Effects Of Dietary Energy Levels On The Intergenerational Energy Metabolism Of Beef Cows During The Transition Period And Placental Sensing Pathway

Cows are the foundation while calves are the key to the beef industry.The problem of low reproductive rate of cows and high mortality rate of calves after birth has long existed in the "small group,large-scale" extensive feeding model of beef cow in China.The transition period,as a critical period for fetal index growth,was often affected by factors such as individual fetal development,which leaded to a decrease in feed intake and insufficient nutrient intake in cows,resulted in negative energy balance and affected the fetal growth and development.The placenta has been studied and confirmed in humans and mice to coordinate the distribution of nutrients and promote healthy fetal development.The effect of dietary energy levels during the transition period on energy metabolism between cow and calf and placental sensing pathway is still unclear.Therefore,this study aimed to investigate the energy levels during the transition period on the growth,energy metabolism,and microbial composition of calves through three experiments,revealing the energy sensing signaling pathways of placenta and providing theoretical and practical support for the integrated cultivation of beef cows and calves.Experiment Ⅰ Effect of dietary energy levels during the transition period on growth performance and blood biochemical indexes of calves18 healthy Simmental crossbred primiparous cows(Simmental bulls× Yunnan local yellow cows: weight was 338.44 ± 16.03 kg,days of pregnancy was 242 ± 4 days)were divided into three treatment groups using a completely randomized block experimental design,namely low energy group(LE,NEm=5.69 MJ/kg,0.9NASEM;TDN=63.43%),medium energy group(ME,NEm=6.28 MJ/kg,1.0 NASEM;TDN=66.01%),and high energy group(HE,NEm=6.90 MJ/kg,1.1 NASEM;TDN=69.74%).Each treatment group had 6replicates,with 1 cow per replicate.The concentrate to roughage ratio of the diet was 40:60,and all basic diets were equal nitrogen diets.The experiment included a prefeeding period of 15 days and a formal feeding period of 60 days,with the formal period ranging from 30 days before delivery to 30 days after delivery.Cows were fed with different energy levels on the 30 days before delivery,and the postpartum dietary energy levels remained consistent(NEm=6.28 MJ/kg;TDN=66.01%).Serum samples were collected from calves on the day 0(before colostrum feeding),day 7,day 14,and day 30 after birth,the results showed as below:(1)The daily average NEm intake in the HE group,ME group,and LE group was 50.92 MJ,46.14 MJ,and 41.82 MJ respectively.The birth weight,day 30 end weight,and ADG(average daily gain)of the HE group calves were significantly higher than those in the LE group(P < 0.05).(2)The serum content of GLU(glucose)in the HE group calves was significantly higher than that in the LE group after birth(P < 0.05),and the serum content of TP(total protein)was significantly lower than that in the ME and LE groups on the day 7(P < 0.05);The serum content of BHBA(β-hydroxybutyric acid)in the LE group calves was significantly higher than that of HE group and ME group on day 0(P < 0.05).(3)The serum contents of GSH-Px(glutathione peroxidase),CAT(catalase),Ret(retinol)in the HE group calves were significantly lower than those in the LE and ME groups on day 0(P < 0.05),while the content of d-ROMs(derivative of reactive oxygen metabolites)was significantly higher than those in the LE group on day 0 and significantly lower than those in the LE and ME groups on day 7(P < 0.05).(4)The serum contents of Cor(cortisol),TNF-α(Tumor Necrosis Factor-α)and IL-6(interleukin-6)in the HE group calves calves were significantly higher than that of the LE group on day 0(P < 0.05),while the content of IL-10(interleukin-10)was significantly lower than that of the LE group(P < 0.05).On day 7,the serum contents of CER(ceruloplasmin)and Cor in the HE group calves were significantly higher than those in the LE and ME groups(P < 0.05).(5)The serum content of IGF-1(insulin-like growth factor 1)in the HE group calves was significantly higher than that in the LE group on days 0,14,and 30(P < 0.05),while the content of FGF-21(fibroblast growth factor 21)in the LE group was significantly higher than that in the HE and ME groups on days 7 and14(P < 0.05).The above results indicated that increasing the energy level of cows during the transition period was more conducive to improving the birth weight and postnatal growth of calves,but there was an increased risk of inflammatory factors and neonatal stress in calves.The NEm level during the periparturient period selected at 6.90 MJ/kg was more conducive to fetal development and postnatal growth of calves.Experiment II Effect