CN112586408B - Method for identifying optimal spawning induction time of small yellow croakers - Google Patents
Method for identifying optimal spawning induction time of small yellow croakers Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention relates to a method for identifying the sexual maturity of small yellow croakers, which comprises the following steps: (1) selecting parent fish, and carrying out anesthesia by using MS-222 anesthetic liquid; (2) after the anesthesia takes effect, weighing and recording the weight; (3) collecting blood, centrifuging and collecting serum for later use; (4) dissecting the female parent fish, taking out the gonad, weighing and recording; (5) calculating the GSI value of female parent fish, measuring the level of estrogen 17 beta-estrol, and identifying whether the gonad is mature; when the female parent fish GSI =14-18 and E2(pg/mL) = 600-800-. By the method, the work of identifying sexual maturity can be completed within 1 day, the best induced spawning time is determined, the success rate of subsequent induced spawning reaches about 93%, a comparable quantitative index of gonad maturity is provided, the time cost is greatly reduced compared with the prior art, and technical parameters are provided for artificial propagation and popularization.
Description
Technical Field
The invention belongs to the technical field of marine fish reproduction, and particularly relates to a method for identifying the sexual maturity of a small yellow croaker, mainly an identification method of the sexual maturity of a female parent fish of the small yellow croaker.
Background
The small yellow croakers are important economic fishes in coastal areas of China, are distributed in the northwest pacific ocean area including China, Korean, and Korean coastal areas, are widely distributed in Bohai sea, south of the yellow sea and east sea in China, have south latitude of 26 degrees in the north and east longitude of 126 degrees of 30 degrees in the east. The small yellow croakers overwinter in deep sea in winter, swim to the coast in spring, lay eggs in 3-6 months, disperse in offshore cable baits, and return to deep sea at the end of autumn. According to different temperatures, salinity and illumination time of living environment, the spawning period of the yellow croaker in the east China sea is from the middle of 2 months to the beginning of 4 months, for example, in the deep sea area such as 50 meters in east and near sea areas of Dachen, leek mountain and Zhoushan, the spawning period is from the middle of 2 months to the beginning of 4 months; ouzin in Wenzhou and the sea area nearby the Ouzin have spawning periods from 2 middle ten days to 4 early months; in the sea area outside the gulf of the three-door and near the Lingjiang mountain, the spawning period is from 3 middle to 4 last days of the month; in the area outside the Yangtze river mouth, the egg laying period is from the first 4 months to the first 5 months. The water depth of the spawning site is 20-50 meters generally, the spawning site is mainly distributed on the inner side of an outer seawater and coastal water intersection sea area, and the larger the salinity level gradient is, the more concentrated the fish school is. The water temperature of the spawning site is slightly changed, and is 11-15 ℃, mainly 12-14 ℃, and the salinity is 24-33 per mill, mainly 28-33 per mill.
After artificial propagation and domestication of the small yellow croakers in Zhejiang for 5 years, the propagation and breeding technology is basically mature, but because the small yellow croakers are widely distributed in China coastal areas, the reproduction time from south to north is not uniform in China, and except that the temperature and the salinity can be used as reference bases, no other sexual maturation technology quantitative quality standards can be referred to. This increases the technical difficulty for popularizing the culture of little yellow croaker and accurately mastering the breeding time.
Under the condition of artificial breeding, the small yellow croaker group can not realize large-scale natural propagation, and large-scale artificial propagation can be realized only by injecting Lrh-A2 or matching with HCG for artificial induced spawning, so as to obtain fertilized eggs. The previous research basically solves the technical problem of artificial spawning induction of the small yellow croakers, the spawning induction rate can reach 88.2 +/-0.2%, the fertilization rate is 78.4 +/-0.1%, and the hatching rate is 76.7 +/-0.1%, however, with the continuous deepening of the research and the production, the best spawning induction time of the small yellow croakers is not consistent every year due to different living temperature, salinity and illumination, from the viewpoint of the spawning induction effect of 5 years continuously, the first spawning time of a whole artificial culture group in Ningbo and Zhoushan sea area is from 26 days to 8 days at the earliest 3 months and from 26 days to 4 months at the latest, the latest spawning time is from 19 days to 3 days at the latest, the fluctuation range of about 10 days is large, technicians are required to continuously perform small spawning induction trials to determine the best opportunity, the work is complicated and the efficiency is low, therefore, how to judge whether the yellow croaker reaches the standard of sexual maturity and how to judge the optimal time for artificially inducing spawning is a key technical problem to be solved urgently. The technical problem is solved, the popularization of the full-artificial breeding technology of the small yellow croakers is facilitated, effective reference is provided for the majority of scientific research technicians, labor and time cost is reduced, and the artificial spawning induction efficiency is improved.
