Tetsuma Murase

Molecular epidemiology of rotaviruses among healthy calves in Japan: Isolation of a novel bovine rotavirus bearing new P and G genotypes

A total of 171 fecal specimens collected from healthy calves on a beef farm in Gifu Prefecture, Japan in 2006-2007 were examined for group A rotaviruses by RT-semi-nested PCR targeting the coding region for VP8*. Nine specimens were positive for rotavirus. G and P genotyping indicated that one strain was G10P[11]-like and six strains were considered to be the same unknown G and P genotypes. Among these six untypeable strains, one strain, AzuK-1, was adapted to cell culture and analyzed. Sequence and phylogenetic analyses of the full lengths of VP4 and VP7 genes revealed that AzuK-1 strain is a novel bovine rotavirus bearing new G21 and P[29] genotypes as confirmed by the RCWG. Furthermore, we detected G21P[29] rotaviruses in fecal specimens collected from healthy calves in Hokkaido, Japan during the period from 1997 to 1998. These findings suggest that novel G21P[29] rotaviruses have been widely prevalent among cattle for over 10 years in Japan.

Sex Identification by Alternative Polymerase Chain Reaction Methods in Falconiformes

Abstract A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z. From sequence analysis, a 3′-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the 3′-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.

Changes in Sex Steroids, Gonadotropins, Prolactin, and Inhibin in Pregnant and Nonpregnant Japanese Black Bears (Ursus thibetanus japonicus)

Abstract We examined changes in the concentrations of serum progesterone (P4), estradiol-17β (E2), FSH, LH, prolactin (PRL), and inhibin to determine their interaction and their effect on the reproductive endocrine controls of pregnant and nonpregnant female Japanese black bears. Fourteen female bears were used in this study over a 2-yr period. In the first year, six of the bears were divided into two groups; a pseudopregnant group and a nonpregnant group. In the second year, the remaining eight bears were also divided into two groups; a pregnant group and a nonpregnant group. Pregnant and pseudopregnant bears had similar P4 trends with both groups exhibiting a significant increase in December, which is the suspected time of implantation in pregnant bears. These trends correlated with an increase in PRL levels, whereas low levels of LH were maintained throughout the year. Nonpregnant bears maintained low concentrations of P4, and compared with pregnant and pseudopregnant bears, they also exhibited a delayed elevation in PRL. Luteinizing hormone activity varied among individual animals, but regardless of reproductive status, fluctuation patterns of E2, FSH, and inhibin did not differ among bears. Our results suggest that PRL may play a luteotropic role in both pregnant and pseudopregnant bears, and is possibly responsible for inducing reactivation of the dormant corpus luteum that precedes implantation in the Japanese black bear.

Characterization of semen collected from beagles and captive japanese black bears ( Ursus thibetanus japonicus )

This study characterized semen collected from the Japanese black bear, Ursus thibetanus aponicus, to provide information on semen cryopreservation for artificial breeding. Preliminary studies using a beagle dog as the model species showed that sperm concentration and total sperm count were lower in semen collected by electroejaculation than in semen collected by digital manipulation, but that sperm motility, viability and morphology were similar. Characterization of semen obtained from Japanese black bears by electroejaculation under general anesthesia revealed that semen volume and total number of spermatozoa collected were lower; but that sperm concentration, motility, viability and morphology were equivalent to those reported in other ursids. When semen was collected via a catheter inserted into the urethra during the stimulation for ejaculation, the sperm concentration, total sperm count and motility were relatively higher than when semen was collected directly in a test tube. Specific normal semen characteristics (mean +/- SEM) were pH, 7.6 +/- 0.0; volume, 0.212 +/- 0.038 mL; sperm concentration, 361 +/- 100 x 10(6)/mL; total sperm count, 84.0 +/- 32.2 x 106; +++ motility, 30 +/- 5%; motility, 77 +/- 3%; viability 77 +/- 2%; and abnormal morphology, 11+/- 2%. These results suggest that semen can be collected from Japanese black bears by electroejaculation.

Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP-dependent hyperactivation in the spermatozoa of Japanese Black bulls.

