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Abstract [Objectives] The effect and mechanism of grape seed and sweet wormwood extract on growth of Monopterus albus were studied.
[Methods] 1 500 healthy M. albus with average mass of 23.8 g/tail were randomly divided into 5 groups. The control group was fed with basal diet and the experimental groups were fed with 10， 20， 30 and 40 g/kg of grape seed and sweet wormwood extract， respectively. The growth performance of the fish was measured after 60 d of feeding at 23-28 ℃.
[Results] The addition of grape seed and sweet wormwood extract in basal diet could increase the body weight gain rate， specific growth rate and proteinefficiency， and decrease the feed coefficient. The activity of digestive enzymes， glutamicoxaloacetic transaminase， glutamicpyruvic transaminase， blood alkaline phosphatase and serum total protein was increased， and the contents of blood glucose and triglyceride were decreased. Compared with the control group， the above indexes showed significant difference （P<0.05） when the dosage was 20 and 30 g/kg （P<0.05）. The difference was not significant （P>0.05） when the dosage was 10 and 40 g/kg （P>0.05）.
[Conclusions] As a feed additive， the plant extract from grape seed and sweet wormwood could increase digestive enzyme activity and regulate blood biochemical parameters of fish body. It had the function of promoting the digestion and absorption， enhancing the metabolic activity of 3 main nutrients， and hastening the growth of fish.
Key words Monopterus albus; Growth promotion; Plant extracts; Grape seed; Sweet wormwood
As a new type of feed additive， plant extracts are favored for their advantages of natural， high efficiency， low toxicity and diversified performance. Plant extract additives can not only prevent and cure aquatic animal diseases， reduce the use of antibiotics and chemical drugs， but also can achieve the effects of improving the digestive enzyme activity[1]， regulateing the blood biochemical index[2-3]and promoting nutrient digestion， absorption and growth[4]. In recent years， the effect of plant extracts on the growth of fish is mostly stayed in the tangerine peel[5]， hawthorn[6]， garlic[7]and other common plant extracts on the evaluation of the feeding effect， while few studies have been conducted on the mechanisms of grape seed， sweet wormwood and others on fish growth. In this study， the effects of grape seed and sweet wormwood extracts on the growth performance， digestive enzyme activity and blood biochemical indexes of M. albus were tested to explore the growth promoting effect and mechanism of the plant extracts. This study provides some theoretical basis for the popularization and application of grape seed and sweet wormwood extracts in green ecological aquaculture. Materials and Methods
Experimental design and feed
A total of 15 test cages were set up in 5 treatment groups with 3 replicates in each group， fed separately the basal diet （control group）， and the diets specially added with 10 g/kg （text group 1）， 20 g/kg （text group 2）， 30 g/kg （text group 3）， and 40 g/kg （text group 4） of the extract from grape seed and sweet wormwood， as shown in Table 1. The basic diet was produced by Huaihua Zhengda Feed Co.， Ltd.， and no additional antibiotics and growthpromoting agents were added as Table 2. The plant extract was composed of grape seed extract and sweet wormwood extract at 4∶1 mass ratio. The main ingredients of grape seed extract are polyphenols， proanthocyanidins， etc.， which are produced by Collier Bioengineering Co.， Ltd. Artemisinin is the main component of sweet wormwood extract， produced by Xian Qingying Bioengineering Co.， Ltd. The plant extract was mixed with the basal diet according to the experimental design before feeding.
Experimental fish and management
1 500 M. albus fingerlings with average weight of 23.8 g， normal body shape， strong physique and regular specifications were randomly divided into 5 groups. The cages with the size of 2.0 m×1.5 m×1.0 m were sewn with 20 mesh polyethylene nodule mesh. The bottom of each cage was covered with 20 cm thick soft mud， and the waterhyacinths were transplanted in the cages. Seven days before breeding， the water and waterhyacinth in the cage were disinfected with 200 g/m3 concentration of quicklime. After the toxicity disappeared， all of M. albus fingerlings were sterilized with 3% salt water and then stored into net cages.
Feeding and management were divided into two stages. After being stocked 3 d， the M. albus was fed with earthworm and chopped fresh fish， then the basic diet was added and mixed with earthworm and fresh fish into dough shape， the amount of basal diet added was from less to more until the M. albus was completely fed on the base diet. This was the first stage. After successful food training， the M. albus with the same size were selected to be kept in separate cages according to the requirements of the experimental design. During the experiment， WT and pH of aquaculture water were measured in the morning， middle and late every day， the daily feed consumption of each group was recorded， and the scientific feeding management was carried out. The breeding site is located in the training base of Huaihua Vocational and Technical College， the breeding time was 60 d from June 1 to July 31， 2012.
