Diurnal Variation Of Ruminal Melatonin And The Influence On Rumen Microorganisms In Lactating Cows

Gut hormones can not only regulate the digestive,absorptive capacity,and immune mechanisms of the intestine through biological rhythms,but also regulate the host by affecting intestinal microorganisms.However,there are still few reports on ruminal hormones and their relationship with rumen microorganisms in ruminants.Therefore,this study investigated the diurnal change of ruminal melatonin and rumen bacteria population structure in lactating cows,and their correlation both in vivo and in vitro.To explore the secretion regulation of ruminal hormones and their effects on ruminal microflora.Experiment 1.Exploration of circadian rhythms of rumen hormones GH,PRL,and MLT in lactating dairy cowsThis part of study investigated the diurnal variation of melatonin?MLT?,growth hormone?GH?and prolactin?PRL?concentrations in rumen fluid of lactating dairy cows,and their contents in serum,saliva.Six mid-term lactating cows with similar weight,parity,and lactation performance were selected.The blood,saliva,and rumen fluid samples were collected at 02:00,10:00,18:00?1st d?and 06:00,14:00,22:00?2nd d?respectively to measure the concentrations of these three hormones.The results showed that the concentration of MLT in serum,saliva and rumen fluid has obvious circadian rhythms,and similar to the change pattern of the Cosine biological clock:the concentration of MLT in dark is significantly higher than the concentration of MLT in light?P<0.05?;PCA analysis of MLT in serum,saliva and rumen fluid showed that MLT in rumen is likely to derive from blood other than saliva.And correlation analysis showed that only MLT in serum had a significant positive correlation with MLT in rumen fluid?R=0.34,P=0.042?.In addition,GH in serum and rumen fluid was not affected by time?P>0.05?,whereas the GH concentration in saliva at 06:00 was significantly higher than that at 02:00,14:00 and 18:00?P<0.05?;PRL concentration in saliva was not influenced by time either?P>0.05?,but PRL concentrations in serum and rumen fluid at different time points are significantly different?P<0.05?and they showed opposite trends.In summary,the rumen fluid contains and MLT,and the diurnal variation of MLT follows the circadian rhythm,which is regulated by the light-dark cycle.Although GH and PRL also exist in rumen fluid,their concentration without obvious rhythm.Experiment 2:Study on the correlation between diurnal change of rumen fermentation parameters and ruminal flora structure of lactating dairy cowsThe main purpose of this part of experiment is to study the diurnal variation of rumen flora in lactating dairy cows,and to screen bacteria that are most relevant to the diurnal variation of MLT in the rumen.The experimental method is same as experiment 1,as six mid-term lactating cows with similar weight,parity,and lactation performance were collected rumen fluid samples at 02:00,10:00,18:00?1st d?and 06:00,14:00,and 22:00?2nd d?respectively.Rumen fluid samples were then determined rumen fermentation parameters and analyzed microflora structure by using 16S amplicon sequencing.Results showed that he rumen flora structure was significantly different between Dark and Light,and the Shannon index at Dark was significantly higher than that at Light?P<0.05?,which complies with Cosine circadian clock rhythm?r2=0.933?;non-parametric test JTK_CYCLE analysis found 4.6%of the identifiable OTU of rumen flora had a significant circadian rhythm?P<0.05?;the rumen fermentation pH,NH3-N and MCP contents showed circadian rhythm,and NH3-N contributed significantly to the rumen flora structure?P<0.05?,MLT and valeric also contributed evidently to the structure of the rumen flora.Differential analysis of microbial abundance found that the relative abundance of Preteobacteria and Succinivibrionaceae in Dark were significantly lower than that in Light,while the relative abundances of Bacteroidetes and Prevotellaceae were significantly higher in Dark than that in Light?P<0.05?;OTU₁,OTU₂823,OTU₂330?all of them are unidentified Succinivibrionaceae?,OTU₄8?Prevotellaceae?,and OTU₁57?undentified Prevotellaceae?have important role in the flora structure,and the diurnal rhythm of relative abundance of OTU₁,OTU₂823,and OTU₂330 are opposite to the ruminal MLT diurnal rhythm.The diurnal variation of relative abundance of OTU₄8 and OTU₁57 is consistent with the