A method for breeding a new line of strong resistance to disease of rice field eel

Abstract

The application discloses a new strain of ricefield eel with strong disease resistance, and relates to the field of ricefield eel breeding. The method comprises the following steps: step one, selection of breeding population; step two, screening of breeding parents; step three, establishment of a basic breeding population; step four, screening of F0 generation of the basic breeding population; step five, establishment and screening of F1 generation; step six, counter-selection of F0 generation; step seven, establishment and screening of F2 generation; step eight, counter-selection of F1 generation; step nine, establishment and screening of F3 generation; and step ten, population verification. The method can not only enhance the immunity and disease resistance of ricefield eel fry and improve the survival rate, but also formulates a technical regulation for ricefield eel fry cultivation, thereby guaranteeing the healthy development of the ricefield eel breeding industry.

Description

Technical Field

[0001] This invention belongs to the field of aquatic breeding technology, specifically relating to a method for breeding a new strain of yellow eel with strong disease resistance. Background Technology

[0002] Yellow eel ( Monopterus albus ), Synbranchiformes, Synbranchidae, genus *Synbranchiformes* Monopterus The yellow eel (Eel spp.) is widely distributed in rice paddies, swamps, and mudflats in East Asia, South Asia, and Southeast Asia. As an important specialty freshwater economic fish, it has seen its aquaculture area expand rapidly in China in recent years due to its delicious flesh, high nutritional value, distinctive flavor, and stable market price, indicating huge market potential. According to the 2024 China Fisheries Statistical Yearbook, domestic yellow eel production has exceeded 355,000 tons, and current supply cannot meet demand, resulting in high prices. However, with the rise of artificial breeding technology, wild eel habitats are decreasing, and their wild germplasm resources are being damaged to varying degrees, making the selection and breeding of superior varieties particularly important.

[0003] Currently, the domestic eel market is dominated by wild-caught eels. Although there have been some technological breakthroughs in artificial breeding in recent years, their share remains relatively small. Since the beginning of eel farming, the most prominent problem has been the high mortality rate caused by low immunity and poor disease resistance in the seedlings. Solving this industry problem through breed selection is urgently needed. Summary of the Invention

[0004] This invention provides a method for breeding a new strain of swamp eel with strong disease resistance, specifically including the following steps:

[0005] Step 1: Selection of breeding populations: RAD-seq sequencing analysis was performed on 137 wild eel samples of different sexes and growth stages collected from 19 populations across the country. Based on the genetic evolutionary relationships of eels across the country, populations from Chongming Island (CM), Weishan Lake (WSH), Huoqiu County (HQ), Jiaoling County (JL), and Lingshui County (LS) were selected from the 19 wild eel populations, with the Chongming Island population serving as the local control population.

[0006] Among them, 19 groups of wild yellow eels are: Baiyangdian (BYD), Chengdu (CD), Chaohu (CH), Chongming Island (CM), Panjin Dagushan (DGS), Dongting Lake (DTH), Ouli Township (EL), Gaoyou Lake (GYH), Haimen City (HM), Huoqiu County (HQ), Hongze Lake (HZH), Jiaoling County (JL), Lingshui County (LS), Pinghu City (PH), Puyang City (PY), Poyang Lake (PYH), Taihu Lake (TH), Weishan Lake (WSH), and Dali, Yunnan (YN);

[0007] Step 2: Selection of breeding parents: The yellow eels from Chongming Island, Weishan Lake, Huoqiu County, Jiaoling County and Lingshui County were systematically screened four times, and those yellow eels that did not meet the standards were eliminated;

[0008] Step 3: Establishment of the breeding base population: Female eels (15.7~18.3 g) and male eels (38.1~42.9 g) of similar size were selected from the above five populations. Liver and serum samples were collected in the laboratory for antioxidant and immunoenzyme activity testing. At the same time, Aeromonas hydrophila was artificially injected into the eels of the five populations. The eels of the five populations were then placed in 90 L blue polyethylene boxes for further culture and hemorrhage was observed. Based on the experimental results, the population with the strongest disease resistance from Huoqiu County (HQ) was selected as the breeding base population.

