Premature capacitation of frozen-thawed spermatozoa from subfertile Japanese black cattle

Artificial insemination (AI) subfertility is an indication of failure of AI with frozen-thawed sperm classified as normal by conventional semen examination. Recently, 8 AI-subfertile Japanese Black cattle (S1-S8) were identified using the routine AI test or in vivo fertilization test, which included AI with frozen-thawed sperm of superovulated females and subsequent non-surgical recovery of presumptive zygotes. In the present study, we assessed capacitation states and in vitro oocyte penetration of frozen-thawed sperm from these bulls to estimate causal factors of AI subfertility. Frozen-thawed sperm from 8 AI-subfertile (S1-S8) and 9 fertile (F1-F9, control) bulls were washed and then used for a chlortetracycline (CTC) staining assay and in vitro fertilization test. The CTC staining assay revealed that approximately 50% of the sperm from 4 of the AI-subfertile bulls (S5-S8) were prematurely progressing into the capacitation state immediately after washing and resuspension in a CaCl(2)-lacking medium. In contrast, most of the sperm from the fertile bulls and other AI-subfertile bulls (S1-S4) remained uncapacitated. Addition of CaCl(2) to the medium effectively promoted a spontaneous acrosome reaction in the sperm samples from the AI-subfertile bulls (S5-S8). Moreover, the in vitro fertilization test showed that rates of sperm penetration into oocytes were significantly lower in sperm samples from the AI-subfertile bulls (S5-S8) than in the control sperm samples from the fertile bulls (F2-F4 and F7-F9). It has previously been suggested that prematurely capacitated sperm undergo a spontaneous acrosome reaction possibly due to uncontrolled influx of calcium ion, and consequently they possess relatively lower in vitro fertilizing ability. It is therefore possible that premature capacitation of sperm used for AI is a causal factor of subfertility of male Japanese Black cattle and a potentially good marker for identification of subfertile bulls for removal from AI programs.     

Transportation of freeze-dried mouse spermatozoa under different preservation conditions

Freeze-dried mouse spermatozoa can be used for normal embryonic development after injection into oocytes, thus indicating that freeze-drying is a useful method for the storage and transportation of genetic materials from animals. We recently reported that storage of freeze-dried mouse spermatozoa requires maintenance at temperatures lower than -80 C for long-term preservation and a pressure of 0.37 mbar at primary drying and that these conditions significantly improve the developmental rate to the blastocyst stage. In this study, we examined the influence of transportation and preservation conditions on freeze-dried spermatozoa. Freeze-dried spermatozoa stored for 2 or 2.5 years at 4 or -80 C were transported round trip overland between Shizuoka and Hokkaido prefectures in Japan or by air between Japan and Belgium. The freeze-drying conditions consisted of primary drying at pressures of 0.04, 0.37 and 1.03 mbar and secondary drying at a pressure of 0.001 mbar. Embryos (2-cell stage) from freeze-dried spermatozoa dried at 0.04 mbar and stored at 4 C for 2 years with and without overland transportation did not develop to term. The development rates of embryos from spermatozoa stored at -80 C for up to 2 years and transported overland, by air and without transportation were 8, 1 and 28%, respectively. The development rates of embryos from spermatozoa without transportation were significantly higher than with transportation (P<0.05). These data indicate that freeze-dried spermatozoa stored at -80 C with and without transportation can retain their ability to generate viable offspring after storage for up to 2 years. However, there are limitations to be considered in the transportation of freeze-dried spermatozoa at ambient temperature.     

Special sugar expression on apoptotic epithelial cells of Peyer's patches and intestinal villi in rat small intestine

