Jin Kobayashi

Feasibility of a nylon-mesh holder for vitrification of bovine germinal vesicle oocytes in subsequent production of viable blastocysts

Abstract To improve the feasibility of nylon-mesh holder for vitrification of bovine cumulus-oocytes complexes (GV-COCs) having germinal vesicle, this study was conducted to demonstrate effects of sugars and protocol of exposure in vitrification on subsequent in vitro maturation, ultrastructural changes, and in vitro development in bovine immature oocytes after cryopreservation using nylon mesh. Before vitrification, GV-COCs were exposed to the cryoprotectant, which was composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40), either by single step or in a stepwise way. The maturation rates in the stepwise exposure with EFS40 or EFT40 were significantly higher (P < 0.05) compared with the corresponding rates in the single step. In the stepwise exposure, few abnormalities were observed compared with the single-step exposure, where most oocytes showed a highly vacuolated cytoplasm with many ruptured mitochondria. Cleavage rates in fertilized oocytes previously exposed stepwise to EFS40 or EFT40 were significantly higher than those exposed by the single-step procedure. The cleaved embryos derived from the stepwise exposure to EFS40 developed to blastocysts. After transfer of blastocysts derived from vitrified GV oocytes, a female calf was born. These results indicate that vitrification of large numbers of bovine GV-COCs using a nylon-mesh holder accompanied with stepwise exposure minimizes structural damage in organelles, resulting in yield of viable blastocysts following in vitro embryo production.

Lactoferrin stimulates A Staphylococcus aureus killing activity of bovine phagocytes in the mammary gland.

Lactoferrin (Lf) may play a key role in the clearance of microorganisms from a host. To study in vitro the bactericidal mechanisms of Lf during nonlactating periods, we investigated whether the effects of Lf were influenced by bovine mammary gland secretory cells (MGSC) and fresh normal bovine serum (NBS) as a source of complement. Phagocytic killing tests demonstrated that a phagocytic mixture of unopsonized Staphylococcus aureus (S. aureus) and MGSC in the presence of Lf reduced bacterial growth, compared with that of unopsonized S. aureus and MGSC without Lf. The opsonization with Lf and fresh NBS together resulted in more than a 95% reduction in CFU. The activation of complement induced by Lf also resulted in increased deposition of C3 on S. aureus, and the phagocytic activity of MGSC was augmented by opsonization with Lf and fresh NBS. Inhibition of C3 deposition by Lf was not induced in the presence of Mg‐EGTA, but was induced by the addition of bovine Lf antiserum. These results strongly suggest that Lf induces the activation of complement in fresh NBS mainly through an alternative pathway. The results demonstrated a Lf‐dependent, antibody‐independent and complement‐mediated phagocytic killing of S. aureus, and implied that Lf was synergistically capable of activating both the alternative pathway of the bovine complement cascade and phagocytosis by phagocytes.

Induction of Nitric Oxide Production Mediated by Tumor Necrosis Factor Alpha on Staphylococcal Enterotoxin C-Stimulated Bovine Mammary Gland Cells

ABSTRACT Mammary gland (MG) secretions (MGS) derived from secretory cows infected with coagulase-negative staphylococci (CoNS) showed somatic cell counts and lactoferrin similar to levels found in the MGS of secretory cows infected with Staphylococcus aureus. However, nitrite and nitrate (NOx) and staphylococcal enterotoxin C (SEC) were found in MGS infected with S. aureus at much higher levels than in cows infected with CoNS. These results suggested that NOx could be intimately correlated with the production of SEC in secretory cows infected with S. aureus. Therefore, we examined the production of NOx and the expression of proinflammatory cytokines and microsomal cytochrome P450 (CYP450) after injection of SEC into the MGS of secretory cows. We were able to detect NOx and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) on MG cells of SEC-injected MGS. It was also found that CYP450 in the MG cells from SEC-injected MGS was down-regulated by approximately one-third in comparison with the cells from phosphate-buffered saline-injected MGS. This in vitro system also showed that NOx could be induced in the culture of bovine macrophage-lined cells (FBM-17) with the supernatants of SEC-stimulated bovine peripheral blood lymphocytes (BoPBLs) but not in the culture of peripheral mononuclear cells with SEC-stimulated BoPBLs. The expression of the mRNA for both inducible nitric oxide synthase and TNF-α in FBM-17 was enhanced by culturing with the supernatant of SEC-stimulated BoPBLs, although CYP450 was down-regulated. These results indicate that the down-regulation of CYP450 was caused by the production of TNF-α in SEC-stimulating MG cells containing macrophages and via NOx production. Therefore, we suggest that NOx released from activated MG cells via the superantigenic activity of SEC caused oxidative damage to the MG in S. aureus-induced mastitis.

