A breeding method for a new high-temperature resistant, fast-growing strain of Penaeus monodon.

Abstract

This invention relates to a method for cultivating a new high-temperature resistant, fast-growing strain of Penaeus monodon, belonging to the field of aquatic breeding. The method involves subjecting juvenile shrimp to extreme high-temperature acute stress for 4-7 days and juvenile shrimp to long-term high-temperature culture stress for 45-60 days. By combining growth parameters, including the average survival time of individuals in the families subjected to extreme high-temperature acute stress, the average survival rate of each family in long-term high-temperature culture, and average body weight, a weighted comprehensive evaluation of the growth and survival rate of different families under high-temperature conditions is conducted. Families with rapid growth and strong high-temperature tolerance are selected for preservation and breeding as parent stock. These parent stock are then subjected to generational and successive selection to obtain a new high-temperature resistant, fast-growing strain of Penaeus monodon.

Description

Technical Field

[0001] This invention relates to the field of aquatic new variety breeding technology, and in particular to a method for breeding a new strain of high-temperature resistant and fast-growing tiger prawn. Background Technology

[0002] The tiger prawn grows rapidly, is large in size, and has a thick shell. It is highly resistant to drying out in air after being removed from water, making it suitable for sale as a live shrimp. As a poikilothermic animal, the tiger prawn's ability to adapt to changes in the external environment (especially water temperature) directly affects its growth, survival, and reproduction. Studies have shown that the optimal growth temperature for tiger prawns is 28-32℃. Water temperatures that are too low or too high significantly affect their feeding, metabolism, and immune function. When the water temperature drops below 20℃ or rises above 35℃, their growth rate and survival rate decrease dramatically. Furthermore, high-temperature stress can also lead to increased oxidative stress in aquatic animals, decreased immunity, resulting in stunted growth, abnormal development, and even death.

[0003] The trend of global warming is becoming increasingly significant. Prolonged periods of high temperatures in summer easily deteriorate water quality, often leading to outbreaks of diseases in shrimp farming, reduced yields, and even total crop failure, posing a challenge to the sustainable development of shrimp farming. Therefore, conducting research on the heat tolerance traits of Penaeus monodon and genetically improving and breeding superior strains adapted to high-temperature summer farming are of great importance. This will enrich the variety of shrimp farmed in my country and promote rotational farming of different species, thereby helping farmers avoid losses, promoting the sustainable development of shrimp farming, and enhancing the efficiency and competitiveness of my country's shrimp farming industry. Summary of the Invention

[0004] This invention overcomes the shortcomings of the prior art and provides a method for cultivating a new strain of high-temperature resistant, fast-growing tiger prawn.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for breeding a new high-temperature resistant and fast-growing strain of Penaeus monodon involves subjecting juvenile shrimp to extreme high-temperature acute stress for 4-7 days and juvenile shrimp to high-temperature long-term culture stress for 45-60 days. The method combines growth parameters, including the average survival time of individuals in the families subjected to extreme high-temperature acute stress, the average survival rate of each family in the long-term culture, and the average body weight, to a weighted comprehensive evaluation of the growth and survival rate of different families under high-temperature conditions. Individuals from rapidly growing families with strong high-temperature tolerance are selected for preservation and breeding as broodstock. These broodstocks are then subjected to subculturing and repeated selection to obtain a new high-temperature resistant and fast-growing strain of Penaeus monodon.

[0007] Furthermore, the extreme high-temperature acute stress temperature is 36-40℃, and the temperature of the culture water is increased by 2℃ every 24 hours from 30-32℃. After the temperature is raised to 36-40℃, the culture is carried out at a constant water temperature for 4-7 days.

[0008] Furthermore, the high-temperature long-term aquaculture stress temperature is 33-35℃.

[0009] Furthermore, the weighted comprehensive evaluation is calculated with growth traits weighted at 70% and heat resistance traits weighted at 30%.

[0010] Furthermore, temperature tolerance tests for each family were conducted in the same aquatic environment. Tests at different stages could be conducted in different bodies of water, but the testing environment had to be kept consistent within the same stage. In the first stage, shrimp (P15-P25) were cultured in different small net cages or small containers in the same body of water.

