262 THE EFFECT OF TRIS AND Botu-Bov^® FOR BOVINE SEXED SPERM CRYOPRESERVATION

Abstract

Sexing of sperm by flow cytometry has been applied worldwide. However, the sorting as well as the cryopreservation process causes damage to the sperm cells. Therefore, the sexed sperm need enhanced protection during the freezing process. Thus, the aim of the present study was to compare the effect of two different extenders for sperm cryopreservation. Two ejaculates from 10 bulls (Bos taurus and Bos indicus) of reproductive age and used in commercial AI programs were used. The ejaculates were collected with an artificial vagina and the semen was analyzed and prepared for separation by flow cytometry. After sorting, the samples were split into two groups for freezing in TRIS-egg yolk or Botu-Bov^® (Biotech Botucatu, Ltda., Sao Paulo, Brazil). Sperm were evaluated by computer-aided semen analysis (CASA) and fluorescent probes to determine plasma and acrosomal membrane integrity. The fertility of the spermwas also tested in an IVF program. Statistical analysis of sperm parameters was achieved using theTukey test with a significant level of P ≤ 0.05. Embryo production data after IVF were analyzed by chi-square test. There was a significant improvement in progressive motility with the use of Botu-Bov, compared with TRIS (67.4% v. 56.7%, respectively). There was also a statistical difference for curvilinear velocity (VCL) and lateral head displacement (ALH), with TRIS showing higher values than Botu-Bov (VCL = 206.6 v. 157.3 µm s^–1, and ALH = 8.0 v. 5.9 µm, respectively). According to the literature, an ALH higher than 7 µm associated with a high VCL characterizes a vigorous and disordered movement indicating hyperactivity. The sperm frozen with the Botu-Bov had a higher straight movement (STR; 85.1%) and linearity (57.4%) than the TRIS group (79.8% and 45.6%, respectively). These features indicate a straighter and more uniform movement when Botu-Bov was used. The morphological analyses using fluorescent probes showed higher proportions of intact plasma and acrosomal membranes for Botu-Bov than for TRIS (50.1% v. 39.4% and 85.3% v. 71.2% for Botu-Bov and TRIS, respectively). Blastocyst formation after IVF was 21% (68/327; blastocysts/matured oocytes) for the TRIS group. This result was statistically different from the 30% (75/252) of blastocysts obtained with Botu-Bov and from the 34% (43/128) obtained in the control group. These results suggest that Botu-Bov provides better conditions for the maintenance of the viability of frozen sexed semen than does TRIS, probably due to the high concentration of essential and nonessential amino acids present in the Botu-Bov extender.

This work was supported by Sexing Technologies – Brazil and HG Lagoa da Serra.