Effects Of Tributyrin And Clostridium Butyricum On Growth Performance And Gastrointestinal Health Of Calves

The immune system and intestinal barrier function of newborn calves are immature,which results in high susceptibility to environmental changes and pathogenic bacteria,and predisposes them to diarrhea.As the main energy substance of intestinal epithelial cells,butyrate can regulate the proliferation of epithelial cells and enhance the intestinal barrier function.However,butyrate has special odor that affects palatability and is easily absorbed by rumen epithelial cells,making it difficult to reach the hindgut.The tributyrin can be gradually decomposed into glycerin and three molecules of butyrate in the intestine,which is more efficient than other butyrate additives.Clostridium butyricum,as an anaerobic bacterium,can produce butyrate in the hindgut and reduce the adhesion of pathogenic bacteria on the intestinal mucosa.The related studies have shown that both tributyrin and Clostridium butyricum can produce butyrate in the hindgut,improve intestinal barrier function,relieve diarrhea,and have the potential to improve growth performance of calves.Therefore,in this study,tributyrin and Clostridium butyricum were supplemented separately or in combination to the milk of calves,and the effects of tributyrin and Clostridium butyricum on calf growth performance and intestine health were comprehensively evaluated by measuring feed intake,body weight and body size,rumen fermentation,blood biochemical index,immune function,fecal scores and rectal microorganisms to provide solutions for the healthy dairy heifer.Exp.1 Effects of tributyrin and Clostridium butyricum on growth performance and rumen fermentation in calvesSixty 2-day-old female Holstein calves with similar body weight were randomly divided into four groups:fed starter and milk（CON group）;fed starter with 2 g/L tributyrin supplemented to milk（TB group）;fed starter with 8 g/d Clostridium butyricum supplemented to milk（CB group）;and fed starter with 2 g/L tributyrin and 8 g/d Clostridium butyricum supplemented to milk（TC group）.All four groups were provided with equal amounts of milk and had free access to starter and water,the experiment period was 90 days.After weaning at 63-day-old,all calves were discontinued from tributyrin and Clostridium butyricum,and 10 calves in each group were randomly followed,with their growth performance,blood biochemical indexes,and rumen fermentation were measured again at 90 days of age.The intake of milk,starter and roughage was recorded daily during the experimental period,and the dry matter intake（DMI）of calves was counted weekly.The body weight and body size of calves were measured at 30,60 and 90 days of age respectively,and their average daily gain（ADG）and feed efficiency（FE）were calculated.At 60 and 90 days of age,jugular venous blood was collected from calves 2 h after morning feeding and bloodβ-hydroxybutyric acid（BHBA）was measured immediately.Subsequently,rumen fluid was collected using a oral cannula,and rumen fluid p H,NH₃-N and volatile fatty acid（VFA）were measured.To determine the apparent digestibility of nutrients,the rectal fecal samples were collected 3 days prior to calf weaning.The results showed that the supplementation of tributyrin and Clostridium butyricum had no significant effect on calf body size,ADG,FE,apparent digestibility of nutrients,rumen fluid p H and NH₃-N and blood BHBA（P>0.050）.Tributyrin significantly reduced the ratio of valerate（P<0.017）and propionate（P<0.043）in the rumen fluid of calves at60 days of age.The supplementation of Clostridium butyricum and the mixed supplementation of both Tributyrin and Clostridium butyricum significantly increased the concentration of acetate in the rumen fluid（P=0.045）,and Clostridium butyricum significantly increased the total dry matter intake and body weight of calves during overall period.（P=0.001）.The above results indicate that the supplementation of tributyrin and Clostridium butyricum to calf milk affected rumen fermentation;while tributyrin had no significant effect on calf growth performance and Clostridium butyricum can improve calf weight.Exp.2 Effect of Tributyrin and Clostridium butyricum on blood biochemical parameters and immune function of calvesThe animal feeding and experimental design were the same as in Exp.1.Blood samples were collected from the jugular vein 2 h after morning feeding at 30,60 and 90days of age,and blood biochemical indices,immune and inflammatory factors and intestinal barrier indices were measured.The results showed that the supplementation of tributyrin and Clostridium butyricum to calf milk had no significant effect on the serum levels of glucose（GLU）,total protein（TP）,albumin（ALB）,globulin（GLO）and the activity of alanine transaminase（ALT）in calves（P>0.050）,and tributyrin had a tendency to reduce the blood levels of blood urea nitrogen（BUN）in 60 days calves（P=0.054）.The tributyrin and Clostridium butyricum significantly reduced the level of blood tumor necrosis factor-α（TNF-α）and Interleukin-6（IL-6）at 90 days of age（P<0.050）,Clostridium butyricum had a tendency to increase the level of blood anti-inflammatory factors interleukin-10（IL-10）at 90 days of age（P=0.094）,and both significantly reduced the levels of diamine oxidase（DAO）,D-lactic acid（D-LA）and Zonulin in the serum of 30days calves（P<0.050）.Indicating that tributyrin and Clostridium butyricum can reduce the level of inflammatory factors and improve intestinal barrier function.Exp.3 Effect of Tributyrin and Clostridium butyricum on diarrhea and rectal microbial composition of calvesThe animal feeding and experimental design were the same as in Exp.1.During the experimental period,fecal scores were conducted daily to calculate the diarrhea rate of calves,and the health status and durg use of calves were recorded.Fecal samples were collected from the rectum of calves at the ages of 30,60,and 90 days to detect the content of VFA in the feces.Sterile swabs were used to collect rectal fecal samples from calves at the age of 60 days for 16S r DNA gene sequencing analysis.The results showed that tributyrin and Clostridium butyricum significantly reduced the diarrhea rate（P=0.011）and drug use（P=0.006）in calves,but had no significant effect on fecal VFA and fecal microbialαdiversity.Nevertheless,Clostridium butyricum significantly affected fecal microbialβdiversity in calves（P<0.050）,resulting in significant enrichment of some short-chain fatty acid producing bacteria,including Olsenella,Clostridium,Faecalibacterium,Bacteroides,and Parabacteroides（P<0.001）.Immune globulins were significantly positively correlated with Subdoligranulum,Alistipes and Phascolarctobacterium（P<0.050）,inflammatory factors were significantly positively correlated with Oscillospiraceae,Erysipelotrichaceae and Phascolarctobacterium（P<0.050）,intestinal barrier index was significantly positively correlated with Faecalitalea,Olsenella and Lachnospiraceae（P<0.050）in Serum of 60 days calves.The results indicating that tributyrin and Clostridium butyricum could relieve diarrhea and promote the enrichment of probiotics in intestine,which was beneficial to intestinal health.This study showed that the tributyrin had no significant effect on calf growth performance,and Clostridium butyricum significantly increased calf body weight during the overall period,and both affected rumen fermentation.Tributyrin and Clostridium butyricum had no significant effect on blood biochemical parameters and fecal VFA of calves,both of which reduced inflammation and decrease diarrhea rates,helped improve intestinal barrier function.Clostridium butyricum significantly increased the abundance of some short-chain fatty acid-producing probiotics in the intestine,which was beneficial to intestinal health.There was no significant combination effect in the mixed addition group.