A method for determining the cryotolerance of semen before cryopreservation by marker and deciding on the freezing method to be applied

Abstract

The present invention relates to a method for optimal freezing of semen in semen production stations by using a marker during the freezing of fresh semen. With the method developed by the invention, the seminal oxidative stress index is determined to predict the freezability of fresh semen in semen production stations and this value is used as a marker for the decision to freeze the semen. If necessary, antioxidants can be added to improve the post-thaw sperm quality.

Description

[0001] DESCRIPTION

[0002] A METHOD FOR DETERMINING THE CRYOTOLERANCE OF SEMEN BEFORE CRYOPRESERVATION BY MARKER AND DECIDING ON THE FREEZING METHOD TO BE APPLIED

[0003] Technical Field

[0004] The present invention relates to a method for optimizing decision-making in semen cry opreservation at semen production stations, utilizing a marker to predict the cryotolerance of fresh semen.

[0005] Prior Art

[0006] In the known state of the art, the motility of fresh semen is measured at semen production stations and if it is found to have a high motility, i.e. a motility value higher than 70%, it is frozen. Fresh semen with a motility value lower than 70% is not frozen and not used.

[0007] The post-thaw quality of some fresh semen with high-motility may be inadequate due to endogenous low antioxidant, high oxidant content and hence high seminal oxidative stress.

[0008] Documents numbered RU2257710C2, WO2007149142A1, CN103120156A and US6054287A describe methods for freezing semen from various animals.

[0009] Aims and Brief Description of the Invention

[0010] The purpose of the present invention is to develop a method for prediction of cryotolerance of fresh semen using the oxidative stress index of seminal plasma as a marker and to apply the freezing process accordingly.

[0011] Another object of the present invention is to develop a method for freezing fresh semen with low motility and low seminal oxidative stress index.

[0012] With this invention, it is possible to determine the oxidative stress index (OSI) value of a small amount of seminal plasma of fresh semen, and if this value is below a certain threshold value, the semen can be frozen and included in production even if the fresh motility value is below the determined rate. On the other hand, if the fresh motility value is high and the seminal OSI value is higher than the threshold value, antioxidants can be added to the semen reconstituent to improve the post-thaw sperm quality. Thus, waste of resources and labor can be minimized.

[0013] With the method of the invention, it is aimed to enhance the efficiency and success of semen preservation by identifying a reliable marker that can accurately forecast the semen's ability to withstand the freezing and thawing process, thereby improving post-thaw semen quality. Thanks to this invention, approximately 400 straws (enough to inseminate 400 cows) of semen can be offered for sale as a result of the production of one ejaculate. This will provide a significant increase in productivity and economic benefits for the stations.

[0014] Detailed Description of the Invention

[0015] The method according to the invention essentially comprises collecting fresh semen, determining the motility of the fresh semen, separating a small amount of seminal plasma from the fresh semen, measuring total antioxidant levels (TAS) and total oxidant levels (TOS) in the separated seminal plasma and calculating an oxidative stress index (OSI) in the form of TOS / TAS, if the motility value is higher than a first threshold value and the OSI value is lower than a second threshold value, add commercial extender to the fresh semen and then freeze the semen; if the motility value is higher than the first threshold value and the OSI value is higher than the second threshold value, add antioxidant to the commercial extender and then freeze the semen; if the motility value is lower than the first threshold value, do not freeze the fresh semen.

[0016] The use of frozen semen can be evaluated by redetermining the post-thaw motility value.

[0017] A sample with a volume of 0.5 ml can be used to determine the oxidative stress index. Seminal plasma can be separated by centrifugation of this sample at 12,000 g for 10 minutes.

[0018] The motility value of fresh semen can be determined by an expert using a computer assisted semen analyzer (CASA) or with the help of a microscope. Antioxidant and oxidant levels can be measured with an automated biochemistry device using commercial spectrophotometric kits.

[0019] Within the scope of the invention, if it is determined that an antioxidant should be added to the semen, the antioxidant can be selected from antioxidants such as ascorbic acid, tocopherol, glutathione, cysteine and mixtures thereof. The dose of antioxidant added is determined according to the OSI rate measured in seminal plasma.

[0020] With the inventive method, seminal plasma oxidative stress index is determined to predict the freezability of fresh semen in semen production stations and this value is used as a marker for the decision to freeze semen. The decision to freeze semen is made according to the second threshold value determined for the oxidative stress index.

[0021] In a preferred embodiment of the invention for the use of bovine (bull) semen, the first threshold value associated with the motility value is 55% and the second threshold value associated with the OSI value is 0.8. By adding antioxidants in the light of the OSI value, it is possible to avoid situations where the post-thaw sperm quality is low, i.e. the post-thaw motility rate is below 40% and cannot be used due to oxidative stress and requires disposal. Thus, in addition to fresh semen with high motility values (greater than 70%), fresh semen with low motility values (between 55% and 70%) can also be frozen and the decline of the post-thaw sperm quality of fresh semen with motility values greater than 70% can be controlled.

[0022] With the inventive method, the freezability (cryotolerance) of fresh semen can be determined by using the OSI value as a marker and, if necessary, antioxidants can be added to improve the post-thaw semen quality. Thus, with the techniques in accordance with the state of the art, it can be ensured that unusable semen can be included in the production, the post-thaw sperm quality for all semen samples can be increased and the semen that is destroyed due to post-thaw sperm quality can be greatly reduced.

Claims

CLAIMS1. A method for the optimal freezing of semen at semen production stations by using a marker during the freezing of fresh semen, including collection of fresh semen, determination of motility of fresh semen, separation of seminal plasma from a sample of fresh semen, measurement of total antioxidant level (TAS) and total oxidant level (TOS) in the separated seminal plasma and calculation of oxidative stress index (OSI) in the form of TOS / TAS, if the motility value is higher than a first threshold value and the OSI value is lower than a second threshold value, commercial extender is added to the fresh semen, followed by freezing of the semen; if the motility value is higher than the first threshold value and the OSI value is higher than the second threshold value, antioxidant is added to the fresh semen together with commercial extender, followed by freezing of the semen; if the motility value is lower than the first threshold value, the fresh semen is not frozen.

2. A method according to claim 1, characterized in that the antioxidant is selected from ascorbic acid, tocopherol, glutathione, cysteine and mixtures thereof.

3. A method according to claim 1 for the use of bovine (bull) semen, characterized in that the first threshold value associated with the motility value is 55% and the second threshold value associated with the OSI value is 0.8.