阅读说明：本技术 一种泉水鱼精子高效超低温冷冻保存方法 (Efficient ultralow-temperature cryopreservation method for spring fish sperms ) 是由 胡鹏 吕云云 刘德 周晓莉 胡燕 谢尚君 于 2021-03-01 设计创作，主要内容包括：本发明属生物技术领域,具体地说是一种泉水鱼精子高效超低温冷冻保存方法。将泉水鱼的精液与抗冻液按体积比1:30的比例混合,混合后经过三步程序降温处理,最终投入到液氮(-196℃),分装保存；抗冻液由稀释液、抗冻剂和添加剂三部分组成。本发明获得的冻精对于泉水鱼人工繁育、种质保存、遗传多样性保护及可持续养殖等方面有着重要意义。(The invention belongs to the technical field of biology, and particularly relates to a spring fish sperm efficient ultralow temperature cryopreservation method. Mixing the sperm of the spring fish with the anti-freezing liquid according to the volume ratio of 1:30, cooling through three steps of procedures after mixing, finally putting into liquid nitrogen (-196 ℃), subpackaging and storing; the antifreezing solution consists of diluent, antifreezing agent and additive. The frozen semen obtained by the invention has important significance for artificial breeding, germplasm preservation, genetic diversity protection, sustainable culture and the like of the spring fish.)

1. A spring fish sperm high-efficiency ultralow-temperature cryopreservation method is characterized by comprising the following steps:

mixing the sperm of the spring fish with the anti-freezing liquid according to the volume ratio of 1:30, performing programmed cooling treatment after mixing, and finally putting into liquid nitrogen (-196 ℃), subpackaging and storing; the antifreeze solution comprises diluent, antifreeze agent and additive, wherein the diluent is composed of NaCl and KCl、CaCl₂·6H₂O、NaHCO₃Glucose and fructose, 30 percent (V/V) methanol as an antifreeze agent and 15 percent (V/V) honey as an additive.

2. The method for the efficient ultra-low temperature cryopreservation of spring fish sperm as claimed in claim 1, wherein the method comprises the following steps: the diluent consists of NaCl, KCl and CaCl₂·6H₂O, glucose and fructose composition: the preparation method comprises collecting NaCl1.5g, KCl0.01g, CaCl₂·6H₂O0.1g，NaHCO₃0.1g, 8.5g of glucose and 3.0g of fructose, dissolving in 800mL of double distilled water, sterilizing by hot pressing, cooling, and fixing the volume to 1000mL to obtain the diluent.

3. The method for the efficient ultra-low temperature cryopreservation of spring fish sperm as claimed in claim 1, wherein the method comprises the following steps: the anti-freezing liquid is prepared by mixing an anti-freezing protective agent and a diluent to prepare a mixed liquid with a methanol (V/V) ratio of 30%, and placing the mixed liquid in a refrigerator at 4 ℃ for later use.

4. The method for the efficient ultra-low temperature cryopreservation of spring fish sperm as claimed in claim 1, wherein the method comprises the following steps: mixing fresh spring fish concentrate and anti-freezing solution uniformly according to the volume ratio of 1:30, and sucking 2mL of mixed solution by a pipette and adding the mixed solution into a 2.5mL freezing tube.

5. The method for the efficient ultra-low temperature cryopreservation of spring fish sperm as claimed in claim 1, wherein the method comprises the following steps: sucking 300 μ L fresh Mel with pipette, adding into freezing tube, mixing, and pre-cooling in refrigerator at 4 deg.C for 5 min.

6. The method for the high-efficiency ultralow-temperature cryopreservation of spring fish sperm as claimed in claim 1, which is characterized in that: cooling the packaged semen by adopting a three-step program cooling method, and then putting the semen into liquid nitrogen for long-term storage, wherein the program cooling program is in a range of 0-minus 40 ℃ and minus 5 ℃/min; -40 to-70 ℃, 10 ℃/min; the temperature is between 70 ℃ below zero and 130 ℃ below zero, and the temperature is 20 ℃/min below zero.