[0009] Step 4: Selection of parent F0 generation for the basic breeding population: Healthy, disease-free, and well-developed female eels (47.2 ± 1.7 g) and male eels (75.8 ± 2.1 g) with well-developed gonads were randomly selected from the population in Huoqiu County (HQ) as parent F0 generation;

[0010] Step 5: Establishment and selection of F1 generation: Using the precise pairing technology of swamp eels, the F0 generation is mated in a 1:1 ratio to obtain the F1 generation. The top 200 female swamp eels of the F1 generation with the fastest growth rate are selected as the parents of the F2 generation.

[0011] Step Six: Reverse Selection of Generation F0: Due to the special physiological phenomenon of sex reversal in swamp eels, the parent female swamp eels of Generation F0 will undergo sex reversal and become male swamp eels after completing reproduction. Therefore, the male swamp eels used as parent male swamp eels of Generation F2 will be backcrossed with the selected female swamp eels of Generation F1.

[0012] Step 7: Establishment and selection of the F2 generation: Using the precise pairing technology of swamp eels, female swamp eels of the F1 generation and male swamp eels of the F0 generation that have completed sex reversal are paired and mated in a 1:1 ratio to obtain the F2 generation. The top 200 female swamp eels of the F2 generation with the fastest growth rate are selected as the parents of the F3 generation.

[0013] Step 8: Reverse selection of F1 generation: Male eels that have undergone reproductive sex reversal in the F1 generation are used as parents for the F3 generation, and they are backcrossed with female eels from the selected F2 generation.

[0014] Step Nine: Establishment and selection of the F3 generation: Using precise eel pairing technology, female eels of the F2 generation and male eels of the F1 generation that have completed sex reversal are paired and mated in a 1:1 ratio to obtain the F3 generation;

[0015] Step 10: Cultivation and Propagation: Large-scale cultivation and propagation of the F3 generation seedlings.

[0016] The disease resistance mentioned above refers to the activities of superoxide dismutase (SOD), acid phosphatase (ACP), malondialdehyde (MDA), and phenol oxidase (PO); and the bleeding situation after challenge with Aeromonas hydrophila.

[0017] Furthermore, the four screening methods in step two are as follows: appearance screening, behavior screening one, behavior screening two, and food screening.

[0018] Furthermore, the four-stage screening method is specifically implemented as follows:

[0019] First screening (appearance screening): Separate the eels by size, check for diseases (discard diseased eels), and put the remaining eels into plastic boxes or screening pools after separating them by size.

[0020] Second screening (behavioral screening one): Observe in the plastic box or screening pool from 12 noon to 2 pm. Eels that climb up the fish tank, raise their heads, and move around alone are of poor quality and are removed and eliminated.

[0021] The third screening (behavioral screening two): Around 6 pm that day, disinfected eels were placed into the eel pond near the feeding platform. The next day at noon, those that came out to move alone, climbed onto the grass, or raised their heads were picked out and eliminated.

[0022] Fourth screening (food screening): If the eels eat more than 1% of their own body weight (calculated based on their body weight) for at least one day in five consecutive days, the batch of eels will be kept; if the eels eat less than 1% of their own body weight for five consecutive days, the batch of eels will be eliminated.

[0023] This invention provides a method for breeding a new strain of swamp eel with strong disease resistance, which has the following beneficial effects:

[0024] 1. The new strain of yellow eel bred in this program can effectively enhance the immunity and disease resistance of its seedlings, and improve the survival rate compared with the parent strain;

[0025] 2. The breeding methods provided in this plan have established technical procedures for the cultivation of eel fry, ensuring the healthy development of the eel farming industry.

[0026] To address the shortcomings of existing technologies, this invention provides a method for breeding a new strain of swamp eel with strong disease resistance, which solves the problem of high mortality caused by low immunity and poor disease resistance in swamp eel seedlings, thus ensuring the healthy development of the swamp eel farming industry. Attached Figure Description

[0027] Figure 1 A flowchart illustrating the breeding method for a new disease-resistant loach strain provided by this invention.

