Infertile Stallions Research Articles

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.

Anti-Müllerian hormone as a biomarker for acute testicular degeneration caused by toxic insults to stallion testes

Recently, anti-Müllerian hormone (AMH) was validated as a reliable marker of testicular damage caused by various chemotherapy drugs in humans and in mice. In horses, the reference values of AMH concentrations in normal stallions, during different seasons of a year, have been recently reported. However, this hormone was not evaluated in subfertile or infertile stallions with testicular damage. Therefore, the objective of this study was to investigate the effects of experimentally induced testicular degeneration on the concentration of AMH in stallions. Severe but transient testicular degeneration was induced in six Miniature horse stallions, in two, separate experiments (three stallions in each experiment), by the administration of a single dose of the contraceptive compound RTI-4587-073(l). Six different stallions served as controls (three stallions in each experiment). Treated and control stallions were switched between the experiments. Concentrations of AMH were determined in 78 samples of blood plasma collected during the first experiment and in 24 samples collected during the second experiment. Furthermore, the expression of AMH in 30 samples of testicular parenchyma, collected from these stallions during the second experiment, was also evaluated, using immunohistochemistry (IHC) and objectively analyzed using computerized methods. During the first experiment, the concentrations of AMH in blood increased significantly in treated stallions (P < 0.05), reaching a 62–151% change from the baseline by day 10 after treatment, before gradually decreasing to the pretreatment levels. There was no change in blood AMH concentration in control stallions. Only a trend to increase AMH concentration was observed in treated stallions during the second experiment (P = 0.055). The labeling for immunoreactive AMH in the Sertoli cells gradually increased after treatment, which was confirmed by the significantly increased IHC optic density score value (P < 0.05) and significantly decreased percentage contribution of negative pixels at fourth week after treatment (P < 0.05). We concluded that AMH is a promising candidate as a biomarker of testicular damage in stallions caused by toxic insults that lead to testicular degeneration.

377 EMBRYO PRODUCTION BY OVUM PICKUP-INTRACYTOPLASMIC SPERM INJECTION-IVC IN AN EQUINE OVUM PICKUP PROGRAM USING SEMEN FROM FERTILE AND INFERTILE STALLIONS

The introduction in equine reproduction of ovum pickup (OPU) combined with intracytoplasmic sperm injection (ICSI), IVC, and embryo transfer, has allowed for the production of offspring from donors and stallions that could not reproduce by conventional techniques. For this reason, we used in our OPU-ICSI-IVC program both fertile stallions and stallions with field records of low or no fertility. Overall, 805 and 584 OPU oocytes were fertilized with sperm from fertile and infertile stallions, respectively. Cleavage rate was statistically lower in the latter group (65.94 v. 59.24%, chi square test; P &lt; 0.05) but embryo development was similar (11.67 v. 8.20% blastocysts/injected oocytes, chi-square test). In order to further investigate the stallion effect on embryo development, we selected 3 stallions with low (A) or no (B, C) fertility in the field and we compared the results of the OPU program with embryo development obtained using oocytes recovered from abattoir ovaries and matured, fertilized, and cultured in vitro as the OPU oocytes. Part of the abattoir oocytes was fertilized with a stallion with known high fertility both in vivo and in vitro (abattoir fertile). Overall, the results (shown in the table) suggest a reduction in the efficiency of stallions A, B, and C compared with to the fertile stallion used as control (10.79, 7.69, and 5.0% v. 17.35%, respectively). For stallions A and B, the efficiency was further reduced in the OPU setting, indicating that the female component can play a role in the overall efficiency of the procedure. In particular, 4 mares out of 8 had a history of no pregnancy and all mares had some rate of inbreeding with the respective stallion used for the ICSI. Instead, the oocytes from the abattoir ovaries were collected in large pools from several mares, representing an average oocyte quality, and the mares were of different breed than the stallions. All data were analyzed by chi-square test and significance was set at P &lt; 0.05. In conclusion, we demonstrated that, for those stallions in which fertility in the field is low or absent, OPU-ICSI-IVP is a suitable choice to obtain embryos, although the efficiency is variable depending not only on the stallion but also on the origin of the oocytes. Table 1.Stallion effect on embryo development of ovum pickup (OPU) and abattoir oocytes This work was supported by Fondazione Cariplo and Regione Lombardia.

