Transcriptome in Liver of Periparturient Dairy Cows Differs between Supplementation of Rumen-Protected Niacin and Rumen-Protected Nicotinamide.

To investigate the difference between rumen-protected niacin (RPN) and rumen-protected nicotinamide (RPM) in the transcriptome of genes relating to the lipid metabolism of the liver of periparturient dairy cows, 10 healthy Chinese Holstein cows were randomly divided into two groups and fed diets supplemented with 18.4 g/d RPN or 18.7 g/d RPM, respectively. The experiment lasted from 14 days before to 21 days after parturition. Liver biopsies were taken 21 days postpartum for transcriptomic sequencing. In addition, human LO2 cells were cultured in a medium containing 1.6 mmol/L of non-esterified fatty acids and 1 mmol/L niacin (NA) or 2 mmol/L nicotinamide (NAM) to verify the expression of the 10 genes selected from the transcriptomic analysis of the liver biopsies. The expression of a total of 9837 genes was detected in the liver biopsies, among which 1210 differentially expressed genes (DEGs) were identified, with 579 upregulated and 631 downregulated genes. These DEGs were associated mainly with lipid metabolism, oxidative stress, and some inflammatory pathways. Gene ontology (GO) enrichment analysis showed that 355 DEGs were enriched in 38 GO terms. The differences in the expression of these DEGs between RPN and RPM were predominantly related to the processes of steroid catabolism, steroid hydroxylase, monooxygenase activity, oxidoreductase activity, hemoglobin binding, and ferric iron binding, which are involved mainly in lipid anabolism and redox processes. The expressions of FADS2, SLC27A6, ARHGAP24, and THRSP in LO2 cells were significantly higher (p < 0.05) while the expressions of BCO2, MARS1, GARS1, S100A12, AGMO, and OSBPL11 were significantly lower (p < 0.05) on the NA treatment compared to the NAM treatment, indicating that NA played a role in liver metabolism by directly regulating fatty acid anabolism and transport, inflammatory factor expression, and oxidative stress; and NAM functioned more as a precursor of nicotinamide adenine dinucleotide (NAD, coenzyme I) and nicotinamide adenine dinucleotide phosphate (NADP, coenzyme II) to participate indirectly in biological processes such as ether lipid metabolism, cholesterol metabolism, energy metabolism, and other processes.

Deubiquitinase UCHL1 regulates estradiol synthesis by stabilizing voltage-dependent anion channel 2.

Lack of estradiol production by granulosa cells blocks follicle development, causes failure of estrous initiation, and results in an inability to ovulate. The ubiquitin-proteasome system plays a critical role in maintaining protein homeostasis and stability of the estrous cycle, but knowledge of deubiquitination enzyme function in estradiol synthesis is limited. Here, we observe that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is more significant in estrous sows and high litter-size sows than in nonestrous sows and low-yielding sows. Overexpression of UCHL1 promotes estradiol synthesis in granulosa cells, and interference with UCHL1 has the opposite effect. UCHL1 binds, deubiquitinates, and stabilizes voltage-dependent anion channel 2 (VDAC2), promoting the synthesis of the estradiol precursor pregnenolone. Cysteine 90 (C90) of UCHL1 is necessary for its deubiquitination activity, and Lys45 and Lys64 in VDAC2 are essential for its ubiquitination and degradation. In vivo, compared with WT and sh-NC-AAV groups, the estrus cycle of female mice is disturbed, estradiol level is decreased, and the number of antral follicles is decreased after the injection of sh-UCHL1-AAV into ovarian tissue. These findings suggest that UCHL1 promotes estradiol synthesis by stabilizing VDAC2 and identify UCHL1 as a candidate gene affecting reproductive performance.

Effects of Alginic Acid on the Porcine Granulosa Cells and Maturation of Porcine Oocytes.

