Effects Of Nisin And Lysozyme On Growth Performance,Rumen Fermentation And Rumen Microbiota Of Hu Sheep

Nowadays,feed shortage and environmental pollution are becoming gradually serious.Therefore,reducing N excretion and methane emission and improving N and energy utilization efficiency in ruminants are of great significance.In rumen,gram-positive bacteria produce metabolites,such as hydrogen,ammonia,and acetate,which could be used to produce methane and urea,contributing to the loss of energy and N.Nisin and lysozyme are two kinds of antimicrobial peptides,which could selectively inhibit the activity of gram-positive bacteria without adverse effects on gram-negative bacteria.Thus,we hypothesized that nisin and lysozyme may have the abilities to improve the energy and nitrogen utilization efficiency in ruminants.In this study,in vitro combined with in vivo experiments were conducted to comprehensively investigate the effects of nisin and lysozyme on growth performance,nitrogen balance,methane production,rumen fermentation,rumen development,and microbial community structure of Hu sheep.1 Effects of nisin and lysozyme on in vitro fermentation,methanogenesis and functional microbial populations of the rumen(1)This study was conducted to evaluate the effects of nisin on in vitro rumen fermentation parameters,methanogenesis and functional microbial populations.FiveμmoI/L Monensin was added as positive control,and the negative control(NC)did not contain any additives.Nisin was added at 3 doses:3(N-3),9(N-9),and 27 mg/100 mL(N-27).Gas and methane production was measured at 0,3,6,9,12,and 24 h after incubation.Samples of culture were collected at 24 h,and used to measure rumen fermentation parameters and functional microbial populations.The results showed that,compared with NC,both nisin and monensin addition dramatically reduced gas and methane production(P<0.05).Nisin addition had no effect on pH,dry matter degradability,and organic matter degradability(P>0.05).Ammonia concentration was reduced by N-9(P<0.05),but was not influenced by N-3 and N-27(P>0.05).In contrast,monensin addition significantly lowered dry matter degradability,organic matter degradability,and ammonia concentration(P<0.05),but had no influence on pH(P>0.05).Compared with NC,both nisin and monensin addition significantly reduced acetate concentration and acetate-propionate ratio(P<0.05),and increased propionate concentration(P<0.05).Quantitative real-time PCR results showed that both nisin and monensin addition had no effects on the populations of total bacteria and Bacteroides(P>0.05).Compared with NC,the populations of protozoa,methanogens,fungi and Firmicutes were not influenced by nisin(P>0.05),but were significantly reduced by monensin addition(P<0.05).Both nisin and monensin addition significantly increased the populations of sulfur-reducing bacteria and Clostridium aminophilum(P<0.05),but had no influence on the population of Clostridium sticklandii(P>0.05).The results indicated that the appropriate concentration of nisin(3 mg/100 mL)could increase propionate concentration,and reduce methane production and ammonia concentration without adverse effect on feed digestion.These changs are probably associated with the variation of rumen functional microbial populations.(2)We studied the effects of lysozyme on in vitro rumen fermentation,methanogenesis and microbial community structure.Lysozyme was added to in vitro ruminal cultures at 5 doses:with 0(L-0,Control),0.1(L-0.1),1(L-1),10(L-10),and 100 mg/100 mL(L-100).Total gas and methane production were measured at different time of incubation.Culture samples were collected at 24 h for analysis of fermentation parameters and functional microbial populations.In addition,samples of L-0,L-1,and L-100 collected at 24 h were also used subjected to metagenomics analysis of bacterial community using Illumina sequencing of 16S rRNA gene amplicons.Compared with control,methane production,ammonia concentration,dry matter digestibility,organic matter digestibility and total volatile fatty acid concentration were not influenced by L-0.1(P>0.05).Methane production and NH3-N concentration were reduced,and propionate concentration was increased by L-1(P<0.05),whereas dry matter digestibility,organic matter digestibility or total volatile fatty acid were not affected(P>0.05).Methane production was reduced,and propionate concentration was increased by L-10 and L-100(P<0.05),but dry matter digestibility and organic matter digestibility were also decreased significantly(P<0.05).The qPCR results showed that total bacteria,fungi and methanogens were significantly reduced by L-100(P<0.05),but were not influenced(P>0.05)by