Effect of seminal plasma removal and environmental temperature on the refrigeration curve and maintenance of the quality of cooled equine spermatozoa

Abstract

Semen refrigeration is a widely used biotechnology in the equine industry. This technique maximizes the use of stallion´s semen, prevents the spread of sexual diseases, eliminates geographical barriers and, most importantly, increases the longevity of the semen. The preservation of cooled semen is achieved through controlled temperature reduction (cooling curve) and consequent reduction of sperm metabolism. However, variations in the environmental temperatures and the presence of seminal plasma can significantly influence the cooling curve and impact sêmen quality. Therefore, the aim of this experiment is to evaluate the effect of seminal plasma removal by measuring the influence of 3 temperatures (5ºC, 22ºC, and 37ºC), and of 2 different extenders on the cooling curve and semen quality. For this study, two ejaculates from four healthy stallions, aged between 5 and 22 years, in a routine collection schedule, will be used. Prior to the study, the stallions will undergo a complete andrological examination. Ejaculates will be collected using an artificial vagina, filtered with a nylon filter, and divided into 4 aliquots. Two of those aliquots will retain seminal plasma, while the other two will have seminal plasma removed through centrifugation (600xg for 10 minutes), for subsequent dilution until reaching a concentration of 50 x 106 spermatozoa/mL in the extenders: BotuSêmen® and BotuSêmenSpecial®. There will be 4 experimental groups: 1) BotuSêmen with seminal plasma, 2) BotuSêmen without seminal plasma, 3) BotuSêmenSpecial® with seminal plasma, and 4) BotuSêmen Special® without seminal plasma (BSS/SP-). After initial processing, the samples will be stored in refrigeration boxes following six different cooling curves: 1) Curve at 5ºC with 1 cold pack; 2) Curve at 22ºC with 1 cold pack; 3) Curve at 37ºC with 1 cold pack; 4) Curve at 5ºC with 2 cold packs; 5) Curve at 22ºC with 2 cold packs; and 6) Curve at 37ºC with 1 cold pack. Seminal samples will be evaluated at the following time: fresh (T0), refrigerated for 8 hours (T8) and 24 hours (T24), regarding sperm kinetics and plasma membrane integrity. Sperm kinetics will be measured using computer-assisted analysis (Hamilton Thorne Research - IVOS 12, Beverly, MA, USA), measuring total sperm motility, progressive motility, curvilinear velocity, linear velocity, average path velocity, and percentage of sperm with rapid movement. Additionally, an aliquot will be alocated for plasma membrane integrity analysis using epifluorescence microscopy. Data will be analyzed using repeated measures ANOVA to compare the effects of treatments (seminal plasma removal, temperatures, andextenders) on sperm kinetics and plasma membrane integrity over the time. Greenhouse-Geisser correction will be applied when necessary to adjust the sphericity of the data and a 5% statistical significance level will be used (P < 0.05).