SCOPE: Alginic acid (AA) from brown algae is a marine organic compound. There is extensive use of AA in the food industry and healthcare, suggesting a high probability of AA exposure. The present study investigates the effects of AA on porcine ovarian granulosa cells (GCs) and oocytes to explore its mechanism in female reproduction because of its adverse effects on reproduction. METHODS AND RESULTS: The study adds 20 µM AA to the porcine primary ovarian GCs medium and porcine oocyte in vitro maturation (IVM) medium. Estrogen and progesterone levels are downregulated in GCs. Reactive oxygen species are excessive, and the antioxidant capacity declines. Then mitochondria-mediated apoptosis pathway is involved in GCs apoptosis. In addition, scores of autophagosomes are found in the experimental cells. Furthermore, AA significantly inhibits the proliferation of GCs around cumulus-oocyte complexes (COCs) accompanied by abnormal spindle assembly, chromosome arrangement disorder, and aberrant cortical granules distribution in oocytes, leading to a decreased oocyte maturation rate. CONCLUSION: These findings suggest that 20 µM AA is toxic to sow reproduction by interfering with estrogen production, oxidative stress, mitochondria-mediated apoptosis, autophagy in GCs of sows, and oocyte maturation.

Methionine influences the profile of intestinal antibiotic resistome through inhibiting the growth of Escherichia coli.

Antibiotic resistance genes (ARGs) are a new type of environmental pollutant. However, studies have mainly focused on the distribution characteristics of ARGs in the livestock environment, lacking of studies on the composition of ARGs in the intestinal tract of animals and the effect of nutrients on intestinal ARGs and microbial communities. Reducing antimicrobial resistance and maintaining optimal animal health and performance are urgently needed. Methionine is an essential amino acid which plays a critical role in the growth and reproductive performance of animals. In this study, feeding experiment, in vitro fermentation and bacterial culture experiment were performed to explore the influence of methionine on the intestinal resistome of sows. We found that dietary 0.2 % methionine supplementation decreased the total abundance of intestinal ARGs, which was further confirmed by in vitro fecal microbial fermentation of sows. Metagenome binning analysis identified that Escherichia coli was the major ARG host, which carried 60-113 ARGs and 134-286 virulence factors, indicating that Escherichia coli in the pig intestine is not only a core ARG host, but also an important pathogen. In addition, we found that methionine supplementation inhibited the growth of Escherichia coli, indicating that dietary methionine may reduce the resistome risk in sow intestine by inhibiting core ARG hosts such as Escherichia coli. These findings reveal that dietary methionine application plays a critical role in intestinal antibiotic resistance, providing a new idea for preventing and controlling environmental pollution by antibiotic-resistant microbes.

Nicotinamide supplementation alters plasma lipidomic profiles of peripartal dairy cows.

Fatty liver syndrome, a common health problem in dairy cows, occurs during the transition from pregnancy to lactation. If the energy supplied to the cow's body cannot meet its needs, a negative energy balance ensues, and the direct response is fat mobilization. Nicotinamide (NAM) has been reported to reduce the nonesterified fatty acid concentration of postpartum plasma. To study the biochemical adaptations underlying this physiologic dysregulation, 12 dairy cows were sequentially assigned to a NAM (45 g/day) treatment or control group. Blood samples were collected on day (D) 1 and D21 relative to parturition. Changes to the plasma lipid metabolism of dairy cows in the two groups were compared using lipidomics. There were significant increases in plasma sphingomyelins d18:1/18:0, d18:1/23:0, d18:1/24:1, d18:1/24:0, and d18:0/24:0 in the NAM group on D1 relative to parturition. In addition, fatty acids 18:2, 18:1, 18:0, 16:1, and 16:0 were obviously decreased on D21 relative to calving. This research has provided insights into how NAM supplementation improves lipid metabolism in perinatal dairy cows.

The Effects of Rumen-Protected Choline and Rumen-Protected Nicotinamide on Liver Transcriptomics in Periparturient Dairy Cows.

