Effects And Mechanism Of Replacing Soybean Meal With Cottonseed Protein Concentrate On The Growth Condition And Intestinal Health Of Nile Tilapia（Oreochromia Niloticus）

The booming development of aquaculture in China has led to the increasing demand for protein,including fish meal and soybean meal.Considering the sustainable development of aquaculture and the need for low-cost dietary formulations,it is urgent to evaluate and use more economical non-food feed protein resources.As a non-food feed protein source,cottonseed protein concentrate has the advantages of higher nutritional value and relatively lower price,so it is a potential high quality plant protein resource.At present,existing studies mainly focus on the effect of cottonseed protein concentrate replacing fish meal,but the research on whether cottonseed protein concentrate can effectively replace soybean meal in omnivorous fish feed is still limited.This study was conducted to investigate the effects of replacing soybean meal with cottonseed protein concentrate on the growth condition and intestinal health of omnivore Nile tilapia（Oreochromia niloticus）and to identify the possible mechanism.The main results of this paper are as follows:1.Effects of replacing soybean meal with cottonseed protein concentrate on the growth condition and intestinal health of Nile tilapiaCottonseed protein concentrate was used to replace 0%（CPC0）,15%（CPC15）,30%（CPC30）,45%（CPC45）,75%（CPC75）and 100%（CPC100）soybean meal in the diet of Nile tilapia,respectively.Nile tilapia with an initial body weight of（70.82 ±0.36g）were randomly divided into 6 treatments with 3 tanks in each treatment and 15 fish per tank for a 10-week experiment.The growth performance,intestinal digestive enzyme activity,intestinal structure and functional indices of Nile tilapia were tested.The results showed that there was no significant difference in survival rate among all treatments.When cottonseed protein concentrate replaced more than 30% soybean meal,the width and height of intestinal villi were significantly reduced.When the replacement ratio reached 45% and above,the weight gain rate and intestinal trypsin activity were significantly decreased.When the replacement rate reached 75% and above,the intestinal transepithelial resistance（TRE）was significantly reduced and the expression of occludin-X1 was significantly increased.In CPC100 treatment,serum LPS content and the expression of claudin-1 were significantly increased.In addition,higher replacement（over 45%）was associated with higher expression of genes of intestinal inflammation and ER stress.In conclusion,the replacement level of soybean meal with cottonseed protein concentrate should not exceed 30%,and higher replacement ratio（45%,75% and 100%）affected the growth condition and intestinal health of Nile tilapia.2.Effects of gossypol on the growth condition,microbial composition and metabolites of Nile tilapiaIn order to explore whether gossypol in cottonseed protein concentrate is the main substance affecting growth and intestinal health,Nile tilapia（average weight 3.38 ±0.03 g）were divided into three treatments: control diet（CON）,control diet supplemented with 150 mg/kg gossypol（ML）and control diet supplemented with 300mg/kg gossypol（MH）.There were 3 replicates in each treatment and 20 fish in each replicate.The 8-week experiment was conducted to measure the growth performance,intestinal digestive enzyme activity,intestinal structure and functional indicators of Nile tilapia.The results showed that the weight gain rate and intestinal lipase activity in ML and MH treatments were significantly decreased compared with the CON treatment,the width and height of intestinal villi and the transepithelial electrical resistance（TER）in ML and MH treatments were significantly decreased,and the m RNA expression of occludin-X1 was significantly increased.16 S r RNA gene sequencing showed that gossypol significantly reduced the richness and diversity of the gut microbiota.Untargeted metabolite analysis revealed that most of the metabolites were downregulated in the gossypol treated groups and riboflavin was the key metabolite with significant difference between CON and gossypol treated groups.In conclusion,gossypol reduced the content of riboflavin in intestinal contents,and caused intestinal inflammation,oxidative stress and apoptosis.3.Intestinal microbiota mediated the gossypol-induced intestinal inflammation,oxidative stress and apoptosis in Nile tilapiaIn order to investigate whether the adverse effects caused by gossypol were mediated by the altered intestinal flora,the intestinal flora of Nile tilapia in CON,ML and MH groups were transplanted into germ-free（GF）zebrafish.Three treatments in the experiment were: GF+CON（GF zebrafish colonized with the gut bacteria of CON treatment）,GF+ML（GF zebrafish colonized with the gut bacteria of ML treatment）and GF+MH（GF zebrafish colonized with the gut bacteria of MH treatment）.The results showed that the expression of inflammatory cytokine genes（il-6,il-8 and tgf-β1）,antioxidant status genes（sod1,sod2 and gpx1a）and apoptosis genes（caspase3 and caspase9）were significantly up-regulated in GF+ML and GF+MH treatments,suggesting intestinal microbiota mediated gossypol-induced intestinal inflammation,oxidative stress and apoptosis in Nile tilapiaIn conclusion,this study investigated the appropriate proportion of cottonseed protein concentrate to replace soybean meal in the diet of Nile tilapia.It was found that gossypol was the main substance in cottonseed protein concentrate that affected the growth condition and intestinal health.In addition,this study also proved that intestinal flora mediated the negative effects induced by gossypol,and the decrease of intestinal riboflavin content may be related to the negative effects induced by gossypol.