Sample collection and index determination
At the end of the experiment， 4 M. albus were randomly selected for each repeat， and blood was extracted from the tail vein of each M. albus sample with the sterile syringe of 2.5 ml. The blood that was collected from the same repetition was placed in asepticcentrifuge tube at 4 ℃ for 8 h， and then centrifuged at 5 000 r/minspeed for 10 min. The supernatant （serum） was kept still at -80 ℃ in the cryogenic refrigerator to prepare for determination. The intestine of each sample was removed respectively， and the water of intestines surface was dried with filter paper. The intestinal wall of each sample removed was cut longitudinally with scissors. The food residue was weighed respectively. According to the weight/volume ratio of 1∶9， the sample homogenate medium of 0.86% normal saline was added and homogenized fully. The suspensions were then centrifuged at 12 000 r/min in a highspeed cryopreservation centrifuge for 10 min. The supernatant （crude enzyme solution） was measured within 24 hours.
On the morning of the beginning and the end of the experiment， the total weight of fish with an empty stomach in each cage was respectively weighed， and the growth indexes such as total weight gain and feed coefficient were calculated. The formula is as follows： weight gain rate is equal to （Wt-W0）/W0×100%; specific growth rate is equal to （lnWt-lnW0）/t×100%; feed coefficient is equal to F/（Wt-Wd-W0）; protein efficiency is equal to （Wt-W0）/（F×P）×100%; survival rate is equal to the final number of surviving fish at the end of the experiment/total number of tested fish×100%. In the formula： Wt represents the total body weight of the fish at the end of the experiment （g）， W0 represents the total body weight of the fish at the beginning of the experiment （g）， Wd represents the total weight of dead fish in experiment （g）， P represents the protein content in the feed （%）， F represents the total weight of the feed （g）， and t represents the total test days （d）.
Digestive enzyme activity determination
Amylase activity was determined by iodinestarch colorimetry， lipase activity by turbidimetric method， trypsin activity by absorbance method.
Blood biochemical index determination
Alkaline phosphatase （ALP） was determined by continuous enzyme assay， glutamicpyruvic transaminase （GPT） and glutamicoxaloacetic transaminase （GOT） by Lais method， total protein （TP） by biuret method， blood glucose （GLU） by glucose oxidase peroxidase assay， triglycerides （TG） and total cholesterol （TCH） by enzyme method， and blood urea nitrogen （BUN） by enzyme coupling rate method. Data treatment
The data were analyzed by SPSS 17.0 software， the Duncans method was used to compare multiply the difference between experimentgroups， and the significant difference was expressed as P<0.05.The test data were expressed as mean±standard （X±SE）.
Results
Effects of Plant Extracts on Growth Performance of M. albus
According to Table 3， the weight gain rate， feed protein efficiency and specific growth rate of each experimental group were significantly higher than those of the control group after 60 d of feeding. Of which， group 2 and group 3 were compared respectively with the control group， there was significant difference （P<0.05），but group 1 and group 4 were compared respectively with the control group， and the difference was not significant （P>0.05）.The feed coefficient of each test group was lower than that of the control group. Of which， test group 2 and test group 3 had respectively significantly difference （P<0.05）， and test group 1 and test group 4 had respectively no significant difference （P>0.05）， in comparison with the control group. The survival rates of M. albus in different treatment groups were similar， and the differences were not significant （P>0.05）. There were no significant differences in growth indexbetween the four test groups （P>0.05）.
Mean with the same or without letters within line were not significantly different （P﹥0.05）; value in the same line with different letters were significantly different （P﹤0.05） in this table; the same as Table 4 and Table 5.
