AANAT transgenic sheep generated via OPS vitrified-microinjected pronuclear embryos and reproduction efficiency of the transgenic offspring

Background The open pulled straw (OPS) vitrification method has been successfully applied in mouse, pig, and goat embryos as well as in buffalo oocytes, but it has not yet been applied to the microinjected embryos. This study examined the effects of OPS vitrification on embryo development and the reproductive capacity of the transgenic offspring in order to establish a method for preservation of microinjected embryos. Methods Ovine pronuclear embryos were microinjected with the exogenous aralkylamine N-acetyltransferase gene (AANAT), frozen by the OPS method, and subsequently thawed for embryo transplantation. Pregnancy rate, lambing rate, survival rate, average birth weight and transgenic positive rate as well as reproduction efficiency and hormone level of the transgenic offspring were investigated to analyze the effect of OPS vitrification on microinjectd pronuclear embryos. Results No significant differences were observed in the birth rate, lamb survival rate and transgenic positive rate between the frozen and non-frozen AANAT-microinjected pronuclear embryos. The average birth weight of the frozen embryos offspring was greater than that of the non-frozen embryos. Importantly, the transgenic offspring that overexpressed the AANAT gene showed improved ovulation efficiency and lambing rate by regulating their hormone levels. Conclusions The OPS vitrification approach may be a valuable method in microinjected- embryo transfer technology, which could reserve embryos and result in fewer unnecessary animal sacrifices. In addition, the AANAT+ transgenic offspring exhibited improved reproductive capacity on account of regulation effect of melatonin on reproductive hormone. These data may provide available references for human-assisted reproduction

Effect of storage time on the silage quality and microbial community of mixed maize and faba bean in the Qinghai-Tibet Plateau

Tibetan Plateau is facing serious shortage of forage in winter and spring season due to its special geographical location. Utilization of forages is useful to alleviate the forage shortage in winter and spring season. Consequently, the current study was aimed to evaluate the influence of storage time on the silage quality and microbial community of the maize (Zea mays L.) and faba bean (Vicia faba L.) mixed silage at Qinghai-Tibet Plateau. Maize and faba bean were ensiled with a fresh weight ratio of 7:3, followed by 30, 60, 90, and 120 days of ensiling. The results showed the pH value of mixed silage was below 4.2 at all fermentation days. The LA (lactic acid) content slightly fluctuated with the extension of fermentation time, with 33.76 g/kg DM at 90 days of ensiling. The AA (acetic acid) and NH3-N/TN (ammonium nitrogen/total nitrogen) contents increased with the extension of fermentation time and no significantly different between 90 and 120 days. The CP (crude protein) and WSC (water soluble carbohydrate) contents of mixed silage decreased significantly (P < 0.05) with ensiling time, but the WSC content remained stable at 90 days. The Proteobacteria was the predominant phyla in fresh maize and faba bean, and Pseudomonas and Sphingomonas were the predominant genera. After ensiling, Lactobacillus was the prevalent genus at all ensiling days. The relative abundance of Lactococcus increased rapidly at 90 days of ensiling until 120 days of fermentation. Overall, the storage time significant influenced the silage fermentation quality, nutrient content, and microbial environment, and it remained stable for 90 days of ensiling at Qinghai-Tibet Plateau. Therefore, the recommended storage time of forage is 90 days in Qinghai-Tibet Plateau and other cool areas

Branched Chain Amino Acids Cause Liver Injury in Obese/Diabetic Mice by Promoting Adipocyte Lipolysis and Inhibiting Hepatic Autophagy

The Western meat-rich diet is both high in protein and fat. Although the hazardous effect of a high fat diet (HFD) upon liver structure and function is well recognized, whether the co-presence of high protein intake contributes to, or protects against, HF-induced hepatic injury remains unclear. Increased intake of branched chain amino acids (BCAA, essential amino acids compromising 20% of total protein intake) reduces body weight. However, elevated circulating BCAA is associated with non-alcoholic fatty liver disease and injury. The mechanisms responsible for this quandary remain unknown; the role of BCAA in HF-induced liver injury is unclear. Utilizing HFD or HFD + BCAA models, we demonstrated BCAA supplementation attenuated HFD-induced weight gain, decreased fat mass, activated mammalian target of rapamycin (mTOR), inhibited hepatic lipogenic enzymes, and reduced hepatic triglyceride content. However, BCAA caused significant hepatic damage in HFD mice, evidenced by exacerbated hepatic oxidative stress, increased hepatic apoptosis, and elevated circulation hepatic enzymes. Compared to solely HFD-fed animals, plasma levels of free fatty acids (FFA) in the HFD + BCAA group are significantly further increased, due largely to AMPKα2-mediated adipocyte lipolysis. Lipolysis inhibition normalized plasma FFA levels, and improved insulin sensitivity. Surprisingly, blocking lipolysis failed to abolish BCAA-induced liver injury. Mechanistically, hepatic mTOR activation by BCAA inhibited lipid-induced hepatic autophagy, increased hepatic apoptosis, blocked hepatic FFA/triglyceride conversion, and increased hepatocyte susceptibility to FFA-mediated lipotoxicity. These data demonstrated that BCAA reduces HFD-induced body weight, at the expense of abnormal lipolysis and hyperlipidemia, causing hepatic lipotoxicity. Furthermore, BCAA directly exacerbate hepatic lipotoxicity by reducing lipogenesis and inhibiting autophagy in the hepatocyte