Effects of radioactive caesium on bull testes after the Fukushima nuclear plant accident

We aimed to investigate the effect of chronic radiation exposure associated with the Fukushima Daiichi Nuclear Plant accident on the testis from 2 bulls. Estimated dose of internal exposure in one bull was 0.7–1.2 mGy (134Cs) and 0.4–0.6 mGy (137Cs) and external exposure was 2.0 mGy (134Cs) and 0.8 mGy (137Cs) (196 days). Internal dose in the other was 3.2–6.1 mGy (134Cs) and 1.8–3.4 mGy (137Cs) and external dose was 1.3 mGy (134Cs) and 0.6 mGy (137Cs) (315 days). Sperm morphology and spermatogenesis were within normal ranges. 134, 137Cs radioactivity was detected but Cs was not detectable in the testis by electron probe microanalysis. Thus, adverse radiation-induced effects were not observed in bull testes following chronic exposure to the above levels of radiation for up to 10 months. Since we could analyse a limited number of testes, further investigation on the effects of ionizing radiation on spermatogenesis should be extended to more animals.

Rapid detection of male-specific DNA sequence in bovine embryos using fluorescence in situ hybridization

An accurate, reliable, and quick (less than an hour) method for determining the sex of bovine embryos was developed using a fluorescence in situ hybridization (FISH), with a probe designed from a bovine Y chromosome specific DNA (BC1.2). First, to improve a protocol of FISH and evaluate an accuracy of the method, lymphocyte nuclei prepared from three bulls, two cows, and one freemartin were tested. We found that 5 min was enough for hybridization. The washing solution adequate for posthybridization was 0.5× SSC at 72°C for 5 min. The whole procedure for FISH can be accomplished in less than an hour. A male‐specific signal was detected, on average, as 97, 0.5, and 83%, respectively, of lymphocytes in males, females, and a freemartin. Using the rapid FISH protocol developed, 28 embryos were divided. According to the presence of the digoxigenin signal, 16 embryos (57.1%) were predicted as male, and 12 embryos (42.9%), predicted as female. Mol. Reprod. Dev. 51:390–394, 1998.

Inflammatory responses of bovine polymorphonuclear neutrophils induced by staphylococcal enterotoxin C via stimulation of mononuclear cells.

ABSTRACT To elucidate the pathological roles of staphylococcal enterotoxin C (SEC) in bovine staphylococcal mastitis, a histopathological analysis of SEC-inoculated mammary glands was performed. SEC-inoculated mammary glands exhibited interstitial inflammation, and the leukocytes that migrated into the gland were predominantly polymorphonuclear neutrophils (PMN). In the gland cistern tissues dissected from SEC-inoculated mammary glands, epithelial cellular degeneration was observed. We also investigated the physiological effects of SEC on PMN in vitro. PMN migration was induced by culture supernatant of SEC-stimulated peripheral blood mononuclear cells (S-PBMC sup) but not by that of nonstimulated PBMC (N-PBMC sup). The concentration of interleukin-8 was significantly (P < 0.05) higher in S-PBMC sup than N-PBMC sup, and a significantly (P < 0.05) higher mRNA expression of growth-regulated oncogenes was detected in SEC-stimulated PBMC than in nonstimulated PBMC. Milk PMN collected from SEC-inoculated mammary glands produced more than 2 times the amount of superoxide at 1 day postinoculation (dpi) than at 0 dpi in the presence of phorbol 12-myristate 13-acetate (PMA). PMN cultured with S-PBMC sup for 24 h also produced significantly (P < 0.05) larger amounts of superoxide than those cultured with N-PBMC sup in the presence of PMA. Moreover, S-PBMC sup induced the long-time survival of PMN. These results indicate that SEC induces the activation of PMN via the stimulation of mononuclear cells.

