Effectiveness of l‐ascorbyl‐2‐monophosphate Na/Ca as a vitamin C source for yellowtail Seriola quinqueradiata juveniles

A feeding experiment was conducted to examine the efficacy of a vitamin C (L‐ascorbic acid, AsA) derivative, l ‐ascorbyl‐2‐monophosphate Na/Ca (AMP‐Na/Ca) by comparing to l ‐ascorbyl‐2‐monophosphate‐Mg (AMP‐Mg) in terms of growth, AsA content in tissues, and hematology in yellowtail Seriola quinqueradiata juvenile (1.10 ± 0.01 g). Furthermore, a stress resistance test for the species fed AMP‐Na/Ca was also conducted. In experiment 1, 11 test pellet diets with different levels of AMP‐Na/Ca (0, 12, 43, 88, 440 and 881 mg AsA kg^?1diet) and AMP‐Mg (0, 16, 52, 106, 595 and 1164 mg AsA kg^?1 diet) were formulated and fed to the yellowtail three times per day for 50 days. In both the vitamin C sources, the survival rates of the fish fed diets without supplemental AsA were lower than 50% at day 20, and more than 50% mortality occurred in fish that fed the diets containing 12 or 16 mg AsA kg^?1 after day 30. However, no significant differences were detected in survival and growth among fish that fed the diets containing more than 43 mg AsA kg^?1. Liver and brain AsA concentrations generally increased with increasing dietary AsA level in both sources. In experiment 2, test diets were formulated to contain 43, 88, 440 and 881 mg AsA kg^?1 using AMP‐Na/Ca, and after 60 days of feeding, yellowtail juveniles were subjected to low salinity and air exposure stress. The fish that received diets with 440 mg AsA kg^?1 showed significantly higher tolerance to low salinity stress and higher survival rate in air exposure stress than those of other groups. The present study demonstrated that yellowtail juveniles could utilize AMP‐Na/Ca as an AsA source like AMP‐Mg, and that supplementation of 43–52 mg AsA kg^?1 diet was optimum for normal growth. However, this study showed that dietary inclusion of 440 mg AsA kg^?1 would be necessary to improve stress resistance of this species.     

Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor

We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca²⁺ mobilization of the cells was evaluated using the Ca²⁺ indicator Fluo3 to monitor Ca²⁺ influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca²⁺ levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.     

Effect of pH on the gelation of walleye pollack surimi and carp actomyosin pastes

ABSTRACT: The effect of pH on thermal gelation and transglutaminase (TGase; EC2.3.2.13)-induced suwari (setting) of surimi and actomyosin pastes was investigated. A strong and elastic gel was produced from walleye pollack surimi paste at pH 7.0 in the presence of Ca²⁺ using a two-step heating method. In contrast, walleye pollack actomyosin paste formed a weak gel under the same conditions as a result of the low concentration of endogenous TGase. In the presence of EGTA [ethyleneglycol bis(2-aminoethylether) tetraacetic acid], weak gels were formed at pH values of 7.0 and 6.0. Non-proteolytic modori (gel weakening) occurred extensively in the course of actomyosin gelation, but not in surimi gelation. Maximum TGase-induced myosin heavy chain cross-linking was observed at a slightly higher pH of 7.5 than at the optimal pH of endogenous TGase activity; the difference being derived from different substrates. Gelation of carp actomyosin paste at pH values of 5.5, 6.0, 6.5 and 7.0 was monitored by measuring storage modulus (G') and loss modulus (G"). A weak gel was formed at all pH values, but a slightly rigid and less elastic gel was obtained at lower pH values. The addition of microbial TGase (MTGase) formed strong elastic gels at pH 7.0 and 6.5. MTGase cross-linked myosin heavy chains even at pH 5.5, but contributed neither to suwari response nor strong gel formation. Overall, results suggest that the optimal pH for the gelation of surimi paste from easy-setting fish species is a compromise between the pH-optima of TGase activity and of preferable actomyosin conformation for myosin cross-linking.     

