Effect And Mechanism Of Intraperitoneal Injection Of Yeast β-glucan During The Suckling Period On Intestinal Health Of Weaned Rabbits

Diarrhea is one of the key factors affecting the early growth and development of meat rabbits,and weaned rabbits are particularly susceptible to infection by various pathogens,which can lead to diarrhea and seriously affect their productive performance after weaning.Therefore,the prevention of infection by complex pathogens during weaning is a crucial task in rabbit breeding.It has been shown that trained organisms produce faster and stronger immune responses to reinfection with homologous and heterologous pathogens,thereby strengthening their resistance to pathogens.Accordingly,the primary objective of this investigation was to assess the impact of intraperitoneal administration of trained immunostimulant(yeast β-glucan)during the lactation period on the intestinal well-being of weaned rabbits and the underlying mechanisms involved.In this study,the control group(C)of rabbits that received intraperitoneal injections of saline was used,while the trained group(T)of rabbits that received intraperitoneal injections of yeast β-glucan solution was also included.The primary focus of this research was to examine the potential impact of yeastβ-glucan-induced trained immunity on the growth performance,intestinal barrier,intestinal immunity,intestinal flora composition and antioxidant capacity of the organism in weaned rabbits were also investigated.Experiment 1: Effect of intraperitoneal injection of yeast β-glucan during the suckling period on the growth performance of weaned rabbitsThe pre-test was conducted in March 2022 at the rabbit farm belonging to Yangling Demonstration Zone,Shaanxi Province,China,for 7 days.The rabbits in the injection group were injected intraperitoneally with yeast β-glucan solution(Concentration: 0.05g/m L,Dose: 65 mg/kg weight)at 22 and 24 days of age,respectively,while the rabbits in the oral group were injected orally with the same dose and concentration of yeastβ-glucan solution at the same age.The control group of rabbits received an equivalent dose of saline injection.Serum samples were collected from both the injection and control groups 12 hours after the second stimulation,whereas in the oral group,serum samples were collected 24 hours after the second stimulation.The formal trial was conducted in April 2022 at the rabbit farm belonging to the Yangling Demonstration Zone of Yangling,Shaanxi Province,China.In this trial,64 suckling rabbits in good health were randomly divided into two groups according to litter size and body weight: the control group(C)and the trained group(T).At 6 and 4 days before weaning(29 and 31 days of age,respectively),the rabbits in both groups were injected intraperitoneally with saline and yeast β-glucan dissolved in saline(Concentration: 0.05 g/m L,Dose: 65 mg/kg weight),respectively.The rabbits were weaned and fed in separate cages at 35 days of age and continuously observed for 14 days.After weaning,the rabbits were scored daily for feces and weighed for daily feed intake.On the day of weaning,7 days after weaning and 14 days after weaning,the rabbits were weighed before morning feeding.At 14 days after weaning,10 rabbits in each group were randomly selected for slaughter sampling.The findings of this study indicate that pre-weaning stimulation with β-glucan resulted in a significant decrease in the incidence of diarrhea during the first week after weaning(P = 0.035)and a tendency to decrease diarrhea during the second week after weaning(P = 0.079)compared to the control group.However,no significant effect on the average daily weight gain,average daily feed intake,or feed-to-weight ratio of the weaned rabbits was observed(P > 0.05).The histopathological analysis revealed that the ileum crypt depth was significantly reduced(P < 0.01)and the VCR was significantly elevated(P < 0.01)in the trained group compared to the control group.Additionally,the apical part of the ileal villi showed slight degradation and flaking in some control groups,while the villi morphology in the trained group was found to be normal.Experiment 2: Effect of intraperitoneal injection of yeast β-glucan during the suckling period on intestinal barrier,immunity and antioxidant capacity of weaned rabbitsAfter slaughter sampling,blood,tissue and mucosa of ileum and appendix were collected and used to determine the expression of intestinal barrier and immune-related genes in the ileum and appendix of rabbits,and serum immunoglobulin and oxidative stress levels were also measured.The present study’s findings