[0011] The beneficial effects of this invention compared with the prior art are as follows: This invention rapidly screens out new varieties of tiger prawns that are resistant to high temperatures and grow quickly by subjecting them to different temperature stresses at two growth stages and combining this with their growth conditions. Detailed Implementation

[0012] The technical solution of the present invention will be further explained below through embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0013] Example 1

[0014] In a preferred embodiment of the present invention, the method for cultivating a new strain of high-temperature resistant, fast-growing tiger prawns is carried out according to the following steps:

[0015] (1) Large-scale family construction and initial screening: Construct no fewer than 20 tiger prawn families with an age difference of ≤3 days, and cultivate each family to the P15 larvae;

[0016] (2) 100-200 P15 juvenile shrimp individuals were randomly selected from each family. The temperature of the culture water was increased by 2°C every 24 hours from 30-32°C to 38±0.5°C to induce acute high temperature stress. Then, the water was kept at a constant temperature for 7 days. The survival time of each individual in each family was recorded. The temperature tolerance test of each family was carried out in the same water environment. In this embodiment, the test was carried out in different small net cages in the same water body to ensure that the culture environment was consistent.

[0017] Table 1 shows the cumulative survival rate and survival time of each family of *Litopenaeus monodon* under 38℃ high-temperature stress. After 25 hours of 38℃ high-temperature stress, no shrimp deaths were observed in any family; after 50 hours, no shrimp deaths were observed in families F0458, M0064, T0001, and S0001; after 100 hours, all shrimp in family M0003 died, and nearly half of the shrimp in families F0416, M0019, M0060, and M0064 died, with survival rates of 48%, 48%, 50%, and 52%, respectively. Survival rates for families F0415, F0432, F0458, and M0008 were significantly lower. Survival rates for families F0442, M0007, M0095, S2028, S0001, and T0001 ranged from 55% to 90%, while survival rates for other families were below 50%. At 125 hours, all shrimp from families F0415, F0430, F0451, M0003, M0019, M0064, and M0080 died, while families T0001 and S0001 still had survival rates of 70% and 64%, respectively. At 150 hours, all shrimp died.

[0018] Survival times ranged from 48 to 150 hours across different families. One-way ANOVA results (Table 2) showed highly significant differences in survival times among families (P < 0.0001). The standard deviations of survival times also differed significantly among families, with the M0003 family exhibiting the lowest standard deviation (11.24) and the M0008 family the highest (30.91). Furthermore, the M0060 and F0441 families had the highest coefficients of variation in survival time (33%), while the M0064 family had the lowest (14%).

[0019] Table 1. Cumulative survival rate and average survival time of different families of Penaeus monodon

[0020]

[0021]

[0022] Table 2 shows the results of a one-way ANOVA of the survival time of each individual from different families of *Litopenaeus monodon* at 38℃. The analysis showed significant differences in survival time among the families, with the F-test indicating extreme significance (P < 0.0001). Further analysis of the multiple comparison test results revealed that the differences in survival time among the experimental families were statistically extremely significant (p < 0.0001), further validating the significant differences in high-temperature tolerance among the families.

[0023] Table 2. Analysis of variance of survival time of different families of Penaeus monodon at 38℃

[0024]

[0025] (3) 1000-1200 P15 juvenile shrimp individuals were randomly selected from each family and cultured for 30-45 days until they reached P45-P60.

[0026] (4) 300-400 uniformly sized P60 juvenile shrimp were randomly selected from each family and fluorescently labeled.

[0027] (5) 30-50 fluorescently tagged individuals were randomly selected from each family and placed in 6 cement ponds of 20 square meters each for long-term culture experiments. There were 3 normal temperature groups and 3 high temperature long-term culture groups. The water temperature was 34±0.5℃.

[0028] (6) After the 56-60 day breeding experiment, the marking color and part of each surviving individual, its family lineage, shell length, body length, weight and other trait data are collected and shown in Tables 3 and 4. The growth, survival rate and other important trait indicators of each family lineage are statistically analyzed based on the test data.

[0029] Table 3. Growth status and overall score of each family in the normal temperature group during the long-term breeding experiment.

[0030]

[0031] Table 4. Growth status and overall score of each family in the high-temperature group of the long-term aquaculture experiment.

