Use of nmda to improve fish growth performance, improve muscle quality and enhance ammonia nitrogen stress resistance

Abstract

The present application relates to the field of feed additives, and discloses a use of N-methyl-D-aspartate for improving growth performance of fish, improving muscle quality and enhancing ammonia nitrogen stress resistance. The present application aims at solving the problems of growth performance reduction, fish muscle quality reduction and vulnerability to water body ammonia nitrogen stress in current fish culture, and provides a use of N-methyl-D-aspartate for improving growth performance of fish, improving muscle quality and enhancing ammonia nitrogen stress resistance, i.e. using NMDA as a feed additive, growth promoter, meat quality improver or immune enhancer to improve fish meat quality, improve fish meat flavor, significantly improve the growth performance of Xiangyun crucian, improve the muscle quality and flavor, enhance the ammonia nitrogen stress resistance, and significantly increase the economic benefits of fish farmers.

Description

Technical Field

[0001] This invention relates to the field of aquaculture, specifically to the use of N-methyl-D-aspartic acid to improve fish growth performance, enhance muscle quality, and strengthen resistance to ammonia nitrogen stress. Background Technology

[0002] N-Methyl-D-aspartic acid (NMDA) is one of the free amino acids found in the brains of mammals and humans. It is an acidic amino acid with two carboxyl groups and one amino group, and has an excitatory effect on the central nervous system. It has been identified as an excitatory neurotransmitter and is therefore called an excitatory amino acid. Studies have found that NMDA is a potent agonist of the neurotransmitters aspartate and glutamate, which regulate pituitary function, and participates in neuroendocrine function, effectively increasing GH levels in peripheral blood. Estienne (1989, 1990) reported that intravenous injection of NMDA significantly increased serum GH levels in castrated ewes and rams; in 1995 and 1996, it was reported that intravenous injection of NMDA in castrated boars increased GH levels by 883%-1095%. Mason (1983) also observed similar effects in adult mice. Xu Zirong et al. (1998) found that feeding 50 mg / kg NMDA could significantly improve the growth rate, serum GH and serum insulin-like growth factor-1 (IGF-1) levels in fattening pigs and significantly improve carcass composition.

[0003] Feng et al. (2003) reported that adding 50 mg / kg NMDA to the diet significantly improved the daily weight gain and feed conversion efficiency of finishing pigs, and markedly improved carcass quality, with serum GH content increasing by 92.54%. Ma Wenqiang and Feng Jie (2008) confirmed that NMDA significantly increased the secretion level of growth hormone in pigs, with an overall increase of 36.91% in the secretion level of growth hormone in castrated boars and 45.95% in the secretion level of growth hormone in finishing sows. However, whether NMDA has the same effect on the growth performance and hormone secretion of fish has not yet been reported in any studies, and further in-depth research is needed on the efficacy and safety of NMDA. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the aforementioned background technology and address the common problems in current fish farming, such as reduced growth performance, decreased fish meat quality, and susceptibility to ammonia nitrogen stress in the water. This invention provides an application of N-methyl-D-aspartic acid (NMDA) to improve fish growth performance, enhance muscle quality, and strengthen resistance to ammonia nitrogen stress. Specifically, the application of this invention uses NMDA as a feed additive, growth promoter, meat quality improver, or immune enhancer to improve fish meat quality and flavor. Compared to ordinary feed, the feeding effect is more significant, noticeably improving the growth performance of Xiangyun crucian carp, enhancing muscle quality and flavor, strengthening resistance to ammonia nitrogen stress, and significantly increasing the economic benefits for fish farmers.

[0005] Furthermore, in some embodiments of the present invention, the intended use is to use NMDA as a feed additive, growth promoter, meat quality improver, or immune enhancer to improve fish growth performance and feed utilization efficiency.

[0006] Furthermore, in some embodiments of the present invention, the intended use is to use NMDA as a feed additive, growth promoter, meat quality improver, or immune enhancer to improve fish fatness and meat yield, and improve fish muscle quality.

[0007] Furthermore, in some embodiments of the present invention, the intended use is to use NMDA as a feed additive, growth promoter, meat quality improver, or immune enhancer to enhance the fish's resistance to ammonia nitrogen stress and increase the content of glutamic acid, a flavor amino acid in fish.

[0008] Furthermore, in some embodiments of the present invention, the intended use is to use NMDA as a feed additive, growth promoter, meat quality improver, or immune enhancer to increase the content of fish flavor nucleotide inosinic acid.

