A method for efficiently preparing abalone tetraploid by timed treatment

Abstract

The application discloses a method for efficiently preparing abalone tetraploid, which comprises the following steps: synchronously fertilizing the ovum of abalone triploid and the sperm of normal diploid to obtain inter-fertility hybridization fertilized eggs; starting timing and synchronously measuring water temperature after fertilization, and recording the first polar body discharge time of the fertilized egg as B and the second polar body discharge time as C; based on the A, B and C, calculating the processing time point (C-B) / 4 and the processing duration (6B-A) / 2, and performing the first polar body discharge inhibition processing under the same fertilization environment. After the processing, the fertilized eggs are resuspended in fresh sand filtered seawater, and are conventionally incubated and cultivated. The application introduces the fertilization water temperature and the embryonic development biology node as quantitative indexes, eliminates the applicable limitations of a single experience threshold in different environments, and can stably and efficiently obtain abalone tetraploid fry, thereby providing a reliable tetraploid parent source for large-scale production of abalone full triploid.

Description

Technical Field

[0001] This invention belongs to the field of shellfish cultivation technology, specifically relating to a timed processing method for efficiently preparing abalone tetraploids. Background Technology

[0002] Triploid shellfish, due to their outstanding advantages such as rapid growth, excellent meat quality, and low fertility, have been widely used in the breeding practices of various economically important shellfish, including oysters, scallops, clams, mussels, pearl oysters, and abalone. Among them, the commercial production of fully triploid oysters is the most mature. The triploid oyster farming industry, represented by Pacific oysters (Crassostrea gigas) and American oysters (Crassostrea virginica), has spread to many countries and regions, including the United States, France, China, Australia, and Mexico. The large-scale production of fully triploid seedlings is based on the biological hybridization of tetraploid and diploid parents. This approach is highly dependent on stable and efficient tetraploid breeding technology and the sustainable maintenance of tetraploid populations.

[0003] Tetraploid induction is mainly achieved through two technical approaches: direct and indirect methods. The direct method involves using chemical or physical means to inhibit meiosis or the first mitosis in diploid eggs, or through gynogenesis, cell fusion, or other pathways to achieve chromosome doubling. Current research shows that while the direct method can yield a certain proportion of tetraploid embryos in Pacific oysters, scallops, mussels, and abalone, tetraploid embryos or larvae are extremely difficult to cultivate to adulthood, resulting in a limited number of surviving adults and making it difficult to establish a stable tetraploid population (Sun Zhenxing et al., 1998; Zhang et al., 2000; Di Guilan et al., CN118696882A). For example, Sun Zhenxing et al. (1998) treated fertilized eggs of *Haliotis discus hannai* with 1 mg / L cytochalasin B (CB) starting 10 min after fertilization and continuing for 15–30 min, achieving a maximum tetraploid embryo rate of 21.9%. Zhang et al. (2000) treated fertilized eggs of *Haliotis discus hannai* with 175 or 225 μmol / L 6-dimethylaminopurine (6-DMAP) for 16–18 min after fertilization (fertilization water temperature 22 ℃), achieving a tetraploid induction rate of 20%–22.5%. The induction rates of these direct methods are generally low, and they also exhibit significant problems such as delayed embryonic development, high malformation rates, and low adult survival rates.

[0004] The indirect method utilizes fertilized eggs produced by hybridization of triploid females and diploid males, employing chemical or physical means to suppress the release of the first polar body (PB1), thereby stably obtaining fertile tetraploid adults to establish a tetraploid base population. This approach has been proven feasible in oysters and other mollusks, but its application in abalone is still in the exploratory stage. Representative studies include: Guo Xirui et al. (CN116806752A) used triploid female and diploid male hybrid fertilized eggs of *Haliotis diversicolor*, treating them with 0.3–1.2 mg / L CB for 15–20 min when the fertilized eggs began to release the first polar body, achieving an induction rate of over 96%; Ke Caihuan et al. (CN114600807A) also used triploid eggs, treating the control group with 20–40 mg / L 6-DMAP for 10–20 min when the first polar body began to release, with the core technical feature being the use of the real-time observed "first polar body release" as an immediate trigger point for chemical inhibition; Yan Zhenglin et al. (2008) treated triploid female hybrid fertilized eggs of *Haliotis diversicolor* with CB, with the treatment starting time based on microscopic observation (approximately 15 s) and lasting for 1.5–8 min, achieving a tetraploid induction rate of 15%–40%. In addition, although Zhang Yuehuan et al. (CN104255586B) proposed a quantitative treatment approach using the developmental stages of fertilized eggs (time periods A, B, and C) as biological indicators in the triploid induction of oysters in Hong Kong, their treatment targets diploid eggs and inhibited the release of the second polar body, with the goal of triploids, which is fundamentally different from the technical route of tetraploid induction in abalone.