Chinese patent publication No. CN106706631A discloses a method for identifying the sexual maturity of parent fish of schizothorax prenanti, which is to judge the sexual maturity by observing the tail handle of the parent fish through a microscope. Since most marine fish do not have obvious secondary characteristics, the method has obvious species specificity and is not suitable for judging the sexual maturity of marine fish, especially small yellow croaker.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for identifying the small yellow croaker sexual maturity, which can accurately judge the small yellow croaker sexual maturity and the optimal spawning induction time by measuring the main sex hormones of the small yellow croaker male and female parent fish and combining with a method for measuring the gonad maturation coefficient (GSI), and can obtain the result on the same day. The artificial spawning induction efficiency is obviously improved.
The invention is realized by the following technical scheme:
the method for identifying the optimal spawning induction time of the small yellow croakers is characterized by comprising the following steps of:
1) in the spawning season of the small yellow croakers, the small yellow croaker parents which have no trauma on the body surface and are healthy and normally eat are randomly selected
The fish is kept in an empty stomach starvation state 8 hours before detection, is kept in an empty stomach state, is beneficial to identifying males and females by using an observation and touch method, is also beneficial to obtaining an accurate GSI value, and is beneficial to identifying parent fish of males and females by using the observation and touch method;
2) sequentially placing the female parent fish identified in the step 1) in anesthetic liquid prepared by sand filtration water, fishing out the female parent fish to a water absorption towel by using a fishing net after the anesthesia takes effect, slightly wiping the female parent fish clean, putting the female parent fish on an electronic scale for weighing after water on the body of the female parent fish is absorbed, and recording the weight of the female parent fish;
3) after weighing, pricking an artery below the vertebra of the female parent fish by using a 1mL syringe to collect blood, wherein the blood collection amount is 95-105 mu L, and at least 50 mu L of whole blood is guaranteed to be collected; about 25 μ L of serum after static separation can be obtained to prepare for at least two concentration gradients to be measured;
4) transferring the collected whole blood into a centrifuge tube from an injector, and in the transferring process, taking down the needle of the injector and slightly pushing the blood into the centrifuge tube of 1.5 mL; otherwise, blood cells are broken, serum turns red, and the final concentration has errors;
5) covering a tube cover of the centrifugal tube, standing for 1 hour, placing the centrifugal tube into a centrifuge at 3000rpm for 5 minutes after obvious layering is seen, sucking supernatant to obtain a serum sample which is transparent light yellow liquid, and transferring the serum sample into another centrifugal tube for numbering for later use;
6) dissecting the female parent fish, taking out the gonad, putting the gonad in an electronic scale to weigh the female parent fish, wherein the precision of the electronic scale is 0.001g, so as to ensure relatively small error and record;
7) calculating the GSI value of the female parent fish, wherein the formula is GSI = gonad weight/body weight multiplied by 100; gonad maturation is identified when the GSI mean +/-SD = 17.69+/-4.37 for first age female parent fish and the GSI mean +/-SD = 14.74+/-4.39 for second age female parent fish; however, when the GSI value of the first-age female parent fish exceeds 20 and the GSI value of the second-age female parent fish exceeds 18, the gonad is over mature and is not suitable for induced spawning;
8) measuring the serum sample in the step 5) by using an estrogen 17 beta-Estrodial (E2) enzyme-linked immunoassay kit, determining the absolute value level of the estrogen 17 beta-Estrodial, and identifying gonad maturation if E2(pg/mL) =718.5 +/-231.0; if the concentration exceeds 1000pg/mL, the gonads are over-mature and begin to degenerate;
9) combining the two results of step 7) and step 8) with production practices, when the female parent fish GSI =14-18, E2(pg/mL) = 600-.
Further, the spawning season of the small yellow croakers in the step 1) refers to the spring after winter, the water temperature of the natural sea area or the cultivation water tends to rise after low temperature in winter, and when the highest temperature reaches more than 15.5 ℃ and lasts for more than 7 days, the photoperiod is 12 hours/day.
