CN118303514A - Tilapia growth-promoting feed additive based on healthy cultivation and application thereof - Google Patents
Tilapia growth-promoting feed additive based on healthy cultivation and application thereof Download PDFInfo
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- CN118303514A CN118303514A CN202410514471.8A CN202410514471A CN118303514A CN 118303514 A CN118303514 A CN 118303514A CN 202410514471 A CN202410514471 A CN 202410514471A CN 118303514 A CN118303514 A CN 118303514A
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- Fodder In General (AREA)
Abstract
The invention provides a tilapia growth-promoting feed additive based on healthy cultivation and application thereof. The sweet orange oil and the yeast culture are compounded and then used as additives for tilapia growth promoting feed. The two components are compatible to produce a positive interaction effect, and the best performance is achieved by the mixture ratio of 0.5% sweet orange oil and 4% yeast culture. The feed additive can improve the growth performance, intestinal digestive enzyme activity and immune activity of tilapia, obviously change the diversity of intestinal flora and metabolites thereof, and improve the nutrition and quality of muscles of tilapia; the stable intestinal microflora structure is more favorable, and the most likely metabolic pathway influenced by the sweet orange oil treatment is metabolism (mainly lipid metabolism and amino acid metabolism).
Description
Technical Field
The invention belongs to the field of feed additives, and particularly discloses a tilapia growth-promoting feed additive based on healthy cultivation and application thereof.
Background
Plant essential oils (PLANT ESSENTIAL Oil, EO) are believed to have potential for development as antibiotic substitutes due to their remarkable antibacterial, growth-promoting effects. EO is provided mainly from the flowers, leaves, stems, seeds, fruits, roots or bark of a part of the plant, and is a secondary metabolite with an aromatic odor. The additive can be quickly absorbed after entering an animal body, has the advantages of high degradation speed, short half-life period, no accumulation in the body and the like, is considered to be a novel green plant feed additive which is nontoxic, harmless and purely natural, and has wide application prospect. Citrus essential oil (citrus essential oils, CEOs) is a natural green, non-toxic aromatic mixture extracted from citrus plants, which is relatively low cost due to its large production capacity and high degree of commercialization, and is more useful for wide application in production.
Researches show that CEOs has selective antibacterial activity, has a large influence on growth kinetics of harmful bacteria such as escherichia coli (ESCHERICHIA COLI), but has a small influence on beneficial bacteria such as Lactobacillus (Lactobacillus), and has important application value in production. The antibacterial and bacteriostatic effects of the anti-oxidation agent are proved in foods, and the anti-oxidation agent has certain application value in the fields of medicines and cosmetics; on livestock and poultry, the feed additive is considered to improve the antioxidant function of the bodies of weaned pigs, can stimulate receptors on digestive tract mucous membranes of the livestock and poultry, plays roles in promoting food and growth, and has good effect on removing stink and harmful gases of livestock and poultry farms. However, CEOs has less application in aquaculture, onlyAcar et al studied the effect of sweet orange peel volatile oil on the growth rate and anti-streptococcicosis ability of oreochromis niloticus (oreochromis mossambieus). The effects of the sweet orange oil on the intestinal health, the muscle nutrition and the like of the aquaculture animals are not reported.
The yeast culture is a microecological product formed by fully anaerobic fermentation of saccharomycetes on a specific culture medium under the control of specific process conditions, and mainly consists of extracellular metabolites of the saccharomycetes, a mutated culture medium after fermentation and a small amount of inactive saccharomycetes cells. Yeast cultures were used as protein supplement feed for ruminants at the earliest, and are now mainly used for ruminant and aquaculture, serving the dual functions of nutrition and health care. A great deal of research at home and abroad proves that the yeast culture has important effects in promoting animal growth, improving feed utilization rate, improving organism immunity, preventing diseases and the like.
The yeast culture is used as a feed additive, and the nutrition effect is mainly to optimize the nutrition value of the feed. The yeast culture is a pure natural feed raw material, and can improve the palatability and the digestibility of the feed. In order to study the addition effect of the yeast culture in fish feed, zeng Hong and the like are subjected to feeding tests by tilapia, and the yeast culture has the tendency of reducing the feed coefficient and improving the weight gain rate; there is evidence that a portion of the beneficial effects of yeast cultures are associated with a type of Mannooligosaccharide (MOS) contained in the yeast cell wall, which can greatly affect the immune system of animals, and that the addition of yeast cultures can reduce morbidity, if researchers inject glucose in the yeast cell wall into salmon, it is found to be a potentially effective immunostimulant, which can greatly reduce mortality in salmon; it has also been reported that the yeast cell wall is a feed immunopotentiator, and can adsorb, phagocytize, destroy and absorb toxic substances such as bacteria, mold, viruses and the like which invade the body, thereby achieving the effect of enhancing the immune function.
