Full Paper. Tsukasa OKANO 1), Tetsuma MURASE 1,2), Sachiko NAKAMURA 1), Takeshi KOMATSU 3), Toshio TSUBOTA 4) and Makoto ASANO 1,5)

Transcription

1 Journal of Reproduction and Development, Vol. 55, No. 2, 2009, Full Paper Normal Sperm Morphology and Changes of Semen Characteristics and Abnormal Morphological Spermatozoa among Peri-mating Seasons in Captive Japanese Black Bears (Ursus thibetanus japonicus) Tsukasa OKANO 1), Tetsuma MURASE 1,2), Sachiko NAKAMURA 1), Takeshi KOMATSU 3), Toshio TSUBOTA 4) and Makoto ASANO 1,5) 1) United Graduate School of Veterinary Sciences, Gifu University, Gifu , 2) Laboratory of Veterinary Theriogenology, Faculty of Applied Biological Sciences, Gifu University, 3) The Institute of Japanese Black Bear in Ani, Akita , 4) Graduate School of Veterinary Medicine, Hokkaido University, Sapporo and 5) Laboratory of Zoo and Wildlife Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu , Japan Abstract. The objectives of this study were to obtain morphological data for normal spermatozoa and to investigate seasonal changes (the early, mid- and post-mating seasons) in abnormal morphology of spermatozoa and the characteristics of semen in Japanese black bears. Semen was collected by electroejaculation from 34 captive male Japanese black bears a total of 74 times. Length of head, width of head, length of midpiece and total length of the spermatozoa were 6.3 ± 0.4, 4.5 ± 0.3, 10.4 ± 0.7 and 69.6 ± 3.1 μm (mean ± SD; 20 semen, 200 spermatozoa), respectively. In the semen collected during the mid-mating season, ejaculate volume, ejaculate ph, sperm concentration, total sperm count, motility, viability and intact acrosomes were 0.46 ± 0.36 ml, 7.3 ± 0.4, 659 ± /ml, 214 ± , 82.9 ± 9.6%, 89.3 ± 9.5% and 97.0 ± 3.2% (mean ± SD; n=21, in ejaculate ph n=8), respectively. Sperm motility and viability in the early (n=7) and mid-mating (n=21) seasons were significantly higher than in the post-mating (n=8) season. The rates of detached heads in the early and mid-mating season were significantly lower than in the post-mating season. The main abnormal morphologies observed (mean ± SD%; n=23) were simply bent tail (19.9 ± 22.6), distal droplets (13.5 ± 11.7), proximal droplets (9.6 ± 7.8), teratoid spermatozoa (6.7 ± 10.7), knobbed acrosome (4.9 ± 8.6), acrosome damage (3.7 ± 2.8) and bent midpiece (3.7 ± 5.1). The data will be useful for artificial breeding and further research on male reproductive physiology in this species. Key words: Electroejaculation, Japanese black bear (Ursus thibetanus japonicus), Semen characteristics, Spermatozoa, Sperm morphology (J. Reprod. Dev. 55: , 2009) he Japanese black bear (Ursus thibetanus japonicus) is a subspecies of Asiatic black bears (Ursus thibetanus) that inhabits the islands of Honshu and Shikoku in Japan [1]. Asiatic black bears are also widely distributed throughout southern Asia, northern China and far eastern Russia [2]. Recently, the number of Asiatic black bears is decreasing, mainly due to habitat loss and over-hunting, and the Asiatic black bear is categorized as Vulnerable on the Red List of the International Union for the Conservation of Nature (IUCN) [2]. The number of Japanese black bears is relatively stable in the Chubu and Tohoku region but is decreasing in Western Japan [1]. Although conservation of wild animal species should ideally be conducted in their own original habitats, such habitats for many wild animals are decreasing very quickly due to human activity [2]. In such cases, it is necessary to conserve these species in ex situ facilities with artificial breeding techniques as well as in their in situ habitats. Accumulation of knowledge concerning spermatozoa and semen is important for successful artificial insemination (AI). For this reason, many studies of semen collection and preservation in ursids have been reported [3 7]. Accepted for publication: December 13, 2008 Published online in J-STAGE: February 5, 2009 Correspondence: M Asano ( asanojr@gifu-u.ac.jp) Japanese black bears are seasonal breeders. With their mating season between mid-june and early August, mating most frequently occurs in mid-july [8]. In male bears, spermatogenesis begins in March during the denning period, and active spermatogenesis is observed between May and July [9 11]; active spermatogenesis almost always ends between October and February [10]. Generally, decrease in semen volume and drop in sperm viability are