Methionine alleviates heat stress-induced ferroptosis in bovine mammary epithelial cells through the Nrf2 pathway.

Heat stress (HS) triggers mammary gland degradation, accompanied by apoptosis and autophagy in bovine mammary epithelial cells, negatively affecting milk performance and mammary gland health. Ferroptosis is iron-mediated regulated cell death caused by over production of lipid peroxides, however, the relationship between ferroptosis and HS in bovine mammary epithelial cells has not been clarified. Methionine (Met) plays a notable role in alleviating HS affecting the mammary glands in dairy cows, but the underlying mechanisms require further exploration. Therefore, we evaluated the regulatory effect and mechanism of Met in alleviating HS-induced ferroptosis by using bovine mammary epithelial cell line (MAC-T) as an in vitro model. The results showed that Met improved cell vitality, restored mitochondrial function; reduced the content of various reactive oxygen species (ROS), especially hydrogen peroxide (H2O2) and superoxide anion (O2·-); had positive effects on antioxidant enzyme activity, namely glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). More importantly, Met reduced labile iron protein (LIP) levels; increased iron storage and simultaneously decreased the levels of lipid reactive oxygen species (lipid ROS) and malondialdehyde (MDA), which all caused by HS in MAC-T. Mechanistically, Met increased the protein expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7, member 11 (SLC7A11) and ferritin heavy chain 1 (FTH1) by activating nuclear factor E2-related factor 2 (Nrf2) expression. Additionally, the protection effect of Met was cut off in MAC-T cells after interference with Nrf2, manifesting in decresing the protein expression levels of GPX4, SLC7A11 and FTH1,and increasing the levels of LIP and lipid ROS. Our findings indicate that Met eases HS-induced ferroptosis in MAC-T through the Nrf2 pathway, revealing that Met produces a marked effect on easing HS-induced bovine mammary gland injury in dairy cows.

Comprehensive Profiling of ceRNA (circRNA-miRNA-mRNA) Networks in Hypothalamic-Pituitary-Mammary Gland Axis of Dairy Cows under Heat Stress.

Heat stress (HS) is directly correlated with mammary gland dysfunction and the hypothalamic-pituitary-mammary gland (HPM) axis is involved in regulating stress responses and lactation in dairy cows. Circular RNAs (circRNAs) play major roles in regulating transcription and post-transcription but their expression in the HPM axis of dairy cows under HS is still unclear. In the present study, we performed RNA sequencing to identify diferentially expressed (DE) circRNAs, DE microRNAs(miRNAs) and DEmRNAs, and performed bioinformatics analysis on those in HPM axis-related tissues of heat-stressed and normal cows. A total of 1680, 1112 and 521 DEcircRNAs, 120, 493 and 108 DEmiRNAs, 274, 6475 and 3134 DEmRNAs were identified in the hypothalamic, pituitary, and mammary gland tissues, respectively. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses indicated that the MAPK signaling pathway is potentially a key pathway. Competitive endogenous RNA (ceRNA) networks related to HS response and lactation regulation were established in three tissues. In conclusion, our results indicate that HS induces differential circRNA expression profiles in HPM axis-related tissues, and the predicted ceRNA network provides a molecular basis for regulating the stress response and lactation regulation in heat-stressed dairy cows.

Identification and bioinformatics analysis of differentially expressed milk exosomal microRNAs in milk exosomes of heat-stressed Holstein cows.

In summer, heat stress is one of the primary reasons for the compromised health and low milk productivity of dairy cows. Hyperthermia affects milk synthesis and secretion in the mammary glands of dairy cows. As molecules for intercellular communication, milk-derived exosomes carry genetic material, proteins, and lipids, playing a crucial role in mammary tissue growth and milk synthesis in dairy cows. The aim of this study was to explore the milk exosomal miRNA profile of heat-stressed and normal Holstein cows. We isolated and identified milk exosomes to screening for differentially expressed miRNAs using small RNA sequencing. Then, TargetScan and miRanda algorithms were used to predict the putative targets of the differentially expressed miRNAs, whereas GO and KEGG pathway enrichment analyses were performed for the differentially expressed miRNA-target genes. Our results showed that 215 miRNAs were significantly differentially expressed in heat-stressed milk exosomes, of which one was upregulated and 214 were significantly downregulated. GO and KEGG enrichment analyses indicated that differentially expressed miRNAs might play a role in apoptosis, autophagy, and the p38 MAPK pathway. qRT-PCR assay verified that the expression of miRNAs was consistent with the sequencing results, warranting further verification of their specific targets of action. In conclusion, changes in the miRNA expression profile of milk exosomes indicated the role of exosomal miRNAs in regulating heat stress resistance and apoptosis in dairy cows. Our results suggested that milk-derived exosomal miRNAs could increase mammary gland resistance to heat stress, thereby enhancing milk synthesis in dairy cows.

