Mechanism In Growth And Rumen Development Alteration By Early Alfalfa Supplementation In Hu Lambs

A healthy rumen is the key for ruminant feeding.Concentrate feeding are essential for the rapid development of rumen epithelium.However,an increased incidence of plaque formation and poor rumen development were observed in calves fed only with concentrate diets during the pre-weaning period.Forage supplementation is reported to increase the ADG and BW and improves rumen health in young calves,while the mechanism was not clear.Therefore,the objectives of the present study were to investigate the effects of early supplementation of chopped alfalfa to starter pellets before weaning on Hu lamb performance and rumen development before and after weaning,and further illustrate the mechanisms of promoting performance and rumen development from rumen microbiota,metabolites,and rumen gene expression level.1 Effects of alfalfa supplementation on growth performance and rumen development of Hu IambsSixty-six healthy male Hu lambs at 5 d of age were purchased from commercial farm.After 5-d-adaptation,six of 66 male Hu lambs were slaughtered at the age of 10 d to serve as a control?CON?.The other 60 lambs were randomly allocated to two dietary treatments:milk replacer and starter pellets without?STA?or with free-choice chopped alfalfa?S-ALF?.The animals were offered 300 g/d of the concentrate mixture and had free access to alfalfa after weaning at the end of week 4?38 d of age?.Feed intake and body weight?BW?were measured two consecutive days each week.The apparent digestibility of diet was measured at week 8.At the end of weeks 1,2,4,5,and 8 of the feeding trial,6 Iambs in each group were randomly selected and sacrificed.Blood samples were collected before sacrifice,weights of carcass,forestomachs and visceral organs were weighted.Rumen content and tissue samples were collected for later measurements.The alfalfa inclusion in the S-ALF group tended to increase the starter intake before weaning,significantly increased the concentrate intake soon after weaning?P<0.05?,and increased the BW?P<0.01?and ADG?P<0.10?in pre-and post-weaning lambs.The S-ALF group had greater carcasses?P<0.05?,rumens?P<0.05?,reticula?P<0.05?,omasums?P<0.10?,abomasums?P<0.05?and visceral organs?P<0.10?than the STA lambs post-weaning.Alfalfa supplementation increased the blood concentration of urea nitrogen,and decreased the molar proportion of propionate one week after weaning?P<0.05?,while pH was not affected.After weaning,S-ALF lambs had higher blood urea nitrogen?P<0.05?.The MCT4 expressions of S-ALF lambs were numericlly higher than STA lambs during pre-weaning period.The mRNA expressions of NHE1,NHE2 and NHE3 were not changed in S-ALF lambs from pre-to post-weaning period,while NHE2 increased at week 2,NHE3 increased at week 4,and NHE1 increased at 4 weeks after weaning in STA lambs?P<0.05?.S-ALF lambs had higher blood concentration of ?-hydroxybutyratc?BHBA?after weaning,and lower incidence of feed plaque,higher rumen muscle thickness at week 4.The mRNA expressions of claudin-1,claudin-4,occludin,IL-1?,IL-6,IL-10,and TGF-/?1 were not changed in S-ALF lambs from pre-to post-weaning period,while claudin-1 increased at week 2,claudin-4,occludin,IL-1?,IL-6,IL-10 and TGF-?1 decreased at one week after weaning?P<0.05?.From the above results,we infer that the free-choice addition of chopped alfalfa to starter diets can improve nutrients intake and absorption,release inflammatory response,relieve weaning stress,and improve the performance of lambs.2 Effects of alfalfa supplementation on rumen microbial development of Hu lambsAs revealed by rumen content microbiota,the relative abundance of Actinobacteria,Bacteroidetes,Firmicuies,and Proteobacteria phyla fluctuated with the intake of the starter and post-weaned diets in STA lambs.With alfalfa intervention,Butyrivibrio,unclassified Clostridiales and unclassified Mogibacteriaceae increased during the intake of alfalfa during the pre-weaning period.After weaning,fewer changes at the genus level were observed in S-ALF lambs compared to in STA lambs.As revealed by rumen epithelial microbiota,the relative abundance of Actinobacteria,Bacteroidetes,Firmicutes,and Proteobacteria phyla also fluctuated with the intake of the starter and post-weaned diets in STA lambs.With alfalfa intervention,unclassified Clostridiales,Mogibacterium,unclassified Ruminococcaceae.Atopobium,Succiniclasticum and Butyrivibrio increased during the pre-weaning period?P<0.05?.After weaning,fewer changes at the genus level were observed in S-ALF lambs compared to in STA lambs.These results suggested that alfalfa intervention reduced the changes in the rumen microbiota during the weaning transition and increased environmental adaptation of the rumen microbiota.3 Effects of alfalfa supplementation on rumen microbial metabolites of Hu lambsCompared with pre-weaning?week 4?,21 metabolites were up-regulated and 25 metabolites were down-regulated significantly in STA lambs?VIP?1.0,|Log2?FC?|?1.0?.These metabolites were enriched in arginine and proline metabolism?P=0.035?,valine,leucine and isoleucine biosynthesis?P=0.038?and D-glutamine and D-glutamate metabolism?impact=1.0?.Compared with pre-weaning,18 metabolites were up-regulated and 19 metabolites were down-regulated significantly in S-ALF lambs?VIP?1.0,|Log2?FC?