7. The method for the efficient ultra-low temperature cryopreservation of spring fish sperm as claimed in claim 1, wherein the method comprises the following steps: thawing spring fish jelly subpackaged and stored in liquid nitrogen, and the specific method comprises the following steps: and taking the cryopreservation tube out of liquid nitrogen by using forceps during thawing, staying in the air for 3-5 seconds, quickly putting the cryopreservation tube into a constant-temperature water bath kettle at 25 ℃ for thawing in a water bath, and taking out when only a little ice core is left by gently shaking.

8. The method for the efficient ultra-low temperature cryopreservation of spring fish sperm as claimed in claim 1, wherein the method comprises the following steps: semen is absorbed, activated in physiological saline, vitality is checked under a microscope, and then indexes such as sperm motility and the like are calculated by a Computer Assisted Sperm Analysis (CASA) system.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a spring fish sperm efficient ultralow temperature cryopreservation method.

Technical Field

A fish belonging to the genus Spirochaeta (Semilabeopochilus) belonging to the order Cyprinidae, the subfamily Viridae, the genus Spirochaeta. Springs are species unique to China. The body is longer, the front part is round, the back part is slightly flat, the front part of the abdomen is flat, and the back part of the head is arc-shaped. Making the anastomosis round and blunt; the cleft mouth is slightly triangular, the upper lip and the lower lip are connected at the corner of the mouth, and a plurality of small cutin bulges which are arranged orderly are arranged on the lips; the lip portion is drawn into the oral cavity, and when the mouth is opened, the lip is turned outward and expanded into a horn shape, thereby being adsorbed on other objects. The posterior labial sulcus is limited to the corners of the mouth. 2 pairs of palps, longer palpus and short and small palpus of jaw. The eyes are small, located slightly above the head. The large, medium scale, small abdominal scale, is trapped under the skin. The dorsal fin has no hard prick. Gray black on the back of the body, gray white on the abdomen, and slight black on each fin; the majority of the body side scales have black edges, the dark patches are thicker from behind the gill hole to in front of the pectoral fin, and thus join into a large black patch.

Spring fish inhabit the middle and lower layers of a water area with larger flow velocity of rivers in the south of China, and are like to live in mountain streams, rock caves with flowing water and places with spring sources of the rivers at ordinary times. The attached animals, plants and other organic substances are often scraped on the rocks at the bottom of the river, and rarely enter a still water body with the stratum being sludge, and the reproductive season moves upstream to lay eggs. Typically, the weight of the plant is 1-1.5 kg to reach sexual maturity. The spawning time is about 3-4 months, and spawns are laid in the stone cracks or the stone caves.

Spring fish are distributed in the trunk and branch of the upper reaches of the Yangtze river and the middle and upper reaches of the Xijiang river of the Zhujiang river water system. The spring fish is common edible fish in Sichuan, Guangxi and Yunnan provinces. The growth rate is relatively slow, about 0.5-1 kg for common people and about 3.5-4 kg for the largest individual. The yield is not large, but the meat is tender and the meat taste is very delicious, and the meat is a rare food rich in fat. Fresh meat is used for medicine. Has effects in invigorating primordial qi and stopping bleeding. It can be used for treating diarrhea, hematemesis, and metrorrhagia, and has high nutritive value. In recent years, due to the continuous expansion of the culture scale and the continuous expansion of the demand for the seed quality of spring fishes, the sperm freezing technology of spring fishes is urgently needed to be established. The ultralow temperature preservation of the fish semen has important significance in aquaculture, genetic breeding and germplasm resource preservation: can realize long-distance transportation of germplasm, and is favorable for hybridization and breeding of fish and protection of gene diversity. With the rapid development of the economy of China and the increasing aggravation of seawater pollution, the preservation of the germplasm of an important freshwater aquaculture excellent variety becomes a problem to be solved urgently in the freshwater aquaculture industry of China. The establishment of the ultra-low temperature freezing technology of the sperm of the spring water fish has urgent needs for protecting the genetic diversity of the sperm, developing the biotechnology breeding and the sustainable development of the freshwater aquaculture industry.

