A kind of artificial breeding method of golden tiger hybrid spot

Abstract

The present invention provides an artificial breeding method for a hybrid grouper of golden tiger. The artificial breeding method comprises the following steps: (1) selecting a healthy brown-spotted grouper aged 3 or above as the female parent; selecting a healthy blue-bodied large-spotted grouper aged 5 or above as the male parent; (2) cultivating the female parent by feeding feed I and feed II daily, wherein feed I is prepared from the following raw materials: soybean powder, vitamin E, scutellaria baicalensis leaf powder, dodder seed, amomum villosum, rehmannia root, and curculigo extract; (3) cultivating the male parent by feeding feed III and feed IV daily; wherein feed III is prepared from the following raw materials: fish meal, choline chloride, pine needle powder, salt algae powder, Morinda officinalis, and Tribulus terrestris extract; (4) after the parent fish reach sexual maturity, collecting eggs from the brown-spotted grouper and semen from the blue-bodied large-spotted grouper, performing artificial insemination, and obtaining fertilized eggs; (5) hatching the fertilized eggs to obtain fry. The breeding method of the present invention effectively increases egg production, and the fertilized eggs are of high quality and the yield of healthy seedlings is large.

Description

Technical Field

[0001] The invention relates to the field of grouper breeding, and in particular to an artificial breeding method for golden tiger hybrid grouper. Background Art

[0002] Grouper is a vital marine fish resource in my country and a pillar of the industry, with an annual demand for fry reaching 1 billion. However, problems such as frequent diseases and low survival rates among grouper fry have become bottlenecks in the industry's development. Through research on the construction of a diversified grouper germplasm bank, the cultivation of hybrid strains with superior traits, phenotypic and genetic trait analysis, genetic analysis of hybrid vigor traits, identification of key genes and molecular markers, and the large-scale propagation and industrial application of superior hybrid germplasm, we are developing technologies for improving grouper varieties and cultivating high-quality germplasm with fast growth and strong stress resistance, thereby addressing the challenges of frequent fry diseases and low survival rates in the industry.

[0003] The "Golden Tiger Hybrid Grouper" is a hybrid bred from a brown-spotted grouper (female) and a blue-bodied grouper (male). Some fry of this hybrid exhibited deformities, impacting the yield of the Golden Tiger Hybrid Grouper. Therefore, further optimizing the breeding process will help improve the yield and quality of Golden Tiger Hybrid Grouper fry and promote the healthy development of the grouper industry, which is of great significance. Summary of the Invention

[0004] In view of this, the present invention proposes an artificial breeding method for golden tiger hybrid spot to solve the above problems.

[0005] The technical solution of the present invention is achieved as follows: an artificial breeding method for golden tiger hybrid spot, comprising the following steps:

[0006] (1) Select healthy brown-spotted grouper over 3 years old as the female parent; select healthy blue-spotted grouper over 5 years old as the male parent;

[0007] (2) In the maternal cultivation, feed I and feed II were fed every day, wherein the feed I was prepared from the following raw materials: soybean powder, vitamin E, scutellaria baicalensis leaf powder, dodder seed, amomum villosum, rehmannia root, and curculigo extract; and the feed II was prepared from the following raw materials: squid, loach, snail meat, and conch meat;

[0008] (3) Male parent cultivation, feeding feed III and feed IV every day; the feed III is made from the following raw materials: fish meal, choline chloride, pine needle powder, salt algae powder, Morinda officinalis, and Tribulus terrestris extract; the feed IV is made from the following raw materials: mackerel, sardines, snail meat, and oysters;

[0009] (4) After the broodstock have reached sexual maturity, eggs of brown-spotted grouper and semen of blue-bodied grouper are collected and artificial insemination is performed to obtain fertilized eggs;

[0010] (5) Hatching the fertilized eggs to obtain fry.

[0011] Furthermore, the feed I is prepared by comprising the following raw materials in parts by weight: 15-25 parts of soybean powder, 4-6 parts of vitamin E, 10-20 parts of scutellaria baicalensis leaf powder, 12-18 parts of dodder seeds, 8-12 parts of amomum villosum, 10-20 parts of rehmannia root, and 15-25 parts of curculigo extract; the preparation method of the feed I comprises the following steps: taking dodder seeds, amomum villosum, and rehmannia root medicinal materials, crushing them, adding water at a material-liquid ratio of 1:8-12 g / ml, decocting for 20-40 minutes, collecting the decoction to obtain a traditional Chinese medicine extract, adding the curculigo extract, scutellaria baicalensis leaf powder, soybean powder, and vitamin E, mixing, and drying to prepare a granular feed.