Effects of Plant Extracts on Digestive Enzyme Activity of M. albus
As shown in Table 4， the activity of amylase， lipase and trypsin in each trial group was higher than those in the control group. The activity of digestive enzymes in the trial group 1， 2 and 3 increased with the increase of the amount of plant extracts. Group 2 and group 3 were compared with the control group respectively. Specifically， the activity of amylase increased by 100% and 133.33%， respectively， the activity of lipase increased by 77.14% and 71.42%， respectively， and the activity of trypsin increased by 66.21% and 71.42%， respectively. And there were significant differences between them （P<0.05）. The digestive enzyme activity of group 4 decreased with the increase of plant extract， but there were no significant differences compared with the control group （P>0.05）.There were no significant differences in digestive enzyme activity between group 2 and group 3 （P>0.05）. Effects of plant extract on blood biochemical indexes of M. albus
Table 5 shows that the activity of ALP， GOT and GPT and the concentrations of TP and BUN were increased， and the contents of GLU， TG and TCH in the M. albus blood were decreased by adding the extracts of grape seeds and sweet wormwood into the basal diet. Compared with the control group， the activity of ALP in test group 2， test group 3 and test group 4 increased respectively by 28.63%， 28.26% and 26.52%， and the activity of GOT in the three groups increased 57.38%， 66.73% and 56.50%， respectively， with significant differences （P<0.05）; the GPT activity of test group 2 and test group 3 increased by 71.39% and 69.09%， respectively， the TG contents of test group 2 and test group 3 decreased by 33.33% and 31.49%， respectively， the GLU contents of test group 2 and test group 3 decreased by 32.28%and 37.83%， respectively， and the differences were significant （P<0.05）; and the concentration of TP in group 2 was significantly higher than that in control group （P<0.05）. Compared with the control group， the concentrations of BUN and TCH in each test group showed an upward or a downward trend， but the differences were not significant （P>0.05）.
Agricultural Biotechnology2019
Effects of plant extracts on growth performance of M. albus
The results showed that the extracts from grape seed and sweet wormwood increased the weight gain rate， protein efficiency， specific growth rate and decreased the feed coefficient of M. albus. The growth performance of M. albus was significantly improved when the dosage was respective 20 and 30 g/kg. This is because the main components such as polyphenols， procyanidins and artemisinin in the extracts of grape seeds and sweet wormwood had a certain effect on the activity of digestive enzymes in the intestinal tract of M. albus and the content of some biochemical indexes in blood. It promoted the digestion and absorption of nutrients and the metabolism of three major substances in M. albus. At the same time， this result may be related to the fact that grape seed extract can increase the activity of antioxidant enzymes such as glutathione peroxidase， superoxide dismutase and so on[8]， and convert toxic substances into lowtoxic or nontoxic substances.
Effects of plant extracts on digestive enzyme activity of M. albus
Fish digestion and absorption of nutrients， maintain normal metabolism， closely related to digestive enzymes. Digestive enzyme activity directly reflects the physiological characteristics of digestion. The results （Table 4） showed that the extract of grape seed and sweet wormwood could significantly increase the digestive enzyme activity of M. albus， and the highest activity of amylase， lipase and trypsin in the four groups were increased 133.33%， 77.14% and 71.42%， respectively. This indicated that some biochemical components of the plant extract could promote the secretion of digestive enzymes， activate the activity of enzymes， accelerate the digestion of the three nutrients and improve the digestibility of feed. At the same time， with the increase of addedamount of the extract， the activity of digestive enzyme in the four experimental groups increased steadily at first and then decreased slightly. The activity of digestive enzyme was the strongest when the dosage reached 30 g/kg and the activity of digestive enzyme showed a decline at the addition of 40 g/kg. There was no significant difference in digestive enzyme activity between test group 2 and test group 3. These results are similar to those reported by Ming et al.[9]. In recent years， there have been many reports on the inhibition of pancreatic lipase activity by plant extracts. Tian et al.[10]indicated that the inhibition rate of grape seed and other plant extracts on pancreatic lipase was over 61.53%， which was inconsistent with the conclusion that this experiment could increase lipase activity， and the reasons need further study.
