Localization of telocytes in rabbits testis: Histological and immunohistochemical approach.

Telocyte (TC) is an interesting unique interstitial cell demonstrated in many human and animal tissues and organs. This study verified, for the first time, the pattern of TC distribution in the testicular tissue of New Zealand White rabbits using histological, immunohistochemical, and electron microscopic tools. Rabbit testicular tissue samples were obtained from three pairs of adult healthy New Zealand white rabbit by surgical procedures. The testicular tissues were stained with hematoxyline-eosin, Crossmon's trichrome and Periodic acid Schiff. The immunohistochemistry was performed using three different antibodies CD34, CD117, and vimentin. The testes were examined by scanning and transmission electron microscopy. Histologically, TCs formed a sheath surrounding the seminiferous tubules. Other TCs were located in the interstitial tissue of the rabbit testis. Immunohistochemically, TCs reacted strongly with CD34, CD117, and vimentin. Scanning electron microscopic findings clearly elucidated the spreading pattern of TCs and their cytoplasmic processes with the interstitial tissue including blood vessels. Both homocellular and heterocellular junctions were demonstrated by transmission electron microscope. On the basis of TCs distribution and connections, the before mentioned data suggested that, TCs may play a potential role in maintaining the testicular construction and regulation. A future work is needed to clarify the actual role played by TCs in monitoring testicular fertility. RESEARCH HIGHLIGHTS: Telocyte (TC) is a unique cell demonstrated in human and animal tissues. TCs formed a sheath surrounding the seminiferous tubules in rabbits and may be located in interstitial tissue. Immunohistochemically, TCs reacted strongly with CD34 and CD117.

Correlation of blood metabolite concentrations and body condition scores with persistent postpartum uterine bacterial infection in dairy cows.

To analyze the relationship of blood metabolite concentrations and body condition score (BCS) with persistent bacterial uterine infection, specifically that caused by Trueperella pyogenes and anaerobic bacteria, uterine bacteriological swabs (n = 128) were collected from 64 Holstein cows at 5 (W5) and 7 (W7) weeks postpartum, and the percentage of neutrophils in the endometrium was evaluated. Blood glucose, total cholesterol (T-cho), blood urea nitrogen (BUN), non-esterified fatty acid (NEFA), and ß-hydroxybutyric acid concentrations were analyzed at 3 weeks (W-3) and 1 week (W-1) prepartum and W3, W5, and W7 postpartum. BCS were evaluated at W-3, W3, and W7. Blood glucose concentrations at W-3 and W-1 in cows with persistent bacterial infection were lower (P = 0.05) than in the rest of the cows. Total BUN concentrations in cows with persistent bacterial infection were lower (P < 0.01) than those in other cows, although the association between the pre or postpartum time and status of infection was not significant. Total NEFA concentrations in cows with persistent bacterial infection were similar to those in uninfected cows and cows positive for infection at W5 but not W7. Total BCS in cows with persistent bacterial infection were lower (P < 0.01) than those in cows positive for infection at both W5 but not W7 and W7 but not W5; however, the association between the pre or postpartum time and status of infection was not significant. Glucose concentrations at W-3 and W-1 negatively correlated with persistent bacterial infection at W5 and W7 (P < 0.01). BUN concentrations at W3 (P < 0.01), W5 (P < 0.05), and W7 (P < 0.05) and BCS at W3 (P < 0.01) negatively correlated with persistent postpartum bacterial infection. Decreased prepartum blood glucose concentrations might be an important risk factor for persistent postpartum bacterial uterine infection in dairy cows.

Persistence of uterine bacterial infection, and its associations with endometritis and ovarian function in postpartum dairy cows.

