Method for completely obtaining high-quality sebastes schlegeli sperms without damage

Abstract

The invention discloses a method for obtaining high-quality sebastes schlegeli sperms in a non-destructive and sufficient manner, and belongs to the technical field of ovovial fish breeding. The method comprises the following steps: (1) carrying out fine culture on male sebastes schlegeli of more than 3 years old, and obtaining sebastes schlegeli of which the genital length is greater than or equal to 1.1 cm as a sperm collection parent; (2) fishing out the sperm-taking parents from the culture water body, and sequentially and immediately carrying out wet towel wrapping fish body airing stress treatment and gill extrusion treatment; and (3) after the sperm parents are subjected to stress stimulation treatment, immediately obtaining sperms by adopting a method of reversely extruding genitals. The sebastes schlegeli sperm collection method has the advantages that high-quality sebastes schlegeli sperms can be efficiently and sufficiently obtained, manual operation steps are reduced, damage of sperm collection to parent fishes is reduced, and breed conservation and cyclic utilization of breeding materials with dominant characters are achieved.

Description

Technical Field

[0001] The invention relates to a method for obtaining fish sperm, in particular to a method for obtaining sufficient high-quality sperm of Scolopendra subspinipes without damage, and belongs to the technical field of ovoviviparous fish breeding. Background Art

[0002] The black carp, also known as black carp, blackhead fish, black rock bass, etc., is mainly distributed in the bottom of the northwest Pacific coast. It is commonly found in the Bohai Sea, Yellow Sea and East China Sea in my country, the waters on both sides of North Korea, the southern waters of Hokkaido and the southern waters of the Sea of ​​Okhotsk in Japan. The black carp has strong resistance to adversity, a wide range of diets and can survive the winter naturally. It is an important economic fish species for cage culture and reproduction and release in northern my country. In recent years, the rapid development of my country's deep-sea cage culture industry has made the black carp increasingly prominent in the fishery economy of my country's northern coastal areas.

[0003] The continuous expansion of the aquaculture industry has made the breeding demand for high-quality fry of Xu's flatfish particularly urgent. However, as an ovoviviparous fish, after the male and female broodstock of Xu's flatfish complete mating in November each year, the sperm is stored in the female fish for 3 months before fertilization and development into fry. In addition, Xu's flatfish has a mating pattern of "one female with multiple males" and "one male with multiple females" during natural mating. It is difficult to track the father of the offspring during the breeding process, and sometimes there is more than one father to track, so the efficiency of breeding is very low.

[0004] In order to solve this problem, the genetic breeding team of Xu's flatfish of Shandong Institute of Marine Resources and Environment developed the "Xu's flatfish artificial insemination technology", which realized the construction of a "one female and one male" full-sib family in breeding work, thus completely breaking the bottleneck of very low efficiency in breeding of good varieties.

[0005] However, in the process of artificial insemination, the method of "killing the father to obtain sperm" is used to obtain sperm from Xu's flatfish, that is, dissecting male broodstock, obtaining testes, cutting up the testes to release sperm. This method cannot preserve the living male parent and will lose breeding materials with excellent traits. After years of technical exploration, it is still not possible to obtain sufficient high-quality sperm without damaging the broodstock of Xu's flatfish. This lack of technology has restricted the genetic breeding and artificial reproduction of Xu's flatfish and hindered the development of the breeding industry. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the object of the present invention is to provide a method for obtaining sufficient amount of high-quality sperm of Scolopendra subspinipes without damage.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for obtaining high-quality sperm of Scolopendra subspinipes without damage, comprising the following steps:

[0009] (1) Male flathead mullets aged 3 years or older were selected for fine culture starting from early September each year, and flathead mullets with a genital length of ≥1.1 cm were obtained as sperm parents;

[0010] (2) subjecting the sperm collection parent to a stress stimulation treatment. Specifically, the sperm collection parent is fished out of the aquaculture water body, and the fish body is immediately wrapped in a wet towel and left to air-dry for a stress treatment of 60s to 90s, and then the gills are immediately squeezed for a treatment time of 10s;

[0011] (3) After the sperm-collecting parent has been subjected to stress stimulation, the sperm is immediately obtained by reversely squeezing the genitals.

