Sperm-bound antisperm antibodies are associated with poor cryosurvival of stallion spermatozoa.

Different stallions exhibit a high level of variation in the ability of their sperm to survive cryopreservation. A large fraction of stallions show poor post-thaw sperm motility, and their semen is not suitable for commercial freezing. In this study, we hypothesized that the presence of sperm-bound antisperm antibodies (ASAs) was associated with poor cryosurvival of stallion sperm. Our objective was to assess the level of ASA binding to stallion sperm, and determine if it was associated with good or poor sperm cryosurvival. In Experiment 1, cooled shipped semen from 27 stallions was frozen using three commercial semen extenders. Sperm motility, membrane integrity, acrosome integrity and apoptosis were evaluated before and after freezing for each aliquot. In addition, the percentage of ASA-bound sperm was evaluated post-thaw. In Experiment 2, semen from 22 stallions was frozen immediately after collection a single formulation of semen extender. Sperm motility and ASA binding were evaluated post-thaw. The results of both experiments showed similar findings. The frequency of ASA-positive samples was higher among stallions with poor sperm cryosurvival (Exp. 1 and 2 = 6/11, 54.5%) than for good sperm cryosurvival (Exp. 1 = 0/16, 0%; Exp. 2 = 1/11, 9.1%). The percentage of IgG- and IgA-bound sperm was also higher in stallions with poor sperm cryosurvival in both experiments (P < 0.05). Post-thaw sperm motility, velocity and distance parameters were lower in ASA-positive than ASA-negative stallions (P < 0.005). No effect of the semen extender used was observed. In addition, stallions with ASAs had a higher percentage of apoptotic sperm than stallions without ASAs. The presence of sperm-bound ASAs was associated with poor cryosurvival for stallion spermatozoa. Thus, it may be beneficial to evaluate stallions for binding of ASAs prior to freezing to offer and indicator of the prognosis for cryosurvival.

Optimization of cryopreservation protocols for cooled-transported stallion semen.

Freezing cooled-transported semen allows veterinarians and breeders to collect and process the semen of stallions on farm, and then ship the semen to a semen freezing center. There, however, is a lack of standardization of shipping and freezing protocols. The objectives were to optimize and simplify protocols to freeze cooled-shipped semen. In Experiment 1, cooled-transported semen was centrifuged at room temperature or 5 °C before freezing. Sperm variables (motility, membrane integrity, acrosome integrity, membrane fluidity) were evaluated before and after freezing. Centrifugation temperature had no effect on post-thaw semen quality. In Experiment 2, cooled-transported semen was centrifuged at room temperature and cryopreserved in three semen freezing extenders. With use of the improved modified French formula, there was less post-thaw total and progressive motility compared with use of Botucrio or the improved lactose-EDTA formula (P<0.0001). Semen cryopreserved in the improved modified French formula also had a lesser percentage of sperm with intact membranes compared with lactose-EDTA, and a greater percentage of sperm with capacitation-like changes compared with Botucrio (P<0.0001). In Experiment 3, semen diluted in each extender was frozen conventionally or placed directly in a -80 °C ultra-freezer. Freezing in the ultra-freezer resulted in a lesser post-thaw sperm motility, but not membrane and acrosome integrity and capacitation-like changes. In conclusion, centrifugation and addition of freezing extender to cooled transported semen can be performed at room temperature or 5 °C. The Botucrio and lactose-EDTA formula are recommended for conventional cryopreservation of cooled-transported stallion semen as compared with the modified French formula.

Administration of enrofloxacin during late pregnancy failed to induce lesions in the resulting newborn foals.

BACKGROUND: A recent study demonstrated that enrofloxacin and ciprofloxacin cross the equine placenta without causing gross cartilage or tendon lesions in the 9-month fetus; however, long-term effects of in utero fluoroquinolone exposure remain unknown. OBJECTIVES: To assess effects of fetal exposure to enrofloxacin on the resulting foal's cartilage and tendon strength. STUDY DESIGN AND METHODS: Healthy mares at 280 days' gestation were allocated into four groups: untreated (n = 5), therapeutic treatment (7.5 mg/kg enrofloxacin, PO × 14 days, n = 6), supratherapeutic treatment (15 mg/kg, PO × 14 days, n = 6) and no mare treatment with treatment of the foals post-partum (n = 2). Mares were allowed to carry pregnancy to term, and foals were maintained on pasture for 5 weeks. After that foals were euthanized, and their articular cartilage and extensor and flexor tendons were examined macroscopically and histologically for lesions. Tendon strength was tested by loading until failure. RESULTS: Administration of enrofloxacin at recommended doses in late gestation did not result in cartilaginous lesions or clinical lameness in any foal by 5 weeks old. Tensile strength was greater in hind tendons than front tendons, but no difference was found between foals born from treated and control mares. Expectedly, osteochondral changes were present both in foals born from enrofloxacin-treated mares and in negative control foals with no apparent association with fluoroquinolone treatment during pregnancy. MAIN LIMITATIONS: Only one time point in gestation was evaluated, and mares treated in the study were healthy at time of treatment. Additionally, it is possible that the assessments performed herein were not sensitive enough to detect subtle or functional changes in the articular cartilage. Further studies are needed to determine if enrofloxacin administration during late pregnancy potentiates osteochondral alterations in the first year of life. CONCLUSIONS: While this study did not assess other stages of gestation or long-term foal outcomes, short-term administration of enrofloxacin to late gestation mares did not result in macroscopic or microscopic lesions in the resulting foals by 5 weeks of age.

Diffusion of fluoroquinolones into equine fetal fluids did not induce fetal lesions after enrofloxacin treatment in early gestation.

While recent work demonstrated that enrofloxacin and ciprofloxacin reach the fetoplacental unit without causing obvious lesions in the 9-month-old equine fetus or resulting foal, many practitioners still hesitate to prescribe a fluoroquinolone during pregnancy. Since early gestation is a critical time for fetal skeletal development, if fluoroquinolones are chondrotoxic to the fetus at any point during gestation, this period would be important. The aim of this study was to assess the effects of 2 weeks' exposure to enrofloxacin on the equine fetus between 46 and 60 days gestation. Twelve pregnancies from nine healthy mares were allocated into two groups: untreated (n=7), or treatment (7.5mg/kg enrofloxacin, PO×14days, n=6). Abortion was induced with prostaglandin 24h after the last enrofloxacin dose, or on the equivalent day of gestation for untreated mares. Four of nine mares were rebred for a second cycle and were assigned to the opposite treatment to serve as their own controls. Fetal fluids from treated mares were analysed for enrofloxacin and ciprofloxacin concentrations. Fetal organs (heart, lungs, spleen, kidney, and liver) and limbs were examined histopathologically. Enrofloxacin and ciprofloxacin diffused to the fetal fluids during early gestation and did not result in detectable abnormalities in the fetus after 14 days of treatment. While current research does not determine long-term foal outcomes, enrofloxacin may be useful for select bacterial infections in pregnant mares.