DNA Methylation of Genes Participating in Hepatic Metabolisms and Function in Fetal Calf Liver Is Altered by Maternal Undernutrition during Gestation.

This study aimed to elucidate the effects of maternal undernutrition (MUN) on epigenetic modification of hepatic genes in Japanese Black fetal calves during gestation. Using a previously established experimental design feeding the dams with 60% (LN) or 120% (HN) of their global nutritional requirements during the 8.5-month gestational period, DNA methylation in the fetal liver was analyzed with reduced representation bisulfite sequencing (RRBS). The promoters and gene bodies in the LN fetuses were hypomethylated compared to HN fetuses. Pathway analysis showed that the genes with DMR in the exon/intron in the LN group were associated with pathways involved in Cushing syndrome, gastric acid secretion, and aldosterone synthesis and secretion. Promoter hypomethylation in the LN group was frequently observed in genes participating in various signaling pathways (thyroid hormone, Ras/Rap1, PIK3-Akt, cAMP), fatty acid metabolism, and cholesterol metabolism. The promoter hypomethylated genes ALPL and GNAS were upregulated in the LN group, whereas the promoter hypermethylated genes GRB10 and POR were downregulated. The intron/exon hypomethylated genes IGF2, IGF2R, ACAD8, TAT, RARB, PINK1, and SOAT2 were downregulated, whereas the hypermethylated genes IGF2BP2, NOS3, and NR2F1 were upregulated. Collectively, MUN alters the promoter and gene body methylation of genes associated with hepatic metabolisms (energy, cholesterol, mitochondria) and function, suggesting an impact of altered gene methylation on the dysregulation of gene expression in the fetal liver.

Expression and differential posttranscriptional regulation of the elongation factor 1 alpha 1 gene in endometrial caruncle and intercaruncle of Japanese Black cattle at early and mid-gestation stages.

The elongation factor 1 alpha 1 (EEF1A1), an isoform of EEF1A, is one of the most abundant cytoplasmic proteins and an important component of the translational machinery. We investigated the relative expression, alternative polyadenylation (APA), and changes in poly(A) tail length of EEF1A1 mRNA in the endometrial caruncle (CAR) and intercaruncle (ICAR) at early and mid-gestation in Japanese Black cattle. The relative EEF1A1 mRNA expression levels in the CAR were the highest on Gestation day 20 and were significantly decreased at mid-gestation. The expression levels in the ICAR were significantly higher than those in the CAR, and the gestation stage had no significant impact. Four different EEF1A1 transcripts with distinct 3' untranslated regions (UTRs) (proximal and distal types) and poly(A) tails (medium and short types) of different lengths were identified. The EEF1A1 mRNAs with distal 3' UTR and medium-length poly(A) tails were specific from the CAR of uterus horn at early gestation. RNA-sequencing data analyses revealed that the HSF1, MZF1, E47, SRF, GATA2, GATA3, GATA6, HNF-3 beta (FOXA2), CPSF1, and Ataxin-2 genes might affect the EEF1A1 gene expression or poly(A) length.

Maternal Nutrient Restriction Disrupts Gene Expression and Metabolites Associated with Urea Cycle, Steroid Synthesis, Glucose Homeostasis, and Glucuronidation in Fetal Calf Liver.

This study aimed to understand the mechanisms underlying the effects of maternal undernutrition (MUN) on liver growth and metabolism in Japanese Black fetal calves (8.5 months in utero) using an approach that integrates metabolomics and transcriptomics. Dams were fed 60% (low-nutrition; LN) or 120% (high-nutrition; HN) of their overall nutritional requirements during gestation. We found that MUN markedly decreased the body and liver weights of the fetuses; metabolomic analysis revealed that aspartate, glycerol, alanine, gluconate 6-phosphate, and ophthalmate levels were decreased, whereas UDP-glucose, UDP-glucuronate, octanoate, and 2-hydroxybutyrate levels were decreased in the LN fetal liver (p ≤ 0.05). According to metabolite set enrichment analysis, the highly different metabolites were associated with metabolisms including the arginine and proline metabolism, nucleotide and sugar metabolism, propanoate metabolism, glutamate metabolism, porphyrin metabolism, and urea cycle. Transcriptomic and qPCR analyses revealed that MUN upregulated QRFPR and downregulated genes associated with the glucose homeostasis (G6PC, PCK1, DPP4), ketogenesis (HMGCS2), glucuronidation (UGT1A1, UGT1A6, UGT2A1), lipid metabolism (ANGPTL4, APOA5, FADS2), cholesterol and steroid homeostasis (FDPS, HSD11B1, HSD17B6), and urea cycle (CPS1, ASS1, ASL, ARG2). These metabolic pathways were extracted as relevant terms in subsequent gene ontology/pathway analyses. Collectively, these results indicate that the citrate cycle was maintained at the expense of activities of the energy metabolism, glucuronidation, steroid hormone homeostasis, and urea cycle in the liver of MUN fetuses.

