Two novel molecular isoforms of band 4.2 in Japanese Sika deer (Cervus nippon yesoensis, Heude) erythrocytes

Two molecular isoforms of band 4.2 were identified in erythrocyte membranes from 25 Japanese Sika deer (Ceryus nippon yesoensis, Heude) based on specific immunorecognition with anti-human band 4.2. These two variants, designated 4.2/78 and 4.2/76, had respective relative molecular weights (Mr) of 78,000 and 76,000 on sodium dodecyl sulfate-polyacrylamide electrophoresis gels and showed similar profiles after limited proteolysis, exhibiting identity in primary structure. 25 adult Sika deer could be divided into two groups according to the 4.2/78:4.2/76 ration, indicating a genetic control in the expression of the molecular isoforms of band 4.2. Both polypeptides were completely retained in cytoskeletal protein-depleted membranes and could be removed by alkaline extraction, suggesting that both proteins contribute to the association of membrane proteins.     

Flow cytometric sexing of X- and Y-chromosome-bearing sperm in Sika deer (Cervus nippon)

The objectives of the study were to determine a practical method of using predetermined sexed semen in Sika deer (Cervus nippon). Semen was collected by electro-ejaculation from two Sika stags and transported to the laboratory and separated into X- and Y-chromosome-bearing sperm after analysis and re-analysis (using a modified high-speed cell sorter), or control (unsorted) semen. Eighty-four Sika hinds were inseminated with 2.8 × 10⁷ unsorted (control) or 2.3 × 10⁶ sorted (X or Y) frozen-thawed semen via intra-uterine laparoscopy 58–66 h after removal of intra-vaginal progesterone-impregnated CIDR devices and the administration of 330 IU PMSG at the time of CIDR removal. No significant differences in the post-thaw motility of control (43.4 ± 4.4%), X- (45.3 ± 4.5%) and Y-sorted (43.5 ± 3.2%) samples were recorded. The sorted frozen-thawed sperm (X, 72.5 ± 6.4%: Y, 75.2 ± 5.5%) recorded significantly (P < 0.05) more intact acrosomes following thawing than the unsorted frozen-thawed (68.2 ± 10.2%) sperm. The individual Sika stags had no effect on the post-thaw sperm motility. Sorted frozen-thawed sperm demonstrated a significantly shorter survival time after thawing than the control sperm (P < 0.05). The number of Sika hinds pregnant following insemination with unsorted or control thawed sperm was significantly higher (33/42; 78.6%) than for hinds inseminated with either X- (5/11; 45.5%) or Y-sorted sperm (15/31; 48.4%). Ultimately 14 out of the 15 calves produced by Sika hinds inseminated with Y-sorted sperm were male (92.9%) and 5/5 calves (100%) from Sika hinds inseminated with X-sorted sperm were female. The sex ratio of the calves born to hinds inseminated with sex-sorted sperm significantly (P < 0.05) deviated for the 48.5% (female, 16/33) and 51.5% (male, 17/33) in the control group. All calves were born between 230 d and 243 d of gestation. Male and female calves in the control group had similar birth and weaning weights as calves from hinds inseminated with X- or Y-sorted sperm. In conclusion it can be said that normal calves of the predicted sex may be produced after intra-uterine insemination conducted by laparoscopy with low numbers of sex-sorted cryopreserved Sika sperm.     

Sika deer (Cervus nippon) velvet antler extract attenuates prostate cancer in xenograft model

The present study determines whether antler extract (AE) possesses inhibitory effects in a prostate cancer (PC) xenograft model and explores the underlying mechanism. After therapeutic intervention for two weeks, AE significantly inhibited prostate cancer xenograft tumor growth by 65.08%, and prostate-specific antigen (PSA) and serum dihydrotestosterone (DHT) levels. However, AE increased the serum testosterone level compared to the vehicle control group. Furthermore, our investigation of the inhibitory effects on angiogenesis and epithelial-to-mesenchymal transition (EMT)-related genes revealed that AE downregulated matrix metalloproteinase 2 (MMP)-2, (MMP)-9, vascular endothelial growth factor (VEGF), zinc finger protein (SNAIL1), twist-related protein 1 (TWIST1), and zinc-finger E-box-binding homeobox 1 (ZEB1) in vivo. In contrast, AE increased tissue inhibitor of MMP (TIMP)-1, (TIMP)-2, and E-cadherin. The results suggest that AE possesses potent anti-PC activity, and this is the first report on the anti-PC effect of AE in vivo.     

