Supplementation of Methionine Dipeptide Enhances the Milking Performance of Lactating Dairy Cows.

Methionine dipeptide (Met-Met) could improve milk protein synthesis in bovine epithelia mammary cells and lactating mice, while the effects of Met-Met on lactation performance, rumen fermentation and microbiota profile in lactating dairy cows have not been explored. For this reason, 60 Chinese lactating Holstein cows were allocated into three treatment groups: control group (CON), 6 g/d methionine dipeptide group (MM), and 6.12 g/d rumen-protected methionine dipeptide group (RPMM). The experiment lasted for 10 weeks to monitor lactation performance, plasma amino acid profile and rumen fermentation parameters and microbiota profile. Results showed that MM increased the energy-corrected milk (ECM), and RPMM increased both milk yield and ECM (p < 0.05). The milk protein concentration and yield were increased by MM and RPMM (p < 0.05). The rumen fermentation showed that RPMM increased total volatile fatty acids, acetate and valerate concentrations (p < 0.05). The relative abundance of Firmicutes, including Succiniclasticum, Selenomonas and Clostridium_XlVa, were enriched and the Prevotella was decreased by RPMM (p < 0.05). In summary, daily supplementing with 6 g of MM or RPMM in lactating dairy cows could improve milk yield and both percentage and yield of milk protein, and RPMM benefited the rumen fermentation and altered the bacterial composition. These results provided the first evidence that Met-Met supplementation can improve lactation performance of dairy cows.

Nicotinamide Supplementation Mitigates Oxidative Injury of Bovine Intestinal Epithelial Cells through Autophagy Modulation.

The small intestine is important to the digestion and absorption of rumen undegradable nutrients, as well as the barrier functionality and immunological responses in ruminants. Oxidative stress induces a spectrum of pathophysiological symptoms and nutritional deficits, causing various gastrointestinal ailments. Previous studies have shown that nicotinamide (NAM) has antioxidant properties, but the potential mechanism has not been elucidated. The aim of this study was to explore the effects of NAM on hydrogen peroxide (H2O2)-induced oxidative injury in bovine intestinal epithelial cells (BIECs) and its potential mechanism. The results showed that NAM increased the cell viability and total antioxidant capacity (T-AOC) and decreased the release of lactate dehydrogenase (LDH) in BIECs challenged by H2O2. The NAM exhibited increased expression of catalase, superoxide dismutase 2, and tight junction proteins. The expression of autophagy-related proteins was increased in BIECs challenged by H2O2, and NAM significantly decreased the expression of autophagy-related proteins. When an autophagy-specific inhibitor was used, the oxidative injury in BIECs was not alleviated by NAM, and the T-AOC and the release of LDH were not affected. Collectively, these results indicated that NAM could alleviate oxidative injury in BIECs by enhancing antioxidant capacity and increasing the expression of tight junction proteins, and autophagy played a crucial role in the alleviation.

Sodium butyrate administration improves intestinal development of suckling lambs.

This study was conducted to investigate the effects of sodium butyrate (SB) supplementation on growth performance, intestinal barrier functions, and intestinal bacterial communities in sucking lambs. Forty lambs of 7 d old, with an average body weight (BW) of 4.46 ±â€…0.45 kg, were allocated into the control (CON) or SB group, with each group having five replicate pens (n = 5). Lambs were orally administered SB at 1.8 mL/kg BW in the SB group or the same volume of saline in the CON group. Treatments were administered from 7 to 35 d of age, when one lamb from each replicate was slaughtered to obtain intestinal tissues and contents. The results showed that supplementation with SB tended to increase the BW (P = 0.079) and the starter intake (P = 0.089) of lambs at 35 d of age. The average daily gain of lambs in the SB group was significantly greater than that in the CON group (P < 0.05). The villus height of jejunum in the SB group was markedly higher (P < 0.05) than that in the CON group. In ileum, lambs in the SB group had lower (P < 0.05) crypt depth and greater (P < 0.05) villus-to-crypt ratio than those in the CON group. Compared with the CON group, the mRNA and protein expressions of Claudin-1 and Occludin were increased (P < 0.05) in the SB group. Supplementation with SB decreased the relative abundances of pathogenic bacteria, including Clostridia_UCG-014 (P = 0.094) and Romboutsia (P < 0.05), which were negatively associated with the intestinal barrier function genes (P < 0.05). The relative abundance of Succiniclasticum (P < 0.05) was higher in the SB group, and it was positively correlated with the ratio of villi height to crypt depth in the jejunum (P < 0.05). Compared with the CON group, the function "Metabolism of Cofactors and Vitamins" was increased in the SB group lambs (P < 0.05). In conclusion, SB orally administration during suckling period could improve the small intestine development and growth performance of lambs by inhibiting the harmful bacteria (Clostridia_UCG-014, Romboutsia) colonization, and enhancing intestinal barrier functions.

