Manipulated Mouse Embryos as Bioassay System for Water Quality Control

Mouse pronuclear stage embryos with intact slit zona pellucida (manipulated) were cultured in vitro until the hatched blastocyst stage in simplex optimized medium with higher K⁺ concentration (KSOM) prepared with three different water types: tap, deionized reverse osmosis (D‐O) water and Milli‐Q system (M‐Q) water. The culture media were supplemented with or without protein and ethylenediaminetetraacetic acid (EDTA, disodium salt). The rates of hatched blastocysts were significantly affected (p < 0.01) by micromanipulation, protein supplement and water source. The water source has no influence (p > 0.05) on development in EDTA‐supplemented protein‐free culture media, whereas in EDTA‐ and protein‐free culture media, the water quality significantly (p < 0.001) affected the rates of development, with higher rates in media prepared with M‐Q water. The micromanipulated embryos showed higher sensitivity to the water quality (p < 0.01). It worth mentioning that the rates of hatched blastocysts in protein‐free culture media were very low (0–7.5%). Furthermore, the three different water types were analysed by measuring the electrical conductivity, inorganic ions, total organic carbon and endotoxins to evaluate the purity. M‐Q water showed the lowest levels of inorganic ion, total organic carbon and endotoxin concentrations. We concluded that manipulated mouse embryos are good system to evaluate the quality of water used in biological system.     

Basal levels and GnRH-induced responses of peripheral testosterone and estrogen in Holstein bulls with poor semen quality

The present study investigated the basal levels and GnRH-induced responses of peripheral testosterone and estrogen in Holstein bulls with poor semen quality. On the basis of semen parameters, bulls (n=5) having poor semen quality were selected as experimental bulls, and good semen quality bulls (n=4) were used as control bulls. Both groups were treated intramuscularly once with GnRH (250 μg of fertirelin acetate). Blood samples were collected at -1 day (d), -30 min and 0 h (treatment) followed by every 30 min for 5 h and 1, 3 and 5 d post-GnRH treatment (PGT), and LH, testosterone and estradiol-17β (E(2)) concentrations were measured. The pretreatment concentrations were used as basal levels. The percentage increments based on the 0-h levels were calculated per bull for each sampling time until 5 h PGT, and differences were compared between the experimental and control groups. The PGT concentrations of testosterone and basal and PGT concentrations of E(2) were significantly lower in the experimental group. The testosterone increment in the experimental group was delayed and significantly lower from 1 to 5 h PGT than those in the control group. It can be suggested that bulls with poor semen quality have delayed and lower GnRH-induced testosterone response and may also have lower estrogen levels.     

Establishment of a large-scale purification procedure for purified recombinant bovine interferon-tau produced by a silkworm-baculovirus gene expression system

We developed a procedure for the large-scale purification of bovine interferon-tau (boIFN-tau) by means of a silkworm-baculovirus gene expression system. Recombinant boIFN-tau (rboIFN-tau) was efficiently produced in the silkworm infected with boIFN-tau cDNA recombinant baculovirus and accumulated in the haemolymph. To establish a purification method suitable for mass production, we tried three crude purification methods, namely, an acidification and neutralization treatment (ANT), silica gel column chromatography (SGCC), and Blue sepharose column chromatography (BSCC) with a combination of Q-sepharose (QSC) and chelating sepharose column chromatographies (CSCC). As a result, the acidification and neutralization treatment was found to be the most efficient and cost effective. With this combination, we obtained 91% pure products. To confirm the applicability of the procedure for mass production, we inoculated 100 silkworms with the recombinant virus, and recovered about 4.55 mg (1.26 x 10(8)U/mg) of 91% pure rboIFN-tau by means of a combination of the ANT, followed by QSC and CSCC.     

