JP2008063235A - Diluted liquid of sperm and method for preserving diluted sperm - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inexpensive diluted liquid of sperm maintaining the sperm activity and cold storable and preservable over a longer period than the conventional diluted liquid of sperm and to provide a method for preserving the diluted sperm. <P>SOLUTION: The diluted liquid of sperm comprises rutin, a specific oil-and-fat or a specified oil and fat emulsified with a polyoxyethylene glycerol fatty acid ester. The method for preserving the diluted liquid of sperm comprises adopting multi-stage cooling. The method for preserving the diluted liquid of sperm comprising the rutin and either one or more of the specific oil-and-fat or that emulsified with the polyoxyethylene glycerol fatty acid ester comprises adopting the multi-stage cooling. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a diluting solution for sperm cryopreservation and a method for cryopreserving sperm.

  In general, livestock semen is diluted and circulated at low temperature or frozen. As a diluted stock solution of pig semen, “modena solution (trade name)”, “androhep solution” and the like are known. The components of the modena solution are glucose 5.50, citric acid NA 1.38, sodium bicarbonate 0.20, EDTA 0.47, citric acid 0.58, and tris 1.13 (g / 200 ml). These diluted stock solutions are medium temperature range stock solutions and can store pig semen diluted at around 16 ° C. for several days.

  Further, as a solution for diluting and storing pork semen, Patent Document 1 describes a “dilution for storing pork semen characterized by adding and mixing 20 parts (parts by weight) of B liquid into 1000 parts of the following A liquid. Solution A—A mixture of the following reagents in 1000 parts in the following proportions: (1) 5.0 parts of sodium citrate (2) 1.0 part of albumin (bovine serum) (3) Sodium β-glycerophosphate 1 0.0 part (4) sodium bicarbonate 1.0 part (5) sodium pyruvate 0.4-0.5 part (6) potassium chloride 0.5 part (7) EDTA-2Na 1.2-1.3 part (8) Amikacin 0.07 to 0.08 part (9) Dibekacin 0.02 to 0.03 part (10) Trishydroxymethylaminomethane 1.5 to 2.0 parts (11) GSH (glutathione) 1.5 ~ 1.6 parts (12) trehalose 45-4 Part (13) Sterilized distilled water remainder B liquid-dimethyl sulfoxide (DMSO) 20 parts dissolved in 0.20-0.25 parts (10 mM) of butylhydroxytoluene (BHT) and heated to 70 ° C or higher with a hot water bath In addition, 80 parts of sterilized distilled water heated in the same manner is added. "A semen dilution for storing pig semen is disclosed.

Patent Document 1 discloses a method for diluting and storing semen of pigs in a refrigerated manner: “Pig semen preservation by adding 20 parts of B solution to 1000 parts of the following A solution at an equal temperature of about 35 ° C. Add about 4 to 5 times the dilution solution for semen, adjust the number of sperm to about 0.5 to 100 million / ml, subdivide the diluted semen and heat to 15 ° C in a thermostatic bath. A method for diluting and storing swine semen characterized in that the semen is stored in a refrigerator by lowering the solution A. A mixture of the following reagents in 1000 parts at a mixing ratio: (1) Sodium citrate 0 parts (2) Albumin (bovine serum) 1.0 part (3) Sodium β-glycerophosphate 1.0 part (4) Sodium bicarbonate 1.0 part (5) Sodium pyruvate 0.4-0.5 part (6) 0.5 parts of potassium chloride (7) EDTA-2Na 1.2-1.3 (8) Amikacin 0.07 to 0.08 part (9) Dibekacin 0.02 to 0.03 part (10) Trishydroxymethylaminomethane 1.5 to 2.0 parts (11) GSH (glutathione) 1.5 -1.6 parts (12) Trehalose 45-46 parts (13) Sterilized distilled water remainder B liquid-dimethyl sulfoxide (DMSO) 20 parts, butylhydroxytoluene (BHT) 0.20-0.25 parts ( 10 mM) dissolved, heated to 70 ° C. or higher with a hot water bath, and then added with 80 parts of sterilized distilled water heated in the same manner.
5. The method for diluting and storing porcine semen according to claim 4, wherein the temperature of the diluted semen of about 35 ° C. is lowered to 15 ° C. in about 2 hours and 30 minutes. Moreover, the diluted semen of about 35 ° C. is further lowered from 15 ° C. to 5 ° C. in about 1 hour and 20 minutes, and the diluted semen is stirred at a predetermined interval ”. Is published.
JP 2000-119101 A

  In addition, there are Patent Documents 2 to 5 as sperm, semen preservation solution, and low-temperature preservation method. Patent Document 2 discloses a sperm low-temperature injury prevention solution containing a long-chain alcohol having 10 to 14 carbon atoms and a lipid, and a long-chain alcohol having 10 to 14 carbon atoms, a lipid, and serum albumin. The contained cryogenic injury prevention solution is described.