It is well known that butyrate and its derivatives have various benefits for the rumen development of ruminants, whereas its effects on the small intestine in preweaned lambs have received little attention. Therefore, the present study investigated the effects of sodium butyrate (SB) supplementation on growth performance, intestinal barrier functions, and intestinal bacterial communities in sucking lambs. The results indicated that SB dietary treatment has beneficial effects on the small intestine development and growth performance of suckling lambs.

Rumen-protected lysine supplementation improved amino acid balance, nitrogen utilization and altered hindgut microbiota of dairy cows.

This study was conducted to evaluate the effects of dietary crude protein (CP) and rumen-protected lysine (RPL) supplementation on lactation performance, amino acid (AA) balance, nitrogen (N) utilization and hindgut microbiota in dairy cows. Treatments were in a 2 × 2 factorial arrangement, and the main effects were CP concentration (16% vs. 18%) and RPL supplementation (with or without RPL at 40 g/cow per day). Forty cows were randomly allocated to 4 groups: low-CP diet (LP), low-CP diet plus RPL (LPL), high-CP diet (HP), high-CP diet plus RPL (HPL). The experiment was conducted for 8 weeks. Results showed that RPL increased the dry matter intake (P < 0.01), milk protein yield (P = 0.04) and energy corrected milk (P = 0.04), and tended to increase milk fat yield (P = 0.06) and fat corrected milk (P = 0.05). Cows in the HP group tended to have higher milk urea N (P = 0.07). Plasma concentrations of Arg, Ile, Lys, Met, Pro, total essential AA and total nonessential AA were increased by RPL (P < 0.05). The total essential AA, total nonessential AA and most AA (except Ile, Phe, Gly and Pro) were increased in the HP group (P < 0.05). N excretion was increased in the HP group through an increase in urea N excretion (P < 0.01) and an upward trend in plasma urea N (P = 0.07). In addition, RPL tended to increase milk protein N secretion (P = 0.08), milk N (P = 0.07) and microbial protein synthesis (P = 0.06), and decreased plasma urea N (P < 0.001). In the hindgut, the bacterial community were different between the LP and LPL groups (P < 0.01). The probiotic abundances of Christensenellaceae_R-7_group and Acinetobacter were increased by RPL (P = 0.03 and 0.03, respectively). The pathogenic abundances of Clostridium_sensu_stricto_1 (P < 0.001) and Turicibacter (P < 0.01) were decreased by RPL. In conclusion, supplementing RPL with low dietary CP could balance AA supply and increase milk protein yield, resulting in an improvement in N utilization efficiency, and altered the composition of the hindgut microbiota to favor the lactation performance of dairy cows.

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.

Effects of milk, milk replacer, and milk replacer plus ethoxyquin on the growth performance, weaning stress, and the fecal microbiota of Holstein dairy calves.

The growth and health statuses of calves during the early stages of development have a significant effect on milk production during their first lactation period. Using appropriate milk replacers helps meet the long-term targets of dairy farmers. This study aimed to examine the effects of milk, milk replacer, and milk replacer plus ethoxyquin on growth performance, antioxidant status, immune function, and the gut microbiota of Holstein dairy calves. A total of 36 neonatal dairy calves were randomly divided into three groups and fed different diets: one group was fed milk, another group was fed milk replacer, and the third group was given milk replacer plus ethoxyquin. The supplementation with ethoxyquin was started on day 35 of the feeding period. The calves were weaned on day 45, and the experiment was conducted until day 49. The blood and fecal samples were collected at the end of the animal experiment. The results showed that milk replacers induced poor growth performance (body weight and average daily gain). However, milk replacer plus ethoxyquin aided in growth performance, enhanced the starter intake and blood antioxidative ability, and elevated the concentration of fecal valeric acid. Moreover, fecal fermentation and 16S rRNA analyses showed that milk replacer plus ethoxyquin altered the microbial composition (reducing Alistipes and Ruminococcaceae and increasing Bacteroides and Alloprevotella). Pearson's correlation assays showed that alterations in fecal microbiota strongly correlated with average daily gain and antioxidative ability. The results indicated the potential of milk replacer plus ethoxyquin in modulating the growth of dairy calves and in enhancing their ability to combat stress.

