Low-loss litopenaeus vannamei growth phenotype determination method

Abstract

The invention relates to the technical field of genetic breeding of aquatic animals, in particular to a low-loss litopenaeus vannamei growth phenotype determination method. The preparation method comprises the following steps: performing anesthesia treatment on a prawn sample to be measured to enable the prawn to enter an anesthetic state, then transferring the prawn into a normal seawater environment, measuring the growth phenotype of the prawn in a window period, and then recovering the normal state of the prawn. According to the method disclosed by the invention, through short-time eugenol anesthesia, mechanical injury and stress reaction caused by jumping and struggling of the prawns in the phenotype determination process are effectively inhibited, and the loss rate of the prawns is obviously reduced. Compared with the prior art, the method has the advantages of simplicity and convenience in operation, extremely low prawn loss, high growth phenotype determination efficiency and the like, is suitable for growth character evaluation in a large-scale genetic breeding process of prawns, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal genetics and breeding technology, specifically to a low-loss method for determining the growth phenotype of Litopenaeus vannamei. Background Technology

[0002] Litopenaeus vannamei ( Litopenaeus vannamei Litopenaeus vannamei is one of the world's largest-scale and most economically valuable aquaculture animals. Its strong adaptability, high feed conversion rate, and rapid growth rate have made it a crucial supporting species for the aquaculture industry in my country and globally. With the advancement of aquaculture technology and the acceleration of industrialization, the market demand for Litopenaeus vannamei continues to grow. The cultivation of high-quality, high-yield, and highly resistant varieties has become a key foundation for ensuring the healthy and sustainable development of the industry.

[0003] In recent years, genetic breeding technology for Litopenaeus vannamei has made continuous progress. Through techniques such as family selection, marker-assisted selection, and whole-genome selection, the efficiency of breeding growth and disease resistance traits in shrimp has been significantly improved. Growth traits, such as body length and weight, are the primary traits for evaluating Litopenaeus vannamei breeding. To date, several domestically bred varieties with growth advantages have been developed. However, compared with imported varieties, the performance of domestically bred varieties still lags significantly, and continuous genetic improvement of growth traits is still needed. In the breeding of growth traits, efficient and accurate determination of growth phenotypes is the foundation for conducting genetic evaluation and selection breeding of growth traits.

[0004] To improve the efficiency of growth trait measurement, researchers have developed a growth phenotypic measurement method based on machine vision and automated recognition (Bao Zhenning, Yu Yang, Li Fuhua. Establishment and application of high-throughput measurement technology of shrimp growth trait phenotype based on Faster R-CNN. Acta Hydrobiologica Sinica, 2023, 47(10), 1576-1584; ZL202010143233.2, etc.). However, in the actual growth trait measurement process, due to the sensitivity of Litopenaeus vannamei to environmental changes and its tendency to jump and struggle, conventional phenotypic measurement methods often result in shrimp jumping, leading to limb injuries and increased stress response, which also reduces the measurement efficiency. Furthermore, shrimp jumping can easily injure the measurement personnel, posing a significant safety hazard. Even if a fixed method is chosen to solve the aforementioned problems, due to the large differences in individual size at different growth stages of shrimp, it is difficult to select a suitable fixed container, and manual fixed operation is complex, time-consuming, and prone to causing injury. Summary of the Invention

[0005] The purpose of this invention is to provide a low-loss method for determining the growth phenotype of Litopenaeus vannamei.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The application discloses a method for measuring the growth phenotype of Litopenaeus vannamei with low loss, wherein the shrimp samples to be measured are subjected to narcotization treatment so that the shrimp enters a narcotization state, and then the shrimp is transferred to a normal seawater environment to realize the measurement of the growth phenotype in a window period, and then the shrimp can recover to a normal state.

[0007] The narcotization treatment is to immerse the shrimp samples to be measured in seawater containing 50 mg / L eugenol for 10 minutes, so that the shrimp body loses the balance ability, turns over, is inactivity and loses the response to stimulation.