|?1.0?.These metabolites were enriched in starch and sucrose metabolism?P=0.012?and D-glutamine and D-glutamate metabolism?impact=1.0?.Compared with STA,21 metabolites were up-regulated and 5 metabolites were down-regulated significantly in S-ALF lambs?VIP?1.0,|Log2?FC?|?1.0?during the pre-weaning period.These metabolites were enriched in Butanoate metabolism?P=0.048?.Eighteen metabolites were up-regulated and 8 metabolites were down-regulated significantly in S-ALF lambs?VIP?1.0,|Log2?FC?|?1.0?during the post-weaning period.These metabolites were enriched in starch and sucrose metabolism?P=0.006?.Uric acid and pantothenic acid were positively correlated with the rumen content Butyrivibrio?P<0.05,R>0.50?.Glycocyamine,phytanic acid,tagatose,benzoic acid and glucuronic acid were positively correlated with the rumen content unclassified Mogibacteriaceae?P<0.05,R>0.50?.Carvone was positively correlated with the rumen epithelial Bifidobacterium?P<0.05,R>0.50?,while salicylic acid and cysteinylglycine were negatively correlated with the rumen epithelial Bifidobacterium?P<0.05,R<-0.50?.Salicylic acid and cysteinylglycine were positively correlated with the rumen epithelial Mogibacterium?P<0.05,R>0.50?.Lactose and cysteinylglycine were positively correlated with the rumen epithelial Succiniclasticum?P<0.05,R>0.50?,while carvone was negatively correlated with the rumen epithelial Succiniclasticum?P<0.05,R<-0.50?.These results suggested that alfalfa intervention changed rumen microbial metabolites,which were functioned to provide energy and promote rumen epithelial development.4 Effects of alfalfa supplementation on rumen transcriptome gene expresseions of Hu lambsCompared with CON,580 and 833 differentially expressed genes?DEgenes?were found in pre-weaned STA lambs,respectively;637 and 939 DEgenes found in pre-weaned S-ALF lambs,respectively.Compared with pre-weaning period,66 and 93 DEgenes were found in STA lambs at one week after weaning,respectively;27 and 31 DEgenes were found in S-ALF lambs at one week after weaning,respectively,and the DEgenes were different from STA lambs.Compared with STA,the expressions of CYP1A1?MYOM1?PDK4 were higher S-ALF lambs,the expression of ZNFX1-AS1₂ was lower in S-ALF lambs in pre-weaning;the expressions of ADIPOQ and C7 were higher in STA lambs,the expressions of FABP4 and GPNMB were lower than STA lambs.These results suggested that alfalfa intervention induced gene expression in pre-weaning period to promote rumen development,and increased the stability of rumen tissue during weaning transition to reduce weaning stress.5 Mechanisms of alfalfa supplementation promoting growth performance and rumen development in Hu lambsDuring the pre-weaning period,alfalfa supplementation increased the relative abundance of rumen content Butyrivibrio,which was associated with higher ability of NDF digestion.The increased relative abundances of rumen epithelial Butyrivibrio and Succiniclasticum could promote the utilization of butyrate and propionate by rumen epithelium.The increased relative abundances of rumen epithelial unclassified Clostridiales,unclassified Ruminococcaceae and Atopobium were associated with rumen health.During the post-weaning period,alfalfa supplementation increased the relative abundance of rumen contentunclassified Clostridiales and unclassified Mogibacteriaceae,to promote a better fiber digestibility,helped lambs to adapted to high fiber diet,released weaning stress,and improved rumen development and growth performance.In the rumen of alfalfa supplemented lambs,the reduction of carvone,and lower expression of ZNFX1-AS1₂,higher expression of MYOM1 were observed.They played different roles in rumen microbial metabolism,rumen cell proliferation and rumen muscle development.Therefore,they could be the biomarkers for alfalfa supplementation promoting rumen development during the pre-weaning period.After weaning,higher concentration of glycocyamine was observed in alfalfa supplemented lambs,which could provide energy during weaning transition.This could be another biomarker for alfalfa supplementation promoting rumen development during the post-weaning period.Therefore,early supplementation of alfalfa increased the relative abundances of rumen content utyrivibrio during the pre-weaning period,rumen content unclassified Clostridiales and unclassified Mogibacteriaceae during the post-weaning period to improved fiber digeatibility of rumen.Alfalfa supplementation increased the relative abundance of rumen epithelial Butyrivibrio,Succiniclasticum,unclassified Clostridiales,unclassified Ruminococcaceae,and Atopobium to promote the health development of rumen.Alfalfa supplementation increased the relative concentration of glycocyamine,maltose and other metabolites to improve energy supplementaiton after weaning.More differentially expressed genes were found during pre-weaning period,while less differentially expressed genes were found during post-weaning period with alfalfa supplementation.Therefore,alfalfa supplementation reduced the inflammatory responses,released weaning stress and improved growth performance.