Temporal Characteristics Of The Milk Flora Of Holstein Cows And Correlation With Milk Composition

In recent years,the demand for dairy products in people’s lives has been increasing,and the health and productivity of cows is directly related to the development of China’s dairy industry.Understanding the changing patterns of dairy flora will allow better control of dairy production and provide more and better dairy products for humans.In addition,breast milk provides not only essential nutrients and a wide range of bioactive compounds,but also beneficial bacteria to meet all the needs of the newborn,and the development of the calf depends on the quantity and quality of the mother’s milk.However,there are relatively few studies using16 S rRNA high-throughput sequencing to analyse the diversity of dairy flora in Holstein cows.In this study,milk samples were collected from six primiparous Holstein cows(0 d),three days(3d),five days(5 d),seven days(7 d),15 days(15 d),21 days(21 d),30 days(30 d)and 60 days(60 d)after parturition from six primiparous Holstein cows.High-throughput sequencing based on the 16 S rRNA V4 variable region was performed on cows in the primiparous and periparous groups,and the dominant phylum was identified at the phylum level with a relative abundance >0.5%,and the dominant genus was identified at the genus level with a relative abundance > 1%,respectively,the dominant genera were selected according to the interrelationship of lactic flora at different times postpartum,characterized by the relative abundance of lactic flora at each time point,construction of a co-occurrence network structure of lactic flora using Spearman’s method and screening of specific flora with potential association with milk components in lactic milk by combining the results of milk composition analysis.Finally,the Tax4 Fun method was used to predict the metabolic function of dairy cattle lactic flora and to select the Top 10 functional genes.The aim was to observe the chronological characteristics of the milk flora of Holstein cows and to analyse the association with milk composition.The main findings are as follows:(1)The composition and structure of the milk flora of Holstein cows varied significantly(P<0.05)depending on the duration of lactation.The diversity and relative abundance of lactic flora from 0 d to 7 d after parturition fluctuated and gradually stabilised from 15 d to 60 d after parturition,and the diversity of lactic flora from 0 d to 7 d was lower than that from 15 d to 60 d in both warren and primiparous Holstein cows.(2)The four dominant phyla with the highest relative abundance in the milk of Holstein cows were Proteobacteria,Firmicutes,Bacteroidota and Actinobacteria in the milk of both groups.The dominant genera for primiparous Holstein cows at all time points were Pseudomonas,UCG-005 and Acinetobacter.The dominant genera at all time points in the warmblooded Holstein cows were UCG-005,Acinetobacter,Vibrio,Bacteroides and Streptococcus.(3)Thirty-four dominant genera were significantly associated with the milk flora of primiparous Holstein cows from 0 d to 7 d postpartum,and 28 dominant genera were significantly associated from 15 d to 60 d postpartum;Forty-two dominant genera were significantly associated with milk flora from 0 d to 7 d postpartum and 33 dominant genera were significantly associated from 15 d to 60 d postpartum in warmblooded Holstein cows.Weissella in the milk of primiparous Holstein cows was positively correlated with the dominant genera of Halogranum,Lactococcus and Parabacteroides,and seven dominant genera of UCG-005 and Marinobacter were negatively correlated.Escherichia-Shigella is positively correlated with the dominant genera of Streptococcus,Brevundimonas and Faecalibacterium in the milk of warmblooded Holstein cows,and the potentially pathogenic bacteria are positively correlated with other pathogenic bacteria.(4)Milk fat content in milk of first-calf Holstein cows was significantly different(P < 0.05)at 30 d compared to other times,and milk protein and urea nitrogen content were significantly different(P < 0.05)at 0 d compared to other times;The milk protein and urea nitrogen milk component content of milk from warmblooded Holstein cows differed significantly(P < 0.05)at0 d compared to other times and correlated with the relative abundance of microorganisms in the milk.Among them,Dubosiella,Ruminococcus,Leuconostoc,Weissella,Candidatus＿Puniceispirillum and OM60(NOR5)＿clade in primiparous Holstein heifers were associated with protein and urea nitrogen content were positively correlated(r ≥0.7).Amylibacter,HIMB11,Candidatus＿Puniceispirillum,SUP05＿cluster,Ileibacterium,Dialister,Colidextribacter and Ralstonia in transhortened Holstein cows were positively correlated with protein and urea nitrogen content(r ≥ 0.7).(5)The main functions of the lactic flora of primiparous and periparturient Holstein cows are carbohydrate,membrane transport,amino acid metabolism,replication and repair,translation and energy metabolism,and the relative abundance of these six functional gene families fluctuates from 0 d to 7 d and gradually stabilises from 15 d to 60 d.In summary,the milk of Holstein cows is rich in flora,with flora significantly associated with milk protein and urea nitrogen content,and the diversity of milk flora and the relative abundance of functional genes is influenced by lactation time and also by litter size.