Further, the observation and the combination of the touch method in the step 1) are used for identifying the male and female parent fishes, and the specific method comprises the following steps:
1) female parent fish with good nutrition status has round, smooth and swollen abdomen and soft hand feeling when touching, the abdomen is clamped by a forefinger and a thumb, and the head is slightly squeezed towards the tail, a small amount of tissue fluid is found to flow out from a cloaca, and the female parent fish is judged to be female;
2) the appearance of the abdomen of the parent fish does not bulge obviously, the hand feeling is hard when the parent fish touches the abdomen, the abdomen is clamped by the forefinger and the thumb, the head is slightly squeezed towards the tail, milky semen is found to flow out from the cloaca along with a small amount of urine, and the parent fish is judged to be male; if the semen can be extruded smoothly, the male enters the breeding period and reaches sexual maturity, the semen producing period of the male is earlier than the egg producing period of the female and has long duration, and the quality of the semen can be maintained when the breeding water temperature is between 13 and 17 ℃. However, when the cultivation temperature exceeds 18 ℃ and lasts for more than 7 days, the spermary can be rapidly degenerated, the sperm quality can also be seriously degenerated, the male sexual maturity is based on whether the semen can be extruded or not, and the method is simple and high in efficiency.
Further, the anesthetic liquid in the step 2) is MS-222 anesthetic liquid, and the concentration of the anesthetic liquid is 20 mg/L.
Further, the effective anesthesia in the step 2) means that the fish body is kept still, the reaction is slow, only the tail part swings slightly, and the fish body cannot escape when touched by hands.
The invention provides a method for identifying the sexual maturity of small yellow croakers, which provides a technical quantitative index for the optimal spawning induction time of the small yellow croakers by measuring the levels of GSI and E2, and can be completed within 1 day. Compared with the prior art, the method has the advantages that whether parent fish groups enter the spawning period and the reproductive season is judged by observing the fertilization situation mainly through small-scale induced spawning experiments, the time is greatly shortened, comparable quantitative indexes exist, the repeatability is high, and the artificial induced spawning efficiency is obviously improved.
By the method, the original identification experiment time is shortened to 8 hours, the best induced spawning time is determined, the subsequent induced spawning success rate reaches about 93%, a comparable gonad maturity quantitative index is provided, important induced spawning parameters are provided for artificial propagation and popularization, and the method has high economic value potential.
Drawings
FIG. 1 shows the length, weight and GSI index of male and female bodies of a yellow croaker during the sexual maturity period;
FIG. 2 shows the variation of the content of E2 in the whole reproductive cycle;
FIG. 3 shows the change of androgen 11-KT content in small yellow croaker during the complete reproductive cycle;
fig. 4 shows the content change of the small yellow croaker Testosterone in the whole reproductive cycle.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be described in further detail with reference to specific examples to better understand the technical solution.
Examples
Preparing materials:
preparing an electronic scale with the precision of 0.001g, a water absorption towel, an operation disc, forceps, 70% disinfection alcohol, a centrifuge, MS222 anesthetic for fish, operation scissors, a plurality of 1mL syringes, a 1.5mL centrifuge tube, an estrogen 17 beta-Estrodial (E2) enzyme-linked immunoassay kit and an enzyme-linked immunosorbent assay, wherein the enzyme-linked immunosorbent assay needs to have a detectable wave band of 400-450 nm; wherein, the estrogen 17 beta-Estrodial (E2) enzyme-linked immunoassay kit can be purchased as a commercial kit product and is measured by following the guiding steps of the kit.
Selecting parent fish:
in the spawning period of the little yellow croaker, when the highest temperature of the natural sea area or the culture water temperature generally reaches more than 15.5 ℃ and lasts more than 7 days, the 6 tails of the female parent fish of the little yellow croaker which has no trauma on the body surface and is healthy and normally eaten are randomly selected and placed in a 300L seawater culture barrel for temporary culture, the surface of the seawater culture barrel is made of toughened glass, the bottom of the seawater culture barrel is conical, and is printed with scale marks and normally oxygenated. The fasting starvation state needs to be kept 8 hours before detection; wherein the weight of the parent fish at one age is generally 50-90g, and the weight of the parent fish at the second age is 190 g; the male and female parent fish is identified by observation combined with a touch method, the parent fish with better nutrition condition has more rounded and swollen abdomen and softer hand feeling when touching, the abdomen is clamped by a forefinger and a thumb, and a small amount of tissue fluid is found to flow out from a cloaca along with urine when gently extruding along the head to the tail; the appearance of the abdomen of the male parent fish does not bulge obviously, the hand feeling is hard when the male parent fish touches the abdomen, the abdomen is clamped by the forefinger and the thumb, milky semen is found along with a small amount of urine flowing out of the cloacae when the male parent fish is gently squeezed from the head to the tail, and if the semen can be squeezed out smoothly, the male parent fish is proved to enter the breeding period and reach the sexual maturity.