[0008] The narcotic agent is eugenol with a purity of 99%, which is mixed and dissolved with 95% alcohol according to a volume ratio of 1:9, and then is added into the seawater, so that the final concentration of the narcotic agent reaches 50 mg / L.

[0009] After the shrimp enters the narcotization state, the sample is transferred to the normal seawater environment, and the growth phenotype is measured by an automatic measurement device in a window period of about 20 minutes, and the sample recovers to a normal swimming state after the measurement.

[0010] Further, (1) a 250L culture water bucket is taken as a narcotization container, 200L of culture seawater is put into the container, and the water temperature is 26-30 DEG C.

[0011] (2) 10ml of eugenol with a purity of 99% (Shanghai Aladdin Bio-Chem Technology Co., Ltd.) is taken as a narcotic agent, 90ml of 95% alcohol (Guo Yao) is used to dissolve the narcotic agent, and then the narcotic agent is added into the narcotization container to configure a narcotic agent solution with a final concentration of 50 mg / L.

[0012] (3) the breeding population for measuring the growth phenotype is taken out from a test pool in batches and is placed into the narcotization container, after narcotization for 10 minutes, the shrimp body loses the balance ability, turns over, is inactivity and loses the response to stimulation, which indicates that the narcotization is successful.

[0013] (4) the narcotized shrimp individuals are placed into normal seawater to recover gradually, at this time, the narcotized shrimps are taken out from the normal seawater respectively, and the growth phenotype is measured by using an automatic phenotype measurement instrument or an electronic scale, and according to the needs, the eye handle ring of each individual is bound, and sampling is performed, since the shrimp is in the narcotization state, the shrimp does not jump during the measurement process, the measurement speed is high, the shrimp is slightly damaged, and the phenotype measurement and sampling speed can be greatly improved.

[0014] (5) the individuals after the measurement are taken out and are placed in the normal seawater for cultivation, and can recover to the normal state after 20 minutes of narcotization.

[0015] The application of the method can be applied to the application of the high-quality shrimp samples screened by the method in shrimp breeding.

[0016] Advantages of the present application: (1) The present application uses a specific amount of eugenol as an anesthetic to anesthetize at a specific time, so that the sample completes the determination in a short window period. After processing in this way, the determination reduces the damage caused by the stress and jumping of shrimps, and improves the reliability and accuracy of the phenotype data. The method of the present application helps to maintain the integrity and health of the experimental sample, and is suitable for the determination of traits in large-scale shrimp genetic breeding process, and has good popularization and application prospect.

[0017] (2) The shrimps anesthetized by the processing method of the present application have fast phenotype determination speed and small damage, which greatly improves the speed of phenotype determination and sampling, reduces the labor cost, improves the breeding efficiency and economic benefit, and provides strong technical support for the rapid selection of shrimp genetic improvement and excellent line. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The effect diagram of the determined shrimp phenotype data, wherein A is the body weight distribution frequency, and B is the body length distribution frequency. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be described clearly and completely below. The present application will be further described in combination with specific embodiments.

[0020] The present application proposes a method of anesthetizing shrimps with anesthetics (adopting optimal concentration selection, anesthesia time control and recovery efficiency), and determining the phenotype of shrimps in the anesthesia gap, and then screening the most suitable anesthetics, anesthetic dose and recovery efficiency for application in shrimp breeding, combining with high-throughput phenotype determination technology, to realize a safe, convenient, low-loss and suitable for automatic equipment operation phenotype determination method, which has important significance for improving the breeding efficiency of shrimps.

[0021] Further, the present application places the Litopenaeus vannamei used for growth phenotype determination in seawater containing 50 mg / L eugenol for 10 minutes, so that the shrimps enter an anesthetized state. The anesthetized shrimps are transferred to normal seawater environment, and the growth phenotype determination is completed within about 20 minutes of the window period. After the determination is completed, the shrimps can quickly recover normal swimming and feeding behaviors, and no obvious damage or death occurs. The method of the present application effectively inhibits the mechanical damage and stress reaction of shrimps caused by jumping and struggling during the phenotype determination process by short-time eugenol anesthesia, and significantly reduces the loss rate of shrimps. Compared with the prior art, the present application has the advantages of simple operation, extremely low shrimp loss, high growth phenotype determination efficiency, and is suitable for growth trait evaluation in large-scale shrimp genetic breeding process, and has broad application prospect.