of dietary energy levels during the transition period on the metabolomics and microbial composition of maternal and calvesOn the basis of experiment 1,LC-MS was used to analyze the serum metabolomics differences of Cow on the day 7 before delivery,on the day 0 after birth(before feeding colostrum)and on the day 30 of calves.RT-q PCR was used to determine the gene expression of placenta development and nutrients transport function.high-throughput sequencing technology(Illumina novaseq 6000)was used to determine the cow bacterial community composition on the day of calving and that of calf meconium and day 30 feces.Correlation of differential metabolites,microorganisms and placental genes were analysised.the results showed as below:(1)The HE group Cows upregulated the tricarboxylic acid cycle,glutamate and glutamine metabolism,and arginine synthesis metabolism pathway.The serum differential metabolic marker contents of L-leucine,L-glutamate,and malic acid in HE group were significantly higher than those in the ME and LE groups(P <0.05).The LE group upregulated glycerol phospholipid metabolism pathway,and the serum differential metabolic marker contents of kynurenine and phosphatidylcholine were significantly higher than those in the ME and HE groups(P < 0.05).High energy level increased energy synthesis and protein metabolism in cows during the transition period,while low energy level increased fat breakdown to generate negative energy balance.(2)On day 0,the HE group calves upregulated retinoic acid metabolism and PI(phosphatidylinositol)signaling pathway,and the serum differential metabolic marker contents of 4-hydroxyretinoic acid and phosphatidylinositol were significantly higher than those in the ME and LE groups(P < 0.05);The LE group upregulated alanine and Aspartate metabolism,and the serum differential metabolic marker content of alanine was significantly higher than that in the ME and HE groups(P < 0.05).on the day 30,HE group calves upregulated steroid hormone synthesis metabolism and purine metabolism,and the serum differential metabolic marker content of taurocholate and adenosine were significantly higher than those in the ME and HE groups(P < 0.05).The serum differential metabolic marker content of serine in the LE group was significantly higher than that in the ME and HE groups(P < 0.05).high energy level during the transition period increased the energy synthesis of calves,while the low energy level increased protein metabolism.(3)The genus abundance of cow fecal Prevotella was significantly decreased while that of Roseburia genus was significantly increased(P < 0.05)in the HE group cows(P < 0.05),while the genus abundance of Bacteroides and Candidatus＿Saccharimonas were significantly increased in the LE group cows(P < 0.05).There were significant differences in lipid metabolism,glycan biosynthesis and metabolism pathways among bacterial communities in cows(P < 0.05).There was no significant difference in microbial composition and diversity between calf meconium(P > 0.05),but the calf meconium of HE group showed an increase in Lactobacillus,Faecalibacterium,and Fusobacterium,and a significant increase in Ruminococcaceae,Bacteroides,and Alloprevotella genera in feces at day 30(P < 0.05).The calf meconium of LE group showed an increase in Clostridium＿sensu＿stricto＿1 and Escherichia-Shigella,and a significant increase in Clostridium＿sensu＿stricto＿1 genera in feces at day 30(P < 0.05).There was significant difference in glycan biosynthesis and metabolism pathway in the bacterial community of fetal calves(P <0.05),and significant differences in glycan biosynthesis and metabolism pathway,Xenobiotics biodegradation and metabolism pathway were observed at day 30 in calves(P < 0.05).Improving the energy level during the transition period could increase the colonization of anti-inflammatory microbiota and microbiota involved in carbohydrate degradation in cows and pro-inflammatory properties in calves,reducing the energy level increased the colonization of nutrient absorption characteristics microbiota in cows and disrupted the intestinal health characteristics microbiota of calves.(4)The m RNA expression levels of GLUT1,GLUT3,IGF-1,IGF-2,VEGFA,SLC38A1(38 member of amino acid transporter solute carrier family 1)in the placenta of cows in HE group were significantly higher than those in LE and ME groups(P < 0.05),and the m RNA expression levels of SOD1(superoxide dismutase 1)and CAT(catalase)were significantly lower than those in LE group(P < 0.05),Improving the energy level of cows during the transition period increased the placental glucose and amino acid transport,but reduced the placental antioxidant capacity.