65 ARE “BAD FREEZER” STALLIONS ALSO “BAD COOLER” STALLIONS?

Abstract

Several factors can interfere with sperm cryopreservation resistance, especially the genetic factors and those related to the plasma membrane composition of the sperm and seminal plasma. However, it is still unclear if the same factors that confer freezing resistance will perform the same role during the cooling process. Thus, the aim of this study was to determine the relation between the resistance to freezing and cooling processes in stallions. Two ejaculates from each of 75 stallions were used. All animals showed good quality of fresh semen (total motility higher than 60% and plasma membrane integrity higher than 50%). After collection, the semen was diluted 1 : 1 with commercial skim milk-based extender (Botu-Semen^TM, Botupharma, Brazil) and then a part was designed to cooling and the another to freezing. The cooled semen was divided into 2 groups: Group PS, in which the semen was diluted with Botu-Semen^TM at a concentration of 50 × 10⁶ sperm mL^–1, and Group SPS, which was subjected to a centrifugation at 600 × g for 10 min and resuspended with Botu-Semen^TM at 50 × 10⁶ sperm mL^–1. Semen samples from both groups were placed in the same cooling passive system for a period of 24 h/5°C. To accomplish the freezing process, the semen sample was subjected to centrifugation at 600 × g for 10 min. The supernatant was discarded, and the pellet was re-suspended in a Botu-Crio^TM. The straws were frozen according to the manufacture. The sperm parameters from fresh semen, cooled semen for 24 h with and without seminal plasma, and frozen semen were evaluated for kinetics by computer-assisted semen analysis and for plasma membrane integrity (IMP%) by epi-fluorescence microscopy. The animals were classified in relation to their resistance to cooling and freezing processes as follow: “bad coolers” – reduction in sperm total motility and in plasma membrane integrity higher than 35% after 24 h of cooling in samples with seminal plasma; “good coolers” – reduction in sperm total motility and in plasma membrane integrity lower than 35% after 24 h of cooling in samples with seminal plasma; “bad freezer” – sperm total motility lower than 40% and progressive motility lower than 20% in seminal sample after thawing; “good freezer” – sperm total motility higher than 60% and progressive motility higher than 30% in seminal sample after thawing. The comparison between the resistance to cooling and freezing processes was performed by Fisher's exact test. The level of significance was 5%. No difference (P < 0.05) between the resistance to cooling and freezing processes was observed. The percentage of stallions “good freezer” and “good cooler” was 54%, “good freezer” and “bad cooler” was 22.6%, “bad freezer” and “good cooler” was 12%, and “bad freezer” and “bad cooler” was 10.6%. Within stallions classified as “good freezer” and “bad cooler,” 52.9% also were “good cooler” when the seminal plasma was removed before the cooling process, and 47.1% remained as “bad cooler.” The result of this study demonstrates that there is a strong relation between the resistance to cooling and freezing processes in stallions. In stallions categorized as “bad cooler,” the seminal plasma presents a major influence on the quality and longevity of cooled semen.