Analysis of bovine sexed sperm for IVF from sorting to the embryo

Abstract

The objective of this study was to characterize bovine semen parameters and determine the best IVF conditions to produce a maximal percentage of blastocysts. Four types of semen were analyzed with CASA and flow cytometry: fresh and frozen non-sexed semen; fresh and frozen sexed semen. Semen was obtained from four Holstein bulls and two ejaculates from each bull were analyzed. Oocytes from slaughterhouse ovaries were matured and fertilized in vitro with all types of semen (for sexed semen, 2, 5 or 10microg/mL heparin was added to the IVF media while for non-sexed semen, 10microg/mL was added in the IVF medium). Presumptive zygotes were co-cultured with Buffalo rat liver cells in Menezo's B2 medium, and cleavage rates at Day 2, and blastocyst rates at Day 7 of culture, were recorded. Sexed semen resulted in fewer blastocysts than non-sexed semen (P<0.05), and certain bulls performed better in IVF. Freezing, and not sexing, had a more significant negative effect on semen quality. Compromised semen quality due to sexing and/or freezing can explain the reduced in vitro blastocyst rates when using frozen-thawed sexed semen. Sexed semen that appeared more capacitated seemed to require less heparin in IVF than sexed semen that appeared less capacitated to produce a maximal percentage of blastocyst. Flow cytometry sorting eliminates spermatozoa that possess compromised DNA, and therefore the reduced fertility seen in vitro is not due to an increased percentage of spermatozoa with compromised DNA. This study describes tools that can monitor semen parameters to optimize IVF conditions and thus obtain maximal blastocyst rates.