[0032] (7) Genetic parameter assessment: The weighted comprehensive assessment method for the extreme high-temperature acute stress (38±0.5℃) experiment was calculated using a mixed linear model. The model formula is y=u+a+e, where: y represents the survival time of individuals in the temperature stress experiment; u is the population mean: the average value of all families on this indicator. For example, if the average survival rate of all families is 60%, then μ=60; a is the additive genetic effect; e is the random residual effect;

[0033] Phenotypic variation is decomposed into two parts: heritable additive effects and non-heritable environmental errors. The heritability (h2) parameter expresses the contribution of additive genetic effects to the overall variation, and its mathematical expression is:

[0034] h 2 =σ a 2 / (σ a 2 +σ e 2 )

[0035] In the formula, σ a 2 σ represents the variation component resulting from the additive effect of genes. e 2This reflects the variation components caused by environmental factors and other non-genetic factors. The Z-test method using the standard normal distribution is employed to calculate the statistic:

[0036]

[0037] Where: h 2 This represents the estimate of heritability. The value represents the standard error. Z (standardized value): Represents the relative performance of a family lineage on a certain indicator (such as survival rate or body weight). The higher the Z value, the better the family lineage performs on that indicator (relative to the average level of other families). When Z ≥ 1.96, the correlation between heritability and phenotype is significant (P < 0.05); when Z ≥ 2.58, it is highly significant (P < 0.01).

[0038] Heritability of heat tolerance trait in tiger prawn h 2 =0.20;

[0039] The heritability h of long-term high-temperature culture stress tolerance at 34±0.5℃ was calculated using a mixed linear model. 2 The heritability h of the growth trait is 0.27. 2 The heritability h of growth traits at room temperature is 0.34. 2 It is 0.25;

[0040] (8) Based on a weighted average of 70% for growth traits and 30% for heat resistance traits, select the top 30% of families with the best comprehensive breeding value for successive generations of breeding and testing to obtain a new strain of high-temperature resistant, fast-growing tiger prawn.

[0041] By assigning weights to the survival rate, growth status, and temperature of each family in the high-temperature aquaculture experiment using the analytic hierarchy process (AHP), a comprehensive score was obtained, and families with better overall traits were selected for breeding.

[0042] Construct a judgment matrix: For each level of criteria, perform pairwise comparisons to assess their importance relative to the next higher-level objective. A 1-9 scale is commonly used to represent the strength of the comparison results.

[0043] 1. Both elements are equally important;

[0044] 3: One element is slightly more important than the other;

[0045] 5: One element is more important than another;

[0046] 7: One element is clearly more important than the other;

[0047] 9: One element is far more important than the other;

[0048] 2, 4, 6, 8: Cases in between.

[0049] The heat tolerance trait was set to be slightly more important than the growth trait. According to the 1-9 scale, the weight of survival rate (heat tolerance) was 0.3 and the weight of body weight was 0.7.

[0050] The standardized formula (Z-score) is: Z = (X - μ) / σ; where X is the actual measured value of a family lineage on a certain indicator (e.g., survival rate of 50% or weight of 10g), μ is the population mean: the average value of all families on that indicator. For example, if the average survival rate of all families is 60%, then μ = 60; σ (standard deviation): the standard deviation of all families on that indicator, reflecting the dispersion of the data. The larger the σ, the greater the difference between families.

[0051] For each sample, the standardized body weight and survival rate data are weighted and summed according to their respective weights, as shown in the following formula: Overall Score = w1 × Z 存活 +w2×Z 重量 Where w1 and w2 are the weight values ​​determined by the analytic hierarchy process (AHP), and in heat-resistant breeding: w1 = 0.3, w2 = 0.7;

[0052] a. Survival rate criteria:

[0053] Room temperature group: Select families with a survival rate of over 70% to ensure good survival ability under standard conditions.

[0054] High-temperature group: Due to the generally low survival rate under high-temperature conditions, families with a survival rate of over 30% are preferred, but this will be relaxed appropriately based on the overall score and body weight.

[0055] b. Weight Standards:

[0056] Normal temperature group: Families with a final weight of 11g or more are given priority, showing good growth traits.

[0057] High temperature group: Due to the impact of high temperature on growth, families with a weight of 7g or more were given priority, showing that they still have a certain growth capacity in high temperature environment.

[0058] c. Overall Score Reference:

[0059] The overall score was calculated using the analytic hierarchy process (AHP), as shown in Tables 5 and 6, with a survival rate weight of 0.3 and a body weight weight of 0.7. Families with an overall score of 0.4 or higher were selected, representing excellent overall growth and heat tolerance.

