Effects Of Heat Stress On Rumen Microbes And Blood Indexes In Holstein Cows At Different Growth Stages

Heat stress has been an important issue in dairy cattle feeding management affecting summer health and economic efficiency.In recent years,global climate change has led to an increase in atmospheric CO₂and average daily temperature,making heat stress a major challenge in dairy farming.This experiment combined 16Sr DNA,metagenomic,metabolomic sequencing and serum index determination to investigate the effects of heat stress on rumen microbiota and serum biochemical,immune,oxidative stress,hormonal in Holstein cows at different stages.In this experiment,10 cows were selected in April and August from growing heifers（monthly age:6.93±0.14）,heifers（monthly age:16.54±0.11）and lactating cows（monthly age:41.02±0.36）.According to the growth stages and temperature and humidity index（THI）,they were divided into 6 treatment groups,namely,growing heifers normal group（CN）,growing heifers heat stress group（C-HS）,heifers normal group（HN）,heifers heat stress group（H-HS）,lactating cow normal group（Dc-N）,lactating cow heat stress group（Dc-HS）.Blood was collected and centrifuged to obtain serum,rumen fluid was collected by a gastric tube tumor fluid sampler,filtered with gauze to obtain rumen fluid.Serum and rumen fluid were immediately transferred to a liquid nitrogen tank for measurement of biochemical parameters,immune,hormonal and oxidative stress indicators and macrogenomics,16Sr DNA,and metabolomics.Results of the current study showed that heat stress and growth stage had significant effects on glucose（GLU）,triglycerides（TG）,total protein（TP）,prolactin（PRL）,glucocorticoids（GC）,triiodothyronine（T₃）,thyroxine（T₄）,Heat Shock Protein 70（HSP70）,insulin（INS）,and cortisol（COR）,it was found that there was an interaction effect whereby heat stress and growth stage had significant effects on GLU,TG,TP,T₃,INS and COR.Further,it was evident that both factors（heat stress and growth stage）,significantly affected blood immunity and indicators of oxidative stress（P<0.001）,but there was no significant interaction among them（P>0.05）.It was noted that heat stress caused a significant increase in Malondialdehyde（MDA）（P<0.001）and a significant decrease in superoxide dismutase（SOD）,Total antioxidant capacity（T-AOC）,and Glutathione peroxidase（GSH-PX）（P<0.001）in cows at all growth stages.Among them,heifers had the lowest MDA content and the highest SOD,T-AOC and GSH-PX content,while lactating cows had the opposite.The heat stress group had a different effect on alpha diversity index of rumen microbes,and a significant increase（P<0.05）in the difference in Chao1 index,Sobs and ACE index between the heat stress group than the normal group was found in growing heifers,while in heifers and lactating cows the difference was not significant（P>0.1）,Good’s Coverage was greater than 0.99 in all groups;dominant phylum in all groups included Firmicutes,Bacteroidota,Proteobacteria.Proteobacteria,Fusobacteria,Acetobacter and Leuconostoc were significantly enriched in groups C-N,Spirochaetes,Planctomycetes,Fibrobacteres,Methanobrevibacter,Ruminococcus as indicator species of C-HS group;Proteobacteria,Acinetobacter,Acetobacter were significantly enriched in the H-N group,Treponema-2,Bifidobacterium,Eubacterium were significantly enriched in the H-HS group,Paenibacillaceae and Deltaproteobacteria were significantly enriched in the Dc-HS group.Replication and repair,glycan biosynthesis and metabolism,and transcription were significantly enriched in the C-HS group（P<0.05）;carbohydrate metabolism was significantly enriched in the H-N group（P<0.05）,and translation was significantly enriched in the Dc-HS group（P<0.05）.Metabolomic results showed that oleamide and prostaglandin D₂were higher in rumen fluid metabolites in the C-HS group than in the C-N group,prostaglandin D₂and 3,14-dihydro Prostaglandin F1αwere higher in rumen fluid metabolites in the H-HS group than in the H-N group,2,3-Dinor prostaglandin in rumen fluid metabolites in the Dc-HS group E1,3-hydroxyvaleric acid,octanedioic acid,and 3-nitrotyrosine were higher in the Dc-N group than in the Dc-HS group.In the serum metabolome,phenylacetylglycine was significantly upregulated in the Cs-N and Hs-N groups,and arachidonic acid was significantly enriched in the Cs-HS and Dcs-HS groups.In summary,growth stage and heat stress affect blood biochemical parameters,hormones,immunity,and oxidative stress indicators in Holstein cows to varying degrees.Oxidative stress,decreased immunity whereas it was also noted that the mammary gland damage can also occur in growing heifers,heifers,and lactating cows exposed to heat stress.Therefore,it was evident that heifers are more resilient to heat stress,whereas lactating cows suffer the most severe damage when exposed to heat stress.Using 16Sr DNA sequencing and metagenomics to analyze the composition and function of rumen microbes,rumen microbes are affected by heat stress and growth stage,with differences in microbiota structure and function between treatment groups.Heat stress led to an increase in the abundance of methanogenic-related flora in growing heifers and lactating cows,a decrease in carbohydrate degradation capacity in growing heifers and heifers compared to the normal group,and more active replication and repair functional pathways in growing heifers.Heat stress affected rumen metabolites and serum metabolites in Holstein cows at different growth stages.Lipid metabolic pathways were significantly enriched and metabolites associated with inflammation were significantly upregulation in lactating cows during heat stress.