RAPID REAL TIME MULTIPOINT PROCEDURE FOR OPTIMIZING SPERM STATE FOR USE IN ASSISTED REPRODUCTIVE TECHNOLOGIES
A method for adjusting the maturation state of mammalian sperm for use in assisted reproductive technologies (ART) is disclosed. A mammalian ejaculate is provided and incubated under controlled conditions. Aliquots of the ejaculate are assayed during incubation period at intervals to determine maturation state and changes in the maturation state by observing the percent positive cells in the aliquot. The assays are repeated with successive aliquots at intervals during incubation to observe real time changes in the maturation state. The ejaculate remaining is processed for the desired ART after the percentage of positive cells in the latest aliquot being assayed begins to decline.
1 . A method for adjusting the maturation state of mammalian sperm for use in an assisted reproductive technology (ART), said method comprising:
providing a mammalian ejaculate;
incubating the ejaculate under controlled conditions;
assaying an aliquot of the ejaculate during incubation period to determine maturation state by observing the percent positive cells in the aliquot;
repeating the assaying step with successive aliquots at intervals during incubation to observe real time changes in the maturation state; and
processing the ejaculate remaining for the desired ART after the percentage of positive cells in the latest aliquot being assayed begins to decline.
2 . The method of claim 1 , wherein the providing step includes collecting the mammalian ejaculate from a mammal using a collection device prewarmed to about the body temperature of the mammal.
3 . The method of claim 2 , wherein the mammal is bovine and the collection device is prewarmed to a temperature in the range of about 30-40° C.
4 . The method of claim 1 , wherein the incubating step includes controlling the temperature of the ejaculate at a temperature in the range of about 40° C. to room temperature.
5 . The method of claim 4 , wherein the mammal is bovine and the incubating step includes controlling the temperature of the ejaculate at a temperature in the range of about 0 to 15° C.
6 . The method of claim 1 , wherein the assaying step includes:
mixing the aliquot with reagents capable of reacting with a marker indicative of sperm cell maturation, wherein the reaction produces fluorescence in connection with a positive reaction with a sperm cell; and
determining a percentage of positive cells.
7 . The method of claim 6 , wherein the reagents include an antibody to the marker and the antibody is labeled with a fluorescent label.
8 . The method of claim 6 , wherein the reagents include a primary and a secondary antibody that is labeled with a fluorescent label.
9 . The method of claim 6 , wherein the reagents include a stabilizer for the sperm cells.
10 . The method of claim 6 , wherein the determination of percent positive cells is made by a method selected from the group consisting of antibody-based, dye-based, motility-based and microscopy based procedures.
agglutination of sperm to themselves or another agent added
11 . The method of claim 1 , wherein processing includes stabilizing the ejaculate remaining for further processing for the desired ART.
12 . The method of claim 11 , wherein the further processing includes making straws having a predetermined amount of sperm cells and freezing the straws for artificial insemination.