The Effect Of Yak Rumen-derived Anaerobic Fungi Orpinomyces Sp. YF3 On In Vitro Fermentation And Microflora

In recent years,people’s material needs have continued to increase,and the number of livestock and poultry breeding in our country has increased correspondingly,but the area of arable land has gradually decreased.With the increase in the use of livestock and poultry feed,the contradiction of "human and animal competition for food" has become increasingly serious.At the same time,a large number of crop straw resources are wasted seriously in our country.Due to the harsh living environment,the yak in our country has strong resistance to rough feeding,and its rumen anaerobic fungi has strong cellulose degradation ability.If the yak rumen anaerobic fungi are added to the rumen of ruminants,it may greatly enhance the ability of rumen microorganisms to decompose lignocellulose.Thereby increasing the amount of crop straw and other non-grain feeds in ruminant diets can achieve the purpose of saving food,reducing the waste of straw resources,and reducing breeding costs at the same time.This experiment first explored the optimal addition amount of the yak rumen anaerobic fungi Orpinomyces sp.YF3 that has a positive effect on rumen fermentation of wheat straw,and then explored the effects of repelling fungi and adding Orpinomyces sp.YF3 on rumen fermentation and microbial flora.This provides a scientific basis for the development and application of yak rumen anaerobic fungi,and for increasing the amount of crop straw and other non-grain feeds in ruminant diets.This experiment consists of two parts:Experiment 1 The effect of exogenous yak rumen anaerobic fungi Orpinomyces sp.YF3 on in vitro fermentationIn this experiment,anaerobic fermentation flasks were used for in vitro fermentation,with 1%(w/v)crushed air-dried wheat straw as the fermentation substrate.4 doses of yak rumen anaerobic fungi Orpinomyces sp.YF3 were added to the fermentation system.The purpose is to determine the optimal additive dosage of Orpinomyces sp.YF3 fermented wheat straw.This experiment adopts a single factor experiment design.Set 4 dosages of Orpinomyces sp.YF3 liquid: 0%,5%,10%,15%,a total of 4 treatments 3 fermentation times(1 d,3 d,5 d).There are 4 repetitions for each fermentation time for each treatment.Take samples after fermentation.Determine the in vitro dry matter disappearance rate of wheat straw,ammonia nitrogen,volatile fatty acid(VFA),xylanase,carboxymethyl cellulase(CMCase),avicelase,acetyl esterase(AE)activities of fermentation liquid.The results showed that: 1)the addition of Orpinomyces sp.YF3 had no significant effect on in vitro dry matter disappearance rate of wheat straw;2)the addition of 15% Orpinomyces sp.YF3 reduced the ammonia nitrogen concentration in the fermentation broth after 3 days of fermentation(P < 0.05);3)The addition of Orpinomyces sp.YF3(10%,15% addition amount)increased the xylanase and AE activities of the fermentation broth after 3 days and 5 days of fermentation(P < 0.05);4)Adding Orpinomyces sp.YF3 increased the ratio of acetate and A : P in the fermentation broth(P <0.05),and the ratio of isobutyric,butyric and isovaleric were reduced(P < 0.05).The results of this experiment show that the addition of the yak rumen anaerobic fungi Orpinomyces sp.YF3 can promote the degradation of lignocellulose,and the optimal dosage is 15%.Experiment 2 The effect of repelling fungi and adding yak rumen anaerobic fungi Orpinomyces sp.YF3 on in vitro fermentation and microbial floraCombined with the results of experiment 1,the anaerobic fermentation flasks were used to carry out in vitro fermentation to explore the effect of repelling fungi and adding yak rumen anaerobic fungi Orpinomyces sp.YF3 on rumen fermentation and microbial flora.The experiment was divided into 4 treatments: control group(C),yak fungi group(CF),fungi repellent group(CA),fungi repellent + yak fungi group(CAF),with 6 replicates for each treatment.This experiment adopts a 2*2 two-factor experiment design,with 1%(w/v)crushed air-dried wheat straw as the fermented substrate.The experiment period contituted 11 days.Cycloheximide(0.25 mg/mL)was added to the fermentation flasks of CA and CAF in d0,and the fermentation was allowed to stand at 39℃ for 2 days to remove the fungi in the fermentation system.During d2-d8,the fermentation broth(50%)from all bottles is drawn every day and transferred to new anaerobic fermentation bottles containing artificial saliva(50%)and wheat straw to remove cycloheximide.During d4-d8,the