Cryopreservation method for sperms of oplegnathus punctatus

Abstract

The invention discloses a method for cryopreserving sperms of oplegnathus punctatus, which comprises the following steps: on the day before the sperms of the oplegnathus punctatus, carrying out drug ripening on male oplegnathus punctatus, on the second day after ripening, collecting seminal fluid by adopting an extrusion method, and collecting the mature seminal fluid into a clean glassware for later use; mPRS diluent is prepared, the MPRS diluent and ethylene glycol are compounded to obtain the anti-freezing liquid, and the mass concentration of the ethylene glycol in the system is 15%; diluting the anti-freezing liquid and the sperm according to a volume ratio of 1: 1 to obtain a sperm mixed liquid; the sperm mixed liquid is balanced for 10 min at the temperature of 4 DEG C, then precooled for 5 min at the position 10 cm away from the liquid nitrogen level, precooled for 5 min above the liquid nitrogen level and finally put into liquid nitrogen for cryopreservation; if the cryopreserved sperm mixed liquid needs to be used, resuscitating and activating the cryopreserved sperm mixed liquid to obtain activated sperms; the survival rate of the sperms after unfreezing and resuscitation is high, and sustainable utilization and development of germplasm resources of the sphagnaceae are better facilitated.

Description

Technical Field

[0001] This invention relates to a method for preserving fish sperm, specifically a method for cryopreserving sperm from spotted rock seabream, belonging to the field of marine biotechnology. Background Technology

[0002] Spotted rock seabream belongs to the order Perciformes ( Perciformes ), Chrysanthemumidae ( Oplegnathidae ), genus *Stonefish* ( Oplegnathus Also known as spotted seabream, it is distributed in the subtropical nearshore waters of China and Korea in the northeastern Pacific Ocean, commonly inhabiting underwater coral reefs or rocky reefs. Although spotted seabream has a wide distribution, it is rare in its natural habitat and is almost never seen in groups. Therefore, cryopreserving the sperm of this valuable fish and establishing a sperm cryopreservation bank is of positive significance for the sustainable utilization and development of valuable fish and high-quality germplasm. Furthermore, it plays an important role in the exploration and innovative utilization of germplasm resources, contributing to the artificial breeding and release of precious species.

[0003] Sperm cryopreservation methods are species-specific. Different species of marine fish live in vastly different environments, resulting in variations in their germplasm characteristics. Although sperm cryopreservation methods have been established for species such as black seabream, yellowfin seabream, and red seabream, no reports have been made for sperm cryopreservation techniques for species of the rock seabream family. Therefore, this invention provides a method for cryopreserving the sperm of spotted rock seabream, providing solid support for the preservation and utilization of marine fish germplasm resources. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the shortcomings of the existing technology by providing a method for cryopreservation of sperm from spotted rock seabream, which has a high sperm viability rate after thawing and recovery, and better supports the sustainable utilization and development of germplasm resources of rock seabream.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for cryopreservation of spotted sea bream sperm, comprising the following steps: (1) Sperm collection: The day before collecting sperm from spotted rock sea bream, the male spotted rock sea bream were given drugs to induce maturity. The day after maturity, the sperm was collected by squeezing and the mature sperm was collected into a clean glass container for later use. (2) Diluting sperm: The semen obtained in step (1) was diluted with antifreeze to obtain a sperm mixture, which was then dispensed into 2ml cryovials for later use. (3) Balancing and cryopreserving sperm: The sperm mixture obtained in step (2) is equilibrated at low temperature for a period of time, then pre-cooled above liquid nitrogen, and finally frozen in liquid nitrogen; if needed, the frozen sperm mixture can be thawed and activated to obtain activated sperm.

[0006] In the above technical solution, in step (1), the male spotted sea bream is a healthy, uninjured, vigorous, and sexually mature six-fish.

[0007] In the above technical solution, in step (1), the drug used for ripening is a luteinizing hormone-releasing hormone analog (LRH-A2), the ripening method is injection, the injection dose is 2.5 μg / kg, and the sperm can be collected the day after the injection.

[0008] In the above technical solution, in step (2), the antifreeze and semen are diluted in a volume ratio of 1:1, and the diluted mixture of semen is dispensed into 2 mL cryopreservation tubes, with each tube containing 0.2 mL.

[0009] In the above technical solution, in step (2), the antifreeze is obtained by compounding MPRS diluent with ethylene glycol, and the mass concentration of ethylene glycol in the system is 5%~15%, preferably 15%.

[0010] In the above technical solution, the MPRS diluent is prepared from glucose, NaCl, NaHCO3, KCl, CaCl2·2H2O, NaH2PO4, MgCl2·6H2O and purified water. The concentrations of each component dissolved in purified water are as follows: glucose 11.01 g / L, NaCl 3.53 g / L, NaHCO3 0.25 g / L, KCl 0.39 g / L, CaCl2·2H2O 0.17 g / L, NaH2PO4 0.22 g / L, MgCl2·6H2O 0.23 g / L.

