Research On The Absorption And Utilization Mechanism Of Dietary Lysozyme In GIFT Tilapia(GIFT, Oreochromis Niloticus)

Lysozyme is one type of natural active compounds which is widespread across the animal and plant community. For its unique antibacterial and regulating functions, it has been applied in many fields like animal production, food preservation and pharmaceutical preparation. Among them, the function of acting as one kind of new-green feed additives has been studied for a long time on livestock and poultry animals, while its applying research on aquatic animals was still relative ly weak, related study restricted to Carassius auratus gibelio and Ctenopharyngodon idellus. According to the published results, we found that the effects of lysozyme were not completely consistent and the main possible reasons for that were the differences of fish species and the in-vivo environment of digestive tract. These differences may consequently make its enzyme activity performance and the ability of absorption and utilization in fish body appear differences, and ultimately led to its different applicative effects. While there has been no published evidence. So in present research, we choose GIFT tilapia as research object and focused on utilization and action mechanism of dietary lysozyme in it. Our study includes six parts: the first one determined the optimum dose of dietary lysozyme in GIFT tilapia through growth pe rformance, immune-antioxidant capacity and serum antibacterial properties; the second part designed the ammonia-nitrogen stress experiment to investigate the fish anti-stress ability on the basis of serum biochemical indices, antibacterial properties and liver antioxidant capacity; the third part was designed for exploring its effects on digestive enzyme activity, morphology structure of liver and intestine, nutrient digestion and absorption in GIFT tilapia and finally analysised the internal relation between fish growth and lysozyme utilization with digestive physiology; the fourth part was conducted to investigate the relative expression levels of endogenous C-type lysozyme and the NF-κB signaling pathway related genes response to exogenous lysozyme addition for GITT tilapia, so as to explore the interrelations between immune genes and their regulating pathway and determined its influence on immune function in the transcription level; In the fifth part, a gastric emptying experiment in four lysozyme additional levels was designed to explore the variation regularity of digesta characteristics in the whole ingestive and digestive process; the sixth part aimed to investigate the optimum active and absorption site of dietary lysozyme in digestive tract. lysozyme resistant ability to liver and gastrointestinal tract was measured in vitro; Meanwhile, effective drug duration of dietary lysozyme was determined in vivo, and also to explore its absorption and metabolism rule in different tissues of the tested fish through FITC-lysozyme tracer technique. Exp. 1To investigate effects of dietary lysozyme supplementation on growth performance, immune-antioxidant capacity and antibacterial properties of GIFT tilapia(Oreochromis niloticus) on the normal feeding conditions, basal diet or basal diet containing 18, 36, 54, 72 or 90 mg/kg lysozyme additive were formulated for a 60-day experiment. The results showed that the 54 mg/kg lysozyme addition group had the best growth and feed efficiency in which weight gain rate and protein efficiency ratio increased and feed conversio n ratio decreased significantly compared with the control group(P<0.05). Lysozyme supplementation decreased hepatosomatic index with significant difference in 90 mg/kg group compared with the control group(P<0.05). Spleen-somatic index was significantly lower in 36 mg/kg and 54 mg/kg groups compared with that of the control group(P<0.05). Crude protein and ash content in whole body in 54 mg/kg group were significantly higher than that of the control group(P<0.05). Lysozyme supplementation in 54 and 72 mg/kg group increased serum and liver superoxide dismutase and catalase activity but significantly decreased malondialdehyde content compared with the control group(P<0.05). Liver lysozyme activity in 54 and 72 mg/kg group was significantly higher than that of the control group, while serum lysozyme activity in groups except L90 group was significantly lower than that of the control group(P<0.05). Serum antibacterial test showed more resistance to Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Vibrio alginolyticus in 54 and 72 mg/kg group compared with the control group but more sensitive to Bacillus subtilis with a 34.71% and 42.21% reduction, respectively. These results indicated that diet lysozyme supplementation(such as 54 mg/kg) can improve GIFT tilapia growth performance by regulating immune-antioxidant capacity of tilapia and improving serum antibacterial properties. On this condition, its optimum adding amount was 54 or 72mg/kg. Exp. 2This study was designed to investigate the effects of dietary lysozyme supplementation on serum biochemical indices, antibacterial properties and liver antioxidant capacity of GIFT tilapia under ammonia-nitrogen stress. Juvenile tilapia with the initial weight of(11.35±0.08) g were fed with six experimental diets(labeled as L0, L18, L36, L54, L72 and L90) which were supplemented with graded lysozyme levels(0, 18, 36, 54, 72 and 90 mg/kg) in basal diet for 60 days. After that, a 24 h ammonia-nitrogen stress test with ammonia chloride was conducted to the experimental fish above. The results showed that:(1) After stress, fish serum biochemical indices were presented a large difference and the fish were showed a different feedback response among groups(P<0.05). The fish in L54 group could reduce damage from ammonia-nitrogen stress through stimulating the immune system and regulating protein metabolism; while mainly through regulating the dynamic variation between high/low density lipoprotein cholesterol, cholesterol and triglyceride in L72 and L90 group.