WO2006072046A2

WO2006072046A2 - Method for sex biasing of artificial insemination

Info

Publication number: WO2006072046A2
Application number: PCT/US2005/047569
Authority: WO
Inventors: Jianmin Liu; Barb Ariel Cohen; Michael Morris
Original assignee: Vicam, L.P.
Priority date: 2004-12-30
Filing date: 2005-12-29
Publication date: 2006-07-06
Also published as: US20060147894A1; WO2006072046A3

Abstract

A method for treating a specimen of semen useful for artificial insemination to increase the conception of female offspring in a mammal is disclosed. The semen is incubated at a predetermined temperature for at least a predetermined period of time. Thereafter, the semen is used for artificial insemination of the mammal. Typically, the predetermined temperature is in the range of about 4°C to about 20°C. Typically, the predetermined period of time if from about 2 hours to about 24 hours.

Description

METHOD FOR SEX BIASING OF ARTIFICIAL INSEMINATION

Field Of The Invention

This invention relates to methods for enhancing the probability of obtaining offspring of a selected sex. More particularly, this invention relates to methods for collection and incubation of spermatozoa prior to artificial insemination to enhance the probability of obtaining offspring of a selected sex.

Background Of The Invention

Farmers and other animal husbandry persons have long recognized the desirability of enhancing the probability of obtaining offspring of a selected sex. In mammals, the male gamete or spermatozoan controls the sex of offspring. Each spermatozoan contains either an X-type or a Y- type sex-determining chromosome.

An X-chromosome spermatozoan creates female offspring after fertilization with an oocyte, while a Y-chromosome spermatozoan creates male offspring after fertilization. Methods have been proposed for increasing the percentage of X- chromosome bearing sperm cells or Y-chromosome bearing sperm cells to thereby achieve a greater chance of achieving female or male offspring, respectively.

Previous methods have included, for example, methods based upon density sedimentation (see, for example, Brandriff, B. F. et al. "Sex Chromosome Ratios Determined by Karyotypic Analysis in Albumin-Isolated Human Sperm," Fertil. Steril., 46, pp. 678-685 (1986)).

U.S. Patent 3,687,806 to Van Den Bovenkamp discloses an immunological method for controlling the sex of mammalian offspring by use of antibodies which react with either X-bearing sperm or Y-bearing sperm and utilizing an agglutination step to separate bound antibodies from unaffected antibodies.

U.S. 4,083,957 to Lang discloses a method for alteration of the sex ratio in animal (including human) offspring by separation of the population of spermatozoa into fractions which are different by reason of the sex-linked electrical charge resident thereon. The separation is carried out by bringing the spermatozoa into close association with an electrostatic charge-bearing material having a charge the sign of which is opposite to the sign of a chosen portion of the spermatozoa, that portion which carries the sex determining character of the unwanted sex, so as to attract and thereby to permit that portion to be isolated, or put to a disadvantage in the fertilization of ova. Concern is expressed with the selection of the charge-bearing material, the adjustment of the pH and particle size thereof, and the control of the surrounding medium in relation to its influence on the charge characteristics of both the charge-bearing material and the spermatozoa. Lang teaches that spermatozoa having respectively male or female sex bearing genetic material also have differing electrostatic charges, normally negative for male and positive for female, and uses this teaching for separation of the male and female spermatozoa with charge bearing materials.

U.S. Patent 4,191 ,749 to Bryant discloses a method for increasing the percentage of mammalian offspring of either sex by use of a male-specific antibody coupled to a solid-phase immunoabsorbant material to selectively bind male- determining spermatozoa, while the female-determining spermatozoa remain unbound in a supernatant.

U.S. Patent 5,021,244 to Spaulding discloses a method for sorting living cells based upon DNA content, particularly sperm populations to produce subpopulations enriched in X-or Y-sperm by means of sex-associated membrane proteins and antibodies specific for such proteins.

U.S. Patent 5,514,537 to Chandler discloses a method and apparatus for the mechanical sorting of mammalian spermatozoa by sex-type, into a fraction enriched in X-chromosome-bearing spermatozoa, and a fraction enriched in Y-chromosome- bearing spermatozoa. Because of their different DNA content, Y-chromosome spermatozoa are on average slightly smaller than X-chromosome spermatozoa. A column is packed with two sizes of beads. The size of the smaller beads is chosen such that, on average, Y-chromosome spermatozoa will readily fit into the interstices between the smaller beads, while X-chromosome spermatozoa, on average, will not readily fit into those interstices. The size of the larger beads is chosen such that the smaller beads will not readily fit into the interstices between the larger beads. A liquid sample containing the sperm is run through a column so that the liquid first encounters the larger beads, and then encounters the smaller beads. The beads act as a sieve, creating a fraction in the larger beads enriched in X- chromosome spermatozoa, and a fraction in the smaller beads enriched in Y- chromosome spermatozoa.