[0028] Figure 2The serum (A, male eel; B, female eel) and liver (C, male eel; D, female eel) SOD activities of five selected breeding populations of swamp eels were measured.

[0029] Figure 3 The serum (A, male eel; B, female eel) and liver (C, male eel; D, female eel) ACP activities of five selected breeding populations of swamp eels were measured.

[0030] Figure 4 The serum (A, male eel; B, female eel) and liver (C, male eel; D, female eel) MDA activities of five selected breeding populations of swamp eels were measured.

[0031] Figure 5 The serum (A, male eel; B, female eel) and liver (C, male eel; D, female eel) PO activities of five selected breeding populations of swamp eels were measured.

[0032] Figure 6 Bleeding after injecting Aeromonas hydrophila into five selected breeding groups of eels. Detailed Implementation

[0033] The following examples are merely illustrative of the invention and should not be construed as limiting it. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. Experimental methods not specifically described in the examples, and reagents and materials not specified in the examples, are performed according to conventional conditions in the art, and all reagents used are commercially available.

[0034] Example 1: Breeding method for a new strain of disease-resistant swamp eel, specifically including the following steps:

[0035] Step 1: Selection of Breeding Population: RAD-seq sequencing analysis was performed on 137 wild swamp eel samples of different sexes and growth stages collected from 19 populations across China (sequencing was conducted by Shanghai Sangon Biotech Co., Ltd.). Based on the genetic evolutionary relationships of swamp eels nationwide, populations from Chongming Island (CM), Weishan Lake (WSH), Huoqiu County (HQ), Jiaoling County (JL), and Lingshui County (LS) were selected from the 19 wild swamp eel populations for subsequent studies, with the Chongming Island population serving as the local control population. (The method for selecting the breeding population is referenced in: Asian Swamp eel Monopterus albus Population Structure and Genetic Diversity in China, Weiwei Lv et al., Frontiers in Genetics.)

[0036] Among them, 19 groups of wild yellow eels are: Baiyangdian (BYD), Chengdu (CD), Chaohu (CH), Chongming Island (CM), Panjin Dagushan (DGS), Dongting Lake (DTH), Ouli Township (EL), Gaoyou Lake (GYH), Haimen City (HM), Huoqiu County (HQ), Hongze Lake (HZH), Jiaoling County (JL), Lingshui County (LS), Pinghu City (PH), Puyang City (PY), Poyang Lake (PYH), Taihu Lake (TH), Weishan Lake (WSH), and Dali, Yunnan (YN);

[0037] Step 2: Selection of breeding parents: The yellow eels from Chongming Island, Weishan Lake, Huoqiu County, Jiaoling County and Lingshui County were systematically screened four times, and those yellow eels that did not meet the standards were eliminated;

[0038] The four screening methods are as follows: appearance screening, behavior screening one, behavior screening two, and food screening.

[0039] First screening (appearance screening): Separate the eels by size, check for diseases (discard diseased eels), and put them into plastic boxes or screening pools after separating by size;

[0040] Second screening (behavioral screening one): Observe in the plastic box or screening pool from 12 noon to 2 pm. If the eels climb up the fish tank, raise their heads, and move around alone, they are fish of poor quality and are removed and eliminated.

[0041] The third screening (behavioral screening two): Around 6 pm that day, disinfected eels were placed into the eel pond near the feeding platform. The next day at noon, those that came out to move alone, climbed onto the grass, or raised their heads were picked out and eliminated.

[0042] Fourth screening (food screening): Eels that eat more than 1% of their body weight for at least one day out of five consecutive days will be kept; eels that eat less than 1% of their body weight for five consecutive days will be eliminated.