(2) Serum antibacterial test showed that there was the maximum resistance ability to Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila in L54 and L72 group and higher inhibiting ability to Vibrio alginolyticus in L36~L90 group(P<0.05), while lysozyme supplementation was helpful for the proliferation of Bacillus subtilis(P<0.05).(3) Liver superoxide dismutase, catalase and glutathione peroxidase activity after ammonia-nitrogen stress were generally enhanced with increased dietary lysozyme level and the highest value was presented in L54 group(P<0.05); Malonaldehyde content in each lysozyme supplemented group was significantly lower than the control group(P<0.05). In summary, our results indicate that ingestion of a basal diet supplemented with 54 and 72 mg/kg lysozyme could produce a most positive and effective regulatory response on serum biochemical indices, antibacterial properties and liver antioxidant indices under ammonia-nitrogen stress, so as to improve fish anti-stress ability. The optimum adding amount in ammonia-nitrogen stress condition was 54 or 72mg/kg. Exp. 3On the process of culturing GIFT, tilapia in Exp. 1, we collected the faeces every 15 days for detecting aparent nutrient digestibility, and detected the digestive enzyme activity and made paraffin section in different parts of digestive tract after 60-day feeding.The results showed as follows:(1) The fish had a different feedback response on the digestive enzyme activity in liver and gastrointestinal tract among groups(P<0.05). The protease activity of stomach, anterior and distal intestine in L36~L72 groups were significantly higher than those in control group(P<0.05), while there were no differences on liver and distal protease activity between groups(P>0.05); The amylase activity of liver, stomach and distal intestine in L72 or L90 group were significantly higher than those in control group(P<0.05), the amylase activity of anterior intestine in L54 group were significantly higher than the control group(P<0.05), and the middle intestine amylase activity in lysozyme additional groups(excepted for L72 group) were also significantly than the control group(P<0.05); The lipase activity in L54 or L72 group were significantly higher than the control group(P<0.05).(2) The intestinal morphology showd that: The anterior and middle intestinal villus density, villus height and villus width in L54 and L72 groups were all significantly larger than the control group(P<0.05), while the muscular thickness had no difference when compared with the control group(P>0.05);The distal intestinal villus density and height in L36~L72 groups were significantly larger than the control group(P<0.05), while there were no differences in villus width(P>0.05) and decreased in muscular thickness in lysozyme additional groups when compared with the control group(P<0.05); The goblet cells in L54 and L72 group were more than the control group(P<0.05).(3) The liver morphology showd that: The liver cells were more voluptuous and denser in L36 and L54 group when compared with the control group, while the health level in L90 group were decreased.(4) For aparent nutrient digestibility, the crude protein digestibility in L36~L90 group were significantly higher than the control group in period I(1~15d) and II(16~30d)(P<0.05), while the crude lipid digestibility had no differences between groups(P>0.05); The digestibility of dry matter, crude protein and crude lipid in L36~L90 group were all significantly higher than control group in period III(31~45d) and IV(46~60d)(P<0.05). From above results, we concluded that 36~72 mg/kg lysozyme could improve GIFT tilapia digestive enzyme activity and promote liver and intestine growth, 36~90 mg/kg lysozyme were benefit for nutrient digestion and absorption. Exp. 4The fish head-kidney, spleen, liver and intestine tissues were samped in L0, L18, L54 and L90 group after a 60-day feeding in Exp. 1, and a quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) technique was carried out to investigate the relative expression levels of endogenous C-type lysozyme and the NF-κB signaling pathway related genes response to exogenous lysozyme addition for GITT tilapia in different tissues. The results showed that:(1) The C1, C2, C3-type lysozyme and NF-κB(P65) m RNA relative expression levels in L54 and L90 group were down-regulated in head-kidney(P<0.05); The three kinds of C-type lysozymes and NF-κB(P65) m RNA relative expression levels in spleen were shown a firstly increasing then decreased tendency with the increasing dietary lysozyme added. The above lysozymes m RNA expression levels in L54 and L90 group were respectively higher or lower than the control group(P<0.05). The NF-κB(P65) m RNA relative expression level in L18 group was significantly higher than the control and other groups(P<0.05).The C1-type lysozyme m RNA in L18 group and the C2, C3-type lysozyme m RNA in L54 group were all significantly up-regulated(P<0.05), while the NF-κB(P65) m RNA relative expression levels in lysozyme added groups(excepted for L18 group) were all significantly lower than the control group(P<0.05); In intestine, the C2, C3-type lysozyme m RNA expression levels in L18 and L54 group were all lower or significantly lower than the control group(P<0.05); The NF-κB(P65)m RNA relative expression levels were shown a firstly increasing then decreased tendency with the increasing dietary lysozyme supplemented level and the L18 group were