Nicotinamide mononucleotide alleviates heat stress-induced oxidative stress and apoptosis in BMECs through reducing mitochondrial damage and endoplasmic reticulum stress.

Heat stress is directly correlated to mammary gland dysfunction in dairy cows, especially in summer. Abnormally high environmental temperature induces oxidative stress and apoptosis in bovine mammary epithelial cells (BMECs). Nicotinamide mononucleotide (NMN) has beneficial effects in maintaining the cellular physiological functions. In this study, we evaluate the protective effect of NMN on heat stress-induced apoptosis of BMECs and explore the potential underlying mechanisms. Our results showed that heat stress considerably decreased cell viability in BMECs, whereas pretreatment of BMECs with NMN (150 µM) for 24 h significantly alleviated the negative effects of heat stress on cells. NMN protected BMECs from heat stress-induced oxidative stress by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing the activity of antioxidant enzymes. It also inhibited apoptosis by reducing the ratio of Bax/Bcl2 and blocking proteolytic the cleavage of Caspase-3 in heat stressed-BMECs. Importantly, NMN treatment could reduce mitochondrial damage through mediating the expression of mitochondrial fission and fusion-related genes, including Dynamin related protein 1 (Drp1), Mitochondrial fission 1 protein (Fis1), and Mitofusin1, 2 (MFN1, 2); and suppress endoplasmic reticulum stress through unfolded protein response regulator Glucose regulated protein 78 (GRP78), and downstream elements Recombinant activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). Above all, our results demonstrate that NMN supplemention attenuates heat stress-induced oxidative stress and apoptosis in BMECs by maintaining mitochondrial fission and fusion, and regulating endoplasmic reticulum stress, which provides the convincing evidence that NMN has valuable potential in alleviating mammary gland injury of dairy cows caused by environmental heat stress.

Effects of zinc-bearing palygorskite on rumen fermentation in vitro.

OBJECTIVE: The aim of the study was to investigate the effect of zinc-bearing palygorskite (Zn-Pal) on rumen fermentation by in vitro gas-production system. METHODS: In trial, 90 incubators were evenly divided into five groups: control (0% Zn-Pal), treatment I (0.2% Zn-Pal), treatment II (0.4% Zn-Pal), treatment III (0.6% Zn-Pal), and treatment IV (0.8% Zn-Pal). The contents of zinc for treatments were 0, 49, 98, 147, 196 mg/kg, respectively. The main chemical composition and microstructure of Zn-Pal was investigated by X-ray diffraction. The physicochemical features were evaluated by Zeta potential analysis, cation-exchange capacity, ethylene blue absorption and specific surface area (the Brunauer-Emmett-Teller method). In vitro gas production (GP) was recorded at 3, 6, 9, 12, 18, 24, 36, 48, 60, and 72 h incubation. Incubation was stopped at 0, 6, 12, 24, 48, and 72 h and the inoculants were tested for pH, microbial protein yield (MCP), NH3-N, volatile fatty acids (VFAs), lipopolysaccharide (LPS). RESULTS: The results showed that the GP in the treatment groups was not significantly different from the control groups (p>0.05). Compared to the control group, pH was higher at 24 h, 48 h (p<0.05), and 72 h (p<0.01) (range 6 to 7). The concentration of NH3-N in the three treatment groups was higher than in the control group at 24 h (p<0.01), meanwhile, it was lower at 48 h and 72 h (p<0.01), except in the treatment IV. The concentration of MCP in treatment I group was higher than in the control at 48 h (p<0.01). Compared with control, the LPS concentration in treatment III became lower at 12 h (p<0.05). Total VFAs in treatments were higher than in the control at 24 h, 48 h (p<0.05). CONCLUSION: These results suggest that the addition of Zn-Pal can improve the rumen fermentation, especially when adding 0.2% Zn-Pal.