Disclosure of Invention

The invention aims to provide a spring fish sperm high-efficiency ultralow-temperature cryopreservation method.

In order to achieve the purpose, the invention adopts the technical scheme that:

mixing the sperm of the spring fish with the anti-freezing liquid according to the volume ratio of 1:30, performing programmed cooling treatment after mixing, and finally putting into liquid nitrogen (-196 ℃), subpackaging and storing; the antifreeze solution comprises diluent, antifreeze agent and additive, wherein the diluent comprises NaCl, KCl and CaCl₂·6H₂O、NaHCO₃Glucose and fructose, the antifreeze is 30 percent (V/V) methanol, and the additive is 15 percent (V/V) honey.

The diluent consists of NaCl, KCl and CaCl₂·6H₂O, glucose and fructose composition: the preparation method comprises collecting NaCl1.5g, KCl0.01g, and CaCl₂·6H₂O 0.1g，NaHCO₃0.1g, 8.5g of glucose and 3.0g of fructose, dissolving in 800mL of double distilled water, sterilizing by hot pressing, cooling, and fixing the volume to 1000mL to obtain the diluent.

The anti-freezing liquid is prepared by mixing an anti-freezing protective agent and a diluent to prepare a mixed liquid with a methanol (V/V) ratio of 30%, and placing the mixed liquid in a refrigerator at 4 ℃ for later use.

Mixing fresh spring fish concentrate and anti-freezing solution uniformly according to the volume ratio of 1:30, and sucking 2mL of mixed solution by a pipette and adding the mixed solution into a 2.5mL freezing tube.

Sucking 300 μ L fresh Mel with pipette, adding into freezing tube, mixing, and pre-cooling in refrigerator at 4 deg.C for 5 min.

Cooling the packaged semen by adopting a three-step program cooling method, and then putting the semen into liquid nitrogen for long-term storage, wherein the program cooling program is in a range of 0-minus 40 ℃ and minus 5 ℃/min; -40 to-70 ℃, 10 ℃/min; the temperature is between 70 ℃ below zero and 130 ℃ below zero, and the temperature is 20 ℃/min below zero.

Thawing spring fish jelly subpackaged and stored in liquid nitrogen, and the specific method comprises the following steps: and taking the cryopreservation tube out of liquid nitrogen by using forceps during thawing, staying in the air for 3-5 seconds, quickly putting the cryopreservation tube into a constant-temperature water bath kettle at 25 ℃ for thawing in a water bath, and taking out when only a little ice core is left by gently shaking.

The semen is sucked, activated in physiological saline, the vitality of the semen is examined under the microscope, the indexes of the sperm motility and the like are calculated by a computer-assisted sperm analysis (CASA) system, the indexes of the sperm motility and the like are calculated by the computer-assisted sperm analysis (CASA) system, 3 visual fields are randomly selected, the number of the sperms in movement is detected to account for the proportion of all the sperms in the visual fields, the difference between the sperm motility and the fresh semen is not obvious after the sperms are detected and thawed, and more than 90 percent of the sperms do rapid linear movement after the sperms are thawed.

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

firstly, methanol is used as an antifreeze agent in the process of cryopreservation, so that the toxicity is reduced while better permeability is kept;

secondly, a 2.5mL freezing tube is adopted in the freezing preservation process, so that the volume is large, and the semen preservation amount is large;

mixing the antifreeze protective agent with the diluent before cooling to prepare a mixed solution, and placing the mixed solution in a refrigerator at 4 ℃ for later use, so that the time is saved, and the operation is simple and easy to implement;

and fourthly, uniformly mixing the fresh spring water fish sperm liquid according to the volume ratio of 1:30, sucking 2mL of mixed liquid by a liquid transfer gun, adding the mixed liquid into a 2.5mL freezing tube, sucking 300 mu L of fresh honey by the liquid transfer gun, adding the fresh honey into the freezing tube, fully and uniformly mixing, and then putting the mixture into a refrigerator at 4 ℃ for precooling for 5 min. Fully and uniformly mixing, then putting into a refrigerator with the temperature of 4 ℃ for precooling for 15min, so that the antifreeze protective agent fully permeates into cells, and the antifreeze protective agent fully permeates into the cells to play a protective role, thereby having important production significance.