observed in seasonal breeders during the non-mating or non-breeding season [12], so frozen preserved semen collected during the non-mating season is considered unsuitable for AI. If high-quality semen can be collected in the non-mating season, more frozen semen can be retained for AI to assure successful artificial breeding. In the bovine and canine, domestic animals, the relationship between abnormal sperm morphology and male infertility has been gradually clarified through study [13, 14]. Sperm morphology is reflected not only in the health of the testes and epididymis but also in the health of the animal itself. In captive Japanese black bear semen collected by the electroejaculation method, relatively many morphologically abnormal spermatozoa are observed [15, 16]. However, the seasonal changes in abnormal sperm morphology in Japanese black bear spermatozoa remain unclear. The objectives of this study were to obtain morphological data

2 BEAR SPERM MORPHOLOGY IN PERI-MATING SEASON 195 for normal spermatozoa and to compare the rates of morphologically abnormal spermatozoa and semen characteristics among 3 seasons (the early, mid- and post-mating seasons) in captive Japanese black bears. Materials and Methods Animals Thirty-four mature male Japanese black bears over 4 years of age at Ani Mataginosato Bear Park (Akita, Japan; N 40, E ) were used as semen donors. Immobilization The animals were isolated in a room and were handled after chemical immobilization by intramuscular administration of zolazepam HCl and tiletamine HCl (Zoletil, Virbac, Carrors, France; 9 mg / kg of the estimated body weight) via a dart blowgun. Semen collection Semen was collected by electroejaculation a total of 74 times (2.2 ± 1.1 times/head) in June-September Electroejaculation was performed according to the method of Okano et al. [5]. Thirty-six of the semen samples from 24 animals that were largely uncontaminated with urine were used for this study. Urine contamination was judged subjectively by the appearance of yellow color. The period of semen collection was divided into 3 seasons, the early mating season (19 June 25 June, n=7), mid-mating season (2 July 31 July, n=21) and post-mating season (29 August 10 September, n=8). Sampling in the same season during the same year from the same bear was performed in bear No. 12 (July 2003, 4 times) and bear No. 22 (July 2003, 2 times). Sampling in different seasons during the same year from the same bear was performed in bear No. 47 (July 2005, September 2005). Measurement of testes volume The bears testes were measured before semen collection in 2005 and The testes data was used in this study if the when semen data was used (successful semen collection). Testes volumes were calculated for 23 animals (early mating season, n=7 [7 animals]; mid-mating season, n=8 [7 animal; one animal was used a second time in a different year]; post-mating season, n=8 [8 animals]). Testes were measured from outside the scrotum, and the testis size was calculated by the formula 3 (length) (width) (depth) (mm) [17]. Semen examination The semen characteristics were examined immediately after collection except for examinations of intact acrosome and abnormal morphology, which were carried out after fixation and transportation to the laboratory. Semen examination was performed according to the method of Okano et al. [5], with some modifications. Briefly, the appearance of the ejaculate was examined, and the ejaculate volume was measured with the aid of a micropipette. Semen ph was measured using ph test paper (Duotest, Macherey- Nagel, Dueren, Germany; n=23, same samples as for measurement of testes volume). Sperm concentration was estimated by hemocytometer. Motility was evaluated by subjective observation under a microscope ( 400) at 38 C, and the results are shown as the percentage of motile (including non-progressive) spermatozoa (% motility). Viability was assessed using a supravital stain with eosin and nigrosin (% viability). Spermatozoa were fixed by mixing with an equal volume of 2% (v/v) glutaraldehyde in M sodium cacodylate/hcl (ph 7.3). Slides were prepared with the glutaraldehyde-fixed sample, and a total of 100 spermatozoa were examined under a phase-contrast microscope ( 1,000) to determine the proportion (%) of spermatozoa with intact acrosomes (% intact acrosomes) according to the method described by Caiza et al. [18] and to obtain morphological data for