Effects of dietary arginine and lysine levels on growth performance and biochemical parameters of juvenile Japanese flounder Paralichthys olivaceus

ABSTRACT: To investigate the effects of disproportionate levels of dietary arginine and lysine on juvenile Japanese flounder Paralichthys olivaceus , growth performance and biochemical parameters were evaluated by feeding five test diets, comprising different levels of arginine and lysine, to triplicate groups of juveniles (initial bodyweight 1.85 g) for 40 days. Crystalline amino acids were supplemented to test diets to correspond to the amino acid pattern found in the whole body protein of the Japanese flounder, except for arginine and lysine. After the feeding trials, plasma arginine, lysine and urea levels, excreted ammonia-N, and liver arginase activity were analysed. Survival, specific growth rate, feed conversion efficiency, and apparent protein retention were adversely affected ( P  <   0.05) by the deficiency in dietary arginine or lysine concentrations. An excess of either lysine or arginine in the diet did not depress growth when the diets contained adequate levels of either arginine or lysine, proving that there is no evidence for an arginine–lysine antagonism. Results for plasma arginine, lysine and urea levels, excreted ammonia, and liver arginase activity also demonstrated that Japanese flounder juveniles are not sensitive to excess dietary levels of lysine and arginine.     

Addition of oxytocin to semen extender improves both sperm transport to the oviduct and conception rates in pigs following AI

Oxytocin (OXT) contained in boar semen is known to produce uterine contraction; therefore, we hypothesized that the co‐injection of OXT with sperm would improve artificial insemination (AI) using liquid or frozen‐thawed boar sperm. We initially examined whether OXT added to semen extender improved sperm transport to the oviduct. Although the addition of OXT did not affect the fresh or frozen‐thawed sperm motility or acrosomal integrity, it significantly increased the number of sperm in the oviduct at 6 h after AI injection with OXT, as compared with the control (P < 0.05). Moreover, some sperm were observed in the sperm reservoir of the isthmus in the OXT treatment group, whereas few sperm were observed in the control. When OXT was added to the semen extender immediately prior to AI, the conception rates were significantly higher in both fresh semen and frozen‐thawed semen than in the control group (P < 0.05: liquid, 87.5% vs. 70.5%; frozen‐thawed, 89.8% vs. 75.0%). From these results, we concluded that the addition of OXT to the semen extender assisted in sperm transportation from the uterus to the oviduct, which resulted in improved reproductive performance.     

The addition of calcium ion chelator,EGTA to thawing solution improves fertilizing ability in frozen–thawed boar sperm

In our previous study, seminal plasma effectively suppressed the induction of sperm to capacitation‐like status and acrosome loss during the thawing process. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, it is necessary to develop a thawing solution without animal‐derived materials. In this study, we focused on the increase of intracellular Ca²⁺ ([Ca²⁺]_i) in sperm after thawing and the negative effects of sperm qualities. After thawing, the fluorescent intensity of [Ca²⁺]_i indicator, Fluo‐3/AM, and the level of phosphorylated tyrosine residue of protein were increased in the sperm. Next, we investigated whether the addition of Ca²⁺ chelators (ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA)) improved post‐thawed sperm motility. When the frozen–thawed sperm were treated with 6 mmol/L EDTA + 6 mmol/L EGTA, sperm motility was significantly increased as compared with control (6 mmol/L EDTA alone) at all incubation periods (P < 0.05). The combinational treatment significantly suppressed the elevation of [Ca²⁺]_i and the tyrosine phosphorylation, which improved the acrosomal status and fertilizing ability in vitro. Furthermore, when the thawing method was applied for artificial insemination, the fertilization rate was significantly higher than control (P < 0.05, 33% vs. 82%). Thus, we conclude that the addition of EDTA + EGTA to thawing solution is a beneficial tool for artificial insemination using frozen–thawed boar sperm.     