fermentation flasks of CF and CAF groups were added 15% Orpinomyces sp.YF3 liquid every day,and then allowed to stand and ferment for 3 days.Sampled at d11 to determine pH,ammonia nitrogen concentration,plant cell wall degradation enzyme activity,VFA,and bacterial diversity of fermentation liquid,and the fungal diversity of the C and CF groups.The experiment results showed that: 1)the addition of Orpinomyces sp.YF3 increased the concentration of ammonia nitrogen(P < 0.001),and had no significant effect on pH;2)the addition of Orpinomyces sp.YF3 increased the CMCase(P < 0.05),and avicelase activity(P= 0.001).Repelling fungi increased the avicelase activity(P < 0.001);3)The addition of Orpinomyces sp.YF3 increased the total VFA concentration and the ratio of acetate,isobutyric,and valeric,and the A:P(P < 0.001).Reduced the ratio of propionate and butyric(P < 0.001).Repelling fungi increased the ratio of acetate and the A : P(P < 0.05),and reduced the concentration of total VFA and the ratio of propionate,isobutyric,isovaleric,and valeric(P <0.05).The addition of Orpinomyces sp.YF3 significantly improved the bacterial diversity,richness and uniformity(chao,shannon and shannonven indexes)of the fermentation broth.Significantly increased the relative abundance of Bacteroidota,Synergistota,Desulfobacterota,Actinobacteria and Fusobacteriota in the fermentation liquid,and reduced the relative abundances of Fibrobacterota and Proteobacteria(P < 0.05).Orpinomyces sp.YF3 increased the relative abundance of Rikenellaceae＿RC9＿gut＿group,unclassified＿f＿＿Rikenellaceae,Ruminococcus,NK4A214＿group,Pyramidobacter,Anaerovorax(P < 0.001)and norank＿f＿＿F082,norank＿f＿＿UCG-010,norank＿f＿＿Bacteroidales＿BS11＿gut＿group,Papillibacter,Schwartzia(P < 0.05),and reduced the relative abundance of Fibrobacter,Succinivibrio,Lachnospiraceae＿UCG-009(P < 0.001),Prevotella,unclassified＿f＿＿Lachnospiraceae,probable＿genus＿10,unclassified＿c＿＿Clostridia,norank＿f＿＿Bacteroidales＿UCG-001(P < 0.05).Analyzing the diversity of fungi in the two groups C and CF found that there are many yeasts in the fermentation broth.The addition of Orpinomyces sp.YF3 had no significant effect on the fungal α diversity of the fermentation broth and the community composition at the phylum level.At genus level,The relative abundance of the four yeasts(Vishniacozyma,Sporidiobolus,Filobasidium,Bulleromyces)are significantly reduced.Repelling fungi has no significant effect on the bacterial α diversity of the fermentation broth.At the phylum level,the relative abundance of the Synergistota and Desulfobacterota were increased(P < 0.05),and the relative abundance of the Fusobacteriota was reduced(P <0.001).At the genus level,repelling fungi increased the relative abundance of norank＿f＿＿F082,Christensenellaceae＿R-7＿group(P < 0.001),Rikenellaceae＿RC9＿gut＿group,norank＿f＿＿Bacteroidales＿BS11＿gut＿group,Anaerovorax,Pyramidobacter(P < 0.05),and reduced the relative abundance of Fusobacterium(P < 0.001),Prevotella,Streptococcus,norank＿f＿＿Bacteroidales＿UCG-001,Eubacterium＿ruminantium＿group(P < 0.05).Correlation analysis between bacteria with plant cell wall degrading enzyme activities and VFA revealed that most of the bacteria with increased relative abundance caused by addition of Orpinomyces sp.YF3 and repelling fungi were positively correlated to CMCase and avicelase activity,total VFA concentration,and the ratio of acetate,and the bacteria with decreased relative abundance are positively correlated with propionate.Correlation analysis between bacteria and significantly different fungi found that there are basically the same correlations.Most of significantly different fungi are significantly positively correlated with Prevotella and Fibrobacter,probable＿genus＿10,and are significantly negatively correlated with Rikenellaceae＿RC9＿gut＿group,unclassified＿f＿＿Rikenellaceae,norank＿f＿＿F082,Ruminococcus,norank＿f＿＿Bacteroidales＿BS11＿gut＿group,NK4A214＿group,Schwartzia,Anaerovorax,Pyramidobacter.This experiment shows that the addition of the yak rumen anaerobic fungus Orpinomyces sp.YF3 can increase the relative abundance of fiber degradation-related bacteria(such as Rikenellaceae＿RC9＿gut＿group,Ruminococcus,NK4A214＿group,Pyramidobacter)and acetic acid-related bacteria(Anaerovorax),and reduce propionic acid-producing bacteria(Succinivibrio,Fibrobacter,Prevotella),increase plant cell wall degrading enzyme activity,promote cellulose degradation,increase total VFA concentration,and acetic acid ratio.This is conducive to improving the utilization rate of ruminant roughage.