WO2017116250A1

WO2017116250A1 - Salmonid sperm cryopreservation method

Info

Publication number: WO2017116250A1
Application number: PCT/PL2016/000050
Authority: WO
Inventors: Joanna NYNCA; Andrzej CIERESZKO
Original assignee: Instytut Rozrodu Zwierzat I Badan Zywnosci Polskiej Akademii Nauk
Current assignee: Instytut Rozrodu Zwierzat I Badan Zywnosci Polskiej Akademii Nauk
Priority date: 2015-12-31
Filing date: 2016-05-09
Publication date: 2017-07-06
Anticipated expiration: 2018-06-30
Also published as: PL231643B1; PL415680A1; EP3397053A1

Abstract

The method of fish sperm cryopreservation, in accordance with the invention, is characterized by the fact that sperm is obtained from prepared males or neo-males, and inspected for quality, then diluted with glucose- methano! extender, and equilibrated for 15 minutes, and subsequently packed into straws which are then closed using a specialist apparatus, labelled with sperm data, and frozen in vapours of liquid nitrogen.

Description

Salmon id sperm cryopreservation method

The object of the invention is fish sperm cryopreservation method, in particular applicable to the Salmonidae and Coregoninae.

Modern hatchery is required to ensure diversified supplies based on the production of Salmonid species, hybrides and lines throughout the year. The main purpose of hybridization is linked with the breeding preferences which aim at producing specimens showing positive qualities resulting from heterosis, such as pace of growth, and immunity to diseases, the latter being especially important in conditions of intense fish breeding. In Salmionids beneficial effects also result from polyploidization, in particular triploidization of hybrides. The necessity to ensure all-year-long supply of fish stands in direct contradiction to the biology of Salmonids, characterized with the highly seasonal reproductive cycle. It is possible to overcome the seasonal character by creating artificial photoperiod conditions aimed at "deceiving" nature, e.g. by creating a year with an altered length, either shorter (approx. 6 months), or longer (approx. 18 months). As a result, the timing of reproductive readiness (spawning period) is moved to a period before or after the natural spawning. The basic problem faced in fish hatching is the necessity to ensure maturity of male and female specimens at the same time. The task is difficult or indeed almost impossible to achieve, e.g. if the periods of parental spawning occurred at significantly different times.

One method developed for Salmonids and used with success was based on sperm pellets processing (Glogowski et ai., 2000). Unfortunately, that method has three drawbacks:

- it is impossible to identify the specific sperm samples (because samples of frozen sperm cannot be marked separately,

- there is a potential risk of transmitting pathogens, in particular viruses, between sperm samples stored in liquid nitrogen (since sperm pellets are not isolated from liquid nitrogen),

- limited possibilities of manipulating with thawed sperm

Other factors significantly reducing chances for successful use of cryopreserved sperm in the practice of fish farming include: poor quality of cryopreserved sperm (approx. 10% motility after thawing) and the recommendation for using the sperm immediately after thawing. Sperm pellet processing is connected with the necessity to activate sperm cell movement immediately after thawing which is a disadvantage due to the extremely short time of motility in sperm cells of Salmonids (only 10-20 s). Due to the above reasons the method of packaging sperm in pellets is no longer used, and the only method used now involves cryopreservation of sperm in straws, i.e. enclosed plastic tubes, most frequently with a capacity of 250 μΙ, yet the quality of the frozen sperm continues to be limited. It is assumed that in order to achieve similar results it is necessary to use 10 x more (i.e. 1000%) cryopreserved spermatozoa in comparison to fresh semen (Billard, 1992), in a period of 20 seconds after thawing, which significantly limits the time required for fertilization (Lahnsteiner, 2000).

The invention is designed to enable cryopreservation at the time when the sperm from males and masculinized females is of the highest quality, establishment of sperm banks for the basic lines cultured at a hatchery, application of cryopreserved sperm for numerous special purposes in fish farming when the roe is of the highest quality.