Fifthly, when the freezing preservation process of the invention is cooled in sections, the packaged semen is cooled by a three-step program cooling method, and then is put into liquid nitrogen for long-term preservation, wherein the program cooling program is 0-40 ℃ range, 5 ℃ below zero/min, 40 ℃ below zero/min to 70 ℃ below zero, 10 ℃ below zero/min, 70 ℃ below zero to 130 ℃ below zero/min. Then directly putting the sperm into liquid nitrogen for long-term preservation until activation, wherein the proper cooling speed is the key point that the sperm can be fully dehydrated and the frozen sperm can safely pass through a dangerous temperature zone, and the whole preservation process has short time; the freezing preservation and cooling procedure is simple and easy to operate, the repeatability and stability are good, the recovery rate of frozen semen after thawing is high, the movement rate after activation is higher than 90%, and the activity difference with fresh semen is not obvious;

and sixthly, after the frozen sperm is taken out from the liquid nitrogen, the frozen sperm stays in the air for 3-5 seconds, then the freezing tube is quickly placed into a constant-temperature water bath kettle at 25 ℃ for water bath for unfreezing, and the frozen sperm is taken out when only a little ice nuclei remain after being gently shaken, so that the sperm can quickly pass through a recrystallization zone, and meanwhile, the structural damage of the sperm caused by overhigh local temperature is avoided.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the invention thereto.

Example 1:

(1)20 live male fishes of spring fishes with good spermary development are captured from the upstream of the Yangtze river in 03 months in 2020, the weight of each fish is 2.5kg-3.0kg, and the abdomen of each fish is lightly squeezed by hands to allow seminal fluid to flow out. Placing the mixture on a dry gauze, washing the genital pore with distilled water for three times until no urine and mucus are left, wiping the mixture with clean toilet paper, slightly pressing the mixture on the abdomen along the direction from the fish head to the fish tail, and collecting the uncontaminated semen in a 50mL centrifuge tube. Detecting sperm motility rate with computer-assisted sperm analysis system (CASA), and selecting sample with motility rate higher than 90% for cryopreservation.

(2) Mixing the sperm of the spring fish with the anti-freezing liquid according to the volume ratio of 1:30, performing programmed cooling treatment after mixing, and finally putting into liquid nitrogen (-196 ℃), subpackaging and storing; the antifreeze solution comprises diluent, antifreeze agent and additive, wherein the diluent comprises NaCl, KCl and CaCl₂·6H₂O、NaHCO₃Glucose and fructose, the antifreeze is 30 percent (V/V) methanol, and the additive is 15 percent (V/V) honey. The diluent consists of NaCl, KCl and CaCl₂·6H₂O, glucose and fructose composition: the preparation method comprises collecting NaCl1.5g, KCl0.01g, CaCl₂·6H₂O 0.1g，NaHCO₃0.1g, 8.5g of glucose and 3.0g of fructose, dissolving in 800mL of double distilled water, sterilizing by hot pressing, cooling, and fixing the volume to 1000mL to obtain the diluent. The antifreeze solution is prepared by mixing antifreeze protective agent and diluent to obtain a mixed solution with a proportion of 30% methanol (V/V), and placing in a refrigerator at 4 ℃ for later use.

(3) Mixing fresh spring fish concentrate and anti-freezing solution uniformly according to the volume ratio of 1:30, and sucking 2mL of mixed solution by a pipette and adding the mixed solution into a 2.5mL freezing tube. Sucking 300 μ L fresh Mel with pipette, adding into freezing tube, mixing, and pre-cooling in refrigerator at 4 deg.C for 5 min.

(4) And (3) carrying out cooling treatment on the subpackaged semen by adopting a three-step program cooling method, and then putting the semen into liquid nitrogen for long-term storage, wherein the program cooling program is 0-40 ℃ below zero, -5 ℃/min, -40-70 ℃ below zero, -10 ℃/min, -70-130 ℃ below zero and-20 ℃/min.