  Patent Document 3 includes a step of providing a sperm sample; a step of cooling the sperm sample to a first temperature; a step of maintaining the sperm sample at the first temperature; a step of cooling the sperm sample to a second temperature; A cryopreservation of sperm is described that includes maintaining the sperm sample at the second temperature; and storing the sperm sample at a third temperature.

  Patent Document 4 describes a method for cryopreserving sex-selected sperm over 1 to 4 hours to 5 ° C. and further cryopreserving a selected sperm cell, which is stored frozen. Yes.

Patent Document 5 describes a sperm cryoprotectant containing 8 to 13 carbon atoms or an aliphatic hydrocarbon having 10 to 13 carbon atoms or decane. Furthermore, a method for preserving sperm is described which includes a step of cooling sperm in the presence of a protective agent containing an aliphatic hydrocarbon having 8 to 13 carbon atoms.
JP-A-10-279401 JP-T-2004-505624 Japanese translation of PCT publication No. 2003-530312 JP-A-8-26901

  However, refrigerated storage of swine diluted semen by “modena solution” at 10 ° C. or lower and artificial insemination using diluted semen stored refrigerated at 10 ° C. or lower are not widespread. This is because it is difficult to secure the sperm necessary for conception because the survival rate of sperm is significantly reduced within a few days after refrigerated storage.

  In addition, the “semen dilution for preserving swine semen” described in Patent Document 1 has an extremely high manufacturing cost of the dilution compared to the existing Modena solution, and in addition, it is difficult to say that the fertilization rate of the stored semen is high. Incidentally, the dilution liquid described in Patent Document 1 costs about three times as much as the modena liquid (85 yen). On the other hand, the semen dilution liquid according to the present invention has the same cost as the modena liquid.

  Furthermore, “diluted preservation method of pig semen” described in Patent Document 1 adds 14 kinds of reagents and performs cold storage. However, these reagents and the temperature drop method are in terms of cost as described above. Not practical.

  Accordingly, an object of the present invention is to provide a semen dilution solution that can maintain sperm activity at low cost and can be refrigerated for a longer period of time than before, and a method for storing the diluted semen.

In order to solve the above-mentioned problems, the present invention has a configuration of a semen dilution liquid characterized by containing rutin,
A semen dilution characterized by containing one or more oils and fats selected from avocado oil, cocoon oil and almond oil,
One or more oils selected from avocado oil, coconut oil, almond oil, pumpkin seed oil, babas oil, borage oil, black currant seed oil, canola oil, castor oil, coconut oil, corn oil, cottonseed oil, evening primrose oil , Grape seed oil, American potato oil, mustard seed oil, olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, safflower oil, sesame oil, shark liver oil, soybean oil, sunflower oil, their hardened oil, those Oil extraction, lecithin, glyceryl tricaproate, glyceryl tricaprylate, glyceryl tricaprate glyceryl triundecanoate, glyceryl trilaurate, glyceryl trioleate, glyceryl trilinoleate, glyceryl trilinolenate, glyceryl tricaprylate / caprate The Seryl tricaprylate / caprate / laurate, glyceryl tricaprylate / caprate / linoleate, glyceryl tricaprylate / caprate / stearate, saturated polyglycolized glycerides, linoleglycerides, caprylic / capric glycerides, modified triglycerides, fractionated The composition of the semen dilution is characterized by containing a mixed fat with one or two or more selected from triglycerides and α-tocophenol,
With a polyoxyethylene glycerin fatty acid ester, the composition of a semen dilution liquid characterized by containing an emulsified oil or fat obtained by emulsifying the oil or mixed oil and fat,
Rutin, the fats and oils, the mixed fats and oils, and the emulsified fats and oils include any two or more of the composition of a semen dilution,
Add the semen dilution to the semen, dilute the semen and cool to a low temperature, the configuration of the method of storing diluted semen,
The cooling is configured as a method for storing diluted semen characterized by multi-stage cooling,
In the multi-stage cooling, the semen and the semen dilution are mixed while being kept at 35 ° C. to 37 ° C., left at room temperature (about 20 ° C.) for 30 minutes to 1 hour, and then stored at an ambient temperature of 16 ° C. for 1 hour. And then stored at 12 ° C. for 2 hours, then stored at 8 ° C. for 4 hours, and then stored at 4 ° C.
The diluted semen is left at room temperature (about 20 ° C.) for 30 minutes to 1 hour, then stored at 16 ° C. ambient temperature for 1 hour, then stored at 12 ° C. ambient temperature for 2 hours, and then at 8 ° C. atmosphere It was set as the structure of the storage method of the diluted semen characterized by storing at temperature for 4 hours, and storing at 4 degreeC after that.