However, these prior art methods often result in insufficient separation of X- and Y-sperm and often damage the sperm, thereby reducing its motility and fertility success rate.

In the commonly owned and assigned U.S. Patents 6,153,373 and 6,489,092, improved methods for sex determination of mammalian offspring are provided using antibodies coupled to magnetic particles for separation of spermatozoa. These methods use magnetic separation to provide gentle separation of populations of spermatozoa.

Lechniak, et al in Reprod Dom Anim 38, 224-227 (2003) describe a study to determine whether sperm pre-incubation prior to fertilization in vitro (IVF) influences the rate of fertilization, embryo development and the sex ratio among blastocysts. Oocyte-cumulus-complexes (OCC) were aspirated from follicles of slaughterhouse ovaries, collected in Hepes-buffered Ham's F- 10, matured in maturation medium under silicone oil for 24 hours at 39°C. Frozen-thawed sperm cells were utilized. After swim-up, the motile fraction of sperm was incubated in Sperm-Talp (no heparin included) for 0, 6 and 24 hours at 39°C. Sperm count was carried out and sperm motility were evaluated. The number of motile sperm cells was kept similar in each experimental group. The motile spermatozoa decreased with time. It was reported by the authors that, when comparisons between groups were made and the actual sex ratios taken into consideration, there were significantly more female hatched blastocysts among the 24 hour group than among those of either the 0- or 6 hour pre- incubation groups. IVF is not a practical procedure for fertilization of large herds.

Summary Of The Invention The invention provides a method for preparing a specimen of semen to increase the relative number of offspring of the female sex in mammals using artificial insemination (AI). Thus, in accord with the present invention, a specimen of semen is collected from a male donor mammal, after collection the specimen is cooled to a predetermined temperature, typically in the range of about 4°C to about 20°C, and the specimen is incubated at that predetermined temperature for a predetermined period of time, typically in the range of from about 2 to about 24 hours. After incubation for the predetermined period of time, the specimen is processed into straws according to conventional procedures and the straws are used for artificial insemination in a corresponding female mammal using conventional procedures. In a preferred embodiment, the straws can be frozen and used in conventional artificial insemination procedures.

By treating the semen as described, it has been found that a significant bias can be obtained in producing offspring of the female sex by artificial insemination in mammals.

Thus, the invention also provides methods for increasing the probability of producing a mammalian offspring of a desired or preferred sex by artificial insemination using semen incubated according to the procedures of the present invention.

As used herein, the term "extender" means a solution prepared for diluting the semen for handling. Several formulations for extenders are available commercially.

In a preferred embodiment of the invention, semen is collected at time zero, the temperature of the semen is reduced to a temperature in the range of about 6°C to about 17°C within about 30 minutes, and the semen is incubated at that temperature for about 4 hours, after which it is processed into straws for artificial insemination. The straws can be frozen. In another preferred embodiment of the invention, the semen is collected in a jacketed container, the jacket comprising a material that holds heat and the material being preheated to a temperature in the range of about 30°C to about 40⁰C.

Detailed Description Of The Invention Including Preferred Embodiments

The present invention provides methods for collection and incubating sperm which are competent (or viable) to fertilize mammalian eggs, e.g., eggs in fertile cows, using standard AI techniques currently employed on farm. As noted above, prior methods of other parties for sperm treatment to bias offspring production often compromise sperm integrity, i.e., their motility and fertilization ability, so that fertilization utilizing such prior art treated sperm requires complicated techniques such as in vitro fertilization (IVF) or ultrasounding of cows during heat to determine side of ovulation, coupled with introduction of a low sperm dose by high uterine horn insemination into the horn attached to the ovary from which the egg is released. It is impossible to use these methods on farm with working dairy herds. The method of the present invention can be utilized for sperm from a variety of mammalian species, including various livestock, such as cattle and sheep, as well as dogs, cats, horses, swine, and other species. The process also is applicable to humans.

