86 EFFECTS OF THREE CRYOPRESERVATION SYSTEMS ON LONGEVITY OF STALLION SPERM AFTER THAWING

Abstract

Use of frozen semen by the horse industry is popular. Several extenders have been used to freeze stallion semen; the most used are EDTA-lactose egg yolk and INRA 82. A new extender named Botu-Crio^® (BioTech, Botucatu, Sao Paulo, Brazil) has been used on a large scale in Brazil. No comparisons of these extenders have been published. The present experiment was designed to compare the ability of these three extenders to preserve sperm longevity after incubation. One ejaculate from each of 13 stallions was used. After collection using an artificial vagina, semen was filtered and diluted 1:1 with a milk-base extender (Botu-Semen^®), split into 3 parts, and then centrifuged (600g/10 min). After removal of the supernatants, each semen pellet was resuspended with an aliquot of one of the extenders (INRA 82, EDTA-Lactose, or Botu-Crio) to a final concentration of 100 × 10⁶ sperm cells mL^–1. Semen was packaged in 0.5-mL French straws, and cooled for stabilization for 20 min at 5°C for Botu-Crio (BC), for 2 h at 5°C for INRA 82 (IR), and without stabilization for EDTA-Lactose (EL) extender. After stabilization, the straws were placed in nitrogen vapor for 15 min and then plunged into liquid nitrogen. Before motility evaluation by CASA using the Hamilton System Analyzer (Hamilton Thorne Biosciences, Beverly, MA, USA), straws were thawed (46°C/20 s) and then incubated at 37°C in a dry block. The patterns of total motility (TM) and progressive motility (PM) were evaluated immediately after thawing (T0), and at 10 (T10), 30 (T30), and 60 (T60) min after incubation at 37°C. Statistical analysis was performed using ANOVA and Tukey's test. Total motility, respectively, for IR, EL, and BC was TM0: 10%, 35%, and 57%; TM10: 25%, 37%, and 68%; TM30: 20%, 21%, and 56%; and TM60: 13%, 10%, and 41%. The respective progressive motility was PM0: 3%, 16%, and 28%; PM10: 11%, 16%, and 32%; PM30: 10%, 8%, and 26%; and PM60: 5%, 2%, and 16%. Motilities were always superior (P < 0.05) at all incubation times with the utilization of Botu-Crio extender. A fertility trial comparing the three extenders is in progress.