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 ± 1.7, %PM 50 ± 2.2, CONT), (%TM 60.3 ± 1.7, % PM 42.5 ± 2.1, LOW), and (%TM 41.3 ± 2, %PM 21.3 ± 1.5, HIGH). Post-thaw motilities for CONT (%TM 54 ± 2.3, %PM 40.8 ± 3.3) and LOW (%TM 51.7 ± 1.8, %PM 36.2 ± 2.1) were not different, but were higher than the HIGH (%TM 31.5 ± 1.2, %PM 17.1 ± 1.0) (p < 0.05). Post-thaw sperm viability was significantly lower in the HIGH (54.7 ± 2.4) than in the CONT (63.8 ± 2.3) or LOW (64.6 ± 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.