We investigated the relationship between the persistence of uterine bacterial infections with cytologically determined endometritis and ovarian function in 65 postpartum Holstein cows. Vaginal mucus discharges were collected, and endometrial smear samples (n = 130) were collected for cytological and bacteriological examinations from the cows at weeks 5 and 7 postpartum (pp). Blood samples were collected at weeks 3, 5 and 7 pp to determine plasma progesterone concentrations to monitor ovarian activity. According to the bacteriological examination, cows were classified into four groups. The first group (n = 32; 49%) comprised cows negative for bacteria at weeks 5 and 7 pp. The second group (n = 11; 17%) comprised cows with bacterial infections at week 5 pp but that were clear of infection at week 7 pp. The third group (n = 12; 19%) comprised cows without bacteria at week 5 pp but that acquired an infection by week 7 pp. The fourth group (n = 10; 15%) comprised cows with bacterial infections at weeks 5 and 7 pp (persistence of infection). A positive correlation (P < 0.001) was noted between the severity of cytologically determined endometritis, purulent vaginal discharge and the persistence of infection. Cows with persistent infections had a significantly (P < 0.01) prolonged luteal phase compared with cows without infection. In conclusion, the prevalence of cytologically determined endometritis and prolonged luteal phase were significantly increased in cows with persistent infections.

Mycoplasma infection in the uterus of early postpartum dairy cows and its relation to dystocia and endometritis.

This study investigated the incidence of mycoplasma infection in the uterus of postpartum Holstein dairy cows and its relationship to the occurrence of endometritis. The genital tracts of 209 cows from three dairy farms in the Iwate Prefecture, Japan, were examined at Weeks 5 and 7 postpartum. The condition of the cervicovaginal mucus was assessed using a Metricheck device and assigned a score from 0 (clear mucus) to 4 (purulent material with fetid odor). Intrauterine samples (N = 418) were collected at Weeks 5 and 7 postpartum using a cytobrush. After its withdrawal, swab samples were placed in mycoplasma culture broth at 37 °C for 72 hours. A novel and rapid polymerase chain reaction was used to detect seven mycoplasma species (Mycoplasma bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens, and M. canadense). The cytobrush was also rolled gently along the length of a glass slide for subsequent polymorphonuclear neutrophil count. The diagnostic criteria for cytological endometritis were 6% or more and 4% or more polymorphonuclear neutrophils at Weeks 5 and 7, respectively. From a subset of cows, additional swabs were rolled against the cytobrush and then placed in transport medium. These samples were then plated on specific agar plates and cultured under aerobic and anaerobic conditions to identify other bacteria present. The incidence of dystocia at the last calving was compared in mycoplasma positive and negative cows. Of the seven mycoplasma species, only M. bovigenitalium was detected; it was detected in 31 of the 418 uterine swabs (7.4%). Twenty-four cows were positive for M. bovigenitalium (eight cows at Week 5, nine cows at Week 7, and seven cows at both Weeks 5 and 7). The incidence of dystocia was higher (P < 0.0001) in mycoplasma positive (7/24; 29.2%) compared with mycoplasma negative (4/185; 2.2%) cows. However, there was no significant association between dystocia at last calving and subsequent uterine infection with other bacteria. In addition, the incidence of cytologic endometritis was higher (P < 0.05) in mycoplasma positive (8/16; 50%) than in mycoplasma negative (47/193; 24.4%) cows at Week 7. Therefore, we concluded that M. bovigenitalium infection in the uterus might be associated with recent dystocia and with cytologic endometritis in postpartum dairy cows.

Neospora caninum and complex vertebral malformation as possible causes of bovine fetal mummification.

Bovine neosporosis, caused by Neospora caninum is a leading cause of abortion in cattle. We postulated that neosporosis could lead to fetal death and mummification. Fifteen mummified fetuses were tested by polymerase chain reaction (PCR) for the mutation in the bovine SLC35A3 gene that causes complex vertebral malformation (CVM) and the pNC-5 gene which identifies N. caninum infection. DNA was extracted from the mummified fetuses and the sex of the mummies was determined by PCR. The CVM mutation was not detected in the mummified fetuses, but 4 fetuses were positive for N. caninum infection. The ages of the mummies with N. caninum infection were 100, 113, 123, and 131 days. Twelve of the 15 mummified fetuses were male. To our knowledge, this is the first detection of N. caninum as a possible cause of bovine fetal mummification.

Effect of total cholesterol, glucose and blood urea nitrogen on embryo quality in post-partum superovulated suckling Japanese Black cattle.

