Effect of rumen microbiota transfaunation on the growth, rumen fermentation, and microbial community of early separated Japanese Black cattle.

This study aimed to investigate the effect of rumen microbiota transfaunation on the growth, rumen fermentation, and the microbial community of Japanese Black cattle that were separated early from their dams. Here, 24 calves were separated from their dams immediately after calving, 12 of which were transfaunated via inoculation with rumen fluid from adult cattle at the age of 2 months while the remaining 12 were kept unfaunated (not-inoculated). Feed efficiency monitoring was performed during 7-10 months of age. Body weight and feed intake were not significantly different between the transfaunated and unfaunated cattle. Transfaunation increased the relative levels of acetate and butyrate but decreased those of propionate, which increased the non-glucogenic/glucogenic short-chain fatty acid ratio. Microbial 16S, 18S, and ITS ribosomal RNA gene amplicon analysis showed that rumen microbial diversity and composition differed between transfaunated and unfaunated cattle; transfaunation increased the abundance of acetate- and butyrate-producing bacteria, and decreased the abundance of bacterial genera associated with propionate production. Transfaunation also increased the abundance of Methanomassiliicoccaceae_group10 (1.94% vs. 0.05%) and Neocallimastix (27.1% vs. 6.8%) but decreased that of Methanomicrobium (<0.01% vs. 0.06%). Our findings indicate that rumen microbiota transfaunation shifts rumen fermentation toward acetate and butyrate production through a change in the rumen microbial composition in Japanese Black cattle.

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.

Prepartum maternal plasma glucose concentrations and placental glucose transporter mRNA expression in cows carrying somatic cell clone fetuses.

In this study, the plasma glucose concentrations of cows carrying a somatic cell clone fetus during late pregnancy and placental glucose transporter (GLUT) mRNA levels at parturition were examined. Parturition was induced using dexamethasone, prostaglandin F(2α) and estriol in cows bearing a clone (Clone) or a fetus fertilized in vivo as a control (DEX). Plasma glucose concentrations were measured in the cows (days 257 and 271 of pregnancy and at parturition) and newborn calves. Cotyledon and caruncle tissues removed just after parturition were used for mRNA extraction. Expression of mRNA was also analyzed in control cows that were induced to undergo parturition without dexamethasone (PG) or that spontaneously delivered (SP). The glucose concentrations of the Clone group were significantly low at all points examined, but those of the calves were normal. The increase in the maternal glucose concentration from day 257 to parturition was significantly lower in the Clone group. Glucose concentrations were negatively correlated with birth weight for clones (day 257; r=-0.584, day 271; r=-0.286, parturition; r=-0.549). There was no difference in mRNA levels in the cotyledons among the animals examined. In the caruncles, the Clone and PG groups showed significantly higher GLUT1 and GLUT3 mRNA levels than the SP group, and the GLUT3 mRNA level was significantly higher in the Clone group than in the DEX group. The glucocorticoid receptor α mRNA level was significantly lower in the SP group than in the DEX group. Although spontaneous parturition and administration of dexamethasone suppressed the placental GLUT mRNA levels, the action was not observed in clone pregnancy. These results raise the possibility of facilitation of glucose transportation through the placenta to meet increased nutritional requirements of overgrown clone fetuses.

Aberrant CpG methylation of the imprinting control region KvDMR1 detected in assisted reproductive technology-produced calves and pathogenesis of large offspring syndrome.

Although somatic cell nuclear transfer (NT) and in vitro fertilization (IVF) have the potential to produce genetically superior livestock, considerable numbers of abnormally large animals, including sheep and cattle affected by "large offspring syndrome" (LOS), have been produced by these assisted reproductive technologies (ART). Interestingly, these phenotypes are reminiscent of Beckwith-Wiedemann syndrome (BWS) in humans, which is an imprinting disorder characterized by pre- and/or postnatal overgrowth. The imprinting control region KvDMR1, which regulates the coordinated expression of growth control genes such as Cdkn1c, is known to be aberrantly hypomethylated in BWS. Therefore, we hypothesized that aberrant imprinting in this region could contribute to LOS. In this study, we analyzed the DNA methylation status of the Kcnq1ot1/Cdkn1c and Igf2/H19 domains on bovine chromosome 29 and examined the coordinated expression of imprinted genes surrounding them in seven calves derived by NT (which showed signs of developmental abnormality), two calves conceived by IVF (both developmentally abnormal), and three conventional calves that died of unrelated causes. Abnormal hypomethylation status at an imprinting control region of Kcnq1ot1/Cdkn1c domain was observed in two of seven NT-derived calves and one of two IVF-derived calves in almost all organs. Moreover, increased expression of Kcnq1ot1 and diminished expression of Cdkn1c were observed by RT-PCR analysis. This study is the first to describe the abnormal hypomethylation of the KvDMR1 domain and subsequent changes in the gene expression of Kcnq1ot1 and Cdkn1c in a subset of calves produced by ART. Our findings provide strong evidence for a role of altered imprinting control in the development of LOS in bovines.