Effect Of Glucose Supply On Metabolism In Mammary Gland Of Dairy Goats And Its Regulatory Mechanism

Lactating ruminant animals need large amounts of glucose to maintain energy requirement for lactation and to synthesize milk components.Therefore,proper supply of the metabolizable glucose for dairy animals is the prerequisite for maintaining their high production and healthy mammary glands.Numerous studies relevant to effect of glucose on milk performance of dairy animals have been conducted with limitited information on the regulation mechanism.Understanding how mammary glucose supply affects lactational function in the mammary gland of dairy animals and its metabolic mechanism will contribute to improve the glucose nutrition for ruminants.Thus,a large cohort of dairy goats was firstly investigated to analyze the correlation between glucose utilization and milk yield in the the current study.Based on the obtained results,further experiments included gradient infusion of glucose in local mammary gland,and in vitro study with dairy goat primary mammary epithelial cells model,in combination with the physiological mechanism exploration and bioinformatics anaysis,aiming to systematically investigate differences of lactation performance and metabolic characteristics of dairy goats with different mammary glucose supply;to further study the changes in dairy goat mammary metabolism with different glucose supply;and finally to analyze the key signal pathway to regulate lactose yield in dairy goats.The result from the current study will be beneficial to establish the nutritional strategy for improving lactose synthesis,and also to provide theoretical basis and technical support for maintaining the high production of lactating dairy animals.1 Metabolic characteristics of dairy goats with difference in mammary glucose utilization and their lactation performance(Part 1)A group of 75 dairy goats(body weight:58.7 ± 1.6 kg,days in milk:132±16,milk yield:1.85 ± 0.68 kg/d)were selected from a farm.Milk yield was recorded,and samples of blood were collected from the carotid artery and mammary vein for the determination of blood parameters,physiological and biochemical indicators.The difference in glucose concentration between carotid artery and mammary vein was calculated to reflect the glucose utilization.The correlation was analyzed between the indicators related to glucose metabolism and lactation performance to investigate the effect of glucose on lactation performance.Then,dairy goats with the highest(n=6)or lowest(n=6)arteriovenous difference in glucose concentration were selected to study how the mammary gland utilization of glucose exerted the influences on glucose metabolism,immunity,mammary function and finally lactation performance.Blood biochemical indicators including concentration of carotid glucose(P<0.01);and glucose(P<0.01),lactate(P<0.01),and BHBA in mammary vein(P=0.01)were significantly correlated with milk yield.Compared those with lower mammary arteriovenous glucose difference(lower utilization).dairy goats with higher arteriovenous glucose difference(higher utilization)had higher milk yield(P<0.01),lactose yield(P<0.01),and venous CO2 concentrtion(P=0.01),but lower venous leucocyte number(P=0.05).The vein transcriptomes showed that the expression of genes in glycolysis/gluconeogenesis pathway was down-regulated in dairy goats with higher mammary glucose utilization,while the expression of genes in NF-κB signaling pathway and NOD-like receptor pathway was up-regulated in those with lower mammary glucose utilization.These results suggested that a close correlation existed between mammary glucose utilization and milk production,which may be related to differences in glucose metabolism,immune status and mammary gland metabolic rate.Changing mammary glucose supply may have regulatory effects on milk production and mammary metabolism.2 Effect and regulation mechanism of mammary glucose supply on milk yield of dairy goats(Part 2)In order to study how the mammary gland responds to the gradient glucose supply at local mammary gland,six dairy goats at middle lactation were installed with external arterial catheterization.They were given a basal diet that met 81%of the energy requirements for lactation,and assigned to one of six doses(0,20,40,60,80,and 100 g/d)of glucose infusion.The experiment was performed in a 6 × 6 Latin square design.Milk yield was recorded.Milk samples were collected for determing milk composition,glucose-related metabolites,index of oxidative stress and concentration