Cryopreservation of the Semen Collected by Electroejaculation from the Hokkaido Brown Bear(Ursus arctos yesoensis).

Abstract

ABSTRACT. Four adult Hokkaido brown bears were used as semen donors, and semen characteristics were examined before freezing and after thawing. A total of 10 electroejaculates were diluted with Tris-egg yolk extender and cooled to 4°C over 90 min. Spermatozoa were equilibrated with 4.7% glycerol for 80 min. Semen packed in 0.25 ml plastic straws were frozen with liquid nitrogen vapor. Percentages (mean ± SD) of motile and live sperm were 96 ± 2 and 86.5 ± 7.2% before freezing, and 43 ± 5 and 67.4 ± 3.9% after thawing, respectively. Although the number of progressively motile sperm after thawing varied among samples (1.8 ± 1.2 × 10 8 cells/ejaculate), frozen semen in the present study might serve for artificial insemination. KEY WORDS: brown bear, freezing, spermatozoa.J. Vet. Med. Sci. 64(4): 373-376, 2002 The Hokkaido brown bears are found only in Hokkaido Island, Japan, and their genetic diversity is known to be low [15]. An assisted reproductive technology like artificial insemination could contribute to the maintainance of their genetic diversity. We previously reported a successful semen collection of the Hokkaido brown bear by electroejaculation [7]. Semen cryopreservation is an essential procedure for assisted breeding; however, there are only a few reports on cryopreservation of the bear semen [2,9]. The objective of the present study was to examine the potential of a freezing procedure used for bull semen with a modification in glycerol concentration.Four adult males, aged 7 to 16 years and maintained at Noboribetsu Bear Park (Noboribetsu, Japan), were used as semen donors. They were kept in male groups and separated from females. A total of 10 trials of electroejaculation were conducted during the breeding season (May to July) in 1996 and 1997. Electroejaculation was conducted as described previously [7]. Briefly, bears were immobilized with tiletamine HClzolazepam HCl (Zoletil; Virbac S. A., Carros, France) in 8 trials, and with midazolam (Dormicum; F. Hoffman-La Roche Ltd., Basel, Switzerland) and ketamine HCl (Ketalar; Sankyo Co., Ltd., Tokyo, Japan) in 2 trials. After immobilization, a handmade bipolar rectal probe with two ring electrodes was inserted into the rectum. The electrical stimulation with 60 Hz alternative current, sine wave and 5 V was repeated till ejaculates were obtained. The semen was assessed by determining ejaculate volume, pH, sperm concentration and the percentage of abnormal sperm as described elsewhere [7]. The percentage of viable spermatozoa was determined by an eosin-nigrosin staining method [7] before and after freezing. Small aliquot of raw and frozen-thawed semen was taken for the assessment of sperm motility. Percentage of sperm in each type of progressive motility status was calculated under a light microscope using a scale of 0 to 4: 0, no movement; 1, slight side-to-side movement with no forward progression; 2, side-to-side movement, slow and steady forward progression; 3, steady, moderate forward progression; 4, steady, rapid forward progression. The total perce...

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

“…The reported sperm concentration of the captive Japanese black bear (mean 361 × 10 6 /ml [16]), Hokkaido brown bear (mean 590 × 10 6 /ml [13]) and Giant panda (mean 645 × 10 6 /ml [22]) was similar to that observed in this study (mean 540 × 10 6 /ml). On the other hand, the reported total ejaculate volume of the captive Japanese black bear (mean 0.2 ml [16]), Hokkaido brown bear (mean 3.7 ml [13]), Giant panda (mean 6.5 ml [22]) and free-ranging Japanese black bear (mean 1.5 ml; this study) differed from each other to some extent.…”

“…On the other hand, the reported total ejaculate volume of the captive Japanese black bear (mean 0.2 ml [16]), Hokkaido brown bear (mean 3.7 ml [13]), Giant panda (mean 6.5 ml [22]) and free-ranging Japanese black bear (mean 1.5 ml; this study) differed from each other to some extent. This might be due not only to species difference but also to the variation in electric stimulation methods and the degree of urine contamination.…”

“…This study showed that frozen-thawed spermatozoa were motile, but the result was not as good as that of other captive ursid [13,22]. Experiments will be required to further improve the post-thaw motility including % +++ motility and % motility, probably by using captive Japanese black bears as a model.…”

“…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.…”

“…As to Ursidae, there are reports on electroejaculation and/or semen cryopreservation in giant pandas [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.…”