The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen‐stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell‐permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine‐phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen‐stored spermatozoa were classified as having a high‐grade acrosome condition before incubation, sperm tyrosine‐phosphorylated proteins, including the 33‐kDa tyrosine‐phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41‐ and 33‐kDa sperm tyrosine‐phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine‐phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen‐stored sperm were classified as having a low‐grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine‐phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine‐phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP‐dependent hyperactivation for the spermatozoa of individual Japanese Black bulls. Mol. Reprod. Dev. 77:910–921, 2010.

Changes in serum inhibin levels and immunolocalization of inhibin/activin subunits during the breeding season in the wild male Japanese black bear (Ursus thibetanus japonicus).

The objective of this study was to investigate the changes in secretion of inhibin and cellular localization of inhibin α and inhibin/activin (βA and βB) subunits during the breeding season in the wild male Japanese black bear. Histological observations of testes were performed and seminiferous tubule diameters were measured. The sections of the testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin α, inhibin/activin βA, and inhibin/activin βB during the breeding season. Serum concentrations of immunoreactive (ir-)inhibin, testosterone, and luteinizin hormone (LH) were measured by radioimmunoassay. Higher values of seminiferous tubule diameters and all types of spermatogenic cells including mature-phase spermatozoa were found during the breeding season. There were seasonal changes in serum concentrations of ir-inhibin, testosterone, and LH. Ir-inhibin was positively correlated with testosterone, and LH. In addition, immunoreactivity of inhibin α, βA, and βB subunits were also detected in Sertoli and Leydig cells during the breeding season. These results suggest that Japanese black bear testes may secrete bioactive inhibins during the breeding season and that the circulating inhibin may be a useful indicator of the testicular function in wild male Japanese black bears.

Roles of extracellular Ca2+ in the occurrence of full-type hyperactivation in boar ejaculated spermatozoa pre-incubated to induce the cAMP-triggered events

There are species differences in the regulatory system for sperm capacitation and subsequent hyperactivation between livestock and laboratory animals. In livestock spermatozoa, it is poorly understood when and how extracellular Ca2+ is necessary for hyperactivation, although it has been demonstrated that the [Ca2+]i increase is indispensable to occurrence of hyperactivation. In this study, we examined necessity of extracellular Ca2+ for the initiation and maintenance of hyperactivation and then sought possible target molecule of Ca2+ that was involved in hyperactivation of boar spermatozoa. Boar ejaculated spermatozoa were pre‐incubated with a cell‐permeable cyclic adenosine monophosphate (cAMP) analog ‘cBiMPS’ and without CaCl2 to induce the cAMP‐triggered events including capacitation‐associated changes. Subsequently, they were incubated with CaCl2 to induce hyperactivation and then used for motility assessment. Many of the spermatozoa after the incubation exhibited full‐type hyperactivation which was characterized by high‐amplitude and extremely asymmetrical beating of whole middle piece and principal piece. The initiation of full‐type hyperactivation required the millimolar concentration of CaCl2 in the medium. However, CaCl2 of the medium was less necessary for maintenance than initiation of full‐type hyperactivation, as hyperactivated spermatozoa were barely affected by the incubation with the Ca2+‐chelating reagent. On the other hand, the pre‐treatment with the inhibitor for Ca2+‐dependent protease ‘calpain 1 and 2’ clearly suppressed the occurrence of CaCl2‐induced hyperactivation without influences on the percentages of motile spermatozoa. Western blotting and indirect immunofluorescence showed distribution of calpain 2 in the middle and principal pieces in which full‐type hyperactivated spermatozoa exhibited extremely asymmetrical beating. On the basis of these results, we conclude that the millimolar concentration of extracellular Ca2+ is necessary for the initiation, but not for the maintenance of full‐type hyperactivation in boar spermatozoa that beforehand undergo the cAMP‐triggered events including capacitation‐associated changes. Moreover, we suggest possible involvement of calpain 2 in the intracellular Ca2+ signal transduction leading to full‐type hyperactivation.