To investigate the effects of rumen-protected choline (RPC) and rumen-protected nicotinamide (RPM) on liver metabolic function based on transcriptome in periparturient dairy cows, 10 healthy Holstein dairy cows with similar parity were allocated to RPC and RPM groups (n = 5). The cows were fed experimental diets between 14 days before and 21 days after parturition. The RPC diet contained 60 g RPC per day, and the RPM diet contained 18.7 g RPM per day. Liver biopsies were taken 21 days after calving for the transcriptome analysis. A model of fat deposition hepatocytes was constructed using the LO2 cell line with the addition of NEFA (1.6 mmol/L), and the expression level of genes closely related to liver metabolism was validated and divided into a CHO group (75 µmol/L) and a NAM group (2 mmol/L). The results showed that the expression of a total of 11,023 genes was detected and clustered obviously between the RPC and RPM groups. These genes were assigned to 852 Gene Ontology terms, the majority of which were associated with biological process and molecular function. A total of 1123 differentially expressed genes (DEGs), 640 up-regulated and 483 down-regulated, were identified between the RPC and RPM groups. These DEGs were mainly correlated with fat metabolism, oxidative stress and some inflammatory pathways. In addition, compared with the NAM group, the gene expression level of FGF21, CYP26A1, SLC13A5, SLCO1B3, FBP2, MARS1 and CDH11 in the CHO group increased significantly (p < 0.05). We proposed that that RPC could play a prominent role in the liver metabolism of periparturient dairy cows by regulating metabolic processes such as fatty acid synthesis and metabolism and glucose metabolism; yet, RPM was more involved in biological processes such as the TCA cycle, ATP generation and inflammatory signaling.

Role of functional fatty acids in modulation of reproductive potential in livestock.

Fatty acids are not only widely known as energy sources, but also play important roles in many metabolic pathways. The significance of fatty acids in modulating the reproductive potential of livestock has received greater recognition in recent years. Functional fatty acids and their metabolites improve follicular development, oocyte maturation and embryo development, as well as endometrial receptivity and placental vascular development, through enhancing energy supply and precursors for the synthesis of their productive hormones, such as steroid hormones and prostaglandins. However, many studies are focused on the impacts of individual functional fatty acids in the reproductive cycle, lacking studies involved in deeper mechanisms and optimal fatty acid requirements for specific physiological stages. Therefore, an overall consideration of the combination and synergy of functional fatty acids and the establishment of optimal fatty acid requirement for specific stages is needed to improve reproductive potential in livestock.

The Combined Use of Medium- and Short-Chain Fatty Acids Improves the Pregnancy Outcomes of Sows by Enhancing Ovarian Steroidogenesis and Endometrial Receptivity.

Fatty acids play important roles in maintaining ovarian steroidogenesis and endometrial receptivity. Porcine primary ovarian granulosa cells (PGCs) and endometrial epithelial cells (PEECs) were treated with or without medium- and short-chain fatty acids (MSFAs) for 24 h. The mRNA abundance of genes was detected by fluorescence quantitative PCR. The hormone levels in the PGCs supernatant and the rate of adhesion of porcine trophoblast cells (pTrs) to PEECs were measured. Sows were fed diets with or without MSFAs supplementation during early gestation. The fecal and vaginal microbiomes were identified using 16S sequencing. Reproductive performance was recorded at parturition. MSFAs increased the mRNA abundance of genes involved in steroidogenesis, luteinization in PGCs and endometrial receptivity in PEECs (p < 0.05). The estrogen level in the PGC supernatant and the rate of adhesion increased (p < 0.05). Dietary supplementation with MSFAs increased serum estrogen levels and the total number of live piglets per litter (p < 0.01). Moreover, MSFAs reduced the fecal Trueperella abundance and vaginal Escherichia-Shigella and Clostridium_sensu_stricto_1 abundance. These data revealed that MSFAs improved pregnancy outcomes in sows by enhancing ovarian steroidogenesis and endometrial receptivity while limiting the abundance of several intestinal and vaginal pathogens at early stages of pregnancy.

