The effects of ergot alkaloids on the breeding stallion reproductive system.

REASONS FOR PERFORMING STUDY: Ergot alkaloids cause a range of pathological conditions in mares. There is no evaluation of the effects of ergot alkaloids from endophyte-infected tall fescue on the stallion breeding soundness examination spermiogram. OBJECTIVES: The objective of this study was to investigate the effect of ergot alkaloids from endophyte-infected tall fescue on the stallion's reproductive functions. STUDY DESIGN: Crossover toxicology experiment. METHODS: Six stallions were fed either toxic endophyte-infected tall fescue seed or a nontoxic endophyte tall fescue seed (Flecha AR-542, MaxQ). The fescue seed content was compounded at 45% of a grain diet and the stallions were fed the grain diet at 1% of their body weight. The stallions were fed the diet for 70 days, then rested for at least 70 days (no fescue seed) and then fed fescue seed for a second 70 days. At regular intervals blood sampling and a breeding soundness examination were performed. RESULTS: The mean time to maximal systemic toxicity was 8.33 h after starting toxic seed ingestion with a mean toxicity level of 49.98 ng alkaloid/mg creatinine. After cessation of feeding toxic seed, the systemic alkaloid concentration fell to control levels within 48 h. There were no significant changes in sperm motility, sperm concentration, sperm cell morphology, total number of sperm cells, number of breeding doses, testicular volume, baseline and human chorionic gonadotropin stimulated testosterone levels. There were no changes in core body temperature and superficial scrotal temperature. The ejaculate from stallions consuming endophyte-infected tall fescue seed had significantly lower gel-free volume (47.5 ± 4.1 ml) than stallions consuming nontoxic endophyte tall fescue seed (62.8 + 4.3 ml, P<0.01). CONCLUSION: Ergot alkaloids decreased the gel-free volume of stallions consuming high levels of ergot alkaloids but statistically significant effects on the spermiogram of adult breeding stallions were not found.

Serum free cortisol fraction in healthy and septic neonatal foals.

BACKGROUND: Relative cortisol insufficiency occurs in septic foals and impacts survival. Serum free (biologically available) cortisol concentration might be a better indicator of physiologic cortisol status than serum total cortisol concentration in foals. HYPOTHESES: In septic foals, (1) low free cortisol concentration correlates with disease severity and survival and (2) predicts disease severity and outcome better than total cortisol concentration. ANIMALS: Fifty-one septic foals; 11 healthy foals; 6 healthy horses. METHODS: In this prospective clinical study, foals meeting criteria for sepsis at admission were enrolled. University-owned animals served as healthy controls. Basal and cosyntropin-stimulated total cortisol concentration and percent free cortisol (% free cortisol) were determined by chemiluminescent immunoassay and ultrafiltration/ligand-binding methods, respectively. Group data were compared by ANOVA, Mann-Whitney U-tests, and receiver operator characteristic curves. Significance was set at P < .05. RESULTS: Basal % free cortisol was highest in healthy foals at birth (58 ± 8% mean ± SD), and was higher (P ≤ .004) in healthy foals of all ages (33 ± 6 to 58 ± 8%) than in adult horses (7 ± 3%). Cosyntropin-stimulated total and free cortisol concentrations were lower (P ≤ .03) in foals with shock (total = 6.2 ± 8.1 µg/dL; free = 3.5 ± 4.8 µg/dL versus total = 10.8 ± 6.0 µg/dL; free = 6.9 ± 3.3 µg/dL in foals without shock) and in nonsurvivors (total = 3.8 ± 6.9 µg/dL; free = 1.9 ± 3.9 µg/dL versus total = 9.1 ± 7.7 µg/dL; free = 5.5 ± 4.4 µg/dL in survivors). Free cortisol was no better than total cortisol at predicting disease severity or outcome in septic foals. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum free cortisol is impacted by age and illness in the horse. There is no advantage to measuring free over total cortisol in septic foals.

Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility.

OBJECTIVE: We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility. DESIGN: A randomised 2 x 3 block design was used. PROCEDURE: Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy in fresh and post-thaw semen. RESULTS: There was a significant difference between the two extenders in the motility of spermatozoa after cryopreservation (48.9% for INRA 96; 38.6% for EZ Mixin OF; P < 0.0001). Glycerol at 4% in freezing medium yielded the highest post-thaw motility, significantly better than 2% (P < 0.05). Three of four stallions had significantly higher post-thaw motility using INRA 96 relative to EZ Mixin OF (P < 0.01), and two of four stallions had significantly higher post-thaw motility using 4% glycerol (P < 0.05). The combination of INRA 96 and 4% glycerol in freezing medium gave the highest average post-thaw motility of 51.5%. CONCLUSION: In this study, INRA 96 combined with 4% glycerol yielded an average recovery of progressively motile sperm consistently above the 35% target.

Hypothalamic-pituitary-adrenal axis assessment in healthy term neonatal foals utilizing a paired low dose/high dose ACTH stimulation test.