Carrying out anesthesia treatment on parent fish:
preparing 15-40L MS-222 anesthetic liquid with the concentration of 20mg/L by using sand filtration water according to the size of a water bucket, sequentially placing temporarily-cultured female parent fishes in the prepared anesthetic liquid, fishing out the female parent fishes by using a fishing net until the anesthesia takes effect, and slightly wiping the female parent fishes by using a water absorption towel; and (4) weighing the clean female parent fish in an electronic scale, and recording the weight. The effective effect of anesthesia is that the fish body is kept still, the reaction is slow, only the tail part swings slightly, and the fish body cannot escape when touched by hands.
Collecting serum:
after weighing, pricking an artery below the vertebra of a female parent fish with a 1mL syringe to collect blood, wherein the blood collection amount is about 200 mu L, at the moment, taking down a syringe needle, slightly pushing the blood into a 1.5mL centrifuge tube, covering a tube cover, standing for 1 hour, allowing obvious layering to be seen, then placing the centrifuge into a 3000rpm, centrifuging for 5 minutes, sucking a supernatant, and transferring the supernatant into another centrifuge tube with the serial number for later use; all actions of this step need to be gentle to avoid causing rupture of red blood cells and affecting the final measurement result.
Parent fish dissection:
dissecting female parent fish, cutting open along abdomen with surgical scissors, taking out other viscera tissue with forceps until gonad is exposed, carefully clamping a section of gonad cloaca with forceps, taking out with surgical scissors, weighing with electronic scale, and recording; all actions in the dissection process need to be careful, granular ova can be obviously seen from the ovaries in the near-mature period, at the moment, the ovarian membranes are thin and easy to be pinched, and if the gonads are not dissected completely, the follow-up calculation errors can be caused.
And (3) gonad maturation identification:
calculating the GSI value of the female parent fish, wherein the formula is GSI = gonad weight/body weight multiplied by 100; gonad maturation is identified when the GSI mean +/-SD = 17.69+/-4.37 for first age female parent fish and the GSI mean +/-SD = 14.74+/-4.39 for second age parent fish; the collected serum samples were measured with estrogen 17 β -Estrodial (E2) elisa kit to determine the absolute level of estrogen 17 β -Estrodial, which was identified as gonadal maturation if E2(pg/mL) =718.5 +/-231.0.
Combining the two results with production practice, the optimal induction time is recommended when the female parent fish GSI =14-18 and E2(pg/mL) = 600-800.
Claims (5)
1. A method for identifying the optimal spawning induction time of small yellow croakers is characterized by comprising the following steps:
1) in the spawning season of the little yellow croakers, the healthy and normally fed parent little yellow croakers without surface trauma are randomly selected, the empty stomach hungry state is kept 8 hours before detection, and the male and female parent fish are identified by utilizing observation and a touch method;
2) sequentially placing the female parent fish identified in the step 1) in anesthetic liquid prepared by sand filtration water, fishing out to a water absorption towel by using a fishing net after the anesthesia is effective, lightly wiping the female parent fish clean, putting the female parent fish on an electronic scale for weighing after water on the body of the female parent fish is absorbed, and recording the weight;
3) after weighing, pricking an artery below the vertebra of the female parent fish by using a 1mL syringe to collect blood, wherein the blood collection amount is 95-105 mu L, and at least 50 mu L of whole blood is guaranteed to be collected;
4) transferring the collected whole blood into a centrifuge tube from an injector, and in the transferring process, taking down the needle of the injector and slightly pushing the blood into the centrifuge tube of 1.5 mL;
5) covering a tube cover of the centrifugal tube, standing for 1 hour, placing the centrifugal tube into a centrifuge at 3000rpm for 5 minutes after obvious layering is seen, sucking supernatant to obtain a serum sample, and transferring the serum sample to another centrifugal tube for numbering for later use;
6) dissecting the female parent fish, taking out the gonad, putting the gonad in an electronic scale to weigh the female parent fish, wherein the precision of the electronic scale is 0.001g, and recording;
7) calculating the GSI value of the female parent fish, wherein the formula is GSI = gonad weight/body weight multiplied by 100; gonad maturation is identified when the GSI mean +/-SD = 17.69+/-4.37 for first age female parent fish and the GSI mean +/-SD = 14.74+/-4.39 for second age female parent fish;
8) measuring the serum sample in the step 5) by using an estrogen 17 beta-estrogenic enzyme-linked immunoassay kit, and determining the absolute value level of the estrogen 17 beta-estrogenic, wherein if the 17 beta-estrogenic (pg/mL) +/-SD =718.5+/-231.0, the gonad is identified as mature;
9) combining the two results of step 7) and step 8) with production practice, the best induction time is obtained when the female parent fish GSI =14-18, 17 beta-Estrodial (pg/mL) = 600-800.