significantly higher than the control group(P<0.05). Considering recent evidence that the NF-κB(P65) and three C-type lysozyme m RNA in liver and spleen were all significantly up-regulated when added with 18 and 54 mg/kg exogenous lysozyme, while there were no direct correlation between NF-κB(P65) and lysozyme m RNA ecpression. Exp. 5To investigate the gastric emptying time of GIFT tilapia fed different lysozyme supplemented diets and explore the variation regularity of digesta characteristics in the whole ingestive and digestive process, in this experinment, a 60-day feeding trial was firstly conducted in GIFT tilapia. The fish were fed with the corresponding diets which were added with 0(L0/the control), 18(L18/low), 54(L54/middle) and 90(L90/high) mg/kg lysozyme, respectively. After tha t, a gastric emptying experiment was carried out by the method of Fe2O3 labeling and continuous sampling with 160±11.23 g GIFT tilapia that were from above- mentioned diet groups. The gastric emptying time was determinged through the changes of total iron contents in stomach digesta, and simultaneously, comparatively analysised the changes of digesta moisture content, p H values, nutrient contents and digestive enzyme avtivity in entire ingestive and digestive procese in each group. The results showed as follows:(1) Exponential model and square root model were all better describing the gastric emptying curve in each group. Trough comparing R2 value, the L0 and L18 group were more suitable for exponential model and the L54, L90 group were more suitable for square root model. The 50% gastric empting time calculated according to the optimal model in L0, L18, L54 and L90 group were 7.97 h, 6.68 h, 6.74 h and 5.49 h, respectively.(2) In the 24 h digestive procees, the digesta moisture of digestive tract in lyszoyme additional groups were overall higher than the control group(P<0.05), and had a faster rising rate(0~12h after feeding) and a long time for peak; The stomach digesta p H value in L54 and L90 group had a faster decreasing rate at 0~6h after feeding, the p H values in distal intestinal digesta in dietary lysozyme groups were also remained the lower level when compared with control group(P<0.05). With the prolonging of feeding time and the passage of food, the digesta p H value in L54 group could quickly restore or close to the original level; For the digestive enzyme specific activity, the L54 and L90 group were all significantly higher than the control group(P<0.05), the anterior intestine protease and lipase specific activity in L54 group were all higher than the control group at 0.5~6h after feeding(P<0.05), while the anterior intestine tissue amylase specific activity had no differences among groups(P>0.05); After 0~12h feeding, the protease and amylase specific activity of middle intestinal digesta in L54 and L90 group were overall higher than the control group(P<0.05), while there were no differences in digesta lipase spec ific activity among groups(P>0.05); the protease specific activity of distal intestinal digesta in L54 group had a little increase when compared with other groups(P>0.05), while there were no differences in amylase and lipid. For nutrient content: the digesta crude protein and crude ash content in L54 and L90 group were significantly higher than the control group(P<0.05), and the crude lipid content was significantly lower than the control group(P<0.05). In summary, our results indicated that the diet supplemented with 54 and 90 mg/kg lysozyme could shorten gastric emptying time of GIFT tilapia and could enhance the ability of digesting nutrient in the feed by promoting moisture ingestion, acidifieding food and boosting the digestive enzyme secretion. Exp. 6The present study was conducted to investigate the optimum active and absorption site and determine its effective drug duration of dietary lysozyme in digestive tract of GIFT tilapia. In this experiment, the in- vitro test was proceeded to measure the resistant ability of dietary lysozyme to liver and gastrointestinal tract, and the in-vivo test was proceeded to detect its absorption and metabolism rule in different tissues of the fish by using FITC-lysozyme tracer technique. The results showed as follows:(1) There was a obvious inhibiting effect on dietary lysozyme activity caused by enzyme extract in GIFT tilapia liver and gastrointestinal tract, and with the extension of reaction time, the relative retention rate was gradually reduced(P<0.05). From high to low order, the relative retention rate of dietary lysozyme in different positions of digestive tract was distal intestine > middle intestine > stomach > liver > anterior intestine. For this reason, dietary lysozyme was shown the best resistance to GIFT tilapia distal intestine and the worst resistance to anterior intestine. In addition, from this vitro test, we also found dietary lysozme in L54 group had the highest relative retention rate in GIFT tilapia liver and stomach when compared with L18, L72, L90 group and L0, L18, L36 group(P<0.05).(2) The FITC-lysozyme tracing showed that: For a single oral administration lysozyme within 48 h, its relative absorptivity in each position of GIFT tilapia from high to low order was middle intestine > anterior intestine > distal intestine = stomach = liver = midkidney = serum > muscle and its main absorption site was upper intestinal tract(P<0.05); In addition, from the concentration-time curve of FITC- lysozyme in serum, we considered the blood drug concentration of lysozyme could maintain 12 h, and then eliminated gradually. In short, the present study verified that distal intestine were the optimum active site; anterior and middle intestine were its main absorption site; its effective drug duration was 12 h.