Electroejaculation and Semen Cryopreservation of Free-Ranging Japanese Black Bears (Ursus thibetanus japonicus)

Abstract

ABSTRACT. The Japanese black bear (Ursus thibetanus japonicus) is endangered for extinction in some areas of Japan, and semen collection and cryopreservation are an important means to preserve genetic resources. The aim of this study was to characterize and c ryopreserve semen of free-ranging Japanese black bears. Semen was collected by electroejaculation procedure from 4 free-ranging Japanese black bears at the capture point in the field. Ejaculates containing motile sperm were recovered from all of the animals and ejaculate volume, total sperm count, % motility (percentage of motile spermatozoa), % viability (percentage of spermatozoa that excluded eosin) and % abnormal morphology (range (mean)) were 0.65-2.20 (1.51) ml, 99-1082 (490) × 10 6 , 5-100 (31), 42-97 (66) and 20-87 (53), respectively. Three of the 4 ejaculates were diluted with an egg yolk-TRIS-citrate-glucose extender and cryopreserved in liquid nitrogen. Motile spermatozoa were observed after freezing and thawing in all cases. This study showed that electroejaculation would be a useful method for collecting semen from free-ranging Japanese black bears and that at least motile spermatozoa would be obtained by fre ezing the thus collected electroejaculates. KEY WORDS: cryopreservation, electroejaculation, free-ranging, Japanese black bear, semen.J. Vet. Med. Sci. 66(11): 1371-1376, 2004 The Japanese black bear (Ursus thibetanus japonicus) is a large mammal that inhabits the islands of Honshu and Shikoku in Japan. In recent years, their habitats have become fragmented and shrunken by human activity, and this species even faces extinction in some areas [7]. What is worse, bears have been killed to protect forestry, agriculture, livestock and humans from damage caused by them.Endangered and threatened wild animals are now attracting attention as a genetic resource and for species conservation, and semen has been recovered from various captive non-domestic animals, including fat-tailed dunnarts, koalas, brushtail possums, long-footed potoroos, northern brown bandicoots and ring-tailed possums [32] [4,20,22,24], Hokkaido brown bears [13,14] and Japanese black bears [16]. However, these studies were done with captive animals, and there are no reports on semen collection and cryopreservation in free-ranging bears. Cryopreservation of semen collected from free-ranging animals is one of the most important methods for preservation of genetic resources and useful for artificial breeding. Moreover, characteristics of semen from wild animals may be essential for understanding reproductive status under field conditions, although studies using captive animals, kept under controlled conditions, may provide valuable data to analogize state under field conditions. It is easy to expect that the state of captive animals may differ from that of free-ranging ones to some extent, because of differences in their living environment and diet. It is difficult to obtain semen from free-ranging Japanese black bears due to limited access to the animals and seasonality of their ...

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

“…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.…”

“…Semen cryopreservation was performed according to the method of our previous study [11,12] with some modifications. Briefly, the diluent used was egg yolk-TRIS-citrateglucose extender [15] supplemented with 0.52 mg/ml of potassium penicillin G and 0.8 mg/ml of streptomycin sulphate (first extender).…”

“…We have customarily used an egg yolk-citrate-based diluent with 8% glycerol in our laboratory for freezing Japanese black bear semen [11,12]. However, we have found percent viability and intact acrosomes of frozen-thawed spermatozoa to be better when freezing semen at lower glycerol concentrations of 4 and 6%.…”

Effects on brown bear (Ursus arctos) spermatozoa freezability of different extender and dilution ratios used for pre-freezing centrifugation

“…Different studies on electroejaculation of bears have indicated an elevated incidence of urospermia, with the consequent loss of semen quality (Okano et al 2004;Chen et al 2007;Kojima et al 2001;Anel et al 2008). Thus, we considered that developing a protocol for washing sperm samples was critical for the success of a sperm recovery plan in the brown bear.…”

In Vitro Maturation of Oocytes Derived from the Brown Bear (Ursus Arctos)

“…There are several reports about techniques for collecting and cryopreserving the semen of ursids [6][7][8][9][10][11][12][13], but these reports mainly focus on comparison of semen research; oocyte collection and in vitro maturation (IVM) have only been reported in one paper [14]. Investigation of oocyte characteristics and development of IVM methods are the next basic steps for application of advanced reproductive technologies, such as in vitro fertilization (IVF), embryo transfer, and somatic cell cloning, in this species.…”