Practical use of percoll density gradient centrifugation on sperm sex determination in commercial dairy farm in Thailand

Our experiment demonstrated an extension of Percoll Density Gradient Centrifugation to use as a selection tool for sperm sexing developed for dairy farmer. We develop a simple protocol for bovine sperm sexing and used for artificial insemination (AI) and in vitro fertilization (IVF) for embryo transfer (ET). The quality of sperm after centrifugation in each layer were significantly different (P < 0.01). There was no significant difference between percentage of live sperm after centrifugation and fresh sperm before centrifugation (P > 0.05). The phenotype and swimming capacity of sperms in each layer were significantly different (P < 0.01). It was found that the most appropriate sperm in the layer after centrifugation and after quinacrine staining was between 65-70% Percoll (R). X-bearing spermatozoa at this layer was 60.75% and motility was 95.86 +/- 0.46%. After insemination, the pregnancy ratio in the experiment was 40% which is similar to the percentage of normal AI. The number of female offspring in the experiment was 71.4% in selected sperm vs 50% in non-selected sperm AI. In the In Vitro fertilization experiment, sperm from our 7 layer Percoll gradient concentration can fertilize and produce blastocyst embryo successfully. The simple 7 layer Percoll gradient concentration protocol is appropriate for dairy farmer to do as a pre-AI step to increase their female offspring in their herd.