Presence of chlorogenic acid during in vitro maturation protects porcine oocytes from the negative effects of heat stress

Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 μM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.     

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 μs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC‐transchromosomic embryos, when the duration of fusion was prolonged to 60 μs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells.     

Metabolomic profiling reveals differential effects of glucagon‐like peptide‐1 and insulin on nutrient partitioning in ovine liver

This study was conducted to identify the insulin‐independent actions of glucagon‐like peptide‐1 (GLP‐1 (7‐36 amide)) in partitioning nutrient metabolism in ovine liver. Four Suffolk wethers (60.0 ± 6.7 kg body weight (BW)) were used in a repeated‐measure design under euglycemic‐‐hyperinsulinemic and hyper ‐GLP‐1 clamps for 150 min with intravenous infusion of insulin (0.5 mU/kg BW/min; from 0 to 90 min), GLP‐1 (0.5 µg/kg BW/min; from 60 to 150 min) and both hormones co‐administered from 60 to 90 min. Liver biopsies were collected at 0, 60, 90 and 150 min to represent the metabolomic profiling of baseline, insulin, insulin plus GLP‐1, and GLP‐1, respectively, and were analyzed for metabolites using Capillary Electrophoresis Time‐of‐Flight Mass Spectrometer. Metabolomics analysis reveals 51 metabolites as being significantly altered (P < 0.05) by insulin and GLP‐1 infusion compared to baseline values. Insulin infusion enhanced glycolysis, lipogenesis, oxidative stress defense and cell proliferation pathways, but reduced protein breakdown, gluconeogenesis and ketogenesis pathways. Conversely, GLP‐1 infusion promoted lipolytic and ketogenic pathways accompanied by a lowered lipid clearance from the liver as well as elevated oxidative stress defense and nucleotide degradation. Despite further research still being warranted, our data suggest that GLP‐1 may exert insulin‐antagonistic effects on hepatic lipid and nucleotide metabolism in ruminants.     

Analyses of leaves from open field-grown transgenic poplars overexpressing xyloglucanase

The transgenic expression of Aspergillus xyloglucanase cDNA (AaXEG2) with 35S promoter in the leaves of open field-grown poplars was studied. The level of xyloglucan in the transgenic poplars was decreased to 15–16% in the non-fertile soil (forest-field soil) and to 21–22% in the fertile soil (farming-field soil) compared with that of the wild-type poplars. The leaves exhibited a smaller surface area with more rounded teeth than those of the wild-type plants, similar to the sun leaf variety that was grown in the incubation room and subsequently greenhoused. The majority of total veins with water-conducting vascular bundles were shorter in the leaves of the transgenic poplars than those of the wild type. This decrease in vein length may result from a decrease in xyloglucan during leaf development, from which large numbers of proteins were markedly downregulated in the leaves of the transgenic plants via proteomic analysis. It seems likely that the leaves of the transgenic poplars came to relax the edges of their tooth rather than extend their veins as a result of the loosening of the xyloglucan cellulose networks in the leaves.     

Effects of (−)‐Epigallocatechin Gallate on the Motility and Penetrability of Frozen–Thawed Boar Spermatozoa Incubated in the Fertilization Medium

Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis) and is known for its antioxidant effects. The objective of the present study was to examine the effects of EGCG during in vitro fertilization (IVF) on the sperm quality and penetrability into oocytes. In the first experiment, the effects of concentration and incubation period of EGCG on the motility and penetrability of spermatozoa were examined. When frozen–thawed spermatozoa were incubated in IVF medium supplemented with 0 (control), 1, 50 and 100 μm EGCG for 1, 3 and 5 h, supplementation with 50 and 100 μm EGCG improved motility of the spermatozoa (p < 0.05), but not viability, as compared with the control group. When frozen–thawed spermatozoa were co‐incubated with in vitro‐matured (IVM) oocytes in IVF medium supplemented with 50 and 100 μm EGCG for 5 h, supplementation of EGCG had positive effects on sperm penetration rates. In the second experiment, the effects of supplementation of EGCG in IVF medium on penetrability of sperm from different boars and development of fertilized oocytes were evaluated. When frozen–thawed spermatozoa from six boars were co‐incubated with IVM oocytes in IVF medium supplemented with 50 μm EGCG, the effect of EGCG on sperm penetration and development of oocytes after fertilization was found to vary with individual boar. Our results indicate that motility and penetrability of boar spermatozoa are improved by co‐incubation with 50 μm EGCG, but the effects vary with individual boars.     