Effects of different combinations of antibacterial compound supplements in calf pellets on growth performance, health, blood parameters, and rumen microbiome of dairy calves.

This study investigated the effects of different combinations of antibacterial compounds (attapulgite, plant essential oils, and chitosan oligosaccharides) on growth performance, blood biochemical parameters, and rumen microbiome of calves. A total of 48 preweaning calves were randomly divided into four groups (n = 12 per group), and fed the following full mixed-ration granule diets for the 67-d-feeding trial: (1) basal diet (control group); (2) basal diet +1,000 g/t attapulgite, plant essential oils, and chitosan oligosaccharide (AEOCO group); (3) basal diet +1,000 g/t attapulgite and chitosan oligosaccharide (ACO group); and (4) basal diet +1,000 g/t attapulgite and plant essential oil (AEO group). The results showed that the daily weight gain of the AEOCO and AEO groups significantly increased (p < 0.05), whereas the feed conversion ratio decreased compared with that of the control group. Among the three treatment groups, AEO group showed the most positive effect, with the diarrhea rate reduced by 68.2% compared with that of the control group. Total protein and globulin levels were lower in the AEO group than in the control group. Albumin levels were higher in the AEOCO and AEO groups than in the control group. Immunoglobulin A, immunoglobulin G, and immunoglobulin M concentrations were higher in the AEOCO group (p < 0.05) than in the control group. The interleukin-6 concentration was lower in the AEOCO and AEO groups than in the control group (p < 0.05). The Chao 1 richness and ACE indices were higher in the AEOCO group than in the control group (p < 0.05). The ACO group had a significantly lower (p < 0.05) relative abundance of Firmicutes than the control group. The relative abundance of Bacteroidetes was the lowest in the control group, whereas that of Spirochaetota and Fibrobacteriota was the highest (p < 0.05). The relative abundance of Succiniclasticum was higher in the ACO and AEO groups (p < 0.05). These findings indicate that the combination of attapulgite, plant essential oils, and chitosan oligosaccharides has ameliorative effects on the growth performance, blood parameters, and rumen microbiome of calves.

Circ_002033 Regulates Proliferation, Apoptosis, and Oxidative Damage of Bovine Mammary Epithelial Cells via the miR-199a-5p-MAP3K11 Axis in Heat Stress.

Heat stress is becoming the major factor regarding dairy cow health and milk quality because of global warming. Circular RNAs (circRNAs) represent a special type of noncoding RNAs, which are related to regulating many biological processes. Nonetheless, little is known concerning their effects on heat-stressed bovine mammary epithelial cells (BMECs). Here, this study found a novel circRNA, circ_002033, using RNA sequencing (RNA-seq) and explored the role and underlying regulatory mechanism in proliferation, apoptosis, and oxidative damage in a heat-stressed bovine mammary epithelial cell line (MAC-T). According to the previous RNA-seq analysis, the abundance of circ_002033 in mammary gland tissue of heat-stressed cows increased relative to nonheat-stressed counterparts. This study found that the knockdown of circ_002033 promoted proliferation and alleviated apoptosis and oxidative damage in heat-stressed MAC-T. Mechanistically, circ_002033 localizes to miR-199a-5p in the cytoplasm of MAC-T to regulate mitogen-activated protein kinase kinase 11 (MAP3K11) expression. Meanwhile, miR-199a-5p and MAP3K11 are also involved in regulating the proliferation and apoptosis of heat-stressed MAC-T. Importantly, circ_002033 knockdown promoted the expression of miR-199a-5p while decreasing that of MAP3K11, thereby enhancing proliferation while alleviating apoptosis and oxidative damage in heat-stressed MAC-T. In summary, we found that circ_002033 regulates the proliferation, apoptosis, and oxidative damage of heat-stressed BMECs through the miR-199a-5p/MAP3K11 axis, providing the theoretical molecular foundation for mitigating heat stress of dairy cows.