[0009] Furthermore, in some embodiments of the present invention, the fish is the Xiangyun crucian carp.

[0010] On the other hand, the present invention provides a feed containing NMDA as a feed additive, growth promoter, meat quality improver, or immune enhancer.

[0011] Furthermore, in some embodiments of the present invention, the feed uses fish meal, soybean meal and rapeseed meal as protein sources and fish oil and soybean oil as fat sources.

[0012] Furthermore, in some embodiments of the present invention, the feed contains fish meal, soybean meal, rapeseed meal, fish oil, soybean oil, flour, choline, multivitamins and minerals, sodium carboxymethyl cellulose (CMC), DMPT, ethoxyquinoline, methionine, calcium dihydrogen phosphate, cellulose, and NMDA.

[0013] Furthermore, in some embodiments of the present invention, the mass ratio of fish meal, soybean meal, rapeseed meal, fish oil, soybean oil, flour, choline, multivitamins and minerals, sodium carboxymethyl cellulose, ethoxyquinoline, methionine, calcium dihydrogen phosphate, and cellulose in the feed is 2-4:30-35:22-27:2-4:2-4:24-28:0.2-0.4:1.7-2.3:2-4:0.04-0.06:0.16-0.24:0.7-1.3:0.9-1.3.

[0014] Furthermore, in some embodiments of the present invention, 0.025-0.4% NMDA is added to the feed, for example, 0.025%, 0.05%, 0.1%, 0.2%, or 0.4% NMDA is added.

[0015] Compared with the prior art, the advantages of the present invention are as follows:

[0016] (1) Adding NMDA to feed helps improve the growth performance, flavor substance accumulation, and resistance to ammonia nitrogen stress of Xiangyun crucian carp. Adding NMDA to feed (especially at a concentration of 0.025%) can significantly improve the growth performance of Xiangyun crucian carp and increase feed utilization efficiency; at the same time, it can increase the condition factor of Xiangyun crucian carp, reduce the weight of its visceral mass, increase the meat yield, and significantly increase the economic benefits of fish farmers.

[0017] (2) In this invention, NMDA is used as a growth promoter and added to Xiangyun crucian carp feed in the form of an additive. As the addition ratio increases, it can be observed that the content of free glutamic acid and inosinic acid in the muscle of Xiangyun crucian carp shows a significant upward trend, and the content of free flavor amino acid glutamic acid in the muscle increases significantly.

[0018] (3) In this invention, NMDA can significantly activate the heat shock protein family genes in Xiangyun crucian carp and reduce the ammonia content in plasma and other body tissues, thereby effectively reducing the toxicity and damage to the body caused by ammonia nitrogen stress. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the effect of NMDA supplementation in feed on the free glutamate content in the muscle of Xiangyun crucian carp.

[0020] Figure 2 This is a schematic diagram showing the effect of NMDA supplementation in feed on the free inosinic acid content in the muscle of Xiangyun crucian carp.

[0021] Figure 3-5 This is a schematic diagram illustrating the effect of NMDA on the relative expression levels of liver defense genes in crucian carp after ammonia nitrogen stress. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Additional aspects and advantages of this invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. It should be understood that the following description is merely illustrative and not intended to limit the invention.

[0023] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0024] The conjunction "composed of..." excludes any unspecified elements, steps, or components. If used in a claim, this phrase makes the claim closed, excluding materials other than those described, except for associated conventional impurities. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following it, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0025] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including the ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0026] Furthermore, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., described below refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example. Moreover, the technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0027] In a specific embodiment of the present invention, the aquaculture experiment was conducted in the Nutrition and Aquaculture Laboratory of Changsha University.

[0028] Example 1

[0029] Experiment on the effect of NMDA on the growth performance of Xiangyun crucian carp

[0030] The Xiangyun crucian carp feed used in this invention contains 0%, 0.025%, 0.05%, 0.1%, 0.2%, and 0.4% NMDA, and the following procedure was used to conduct indoor culture experiments on Xiangyun crucian carp.

[0031] (1) The Xiangyun crucian carp feed of the present invention uses fish meal, soybean meal and rapeseed meal as the main protein sources and fish oil and soybean oil as the fat sources. Six experimental feeds with NMDA content of 0%, 0.025%, 0.05%, 0.1%, 0.2% and 0.4% were designed as the basic feed (see Table 1). All components in the feed were separated by a 20-mesh sieve, and then the feed was stirred for 20 minutes by a feed mixer. The feed was then granulated to 2.0 mm using a laboratory pellet mill, dried at 60°C using a blower dryer, and then sealed and stored at -20°C.