  Since this invention is the above structure, the following effects are acquired. A semen dilution can be easily created at low cost. In addition, by using this diluted solution, the spermatozoa in the diluted semen can be kept in an extremely high state as compared with the conventional diluted solution. Furthermore, the refrigerated storage days can be significantly extended (from 7 days to 10 days) compared to the conventional (4 days to 5 days).

  Therefore, it contributes to the spread of artificial insemination in pigs. As a result, the number of breeding male pigs can be reduced, and the economic effects such as drastically reducing the labor of pig farmers are great. Furthermore, it is also effective for diluting and refrigerated storage of semen such as mammals other than pigs, for example, livestock animals such as cattle and horses, and other rare wild animals.

  For the purpose of diluting semen and storing it in a liquid state for a long time, rutin, one or more types of fats and oils selected from avocado oil, cocoon oil and almond oil (which may contain the following other fats and oils), and polyoxyethylene A semen dilution containing emulsified fats and oils emulsified with glycerin fatty acid ester is mixed with semen while maintaining at 35 ° C. to 37 ° C., and left at room temperature (about 20 ° C.) for 30 minutes to 1 hour, followed by 16 ° C. It was realized by storing at ambient temperature for 1 hour, then at ambient temperature of 12 ° C. for 2 hours, then at ambient temperature of 8 ° C. for 4 hours, and then stored at 4 ° C. The other oils and fats are pumpkin seed oil, babas oil, borage oil, black currant seed oil, canola oil, castor oil, coconut oil, corn oil, cottonseed oil, evening primrose oil, grape seed oil, red pepper oil, mustard seed oil, olive oil, Palm oil, palm kernel oil, peanut oil, rapeseed oil, safflower oil, sesame oil, shark liver oil, soybean oil, sunflower oil, hardened oil thereof, fractionated oil thereof, lecithin, glyceryl tricaproate, glyceryl tricaprylate Glyceryl tricaprate glyceryl triundecanoate, glyceryl trilaurate, glyceryl trioleate, glyceryl trilinoleate, glyceryl trilinolenate, glyceryl tricaprylate / caprate, glyceryl tricaprylate / caprate / laurate, g 1 selected from seryl tricaprylate / caprate / linoleate, glyceryl tricaprylate / caprate / stearate, saturated polyglycolized glyceride, linole glyceride, caprylic / capric glyceride, modified triglyceride, fractionated triglyceride, α-tocophenol Species or two or more species.

  Hereinafter, the semen dilution which is this invention is demonstrated in detail based on an accompanying drawing. FIG. 1 is a diagram showing the results of evaluating the overactive state of sperm when semen is diluted and stored with a semen dilution containing rutin. FIG. 1 (A) shows the evaluation results of the sperm overactivity state. FIG. 1 (B) is a table showing a method for evaluating an overactive state of sperm. FIG. 1C is a graph of the evaluation results of FIG.

  Test method: 50 ml of semen immediately after collection (Duroc), 950 ml of diluted solution containing 0.01 g or 0.005 g of rutin in 1000 ml of modena solution, and maintained at a temperature of 35 ° C. to 37 ° C. The diluted semen was prepared by diluting and mixing so that the semen was about 20 times. Rutin was manufactured by Joban Plant Science Institute. The content is 99.7%.

  Then, it hold | maintained at 16 degreeC incubator, and after about 3 hours passed, it heated for 10 minutes with the water bath hold | maintained at about 37 degreeC. Subsequently, 15 μl of the diluted semen was dropped on a slide glass maintained at 37 ° C., and the number of spermatozoa in an overactive state was observed under a microscope while comparing with the non-addition group, according to the criteria of FIG. It was judged. Microscopic observation of these same semen dilutions was repeated three times.