Aim: This study was conducted to examine the effect of blood metabolites on embryo quality in post-partum suckling Japanese Black cattle. Methods: Blood samples were taken from 23 cows 30 days before, at and 30 days after parturition. Cows were synchronized 40 or 41 days after calving (day 0) and divided into three groups: control (n = 6), gonadotropin-releasing hormone ([GnRH]n = 10) and estradiol benzoate ([EB]n = 7). All groups received a controlled internal drug release (CIDR) device intravaginally together with 2 mg EB i.m. on day 0 and superovulation was induced in all groups from days 5-7 with a gradually decreasing dose of follicle-stimulating hormone (FSH). Two milligrams of EB was given on day 8 and GnRH (0.1 mg) was given on day 9 of insertion of the CIDR in the EB and GnRH groups. Cows were inseminated twice after the onset of estrus and embryos were recovered 7-8 days after artificial insemination. Results: The number of corpus luteum detected by ultrasonography in the EB group was significantly higher (P < 0.05) than that in the GnRH group. The number and rate of transferable and freezable embryos did not differ significantly among the groups. Regardless of the treatments, the total cholesterol level from parturition until 30 days after parturition was significantly higher (P < 0.01) in the good category than in the poor category of cows. Conclusions: The number of transferable embryos produced by post-partum superovulated suckling Japanese Black cattle was affected by the level of total cholesterol from parturition until 30 days after parturition. Moreover, administration of EB in CIDR-treated cows increased the numbers of corpus luteum and yielded better rates of transferable and freezable embryos. (Reprod Med Biol 2008; 7: 55-62).

Complex vertebral malformation in Holstein cows in Japan and its inheritance to crossbred F1 generation.

Complex vertebral malformation (CVM) is an autosomal recessive inherited defect in the Holstein breed. It causes intra-uterine mortality leading to repeat breeding and involuntary culling of cows. This study was carried out to show the prevalence of CVM carriers among Holstein cows in five dairy farms in Hiroshima Prefecture, South Western Japan and whether the defect could be inherited to crossbred F1 generation, when Japanese Black semen was used to inseminate a Holstein cow. Two hundreds Holstein cows were used in the study. Blood samples were collected from all cows in a heparinized tubes and genomic DNA was extracted with a commercial kit. Apart of the bovine solute carrier family 35 member 3 (SLC35A3) gene around the suspected mutation (G to T) was amplified with an allele specific polymerase chain reaction (AS-PCR). For DNA sequencing, PCR products of 522 bp were obtained from the genomic DNA of the cows. Out of 200 examined cows, 26 cows (13.0%) were found to be CVM carriers. Wild type cows showed amplification with the G-specific primer pair but not with the T-specific primer pair, while carrier ones showed amplification of both the G- and T-specific primers, exhibited two DNA bands of 395 bp. Based on the clinical history of the cows, lowered reproductive performance was noticed in carrier ones. Out of four crossbred F1 calves (Holstein x Japanese Black), two were CVM carrier. Crossbred F1 could inherit the CVM defect when Japanese Black semen used to inseminate a carrier Holstein cow. In conclusion, the study reports the occurrence of CVM among Holstein cows in Hiroshima Prefecture, Japan; moreover, it describes the first cases of CVM in crossbred F1 generation.

Deficiency of uridine monophosphate synthase (DUMPS) and X-chromosome deletion in fetal mummification in cattle.

Ten mummified fetuses were tested for the deficiency of uridine monophosphate synthase (DUMPS), which is known to contribute to the embryonic and fetal mortality in cattle. Genomic DNAs of the mummified fetuses were extracted from tissue samples collected from the mummies and were amplified by GenomiPhi DNA amplification kit. UMPS gene of the mummies was amplified by polymerase chain reaction (PCR) with DUMPS primers. Out of ten mummies examined, two fetuses were heterozygous (carriers) for DUMPS as indicated by the presences of three bands of 89, 53 and 36 bp. Estimated stage of gestation when the death occurred in the two mummies was 3.5 and 2.5 months, respectively. The other fetuses exhibited only two bands of 53 and 36 bp on the polyacrylamide gel indicated that they were normal. On the other hand, all the mummies were sexed using AMX/Y primers. Specific regions of Y and X chromosomes were amplified by PCR using AMX/Y. The expected 280 bp fragment in the female sample and the 280 and 217 bp in the male sample were observed. Nine mummies had a normal X and Y chromosome bands; however, the other mummified fetus exhibited only Y chromosome band, while the constitutive X chromosome fragment was missing. The estimated stage of gestation when the death occurred in this mummified fetus was 100 days. This might be the first report of DUMPS and X-chromosome deletion at the amelogenin gene in bovine-mummified fetuses in Japan.

