CN110720410B - Method for improving fertilization rate of zebra fish - Google Patents

Method for improving fertilization rate of zebra fish Download PDF

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CN110720410B
CN110720410B CN201910974679.7A CN201910974679A CN110720410B CN 110720410 B CN110720410 B CN 110720410B CN 201910974679 A CN201910974679 A CN 201910974679A CN 110720410 B CN110720410 B CN 110720410B
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zebra fish
fish
box body
water
fertilization rate
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CN110720410A (en
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代小新
赵波
牟毅
高阳
储张杰
刘志强
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Zhejiang Ocean University ZJOU
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract

The invention relates to the technical field of aquatic animal breeding, and aims to further improve the fertilization rate of zebra fish, and a technical staff group of the application develops deep research on the basis of earlier stage research, and provides a method for improving the fertilization rate of zebra fish.

Description

Method for improving fertilization rate of zebra fish
Technical Field
The invention relates to the technical field of aquatic animal breeding, in particular to a method for improving the fertilization rate of zebra fish.
Background
The zebra fish is a subtropical freshwater fish, is small in size, is slightly spindle-shaped due to prolonged body, has 87% of high homology with human genes, has prominent advantages as a model organism, has high scientific research value, and is utilized in aspects of retinal repair research, auditory repair research, environmental monitoring and the like.
The zebra fish is periodically laid eggs, fertilized in vitro and developed in vitro. The fish roe of the zebra fish is sticky and can sink to the water bottom, so that the zebra fish parent fish can not eat the roe. In the previous research on fertilization and propagation of zebra fish, in order to prevent the problem of low propagation rate caused by egg eating of parent zebra fish, the applicant develops a breeding box for separating fish eggs from parent fish, see Chinese patent CN 201711320960.6' a device and a method for improving fertilization rate of zebra fish. However, unfertilized fish eggs are too scattered during sinking and have less chance of contacting sperm, so that fertilization cannot be achieved once they sink to the bottom of the tank.
Meanwhile, the applicant disclosed in the above patent documents a technical means for increasing the fertilization rate by injecting melatonin. However, factors influencing the quality and fertilization rate of the zebra fish roes are various, and a method for improving the fertilization rate by singly adopting melatonin still has great improvement space.
Disclosure of Invention
In order to further improve the fertilization rate of the zebra fish, a technical staff group develops deep research on the basis of earlier-stage research, and provides a method for improving the fertilization rate of the zebra fish, compared with the patent CN201711320960.6, more factors influencing the fertilization of the zebra fish are considered, so that the aim of improving the fertilization rate of the zebra fish is fulfilled.
The invention provides the following technical scheme:
a method for improving the fertilization rate of zebra fish comprises the following steps:
(1) carrying out conventional nutrition-enriched cultivation on female zebra fish and male zebra fish of 10-12 weeks old separately, keeping the illumination time to be prolonged to 12-14 hours/day, and keeping the rest time in a dark state;
(2) adding female zebra fish and male zebra fish into a culture box capable of separating fish eggs according to the ratio of female to male being 1: 2-3 when the zebra fish is 20-21 weeks old, adding melatonin into a water body of the culture box, and keeping the concentration of the melatonin in the water body at 5-10 mug/L;
(3) keeping the illumination, quickly raising the water temperature by 1-2 ℃ within 30-60 min and keeping for 2-3 hours, enabling male zebra fish to chase and stimulate female zebra fish to lay eggs, and then recovering the water temperature;
(4) and separating the fish eggs from the parent fish, and calculating the fertilization rate of the fish eggs.
According to the method for improving the fertilization rate of the zebra fish, on the basis of early research, direct injection of melatonin into the body of the female zebra fish is considered, and although the melatonin can achieve the effect of strengthening and improving the fertilization rate, the method has large stimulation on parent fish. Therefore, the method strengthens the nutrition of the parent fish at a certain temperature and under certain illumination intensity, adds the melatonin in the water body after the parent fish is matured, utilizes the strong oxidizing property of the melatonin to directly act on the egg cells discharged into the water body, prevents the aging of ovulated eggs, and is simpler to operate compared with the method of injecting the melatonin in the earlier stage patent so as to improve the fertilization rate.