Effect of Methionine Hydroxy Analog on Hu Sheep Digestibility, Rumen Fermentation, and Rumen Microbial Community In Vitro.

This experiment was conducted to evaluate the effects of a methionine hydroxy analog (MHA) on in vitro gas production, rumen fermentation parameters, and rumen microbiota. Two different MHA, 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester (HMBi) and the calcium salt of the hydroxy analog of methionine (MHA-Ca), were selected for in vitro experiments. The treatments were the Control group (0% of MHA), HMBi group (2%HMBi), and MHA-Ca group (2%MHA-Ca). Dry matter digestibility was measured after 12 h and 24 h of fermentation, and fermentation parameters and microbial composition were analyzed after 24 h. HMBi and MHA-Ca showed increased (p = 0.001) cumulative gas production in 3 h. The total volatile fatty acids, microbial protein (MCP) concentration, acetate, and acetate to propionate ratio in the HMBi and MHA-Ca groups were significantly higher than those in the Control group (p = 0.006, p = 0.002, p = 0.001, p = 0.004), and the NH3-N concentrations in the HMBi and MHA-Ca groups were significantly lower than those in the Control group (p = 0.004). The 16S rRNA sequencing revealed that the HMBi group had a higher (p = 0.039, p = 0.001, p = 0.027) relative abundance of Bacteroidetes, Firmicutes, and Synergistetes and a lower relative abundance of Proteobacteria (p = 0.001) than the Control group. At the genus level, Prevotella abundance was higher (p = 0.001), while Ruminobacter abundance was lower (p = 0.001), in the HMBi and MHA-Ca groups than in the Control group. Spearman's correlation analysis showed that the relative abundance of Prevotella_1, Streptococcus, and Desulfovibrio was positively correlated with dry matter digestibility, MCP, and fermentation parameters. MHA, thus, significantly increased gas production and altered the rumen fermentation parameters and microbiota composition of sheep.

Preliminary Transcriptome Analysis of Long Noncoding RNA in Hypothalamic-Pituitary-Mammary Gland Axis of Dairy Cows under Heat Stress.

Heat stress (HS) is directly correlated to mammary gland dysfunction in dairy cows, especially in summer. The hypothalamic-pituitary-mammary gland axis (HPM axis) plays an important role in the regulation of stress response and lactation physiology in heat-stressed dairy cows. The aim of this study was to explore the lncRNA profile, and the competitive endogenous RNA (ceRNA) regulatory network in hypothalamus, pituitary, and mammary gland tissues of heat-stressed and normal dairy cows. We performed RNA sequencing (RNA-seq) to identify differentially expressed (DE) lncRNAs, and the ceRNA regulatory network was established in HPM-axis-related tissues. Our results showed that a total of 13, 702 and 202 DE lncRNAs were identified in hypothalamus, pituitary, and mammary glands, respectively. Of lncRNAs, 8, 209 and 45 were up-regulated, and 5, 493 and 157 lncRNAs were down-regulated. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that DE lncRNAs target genes that might play a role in hormone synthesis, secretion and action, apoptosis, mitogen-activated protein kinase (MAPK), AMP-activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR) signaling pathway. Moreover, the ceRNA regulatory network associated with the MAPK signaling pathway in HPM-axis-related tissues contains 3286 lncRNA-mRNA pairs. Furthermore, the ceRNA regulatory network associated with apoptosis, prolactin, AMPK, and mTOR signaling pathway in the mammary gland contains 772 lncRNA-mRNA pairs. Thus, some lncRNAs may be involved in the regulation of stress response and the physiological process of lactation. The changes in lncRNA expression profiles and ceRNAs (lncRNA-miRNA-mRNA) in HPM-axis-related tissues are the key to affect the stress response and lactation physiology of dairy cows under HS, which provide a theoretical basis for the molecular mechanism in the stress response of HPM-axis-related tissues in dairy cows under HS.

Dietary Palygorskite-Based Antibacterial Agent Supplementation as an Alternative to Antibiotics Improves Growth Performance, Blood Parameters, and Rumen Microbiota in Sheep.