Plasma concentrations and effects of glucagon-like peptide-1 (7-36) amide in calves before and after weaning

Glucagon-like peptide-1 (7-36) amide (GLP-1), secreted by the small intestine, has insulinotropic and glucose-lowering action. Basal plasma GLP-1 concentrations were measured in calves around the weaning period, the effect of short-chain fatty acids (SCFA) on plasma GLP-1 concentrations was examined, and the effects of GLP-1 administration on plasma insulin, glucagon, and glucose concentrations were measured. Thirteen Holstein bull calves were fed whole milk and solid feed and weaned at 7 wk of age. Preprandial plasma samples were obtained from 5 calves once a week from week 0 to 13 to measure basal concentrations of plasma GLP-1 and insulin (experiment 1). Four calves were intravenously administered with a mixed solution of SCFA (2.4 mmol/kg body weight [BW]) in week 2 and 11 to measure plasma GLP-1 concentrations (experiment 2). Another 4 calves were intravenously injected with GLP-1 (1.0 μg/kg BW) to elucidate the response of plasma insulin, glucagon, and glucose concentrations in week 1, 2, 4, 6, 7, 9, 11, and 13 (experiment 3). In experiment 1, age and weaning did not affect preprandial basal concentrations of plasma GLP-1 throughout the experimental period. Preprandial insulin concentrations increased after weaning (P < 0.05), and GLP-1 and insulin were more strongly correlated postweaning than preweaning. In experiment 2, intravenous treatment with SCFA increased plasma GLP-1 concentrations in both week 2 and 11 (P < 0.05.) In experiment 3, intravenous GLP-1 treatment decreased plasma glucose concentrations throughout the experiment (P < 0.05), but increased plasma insulin concentrations only after weaning (P < 0.05). Treatment with GLP-1 did not affect plasma glucagon concentrations, regardless of age. These results indicate that preprandial basal concentrations of plasma GLP-1 in calves are not changed by weaning, but SCFA stimulate GLP-1 secretion. The insulinotropic action of GLP-1 is detected only after weaning, but the glucose-lowering action of GLP-1 is not affected by weaning.     

Morphological changes in the rat endocrine pancreas within 12 h of intravenous streptozotocin administration

We examined early morphological changes in pancreatic endocrine cells within 12 h of intravenous streptozotocin (STZ) administration (60 mg/kg). Thirty rats were allocated either to a control group (vehicle alone) or to one of four experimental groups tested after 3, 6, 9 and 12 h. Karyopyknosis and cytoplasmic vacuoles were first observed in beta-cell cytoplasm 3 h after STZ administration (STZ-3 h), and the most severe damage was found in beta cells at STZ-12 h. Insulin-positive non-islet cells were observed near the intercalated duct (ICD) and/or centroacinar (CA) cells at STZ-6 h and their numbers peaked at STZ-6 h. The distribution patterns of the insulin-positive cells and those of nestin and insulin-like growth factor-1 were similar and their nuclei were positive for proliferating cell nuclear antigen. Thus, ICD cells and/or CA cells reacted immediately to transform into insulin-secreting cells to replace injured beta cells (or to compensate for the lack of beta cells) within 12 h of STZ administration.     

Neutralizing test of hemagglutinating encephalomyelitis virus (HEV) in FS-L3 cells cultured without serum

FS-L3 cells, originating from porcine kidney, were used for propagation of Hemagglutinating encephalomyelitis virus (HEV) and development of a virus neutralizing (VN) test. Sera of pigs, rats, cows and dogs had VN activities to HEV. On the other hand, sera of mice, rabbits, goats, sheep, horses, cats, chickens, hamsters and human did not have measurable VN activities, although these sera had high HI activities. Our results support the idea that the VN is a more reliable measure of HEV infection than the conventionally used HI test.