[0022] Source of anesthetics: Eugenol (C10 H 12 O2, 99%>, purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China).

[0023] Example 1: Determination of the optimal anesthetic dose and time in Penaeus vannamei phenotyping The method of high-throughput determination of growth phenotype after anesthetizing shrimp with anesthetics first needs to determine the optimal anesthetic and its optimal anesthetic dose and treatment time, so the exploration of the optimal anesthetic dose and time is first determined.

[0024] 1.1 Preparation of anesthetic container The experiment was carried out in the Wenchang Breeding Base of Xingbang Marine Biotechnology Co., Ltd. One 250L water bucket was taken as an anesthetic container, which was cleaned and disinfected, and then 200L of seawater with the same salinity as the parent shrimp culture pond was added, with a salinity of 30‰ and a water temperature of 28 degrees Celsius.

[0025] 1.2 Different anesthetic dose and time treatment groups The anesthetic selected was the commonly used water product anesthetic eugenol (Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China). In order to determine the optimal anesthetic dose and anesthetic time, three doses of 50mg / L, 100mg / L and 200mg / L were set, each dose was treated for 5 minutes and 10 minutes, a total of 6 treatment groups, and the survival rate of shrimp and anesthetic recovery time in different treatment groups were compared to determine the optimal dose and time for breeding phenotype determination (see Table 1); specifically: 1) Anesthetic preparation: 10ml, 20ml and 40ml of 99% liquid eugenol were taken respectively, and 90ml, 180ml and 360ml of 95% concentrated anhydrous ethanol were added in turn for dissolution, and then added to the anesthetic container in 1.1 above to prepare anesthetic water with a final concentration of 50mg / L, 100mg / L and 200mg / L, and three buckets were set for each anesthetic concentration.

[0026] 2) 20 individuals for determination were put into the 50mg / L, 100mg / L and 200mg / L anesthetic concentration buckets, with a body weight of about 40g, and the timing started from the shrimp being put into the bucket.

[0027] 3) During the anesthetic process, the state of the shrimp was observed, and when the shrimp lost the ability to balance and turned on its side, was inactive and lost the response to stimulation, it was indicated that the Penaeus vannamei shrimp had been anesthetized. If the shrimp began to swim and had obvious avoidance behavior to the net, it was considered to have recovered to normal.

[0028] 4) After 5 minutes of treatment with different concentrations of eugenol, observe the anesthesia state, and take out 10 individuals from each bucket and place them in normal seawater for recovery. Observe the recovery time and survival rate under different concentrations of treatment.

[0029] 5) After 10 minutes of treatment, observe the anesthesia of the remaining 10 shrimps in each bucket, then take them out and observe the recovery time and mortality rate.

[0030] 1.3 Comparison of effects of different concentrations and anesthesia times The recovery time and survival rate of the shrimps treated above were statistically analyzed, and the survival rate was observed in the culture pond for 3 days. The results are shown in Table 1. At a concentration of 50 mg / L, the shrimps can recover in 5 minutes after 5 minutes of anesthesia, and the time window for phenotype determination is short. The recovery time is 10 minutes at a concentration of 100 mg / L for 5 minutes, and the stimulation of 200 mg / L concentration on the shrimps is too large, which can cause the death of the shrimps. Prolonging the anesthesia time will also increase the recovery time. In the group treated with 50 mg / L concentration for 10 minutes, the recovery time of the shrimps is 20 minutes, and there is no death of the shrimps, which is a more appropriate anesthesia concentration.

[0031] Table 1 Anesthesia time and recovery of eugenol anesthesia at different concentrations

[0032] Example 2: A low-loss Litopenaeus vannamei growth phenotype determination method and its breeding application The experiment was conducted at Xinfang Marine Biological Technology Co., Ltd. in Wenchang Breeding Base in December 2023. The growth phenotype of mixed breeding family materials was determined, and samples were taken for subsequent genotyping.