Desalted and lyophilized bovine seminal plasma delays induction of the acrosome reaction in frozen-thawed bovine spermatozoa in response to calcium ionophore

Cryopreservation is partially damaging and induces capacitation-like changes in spermatozoa. Seminal plasma (SP) contains a variety of biochemical components, such as protein and lipids, which are specific for the regulation of sperm cell function including those effective for decapacitation of spermatozoa. Therefore, this study tested the hypothesis that desalted and lyophilized SP could prevent premature capacitation (cryocapacitation) of Japanese Black bull spermatozoa. Seminal plasma was desalted by using Sephadex G-25 desalting column and lyophilized before added to semen extender at final concentrations 0, 2.5, 12.5, and 25 mg/mL. Frozen-thawed sperm progressive motility, acrosomal integrity, abnormal morphology, and the calcium ionophore A23187-induced acrosome reaction were assessed. Protein and lipid compositions in SP were analyzed by SDS-PAGE and thin-layer chromatography, respectively. The results revealed that progressive motility, intact acrosome, and abnormal morphology were not substantially modified by addition of SP. Stimulation of spermatozoa with calcium ionophore A23187 resulted in a time-dependent induction of the acrosome reaction, which was delayed by the desalted and lyophilized SP. There was no difference in the protein profile of SP before and after gel filtration. In total, 19 protein bands with molecular masses ranging from 5.2 to 185.8 kDa were detected and those of 185.8, 80, 34, 20.8, 18.8, 17.5, and 10 kDa were considered as novel proteins. Neutral lipids and phospholipids before and after gel filtration were the same, and the detected neutral lipid spots were monoacylglycerol, cholesterol, 1,2- and 1,3-disaturated diacylglycerol, 1,2- and 1,3-saturated, unsaturated diacylglycerol, whereas the detected phospholipid spots were sphingomyelin, phosphatidylcholine, phosphatidylserine, and three species of phosphatidylinositol, phosphatidylethanolamine, cerebroside, and polyglycerol phosphatide. The results suggest that premature capacitation during freeze-thaw processes could be reduced by adding desalted and lyophilized SP.

Hematological and Biochemical Reference Values for the Endangered Kiso Horse

To establish blood and biochemical references for the endangered Kiso horse, blood samples were collected from 111 adult Kiso horses, 74.5% of the existing breed. The samples were analyzed for 23 hematological and biochemical parameters to determine their means and standard deviations (SD). We compared the mean ± 2SD with the reference values cited in one of the most commonly used veterinary textbooks in Japan. The hematology of Kiso horses is characterized by lower erythrocyte count and hematocrit and hemoglobin levels. In addition, their serum biochemistry showed lower levels of aspartate transaminase, alkaline phosphatase, and γ-glutamyl transferase. Whether these propensities are attributed to breed-specific factors or are acquired factors remains unclear. Nevertheless, this study provides useful diagnostic indices for the endangered Kiso horse.

Methodological factors affecting the results of staining frozen–thawed fertile and subfertile Japanese Black bull spermatozoa for acrosomal status

In the present study, some methodological factors affecting the acrosomal staining of frozen-thawed Japanese Black bull spermatozoa were investigated by examining; the effect of fixation/permeabilization procedure on intact acrosome percentage after fluorescein isothiocyanate peanut agglutinin (FITC-PNA) staining, the acrosomal staining patterns by using two types of fluorescent probes FITC-PSA (Pisum Sativum Agglutinin) and FITC-PNA and the effect of staining methods, either smear or vial, on intact acrosome percentage. Then intact acrosome percentage was compared between the samples stained by thus established method and those simply fixed with glutaraldehyde (glutaraldehyde fixation method). A possibility that FITC-PNA staining or the glutaraldehyde fixation methods could detect any difference in intact acrosome percentage or acrosomal staining patterns between fertile and subfertile bulls was also examined. The results showed that (1) 4% paraformaldehyde fixation plus 1% Triton X-100 permeabilization was better than absolute ethanol alone, (2) FITC-PNA acrosomal labeling was more specific than FITC-PSA, (3) sperm suspensions should be smeared and gently processed before acrosomal staining rather than spotted onto glass slides after staining in vial in order to avoid excessive mechanical damage of the sperm acrosome, and (4) staining spermatozoa with FITC-PNA had no major advantages over examination of simply glutaraldehyde fixed sperm samples and both failed to detect any significant difference in intact acrosome percentage between the fertile and the subfertile bulls used here. The present study demonstrates important methodological considerations which need to be taken into account in order to design a reliable and reproducible protocol for the study of the acrosome.