[0012] Preferably, in step (1), the fine culture method is specifically as follows: (i) selection and feeding of bait: fresh sand croaker or small yellow croaker is selected as bait fish, and a mixture of multivitamins, fish oil, arginine, lysine and minerals is stuffed into the gastrointestinal tract of the bait fish, wherein the amounts of the multivitamins, fish oil, arginine, lysine and mineral mixture are 3wt%-5wt%, 5wt%-8wt%, 2%-3%, 2%-3% and 1%-2% of the weight of the bait fish, respectively; After the bait is prepared, it can be fed to the male broodstock, once a day, and the feeding amount is 3-5% of the weight of the male broodstock; (ii) Cultivation light requirements: light intensity 300lx, light cycle is the same as the natural light cycle; (iii) Cultivation water temperature requirements: the water temperature slowly drops from 24°C to 16°C, with an average daily drop of 0.1°C; (iv) Other cultivation condition requirements: dissolved oxygen ≥6mg / L, ammonia nitrogen content ≤0.05mg / L, salinity 28-30, harmful bacteria content ≤10 3 CFU / m 3 , change the water twice a day, each time changing more than 70%.

[0013] Preferably, in step (2), the method of wrapping the fish body with a wet towel and leaving it to air for stress treatment is specifically as follows: wrap the body of the sperm collection parent with a wet towel, exposing the head and tail, and place it on a mesh to air; the method of squeezing the gills is specifically as follows: wrap the entire head of the sperm collection parent with a wet towel, and gently squeeze the gills to restrict breathing.

[0014] Preferably, in step (3), the method of reverse squeezing the genitals is specifically as follows: invert the sperm collection parent with its head facing downward and its tail facing upward, wrap the head as a whole with a wet towel, fix the head and tail, wipe the area around the genitals with a dry towel, pinch the thumb and index finger from the pectoral fin to 1 / 2 of the genitals, gently squeeze while slowly moving toward the genitals, move both fingers to the side of the genitals close to the tail and then reverse squeeze the genitals; if semen flows out after one squeeze, collect the semen and the squeezing operation is completed; otherwise, repeat the reverse squeezing operation until semen flows out or the genitals are damaged.

[0015] The present invention is beneficial in that:

[0016] (1) It can efficiently obtain high-quality sperm from the flatfish, reduce the number of manual operations, and reduce the damage to the broodstock during sperm extraction;

[0017] (2) Sufficient high-quality sperm can be obtained from the flatfish. The number of sperm obtained from one male fish can reach 10 8 -10 9 The sperm quantity can meet the needs of artificial insemination of at least 3 female fish at the same time, and can also meet the requirements of sperm cryopreservation, stem cell transplantation, directional breeding and other research and application requirements;

[0018] (3) The sperm obtained was of high quality and free of impurities. Under a microscope, it was found to be structurally intact and vigorous, with an activity rate of more than 95%, which could meet the needs of downstream research and production.

[0019] (4) While obtaining sufficient amounts of high-quality sperm from the flatfish, the living parental material of the flatfish was retained, thus achieving the conservation and recycling of breeding materials with superior traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a picture of the reproductive organs of a mature male broodstock (the parent from which sperm is collected);

[0021] Figure 2 This is a schematic diagram of the squeezing starting point of the thumb and index finger when performing reverse squeezing of the genitals;

[0022] Figure 3 It is a schematic diagram of the squeezing process of the thumb and index finger when performing reverse squeezing of the genitals;

[0023] Figure 4 This is a picture of the testis of the flatfish with a genital length of ≤0.6cm;

[0024] Figure 5 This is a picture of the testis of the flatfish with a genital length ranging from 0.6 cm (not included) to 1.1 cm (not included);

[0025] Figure 6 This is a picture of the testis of the flatfish with a genital length of ≥1.1cm;