Cesium radioactivity in peripheral blood is linearly correlated to that in skeletal muscle: Analyses of cattle within the evacuation zone of the Fukushima Daiichi Nuclear Power Plant

The accident at the Fukushima Daiichi Nuclear Power Plant (FNPP) released a large amount of radioactive substances into the environment. Furthermore, beef contaminated with radioactive cesium above the 500 Bq/kg safety standard was circulated in the food chain in 2011. Japanese consumers remain concerned about the safety of radioactively contaminated food. In our previous study, we detected a linear correlation between radioactive cesium ((137) Cs) activity in blood and muscle around 500 to 2500 Bq/kg in cattle. However, it was unclear whether the correlation was maintained at a lower radioactivity close to the current safety standard of 100 Bq/kg. In this study, we evaluated 17 cattle in the FNPP evacuation zone that had a (137) Cs blood level less than 10 Bq/kg. The results showed a linear correlation between blood (137) Cs and muscle (137) Cs (Y = 28.0X, R(2)  = 0.590) at low radioactivity concentration, indicating that cesium radioactivity in the muscle can be estimated from blood radioactivity. This technique would be useful in detecting high-risk cattle before they enter the market, and will contribute to food safety.

Electron probe X-ray microanalysis of boar and inobuta testes after the Fukushima accident

We aimed to investigate the effect of chronic radiation exposure associated with the Fukushima Daiichi Nuclear Power Plant (FNPP) accident on the testes of boar and inobuta (a hybrid of Sus scrofa and Sus scrofa domestica). This study examined the contamination levels of radioactive caesium (Cs), especially 134Cs and 137Cs, in the testis of both boar and inobuta during 2012, after the Fukushima accident. Morphological analysis and electron-probe X-ray microanalysis (EPMA) were also undertaken on the testes. The 134Cs and 137Cs levels were 6430 ± 23 and 6820 ± 32 Bq/kg in the boar testes, and 755 ± 13 and 747 ± 17 Bq/kg in the inobuta testes, respectively. The internal and external exposure of total 134Cs and 137Cs in the boar testes were 47.1 mGy and 176.2 mGy, respectively, whereas in the inobuta testes, these levels were 6.09 mGy and 59.8 mGy, respectively. Defective spermatogenesis was not detected by the histochemical analysis of radiation-exposed testes for either animal. In neither animal were Cs molecules detected, using EPMA. In conclusion, we showed that adverse radiation-induced effects were not detected in the examined boar and inobuta testes following the chronic radiation exposure associated with the FNPP accident.

Software development for estimating the concentration of radioactive cesium in the skeletal muscles of cattle from blood samples

The 2011 earthquake severely damaged the Fukushima Daiichi Nuclear Power Plant (FNPP), resulting in the release of large quantities of radioactive material into the environment. The deposition of these radionuclides in rice straw as livestock feed led to the circulation of contaminated beef in the market. Based on the safety concern of the consumers, a reliable method for estimating concentrations of radioactive cesium in muscle tissue is needed. In this study, we analyzed the concentrations of radioactive cesium in the blood and skeletal muscle of 88 cattle, and detected a linear correlation between them. We then developed software that can be used to estimate radioactive cesium concentrations in muscle tissue from blood samples. Distribution of this software to the livestock production field would allow us to easily identify high-risk cattle, which would be beyond the safety regulation, before shipping out to the market. This software is planned to be released as freeware. This software would contribute to food safety, and aid the recovery of the livestock industry from the damage creacted by the 2011 Tohoku earthquake and tsunami.

Collection and frozen storage of semen for artificial insemination in red foxes ( Vulpes vulpes )

This study was conducted on red foxes to determine the appropriate voltage in electroejaculation for semen collection from stud males, and to confirm whether frozen semen with bovine semen extender can be used for artificial insemination. The proper load voltage for electroejaculation was 3–4 V based on semen collection rates and concentrations of spermatozoa. Frozen semen was prepared according to the known procedure for cows. In frozen-thawed semen, a relatively high conception rate (81.3%) was obtained in vixens, in which the optimum insemination time was detected by vaginal electrical resistance. These findings demonstrate that the restricted condition for semen collection by electroejaculation with cryopreservation of semen using bovine semen extender can be applied to artificial insemination of red foxes.