Evaluation of the effectiveness of semen processing techniques to remove bovine viral diarrhea virus from experimentally contaminated semen samples.

The aim of this study was to evaluate the capacity of three semen processing techniques, Percoll gradient centrifugation, Swim-up and a combination of Swim-up and Percoll gradient centrifugation, to reduce the viral load of bovine viral diarrhea virus (BVDV) in experimentally infected semen samples. The evaluation was performed using two approaches: first, searching for the presence of virus in the processed samples (via virus titration and RT-PCR) and second, ascertaining the possible interference on in vitro embryo production. The sperm count and DNA integrity (Comet assay) of the processed samples were analyzed (Experiment 1). The amount of virus in the processed samples was determined by titration in cell culture (Experiment 2). The samples processed by Swim up/Percoll gradient centrifugation were utilized for in vitro embryo production, and the embryos produced were tested for BVDV by RT-PCR (Experiment 3). Sperm concentration, Comet assay and embryo production were analyzed by chi-squared tests (P<0.05). There was a significant difference between sperm separation techniques when the sperm count and Comet assay were analyzed. The sperm count obtained from the Swim up/Percoll gradient centrifugation group was lower than that obtained in either of the two other groups (Swim up and Percoll gradient centrifugation), and the Comet assay showed that the combination of the two semen processing techniques (Swim up/Percoll gradient) produced a 1.1% prevalence of Comet level 2, which was not observed in the other groups. The BVDV titer (10(6.68)TCID(50)/mL) added to experimentally infected semen samples decreased after Percoll gradient centrifugation to 10(2.3)-10(1)TCID(50)/mL; for the Swim up group, the titer range was 10(3.3)-10(1.87)TCID(50)/mL, and in the Swim up/Percoll gradient centrifugation group, BVDV was undetectable. The decreases in titer varied from 99.9% in the Swim up-processed group to 100% in the Swim up/Percoll gradient centrifugation group. In vitro embryo production displayed similar blastocyst development rates among all groups, and RT-PCR was negative for the produced embryos. The data showed that the combination of Swim up/Percoll gradient centrifugation promoted the elimination of BVDV from the semen samples without damaging spermatozoa cells and also allowed successful in vitro embryo production free of BVDV. Hence, the risk of BVDV contamination is negligible for the embryo recipient.

Genetic polymorphisms at the leptin receptor gene in three beef cattle breeds

The genetic diversity of a single nucleotide polymorphism (SNP) at the exon 20 (T945M) of the leptin receptor gene (LEPR) and of three short tandem repeats (STRs BM7225, BMS694, and BMS2145) linked to LEPR was investigated in three beef cattle herds (Brangus Ibagé, Charolais, and Aberdeen Angus). A cheap and effective new method to analyze the T945M polymorphism in cattle populations was developed and the possible role of these polymorphisms in reproduction and weight gain of postpartum cows was evaluated. High levels of genetic diversity were observed with the average heterozygosity of STRs ranging from 0.71 to 0.81. No significant association was detected between LEPR markers and reproductive parameters or daily weight gain. These negative results suggest that the LEPR gene polymorphisms, at least those herein described, do not influence postpartum cows production.