[0026] Figure 7 This is a schematic diagram of wrapping a fish body with a wet towel when performing a stress treatment of wrapping a fish body with a wet towel and leaving it to air-dry;

[0027] Figure 8 This is a picture of the semen obtained from a mature male broodstock (the parent from which the semen is collected). DETAILED DESCRIPTION

[0028] The method for obtaining a sufficient amount of high-quality sperm of Xu's flatfish without damage provided by the present invention comprises three steps: first, finely culturing Xu's flatfish to obtain mature male broodstock; second, subjecting the mature male broodstock to stress stimulation treatment; and third, obtaining a sufficient amount of high-quality sperm of Xu's flatfish from the male broodstock subjected to stress stimulation treatment by adopting a method of reversely squeezing the genitals.

[0029] The above three steps are specifically introduced below with reference to the accompanying drawings.

[0030] 1. Carefully cultivate the flathead mullet to obtain mature male broodstock

[0031] 1. Fine cultivation of Xu's flathead mullet

[0032] Select male 3-year-old flathead scorpionfish as broodstock and start fine cultivation from early September each year. The method of fine cultivation is as follows:

[0033] (1) Selection and feeding of bait: fresh sand croaker (or small yellow croaker) is selected as bait fish, and a mixture of multivitamins, fish oil, arginine, lysine and minerals is stuffed into the gastrointestinal tract of the bait fish. The amounts of the multivitamins, fish oil, arginine, lysine and mineral mixture are 3wt%-5wt% (4% in this embodiment), 5wt%-8wt% (6% in this embodiment), 2%-3% (2.5% in this embodiment), 2%-3% (2.5% in this embodiment), and 1%-2% (1.5% in this embodiment) of the weight of the bait fish, respectively. Among them:

[0034] Each 1kg of multivitamin contains: vitamin A acetate 0.73g, vitamin B120.003g, vitamin C121.2g, vitamin D30.003g, DL vitamin E tocopheryl acetate 18.8g, vitamin K1.8g, thiamine hydrochloride 2.7g, pyridoxine hydrochloride 1.8g, niacin 27.8g, folic acid 0.68g, riboflavin 9.1g, calcium pantothenate 12.7g, inositol 181.8g and biotin 0.27g, and the rest is cellulose;

[0035] Each 1kg of the mineral mixture contains: 80g of magnesium sulfate, 370g of sodium dihydrogen phosphate, 130g of potassium chloride, 40g of ferric citrate, 20g of zinc sulfate, 0.2g of cuprous chloride, 0.15g of aluminum chloride, 0.15g of potassium iodide, 0.01g of sodium selenite, 2.0g of manganese methionine and 1.0g of cobalt chloride, and the rest is cellulose.

[0036] After the bait is prepared, it can be fed to the male broodstock once a day, and the feeding amount is 3-5% of the weight of the male broodstock (the feeding amount in this embodiment is 4%). (2) Cultivation light requirements: light intensity 300lx, light cycle is the same as the natural light cycle;

[0037] (3) Cultivation water temperature requirements: The water temperature should be slowly reduced from 24°C to 16°C, with an average daily drop of 0.1°C, to avoid acute and drastic temperature drops;

[0038] (4) Other cultivation conditions: dissolved oxygen ≥ 6 mg / L, ammonia nitrogen content ≤ 0.05 mg / L, salinity 28-30, harmful bacteria content ≤ 10 3 CFU / m 3 , change the water twice a day, each time changing more than 70%.

[0039] As a control, male Scorpionfish aged 3 years and above were selected as broodstock and routine culture was carried out from the beginning of September each year. The specific method of routine culture is as follows:

[0040] (1) Selection and feeding of bait: Fresh sand mullet (or small yellow croaker) is selected as bait fish to feed male broodstock, and the feeding amount is 3%-5% of the weight of male broodstock (the feeding amount in this embodiment is 4%) once a day;

[0041] (2) Cultivation lighting requirements: Turn on the lights during daily management such as feeding and water changes, and turn off the lights after the work is completed;

[0042] (3) Cultivation water temperature requirements: the same as the natural water temperature changes in the sea area;

[0043] (4) Other cultivation conditions: Normal water change, twice a day, each time changing more than 70%.

