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 ...

ABSTRACT. Twenty-one wild male Japanese black bears ( Ursus thibetanus japonicus) were captured in the summer-autumn of 1998-2000 in the vicinity of Neo Village, Gifu Prefecture. Testes were measured, and testicular samples were biopsied and observed histol ogically. Four steroidogenic enzymes, i.e., cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), 17-α hydroxylase cytochrome P450 (P450c17), and aromatase cytochrome P450 (P450arom) were immunolocalized. Serum testosterone concentrations were measured by radioimmunoassay. Testis size changed little from 1-3 years of age, increased rapidly at 4 years, and attained its peak at 5 years. Serum testosterone concentrations ranged from 0.05 to 1.78 ng/ml, and the mean ± standard deviation was 0.43 ± 0.48 ng/ml. Age of sexual maturation in wild male Japanese black bears was estimated to be 3-4 years. Seasonal changes in spermatogenesis were obvious; active in June, July and August, degenerated by September. Leydig cells, Sertoli cells and germ cells have the capability of synthesizing androgen, and Leydig cells, Sertoli cells, spermatids and spermatogonia have the capability of synthesizing estrogen in Japanese black bears. KEY WORDS: Japanese black bear, spermatogenesis, steroidogenic enzyme, testosterone, Ursus thibetanus japonicus.J. Vet. Med. Sci. 65(10): 1093-1099, 2003 In order to enhance the conservation and management of wild animals, we have to know the population dynamics of breeding-age animals. For this purpose, age at sexual maturation and seasonal changes in reproductive activity such as gonadal morphology profiles and sexual behavior in the population need to be determined [11]. With this in mind ecological investigations and physiological examinations of the Japanese black bear (Ursus thibetanus japonicus) are being performed in many study areas, and reproductive physiology in the male Japanese black bear has been reported recently from this point of view [11,12,22].The age of sexual maturation (puberty) in male Japanese black bears is estimated to be 2-4 years with some variations among studies based on the histology of testes, their size and weight, and the diameter of seminiferous tubules [11,12,16,22]. It is known that the reproductive physiology of the male Japanese black bear shows notable seasonal changes [25] with mating occurring from June to August [29]. In relation to this, testes indicate physiological and morphological changes, and the period of high spermatogenic activity is limited to several months, including the mating season [13].The aim of this study was to determine the age of sexual maturation and the seasonal changes in spermatogenesis and testicular steroidogenesis in wild male Japanese black bears. MATERIALS AND METHODS Capture and handling:Wild male Japanese black bears that inhabit Neo Village, Gifu Prefecture, were caught by barrel-type traps from 26 June 1998 to 13 November 1998, from 15 May 1999 to 12 November 1999, and from 15 July 2000 to 31 August 2000. Capture of bears was...

The Fukushima Daiichi Nuclear Power Plant (FDNPP) accident that occurred after the Great East Japan Earthquake in March 2011 released large quantities of radionuclides to the environment. The long-term effects of radioactive cesium (Cs) on biota are of particular concern. We investigated the accumulation of radioactive Cs derived from the FDNPP accident, and chronic effects of environmental radionuclides on male reproduction, in the large Japanese field mouse (Apodemus speciosus). In 2013 and 2014, wild mice were captured at 2 sites in Fukushima Prefecture and at 2 control sites that were distant from Fukushima. Although the median concentrations of 134Cs and 137Cs in the mice from Fukushima exceeded 4,000 Bq/kg, there were no significant differences in the apoptotic cell frequencies or the frequencies of morphologically abnormal sperm among the capture sites. Thus, we conclude that radiation did not cause substantial male subfertility in Fukushima during 2013 and 2014, and radionuclide pollution levels in the study sites would not be detrimental to spermatogenesis of the wild mice in Fukushima.

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...

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...

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The objectives of the present study were to observe the temporal pattern of avian influenza virus (AIV) introduction into Japan and to determine which migratory birds play an important role in introducing AIV. In total, 19,407 fecal samples from migratory birds were collected at 52 sites between October 2008 and May 2015. Total nucleic acids extracted from the fecal samples were subjected to reverse transcription loop–mediated isothermal amplification to detect viral RNA. Species identification of host migratory birds was conducted by DNA barcoding for positive fecal samples. The total number of positive samples was 352 (prevalence, 1.8%). The highest prevalence was observed in autumn migration, and a decrease in prevalence was observed. During autumn migration, central to southern Japan showed a prevalence higher than the overall prevalence. Thus, the main AIV entry routes may involve crossing the Sea of Japan and entry through the Korean Peninsula. Species identification was successful in 221 of the 352 positive samples. Two major species sequences were identified: the Mallard/Eastern Spot-billed duck group (115 samples; 52.0%) and the Northern pintail (61 samples; 27.6%). To gain a better understanding of the ecology of AIV in Japan and the introduction pattern of highly pathogenic avian influenza viruses, information regarding AIV prevalence by species, the prevalence of hatch-year migratory birds, migration patterns and viral subtypes in fecal samples using egg inoculation and molecular-based methods in combination is required.

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