[0012] Furthermore, the feed II is prepared by including the following raw materials in parts by weight: 15-25 parts of squid, 20-30 parts of loach, 10-20 parts of snail meat, and 35-45 parts of conch meat.

[0013] Furthermore, the feed III is prepared by including the following raw materials in parts by weight: 15-25 parts of fish meal, 4-6 parts of choline chloride, 8-12 parts of pine needle powder, 15-25 parts of salina powder, 18-22 parts of Morinda officinalis, and 20-30 parts of Tribulus terrestris extract; the preparation method of the feed III comprises the following steps: taking Morinda officinalis medicinal material, crushing it, adding 70%-80% v / v ethanol solution at a material-liquid ratio of 1:4-6 g / ml, and extracting for 8-12 hours at a soaking temperature of 50-60°C, collecting the extract, adding Tribulus terrestris extract, salina powder, pine needle powder, fish meal, and choline chloride, mixing, and drying to prepare a granular feed.

[0014] Furthermore, the feed IV is prepared by comprising the following raw materials in parts by weight: 15-25 parts of mackerel, 20-30 parts of sardines, 20-30 parts of snail meat, and 25-35 parts of oysters.

[0015] Furthermore, the preparation of the Curculigo extract is as follows: take Curculigo, crush it into 100 mesh, add 75%-85% v / v ethanol solution at a solid-liquid ratio of g / ml of 1:8-12, soak for 0.5-1h, reflux extraction 2-3 times, each time for 1-2h, filter, combine the extracts, and concentrate under reduced pressure to recover ethanol to obtain the Curculigo extract.

[0016] Furthermore, the preparation of the Tribulus terrestris extract is as follows: the Tribulus terrestris raw material is taken, soaked in 3%-5% v / v acetic acid solution for 1-2 hours, the Tribulus terrestris is taken out, ethyl acetate is added at a solid-liquid ratio of g / ml of 1:5-6, and extracted for 1-2 hours, filtered to obtain the extract, and concentrated under reduced pressure to obtain the Tribulus terrestris extract.

[0017] Furthermore, feed I and feed II are fed 2-3 times a day, with each feeding amount being 1% to 2% of the parent fish body weight, and the feeding weight ratio of feed I to feed II is 1:8-12.

[0018] Furthermore, feed III and feed IV are fed 2-3 times a day, with each feeding amount being 2% to 3% of the parent fish body weight, and the feeding weight ratio of feed III to feed IV is 1:14-16.

[0019] Furthermore, the specific operation of the artificial insemination is as follows: add seawater to the container containing the eggs, and then add semen, with the ratio of eggs, semen and seawater being 1kg:1-1.5mL:1-1.5mL, mix well and let it stand for 3-4 minutes.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The breeding method of the present invention effectively increases the amount of eggs laid, has a good fertilization rate, and the quality of the fertilized eggs is high, has a high hatching rate, the fry are free of deformities, and the yield of healthy seedlings is large. Among them, feeding feed I and feed II every day in the maternal cultivation effectively increases the amount of eggs laid and the quality of eggs laid, and feeding feed III and feed IV every day in the paternal cultivation greatly improves the quality of semen, comprehensively improves the quality of fertilized eggs, reduces or even avoids fry deformities, increases the yield of healthy seedlings, is conducive to improving the survival rate and growth rate of fry, has good stress resistance traits, and improves breeding efficiency and the output rate of excellent varieties.

[0022] (2) In the maternal cultivation of the present invention, feed I prepared from soybean powder, vitamin E, Glechoma longituba leaf powder, Cuscuta chinensis, Amomum villosum, Rehmannia glutinosa and Curculigo extract, combined with feed II prepared from squid, loach, snail meat and conch meat, not only improves the physical fitness and immunity of female brown-spotted grouper, but also effectively improves the spawning quality of female fish; in the paternal cultivation of the present invention, feed III prepared from fish meal, choline chloride, pine needle powder, salt algae powder, Morinda officinalis and Tribulus terrestris extract, combined with feed IV prepared from mackerel, sardines, snail meat and oyster, helps to increase the amount of semen, improve the sperm quality, and enhance the reproduction and breeding ability. DETAILED DESCRIPTION

[0023] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0024] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0025] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0026] Preparation Example 1-Feed I

[0027] (1) Preparation of Curculigo extract: Take Curculigo medicinal material, grind it into 100 mesh, add 80% v / v ethanol solution at a solid-liquid ratio of g / ml of 1:10, soak for 0.5 h, reflux extraction twice, each time for 1.5 h, filter, combine the extracts, and concentrate under reduced pressure to recover ethanol to obtain Curculigo extract.