[0033] 2.1 Preparation of anesthesia container Two 250L water buckets were used as anesthesia containers, which were cleaned and disinfected, then 200L of seawater with the same salinity as the parent shrimp culture pond was added, with a salinity of 30‰ and a water temperature of 28°C.

[0034] The anesthetic was eugenol (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China), with a concentration of 50 mg / L. Specifically, 10 ml of 99% pure eugenol was dissolved with 90 ml of 95% alcohol (China Pharmaceutical Group) and then added to the anesthesia container to prepare a 50 mg / L anesthetic.

[0035] 2.2 Shrimp anesthesia The experiment is started in the laboratory. 20 prawns are taken out of the breeding pond at a time for anesthesia. After 10 minutes of anesthesia, they are taken out and placed in a normal seawater barrel to wait for phenotype determination. These anesthetized prawns are also placed in another anesthetizing barrel for 20 prawns to be anesthetized at the same time. After 10 minutes of anesthesia, they are taken out and placed in a normal seawater barrel to wait for phenotype determination. The prawn population for determination is anesthetized in turn, and determination is carried out in turn to ensure that the phenotype determination process is not interrupted and can be carried out continuously.

[0036] 2.3 Prawn growth phenotype determination The prawns are placed on the prawn phenotype high-throughput determination device to determine the weight, length, carapace, and length, width, and height of each abdominal segment. Since the prawns are in an anesthetized state, the prawns will not jump when they are taken out and placed on the determination device. The entire process from taking out the prawns to completing the measurement can be completed within 10 seconds, greatly improving the speed of phenotype determination.

[0037] Since the individual needs to be genotyped for whole-genome selection breeding research, the prawns for determination must be taken out for subsequent DNA extraction and bound with eye handle rings for individual differentiation. After the above operations are completed, they are placed back into the original breeding pond, and the survival rate is observed. A total of 1664 individual phenotype data are determined by this method. No prawns died during the determination process, and the determination was completed 20 minutes after being taken out after anesthesia. After completion, the prawn samples in the breeding pond recovered and woke up. After being placed in the breeding pond for 3 days, only 2 individuals died due to molting, and the overall survival rate was 99.9%.

[0038] 2.4 Prawn growth phenotype data statistics The determined prawn phenotype data are shown in Table 1. Figure 1 The average weight is 18.10±4.17, and the average length is 122.23±10.16. The above phenotype data are combined with the genotyping data to calculate the GEBV of each individual's weight and length traits, and the best individuals are selected for breeding.

[0039] Finally, it should be noted that the above description is only a preferred embodiment of the present application and does not limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some technical features. Any modification, equivalent substitution, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A low-loss method for determining the growth phenotype of Litopenaeus vannamei, characterized in that: The shrimp samples to be tested were anesthetized to induce anesthesia. The shrimp were then transferred to a normal seawater environment, and their growth phenotype was measured during the window period. Afterward, the shrimp could recover to normal.

2. The low-loss method for determining the growth phenotype of Litopenaeus vannamei according to claim 1, characterized in that: The anesthesia treatment involved immersing the shrimp sample to be tested in seawater containing 50 mg / L eugenol for 10 minutes. The shrimp lost their balance, rolled over, became inactive, and lost their response to stimuli.

3. The low-loss method for determining the growth phenotype of Litopenaeus vannamei according to claim 2, characterized in that: The anesthetic agent is eugenol with a purity of 99%. The anesthetic agent is mixed and dissolved with 95% alcohol at a volume ratio of 1:9, and then added to seawater to achieve a final concentration of 50 mg / L.

4. The low-loss method for determining the growth phenotype of Litopenaeus vannamei according to any one of claims 1-3, characterized in that: After entering anesthesia, the sample is transferred to a normal seawater environment, and growth phenotype is measured using an automated measuring device within a window period of approximately 20 minutes. After the measurement, the sample returns to a normal swimming state.

5. An application of the method according to claim 1, characterized in that: The method described above determines the application of high-quality shrimp samples in shrimp breeding.

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