110 COMPARISON OF ANDROMED®, BIOXCELL®, AND BOTU-BOV® EXTENDERS FOR CRYOPRESERVATION OF BULL SEXED SEMEN

Abstract

Sexing semen has become a worldwide technology now available in many countries through the use of flow cytometry for sexing mammal sperms (Johnson and Welch 1999 Theriogenology 52, 1323–1341). Because straws containing sexed semen have a low concentration, any condition that either improves or decreases freezing capabilities will considerably change semen quality. During cryopreservation, spermatozoa have been described as undergoing many changes that lead to membrane damage, which may result in decreased fertility (Watson 2000 Reprod. Fertil. Dev. 6 (Suppl 1), 481). Since many cryoprotectants are available on the market, the objective of the present study was to compare 3 different extenders for freezing sex-sorted semen. For this study, 25 ejaculates were collected from 8 bulls of different breeds, diluted, then dyed with Hoechst 33342 (Schenk et al. 1999 Theriogenology 52, 1375–1391), and sexed by flow cytometry (SX MoFlo®; DaKoCytomation, Inc., Fort Collins, CO, USA). After being cooled at 4°C for 1 h and 30 min, the sexed semen was centrifuged and diluted in AndroMed® (Minitüb, Tiefenbach, Germany), Bioxcell® (IMV, Aigle, France), or Botu-Bov® (Biotech Botucatu, Ltda., Sao Paulo, Brazil); the semen was packaged at 3 million total sperm in 0.25-mL straws and frozen in an automatic freezer (Digit cool 5300® IMV). To evaluate the freezing quality, the straws were thawed and incubated at 35°C for 15 min. The progressive motility was observed through an optical microscope (Coleman 200T). The statistical analyses were done using the SAS program (SAS Institute, Inc., Cary, NC, USA) and the Tukey test (P ≤ 0.05). Results show that there was no statistical difference between Bioxcell and AndroMed extenders (P ≤ 0.05). However, Botu-Bov extender showed a significant difference when compared with Bioxcell and AndroMed (see Table 1). It is also important to point out that 40% of the samples frozen with AndroMed showed non-aligned movement. Even though few ejaculates were used for this study, preliminary results showed that Bioxcell seemed to be the most suitable for freezing bull sexed semen. Table 1. Percentage of progressively motile spermatozoa after thawing