have identified endocrine factors and mechanisms important for normal reproductive function, and demonstrated specific hormonal changes in pituitary and testicular function between fertile, subfertile and infertile stallions. Recent evidence suggests that the primary defect is at the level of the testes. The nature of the dysfunction does not appear to involve changes in LH receptor binding kinetics but may be related to post-receptor mechanisms.

Androgen and oestrogen response to a single injection of hCG in cryptorchid horses

Androgen (testosterone and androstenedione) and oestrogen (oestradiol -17 beta and oestrone) concentrations were measured by radio-immunoassay in the peripheral plasma of two geldings (five-years-old), three bilateral cryptorchids (two, two and a half, and five-years-old) and three normal intact stallions (four, five and five and a half-years-old) before and after a single injection of 10,000 iu human chorionic gonadotrophin (hCG). In the stallions, hCG administration resulted in an immediate sharp increase of conjugated oestrogens and a more gradual increase of unconjugated androgens. In the cryptorchids, the unconjugated androgens increased following a similar pattern to that observed in the stallions, but reached lower peak values, whereas the conjugated oestrogens showed only a very slight increase. In the stallions and cryptorchids, the maximum oestrogen levels were reached two days after injection, whereas the maximal levels for androgens were reached a day later. In the geldings, hCG injection had no effect on plasma steroid levels. It is suggested that the measurement of unconjugated androgens (testosterone or/and androstenedione) before and three days after intravenous injection of 10,000 iu hCG may prove useful for the diagnosis of cryptorchidism or exploration of testicular function in stallions.