the spermatozoa (n=23, same samples in measurement of testes volume). Spermatozoa morphology was divided into 3 categories, normal morphology, major defects and minor defects, and then the morphologically abnormal spermatozoa were divided into the following 4 categories: head, midpiece and neck, tail and other defects [13, 14, 19] (Table 1). A full listing of the abnormal morphologies is presented in Table 1. Normal spermatozoa were observed in 20 randomly-selected semen samples (early mating season, n=9; mid-mating season, n=4; post-mating season, n=7). About 10 fixed spermatozoa in these samples (a total of 200) were digitally photographed under a phasecontrast microscope ( 400 or 1,000) and measured with image analysis software (Aqua-Lite, Hamamatsu Photonics, Hamamatsu, Japan) to obtain the head length, head width, midpiece length and total length. Statistics Data are presented as means ± SD. The differences in the characteristics among the three seasons were tested by one-factor ANOVA. Differences found by ANOVA were tested with Fisher s protected least significant difference post-hoc test. Differences of P<0.05 were regarded as statistically significant. Results Normal morphology of spermatozoa The normal morphology of the spermatozoon is shown in Fig. 1. Length of head, width of head, length of midpiece and total length of the spermatozoa were 6.3 ± 0.4, 4.5 ± 0.3, 10.4 ± 0.7 and 69.6 ± 3.1 μm, respectively. Semen characteristics in the different collection seasons The semen characteristics in the early, mid- and post-mating seasons are shown in Table 2. There were no statistically significant differences in body weight, testes volume, ejaculate volume, ejaculate ph, sperm concentration, total sperm count or intact acrosomes among the three seasons. Sperm motility and viability in the early and mid-mating seasons were significantly higher than in the post-mating season. Abnormal morphologies in the different collection seasons The abnormal morphologies observed during the early, mid- and post-mating seasons are shown in Table 1. The rates of the abnormal (except detached head) and normal morphologies examined

3 196 OKANO et al. Table 1. Abnormal morphology rates (%) of Japanese black bear spermatozoa in different seasons (mean ± SD) Early mating season Mid-mating season Post-mating season No. of males No. of samples Normal morphology 39.9 ± ± ± 22.5 Major defects (%) 34.3 ± ± ± 14.0 Minor defects (%) 38.1 ± ± ± 28.2 Head defects: Major Double heads 0.1 ± ± ± 0.4 Amorphous head 1.3 ± ± ± 0.5 Pear-shaped head 0.7 ± ± ± 0.0 Knobbed acrosome 4.3 ± ± ± 3.6 Nuclear vacuoles 0.1 ± ± ± 0.0 Minor Microcephalic 2.0 ± ± ± 0.5 Macrocephalic 0.1 ± ± ± 0.0 Damaged or reacted acrosome 2.9 ± ± ± 2.3 Overall 11.6 ± ± ± 4.7 Midpiece or neck defects: Major Double midpieces 0.1 ± ± ± 0.4 Kinked midpiece 0.1 ± ± ± 0.7 Short midpiece 0.0 ± ± ± 0.4 Thin midpiece 0.3 ± ± ± 0.5 Thick midpiece 1.6 ± ± ± 0.8 Midpiece with irregular debris 0.1 ± ± ± 0.0 Double necks 0.0 ± ± ± 0.4 Proximal droplets 12.7 ± ± ± 8.9 Minor Bent midpiece 2.0 ± ± ± 7.4 Bent neck 1.0 ± ± ± 3.4 Distal droplets 8.4 ± ± ± 12.2 Overall 26.4 ± ± ± 20.7 Cytoplasmic droplets 21.1 ± ± ± 20.0 Tail defects: Major Double tails 0.4 ± ± ± 0.4 Dag defect (tightly bent or coiled) 1.7 ± ± ± 3.0 Short tail 0.6 ± ± ± 0.7 Minor Simply coiled tail 2.6 ± ± ± 1.4 Terminally coiled tail 1.6 ± ± ± 5.4 Simply bent tail 15.6 ± ± ± 27.2 Overall 22.4 ± ± ± 34.5 Other defects: Major Teratiod spermatozoa 10.0 ± ± ± 8.1 Minor Detached tail 1.1 ± ± ± 4.8 Detached head 0.9 ± 0.9 a 1.5 ± 0.8 a 3.1 ± 2.2 b Groups with different superscripts in the same row are significantly different (P<0.05). The sum of normal morphology and major and minor defects is over 100% because some spermatozoa contained more than one defect. did not show any statistically significant differences among the three seasons. The rates of detached head in the early and mid-mating seasons were significantly lower than in the post-mating season. The main abnormal morphologies observed were simply bent tail (19.9 ± 22.6%, Fig. 2a), distal droplets (13.5 ± 11.7%, Fig. 2b), proximal droplets (9.6 ± 7.8%, Fig. 2c), teratoid spermatozoa (6.7 ± 10.7%, Fig. 2d f), knobbed acrosome (4.9 ± 8.6%, Fig. 2g), damaged or reacted acrosome (3.7 ± 2.8%, Fig. 2h) and bent midpiece (3.7 ± 5.1%, Fig. 2b). Fig. 1. Normal morphology of the Japanese black bear spermatozoon. a) Whole, b) Anterior half.