NR1D1 targeting CYP19A1 inhibits estrogen synthesis in ovarian granulosa cells.

The circadian system performs an important role in mammalian reproduction with significant effects on hormone secretion. Nuclear receptor subfamily 1 group D member 1 (NR1D1) functions as a transcriptional repressor in the circadian system and affects granulosa cells (GCs), but how it regulates estrogen synthesis has not been clarified. We investigated the effect of NR1D1 on estrogen synthesis and found that NR1D1 was highly expressed in GCs, mainly in cell nuclei. Additionally, the expression of NR1D1 and estrogen synthesis key genes CYP19A1, CYP11A1 and StAR showed rhythmic changes in porcine ovarian GCs. Activation of NR1D1 enhances its ability to inhibit the transcriptional activity of CYP19A1 by binding to the RORE on the CYP19A1 promoter, resulting in a decrease in estradiol content. Interference with NR1D1 can eliminate the transcriptional inhibition of CYP19A1 and promote the synthesis of estradiol. The results suggest that the hormone secretion of the ovary itself is also regulated by the biological clock, and any factors that affect the circadian rhythm can affect the endocrine and reproductive performance of sows, so the natural rhythm of sows should be maintained in production.

Physically effective neutral detergent fiber improves chewing activity, rumen fermentation, plasma metabolites, and milk production in lactating dairy cows fed a high-concentrate diet.

Subacute ruminal acidosis (SARA) continues to be a common and costly metabolic disorder in high-producing dairy cows worldwide. The objective of this study was to evaluate if increasing the concentration of physically effective neutral detergent fiber (peNDF) in diets can reduce the risk of SARA in cows fed a high-concentrate diet. Thirty second-parity Holstein cows in mid lactation (131 ± 8.3 d in milk) were randomly allocated to 3 dietary treatments (10 dairy cows per group): high (11.3%, high peNDF8.0), medium (10.6%, medium peNDF8.0), or low (9.0%, low peNDF8.0) concentration of peNDF8.0. The diets were prepared by mixing the same total mixed ration (57% concentrate and 43% roughages) for 10, 18, or 60 min, respectively. The treatments were fed for 36 d with 21 d for adaptation and 15 d for sampling. The peNDF8.0 intake was positively correlated with the peNDF8.0 concentration. Chewing and ruminating times adjusted for dry matter intake and NDF intake were linearly increased with the increased dietary peNDF8.0 concentration. The high peNDF8.0 diet decreased the number of meals per day. The increased dietary peNDF8.0 concentration linearly increased the rumen fluid pH, the molar percentage of acetate and isobutyrate, acetate-to-propionate ratio, and ammonia nitrogen concentration, but linearly decreased the molar percentages of propionate and valerate. The total VFA concentration and the molar percentages of butyrate and isovalerate remained unchanged. Meanwhile, the increase in the peNDF8.0 concentration of the diet linearly increased the activities of carboxymethyl cellulase, avicelase, ß-glucanase, and ferulic acid esterase in rumen fluid, but did not affect the activities of xylanase. Total plasma antioxidant capacity, γ-glutamyl transpeptidase activity, and plasma concentrations of total protein, albumin, creatinine, and malondialdehyde were linearly decreased by the increased dietary peNDF8.0 concentration. The increase in peNDF8.0 concentration raised the plasma concentrations of glucose, triglyceride, cholesterol, and blood urea nitrogen. Somatic cell counts in the milk were positively correlated with the dietary peNDF8.0 concentration. The feed and milk energy efficiencies were unaffected by the treatments. Shortening the total mixed ration mixing time may be a practical strategy to increase the peNDF8.0 concentration and reduce the risk of SARA in dairy cows fed high-concentrate diets.

Decreased amylolytic microbes of the hindgut and increased blood glucose implied improved starch utilization in the small intestine by feeding rumen-protected leucine in dairy calves.