[0060] Example 2

[0061] The F2 generation family selected in Example 1 was subjected to a high-temperature tolerance culture experiment in an indoor cement tank. Healthy, high-temperature tolerant, fast-growing F2 generation tiger prawns with a body length of approximately 4-5 cm were selected, while ordinary tiger prawns of the same size were selected as a control. Each experimental group consisted of 30 prawns, and each group was divided into 3 parallel groups. The culture environment was the same. During the culture process, a high-temperature stress experiment was conducted. The mixed growth characteristics of the F2 generation family in the cement tank after 2 months were as follows: In the normal temperature group (30℃), the average weight of the F2 generation family ranged from 9.25 to 16.63 g, with an average weight of 11.93 ± 1.74 g, while the average weight of the control group was 10.59 ± 3.87 g, and the growth rate was 12.65% higher than that of the control group; In the high temperature group (34℃), the average weight of the F2 generation family ranged from 5.58 to 9.43 g, with an average weight of 7.19 ± 1.06 g, while the average weight of the control group was 5.96 ± 2.27 g, and the growth rate was 20.64% higher.

[0062] Example 3

[0063] The F2 generation families selected in Example 1 were subjected to a summer high-temperature tolerant culture experiment in outdoor high-level ponds in Shenzhen. 5000 healthy, high-temperature tolerant, fast-growing F2 generation juveniles of the new tiger prawn variety, with a body length of approximately 4-5 cm, were fluorescently labeled. Simultaneously, 15000 juveniles of the same size common tiger prawn were selected as a control. The stocking density was 20,000 shrimp per acre, mixed in the same pond for growth testing. Two parallel groups were set up and cultured for 130 days. In pond 13, the average weight of the broodstock from each family ranged from 21.43 to 44.8 g, with an average weight of 34.15 ± 5.56 g. The average weight of the wild-type control group ranged from 29.15 ± 5.56 g. The F2 generation's growth rate was 15.68% higher than the control group on average. In pond 14, the average weight of the broodstock from each family ranged from 35.56 ± 7.94 g, while the average weight of the control group ranged from 30.84 ± 7.84 g. The F2 generation's growth rate was 15.31% higher on average.

[0064] Example 4

[0065] The F2 generation family selected in Example 1 was subjected to a high-temperature tolerance production test in aquaculture experiments (9 ponds covering an area of ​​20 mu). Healthy, high-temperature tolerant, fast-growing F2 generation tiger prawn larvae with a body length of approximately 1.5 cm were selected, while ordinary tiger prawns of the same size were selected as a control. On August 3, 930,000 F2 generation larvae and 450,000 wild control larvae were stocked at a stocking density of 100,000-120,000 larvae / mu. After 102 days of culture, the average body length of the F2 generation was 12.52±1.03 cm, the average weight was 27.78±7.13 g, and the average yield was 2000-2125 kg / mu. The average body length of the control group was 12.0±1.11 cm, the average weight was 25.01±7.12 g, and the average yield was 1650-1750 kg / mu. The average weight growth rate increased by 11.08%, the survival rate increased by 6%, and the yield increased by 21%.

Claims

1. A method for cultivating a new high-temperature resistant, fast-growing strain of Penaeus monodon, characterized in that, By subjecting juvenile shrimp families to extreme high-temperature acute stress and juvenile shrimp families to long-term high-temperature culture stress, and combining growth parameters including the average survival time of individuals in the extreme high-temperature acute stress families and the average survival rate and average weight of each family in the long-term high-temperature culture, a weighted comprehensive evaluation of the growth and survival rate of different families under high-temperature conditions was conducted. Families with rapid growth and strong high-temperature tolerance were selected for preservation and breeding into broodstock, and then subjected to subculturing and successive generations of selection to obtain a new strain of high-temperature resistant and fast-growing tiger prawn. The extreme high-temperature acute stress was defined as the culture water temperature being increased by 2°C every 24 hours from 30-32°C to 38±0.5°C, followed by constant water temperature culture for 7 days. The aforementioned high-temperature long-term aquaculture stress refers to a water temperature of 34±0.5℃ for 60 days.

2. The method according to claim 1, characterized in that, The weighted comprehensive evaluation is calculated with growth traits weighted at 70% and heat resistance traits weighted at 30%.

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