Yukio Kanai

Difference in tenderness and pH decline between water buffalo meat and beef during postmortem aging.

The objective of this research was to determine the difference in tenderness and some characteristics of water buffalo meat and beef during postmortem aging. Five female crossbred water-buffalo (Philippine Carabao×Bulgarian Murrah) and five female crossbred cattle (Brahman×Philippine Native), were finished on the same diet for 6 months and slaughtered at 30 months of age. The muscle pH was measured at 40min, 3h, 7h, 24h, and 48h postmortem. Longissimus thoracis (LT) and semimembranosus (SM) muscles were excised at 2d postmortem, and shear force was measured at 2, 4, 7, and 14d postmortem. Glycogen and lactate concentrations were determined from 0, 2, and 4d LT samples, and myosin heavy chain type of buffalo and cattle LT was determined by ELISA methods. Myofibrillar protein degradation was also observed by SDS-PAGE and Western blotting of fast-type troponin T. Results showed that the buffalo meat had significantly lower shear force values compared to beef for LT and SM muscles, which was supported by a difference in troponin T degradation. Postmortem pH decline of buffalo meat was significantly slower than that of beef, which was confirmed by lactic acid concentrations, but was not explained by glycogen content. In addition, there was no significant difference in the ratio of slow to fast type muscle fibers in buffalo and cattle, indicating that myosin heavy chain type was not responsible for the difference in pH decline and tenderness between the buffalo meat and beef. This study demonstrated that the tenderness of water buffalo meat was superior to that of Brahman beef, which may have been due to the difference in pH decline and the subsequent effect on muscle protease activity.

Assessment of DNA damage in individual hamster embryos by comet assay

DNA damage induced by either light exposure or oxidative stress likely contributes to the compromised development in vitro of cultured preimplantation embryos. Using the comet assay, which entails microgel electrophoresis that can readily detect single‐strand breaks in DNA, a significant increase in DNA damage was detected in individual one‐cell hamster embryos that were treated with either ultraviolet light or hydrogen peroxide. In addition, an increase in DNA damage also was observed following exposure of one‐cell embryos to visible light. When the embryos were placed in drops of culture medium that were covered with mineral oil and the dishes then placed in a portable incubator containing 5% CO2 in air at 37°C, visible and UV light irradiation for 30 min still induced extensive DNA damage when compared to control embryos that were kept in the dark. In contrast, infrared irradiation did not induce an increase in DNA damage. DNA damage also was measured in individual one‐ and two‐cell stage embryos developed in vivo or in vitro. The extent of DNA damage in the cultured embryos was significantly greater than in embryos that developed in vivo. These results highlight the usefulness of the comet assay to assess DNA damage in individual preimplantation embryos and how the assay can be used to monitor culture conditions in vitro. Mol. Reprod. Dev. 54:1–7, 1999.

Vitrification of bovine oocytes and its application to intergeneric somatic cell nucleus transfer

We determined the efficacy of a microdrop vitrification procedure for cryopreservation of bovine oocytes, using vitrified oocytes as cytoplasts for intraspecies and intergeneric somatic cell nucleus transfer (NT). In vitro matured bovine MII oocytes were vitrified in microdrops with a vitrification solution containing 35% ethylene glycol, 5% polyvinyl pyrrolidone, and 0.4 M trehalose. After warming, approximately 80% of the vitrified oocytes were morphologically normal, and their enucleation rate was similar to that of fresh oocytes. The NT embryos constructed with bovine cumulus cells and the vitrified oocytes developed similar to blastocysts constructed with fresh oocytes, although the cell number of NT blastocysts originating from vitrified oocytes was lower than that of the fresh control. In a second experiment, we examined the development of NT embryos constructed with vitrified bovine oocytes and bovine fibroblasts (intraspecies NT embryos) or swamp buffalo fibroblasts (intergeneric NT embryos). There were no differences between the intraspecies and intergeneric NT embryos in fusion, cleavage and development to blastocysts, except for lower cell numbers in the intergeneric NT blastocysts. In conclusion, the efficacy of this microdrop vitrification procedure and the production of swamp buffalo NT blastocysts using vitrified bovine oocytes was demonstrated.