  The “additive” in FIG. 1A is the amount (g) of rutin added to 1000 ml of Modena solution. Therefore, “rutin 0.01 g” means that the stock solution of semen is diluted 10 to 20 times with a semen dilution having a concentration of rutin 0.01 g / modena solution 1000 ml, and “rutin 0.005 g” The semen stock solution is diluted 10 to 20 times with a semen dilution solution with a concentration of 0.005 g of rutin / 1000 ml of modena solution, and the “no addition group” means that the semen stock solution is 10 to 20 times with the modena solution. It means diluted semen that has been diluted.

  The “average value” in FIG. 1A is an activated state when the low temperature storage test is repeated five times, the number of sperm in an overactive state is measured as described above, and the rutin-free group is set to 1. It is an average value of evaluation points when the frequency of sperm is determined based on the criterion of FIG.

  In addition, since the “standard deviation” of the average value obtained by each repeated test was “0.33” for rutin 0.01 g and “0.50” for 0.005 g rutin, It can be seen that there is reproducibility between them.

  The result of FIG. 1 shows that when the semen is diluted 10 to 20 times with the semen dilution solution in which rutin is added to the conventional modena solution, it is overactive compared to the conventional modena solution (no addition group). It shows that there are very many sperm in the state.

  Here, only the result of diluting the semen 10 to 20 times with the semen dilution solution in which 0.01 g and 0.005 g of rutin are added to 1000 ml of the modena solution used conventionally is shown. The overactive state of sperm in semen was good with significant difference even when 0.001 g of rutin was added to 1000 ml of modena solution. Moreover, when the addition amount was increased stepwise, no change was observed in the increasing tendency of the overactive state when the amount of rutin was 0.1 g or more with respect to 1000 ml of the modena solution.

  That is, in order to improve the overactive state of sperm in refrigerated storage with rutin, 0.001 g to 0.1 g of rutin is preferably added to 1000 ml of a semen dilution such as modena solution. At concentrations lower than 0.001 g / diluent 1000 ml, the overactive state is similar to that without addition of rutin, and at concentrations higher than 0.1 g / diluent 1000 ml, the overactive state is 0.1 g / diluent in the overactive state. There is no difference from the case of 1000 ml, and the manufacturing cost of the diluting solution increases, which is not preferable.

  Therefore, the addition amount of rutin is more preferably 0.005 g to 0.01 g of rutin with respect to 1000 ml of semen dilution such as modena solution.

  From the results shown in FIG. 1, the semen diluted solution containing rutin according to the present invention has an extremely large amount of initial overactive sperm compared with the conventional modena solution diluted and refrigerated storage. It can be said that refrigerated storage is possible. Moreover, in contrast to the conventional semen preservation diluent (Patent Document 1) diluted 4 to 5 times, in the present invention, it is diluted 20 times, and the fertilization rate is still in a practical range, It can be said that it is possible to reduce the number of breeding breeders.

  FIG. 2 shows the results of observing the survival rate of sperm when the semen was diluted with the semen dilution of the present invention and stored refrigerated. FIG. 2 (A) shows the results of observing the survival rate of sperm when various additives are added to the Modena solution, cooled by various cooling methods, and stored refrigerated for up to 10 days. FIG. 2B is a graph of the result of FIG. The vertical axis SR (%) is the survival rate, and the horizontal axis day is the date when the storage period and the survival rate were measured.

  T1 to T6 in FIG. 2 (A) are each test group, and each test group diluted semen by the following method and stored at 4 ° C. until 0 day (0 in the figure) to 10 days (10D in the figure). On day 0 (1 hour after dilution), day 1 (1D in the figure), day 4 (4D), day 7 (7D), day 10 (10D), viable sperm were observed by the following method. Survival rate was calculated by the method.

  In addition, survival rate was described as an average value when the same test was repeated 5 times. Moreover, ± represents the difference from the average value to the lowest value and the highest value among all the test values.

