Apoptosis and developmental capacity of vitrified parthenogenetic pig blastocysts.

This study was conducted to evaluate whether the poor developmental capacity of pig embryos after vitrification was related to the occurrence of apoptosis. Parthenogenetic blastocysts were used as the research material. The blastocoel recovery rate, mitochondrial membrane potential (ΔΨm), amount of early apoptosis, activities of several caspases, and relative abundance of mRNA of apoptosis-related genes involved in mitochondria and death receptor apoptotic pathways were detected before or after vitrification. The results indicate that the blastocoel recovery rate (31.0%) and total cells (31.8) of vitrified blastocysts were less than those of fresh blastocysts (100% and 38.2, P < 0.05). The ΔΨm of vitrified blastocysts was 0.46, which was less than that of fresh blastocysts (1.02, P < 0.05). The rate of apoptotic cells in vitrified blastocysts (8.1%) after TUNEL (TdT-mediated dUTP Nick-End Labeling) assay was markedly greater than that in fresh blastocysts (3.9%, P < 0.05). The pan-caspase, caspase-3, caspase-8 and caspase-9 activities of vitrified blastocysts (20.7, 20.6, 17.6 and 19.9) were markedly greater than those of fresh blastocysts (7.4, 6.5, 5.5 and 6.3, P < 0.05). The real-time PCR results indicated that relative abundance of caspase-8 and TNF-α mRNA from death receptor apoptotic pathway and caspase-9 for the mitochondrial apoptotic pathway genes in the vitrified group were greater than those in the fresh group P < 0.05). The relative abundance of Bcl-2 and SOD-1 mRNA for the mitochondrial pathway genes in the vitrified group was less than those in the fresh group (P < 0.05). In conclusion, the poor developmental capacity of vitrified parthenogenetic pig blastocysts was closely related with apoptosis. Both mitochondria and death receptor-mediated apoptotic pathways participated the occurrence of this apoptosis.

Effects of nutrient restriction and arginine treatment on oxidative stress in the ovarian tissue of ewes during the luteal phase.

The aim of this study was to determine whether nutrient restriction and arginine treatment affect energy metabolism changes and oxidative stress through the mitochondrial pathway in the ovarian tissue of ewes during the luteal phase. On days 6-15 of the estrous cycle, 24 multiparous Hu sheep (BW = 43.56 ±â€¯1.53 kg) were randomly assigned to three groups: control group (CG; n = 6), restriction group (RG; n = 9), and l-arginine group (AG; n = 9) administered Arg treatment (or vehicle) three times per day. The ewes were slaughtered at the end of treatment, and blood samples and ovaries were collected for analysis. In this study, the expression levels of antioxidase enzymes (SOD2, CAT and GPX1) and mitochondrial biogenesis-related genes (ESRRA and TFAM), as well as antioxidase activity and mitochondrial function were examined in ovarian tissue. Nutrient restriction resulted in activation of ESRRA and TFAM and an increase in relative mtDNA copy number, whereas arginine treatment led to a pronounced recovery of ovarian tissue. In addition, we observed increased AMPK phosphorylation at Thr172 and SIRT3 levels in nutrient restricted ewes, and these effects decreased with arginine treatment. In conclusion, the present results indicated that short-term nutritional restriction led to changes in energy metabolism and oxidative stress. These changes disrupted the redox balance, thus leading to apoptosis through the mitochondria-dependent apoptosis pathway. Arginine treatment altered gene expression in ovarian tissue and increased the resistance to oxidative stress and the anti-apoptosis capacity. The results presented here suggest a potential method to increase agricultural productivity and economic benefits in the sheep industry by using dietary supplementation with arginine to decrease temporary undernutrition of ewes.

Non-invasive assessment of culture media from goat cloned embryos associated with subjective morphology by gas chromatography - mass spectroscopy-based metabolomic analysis.

Pre-implantation embryo metabolism demonstrates distinctive characteristics associated with the development potential of embryos. We aim to determine if metabolic differences correlate with embryo morphology. In this study, gas chromatography - mass spectroscopy (GC-MS)-based metabolomics was used to assess the culture media of goat cloned embryos collected from high-quality (HQ) and low-quality (LQ) groups based on morphology. Expression levels of amino acid transport genes were further examined by quantitative real-time PCR. Results showed that the HQ group presented higher percentages of blastocysts compared with the LQ counterparts (P < 0.05). Metabolic differences were also present between HQ and LQ groups. The culture media of the HQ group showed lower levels of valin, lysine, glutamine, mannose and acetol, and higher levels of glucose, phytosphingosine and phosphate than those of the LQ group. Additionally, expression levels of amino acid transport genes SLC1A5 and SLC3A2 were significantly lower in the HQ group than the LQ group (P < 0.05, respectively). To our knowledge, this is the first report which uses GC-MS to detect metabolic differences in goat cloned embryo culture media. The biochemical profiles may help to select the most in vitro viable embryos.

Effects of diet and arginine treatment during the luteal phase on ovarian NO/PGC-1α signaling in ewes.