Preferably, the incubator capable of separating fish eggs in step (2) comprises an upper incubator body, a lower incubator body and a middle passage communicating the upper incubator body and the lower incubator body, wherein a plurality of partition plates are arranged between the middle passage and the upper incubator body side by side, adjacent partition plates form a space capable of blocking female zebra fish and male zebra fish from passing through but allowing fish eggs to pass through, the middle passage is connected with the upper end part of the upper incubator body and the lower end part of the middle passage connected with the lower incubator body is in a horn shape, the caliber of the horn shape gradually decreases towards the middle part of the middle passage, the middle part of the middle passage is in a necking passage, a water outlet is arranged on the side surface of the lower end part, and a water inlet is arranged at the port of the upper incubator body.
The culture box is provided with the upper box body, the middle channel and the lower box body, when fish eggs sink to the middle channel from the upper box body, the fish eggs are gathered in a too-dispersed state before changing due to the fact that the caliber of the middle channel is reduced, so that the contact chance of unfertilized fish eggs and sperms is increased, the water diversion outlet is formed in the side face of the lower end portion of the middle channel, so that water flow from top to bottom is formed in the culture box, and the fish eggs and the like in the upper box body are driven to flow downwards. And the diversion export sets up on the lower tip of well passageway, can make rivers keep longer route on the one hand, and on the other hand can make the water in the lower box remain stable basically, and the roe that sinks like this in the lower box can avoid disturbance once more and diversion export outflow, has perhaps avoided appearing the roe when setting up the diversion export on the lower box and has piled up the problem of plug diversion export.
Preferably, the water diversion outlet is arranged at the lower end part close to the necking channel, an inclined guide plate for shielding the water diversion outlet is further arranged on the inner side of the lower end part, the joint of the inclined guide plate and the lower end part is arranged above the water diversion outlet, and the included angle between the cross section of the inclined guide plate and the longitudinal section of the side face of the lower end part is an acute angle. The oblique baffle can prevent that the roe in the well passageway from following the diversion export and flowing, and the oblique baffle of slope can increase the water flow path simultaneously, leans on under the condition that sets up as far as possible at the diversion export and drives the water circulation.
Preferably, the surface of the partition board in the upper box body is provided with a diversion slope with a circular arch or triangle cross section. Therefore, under the drive of water flow, when the fish eggs fall on the partition board, the fish eggs can easily slip off, and the fish eggs are prevented from being deposited on the partition board. And through comparison, the water guiding effect of the circular arch-shaped water guiding slope is better, which may be because the slopes of any point on the circular arch-shaped water guiding slope are different, so that the fish roes are difficult to be in a balanced static state under the driving of water flow, or the chance of adjusting to the balanced static state is lacked, and the fertilization rate of the zebra fish adopting the circular arch-shaped water guiding slope can be improved by 2% -3% compared with that of the zebra fish adopting the triangular water guiding slope.
Preferably, the upper box body is also provided with an air inlet pipe and an air pump connected with the air inlet pipe, the air outlet port of the air inlet pipe is connected with the shower head, and the air outlet port is positioned above the upper box body and extends into the water surface of the upper box body. Adopt to drench the fluffy head and admit air, the air is more dispersed, compares with direct with loudspeaker form air inlet and admits air gentler moreover, can reduce the impact to the zebra fish when reinforceing down rivers, reduces the environmental stress reaction of zebra fish.
Preferably, in the steps (2) to (4), the pH value of the water body in the cultivation box is controlled to be 7-8, and the illumination intensity is controlled to be 100-320 lx. The alkalescence of the water body is controlled, the sufficient illumination intensity is kept, and the zebra fish is promoted to lay eggs.
