Compositions and methods for processing sperm
View Patent ↗The present invention relates to compositions and methods for the handling of processed sperm including samples that are freshly collected, those transported as fresh samples, 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 cell. Trauma can reduce the motility, fertility, viability and overall integrity of the sperm and reduce the ability to fertilize, produce an embryo and 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 treating sperm cells during processing to improve cell integrity comprising the steps of:
a) forming a sperm cell composition comprising contacting the sperm cells and a media comprising vitamin B12, or a vitamin B12 vitamer, in a concentration range of 0.01 mg/ml to 1 mg/ml; and
b) processing the sperm cell composition.
2. The method as claimed in claim 1 , further comprising the step of holding the sperm cell composition at a holding temperature without freezing for a period of time in the range of about 2 seconds to about a week.
3. The method as claimed in claim 2 , wherein the holding period is selected from a range selected from the group of: about 2 sec to about 3 min; about 3 min to about 15 min; about 15 min to about 1 hr; about 1 hr to about 8 hrs; about 8 hrs to about 12 hrs; about 12 hrs to about 18 hrs; about 8 hrs to about 24 hrs; about 24 hrs to about 48 hrs; about 48 hrs to about 72 hrs; about 72 hrs to about 96 hrs; about 96 hrs to about 120 hrs; about 120 hrs to about 144 hrs; and about 144 hrs to about 168 hrs.
4. The method of claim 2 , wherein the holding temperature is in the range of 4° C. and 39° C.
5. The method of claim 4 , wherein the holding temperature is in the range of about 4° C. to 16° C.
6. The method as claim in claim 1 further comprising the step of transporting the sperm cell composition from a first location to a second location.
7. The method as claimed in claim 1 , wherein the step of processing the sperm cell composition comprises two or more processing steps selected from the group consisting of: holding; transporting; buffering; chilling; warming; staining; diluting; concentrating; exciting with a laser; electronic charging; deflecting; ablating; collecting; shaking; oscillating; magnetically separating; oxygenating; labeling; precipitating; centrifuging; resuspending; mixing; dialyzing; cryostabilizing; freezing; thawing; vitrifying; culturing; inseminating; microinjecting; microfluidic processing; microchip processing; jet and air processing and flow cytometry processing.
8. The method as claimed in claim 1 , wherein the sperm cells have been frozen and thawed prior to the step of processing.
9. The method as claimed in any one of claim 1 - 7 or 8 , wherein the method further comprises the step of freezing the sperm cell composition to form a frozen sperm cell composition.
10. The method as claimed in claim 1 , wherein the concentration of the vitamin B12 or the vitamin B12 vitamer in the sperm cell composition is in the range selected from the group consisting of: 0.01 to 0.25 mg/ml; 0.01 to 0.5 mg/ml; 0.05 to 0.1 mg/ml; 0.05 to 1.0 mg/ml; 0.1 to 0.25 mg/ml; 0.1 to 0.5 mg/ml; 0.1 to 1 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; 0.35 to 0.5 mg/ml; 0.35 to 1 mg/ml; and 0.5 to 1 mg/ml.
11. The method as claimed in claim 10 , wherein the concentration of the vitamin B12 or vitamin B12 vitamer in the first 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.
12. The method as claimed in claim 11 , wherein the concentration of the vitamin B12 or vitamin B12 vitamer in the sperm cell composition is 0.15 mg/ml 0.25 mg/ml or 0.35 mg/ml.
13. The method as claimed in claim 1 , wherein the sperm cell sample contains unwanted cells.
14. The method as claimed in claim 13 , wherein the sperm cell sample is manually ablated to kill unwanted cell types within the sperm cell sample.
15. The method as claimed in claim 1 , wherein the sperm cell composition comprises a cryoprotectant.
16. The method as claimed in claim 1 , wherein the sperm cells are obtained from a human, bovine, swine, ovine, equine, deer, elk, buffalo, canine, feline, chimpanzee, gorilla, or a whale, dolphin or other marine mammal.
17. The method as claimed in claim 1 , wherein the vitamin B12, or vitamin B12 vitamer, is at a concentration of 0.25 mg/ml.
18. The method as claimed in claim 1 , wherein the vitamin B12, or vitamin B12 vitamer, is at a concentration in the range of 0.15 mg/ml to 0.5 mg/ml.
19. The method as claimed in claim 1 , wherein the sperm cell composition further comprises alpha-ketoglutarate (AKG), or an AKG derivative, at a concentration in the range of 0.15 mg/ml to 0.5 mg/ml.
20. The method of claim 1 , further comprising the step of forming a second sperm cell composition comprising contacting sperm cells with a second media, the second media comprising vitamin B12, or a vitamin B12 vitamer, in a concentration range of 0.01 mg/ml to 1 mg/ml.