Alleviation of maternal hyperthermia-induced early embryonic death by administration of melatonin to mice

Abstract:  Maternal hyperthermia induces early embryonic death via increased oxidative stress to the embryo. In this study, we examined whether melatonin administered to heat‐stressed pregnant mice would reduce hyperthermia‐induced embryonic death. Mice were heat stressed (12 hr at 35°C, 60% relative humidity) on the day of mating and melatonin (3 mg/kg body weight) was injected subcutaneously every 2 hr during heat exposure. Thereafter, zygotes were collected, and in vitro developmental ability and intracellular glutathione (GSH) content were assessed. In addition, reactive oxygen species (ROS) levels and free radical scavenging activity (FRSA) in the oviduct as well as lipid peroxidation in the liver were measured. Melatonin administration was associated with a tendency for higher intracellular GSH content in zygotes (1.67 pmol/zygote) and a significantly higher percentage of embryos that developed to the morula or blastocyst stage (47.91%; P < 0.01) compared with the parameters in heat‐stressed mice that were administered a placebo (1.48 pmol GSH/zygote and 14.78% development). Lipid peroxidation levels in the liver and ROS levels in the oviduct were the same in melatonin‐treated stressed mice and the controls, while these parameters were significantly higher in heat‐stressed mice that were not treated with melatonin. Furthermore, FRSA in the oviduct was significantly (P < 0.05) higher in the melatonin‐treated mice than in the controls. These results suggest that administration of melatonin to heat‐stressed mice alleviates hyperthermia‐induced early embryonic death and that this is accomplished in part by maintaining a neutral redox status within the mother.

Redox Status of the Oviduct and Cdc2 Activity in 2-Cell Stage Embryos in Heat-Stressed Mice

Abstract Mammalian preimplantation embryos are vulnerable to heat stress. However, the mechanisms by which maternal heat stress compromises embryonic development are unclear. We hypothesized that the loss of developmental competence in maternally heat-stressed embryos results from enhanced oxidative stress in the oviducts. In experiment 1, oviducts and zygotes were collected from mice that were heat-stressed at 35°C and 60% relative humidity for 12 h on the day of pregnancy as well as from control mice. The zygotes were cultured for 84 h to assess their development, and the H2O2 level, glutathione concentration, and free radical scavenging activity (FRSA) were measured in the oviduct. In experiment 2, zygotes were cultured for 22 h to reach the late G2 phase in the 2-cell stage, and Cdc2 activity was assessed using immunoblotting. A high percentage (87.6%) of control embryos developed to morulae or blastocysts, whereas the majority (67.4%) of the heat-stressed group arrested at the 2-cell stage. Although heat stress did not alter the FRSA or glutathione concentration in the oviducts, the H2O2 level (P < 0.01) and its ratio to the FRSA (P < 0.05) significantly increased in the heat-stressed group. The Cdc2 activation at the 2-cell stage, as shown by the ratio of the dephosphorylated form to the phosphorylated form, was evident in control embryos but absent in heat-stressed embryos, and the level was similar to that in embryos blocked at the 2-cell stage (positive control). These results indicate that maternal heat stress enhances oxidative stress in the oviducts and that loss of developmental competence in maternally heat-stressed embryos correlates with a defect in Cdc2 activity at the 2-cell stage.

Characteristics of the estrous cycle of the swamp buffalo under temperate conditions

Estrous cycle, duration of estrus and time of ovulation of eight cyclic buffaloes were examined during a period of one year. Animals were kept under loose-housing conditions and fed according to the Japanese Feeding Standards for dairy cattle. All the animals were observed for the occurrence of estrus twice daily by using a vasectomized bull, and ovarian cycles of each animal were monitored by weekly rectal palpation. Duration of estrus and time of ovulation were determined in 32 estrous periods from eight animals. Animals came in estrus throughout the year. The estrous cycles corresponding to single ovarian cycles ranged from 11 to 38 days with a mode interval of 20 days, averaged 21.5+/-4.7 days. Percentage of the cycles within a range of a mean+/-1 SD (17-26 days) was 79.2%, whereas that of cycles shorter or longer than the expected range was 9.4% and 11.4%, respectively. Estrus took place regardless of the time of day and lasted 9 to 27 hr (19.9+/-4.4 hr). Ovulation occurred 6 to 21 hr (13.9+/-3.4 hr) after the end of estrus, with a mode interval of 12 hr. There were no significant seasonal variations in the estrus characteristics studied.

Comparison of the thermoregulatory response of buffaloes and tropical cattle, using fluctuations in rectal temperature, skin temperature and haematocrit as an index

In previous comparative studies of buffaloes and temperate cattle, a greater increase in rectal temperature (RT) and skin temperature (ST), and a greater decrease in haematocrit (Ht) have been observed in buffaloes than in temperate cattle with an increase in ambient temperature (AT). Our series of previous experiments suggested that great changes in RT, ST and Ht are induced in buffaloes by a marked increase in blood flow from the body core to the surface, which accelerates dissipation of heat from the skin surface. On the basis of these suggestions, the present study was undertaken to compare fluctuations in RT, ST and Ht between buffaloes and tropical cattle. Fluctuations in the aforementioned parameters, particularly RT and Ht, were greater in buffaloes than in cattle. Moreover, the correlation for RT or Ht v. AT was significant for buffaloes (r=0.33 and -0.37, respectively) but not for cattle. The correlation coefficient for ST v. AT was significant in both species, but was greater in buffaloes (r=0.63) than in cattle (r=0.56). These results demonstrate that with changes in ambient temperature, RT, ST and Ht fluctuate much more in buffaloes than in tropical cattle, as found previously for temperate cattle. Therefore, the distinctive thermoregulatory responses of buffalo are confirmed as being specific to this species.

