CN111248115A - Indoor culture method for obtaining high-quality fertilized eggs of seawater medaka - Google Patents

Abstract

The invention relates to an indoor breeding method for obtaining high-quality fertilized eggs of medaka in seawater, which belongs to the field of aquaculture.

Description

Indoor culture method for obtaining high-quality fertilized eggs of seawater medaka

Technical Field

The invention relates to the field of fish breeding, in particular to a method for obtaining high-quality zygotes of ocean-mode organism medaka.

Background

Medaka (Oryzias melastinma), also called medaka, medaka in sea, originally produced in Pakistan, India, Burmese and coastal waters of thailand. Taxonomically, the ocean medaka is of the same genus as Japanese medaka, phylum chordata, class Phillipes, order Argochaetales, family Guapidae, genus medaka. The seawater medaka has the characteristics of small individual, easy sex distinction, short generation cycle, high egg laying rate, wide salinity adaptation range and the like, and is recommended to be an ideal ocean model fish species at present.

The seawater medaka is more and more widely used for the research of marine ecotoxicology due to the sensitivity to the intervention of pollutants, and the embryo of the seawater medaka is considered as an important tool for the research of toxicology by the research institute of health and environmental science of the international society for life science. In addition, the fertilized eggs of the seawater medaka can also be used for gene knockout test research and are excellent materials for researching gene editing and simulating human diseases. Research has shown that: the seawater medaka embryo is used as a toxicology and gene editing research model and has the advantages that (1) fish eggs are easy to obtain, the generation period is short (sexual maturity can be achieved after 3 months of age), the conditions are suitable, the eggs can be laid every day, and the egg laying amount is large; (2) the fish roe has large volume, transparent color and difficult damage, changes obviously at each development stage, and is easy to carry out various experimental observations and operations; (3) the roe is sensitive to the intervention of various pollutants in the environment, and can quickly reflect the environment pollution condition.

Whether the method is used for toxicology or gene editing research, a large amount of good-developed seawater medaka zygotes with high fertilization rate need to be provided. However, at present, related patents of seawater medaka mainly focus on the development of cultivation equipment, hatching of fertilized eggs, and the like. The patents on the indoor breeding method are only 'a laboratory anniversary breeding method of medaka in seawater (publication number: 102578006A)' and 'a medaka efficient breeding and hatching method (publication number: 105900879A)'. The former prescribes the culture parameters of the seawater medaka such as the culture salinity, temperature, density, illumination and the like, and solves the problem of indoor annual culture of the seawater medaka. The latter designs an incubator, and fertilized eggs obtained by induced spawning are placed in the incubator for incubation, so that higher survival rate and hatchability are obtained. The existing method cannot well solve the problem of how to obtain a large amount of seawater medaka fish eggs with high fertilization rate in a short time. Therefore, an indoor culture method capable of rapidly and efficiently obtaining high-quality fertilized eggs of medaka in seawater is needed.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to design and provide an indoor culture method for quickly and efficiently obtaining high-quality fertilized eggs of medaka.

The technical solution adopted by the invention is as follows:

an indoor culture method for obtaining high-quality fertilized eggs of medaka in seawater comprises the following specific steps:

(1) physical and chemical conditions for parent fish culture

a. Temperature: 26-28 ℃;

b. humidity: 40-60 percent;

c. light intensity: 63-125 μmol. m^-2·s^-1；

d. The illumination period is as follows: light-to-dark ratio of 14: 10;

e. culturing water: the moving water body of each fish is about 5L;

(2) parent fish selection

a. The age of the month: 6-8 months old;

b. body length: 25-28 mm;

c. the advantages and characteristics of the male fish: the ratio of the hip fin length to the body length is about 0.18-0.25, male fish with longer hip fin lines is selected as parent fish, and the larger the ratio of the hip fin length to the body length is, the stronger the fertility is. 2-3 hip fin lines close to the abdomen are extended to be 1 time of other hip fin lines, the ratio of the hip fin lines to the body length is 0.4-0.6, and the reproductive capacity of the male fishes is strong;

d. the female fish has the advantages that: the ratio of body height to body length is 0.24-0.38, female fish with larger abdomen is selected as parent fish, the ratio of body height to body length is 0.24-0.38, the ratio of body height to body length is larger, and the reproductive capacity is stronger.

(3) Male to female ratio

The ratio of the female fish to the male fish is 1: 1;

(4) bait throwing

Periodically feeding 2 nauplii hatched for the first time in the morning and afternoon, and matching 1 time of artificial bait in the period.

Compared with the prior art, the invention has the beneficial effects that:

the method of the invention selects the development characteristics of female fish and male fish in the cultivation practice to obtain a set of method for selecting high-quality male and female parent fish, and can obtain fertilized eggs with high fertilization rate in a short time by continuously improving and adjusting the cultivation conditions, thereby providing experimental materials for toxicology and gene editing technology research.

Detailed Description

The invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention in any way.

