Studies On Dietary Supplementation With Vitamin B-Complex On Gastrointestinal Microflora Composition And Growth Performance Of Growing Goats

Nowadays,the requirements for higher quality of animal products have gradually increased in the world.In practice,in order to enhance the productivity and quality,some feed additives are added into basal diets especially in the intensive farming system.Vitamin B including vitamin B1,B2,B6,B12 and etc.is water-soluble substance,which plays a synergistic role in regulating metabolism and also acts as an essential nutrient for animal development and growth.Previous studies have shown that vitamin B could be synthesized by microorganisms in the rumen of the ruminants,and the amount is sufficient to meet the ruminants’ growth.Therefore,it was considered no necessary to extra-supplemented with dietary vitamin B for the ruminants.In recent years,it’s reported that vitamin B should be supplemented when the roughage can not be fully digested in young ruminants with not fully developed rumination and fast growing.However,till now research about the optimal dosage of vitamin B supplemented in the basal diet is still unknown,and the effect of vitamin B on ruminant mircobiota composition in the digestive tracts and growth performance is still not clear.In this study,young female goats were used as animal models,and different doses of vitamin B-complex were added into the diet.The aim of this study was to explore the optimal dosage of vitamin B in the diet of young goats,and as well as the effect of vitamin Bcomplex on growth performance and the relevant mechanism.1.Effects of vitamin B-complex on growth performance in goats and its optimal dosageIn the first experiment,24 crossbred boer goats(three-month-old)with body weight of(16.78±1.52 kg)were fed with the basal diet supplemented with 0 mg/kg vitamin B-complex(control group,C),low dosage of vitamin B complex(1.2 mg vitamin B1,2.4 mg vitamin B2,2.4 mg vitamin B3,2.4 mg vitamin B5,1.2 mg vitamin B6,8 ug vitamin B12)per kg diet(low-dose group,L),moderate dosage of vitamin B with 5 times of those in L group(moderate group,M),and high-dose vitamin group with 10 times of those in L group(high dosage group,H),respectively.All animals were free access to water and food during the experimental period of 56 days.Body weight and feed intake were recorded.Blood and fresh feces were collected for biochemical parameters analysis at the end of the experiment.High-throughput sequencing based on 16S rRNA was used to measure fecal microflora composition in feces.The results showed that compared to control group,H group showed a significant increase in body weight gain and the ratio of feed conversion(P<0.05),while there were no significant changes in group L and M compared to the control group.The results of plasma biochemical parameters showed that the total protein(TP)and globulin(GLOB)concentrations in plasma were significantly decreased in H group(P<0.05)compared to the control group,however,there was no significant changes among C,L and M groups(P>0.05).The level of vitamin B in plasma were measured by HPLC method.The results showed that plasma VB1 and VB2 concentrations in group H were slightly increased,however,the total amount of plasma B vitamins in both H and M group was increased significantly compared to the control group(P<0.05).High throughput sequencing results showed that Firmicutes and Bacteroidetes were the dominant bacteria in the feces of goats.Alpha diversity analysis showed that the relative abundance and ACE index of species in H group were significantly lower than that in C group(P<0.05).The relative abundance of beneficial bacteria such as Prevotella copri and Bacteroides thetaiotaomicron was significantly higher in H group compared to control(P<0.05),but no significant changes were observed in group M(P>0.05).Additionally,the relative abundance of Ruminococcus bromii and Escherichia coli in groups M and H was significantly lower than that in C group.The above results indicate that dietary supplementation of vitamin B-complex can improve the growth performance of goats which maybe related to the increase of beneficial bacteria such as Prevotella copri and etaiotaomicron as well as the decrease of harmful bacteria in the intestinal.2.Effects of high dose vitamin B-complex on growth performance and muscle growth in goatsIn the second experiment,ten three-month-old female goats(16.78 ± 1.52 kg)were employed and randomly divided into control group(CON)and experimental group(VB),respectively.The dose of vitamin B added in the CON and VB groups was the dose of group C and group H as in the first experiment.The whole experiment lasted for 77 days.The results showed that compared with the control group,the body weight in VB group was