Based on the beneficial research results, the yeast culture and the sweet orange oil are combined, the tilapia is taken as a test object, and the related test is carried out, and the results show that the positive interaction effect is generated after the combination of the yeast culture and the sweet orange oil, namely the 0.5% sweet orange oil and the 4% yeast culture, is the best in proportion.
Disclosure of Invention
The invention aims to provide a tilapia growth-promoting feed additive based on healthy cultivation and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
A growth promoting feed additive for tilapia based on healthy cultivation is prepared by adding 0.3-0.5% of sweet orange oil and 2-4% of yeast culture per kg of tilapia base material. Preferably, 0.5% orange oil and 4% yeast culture by mass per kg of tilapia base material are added.
1 Materials and methods
The tilapia for the test is Nile tilapia, and is provided by a bridge pilot scale of the institute of fresh water and aquatic products of Fujian province, and the average weight is about 170 g.
1.1.2 Sweet orange oil and Yeast cultures
Sweet orange oil (MIC, origin: brazil) was purchased from Shanghai Jiuhuai industries, inc., product model: 8008-57-9, which is obtained from cold pressed fresh orange peel, is a yellowish-brown liquid with strong and heavy aroma, and has special orange smell and mild aromatic taste. Yeast cultures were purchased from Yao Biotechnology (salt city) Co., ltd., product model: q/320981YYK 004-2021.
1.1.3 Test feeds and groups
The basic feed comprises the following components: 3% of flour, 2.5% of lysine residue, 19.5% -24% of rice bran, 34% of bean pulp, 21% of vegetable pulp, 6.5% of fish meal, 1.3% of soybean oil, 2% of attapulgite powder, 3% of calcium dihydrogen phosphate, 0.7% of methionine and 2% of vitamin-mineral premix.
0, 0.3 Percent, 0.5 percent of sweet orange oil and 0, 2 percent and 4 percent of yeast culture (the mass percentages of all raw materials are respectively added into each kilogram of base material (see table 1 for details), the sum of the mass percentages of all raw materials is 100 percent, and after the raw materials are uniformly mixed, 7 kinds of granular feed with the same particle size are prepared by a soft granulator, and the granular feed is stored for standby after being dried in the air.
Table 1 test group
1.2 Test methods
1.2.1 Test fish culture management
The test was performed at the banyan bridge test base of the institute of fresh water aquiculture, fujian province. After stopping feeding for 24 hours, the tilapia with basically consistent specification and healthy and intact tilapia is selected from a cement pond and put into an indoor circulating water system (the diameter of a test barrel is 1.0m, the water depth is 0.8 m), and 15 tilapia are put into each barrel. The tests were divided into 7 groups (1 control group, 6 test groups) of 3 replicates each. The test water is aerated tap water, the water temperature is 25-28 ℃ during the test, and the dissolved oxygen is not less than 5.0mg/L, pH 7.0.0-7.5. The cycle efficiency is 2 h/full cycle, the sewage is discharged 1 time every 1 week in the early stage, and 1 time every week in the later stage. Feeding is carried out by feeding for 2 times/d (8:00-9:00 am, 4:00-5:00 pm) until the feeding is completed within 30 min. The test period was 30 days.
1.2.2 Growth indicator determination
After the test, each growth index is measured after the test fish stops feeding for 24 hours:
Weight gain rate (WGR,%) = (W f—Ws)/Ws ×100%
Survival rate (SR,%) =n f/Ns ×100%
Wherein W s is the initial fish mass, and W f is the fish mass at the end of the test; the N s test starts the fish mantissa and the N f test ends the fish mantissa.
2 Results
The test results are shown in Table 2. Compared with the control group A, the weight gain rate of the test B, E group is higher than that of the control group, but the difference is not obvious (P is more than 0.05), and the weight gain rate of the other test groups is obviously higher than that of the control group (P is less than 0.05); no significant difference (P > 0.05) was found between trial D and F, significantly higher than the other groups (P < 0.05) except trial G; the highest weight gain rate of the test group G is up to 48.16%, which is obviously higher than that of other groups (P is less than 0.05), and the best growth performance of the test tilapia of the compatibility combination can be obtained under the test condition.
TABLE 2 Effect of different compatibility ratios of orange oil and Yeast cultures on Tilapia growth Performance
Note that: the same row of superscript letters indicates significant differences (P < 0.05), and the same row of superscript letters indicates insignificant differences (P > 0.05) or no superscript letters.
