Multiple sexed embryo production system for bovine mammals
First Claim
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1. A method of producing multiple embryos from a female bovine mammal comprising:
- a. creating superovulation in said female bovine mammal to create at least two eggs in each of said female bovine mammal comprising the step of using an ovulatory pharmaceutical to cause multiple eggs to be produced in said female bovine mammal further comprising the steps of;
i) injecting a dosage of follicle stimulating hormone in each of said female bovine mammal a plurality of times a day;
ii) administering said dosage of follicle stimulating hormone with prostaglandin F-2-alpha;
and wherein said step of injecting said dosage of follicle stimulating hormone in each of said female bovine mammal a plurality of times a day comprises injecting said follicle stimulating hormone in approximately half day increments at a dosage level of 6, 6, 4, 4, 2, 2, 2, and 2 mg between days 9 and 12 inclusive of the estrus cycle and wherein administering said dosage of follicle stimulating hormone with prostaglandin F-2-alpha comprises supplementing 25 and 12.5 mg of prostaglandin F-2-alpha on the sixth and seventh dosages, respectively, of said follicle stimulating hormone;
b. collecting sperm cells from at least one male bovine mammal;
c. staining said collected sperm cells with Hoechst 33342;
d. sorting the stained sperm cells with a flow cytometer to yield live sperm cells of a desired sex;
e. concentrating said live sperm cells of a desired sex;
f. establishing an insemination sample having in the range of 1×
105 to 6×
105 live sperm cells of a desired sex;
g. inserting said insemination sample into said female bovine mammal, half the dose into each uterine horn of said female bovine mammal, 20 to 24 hours post-onset of estrus for said female bovine mammal;
h. fertilizing a plurality of said eggs in said female bovine mammal; and
i. producing at least two embryos of a desired sex from said female bovine mammal in which a plurality of said eggs were fertilized.
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Abstract
Improved insemination systems particularly adapted to use for sex-selected sperm sorting include systems which achieve superovulation and then multiple embryo production with sexed embryos. These systems combine with other techniques, including techniques for enhanced sheath fluid and other strategies which minimize stress on the sperm cells, and, potentially, a 2.9 percent sodium citrate sheath solution for bovine species and a hepes bovine gamete media for equine species. Improved collection systems and techniques for the process are described so that commercial application of sperms samples as well as the resulting animals may be achieved.
557 Citations
9 Claims
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1. A method of producing multiple embryos from a female bovine mammal comprising:
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a. creating superovulation in said female bovine mammal to create at least two eggs in each of said female bovine mammal comprising the step of using an ovulatory pharmaceutical to cause multiple eggs to be produced in said female bovine mammal further comprising the steps of; i) injecting a dosage of follicle stimulating hormone in each of said female bovine mammal a plurality of times a day; ii) administering said dosage of follicle stimulating hormone with prostaglandin F-2-alpha; and wherein said step of injecting said dosage of follicle stimulating hormone in each of said female bovine mammal a plurality of times a day comprises injecting said follicle stimulating hormone in approximately half day increments at a dosage level of 6, 6, 4, 4, 2, 2, 2, and 2 mg between days 9 and 12 inclusive of the estrus cycle and wherein administering said dosage of follicle stimulating hormone with prostaglandin F-2-alpha comprises supplementing 25 and 12.5 mg of prostaglandin F-2-alpha on the sixth and seventh dosages, respectively, of said follicle stimulating hormone; b. collecting sperm cells from at least one male bovine mammal; c. staining said collected sperm cells with Hoechst 33342; d. sorting the stained sperm cells with a flow cytometer to yield live sperm cells of a desired sex; e. concentrating said live sperm cells of a desired sex; f. establishing an insemination sample having in the range of 1×
105 to 6×
105 live sperm cells of a desired sex;g. inserting said insemination sample into said female bovine mammal, half the dose into each uterine horn of said female bovine mammal, 20 to 24 hours post-onset of estrus for said female bovine mammal; h. fertilizing a plurality of said eggs in said female bovine mammal; and i. producing at least two embryos of a desired sex from said female bovine mammal in which a plurality of said eggs were fertilized. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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Specification