diurnal variation of rumen MLT concentrations.In conclusions,some rumen bacteria and fermentation parameters also behave circadian rhythm,and light-dark cycle or MLT have a significant impact on the relative abundance of Prevotellaceae and Succinivibrionaceae family.Among them,the diurnal rhythm of relative abundance of Succinivibrionaceae family is opposite to that of MLT,whereas the circadian rhythm of relative abundance of Prevotellaceae family is consistent with MLT circadian rhythm.Experiment 3.Effects of melatonin on rumen flora structure and fermentation parameters in vitro fermentationThe main purpose of this part of experiment is to verify the relationship between MLT and rumen flora by in vitro simulated rumen fermentation techniques.Artificial saliva+standard melatonin solution?CK0 group?,artificial saliva+rumen fluid?CK1 group?and artificial saliva+rumen fluid+standard melatonin solution?MLT group?were cultured in vitro for 24 h respectively.The culture rumen fluid samples were collected at 0,4,8,12,16,20,24 h to analyze the MLT concentration,and the rumen fermentation parameters and rumen flora structure in the CK1 and MLT groups.Results showed that both the culture time and treatment had a significant effect on the concentration of MLT in the culture medium?P<0.01?,and the degradation rate of MLT is faster in CK1 and MLT groups than in CKO group?P<0.05?;The differences of ? and ? diversity and rumen fermentation parameters between the MLT and CK1 group were not significant,but the shannon index were the lowest at 12 h,which also conforms to the Cosine circadian rhythm?r2 is 0.872 and 0.910 respectively?;the difference analysis of the flora revealed that the abundance of Batroidetes,Bacteroidia,Bacteroidiales,and Prevotellal were significantly higher than those in CK1 group?P<0.05?,while Kiritimatiellaeota,Kiritimatiellae and WCHB1₄1 in CK1 group were significantly higher than those in MLT group?P<0.05?;random forest analysis showed most of the bacteria which could represent MLT were belong to Bacteroidales in groups CK1 and MLT,and OTU395 as well as OTU365,which both in this two groups were belong to Prevotellaceae.Correlation analysis showed that there was a significant positive correlation between Prevotellaceae or Prevotella 1 and MLT?P<0.05?,and MLT significantly increased the relative abundance of Prevotellaceae and Prevotella 1,whereas reduced the relative abundance of Succinivibrionaceae,unidentified Succinivibrionaceae,Ruminobacter and Succinivibrionaceae UCG-002?P<0.05?;in flora function prediction analysis,MLT was significant decreased the flora functions of cell motility and secretion,atrazine degradation,phosphate metabolism and protein processing;Bacteroidetes,Bacteroidia,Bacteroidales,Prevotellaceae,Prevotella 1 and Succinivibrioaceae also have significant correlations with these pathways?P<0.05?.In summary,MLT has no impacts on the ? and ? diversity of rumen flora in vitro culture,and the bacteria may have a biological rhythm when cultured in vitro;MLT has a great impact on Bacteroidetes,and MLT could enhance the relative abundance of Prevotellaceae,whereas decrease the relative abundance of Succinivibrionaceae.Conclusions:1)Lactating cows have certain concentrations of melatonin in the rumen,and the contents of melatonin conforms to the circadian rhythm;the pH value,ammonia nitrogen concentration and rumen microbial protein content in rumen fluid and 4.6%rumen bacteria showed circadian rhythm.2)In vitro experiment showed that the rumen flora and its fermentation parameters were not influenced by the concentration of MLT,but some rumen bacteria may have biological rhythm even in vitro.3)Both in vivo and in vitro experiments have shown that ruminal MLT exist relationship with Prevotellaceae and Succinivibrionaceae,as MLT can increase the relative abundance of Prevotellaceae and Prevotella 1,while reducing the relative abundance of Succinivirobrionaceae,unidentified Succinivirobrionaceae,Ruminobacter and Succinivirobrionaceae UCG-002,the specific mechanism still needs further research.4)The light and dark cycle may influence the abundance of Bacteroidetes,especially Prevotella and Succinivibrionaceae,thereby regulate the rumen flora function of atrazine degradation,cell motility and secretion,phosphate metabolism,protein folding and associated processing as well as lysine biosynthesis,and finally regulate milking performance of dairy cows.