JP2000239101A - Rat sperm cryopreservation method - Google Patents

Abstract

(57) [Summary] [Objective] To provide a cryopreservation method for rat sperm. [Constitution] pH of sperm collected from rat at room temperature
, An osmotic pressure adjusting agent that keeps the pH in the range of 6.8-7.3,
Suspension is carried out in a primary diluent containing an electrolyte, a nutrient source, a sperm membrane protective component, and a bacteriostatic agent.
Leave for at least 0 minute within 24 hours and at 2-10 ° C. for at least 10 minutes within 24 hours, and then add the secondary diluent containing a protective agent to prevent freezing damage of the cells with the sperm suspension primary diluent. Cool to the same temperature of 2-10 ° C., add this secondary diluent to the sperm suspension primary diluent,
A method of cryopreservation of rat spermatozoa in which the solution after stirring is frozen by a straw method or a pellet method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for cryopreserving rat spermatozoa. In particular, the present invention relates to a cryopreservation method for rat sperm that is not subjected to centrifugation and washing before primary dilution.

[0002]

2. Description of the Related Art Long-term preservation methods for semen include 1) liquid preservation method and 2) cryopreservation method. The latter method allows semipermanent preservation of semen because catabolic metabolism of semen is almost zero. To Cryopreservation of mammalian sperm such as cows, pigs, sheep, and mice is performed by haploid genome (haploid geometry).
nome) is very important as a method for preserving genetic resources, such as efficient storage of strains such as transgenics and rare species,
Applied to multiplication. It has also been established as a fertility treatment in humans. On the other hand, transgenic rats are very important as experimental animals in the medical field, particularly in the fields of physiology and nutrition, and are also valuable animals for disease models such as diabetes and hypertension. Because no preservation method has been developed, rats having these particular traits had to be utilized while being bred, bred and maintained.

In the conventional cryopreservation of livestock semen, 500-1000 g of semen is collected after collection of sperm because components in the seminal plasma have an adverse effect on the viability of sperm after freeze-thawing. A treatment for removing sperm by centrifugation to wash sperm is performed. Further, in order to replace the washing liquid used for this washing with the semen diluting liquid (primary diluting liquid) at the time of freezing, a similar centrifugation treatment is applied to the semen. Thus, in the process of cryopreservation of semen, there are many opportunities to apply physical stimuli and shocks to the semen, but there is little effect on sperm motility in cattle, pigs, sheep, mice, etc. There is no problem in mobility. However, it has been thought that sperm cannot be preserved in rats by the cryopreservation method employed for spermatozoa of the above-mentioned animals, and cryopreservation of sperm cannot be performed in rats. There is no report on cryopreservation of rat sperm.

[0004]

Accordingly, it is an object of the present invention to establish and provide a method for cryopreserving rat sperm.

[0005]

The present inventors have studied that the spermatozoa of rats are more sensitive to physical stimuli such as centrifugation and shocks than spermatozoa of the above-mentioned animals. It is surprising to try the freezing and thawing treatment, which eliminates the centrifugation and washing treatments, which gives a physical stimulus and impact, to the above-mentioned method usually used in cryopreservation of conventional sperm. Fortunately, it has been found that cryopreservation of rat sperm is possible. The gist of the present invention is that sperm collected from a rat is adjusted to pH 6.
The suspension is kept in a primary diluent containing a buffer, an osmotic pressure regulator, an electrolyte, a nutrient, a sperm membrane protective component, and a bacteriostatic agent, which are kept in the range of 8-7.3, and the suspension is placed in a 15 ° C incubator for 10 minutes. Not less than 24 hours and not less than 10 minutes at 2-10 ° C
Leave for 4 hours or less, then cool the secondary diluent to which a protective agent for preventing cell freezing damage has been added to 2-10 ° C. at the same temperature as the primary diluent for sperm suspension. Add the primary diluent to the sperm suspension primary diluent, stir,
A cryopreservation method for rat spermatozoa characterized by freezing the liquid after stirring by a straw method or a pellet method, and preferably, a container filled with the liquid after stirring and holding the liquid after stirring the sperm suspended therein. 1-4c from the surface of liquid nitrogen
m for 2 to 60 minutes after leaving at a distance in the range of m, and then freeze-preserving the rat spermatozoa into liquid nitrogen, or dropping a sperm-floating agitated liquid into a small dry ice hole and cooling to -80. And cooled to 2-20 ° C.
This is a method for cryopreservation of the rat spermatozoa, which is left in liquid nitrogen after standing for minutes. The present inventors have solved the above-mentioned problem by eliminating the centrifugation treatment and the washing treatment, and appropriately combining the composition of the diluent, the antibiotic to be added, the cooling condition, the freezing condition, and the like.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail. About collection of rat sperm. Spermatozoa are mature male rats aged 12-15 weeks, especially SD
Strain: Collected from the epididymis of Spraque-Dawley rats. For the collection method, for example, anesthetize the male rat,
After killing, the epididymis was removed and kept at room temperature (23-25).
℃) in a primary diluent to release sperm,
In addition, there is a method in which a sperm is collected without killing the male by using the phenomenon of ejaculation when anesthetizing a male rat with ether.
As the above-mentioned primary diluent, the yolk liquid shown in Table 1 below is preferable. In addition, a liquid for tissue culture, in vitro fertilization, or cryoserum dilution of human, bovine, mouse and the like can be used.