Detection of Lyme disease spirochetes in the skin of naturally infected wild sika deer (Cervus nippon yesoensis) by PCR.

We demonstrated the presence of Borrelia burgdorferi sensu lato DNA in the skin tissues of naturally infected wild sika deer, using PCR. The risk of transmission of B. burgdorferi sensu lato is recognized in sika deer.     

Seroprevalence of fasciolosis in Hokkaido sika deer (Cervus nippon yesoensis) from Hokkaido Prefecture,Japan revealed by ELISA using recombinant cathepsin L1

Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola, has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis.     

Hybridization between Red deer (Cervus elaphus) and Sika deer (Cervus nippon) with particular reference to stocks in N.W. England

Sika deer ( Cervus nippon Temminck) from Japan were introduced into Great Britain and Ireland first in 1860, and, for a time, were much sought after by owners of deer parks. Later, they were introduced or allowed to escape into various parts of the country, and feral populations have become established in Scotland, England and Ireland.
Hybridization between Sika and Red deer ( Cervus elaphus L.) has occurred frequently in deer parks but has been reported only recently from feral and wild stocks.
In part of north-west England, there have been reports of hybrids from time to time ever since the 1920s, but these have become commonplace only recently. Since the Red deer inhabiting other parts of the region are all of the native race ( Cervus elaphus scolicus Lönnberg) and no other population of this race exists elsewhere in England, work was begun to assess the threat to the survival of both the race and the species posed by Sika deer and, more particularly, by the hybrids on Cartmel Fell.
Using multivariate methods of analysis on a range of skull measurements (see page 556) obtained from samples of both species and their hybrids, an attempt was made to find some means of distinguishing the hybrids from the species irrespective of the extent to which introgression had occurred. This was largely successful using canonical variate analysis, but only when the sets of data used to construct the basic matrix were both homogeneous and biologically relevant, i.e. of the same age and sex, and where the samples were obtained from the races involved in the hybridization. The present classification of the subspecies of Sika deer received little support from this study. Many of the subspecies as described in the literature were found to be indistinguishable from some of the hybrids, which suggests that some revision may be necessary.     

四川梅花鹿春季昼夜活动节律与时间分配

1997年4月,在四川铁布自然保护区用直接观察法对220头次四川梅花鹿群的昼夜活动节律和时间分配进行了观察。结果表明,鹿群春季昼夜活动的规律性较强。白昼,鹿群活动呈现明显的双峰型,2个高峰时段为08：30和19：30前后,活动频率分别为71．9％和94．13％,其他时间多处于休息状态;鹿群夜间活动强于昼间,活动频率均在6l％以上,仅在半夜01：30前后有一个相对不活跃期。同时发现,春季活动高峰期约90％的活动时间被四川梅花鹿用于采食和移动。这可能与光照、食物及人类活动的影响有关。     

Helminthes detected from wild sika deer (Cervus nippon centralis) in Kanto-Chubu region,Japan

The helminth fauna of 105 sika deer (Cervus nippon centralis) captured in Yamanashi, Kanagawa and Nagano Prefectures, Japan was investigated during 2014–2019. As a result, 12 helminthes, i.e. 3 digeneans (Ogmocotyle sikae, Dicrocoelium chinensis and D. dendriticum), 8 nematodes (Gongylonema pulchrum, Dictyocaulus sp., Pygarginema sp., Spiculopteragia houdemeri, Chabaudstrongylus ninhae, Trichuris discolor, Oesophagostomum sikae and Oes. asperum), and 1 cestode (Moniezia sp.) were detected. To our knowledge, this is the first report of Pygarginema sp., Cha. ninhae, and Oes. asperum from sika deer in Japan. Some helminthes detected in the present study can infect livestock. Considering the possibility of the spread of the helminthes to livestock through deer excrement, it is important to promote understanding the parasite fauna in wild deer.     