Effects of plant extracts on blood biochemical indexes of M. albus
The indexes of ALP， GOT， GPT， GLU， TCH， TG， TP and BUN in blood are important blood biochemical indexes to evaluate fish health status， nutritional status and adaptation to the environment. The changes of these indexes can reflect the physiological state of the body[11]. Serum ALP is an isoenzyme with genetic markers， and its activity can reflect growth rate and growth performance. In the experiment， plant extracts could significantly improve the activity of ALP， which coincided with the results of the improvement of growth performance. GOT， GPT and TP in serum reflect the protein metabolism and nutritional status. GPT is involved in the transaminative action of the body and is closely related to nitrogen utilization. The increase of GOT and GPT concentrations may indicate an increase in protein synthesis[12]. If GPT activity is high， protein synthesis and metabolism will be exuberant， which is beneficial to nitrogen accumulation. If GOT activity is high， urea will be produced quickly and the toxicity could be alleviated. TP in serum mainly reflects liver function and has good parallelism with activity of GPT and GOT. The results showed that the extracts of grape seed and sweet wormwood could significantly increase the activity of GOT and GPT and the content of TP. This indicates that the plant extract can improve the utilization rate of field eel to feedprotein， and has the function of accelerating protein metabolism and promoting growth. The high concentration of BUN in the test groups may be due to the high protein metabolism and rapid urea production in the test group， and the relatively slow rate of penetration and diffusion of urea through the skin. This is inconsistent with the results of Gu et al.[13]， and the specific reasons need to be further studied.
Blood GLU is an important index to reflect glucose metabolism， tissue cells and liver function[14]. Carbohydrates in food are digested into the blood， and GLU in the blood is rapidly transferred to the liver to form the liver glycogen to maintain the bodys energy needs. The blood GLU content is lower when liver function is normal. In recent years， Guo et al.[15]has reported that the yellowginseng polysaccharide has hypoglycemic effect. The results showed that the extract from grape seed and sweet wormwood can decrease blood GLU concentration of M. albus. This indicated that the extract can relieve liver burden， enhance liver function and promote carbohydrate metabolism. Fat synthesized from the liver enters the blood， and TG transports lipids in the form of lipoproteins to extrahepatic tissues for use. The amount of TCH reflects the absorption of lipids， and the reduction of cholesterol can be achieved by reducing the activity of cholesterol synthase， reducing the absorption of cholesterol and promoting the excretion and transport of lipids. There are many reports that the feed additive of plant extract has lipid lowering effect[16]. The results showed that compared with the control group， the contents of TG and TCH in the four test groups decreased steadily and then increased slightly， and the TG content decreased most significantly when the dosage of 20 and 30 g/kg. Adding the extract to the basal diet can reduce the content of TG and TCH， which may be due to that the plant extract can protect the liver， enhance the liver function and accelerate the excretion in order to reduce the absorption of lipids under the condition of good liver function. It may also due to that the active substances such as proanthocyanidin etc. can increase the activity of lecithin cholesterol acyltransferase， accelerate the decomposition and transport of cholesterol， and show the effect of reducing the content of TG and TCH.
Conclusion
The plant extract from grape seed and sweet wormwood can not only increase the activity of amylase， lipase and trypsin in intestinal tract of M. albus， promote the digestion and absorption of nutrients， and improve the utilization rate of feed， but also can increase the activity of ALP， GOT， GPT and the content of TP in blood， decrease the contents of GLU TG and TCH， and make the metabolism of protein， fat and carbohydrate more vigorous， thus promoting the growth of fish body.
References
[1] WINDISCH W， SCHEDLE K， PLITZNER C. Use of phytogenic products as feed additives for swine and poultry[J]. Journal of animal science， 2008， 86： 140-148.
[2] TIAN XX， LI P， SUN YF. Progress of applying of plant extract in poultry production[J]. Tianjin Agricultural Sciences， 2015， 21（07）： 31-34.
[3] ZHANG T. Effect of oregano essential oil on reducing transport stress and improving meat quality in pigs[D]. Huangzhong Agricultural University， 2016.
[4] QI SL， LALLES JP， BOSI P. Effects of bioactive substances on gastrointestinal tract and growth performance of weaned piglets[J]. Animal Science Abroad （Pigs and Poultry）， 2010， 30（5）： 36-38.
[5] JI H， LI HM， ZHOU JS. Feeding attraction of four kinds of plantcrude extraction to goldfish （Carassius auratus）[J]. Fisheries Science， 2008， 27（2）： 67-70. [6] MIAO YT， WANG AL， LIN HZ. Feeding attractive functions of eight chinese herbal medicines for Pangasius sutchi[J]. Journal of Hydroecology， 2008， 1（1）： 112-114.
[7] LI XX， LEN XJ， LI XQ. On the effects of different feed attractants on hybridized prussian carp and Sarotherodon Sp[J]. Cereal & Feed Industry， 2006， （11）： 37-39.
[8] MIAO ZR， YUAN X， LIAO LN. Experimental study on antioxidant function of proanthocyanidinphospholipid complex from grape seed extract[J]. Medical and Health Technology， 2013： 46-49.
