Avaliação de características espermáticas após desafio laboratorial que mimetiza condições uterinas e sua relação com resultados de fertilidade em campo

Abstract

The aim of this study was to evaluate in vitro resistance of frozen-thawed bovine semen after being submitted to a laboratory challenge that mimics conditions of the cow´s reproductive tract, and to compare in vitro semen characteristics with in vivo fertility of the bulls evaluated. Semen doses from 6 bulls were used, with one batch of each bull and three replications per batch being evaluated (n = 18). For each semen batch, the following procedure was performed: initially, the dose of semen (500 μL) was thawed at 37ºC for 30 seconds (Control Group) and the following parameters were evaluated: motility, vigor, concentration, sperm morphology, plasma membrane functionality (hyposmotic test; HOST), computerized analysis of sperm ki-netics (CASA) and analysis of chromatin integrity and sperm morphometry by the toluidine blue stain. Then, an aliquot of 200 μL of the thawed sample was submitted to the in vitro sem-inal resistance test proposed here (called laboratory challenge). The 200 μL aliquot was depos-ited on a discontinuous Percoll gradient (250 μL 45% Percoll + 250 μL 90% Percoll) and cen-trifuged at 410x g for 10 min. After centrifugation, the supernatant was removed and, in the pellet formed, concentration was adjusted in TALP medium (5x10 6 sptz/ml) and heparin (5 μg/ml) was added. This sample was then incubated at 38.5ºC for 2h at controlled atmosphere with 5% CO2, 5% O2 and 90% N2. At the end of the laboratory challenge, the same semen analyzes described for the control group were performed (Challenge Group). Finally, the in vitro semen characteristics were compared with the in vivo fertility results of these semen batches. Field data were analyzed by logistic regression. The laboratory results were analyzed by ANOVA. For all tests, a level of 5% significance was considered by the R software. The total conception rate (CR) for the six bulls was 49% (n= 890 cows). The bull effect was the most important factor (P = 0.007) in the overall model of CR at 30 days after insemination. No effect on CR was observed for Body Condition Score (P = 0.459), AI Technician (P = 0.562) or female lot (P = 0.398), nor their interactions (P > 0.05). Higher (P < 0.05) field fertility was detected in bull 6 compared to bulls 1 and 2. Regarding to the semen parameters after thawing, greater (P = 0.0051) membrane integrity (HOST) was observed for bull 1 compared to bull 6. Additionally, bull 1 demonstrated higher (P = 0.0001) Form Factor, lower (P = 0.0025) Antero-posterior Symmetry and larger (P = 0.0141) Fourier 1 than bull 6. In the post-challenge evalu-ation, greater visual motility (P = 0.0002) and vigor (P = 0.0005) were observed for bull 6 compared to bull 1. Only for the morphometric sperm trait Form Factor the differences between bulls 1 and 6 remained present, inferring that this sperm characteristic is intrinsic to bull 1. Regarding to the DNA integrity, no differences were observed between the bulls evaluated, before or after challenge. It was concluded that the laboratory challenge here proposed allowed to identify differences in the in vitro resistance of semen samples from different bulls, consid-ering the parameters visual motility and vigor, plasma membrane functionality and total and progressive motility of CASA. However, further studies are needed to confirm the real capacity of the present laboratory challenge to identify bulls with higher and/or lower conception rates.