[0005] However, existing tetraploid induction techniques for abalone still have significant technical defects and biases.

[0006] First, the direct method has a low induction rate (generally 10%–35%) and difficulty in adult survival, making it difficult to meet the needs of population construction. Although the indirect method can achieve a higher induction rate under specific conditions, it heavily relies on manual real-time microscopic observation. Due to the lack of quantitative methods, operators must continuously observe under a microscope to determine the moment of "first release of the first polar body" as the treatment start point. This instantaneous triggering mode suffers from severe human operation lag and subjective judgment errors. Furthermore, due to the extremely narrow dosing window (usually only a few minutes), in large-scale breeding practices, operators find it difficult to simultaneously achieve real-time monitoring and instantaneous induction treatment of multiple fertilization hatching ponds, limiting this technology to small-scale trials with "fixed personnel and single batches".

[0007] Secondly, as a typical stenothermic molluskaloid, the fertilized eggs of the wrinkled abalone are extremely sensitive to temperature changes. Fluctuations in the fertilization water temperature directly lead to a significant shift in the absolute time of polar body release. Although existing methods provide empirical treatment duration ranges (e.g., 10–20 min), these fixed threshold ranges do not fully consider the dynamic regulatory effect of temperature on embryonic development rate. This single static empirical threshold has limitations in the application of non-constant fertilization environments, especially in the marginal temperature ranges deviating from the optimal incubation temperature (e.g., around 17°C), which may lead to the inability to correctly anchor the biological window for polar body release. Furthermore, the fixed range makes it difficult to simultaneously consider timely termination of treatment to reduce the negative impact of potential drug toxicity on embryonic development. This results in significant fluctuations in induction rates in actual multi-batch applications, a substantial decrease in induction efficiency and larval survival rates, and difficulty in consistently obtaining tetraploid seedlings suitable for subsequent triploid hybridization.

[0008] It is important to note that a long-standing technological bias in this field holds that "an immediate triggering mode using the release of the first polar body as the starting point of treatment" or "using a rigid, fixed time window" is sufficient to meet the induction requirements, while neglecting the dynamic interference of environmental temperature on embryonic development. Furthermore, it overlooks the need to construct a formulaic correlation between environmental temperature measurement indicators and biological developmental milestones to replace traditional, single, substantive treatment nodes and fixed empirical thresholds. This bias has resulted in the critical time points for tetraploid induction remaining empirical and non-quantitative for a long time, hindering the automation, standardization, and industrial application of abalone tetraploid breeding technology. Summary of the Invention

[0009] The purpose of this invention is to overcome the defects and technical biases in the prior art and to provide an efficient timed processing method for preparing abalone tetraploids.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A timed processing method for efficiently preparing abalone tetraploids includes the following steps:

[0012] (1) The triploid oocytes of the preceding batch used to calibrate the treatment conditions were simultaneously fertilized with normal diploid sperm to obtain interploid hybrid fertilized eggs;

[0013] (2) After fertilization, start timing and measure the water temperature simultaneously. The water temperature is recorded as A, and the unit is ℃. At the same time, observe and record the time of expulsion of the first polar body of the interploid hybrid fertilized egg, recorded as B, and the time of expulsion of the second polar body, recorded as C. The time unit is min, accurate to s.

[0014] (3) Based on the measured A, B and C, the treatment time point is calculated as (CB) / 4, and the treatment duration is calculated as (6B-A) / 2, where A is converted to 1 min (60 s) at 1 ℃ and then used in the calculation to achieve linear correction of temperature on the embryonic development process, accurate to s;

[0015] (4) Under the same fertilization environment, the triploid eggs and normal diploid sperm of subsequent batches of abalone were synchronously fertilized to obtain interploid hybrid fertilized eggs. The first polar body of the interploid hybrid fertilized eggs was suppressed at the treatment time point (CB) / 4 calculated in step (3). The treatment duration was (6B-A) / 2 calculated in step (3). Then the treated fertilized eggs were resuspended in fresh sand-filtered seawater and conventionally incubated and cultivated.