Effects of hydrogen peroxide and l-tryptophan on antioxidative potential, apoptosis, and mammalian target of rapamycin signaling in bovine intestinal epithelial cells.

Amino acids are primarily absorbed in the ruminant small intestine, and the small intestine is a target organ prone to oxidative stress, causing intestinal disfunction. Previous study suggested that l-Trp could benefit intestinal function and production performance. This study aimed to explore the effects of l-Trp on hydrogen peroxide (H2O2)-induced oxidative injury in bovine intestinal epithelial cells (BIEC) and the potential mechanism. The effects of l-Trp on cell apoptosis, antioxidative capacity, AA transporters, and the mammalian target of rapamycin (mTOR) signaling pathway were evaluated in BIEC treated with 0.8 mMl-Trp for 2 hours combined with or without H2O2 induction. In addition, to explore whether the effects of 0.8 mMl-Trp on oxidative stress were related to mTOR, an mTOR-specific inhibitor was used. The percentage of apoptosis was measured using flow cytometry. The relative gene abundance and protein expression in BIEC were determined using real-time PCR and Western blot assay, respectively. Results showed l-Trp at 0.4 and 0.8 mM enhanced the cell viability, and it was inhibited by l-Trp at 6.4 mM. l-Tryptophan at 0.4, 0.8, and 1.6 mM remarkably decreased the percentage of apoptosis and enhanced antioxidative capacity in H2O2-mediated BIEC. Moreover, l-Trp at 0.8 mM increased the relative gene abundance and protein expression of antioxidative enzymes and AA transporters, and the mTOR signaling pathway. The mTOR inhibitor lowered the protein expression of large neutral amino acid transporter 1, but the inhibition of mTOR did not alter the activities of catalase and superoxide dismutase or protein expression of alanine-serine-cysteine transporter 2 with or without H2O2 induction. l-Tryptophan increased catalase and superoxide dismutase activities in H2O2-mediated BIEC, although not with a present mTOR inhibitor. l-Tryptophan increased the protein expression of large neutral amino acid transporter 1 and alanine-serine-cysteine transporter 2 in H2O2-mediated BIEC with or without the presence of an mTOR inhibitor. The present work suggested that l-Trp supplementation could alleviate oxidative injury in BIEC by promoting antioxidative capacity and inhibiting apoptosis, and the mTOR signal played vital roles in the alleviation.

Leucine protects bovine intestinal epithelial cells from hydrogen peroxide-induced apoptosis by alleviating oxidative damage.

BACKGROUND: The present study aimed to investigate whether leucine (Leu) alleviates oxidative injury in bovine intestinal epithelial cells (BIECs) induced by hydrogen peroxide (H2 O2 ), as well as the underlying molecular mechanisms. RESULTS: BIECs were treated with H2 O2 (1 mmol L-1 ) and/or Leu (0, 0.9, 1.8 or 3.6 mmol L-1 ) for 2 h. Leu increased cell viability (P < 0.05) and decreased the release of lactate dehydrogenase (P < 0.05) in BIECs challenged by H2 O2 . Then, the cells were treated with H2 O2 (1 mmol L-1 ) and/or Leu (1.8 mmol L-1 ) for 2 h. Compared with the H2 O2 group, cells treated with Leu and Leu + H2 O2 exhibited increased (P < 0.05) mRNA and protein expression of superoxide dismutase 2 (SOD2), catalase (CAT), glutathione peroxidase 1 (GPx1), heme oxygenase 1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2). BIECs treatment with Leu significantly reduced (P < 0.05) apoptosis induced by H2 O2 . BIECs were transfected with Nrf2 small interfering RNA (siRNA) for 48 h and/or treated with H2 O2 (1 mmol L-1 ) and/or Leu (1.8 mmol L-1 ) for another 2 h. Transfection with Nrf2 siRNA abrogated the protective effect of Leu against H2 O2 -induced apoptosis and the mRNA and protein expression of SOD2 (P < 0.05). CONCLUSION: These results indicate that Leu promotes the relative expression of antioxidant enzymes (SOD2, CAT and GPx1) and phase II detoxification enzymes (HO-1) by upregulating nuclear Nrf2 and activating the Nrf2 signaling pathway, thus enhancing the antioxidant capacity of cells. © 2022 Society of Chemical Industry.