The invention has the advantages that:
(1) The sweet orange oil and the yeast culture are compounded and then used as additives for tilapia growth promoting feed. The two components are compatible to produce a positive interaction effect, and the best performance is achieved by the mixture ratio of 0.5% sweet orange oil and 4% yeast culture.
(2) The sweet orange oil is added into the basic feed, so that the growth performance and the immunocompetence of the tilapia mossambica can be improved, the diversity of intestinal flora and metabolic products thereof are obviously changed, the stability of the intestinal microbial community structure is better maintained, and the most likely metabolic pathway influenced by the sweet orange oil treatment is metabolism (mainly lipid metabolism and amino acid metabolism). The ratio of sweet orange oil added in the feed in the study is preferably 0.5%.
(3) The influence of sweet orange oil on the growth performance, digestive enzyme activity, muscle nutrition, immune index, intestinal flora diversity and metabonomics of tilapia is researched by adopting experimental ecology and biochemical analysis methods, so that technical reference and theoretical basis are provided for the application of natural plant essential oil in tilapia compound feed and the development of no anti-green aquatic compound feed additive. The results show that: in the case of embodiment 1, the Weight Gain Rate (WGR), specific Growth Rate (SGR) and Feed Efficiency (FE) of tilapia in each test group gradually increased with increasing ratio of orange oil addition, with group D being highest, and increased by 13.97%, 9.72% and 15.77% respectively compared to the control group; the alpha-Amylase (AMS), lipase (LPS) and Trypsin (TPS) activities of the intestinal tracts of the test fishes gradually rise along with the increase of the addition proportion of sweet orange oil, and the D group is the highest, wherein the TPS activity (1270.95U/ml) of the D group is obviously higher than that of other groups (P < 0.05); the crude fat content of the tilapia muscle of each test group is obviously lower than that of the control group (P < 0.05), the Essential Amino Acid (EAA) content, the semi-essential amino acid (HEAA) content and the Essential Amino Acid Index (EAAI) of the muscle of each test group show a trend of increasing along with the increase of the addition proportion of sweet orange oil, and the total amino acid amount (TAA), EAA, HEAA and umami amino acid (DAA) are all the highest in the D group. In conclusion, the sweet orange oil is added into the basic feed, so that the growth performance and intestinal digestive enzyme activity of tilapia can be improved, and meanwhile, the muscle nutrition and the quality of tilapia are improved to a certain extent, and the addition proportion is preferably 0.5%.
Drawings
FIG. 1 is a PCA analysis;
FIG. 2 shows the intestinal microbial composition at the portal classification level;
FIG. 3 is a graph showing intestinal microbial composition at a genus classification level;
FIG. 4-1/4-2/4-3/4-4 is a volcanic plot of all differential metabolites in positive and negative ion mode;
FIG. 5 is a thermal diagram of a cluster analysis of metabolites;
FIG. 6 is a KEGG pathway analysis of differential metabolites.
Description of the preferred embodiments 1
1 Materials and methods
1.1 Test materials
1.1.1 Test fish
The tilapia for the test is Nile tilapia, and is provided by a bridge pilot scale of the institute of fresh water and aquatic products of Fujian province, and the average weight is about 240 g.
1.1.2 Sweet orange oil
Sweet orange oil (MIC, origin: brazil) is purchased from Shanghai Jiuhuai industries, inc., and is obtained from cold-pressed fresh sweet orange peel, and is a yellowish-brown liquid with intense and heavy aroma, and has special orange smell and mild aromatic taste.
1.1.3 Test feed
0, 0.1%, 0.3% And 0.5% of sweet orange oil (A, B, C, D groups respectively) are added into each kilogram of base material respectively, the mixture is uniformly mixed, 4 kinds of granulated feeds with the same particle size are prepared by a soft granulator, and the granulated feeds are stored for standby after being dried in the air. The conventional nutritional ingredients of the test feeds are shown in table 1.
Table 1 test feed composition and nutrient level (dry matter basis)%
1) The vitamin-mineral premix is prepared by providing :VA 80000IU,VD 40000IU,VE 1000mg,VK 100mg,VB1100 mg,VB2200 mg,VB6150 mg,VB122 mg,VC esters 1000mg, folic acid 80mg, nicotinic acid 800mg, calcium pantothenate 400mg, biotin 3mg, inositol per kg of feed 1000mg,Mg(as magnesium sulfate)1000mg,Mn(as manganese sulfate)100mg,Zn(as zinc sulfate)800mg,Cu(as copper sulfate)200mg,Fe(as ferrous sulfate)1000mg,Se(as sodium selenite)5mg,Co(as cobalt chloride)30mg,I(as potassium iodide)15mg.
2) The feed nutrition level is the actual measurement value.