This research aimed to investigate the effects of a palygorskite-based antibacterial agent (PAA) as an alternative to antibiotics on growth performance, blood parameters, and rumen microbiota in sheep. A total of 120 sheep were randomly divided into five groups of six replicates with four sheep each. Sheep were fed a basal diet, an antibiotic diet supplemented with 500 g/t chlortetracycline (CTC), and a basal diet supplemented with 500, 1000, and 2000 g/t PAA for 80 d, respectively. Supplementation with 2000 g/t PAA and 500 g/t CTC increased the average daily gain (ADG) of sheep compared with the control group (p < 0.05). Diets supplemented with 2000 g/t PAA and 500 g/t CTC reduced (p < 0.05) the feed:gain ratio (F/G ratio) in the overall periods. Dietary supplementation with 1000 g/t PAA significantly increased albumin and total protein (p < 0.05). A significant positive correlation was found between growth hormone concentration and PAA supplementation (p < 0.05). In addition, compared to the control group, the CTC group had higher growth hormone concentration and lower lipopolysaccharide concentration (p < 0.05). No difference was observed between the five groups in terms of rumen fermentation characteristics (p > 0.05). At the phylum level, the relative abundance of Proteobacteria was lower in the PAA 2000 and CTC 500 groups than in the control and PAA 500 groups (p < 0.05). At the genus level, a significant decrease (p < 0.05) in the relative abundance of RuminococcaceaeUCG-010 was observed in the PAA 1000, PAA 2000, and CTC 500 groups compared with that in the control group. In addition, the relative abundance of Prevotella1 (p < 0.05) was higher in the PAA 2000 group than in the control group. These findings indicate that dietary supplementation with PAA has ameliorative effects on growth performance, blood parameters, and rumen microbiota, with an optimal dosage of 2000 g/t for sheep.

Comparison of ruminal microbiota, metabolomics, and milk performance between Montbéliarde×Holstein and Holstein cattle.

Holstein cattle are well known for their high average milk yield but are more susceptible to disease and have lower fecundity than other breeds of cattle. The purpose of this study was to explore the relationship between ruminal metabolites and both milk performance and ruminal microbiota composition as a means of assessing the benefits of crossbreeding Montbéliarde and Holstein cattle. This experiment crossbred Holstein with Montbéliarde cattle, aimed to act as a reference for producing high-quality dairy products and improving the overall efficiency of dairy cattle breeding. Based on similar age, parity and lactation time, 46 cows were selected and divided into two groups (n = 23 per group) for comparison experiment and fed the same formula: Montbéliarde×Holstein (MH, DIM = 33.23 ± 5.61 d), Holstein (H, DIM = 29.27 ± 4.23 d). Dairy herd improvement (DHI) data is an important basis for evaluating the genetic quality of bulls, understanding the quality level of milk, and improving feeding management. We collected the DHI data of these cows in the early lactation, middle lactation and late lactation period of 10 months. The results showed that the average milk production and protein content in Montbéliarde×Holstein were 1.76 kg (34.41 kg to 32.65 kg, p > 0.05) and 0.1% (3.54 to 3.44%, p < 0.05) higher than in Holstein cattle. Moreover, milk from Montbéliarde×Holstein cattle had lesser somatic cell score (1.66 to 2.02) than Holstein cattle (p < 0.01). A total of 10 experimental cattle in early lactation were randomly selected in the two groups (Lactation time = 92.70 ± 6.81), and ruminal fluid were collected by oral gastric tube. Using 16S rRNA microbial sequencing, we compared the ruminal microbiota composition and found that Montbéliarde×Holstein cattle had a lower abundance of Alphaproteobacteria (p < 0.05) and higher abundance of Selenomonas than Holstein cattle (p < 0.05). These bacteria play roles in protein degradation, nitrogen fixation and lactic acid degradation. The abundance of Succiniclasticum was also greater in Montbéliarde×Holstein cattle (p = 0.053). Through ruminal metabolome analysis, we found that the levels of trans-ferulic acid, pyrrole-2-carboxylic acid, and quinaldic acid were significantly increased in Montbéliarde×Holstein cattle, while that of lathosterol was significantly decreased. The changes in the levels of these metabolites could confer improved antioxidant, anti-inflammatory, and antibacterial activities.