L-0.1,L-1,or L-10.Principal Component Analysis of the sequencing data showed clear differences in the composition of the ruminal bacterial community between the control and the lysozyme treatments,demonstrating evident impact of the lysozyme addition.The abundance of propionate-producing bacteria(e.g.,Selenomonas and Succinivibrio)was increased by lysozyme,resulting in more hydrogen being directed to production of propionate instead of methane.Moreover,the reduced ammonia concentration in L-1 was probably due to the lower abundance of proteolytic bacteria(e.g.,Prevotella and Bacteroides)inhibited by lysozyme.Appropriate lysozyme addition(1 mg/100mL)can be used to modulate ruminal microbial ecology and reduce methanogenesis and ammoniagenesis by rumen microbiome without adversely affecting feed digestion or fermentation in short-term.2 Effects of nisin and lysozyme on growth performance,rumen fermentation,and nitrogen balance of Hu sheepTo further validate the in vitro results,this trial was conducted to evaluate the effects of nisin and lysozyme on growth performance,rumen fermentation and nitrogen balance.Thirty-six two-month-old male Hu sheep(22.8±1.79 kg)were assigned to three groups based on a randomly block design,with each group composed of four units of three lambs.The feeding trial was devided into two periods:period 1 was from week 1 to 6,and period 2 was from week 7 to 12.The control group was offered basal diet in the two periods.The nisin group was fed basal diets with 450 mg/d nisin and 1350 mg/d nisin each sheep in period 1 and 2,respectively.The lysozyme group was fed basal diets added with 100 mg/d lysozyme and 500 mg/d lysozyme each sheep in period 1 and 2,respectively.The results showed that DMI of nisin group was significantly higher than control(P<0.05),but the DMI was not affected in lysozyme group(P>0.05).Compared with control,nisin and lysozyme addition had no influence on body weight gain,feed conversion rate,and nutrient digestibility of sheep(P>0.05).Both nisin and lysozyme reduced the concentrations of ammonia,isobutyrate,and TBCVFA significantly(P<0.05),but nisin or lysozyme had no effects on the concentrations of TVFA,acetate,propionate,butyrate and acetate/propionate ratio(P>0.05).Both nitrogen retention(g/d)and nitrogen retention rate(%of N intake)in nisin group and lysozyme group were numerical higher than that in the control,but not significant(P>0.05).Overall,nisin and lysozyme could increase DMI,improve rumen fermentation,and reduce production of BCVFA and ammonia,which contributed to the improvement of nitrogen metabolism of Hu sheep.3 Effects of nisin and lysozyme on rumen fermentation,rumen development,and rumen microbial community structure of Hu sheepThis trial was conducted to evaluate the effects of nisin and lysozyme on rumen fermentation,rumen development,and rumen microbial community structure of Hu sheep.Sheeps were slaughtered for sample collection at the end of feeding experiment.The results showed that nisin and lysozyme significantly improved the weight of liver but have no effect on rumen papillae development.Compared with control,nisin and lysozyme addition had no influence on pH value,TVFA,acetate,propionate,branched chain VFA concentration,and acetate-propionate ratio(P>0.05).However,the supplemention of nisin and lysozyme significantly decreased the ammonia concentration(P<0.05).The qPCR results showed that nisin and lysozyme did not affect the population of total bacteria,fungi,protozoa,and methanogens compared with the control(P>0.05).In nisin and lysozyme group,the number of Clostridiu.aminophilum,a hyper ammonia-producing bacteria(HAB),is numerical lower than that in control group,but not significant(P>0.05).However,Clostridium sticklandii,another HAB,was not detected.High-throughput MiSeq sequencing results showed that shannon index increased significantly in lysozyme group(P<0.05),but was not influenced by nisin(P>0.05).Multi-comparison on phylum,genus and OTU level demonstrated that the distribution patterns of bacteria among the control,nisin group and lysozyme group were not different.The relative abundance of genus Methanosphaera in nisn and lysozyme group was significantly lower than that in the control without adverse effect on the major predominate fibrolytic bacteria and proteolytic bacteria(P<0.05).In conclusion,nisin and lysozyme could increase liver weight and reduce ruminal ammonia concentration and the relative abundance of genera Methanosphaera without adverse effect on the major predominate fibrolytic bacteria and proteolytic bacteria.These modifications might be related to the increase of energy and nitrogen utilization.