人工诱导黄颡鱼多倍体技术研究
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摘要
黄颡鱼(Pelteobagrus fulvidraco)是我国重要的淡水名优经济鱼类,其肉质细嫩,味道鲜美,营养丰富,深受广大消费者喜爱,现已成为重要的养殖对象。但其性成熟时间早,人工养殖条件下一冬龄即可达到性成熟。性成熟过早将导致生殖能耗加大,一定程度上影响了其生长性能,造成个体规格普遍偏小。为了提高养殖效益,就需要尽可能地推迟性成熟的时间或者抑制其性腺发育,以达到快速生长的目的。由于三倍体鱼潜在不育性,而四倍体和二倍体杂交又可得到大量的三倍体,运用染色体操作技术进行黄颡鱼的多倍体诱导无疑是解决这一养殖困境的有效手段。为此我们于去年5-8月进行了黄颡鱼多倍体的人工诱导试验,现将主要结果报道如下：
(1)采用冷休克、热休克和静水压休克三种方法进行了黄颡鱼的三倍体诱导试验,并对适宜条件下三种方法的诱导效果进行比较。结果表明：①冷休克、热休克和静水压休克都能成功地诱导出黄颡鱼三倍体。25℃受精水温条件下,冷休克法适宜诱导条件为受精后3min,在4℃冷水中处理10min,胚胎三倍体化率和相对出苗率分别为58%和53%；热休克法适宜诱导条件为受精后2min,在40.5℃热水中处理2min,胚胎三倍化率和相对出苗率分别为59%和36%；静水压休克法适宜诱导条件为受精后3min,在550kg／cm2压力条件下持续处理3min,胚胎三倍化率和相对出苗率分别为55%和54%。三倍体诱导率受处理起始时刻、处理温度(或压力)和处理持续时间3因素的影响。②综合考虑胚胎三倍化率、相对出苗率以及成鱼期诱导率,静水压法诱导效果要优于冷休克法和热休克法；冷休克法和热休克法效果差不多,但有各自的优点,前者表现为较高的孵化率,而后者表现为较高的诱导率。
(2)采用热休克阻止受精卵第一次卵裂的方法诱导黄颡鱼四倍体。运用正交试验对影响热休克诱导效果的主要三个因素,即处理起始时间(42-57min每隔3min设一个处理组)、处理温度(39、40和41℃)及处理持续时间(1、1.5和2min)进行了筛选。结果表明：胚胎的四倍化率为0%-42.11%,相对出苗率为0%-50.57%。诱导黄颡鱼四倍体较适宜条件为受精后45min和54min左右以40.4±0.2℃处理1.5-1.8min。
(3)在受精后42-58min采用诱导黄颡鱼三倍体的适宜温度40.5℃和持续时间2min处理受精卵进行黄颡鱼四倍体的大批量诱导。结果表明：原肠期胚胎四倍化率为10.61%-62.16%,出膜期胚胎四倍化率为11.11%-75%,相对出苗率为9.77%-36.05%。其中以受精后45min和54min这两个处理组的四倍化率为最高。
(4)运用鱼类胚胎制备高质量的染色体目前已经广泛的应用在鱼类多倍体育种、杂交育种、核型分析和性别控制等过程中。但一直以来胚胎染色体制片都存在操作繁琐、取样时间长和稳定性差等弊端,使得对于开展大批量的实验研究极为不利。本试验通过对常规胚胎染色体制片方法加以改良,建立了一种快速、简单、稳定性好的鱼胚染色体永久标本制备方法。
The yellow catfish Pelteobagrus fulvidraco, one of the commercial freshwater species in China, with tender flesh, delicious taste, high value of nutritional, has been widely cultured in our country due to the success of artificial propagation in 2000s. However, sexual maturation occurs usually less than one years old yellow catfish under culture conditions, before commercial size is reached. This is considered a major problem because sexual maturation is accompanied by a decrease in growth rate and flesh quality. In order to improve the breeding benefit and accelerate the growth rate of yellow catfish, we need to delay puberty or permanently inhibiting gonad development of yellow catfish as far as possible. A solution to this problem may be found in the production of triploid fish that are functionally sterile from the odd number of chromosomes that cause disruption of meiosis in the gametes. In present study, several shock methods such as cold shock, heat shock and pressure shock are used to induce triploidy and tetraploidy in yellow catfish during last May to August. The main results are summarized as follows:
(1) The optimal conditions for triploidy yellow catfish induction were investigated by altering the timing, and the intensity and duration of application of cold, heat and pressure shocks and the efficiency of three methods were also compared. The results shows:①The triploidy yellow catfish could be obtained by cold, heat and hydrostatic pressure shock and triploid rates in experimental groups were mainly affected by the timing, intensity and duration of shocks. In July, at the fertilization temperature of 25℃, treatment optimal for cold shock was 4℃for 10min at 3min after fertilization with 58% triploid embryos at gastrul stage and 53%viable fry to control at hatching; and for heat shock was 40.5℃for 2min at 2min after fertilization with 59% triploid embryos at gastrul stage and 36% viable fry to control at hatching; and for pressure shock was 550kg/cm2 for 3min at 3min after fertilization with 55% triploid embryos at gastrul stage and 54% viable fry to control at hatching.②Comparing the rate of triploid at gastrul stage, the rate of viable fry to control, and the rate of triploid at adulthood synthetically, it is suggested that the efficiency of pressure shock was much higher than the other two methods. Cold shock and heat shock resulted in more or less the same effect, but both have their respective merits. Cold shock has a relatively high rate of viable fry to control, and heat shock has a relatively high rate of triploid at adulthood.
