Characteristics of Frozen-Thawed Spermatozoa Cryopreserved with Different Concentrations of Glycerol in Captive Japanese Black Bears (Ursus thibetanus japonicus)

Abstract

ABSTRACT. Seven mature Japanese black bears were used as semen donors, and a total of 7 semen samples collected from the animals by the electroejaculation method were cryopreserved in liquid nitrogen. Egg yolk-TRIS-citrate-glucose extender was used, and the effects of different final concentrations of glycerol, at 4-12% (v/v), on frozen-thawed spermatozoa were examined. No significant difference was observed in percent motility or percent abnormal morphology of frozen-thawed spermatozoa among the different glycerol concentrations. Percent viability and percent intact acrosomes of spermatozoa cryopreserved with 4 and 6% glycerol were significantly higher than those with 10 and 12% glycerol. These results suggest that a suitable glycerol concentration for freezing Japanese black bear semen within the range tested would be 4-6%. KEY WORDS: glycerol, Japanese black bear, semen cryopreservation.J. Vet. Med. Sci. 68(10): 1101-1104, 2006 The Japanese black bear (Ursus thibetanus japonicus) is an endangered species in some areas of Japan [5]. Semen cryopreservation is one of the most important techniques for species conservation as a genetic resource. There are reports of semen cryopreservation in ursids, including giant pandas (Ailuropoda melanoleuca) [13,14,18], Hokkaido brown bears (Ursus arctos yesoensis) [7], and Japanese black bears [11,12]. However, although efforts to establish an optimal technique are important, no comparative studies of different glycerol concentrations of diluents for bear semen cryopreservation have been reported. Glycerol has been widely used as a cryoprotectant in semen cryopreservation in domestic and non-domestic animals [19], although optimal glycerol concentrations vary among species [1]. Glycerol, a cryoprotective agent, exerts toxic effects on spermatozoa, such as plasma membrane rupture and acrosomal damage [6,10].The objective of the present study was to examine the effect of different glycerol concentrations of diluents for semen cryopreservation in Japanese black bears.Seven mature male Japanese black bears over 4 years of age at Ani Mataginosato Bear Park, Akita, Japan (N 40°, E 140.4°), were used as semen donors. They had been kept with 6 females and approximately 30 other males and had been allowed to mate freely with the females. Semen was collected once from each animal by electroejaculation during the mating season in June and July 2005. Electroejaculation was conducted according to the method of Kojima et al. [9] and our previous study [12] with some modifications.Briefly, the animals were immobilized by intramuscular administration of zolazepam HCl and tiletamine HCl (Zoletil, Virbac, Carrors, France; 9 mg/kg of the estimated body weight). After immobilization, the penis was cleaned, and the bladder was then emptied and flushed with sterile physiological saline by catheterization (5-fr. polypropylene urinary catheter; Sovereign, Sherwood Medical, MO, U.S.A.). A collection catheter (8-fr. Sovereign) whose tip had been cut off to widen the hole was inserted into the urethra...

Cryoprotective effect of different glycerol concentrations on domestic cat spermatozoa

“…Additionally, 3% glycerol resulted in higher percentage of intact acrosomes in dead cells compared with 5% and 7% glycerol. Thus, a detrimental effect on acrosome integrity due to high glycerol concentrations seems to exist in domestic cat spermatozoa as already reported in other species [16,34,37].…”

Effect of colloid (Androcoll-Bear, Percoll, and PureSperm) selection on the freezability of brown bear (Ursus arctos) sperm

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The antioxidant effects of soybean lecithin- or low-density lipoprotein-based extenders for the cryopreservation of brown-bear (Ursus arctos) spermatozoa

“…Ishikawa et al (2002) reported the use of 15% egg yolk for freezing semen from Hokkaido brown bears. Okano et al (2004Okano et al ( , 2006aOkano et al ( , 2006b cryopreserved semen from Japanese black bears at a final eggyolk concentration of 20%. Giant panda spermatozoa have been cryopreserved using 20% egg yolk (Spindler et al 2004).…”