Equine epididymal spermatozoa: A practical guide to collection, processing, and laboratory techniques

Abstract

Equine epididymal spermatozoa are the last source for gamete rescue in case of sudden death or emergency castration of a valuable stallion. This study aimed to compare the concentration and kinematic parameters of equine epididymal spermatozoa under different protocols. We therefore tested the influence of general and local anesthesia, transport, harvesting technique, refrigeration, seasonality, and age. We hypothesized that all these parameters might influence the quality of spermatozoa and that a standard protocol would be helpful for the practitioner. We analyzed 142 samples after elective orchiectomy using a series of comparative protocols. First, we looked at the effect of general and local anesthesia and compared a TIVA (total intravenous anesthesia) vs PIVA (partial intravenous anesthesia), with or without local lidocaine. We then looked at the influence of transport length and temperature. Once arrived at the laboratory we tested the retrograde flush technique as well as flotation to harvest spermatozoa, we tested two commercial extenders, centrifugation, and refrigeration effect. Finally, we looked at the seasonality and age influence over the study parameters to formulate prognostic characteristics for the owners. All samples collected were analyzed using computer-assisted sperm analysis (SCA^ⓇCASA). The results of this study show that PIVA (partial intravenous anesthesia) has an impact on motility parameters when compared to a TIVA (total intravenous anesthesia) protocol. Local lidocaine did not affect the kinematic characteristics of epididymal spermatozoa. Kinematic characteristics decreased with transport at 5 degrees Celsius but viable spermatozoa were still successfully harvested after 72 hours. The retrograde flush technique was superior for harvesting spermatozoa and centrifugation did not improve any of the parameters tested. Nonetheless, age and season influenced the kinematic characteristics of epididymal spermatozoa. This study looked at healthy stallions that underwent elective orchiectomy therefore the results might indicate more of the characteristic of equine spermatozoa before ejaculation than of epididymal spermatozoa of stallions during emergency castration such as in the case of scrotal hernia or sudden death. However, the results of this study show that epididymal spermatozoa maintain proper viability after testes are removed and that their kinematic characteristics are preserved during general and local anesthesia. During laboratory processing, the retrograde flush technique seems superior, and centrifugation is not necessary. Older stallions are expected to have reduced motility parameters and concentration as well as stallions outside of reproduction season.