[0043] Step 3: Establishment of the breeding base population: From the five populations mentioned above, 10 female eels (15.7 ~ 18.3 g) and 10 male eels (38.1 ~ 42.9 g) of similar size were selected. Liver and serum samples were collected in the laboratory for antioxidant and immunoenzyme activity testing. Simultaneously, from each of the five populations, 50 eels (25 females and 25 males) were artificially injected with Aeromonas hydrophila. The eels from each of the five populations were then placed in 90 L blue polyethylene tanks for further rearing, and bleeding was observed. Based on the experimental results, the population from Huoqiu County (HQ) was ultimately selected as the breeding base population. The specific experimental steps and results are as follows:

[0044] (1) Detection of superoxide dismutase (SOD) activity in five breeding populations.

[0045] Ten female (15.7–18.3 g) and ten male (38.1–42.9 g) eels of similar size were randomly selected from five breeding populations: Chongming Island (CM), Weishan Lake (WSH), Huoqiu County (HQ), Jiaoling County (JL), and Lingshui County (LS). Liver tissue and serum were obtained from dissection. The tissues were thoroughly ground and diluted with physiological saline to obtain a 10% tissue homogenate. SOD activity assay kits (catalog number: A001-3, brand: Nanjing Jiancheng) were used to detect the activity of the liver and serum.

[0046] Experimental results are as follows Figure 2 As shown, serum SOD activity in male eels from the WSH and HQ populations was significantly higher than that in other populations (P<0.05). Serum SOD activity in female eels from the WSH population was significantly higher than that in the CM and LS populations (P<0.05), while SOD activity in female eels from the HQ and JL populations was not significantly different from that in the other three populations (P>0.05). Liver SOD activity in male eels from the HQ population was significantly higher than that in the CM and LS populations (P<0.05), while liver SOD activity in male eels from the WSH and JL populations was not significantly different from that in the other three populations (P>0.05). Liver SOD activity in female eels from the HQ population was significantly higher than that in the other four populations (P<0.05). Serum SOD activity in male and female eels from the WSH population was 65.9% and 114.3% higher than that in the local population (CM), respectively; serum SOD activity in male and female eels from the HQ population was 85.9% and 64.8% higher than that in the local population (CM), respectively; liver SOD activity in male and female eels from the WSH population was 71.6% and 21.6% higher than that in the local population (CM), respectively; liver SOD activity in male and female eels from the HQ population was 32.8% and 104.8% higher than that in the local population (CM), respectively.

[0047] (2) Detection of acid phosphatase (ACP) activity in five selected breeding populations.

[0048] Ten female (15.7–18.3 g) and ten male (38.1–42.9 g) eels of similar size were randomly selected from five breeding populations: Chongming Island (CM), Weishan Lake (WSH), Huoqiu County (HQ), Jiaoling County (JL), and Lingshui County (LS). Liver tissue and serum were obtained from dissection. The tissues were thoroughly ground and diluted with physiological saline to obtain a 10% tissue homogenate. The liver and serum samples were tested using an ACP enzyme activity assay kit (catalog number: A060-2, brand: Nanjing Jiancheng).

[0049] Experimental results are as follows Figure 3As shown, serum ACP activity in male and female eels from the WSH and HQ groups was significantly higher than that in the other three groups, while serum ACP activity in female eels from the CM group was significantly higher than that in the JL and LS groups (P<0.05). Liver ACP activity in both male and female eels from the HQ group was significantly higher than that in the other four groups (P<0.05), while liver ACP activity in female eels from the CM and WSH groups was significantly higher than that in the JL and LS groups (P<0.05). Serum ACP activity in male and female eels from the HQ group was 63.1% and 98.3% higher than that in the local CM group, respectively; serum ACP activity in male and female eels from the WSH group was 93.0% and 190.7% higher than that in the local CM group, respectively; liver ACP activity in male and female eels from the HQ group was 168.4% and 76.7% higher than that in the local CM group, respectively.

[0050] (3) Detection of malondialdehyde (MDA) activity in five breeding populations.

[0051] Ten female (15.7–18.3 g) and ten male (38.1–42.9 g) eels of similar size were randomly selected from five breeding populations: Chongming Island (CM), Weishan Lake (WSH), Huoqiu County (HQ), Jiaoling County (JL), and Lingshui County (LS). Liver tissue and serum were obtained from dissection. The tissues were thoroughly ground and diluted with physiological saline to obtain a 10% tissue homogenate. Liver and serum samples were tested using an MDA activity assay kit (catalog number: A003-1, brand: Nanjing Jiancheng).

