Mapping QTL for fat area ratios and serum leptin concentrations in a Duroc purebred population

The reduction of extra subcutaneous, intermuscular and abdominal fat is important to increase the carcass lean percentage of pigs. Image analyses of fat area ratios were effective for estimation of separated fat in pig carcasses. Serum concentrations of leptin are useful as physiological predictors of fat accumulation in pigs. The objectives of the present study were to perform a quantitative trait locus (QTL) analysis for fat area ratios and serum leptin concentrations in a Duroc purebred population. Pigs (n = 226 to 538) were measured for fat area ratios of carcass cross‐sections at the fifth to sixth thoracic vertebrae, half body length and last thoracic vertebra using an image analysis system, and serum leptin concentration. In total, animals were genotyped for 129 markers and used for QTL analysis. For fat area ratios, four significant and 12 suggestive QTLs were detected on chromosomes 1, 6, 7, 8, 9, 12 and 13. Significant QTLs were detected on the same region of chromosome 6, which was located near a leptin receptor gene. For serum leptin concentrations, two significant and two suggestive QTLs were detected on chromosomes 6, 9, and 16, and the QTLs on chromosome 6 were also in the same region for fat area ratios.     

Purification and characterization of polyphenol oxidase from garland chrysanthemum (Chrysanthemum coronarium L.)

Polyphenol oxidase (PPO) of garland chrysanthemum (Chrysanthemum coronarium L.) was purified approximately 32-fold with a recovery rate of 16% by ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme appeared as a single band on PAGE and SDS-PAGE. The molecular weight of the enzyme was estimated to be about 47000 and 45000 by gel filtration and SDS-PAGE, respectively. The purified enzyme quickly oxidized chlorogenic acid and (-)-epicatechin. The K(m) value (Michaelis constant) of the enzyme was 2.0 mM for chlorogenic acid (pH 4.0, 30 degrees C) and 10.0 mM for (-)-epicatechin (pH 8.0, 40 degrees C). The optimum pH was 4.0 for chlorogenic acid oxidase (ChO) and 8.0 for (-)-epicatechin oxidase (EpO). In the pH range from 5 to 11, their activities were quite stable at 5 degrees C for 22 h. The optimum temperatures of ChO and EpO activities were 30 and 40 degrees C, respectively. Both activities were stable at up to 50 degrees C after heat treatment for 30 min. The purified enzyme was strongly inhibited by l-ascorbic acid and l-cysteine at 1 mM.     

Behaviors of D- and L-lactic acids during the brewing process of sake (Japanese rice wine)

The amounts of D- and L-lactic acids during the brewing process of sake were determined by capillary electrophoresis using 2-hydroxypropyl-beta-cyclodextrin as a chiral selector. Because L-lactic acid, which prevents the growth of nonuseful microorganisms, is a raw material of sake, the ratio of L-lactic acid to total lactic acid is almost 1.0 at the initial stage of sake brewing. During brewing, the ratio decreased gradually and finally reached 0.39. Yeast (Saccharomyces cerevisiae) for sake brewing produced D-lactic acid, but not L-lactic acid in a culture medium. These results suggest that the decrease in the ratio of L-lactic acid to total lactic acid during sake brewing resulted in D-lactic acid production by yeast. The ratios in 18 brands of sake obtained commercially ranged from 0.23 to 0.78. The levels of D-lactic acid in sake (140-274 mg/L) were in a narrower range than those of L-lactic acid (61-461 mg/L). Although the D-lactic acid level in sake did not correspond to total lactic acid level, the L-lactic acid level correlated well with total lactic acid level (R(2) = 0.867). These results suggest that the ratio of L-lactic acid to total lactic acid in sake reflected the amount of L-lactic acid added at the initial stage of sake brewing.     

In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop

The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.     

Arabidopsis thaliana PRL1 is involved in low-calcium tolerance

Calcium (Ca) deficiency symptoms in plants often occur in agriculture; however, little is known about the mechanisms for adaptation to low-Ca conditions. To understand the mechanisms, we screened for Arabidopsis thaliana (L.) Heynh mutants sensitive to low Ca. Here, we describe one of the mutants, lcs1-1, isolated from the screen. The relative shoot growth of the mutant was reduced under the low-Ca conditions compared with the wild-type plants. Genetic mapping and genome resequencing revealed that lcs1-1 has one nonsynonymous mutation in the region of the chromosome responsible for the phenotype. The mutation is in Pleiotropic Regulatory Locus 1 (PRL1). An allelism test between lcs1-1 and a T-DNA inserted allele of prl1 demonstrates that the causal gene of lcs1-1 is PRL1. It has been reported that PRL1 is involved in sugar metabolism; however, the involvement of PRL1 in low-Ca tolerance has not been reported. Our results suggest a new insight connecting sugar metabolism with a mechanism for low-Ca tolerance in plants.     

Evaluation of readily available nonexchangeable potassium in soil by sequential extractions with 0.01 molar hydrochloric acid