Enhanced type X collagen expression in the extruded nucleus pulposus of the chondrodystrophoid dog

The causes of early degeneration and calcification of the nucleus pulposus in the chondrodystrophoid dog are poorly understood, and the underlying molecular mechanism of this process has not yet been clearly defined. Type X collagen is one of the key molecules in endochondral bone growth and development, especially matrix calcification. The relationship between type X collagen and disc degeneration and calcification in chondrodystrophoid dogs has not yet been studied. We analyzed the expression of type X collagen in degeneration and calcification of the intervertebral disc in chondrodystrophoid dogs, using type X collagen immunohistochemistry. Control intervertebral discs were collected from five dogs (4 female, 1 male, average age 1.3 years, beagle breed). Degenerated intervertebral discs were surgically removed from 11 canine patients with intervertebral disc extrusion (1 female, 10 male, average age 5.1 years, dachshund breed) in Nippon Veterinary and Animal Science University. All extruded disc samples showed hypertrophic changes and clustering of cells, typical features observed in the degenerated nucleus pulposus. The relative expression of type X collagen in the degenerated nucleus pulposus (84.3 +/- 11.0%) was significantly increased compared to the control nucleus pulposus (5.4 +/- 5.4%). Our findings suggest that type X collagen might contribute to the development of degeneration or calcification in the nucleus pulposus of the chondrodystrophoid dog.     

Interactive effect of two methyl‐group sources on the growth and phospholipids’ content of kuruma shrimp Marsupenaeus japonicus Bate post‐larvae

Six micro‐bound diets were formulated to contain three levels of choline chloride (CC) (0.0, 0.6 and 1.2 g kg⁻¹) and 2 levels of methionine (Met) (0 and 15 g kg⁻¹). Soybean protein isolates (SPI) were used as the main protein source for its limited Met content. A significant (P < 0.05) interaction was determined between CC and Met on the survival (S %), weight gain (WG%), specific growth rate (SGR % day⁻¹), feed efficiency ratio (FER), protein efficiency ratio (PER) and phosphatidylcholine (PC), phoshphatidylethanolamine (PE) and Met contents of the whole body of shrimp. The shrimp group did not receive either supplemental CC or Met showed lower (P < 0.05) values of the above‐mentioned parameters than other shrimp groups fed with 0.6 and 1.2 g kg⁻¹ supplemental CC with or without Met supplementation. The present study showed that supplementation of 1.2 g kg⁻¹ CC in the diets could compensate shrimp post‐larvae with the needed methyl group when received Met‐deficient diets. The study also assumed that the biosynthesis of PC in the shrimp’s body can be achieved by the methylation of PE through the S‐adenosylmethionine (SAM) pathway and/or through the cytosine di‐phosphoryl (CDP) choline pathway directly from dietary choline.     

Coordination of crown structure, leaf plasticity and carbon gain within the crowns of three winter-deciduous mature trees

We examined the vertical profiles of leaf characteristics within the crowns of two late-successional (Fagus crenata Blume and Fagus japonica Maxim.) and one early-successional tree species (Betula grossa Sieb. et Zucc.) in a Japanese forest. We also assessed the contributions of the leaves in each crown layer to whole-crown instantaneous carbon gain at midday. Carbon gain was estimated from the relationship between electron transport and photosynthetic rates. We hypothesized that more irradiance can penetrate into the middle of the crown if the upper crown layers have steep leaf inclination angles. We found that such a crown has a high whole-crown carbon gain, even if leaf traits do not change greatly with decreasing crown height. Leaf area indices (LAIs) of the two Fagus trees (5.26-5.52) were higher than the LAI of the B. grossa tree (4.50) and the leaves of the F. crenata tree were more concentrated in the top crown layers than were leaves of the other trees. Whole-crown carbon gain per unit ground area (micromol m(-2) ground s(-1)) at midday on fine days in summer was 16.3 for F. crenata, 11.0 for F. japonica, and 20.4 for B. grossa. In all study trees, leaf dry mass (LMA) and leaf nitrogen content (N) per unit area decreased with decreasing height in the crown, but leaf N per unit mass increased. Variations (plasticity) between the uppermost and lowermost crown layers in LMA, leaf N, the ratio of chlorophyll to N and the ratio of chlorophyll a to b were smaller for F. japonica and B. grossa than for F. crenata. The light extinction coefficients in the crowns were lower for the F. japonica and B. grossa trees than for the F. crenata tree. The leaf carbon isotope ratio (delta(13)C) was higher for F. japonica and B. grossa than for F. crenata, especially in the mid-crown. These results suggest that, in crowns with low leaf plasticity but steep leaf inclination angles, such as those of F. japonica and B. grossa trees, irradiance can penetrate into the middle of the crowns, thereby enhancing whole-crown carbon gain.