Factor XI mutation in a Holstein cow with repeat breeding in Japan.

Factor XI deficiency is an autosomal recessive coagulopathy in Holstein cattle. Affected cows have a tendency to show repeat breeding. Forty repeat breeding Holstein Friesian cows were selected and tested for the Factor XI mutation. Genomic DNA was isolated from the blood of the cows (n=40). Exon 12 of the Factor XI gene of the cows was amplified by PCR. One repeat breeding cow was heterozygous to the Factor XI mutation as indicated by the presence of two DNA fragments of 320 bp and 244 bp. The insertion of the 76 bp in the heterozygous cow was confirmed by DNA sequencing. The heterozygous cow was in her fourth lactation. She gave birth to male twins at the last calving. She was inseminated artificially four times after the last calving. Factor XI deficiency in cattle has been reported in different countries. However, no case was reported in Japan. This might be the first to report Factor XI mutation in Holstein cattle in Japan.

DNA extraction from bovine mummified fetuses and detection of factor XI gene deficiency in the mummies.

Genomic DNA extracted from bovine mummified tissue is valuable material for detection of some genes that may contribute to fetal abnormalities. In this study bovine genomic DNA was extracted from the hardened tissue samples of ten bovine mummified fetuses. The amount of genomic DNA extracted from 2 g of the mummified tissues by the phenol/chloroform-ethanol method was low (less than 4 microg/ml) for all samples. The extracted DNA was then amplified by the GenomiPhi DNA amplification system. After amplification, the amount of DNA was increased to more than 100 microg/ml for all samples. This amplification system was shown to be a good tool for amplifying the genomic DNA of the mummified fetuses. The amplified genomic DNA was used for testing the mummies for Factor XI gene deficiency, an autosomal recessive deficiency involved in the early stages of the intrinsic blood coagulation pathway. Exon 12 of the Factor XI gene of the mummies was amplified by PCR. Two of the ten mummified fetuses were heterozygous for the Factor XI gene as indicated by the presence of two amplified DNA fragments of 320 bp and 244 bp. Factor XI deficiency has already been described in Holstein cattle. However, no report is available for bovine fetus. In this study, DNA was extracted and amplified from the bovine mummified fetuses, and the samples were successfully tested for Factor XI gene deficiency in the mummies.

A case of freemartin with atresia recti and ani in Japanese Black calf.

A female Japanese Black calf was born on 25 March 2003 at Hiroshima University Farm as a co-twin to a male Japanese Black calf. The male calf showed no external urogenital abnormalities. The absence of anal opening and external features of freemartinism were observed in the female. A small opening to the vulva (about 1.5 cm in length) with fused lips and a prominent clitoris were seen. The hair around the vulva was 3.5 cm in length and was heavy and dense. The distance from the vulva to the atretic anus was 9.0 cm. There were no other detectable abnormalities on physical examination. The PCR-based DNA test showed male-specific sequences confirming the calf to be freemartin. At autopsy 1 day after the calf birth, the gonads were found to be small and hard and the left uterine horn showed segmental aplasia near its proximal end. Two seminal glands (remnants of mesonephric duct) were located on both sides of the uterine body. A cervix was absent. The vagina was underdeveloped and looked like a tubual structure. The rectal end was closed, while the distance from the end of the atretic rectum to the absent anal opening was about 4.0 cm. On histological examination, the gonads exhibited extensive morphologic alteration; there was no cortex with the absence of ovarian structures. The seminal glands consisted of hypoplastic glandular tissue surrounded by extensive fibrous connective tissue. In conclusion, this is a case report of a freemartin with atresia recti and ani.