USE OF DIFFERENT EXTENDERS TO CRYOPRESERVATION OF MANGALARGA MARCHADOR SPERM

Abstract

The techniques applied to animal reproduction such as artificial insemination, transfer and in vitro production of embryos, heat synchronization and induction, and gametes cryopreservation have been more utilized in veterinary practice each day. Nevertheless, some techniques have not achieved their full technical capacity within the equine reproduction field, such as semen cryopreservation.  The aim of this study was to evaluate the characteristics of post-thawing semen (total motility, strength, plasmatic and acrosomal membrane integrity) of Mangalarga Marchador breed stallions, using three different extenders (Botucrio, FR5 and FR6).  Ejaculates from five stallions were collected and the gel-free semen was diluted in a 1:1 dilution in skim milk extender, and centrifuged at 600 g for 10 minutes. After the centrifugation the supernatant was removed and sperm pellet was divided and re-suspended using three different extenders to a concentration of 200 x 10⁶ cells/mL. The samples were packed into 0.5 mL straws, placed in a stainless steel support and kept inside the refrigerator (5 ^oC) for one hour. Subsequently, these straws were kept at a height of 6 cm from liquid nitrogen for 15 minutes in an isotherm box and, after that, plunged into liquid nitrogen (-196 ^oC) and stored in a liquid nitrogen holding tank. There were no differences in the parameters evaluated when extenders using mixed amides and glycerol (Botucrio and FR6) were used (P > 0.05). All parameters evaluated were lower for the extender containing only glycerol (P<0.05). The use of cryoprotectants (methylformamide and dimethylformamide)  in association with glycerol concentrations around 1 to 2% is an alternative for semen cryopreservation of Mangalarga Marchador breed stallions.

Keywords: amides; cryoprotectant; equine; frozen semen; glycerol.