Effects of oxygen tension in the gas atmosphere during in vitro maturation, in vitro fertilization and in vitro culture on the efficiency of in vitro production of mouse embryos

Effects of oxygen (O2) tension in the gas atmosphere during in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) on the efficiency of in vitro production of mouse embryos were examined. Mouse oocytes recovered from large antral follicles were subjected to IVM in Waymouth medium for 15, 16 and 17 hr under 5 or 20% O2 and then subjected to IVF and IVC under 5 or 20% O2 tension. Lowering the O2 tension in the gas atmosphere for IVM from 20 to 5% improved the cleavage rate after IVF when the oocytes were subjected to IVM for 15 hr; however, no improvement in the cleavage rate was observed when the culture period for IVM was extended to 16 and 17 hr. Lowering the O2 tension to 5% for IVM and IVC improved the development of the cleaved oocytes to the blastocyst stage, regardless of the culture period for IVM. However, the O2 tension for IVF had no remarkable effect on the subsequent embryonic development. These results demonstrate that 5% O2 is superior to 20% O2 for IVM and IVC, and suggest that 20% O2 for IVM may delay oocyte maturation and/or the acquisition of fertilizability and impair the developmental competence of oocytes.     

Establishment of a Model of Spontaneously-Running-Tokushima-Shikoku Rats with Left Atrial Thrombosis

Studies that investigate the underlying mechanisms of disease and treatment options typically require the use of a suitable animal model. Few suitable animal models exist for left atrial thrombosis. Here, we demonstrated that the Spontaneously-Running-Tokushima-Shikoku (SPORTS) rat — a Wistar strain known for its running ability—is predisposed to the development of thrombi in the left atrium. We investigated the incidence of left atrial thrombosis in male (n = 16) and female (n = 17) SPORTS rats and observed organized atrial thrombosis in 57% and 38% of males and female rats, respectively. In the male rats, systolic blood pressures and heart rates were significantly higher in SPORTS rats than in control Wistar rats. We could not find any evidence of arrhythmias, such as atrial fibrillation, during electrocardiographic examination of SPORTS rats. We believe that the SPORTS rat could serve as a new research model for left atrial thrombosis; further, it may be suitable for research investigating the development of new antithrombotic approaches for the control of atrial thrombosis or familial thrombophilia in humans.     

A case of feline pancreatitis

A 14-year-old, spayed female, domestic shorthair cat was referred to us with anorexia, pyrexia, and jaundice. Total bilirubin (TBIL) and feline trypsin-like immunoreactivity (fTLI) levels were remarkably high. Based on laparoscopic biopsy of the pancreas, the cat was diagnosed as having pancreatitis. As a result of treatment with a synthetic protease inhibitor and corticosteroid, the TBIL and fTLI values returned to normal and the clinical course was good.     

A cross-protection experiment in pigs vaccinated with Haemophilus parasuis serovars 2 and 5 bacterins, and evaluation of a bivalent vaccine under laboratory and field conditions.

Cross-protection between Haemophilus parasuis serovars 2 and 5 was examined in pigs using a bacterin based vaccine, and subsequently the safety and efficacy of a bivalent vaccine were evaluated. Upon intratracheal challenge of a serovar 2 or 5 strain, pigs immunized with a monovalent vaccine were protected against challenge with a homologous serovar strain, but not with a heterologous serovar strain. Immunization with a bivalent vaccine containing both serovars 2 and 5 bacterins conferred protection in pigs against lethal challenge with each of the serovar strains. A total of 86 pigs from two SPF herds were injected with the bivalent vaccine intramuscularly twice at a four-week interval. No adverse reactions following the vaccination were observed. On day 7 after the second vaccination, vaccinated and non-vaccinated control pigs from herd A were transferred to herd B, where Glasser's disease had broken out. Pigs in the control group developed clinical signs of the disease, and 6 of 8 (75%) pigs died until slaughter, in contrast with only 4 of 46 (9%) pigs in the vaccinated group. In herd C, where there was no outbreak of Glasser's disease, complement fixation antibody titer was raised only in the vaccinated group. A challenge experiment on days 20 and 79 after the second vaccination showed that only the vaccinated pigs were protected. From these findings, the safety and efficacy of the bivalent vaccine were confirmed under laboratory and field conditions.     

Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.