The effects analysis of two neonicotinoid insecticides on in vitro maturation of porcine oocytes using hanging drop monoculture method

Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure.     

Differences in Muscle Fiber Recruitment Patterns between Continuous and Interval Exercises

We evaluated differences in muscle fiber recruitment patterns between continuous and interval training to develop an optimal training program for Thoroughbred horses. Five well trained female thoroughbred horses (3–4 years old) were used. The horses performed two different exercises on a 10% inclined treadmill: 90%VO₂ max for 4 min (continuous) and 90% VO₂ max for 2 min × 2 times with 10-min interval (interval). Muscle samples were obtained from the middle gluteal muscle before and immediately after the exercises. Four muscle fiber types (type I, IIA, IIA/X, and IIX) were immunohistochemically identified, and the optical density of periodic acid Schiff staining (OD-PAS) in each fiber type and glycogen content of the muscle sample were determined by quantitative histochemical and biochemical procedures, respectively. No significant differences were found in the OD-PASs and glycogen contents between the continuous and interval exercises, but the decreases in OD-PAS of fast-twitch muscle fibers were obvious after interval as compared to continuous exercise. Interval exercise may be a more effective training stimulus for the glycolytic capacity of fast-twitch muscle fiber. The data about muscle fiber recruitment can provide significant insights into the optimal training program not only for thoroughbred horses, but also for human athletes.     

Sarcoplasmic Reticulum Ca2+-ATPase Activity and Glycogen Content in Various Fiber Types after Intensive Exercise in Thoroughbred Horses

To find a new parameter indicating muscle fitness in Thoroughbred horses, we examined time-dependent recovery of glycogen content and sarcoplasmic reticulum (SR) Ca²⁺-ATPase activity of skeletal muscle after intensive treadmill running. Two repeated 50-sec running sessions (13 m/sec) were performed on a flat treadmill (approximately 90%VO₂max). Muscle samples of the middle gluteal muscle were taken before exercise (pre) and 1 min, 20 min, 60 min, and 24 hr after exercise. Muscle fiber type composition was determined in the pre muscle samples by immunohistochemical staining with monoclonal antibody to myosin heavy chain. SR Ca²⁺-ATPase activity of the muscle and glycogen content of each muscle fiber type were determined with biochemical analysis and quantitative histochemical staining, respectively. As compared to the pre value, the glycogen content of each muscle fiber type was reduced by 15–27% at 1 min, 20 min, and 60 min after the exercise and recovered to the pre value at 24 hr after exercise test. These results indicate that 24 hr is enough time to recover glycogen content after short-term intensive exercise. The mean value of the SR Ca²⁺-ATPase activity showed a slight decrease (not significant) immediately after exercise, and complete recovery at 60 min after exercise. There were no significant relationship between the changes in glycogen content of each muscle fiber type and SR Ca²⁺-ATPase. Although further studies are needed, SR Ca²⁺-ATPase is not a useful parameter to detect muscle fitness, at least in Thoroughbred horses.     

Effect of Treadmill Exercise and Hydrogen-rich Water Intake on Serum Oxidative and Anti-oxidative Metabolites in Serum of Thoroughbred Horses

The present study aimed to clarify changes of oxidative stress and antioxidative functions in treadmill-exercised Thoroughbred horses (n=5, 3 to 7 years old), using recently developed techniques for measurement of serum d-ROMs for oxidative stress, and BAP for antioxidative markers. Also, the effect of nasogastric administration of hydrogen-rich water (HW) or placebo water preceding the treadmill exercise on these parameters was examined. Each horse was subjected to a maximum level of treadmill exercise in which the horses were exhausted at an average speed of 13.2 ± 0.84 m/sec. Blood samples were taken 4 times, immediately before the intake of HW or placebo water at 30 min preceding the treadmill exercise, immediately before the exercise (pre-exercise), immediately after the exercise (post-exercise) and at 30 min following the exercise. In all horses, both d-ROMs and BAP values significantly increased at post-exercise. The increase in d-ROMs tended to be lower in the HW trial, as compared to the placebo trial at pre-exercise. The increase in BAP was considerable at approximately 150% of the pre-exercise values in both the HW and placebo treatment trials. The BAP/d-ROMs ratio was significantly elevated at post-exercise in both treatment trials, while a significant elevation was also observed at pre-exercise in the HW trial. BAP, d-ROM, and the BAP/d-ROM ratio tended to decline at 30 min after the exercise, except BAP and BAP/d-ROMs in the placebo trial. These results demonstrate that the marked elevation of oxidative stress and anitioxidative functions occurred simultaneously in the intensively exercised horses, and suggest a possibility that HW has some antioxidative efficacy.     

Selection of In Vitro-Matured Porcine Oocytes Based on Localization Patterns of Lipid Droplets to Evaluate Developmental Competence

Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.     

Annual changes in the hatching period of the dominant cohort of larval and juvenile ayu Plecoglossus altivelis altivelis in the Shimanto Estuary and adjacent coastal waters during 1986–2001

ABSTRACT:   Larval and juvenile ayu from seven year-groups (1986, 1987, 1992, 1995, 1996, 1999 and 2000) were collected in the Shimanto Estuary and adjacent coastal waters. The present study focuses on the variation among year-groups in the hatching period estimated by age determination using the otolith. The hatching period of the dominant cohorts in the 1986, 1987 and 1992 year-groups occurred from late October to mid-November. A delay of the hatching period of the dominant cohort was observed from the 1995 year-group, which occurred in late November to early December, and the dominant periods in the 1996, 1999 and 2000 year-groups were observed from early to late December. It appears that the delay of the hatching period of dominant cohorts was not due to a delay of spawning, but a high mortality of early hatched larvae. The water temperature in autumn in the coastal waters adjacent to the Shimanto River has tended to rise over the past 20 years and this trend was especially notable in the late 1990s. The delay in the hatching period of the dominant cohort observed from the 1995 year-group was likely to be related to the rise in seawater temperatures in autumn.     

Free Radical Formation after Intensive Exercise in Thoroughbred Skeletal Muscles

Although high oxygen consumption in skeletal muscle may result in severe oxidative stress, there are no direct studies that have documented free radical production in horse muscles after intensive exercise. To find a new parameter indicating the muscle adaptation state for the training of Thoroughbred horses, we examined free radical formation in the muscle by using electron paramagnetic resonance (EPR). Ten male Thoroughbred horses received conventional training for 18 weeks. Before and after the training period, all horses performed an exhaustive incremental load exercise on a 6% incline treadmill. Muscle samples of the middle gluteal muscle were taken pre-exercise and 1 min, 1 hr, and 1 day after exercise. Muscle fiber type composition was also determined in the pre-exercise samples by immunohistochemical staining with monoclonal antibody to myosin heavy chain. We measured the free radical in the muscle homogenate using EPR at room temperature, and the amount was expressed as relative EPR signal intensity. There was a significant increase in Type IIA muscle fiber composition and a decrease in Type IIX fiber composition after the training period. Before the training period, the mean value of the relative EPR signal intensity showed a significant increase over the pre-exercise value at 1 min after the exercise and an incomplete recovery at 24 hr after the exercise. While no significant changes were found in the relative EPR signal intensity after the training period. There was a significant relationship between percentages of Type IIA fiber and change rates in EPR signal intensity at 1 min after exercise. The measurement of free radicals may be useful for determining the muscle adaptation state in the training of Thoroughbred horses.