[0052] Experimental results are as follows Figure 4 As shown, serum MDA activity in male and female eels from the HQ group was significantly higher than that in the WSH, JL, and LS groups (P<0.05), while serum MDA activity in male eels from the CM group was not significantly different from that in the other four groups (P>0.05). Liver MDA activity in male and female eels from the WSH and HQ groups was significantly higher than that in the CM, JL, and LS groups (P<0.05). Serum MDA activity in male and female eels from the HQ group was 50.0% and 97.5% higher than that in the local CM group, respectively; liver MDA activity in male and female eels from the WSH group was 93.1% and 98.6% higher than that in the local CM group, respectively; and liver MDA activity in male and female eels from the HQ group was 159.2% and 76.6% higher than that in the local CM group, respectively.

[0053] (4) Detection of phenol oxidase (PO) activity in five selected breeding populations.

[0054] Ten female (15.7–18.3 g) and ten male (38.1–42.9 g) eels of similar size were randomly selected from five breeding populations: Chongming Island (CM), Weishan Lake (WSH), Huoqiu County (HQ), Jiaoling County (JL), and Lingshui County (LS). Liver tissue and serum were obtained from dissection. The tissues were thoroughly homogenized and diluted with physiological saline to obtain a 10% tissue homogenate. The liver and serum were tested using a PO activity assay kit (catalog number: H247-1, brand: Nanjing Jiancheng).

[0055] Experimental results are as follows Figure 5 As shown, there were no significant differences in serum and hepatic PO activities among female and male eels in the five groups (P>0.05). Regarding serum PO activity, the serum PO activity of male eels in the WSH group and female eels in the LS group was slightly higher than that of the other groups. Regarding hepatic PO activity, the hepatic PO activity of both female and male eels in the JL group was significantly higher than that of the other groups. The serum PO activity of female eels in the WSH and HQ groups was 60.0% higher than that of the local population (CM); the hepatic PO activity of male eels in the WSH and HQ groups was 33.2% and 42.4% higher than that of the local population (CM), respectively.

[0056] (5) Detection of hemorrhage in five selected breeding groups challenged with Aeromonas hydrophila.

[0057] From five breeding populations—Chongming Island (CM), Weishan Lake (WSH), Huoqiu County (HQ), Jiaoling County (JL), and Lingshui County (LS)—25 female and 25 male eels of similar size were randomly selected from each population and artificially injected with Aeromonas hydrophila. Aeromonas hydrophila (Purchased from Beina Biotechnology). Before injection, the strain was cultured on LB medium at 28°C and 150 rpm for 24 h. Afterward, it was centrifuged at 6000 rpm for 5 min using a low-temperature centrifuge, then washed with PBS and centrifuged again. This process was repeated twice. After plate counting, the bacterial suspension concentration was adjusted to 1 × 10⁻⁶ using PBS. 6 After injecting each eel with 200 μL of Aeromonas hydrophila suspension (cfu / mL), the eels were placed in a 90 L blue polyethylene box for continued rearing, and bleeding was observed.

[0058] Experimental results are as follows Figure 6 As shown, in the CM, LS, and JL populations, all eels hemorrhaged and eventually died 3, 4, and 5 days after Aeromonas hydrophila challenge, respectively. In the HQ population, only 18 eels hemorrhaged and died 5 days after Aeromonas hydrophila challenge, and 15 eels survived 7 days later. In contrast, the WSH and HQ populations had 41 and 35 eels hemorrhaging 7 days after Aeromonas hydrophila challenge, respectively.