1.2 Test methods
1.2.1 Test fish culture management
The test was performed at the banyan bridge test base of the institute of fresh water aquiculture, fujian province. After stopping feeding for 24 hours, the tilapia with basically consistent specification and healthy and intact tilapia is selected from a cement pond and put into an indoor circulating water system (the diameter of a test barrel is 1.0m, the water depth is 0.8 m), and 15 tilapia are put into each barrel. The tests were divided into 4 groups (1 control group, 3 test groups) of 3 replicates each. The test water is aerated tap water, the water temperature is 25-30 ℃ during the test, and the dissolved oxygen is not less than 5.0mg/L, pH 6.9.9-7.2. The cycle efficiency is 2 h/full cycle, the sewage is discharged 1 time every 1 week in the early stage, and 1 time every week in the later stage. Feeding is carried out by feeding for 2 times/d (8:00-9:00 am, 4:00-5:00 pm) until the feeding is completed within 30 min. The test period was 50 days.
1.2.2 Growth indicator determination
After the test, each growth index is measured after the test fish stops feeding for 24 hours:
weight gain rate (WGR,%) = (W f—Ws)/Ws ×100%;
Specific growth rate (SGR,%) = (lnW f—lnWs)/t×100%
Feed Coefficient (FCR) =b t/(Wf—Ws);
feed efficiency (FE,%) =w t/Bt ×100%;
Survival rate (SR,%) =n f/Ns ×100%;
Dirty volume ratio (VSI,%) =w v/Wo ×100%;
liver volume ratio (HSI,%) =w h/Wo ×100%;
fullness (CF) =w o/L3 ×100.
Wherein W s is the initial fish mass, and W f is the fish mass at the end of the test; w v is viscera weight, W h is liver pancreas weight and L is body length, W o is fish body mass; b t is total feed intake in the test process, N s test starts fish mantissa, N f test ends fish mantissa, and T is test period.
1.2.3 Intestinal digestive enzyme Activity assay
And (3) respectively measuring alpha-Amylase (AMS), lipase (LPS) and Trypsin (TPS) in the intestinal tracts of the test fish by using a digestive enzyme test box (built in Nanjing), wherein the specific method refers to the use instruction of the test box.
1.2.4 Muscle nutrition assay
After the feeding test, 5 fish were taken from each test group, 40g of back muscle on each lateral line was peeled off, and frozen at-20℃for use. Muscle nutrition: the water content is measured by a GB 5009.3-2016 constant pressure constant temperature drying method, the crude ash content is measured by a GB 5009.4-2016550 ℃ dry ash separation method, the crude protein content is measured by a GB 5009.5-2016 Kjeldahl nitrogen determination method, and the crude fat content is measured by a GB 5009.6-2016 Soxhlet extraction method; the amino acid composition and content (excluding tryptophan) were determined by means of an amino acid autoanalyzer in accordance with the hydrochloric acid method of GB 5009.124-2016.
1.3 Data processing
The statistical values are expressed as "mean ± standard deviation" using Excel and SPSS software treatments. At a significant level of 0.05, the test data were subjected to one-way analysis of variance and Duncan's multiple range method to examine their significant differences.
2 Results
2.1 Effect of sweet orange oil on Tilapia growth Performance
The effect of orange oil on tilapia growth performance is shown in table 2. The weight gain rate, specific growth rate and feed efficiency of each test group were gradually increased with increasing ratio of orange oil addition, wherein group D was highest, and increased by 13.97%, 9.72% and 15.77% respectively compared to control group a; the feed coefficient was gradually decreased, with group D being the lowest (1.75), by 13.79% compared to control group a. The viscera volume ratio and the liver volume ratio of each group are the largest in the group D, and the viscera volume ratio and the liver volume ratio among the other groups are not obviously different (P is more than 0.05) except that the viscera volume ratio of the group B is obviously lower than that of each group (P is less than 0.05); the fullness of each test group was lower than the control group a, and the test groups B and C were significantly lower than group a (P < 0.05). The survival rate of the tilapia mossambica in each group reaches 100 percent.
TABLE 2 Effect of sweet orange oil on Tilapia growth Performance
Note that: the same row of superscript letters indicates significant differences (P < 0.05), and the same row of superscript letters indicates insignificant differences (P > 0.05) or no superscript letters. The following is the same.
2.2 Effect of sweet orange oil on Tilapia digestive enzyme Activity
As can be seen from table 3, the activities of alpha-Amylase (AMS), lipase (LPS) and Trypsin (TPS) in the intestinal tract of the tilapia in the test all increased with increasing ratio of sweet orange oil, and reached 10.70U/mg, 0.99U/g and 1270.95U/ml respectively, with the highest D group, wherein TPS activities were significantly higher than that of the other groups (P < 0.05) by 2.96 times that of the control group a, 2.50 times that of the control group B and 1.52 times that of the control group C; however, there was no significant difference in AMS and LPS activity between groups (P > 0.05).