Starch digestion in the small intestine in ruminants is relatively lower compared with that in monogastric animals, likely due to low pancreatic α-amylase secretion. Previous studies suggested that leucine could increase pancreatic α-amylase secretion in the small intestine of heifers cannulated with abomasal, duodenal, and ileal catheters. However, the surgical procedures probably have an effect on pancreatic function. Thus, we used rumen-protected leucine (RP-Leu) to explore its effect on small intestinal digestion of starch in calves without any surgery in 3 experiments. The first experiment was to explore whether RP-Leu could improve post-ruminal starch digestion in 5-mo-old calves (158 ± 19 kg body weight ± standard deviation). We found that RP-Leu did not affect rumen fermentation profile or whole-tract starch digestibility, but it increased blood glucose concentration and fecal pH and decreased fecal propionate molar proportion. Additionally, RP-Leu increased fibrolytic genera Ruminiclostridium and Pseudobutyrivibrio and decreased the amylolytic genus of Faecalibacterium. The second experiment compared RP-Leu and rumen-protected lysine (RP-Lys) for their effects on post-ruminal starch digestion in 6-mo-old calves (201 ± 24 kg body weight). The responses of blood glucose concentration, fecal pH, fecal propionate proportion, and starch digestibility to RP-Leu supplementation were similar to those observed in experiment 1. Cellulolytic family Ruminococcaceae and Bacteroidales BS11 gut group tended to be increased by RP-Leu. In contrast, RP-Lys showed no significant influence on the above measurements. The third experiment determined the interaction between RP-Leu and rumen-escape starch (RES) on the small intestinal digestion of starch in 8-mo-old calves (289 ± 26 kg body weight). An interaction between RP-Leu and RES levels was observed in fecal butyrate concentration and the relative abundance of family Bacteroidaceae, and genera Ruminococcaceae UCG-005 and Bacteroides. We found that RP-Leu tended to increase the abundance of fecal Firmicutes and decrease Spirochaetae. In conclusion, RP-Leu, but not RP-Lys, increased blood glucose concentration and decreased the amount of starch fermented in the hindgut in a RES dose-dependent manner, suggesting that RP-Leu might stimulate starch digestion in the small intestine.

Choline and methionine regulate lipid metabolism via the AMPK signaling pathway in hepatocytes exposed to high concentrations of nonesterified fatty acids.

High concentrations of nonesterified fatty acids (NEFAs) and ß-hydroxybutyric acid (BHBA) induce lipid peroxidation, resulting in liver damage. Choline and methionine (Met) can promote energy balance and benefit liver health in transition dairy cows; however, the regulating mechanism remains unclear. In the present study, we established the hepatocyte damage model by 1.5 mM NEFAs or BHBA treatment, and examined lipid metabolism in hepatocytes. The results showed that 1.5 mM NEFAs and 1.5 mM BHBA significantly decreased the messenger RNA (mRNA) expression of AMP-activated protein kinase (AMPK)-α as well as its target genes carnitine palmitoyltransferase-1α (CPT-1α), acetyl-CoA carboxylase, fatty acid synthetase, and Apolipoprotein B100 (ApoB100). Choline and Met upregulated the phosphorylation level of AMPK-α, which was blocked by BML (an AMPK-α inhibitor). The mRNA expression level of peroxisome proliferator-activated receptor-α (PPAR-α), CPT-1α, and ApoB100 showed a similar trend. The expressions of liver X recptoer α (LXR-α) and sterol regulatory element-binding protein 1c (SREBP-1c) were decreased by choline and Met, while only the decrease of LXR-α was blocked by BML. These findings indicate that the high-level NEFAs and BHBA weaken the lipid metabolism by impairing the fatty acid oxidation, synthesis, and transport proteins. Choline and Met regulate PPAR-α and LXR-α transcriptional activity through AMPK-α phosphorylation and regulate SREBP-1c independently of AMPK-α to promote lipid oxidation and transport in NEFAs-treated hepatocytes.

Specific enrichment of microbes and increased ruminal propionate production: the potential mechanism underlying the high energy efficiency of Holstein heifers fed steam-flaked corn.