4 BEAR SPERM MORPHOLOGY IN PERI-MATING SEASON 197 Table 2. Semen characteristics, body weights and testes volumes of Japanese black bears in different seasons (mean ± SD) Early mating season Mid-mating season Post-mating season No. of males 7 16 (8)* 7 No. of samples 7 21 (8)* 8 Body weight (kg) 105 ± ± ± 21 Testes volume (mm) 38.6 ± ± ± 2.5 Ejaculate volume (ml) 0.57 ± ± ± 0.30 Ejaculate ph 7.2 ± ± ± 0.5 Sperm concentration ( 10 6 /ml) 316 ± ± ± 431 Total sperm count ( 10 6 ) 149 ± ± ± 200 Motility (%) 80.0 ± 11.5 a 82.9 ± 9.6 a 60.6 ± 30.3 b Viability (%) 94.6 ± 2.6 a 89.3 ± 9.5 a 78.5 ± 13.7 b Intact acrosomes (%) 97.1 ± ± ± 2.3 *: Eight samples were collected for testes volume and ejaculate ph in the mid-mating season. Groups with different superscripts in the same row are significantly different (P<0.05). Fig. 2. Abnormal morphology of Japanese black bear spermatozoa Bar, 5 μm. a) Simply bent tail. b) Bent midpiece and distal droplet. c) Proximal droplet. d f) Teratoid spermatozoa. g) Double neck/ midpiece and knobbed head. h) Damaged or reacted acrosome. Discussion In the present study, the morphological parameters of normal spermatozoa in Japanese black bears were clarified. It remains possible that there are seasonal changes in these morphological parameters. Accumulation of morphological data for normal spermatozoa is important, and provides a useful index for examination of abnormal spermatozoa. Spermatozoa morphology varies among species. The heads of sperm are flat and oval-shaped in domestic mammals and humans, gooseneck in shape in rats and mice and corded in roosters [12, 20]. The sizes of sperm heads (length/ width) in bulls, boars and dogs are / , /3.6 and 6.5/ μm, respectively [20]. In carnivores, species closely related to ursids (i.e, dogs, lions, Panthera leo, and minks, Mustela vison), the sperm head is generally thicker, shorter and wider and thus rounder than in other domestic mammals [20]. In the present study, we found that the sperm heads of the Japanese black bears were rounder than in dogs. In giant panda, Ailuropoda melanoleuca, dogs and cats, which are closely related to Japanese black bears, the total length of the sperm is 51.2, 55.3 and 59.3 μm, respectively [21]. The total length of the sperm from the Japanese black bears in the present study was similar to those of these related species. Sperm motility and viability in the early and mid-mating seasons were significantly higher than those in the post-mating season. Reproductive function of Japanese black bears shows seasonal changes. The mating season is from mid-june to early August [8], and spermatogenesis is active in summer [9 11]. In September, shortly after mating season, few spermatozoa are observed in the seminiferous tubules [9, 11]. However, it has been reported that many spermatozoa are present in the epididymal duct in September [22]. Maturation and storage of spermatozoa takes place in the epididymis. Spermatozoa take about 10 days to pass through the epididymis in bulls, goats and rabbits and are stored in the cauda epididymis for a few weeks while maintaining their fertility [23, 24]. Thus, the semen collected in the post-mating season would have been stored for a few weeks in the epididymis. In the postmating season, because new spermatozoa are not replenished, the semen characteristics deteriorate. For this reason, the characteristics of semen collected in the post-mating season are considered to be inferior to those in the mating season. On the other hand, in the horse, another seasonal breeder, the rates of sperm motility and normal morphological sperm in winter (non-mating season) are reported to be significantly higher than in May (mating season) [25]. Collection of semen from Japanese black bears in the nonmating season is needed in order to study its effectiveness in AI. The rates of the abnormal sperm morphologies examined, except for detached heads, were not statistically different among the three seasons, and no clear seasonal changes in abnormal morphologies were observed in the 3 seasons examined in this study. The rates of detached heads in the early and mid-mating seasons were significantly lower than in the post-mating season. Detached heads are caused not only artificially by unsuitable sperm manipulation but also by aging of spermatozoon itself [13]. Such a change, i.e., deterioration of semen after the mating season, has already been