significantly increased after 70 days of treatment(P<0.05),and the food intake was significantly increased at 70 d(P<0.05).Serum biochemical indexes showed that VB supplementation significantly decreased the level of total protein(TP)and globulin(GLOB)at 28 d(P<0.05).The VB content in the VB group was significantly changed in the plasma and ruminal epithelial tissues(P<0.05).In addition,VB content in the rumen fluid was tended to increase in VB group compared to CON(0.05<P<0.1).HE staining showed that the cross-sectional area of muscle fibers in VB group had a tendency to increase compared to CON(0.05<P<0.1).Growth related factors and AMPK/m-TOR signaling pathway-related genes were detected by real-time PCR.The results showed that vitamin B-complex supplementation significantly decreased Myf5 mRNA expression(P<0.05).Key genes ofAMPK/m-TOR signaling pathway including IGF1,AMPK,P-AMPK and S6K1 in VB group were significantly down-regulated in longissimus(P<0.05).PAX7 gene expression was significantly increased(P<0.05).The results of FOXO signaling pathway-related genes showed that compared with the control group,FoxO1(P<0.05)gene and protein expression,Fox03 and Fox04(P<0.01)genes expression were significantly down-regulated by VB treatment.The ubiquitin proteasome system related genes such as Atrogin and NF-κB mRNA expression was significantly down regulated in VB group(P<0.05).The expressions of Myostatin and MuRFl genes were also significantly down regulated by VB(P<0.01).In the lysosomal pathway,the levels of LC3B,ATG5,ATG7,ATG9,ATG12,and ATG16L genes was significantly down regulated in VB group compared with control(P<0.01),however,there was no significant change in LC3B protein expression(P>0.05).The results showed that there were no significant changes between VB and CON groups(P>0.05).The above results suggest that dietary vitamin B supplementation could significantly increase the body weight and feed intakeThe inhibition of the ubiquitin proteasome system and autophagylysozyme somatic pathway maybe involved in the effect of VB promoting growth in goats.3.Effects of vitamin B-complex on intestinal epithelial morphology and microbiota composition in the digestive tractsThe experimental design is the same as the second chapter.The results showed that the relative abundance of ruminal microbiota did not change significantly by VB(P>0.05).There were no significant differences in the indices of Observed species,Chao 1,ACE and PD whole tree in the VB group(P>0.05).The genus level of rumen bacterias have no significant change(P>0.05).Subsequently,we analyzed the morphological structure and microflora composition of the cecum and colon.The results showed that compared with the control group,the intestinal villi height of the jejunum in VB group increased significantly(P<0.01),and the V/C(villi height/crypt depth)ratio increased significantly(P<0.05).There were no significant changes in villous height and V/C ratio of the ileum epithelium(P>0.05),but the crypt depth was relatively reduced(P<0.05).The relative abundance of microbiota composition the cecum and colonic digesta showed that Firmicutes and Bacteroidetes were the predominant bacteria in phylum levels.Compared to the control group,the ratio of Firmicutes to Bacteroidetes had a tendency to decrease(0.05<P<0.1).However,there was no significant change in the colon contents of the Firmicutes and Bacteroidetes ratio(P>0.05).The ACE,Chaol and Shannon index of microflora in the cecum and the conolic digesta in VB group were not altered by VB treatment(P>0.05).At the genus level,the abundance of Unidentified Christensenellaceae genus in VB group had a tendency to decrease compared with the control group(0.05<P<0.1).In contrast,the abundance of Intestinimonas bacteria showed an increasing trend(0.05<P<0.1).The results of colon bacterial genus levels showed that,similar to the cecum,the abundance of the Unidentified Christensenellaceae genus in VB group was significantly lower than that in control group(P<0.05).The results of genes and proteins in the cecum and colon showed that there was no significant change in the expression of tight junction genes in the cecum in the VB group compared with the control group(P<0.05).However,the gene expressions of EGFR,Occludin,MKI67 and CCND in the colon were significantly up-regulated(P<0.05),the protein expression of ZO-1 was also significantly up-regulated(P<0.05).The above results suggest that the beneficial effects of dietary vitamin B-complex supplementation on growth performance might be related to the increase of ruminal microflora abundance,and the increasing of beneficial bacteria as well as the reduction of the Pathogenic bacteria in the gut.At the same time,it can promote the expression of tight junction genes and proteins in colon.