Serological survey for Lyme disease in sika deer (Cervus nippon yesoensis) by enzyme-linked immunosorbent assay (ELISA).

Seventy-six wild sika deer (Cervus nippon yesoensis) from areas endemic for Borrelia burgdorferi during 1988 to 1989 had the IgG antibody to the bacteria in enzyme-linked immunosorbent assay. The percentage of seropositive deer was 25.0% to strain HO14 and 22.4% to strain HP3, respectively. Specific IgG antibody titers were high in summer but low in winter. In summer, seropositive deer were 75.0%. Similar results were obtained in serum specimens obtained monthly from 4 farmed deer. It was suggested that the sika deer could be one of the wild reservoirs for B. burgdorferi in Hokkaido, Japan. The transmission risk of B. burgdorferi is considered to be greatest during late spring to early summer.     

Prevalence and phylogenetic analysis of Cryptosporidium infections in Yezo sika deer (Cervus nippon yesoensis) in the Tokachi sub-prefecture of Hokkaido,Japan

The Yezo sika deer (Cervus nippon yesoensis) on the island of Hokkaido, Japan are currently recognized as overabundant. Hunting is used to control the deer population, and this has increased the supply of game meat, which is associated with a high risk of various food-borne infections. Additionally, the sub-prefecture Tokachi has a dense population of livestock, which are potentially at risk of cross-species infections from the deer. In this study, we undertook the first analysis of the incidence of Cryptosporidium infection in the Yezo sika deer in the Tokachi area using polymerase chain reaction testing and phylogenetic analysis. Polymerase chain reaction analysis showed Cryptosporidium species present in 7.5% of fecal samples (13/173) collected from deer hunted between 2016 and 2017. However, the zoonotic Cryptosporidium paruvm parasite was not detected in the phylogenetic analysis; when sequenced, all species in the positive samples matched the Cryptosporidium deer genotype. However, deer may act as a reservoir of the zoonotic Cryptosporidium parvum parasite, which affects both humans and livestock. Therefore, we recommend the continuation of surveys of the incidence of Cryptosporidium infections in Yezo sika deer.     

Detection and identification of Theileria infection in sika deer ( Cervus nippon ) in China

The sika deer ( Cervus nippon ) is a first-grade state-protected animal in China and designated a threatened species by the World Conservation Union. To detect hemoparasite infection of sika deer, blood samples were collected from 24 animals in the Hubei Province Deer Center. Genomic DNA was extracted, and the V4 hypervariable region encoding 18S rRNA was analyzed by reverse line blot hybridization assay. PCR products hybridized with Babesia / Theileria genus-specific probes but failed to hybridize with any of the Babesia or Theileria species-specific probes, suggesting the presence of a novel, or variant, species. Here 18S rRNA and internal transcribed spacer (ITS) genes were amplified, cloned, and sequenced from 7 isolates. Alignment and BlastN of the cloned sequences revealed high similarities to the homologous 18S rRNA genes and ITS genes of Theileria cervi (AY735122), Theileria sp. CNY1A (AB012194), and Theileria sp. ex Yamaguchi (AF529272). Phylogenetic analysis based on the 18S rRNA gene and ITS sequences showed that all cloned sequences were grouped within the Theileria clade. Phylogeny based on the 18S rRNA gene divided the organisms into 2 groups. Group 1 was closest to Theileria sp. ex Yamaguchi (AF529272), and group 2 was distinct from all other identified Theileria and Babesia species. These results suggest the existence of Theileria sp. infection in sika deer in China. To our knowledge, this is the first report of cervine Theileria sp. in China.     