An indoor cultivation method for obtaining high-quality fertilized eggs of medaka (Oryzias melastigma), which comprises the following conditions:

water for parent fish cultivation: the seawater is taken from the non-polluted wild seawater, is filtered by precipitated sand and absorbent cotton before use, and the physical and chemical conditions for parent fish culture are as follows:

controlling the salinity to be 31 +/-1;

culturing temperature: 26-28 ℃;

air humidity: 40-60 percent;

cultivation light intensity: 63-125 μmol. m^-2·s^-1；

The illumination period is as follows: light-to-dark ratio of 14: 10, lighting period from 6 am to 8 am;

culturing water: ensuring that the moving water body of each parent fish is more than 5L;

after the gonad of the parent fish is mature, the parent fish is screened under the following conditions:

the method comprises the following steps of selecting parent fish of 6-8 months old, wherein the body length is 25-28mm, the female fish and the male fish are similar in month old, the body shape of the male fish is slightly larger than that of the female fish, and hip fins of the male fish can well wrap reproductive pores of the female fish, so that a transient and stable breeding microenvironment is provided for the parent fish, and the fertility rate is improved; under the precondition that the hip fin length ratio is 0.18-0.25, male fish with longer hip fin strips is selected as parent fish, and the reproductive capacity is stronger when the hip fin length-body length ratio is larger. 2-3 hip fin lines close to the abdomen are extended to 1 time of other hip fin lines, the proportion of the hip fin lines to the body length is close to 0.5, and the reproductive capacity of the male fish is strong; the ratio of the height to the length of the female fish is 0.24-0.38, the female fish with larger abdomen needs to be selected as parent fish, and the larger the ratio of the height to the length of the female fish is, the stronger the fecundity is; the male-female ratio is 1: the optimal culture density in 1, 20L water is 2 female and 2 male.

The feeding strategy of the indoor parent fish culture method is as follows:

the nauplii are fed with artificial bait for 1 time of Seger (Qingdao Seger bioengineering Co., Ltd.) after 2 times of primary incubation at 8 am and 5 pm every day. Hatching the artemia cysts in a hatching barrel in the previous day, and collecting nauplii after 28-32 hours for feeding parent fishes. The feeding amount must ensure that a small amount of living artemia still remain in the water body before the next feeding, but the feeding amount is not too high, so that the artemia are prevented from dying and consuming oxygen in a large amount.

Fertilized eggs of the indoor culture method are collected as follows:

during the process of parent fish culture, the bottom suction, egg picking, water change, excrement removal and residual bait removal are carried out every day, and the water change amount is 1/2. Sucking seawater at the bottom of the fish tank into a 1-2L big beaker, standing, and removing the upper seawater layer when roe sinks to the bottom of the beaker. The fertilized eggs were carefully aspirated with a plastic pipette, transferred to a petri dish, and used directly for embryo observation. After the observation, the fish is transferred to a 500mL beaker again by a straw, and the seawater is filtered for culture, and the fish is used for incubation culture of the larvae.

Through experimental observation of 60 days, under the same culture conditions, the average egg laying amount of female fishes in the two-female and two-male experimental group is the highest and is 29 fishes/day, and the average egg laying amount of the female fishes in the two-female and two-male experimental group is 28 fishes/day and is obviously higher than that of the three-female and one-male experimental group (21 fishes/day). In the two-female two-male experimental group, a single female fish can produce 45 eggs in one day.

The spawning fertilization rate of the experimental group of the two-female two-male parent fish is obviously higher than that of the experimental group of three-female one-male and the experimental group of three-male one-female. The average fertilization rate of the experimental groups of the two females and two males is as high as 92%, while the fertilization rates of the other two groups are only 81% and 72%.

Claims (1)

1. An indoor culture method for obtaining high-quality fertilized eggs of medaka in seawater is characterized by comprising the following specific steps:

(1) physical and chemical conditions of cultivation

a. Temperature: 26-28 ℃;

b. humidity: 40-60 percent;

c. light intensity: 63-125 μmol. m^-2·s^-1；

d. The illumination period is as follows: light-to-dark ratio of 14: 10;

e. culturing water: the moving water body of each fish is about 5L;

(2) parent fish selection

a. The age of the month: 6-8 months old;

b. body length: 25-28 mm;

c. the advantages and characteristics of the male fish: male fish with hip fin length accounting for 0.18-0.25 of body length and longer hip fin lines is selected as parent fish, 2-3 hip fin lines close to the abdomen are extended to be 1 time of other hip fin lines, and the ratio of the length to the body length is 0.4-0.6, so that the male fish has stronger reproductive capacity;

d. the female fish has the advantages that: selecting female fish with height-to-length ratio of 0.24-0.38 and large abdomen as parent fish, wherein the height-to-length ratio is in the range of 0.24-0.38, and the larger the height-to-length ratio is, the stronger the fecundity is;

(3) male to female ratio

The ratio of the female fish to the male fish is 1: 1;

(4) bait throwing

Periodically feeding 2 nauplii hatched for the first time in the morning and afternoon, and matching 1 time of artificial bait in the period.