TABLE 3 Effect of sweet orange oil on Tilapia digestive enzyme Activity
2.3 Effect of sweet orange oil on muscle nutrition of Tilapia mossambica
2.3.1 Effect of sweet orange oil on conventional nutritional ingredients of Tilapia muscle
The sweet orange oil with different proportions is added into the basic feed, the water content of the tilapia muscles is increased to different degrees, wherein the water content of the group B is obviously higher than that of the group A (P is less than 0.05) of the control group, and the difference among other groups is not obvious (P is more than 0.05); the crude protein content of the tilapia muscle of each test group is firstly reduced and then increased along with the increase of the addition proportion of the sweet orange oil, and the crude protein content of the B group is obviously lower than that of the control group (P is less than 0.05), and no obvious difference exists among other groups (P is more than 0.05); the crude fat content of each test group was significantly lower than that of control group A (P < 0.05); the sweet orange oil has no obvious effect on the crude ash content of tilapia muscle (P is more than 0.05). The results are detailed in Table 4.
TABLE 4 Effect of sweet orange oil on conventional nutritional ingredients of Tilapia muscle (%)
2.3.2 Effect of sweet orange oil on muscle amino acid content of Tilapia mossambica
The research results show that the sweet orange oil also has a certain influence on the amino acid content of tilapia muscles (Table 5).
TABLE 5 Effect of sweet orange oil on muscle amino acid content of Tilapia (%)
The content of most amino acids in the tilapia muscle of each test group is basically consistent with that of a control group, wherein the content of Phe, thr, val and Arg in the D group is obviously higher than that in the control group (P is less than 0.05); the EAA and HEAA contents and EAAI of the muscles between each group showed a tendency to increase with increasing ratio of orange oil addition, while TAA, EAA, HEAA, DAA and EAAI were highest in group D, respectively 19.14%, 7.76%, 1.83%, 7.33% and 88.14.
Discussion 3
3.1 Effect on Tilapia growth Properties
Because the plant essential oil smell is aromatic, a proper amount of the plant essential oil smell can inhibit unpleasant smell in the feed, and the feed has the functions of feeding and improving the feed intake of animals, thereby improving the growth performance of the cultured animals. The research shows that the feed intake of weaned pigs and growing pigs is obviously increased by increasing the addition amount of the plant essential oil in a proper range. In the study, with the increasing of the addition ratio of the sweet orange oil, the weight gain rate, the specific growth rate and the feed efficiency of each test group are gradually increased, wherein the D group is respectively increased by 13.97%, 9.72% and 15.77% compared with the control group, the feed coefficient is gradually decreased by 13.79% compared with the control group, and the ingestion amount among the groups is basically consistent. It is explained that the growth promoting effect of the sweet orange oil on the tilapia is mainly realized by improving the feed conversion efficiency rather than the feed intake. The reasons for this difference may be related to the variety of the test animal, the type of basal diet, the nature and dosage of the plant essential oil, etc.
3.2 Effect on Tilapia digestive enzyme Activity
In the test, the AMS, LPS and TPS activities of the intestinal tracts of the tilapia are positively correlated with the addition proportion of the sweet orange oil, and the AMS and LPS activities of the groups are not obvious, but the TPS activity (1270.95U/ml) of the group D is obviously higher than that of the other groups and is nearly 3 times that of the control group. The orange oil has a certain improvement effect on the intestinal AMS and LPS activities of tilapia, and can obviously improve the TPS activity. It is presumed that the sweet orange oil can improve the balance of intestinal flora, the activity of digestive enzymes and the digestion utilization rate of feed by inhibiting and even killing intestinal harmful microorganisms and promoting the propagation and growth of beneficial bacteria, thereby achieving the purpose of improving the growth performance of tilapia.
3.3 Effect on muscle nutrition of Tilapia
The test results show that the addition of the sweet orange oil to the basal feed significantly reduces the crude fat content in the tilapia muscle, while the EAA and HEAA contents and EAAI in the muscle all show a tendency to gradually increase with the increase of the addition proportion of the sweet orange oil, and both TAA, EAA, HEAA, DAA and EAAI are highest in group D. The orange oil has a certain improvement effect on the muscle nutrition and quality of tilapia mossambica. The orange oil has the potential of developing as a fish growth promoter, and has the potential of developing as a fish meat quality improvement functional feed additive, for example, fish with special aromatic smell of muscles can be produced by feeding compound feed containing a certain proportion of plant essential oil, so that the consumer can increase the consumption of the basket for common people.