[0011] In the above technical solution, in step (3), the low temperature equilibrium refers to equilibration at 4°C for 10 minutes; the pre-cooling refers to pre-cooling 10 cm above the liquid nitrogen surface for 5 minutes and pre-cooling above the liquid nitrogen surface for 5 minutes.

[0012] In the above technical solution, step (3) refers to taking out the frozen sperm mixture, thawing the sperm in a water bath at 37°C, and then activating the sperm with seawater at room temperature.

[0013] Compared with existing technologies, it has the following characteristics: (1) The method is simple to operate and requires minimal equipment; only an insulated foam box and a liquid nitrogen tank are needed to achieve on-site cryopreservation of spotted sea bream germplasm resources. (2) Using 2 mL cryotubes for preservation, a large-scale frozen sperm bank of spotted sea bream can be quickly established; (3) Cryopreservation has good effect, low cost, and can preserve the sperm of spotted sea bream for a long time, so as to realize the long-term stable preservation of spotted sea bream germplasm resources. It can also collect and preserve the germplasm resources of high-quality spotted sea bream parents for breeding and expansion. Detailed Implementation

[0014] The following describes in detail the specific embodiments of the technical solution of the present invention, but the present invention is not limited to the following description: Screening Example 1: Selecting the optimal type and ratio of antifreeze: 1. Experimental materials: All spotted sea bream used in the experiment were provided by Laizhou Mingbo Aquatic Products Company. Six healthy, sexually mature male sea bream with no external injuries were selected as experimental materials. Semen was collected using the squeezing method. The male sea bream were injected with a luteinizing hormone-releasing hormone analog (LRH-A2) at 2.5 μg / kg the day before collection to induce maturation. Semen collection could be performed the day after injection. During collection, the male sea bream were removed from the water, their abdomens were dried with a towel, and the abdomens were gently squeezed. The mature semen flowed out naturally and was collected using a pipette into a clean glass container. Care was taken to prevent contamination by water, blood, body mucus, urine, and feces. Water and body mucus could be removed simply by wiping with a towel before collection. If the squeezing site was incorrect, urine and feces could easily be squeezed out, especially urine, which may be difficult to detect in small amounts. Therefore, the collection site must be accurate. If the squeezed semen is light yellow or yellow, it is likely contaminated with urine and should be discarded.

[0015] 2. Preparation of sperm diluent: Sperm diluents MPRS and Cortland were prepared in purified water using glucose, NaCl, NaHCO3, KCl, CaCl2·2H2O, NaH2PO4, MgCl2·6H2O, and MgSO4·7H2O. The specific formulas are shown in Table 1. Table 1. Composition of the diluent used for cryopreservation of spotted sea bream sperm. Table 1. Composition of diluent for cryopreservation of ethylene glycol nathuspunctatus (g / L) ingredients MPRS Cortland glucose 11.01 1.0 NaCl 3.53 7.25 NaHCO3 0.25 1.0 KCl 0.39 0.38 CaCl2·2H2O 0.17 0.18 NaH2PO4 0.22 0.41 MgCl2·6H2O 0.23 - MgSO4·7H2O - 0.23 3. Screening of antifreeze agents and their concentrations To screen for the optimal combination of diluent and cryoprotectant for cryopreservation of spotted sea bream sperm, ethylene glycol, the most common permeable cryoprotectant, was used to prepare cryoprotectants with MPRS and Cortland diluents, respectively, at concentration gradients of 5%, 10%, and 15%. The cryoprotectant was diluted with semen at a 1:1 volume ratio, and the diluted semen was aliquoted into 2 mL cryovials, 0.2 mL per tube. The tubes were then equilibrated at 4°C for 10 min, pre-cooled at 10 cm above the liquid nitrogen surface for 5 min, and then pre-cooled above the liquid nitrogen surface for 5 min before being immersed in liquid nitrogen for cryopreservation. The cryopreserved sperm mixture was thawed in a water bath at 37°C, and then activated with seawater at room temperature. Sperm motility was immediately observed and measured under a microscope. This freezing-thawing and activation process was repeated to determine the optimal cryoprotectant and concentration.

[0016] 4. Results: Table 2 shows the sperm reactivation rate and rapid motility time when the ethylene glycol content in the cryoprotectant is different: Table 2. Resurrection rate and rapid motility time of spotted sea bream sperm after cryopreservation. ratio survival rate (%) fast swimming time (s) MPRS + 5% ethylene glycol 10%±(0%) 39.72±6.74 MPRS + 10% ethylene glycol 70%±(13.33%) 81.38±29.52 MPRS + 15% ethylene glycol 70%±(1.73%) 126.78±46.82 Cortland + 5% ethylene glycol 26.66%±(4.44%) 51.67±3.58 Cortland + 10% ethylene glycol 33.33%±(11.11%) 48.30±2.96 Cortland + 15% ethylene glycol 65%±(10%) 84.08±9.32 fresh sperm 91.67 ± 2.88 139.95 ± 9.17 As shown in Table 2, since the recovery viability of MPRS+15% ethylene glycol reached (70±1.73)% and the rapid motility time reached (126.78±46.82)s, there was no significant difference compared with fresh semen; therefore, MPRS+15% ethylene glycol was selected as the cryopreservation solution for spotted sea bream sperm.