[0007]

[Table 1]

[0008] Since the diluent or preservation solution is basically an in vitro storage medium for sperm instead of seminal plasma, it has pH, osmotic pressure, buffering action, electrolytes, nutrients of seminal plasma, and sperm. Contains lipoprotein components such as egg yolk for membrane protection (milk or skim milk powder may be used in place of egg yolk), and antibiotics as bacterial inhibitors. For cryopreservation, it is necessary to incorporate a secondary diluent to which a protective agent for preventing cell freezing damage, for example, glycerin, is added.

In place of the glycerin added to the secondary diluent, other cryopreservatives such as DMSO, ethylene glycol, propanediol, ethanol, acetamide, sucrose, trehalose, polyvinylpropidone, raffinose, glucose , Skim milk, serum,
What added BSA etc. at an appropriate concentration is used.
Various antibiotics such as penicillin and streptomycin can be used as the bacterial inhibitor. The composition used in the primary diluent can be adopted as the basic composition of the secondary diluent, and the blending amount of the secondary diluent is almost equivalent to that of the primary diluent.

[0010] Freezing operation. In order to freeze and store the semen, the sperm is cooled to around 0 ° C., passes through 0 ° C. or less at which the medium freezes without impairing the motility and fertility at the time of collecting the sperm, and the liquid nitrogen is discharged at −196 ° C.
Need to be cooled down. Therefore, in the freezing operation of rat semen, in order to protect spermatozoa from the low-temperature shock during cooling and the formation of ice crystals during freezing, the composition of the diluent, for example, conventionally used for cryopreservation of pig spermatozoa. It is necessary to select the composition of the diluted solution, and appropriately select the cooling conditions and the freezing conditions. Also, as a storage method,
There are a straw method and a pellet method.
Plastic straw, ie 0.25 ml, 0.5
The solution is inhaled into a straw of 1.0 ml or 1.0 ml, a space is provided, and sealed with straw powder or the like. In addition, in this specification, a straw means a container which can contain a liquid formed by mixing and mixing a secondary diluent for freezing, and examples thereof include a capsule, a plastic straw, and a glass tube. . The enclosed straw can be selected 2 cm (1 to 4 cm) from the liquid nitrogen level.
After leaving it for 2 minutes (2-60 minutes), it is poured into liquid nitrogen. In the pellet method, a small amount of the final diluent after addition of the secondary diluent, for example, 0.01 to 2.0 ml, is dropped into a small hole of dry ice and cooled to −80 ° C., and the temperature is reduced to several minutes. (2-20 minutes) After standing, put into liquid nitrogen.

[0011]

EXAMPLES Example 1 After SD male adult rats were anesthetized and killed, their epididymis was excised and chopped in a primary diluent at room temperature to suspend sperm. Put the sperm suspension in a thermostat at 15 ° C for 30 minutes.
And then put in a refrigerator at 4-5 ° C for 30 minutes.
Allowed to cool for minutes. In addition, this 15 degreeC and 4-5
The standing time at ° C may be 10 minutes or more and 24 hours or less. About 6% (v
/ V) glycerin (can be selected in the range of 1-10%) and OEP (obverse) of about 1.4% (v / v) (can be selected in the range of 0.5-4.0%). Es paste)
(Surfactant from NOVA CHEMICAL SALES, INC., MA, USA)
Is cooled in a refrigerator at 4-5 ° C. By doing so, the motility of sperm after freeze-thaw can be improved. Then, dilute the sperm suspension dilution at 4-5 ° C.
After cooling, the secondary diluent at 4-5 ° C. was added in the same amount as the sperm floating primary diluent and stirred. This solution was added to 0.25 and 0.
The mixture was sealed in a 5 ml plastic straw and kept at 1-4 cm from the liquid nitrogen level for several minutes to 20 minutes (straw method) or cooled by dripping into a small hole of dry ice (bellet method), and then cooled. The beret on the straw or on the dry ice is immersed in liquid nitrogen and stored frozen.