Conclusion 4
The addition of a proper amount of sweet orange oil into the basic feed can improve the growth performance and intestinal digestive enzyme activity of tilapia, and has a certain improvement effect on the nutrition and quality of muscles, and the addition proportion is preferably 0.5%.
Description of the preferred embodiments 2
1 Materials and methods
1.1 Test materials
1.1.1 Test fish
The tilapia for the test is provided by a bridge pilot scale of the institute of fresh water and aquatic products of Fujian, and the average weight is about 240 g.
1.1.2 Sweet orange oil
Sweet orange oil (MIC, origin: brazil) is purchased from Shanghai Jiuhuai industries, inc., and is obtained from cold-pressed fresh sweet orange peel, and is a yellowish-brown liquid with intense and heavy aroma, and has special orange smell and mild aromatic taste.
1.1.3 Test feed
0, 0.1%, 0.3%, 0.5% And 0.7% of sweet orange oil (A, B, C, D, E groups respectively) are added into each kilogram of base material respectively, and 5 kinds of granular feed with the same particle size are prepared by a soft granulation machine after uniformly mixing, and are stored for standby after airing. The conventional nutritional ingredients of the test feeds are shown in table 1.
Table 1 test feed composition and nutrient level (dry matter basis)%
1) The vitamin-mineral premix is prepared by providing :VA 80000IU,VD 40000IU,VE 1000mg,VK 100mg,VB1100 mg,VB2200 mg,VB6150 mg,VB122 mg,VC esters 1000mg, folic acid 80mg, nicotinic acid 800mg, calcium pantothenate 400mg, biotin 3mg, inositol per kg of feed 1000mg,Mg(as magnesium sulfate)1000mg,Mn(as manganese sulfate)100mg,Zn(as zinc sulfate)800mg,Cu(as copper sulfate)200mg,Fe(as ferrous sulfate)1000mg,Se(as sodium selenite)5mg,Co(as cobalt chloride)30mg,I(as potassium iodide)15mg.
2) The feed nutrition level is the actual measurement value.
1.2 Test methods
1.2.1 Test fish grouping and farming
The test was performed at the banyan bridge test base of the institute of fresh water aquiculture, fujian province. After stopping feeding for 24 hours, the tilapia with basically consistent specification and healthy and intact tilapia is selected from a cement pond and put into an indoor circulating water system (the diameter of a test barrel is 1.0m, the water depth is 0.8 m), and 15 tilapia are put into each barrel. The tests were divided into 5 groups (1 control group, 4 test groups) of 3 replicates each. The test water is aerated tap water, the water temperature is 25-30 ℃ during the test, and the dissolved oxygen is not less than 5.0mg/L, pH 6.9.9-7.2. Feeding is carried out by feeding for 2 times/d (8:00-9:00 am, 4:00-5:00 pm) until the feeding is completed within 30 min. The test period was 8 weeks.
1.2.2 Growth indicator determination
After the test, each growth index is measured after the test fish stops feeding for 24 hours:
weight gain rate (WGR,%) = (W f—Ws)/Ws ×100%;
Specific growth rate (SGR,%) = (lnW f—lnWs)/t×100%
Feed Coefficient (FCR) =b t/(Wf—Ws);
feed efficiency (FE,%) = (W f—Ws)/Bt ×100%;
Survival rate (SR,%) =n f/Ns ×100%;
Dirty volume ratio (VSI,%) =w v/Wo ×100%;
liver volume ratio (HSI,%) =w h/Wo ×100%;
fullness (CF) =w o/L3 ×100.
Wherein W s is the initial fish mass, and W f is the fish mass at the end of the test; w v is viscera weight, W h is liver pancreas weight and L is body length, W o is fish body mass; b t is total feed intake in the test process, N s test starts fish mantissa, N f test ends fish mantissa, and T is test period.
1.2.3 Serum Biochemical index determination
5 Fish were randomly collected from each test barrel, blood was collected from the tail vein, and serum was collected from the test barrels after centrifugation at 4℃for 10min (3000 r/min) and stored in a refrigerator at-80℃for testing. The activity of acid phosphatase (ACP) in serum is determined by using a kit produced by Nanjing built biological engineering research, the activity of alkaline phosphatase (AKP) is determined by using a kit produced by Shenzhen Leidu life sciences Co., ltd.) for the total Cholesterol (CHO), triglyceride (TG), high-density protein cholesterol (HDL-C) and low-density protein cholesterol (LDL-C) content.