[9] MING JH， LIU B， ZHOU QL. Application of functional oligosaccharide in feed in aquatic animals[J]. Fisheries Science， 2008， 27（9）： 490-493.
[10] TIAN Q， WU ZJ， HUANG DR. Extraction process of pancreatic lipase inhibitor from grape seed[J]. food research and development， 2010， 31（4）： 41-44.
[11] ZHOU CP， LIU B， WANG GY. Effects of the compound of oligosaccharide and Chinese medicines and flavomycin on growth and disease resistance of allogynogenetic crucian carp[J]. Freshwater Fisheries， 2009， 39（3）： 46-51.
[12] HUANG XX. Effects of IUGR on immune function development of the liver and spleen in newborn piglet[D]. Nanjing： Nanjing Agricultural University， 2011.
[13] GU LF， HOU YQ， DING BY. Effects of several plant extracts on growth performance and blood biochemical indices in Carassius auratus Gibelio[J]. Fisheries Science， 2008， 38（2）： 23-26.
[14] ZHAO WP， LIU YJ， PAN Q. Changes of blood sugar and liver glycogen levels after feeding in grass carp[J]. Acta Scientiarum Naturalium Universitatis Sunyatseni， 2002， 41（3）：64-67.
[15] GUO J， ZHANG J， WANG JL. Protective effect of polysaccharides from Radix Ginseng on islet cells injured by alloxan[J]. Gansu Science and Technology， 2013， 29（7）：124-126.
[16] WANG JY， GAO HW， WANG YP. Effects of some Chinese medical herbs on blood biochemical index of chicken[J]. Animal Husbandry & Veterinary Medicine， 2003， 35（11）： 8-10.
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
[Methods] 1 500 healthy M. albus with average mass of 23.8 g/tail were randomly divided into 5 groups. The control group was fed with basal diet and the experimental groups were fed with 10， 20， 30 and 40 g/kg of grape seed and sweet wormwood extract， respectively. The growth performance of the fish was measured after 60 d of feeding at 23-28 ℃.
[Results] The addition of grape seed and sweet wormwood extract in basal diet could increase the body weight gain rate， specific growth rate and proteinefficiency， and decrease the feed coefficient. The activity of digestive enzymes， glutamicoxaloacetic transaminase， glutamicpyruvic transaminase， blood alkaline phosphatase and serum total protein was increased， and the contents of blood glucose and triglyceride were decreased. Compared with the control group， the above indexes showed significant difference （P<0.05） when the dosage was 20 and 30 g/kg （P<0.05）. The difference was not significant （P>0.05） when the dosage was 10 and 40 g/kg （P>0.05）.
[Conclusions] As a feed additive， the plant extract from grape seed and sweet wormwood could increase digestive enzyme activity and regulate blood biochemical parameters of fish body. It had the function of promoting the digestion and absorption， enhancing the metabolic activity of 3 main nutrients， and hastening the growth of fish.
Key words Monopterus albus; Growth promotion; Plant extracts; Grape seed; Sweet wormwood
As a new type of feed additive， plant extracts are favored for their advantages of natural， high efficiency， low toxicity and diversified performance. Plant extract additives can not only prevent and cure aquatic animal diseases， reduce the use of antibiotics and chemical drugs， but also can achieve the effects of improving the digestive enzyme activity[1]， regulateing the blood biochemical index[2-3]and promoting nutrient digestion， absorption and growth[4]. In recent years， the effect of plant extracts on the growth of fish is mostly stayed in the tangerine peel[5]， hawthorn[6]， garlic[7]and other common plant extracts on the evaluation of the feeding effect， while few studies have been conducted on the mechanisms of grape seed， sweet wormwood and others on fish growth. In this study， the effects of grape seed and sweet wormwood extracts on the growth performance， digestive enzyme activity and blood biochemical indexes of M. albus were tested to explore the growth promoting effect and mechanism of the plant extracts. This study provides some theoretical basis for the popularization and application of grape seed and sweet wormwood extracts in green ecological aquaculture. Materials and Methods
Experimental design and feed
A total of 15 test cages were set up in 5 treatment groups with 3 replicates in each group， fed separately the basal diet （control group）， and the diets specially added with 10 g/kg （text group 1）， 20 g/kg （text group 2）， 30 g/kg （text group 3）， and 40 g/kg （text group 4） of the extract from grape seed and sweet wormwood， as shown in Table 1. The basic diet was produced by Huaihua Zhengda Feed Co.， Ltd.， and no additional antibiotics and growthpromoting agents were added as Table 2. The plant extract was composed of grape seed extract and sweet wormwood extract at 4∶1 mass ratio. The main ingredients of grape seed extract are polyphenols， proanthocyanidins， etc.， which are produced by Collier Bioengineering Co.， Ltd. Artemisinin is the main component of sweet wormwood extract， produced by Xian Qingying Bioengineering Co.， Ltd. The plant extract was mixed with the basal diet according to the experimental design before feeding.