[0016] In a preferred embodiment of the present invention, the abalone is a wrinkled abalone or a green abalone.

[0017] In a preferred embodiment of the present invention, the temperature for synchronous fertilization is 17–24 °C.

[0018] In a preferred embodiment of the present invention, the method for inhibiting the emission of the first polar body of the interploid hybridization zygote is to treat the zygote with a chemical drug solution.

[0019] More preferably, the chemical drug is cytochalasin B.

[0020] More preferably, the concentration of the chemical drug solution is 1 to 2 mg / L.

[0021] More preferably, the chemical drug is 6-dimethylaminopurine.

[0022] More preferably, the concentration of the chemical drug solution is 25–35 mg / L.

[0023] The beneficial effects of this invention are:

[0024] 1. This invention provides a timed treatment method that integrates fertilization temperature (an environmental indicator) with the expulsion times of the first and second polar bodies (biological indicators). It establishes a formulaic scheme based on both environmental and biological factors, improving upon the traditional instantaneous triggering mode of "using the release of the first polar body as the treatment start point" into an adaptive timed treatment mode that can be precisely quantified in advance. This scheme effectively avoids polar body expulsion time drift caused by environmental temperature fluctuations, ensuring that the drug treatment window correctly covers the target developmental stage, achieving precise quantification and temperature adaptation of key time points for tetraploid induction, and eliminating excessive reliance on human experience. Therefore, this invention effectively overcomes the shortcomings of existing technologies, such as reliance on a single fixed time point or non-quantitative observation points targeting the release of the first polar body, and the poor operational stability and low repeatability caused by using empirical ranges for treatment duration.

[0025] 2. This invention, through a standardized scheme using (CB) / 4 as the treatment start point and (6B-A) / 2 as the treatment duration, significantly improves the accuracy and controllability of tetraploid induction. This standardized induction scheme effectively reduces the risk of induction failure due to temperature fluctuations or differences in embryonic development, ensuring the stability of induction efficiency across multiple batches. According to experimental data (see Table 1 of Example 3), compared to the passive instantaneous triggering mode using the first polar body release as the treatment start point adopted by Ke Caihuan et al., the technical solution of this invention exhibits better robustness in the marginal temperature range deviating from the optimal water temperature. For example, in a low water temperature environment of 17.0℃, the induction rate of this method decreases to 86.01%, while the induction rate of this invention remains stable at over 90% within a wide temperature range of 17.0℃ to 23.5℃, with low variability across multiple batches. This invention, while ensuring the induction rate, effectively improves technical reproducibility, standardization, and stability of production across the entire temperature range.

[0026] 3. This invention is simple to operate, operates under mild conditions, and has good process repeatability and industrialization promotion value.

[0027] 4. The technical solution of the present invention has significant technical progress, breaking through the long-standing technical prejudice in the field that "the induction requirements can be met by taking the release of the first polar body as the starting point of the treatment". It provides a scientific and quantitative timed optimization paradigm for tetraploid breeding of narrow-temperature mollusks, effectively avoiding the defects of over-reliance on human experience and significantly improving the stability of the induction batch. Attached Figure Description

[0028] Figure 1 This is a DNA content analysis diagram of the wrinkled abalone larvae obtained in Example 1 of the present invention (2N: diploid, 4N: tetraploid).

[0029] Figure 2This is a DNA content analysis diagram of the green abalone larvae obtained in Example 2 of the present invention (2N: diploid, 4N: tetraploid). Detailed Implementation

[0030] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0031] Example 1

[0032] (1) The triploid oocytes of the previous batch of wrinkled disc abalone used to calibrate the treatment conditions were simultaneously fertilized with the sperm of normal diploids to obtain interploid hybrid fertilized eggs;

[0033] (2) Timing was started after fertilization and the water temperature was measured simultaneously. The water temperature was recorded as A = 18.0 ℃. The time of expulsion of the first polar body of the fertilized egg was recorded as B = 9 min 0 s and the time of expulsion of the second polar body was recorded as C = 39 min 0 s.