Low-dose insemination—Why, when and how

The typical dose for insemination into the uterine body of the mare is >300 × 10 6 progressively motile spermatozoa (PMS) and an insemination dose of >200 × 10 6 PMS is recommended for frozen–thawed semen. Low-dose insemination techniques allow for a drastic reduction in the numbers of spermatozoa required to achieve pregnancy. Acceptable pregnancy rates can be achieved with doses ranging from 1 to 25 × 10 6 PMS in volumes ranging from 20 to 1000 μL. Two techniques have been described: hysteroscopic insemination and transrectally guided deep horn insemination using a pipette. Similar pregnancy rates can be attained by either method when 5 × 10 6 PMS are used. Hysteroscopic insemination may provide an advantage when the dose is 1–3 × 10 6 PMS. These techniques have the potential to make more efficient use of frozen–thawed or sex-sorted semen from certain stallions. The use of low-dose insemination to improve fertility of infertile stallions warrants further investigation.

Advanced fertility diagnosis in stallion semen using transmission electron microscopy

Routine semen analysis of stallions is based on light microscopy (LM). However, there are still a number of animals that are subfertile or even infertile not being identified with conventional semen analysis. The objective of this study was to investigate the suitability of transmission electron microscopy (TEM) for advanced fertility diagnosis in stallion. We examined ejaculates of 46 stallions with known fertility. Animals were divided into three different groups: group 1, fertile stallions (pregnant mares ≥ 70%, n = 29); group 2, subfertile stallions (pregnant mares 10–69%, n = 14); group 3, infertile stallions (pregnant mares < 10%, n = 3). Ejaculates were collected in spring 2002. Conventional semen analysis (volume, sperm concentration, motility, live:dead ratio and percentage of morphologically normal sperm) was immediately performed after semen collection. Ultrastructural analysis included the evaluation of 200 acrosomes, heads, midpieces and cross-sections of tails as well as 100 longitudinal sections of tails from every ejaculate. Using LM, we found a significant increase of morphological deviations from 24.5% ( x ˜ ) in group 1 to 34.5% in group 2 and 73.5% in group 3. Using TEM, we found a significant increase of detached acrosomes from 6.1% in group 1 to 7.6% in group 2 and 21.4% in group 3. Deviations in tubule pattern were also increased (but not significant) from 2.7% in fertile and 2.8% in subfertile to 11.4% in infertile stallions as well as multiple tails from 1.9% in fertile to 2.0% in subfertile and 8.9% in infertile. Our data indicate that TEM is suitable for advanced fertility diagnostic in stallions, giving a connection between fertility and morphology. It suggests that the most likely reason for sub- and infertility in stallion in case of increased LM pathomorphology of semen are acrosomal alterations, especially detached acrosomes.

Different CREM-isoform gene expression between equine and human normal and impaired spermatogenesis

Histone-to-protamine exchange causes chromatin condensation ceasing gene expression in elongating spermatids. Gene expression of protamines is regulated by the transcription factor cAMP-responsive element modulator (CREM). Altered CREM expression results in male infertility, as shown by CREM-knock-out mice being sterile due to round spermatid maturation arrest and patients exhibiting round spermatid maturation arrest revealing a lack or substantial reduction of both CREM-mRNA and CREM-protein. Similar defects in histone-to-protamine exchange have been suggested in infertile stallions exhibiting enlarged sperm heads. The CREM-gene consists of 14 exons. Alternative exon splicing results in the production of both activator and repressor proteins. To further clarify the role of different CREM-isoforms for male infertility, the expression pattern of various CREM-isoforms during equine and human normal and impaired spermatogenesis was investigated by RT-PCR. Stallions with normal spermatogenesis expressed six activators and three repressors. In men three activators and seven different repressors were detected. In one stallion and patients with impaired spermatogenesis, only repressors were found. It is concluded that (i) stallion and man reveal a different CREM expression pattern, (ii) the expression of CREM activators is a prerequisite for normal spermatogenesis, and (iii) the lack of CREM activator expression results in male infertility.

An overview of low dose insemination in the mare.

The need for relatively high numbers of spermatozoa for artificial insemination limits our application of recently available technologies such as sex-sorted semen. The fertility of two different methods of low dose insemination using fresh, frozen and sex-sorted semen are compared in this overview. Satisfactory conception rates are described using very low doses of spermatozoa inseminated by either hysteroscopic or deep uterine insemination methods, proving the stallion is fully fertile. The hysteroscopic method appears to give higher conception rates when inseminating fewer than 5 x 10(6) spermatozoa and is therefore, the preferred method of insemination for sex-sorted spermatozoa. However, hysteroscopic deposition of low numbers of spermatozoa from infertile stallions does not appear to improve their fertility.

