Contamination Investigation Of Maize Silage AFT-B1 And Its Effect On Rumen Environment In Henan Province English Title

Henan,located in Zhongyuan,is a large province of agriculture as well as animal husbandry.In recent years,with the support of favorable national policies,the cattle and sheep industry has developed rapidly,and the amount of cattle and sheep has become an important support for the development of the livestock industry,ranking in the forefront of the country for many consecutive years.Feed material is the material basis for the development of grass and livestock industry.Whole corn silage accounts for 60%～70%of dairy cows’diet,and its quality directly affects dairy cows’performance（weight gain,yield increase,reproductive rate,etc.）and their health.Aflatoxin B₁（AFB₁）is a common mycotoxin in feed,which affects the health of dairy cows and milk quality,causes losses in dairy farming,and harms calves and human health.To understand the contamination of maize silage AFB₁in Henan province and its effect on rumen environment of dairy cows,and to provide reference for dairy production and disease prevention and control.From October 2020 to October 2021,more than 200 samples of whole corn silage were collected from more than 200 dairy farms in 15 cities of 5 regions in Henan Province for two consecutive years.The content of AFB1 was detected by enzyme-linked immunosorbmetry（ELISA）,and the double flow artificial rumen simulation system and macrogenomic sequencing were used.The effect of AFB₁on rumen environment was studied.The results of the investigation on AFB₁contamination of maize silage in Henan province in 2020 and 2021 showed that the detection rate of aflatoxin B₁was slightly different in different regions of Henan Province.The average detection rate of aflatoxin B₁was lower than the limit value by 30μg/kg in each region,but the detection rate of aflatoxin B₁was higher in each region,and the maximum value exceeded the limit value by 3 times.The results of the survey in 2020 showed that the detection rate of whole corn silage samples in Henan province was 65.14%,and the sample overstandard rate was 4.59%.There were significant differences in the sample detection rate and over standard rate in different regions of Henan Province（P<0.05）.The detection rates in the five regions were 36.84%in western Henan,57.89%in central Henan,58.33%in southern Henan,75.00%in northern Henan and92.30%in eastern Henan.The over-standard rates of samples were 0%in western Henan,0%in southern Henan,5.26%in central Henan,6.81%in northern Henan and 7.69%in eastern Henan.The results of 2021 showed that the detection rate of whole corn silage samples in Henan province was 97.16%,and the sample overstandard rate was 0.The detection rates of samples in different areas of Henan province were not significant（P>0.05）.The detection rates of samples in five areas were 95%in north henan,95%in central Henan,100%in east Henan,100%in west Henan and 100%in south Henan.The detection rates of samples were all relatively high,and the over standard rates of samples were all 0.The results of the two years’investigation showed that the pollution of AFB1 in silage of dairy farms in five regions of Henan province was widespread,but the overall pollution degree was relatively light,and the pollution in eastern Henan,northern Henan and central Henan was relatively serious.The effects of AFB₁supplementation with different concentrations on rumen environment of Holstein dairy cows were studied by in vitro artificial rumen simulation and macrogenomic sequencing.Five different aflatoxin B₁diet groups were designed by the balanced 5-5 Latin method,which was divided into 5 cycles,and each cycle was cultured for10 days.Before feeding in the morning and evening,two batches of fermentation broth inoculated for 3,8,9 and 10 days in each artificial rumen fermentation tank were collected,one of which was used for pH value determination and the count of parasites.The other sample was cryopreserved at-20℃to measure ammonia nitrogen concentration and extract DNA for macro factor sequencing.PH value determination,protozoa technology,ammonia nitrogen concentration determination and metagenomic sequencing analysis of 16S r RNA V3-V4 region and 18S r DNA V4 region showed that:1)AFB1 had certain effects on rumen physical and chemical indexes.PH value of AFB1 diet was lower than that of blank control group,and there was no significant difference in pH value between groups（P>0.05）,indicating that AFB₁supplementation reduced rumen pH value and increased rumen acidity.Artificial rumen simulation had a significant effect on the ammonia-nitrogen concentration of the samples（P<0.05）,and the ammonia-nitrogen concentration of the samples in the blank control group decreased first and then increased,showing an overall upward trend;There was no significant difference in ammonia nitrogen concentration between AFB₁group and non-AFB₁group（P>0.05）,and the main factor that reduced ammonia nitrogen concentration was artificial rumen simulation technology.2)AFB₁significantly affected some microorganisms in rumen,and compared with the blank control group,the relative abundance of prevotellaceae,unclassified Bacteroidetes and unclassified firmicutes was significantly decreased（P<0.05）,while the relative abundance of proteobacteria was significantly increased（P<0.05）.Compared with the blank control group,the number of rumen protozoa was significantly decreased（P<0.05）.AFB₁could inhibit the growth of rumen protozoa side stomata and Chytrid fungi,and promote the growth of Trichoderma fungi.There was no significant difference in rumen microbial population among groups supplemented with different concentrations of AFB₁（P>0.05）.Overall,the higher the AFB1concentration,the greater the influence.3)artificial rumen simulation technology to the influence of population structure in the rumen bacteria significantly,and the original rumen liquid ratio,fan of thick wall door,the door deformation bacteria and trace the relative abundance of door was lower（P<0.05）,door germs bacteroidetes,warts and glue the relative abundance of the spherical bacteria door significantly increased（P<0.05）,the advantage of eukaryotic species changed significantly,The relative abundance of rumen fungi was high,while rumen protozoa was low.4)The microbial community structure in artificial rumen changed with the extension of cultured time,and the relative abundance of Clostridium was significantly decreased compared with cultured days 3,8,9 and 10 in vitro（P<0.05）.The relative abundance of unclassified＿Bacteroidetes,Treponema and Candidatus＿Endomicrobium was significantly increased（P<0.05）,the relative abundance of Butyrivibrio was decreased first and then increased,and the relative abundance of Trichophiloides was significantly decreased（P<0.05）.5)AFB₁significantly decreased（P<0.05）the relative abundance of bacteroidetes in artificial rumen,leading to decreased protein hydrolytic ability in rumen,compared with dietary groups supplemented with different AFB1 concentrations.The prolongation of artificial culture time in vitro significantly increased（P<0.05）the relative abundance of butyrivibrio and Bacteroidetes in artificial rumen,and improved the capacity of hydrolyzed protein and acid production in the rumen.In conclusion,AFB₁reduced the abundance of bacteria in Bacteroidetes phylum,resulting in decreased rumen protein hydrolytic ability.The prolongation of artificial culture time in vitro significantly increased the relative abundance of butyrivibrio and Bacteroidetes,and thus increased the capacity of rumen hydrolysate and acid production.AFB1 had a certain effect on rumen eukaryotic population diversity,but AFB1 did not change the dominant species,but only had a certain effect on species richness.At the same time,there were many unannotated fungi and protozoa in the sequencing results of this study,indicating that the current research on rumen eukaryotes is not comprehensive enough and further research and exploration are needed.