171 IN VITRO FERTILIZATION USING FROZEN - THAWED SEXED SEMEN TREATED WITH RECOMBINANT HEPARIN-BINDING PROTEINS
Authors: Ordonez-Leon E. A. , Kjelland M. E. , Moreno J. F. , Welsh T. H. , Randel R. D. , Lammoglia M. A. , Ducolomb Y. , Romo S. 
Published: December 2011

The decrease in the fertility of males can have a negative economic impact on the production of milk and meat in cattle. As a result, interest exists in evaluating diverse proteins that might be able to increase the fertility of sperm. This is the case of the heparin-binding proteins (HBP), specifically the fertility-associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), which act to favour capacitation and the acrosome reaction, perhaps by modulating the immune system response toward the sperm. The objective of this study was to evaluate the effect of adding recombinant FAA and recombinant TIMP-2 to Hereford sexed semen (X-chromosome-bearing sperm) for use in an IVF program. For IVF, X-sorted semen was prepared both with and without HBP. For this, 25 μg of FAA and 25 μg of TIMP-2 were added per milliliter of semen and were left to adsorb for 20 min. The semen was then diluted, packaged in straws and cryopreserved. Ovaries were obtained from a slaughterhouse and transported to the laboratory to obtain cumulus–oocyte complexes by follicular aspiration and were cultured in maturation medium (TCM-199 supplemented with FSH, LH and oestradiol) for 24 h. All incubations were performed at 38.5°C in a humidified atmosphere of 5% CO2 in air. For insemination, frozen-thawed semen was washed by centrifugation in 2 concentration gradients of a silica-based colloid solution. The sperm concentration used for IVF was 1 × 106 spermatozoa mL–1. Gametes were co-incubated for 22 h in fertilization medium (TALP added with BSA, heparin, penicillamine, hypotaurine and epinephrine). For IVD, presumptive zygotes were incubated for 7 days in a modified IVD medium (SOF) supplemented with FCS and BSA. A total of 363 in vitro-matured oocytes were used in the control vs 405 oocytes in the treatment group. A Pearson chi-square test was used to determine the statistical significance. The cleavage rates for the control (61.4%) and treatment (83.7%) groups were different (P < 0.001). However, the numbers of blastocysts, expanded blastocysts and hatched blastocysts produced in the control (10.2, 6.3 and 0%, respectively) and treatment (7.4, 7.4 and 0.7%, respectively) groups were similar (all P > 0.1). The fact that there was an increase in the early embryonic development rates and not in the development of the embryos to the blastocyst stage suggests that HBP could play a role in fertilization, but not in embryo development. The results also demonstrate that the addition of the recombinant proteins to the semen increased the fertilizing capacity of the sperm at a concentration of 1 × 106 sperm cells mL–1 of fertilization medium. To further elucidate the effect of the HBP, an evaluation of sperm penetration and pronuclei formation is proposed as a complement to these results.
We thank Tod C. McCauley, Roy L. Ax and the staff at TMI Laboratories International Inc. for their assistance. We also thank Richard W. Lenz and the staff at Sexing Technologies for their assistance.