[0032] Table 16 Feed Formulas and Basic Components

[0033]

[0034]

[0035] (2) Before the start of the breeding experiment, Xiangyun crucian carp were temporarily kept in four glass fiber tanks (1500L) for two weeks. A recirculating aquaculture system was used. During the temporary keeping period, the Xiangyun crucian carp were fed at two time points at 8:00 and 14:00 every day. The feed fed was the control group feed (i.e. feed containing 0% NMDA).

[0036] (3) After the Xiangyun crucian carp reached stable growth, they were divided into tanks. 360 Xiangyun crucian carp of similar size and shape (17.03±0.06g) were weighed in batches and randomly assigned to 18 100L fiberglass tanks. There were 6 treatments in total, with 3 replicates per treatment and 20 Xiangyun crucian carp per replicate. During the experiment, the Xiangyun crucian carp were fed at 8:00 and 14:00 daily until they appeared to be satiated. The water flow rate in the culture system was 1300mL / min, the ammonia nitrogen level was maintained below 0.1mg / kg, the pH was 6.5-7.0, and the light duration was from 8:00 to 20:00. The culture experiment lasted for 60 days.

[0037] (4) Record the water temperature of the fish tank, the amount of food given to each fish tank and the feeding situation every day.

[0038] (5) After the rearing period, the Xiangyun crucian carp were fasted for 24 hours. All fish from each tank were removed and anesthetized with 50 mg / L MS-222 (ethyl m-aminobenzoate methanesulfonate). The fish were then weighed per tank to calculate the final body weight, weight gain rate, specific growth rate, and feed efficiency, among other growth performance indicators. For each replicate, six fish were randomly selected to measure body length, body weight, and visceral weight, and to test body shape indicators such as condition factor, liver-to-body ratio, and visceral-to-body ratio. The formulas used in the experiment are as follows:

[0039] Weight gain rate, % = (Wf - Wi) / Wi × 100

[0040] Specific growth rate, % / d = [ln(Wf) - ln(Wi)] / d × 100

[0041] Feed efficiency, % = (Wf - Wi) / FI × 100

[0042] Feed intake rate, %BW / d = FI / [d×(Wf+Wi)]×100

[0043] fatness, g / cm 3 =W / L 3 ×100

[0044] Liver body weight ratio, % = Wh / W × 100

[0045] Organ-to-body ratio, % = Wv / W × 100

[0046] Where Wi and Wf are the initial and final body weights (g) of the experimental fish, respectively, d is the number of days of culture (60 days), FI represents the amount of food consumed by the experimental fish, and Wv, Wh, W and L represent the visceral weight (g), liver weight (g), body weight (g) and body length (cm) of a single fish, respectively.

[0047] All data in this embodiment were statistically analyzed using SPSS 19.0 software, and the data are expressed as mean ± standard error. Each relevant indicator was first tested for homogeneity of variance; after confirming homogeneity of variance, one-way ANOVA was used for one-way ANOVA; if the differences between experimental groups were significant (P < 0.05 indicates significant difference), Duncan's method was used for multiple comparisons.

[0048] The effects of NMDA on the growth performance of Xiangyun crucian carp are shown in Table 2 below.

[0049] Table 2. Effects of NMDA Addition to Feed on Growth Performance of Xiangyun Crucian Carp

[0050]

[0051] Note: Data are expressed as mean ± standard error. Superscripts in the same column with the same lowercase letter or no letter indicate no significant difference (P>0.05), while superscripts with different lowercase letters indicate significant difference (P<0.05).

[0052] Table 2 shows that with the increase of NMDA supplementation level, the weight gain rate, specific growth rate, and feed efficiency of the experimental fish first increased and then gradually decreased. Among them, the experimental fish in the 0.025% NMDA supplementation group showed significantly better growth performance than the control group and other supplementation groups. The feed intake rate in the 0.025%, 0.2%, and 0.4% NMDA supplementation groups was significantly higher than that in the 0.1% NMDA supplementation group.

[0053] The effects of NMDA on the body shape indicators of Xiangyun crucian carp are shown in Table 3 below.

[0054] Table 3. Effects of NMDA Addition to Feed on Body Morphology of Xiangyun Crucian Carp

[0055]

[0056] Note: Data are expressed as mean ± standard error. Superscripts in the same column with the same lowercase letter or no letter indicate no significant difference (P>0.05), while superscripts with different lowercase letters indicate significant difference (P<0.05).