The sperm of the rat frozen by each of the above methods was thawed, and the fertilization ability of the frozen and thawed spermatozoa was examined. In the straw method, a straw stored in liquid nitrogen was melted by shaking in warm water at 35 ° C. for 10 seconds. After thawing, a sperm diluent that can appropriately dilute about 1 ml of sperm sperm
It is called frozen sperm thawing solution. ), For example, RIECM; Miyo
shi et al. 1993, a sperm suspension in a medium hestro may be injected, which may be a common embryo culture or tissue culture. In the pellet method, 0.05 ml of frozen semen pellet was directly added to 1 ml of frozen sperm melt at 38 ° C. to be thawed. Thawed sperm immediately after thawing, 30 minutes later,
After 1 hour, 2 hours, 3 hours, and 4 hours, the vitality was observed microscopically with a semen property inspection panel (manufactured by Fujiheisha), and the sperm survival index was determined. The sperm survival index is obtained by giving ++, ++, +, ± to the sperm's motility.
The coefficient is determined by giving a coefficient of + to 100, 75 to +, 60 to +, and 25 to ±, multiply the respective survival rates, and sum the obtained numerical values and divide by 100. The results are shown in Table 2 for the straw method and in Table 3 for the pellet method (however, data up to 30 minutes after melting).

[0013]

[Table 2]

[0014]

[Table 3]

Results and discussion: It is understood from Tables 2 and 3 that spermatozoa exhibiting motility can be obtained after freeze-thawing in both the straw method and the pellet method.

Confirmation of fertilization ability of frozen-thawed spermatozoa. In order to determine the fertilizing ability of the frozen and thawed spermatozoa of the rat semen cryopreserved by the straw method, 0.05 m was injected into the uterus of a female rat (10-20 weeks old) in which pseudopregnancy was induced.
One liter of frozen-thawed semen (total sperm count: 3-5 × 10 ⁶ ) was surgically injected. On days 19-20 post-semen injection, these females were anesthetized and laparotomized to confirm the presence of fetuses in the uterus.
Table 4 shows the results.

[0017]

[Table 4]

As shown in Table 4, whether or not the frozen-thawed sperm had normal fertility was examined by artificial insemination by injection into the uterus, and a viable morphologically normal fetus was obtained. From the above results, it was revealed that the cryopreservation method of rat spermatozoa of the present invention shows that sperm of rats after freeze-thawing has normal fertility and develops into 19-20 day-old fetuses. It can be seen that the rat sperm cryopreservation method of the present invention is useful.

Further, an experiment shown in Table 5 was conducted in order to examine the effect of physical stimulation by centrifugation of rat sperm on rat sperm. Rat sperm were found to be vulnerable to physical stimuli, reducing their motility.

[0020]

[Table 5]

By the way, when the influence of the centrifugation treatment of porcine spermatozoa was examined under the same conditions as that of the rat spermatozoa, the sperm survival index was lower without the spermatozoa. This indicates that the effect of seminal plasma is greater than in rats.

[0022]

As described above, the discovery that cryopreservation of rat sperm is possible and the establishment and provision of a method for cryopreservation of rat sperm are described in the medical field, especially in physiology and nutrition. As an experimental animal in the field of
In addition, the present invention has an effect of facilitating maintenance of a rat that is valuable as a disease model animal such as diabetes and hypertension, and facilitating the supply of the rat.

Claims (3)

[Claims] The present invention comprises a buffer, an osmotic pressure regulator, an electrolyte, a nutrient, a sperm membrane protective component, and a bacteriostatic agent that maintain the pH of a sperm collected from a rat at room temperature in the range of 6.8 to 7.3. Float in the primary diluent, leave it in a 15 ° C incubator for 10 minutes or more and 24 hours or at 2-10 ° C for 10 minutes or more and 24 hours, and then add a protective agent to prevent cell freezing damage Then, a secondary diluent cooled to 2-10 ° C., which is the same temperature as the sperm floating primary diluent, is added to the sperm floating primary diluent and stirred, and the liquid after the stirring is frozen by a straw method or a pellet method. A cryopreservation method for rat spermatozoa. 2. A container filled with a sperm-floated stirred liquid while maintaining a space, is left within a range of 1-4 cm from the surface of liquid nitrogen, left for 2-60 minutes, and then poured into liquid nitrogen. The method for cryopreserving rat spermatozoa by the straw method according to claim 1. 3. The liquid after stirring, in which the sperm is suspended, is dropped into a small hole of dry ice, cooled and cooled to −80 ° C., left at that temperature for 2 to 20 minutes, and then poured into liquid nitrogen. The method for cryopreserving rat spermatozoa according to claim 1.