Protease activity higher in postmortem water buffalo meat than Brahman beef

We previously demonstrated that postmortem water buffalo meat had higher tenderness than Brahman beef. In order to explain this difference in tenderness, the objective of the current study was to investigate the protease activity in these two meats. Five female crossbred water buffalo (Philippine Carabao×Bulgarian Murrah) and five female crossbred cattle (Brahman×Philippine Native) were slaughtered at 30months of age, followed by immediate sampling of Longissimus thoracis muscle for measurement of protease activity. Results showed that buffalo meat had significantly higher protease activity compared to beef (P<0.05). Furthermore, calpain inhibitor 1, a specific inhibitor of calpains 1 and 2, was the most effective inhibitor of protease activity. There was no difference in calpastatin activity, and no major differences were observed in calpains 1, 2, and calpastatin expression by Western blotting. This study suggests that higher calpain activity in early postmortem buffalo meat was responsible for the increased tenderness of water buffalo meat compared to beef.

Possible roles of myostatin and PGC-1α in the increase of skeletal muscle and transformation of fiber type in cold-exposed chicks: Expression of myostatin and PGC-1α in chicks exposed to cold

This study examined the hypothesis that myostatin and PGC-1alpha are involved in the increase in skeletal muscle mass and transformation of fiber type in cold-exposed chicks. One-week-old chicks were exposed to acute (24h) or long-term (8d) cold at 4 degrees C or kept warm at 30 degrees C. Acute cold exposure induced a significant increase in the skeletal muscle weight and the ratio of slow- to fast-fiber specific troponin I expression (sTnI/fTnI), accompanied by a significant decrease in lactate dehydrogenase activity. Expression of myostatin mRNA in the muscle was significantly lower in cold-exposed chicks than in the controls, whereas PGC-1alpha mRNA expression was significantly enhanced. These changes in the gene expression rapidly returned to the levels of the control chicks after the end of cold exposure, whereas the changes in fiber type and enzymatic activity were not resumed within 24h after removal of cold exposure. On the other hand, long-term exposure to cold resulted in a remarkable increase in skeletal muscle weight, accompanied by a significant increase in the ratio of sTnI/fTnI and the enzymatic activities of cytochrome oxidase and lactate dehydrogenase. However, the expression level of myostatin mRNA in cold-exposed chicks was not different from that in their age-matched control chicks and that of PGC-1alpha mRNA was significantly lower than in the controls. These results indicate that myostatin and PGC-1alpha expression in the skeletal muscle rapidly change in response to acute cold, suggesting the possibility that these two genes could be involved in the increase in muscle mass and transformation of fiber type, respectively, at the initial stage of adaptation in cold-exposed chicks.

Possible roles of myostatin and PGC-1alpha in the increase of skeletal muscle and transformation of fiber type in cold-exposed chicks: expression of myostatin and PGC-1alpha in chicks exposed to cold.

This study examined the hypothesis that myostatin and PGC-1alpha are involved in the increase in skeletal muscle mass and transformation of fiber type in cold-exposed chicks. One-week-old chicks were exposed to acute (24h) or long-term (8d) cold at 4 degrees C or kept warm at 30 degrees C. Acute cold exposure induced a significant increase in the skeletal muscle weight and the ratio of slow- to fast-fiber specific troponin I expression (sTnI/fTnI), accompanied by a significant decrease in lactate dehydrogenase activity. Expression of myostatin mRNA in the muscle was significantly lower in cold-exposed chicks than in the controls, whereas PGC-1alpha mRNA expression was significantly enhanced. These changes in the gene expression rapidly returned to the levels of the control chicks after the end of cold exposure, whereas the changes in fiber type and enzymatic activity were not resumed within 24h after removal of cold exposure. On the other hand, long-term exposure to cold resulted in a remarkable increase in skeletal muscle weight, accompanied by a significant increase in the ratio of sTnI/fTnI and the enzymatic activities of cytochrome oxidase and lactate dehydrogenase. However, the expression level of myostatin mRNA in cold-exposed chicks was not different from that in their age-matched control chicks and that of PGC-1alpha mRNA was significantly lower than in the controls. These results indicate that myostatin and PGC-1alpha expression in the skeletal muscle rapidly change in response to acute cold, suggesting the possibility that these two genes could be involved in the increase in muscle mass and transformation of fiber type, respectively, at the initial stage of adaptation in cold-exposed chicks.