Here, the used semen was pork (Duroc species) semen, and the one immediately after collection was used. The modena liquid has the composition described above. For the measurement of the viable sperm, the one that was moving forward during observation under the microscope was determined as the viable sperm. The survival rate was calculated by the following formula (1).
Survival rate (%) = (number of surviving sperm / number of observed sperm) × 100 (1)

  “T1” refers to lecithin (equivalent to 0.4 g) emulsified with polyoxyethylene glycerin fatty acid ester (0.1% by weight per oil, the remaining distilled water) while keeping the collected semen at 35 ° C. to 37 ° C. It diluted 20 times with the semen dilution liquid (35 degreeC-37 degreeC) which is this invention added to 1000 ml of modena liquids. In addition, lecithin is egg yolk lecithin, and the product number PL-30S made from Kewpie Co., Ltd. was used. same as below. Here, egg yolk lecithin is added as fat.

  Next, in the method of (A) shown in FIG. 3, that is, the 20-fold diluted semen is allowed to stand at room temperature (about 20 ° C.) for 30 minutes to 1 hour, and then stored at an ambient temperature of 16 ° C. for 1 hour. Next, it was stored at an ambient temperature of 12 ° C. for 2 hours, then subjected to multi-stage cooling that was stored at an ambient temperature of 8 ° C. for 4 hours, and finally held in an incubator at 4 ° C. for 10 days.

  The emulsification was performed at 2500 rpm for 5 minutes using a high-pressure homomixer. same as below. As the polyoxyethylene glycerin fatty acid ester, product number BREDOL694 manufactured by Acnobel Co., Ltd. was used.

  “T2” is a multi-stage cooling by the method of (A) shown in FIG. 3 by diluting 20 times with a modena solution (35 ° C. to 37 ° C.) while keeping the collected semen at 35 ° C. to 37 ° C. And finally kept in an incubator at 4 ° C. for 10 days.

  “T3” is a modena solution of lecithin (equivalent to 0.4 g) emulsified with sodium lauryl sulfate (0.1% by weight per lecithin, remaining distilled water) while maintaining the collected semen at 35 ° C. to 37 ° C. Dilute 20 times with semen diluted solution (35 ° C to 37 ° C) added to 1000 ml, perform multi-step cooling by the method of (A) shown in Fig. 3, and finally hold in an incubator at 4 ° C for 10 days did. In addition, sodium lauryl sulfate used by Wako Pure Chemical Industries, Ltd. and for biochemistry was used.

  In “T4”, lecithin (equivalent to 0.4 g) emulsified with OEP (0.1% by weight per lecithin, remaining distilled water) is maintained in 1000 ml of Modena solution while keeping the collected semen at 35 ° C. to 37 ° C. It diluted 20 times with the added semen dilution liquid (35 degreeC-37 degreeC), the multistep cooling was performed by the method of (A) shown in FIG. 3, and it hold | maintained in the 4 degreeC incubator for 10 days finally. In addition, OEP is Miyazaki Chemical Co., Ltd. and EQUEX STM.

  For “T5”, the semen diluted solution prepared in “T1” was immediately diluted with a 4 ° C. incubator without being subjected to multi-step cooling, and held for 10 days.

  For “T6”, the semen diluted solution prepared in “T2” was immediately cooled after being diluted in a 4 ° C. incubator without being subjected to multi-step cooling and held for 10 days.

  The result of FIG. 2 shows that lecithin was added to the modena solution compared to the case where the semen was diluted with the conventional semen dilution mode, which was diluted, and cooled immediately without multi-step cooling (T6). The semen dilution (T5) of the present invention shows that the survival rate was about 1.5 times on all the survival rate measurement days.

  Further, in the case where the multi-stage cooling shown in FIG. 3 is employed (T1), the conventional modena solution (T6) decreases to a survival rate of almost 50% in one day, whereas on the tenth day However, the survival rate was more than 60%. Furthermore, in the result of T1, the survival rate exceeded all the survival rate measurement days compared with sodium lauryl sulfate (T3) and OEP (T4) conventionally used as a pig semen dilution liquid.

  In addition, when the semen is diluted with the conventional Modena solution and the multi-stage cooling shown in FIG. 3 is performed (T2), the survival rate is reduced to 50% when it is immediately cooled to 4 ° C. after the dilution (T6). Compared with the number of storage days, T6 decreased to a survival rate of 50% on the first day after storage at 4 ° C., whereas T2 did not decrease to a survival rate of 50% until 10 days after storage at 4 ° C.

  In order for the diluted semen of pigs to withstand practical use, a survival rate of approximately 70% is required. Here, since the semen dilution rate is 20 times, the survival rate may be reduced by 70%. However, by appropriately selecting the semen dilution rate, a fertilization rate equivalent to the survival rate of 70% is maintained, and 10% It is possible to store for 4 days at 4 ° C for artificial insemination.