[0044] 2. Obtain mature male broodstock

[0045] After 80 days of culture, 20 male broodstock of finely cultured flathead mullet and 20 male broodstock of conventionally cultured flathead mullet were taken, and the genital length of each male broodstock was measured. Then the male broodstock were dissected to observe the development degree of gonads (testis) and sperm motility, and the sperm density was counted (the gonads were mixed with diluent in a volume ratio of 1:1, the gonads were cut into pieces to release sperm, the mixture was filtered through a 300-mesh silk sieve to obtain semen, and the sperm density was counted under a microscope). The results are shown in Table 1.

[0046] Table 1 Statistics of male broodstock reproductive organ length, testicular development and sperm motility

[0047]

[0048] Among them, in the fine culture group, the testicular development of the flatfish with a genital length of ≤0.6cm was Figure 4 The testicular development of the scorpionfish with a genital length ranging from 0.6 cm (not included) to 1.1 cm (not included) is shown in Figure 5 The testicular development of the flatfish with a genital length of ≥1.1 cm is shown in Figure 6 .

[0049] From Table 1 and Figure 4 , Figure 5 and Figure 6 It can be seen that after the above-mentioned fine cultivation, the genital length, testicular development level and sperm motility of male broodstock of Xu's flathead fish have been significantly improved.

[0050] Since the testicular development and sperm motility of male broodstock with genital length ≥ 1.1 cm are particularly outstanding, they are selected as the parents for sperm collection.

[0051] The reproductive morphology of mature male broodstock (sperm collection parent) can be seen in Figure 1 .

[0052] 2. Stress treatment of mature male broodstock (sperm collection parents)

[0053] 1. The method of coercive stimulation

[0054] The stress stimulation treatments for mature male broodstock (sperm collection parents) specifically include the following three treatment methods: squeezing the gills, wrapping the fish body with a wet towel and leaving it in the air to stress, and cooling stimulation.

[0055] The operation process of the above three processing methods is as follows:

[0056] (1) Squeeze the gills (referred to as treatment A): Wrap the entire head of the sperm collection parent with a wet towel and gently squeeze the gills (restrict breathing) for a certain period of time;

[0057] (2) Wrapping the fish with a wet towel and leaving it in the air to stress (referred to as treatment B): Figure 7 As shown, wrap the body of the sperm collection parent with a wet towel, exposing the head and tail, and place it on a drain net to dry for a certain period of time;

[0058] (3) Cooling stimulation (referred to as treatment C): The sperm collection parent was removed from the culture water and placed in water 5°C lower than the culture water temperature for a certain period of time.

[0059] 2. The process of coercive stimulation

[0060] 180 male broodstock with reproductive organ length ranging from 1.1cm to 1.5cm were selected from the sperm collection parents and randomly divided into 9 groups, with 20 broodstock in each group. The 9 groups of sperm collection parents were subjected to stress stimulation according to the following 9 stress stimulation processes.

[0061] The process of the nine types of stress stimulation is as follows:

[0062] Type 1 (Group 1): No stress stimulation treatment (control group);

[0063] Type 2 (Group 2): The sperm-collecting parent was removed from the aquaculture water and immediately subjected to treatment A for 10 seconds;

[0064] Type 3 (Group 3): The sperm-collecting parent was removed from the aquaculture water and immediately subjected to treatment B for 90 seconds;

[0065] Type 4 (Group 4): The sperm-collecting parent was removed from the aquaculture water and immediately subjected to C treatment for 90 seconds;

[0066] Type 5 (Group 5): The sperm collection parent was removed from the aquaculture water and immediately subjected to treatment B for 30 seconds, followed by treatment A for 10 seconds;