[0028] (2) The preparation method of feed I comprises the following steps: taking Cuscuta chinensis, Amomum villosum and Rehmannia glutinosa medicinal materials respectively according to Table 1, crushing them, adding water at a material-liquid ratio of 1:10 g / ml, decocting for 30 min, collecting the decoction to obtain a Chinese medicine extract, adding Curculigo extract, Glechoma longituba leaf powder, soybean powder and vitamin E, mixing, drying and preparing granular feed, respectively, to obtain feed I-A, feed I-B and feed I-C.

[0029] Table 1-Feed I Ingredients

[0030]

[0031] Preparation Example 2-Feed II

[0032] According to the ingredients in Table 2, take the raw materials, mix them, mince the meat and set aside.

[0033] Table 2-Feed II Ingredients

[0034]

[0035] Preparation Example 3-Feed III

[0036] (1) Preparation of Tribulus terrestris extract: Take the raw material of Tribulus terrestris, soak it in 4% v / v acetic acid solution for 1.5 hours, take out the Tribulus terrestris, add ethyl acetate at a solid-liquid ratio of g / ml of 1:5, and soak it for 1.5 hours. Filter to obtain the extract, and concentrate under reduced pressure to obtain the Tribulus terrestris extract.

[0037] (2) The preparation method of feed III comprises the following steps: taking Morinda officinalis medicinal materials according to Table 3, crushing them, adding 75% v / v ethanol solution at a material-liquid ratio of g / ml of 1:5, and soaking for 10 hours at a soaking temperature of 55℃±5℃, collecting the extract, adding Tribulus terrestris extract, Dunaliella salina powder, pine needle powder, fish meal, and choline chloride, mixing, drying, and making granular feed.

[0038] Table 3 - Feed III ingredients

[0039]

[0040] Preparation Example 4-Feed IV

[0041] According to the ingredients in Table 4, take the raw materials, mix them, mince the meat and set aside.

[0042] Table 4-Feed IV Ingredients

[0043]

[0044] Example 1 - Artificial breeding method of golden tiger hybrid spot

[0045] (1) Select healthy brown-spotted grouper over 3 years old as the female parent; select healthy blue-spotted grouper over 5 years old as the male parent; the male-female ratio is 1:0.5;

[0046] (2) For parent cultivation, feed I-A and feed II-A were fed every day. I-A and feed II-A were fed twice a day. The feeding amount each time was 1% to 2% of the parent fish body weight. The feeding weight ratio of feed I-A to feed II-A was 1:10.

[0047] (3) For male parent cultivation, feed III-A and feed IV-A were fed every day; feed III-A and feed IV-A were fed three times a day, and the feeding amount each time was 2% to 3% of the parent fish body weight, and the feeding weight ratio of feed III-A to feed IV-A was 1:15.

[0048] (4) When the broodstock reached sexual maturity, eggs of brown-spotted grouper and semen of blue-bodied grouper were collected and artificial insemination was performed. The specific operation of artificial insemination was as follows: seawater was added to the container containing eggs, and then semen was added. The ratio of eggs, semen and seawater was 1 kg:1 mL:1 mL. After mixing, the mixture was allowed to stand for 3 minutes to obtain fertilized eggs.

[0049] (5) The fertilized eggs were removed and placed in a culture pond for incubation. The water in the pond was slightly aerated, with a water temperature of 25°C ± 2°C, a salinity of 30‰, and a pH of 8.0 ± 0.1 to obtain fry.

[0050] Example 2 - Artificial breeding method of golden tiger hybrid spot

[0051] (1) Select healthy brown-spotted grouper over 3 years old as the female parent; select healthy blue-spotted grouper over 5 years old as the male parent; the male-female ratio is 1:0.5;

[0052] (2) For parent cultivation, feed I-B and feed II-B were fed every day. Feed I-B and feed II-B were fed twice a day. The feeding amount each time was 1% to 2% of the parent fish body weight. The feeding weight ratio of feed I-B to feed II-B was 1:10.