Corn grain has a high starch content and is used as main energy source in ruminant diets. Compared with finely ground corn (FGC), steam-flaked corn (SFC) could improve the milk yield of lactating dairy cows and the growth performance of feedlot cattle, but the detailed mechanisms underlying those finding are unknown. The rumen microbiome breaks down feedstuffs into energy substrates for the host animals, and contributes to feed efficiency. Therefore, the current study was conducted to investigate the ruminal bacterial community changes of heifers fed differently processed corn (SFC or FGC) using 16S rRNA sequencing technologies, and to uncover the detailed mechanisms underlying the high performance of ruminants fed the SFC diet. The results revealed that different processing methods changed the rumen characteristics and impacted the composition of the rumen bacteria. The SFC diet resulted in an increased average daily gain in heifers, an increased rumen propionate concentration and a decreased rumen ammonia nitrogen concentration. The relative abundance of the phylum Firmicutes and Proteobacteria were tended to increase or significantly increased in the heifers fed SFC diet compared with FGC diet. In addition, the relative abundance of amylolytic bacteria of the genera Succinivibrio, Roseburia and Blautia were elevated, and the cellulolytic bacteria (Ruminococcaceae_UCG-014 and Ruminococcaceae_UCG-013) were decreased by the steam flaking method. Spearman correlation analysis between the ruminal bacteria and the microbial metabolites showed that the rumen propionate concentration was positively correlated with genera Succinivibrio and Blautia abundance, but negatively correlated with genera Ruminococcaceae_UCG-014 abundance. Evident patterns of efficient improvement in rumen propionate and changes in rumen microbes to further improve feed conversion were identified. This observation uncovers the potential mechanisms underlying the increased efficiency of the SFC processing method for enhancing ruminant performance.

Leucine Regulates the Exocrine Function in Pancreatic Tissue of Dairy Goats In Vitro.

This study aimed to investigate the effects of leucine (Leu) on the synthesis and secretion of digestive enzymes in cultured pancreatic tissue of dairy goats and on the signaling molecules. Fresh pancreatic tissue from dairy goats was cut into approximately 2 mm × 2 mm pieces and incubated in oxygenated Krebs-Ringer bicarbonate buffer containing 0 (the control), 0.40, 0.80, or 1.60 mM Leu at 39°C in a CO2 incubator for 180 min. The results showed that Leu increased the release of α-amylase, trypsin, and chymotrypsin in the buffer and tissue, as well as the total activity (P < 0.05), especially at 0.40 and 0.80 mM. Compared with the control, 1.60 mM Leu increased the release of α-amylase and the total activity of trypsin and chymotrypsin (P < 0.05) but had no effect on the tissue concentration of α-amylase, trypsin, and chymotrypsin or the total activity of α-amylase (P > 0.05). Leu improved the mRNA expression of α-amylase, trypsin, and chymotrypsin (P < 0.05), especially at 0.80 and 1.60 mM. The activity and mRNA expression of lipase were not affected (P > 0.05). Compared with the control, 0.40 and 0.80 mM Leu increased the expression of the γ isoform of 4EBP1 (P < 0.05), implying increased phosphorylation of 4EBP1. Leu increased the phosphorylation of S6K1 (P < 0.05). Compared with the control, 0.40 and 0.80 mM Leu decreased the eEF2 phosphorylation level (P < 0.05). Conclusively, these results suggested that Leu could regulate the synthesis of pancreatic enzymes by increasing the mRNA expression and phosphorylation level of protein factors in the mammalian target of rapamycin pathway and the optimal Leu level in this experiment was 0.80 mM.

Isoleucine Regulates the Synthesis of Pancreatic Enzymes via the Activation of mRNA Expression and Phosphorylation in the Mammalian Target of Rapamycin Signalling Pathways in Pancreatic Tissues.

