Separation of X spermatozoa from cauda of epididymis prior to selection of female embryos in local goat

Abstract

The objective of this study is the selection of X epididymal spermatozoa of local buck by discontinuous ficoll density gradient and using these spermatozoa for in vitro fertilization and identify the ratio of produced female embryos after identification by polymerase chain reaction (PCR). Epididymal spermatozoa were harvested from the cauda epididymis by slicing, and then spermatozoa were estimated and submitted to in vitro maturation and capacitation prior to separation of X from Y bearing spermatozoa and use for in vitro fertilization. Modified discontinuous ficoll density gradient method used for the separation of X and Y-bearing sperm by using 3 layers of discontinuous ficoll (60, 40, and 20%) or 4 layers of discontinuous ficoll (80, 72, 64, and 50%). The centrifugation applied at 200×g or 300×g. In the protocol of 3 layers of discontinuous ficoll (60, 40, and 20%) and centrifugation at 200×g and 300×g the results showed that the mean of the sperms at the 3rd was 79.014 ± 3.12 and 79.39 ± 2.11 respectively and this show the sperms was not completely separated. In the protocol of 4 layers of discontinuous ficoll (80, 72, 64, and 50%) and centrifugation at 200×g showed that the mean of the sperms at the 4th layer was (42.79 ± 1.38) as compared with the first three layers and with the protocol centrifugation at 300×g. The mean of the sperm lost showed (7.066 ± 0.56) and a little percent from centrifugation at 300×g showed 11.28 ± 1.55. Oocytes collected from ovaries which obtained from the slaughter house then matured oocyte was applied. The maturation rate for grade A and B used for IVF by spermatozoa after ficoll density gradient it was 67.60% and 52.74%, while the fertilization rate for the grade A and B was 70.68% and 78.26% respectively. The sex of embryos was determined by polymerase chain reaction (PCR) using specific primers for detection of SRY gene. The percentage of goat embryos obtained by IVF by sperms selected with ficoll density techniques after centrifugation at 200×g at different stage of development was 70.13%. The percentage of the male embryos was 16.67% while the female embryos was 83.33%. The results showed that the best method for selection of female embryos was by using 4 layers of discontinuous ficoll gradient method (80, 72, 64, and 50 %) with centrifugation at 200×g.