Experimental fish and management
1 500 M. albus fingerlings with average weight of 23.8 g， normal body shape， strong physique and regular specifications were randomly divided into 5 groups. The cages with the size of 2.0 m×1.5 m×1.0 m were sewn with 20 mesh polyethylene nodule mesh. The bottom of each cage was covered with 20 cm thick soft mud， and the waterhyacinths were transplanted in the cages. Seven days before breeding， the water and waterhyacinth in the cage were disinfected with 200 g/m3 concentration of quicklime. After the toxicity disappeared， all of M. albus fingerlings were sterilized with 3% salt water and then stored into net cages.
Feeding and management were divided into two stages. After being stocked 3 d， the M. albus was fed with earthworm and chopped fresh fish， then the basic diet was added and mixed with earthworm and fresh fish into dough shape， the amount of basal diet added was from less to more until the M. albus was completely fed on the base diet. This was the first stage. After successful food training， the M. albus with the same size were selected to be kept in separate cages according to the requirements of the experimental design. During the experiment， WT and pH of aquaculture water were measured in the morning， middle and late every day， the daily feed consumption of each group was recorded， and the scientific feeding management was carried out. The breeding site is located in the training base of Huaihua Vocational and Technical College， the breeding time was 60 d from June 1 to July 31， 2012.
Sample collection and index determination
At the end of the experiment， 4 M. albus were randomly selected for each repeat， and blood was extracted from the tail vein of each M. albus sample with the sterile syringe of 2.5 ml. The blood that was collected from the same repetition was placed in asepticcentrifuge tube at 4 ℃ for 8 h， and then centrifuged at 5 000 r/minspeed for 10 min. The supernatant （serum） was kept still at -80 ℃ in the cryogenic refrigerator to prepare for determination. The intestine of each sample was removed respectively， and the water of intestines surface was dried with filter paper. The intestinal wall of each sample removed was cut longitudinally with scissors. The food residue was weighed respectively. According to the weight/volume ratio of 1∶9， the sample homogenate medium of 0.86% normal saline was added and homogenized fully. The suspensions were then centrifuged at 12 000 r/min in a highspeed cryopreservation centrifuge for 10 min. The supernatant （crude enzyme solution） was measured within 24 hours.
On the morning of the beginning and the end of the experiment， the total weight of fish with an empty stomach in each cage was respectively weighed， and the growth indexes such as total weight gain and feed coefficient were calculated. The formula is as follows： weight gain rate is equal to （Wt-W0）/W0×100%; specific growth rate is equal to （lnWt-lnW0）/t×100%; feed coefficient is equal to F/（Wt-Wd-W0）; protein efficiency is equal to （Wt-W0）/（F×P）×100%; survival rate is equal to the final number of surviving fish at the end of the experiment/total number of tested fish×100%. In the formula： Wt represents the total body weight of the fish at the end of the experiment （g）， W0 represents the total body weight of the fish at the beginning of the experiment （g）， Wd represents the total weight of dead fish in experiment （g）， P represents the protein content in the feed （%）， F represents the total weight of the feed （g）， and t represents the total test days （d）.
Digestive enzyme activity determination
Amylase activity was determined by iodinestarch colorimetry， lipase activity by turbidimetric method， trypsin activity by absorbance method.
Blood biochemical index determination
Alkaline phosphatase （ALP） was determined by continuous enzyme assay， glutamicpyruvic transaminase （GPT） and glutamicoxaloacetic transaminase （GOT） by Lais method， total protein （TP） by biuret method， blood glucose （GLU） by glucose oxidase peroxidase assay， triglycerides （TG） and total cholesterol （TCH） by enzyme method， and blood urea nitrogen （BUN） by enzyme coupling rate method. Data treatment
The data were analyzed by SPSS 17.0 software， the Duncans method was used to compare multiply the difference between experimentgroups， and the significant difference was expressed as P<0.05.The test data were expressed as mean±standard （X±SE）.