Maternal Undernutrition during Pregnancy Alters Amino Acid Metabolism and Gene Expression Associated with Energy Metabolism and Angiogenesis in Fetal Calf Muscle.

To elucidate the mechanisms underlying maternal undernutrition (MUN)-induced fetal skeletal muscle growth impairment in cattle, the longissimus thoracis muscle of Japanese Black fetal calves at 8.5 months in utero was analyzed by an integrative approach with metabolomics and transcriptomics. The pregnant cows were fed on 60% (low-nutrition, LN) or 120% (high-nutrition, HN) of their overall nutritional requirement during gestation. MUN markedly decreased the bodyweight and muscle weight of the fetus. The levels of amino acids (AAs) and arginine-related metabolites including glutamine, gamma-aminobutyric acid (GABA), and putrescine were higher in the LN group than those in the HN group. Metabolite set enrichment analysis revealed that the highly different metabolites were associated with the metabolic pathways of pyrimidine, glutathione, and AAs such as arginine and glutamate, suggesting that MUN resulted in AA accumulation rather than protein accumulation. The mRNA expression levels of energy metabolism-associated genes, such as PRKAA1, ANGPTL4, APLNR, CPT1B, NOS2, NOS3, UCP2, and glycolytic genes were lower in the LN group than in the HN group. The gene ontology/pathway analysis revealed that the downregulated genes in the LN group were associated with glucose metabolism, angiogenesis, HIF-1 signaling, PI3K-Akt signaling, pentose phosphate, and insulin signaling pathways. Thus, MUN altered the levels of AAs and expression of genes associated with energy expenditure, glucose homeostasis, and angiogenesis in the fetal muscle.

Effects of low and high levels of maternal nutrition consumed for the entirety of gestation on the development of muscle, adipose tissue, bone, and the organs of Wagyu cattle fetuses.

This study aimed to investigate the effects of high and low levels of energy intake during the entire gestation period on the skeletal muscle development, organ development, and adipose tissue accumulation in fetuses of Wagyu (Japanese Black) cows, a breed with highly marbled beef. Cows were allocated to a high-nutrition (n = 6) group (fed 120% of the nutritional requirement) or low-nutrition (n = 6) group (fed 60% of the nutritional requirement). The cows were artificially inseminated with semen from the same sire, and the fetuses were removed by cesarean section at 260 ± 8.3 days of fetal age and slaughtered. The whole-body, total muscle, adipose, and bone masses of the fetal half-carcasses were significantly higher in the high-nutrition group than the low-nutrition group (p = 0.0018, 0.009, 0.0004, and 0.0362, respectively). Fifteen of 20 individual muscles, five of six fat depots, nine of 17 organs, and seven of 12 bones that were investigated had significantly higher masses in the high-nutrition group than the low-nutrition group. The crude components and amino acid composition of the longissimus muscle significantly differed between the low- and high-nutrition groups. These data indicate that maternal nutrition during gestation has a marked effect on the muscle, bone, and adipose tissue development of Wagyu cattle fetuses.

Maternal Nutrition During Gestation Alters Histochemical Properties, and mRNA and microRNA Expression in Adipose Tissue of Wagyu Fetuses.