(5) Thawing spring fish jelly subpackaged and stored in liquid nitrogen, and the specific method comprises the following steps: and taking the cryopreservation tube out of liquid nitrogen by using forceps during thawing, staying in the air for 3-5 seconds, quickly putting the cryopreservation tube into a constant-temperature water bath kettle at 25 ℃ for thawing in a water bath, and taking out when only a little ice core is left by gently shaking.

(6) Semen is absorbed, activated in physiological saline, vitality is checked under a microscope, and then indexes such as sperm motility and the like are calculated by a Computer Assisted Sperm Analysis (CASA) system. After activation, the motility rate of frozen semen is more than 90 percent and is close to the level of fresh semen by using a microscope to detect.

Example 2

(1) In 2019, in 3-4 months, in the reproductive period of spring fish, in the Xichuang spring fish culture base of Jiangchang, Sichuan province and the parent fish culture workshop of spring fish, male fish are fished out from the culture pond and placed on sponge, the reproductive hole is washed by distilled water for three times, the male fish is wiped by a paper towel, and the belly is slightly squeezed from the back to the front to obtain fresh essence. Collecting 40 fish tails of the spring fish, counting 80mL of fresh essence, detecting the activity of the spring fish by a microscope to be higher than 80%, and putting the spring fish in an ice box to return to a laboratory for freezing storage;

(2) mixing the sperm of the spring fish with the anti-freezing liquid according to the volume ratio of 1:30, performing programmed cooling treatment after mixing, and finally putting into liquid nitrogen (-196 ℃), subpackaging and storing; the antifreeze solution comprises diluent, antifreeze agent and additive, wherein the diluent comprises NaCl, KCl and CaCl₂·6H₂O、NaHCO₃Glucose and fructose, the antifreeze is 30 percent (V/V) methanol, and the additive is 15 percent (V/V) honey. The diluent consists of NaCl, KCl and CaCl₂·6H₂O, glucose and fructoseConsists of the following components: the preparation method comprises collecting NaCl1.5g, KCl0.01g, CaCl₂·6H₂O 0.1g，NaHCO₃0.1g, 8.5g of glucose and 3.0g of fructose, dissolving in 800mL of double distilled water, sterilizing by hot pressing, cooling, and fixing the volume to 1000mL to obtain the diluent. The antifreeze solution is prepared by mixing antifreeze protective agent and diluent to obtain a mixed solution with a proportion of 30% methanol (V/V), and placing in a refrigerator at 4 ℃ for later use.

(3) Mixing fresh spring fish concentrate and anti-freezing solution uniformly according to the volume ratio of 1:30, and sucking 2mL of mixed solution by a pipette and adding the mixed solution into a 2.5mL freezing tube. Sucking 300 μ L fresh Mel with pipette, adding into freezing tube, mixing, and pre-cooling in refrigerator at 4 deg.C for 5 min.

(4) Cooling the packaged semen by adopting a three-step program cooling method, and then putting the semen into liquid nitrogen for long-term storage, wherein the program cooling program is in a range of 0-minus 40 ℃ and minus 5 ℃/min; -40 to-70 ℃, 10 ℃/min; the temperature is between 70 ℃ below zero and 130 ℃ below zero, and the temperature is 20 ℃/min below zero.

(5) Thawing spring fish jelly subpackaged and stored in liquid nitrogen, and the specific method comprises the following steps: and taking the cryopreservation tube out of liquid nitrogen by using forceps during thawing, staying in the air for 3-5 seconds, quickly putting the cryopreservation tube into a constant-temperature water bath kettle at 25 ℃ for thawing in a water bath, and taking out when only a little ice core is left by gently shaking.

(6) Semen is absorbed, activated in physiological saline, vitality is checked under a microscope, and then indexes such as sperm motility and the like are calculated by a Computer Assisted Sperm Analysis (CASA) system. After activation, the motility rate of frozen semen is more than 90 percent and is close to the level of fresh semen by using a microscope to detect.

(7) The frozen semen is used for artificial insemination, the fertility rate and the hatchability are both more than 80%, and the survival rate of the offspring seeds is not obviously different from that of the fresh semen.