The method of fish sperm cryopreservation, in accordance with the invention, is characterized by the fact that sperm is obtained from prepared males or neo-males, and inspected for quality, then diluted with glucose- methanol extender, and equilibrated for 15 minutes, and subsequently packed into straws which are then closed using a specialist apparatus, labelled with sperm data, and frozen in vapours of liquid nitrogen.

Preparation of males or neo-males at the optimal stage of sexual maturity is of profound importance for the success of cryopreservation. Due to the fact that cryopreservation, by its definition, causes damage in cells, good quality of the input material (sperm) is of key importance.

Sperm acquisition and quality control involves inspection of the sperm concentration and motility. Concentration is assessed with calorimetric measurement and motility with an objective method of Computer-assisted sperm analysis (CASA). As a result of these tests sperm will be qualified for freezing.

Diluting the sperm with giucose-methanol extender and short (15 minute long) equilibration. Filling in and closing the straws with a specialist apparatus, and at the same time imprinting information onto the straws with a specialist apparatus.

Freezing in vapours of liquid nitrogen.

Quality control of the freezing process. Sperm is assessed for quality taking into account its post-thawing motility, and based on that it is qualified for storage. Storage of sperm in liquid nitrogen and its use to meet production targets.

The findings acquired in the season from autumn 2013 to spring 2014 show universal applicability of the developed cryopreservation method for Salmonids. High quality sperm was obtained after thawing material from all the investigated species of both genera: Salmo and Salvelinus. Likewise, the results acquired in the case of European whitefish sperm are twice as high as those obtained recently, which has been presented in the following table.

Percentage of sperm cells and fertilizing capacity of semen cryopreserved with the use of glucose- methanol extender (mean ± SD). The chart shows absolute data for fertilization rate, amounting to 80 - 100% of the control values.

The first experiments were conducted to re-scale the method for the needs of application in fertilizing a larger amount of roe. Experiments of this type are of key importance for practical implementation of the method. The initial findings show high fertilization rates (83 - 90%) for the number of roe grains in the range of 100 - 1600, which means a 16-fold increase in comparison with the previously applied procedure (Fig. 7). This may be a breakthrough in attempts towards re-scaling the method. Contrary to the common opinions, the presented high fertilization rates were obtained for sperm of masculinized females from spring spawn (May). Up until now it was assumed that sperm from this period cannot be successfully frozen. In another example, in production conditions three portions of roe, each with 10,000 grains, were successfully fertilized. The obtained results confirm effectiveness and universal applicability of the developed method. The invention enabled initial progress in attempts towards re-scaling, which is shown in the Figure 1. The object of the invention has been presented as an executed example whereby the cryopreservation method was used for sperm of rainbow trout. The example has shown usefulness of four extenders (I methanol, glucose; II DMSO, glucose; III methanol, sucrose with addition of yolk, IV methanol and sucrose) for cryopreservation of rainbow trout sperm in straws (Fig. 1 , Nynca et al., 2013). Extenders I and II proved to be most effective. In spite of decreased motility and life span of spermatozoa frozen with the use of extender I and II, the findings show that frozen sperm retained similar fertilizing capacity to that of fresh sperm. It should be emphasized that the ratio of sperm cells to an egg at the time of fertilization was 2 million, which is less by approx. 3 million sperm cells/egg recommended in fertilization procedures using cryopreserved sperm (Billard 1992). In connection with this the assumed goal was achieved whereby method of packaging rainbow trout sperm in straws was initially developed as an alternative to the traditional procedure of cryopreservation of sperm pellets. The method based on the use of methanol and glucose was a starting point for further works aimed at improving cryopreservation of rainbow trout sperm.

Cryopreserved sperm will be used for: preserving genetic material of valuable lines for fish culture, applying sperm in stimulation of gynogenesis, producing desired commercial cross-breeds and for preserving unused excess sperm.

Claims

Patent claim

The method of fish sperm cryopreservation, based on freezing the material, characteristic in the fact that sperm is obtained from prepared males or neo-males, and inspected for quality, then diluted with glucose-methanol extender, and equilibrated for 15 minutes, and subsequently packed into straws which are then dosed using a specialist apparatus, labelled with sperm data, and frozen in vapours of liquid nitrogen.