Preferably, the step (3) is kept under light, which means that the state is changed from dark state to light for 1-2 hours. 1 ~ 2 hours after turning to the illumination from the dark state is the time of estrus activity and spawning of zebra fish, and the temperature that rises fast this moment provides environmental stimulation, can increase zebra fish's the egg laying volume, increases the fertilization probability.
Preferably, the method also comprises the step (1) of adding a sunflower pollen extract into the water body in which the male zebra fish is positioned, wherein the concentration is 30-40 mu g/L; the preparation process of the sunflower pollen extract comprises the following steps: grinding and crushing sunflower pollen, sieving the sunflower pollen with a sieve of 100-200 meshes, then placing the sunflower pollen in ethanol for reflux extraction at 23-28 ℃ for 72-96 hours, and then carrying out ventilation drying at 0-4 ℃ to obtain the sunflower pollen extract. The sunflower pollen is a male germ cell of sunflower, contains rich amino acids, trace elements, microorganisms, flavonoid compounds and the like, is added into a water body in which male zebra fish are positioned, promotes the development of sexual glands of the male zebra fish and improves the sperm quality, so that the effect of melatonin on the zebra fish egg cells is cooperated, and the fertilization rate is improved. The concentration of sunflower pollen is not suitable to be too high, because ethanol extraction is adopted, ethanol volatilization cannot be completely ensured, so that excessive addition can cause environmental stress reaction to male zebra fish, the extraction temperature is not suitable to be too high, and pollen is easy to lose efficacy.
The invention has the following beneficial effects:
the method for improving the fertilization rate of the zebra fish prevents the ovum from aging after ovulation by adding melatonin into the water body, reduces stimulation to female parent fish, simultaneously adds sunflower pollen extract into the water body where the male parent fish is located, promotes the development of male zebra fish sexual glands and improves sperm quality, and is provided with a specific breeding box to increase the fertilization chance.
Drawings
FIG. 1 is a structural view of the habitat of the invention.
Fig. 2 is an enlarged view at a in fig. 1.
Fig. 3 is another embodiment of the guide ramp.
In the figure, the water spraying device comprises an upper box body 1, an upper box body 11, a water inlet 12, a spraying nozzle 2, a middle channel 21, an upper end 22, a lower end 23, a necking channel 24, a water diversion outlet 25, an inclined guide plate 3, a lower box body 4, a partition plate 41, a circular arch-shaped flow guide slope 41' and a triangular flow guide slope.
Detailed Description
The following further describes the embodiments of the present invention.
The starting materials used in the present invention are commercially available or commonly used in the art, unless otherwise specified, and the methods in the following examples are conventional in the art, unless otherwise specified.