of amino acids.Blood samples were collected to determine blood biochemical parameters,insulin,glucagon and amino acid.Milk yield,and lactose yield increased in a quadratic manner(P<0.01)with the increasing glucose infusion and reached the highest at infusion level of 60 g/d.Mammary blood flow(P<0.05),mammary artery glucose supply(P<0.05)increased linearly with the increasing infusion of glucose from 0 to 60 g/d,and then levelled off.Glucose utilization decreased significantly(P<0.01)with the increasing infusion of glucose.The milk concentration of glucose(P=0.03)and radical oxygen absorbance capacity(P=0.05)increased in a quadratic manner,whereas milk glucose-6-phosphate(P<0.01)and reactive oxygen species(P<0.01)decreased in a quadratic manner.The ratio of glucose-6-phosphate to glucose in milk tended to decrease in a quadratic manner(P=0.07).Lactate concentration increased linearly(P=0.02),while milk malondialdehyde tended to decrease(P=0.07)with increasing infusion of glucos.Glucose infusion caused an increased blood insulin in all infusion groups(P<0.01),while the dynamic changes of glucagon were inversely proportional to insulin.With the increasing infusion of glucose,revised quantitative insulin sensitivity check index decreased in a quadratic manner(P<0.01).The result indicate that mammary glucose supply can regulate lactation performance in a dose-dependent manner vir mammary blood flow,glucose utilization and metabolism.Excess supply of glucose may induce acute glycolysis,causing the accumulation of reactive oxygen species in the mammary gland.This may be the direct cause for the decreased lactose synthesis and adverse mammary health under excess mammary supply of glucose.3 Changes in mammary metabolism induced by glucose and its molecular mechanism(Part3)Based on the results of the above gradient glucose infusion,three glucose doses including 0,60.and 100 g/d were selected to simulate three mammary glucose states:glucose deficiency,glucose optimum,and glucose excess.Gradient infusion was performed again in a repeated 3 × 3 Latin square design,with 12 days each period,including 5 d transition and 7 d infusion.The mammary tissue was biopsied on the last day of each period for analysis of transcriptome.Dynamic network biomarkers sensitive to glucose dose were screened by differential co-expression analysis and network topology analysis.In addition,key driver analysis was performed.We obtained the molecular network of mammary glycolysis induced by excess glucose and found several key driver molecules that control the subnetworks of mammary metabolism altered by glucose excess,including NFKB2,HSPA8,MAP3K14,HEXB,and HSPA5.The NFKB2 was predicted to be the main key driver through the global differential co-expression network.In order to verify the selected DNB and key driving genes,the primary mammary epithelial cells of lactating goats were incubated in vitro at different concentrations(5,10,20 mmol/L)of glucose,and the expression levels of key driver genes in the cells were analyzed at 24 h after treatment.In vitro results were consistent with those in vivo.indicating the accuracy of dynamic network biomarker screening and key driver molecules.On this basis,two types of glucose metabolism modulators(Genoderic acid A,and Galloflavin),NF-κB2 activator(Phorbol 12-myristate 13-acetate)and NF-κB2 inhibitor(4-N-[2-(4-phenoxyphenyl)ethyl]-4,6-quinazolinediamine)were added to medium containing high doses of glucose to determine their ability to regulate glucose metabolism and their effect on NF-κB2.The NF-κB2 played a key role in glycolysis induced by high glucose.By simultaneously blocking NF-κB2,reducing the downstream glycolysis and increasing intracellular ROS scavenging,the metabolic shift towards glycolysis induced by high glucose was inhibited and the harm to mammary health was reduced.In summary,the current study discovered that the effects of mammary glucose supply on milk yield and lactose synthesis are not dependent on the energy balance,but related to glucose utilization rate and intracellular glucose metabolism pathway.Excessive mammary glucose supply can lead to the increased activity of glycolysis and accumulation of free radicals in the mammary gland by NF-κB2,and eventually caused reduced milk yield and lactose yield.By simultaneously inhibiting NF-κB2,targeting the inhibition of glycolysis and accelerating intracellular ROS clearance,the efficiency of lactose synthesis can be increased and the mammary health can be maintained even under excess mammary glucose supply.