Effects of age, season, and fertility status on plasma and intratesticular immunoreactive (IR) inhibin concentrations in stallions

The nature of the relationship between inhibin and reproductive function in the stallion is yet to be elucidated. Blood and testes from 51 light horse stallions ranging in age from 2 mo to 25 years were collected during the breeding and nonbreeding seasons to study the effects of testicular maturation, aging, season, and fertility status on peripheral and intratesticular concentrations of Ir inhibin and other reproductive hormones. Of the 51 stallions, 12 age-matched stallions (6 fertile, 3 subfertile, and 3 infertile) were used in the fertility study. Blood samples were taken before castration and plasma stored at −20°C for analysis of Ir inhibin, luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), estradiol (E 2), and estrogen conjugates (EC) by radioimmunoassay (RIA). Testes were homogenized and testicular extracts prepared and frozen at −70°C for analysis of Ir inhibin, T, E 2, and EC by RIA. Plasma concentrations of Ir inhibin, LH, FSH, T, E 2, and EC and intratesticular concentrations of Ir inhibin, T, E 2, and EC increased with age (P < 0.01). The most dramatic effect appeared to be during testicular maturation. An aging effect was not observed in adult stallions. A seasonal effect was not detected for any of the plasma hormones, whereas for the intratesticular hormones the only change noted was an increase in T in the nonbreeding season (P < 0.05). Plasma Ir inhibin, E 2, and EC were lower (P < 0.01) and gonadotropins higher (P < 0.05) in infertile stallions. Plasma T levels did not change. Intratesticular Ir inhibin concentrations tended to be lower (P < 0.1) in subfertile stallions and significantly lower (P < 0.01) in infertile stallions, whereas intratesticular steroid levels were not different among the three groups. In conclusion, plasma and intratesticular Ir inhibin concentrations seem to be affected by testicular maturation and fertility status.

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.

HCG binding to the testicular LH receptor is similar in fertile, subfertile, and infertile stallions.

Recent evidence in our laboratory suggests that the cause of idiopathic subfertility/infertility in breeding stallions may originate in the testes at the luteinizing hormone (LH) receptor or postreceptor level. The objective of this research was to determine if LH receptor binding activity is altered in subfertile and infertile stallions. Six fertile, three subfertile, and three infertile stallions, ages 11-23 years, were classified according to normal semen parameters and pregnancy rates and then castrated in the breeding season. Blood was collected prior to castration, and plasma was stored until analyzed for LH, follicle stimulating hormone (FSH), estrogen conjugates (EC), estradiol (E2), testosterone (T), and inhibin (I) by radioimmunoassay (RIA). Testicular cell membranes were prepared and snap-frozen until analyzed for LH binding activity by radioreceptorassay (RRA) using increasing amounts of I125 human chorionic gonadotropin (hCG). Luteinizing hormone receptor numbers and affinity constants were determined by Scatchard analysis. Plasma LH, FSH, EC, E2, and T levels did not differ between fertile and subfertile stallions, but LH and FSH were significantly higher (P < 0.05) and EC, E2, T, and I levels were significantly lower (P < 0.05) in infertile stallions as compared to fertile and subfertile stallions. Receptor number (Rt) and affinity constants Ka were similar (P > 0.05) between fertile (Rt = 9.44 x 10(-11) M, Ka = 0.300 x 10(10) M-1), subfertile (Rt = 13.02 x 10(-11) M, Ka = 0.194 x 10(10) M-1), and infertile (Rt = 7.65 x 10(-11) M, Ka = 0.380 x 10(10) M-1) stallions. In conclusion, these data suggest that an endocrine dysfunction in the testes of stallions with poor fertility may not be due to a LH receptor disorder but may be due to a postreceptor malfunction.

Endocrine Regulation of Reproductive Function in Fertile, Subfertile and Infertile Stallions

Endocrine Regulation of Reproductive Function in Fertile, Subfertile and Infertile Stallions

Endocrine Profiles in Fertile, Subfertile, and Infertile Stallions: Testicular Response to Human Chorionic Gonadotropin in Infertile Stallions1

Endocrine Profiles in Fertile, Subfertile, and Infertile Stallions: Testicular Response to Human Chorionic Gonadotropin in Infertile Stallions1

Serum levels of testosterone and oestrogens in normal and infertile stallions

The serum concentrations of testosterone and oestrogens were determined in stallions classified as geldings, normal (according to age) or infertile (azoospermic). There were significant differences in testosterone and oestrogen levels between the groups. Normal concentrations of testosterone and total oestrogens were attained after 16 months of age. Castrated and immature horses (< 15 months of age) had lower levels of testosterone and total oestrogens than did mature and infertile stallions. Azoospermic stallions had lower levels of testosterone and total oestrogens than did normal mature horses except in one case (B4) in which the level of total oestrogens was increased markedly. Critical ellipses for testosterone and total oestrogens were obtained for both groups (6-15-month-old colts and stallions aged > 4 years). The results suggest that serum levels of testosterone and total oestrogens may provide a sensitive index of the endocrine function of the testis in male horses.