1.2.4 Analysis of intestinal flora diversity
Stopping feeding for 24 hours after the test is finished, taking 3 test fishes randomly from each group, wiping the body surface with 75% ethanol, gently separating intestinal tracts from the abdominal cavity with sterile scissors and forceps, washing with PBS buffer solution, placing in a 5mL sterile freezing tube, and storing at-80 ℃ for analysis. Intestinal samples were labeled as group a (control) and group B, group C, group D, group E (test), respectively.
The qualified sample is subjected to PCR amplification by using 16S rDNA universal primers 338F (ACTCCTACGGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT), PCR products are extracted by using a DNeasy PowerSoil Pro Kit (QIAGEN, germany) kit, 16S rDNA high-throughput sequencing is carried out by entrusted Shanghai Meiji biological medicine technology Co., ltd, and the obtained data are subjected to biological informatics analysis such as Alpha index analysis, flora structure and composition, function prediction and the like.
1.2.5 Enterobacteria metabonomics
Stopping feeding for 24 hours after the test is finished, taking 3 test fishes randomly, taking 75% ethanol to wipe the body surface, then gently separating intestinal tracts from the abdominal cavity by using sterile scissors and tweezers, selecting the intestinal tracts at the same position, rapidly sucking residual body fluid by using dust-free absorbent paper, shearing intestinal tissues into small blocks with the side length of 0.5cm, precisely weighing, quick-freezing the intestinal tracts in a tinfoil groove containing liquid nitrogen, then putting the quick-frozen intestinal tracts into a liquid nitrogen precooled screw freezing tube, rapidly carrying out liquid nitrogen quick-freezing for more than 5 minutes, and then transferring the quick-frozen intestinal tracts to a refrigerator at the temperature of minus 80 ℃ for freezing. Samples were commissioned for metabonomics analysis by Shanghai Meiji Biotechnology Co.Ltd.
1.3 Data processing
The statistical values are expressed as "mean ± standard deviation" using Excel and SPSS software treatments. At a significant level of 0.05, the test data were subjected to one-way analysis of variance and Duncan's multiple range method to examine their significant differences.
2 Results
2.1 Effect of sweet orange oil on Tilapia growth Performance
The effect of orange oil on tilapia growth performance is shown in table 2. The powder quality (W f), the Weight Gain Rate (WGR) and the Specific Growth Rate (SGR) of the tilapia are all increased along with the increase of the addition proportion of the sweet orange oil, the W f of the test group D and the test group E are obviously higher than those of the control group A and the test group B (P < 0.05), the difference between the group C and the other groups is not obvious (P > 0.05), and the WGR and the SGR between the groups are not obvious (P > 0.05); the Feed Efficiency (FE) and the liver volume ratio (HSI) both show a trend of increasing firstly and then decreasing along with the increase of the addition proportion of the sweet orange oil, and the change trend of the Feed Coefficient (FCR) is opposite to the trend of increasing firstly and then decreasing, namely the FE is highest, the HSI is largest and the FCR is lowest under the addition level of the group D; there were no significant differences in the volume to volume ratio (VSI), fullness (CF) and Survival (SR) between groups (P > 0.05).
TABLE 2 Effect of sweet orange oil on Tilapia growth Performance
Note that: the same row of superscript letters indicates significant differences (P < 0.05), and the same row of superscript letters indicates insignificant differences (P > 0.05) or no superscript letters. The following is the same.
2.2 Effect of sweet orange oil on serum Biochemical index of Tilapia
The effect of sweet orange oil on serum biochemical index of tilapia is shown in Table 3. The activity of acid phosphatase (ACP) and alkaline phosphatase (AKP) of test fish serum is increased and then reduced with the increase of the addition proportion of sweet orange oil, the addition level of the test fish serum reaches the maximum value in the group D, namely 5.73 Kirschner units/100 mL and 22.36 Kirschner units/100 mL, and the AKP activity of the group D is obviously higher than that of the control group A, the test group B and the group C (P is less than 0.05); total Cholesterol (CHO), triglyceride (TG) and high density protein cholesterol (HDL-C) in serum all show a trend of increasing and then decreasing with increasing addition proportion of sweet orange oil, the addition level of the serum also reaches the maximum value in the group D, and the HDL-C content of the group D is obviously higher than that of the group A, the group B and the group C (P < 0.05); the low-density protein cholesterol (LDL-C) content gradually decreases along with the increasing addition proportion of the sweet orange oil, the LDL-C content of the E group is obviously lower than that of the control A group, the test B group and the C group (P < 0.05), and the difference among the other groups is not obvious (P > 0.05).