Enhancement of BCAT2-Mediated Valine Catabolism Stimulates ß-Casein Synthesis via the AMPK-mTOR Signaling Axis in Bovine Mammary Epithelial Cells.

Valine, a kind of branched-chain amino acid, plays a regulatory role beyond that of a building block in milk protein synthesis. However, the underlying molecular mechanism through which valine stimulates ß-casein synthesis has not been clarified. Therefore, our study aimed to evaluate the effect of valine on ß-casein synthesis and shed light into the molecular mechanism using an in vitro model. Results showed that valine supplementation significantly increased ß-casein synthesis in bovine mammary epithelial cells (BMECs). Meanwhile, the supplementation of valine resulted in high levels of branched-chain aminotransferase transaminase 2 (BCAT2), TCA-cycle intermediate metabolites, and ATP, AMP-activated protein kinase (AMPK) inhibition, and mammalian target of rapamycin (mTOR) activation. Furthermore, the inhibition of BCAT2 decreased the ß-casein synthesis and downregulated the AMPK-mTOR pathway, with similar results observed for AMPK activation. Together, the present data indicate that valine promotes the synthesis of ß-casein by affecting the AMPK-mTOR signaling axis and that BCAT2-mediated valine catabolism is the key target.

Effect of methionine hydroxy analog feed supplements: Significant alteration and enrichment of rumen microbiota and metabolome in Hu sheep.

Methionine hydroxy analogs (MHA) are widely used as the main sources of methionine in ruminant feed production. The purpose of this study was to explore the effect of using MHA supplements such as MHA as a salt of calcium (MHA-Ca) and 2-hydroxy-4-(methylthio)-butanoic acid isopropyl ester (HMBi) as sources of methionine on the rumen microbiota and metabolome in Hu sheep. Seventy-two healthy Hu sheep were randomly assigned to three dietary treatment groups: control, MHA-Ca, and HMBi groups. The results showed that the concentrations of total volatile fatty acids, acetate, and propionate were higher in the HMBi group than in the control group. The HMBi and MHA-Ca groups had higher alpha diversity values than those in control group. We compared the rumen microbiota by using 16S rRNA gene sequencing. At the phylum level, the HMBi group had a higher relative abundance of Firmicutes and a lower relative abundance of Synergistetes than did the control group. At the genus level, the control group had a higher relative abundance of Treponema_2 than did the HBMi group and a higher relative abundance of Prevotellaceae_UCG_004 than did the MHA-Ca group. Metabolomic analyses revealed that fatty acids, amino acids, lipids, organic acids, sugars, amines, and nucleosides were significantly altered in both MHA-Ca and HMBi groups. Metabolites with significant differences were enriched in amino acid and carbohydrate metabolisms, such as phenylalanine metabolism, biosynthesis of amino acids, tryptophan metabolism, galactose metabolism, and tyrosine metabolism. Above all, the findings presented in this study indicate that MHA alter the rumen microbiota and metabolites and that different forms of MHA have different impacts. The results of our study contribute to a better understanding of the effects of MHA.

Characteristics of ruminal microbiota and metabolome in Holstein cows differing in milk protein concentrations.

The rumen is a vital organ containing vast amounts of microbes that play a key role in the digestion of nutrients and affect the production performance of ruminants. However, few studies have focused on the characterization of the ruminal microbiota composition and function in cows with long-term difference milk protein concentrations, and the relationship between milk protein concentration and ruminal microbiota remains elusive. In this study, we collected the data of milk protein concentrations of 1,025 Holstein cows for 10 mo on a commercial farm. Based on the milk protein concentrations, 30 cows were selected and divided into three groups (n = 10 per group): low milk protein group (LMP, milk protein concentration < 3.1%), medium milk protein group (MMP, 3.1% ≤ milk protein concentration < 3.4%), and high milk protein group (HMP, milk protein concentration ≥ 3.4%). The ruminal microbiome, metabolome, VFA concentrations and proportions, and amino acid profiles of the three groups were analyzed. The data showed that free amino acid (FAA) levels were lower in the rumen and higher in the plasma of HMP cows (P < 0.05). In addition, lower NH3 concentrations were observed in the rumen, plasma, and milk of the HMP cows (P < 0.05). Protease activity and isobutyric acid molar proportion in the rumen were lower in the HMP group (P < 0.05). Microbiome analysis showed that HMP cows had lower microbial diversity (represented as Shannon and Simpson indices) than LMP cows. At the genus level, lower relative abundances of Prevotella_1 and Ruminococcaceae_UCG_005 were observed in the HMP group (P < 0.05). At the operational taxonomic unit (OTU) level, a lower relative abundance of OTU3 (Prevotella ruminicola) was observed in the HMP group (P < 0.05). We found that the relative abundances of ruminal Prevotella_1 and OTU3 (Prevotella ruminicola) were negatively correlated with milk protein concentration (P < 0.05). These findings suggested that the cows with long-term high milk protein concentrations had lower microbial diversity and weaker protein degradation ability in the rumen. Furthermore, our observations identified a correlation between the milk protein concentration and ruminal microbiota.