  For example, 8 to 9-fold dilution is sufficient for T1. In addition, the fertilization rate is sufficiently maintained at about 2 to 5 times in T2, and about 2 times in T5, and it is practically used. Furthermore, in consideration of the current distribution situation, if the liquid refrigerated storage at 4 ° C. for 7 days is possible, the semen diluted solution according to the present invention and the method for storing diluted semen by multistage cooling according to the present invention were prepared. It can be said that chilled storage diluted semen is sufficiently practical.

  Furthermore, by adding the above-mentioned concentration of rutin to the present invention, it is of course possible to increase the survival rate, increase the number of overactive sperm, and extend the storage days. In addition, it is needless to say that the semen dilution and the method for storing diluted semen according to the present invention can be applied to dilution and refrigeration storage of semen not only for pig sperm but also for mammals and livestock.

  Therefore, even after storage at 4 ° C for 10 days, the refrigerated storage technology of diluted semen has advanced so much that it can withstand practical use, thereby solving the problem of artificial insemination of pigs due to the number of days of refrigeration and the conception rate, It can be said that the rate of artificial insemination in pigs can be improved. As a result, the number of breeding male pigs is reduced, and the economic effects such as reduction of the labor of pig farmers are extremely large.

The additive here was “lecithin 0.4 g equivalent / 1000 ml modena solution” emulsified with polyoxyethylene glycerin fatty acid ester, but almond oil emulsified with polyoxyethylene glycerin fatty acid ester 0.4 g / 1000 ml. Modena liquid,
Even a semen diluted 10 to 20 times with 0.4 g of koji oil / 1000 ml modena solution or avocado oil 0.4 g / 1000 ml modena solution was emulsified with polyoxyethylene glycerin fatty acid ester “equivalent to 0.4 g of lecithin / 1000 ml modena solution. The spermatozoa can be stored in a refrigerated state (both rapid cooling and multi-stage cooling) while maintaining the sperm overactivity, survival rate, and fertilization rate equivalent to semen diluted 10 to 20 times. Details will be described later with reference to FIG.

  Hereinafter, a method for storing diluted semen according to the present invention will be described in detail with reference to the accompanying drawings. FIG. 3 is a diagram showing an example of an embodiment of the method for storing diluted semen according to the present invention.

  The vertical axis (temp.) In the figure is the temperature of the outside air when the diluted semen is cooled, and the unit is ° C. The horizontal axis (time) is the cooling time of the diluted semen.

  (A) in a figure is an example of the cooling method of the diluted semen which is this invention. (B) in the figure is the cooling method disclosed in Patent Document 1. (C) in the figure is the cooling method disclosed in Patent Document 3.

  An example of the method for storing diluted semen (A) according to the present invention is to mix semen and semen diluted solution while maintaining at 35 ° C. to 37 ° C., and leave it at room temperature (about 20 ° C.) for 30 minutes to 1 hour. Cool, then allow the incubator to reach 16 ° C. at a temperature change rate of 0.75 ° C./min and store at that ambient temperature for 1 hour, then the incubator at 12 ° C. at a temperature change rate of 0.75 ° C./min. The temperature is allowed to reach 0 ° C. and stored at that ambient temperature for 2 hours, then the incubator is allowed to reach 8 ° C. at a temperature change rate of 0.75 ° C./min and stored at that ambient temperature for 4 hours, after which the incubator is stored at 0 ° C. Allow the temperature to reach 4 ° C. at a temperature change rate of 75 ° C./min, and store the diluted semen at 4 ° C. It is characterized by such multi-stage cooling.

  Note that the multi-stage cooling is limited to the temperature and time shown in FIG. 3 as long as it takes about 6 to 12 hours and cools slowly (including the case where a smooth curve is drawn). Therefore, it is possible to maintain an equivalent sperm overactivity state and to refrigerate for a long time (about 10 days at 4 ° C.).

  In addition, the diluted semen used for multi-stage cooling includes the semen dilution of the present invention, the conventionally known semen dilution, dilutions obtained by adding other reagents to them, and other dilutions. Diluted semen diluted for storage shall also be included.

  On the other hand, in the cooling method (B) shown in Patent Document 1, the temperature of diluted semen is lowered from 35 ° C., which is the semen dilution temperature, to 16 ° C. over about 2 hours and 30 minutes, and then about 1 hour and 20 minutes. Reduce the temperature to 5 ° C over a long time and store in the refrigerator at that temperature. As described above, it is difficult to say that the method of Patent Document 1 can realize refrigerated storage that can withstand practical use.