[0057] Table 3 shows that the condition factor (BFF) of the 0.025% NMDA-added group was significantly lower than that of the control group, and the liver-to-body ratio (HB / BFF) of the 0.025% NMDA-added group also tended to be lower than that of the control group. As the proportion of NMDA added to the feed increased, the HB / BFF of the experimental fish gradually decreased. These results indicate that adding 0.025% NMDA to the feed can significantly improve the growth performance of Xiangyun crucian carp, increase feed utilization efficiency, and simultaneously improve the condition factor, reduce the weight of the viscera mass, and increase the meat yield.

[0058] Example 2

[0059] Experiment on the effect of NMDA on the muscle flavor of Xiangyun crucian carp

[0060] In this embodiment, the experimental materials and methods, experimental location, and feeding management plan are the same as those described in Example 1. After the 60-day breeding experiment, three fish close to their average weight were randomly selected from each tank. The dorsal muscle of the Xiangyun crucian carp was taken from the ice, frozen at -80℃, and then transferred to a freeze dryer for thorough drying. The content of free flavor amino acid glutamic acid and flavor nucleotide inosinic acid in the freeze-dried muscle samples was determined. The determination method is as follows:

[0061] Determination of free amino acids: Accurately weigh 2-4 g (accurate to 0.0001 g) of fish muscle sample into a 10 mL volumetric flask, add 0.02 mol / L hydrochloric acid to dissolve and dilute to volume. After sonicating the solid sample for 20 min, centrifuge at 6000 r / min for 5 min, and collect the supernatant for purification.

[0062] Activate the C18 purification column (SHISEIDO, 4.6mm*250mm*5μm) with 5mL methanol and 5mL water respectively. Add 2.5mL of sample and 1.5mL of 0.02mol / L hydrochloric acid. Accurately transfer 100μL of the sample after column chromatography into a 15mL centrifuge tube and place it in a vacuum drying oven at 60℃ for 2h (to completely dry the solvent). Purge the centrifuge tube with nitrogen and accurately add 50μL of derivatizing reagent, i.e., ethanol: phenyl isothiocyanate: water: triethylamine = 7:1:1:1 (prepare fresh and purge with nitrogen during preparation). Derivatize at room temperature for 30min. Add 0.45mL of mobile phase A (0.1mol / L anhydrous sodium acetate: acetonitrile = 97:3), mix well, and pass through a 0.45μm organic membrane liquid chromatograph (Agilent, 1260).

[0063] Determination of free nucleotides: Accurately weigh the well-mixed sample into a centrifuge tube, add 10 mL of 10% perchloric acid, mix well and sonicate for 30 min, centrifuge and take out the supernatant, extract the residue again with 5% perchloric acid, combine the supernatants, adjust the pH to 6.5 with potassium hydroxide, make up to 50 mL, mix well and filter through a 0.22 μm microporous membrane before determination by liquid chromatography (Agilent, 1260).

[0064] All data in this embodiment were statistically analyzed using SPSS 19.0 software, and the data are expressed as mean ± standard error. Each relevant indicator was first tested for homogeneity of variance; after confirming homogeneity of variance, one-way ANOVA was used for one-way ANOVA; if the differences between experimental groups were significant (P < 0.05 indicates significant difference), Duncan's method was used for multiple comparisons.

[0065] The content of free glutamic acid and inosine acid in the muscle of each group of Xiangyun crucian carp is as follows: Figure 1 As shown, from Figure 1 As can be seen, with the gradual increase of NMDA content in the feed, the contents of free glutamic acid and inosinic acid in the muscle showed a significant upward trend. The content of free flavor amino acid glutamic acid in the muscle was higher in the NMDA-added groups than in the control group, and significantly higher in the 0.1% and 0.4% NMDA-added groups. The content of muscle flavor nucleotide inosinic acid showed a similar increasing trend, and was significantly higher in the 0.05%-0.02% NMDA groups than in the control group. These results indicate that adding an appropriate amount of NMDA to the feed can increase the content of flavor substances in the muscle of Xiangyun crucian carp and improve its flavor quality.

[0066] Example 3

[0067] Experiment on the effect of NMDA on liver defense genes of crucian carp after ammonia nitrogen stress

[0068] In this embodiment, the experimental materials and methods, experimental location, and husbandry management plan are the same as those described in Example 1. After the 60-day culture experiment, once the sampling for Examples 1 and 2 was completed, all experimental fish were returned to their original experimental tanks for ammonia nitrogen stress experiments. The ammonia nitrogen stress treatment was as follows: using ammonium chloride (NH4Cl) at 10 g / L as the stock solution, the NH4Cl concentration in each experimental tank was adjusted to 20 mg / L for acute ammonia nitrogen stress treatment. 24 hours after the challenge, three fish were randomly selected from each tank, and their liver tissue was dissected on ice, quickly frozen in liquid nitrogen, and then transferred to a -80°C freezer for storage, pending genetic testing and analysis.