[0034] (3) Based on the measured A=18.0 ℃ (converted to 18 min 0 s), B=9 min 0 s and C=39 min 0 s, the processing time point is calculated as (CB) / 4=7 min 30 s, and the processing duration is calculated as (6B-A) / 2=18 min 0 s;

[0035] (4) Under the same fertilization environment, the triploid eggs of subsequent batches of abalone and the sperm of normal diploids were synchronously fertilized to obtain interploid hybrid fertilized eggs. The fertilization water temperature was 18.0 ℃. The treatment time point was 7 min 30 s. The interploid hybrid fertilized eggs obtained after synchronous fertilization were placed in 30 mg / L 6-dimethylaminopurine (6-DMAP) solution to inhibit the release of the first polar body of the fertilized eggs. The treatment duration was 18 min 0 s. After the treatment, the fertilized eggs were resuspended in fresh sand-filtered seawater and hatched and cultivated according to conventional methods to obtain the tetraploid larvae of wrinkled disc abalone.

[0036] (5) During the vesicular larval stage, ploidy was determined by flow cytometry. The results showed that after timed treatment combining fertilization temperature and the expulsion time of the first and second polar bodies, the tetraploid induction rate reached 97.80±3.62%. Figure 1 As shown, the ploidy peak of the induced larvae was obvious, with a concentrated and relatively uniform peak distribution. The DNA index was within the range of 2.00 ± 0.1, consistent with tetraploid characteristics. The results indicate that timed treatment can efficiently obtain a population with a high proportion of tetraploids, which is beneficial for the efficient screening of high-quality tetraploid abalone seedlings.

[0037] Example 2

[0038] (1) The triploid oocytes of green abalone used to calibrate the treatment conditions were simultaneously fertilized with normal diploid sperm to obtain interploid hybrid fertilized eggs;

[0039] (2) Timing was started after fertilization and the water temperature was measured simultaneously. The water temperature was recorded as A = 23.0 ℃. The time of expulsion of the first polar body of the fertilized egg was recorded as B = 7 min 30 s and the time of expulsion of the second polar body was recorded as C = 32 min 30 s.

[0040] (3) Based on the measured A=23.0 ℃ (equivalent to 23 min 0 s), B=7 min 30 s and C=32 min 30 s, the processing time point is calculated as (CB) / 4=6 min 15 s, and the processing duration is calculated as (6B-A) / 2=11 min 0 s;

[0041] (4) Under the same fertilization environment, the triploid eggs of subsequent batches of abalone and the sperm of normal diploids were synchronously fertilized to obtain interploid hybrid fertilized eggs. The fertilization water temperature was 23.0 ℃. The treatment time point was 6 min 15 s. The interploid hybrid fertilized eggs obtained after synchronous fertilization were placed in a 1.50 mg / L cytochalasin B (CB) solution to inhibit the release of the first polar body of the fertilized eggs. The treatment duration was 11 min 0 s. After the treatment, the fertilized eggs were resuspended in fresh sand-filtered seawater and hatched and cultivated according to conventional methods to obtain green abalone tetraploid larvae.

[0042] (5) During the vesicular larval stage, ploidy was determined by flow cytometry. The results showed that after timed treatment combining fertilization temperature and the expulsion time of the first and second polar bodies, the tetraploid induction rate could reach 90.18±5.85%. Figure 2 As shown, the ploidy peak of the induced larvae was obvious, with a concentrated and relatively uniform peak distribution. The DNA index was within the range of 2.00 ± 0.1, consistent with tetraploid characteristics. The results indicate that timed treatment can efficiently obtain a population with a high proportion of tetraploids, which is beneficial for the efficient screening of high-quality tetraploid green abalone seedlings.

[0043] Example 3

[0044] Method 1: Triploid oocytes of the wrinkled disc abalone and normal diploid sperm were synchronously fertilized at different temperatures to obtain interploid hybrid fertilized eggs. The treatment time point was the appearance of the first polar body in the fertilized egg. The fertilized eggs were placed in 35 mg / L 6-DMAP solution to inhibit the release of the first polar body. The treatment duration was 10 min 0 s.

[0045] Method 2:

[0046] (1) Triploid oocytes of the previous batch of abalone used for calibration treatment conditions and normal diploid sperm were fertilized synchronously at different temperatures to obtain interploid hybridization fertilized eggs;

[0047] (2) Timing and water temperature measurement should begin immediately after fertilization, specifically including:

[0048] A. When the water temperature is A=17.0 ℃ (equivalent to 17 min 0 s), the observation and recording time of the first polar body expulsion of the fertilized egg is B=10 min 0 s and the time of the second polar body expulsion is C=45 min 40 s. Based on the measured A, B and C, the treatment time point is calculated as (CB) / 4=8 min 55 s, and the treatment duration is calculated as (6B-A) / 2=21 min 30 s.