5 198 OKANO et al. described. In addition, detached heads may be induced in testicular hypoplasia [13]. The testes of Japanese black bears shrink after September due to physiological seasonal changes [11]. Such a physiological status may be similar to testes hypoplasia. On the other hand, the rate of detached tails did not differ among the seasons statistically, although it tended to be higher in the post-mating season (P=0.17). The rate of normal morphology, major defects and minor defects did not differ significantly among the seasons examined. The rates of major (27.1 ± 18.0%) and minor (53.6 ± 30.3%) defects were high throughout the seasons. In domestic mammals, the rate of abnormal morphology is usually under 10%, and if it is more than 20 30%, the conception rate generally falls [12]. In the bear park used for this study, bear breeding is accomplished easily due to natural mating. Thus, the abnormal morphology rate shown in this study may be in within the normal range for this species. It is also possible that the high abnormal morphology rate in this study was due to the collection method, electroejaculation. The abnormal morphology most frequently observed in the present study was simply bent tail, and this morphology accounted for about one-quarter of all the abnormal morphologies. Generally, bent tail is the most common abnormal morphology [26]. Hypoosmotic conditions are considered to induce a simply bent tail [13]. Dilution of semen with physiological saline reportedly induces this change [13]. In the present study, we used physiological saline to dilute semen for fixing of spermatozoa, and this may sometimes induce this kind of abnormal morphology. In the future, countermeasures will be necessary to prevent this. Another reason for the simply bent tails may be contamination of the samples with urine or the physiological saline used in washing the urinary bladder. The rate of spermatozoa with cytoplasmic droplets did not differ significantly among the seasons examined. The presence of cytoplasmic droplets indicates that spermatozoa are immature [12]. Such droplets are classified as proximal or distal according to the site of their attachment to the midpiece. As it matures, the cytoplasmic droplet moves from the proximal to distal part of the midpiece [13]. Thus, spermatozoa with proximal droplets are less mature. The incidence of proximal droplets is a sign of spermatogenesis abnormality [13], whereas the incidence of distal droplets does not indicate a serious defect [13]. In the present study, many spermatozoa with cytoplasmic droplets were observed throughout the seasons. Because many immature spermatozoa with cytoplasmic droplets were observed during the post-mating season, it is possible that spermatozoa are produced after the mating season until some later date. However, in previous studies, few spermatozoa were histologically observed in the bear testis in September [9, 11]. It is unclear why the rate of cytoplasmic droplets is so high shortly after the mating season. On the other hand, many cytoplasmic droplets are also observed throughout the mating season. It is not surprising that there are many immature spermatozoa in the early mating season, but it is unclear why many cytoplasmic droplets are observed in the mid-mating season. It may be the result of the high frequency of natural ejaculation in addition to the electroejaculations performed in this study. In the mid-mating season, many mature male bears copulate more than once almost every day [8]. When the frequency of semen collection is high, the collected spermatozoa contain more cytoplasmic droplets [12]. In the present study, many teratoid spermatozoa were observed throughout the seasons. Teratoid spermatozoa contain a very severe structural aberration, and the cell is barely recognizable as a sperm cell [13]. In the semen of normal bulls, the incidence of teratoid spermatozoa is usually below 1% [13]. Frequent incidence of this malformation indicates the occurrence of abnormal spermatogenesis in an animal. As stated above, the studied animals produced many spermatozoa with major defects throughout the seasons. Generally, major defects in spermatozoa are induced by genetic and environmental factors, so it is unclear why major defects from abnormal spermatogenesis were observed frequently in the spermatozoa of the animals examined. In cheetahs (Acinonyx jubatus), a high abnormal morphology rate and low reproductive rate has been observed due to inbreeding [27 29]. In the bear park used in the present study, inbreeding has not advanced among the bears so far. In conclusion, we obtained normal morphological data for spermatozoa in Japanese black bears. The sperm motility and viability rates in the early and mid-mating seasons are significantly higher than in the post-mating season. Moreover, the rates of detached heads in the early and mid-mating seasons are significantly lower than in the post-mating season. Acknowledgements The authors wish to thank the student members of the Laboratory of Zoo and Wildlife Medicine, Gifu University, the members of the Student Chapter of the Japanese Society of Zoo and Wildlife Medicine and Dr. R Nakashita (Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo, Japan) for their valuable assistance in semen collection. This work could not have been conducted without the kind cooperation of Messrs. A Izumi, M Suzuki and S Kikuchi of the Ani Mataginosato Bear Park (Akita, Japan). 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