This study aimed to assess the ruminal microbiome, metabolome, volatile fatty acid concentrations, and amino acid profiles of Holstein cows with different milk protein concentrations. Previous studies have reported that ruminal microbiota can affect the lactation performance of dairy cows. However, little is known about the composition and function of ruminal microbiota in dairy cows differing in milk protein concentrations. In this study, we collected the milk protein concentrations data of 1,025 Holstein cows for 10 mo on a commercial farm. Three groups of cows (n = 10 per group) with low, medium, and high milk protein concentrations were selected. We found that cows with long-term high milk protein concentrations had lower microbial diversity, relative abundances of specific ruminal microbiota, protease activity, and amino acid concentration in the rumen compared to the cows with long-term low milk protein concentration. Meanwhile, cows with long-term high milk protein concentration showed higher amino acid concentrations in the plasma and lower ammonia levels in rumen, plasma and milk than cows with low milk protein concentration. Our findings revealed the correlation between milk protein concentration and specific ruminal microbiota, and proposed a possibility that ruminal microbiota affected milk protein concentration by altering host amino acid profile.

SIRT3 protects bovine mammary epithelial cells from heat stress damage by activating the AMPK signaling pathway.

With global warming, heat stress has become an important challenge for the global dairy industry. Sirtuin 3 (SIRT3), an important mitochondrial NAD+dependent decarboxylase and a major regulator of cellular energy metabolism and antioxidant defense, is integral to maintaining normal mitochondrial function. The aim of this study was to assess the protective effect of SIRT3 on damage to bovine mammary epithelial cells (BMECs) induced by heat stress and to explore its potential mechanism. Our results indicate that SIRT3 is significantly downregulated in heat-stressed mammary tissue and high-temperature-treated BMECs. SIRT3 knockdown significantly increased the expression of HSP70, Bax, and cleaved-caspase 3 and inhibited the production of antioxidases, thus promoting ROS production and cell apoptosis in BMECs. In addition, SIRT3 knockdown can aggravate mitochondrial damage by mediating the expression of genes related to mitochondrial fission and fusion, including dynamin-related protein 1, mitochondrial fission 1 protein, and mitochondrial fusion proteins 1and 2. In addition, SIRT3 knockdown substantially decreased AMPK phosphorylation in BMECs. In contrast, SIRT3 overexpression in high-temperature treatment had the opposite effect to SIRT3 knockdown in BMECs. SIRT3 overexpression reduced mitochondrial damage and weakened the oxidative stress response of BMECs induced by heat stress and promoted the phosphorylation of AMPK. Taken together, our results indicate that SIRT3 can protect BMECs from heat stress damage through the AMPK signaling pathway. Therefore, the reduction of oxidative stress by SIRT3 may be the primary molecular mechanism underlying resistance to heat stress in summer cows.

Combined signature of rumen microbiome and metabolome in dairy cows with different feed intake levels.