Our previous study clarified that the apical regions of both the follicle-associated epithelium (FAE) of Peyer's patches and the intestinal villi are the only adhesion sites of indigenous bacteria in rat jejuno-ileum. To survey the ligands against bacterial lectins, sugar expression patterns on epithelial cells were lectin-histochemically investigated using 21 lectins in the jejuno-ileal Peyer's patches of rats. As a result, (D-glcNAc)(2-4), detected by Solanum tuberosum (STL) and by Lycopersicon esculentum (LEL), and beta-D-gal(1-3)-D-galNAc detected by Peanut agglutinin (PNA), were strongly expressed on the brush borders of the apical regions of the FAE and the intestinal villi. On the other hand, neither sugar was expressed on the brush borders of the basal regions of both FAE and intestinal villi. The positive intensities for the lectins correlated with the progression of epithelial apoptosis in the FAE and in the intestinal villi. Moreover, the double staining with lectin histochemical method and the in situ nick end-labeling method could simultaneously detect the strong expression of both sugars and nuclear DNA fragmentation in epithelial cells at the late apoptotic stage. Other sugar expression patterns in the intestinal villi were similar with those in the FAE. There were no lectins specific for M cells in the FAE. From these findings, the possible sugars of ligands against some indigenous bacterial lectins, expressing specially on the apoptotic epithelial cells, might be narrowed down in rat jejuno-ileum.     

The infectivity and pathogenicity of a group 2 bovine coronavirus in pups

Canine respiratory coronavirus (CRCoV), which is more closely related to the bovine coronavirus (BCoV), has recently been detected in dogs. In this study, we examined whether BCoV was capable of infecting and exhibiting pathogenicity in dogs. Three 1-month-old pups were oronasally given field isolates of BCoV, and were kept together with 2 control animals. As a result, increases in BCoV-neutralizing antibody titers were confirmed in all pups in the challenged and control groups. Moreover, the virus gene was also detected in oral and rectal swabs by RT-PCR. These results indicate that BCoV infects dogs, and easily infects other dogs that are kept together. However, no clinical symptoms such as respiratory symptoms and diarrhea were observed.     

A rapid agglutination assay for canine brucellosis using antigen coated beads

Brucella canis is the causative agent of canine brucellosis and facultative intracellular pathogen. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. In this study, crude antigens were extracted from B. canis using hot saline, coated on to latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. Mixing the antigen coated latex beads with the sera of dogs infected with B. canis produced clear agglutination, but this was not so for B. canis free dog sera. N-terminal amino acid sequence analysis of the crude hot saline extracts, showed that they contained copper-zinc superoxide dismutase, ribose ABC transporter and hypothetical protein of Brucella as antigens. A serological survey of canine serum samples conducted by means of an agglutination test using the antigen coated latex beads, showed that this method was more specific than the tube agglutination test using whole bacterial cell antigens. Although these results suggest that our method in which crude hot saline extracted antigens are coated on to latex beads would be useful in the serological diagnosis of canine brucellosis, we need further investigation using more serum samples to confirm the usefulness of our method.     

Controlling highly prevalent Staphylococcus aureus mastitis from the dairy farm

In 57 Holstein cows where the dairy farm uses a milking parlor system, the somatic cell count (SCC) increased persistently in the bulk milk (monthly mean 52.3 x 10(4) cells/ml; range 21 to 94 x 10(4) cells/ml). We detected S. aureus in 24 (41.2%) of the 54 lactating cows and in 29 (12.8%) of 227 quarters of the 57 milking cows in the herd. A control program was implemented in an effort to eradicate S. aureus mastitis from this dairy farm. The control plan established improved handling of the lactating cows, improved milking procedures, dry-cow therapy, and culling of infected cows. The program was monitored for 3.5 years by frequent checkups on the rate of S. aureus infection, the SCC, and the changes in milk composition. Eighteen months after the control program was started, the rate of S. aureus infection in the quarter milk decreased dramatically, and no S. aureus isolates were found in the milk of the remaining cows. The SCC in the bulk milk of the herd dropped to a monthly mean of <20 x 10(4) cells/ml. In conclusion, the control program was effective for controlling persistent S. aureus mastitis in this dairy herd.     