We hypothesized that maternal low or high nutrition would give unique effects to morphological and molecular dynamics in adipose tissue of fetus of fatty breed Wagyu (Japanese Black) cattle which produce highly marbled beef. This study aimed to determine the effects of maternal energy intake in Wagyu cows, during gestation on fetal adipose tissue development, histochemical properties, and gene and microRNA (miRNA) expression. Cows were allocated to one of two nutritional energy groups: 120% (HIGH) or 60% nutritional requirements of (LOW). Fetuses (n = 6 per treatment) were removed from pregnant cows by cesarean section at fetal age 260 ± 8 days and euthanized. Subcutaneous adipose tissue (SAT), thoracic cavity visceral adipose tissue (TVAT), and perirenal adipose tissue (PAT) were collected for analysis. In histochemical analysis, in SAT and PAT, HIGH fetuses had greater diameter of adipocytes than LOW fetuses (P<0.05). Only in SAT, LOW fetuses had more Leptin (LEP) mRNA and tended to have more Peroxisome Proliferator-Activated Receptor gamma (PPARG) CCAAT-enhancer-binding proteins alpha (CEBPA) and Glucose transporter (GLUT) 4 mRNA(P<0.10). In all SAT, TVAT, and PAT, LOW fetuses had higher levels of the brown adipose tissue (BAT) biomarkers Uncoupling Protein (UCP) 1 and PPARG coactivator (PGC) 1α mRNA than HIGH fetuses (P<0.08). Meanwhile, in the other adipose tissue, LOW fetuses had lower PPARG, CEBPA, and Zinc Finger Protein (ZFP) 423 (in TVAT and PAT), FASN (in TVAT), LEP and GLUT4 mRNA (in PAT; P<0.10). In particular, in TVAT and PAT, LOW fetuses exhibited lower expression of WAT biomarkers (PPARG and ZFP423). Differential expression of various miRNAs related to adipogenesis between the LOW and HIGH fetuses was detected in an adipose tissue-specific manner (P<0.10). Based on adipose tissue-specific effects of maternal nutrition, these findings suggested that poor maternal nutrition in Wagyu cattle increased BAT development in SAT, TVAT and PAT, while elevated maternal nutrition stimulated fetal SAT development compared with that of TVAT and PAT.

Effect of restricted suckling on the superovulatory response and reproductive performance in postpartum Japanese black cows.

The reproductive performance of postpartum cows is affected by factors such as suckling and nutrition. We investigated the effect of a restricted suckling period on the superovulatory response and the fertility after flushing in postpartum Japanese Black cows. Forty-seven postpartum cows were used in this study. At 7 days postpartum, the cows were divided into 2 groups: (1) continuous access to calves from birth to weaning at 3 months postpartum (ad libitum suckling group; n=20); and (2) twice daily suckling to the calves penned adjacent to them (restricted suckling group; n=27). All cows were initiated a superstimulatory treatment with a controlled internal drug releasing device and follicle stimulating hormone at 40 days postpartum. Embryos were nonsurgically collected at 7 or 8 days after estrus. After uterine flushing, the cows were again used for reproduction. There were no significant differences between the ad libitum and restricted suckling groups in terms of the numbers of transferable (6.7 ± 5.4 versus 7.9 ± 7.0) and freezable embryos (5.5 ± 4.9 versus 6.2 ± 7.0). In contrast, the interval to the first estrus after flushing in the restricted suckling group was lower (P<0.05) than that in the ad libitum suckling group (8.9 ± 5.7 days versus 27.9 ± 24.2 days). These results suggest that restricted suckling in postpartum Japanese Black cows does not affect the superovulatory response and embryo quality; however, it improves their fertility after flushing.

Concentration of macrophage colony-stimulating factor (M-CSF) in bovine peripheral blood during pregnancy.

Macrophage colony-stimulating factor (M-CSF) is a hemopoietic cytokine with a primary role in placental physiology. Gene expression of M-CSF in the bovine endometrium shows a temporal upward trend during early and mid pregnancy. This study determined the plasma M-CSF levels during pregnancy using ELISA. In experiment 1, to investigate the relationship between the concentration of M-CSF in peripheral blood and pregnancy, the plasma M-CSF levels were determined in 125 pregnant and 21 non-pregnant Japanese Black cows. The pregnant animals were divided into nine groups based on the month of pregnancy. An ELISA for bovine M-CSF established previously was used according to the authors' instructions. In experiment 2, the plasma M-CSF level was determined to investigate the temporal changes in its concentration in the peripheral blood during pregnancy. In experiment 1, the plasma M-CSF level varied from month to month during pregnancy; the mean level in the first-month of pregnancy was significantly higher than those in the third and last months of pregnancy and non-pregnancy (P<0.05). In experiment 2, the plasma M-CSF level varied with the day of pregnancy (P<0.05). The mean level of plasma M-CSF decreased gradually until 6 weeks of pregnancy; it appeared to increase during weeks 7-9, then varied with several small peaks until 27 weeks of pregnancy and finally decreased gradually until parturition. These results suggest that the plasma M-CSF level may be related to changes in the uterus and placenta as pregnancy progresses.

