Noboru Fujihara

Accessory reproductive fluids and organs in male domestic birds

Accessory reproductive fluids and organs of male domestic fowl, turkeys, ducks, quail, guinea fowl and pigeons were examined. Male domestic fowl, turkeys and ducks produced different quantities of ...

Passive Immunization with Milk Produced from an Immunized Cow Prevents Oral Recolonization by Streptococcus mutans

ABSTRACT Cell surface protein antigen (PAc) and water-insoluble glucan-synthesizing enzyme (GTF-I) produced by cariogenicStreptococcus mutans are two major factors implicated in the colonization of the human oral cavity by this bacterium. We examined the effect of bovine milk, produced after immunization with a fusion protein of functional domains of these proteins, on the recolonization of S. mutans. To prepare immune milk, a pregnant Holstein cow was immunized with the fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc and the glucan-binding (GB) domain of GTF-I. After eight adult subjects received cetylpyridinium chloride (CPC) treatment, one subgroup (n = 4) rinsed their mouths with immune milk and a control group (n = 4) rinsed with nonimmune milk. S. mutans levels in saliva and dental plaque decreased after CPC treatment in both groups. Mouth rinsing with immune milk significantly inhibited recolonization of S. mutans in saliva and plaque. On the other hand, the numbers of S. mutans cells in saliva and plaque in the control group increased immediately after the CPC treatment and surpassed the baseline level 42 and 28 days, respectively, after the CPC treatment. The ratios ofS. mutans to total streptococci in saliva and plaque in the group that received immune milk were lower than those in the control group. These results suggest that milk produced from immunized cow may be useful for controlling S. mutans in the human oral cavity.

Prevention of the production of lipid peroxide in rooster spermatozoa

Abstract The effects on lipid peroxide in chicken sperm of seminal plasma, Ringer-Phosphate-Medium and some chemical agents such as membrane alterants (bovine serum albumin, cholesterol), chelating agents (calcium, magnesium), cryoprotectants (glycerol, dimethyl-sulfoxide) and citrate were examined. Seminal plasma, Ringer-Phosphate-Medium, calcium and citrate reduced peroxide production and provided minimal protection against it. On the contrary, glycerol, dimethylsulfoxide and magnesium partly inhibited the production of lipid peroxide, and bovine serum albumin and cholesterol had no inhibiting effect. These results suggest that a certain amount of seminal plasma, calcium and citrate can inhibit the endogenous lipid peroxidation in sperm and maintain fertilizing ability.

Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts

A series of five experiments measured the high survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. The vitrification solution (designated VS) contained 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline. Embryos developed in vitro at Days 7 and 8 (Day 0 = insemination day) were exposed in one step to VS for 1 min or two steps with 10% ethylene glycol for 5 min and then VS for 1 min. In both cases, the embryos were finally cryopreserved in liquid nitrogen. After the embryos were warmed rapidly and the VS solution diluted, the survival rates were assessed by monitoring hatching rate in vitro. They were 13.0% for the one-step and 72.7% for the two-step procedures (P < 0.001). When embryos were exposed to individual solutions containing 6% (w/v) of each of 4 macromolecules (polyethylene glycol, BSA, polyvinylpyrrolidone or Ficoll) in the two-step protocol and then cryopreserved, the survival rates were 79.3, 34.8, 41.4 and 57.1%, respectively. After embryos had been exposed to the VS in two steps and then cryopreserved, there were no significant differences in survival rates when the solutions were diluted with or without sucrose. These results indicated that a vitrification solution containing polyethylene glycol can be used for cryopreservation of bovine blastocysts produced in vitro, and that a two-step addition of VS improved the in vitro survival of post-warming embryos. It was also shown to be possible to dilute post-warming embryos directly without the use of sucrose solution.

Expression of endothelial nitric oxide synthase in the porcine oocyte and its possible function

The present study was designed to investigate the localization of endothelial nitric oxide synthase (eNOS) in porcine oocytes and its possible function during in vitro development. RT-PCR and immunoblotting analyses revealed the presence of eNOS in the oocytes prepared from small follicles, with an amplified product of 456 bp and an apparent mol wt of 130 kDa, respectively. The synthesis of oocyte NO was suppressed during a 72-h culture of cumulus-oocyte complexes in the presence of follicle-stimulating hormone (FSH), but not luteinizing hormone (LH). However, the decrease in NO synthesis did not result from the levels of eNOS mRNA and its protein, as revealed by analyses of RT-PCR and Western blot analysis, suggesting that expression of oocyte eNOS is not dependent upon gonadotropin stimulation. In proliferated cumulus cells, LH receptor mRNA expression was detected after a 48-h culture with FSH, as revealed by RT-PCR analysis. mRNA expression was inhibited by an NO-releasing agent (S-nitroso-N-acetyl-DL-penicillamine) after an additional 24-h culture. These results suggest that oocytes may release eNOS-derived NO as a signal for somatic cells to steadily suppress the development of cumulus cells, if not FSH stimulation. Conversely, the synthesis of NO is suppressed during the action of FSH on the cumulus cells with no changes in eNOS expression.

Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro

Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar.

Ultrastructure of bovine in vitro-produced blastocysts cryopreserved by vitrification.

The objective of this study was to examine ultrastructural aspects of bovine in vitro-produced blastocysts associated with cryopreservation by vitrification. Morphologically good embryos were used and treated with ethylene-glycol-based vitrification solution (VS). The untreated embryos had conventional fine structure. The post-warming embryos treated with direct exposure to VS (one-step procedure) showed cellular damage structurally by cryopreservation, which included loss of microvilli, disruption of the plasma membrane, mitochondrial changes and swelling of the endoplasmic reticulum. However, nuclei and junctional regions seemed to be resistant to cryoinjury. In contrast, the post-warming embryos pre-equilibrated with 10% ethylene glycol for 5 min and subsequent exposure to VS (two-step procedure) showed less damage than those treated by the one-step procedure. Post-warming embryos treated by the two-step procedure were cultured in vitro for 18 h. Some embryos survived and their structures re-formed to the former state, while other embryos showed serious injuries and could not reconstitute the blastocoele. Three post-warming embryos treated by the two-step procedure that survived after in vitro culture were transferred to three recipients and one of these resulted in pregnancy. These results indicate that cryopreservation by vitrification can damage membranous structures of the cells of bovine embryos, the extent and nature of this damage being dependent on the vitrification procedure.

Gene Transfer to Chicken Blastoderm by Lipofection or Electroporation

Abstract Furuta, H., Kim, K.B. and Fujihara, N. 2000. Gene transfer to chicken blastoderm by lipofection or electroporation. J. Appl. Anim. Res., 17: 209–216. It was proved that an exogenous gene was successfully transferred to chicken embryos by the injection of the gene into blastoderm of freshly oviposited fertilized eggs. In this study, green fluorescent protein (GFP) was used as a marker gene. Tlie GFP dissolved in DOTAP Liposomal Transfection Reagent was introduced into the blastoderm by the method of lipofection or electroporation. After the injection of gene, the eggs were incubated in routine manner until developmental stage 11 to 15. Survival rate of the treated embryos at the stage 11 was 46 per cent (27/59) for the lipofection and 49 per cent (81/166) for the electroporation, respectively. Expression percentages of the GFP for the treated eggs were 74 per cent (20/27) for the lipofection and 5 per cent (4/81) for the eleclroporation, respectively. The GFP was observed in several sites of embryonic tissues and in some of the primordial germ cells (PGCs) in the former method only. Thus lipofection method was considered, slightly superior to the electroporation method for the introduction of exogenous gene to chicken embryos.

Genetic Diversity of the Giant Panda (Ailuropoda melanoleuca) Between Big and Small Populations

Abstract Fang, S.G., Wan, Q.H. and Fujihara, N. 2002. Genetic diversity of the giant panda (Ailuropoda melanoleuca) between big and small populations. J. Appl. Anim. Res., 21: 65–74. Genetic diversity of the giant panda was examined by DNA fingerprinting method in order to determine genetic distances between two groups, big and small populations. Results obtained suggest that small population may induce the reduction of genetic diversity of the giant panda and result in extinction of the species. These results suggest the need for reintroduction of individuals into small population for preventing the reduction of genetic diversity in giant panda population.

An attempt to produce transgenic chicken mediating sperm cells as vectors

Abstract Hasebe, M., Soh, T., Hattori, M. and Fujihara, N. 1998. An attempt to produce transgenic chicken mediating sperm cells as vectors. J. Appl. Anim. Res., 14: 143–150. An attempt was made to produce possible transgenic chicken via sperm cells as vectors. In vitro incubation of ejaculated chicken spermatozoa with exogenous gene (MiwZ DNA) showed a possibility of the incorporation of foreign gene into sperm cells, which was examined by the PCR analysis. The embryos obtained from the eggs inseminated with DNA-treated spermatozoa indicated an existence of transferred exogenous gene in the blastodermal cells of the incubated fertile eggs. Exogenous gene injected into rooster testes was neither detected in the sperm cells nor in the blastodermal cells of fertilized eggs, which were inseminated with spermatozoa obtained from the testes-injected rooster. Sperm head, tail and cell membrane separated from DNA-treated spermatozoa revealed to contain foreign DNA, suggesting a possibility of incorporation of exo...