IN-VITRO CHARACTERISTICS OF CANINE SPERMATOZOA SUBJECTED TO 2 METHODSOF CRYOPRESERVATION

The in vitro Viability of canine spermatozoa was evaluated after freez ing-thawing using the Andersen method, and the commercial CLONE method . These methods differ in the extenders used, number of dilution steps , and equilibration times as well as in both freezing and thawing tech niques and rates. Insemination with semen frozen-thawed by either meth od gives high whelping rates in practice, implying that dog spermatozo a can retain their fertilizing ability after being subjected to widely different preservation methods. The in vitro viability of spermatozoa processed by these methods has not been previously evaluated in detai l. Three ejaculates were collected from each of 5 fertile dogs. Each e jaculate was divided into 2 parts and frozen in medium straws accordin g to the 2 methods. Two straws were thawed and examined from each free zing batch. Sperm motility was assessed in the undiluted semen, and in frozen-thawed semen immediately after thawing, and after storage for 3, 6 and 24 h at room temperature (Straw 1) or 1, 2 and 3 h at 37 degr ees C (Straw 2, thermoresistance test). The integrity of the sperm pla sma membrane was evaluated in undiluted, in equilibrated (diluted and chilled), and in frozen-thawed spermatozoa using fluorophore probes. T he acrosome morphology of frozen-thawed spermatozoa was assessed using a commercial stain (Spermac(R)). Motility immediately after thawing w as significantly higher with the CLONE method (75.3% [SD=4.0] for Stra w 1 and 73.7% [SD=3.2] for Straw 2) than with the Andersen method (70. 0% [SD=5.1] and 69.7% [SD=3.2]). Motility decreased during storage aft er thawing. Spermatozoa frozen-thawed using the CLONE method showed a significantly lower thermoresistance. The proportion of spermatozoa wi th intact plasma membrane was not affected by the equilibration proced ure used with either method but was significantly decreased (P<0.001) after thawing with both methods. The percentage of spermatozoa exhibit ing changes thought to represent different stages of acrosomal degrada tion, was 45.7% (SD=5.3) using the Andersen method and 44.1% (SD=9.2) using the CLONE method. Both cryopreservation methods thus resulted in high initial post-thaw sperm motility and membrane integrity but low thermoresistance, and under both methods a large proportion of sperm c ells were undergoing acrosomal degradation. The methods differed signi ficantly in terms of their effect on sperm motility but not on plasma membrane integrity or acrosomal morphology. (C) 1997 by Elsevier Scien ce Inc.