This study aimed to investigate the effects of isoleucine (Ile) on the synthesis and secretion of digestive enzymes and cellular signalling in the pancreatic tissue of dairy goats. The pancreatic tissues were incubated in buffer containing 0, 0.40, 0.80, and 1.60 mM Ile. High levels of Ile significantly increased the buffer release and total concentration of ɑ-amylase in the tissues (P < 0.001). The total trypsin and chymotrypsin concentrations in each of the Ile groups were significantly higher than those in the control group (P < 0.05); however, lipase was not affected. High levels of Ile significantly increased ɑ-amylase mRNA expression (P < 0.001) but had no effect on the mRNA expression of trypsin, chymotrypsin, or lipase. Ile did not affect S6K1 phosphorylation levels. High levels of Ile significantly increased the expression of the γ isoform of 4EBP1 (P < 0.001), which indicated that the phosphorylation of 4EBP1 was significantly increased. The phosphorylation level of eEF2 gradually decreased with the addition of Ile (P < 0.001). These results suggested that high doses of Ile can regulate the excretion of enzymes, especially ɑ-amylase, in the pancreatic tissues of dairy goats by modulating mTOR signalling, and this regulation is independent of the mTOR-S6K1 pathway.

Molecular mechanisms relating to amino acid regulation of protein synthesis.

Some amino acids (AA) act through several signalling pathways and mechanisms to mediate the control of gene expression at the translation level, and the regulation occurs, specifically, on the initiation and the signalling pathways for translation. The translation of mRNA to protein synthesis proceeds through the steps of initiation and elongation, and AA act as important feed-forward activators that are involved in many pathways, such as the sensing and the transportation of AA by cells, in these steps in many tissues of mammals. For the translation, phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) is a critical molecule that controls the translation initiation and its functions can be regulated by some AA. Another control point in the mRNA binding step in the translation initiation is at the regulation by mammalian target of rapamycin, which requires a change of phosphorylation status of ribosomal protein S6. In fact, the change of phosphorylation status of ribosomal protein S6 might be involved in global protein synthesis. The present review summarises recent work on the molecular mechanisms of the regulation of protein synthesis by AA and highlights new findings.

Selection of suitable reference genes for core clock gene expression analysis by real-time qPCR in rat ovary granulosa cells.

Selection of a suitable endogenous reference gene is essential for investigating expression of clock genes Bmal1, Clock, Pers, Crys, Rev-erbα/ß, and RORα/ß/γ involved in the circadian system. In this study, we treated rat ovary granulosa cells with dexamethasone to synchronize circadian oscillation in vitro and determined expression levels of Bmal1 and Per2 and six candidate reference genes (Actb, Beta actin; B2m, Beta-2-microglobulin; Ppia, Cyclophilin A; Gapdh, Glyceraldehyde-3-phosphate dehydrogenase; Hprt, Hypoxanthine guanine phosphoribosyl transferase and Tbp, TATA-box-binding protein) using quantitative real-time PCR. We then employed three software programs, GeNorm, NormFinder, and BestKeeper, to analyze the expression data for the selection of the best reference gene. According to GeNorm, Tbp and B2m were assessed as the most stable reference genes; Tbp and Hprt were best by NormFinder and BestKeeper, respectively. Thus, we recommend Tbp as the most suitable reference gene for studying clock genes expression in rat ovary granulosa cells in vitro.

Leucine-induced promotion of post-absorptive EAA utilization and hepatic gluconeogenesis contributes to protein synthesis in skeletal muscle of dairy calves.

The high rate of protein synthesis in skeletal muscle of dairy calves can benefit their first lactation even lifetime milk yield. Since the rate of protein synthesis is relatively low in the post-absorptive state, the aim of this research was to determine whether leucine supplementation could increase the post-absorptive essential amino acid (EAA) utilization and protein synthesis in the skeletal muscle. Ten male neonatal dairy calves (38 ± 3 kg) were randomly assigned to either the control (CON, no leucine supplementation, n = 5) or supplementation with 1.435 g leucine/L milk (LEU, n = 5). Results showed that leucine significantly increased the length and protein concentration in longissimus dorsi (LD) muscle, whereas it decreased creatinine concentration and glutamic-oxalacetic transaminase (GOT) activity. Compared to the control group, leucine supplementation also reduced the glutamic-pyruvic transaminase (GPT) activity. Supplementation of leucine improved the phosphorylation of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E-binding protein 1 (4EBP1) and substrates ribosomal protein S6 kinase 1 (p70S6K). Supplementation of leucine resulted in increased concentrations of glucose, methionine, threonine, histidine and EAAs and decreased concentration of arginine in serum. Liver glucose concentration was higher and pyranic acid was lower in LEU compared to CON. In conclusion, leucine supplementation can promote post-absorptive EAA utilization and hepatic gluconeogenesis, which contributes to protein synthesis in skeletal muscle of dairy calves.

Effects of dietary leucine and phenylalanine on gastrointestinal development and small intestinal enzyme activities in milk-fed holstein dairy calves.

This study was investigated the effects of dietary supplementation of leucine and phenylalanine on the development of the gastrointestinal tract and the intestinal digestive enzyme activity in male Holstein dairy calves. Twenty calves with a body weight of 38 ± 3 kg at 1 day of age were randomly divided into four groups: a control group, a leucine group (1.435 g·l-1), a phenylalanine group (0.725 g·l-1), and a mixed amino acid group (1.435 g·l-1 leucine plus 0.725 g·l-1 phenylalanine). The supplementation of leucine decreased the short-circuit current (Isc) of the rumen and duodenum (P<0.01); phenylalanine did not show any influence on the Isc of rumen and duodenum (P>0.05), and also counteracted the Isc reduction caused by leucine. Leucine increased the trypsin activity at the 20% relative site of the small intestine (P<0.05). There was no difference in the activity of α-amylase and of lactase in the small intestinal chyme among four treatments (P>0.05). The trypsin activity in the anterior segment of the small intestine was higher than other segments, whereas the α-amylase activity in the posterior segment of the small intestine was higher than other segments. Leucine can reduce Isc of the rumen and duodenum, improve the development of the gastrointestinal tract, and enhance trypsin activity; phenylalanine could inhibit the effect of leucine in promoting intestinal development.

Metabolomics for the Effect of Biotin and Nicotinamide on Transition Dairy Cows.

The objective of this study was to evaluate alterations in serum metabolites of transition dairy cows affected by biotin (BIO) and nicotinamide (NAM) supplementation. A total of 40 multiparous Holsteins were paired and assigned randomly within a block to one of the following four treatments: control (T0), 30 mg/day BIO (TB), 45 g/day NAM (TN), and 30 mg/day BIO + 45 g/day NAM (TB+N). Supplemental BIO and NAM were drenched on cows from 14 days before the expected calving date. Gas chromatography time-of-flight/mass spectrometry was used to analyze serum samples collected from eight cows in every groups at 14 days after calving. In comparison to T0, TB, TN, and TB+N had higher serum glucose concentrations, while non-esterified fatty acid in TN and TB+N and triglyceride in TB+N were lower. Adenosine 5'-triphosphate was significantly increased in TB+N. Both TN and TB+N had higher glutathione and lower reactive oxygen species. Moreover, TB significantly increased inosine and guanosine concentrations, decreased ß-alanine, etc. Certain fatty acid concentrations (including linoleic acid, oleic acid, etc.) were significantly decreased in both TN and TB+N. Some amino acid derivatives (spermidine in TN, putrescine and 4-hydroxyphenylethanol in TB+N, and guanidinosuccinic acid in both TN and TB+N) were affected. Correlation network analysis revealed that the metabolites altered by NAM supplementation were more complicated than those by BIO supplementation. These findings showed that both BIO and NAM supplementation enhanced amino acid metabolism and NAM supplementation altered biosynthesis of unsaturated fatty acid metabolism. The improved oxidative status and glutathione metabolism further indicated the effect of NAM on oxidative stress alleviation.