BACKGROUND: Hypothalamic-pituitary-adrenal (HPA) axis function is dynamic in the neonatal foal. The paired low dose/high dose cosyntropin (ACTH) stimulation test allows comprehensive HPA axis assessment, but has not been evaluated in neonatal foals. HYPOTHESIS: Foal age will significantly affect cortisol responses to a paired 10 and 100 microg dose cosyntropin stimulation test in healthy neonatal foals. ANIMALS: Twenty healthy neonatal foals. METHODS: HPA axis function was assessed in 12 foals at birth and at 12-24, 36-48 hours, and 5-7 days of age. At each age, basal cortisol and ACTH concentrations were measured and cortisol responses to 10 and 100 microg cosyntropin were assessed with a paired ACTH stimulation test protocol. Eight additional 36-48-hour-old foals received saline instead of 10 microg cosyntropin in the same-paired ACTH stimulation test design. RESULTS: At birth, foals had significantly higher basal cortisol and ACTH concentrations and higher basal ACTH : cortisol ratios compared with foals in all other age groups. A significant cortisol response to both the 10 and 100 microg doses of cosyntropin was observed in all foals. The magnitude of the cortisol response to both doses of cosyntropin was significantly different across age groups, with the most marked responses in younger foals. There was no effect of the paired ACTH stimulation test design itself on cortisol responses. CONCLUSIONS AND CLINICAL IMPORTANCE: A paired 10 and 100 microg cosyntropin stimulation test can be used to evaluate HPA axis function in neonatal foals. Consideration of foal age is important in interpretation of HPA axis assessment.

Advancements in large animal embryo transfer and related biotechnologies.

Embryo transfer has been an inherent part of cattle breeding for more than 35 years and has also gained remarkable interest from the equine industry after several breeds allowed registration of more than one foal per year. In both large animal species, non-surgical embryo recovery and transfer are well-established techniques. However, success rates after superovulation and cryopreservation of embryos in horses are still lagging behind those of cattle, and more research is needed to address these areas. To address the problem of freezing large equine embryos, we offer a preliminary demonstration of a new cryopreservation method which involves reduction of the blastocoelic volume and microinjection of cryopreservative. Successful cryopreservation will improve the ability of practitioners to preserve and implant embryos in recipient mares. Recent advances in the use of equine FSH to induce superovulation in mares brings to the forefront the issue of how to best preserve the large number of embryos that are produced. Finally, the use of sexed semen after superovulation will provide the bovine and equine breeding industry the offspring of the desired sex.

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility.

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies.

Extraction and quantification of acrosin, beta-N-acetylglucosaminidase, and arylsulfatase-A from equine ejaculated spermatozoa.

Acrosin, Arysulfatase A, and beta-N-acetylglucosaminidase are three key enzymes localized within the mammalian acrosome that play a pivotal role in the penetration of the oocyte. The objectives of this study were to compare two methods of enzyme extraction based on the activities of these enzymes from equine spermatozoa. Method A utilized a 0.5 M Tris-maleate buffer containing 0.1% Triton X-100 and Hyamine 2389. Method B used 0.05 M Tris-HCl, 0.05 M MgCl2 in 0.05 M Tris-maleate, followed by 0.05 M Tris-maleate containing 0.1% Triton X-100. Results indicated that acrosin was initially bound in an acrosin-acrosin inhibitor complex; this complex was dissociated after incubating the extract in 2 mM HCl. Significant (P < 0.001) increases in acrosin activity were found after-acid extraction from 0.076 U/mg after Method B to 0.327 U/mg after Method A. Arylsulfatase A activity was found to have a higher mean activity (P < 0.03) after Method A (0.012 U/mg) as opposed to Method B (0.007 U/mg). Similarly, beta-N-acetylglucosaminidase was found to have a higher mean specific activity (P < 0.001) after Method A (0.037 U/mg) as compared to Method B (0.008 U/mg). This is the first report of the quantification of these enzymes from equine spermatozoa which can ultimately be used as an index of acrosomal damage in cryopreserved semen, and provide additional insight into biochemical alterations between normal vs. abnormal semen.

Influence of endophyte-infected tall fescue on serum prolactin and progesterone in gravid mares.

Thirty mares in late gestation were used in a 3-yr study to assess effects of the tall fescue endophyte Acremonium coenophialum on serum prolactin (PRL) and progesterone. Two paddocks of each treatment, 0 or 100% infected 'Kentucky 31' tall fescue, were grazed by the mares for 21 d. Blood was collected three times per week until parturition. At 7-d intervals, mares were challenged with thyrotropin-releasing hormone (TRH) while grazing and blood was collected postinjection. Mares grazing 100% infected tall fescue (E+) had decreased serum PRL compared with mares grazing the 0% infected tall fescue (E-) in 2 of 3 yr. Within 8 d postgrazing, serum PRL for E+ mares equaled or surpassed values of the E- mares. Serum PRL was not different during the 3rd yr. In response to TRH, serum PRL rate of increase was similar between treatments but remained elevated (P less than .01) in the E+ mares at the 180-, 240-, and 300-min sample times. Serum progesterone was lowered (P less than .05) by E+ but increased to control values within 10 d postgrazing. It is concluded that serum PRL and progesterone in the gravid mare were decreased by the presence of A. coenophialum in 'Kentucky 31' tall fescue grass but normal levels were reestablished within 2 to 3 wk.