Sex-sorting of spermatozoa affects developmental competence of in vitro fertilized oocytes in a bull-dependent manner

Abstract

The aim of the present study was to clarify if flow-cytometric sex-sorting of bovine sperm affected in vitro blastocyst production in different bulls, either in terms of its ability to fertilize the oocyte or by interfering with post-fertilization embryo development. We performed in vitro fertilization (IVF) using both commercially available frozen-thawed X-sorted and non-sorted sperm of 4 Holstein bulls at 3 concentrations (1 × 10⁶, 2 × 10⁶, and 5 × 10⁶ sperm/ml). When fertilization rates were compared, a variation in fertilization rates among different sperm concentrations was detected in 2 bulls, with similar results for X-sorted and non-sorted sperm. However, we found no evidence that the fertilization rates were affected by the sorting process. To investigate effects on embryo development, we determined the optimum sperm concentration for IVF in each bull, which resulted in similar fertilization rates among bulls. We next performed IVF using both X-sorted and non-sorted sperm of the 4 bulls at their optimum sperm concentration and compared in vitro embryo development. Cleavage rates with X-sorted sperm were similar to their non-sorted counterparts. However, significantly reduced blastocyst development was associated with the use of X-sorted sperm in one bull, whereas in the other three bulls, blastocyst development after IVF with X-sorted and non-sorted sperm was similar. In conclusion, in our system, X-sorting affects in vitro blastocyst production by reducing the developmental competence of fertilized oocytes rather than affecting the fertilization ability of the sperm. However, the occurrence of this phenomenon varies among bulls.

Fig. 1.

The frequencies of total and normal fertilization after IVF with non-sorted and X-sorted sperm of 4 different bulls at different sperm concentrations. Data are presented as mean ± SEM. Within the same bull, a and b differ significantly at P < 0.05. Dotted line (Ave) denotes the mean level for the total average value for fertilization or normal fertilization rates from the four experimental replication calculated from all fertilization groups irrespective of bull and sperm concentration.

Fig. 2.

Comparison of normal fertilization rates among bulls within X-sorted and non-sorted groups (A) and between X-sorted and non-sorted sperm within each bull (B) after IVF with sperm concentrations optimized for each bull. Data are presented as mean ± SEM. Significant differences were not detected (P > 0.05).

Fig. 3.

Comparison of embryo development between X-sorted and non-sorted sperm within each bull after IVF with sperm concentrations optimized for each bull. Data are presented as mean ± SEM. a and b differ significantly at P < 0.05.

Fig. 4.

Comparison of embryo development among bulls within X-sorted and non-sorted groups after IVF with sperm concentrations optimized for each bull. Data are presented as mean ± SEM. Within the same category, a and b differ significantly at P < 0.05.