Assessment LOPU-IVF in Japanese sika deer (Cervus nippon nippon) and application to Vietnamese sika deer (Cervus nippon pseudaxis) a related subspecies threatened with extinction

In mammals, recovery of oocytes by laparoscopic ovum pick-up (LOPU) coupled with in vitro production (IVP) of embryos represents a promising strategy for both amplification and genetic management of sparse animals from captive endangered wild species. As integrated technique developed mainly for domestic livestock, LOPU-IVP requires several studies to set up protocols for follicular stimulation or optimization of IVP before envisaging successful transposition to wild species. In deer, many endangered subspecies would be potentially concerned by applying such an approach using common subspecies for protocols optimization. The aim of the present study was to assess efficiency of follicle stimulation using ovine FSH (oFSH) for recovery of oocytes by LOPU in common sika deer (Cervus nippon nippon) before transposition of an optimized methodology for IVP of embryos from endangered Vietnamese sika deer hinds (Cervus nippon pseudaxis). In common sika deer, two doses of oFSH (0.25 and 0.5 U) and two frequencies of administration (12 and 24 h) were compared by monitoring of subsequent ovarian response, quality of oocytes recovered by LOPU, and in vitro developmental competence. In a first experiment, the dose of oFSH administered did not significantly affect the total number of follicles aspirated per hind per session (8.6 ± 1.0 vs. 8.2 ± 1.6 with 0.5 vs. 0.25 U oFSH, respectively; not significant). In a second experiment, frequency of 0.25 U oFSH administration did not affect ovarian response. Efficiency of IVP determined on blastocysts rates after in vitro maturation, fertilization, and development in oviduct epithelial cells coculture was increased when FSH was administered at 12-h intervals. Immune response after several follicular stimulations was detected against exogenous oFSH in plasma from the majority of sika deer hinds but was not associated with decreased ovarian response. When 0.25 U oFSH was administered at 12-h intervals to Vietnamese sika deer (N = 4), good quality cumulus oocyte complexes with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Development to the blastocyst stage occurred in a high proportion (30% of oocytes) after coculture with ovine epithelial cells allowing cryobanking of transferable embryos from Vietnamese sika deer. These results confirm that LOPU-IVF after ovarian stimulation with oFSH may be a successful tool for cryobanking transferable embryos from endangered sika deer subspecies.     

Molecular detection of tick-borne protozoan parasites in sika deer (Cervus nippon) from western regions of Japan

The sika deer (Cervus nippon) is one of the most common species of wildlife in Japan. This study aimed to reveal the prevalence of tick-borne protozoan parasites in wild sika deer living in western Japan. We used nested polymerase chain reaction (PCR) to detect the 18S rRNA gene of tick-borne apicomplexan parasites (Babesia, Theileria, and Hepatozoon spp.) from 276 blood and liver samples from sika deer captured in the Yamaguchi, Oita, Kagoshima, Okayama, Ehime, Kochi, and Tokushima Prefectures. In total, 259 samples (259/276; 93.8%) tested positive in the nested PCR screening. Gene sequencing revealed that 99.6% (258/259) of positive samples contained Theileria sp. (sika 1), while Theileria sp. (sika 2), another Theileria species, was detected in only 3 samples. We also found that one sample from a sika deer captured in Kagoshima contained the gene of an unidentified Babesia sp. related to Babesia sp. Kh-Hj42, which was previously collected from tick in western Siberia. In conclusion, we found a high prevalence of piroplasms in sika deer from western Japan, and DNA analysis revealed that Theileria sp. (sika 1) had the highest infection rate.     

