Effects of Final Dilution Rate, Sperm Concentration and Times for Cooling and Glycerol Equilibration on Post-Thaw Characteristics of Canine Spermatozoa

Abstract

ABSTRACT. This study re-evaluated a protocol for cryopreservation of canine semen. Semen from 4 beagle dogs was pooled, concentrated by centrifugation and adjusted to increasing sperm concentrations by adding back seminal plasma. The prepared or original semen was diluted with an extender (Egg yolk-Tris-citrate-glucose) and cooled to 4°C (cooling), followed by a second dilution with the same extender including glycerol, equilibrated at 4°C (equilibration), then stored in liquid nitrogen. The semen was diluted for frozen samples having a fixed sperm concentration with increasing dilution rates or for those having the reverse combinations. Various dilution rates of 2.5-10 folds or sperm concentrations of 0.25-2.5 × 10 8 /ml had no significant effect on post-thaw sperm characteristics. When cooling was done for different times (0-26 hr) with glycerol equilibration for 1 hr, post-thaw characteristics were better at 2 and 3 hr of cooling, while various times for equilibration (0-4 hr) with cooling for 3 hr had no effect. These results suggest that different dilution rates and sperm concentrations within the ranges tested may not affect the post-thaw sperm characterisitics and that sufficient time for cooling may be essential but a specific equilibration time may not necessarily be required. KEY WORDS: artificial insemination, canine, cryopreservation, semen, spermatozoa.J. Vet. Med. Sci. 66(11): 1359-1364, 2004 Biotechnological studies may contribute much to the conservation of endangered wildlife species, and assisted reproduction techniques in domestic dogs may be applicable to non-domesticated canid species [11,14]. Indeed, techniques of sperm cryopreservation developed for domestic dogs have been applied to captive red wolves [10]. On the other hand, if semen were collected from wild animals in the field, manipulation of the ejaculate would have to be carried out under unfavorable conditions. Thus, modifications would be needed for the application of techniques developed for dogs to wild animals.Successful artificial insemination with frozen canine semen has been well documented [18,22,23,25,27] since the first conception was reported by Seager [21], and recently, the freezing protocol for canine semen has been improved [1,3,20,[28][29][30]. If semen is diluted to a constant final sperm concentration, the dilution rates may differ. Alternatively, if the dilution rate is fixed, the final sperm concentration might fluctuate according to the sperm concentration measured in the collected semen. Systematic studies examining the effects of the final dilution rate and the final sperm concentration on post-thaw sperm characteristics are lacking, except for limited information provided by Peña and Linde-Forsberg [17].In most cases, canine semen is diluted, equilibrated with a cryoprotectant such as glycerol, and stored frozen in liquid nitrogen. In some reports, collected semen is directly diluted with an extender including glycerol and then equilibrated for hours before freezing [1,7,9,13,18,20,23,26].In other reports, s...

Spermatozoa recovery and post-thawing quality of brown bear ejaculates is affected for centrifugation regimes

“…It is known that there is a great variability among species regarding sensitivity to injury caused by centrifugation. Thus, spermatozoa from rat (Cardullo and Cone 1986), human (Ng et al 1992;Alvarez et al 1993;Aitken and Clarkson 1988) and mouse (Katkov and Mazur 1998) are sensitive to centrifugal forces, but spermatozoa from other species are not (dog, Okano et al 2004b;stallion, Cochran et al 1984;Sieme et al 2004;Brinsko et al 2000;Crockett et al 2001;Waite et al 2008;bull, Pickett et al 1975;and boar Carvajal et al 2004;Matas et al 2007).…”

The Positive Effects of Seminal Plasma During the Freezing Process on Cryosurvival of Sperm with Poor Freezability in the Rhesus Macaque (Macaca mulatta)

“…Previous study compared the effects of low (5 × 10 6 sperm/ml) and high sperm concentrations (5 × 10 7 and 5 × 10 8 sperm/ml) on the cryosurvival of sperm from rhesus macaques and revealed that post-thaw sperm motility was not affected when sperm were cryopreserved at high concentrations [26]. Another study showed that different sperm concentrations within a tested range (up to 10 times) did not affect the post-thaw characteristics of canine sperm [27]. In our study, all of the rhesus macaque sperm samples were frozen at high sperm concentrations (ranging from 1.9 × 10 7 to 2.0 × 10 8 sperm/ml, calculated from Table 1).…”

The Comparative Cryobiology of Preimplantation Embryos from Domestic Animals

“…Many investigations into the cryobiology of spermatozoa in dogs have been reported (Bouchard et al, 1990;Farstad, 1996;Hay et al, 1997;Okano et al, 2004;Rota et al, 1995;Songsasen et al, 2002;Thirumala et al, 2003). In addition, reports on oocyte maturation, in vitro fertilization, and embryo culture have been published (Renton et al, 1991;Yamada et al, 1992;Yamada et al, 1993).…”