Example 1
As shown in figure 1, the culture box capable of separating fish and eggs is sequentially provided with an upper box body 1, a middle channel 2 and a lower box body 3 from top to bottom, and the upper box body, the lower box body and the middle channel are respectively provided with a circular cross section for convenient processing and placement. The upper box body is provided with a water inlet 11, the joint of the middle channel and the upper box body is provided with the clapboards 4 which are arranged side by side and used for separating the upper box body from the middle channel, and the adjacent clapboards are provided with intervals, so that the parent zebra fish of the upper box body can be prevented from entering the middle channel, but the roes are allowed to enter the middle channel from the upper box body. As shown in fig. 2, the baffle is provided with a circular arc diversion slope 41 on the surface of the upper box body, and the cross section of the baffle is circular arc, so that roe can be prevented from depositing on the upper surface of the baffle. Of course, an inclined ramp is preferably used at the partition immediately to the side of the upper tank. The diameters of the ports of the upper end part 21 of the middle channel connected with the upper box body and the lower end part 22 of the middle channel connected with the lower box body are respectively gradually reduced towards the middle part of the middle channel, so that the upper end part and the lower end part of the middle channel are in a horn shape, and the middle part of the middle channel is in a necking channel 23 capable of collecting roes and increasing contact between the roes and sperms. The side surface of the lower end part of the middle channel is provided with a water outlet 24, so that water in the culture box is allowed to flow downwards and flow out from the water outlet, water flow from top to bottom is formed in the culture box, fish eggs in the upper box body are driven to enter the middle channel and the lower box body in an accelerated manner, and the probability of being eaten by parent fish is reduced. The water outlets are circumferentially and uniformly distributed at the lower end part of the middle channel, so that the water flow is more dispersed and uniform. The inboard of the lower tip of well passageway still is equipped with oblique baffle 25, and the diversion export is close to the lower tip and is set up with necking down passageway junction, and the junction of oblique baffle and lower tip is located the top of diversion export, and oblique baffle is equipped with circular cross section, and the vertical section of oblique baffle on vertical direction and the vertical section's of lower tip side contained angle is the acute angle. During the use, breed the water of incasement and flow and collect from leading the water outlet and manage the pipeline, form decurrent rivers like this in breeding the incasement, drive the roe and flow downwards and through the necking down passageway, increase the fertilization chance, the water inlet of box is intake in the simultaneous control, keeps breeding incasement water level stable.
Compared with the cultivation box in the specific embodiment of the patent application, the cultivation box in the embodiment has the advantage that the fertilization rate of the zebra fish is 5.384-6.913% higher than that of the zebra fish tested under the experimental conditions of the blank control group of the patent application.
Example 2
The cultivation box of the embodiment is further modified on the cultivation box of the embodiment 1, as shown in fig. 3, a triangular diversion slope 41' is arranged on the surface of the partition board positioned on the upper box body, and of course, an inclined slope is still adopted at the partition board close to the side surface of the upper box body. An air inlet pipe and an air pump are also arranged, the air inlet pipe is connected with the air pump, an air outlet port of the air inlet pipe is positioned above the upper box body, a shower head 12 is arranged at the air outlet port of the air inlet pipe, and the shower head extends into the water surface of the upper box body and then ventilates downwards.
Example 3
A method for improving the fertilization rate of zebra fish comprises the following steps:
(1) separately and conventionally culturing female zebra fish and male zebra fish of 10 weeks old in a nutrition-enriched way, and keeping the illumination time to be prolonged to 12 hours/day, the illumination intensity to be 100lx and the rest time to be kept in a dark state;
(2) adding female zebra fish and male zebra fish into a culture box capable of separating fish eggs according to the ratio of female to male of 1:2 when the zebra fish is 20 weeks old, and adding melatonin into a water body of the culture box to keep the concentration of the melatonin in the water body at 5 mug/L;
(3) after the dark state is changed into illumination for 2 hours, quickly raising the water temperature to 1 ℃ within 30min and keeping for 2 hours, enabling male zebra fishes to chase and stimulate female zebra fishes to lay eggs, and then recovering the water temperature;
(4) and separating the fish eggs from the parent fish, and calculating the fertilization rate of the fish eggs.
Example 4
A method for improving the fertilization rate of zebra fish comprises the following steps:
(1) separating female zebra fish and male zebra fish of 11 weeks old by conventional nutrition-enriched cultivation, and keeping the illumination time to be prolonged to 14 hours/day, the illumination intensity to be 280lx, and keeping the rest time in a dark state;
(2) adding female zebra fish and male zebra fish into a culture box capable of separating fish eggs according to the ratio of female to male being 1:3 when the zebra fish is 20 weeks old, and adding melatonin into a water body of the culture box to keep the concentration of the melatonin in the water body at 8 mug/L;
(3) after the dark state is changed into illumination for 1 hour, quickly raising the water temperature by 2 ℃ within 45min and keeping for 3 hours, enabling male zebra fish to chase and stimulate female zebra fish to lay eggs, and then recovering the water temperature;
(4) and separating the fish eggs from the parent fish, and calculating the fertilization rate of the fish eggs.