Results
Effects of Plant Extracts on Growth Performance of M. albus
According to Table 3， the weight gain rate， feed protein efficiency and specific growth rate of each experimental group were significantly higher than those of the control group after 60 d of feeding. Of which， group 2 and group 3 were compared respectively with the control group， there was significant difference （P<0.05），but group 1 and group 4 were compared respectively with the control group， and the difference was not significant （P>0.05）.The feed coefficient of each test group was lower than that of the control group. Of which， test group 2 and test group 3 had respectively significantly difference （P<0.05）， and test group 1 and test group 4 had respectively no significant difference （P>0.05）， in comparison with the control group. The survival rates of M. albus in different treatment groups were similar， and the differences were not significant （P>0.05）. There were no significant differences in growth indexbetween the four test groups （P>0.05）.
Mean with the same or without letters within line were not significantly different （P﹥0.05）; value in the same line with different letters were significantly different （P﹤0.05） in this table; the same as Table 4 and Table 5.
Effects of Plant Extracts on Digestive Enzyme Activity of M. albus
As shown in Table 4， the activity of amylase， lipase and trypsin in each trial group was higher than those in the control group. The activity of digestive enzymes in the trial group 1， 2 and 3 increased with the increase of the amount of plant extracts. Group 2 and group 3 were compared with the control group respectively. Specifically， the activity of amylase increased by 100% and 133.33%， respectively， the activity of lipase increased by 77.14% and 71.42%， respectively， and the activity of trypsin increased by 66.21% and 71.42%， respectively. And there were significant differences between them （P<0.05）. The digestive enzyme activity of group 4 decreased with the increase of plant extract， but there were no significant differences compared with the control group （P>0.05）.There were no significant differences in digestive enzyme activity between group 2 and group 3 （P>0.05）. Effects of plant extract on blood biochemical indexes of M. albus
Table 5 shows that the activity of ALP， GOT and GPT and the concentrations of TP and BUN were increased， and the contents of GLU， TG and TCH in the M. albus blood were decreased by adding the extracts of grape seeds and sweet wormwood into the basal diet. Compared with the control group， the activity of ALP in test group 2， test group 3 and test group 4 increased respectively by 28.63%， 28.26% and 26.52%， and the activity of GOT in the three groups increased 57.38%， 66.73% and 56.50%， respectively， with significant differences （P<0.05）; the GPT activity of test group 2 and test group 3 increased by 71.39% and 69.09%， respectively， the TG contents of test group 2 and test group 3 decreased by 33.33% and 31.49%， respectively， the GLU contents of test group 2 and test group 3 decreased by 32.28%and 37.83%， respectively， and the differences were significant （P<0.05）; and the concentration of TP in group 2 was significantly higher than that in control group （P<0.05）. Compared with the control group， the concentrations of BUN and TCH in each test group showed an upward or a downward trend， but the differences were not significant （P>0.05）.
Agricultural Biotechnology2019
Effects of plant extracts on growth performance of M. albus
The results showed that the extracts from grape seed and sweet wormwood increased the weight gain rate， protein efficiency， specific growth rate and decreased the feed coefficient of M. albus. The growth performance of M. albus was significantly improved when the dosage was respective 20 and 30 g/kg. This is because the main components such as polyphenols， procyanidins and artemisinin in the extracts of grape seeds and sweet wormwood had a certain effect on the activity of digestive enzymes in the intestinal tract of M. albus and the content of some biochemical indexes in blood. It promoted the digestion and absorption of nutrients and the metabolism of three major substances in M. albus. At the same time， this result may be related to the fact that grape seed extract can increase the activity of antioxidant enzymes such as glutathione peroxidase， superoxide dismutase and so on[8]， and convert toxic substances into lowtoxic or nontoxic substances.