Borrelia burgdorferi Sensu Lato in an Endemic Environment: Wild Sika Deer (Cervus nippon yesoensis) with Infected Ticks and Antibodies

Ticks and blood samples were collected from wild sika deer (Cervus nippon yesoensis) during a hunting season (August to October) of 1991 at a selected location in Hokkaido, Japan. Ixodes persulcatus (adult and nymph) and I. ovatus (adult) were the common ticks on sika deer. Spirochetes were detected in the midgut of the ticks by the indirect peroxidase-conjugated antibody staining method and by dark-field microscopy after cultivation. By the reactive pattern of monoclonal antibodies, isolates were considered to belong to Borrelia garinii or B. japonica. In an antibody test, the percentage of seropositive deer was 69.0%. Most of the adult sika deer were positive for antibodies to the spirochetes. There are significant age-dependency in antibody level and seropositive rate. The surveillance of deer should be valuable in monitoring the transmission risk of B. burgdorferi sensu lato in nature.     

Successful in vitro production of embryos in the red deer (Cervus elaphus) and the sika deer (Cervus nippon)

The aim of our study was to define the conditions for IVM and IVF of oocytes in 2 common deer species as models for endangered related subspecies. Immature oocytes were recovered during the breeding season from postmortem ovaries (red deer) or by repeated laparoscopic follicular aspiration (sika deer). Oocytes were cultured for 24 h in IVM medium supplemented with EGF or FSH and follicular fluid. Stag semen was collected by electroejaculation (both species) or by epididymal flushing (red deer) and cryopreserved. For IVF, oocytes were exposed to different concentrations of thawed spermatozoa in a modified Tyrode albumin lactate pyruvate medium supplemented with 20% (v/v) estrus sheep serum for 18 h. After IVF, presumptive zygotes were allowed to develop in vitro for 7 days in synthetic oviduct fluid (SOF) supplemented with fetal calf serum (10%, v/v). In both species, the presence of ovine FSH and follicular fluid improved the in vitro maturation rate. In the sika deer, the optimal sperm concentration for IVF was 10(6)/mL and some fertilized oocytes reached the early morula stage (20 to 25 cells). In the red deer, after IVF with ejaculated or epididymal spermatozoa (2.0 x 10(6)/mL), 20% of zygotes developed to the blastocyst stage (50 to 80 cells).     

珍稀动物--梅花鹿及其研究

梅花鹿曾广泛分布在亚洲东北部,具有非常高的经济价值。茸角、鹿血、鹿胎等是传统的名贵药材、长期以来梅花鹿受到严重的狩猎压力,国内的野生梅花鹿种群大部分已经绝灭,仅在四川、江西和东北的少数几个地方还残存几个数量非常少的种群,是我国的一级重点保护动物。我国饲养梅花鹿的历史较长。已有的研究工作主要集中在鹿产品的理化特性、化学成分、炮制方法和药效等方面。我们对于野生梅花鹿的生物学和生态学知识还很匮乏。国外的研究工作主要集中在对野生种群生态学的研究方法,日本科学家进行了大量的研究。梅花鹿的生物学特性,鹿产品的经济价值以及国内外对于梅花鹿的研究进展知识,有助于我们对梅花鹿的了解、保护和进一步的开发利用。     

Pedigree Analysis of the Sika Deer (Cervus nippon) using Microsatellite Markers

The usefulness of microsatellite markers in pedigree analysis of the sika deer (Cervus nippon) was tested in a herd in which the maternal lineages were recorded. Eighteen sets of microsatellite primers originally designed for bovine, ovine, and cervine loci successfully amplified polymorphic DNA in the deer. The numbers of alleles per locus ranged from two to seven, and the observed heterozygosity ranged from 0.350 to 0.900. The resolution power of the markers in paternity testing was then determined by calculating exclusion probabilities and paternity indices. Parentages of the study population were efficiently discriminated by genotyping 17 microsatellite loci. The microsatellite data were also used to calculate the genetic relatedness between individuals, which significantly correlated with coancestry coefficients for the pairs. Our results demonstrate that the microsatellite markers are efficient tools in studying the social structure and behavior of the sika deer, as well as in monitoring the inbreeding status.