[0069] The ammonia stress resistance genes selected for detection in this embodiment are heat shock protein 70 (Hsp70), Rhag, and Rhcg2. Heat shock protein 70 is a widely distributed heat protein in animals that protects the body from external stress. Rhag and Rhcg2 are members of the Rh protein family in animals and act as ammonia transporters in aquatic animals, mediating and promoting the transport and metabolism of ionized ammonia, thereby reducing the damaging effects of excessive ammonia on the body.

[0070] First, total RNA was extracted from liver tissue using TRlzol reagent. Subsequently, spectrophotometry and agarose gel electrophoresis were used to determine the quality and concentration of the RNA. Then, the RNA was reverse transcribed into a complementary DNA strand using a PrimeScrip RT kit (TaKaRa, Dalian, China). Real-time quantitative PCR of relevant genes was performed using a CFX96 Real-time PCR instrument (Bio-Rad, USA). The reaction system was as follows: 2 μL cDNA, 0.8 μL upstream and downstream primers, 10 μL SYBR Premix ExTaq (TaKaRa), and 6.4 μL double-distilled water. The cycling conditions were as follows: denaturation at 95℃ for 3 minutes, 40 cycles at 95℃ for 10 seconds, 20 seconds at 60℃, and 10 seconds at 72℃. β-actin was used as an internal reference gene to detect the relative expression level of the target gene. Relative quantification was performed using the contrast CT value method (2...). -ΔΔCt (Method). Expression levels of relevant genes in the liver of Xiangyun crucian carp after ammonia nitrogen stress, such as... Figure 3-5 As shown.

[0071] All data in this embodiment were statistically analyzed using SPSS 19.0 software, and the data are expressed as mean ± standard error. Each relevant indicator was first tested for homogeneity of variance; after confirming homogeneity of variance, one-way ANOVA was used for one-way ANOVA; if the differences between experimental groups were significant (P < 0.05 indicates significant difference), Duncan's method was used for multiple comparisons.

[0072] Depend on Figure 3-5 The results show that with increasing NMDA supplementation levels in the feed, the expression level of the Hsp70 gene in the liver of Xiangyun crucian carp significantly increased, with the Hsp70 gene expression in the 0.1-0.4% NMDA supplementation group being significantly higher than that in the control group. The relative expression levels of Rhag and Rhcg2 genes gradually decreased with increasing NMDA supplementation. Specifically, the Rhag gene expression in the 0.1% NMDA supplementation group was significantly lower than that in the control group, while the Rhcg2 gene expression was significantly downregulated in the 0.2% and 0.4% NMDA supplementation groups. These results indicate that NMDA can significantly activate the heat shock protein family genes in Xiangyun crucian carp, reducing ammonia content in plasma and other body tissues, thereby effectively reducing the toxicity and damage caused by ammonia nitrogen stress. Reduced ammonia nitrogen toxicity leads to decreased ammonia transport by Rhag and Rhcg2.

[0073] Considering the overall performance of Xiangyun crucian carp in terms of growth, muscle flavor compounds, and liver resistance gene expression under ammonia nitrogen stress, NMDA supplementation in feed can help improve the growth performance, flavor compound accumulation, and resistance to ammonia nitrogen stress in Xiangyun crucian carp. In summary, adding 0.025% NMDA results in optimal growth, flavor, and health performance for Xiangyun crucian carp.

[0074] Those skilled in the art will readily understand that the above description is merely an embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An application of N-methyl-D-aspartic acid, characterized in that the application is as a feed additive to enhance the resistance of fish to ammonia nitrogen stress and increase the content of glutamic acid, a flavor amino acid in fish; wherein the fish is Xiangyun crucian carp.

2. An application of N-methyl-D-aspartic acid, characterized in that the application is as a growth promoter, meat quality improver, or immune enhancer to enhance the resistance of fish to ammonia nitrogen stress and increase the content of glutamic acid, a flavor amino acid in fish; wherein the fish is Xiangyun crucian carp.

3. An application of N-methyl-D-aspartic acid, characterized in that the application is to increase the content of inosinic acid, a flavor nucleotide in fish; the fish being crucian carp.

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