184 THE EFFECT OF A NOVEL SEMEN DISINFECTION TREATMENT ON THE VIABILITY AND FERTILIZING CAPACITY IN VIVO OF BOVINE SPERMATOZOA
Authors: de la Rey M. , Morfeld K.A. , Treadwell R. , Loskutoff N.M. 
Published: December 2004

The goal of this investigation was to determine the effect of a novel procedure, consisting of an initial incubation in antibiotic cocktail followed by centrifugation through a trypsinized Percoll gradient, on the viability and fertilizing capacity in vivo of bovine sperm as compared to standard processing methods. Exp 1: Semen collected by electroejaculation from 12 bulls were aliquoted into four treatment groups: (1) control (standard method) using Biladyl A (Minitübe, Tiefenbach, Germany) at a 1:4 (semen:diluent) dilution; (2) antibiotic cocktail (gentamicin, spectinomycin, lincomycin, tylosin, and kanamycin at 250, 300, 150, 200, and 1000 μg/mL, respectively) at a 1:9 (semen:cocktail) dilution and incubation (38°C) for 15 min; (3) trypsinized Percoll (Sigma, St. Louis MO, USA) gradient treatment by layering 1 mL semen on the top of a 90, 45, and 30% Percoll (bottom to top) gradient, with the 90 and 30% segments of the column containing 10 μg/mL soy-based trypsin inactivator (Sigma) and the 45% segment containing 0.25% trypsin (Sigma), and then centrifuging at 700g for 30 min; and (4) a combination of treatments 2 and 3 by concentrating the sperm by gentle centrifugation (300g for 10 min) after incubation in antibiotic cocktail, and then layering 1 mL of the sperm suspension on the Percoll gradients. In treatments 2–4, the treated sperm pellets were resuspended in Biladyl A (1:4), and all treatments were refrigerated (4°C) and examined at 0, 24, 48, and 72 hours for progressive motility, viability, and acrosomal integrity. The results were analyzed statistically using the Student's t-test (P < 0.05). Exp 2: Semen samples collected from two bulls were treated with either the control method or the combination antibiotic cocktail and trypsinized Percoll methods (1 and 4 above), and then used to AI a total of 5 (control) and 6 (combined treatment) superovulated cows three times at 12-h intervals. Day 7 embryos were recovered and assessed for stage and morphological quality. The results for Exp 1 indicated that sperm treated with either the trypsinized Percoll alone or in combination with the antibiotic cocktail had significantly higher (P < 0.05) progressive motility than the control at 0 (87.5 and 89.2 vs. 75.4%), 24 (87.1 and 87.9 vs. 73.2%), 48 (87.1 and 86.7 vs. 70.7%), and 72 h (82.1 and 80.4 vs. 64.6%, all respectively) post-treatment. Likewise, treated sperm had significantly greater viable and acrosome-intact sperm than the control at 0 to 72 h post-treatment. Although not statistically significant, there were more transferable-quality embryos recovered from cows inseminated using sperm treated with the combined method as compared to the control (58.9 vs. 43%, respectively). In conclusion, the trypsinized Percoll gradient method for processing bovine semen (alone or in combination with antibiotic cocktail) improved the quality of bovine sperm refrigerated for up to 72 h and had no detrimental affect on the number of transferable-quality embryos collected after AI.