Nonexchangeable potassium (K-ne), i.e. 1 M NH₄OAc-nonexchangeable K, often contributes significantly to plant nutrition. However conventional extraction methods often extract much more K-ne than plants even after intensive cropping, suggesting the difficulty in evaluating the amount of readily available soil K-ne. In this study, we used a milder extraction method (0.01 M HCl method) to examine its applicability to evaluate the amount of readily available K-ne in soil. In the first experiment, the concentration of K-ne in twenty surface soils sampled from agricultural fields in Japan and K-bearing minerals was determined by the 0.01 M HCl method, i.e. sequential extraction with 0.01 M HCl over a period of 10 d after removal of exchangeable K, and by conventional methods. The average percentage of the soil K-ne extracted by the 0.01 M HCl method amounted to 0.66% of the total K amount, and was much lower than that by a single extraction with 1 M HNO₃ (2.0%) or with 0.2 M sodium tetraphenylboron for 2 d (22%). In the second experiment, the amount of K-ne removed by chemical extractions was compared with that of K-ne removed by maize plants grown for 29 d in five of the above soils. The amount of the K-ne evaluated by the 0.01 M HCl method gave the highest correlation (p < 0.05) with that of the K-ne utilized by plants among the extraction methods applied. The amount of soil K-ne extracted by the 0.01 M Hel method could therefore become a suitable index of the amount of readily available K-ne in soil. Extraction of K-ne in soils after maize planting further indicated that plants had removed K-ne more intensively than the 0.01 < HCl method probably only from the rhizosphere, although a high correlation was observed between the amount of K-ne removed by the 0.01 M Hel method and that by plants. This implies that the estimation of the amount of K-ne utilized by plants requires not only soil chemical analysis but also the evaluation of the percentage of the soil volume where the plant-induced release of K-ne actually occurs.     

Isolation of lignin–carbohydrate bonds in wood. Model experiments and preliminary application to pine wood

A novel method for analysis of benzylic ether type lignin–carbohydrate bonds has been developed by using model compounds. Four diastereomers of model compound 3-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-3-(methyl -d-glucopyranoside-6-O-yl)-1-propanol (GGMGP), were ozonized in acetic acid/water/methanol 16:3:1 for 1h at 0°C. The product from ozonation of each diastereomer was saponified and the corresponding -etherified tetronic acid (TAMGP) was isolated using ion exchange chromatography. Minor amounts of methyl -d-glucopyranoside (MGP) and small amounts of a gluconic acid etherified with tetronic acid (TAGLCA), tetronic acid, gluconic acid, and glyceric acid were detected in the product mixture of ozonated benzylic ether type model compounds. The results suggest that a benzyl ether bond between lignin and carbohydrate is rather stable during the ozone treatment. Acid treatments with sulfuric acid or trifluoroacetic acid of the derived TAMGP led to cleavage of the glucosidic bond but only a small amount of products (tetronic acid and glucose) resulting from cleavage of the -ether bond were formed. The successful chemical treatments were used for studies of benzylic ether bonds in Japanese red pine. The results suggest the presence of benzylic ether bonds to polysaccharides in the wood.Part of this paper was presented at the 10th International Symposium of Wood and Pulping Chemistry (ISWPC), Yokohama, 1999; the 11th ISWPC, Nice, 2001; and as a rapid comunication in J Wood Sci (2000) 46:263–265     

Effect of VRTN gene polymorphisms on Duroc pig production and carcass traits,and their genetic relationships

The thoracic vertebral number is associated with body length and carcass traits, and represents one of the most important traits in the pig industry. Recent studies have shown that vertnin (VRTN) gene is associated with variations in the vertebral number in commercial European pigs. However, the genetic relationships and effect of this VRTN gene in pig production and carcass traits remain uncertain. Therefore, we investigated the genetic relationships among traits such as vertebral numbers, carcass weight and length‐related traits, and meat production traits, and the effect of VRTN gene polymorphisms on these traits in a Duroc purebred population selected for its meat production traits. Highly positive genetic correlations were obtained between the thoracic vertebral numbers and length‐related traits (0.56 to 0.84), whereas low correlations were obtained with production traits and carcass weight (?0.16 to 0.05). VRTN gene polymorphisms indicated that the number of thoracic vertebrae and length‐related traits were significantly associated with the VRTN genotype, but had no significant effect on production traits and carcass weight. The results indicate that VRTN gene may be used as an effective selection marker to obtain pigs with high thoracic vertebral numbers and length‐related traits, without adversely affecting meat production traits.     

Effects of purple sweet potato anthocyanins on development and intracellular redox status of bovine preimplantation embryos exposed to heat shock

The development of cleavage stage preimplantation embryos is disrupted by exposure to heat shock, such as high temperatures in the summer season. In this study, we investigated whether addition of anthocyanins, which are strong scavengers of reactive oxygen species (ROS), improves development and intracellular redox status of heat-exposed bovine preimplantation embryos by reduction of heat shock-derived oxidative stress. After in vitro fertilization (IVF), embryos were cultured at 38.5 C through Day 8 (Day 0=day of IVF) with 0, 0.1, 1 and 10 microg/ml anthocyanins (non-heat-shocked group). On Day 2, embryos were cultured at 41.5 C for 6 h with 0, 0.1, 1 and 10 microg/ml anthocyanins followed by culture at 38.5 C until Day 8 (HS group). After exposure to heat shock, the intracellular ROS and glutathione (GSH) contents of individual embryos were measured in the non-heat-shocked and HS groups using fluorescent probes. On Day 8, the blastocysts formation rates of the embryos and total cell numbers of blastocysts were evaluated. Embryos exposed to heat shock without anthocyanins showed a significant decrease in blastocyst formation rate and GSH content (P<0.05) and an increase in intracellular ROS (P<0.05) compared with non-heat-shocked embryos. In contrast, addition of 0.1 microg/ml anthocyanins significantly (P<0.05) improved the blastocyst formation rate of the heat-shocked embryos. Addition of any dose of anthocyanins produced a significant decrease in the ROS levels (P<0.05) and tended to increase the GSH levels under heat-shock conditions. However, addition of higher concentrations (1 and 10 microg/ml) of anthocyanins to the culture media under heat shock did not improve the development of embryos. These results indicate that anthocyanins maintain the intracellular redox balance of heat-shocked bovine embryos by reducing intracellular oxidative stress and increasing the GSH levels. Thus, alterations of the redox state using natural antioxidative polyphenols is a useful approach for reducing heat shock-derived oxidative stress.