| Glycerin-Tributylat,as a class of feed additives with antimicrobial properties,has the efficacy of preventing intestinal inflammation,protecting intestinal mucosa,and reducing oxidative stress,etc.It has been widely used in livestock feed,especially in poultry and piglets,and the direction of application in lactating calves is still to be further explored.In this test,the effect of glycerol Glycerin-Tributylat on the prevention and treatment of mice infected with E.coli of bovine origin was investigated based on the diarrhea model of E.coli O157:H7 of bovine origin;on the basis of this test,the effect of glycerol Glycerin-Tributylat on the growth,blood immunity,oxidative index and intestinal microorganism of lactating calves was investigated.The main contents and results of this study are as follows.(1)Establishment of a mouse model of enterohemorrhagic E.coli diarrhea:using a completely randomized test,30 female KM mice with an initial weight of(20±1)g at SPF level were selected and divided into 5 groups of 6 mice each;the concentration of the bacterial solution was tested using a 600 nm OD ultraviolet spectrometer,and the solution was diluted with saline and adjusted to:0.4×10~8CFU/mL、0.8×10~8CFU/mL、1.0×10~8CFU/mL、1.6×10~8CFU/mL、2.4×10~8CFU/mL,The results showed that the diarrhea rate reached 83.33%at the concentration of 1.0×108 CFU/mL,and the mortality rate was 0,which was determined as the most suitable modeling dose.This concentration was determined to be the most suitable modeling dose.(2)Effect of glyceryl Glycerin-Tributylat on the intestine of mice with enterohemorrhagic E.coli diarrhea:120 female 4-week-old SPF-grade KM mice with an initial weight of(20±1)g were selected and divided into 6 groups of 20 mice each after the pre-feeding period,and saline was administered to the control and self-healing groups before daily morning feeding at the first13 d of the test period,respectively;test groupsⅠ,Ⅱ,ⅢandⅣwere The following ratios of 1.0g/kg,2.5 g/kg,3.5 g/kg and 5.0 g/kg of triglyceride(mass of triglyceride/mouse body weight)were administered to the control and self-healing groups,respectively,before each morning feeding.By the 14th day of the test,mice in each group were deprived of food but not water for8 h,after which the control group was injected intraperitoneally with 0.2 mL of sterilized saline,and the self-healing group and test Ⅰ,Ⅱ,Ⅲ and Ⅳ groups were injected intraperitoneally with0.2 mL of 1.0×10~8CFU/mL EHEC O157:H7 bacterial solution.After the attack,the control and self-healing groups were given saline before the daily morning feeding,and the four testal groups were given the previous corresponding triglyceride before the daily morning feeding until28 d.Five mice were randomly sampled on days 1,7 and 14 after the infection for 28 days.The difference in mean daily weight gain between the groups was highly significant(P<0.05)from day 1 to 7 after the attack,and the greatest decrease in mean daily weight gain was observed in the self-healing group;from day 8 to 14 after the attack,the mean daily weight gain of mice with diarrhea in test groups Ⅱ and Ⅲ was significantly higher(P<0.05).On days 1 and 7 after the attack,IL-1βwas significantly higher in the self-healing group(P<0.05),with little change between the control group and the four test groups;on day 1 after the attack,IL-4 was lower in the self-healing group(P<0.05),and IL-4 in test group Ⅲ was higher than that in the remaining three test groups and the control group;on days 1 and 7 after the attack,IL-10 was lower in the self-healing group(P<0.05),and IL-10 in test group Ⅲ was higher than that in the remaining three test groups.At days 1 and 14 post-attack,TNF-αwas significantly higher in the self-healing group(P<0.05),while there was little change between the control group and the four testal groups.At days 1 and 7 after the attack,IgA was reduced in the self-healing group(P<0.05),while IgA was higher in test groups Ⅱ,Ⅲ and Ⅳ than in the control group and test group Ⅰ on day 1 after the attack,and higher in test group Ⅱ than in the control group and the remaining three test groups on day 7 after the attack;at days 1 and 7 after the attack,IgG was significantly lower in the self-healing group with diarrhea(P<0.05),and IgG in test group Ⅲ was higher than in the remaining three test groups.The IgM in the self-healing group was significantly lower(P<0.05)on the 1st and 7th days after the attack,while the IgM in the diarrhea mice of the test group Ⅲ was significantly higher than that in the remaining three test groups.(3)Effect of different levels of Glycerin-Tributylat on growth performance and blood indexes of lactating calves:the difference in body weight of lactating calves in each test group during the test period was not significant(P>0.05);on day 20 of the test period,the body height of lactating calves in test groups Ⅱ and Ⅲ was significantly higher than that of control group and test group I(P<0.05).On days 15 and 30 of the test period,MDA levels in the blood of lactating calves in test group Ⅲ were significantly lower(P<0.05);on day 30 of the test period,GSH levels in the blood of lactating calves in all three test groups were significantly higher(P<0.05);CAT levels in the blood of lactating calves in all three test groups were significantly higher(P<0.05).The concentration of TNF-αwas significantly lower in lactating calves of test groups Ⅱ and Ⅲ at 15 days of the test period(P<0.05);the concentration of IL-4 was significantly higher in lactating calves of test groups Ⅱ and Ⅲ at 15 and 30 d of the test period(P<0.05);and the concentration of IL-10 was significantly higher in lactating calves of the three test groups at 30 days of the test period(P<0.05).(4)Effect of Glycerin-Tributylat on fecal microbial diversity in lactating calves:Before morning feeding on days 0,10,20 and 30 of the formal test phase,three test cows were selected from four groups and 30 g of rectal end fecal samples were scraped and tested for 16 S rRNA.48samples yielded 3155435 valid genes and 8520 OTUs were obtained in 41 phyla,256 orders,438orders,99 families,981 genera and 612 species.phylum,256 phyla,438 orders,99 families,981genera and 612 species.From day 0 to day 10,the changes in Chao1,Shannon and Simpson indices did not show significance(P>0.05).However,when time lapsed to d 20,the Chao1index was significantly higher(P<0.05)in test Ⅱ and Ⅲ groups than in the control group,while the changes in Shannon and Simpson indices were less significant(P>0.05).At d 30,Chao1,Shannon,and Simpson indices were higher in test groups Ⅱ and Ⅲ than in the control group(P<0.05).There was no significant difference in the dominant flora of each test group from day 0 to day 10 just after the start of the test(P>0.05).On the 20th day,the number of Aspergillus phylum increased significantly in test groups Ⅱ and Ⅲ(P<0.05).At day 30,the number of the phylum Anaplasma was also increased in both test groups Ⅱ and Ⅲ.In summary,in the diarrhea mouse test,glyceryl Glycerin-Tributylat significantly increased the serum IgA,IgG and IgM levels;decreased the concentrations of IL-1βand TNF-α;increased the levels of anti-inflammatory cytokines such as IL-4 and IL-10,and 2.5 g/kg～3.5 g/kg of glyceryl Glycerin-Tributylat had the best effect on diarrhea mice.In the calf test,glyceryl Glycerin-Tributylat could significantly increase the concentration of immunoglobulin and decrease the concentration of pro-inflammatory factors in lactating calves;reduce the concentration of MDA in calves and regulate the concentration of GSH-Px and CAT in calf serum;it could increase the diversity of floraa and also increase the relative abundance of the phylum Bacillus mimicus,improve the intestinal environment and enhance immunity.The best effect of adding 3.5 g/kg triglyceride to calves was achieved in test group Ⅲ. |