[0053] (3) For male parent cultivation, feed III-B and feed IV-B were fed every day; feed III-B and feed IV-B were fed three times a day, and the feeding amount each time was 2% to 3% of the parent fish body weight, and the feeding weight ratio of feed III-B to feed IV-B was 1:15.

[0054] (4) When the broodstock reached sexual maturity, eggs of brown-spotted grouper and semen of blue-bodied grouper were collected and artificial insemination was performed. The specific operation of artificial insemination was as follows: seawater was added to the container containing eggs, and then semen was added. The ratio of eggs, semen and seawater was 1 kg:1 mL:1 mL. After mixing, the mixture was allowed to stand for 3 minutes to obtain fertilized eggs.

[0055] (5) The fertilized eggs were removed and placed in a culture pond for incubation. The water in the pond was slightly aerated, with a water temperature of 25°C ± 2°C, a salinity of 30‰, and a pH of 8.0 ± 0.1 to obtain fry.

[0056] Example 3 - Artificial breeding method of golden tiger hybrid spot

[0057] (1) Select healthy brown-spotted grouper over 3 years old as the female parent; select healthy blue-spotted grouper over 5 years old as the male parent; the male-female ratio is 1:0.5;

[0058] (2) For parent cultivation, feed I-C and feed II-C were fed every day. Feed I and feed II were fed twice a day. The feeding amount each time was 1% to 2% of the parent fish body weight. The feeding weight ratio of feed I-C to feed II-C was 1:10.

[0059] (3) For male parent cultivation, feed III-C and feed IV-C were fed every day; feed III and feed IV were fed three times a day, and the feeding amount each time was 2% to 3% of the parent fish body weight, and the feeding weight ratio of feed III-C to feed IV-C was 1:15.

[0060] (4) When the broodstock reached sexual maturity, eggs of brown-spotted grouper and semen of blue-bodied grouper were collected and artificial insemination was performed. The specific operation of artificial insemination was as follows: seawater was added to the container containing eggs, and then semen was added. The ratio of eggs, semen and seawater was 1 kg:1 mL:1 mL. After mixing, the mixture was allowed to stand for 3 minutes to obtain fertilized eggs.

[0061] (5) The fertilized eggs were removed and placed in a culture pond for incubation. The water in the pond was slightly aerated, with a water temperature of 25°C ± 2°C, a salinity of 30‰, and a pH of 8.0 ± 0.1 to obtain fry.

[0062] Comparative Example 1

[0063] The main difference from Example 1 is that the feed I was used without adding Cuscuta seeds and Curculigo extracts, and the amount of Patella datura leaf powder, Amomum villosum, and Rehmannia root was increased.

[0064] Table 5 - Ingredients

[0065] raw material Dosage / g soybean flour 20 Vitamin E 5 Cistanche deserticola leaf powder 30 Cuscuta australis 0 Amomum villosum 20 Rehmannia root 25 Curculigo Extract 0

[0066] Other operations are consistent with those in Example 1.

[0067] Comparative Example 2

[0068] The main difference from Example 1 is that the feed III does not contain the Tribulus terrestris extract, but is replaced by the Cistanche deserticola extract.

[0069] Table 6 - Ingredients

[0070] raw material Dosage / g fishmeal 20 Choline chloride 5 pine needle powder 10 Dunaliella powder 20 Morinda officinalis 20 Cistanche deserticola extract 25

[0071] Other operations are consistent with those in Example 1.

[0072] Test example

[0073] In January 2023, at the Hainan Oriental grouper breeding farm, golden tiger hybrid groupers were bred in indoor cultivation ponds according to the artificial breeding methods of Examples 1-3 and Comparative Examples 1-2, respectively. Test groups 1-5 were set, and the number of female healthy brown-spotted groupers (over 3 years old) in each group was 250. The test groups 1-5 were bred according to the artificial breeding methods of Examples 1-3 and Comparative Examples 1-2, respectively, to obtain golden tiger hybrid grouper fry. The egg production, fertilization rate, hatching rate, and fry deformity rate of each of the above Examples 1-3 and Comparative Examples 1-2 were measured respectively.

[0074] The average egg weight per female broodstock = total egg weight / number of female broodstock

[0075] The results are shown in Table 7.

[0076] Table 7 Artificial breeding of hybrid golden tiger spots

[0077]

[0078] The above results show that the breeding methods of Examples 1-3 of the present invention effectively increase the egg production, have a good fertilization rate, and the fertilized eggs are of high quality and have a high hatching rate. The fry have no deformities and the yield of healthy fry is large.