Example 5
A method for improving the fertilization rate of zebra fish comprises the following steps:
(1) separating female zebra fish and male zebra fish of 12 weeks old by conventional nutrition-enriched cultivation, and keeping the illumination time to be prolonged to 14 hours/day, the illumination intensity to be 320lx, and keeping the rest time in a dark state;
(2) adding female zebra fish and male zebra fish into a culture box capable of separating fish eggs according to the ratio of female to male being 1:3 when the zebra fish is 21 weeks old, and adding melatonin into a water body of the culture box to keep the concentration of the melatonin in the water body at 10 mug/L;
(3) after the dark state is changed into illumination for 1 hour, the water temperature is rapidly raised to 2 ℃ within 60min and kept for 3 hours, male zebra fish chase stimulates female zebra fish to lay eggs, and then the water temperature is recovered;
(4) and separating the fish eggs from the parent fish, and calculating the fertilization rate of the fish eggs.
Example 6
A method for improving the fertilization rate of zebra fish is different from the embodiment 4 in that the step (1) further comprises the steps of adding a sunflower pollen extract into a water body in which male zebra fish is located, enabling the sunflower pollen extract to be 36 mu g/L, grinding and crushing the extract, sieving the ground extract with a 120-mesh sieve, then placing the extract in ethanol, carrying out reflux extraction at 26 ℃ for 80 hours, and carrying out ventilation drying at 0 ℃ to obtain the sunflower pollen extract.
Example 7
A method for improving the fertilization rate of zebra fish is different from the embodiment 4 in that the step (1) further comprises the steps of adding a sunflower pollen extract into a water body in which male zebra fish is located, enabling the sunflower pollen extract to be 30 microgram/L in concentration, grinding and crushing the extract through sunflower pollen, sieving the ground extract through a 200-mesh sieve, then placing the extract into ethanol, extracting the extract for 96 hours under reflux at 23 ℃, and then drying the extract in a ventilation mode at 4 ℃ to obtain the sunflower pollen extract.
Example 8
A method for improving the fertilization rate of zebra fish is different from the embodiment 4 in that the step (1) further comprises the steps of adding a sunflower pollen extract into a water body in which male zebra fish is located, enabling the sunflower pollen extract to be 40 mu g/L, grinding and crushing the extract, sieving the ground extract by a 100-mesh sieve, then placing the extract in ethanol, carrying out reflux extraction at 28 ℃ for 72 hours, and carrying out ventilation drying at 0 ℃ to obtain the sunflower pollen extract.
Control group 1
The blank control group of patent CN201711320960.6 was used, except that the cultivation box of example 1 of the present application was used in place of the device mentioned in the blank control group.
Control group 2
The difference from the example 3 is that melatonin of 5 mug/L is injected into the female zebra fish in the step (2) instead of adding the melatonin into the water body.
Control group 3
The difference from example 3 is that in step (3), the water temperature was raised to 1 ℃ and maintained for 5 hours after shifting from the dark state to the light state and then for 2 hours.
Control group 4
The difference from example 6 is that the sunflower pollen extract was added at a concentration of 100. mu.g/L.
Control group 5
The difference from example 6 is that the sunflower pollen extract was added at a concentration of 10. mu.g/L.
Control group 6
The difference from example 6 is that the sunflower pollen extract is prepared at an ethanol extraction temperature of 60 ℃.
Control group 7
The difference from example 6 is that the sunflower pollen extract is prepared at an ethanol extraction temperature of 10 ℃.
Statistics are carried out on the fertilization rates of the zebra fish in the examples 3-8 and the control groups 1-7, and the results are shown in the following tables 1-2.