[0059] Five experiments showed that the antioxidant and immunomodulatory enzyme activities of both female and male eels from the Huoqiu County (HQ) population were generally higher. Furthermore, the activities of most enzymes in the Huoqiu County (HQ) population were 5% higher than those in the local Chongming Island (CM) population. Meanwhile, after injection of Aeromonas hydrophila, all eels from the Chongming Island (CM), Jiaoling County (JL), and Lingshui County (LS) populations showed hemorrhage within 5 days, while some eels from the Weishan Lake (WSH) and Huoqiu County (HQ) populations did not show hemorrhage even 7 days after infection. Therefore, it can be preliminarily concluded that the eels from the Weishan Lake (WSH) and Huoqiu County (HQ) populations have stronger resistance to hemorrhage disease, and the Huoqiu County (HQ) population, exhibiting the best performance, should be selected as the base population for breeding.

[0060] Experimental data are presented as mean ± standard error (mean ± SE). Data were calculated using Microsoft Excel and analyzed using SPSS 22.0 software. Significant differences between groups were assessed using one-way ANOVA and Duncan's multiple comparisons; a p-value <0.05 was considered statistically significant.

[0061] Step 4: Selection of the F0 generation of parental eels for the basic breeding population: 150 healthy, disease-free, and well-developed female eels (47.2 ± 1.7 g) and 150 male eels (75.8 ± 2.1 g) were randomly selected from the population in Huoqiu County (HQ) as the F0 generation of parental eels;

[0062] Step 5: Establishment and selection of F1 generation: Using the precise pairing technology of swamp eels, the F0 generation is mated in a 1:1 ratio to obtain the F1 generation. The top 200 female swamp eels of the F1 generation with the fastest growth rate are selected as the parents of the F2 generation.

[0063] Step Six: Reverse Selection of Generation F0: Due to the special physiological phenomenon of sex reversal in swamp eels, the parent female swamp eels of Generation F0 will undergo sex reversal and become male swamp eels after completing reproduction. Therefore, the male swamp eels used as parent male swamp eels of Generation F2 will be backcrossed with the selected female swamp eels of Generation F1.

[0064] Step 7: Establishment and selection of the F2 generation: Using the precise pairing technology of swamp eels, female swamp eels of the F1 generation and male swamp eels of the F0 generation that have completed sex reversal are paired and mated in a 1:1 ratio to obtain the F2 generation. The top 200 female swamp eels of the F2 generation with the fastest growth rate are selected as the parents of the F3 generation.

[0065] Step 8: Reverse selection of F1 generation: Male eels that have undergone reproductive sex reversal in the F1 generation are used as parents for the F3 generation, and they are backcrossed with female eels from the selected F2 generation.

[0066] Step Nine: Establishment and Screening of Generation F3: Using precise eel pairing technology, female eels from Generation F2 and male eels from Generation F1 that have completed sex reversal are paired and mated in a 1:1 ratio to obtain Generation F3, which is a new eel strain with strong disease resistance.

[0067] Step 10: Cultivation and Propagation: Large-scale cultivation and propagation of the F3 generation seedlings.

[0068] Example 2: Verification of disease resistance of the new eel strain bred in Example 1.

[0069] The experimental method is the same as step three in Example 1, and the experimental results are shown in Table 1 below:

[0070] Table 1. Results of activity detection of several enzymes in serum and liver of new strain of yellow eel.

[0071]

[0072] Seven days after challenge with Aeromonas hydrophila, the number of hemorrhagic eels was 24, representing a 31.4% reduction in morbidity compared to the parent strain.

[0073] Three other groups of newly bred and propagated eel strains were randomly selected, and the activities of SOD, ACP, MDA, and PO were tested. The results showed that the differences from the data in Table 1 above were within 10%. This indicates that the new eel strains selected and harvested in Example 1 have stable disease resistance after large-scale cultivation and propagation.