  Moreover, the cooling method (C) shown in Patent Document 3 decreases the temperature of diluted semen from 0 ° C. to 10 ° C. at a temperature change rate of 0.2 ° C. to 0.5 ° C./min. Held for 4 to 21 hours, followed by cooling to −40 ° C. to −100 ° C., holding at that temperature for 7 to 20 minutes, and further cooling to −190 ° C. to −200 ° C. Store frozen.

  In the cooling method (C), firstly, fats and oils (lecithin) having an emulsifying action are added, but since other fats and oils are not completely emulsified, the particles of lecithin are not observed when observed with a microscope. May be confused with impurities. Second, there is a drawback that it takes time to complete the product as compared with the production of diluted semen (modena solution) for storage at 16 ° C. that is currently distributed.

  FIG. 4 is a diagram showing the results of evaluating the overactive state of sperm when semen was diluted and stored with a semen dilution containing specific fats and oils.

  FIG. 4 (A) shows the results of observing the survival rate of sperm when the above fats and oils were added to the Modena solution, cooled by various cooling methods, and stored refrigerated for up to 10 days. FIG. 2B is a graph of the result of FIG. The vertical axis SR (%) is the survival rate, and the horizontal axis day is the date when the storage period and the survival rate were measured.

  N1 to N6 in FIG. 4 (A) are each test group, and each test group dilutes semen by the same method as in Example 1, and it is 4 until 0 day (0 in the figure) to 10 days (10D in the figure). Stored at 0 ° C., live sperm explained in Example 1 on day 0 (1 hour after dilution), day 1 (1D in the figure), day 4 (4D), day 7 (7D), day 10 (10D) The survival rate was calculated by the method of Example 1. In this case, avocado oil, cocoon oil, and almond oil were mainly examined instead of rutin.

  “N1” is the semen dilution according to the present invention in which almond oil (equivalent to 0.3 g) emulsified with lecithin (equivalent to 0.1 g) is added to 1000 ml of modena solution while keeping the collected semen at 35 ° C. to 37 ° C. It diluted 20 times with the liquid (35 to 37 degreeC).

  Next, in the method of (A) shown in FIG. 3, that is, the 20-fold diluted semen is allowed to stand at room temperature (about 20 ° C.) for 30 minutes to 1 hour, and then stored at an ambient temperature of 16 ° C. for 1 hour. Next, it was stored at an ambient temperature of 12 ° C. for 2 hours, then subjected to multi-stage cooling that was stored at an ambient temperature of 8 ° C. for 4 hours, and finally held in an incubator at 4 ° C. for 10 days. In addition, the product number A2154 by Sigma-Aldrich Japan Co., Ltd. was used for almond oil.

  “N2” is a semen dilution according to the present invention in which avocado oil (equivalent to 0.3 g) emulsified with lecithin (equivalent to 0.1 g) is added to 1000 ml of modena solution while keeping the collected semen at 35 ° C. to 37 ° C. The solution (35 ° C. to 37 ° C.) was diluted 20-fold, subjected to multi-stage cooling by the method (A) shown in FIG. 3, and finally held in an incubator at 4 ° C. for 10 days. As the avocado oil, edible avocado oil was used.

  “N3” is a diluted semen solution (35 ° C. to 37 ° C.) obtained by adding coconut oil (equivalent to 0.3 g) emulsified with lecithin (equivalent to 0.1 g) to 1000 ml of modena solution while keeping the collected semen at 35 ° C. to 37 ° C. 37 degreeC), it diluted 20 times, and the multistage cooling was performed by the method of (A) shown in FIG. 3, and finally it hold | maintained for 10 days in a 4 degreeC incubator. In addition, the cocoon oil used the Yamagata industrial company make.

  “N4” is diluted 20 times with a semen dilution (35 ° C. to 37 ° C.) in which lecithin (equivalent to 0.1 g) is added to 1000 ml of modena solution while keeping the collected semen at 35 ° C. to 37 ° C. Then, multistage cooling was performed by the method (A) shown in FIG. 3, and finally, it was kept in an incubator at 4 ° C. for 10 days.

  “N5” is a multi-stage cooling by the method (A) shown in FIG. 3 by diluting the collected semen 20-fold with modena solution (35 ° C.-37 ° C.) while maintaining the collected semen at 35 ° C.-37 ° C. And finally kept in an incubator at 4 ° C. for 10 days.

