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.

Evaluation of mare endometrial cytology using the novel cytotape technique.

The cytobrush is considered the method of choice to obtain endometrial samples. Rigid brush fibers, however, may induce endometrial irritation and bleeding, or cell fragmentation, decreasing quality and diagnostic value of the samples. It was hypothesized that samples collected using a novel cytotape would provide sample smears of greater quality and less blood contamination than the cytobrush. Endometrial samples were collected with a cytotape and a cytobrush from ten mares without endometritis. Endometritis was then induced with artificial insemination, and samples were again collected 6 h after insemination. A cytology smear and bacterial culture were prepared from each sample. The collection methods and times were compared in terms of number and integrity of endometrial cells; number, integrity, and percentage of neutrophils; number of red blood cells, and number of colony-forming units. Frequency of positive cytology and culture was compared when there was use of each technique. The sensitivity, specificity, positive predictive value, and negative predictive value of cytology and culture for each technique was calculated using endometrial biopsy as the gold standard. While all samples had adequate and comparable cellularity and cell integrity, cytotape samples had less red blood cell contamination compared to cytobrush samples (P < 0.05). The number and percentage of PMNs, frequency of positive cytology diagnosis, number of colony-forming units and frequency of positive cultures did not differ between collection methods. In conclusion, the cytotape is a rapid, easy, and practical technique that can provide endometrial samples with similar diagnostic value to the cytobrush, but with less blood contamination.

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.

Pharmacokinetics of intravenous and oral administration of enrofloxacin to the late-term pregnant and non-pregnant mares.

BACKGROUND: Enrofloxacin may be an alternative antimicrobial for unresponsive cases of severe bacterial infections in pregnant mares. As pregnancy may affect drug bioavailability, distribution, metabolism and excretion, dose adjustment might be necessary. OBJECTIVES: To determine the disposition of orally and intravenously administered enrofloxacin in pregnant and non-pregnant mares. STUDY DESIGN: Randomised cross-over experiment. METHODS: Six light-breed, healthy pregnant mares (260 days gestation) were given a single dose of either intravenous (5 mg/kg bwt) or oral compounded (7.5 mg/kg bwt) enrofloxacin, with the opposite dose administered after a 7-day washout. The protocol was repeated 45-60 days post-partum, 15-30 days after foals were weaned. Plasma samples were obtained via venepuncture at 0, 5, 10, 20, 30, 45, 60, 90 min, and 2, 3, 4, 6, 8, 12, 24, 36, 48 and 72 h after enrofloxacin administration. Enrofloxacin and ciprofloxacin concentrations were measured by LC-MS/MS. Concentration versus time data were analysed based on non-compartmental pharmacokinetics. RESULTS: Enrofloxacin AUC0-∞ was significantly higher in pregnant mares than non-pregnant mares after PO administration and tended to be higher after i.v. administration. Ciprofloxacin maximum plasma concentration (Cmax ) and concentration at 24 h (C24h ) were higher, and half-life of the terminal phase (t½λz ) was longer in pregnant mares than non-pregnant mares after oral administration. Similarly, ciprofloxacin C24h was higher in pregnant mares with intravenous administration. Oral bioavailability did not differ based on pregnancy status. MAIN LIMITATIONS: Only six healthy light breed mares were assessed. Disease or horse breed may affect the endpoints evaluated. A lack of established enrofloxacin AUC/MIC targets for equine pathogens limits pharmacokinetic-pharmacodynamic conclusions. CONCLUSIONS: The oral form of enrofloxacin was well absorbed, and oral bioavailability was comparable to previous studies. While differences in enrofloxacin and ciprofloxacin pharmacokinetics were seen between pregnant and non-pregnant mares, the recommended drug dose and dose intervals are appropriate for MIC <0.25 µg/mL. Dosages may need to be adjusted for bacteria with a MIC >0.25 µg/mL.

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.

Diffusion of enrofloxacin to pregnancy fluids and effects on fetal cartilage after intravenous administration to late pregnant mares.