Claims

1. A method for breeding a new strain of yellow eel with strong disease resistance, characterized in that... Includes the following steps: Step 1: Selection of breeding population: RAD-seq sequencing analysis was performed on 137 wild eel samples of different sexes and growth stages collected from 19 populations across the country; based on the genetic evolutionary relationship of eels across the country, the Chongming Island CM, Weishan Lake WSH, Huoqiu County HQ, Jiaoling County JL and Lingshui County LS populations were selected from the 19 wild eel populations, with the Chongming population serving as the local control population; Among them, 19 groups of wild yellow eels are: Baiyangdian (BYD), Chengdu (CD), Chaohu (CH), Chongming Island (CM), Panjin Dagushan (DGS), Dongting Lake (DTH), Ouli Township (EL), Gaoyou Lake (GYH), Haimen City (HM), Huoqiu County (HQ), Hongze Lake (HZH), Jiaoling County (JL), Lingshui County (LS), Pinghu City (PH), Puyang City (PY), Poyang Lake (PYH), Taihu Lake (TH), Weishan Lake (WSH), and Dali, Yunnan (YN); Step 2: Selection of breeding parents: The yellow eels from Chongming Island, Weishan Lake, Huoqiu County, Jiaoling County and Lingshui County were systematically screened four times, and those yellow eels that did not meet the standards were eliminated; Step 3: Establishment of the breeding base population: From the above five populations, female eels of similar size (15.7-18.3 g) and male eels (38.1-42.9 g) were selected. Liver and serum samples were collected in the laboratory for antioxidant and immunoenzyme activity testing. At the same time, Aeromonas hydrophila was artificially injected into the eels from the five populations. The eels were then placed in blue polyethylene boxes for further rearing, and bleeding was observed. Based on the experimental results, the HQ population from Huoqiu County, which had the strongest disease resistance, was selected as the breeding base population. Step 4: Selection of parent F0 generation for the basic breeding population: 47.2 ± 1.7 g of healthy, disease-free, and well-developed female eels and 75.8 ± 2.1 g of male eels were randomly selected from the HQ population in Huoqiu County as parent F0 generation; Step 5: Establishment and selection of F1 generation: Using the precise pairing technology of swamp eels, the F0 generation is mated in a 1:1 ratio to obtain the F1 generation. The top 200 female swamp eels of the F1 generation with the fastest growth rate are selected as the parents of the F2 generation. Step Six: Reverse Selection of Generation F0: Due to the special physiological phenomenon of sex reversal in swamp eels, the parent female swamp eels of Generation F0 will undergo sex reversal and become male swamp eels after completing reproduction. Therefore, the male swamp eels used as parent male swamp eels of Generation F2 will be backcrossed with the selected female swamp eels of Generation F1. Step 7: Establishment and selection of the F2 generation: Using the precise pairing technology of swamp eels, female swamp eels of the F1 generation and male swamp eels of the F0 generation that have completed sex reversal are paired and mated in a 1:1 ratio to obtain the F2 generation. The top 200 female swamp eels of the F2 generation with the fastest growth rate are selected as the parents of the F3 generation. Step 8: Reverse selection of F1 generation: Male eels that have undergone reproductive sex reversal in the F1 generation are used as parents for the F3 generation, and they are backcrossed with female eels from the selected F2 generation. Step Nine: Establishment and selection of the F3 generation: Using precise eel pairing technology, female eels of the F2 generation and male eels of the F1 generation that have completed sex reversal are paired and mated in a 1:1 ratio to obtain the F3 generation; Step 10: Cultivation and Propagation: Large-scale cultivation and propagation of the F3 generation seedlings; The four screening methods in step two are as follows: appearance screening, behavior screening one, behavior screening two, and food screening. Physical screening: Separate the eels by size, check for diseases, discard diseased eels, and separate the remaining eels by size and put them into plastic boxes or screening tanks; Behavioral screening method 1: Observe in plastic boxes or screening pools from 12 noon to 2 pm. Eels that climb up the fish tank, raise their heads, and move around alone are of poor quality and should be removed and eliminated. Behavioral screening method 2: Around 6 pm on the same day, disinfected eels are placed into the eel pond near the feeding platform. The next day at noon, those that come out to move alone, climb onto the grass, or raise their heads are picked out and eliminated. Food selection: If the eels eat more than 1% of their body weight on at least one day out of five consecutive days, that batch of eels will be kept. Those that eat less than 1% of their own body weight for five consecutive days will be eliminated.

Images