2. The method as claimed in claim 1, wherein the spawning season of the small yellow croakers in step 1) refers to spring after winter, the natural sea area or cultivation water temperature is increased after winter, the maximum temperature is above 15.5 ℃ and lasts for more than 7 days, and the photoperiod is 12 hours/day.
3. The method for identifying the optimal spawning induction time of yellow croakers according to claim 1, wherein the step 1) of identifying the male and female parent fish by observing and combining a touch method comprises the following steps:
1) female parent fish with good nutrition status has round, smooth and swollen abdomen and soft hand feeling when touching, the abdomen is clamped by a forefinger and a thumb, and the head is slightly squeezed towards the tail, a small amount of tissue fluid is found to flow out from a cloaca, and the female parent fish is judged to be female;
2) the appearance of the abdomen of the parent fish does not bulge obviously, the hand feeling is hard when the parent fish touches the abdomen, the abdomen is clamped by the forefinger and the thumb, the head is slightly extruded towards the tail, milky semen is found to flow out from the cloacae along with a small amount of urine, and the parent fish is judged to be male; if the semen can be extruded smoothly, the male enters the reproductive stage and reaches sexual maturity, the sperm producing period of the male is earlier than the egg producing period of the female and has long duration, and the sperm quality can be maintained when the culture water temperature is between 13 and 17 ℃.
4. The method for identifying the best oxytocic time for yellow croaker as claimed in claim 1, wherein the anesthetic solution in step 2) is MS-222 anesthetic solution with a concentration of 20 mg/L.
5. The method for identifying the best spawning induction time of yellow croaker according to claim 1, wherein the anesthesia effect in step 2) means that the fish body is kept still, the reaction is slow, and only the tail part swings slightly, so that the fish body cannot escape when touched by hand.
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| CN1601275A (en) * | 2003-09-28 | 2005-03-30 | 中国水产科学研究院东海水产研究所 | Analyzing method of fish sexual gland |
| CN101776693A (en) * | 2010-01-20 | 2010-07-14 | 湖南省水产科学研究所 | Method for testing maturity of giant salamander |
| CN106706631A (en) * | 2016-03-02 | 2017-05-24 | 毕节市水产技术推广站 | Method for identifying sexual maturity of parent fish of schizothorax |
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| CN104904636B (en) * | 2015-06-10 | 2018-06-19 | 浙江省海洋水产研究所 | A kind of induced spawning method of little yellow croaker |
| CN106172117B (en) * | 2016-07-14 | 2019-08-06 | 浙江省海洋水产研究所 | Little yellow croaker raun and Larimichthys crocea milter cross breeding method |
| CN107736280A (en) * | 2017-11-29 | 2018-02-27 | 安徽金阳光农业服务有限公司 | A kind of Pelteobagrus fulvidraco parent population reinforced cultivating and biological control technology |
| CN108077120A (en) * | 2017-12-18 | 2018-05-29 | 浙江省海洋水产研究所 | A kind of induction feminisation method of the undifferentiated little yellow croaker of sexual gland |
| CN108142328B (en) * | 2018-01-11 | 2020-05-05 | 浙江省海洋水产研究所 | Early propagation and rapid seedling raising method for small yellow croakers |
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| CN1601275A (en) * | 2003-09-28 | 2005-03-30 | 中国水产科学研究院东海水产研究所 | Analyzing method of fish sexual gland |
| CN101776693A (en) * | 2010-01-20 | 2010-07-14 | 湖南省水产科学研究所 | Method for testing maturity of giant salamander |
| CN106706631A (en) * | 2016-03-02 | 2017-05-24 | 毕节市水产技术推广站 | Method for identifying sexual maturity of parent fish of schizothorax |
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