[0067] Type 6 (Group 6): The sperm-collecting parent was removed from the aquaculture water and immediately subjected to treatment B for 60 seconds, followed by treatment A for 10 seconds;

[0068] Type 7 (Group 7): The sperm-collecting parent was removed from the aquaculture water and immediately subjected to treatment B for 90 seconds, followed by treatment A for 10 seconds;

[0069] Type 8 (Group 8): The sperm collection parent was removed from the aquaculture water and immediately subjected to treatment B for 90 seconds, followed by treatment C for 90 seconds;

[0070] Type 9 (Group 9): The sperm collection parent was fished out of the aquaculture water, and immediately subjected to treatment C for 90 seconds, and then immediately subjected to treatment A for 10 seconds.

[0071] 3. Obtaining sperm from male broodstock subjected to stress stimulation by reverse extrusion of genitalia

[0072] After the above 9 groups of sperm collection parents were subjected to the coercion stimulation treatment, the sperm was immediately obtained by the method of reverse extrusion of the genitals. The method of reverse extrusion of the genitals is as follows:

[0073] Turn the sperm collection parent upside down (head down, tail up), wrap the head with a wet towel, fix the head and tail, and wipe the genital area with a dry towel. Figure 2 As shown, pinch your thumb and index finger between the pectoral fin and the genitals, gently squeeze and move slowly toward the genitals, as shown in the figure. Figure 3 As shown, move the two fingers to the tail side of the genitals (at Figure 3After squeezing the genitals (the middle is the right side of the genitals), squeeze the genitals in the reverse direction (towards the pectoral fin direction). If semen flows out after one squeezing, collect the semen and the squeezing operation is completed; otherwise, repeat the reverse squeezing operation until semen flows out or the genitals are damaged.

[0074] After the reverse squeezing of the genitals was completed, the number of broodstock from which sperm was obtained in each group was recorded, the vitality and genital status of the sperm-collecting parents after the stress stimulation treatment were observed and recorded, and the vitality and quantity of the obtained sperm were further tested under a microscope.

[0075] The results of obtaining sperm of S. xu's flathead mullet using 9 stress stimulation combinations are shown in Table 2.

[0076] Table 2 Comparison of sperm obtained from Scorpio hussini using different stress stimulation treatments

[0077]

[0078] From Table 2 we can see that:

[0079] (1) Except for Groups 3, 6, 7, and 8, no sperm was obtained from other groups. In some groups, the genitals of the parent fish were injured and bleeding due to excessive squeezing of the genitals.

[0080] (2) The success rate of obtaining sperm from S. philippinarum in Groups 6 and 7 reached 100%. After obtaining sperm, the reproductive organs of the parent fish were not injured and the vitality of the parent fish was normal, which did not bring any adverse effects to the next sperm collection. Figure 8 As shown, the semen is milky white and sticky droplets can be observed with the naked eye, and the sperm can be observed to be sufficient in number and good in motility under a microscope;

[0081] (3) The success rate of obtaining sperm from Group 3 and Group 8 was only 60% and 40%, which was much lower than that of Group 6 and Group 7. In addition, some broodstock showed genital injuries and bleeding due to excessive squeezing of the genitals.

[0082] Analyzing the above results, we can find that:

[0083] (1) Different stress stimulation treatments (Group 2, Group 3, and Group 4) had a significant impact on the semen collection of the flathead mullet. Among the three stress stimulations, namely, squeezing the gills for 10 seconds, wrapping the fish with a wet towel and leaving it in the air for 90 seconds, and cooling for 90 seconds, the flathead mullet was most sensitive to the stress of wrapping the fish with a wet towel and leaving it in the air for 90 seconds (Group 3).

[0084] (2) The treatment time of wrapping the fish body with a wet towel and leaving it in the air to force stress had a great impact on the semen collection of the flatfish. If the treatment time was too short, it would not effectively stimulate the ejaculation of the flatfish (Group 5, 30 seconds). The appropriate treatment time was 60 seconds to 90 seconds (Groups 6 and 7).