(5)The m RNA expression levels of placental GLUT1,GLUT3,SLC38A1,IGF-1,VEGFA,and NOS3(nitric oxide synthase)were positively correlated with calf birth weight and day 30 body weight(r=0.60 to0.77,P < 0.05).The m RNA expression levels of placental GLUT1 and GLUT3 were positively correlated with the serum GLU and LEP concentrations of calves at day 0 and day 30(r=0.60 to 0.89,P < 0.05).The m RNA expression levels of placental SOD1 and CAT were positively correlated with serum GSH-Px and IL-10 levels at day 0 in calves(r=0.63 to 0.76,P < 0.05).The expression of placental promoting nutrient transport genes was positively correlated with calf growth,while the expression of placental antioxidant genes was positively correlated with the antioxidant capacity of calves.(6)The differential metabolites L-leucine,L-glutamic acid,and malic acid in cows serum were positively correlated with phosphatidylinositol in calves serum at day 0(r=0.61-0.83,P < 0.05),while Lleucine in cow serum was positively correlated with 4-hydroxyretinoic acid in calf serum at day 0(r=0.70,P< 0.05).L-leucine,L-glutamic acid,and phosphatidylinositol were positively correlated with placental GLUT1 and GLUT3 gene m RNA expression(r=0.61 to 0.76,P < 0.05).The high energy level of cows promoted energy synthesis of calves by enhancing the expression and transmission of placental glucose transporters.(7)The three dominant genera of Faecalibacterium,Lactobacillus,and Fusobacterium in meconium were positively correlated with the levels of GLU and d-ROMs in fetal calf serum(r=0.65 to 0.90,P < 0.05),as well as with the expression levels of GLUT1,GLUT3,and IGF-1 in placenta(r=0.62 to 0.83,P < 0.05).The above results indicated that the energy levels of cows during the transition period affected the energy metabolism pathway of cows and calves,as well as the microbial composition involved in the energy metabolism pathway.The GLUT1 and GLUT3 genes in the placenta mediated the energy metabolism transfer between cows and calves and regulated the growth metabolism of calves.Experiment III Study on the energy sensing signal pathway of PlacentaBased on the Experiment I and Experiment II,Experiment III used the bovine placental trophoblast cell line as a model,and added in culture media containing glucose concentrations of 1,2,4,8,and 16 mg/m L,respectively.After 24 hours of treatmented,detection and analysis were conducted to study the adaptive change and sensing signal pathway of the placenta in response to glucose levels.The results showed as below:(1)After subculture of placental trophoblast cell lines,the cell morphology and growth curve,CK-7protein immunofluorescence,PL(placental prolactin)and CG(chorionic gonadotropin)secretion were identified,and the cell function was normal.Subsequent experiments can be conducted.(2)Compared to the 1 mg/m L treatment group,increasing glucose levels to 8 mg/m L significantly increased ATP production and GLUT1,GLUT3,and GLUT4 m RNA expression(P < 0.05).When the glucose level exceeded 8 mg/m L,the The above genes m RNA expression significantly decreased(P < 0.05).(3)the m RNA expression levels of AMPK(AMP activated protein kinase),m TOR(mammalian rapamycin target protein),HIF-1α(Hypoxia inducible factor-1α)、 Akt(protein kinase B)and PI3K(phosphatidylinositol kinase)in placental trophoblast cells significantly increased when Glucose levels exceeding 2 mg/m L(P < 0.05).when the glucose level exceeded 8 mg/m L,The above genes expression significantly decreased(P < 0.05).(4)Under the treatment of 8 mg/m L glucose level in placental trophoblast cells and the addition of m TOR inhibitor Rapa,the expression level of GLUT1,GLUT3,AMPK,m TOR,HIF-1α m RNA expression level significantly decreased(P < 0.05),but the m RNA expression level of AMPK was still significantly higher than that of the 1mg/m L glucose treatment group(P < 0.05).The relative density of m TOR,p-m TOR,pHIF-1α,p-AMPK protein was significantly reduced when Rapa added(P < 0.05).The above results indicated that glucose could mediate expression of glucose transporters and affected energy transfer in bovine placental trophoblast cells through the AMPK/PI3K-m TOR sensing signaling pathway.In summary,increasing the energy level during the transition period improved the birth weight and daily gain of calves.The energy level during the transition period affected the energy metabolism pathway of cows and calves,as well as the composition of fecal microorganisms involved in the energy metabolism pathway.glucose mediated the regulation of placental GLUT1 and GLUT3 gene expression through the AMPK/PI3 K m TOR energy sensing signaling pathway.The expression of placental glucose transporters mediated energy metabolism transfer between cows and calves,affecting fetal calves development and postnatal growth in calves.NEm=6.90 MJ/kg during the periparturient period is more conducive to the development of fetal calves and calf growth.