The aim of this study was to determine whether arginine (Arg) supplementation of malnourished ewes affects the expression of key NO/PGC-1α signaling pathway genes in the ovary. On Day 6-15 of the estrous cycle, 24 multiparous Hu sheep (BW = 43.56 ± 1.53 kg) were randomly assigned to three groups: control group (CG; n = 6), restriction group (RG; n = 9) and l-arginine group (AG; n = 9), and administered Arg treatment (or vehicle) three times per day. The ewes were slaughtered at the end of treatment, and blood samples and ovaries were collected for analysis. The results of our analyses showed that both short-term feed-restriction and/or supplementation with L-Arg-HCl affected the number of different size follicles observed in the ovary, and the relative day of estrus behavior initiation of ewes. Specifically, the relative day of estrus behavior initiation was significantly advanced in AG compared with that in RG ewes (P < 0.05). Both the number of ≤2 mm-ovarian follicles (P < 0.05) and the total number of ovarian follicles (P < 0.05) were significantly increased in the RG and AG compared with that in the CG ewes. RG ewes exhibited a higher proportion of ≤2 mm (P < 0.05), but a lower proportion of >5 mm follicles than did CG ewes (P < 0.05). The mean number of corpus lutea ≥5 mm was significantly increased in AG as compared to that in either CG or RG ewes. Furthermore, the expression of eNOS, nNOS, iNOS, PDE5A, PDE9A, PRKG2, and PPARGC1A varied significantly among the treatment groups (P < 0.05). GUCY1A3 mRNA levels were significantly increased in RG and AG as compared to those in CG ewes (P < 0.05), whereas conversely, GUCY1B3 mRNA levels were significantly decreased in CG and RG as compared to those in AG ewes (P < 0.05). P53 mRNA levels were found to vary significantly among the three experimental treatment groups (P < 0.05), and similarly, the relative expression levels of P53 were greater in AG and RG than in CG ewes (P < 0.05). The levels of eNOS protein were significantly higher in RG than in either CG or AG ewes (P < 0.05). The relative expression levels of PGC-1α were significantly higher in RG (P < 0.05) and significantly lower in AG ewes (P < 0.05) than in CG ewes. In conclusion, the results of the present study indicate that feed-restriction negatively affects follicular development, and that Arg-supplementation may modulate the expression of key NO/PGC-1α signaling pathway genes in the ovary and thereby accelerate ovulatory processes and the estrous rate. Elucidation of mechanisms underlying these effects of Arg on gene expression in the ewe ovary requires further investigation.

(1)H-Nuclear Magnetic Resonance-Based Plasma Metabolic Profiling of Dairy Cows with Fatty Liver.

Fatty liver is a common metabolic disorder of dairy cows during the transition period. Historically, the diagnosis of fatty liver has involved liver biopsy, biochemical or histological examination of liver specimens, and ultrasonographic imaging of the liver. However, more convenient and noninvasive methods would be beneficial for the diagnosis of fatty liver in dairy cows. The plasma metabolic profiles of dairy cows with fatty liver and normal (control) cows were investigated to identify new biomarkers using (1)H nuclear magnetic resonance. Compared with the control group, the primary differences in the fatty liver group included increases in ß-hydroxybutyric acid, acetone, glycine, valine, trimethylamine-N-oxide, citrulline, and isobutyrate, and decreases in alanine, asparagine, glucose, γ-aminobutyric acid glycerol, and creatinine. This analysis revealed a global profile of endogenous metabolites, which may present potential biomarkers for the diagnosis of fatty liver in dairy cows.

Prenatal smoking exposure and dopaminergic genotypes interact to cause a severe ADHD subtype.

BACKGROUND: In utero exposure to smoking and alcohol are common risk factors that have been associated with attention-deficit/hyperactivity disorder (ADHD) in human beings and animal models. Furthermore, molecular studies have focused on the association between ADHD and DNA polymorphisms in dopamine pathway-related genes. We examined the joint effects of genetic and prenatal substance exposures on DSM-IV and population-defined subtypes of ADHD. METHODS: Logistic regression was used to assess the relationship between ADHD subtypes, DAT1 and DRD4 polymorphisms, and prenatal substance exposures in a birth-record sample of male and female twin pairs, aged 7-19 years. RESULTS: Interactions between prenatal exposure to smoking and variations in the DAT1 and DRD4 loci were observed in children with either the DSM-IV or population-defined ADHD combined subtypes. The odds of a diagnosis of DSM-IV combined subtype was 2.9 times greater in twins who had inherited the DAT1 440 allele and who were exposed, than in unexposed twins without the risk allele. The OR was 2.6 in the population-defined subtype. Odds ratios for the DRD4 seven-repeat allele were 3.0 (2.8) in the population-defined (DSM-IV) combined ADHD subtypes. The OR for exposed children with both alleles was 9.0 (95% confidence interval=2.0-41.5) for the population-defined combined subtypes. CONCLUSIONS: Results indicate that smoking during pregnancy is associated with specific subtypes of ADHD in genetically susceptible children.

Estimation of prevalence of DSM-IV and latent class-defined ADHD subtypes in a population-based sample of child and adolescent twins.

The goal of this study is to determine the prevalence and age of onset of Diagnostic and Statistical Manual of Mental Disorders and latent class-derived attention deficit/hyperactivity disorder (ADHD) subtypes in a population-based twin sample of boys and girls. Missouri birth records identified families with a twin pair 7 to 18 years of age. Telephone screening interviews for ADHD symptoms were completed for 5007 families. Diagnostic assessments were administered to 564 families with at least one twin meeting screening criteria, plus 183 control families. Prevalence and age of onset for both ADHD nosologies were calculated by sex and zygosity from parent report data. The prevalence of any DSM-IV ADHD was 6.2% overall, 7.4% in boys and 3.9% in girls. The inattentive subtype was most common in boys; the combined subtype was most common in girls. The mean age of onset of symptoms in children with any DSM-IV ADHD was 3.5 years, with no significant differences between boys and girls. Prevalences of latent class defined ADHD subtypes also varied by sex with the severe inattentive and combined classes more common in boys than girls. The age of onset of symptoms did not differ between boys and girls but were higher than in the DSM-IV subtypes. Findings in this twin sample showed that clinically significant ADHD, defined by either DSM-IV or latent class criterion, has an early age of onset and is more common in boys than girls. As clinical samples are most commonly composed of male combined subtypes, the inattentive subtype of both sexes in the general population is an under-treated segment of the general population.