indicate that the trained group exhibited a significantly higher relative expression of TNF-α and IL-10 genes in appendix than the control group(P < 0.01).Additionally,the control group showed a significantly lower relative expression of the IL-6 gene(P < 0.05).Conversely,no significant differences in the relative expression of IFN-β,TLR2,TLR3,and TLR4 genes were observed between the two groups.Notably,the relative expression of TLR2 in ileal tissue of the trained group demonstrated a tendency to decrease(P = 0.074),and the relative expression of TNF-α,IL-10,IFN-β and IL-6 genes was not statistically different.Importantly,the results indicated that the trained group showed a significantly higher level of ileal mucosal LYZ(P < 0.01),while the level of LYZ in appendix mucosa showed a tendency to increase(P= 0.055).Moreover,a trend towards increased Muc2 levels was observed in the ileal mucosa of rabbits in the trained group(P = 0.058).Additionally,the serum Ig A and Ig G levels in the ileal mucosa of rabbits in the trained group were significantly elevated(P <0.01),along with significantly increased levels of mucosal s Ig A in their ileal mucosa(P <0.01).However,no statistical difference was observed in s Ig A levels in the appendix.Serum MDA levels(P < 0.01)and hydroxyl radical levels were significantly decreased in the trained group(P < 0.01),but did not affect T-AOC levels(P = 0.723).Experiment 3: Effect of intraperitoneal injection of yeast β-glucan during the suckling period on the structure of ileal mucosal flora of weaned rabbitsTo investigate the effect of β-glucan-induced trained immunity on the intestinal flora structure of weaned rabbits,16 S r DNA sequencing was performed on ileal mucosal samples to analyze the effect of β-glucan-induced training immunity on the intestinal flora structure of the rabbits and its microbial mechanism for reducing the rate of diarrhea in the weaning period.The study’s findings revealed no significant difference in the Shannon index of ileal mucus flora between the two groups of rabbits.The β-diversity analysis of the flora showed that the two groups of ileal mucus samples showed different clustering distributions(P < 0.01).Compared to the control group,the proportion of the Firmicutes in the ileal mucus of the rabbits in the trained group was reduced(P < 0.05).At the family level,the study observed a significant decrease(P < 0.05)in the relative abundance of Lachnospiraceae in the trained group,while Oscillospiraceae and Enterobacteriaceae exhibited significantly higher abundance(P < 0.05).At the genus level,the relative abundances of Clostridia＿vadin BB60＿group exhibited significantly higher abundance(P< 0.05).The relative abundance of Christensenellaceae R-7 group,Eubacterium＿xylanophilum＿group,Clostridia＿vadin BB60＿group,Clostridia＿UCG-014,and Tyzzerella showed significant positive correlation with serum hydroxyl radical level and ileal crypt depth(r > 0.48,P < 0.05),and significant negative correlation with serum Ig G level,earthworm TNF-α gene expression level,and ileal VCR(|r| > 0.45,P < 0.05).The relative abundance of Romboutsia showed a positive correlation with ileal LYZ gene expression level(r > 0.24,P < 0.05)and a negative correlation with serum MDA level(|r| > 0.45,P < 0.05).In this experiment,we further established the microbial genus level interaction network map,and the trained group ileal mucosa microbial genus level interaction network map showed positive interaction between Nitrospira,Acidibacter and many unnamed genera and other genera,and negative interaction between Nitrospira and Acidibacter.The control ileal mucosal organism genus level interaction network map showed that Coriobacteriaceae＿UCG-002 had mainly positive interactions with other genera and positive interactions with 15 genera.Among them,Proteobacteria was mainly present in the negative correlation network in the control group,but in the reciprocal network of the trained group,Proteobacteria showed positive correlations with other bacteria.In this experiment,the intraperitoneal injection of yeast β-glucan in young rabbits during the suckling period effectively reduced the diarrhea rate after weaning,maintained the normal intestinal morphology and structure,enhanced the intestinal and body immune response level,enhanced the intestinal mucosal barrier and reduced the oxidative stress level of the body,and at the same time was beneficial to maintain the stability of intestinal flora.In conclusion,injection of yeast β-glucan-induced training immunity during the suckling period was beneficial in maintaining intestinal health in weaned rabbits.