Effect of total cholesterol, glucose and blood urea nitrogen on embryo quality in post-partum superovulated suckling Japanese Black cattle.

Aim: This study was conducted to examine the effect of blood metabolites on embryo quality in post-partum suckling Japanese Black cattle. Methods: Blood samples were taken from 23 cows 30 days before, at and 30 days after parturition. Cows were synchronized 40 or 41 days after calving (day 0) and divided into three groups: control (n = 6), gonadotropin-releasing hormone ([GnRH]n = 10) and estradiol benzoate ([EB]n = 7). All groups received a controlled internal drug release (CIDR) device intravaginally together with 2 mg EB i.m. on day 0 and superovulation was induced in all groups from days 5-7 with a gradually decreasing dose of follicle-stimulating hormone (FSH). Two milligrams of EB was given on day 8 and GnRH (0.1 mg) was given on day 9 of insertion of the CIDR in the EB and GnRH groups. Cows were inseminated twice after the onset of estrus and embryos were recovered 7-8 days after artificial insemination. Results: The number of corpus luteum detected by ultrasonography in the EB group was significantly higher (P < 0.05) than that in the GnRH group. The number and rate of transferable and freezable embryos did not differ significantly among the groups. Regardless of the treatments, the total cholesterol level from parturition until 30 days after parturition was significantly higher (P < 0.01) in the good category than in the poor category of cows. Conclusions: The number of transferable embryos produced by post-partum superovulated suckling Japanese Black cattle was affected by the level of total cholesterol from parturition until 30 days after parturition. Moreover, administration of EB in CIDR-treated cows increased the numbers of corpus luteum and yielded better rates of transferable and freezable embryos. (Reprod Med Biol 2008; 7: 55-62).

The development of a sensitive enzyme immunoassay for the determination of estrone and estradiol-17beta in bovine blood plasma based on the same homologous combination with antiserum and steroid-enzyme conjugate.

The development of a sensitive enzymeimmunoassay (EIA) for the determination of estrone (E1) and estradiol-17beta (E2 beta) in bovine plasma is described. The assay is a homologous double-antibody EIA with E2beta 17hemisuccinate (HS) as hapten for the immunoreactive reagent. The antiserum was raised against E2beta 17HS bovine serum albumin conjugate in the rabbit, and E2beta 17HS-horseradish peroxidase was used as steroid-enzyme conjugate. Each estrogen EIA was distinguished only by using the each working standard and sample for the EIA. Bovine plasma E1 and E2beta were extracted and purified before EIA. The antiserum was used at 1:1,750,000 dilutions for EIA. Estrone and E2beta showed high cross-reactivity with the antiserum (E1: 350.7%, E2beta:100%). The sensitivities were <0.03 pg/well for E1 and <0.12 pg/well for E2beta. Recovery rates of E1 and E2beta added to bovine blood plasma were 94.5% and 93.9%, respectively. The precision for EIA of estrogens was below 9.7%. The profiles of either estrogen as determined by EIA corresponded closely well with follicle dynamics in the cow during the estrous cycles and with placental function in pregnant animals. In conclusion, our new EIA can be applied with sufficient sensitivities, recovery and precision for the routine analysis of E1 and E2beta concentrations in bovine plasma.

The bovine Mx1 gene: characterization of the gene structure, alternative splicing, and promoter region.

The Mx gene encodes an antiviral protein and is induced by type 1 interferons (IFNs). In this study, a new bovine Mx gene (designated Mx1B) was isolated from the endometrial cDNA library of the early pregnant cow. Although the Mx1B cDNA contained a single open reading frame (ORF) the same as the known Mx1, the 5' untranslated region (UTR) and 5' coding region of Mx1B were rather different from the corresponding regions of Mx1. Genomic structure analysis revealed that bovine Mx1B was an alternative splicing variant of Mx1 and had transcription regulatory sequences in the upstream region. RT-PCR and its sequencing identified another Mx1 splicing variant and demonstrated that these bovine Mx1 splicing variants were ubiquitously expressed in various tissues. Furthermore, it was found that all the bovine breeds investigated had identical splice sites of Mx1 and Mx1B. It is speculated that cattle have at least two functional Mx isoforms that might provide strong natural resistance to specific viruses.