TABLE 1 test results of examples 3 to 8 and control groups 1 to 2
TABLE 1 test results for examples 3-8 and control 1
Figure BDA0002233210740000061
TABLE 2 test results of control 2 to control 7
Index (I) Control group 2 Control group 3 Control group 4 Control group 5 Control group 6 Control group 7
Fertilization rate/%) 84.01±1.664 85.24±1.825 84.92±1.006 85.67±1.768 85.22±1.322 85.82±2.062

Claims (8)

1. A method for improving the fertilization rate of zebra fish is characterized by comprising the following steps:
(1) carrying out conventional nutrition-enriched cultivation on female zebra fish and male zebra fish of 10-12 weeks old separately, keeping the illumination time to be prolonged to 12-14 hours/day, and keeping the rest time in a dark state;
(2) adding female zebra fish and male zebra fish into a culture box capable of separating fish eggs according to the ratio of female to male being 1: 2-3 when the zebra fish is 20-21 weeks old, adding melatonin into a water body of the culture box, and keeping the concentration of the melatonin in the water body at 5-10 mug/L;
(3) keeping the illumination, quickly raising the water temperature by 1-2 ℃ within 30-60 min and keeping for 2-3 hours, enabling male zebra fish to chase and stimulate female zebra fish to lay eggs, and then recovering the water temperature;
(4) and separating the fish eggs from the parent fish, and calculating the fertilization rate of the fish eggs.
2. The method for improving fertilization rate of zebra fish according to claim 1, wherein the cultivation box capable of separating fish eggs in step (2) comprises an upper box body, a lower box body and a middle channel communicating the upper box body and the lower box body, wherein a plurality of baffles are arranged between the middle channel and the upper box body side by side, adjacent baffles form a space which can block female zebra fish and male zebra fish from passing through but allow fish eggs to pass through, the middle channel is connected with the upper end part of the upper box body and the lower end part of the middle channel is connected with the lower box body and is in a trumpet shape with the caliber gradually reduced towards the middle part of the middle channel, the middle part of the middle channel is in a necking channel, a water outlet is arranged on the side surface of the lower end part, and a water inlet is arranged at the port of the upper box body.
3. The method for improving the fertilization rate of the zebra fish according to claim 2, wherein the water outlet is arranged at the lower end part near the necking channel, an inclined guide plate for shielding the water outlet is further arranged at the inner side of the lower end part, the joint of the inclined guide plate and the lower end part is arranged above the water outlet, and the included angle between the cross section of the inclined guide plate and the longitudinal section of the side surface of the lower end part is an acute angle.
4. The method for improving fertilization rate of zebra fish according to claim 2 or 3, wherein the surface of the partition board in the upper box body is provided with a diversion slope with a circular arch or triangle cross section.
5. The method for improving the fertilization rate of the zebra fish according to claim 2 or 3, wherein an air inlet pipe and an air pump connected with the air inlet pipe are further arranged in the upper box body, a shower head is connected to an air outlet port of the air inlet pipe, and the air outlet port is located above the upper box body and extends into the water surface of the upper box body.
6. The method for improving fertilization rate of zebra fish according to claim 1, wherein the pH value of the water in the cultivation box is controlled to be 7-8 in steps (2) - (4), and the illumination intensity is controlled to be 100-320 lx.
7. The method for improving fertilization rate of zebra fish according to claim 1, wherein the step (3) of maintaining the light is performed after the state of darkness is changed to light for 1-2 hours.
8. The method for improving fertilization rate of zebra fish according to claim 1, further comprising the step of (1) adding sunflower pollen extract to the water body in which the male zebra fish is located, wherein the concentration of the sunflower pollen extract is 30-40 μ g/L; the preparation process of the sunflower pollen extract comprises the following steps: grinding and crushing sunflower pollen, sieving the sunflower pollen with a sieve of 100-200 meshes, then placing the sunflower pollen in ethanol for reflux extraction at 23-28 ℃ for 72-96 hours, and then carrying out ventilation drying at 0-4 ℃ to obtain the sunflower pollen extract.
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