  The results in FIG. 4 show that lecithin, almond oil emulsified with lecithin, avocado oil, and salmon oil are added as compared with the case where semen is diluted with modena solution, which is a conventionally used semen dilution, and cooled in multiple stages. Multi-stage cooling of the semen dilution diluted with the modena solution showed that the survival rate was high on all the survival measurement days.

  Furthermore, compared with lecithin addition (N5), the preservation | save effect more than equivalent was seen by the dilution and preservation | save by the semen dilution liquid containing almond oil, avocado oil, and a bran oil. Therefore, it can be said that almond oil, avocado oil, and cocoon oil have an effect of suppressing the decrease in sperm activity.

  In addition, content of these almond oil, avocado oil, and cocoon oil is 0.05g-0.2g independently per 1000 ml of dilution liquid, More preferably, it is 0.1g. Furthermore, when adding almond oil, avocado oil, and cocoon oil each independently, it is more desirable to emulsify 0.1 g with lecithin 0.3 g.

  In addition, almond oil, avocado oil, and camellia oil alone, even if an amount less than 0.05 is added to the Modena solution, there is no effect of improving the survival rate of sperm. On the contrary, it acted fatally on sperm.

It is a figure which shows the result of having evaluated the overactive state of the sperm when a semen is diluted with the semen dilution liquid containing a rutin and preserve | saved. It is the result of having observed the sperm survival rate when diluting semen using the semen dilution liquid which is this invention, and refrigerated storage. It is a figure which shows an example of embodiment of the storage method of the diluted semen which is this invention. It is a figure which shows the result of having evaluated the overactive state of the sperm when diluting and storing a semen with the semen dilution liquid containing specific fats and oils.

Claims (9)

  A semen dilution characterized by containing rutin.   A semen dilution, comprising one or more oils and fats selected from avocado oil, camellia oil, and almond oil.   One or more oils selected from avocado oil, coconut oil, almond oil, pumpkin seed oil, babas oil, borage oil, black currant seed oil, canola oil, castor oil, coconut oil, corn oil, cottonseed oil, evening primrose oil , Grape seed oil, American potato oil, mustard seed oil, olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, safflower oil, sesame oil, shark liver oil, soybean oil, sunflower oil, their hardened oil, those Oil extraction, lecithin, glyceryl tricaproate, glyceryl tricaprylate, glyceryl tricaprate glyceryl triundecanoate, glyceryl trilaurate, glyceryl trioleate, glyceryl trilinoleate, glyceryl trilinolenate, glyceryl tricaprylate / caprate The Seryl tricaprylate / caprate / laurate, glyceryl tricaprylate / caprate / linoleate, glyceryl tricaprylate / caprate / stearate, saturated polyglycolized glycerides, linoleglycerides, caprylic / capric glycerides, modified triglycerides, fractionated A semen dilution, comprising a mixed fat with one or more selected from triglycerides and α-tocophenol.   4. The semen dilution according to claim 2, comprising an emulsified fat and oil in which the fat is emulsified with a polyoxyethylene glycerin fatty acid ester. 5.   A semen dilution, comprising two or more of rutin, the fat and oil of claim 2, the mixed fat and oil of claim 3, and the emulsified fat and oil of claim 4.   A method for preserving diluted semen, comprising adding the semen dilution of claim 1, 2, 3 or 4 to the semen, diluting the semen, and cooling to low temperature.   6. The method for storing diluted semen according to claim 5, wherein the cooling is multistage cooling.   Multi-stage cooling mixes semen and semen dilution while maintaining at 35 ° C to 37 ° C, leave at room temperature (about 20 ° C) for 30 minutes to 1 hour, and then store at ambient temperature of 16 ° C for 1 hour. 7. The method for preserving diluted semen according to claim 6, wherein the method is then stored for 2 hours at an ambient temperature of 12 ° C., then stored for 4 hours at an ambient temperature of 8 ° C., and then stored at 4 ° C.   The diluted semen is left at room temperature (about 20 ° C.) for 30 minutes to 1 hour, then stored at 16 ° C. ambient temperature for 1 hour, then stored at 12 ° C. ambient temperature for 2 hours, and then at 8 ° C. atmosphere A method for preserving diluted semen, characterized by storing at temperature for 4 hours and then storing at 4 ° C.

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