[0085] (3) On the basis of the stress of wrapping the fish body with a wet towel and leaving it in the air, superimposing the gill squeezing stimulation can significantly increase the success rate of sperm extraction (Group 6 and Group 7), while superimposing the cooling stimulation can reduce the success rate of sperm extraction (Group 8). Cooling is not conducive to the ejaculation of semen in the flatfish.

[0086] (4) After cooling stimulation for 90 seconds, superimposed gill squeezing stimulation still could not promote the ejaculation of sperm in the flatfish (Group 9). Cooling is not conducive to the ejaculation of sperm in the flatfish.

[0087] In summary, after the sperm-collecting parent is fished out of the aquaculture water, the fish body should be immediately wrapped with a wet towel and left to air-dry for 60s-90s, and then the gills should be squeezed immediately for 10s. Finally, the genitals should be squeezed immediately by reverse squeezing. This is the best way to obtain sufficient high-quality sperm of Xu's flatfish without damage.

[0088] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for obtaining high-quality sperm of Scorpionfish xu's without damage, characterized in that: The following steps are involved: (1) Male flathead mullets aged 3 years or older were selected for fine culture starting from early September each year, and flathead mullets with a genital length of ≥1.1 cm were obtained as sperm parents; (2) subjecting the sperm collection parent to a stress stimulation treatment. Specifically, the sperm collection parent is fished out of the aquaculture water body, and the fish body is immediately wrapped in a wet towel and left to air-dry for a stress treatment of 60s to 90s, and then the gills are immediately squeezed for a treatment time of 10s; (3) After the sperm-collecting parent has been subjected to stress stimulation, the sperm is immediately obtained by reversely squeezing the genitals.

2. The method according to claim 1, characterized in that: In step (1), the fine cultivation method is specifically: (i) Selection and feeding of bait: fresh sand mullet or small yellow croaker is selected as bait fish, and a mixture of multivitamins, fish oil, arginine, lysine and minerals is stuffed into the gastrointestinal tract of the bait fish, wherein the dosage of the multivitamins, fish oil, arginine, lysine and mineral mixture is 3wt%-5wt%, 5wt%-8wt%, 2%-3%, 2%-3% and 1%-2% of the weight of the bait fish, respectively. After the bait is prepared, it can be fed to the male broodstock, and the feeding is once a day, and the feeding amount is 3-5% of the weight of the male broodstock; (ii) Cultivation light requirements: light intensity 300 lx, light cycle is the same as natural light cycle; (iii) Cultivation water temperature requirement: the water temperature slowly drops from 24°C to 16°C, with an average daily drop of 0.1°C; (iv) Other cultivation conditions: dissolved oxygen ≥ 6 mg / L, ammonia nitrogen content ≤ 0.05 mg / L, salinity 28-30, harmful bacteria content ≤ 10 3 CFU / m 3 , change the water twice a day, each time changing more than 70%.

3. The method according to claim 1, characterized in that In step (2), the method of wrapping the fish body with a wet towel and leaving it in the air for stress treatment is specifically as follows: Wrap the body of the sperm collection parent with a wet towel, exposing the head and tail, and place it on a mesh screen to dry.

4. The method according to claim 1, characterized in that: In step (2), the method of squeezing the gills is specifically as follows: Wrap the entire head of the sperm collection parent with a wet towel and gently squeeze the gills to restrict breathing.

5. The method according to claim 1, characterized in that In step (3), the method of reversely squeezing the genitals is specifically as follows: Turn the parent to be collected for sperm upside down with its head facing downward and its tail facing upward, wrap the entire head with a wet towel to fix the head and tail, wipe the area around the genitals with a dry towel, pinch the thumb and index finger between the pectoral fin and 1 / 2 of the genitals, gently squeeze and move slowly toward the genitals, move both fingers to the side of the genitals near the tail and squeeze the genitals in the opposite direction. If semen flows out after one squeeze, collect the semen and the squeezing operation is over; otherwise, repeat the reverse squeezing operation until semen flows out or the genitals are damaged.

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