Comprehensive evaluation method for saline-alkali tolerance of carassius auratus gibelio

Abstract

The invention discloses a comprehensive evaluation method for saline-alkali tolerance of carassius auratus gibelio, which comprises the following steps: performing acute saline-alkali stress on the carassius auratus gibelio and carassius auratus in an embryonic stage, a larva stage and a juvenile stage, performing saline-alkali domestication on a fingerling stage at the same time, increasing salinity and alkalinity once every 10 days in the saline-alkali domestication stage, keeping the up-regulation concentration at a stable maximum value according to fingerling physiological indexes, and obtaining the saline-alkali tolerance of the carassius auratus gibelio. Counting the evaluation value of the saline-alkali tolerance of the carassius auratus gibelio in each stage, determining the weight coefficient of the evaluation value in each stage, then calculating the comprehensive evaluation value of the saline-alkali tolerance in each stage, and comprehensively evaluating the saline-alkali tolerance of the carassius auratus gibelio according to the total value after addition. According to the established saline-alkaline tolerance evaluation system for the carassius auratus gibelio, multiple stages of saline-alkaline tolerance index values are selected for comprehensive weight analysis, the evaluation method is superior to a traditional single-index evaluation method, the tolerance level of the carassius auratus gibelio to saline-alkaline stress can be comprehensively reflected, and basic data are provided for comprehensive evaluation of new germplasm characteristics of the carassius auratus gibelio.

Description

Technical Field

[0001] This invention relates to the field of fish salt tolerance testing technology, specifically to a comprehensive evaluation method for salt tolerance of crucian carp without intermuscular spines. Background Technology

[0002] China has 99 million hectares of saline-alkali land, of which 46 million hectares are low-lying saline-alkali waters, accounting for approximately 55% of the country's total lake area. Saline-alkali waters are characterized by high carbonate alkalinity, high pH, ​​and complex ionic composition, resulting in poor biological resources and low fishery productivity. However, many medium- and high-alkalinity saline-alkali lakes worldwide still have fish species that have long inhabited and formed dominant populations. Experiments have shown that "using fish to control alkalinity" is an effective way to develop and utilize saline-alkali waters, achieving a win-win situation for both economic and ecological benefits. Fish species that can live in high-salinity, high-pH waters are undoubtedly key to "using fish to control alkalinity." Therefore, selecting or breeding improved species suitable for medium- and high-alkalinity saline-alkali waters for aquaculture can rationally and sustainably utilize saline-alkali waters and develop saline-alkali water fisheries.

[0003] Currently, salt and alkali tolerance assessment in fish often involves static acute toxicity tests at a specific stage, such as the embryonic, larval, or juvenile stage, using the salt and alkali tolerance performance of a single factor at a specific stage as an evaluation index for the fish species' salt and alkali tolerance. Studies have shown that salt and alkali tolerance varies across different growth and developmental stages of fish. The LC50 of salinity and alkalinity at 96 h for juvenile silver carp (3.0 cm in length) is also discussed. 50 The concentrations were 8.6 g / L and 64.2 mmol / L, respectively, while the LC50 values ​​for salinity and alkalinity of silver carp (body length 8.0 cm) over 96 h were 8.6 g / L and 64.2 mmol / L, respectively. 50 The alkalinity tolerance levels were 13.8 g / L and 70.4 mmol / L, respectively. The suitable salinity for the survival of large-scaled barbel larvae, fry, and fingerlings ranged from 5.0 g / L to 10.0 g / L, with varying alkalinity tolerance at each stage, exhibiting a similar trend. Using a single method to assess salt and alkali tolerance at only one stage of fish growth may lead to inaccurate results. Therefore, how to comprehensively assess salt and alkali tolerance at multiple growth stages of fish is a problem that urgently needs to be solved by those skilled in the art.

[0004] The spiny crucian carp without intermuscular spines is a new germplasm created by the Cyprinidae Genomics Innovation Team of the Heilongjiang Fisheries Research Institute using gene editing technology. To improve the evaluation data of this spiny crucian carp and advance its industrialization, its salt tolerance performance needs to be comprehensively assessed. Summary of the Invention

[0005] This invention provides a comprehensive evaluation method for the salt and alkali tolerance of crucian carp without intermuscular spines. The method assesses the salt and alkali tolerance of crucian carp without intermuscular spines and crucian carp during the embryonic, larval, and juvenile stages, as well as during salt and alkali acclimatization. The obtained data are then standardized, weighted, and comprehensively evaluated to accurately determine the tolerance level of crucian carp without intermuscular spines to salt and alkali stress.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A comprehensive evaluation method for salt and alkali tolerance of crucian carp without intermuscular spines includes identifying the salt and alkali tolerance of crucian carp without intermuscular spines, measuring salt and alkali tolerance at different stages such as embryonic period, larval period, juvenile period and salt and alkali acclimatization period, standardizing and weighting the obtained data, calculating the comprehensive evaluation value of each stage and adding them together, and evaluating the comprehensive salt and alkali tolerance of crucian carp without intermuscular spines based on the total value after addition.