TABLE 3 Effect of sweet orange oil on serum Biochemical index of Tilapia
2.3 Effect of sweet orange oil on Tilapia intestinal flora
To determine the effect of orange oil on intestinal flora, 16S ribosomal RNA (rRNA) gene sequencing was performed. The alpha-diversity index shows that sweet orange oil has no significant effect (P > 0.05) on gut microbiota OUT, richness (Chao 1 and Ace index) and diversity (Shannon and Simpson index).
The results of Principal Component Analysis (PCA) showed that the samples of the sweet orange oil treatment (B, C, D, E) group and the control A group were interdigitated but non-overlapping, indicating that the sweet orange oil treatment was able to affect the colonic structure of tilapia intestinal microorganisms. In addition, the samples in group D were most concentrated, indicating that 0.5% orange oil was more beneficial for maintaining the stability of the intestinal microbiota structure.
At the phylum classification level, the intestinal microbiota of tilapia is mainly composed of Proteus (Proteus), actinomycetes (Actinobacteriota), bacteroides (Bacteroidota), fusobacterium (Fusobacteriota) and Thick-walled bacteria (Firmicutes), accounting for more than 90% of the total relative abundance of the entire microbiota. The relative abundance of Proteus (Proteus) decreased in groups B and C compared to control group A, while the relative abundance of actinomycetes (Actinobacteriota), bacteroidetes (Bacteroidota), fusobacterium (Fusobacteriota) and Thick-walled (Firmicutes) increased. The relative abundance of actinomycota (Actinobacteriota) in test group D tended to rise and the relative abundance of firmicutes (Firmicutes) tended to decrease compared to control group a. The relative abundance of the fusobacterium gate (Fusobacteriota) in test group E tended to rise and the relative abundance of the firmicutes gate (Firmicutes) tended to decrease compared to control group a.
At the genus classification level, the addition of 0.1% (group B) and 0.3% (group C) orange oil reduced the relative abundance of Achromobacter (Achromobacter) and geobacillus (Pedobacter) in the gut, and increased the relative abundance of whale (Cetobacterium) and Mycobacterium (Mycobacterium) in the gut. The addition of 0.5% (group D) orange oil reduced the relative abundance of streptococcus (unclassified _f_ Peptostreptococcus) in the gut and increased the relative abundance of Mycobacterium (mycrobacterium) in the gut. The addition of 0.7% (group E) orange oil reduced the relative abundance of Streptococcus (unclassified _f_ Peptostreptococcus) in the gut and increased the relative abundance of Celebracella (Cetobacterium) in the gut.
TABLE 4 alpha-diversity index
2.4 Effect of sweet orange oil on Tilapia Metabolic group
The influence of sweet orange oil on intestinal metabolites is explored by adopting a metabonomics method. In this experiment, there was a clear difference in intestinal metabolite content for each group of tilapias. The intestinal metabolites were significantly altered in the orange oil treated (B, C, D, E) group compared to the control a group. In positive ion mode, the down-regulated amount of group B metabolites was about 2 times the up-regulated amount, the down-regulated amount of group D metabolites was about 3 times the up-regulated amount, the down-regulated amount of group E metabolites was substantially identical to the up-regulated amount, and the up-regulated amount of group C metabolites was about 2 times the down-regulated amount, as compared to the control group a. In negative ion mode, the amount of down-regulated and up-regulated metabolites of group B, D were substantially identical to the amount of up-regulated metabolites of group C, which was approximately 2-fold the amount of down-regulated, and the amount of up-regulated metabolites of group E, which was approximately 2.7-fold the amount of down-regulated, compared to control group A.
Total 50 different metabolites were found between the three treatments by coacervation hierarchical clustering analysis. The KEGG metabolic pathways of the differential metabolites were analyzed to obtain the six most likely metabolic pathways affected by orange oil treatment, respectively: metabolism (mainly lipid metabolism and amino acid metabolism), biological systems, diseases, environmental information processing, cellular processes, and genetic information processing.
Results 4 results
The addition of a proper amount of sweet orange oil into basic feed can improve the growth performance and the immunocompetence of tilapia, obviously change the diversity of intestinal flora and metabolic products thereof, and simultaneously is more beneficial to maintaining the stability of the intestinal microbial community structure, and the most likely metabolic pathway influenced by the sweet orange oil treatment is metabolism (mainly lipid metabolism and amino acid metabolism). The ratio of sweet orange oil added in the feed in the study is preferably 0.5%.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (2)
1. A tilapia growth promoting feed additive based on healthy cultivation, which is characterized in that 0.3-0.5% of sweet orange oil and 2-4% of yeast culture are added into each kilogram of tilapia base material.
2. The health-farming-based tilapia growth-promoting feed additive according to claim 1, wherein 0.5% by mass of orange oil and 4% by mass of yeast culture are added per kg of tilapia base material.
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