(2) Orthogonal composite designs were used in experiments. Eggs were hand-stripped and fertilized artificially. First division was visible and occurred about 57min after fertilization. Zygotes were developed in freshwater at 25℃. Using heat shock treatment for blocking first division of fertilized eggs, tetraploidy were indued by application of 39,40 and 41℃heat shocks to eggs for 1,1.5 and 2min at 42,45,48,51, 54 and 57min AF in yellow catfish. The results showed that the tetraploid rates of embryos were range from 0%to 42.11%, and the viable fry rates to control were were range from 0%to50.57%. The suitable factors of heat shock treatments for inducing tetraploid in yellow catfish were 40±0.2℃for 1.5-1.8min at 45 or 54min AF.
(3) A heat shock of 40.5℃for 2min that was suitable for triploidy produced was applied to induce tetraploidy at 42-58min AF. The results showed that the tetraploid rates of embryos at gastrul stage were range from 10.61%to 62.16%, and the tetraploid rates of embryos at hatching stage were range from 11%to 75%, and the viable fry rates to control were range from 9.77%to 36.05%. Shocks applied at 44min and 54min AF resulted in the highest tepraploid rates of embryos in all of the treatment groups.
(4) In fish, the high quality mitotic chromosome made from embryos is widely applied in the study of polyploid breeding, hybrid Breeding, karyotype analysis and sex control. It is disadvantage to use conventional preparation for chromosome methods in experiment due to taking a very long time, having fussy steps and lacking stability. In this paper, a rapid, simple and stable method for obtaining permanent chromosome preparation from embryos of fish was established by improving the conventional method.
(1)采用冷休克、热休克和静水压休克三种方法进行了黄颡鱼的三倍体诱导试验,并对适宜条件下三种方法的诱导效果进行比较。结果表明：①冷休克、热休克和静水压休克都能成功地诱导出黄颡鱼三倍体。25℃受精水温条件下,冷休克法适宜诱导条件为受精后3min,在4℃冷水中处理10min,胚胎三倍体化率和相对出苗率分别为58%和53%；热休克法适宜诱导条件为受精后2min,在40.5℃热水中处理2min,胚胎三倍化率和相对出苗率分别为59%和36%；静水压休克法适宜诱导条件为受精后3min,在550kg／cm2压力条件下持续处理3min,胚胎三倍化率和相对出苗率分别为55%和54%。三倍体诱导率受处理起始时刻、处理温度(或压力)和处理持续时间3因素的影响。②综合考虑胚胎三倍化率、相对出苗率以及成鱼期诱导率,静水压法诱导效果要优于冷休克法和热休克法；冷休克法和热休克法效果差不多,但有各自的优点,前者表现为较高的孵化率,而后者表现为较高的诱导率。
(2)采用热休克阻止受精卵第一次卵裂的方法诱导黄颡鱼四倍体。运用正交试验对影响热休克诱导效果的主要三个因素,即处理起始时间(42-57min每隔3min设一个处理组)、处理温度(39、40和41℃)及处理持续时间(1、1.5和2min)进行了筛选。结果表明：胚胎的四倍化率为0%-42.11%,相对出苗率为0%-50.57%。诱导黄颡鱼四倍体较适宜条件为受精后45min和54min左右以40.4±0.2℃处理1.5-1.8min。
(3)在受精后42-58min采用诱导黄颡鱼三倍体的适宜温度40.5℃和持续时间2min处理受精卵进行黄颡鱼四倍体的大批量诱导。结果表明：原肠期胚胎四倍化率为10.61%-62.16%,出膜期胚胎四倍化率为11.11%-75%,相对出苗率为9.77%-36.05%。其中以受精后45min和54min这两个处理组的四倍化率为最高。
(4)运用鱼类胚胎制备高质量的染色体目前已经广泛的应用在鱼类多倍体育种、杂交育种、核型分析和性别控制等过程中。但一直以来胚胎染色体制片都存在操作繁琐、取样时间长和稳定性差等弊端,使得对于开展大批量的实验研究极为不利。本试验通过对常规胚胎染色体制片方法加以改良,建立了一种快速、简单、稳定性好的鱼胚染色体永久标本制备方法。
The yellow catfish Pelteobagrus fulvidraco, one of the commercial freshwater species in China, with tender flesh, delicious taste, high value of nutritional, has been widely cultured in our country due to the success of artificial propagation in 2000s. However, sexual maturation occurs usually less than one years old yellow catfish under culture conditions, before commercial size is reached. This is considered a major problem because sexual maturation is accompanied by a decrease in growth rate and flesh quality. In order to improve the breeding benefit and accelerate the growth rate of yellow catfish, we need to delay puberty or permanently inhibiting gonad development of yellow catfish as far as possible. A solution to this problem may be found in the production of triploid fish that are functionally sterile from the odd number of chromosomes that cause disruption of meiosis in the gametes. In present study, several shock methods such as cold shock, heat shock and pressure shock are used to induce triploidy and tetraploidy in yellow catfish during last May to August. The main results are summarized as follows:
(1) The optimal conditions for triploidy yellow catfish induction were investigated by altering the timing, and the intensity and duration of application of cold, heat and pressure shocks and the efficiency of three methods were also compared. The results shows:①The triploidy yellow catfish could be obtained by cold, heat and hydrostatic pressure shock and triploid rates in experimental groups were mainly affected by the timing, intensity and duration of shocks. In July, at the fertilization temperature of 25℃, treatment optimal for cold shock was 4℃for 10min at 3min after fertilization with 58% triploid embryos at gastrul stage and 53%viable fry to control at hatching; and for heat shock was 40.5℃for 2min at 2min after fertilization with 59% triploid embryos at gastrul stage and 36% viable fry to control at hatching; and for pressure shock was 550kg/cm2 for 3min at 3min after fertilization with 55% triploid embryos at gastrul stage and 54% viable fry to control at hatching.②Comparing the rate of triploid at gastrul stage, the rate of viable fry to control, and the rate of triploid at adulthood synthetically, it is suggested that the efficiency of pressure shock was much higher than the other two methods. Cold shock and heat shock resulted in more or less the same effect, but both have their respective merits. Cold shock has a relatively high rate of viable fry to control, and heat shock has a relatively high rate of triploid at adulthood.
(2) Orthogonal composite designs were used in experiments. Eggs were hand-stripped and fertilized artificially. First division was visible and occurred about 57min after fertilization. Zygotes were developed in freshwater at 25℃. Using heat shock treatment for blocking first division of fertilized eggs, tetraploidy were indued by application of 39,40 and 41℃heat shocks to eggs for 1,1.5 and 2min at 42,45,48,51, 54 and 57min AF in yellow catfish. The results showed that the tetraploid rates of embryos were range from 0%to 42.11%, and the viable fry rates to control were were range from 0%to50.57%. The suitable factors of heat shock treatments for inducing tetraploid in yellow catfish were 40±0.2℃for 1.5-1.8min at 45 or 54min AF.
(3) A heat shock of 40.5℃for 2min that was suitable for triploidy produced was applied to induce tetraploidy at 42-58min AF. The results showed that the tetraploid rates of embryos at gastrul stage were range from 10.61%to 62.16%, and the tetraploid rates of embryos at hatching stage were range from 11%to 75%, and the viable fry rates to control were range from 9.77%to 36.05%. Shocks applied at 44min and 54min AF resulted in the highest tepraploid rates of embryos in all of the treatment groups.
(4) In fish, the high quality mitotic chromosome made from embryos is widely applied in the study of polyploid breeding, hybrid Breeding, karyotype analysis and sex control. It is disadvantage to use conventional preparation for chromosome methods in experiment due to taking a very long time, having fussy steps and lacking stability. In this paper, a rapid, simple and stable method for obtaining permanent chromosome preparation from embryos of fish was established by improving the conventional method.
引文
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