Crosstalk between Endoplasmic Reticulum Stress and Oxidative Stress in Heat Exposure-Induced Apoptosis Is Dependent on the ATF4-CHOP-CHAC1 Signal Pathway in IPEC-J2 Cells.

The intestinal epithelium is susceptible to heat stress (HS), which leads to gut leakage and inflammation. However, the mechanisms underlying HS-induced intestine dysfunction have yet to be elucidated. We established an in vitro chronic heat exposure-induced intestinal injury of intestinal porcine epithelial cells (IPEC-J2) exposed to high temperatures (43 °C) for 12 h. The results revealed that HS increased reactive oxygen species (ROS) generation and decreased superoxide dismutase 2 (SOD2) expression, leading to oxidative stress. Western blotting analysis demonstrated that HS induced apoptosis as evidenced by increased cytochrome c (Cyt c) release in the cytoplasm and caspase 3 activation. Transcriptome sequencing analysis revealed that HS activated the endoplasmic reticulum stress (ERS) response/unfolded protein response (UPR) but inhibited glutathione metabolism. Specifically, HS triggered the pro-apoptotic activating transcription factor 4 (ATF4)/CEBP-homologous protein (CHOP) branch of the UPR. Interestingly, glutathione-specific gamma-glutamylcyclotransferase1 (CHAC1) involved in glutathione degradation was upregulated due to heat exposure and was proved to be downstream of the ATF4-CHOP signal pathway. Knockdown of CHAC1 attenuated the HS-induced decrease in glutathione level and cell apoptosis. These studies suggest that crosstalk between ERS and oxidative stress in HS-induced apoptosis might be dependent on the ATF4-CHOP-CHAC1 signal pathway in IPEC-J2 cells.

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.

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.

1,25-Dihydroxyvitamin D3 modulates calcium transport in goat mammary epithelial cells in a dose- and energy-dependent manner.

BACKGROUND: Calcium is a vital mineral and an indispensable component of milk for ruminants. The regulation of transcellular calcium transport by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3, the active form of vitamin D) has been confirmed in humans and rodents, and regulators, including vitamin D receptor (VDR), calcium binding protein D9k (calbindin-D9k), plasma membrane Ca(2+)-ATPase 1b (PMCA1b), PMAC2b and Orai1, are involved in this process. However, it is still unclear whether 1,25-(OH)2D3 could stimulate calcium transport in the ruminant mammary gland. The present trials were conducted to study the effect of 1,25-(OH)2D3 supplementation and energy availability on the expression of genes and proteins related to calcium secretion in goat mammary epithelial cells. METHODS: An in vitro culture method for goat secreting mammary epithelial cells was successfully established. The cells were treated with different doses of 1,25-(OH)2D3 (0, 0.1, 1.0, 10.0 and 100.0 nmol/L) for calcium transport research, followed by a 3-bromopyruvate (3-BrPA, an inhibitor of glucose metabolism) treatment to determine its dependence on glucose availability. Cell proliferation ratios, glucose consumption and enzyme activities were measured with commercial kits, and real-time quantitative polymerase chain reaction (RT-qPCR), and western blots were used to determine the expression of genes and proteins associated with mammary calcium transport in dairy goats, respectively. RESULTS: 1,25-(OH)2D3 promoted cell proliferation and the expression of genes involved in calcium transport in a dose-dependent manner when the concentration did not exceed 10.0 nmol/L. In addition, 100.0 nmol/L 1,25-(OH)2D3 inhibited cell proliferation and the expression of associated genes compared with the 10.0 nmol/L treatment. The inhibition of hexokinase 2 (HK2), a rate-limiting enzyme in glucose metabolism, decreased the expression of PMCA1b and PMCA2b at the mRNA and protein levels as well as the transcription of Orai1, indicating that glucose availability was required for goat mammary calcium transport. The optimal concentration of 1,25-(OH)2D3 that facilitated calcium transport in this study was 10.0 nmol/L. CONCLUSIONS: Supplementation with 1,25-(OH)2D3 influenced cell proliferation and regulated the expression of calcium transport modulators in a dose- and energy-dependent manner, thereby highlighting the role of 1,25-(OH)2D3 as an efficacious regulatory agent that produces calcium-enriched milk in ruminants when a suitable energy status was guaranteed.