[0049] B. When the water temperature is A=19.0 ℃ (equivalent to 19 min 0 s), the observation and recording of the first polar body expulsion time of the fertilized egg is B=9 min and the second polar body expulsion time is C=38 min. Based on the measured A, B and C, the treatment time point is calculated as (CB) / 4=7 min 15 s, and the treatment duration is calculated as (6B-A) / 2=17 min 30 s.

[0050] C. At a water temperature of A = 23.5 ℃ (equivalent to 23 min 30 s), the observation and recording of the first polar body expulsion time of the fertilized egg is B = 7 min 15 s and the second polar body expulsion time is C = 30 min 55 s. Based on the measured A, B and C, the treatment time point is calculated as (CB) / 4 = 5 min 55 s, and the treatment duration is calculated as (6B-A) / 2 = 10 min 0 s.

[0051] (3) Under the three fertilization environments obtained in step (2), the triploid oocytes and normal diploid sperm of the subsequent batches of abalone were synchronously fertilized to obtain interploid hybrid fertilized eggs. The fertilized eggs were placed in 35 mg / L 6-DMAP solution to inhibit the release of the first polar body of the fertilized eggs at the corresponding (CB) / 4 treatment time point. The treatment duration was the corresponding (6B-A) / 2.

[0052] After treatment using methods one and two, the fertilized eggs were resuspended in fresh sand-filtered seawater and incubated using conventional methods to obtain tetraploid larvae of the wrinkled abalone.

[0053] During the larval stage of the vegetative fin, ploidy was determined by flow cytometry. Table 1 shows the experimental results of inducing tetraploids of the wrinkled disc abalone using two methods. Compared with the single empirical treatment time point and fixed treatment duration used in the immediate treatment method (Method 1) which uses the expulsion of the first polar body as the trigger point, the timed treatment method (Method 2) described in this invention is less affected by water temperature fluctuations and the subjective judgment of operators, and has higher stability and repeatability. It is more suitable for the standardized production and automated expansion of polyploid seedlings.

[0054] Table 1

[0055]

[0056] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A timed processing method for efficiently preparing abalone tetraploids, characterized in that: Includes the following steps: (1) The triploid oocytes of the preceding batch used to calibrate the treatment conditions were simultaneously fertilized with normal diploid sperm to obtain interploid hybrid fertilized eggs; (2) After fertilization, start timing and measure the water temperature simultaneously. The water temperature is recorded as A, and the unit is ℃. At the same time, observe and record the time of expulsion of the first polar body of the interploid hybrid fertilized egg, recorded as B, and the time of expulsion of the second polar body, recorded as C. The time unit is min, accurate to s. (3) Based on the measured A, B and C, the treatment time point is calculated as (CB) / 4, and the treatment duration is calculated as (6B-A) / 2, where A is converted to 1 min (60 s) at 1 ℃ and then used in the calculation to achieve linear correction of temperature on the embryonic development process, accurate to s; (4) Under the same fertilization environment, the triploid eggs and normal diploid sperm of subsequent batches of abalone were synchronously fertilized to obtain interploid hybrid fertilized eggs. The first polar body of the interploid hybrid fertilized eggs was suppressed at the treatment time point (CB) / 4 calculated in step (3). The treatment duration was (6B-A) / 2 calculated in step (3). Then the treated fertilized eggs were resuspended in fresh sand-filtered seawater and conventionally incubated and cultivated.

2. The method as described in claim 1, characterized in that: The abalone in question is either wrinkled abalone or green abalone.

3. The method as described in claim 1, characterized in that: The temperature for synchronous fertilization is 17–24 °C.

4. The method as described in claim 1, characterized in that: The method for inhibiting the emission of the first polar body from interploid hybridization zygotes is to treat the zygotes with a chemical drug solution.

5. The method as described in claim 4, characterized in that: The chemical drug in question is cytochalasin B.

6. The method as described in claim 5, characterized in that: The concentration of the chemical drug solution is 1–2 mg / L.

7. The method as described in claim 4, characterized in that: The chemical drug is 6-dimethylaminopurine.

8. The method as described in claim 7, characterized in that: The concentration of the chemical drug solution is 25–35 mg / L.

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