Feed intake is a major factor in maintaining the balance between ruminal fermentation and the microbial community of dairy cows. To explore the relationship among feed intake, microbial metabolism, and ruminal fermentation, we examined the combined signatures of the microbiome and metabolome in dairy cows with different feed intake levels. Eighteen dairy cows were allocated to high feed intake (HFI), medium feed intake (MFI), and low feed intake (LFI) groups according to their average daily feed intake. 16S rDNA sequencing results revealed that the relative abundance of Firmicutes in the HFI group was significantly higher than that in the MFI and LFI groups (P < 0.05). The ratio of Bacteroidetes to Firmicutes was significantly lower in the HFI group than in the MFI and LFI groups (P < 0.05). The relative abundance of Lachnospiraceae_unclassified, Veillonellaceae_unclassified, and Saccharofermentants was significantly higher in the HFI group than in the LFI and MFI groups (P < 0.05). The relative abundance of Erysipelotrichaceae_unclassified and Butyrivibrio was significantly higher in the HFI group than in the MFI and LFI groups (P < 0.05). Ultra high performance liquid chromatography-mass spectrometry revealed five key pathways, including the linoleic acid metabolism pathway, alpha-linolenic acid metabolism, arginine and proline metabolism, glutathione metabolism, and valine, leucine, and isoleucine biosynthesis, which are closely related to energy and amino acid metabolism. Linoleic acid, glutamate, alpha-linolenic acid, l-methionine, and l-valine levels were significantly lower in the HFI group than in the MFI and LFI groups (q < 0.05), while the relative content of glutamate was significantly lower in the MFI group than in the LFI group (q < 0.05). Stearic acid content was significantly higher in the HFI group than in the LFI group (q < 0.05). Our findings provide insight into the rumen microbiome of dairy cows with different feed intake and the metabolic pathways closely associated with feed intake in early-lactating cows. The candidates involved in these metabolic pathways may be useful for identifying variations in feed intake. The signatures of the rumen microbiome and metabolome in dairy cows may help make decisions regarding feeding.

Analysis of Astragalus Polysaccharide Intervention in Heat-Stressed Dairy Cows' Serum Metabolomics.

This experiment was conducted to investigate the effects of astragalus polysaccharides (APS) on serum metabolism of dairy cows under heat stress. Thirty healthy Holstein dairy cows were randomly divided into three groups (10 cows in each group). In the experimental group, 30 mL/d (Treatment I) and 50 mL/d (Treatment II) of APS injection were injected into the neck muscle respectively. Each stage was injected with APS for 4 days (8:00 a.m. every day) and stopped for 3 days. Serum hormone and antioxidant indexes of dairy cows were investigated. Through repeated measurement analysis of variance, the results have shown that cortisol (COR) (F = 6.982, p = 0.026), triiodothyronine (T3) (F = 10.005, p = 0.012) and thyroxine (T4) (F = 22.530, p = 0.002) at different time points were significantly different. COR showed a downward trend, T3 and T4 showed an upward trend. At each time point, different concentrations of APS have significant effects on COR (F = 30.298, p = 0.000 < 0.05), T3 (F = 18.122, p = 0.001), and T4 (F = 44.067, p = 0.000 < 0.05). However, there were no significant differences in serum insulin (INS), glucagon (GC) and heat shock protein 70 (HSP70) between different time points (p > 0.05) and at each time point (p > 0.05). Additionally, the results have also shown that there were also no significant differences in serum Superoxide dismutase (SOD), malondialdehyde (MDA) and lactate dehydrogenase (LDH) between different time points (p > 0.05) and at each time point (p > 0.05). However, the injection of APS had a significant impact on glutathione peroxidase (GSH-Px) (F = 9.421, p = 0.014) at different times, and showed a trend of rising first and then falling. At each time point, APS of different concentrations had no significant effect on GSH-Px (p > 0.05). Furthermore, we used gas chromatography-mass spectrometry (GC-MS) non-targeted metabolomics to determine the potential markers of APS for heat-stressed dairy cows. Twenty metabolites were identified as potential biomarkers for the diagnosis of APS in heat-stressed dairy cows. These substances are involved in protein digestion and absorption, glutathione metabolism, prolactin signaling pathway, aminoacyl-tRNA biosynthesis, pentose and glucuronate interconversions, and so on. Our findings suggest that APS have an effect on the serum hormones of heat-stressed dairy cows, and regulate the metabolism of heat-stressed dairy cows through glucose metabolism and amino acid metabolism pathways.