BACKGROUND: In selective cases, enrofloxacin may be an alternative antibacterial agent to treat unresponsive infections in pregnant mares. Supratherapeutic doses of enrofloxacin are toxic to adult horses and also to newborn foals, however, it is unknown if enrofloxacin crosses the equine placenta or if it is toxic to the fetus. OBJECTIVES: To assess the diffusion of enrofloxacin and its metabolite to fetal fluids and its effects on fetal cartilage when administered to pregnant mares. STUDY DESIGN: In vivo and terminal controlled experiment. METHODS: Healthy mares at 260 days of gestation were allocated into three groups: untreated (n = 3), therapeutic treatment (5 mg/kg enrofloxacin, i.v., n = 7) or supratherapeutic treatment (10 mg/kg, i.v., n = 6) for 11 days. Fetal fluids were collected on days 1, 5 and 11 of treatment. Premature delivery was induced on day 11 with oxytocin and fetal fluids and plasma were collected during delivery. Plasma and fetal fluid enrofloxacin and ciprofloxacin concentrations were measured by liquid chromatography-mass spectrometry. Fetal articular cartilage was examined macroscopically and histologically for lesions. RESULTS: Enrofloxacin and ciprofloxacin reached the minimum inhibitory concentrations for common pathogens in all fluids. Ciprofloxacin did not increase with the double enrofloxacin dose in maternal plasma, but allantoic fluid showed a 10-fold increase relative to fetal trough plasma concentrations. Administration of enrofloxacin at recommended doses did not result in cartilaginous lesions in fetuses. MAIN LIMITATIONS: Only one time point in gestation was evaluated and mares treated in the study were healthy at the time of treatment. It remains to be determined if enrofloxacin shows toxicity at other stages of pregnancy, after a longer duration of treatment, or once the foals are delivered and articular surfaces are weightbearing. CONCLUSIONS: Short-term administration of enrofloxacin to late gestation mares resulted in detectable enrofloxacin and ciprofloxacin concentrations in fetal fluids and did not result in macroscopic or microscopic lesions in the fetus. While further research is needed to address long-term foal outcomes, enrofloxacin may be useful for select bacterial infections in pregnant mares.

Effect of urine contamination on stallion semen freezing ability.

Urospermia is a common ejaculatory dysfunction of stallions. Current practice suggests that urine contaminated semen should not be used for cryopreservation. The aim of this study was to determine effects of urine contamination on semen freezing. Sixty-five ejaculates from eight stallions were divided into no urine (CONT), low (20% urine, LOW), and high (50% urine, HIGH) samples. Semen was extended with a commercial cooling extender, cushion-centrifuged, resuspended to 200 million/mL in a commercial egg-yolk based extender, and cryopreserved in liquid nitrogen. A subset of ejaculates (n = 20) were split in two after cushion-centrifugation, and one half of the ejaculate was submitted to a single-layer gradient centrifugation before cryopreservation. Sperm motility parameters were assessed pre- and post-freezing with an automated sperm analyzer. Semen pH, creatinine, and urea concentrations were assessed in raw samples, after urine contamination and after centrifugation and extension. Statistical analyses were performed with ANOVA and Tukey's posthoc. There were significant reductions in total and progressive sperm motilities (i.e., %TM and %PM, respectively) with increasing urine contamination pre-freezing (%TM 67 ±â€¯1.7, %PM 50 ±â€¯2.2, CONT), (%TM 60.3 ±â€¯1.7, % PM 42.5 ±â€¯2.1, LOW), and (%TM 41.3 ±â€¯2, %PM 21.3 ±â€¯1.5, HIGH). Post-thaw motilities for CONT (%TM 54 ±â€¯2.3, %PM 40.8 ±â€¯3.3) and LOW (%TM 51.7 ±â€¯1.8, %PM 36.2 ±â€¯2.1) were not different, but were higher than the HIGH (%TM 31.5 ±â€¯1.2, %PM 17.1 ±â€¯1.0) (p < 0.05). Post-thaw sperm viability was significantly lower in the HIGH (54.7 ±â€¯2.4) than in the CONT (63.8 ±â€¯2.3) or LOW (64.6 ±â€¯3.4) groups. Semen creatinine and urine levels were significantly higher with increasing urine contamination and were significantly decreased after centrifugation and resuspension in freezing extender. Pre-treatment semen pH was significantly lower than semen contaminated with low or high amounts of urine, and pH decreased significantly after centrifugation and resuspension. Gradient centrifugation did not improve %TM in the control group, but it did improve pre-freeze %TM and %PM in the low and high groups and improved significantly post freezing %TM and %PM in the high urine contaminated group. Semen contaminated with a small amount of urine may be suitable for freezing, whereas highly contaminated semen might not be usable. Although urine was mostly removed in this fashion, the initial exposure to high quantities was sufficient to decrease sperm motility pre- and post-freezing, whereas low urine contamination was not as detrimental.