[0008] The overall salt tolerance capacity is obtained from the overall evaluation value of different stages: embryonic period, larval period, juvenile period, and salt acclimatization period.

[0009] During the salinity and alkalinity acclimatization period, the salinity and alkalinity were adjusted upwards every 10 days, with the adjustment concentration based on the highest value that kept the fish's physiological indicators stable.

[0010] The calculation process of the overall evaluation at different stages of embryonic period, larval period, juvenile period and saline-alkali acclimatization period, the sum of the saline-alkali evaluation values ​​of each stage is the comprehensive evaluation value.

[0011] Calculation of the comprehensive evaluation value of salt and alkali tolerance at each stage:

[0012] The result of standardizing the data of the i-th evaluation indicator, a i :

[0013]

[0014] Weighting coefficients w for each evaluation indicator j :

[0015]

[0016] , 1 /

[0017]

[0018]

[0019] Comprehensive salt and alkali tolerance evaluation value y:

[0020]

[0021] In the formula, x i Let x represent the value of the i-th evaluation index. max Let x represent the maximum value of the i-th comprehensive index. min p represents the minimum value of the i-th comprehensive index; ij e represents the proportion of the i-th indicator value under the j-th indicator.j The information entropy of the evaluation index, d j represents the information utility value, and m represents the number of evaluation index stages.

[0022] The beneficial effects of this invention are:

[0023] This invention establishes an evaluation system for the salt and alkali tolerance of crucian carp without intermuscular spines. It uses multiple stages of salt and alkali tolerance index values ​​for weighted comprehensive analysis, which is superior to the traditional single index evaluation method. It can comprehensively reflect the tolerance level of crucian carp without intermuscular spines to salt and alkali stress and provide basic data for the comprehensive evaluation of the characteristics of new germplasm of crucian carp without intermuscular spines. Detailed Implementation

[0024] The technical solution of the present invention will be further described below through specific embodiments. Without departing from the concept of the present invention, those skilled in the art can make simple modifications or equivalent substitutions to the present invention, all of which fall within the protection scope of the present invention.

[0025] Example 1: A comprehensive evaluation method for salt and alkali tolerance in crucian carp without intermuscular spines

[0026] Acute salt-alkali stress was applied to the embryonic, larval, and juvenile stages of both *Carassius acutissima* and *Carassius spp.*, while a salt-alkali acclimatization experiment was conducted on the fry stage. Salt-alkali tolerance evaluation values ​​were obtained for each stage, and weighting coefficients for each stage were calculated. The overall salt-alkali tolerance evaluation value for each stage was then statistically summed, and the salt-alkali tolerance performance of *Carassius acutissima* was assessed based on the summed value. A weighted comprehensive scoring method was used to calculate the overall salt-alkali tolerance evaluation value for each stage.

[0027] The specific process of determining salt tolerance during the embryonic period is as follows:

[0028] (1) Select fertilized eggs of crucian carp and crucian carp without intermuscular spines; select artificially fertilized eggs of crucian carp and crucian carp that have developed to the late blastocyst stage for salt tolerance assessment during the embryonic stage; select floating and swimming fry with a total length of 6.5 mm or more for salt tolerance assessment during the larval stage; select juvenile fry that have been raised in ponds for 1 month with a total length of 2.5 cm or more for salt tolerance assessment during the domestication stage; select 1-year-old fish with a body length of 8.0 cm or more for salt tolerance assessment during the acclimatization period.

[0029] (2) The embryos were incubated in water with different concentrations of salinity and alkalinity, and the survival rate and hatching rate of the embryos were recorded and standardized.

[0030] Three biological replicates were set up for both crucian carp without intermuscular spines and crucian carp. The embryonic salt and alkali tolerance assessment test was conducted in 500 mL plastic bowls. The salinity stress concentration was 3‰ NaCl, and the alkalinity stress concentration was 10 mmol / L NaHCO3. Thirty fertilized eggs were placed in each experiment, and the acute stress period was 96 h. The embryo hatching rate was recorded.

[0031] The salinity and alkalinity tolerance assessment test for larvae was conducted in a stainless steel basin (38 cm in diameter) with 5 L of water. The salinity and alkalinity stress concentrations were the same as those used in the embryonic stage test. Thirty larvae were used in each test, and the survival rate of the larvae under stress for 96 hours was recorded.

[0032] The salt tolerance test for juvenile fish was conducted in a glass aquarium (85 cm × 52 cm × 60 cm) with 20 L of water. The salinity and alkalinity stress concentrations were 12‰ NaCl and 60 mmol / L NaHCO3, respectively. Thirty juvenile fish were tested, and their survival rate was recorded after 96 h.