[0079] Among them, in Comparative Example 1, the dodder and curculigo extracts were not added, and not only the egg production was significantly reduced, but also the quality of the fertilized eggs was greatly affected. In Comparative Example 2, the dodder extract was not added, and the fertilization rate and hatching rate were significantly reduced, and the deformity rate of the fry was affected.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An artificial breeding method for golden tiger hybrid spot, characterized in that, The following steps are involved: (1) Select healthy brown-spotted grouper over 3 years old as the female parent; select healthy blue-spotted grouper over 5 years old as the male parent; (2) Cultivation of mother plants: feed I and feed II are fed every day. Feed I is prepared by the following raw materials in parts by weight: 20-25 parts of soybean powder, 5-6 parts of vitamin E, 10-15 parts of Curculigo leaf powder, 12-15 parts of Cuscuta seeds, 10-12 parts of Amomum villosum, 15-20 parts of Rehmannia root, and 20-25 parts of Curculigo extract. Preparation of feed I: take Cuscuta seeds, Amomum villosum, and Rehmannia root, grind them, add water at a material-liquid ratio of 1:8-12 g / ml, boil for 20-40 minutes, collect the decoction to obtain the Chinese medicine extract, add Curculigo extract, Curculigo leaf powder, soybean powder, and vitamin E, mix, dry, and make pellet feed. Feed II is prepared by including the following raw materials in parts by weight: 15-20 parts of squid, 25-30 parts of loach, 10-15 parts of snail meat, and 40-45 parts of conch meat; Preparation of Curculigo extract: Take Curculigo, grind it into 100 mesh, add 75%-85% v / v ethanol solution at a solid-liquid ratio of g / ml of 1:8-12, soak for 0.5-1 hour, reflux extraction 2-3 times, each time for 1-2 hours, filter, combine the extracts, and concentrate under reduced pressure to recover ethanol to obtain Curculigo extract; (3) Male parent cultivation, feeding feed III and feed IV every day; feed III is made from the following raw materials in parts by weight: 15-20 parts of fish meal, 5-6 parts of choline chloride, 10-12 parts of pine needle powder, 15-20 parts of Dunaliella powder, 20-22 parts of Morinda officinalis, and 25-30 parts of Tribulus terrestris extract; preparation of feed III: take Morinda officinalis medicinal material, crush it, add 70%-80% v / v ethanol solution at a material-liquid ratio of 1:4-6, and extract for 8-12 hours at a soaking temperature of 50-60°C, collect the extract, add Tribulus terrestris extract, Dunaliella powder, pine needle powder, fish meal, and choline chloride, mix, dry, and make granular feed; feed IV is made from the following raw materials in parts by weight: 15-20 parts of mackerel, 25-30 parts of sardines, 20-25 parts of snail meat, and 30-35 parts of oysters; Preparation of Tribulus terrestris extract: Take Tribulus terrestris raw material, soak it in 3%-5% v / v acetic acid solution for 1-2 hours, remove the Tribulus terrestris, add ethyl acetate at a solid-liquid ratio of 1:5-6 g / ml, and extract for 1-2 hours, filter to obtain the extract, and concentrate under reduced pressure to obtain the Tribulus terrestris extract; (4) When the broodstock reach sexual maturity, collect eggs from brown-spotted grouper and semen from blue-bodied grouper, perform artificial insemination, and obtain fertilized eggs; (5) Hatching the fertilized eggs to obtain fry.

2. The artificial breeding method of a golden tiger hybrid spot according to claim 1, wherein Feed I and Feed II are fed 2-3 times a day, and the feeding amount each time is 1%~2% of the broodstock body weight. The feeding weight ratio of Feed I and Feed II is 1:8-12.

3. The artificial breeding method of a golden tiger hybrid spot according to claim 1, wherein Feed III and Feed IV are fed 2-3 times a day, and the feeding amount each time is 2%~3% of the broodstock body weight. The feeding weight ratio of Feed III to Feed IV is 1:14-16.

4. The artificial breeding method of a golden tiger hybrid spot according to claim 1, wherein The specific operation of artificial insemination is as follows: add seawater to the container containing eggs, and then add semen. The ratio of eggs, semen and seawater is 1kg: 1-1.5mL: 1-1.5mL. After mixing, let it stand for 3-4 minutes.

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