Compositions and methods for improving the quality of processed sperm
The present invention generally relates to compositions and methods for the handling of processed sperm populations including samples that are freshly collected, those transported as fresh samples, as well as samples that are frozen and thawed, those sorted into one or more subpopulations, and those that are otherwise processed or handled that impose trauma on the sperm cell. Such trauma can reduce the motility, fertility, viability and overall integrity of the sperm and reduce the sperm's ability to fertilize an egg, grow into a health embryo and produce a healthy offspring. The present invention relates to novel compounds that can be added to the sperm cell sample to reduce the traumatic effects of physical stress during mild as well as extensive sperm cell processing, methods of using the compounds in standard sperm processing procedures, the end products made from these methods including sperm and embryos, as well as methods of using those end products in assisted reproductive biology techniques in animals.
1. A method of freezing sperm comprising:
diluting a semen sample in a media comprising vitamin B12 and alpha-ketoglutarate to form a sperm cell composition, wherein the final concentration of the vitamin B12 in the sperm cell composition is 0.01 mg/ml to 1 mg/ml;
cooling the sperm cell composition;
adding a cryoextender to the sperm cell composition to form a cryoextended sperm cell composition; and
freezing the cryoextended sperm cell composition.
2. The method of claim 1 , wherein the final concentration of the alpha ketoglutarate in the sperm cell composition is selected from the group consisting of: 0.01 to 5.0 mg/ml; 0.01 to 0.25 mg/ml; 0.01 to 0.5 mg/ml; 0.01 to 1 mg/ml; 0.01 to 2.5 mg/ml; 0.01 to 5 mg/ml; 0.05 to 0.1 mg/ml; 0.05 to 1.0 mg/ml; 0.05 to 2.5 mg/ml; 0.1 to 0.25 mg/ml; 0.1 to 0.5 mg/ml; 0.1 to 1 mg/ml; 0.1 to 2.5 mg/ml; 0.1 to 5 mg/ml; 0.15 to 0.45 mg/ml; 0.15 to 0.5 mg/ml; 0.25 to 0.35 mg/ml; 0.25 to 0.5 mg/ml; 0.25 to 1 mg/ml; 0.25 to 2.5 mg/ml; 0.25 to 5 mg/ml; 0.35 to 0.5 mg/ml; 0.35 to 1 mg/ml; 0.35 to 2.5 mg/ml; 0.35 to 5 mg/ml; 0.5 to 1 mg/ml; 0.5 to 2.5 mg/ml; 0.5 to 5 mg/ml; 1 to 2.5 mg/ml; and 1 to 5 mg/ml.
3. The method of claim 1 , wherein the final concentration of the vitamin B12 or alpha ketoglutarate in the sperm cell composition is selected from the group consisting of: 0.05 mg/ml; 0.1 mg/ml; 0.15 mg/ml; 0.25 mg/ml; 0.35 mg/ml; 0.45 mg/ml; and 0.5 mg/ml.
4. The method of claim 1 , wherein the final concentration of the vitamin B12 or alpha ketoglutarate in the sperm cell composition is selected from the group consisting of: 0.15 mg/ml; 0.25 mg/ml; and 0.35 mg/ml.
5. The method of claim 1 , wherein the media is a Tris-based media.
6. The method of claim 1 , wherein the cryoextender is a glycerol-based cryoextender.
7. The method of claim 1 , wherein the step of cooling comprises cooling the sperm cell composition to 4° C.
8. The method of claim 1 , further comprising the step of holding the cooled sperm cell composition for a period of time prior to the step of adding a cryoextender.
9. The method of claim 1 , wherein the cooled sperm cell composition is held for a period of 90 minutes.
10. The method of claim 1 , wherein the semen sample is derived from a human, bovine, swine, ovine, equine, deer, elk, buffalo, canine, feline, chimpanzee or gorilla, or whale, dolphin or other marine mammal.