[0017] Establishment of a sperm cryopreservation bank: Record relevant information such as the geographical location, population, age, cryopreservation solution used, dilution ratio, freezing steps, freezing date, and storage location of the collected parent fish, establish a sperm cryopreservation file, and place 100 samples of spotted sea bream semen in the sperm cryopreservation bank for storage.

[0018] The present invention will now be described in conjunction with specific embodiments: Example 1: (1) Sperm collection: The method for collecting sperm is the same as in Example 1: six healthy, sexually mature male fish with no external injuries are selected as the collection targets. One day before collecting sperm from the spotted rock seabream, the male spotted rock seabream are injected with a luteinizing hormone-releasing hormone analog (LRH-A2) at a dose of 2.5 μg / kg. The sperm can be collected by squeezing the day after the injection and the mature semen is collected into a clean glass container for later use.

[0019] (2) Diluting sperm: MPRS diluent was prepared using the same formula as in Screening Example 1; antifreeze was prepared by mixing MPRS diluent with ethylene glycol, with the mass concentration of ethylene glycol in the system being 15%; the antifreeze and semen were diluted at a volume ratio of 1:1 to obtain a sperm mixture. (3) Balancing and cryopreserving sperm: The sperm mixture obtained in step (2) was equilibrated at 4°C for 10 min; then, it was pre-cooled at 10 cm above the liquid nitrogen surface for 5 min, then pre-cooled above the liquid nitrogen surface for 5 min, and finally frozen in liquid nitrogen.

[0020] If needed, the frozen sperm mixture can be thawed and activated to obtain activated sperm: take out the frozen sperm mixture, thaw the sperm in a water bath at 37°C, and then activate the sperm with seawater at room temperature; observe and count the sperm motility under a microscope immediately. In this example, the sperm revival rate was 70.33 ± 2.52%, and the rapid motility time reached 125.09 ± 25.73 s.

[0021] The above examples are merely illustrative of the technical concept and features of the present invention and should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the essence of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for cryopreservation of sperm of Oplegnathus fasciatus, characterized in that, The method comprises the following steps: (1) collecting sperm: The day before collecting sperm of Oplegnathus punctatus, the male fish is subjected to drug-induced maturation, and the day after maturation, the sperm is collected by squeezing method and collected into a clean glass container for use; (2) diluting sperm: The sperm obtained in step (1) is diluted with an antifreeze solution, and the sperm mixture is divided into 2ml cryopreservation tubes for standby; (3) balancing and freezing sperm: The sperm mixture obtained in step (2) is balanced at low temperature for a period of time, then pre-cooled above liquid nitrogen, and finally frozen in liquid nitrogen; if needed, the frozen sperm mixture is recovered and activated to obtain activated sperm.

2. The method for cryopreservation of sperm of Sparus aurata according to claim 1, characterized in that, In step (1), the Oplegnathus punctatus male fish is healthy, without injury on the body surface, strong vitality and mature.

3. The method for cryopreservation of spotted sea bream sperm according to claim 1, characterized in that, In step (1), the drug for drug-induced maturation is luteinizing hormone-releasing hormone analogue LRH-A2, and the injection amount is 2.5 μg / kg, and the sperm can be collected the day after injection.

4. The method for cryopreservation of spotted sea bream sperm according to claim 1, characterized in that, In step (2), the antifreeze solution and the sperm are diluted at a volume ratio of 1:1, and the diluted mixture is divided into 2 mL cryopreservation tubes, 0.2 mL per tube.

5. The method for cryopreservation of spotted sea bream sperm according to claim 1, characterized in that, In step (2), the antifreeze solution is obtained by compounding MPRS diluent and ethylene glycol, and the mass concentration of ethylene glycol in the system is 5%-15%.

6. The sperm freezing method of Sparus aurata according to claim 5, characterized in that, The mass concentration of ethylene glycol in the system is 15%.

7. The sperm freezing method of Sparus aurata according to claim 5, characterized in that, The MPRS diluent is prepared from glucose, NaCl, NaHCO3, KCl, CaCl2·2H2O, NaH2PO4, MgCl2·6H2O and pure water, and the concentrations of the components dissolved in pure water are: glucose 11.01 g / L, NaCl 3.53 g / L, NaHCO3 0.25 g / L, KCl 0.39 g / L, CaCl2·2H2O 0.17 g / L, NaH2PO4 0.22 g / L, MgCl2·6H2O 0.23 g / L.

8. The method for cryopreservation of spotted sea bream sperm according to claim 1, characterized in that, In step (3), the low-temperature balancing refers to balancing at 4°C for 10 minutes; the pre-cooling refers to pre-cooling at a distance of 10 cm above the liquid nitrogen surface for 5 minutes, and pre-cooling above the liquid nitrogen surface for 5 minutes.

9. The sperm freezing method of Sparus aurata according to claim 1, characterized in that, In step (3), the recovery and activation refer to taking out the frozen sperm mixture, thawing the sperm in a 37°C water bath, and activating the sperm at room temperature after thawing.