Ultrastructural study on the differentiation and the fate of M cells in follicle-associated epithelium of rat Peyer's patch

The differentiation process of immature microvillous epithelial cells to M cells and the fate of M cells in the follicle-associated epithelium (FAE) of the mucosa-associated lymphoid tissues are still unclear. In this study, the differentiation process and the fate of M cells were clarified in rat Peyer's patches under a transmission electron microscope. Almost all immature epithelial cells were found to possess long, slender microvilli, which gradually shortened, thickened and dispersed as the immature epithelial cells migrated away from the crypt orifices. These morphological changes started in the centers and moved to the peripheries of the apical surfaces of epithelial cells, accompanied by the protrusion of apical cytoplasm out of the terminal web. During these changes, the bundles of microfilaments of microvilli never shortened, and both small vesicles in the apical cytoplasm and tiny invaginations of the apical membranes were found. The intraepithelial migrating cells gradually accumulated to form typical intraepithelial pockets. In all FAE, there was no morphological sign of cell death in M cells. The rearrangement of microfilament bundles, the reconstruction of microvilli and the disappearance of pockets resulted in the transformation of M cells into microvillous epithelial cells. These serial ultrastructural changes suggest that M cells are a temporal and transitional cell type caused by the active engulfment of luminal substances and that when the engulfment ceases, the M cells transform into mature microvillous epithelial cells.     

Fatal winter dysentery with severe anemia in an adult cow

An adult dairy cow fatally affected with winter dysentery was investigated pathologically and virologically. The cow had severe anemia and diarrhea with massive blood. Pathologically, the loss of surface epithelial cells and necrosis of crypt epithelial cells in the large intestine were observed. Bovine coronavirus (BCV) antigen was observed in necrotic crypt epithelial cells of the large intestine. Virus particles were found in the necrotic epithelial cells of the large intestine. Virologically, BCV was isolated from the feces of the dead cow. The dead cow had no serum antibody against BCV although the co-habitants did. These suggest that severe infection of BCV in the cow without the BCV antibody accompanied by severe hemorrhagic anemia resulted in the cow's death.     

Phagocytotic activation of muscularis resident macrophages inhibits smooth muscle contraction in rat ileum

Intestinal muscularis resident macrophages distributed in myenteric region may play an important role in the immunological host defense against infection. In this study, we investigated the phagocytic stimulation of resident macrophages on cyclooxygenase-2 (COX-2) expression and smooth muscle contraction in the small intestine of rat. After the injection of FITC-dextran to rat, phagocytosed macrophages could be detected in the myenteric plexus. FITC-positive macrophages were also immunostained with COX-2 antibody. The number of COX-2 immunopositive cells increased in a time-dependent manner reaching its maximum at 4 hr after the injection, which then decreased gradually but considerable number of cells were still remained on 7 days. The injection of FITC-dextran, however, did not change the population of ED2-positive resident macrophages even on 7 days. Production of PGE2 was significantly higher in the dextran treated tissue as compared to control tissue. In the smooth muscle tissue phagocytosed dextran, carbachol-induced contraction was significantly decreased. The suppression of the carbachol-induced contraction was completely restored by COX inhibitor, indomethacin. Finally we demonstrated that, in freshly isolated macrophage cells, addition of dextran induced a slow and sustained increase in intracellular Ca2+ concentration. These results indicate that phagocytotic activation of muscularis resident macrophages induces COX-2 gene expression and then results in production of PGE2 to suppress the smooth muscle contractile activity.     

Development of phototaxis in the early life stages of Pacific bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis>

We investigated the development of phototaxis in larval and early juvenile stages of Pacific bluefin tuna Thunnus orientalis to detail behavior development in relation to light. We observed the distribution of Pacific bluefin tuna in an illumination gradient tank from 1 to 26 days after hatching (DAH). Two long rectangular tanks (100 × 10 × 10 cm) were used for experiments; each tank was divided into four sections for observations and one tank was equally illuminated at 1.0 × 10³ lx as the control tank, and the other was at 4.0 × 10², 1.0 × 10³, 1.0 × 10⁴ and 7.0 × 10⁴ lx as the illumination gradient tank. Laboratory-reared larvae and juvenile were released into each tank and acclimatized for 10 min. Thereafter, the number of individuals in each section was counted for 5 times with 10-min intervals. Until 2 DAH (3.6 ± 0.1 mm BL), larvae distributed homogeneously in each section in both tanks. After 3 DAH (3.7 ± 0.1 mm BL) when the eyes of the larvae were pigmented, most fish moved into the 7.0 × 10⁴ lx section in the illumination gradient tank, while fish in the control tank maintained a uniform distribution during the experimental period (Chi-square test, P < 0.05). The results suggest that the post-larvae and juvenile stages show strong positive phototaxis.