Significant heparin effect on bovine embryo development during sexed in vitro fertilization

Abstract

The aim of this study was to investigate the effect of different heparin concentrations in the course of sexed in vitro fertilization (IVF), on bovine embryonic development and development to term following embryo transfer (ET). With a total of 9156 oocytes for IVF, sorted as well as unsorted sperm from four bulls had different heparin requirements for achieving the highest rate of development in vitro. However, when optimal heparin concentrations were used (40 to 80 µg/ml), the performance of X-sorted sperm (0.3 × 10⁶/ml/IVF droplet) from all four bulls, as judged by blastocyst development (Bulls A, B, C, and D: 25.2, 19.7, 25.1, and 9.8%, respectively), was significantly increased, and the blastocyst rate was comparable to that observed with unsorted sperm at certain heparin concentrations within the four bulls. We determined that near-optimal blastocyst development was possible with sorted sperm from all four bulls, when a heparin concentration of 40 µg/ml was used. Pregnancy rates at d 70 post ET ranged from 39.1 to 40.3% (P > 0.05), and the calving rates ranged from 34.4 to 35.1% (P > 0.05), when heparin was used at a concentration of 10 μg/ml (n = 236), 20 μg/ml (n = 189), and 40 μg/ml (n = 305), respectively. Our study demonstrates that, although the sorted sperm of different bulls performed optimally over a range of heparin concentrations, a generally accepted heparin concentration of 40 µg/ml can be set for sexed IVF. This improvement is beneficial when sexed embryo production by ovum pickup and IVF is an essential component of genetic breeding programs.

Keywords: Bovine; Embryo transfer; Embryos; In vitro fertilization (IVF); Sorted sperm.

Fig. 1.

The effect of heparin concentration on fertilization status in IVF with bovine X-sorted sperm cells. Heparin concentrations ranged from 10 to 80 µg/ml during the IVF process. U, unsorted control; S, sorted sperm.

Fig. 2.

Bull-specific effect of different heparin concentrations on blastocyst development post-IVF with X-sorted sperm. Embryo development to the blastocyst stage was recorded on d 7 after IVF. (a) The significantly higher rate of blastocyst development is labeled as ▲ (unsorted) and * (sorted) in the same bull, respectively. Significance is indicated as non-overlapping error bars at the same heparin concentration by a direct comparison between sorted and unsorted group. As shown in Bull A, sorted sperm gave rise to a significantly higher rate of blastocyst development when the heparin concentration was increased to 40 µg/ml. By pair-wise comparison, there was no significant difference in blastocyst development between X-sorted sperm and conventional controls after increasing the heparin concentration during IVF. Sorted sperm from Bull B showed a significant increase in blastocyst development when heparin concentration was increased to 10 µg/ml and higher. The highest blastocyst rates in unsorted IVF compared to sorted groups were found when the heparin concentration was higher than 60 µg/ml (60–100 µg/ml). Sorted sperm cells from Bull C gave lower results when compared to the unsorted controls during IVF; however, a comparable blastocyst development rate was reached between groups of sorted and unsorted sperm in Bull C with a heparin concentration of 10 µg/ml. A similar tendency was observed for Bull D (sorted vs. unsorted). Nevertheless, in Bull D, a significant increase in blastocyst development was observed when the heparin concentration was increased to 20 µg/ml, and remained level with the rate of development even when heparin was increased from 20 to 100 µg/ml. Solid line, sorted sperm; dotted line, unsorted control. (b) Deduced heparin concentration for optimally sexed IVF was 40 µg/ml.