[0033] The salinity and alkalinity tolerance assessment test during the acclimatization period was conducted in a glass aquarium (85 cm × 52 cm × 60 cm) with a water volume of 90 L. The initial salinity and alkalinity acclimatization concentrations were the safe concentrations of NaCl and NaHCO3 after 96 h, respectively. The salinity and alkalinity were adjusted every 10 days, with the adjustment concentrations reaching the highest values ​​that maintained stable physiological indicators in the fish. Thirty one-year-old fish were used in the test, and the salinity and alkalinity concentrations were recorded at each adjustment.

[0034] The relative values ​​of each measurement data were calculated, and the relative values ​​of each trait index are shown in Table 1.

[0035] Table 1. Relative values ​​of indicators at different stages of crucian carp salt tolerance assessment

[0036]

[0037] (3) Calculate the weighting coefficient of embryo hatching rate using the entropy method

[0038] The result of standardizing the data of the i-th evaluation indicator, a i :

[0039]

[0040] Weighting coefficients w for each evaluation indicator j :

[0041]

[0042] , 1 /

[0043]

[0044]

[0045] Comprehensive salt and alkali tolerance evaluation value y:

[0046]

[0047] In the formula, x i Let x represent the value of the i-th evaluation index. max Let x represent the maximum value of the i-th comprehensive index. min p represents the minimum value of the i-th comprehensive index; ij e represents the proportion of the i-th indicator value under the j-th indicator. j The information entropy of the evaluation index, d j represents the information utility value, and m represents the number of evaluation index stages (4).

[0048] The weight coefficients of each evaluation indicator were calculated, and the calculation process data are shown in Tables 2-4. Table 2 shows the normalized results (p) for each evaluation indicator. ij Table 3 shows the k value, credit utility value d, and weight coefficient w calculated according to the formula.

[0049] Table 2. Proportion of Sample Values ​​for Each Indicator in the Evaluation of Salt and Alkali Tolerance of Crucian Carp

[0050]

[0051] Table 3. Weight values ​​of each indicator in the evaluation of crucian carp's salt and alkali tolerance.

[0052]

[0053] (4) Obtain the embryonic period salt and alkali tolerance evaluation value by multiplying the standardized value by the weighting coefficient.

[0054] The process for assessing salt tolerance during the larval, juvenile, and acclimatization stages is similar to that during the embryonic stage. The sum of the salt tolerance evaluation values ​​at each stage is the comprehensive evaluation value.

[0055] The comprehensive evaluation method of this invention was used to comprehensively evaluate the salt and alkali tolerance of crucian carp without intermuscular spines and crucian carp during the embryonic, larval, juvenile, and saline-alkali acclimatization stages. The results are shown in Table 4. The higher the comprehensive evaluation value of each experimental group, the stronger the salt and alkali tolerance. The comprehensive evaluation values ​​of salt and alkali tolerance for crucian carp without intermuscular spines and crucian carp were 0.4719 and 0.4999, respectively, with no significant difference between the two, indicating that crucian carp without intermuscular spines and crucian carp had the same salt and alkali tolerance.

[0056] Table 4. Comprehensive evaluation values ​​of crucian carp's salt and alkali tolerance

[0057]

Claims

1. A comprehensive evaluation method for salt and alkali tolerance in crucian carp without intermuscular spines, characterized in that, Includes the following steps: Salt and alkali tolerance of crucian carp without intermuscular spines was assessed by measuring salt and alkali tolerance at different stages, including the embryonic stage, larval stage, juvenile stage, and salt and alkali acclimatization stage. The obtained data were standardized, weighted, and the comprehensive evaluation values ​​for each stage were calculated and summed. The overall salt and alkali tolerance of crucian carp without intermuscular spines was evaluated based on the summed value.

2. The comprehensive evaluation method for salt and alkali tolerance of crucian carp without intermuscular spines according to claim 1, characterized in that, The overall salt tolerance capacity is obtained from the overall evaluation value of different stages: embryonic period, larval period, juvenile period, and salt acclimatization period.

3. The comprehensive evaluation method for salt and alkali tolerance of crucian carp without intermuscular spines according to claim 1, characterized in that, During the salinity and alkalinity acclimatization period, the salinity and alkalinity were adjusted upwards every 10 days, with the adjustment concentration based on the highest value that kept the fish's physiological indicators stable.

4. The comprehensive evaluation method for salt and alkali tolerance of crucian carp without intermuscular spines according to claim 2, characterized in that, The calculation process for the overall evaluation of different stages—embryonic period, larval period, juvenile period, and saline-alkali acclimatization period—is as follows: The result of standardizing the data of the i-th evaluation indicator, a i : ； Weighting coefficients w for each evaluation indicator j : ； , 1 / ； ； ； Comprehensive salt and alkali tolerance evaluation value y: In the formula, x i Let x represent the value of the i-th evaluation index. max Let x represent the maximum value of the i-th comprehensive index. min p represents the minimum value of the i-th comprehensive index; ij e represents the proportion of the i-th indicator value under the j-th indicator. j The information entropy of the evaluation index, d j represents the information utility value, and m represents the number of evaluation index stages.

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