Effects of plant extracts on digestive enzyme activity of M. albus
Fish digestion and absorption of nutrients， maintain normal metabolism， closely related to digestive enzymes. Digestive enzyme activity directly reflects the physiological characteristics of digestion. The results （Table 4） showed that the extract of grape seed and sweet wormwood could significantly increase the digestive enzyme activity of M. albus， and the highest activity of amylase， lipase and trypsin in the four groups were increased 133.33%， 77.14% and 71.42%， respectively. This indicated that some biochemical components of the plant extract could promote the secretion of digestive enzymes， activate the activity of enzymes， accelerate the digestion of the three nutrients and improve the digestibility of feed. At the same time， with the increase of addedamount of the extract， the activity of digestive enzyme in the four experimental groups increased steadily at first and then decreased slightly. The activity of digestive enzyme was the strongest when the dosage reached 30 g/kg and the activity of digestive enzyme showed a decline at the addition of 40 g/kg. There was no significant difference in digestive enzyme activity between test group 2 and test group 3. These results are similar to those reported by Ming et al.[9]. In recent years， there have been many reports on the inhibition of pancreatic lipase activity by plant extracts. Tian et al.[10]indicated that the inhibition rate of grape seed and other plant extracts on pancreatic lipase was over 61.53%， which was inconsistent with the conclusion that this experiment could increase lipase activity， and the reasons need further study.
Effects of plant extracts on blood biochemical indexes of M. albus
The indexes of ALP， GOT， GPT， GLU， TCH， TG， TP and BUN in blood are important blood biochemical indexes to evaluate fish health status， nutritional status and adaptation to the environment. The changes of these indexes can reflect the physiological state of the body[11]. Serum ALP is an isoenzyme with genetic markers， and its activity can reflect growth rate and growth performance. In the experiment， plant extracts could significantly improve the activity of ALP， which coincided with the results of the improvement of growth performance. GOT， GPT and TP in serum reflect the protein metabolism and nutritional status. GPT is involved in the transaminative action of the body and is closely related to nitrogen utilization. The increase of GOT and GPT concentrations may indicate an increase in protein synthesis[12]. If GPT activity is high， protein synthesis and metabolism will be exuberant， which is beneficial to nitrogen accumulation. If GOT activity is high， urea will be produced quickly and the toxicity could be alleviated. TP in serum mainly reflects liver function and has good parallelism with activity of GPT and GOT. The results showed that the extracts of grape seed and sweet wormwood could significantly increase the activity of GOT and GPT and the content of TP. This indicates that the plant extract can improve the utilization rate of field eel to feedprotein， and has the function of accelerating protein metabolism and promoting growth. The high concentration of BUN in the test groups may be due to the high protein metabolism and rapid urea production in the test group， and the relatively slow rate of penetration and diffusion of urea through the skin. This is inconsistent with the results of Gu et al.[13]， and the specific reasons need to be further studied.
Blood GLU is an important index to reflect glucose metabolism， tissue cells and liver function[14]. Carbohydrates in food are digested into the blood， and GLU in the blood is rapidly transferred to the liver to form the liver glycogen to maintain the bodys energy needs. The blood GLU content is lower when liver function is normal. In recent years， Guo et al.[15]has reported that the yellowginseng polysaccharide has hypoglycemic effect. The results showed that the extract from grape seed and sweet wormwood can decrease blood GLU concentration of M. albus. This indicated that the extract can relieve liver burden， enhance liver function and promote carbohydrate metabolism. Fat synthesized from the liver enters the blood， and TG transports lipids in the form of lipoproteins to extrahepatic tissues for use. The amount of TCH reflects the absorption of lipids， and the reduction of cholesterol can be achieved by reducing the activity of cholesterol synthase， reducing the absorption of cholesterol and promoting the excretion and transport of lipids. There are many reports that the feed additive of plant extract has lipid lowering effect[16]. The results showed that compared with the control group， the contents of TG and TCH in the four test groups decreased steadily and then increased slightly， and the TG content decreased most significantly when the dosage of 20 and 30 g/kg. Adding the extract to the basal diet can reduce the content of TG and TCH， which may be due to that the plant extract can protect the liver， enhance the liver function and accelerate the excretion in order to reduce the absorption of lipids under the condition of good liver function. It may also due to that the active substances such as proanthocyanidin etc. can increase the activity of lecithin cholesterol acyltransferase， accelerate the decomposition and transport of cholesterol， and show the effect of reducing the content of TG and TCH.
Conclusion
The plant extract from grape seed and sweet wormwood can not only increase the activity of amylase， lipase and trypsin in intestinal tract of M. albus， promote the digestion and absorption of nutrients， and improve the utilization rate of feed， but also can increase the activity of ALP， GOT， GPT and the content of TP in blood， decrease the contents of GLU TG and TCH， and make the metabolism of protein， fat and carbohydrate more vigorous， thus promoting the growth of fish body.
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Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU