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.

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.

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.

Phenylalanine regulates initiation of digestive enzyme mRNA translation in pancreatic acinar cells and tissue segments in dairy calves.

As new nutritional strategies for ruminant are designed to change production efficiency by improving the supply of rumen protect protein, lipid, and even starch, the digestive system must fit to utilize these increased nutrient supplies, especially the pancreas. The objective of this study was to investigate the effects of phenylalanine (Phe) on digestive enzymes synthesis or secretion and cellular signaling in pancreatic acinar (PA) cells of dairy calves. The PA cells isolated from fresh pancreas of dairy calves, and cultured in completed RIPA 1640 medium with no fetal serum but 0, 0.15 and 0.45 mM Phe at 37°C in CO2 incubator for 120 min. The pancreatic tissue segments (PTS) was cut approximately 2 × 2 mm from the fresh pancreas, and incubated in oxygenated Krebs-Ringer bicarbonate (KRB) buffer containing 0 or 0.35 mM Phe at 39°C for 180 min, and the samples were collected every 60 min after incubation. In PA cells, Phe increased (P < 0.05) the α-amylase secretion and mRNA expression, the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). In PTS, the Phe increased (P < 0.05) α-amylase and trypsin synthesis, secretion and mRNA expression, as well as the phosphorylation of S6K1 and 4EBP1. Conclusively, these results suggested that Phe regulates the synthesis or secretion of α-amylase, trypsin and lipase through mRNA translation initiation factors - S6K1 and 4EBP1.

Duodenal infusions of isoleucine influence pancreatic exocrine function in dairy heifers.

Four healthy Holstein heifers (235 ± 12 kg) fitted with duodenal and pancreatic cannulas were used to investigate infusion of isoleucine (Ile) on the pancreatic exocrine function in a 4 × 4 Latin square design. Three doses of Ile, 10, 20 and 30 g in 2500 ml water, respectively, were infused into the duodenum over a period of 12 h in Experiment (Exp) 1 and over 10 d in Exp 2. Hourly pancreatic juice and jugular blood were taken during the infusion period in Exp 1, and the blood samples were taken in 2-h intervals over the last 2 d in Exp 2. Compared with no Ile infusion, the Ile infusions in both experiments increased the concentration and secretion rate of the protein, activity of ɑ-amylase and trypsin and plasma cholecystokinin. The secretion rate of ɑ-amylase and the activity of trypsin linearly increased with the Ile doses. The pancreatic juice secretion linearly increased with Ile in Exp 2 but not in Exp 1. Isoleucine linearly increased plasma insulin in Exp 1, but not in Exp 2. No effects of Ile on pH of pancreatic juice, the activity of chymotrypsin and lipase and plasma glucose were found. In conclusion, duodenal Ile infusion could increase the pancreatic exocrine function of Holstein heifers, especially ɑ-amylase, and the increment appeared to be dose and time dependent.

Regulation of Nutritional Metabolism in Transition Dairy Cows: Energy Homeostasis and Health in Response to Post-Ruminal Choline and Methionine.

This study investigated the effects of rumen-protected methionine (RPM) and rumen-protected choline (RPC) on energy balance, postpartum lactation performance, antioxidant capacity and immune response in transition dairy cows. Forty-eight multiparous transition cows were matched and divided into four groups: control, 15 g/d RPC, 15 g/d RPM or 15 g/d RPC + 15 g/d RPM. Diet samples were collected daily before feeding, and blood samples were collected weekly from the jugular vein before morning feeding from 21 days prepartum to 21 days postpartum. Postpartum dry matter intake (DMI) was increased by both additives (P < 0.05), and energy balance values in supplemented cows were improved after parturition (P < 0.05). Both RPC and RPM decreased the plasma concentrations of non-esterified fatty acids (NEFA), ß-hydroxybutyric acid (BHBA), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (P < 0.05), but increased the plasma levels of glucose, very-low-density lipoprotein (VLDL) and apolipoprotein B100 (ApoB 100, P < 0.05). The supplements improved milk production (P < 0.05), and increased (P < 0.05) or tended to increase (0.05 < P < 0.10) the contents of milk fat and protein. The post-ruminal choline and methionine elevated the blood antioxidant status, as indicated by total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px) activity and the vitamin E concentration (P < 0.05), and reduced the plasma malondialdehyde (MDA) level (P < 0.05). Furthermore, RPM and RPC elevated the plasma interleukin 2 (IL-2) concentration and the CD4+/CD8+ T lymphocyte ratio in peripheral blood (P < 0.05). Alternatively, the levels of tumor necrosis factor-α (TNF-α) and IL-6 were decreased by RPM and RPC (P < 0.05). Overall, the regulatory responses of RPC and RPM were highly correlated with time and were more effective in the postpartum cows. The results demonstrated that dietary supplementation with RPC and RPM promoted energy balance by increasing postpartal DMI and regulating hepatic lipid metabolism, improved postpartum lactation performance and enhanced antioxidant capacity and immune function of transition dairy cows.