To practice the present invention, first semen is collected from a male donor, e.g., a proven artificial insemination bull. The semen ejaculate is collected into a collection tube. The size of the collection tube is adjusted for the particular mammal and donor. Typically, for a bull, a 15 ml collection tube can be used. Preferably, the collection tube is jacketed with a material to control the temperature of the ejaculate. Various matrials such as insulating materials can be used. Alternatively, a material having a high heat capacity can be used. Typically, such material is conditioned to a desired temperature prior to collection. Conveniently, the collection tube is immersed in a second container (or jacket) containing freezer pack gel that has been preconditioned to a desired temperature. In one embodiment of the invention, before collection of the ejaculate, the jacket material is preheated to a temperature in the range of from about 30°C to about 4O⁰C, preferably in the range of about 32°C to about 35°C. In an alternative embodiment of the invention, the jacket material is precooled to a temperature in the range of from about 4⁰C to about 20⁰C, preferably in the range of about 6°C to about 17°C.

Any suitable material can be used in the jacket as long as it has sufficient heat capacity to maintain the desired temperature during the semen collection step and is suitable for immersing the collection tube into the material or coating the collection tube with the material, e.g. a viscous gel layer preferably with an outer protective layer for handling without affecting the gel layer. Alternatively, the collection tube can be coated with a material of suffient heat capacity and stability for handling.

Also, alternatively, the collection tube can be promptly placed into a larger container of water at the desired collection temperature, e.g., a 250 ml beaker of water at 32°C.

Next, the collection tube (with or without jacket, but preferably with jacket) is placed promptly into a temperature controlled device and cooled, if necessary, to a predetermined temperature in the range of from about 4°C to about 20°C, preferably in the range of about 6°C to about 17°C. Preferably, the collection tube is placed in a circulating water bath within no more than about 5 minutes of collection, more preferably in no more than about 3 minutes, most preferably in no more than about 1 minute. In addition, it is preferred to cool the collection tube with insulating jacket (e.g., gel filled second container) in place. For example, the jacketed collection tube is placed directly into the water bath. Preferably, the semen temperature is reduced to the predetermined temperature in about 30 minutes or less.

If a non-jacketed collection tube is used and the tube placed into a beaker of water aftter collection, then, preferably, the beaker with the collection tube therein is placed in a temperature controlled device where the incubation temperature is reached and maintained for the desired time period.

The collected semen is then incubated at the predetermined temperature for a predetermined period of time, typically from about 2 to about 24 hours, preferably from about 2 to about 12 hours, more preferably from about 2 to about 8 hours, most preferably from about 4 to about 6 hours. After incubation, the semen is extended to the desired volume and straws are prepared according to conventional procedures. Typically, an egg yolk extender is used for volume dilution. A typical extender without glycerol is made by separating the egg yolks from the egg white of fresh eggs, removing the yolk membrane by rolling on a filter paper and mixing with 2.9% sodium citrate buffer (preferably with antibiotics) at a ratio of 200 ml egg yolk to 800 ml citrate buffer. A typical glycerol extender is made by adding glycerol to 14% by volume. Commercially available extenders that can be used include, for example, Biladyl^®, Triladyl^® and Biociphos Plus™, BioXcell A™ and BioXcell B™. The cells then were transferred to straws and frozen using standard freezing techniques. Other non egg yolk extenders known to those skilled in the art also can be used.

The straws are used for artificial insemination according to conventional methods. The straws can be frozen and stored prior to use for artificial insemination using conventional methods. Typically, the straws are thawed and semen from a straw deposited in the uterus just beyond the cervix.

In certain embodiments of the invention, the semen is extended with a non- glycerol extender after collection prior to incubation. Typically, about 2 ml to about 10 ml of extender is used per ml of semen. Final extension to the desired dose of cells per unit volume is made typically using a glycerol extender prior to preparing and freezing straws.

If not constrained by loss of sperm integrity during incubation and/or storage, artificial insemination techniques can use either "high dose" or "low dose" methods (reflecting the relative amounts of spermatozoa (per straw) used for insemination); the methods of the invention are applicable with any amount of spermatozoa (i.e., including both high dose and low dose methods).

In certain embodiments of the methods using spermatozoa collected using the methods of the present invention, a relatively high dose is used, e.g., greater than about 10 million cells are used for insemination. In these embodiments, the number of spermatozoa administered preferably is at least about 20 million, more preferably at least about 30 million, still more preferably at least about 40 million, and yet more preferably at least about 50 million. A typical range for the high dose is from about 10 million to 20 million sperm cells per straw. For young bulls, the amount of cells per straw is increased typically from about 25% to about 50%.

In other embodiments of the methods using spermatozoa collected using the methods of the present invention, a relatively low dose is used, e.g., less than about 10 million cells are used for insemination. In these latter embodiments, the number of spermatozoa administered preferably is less than about 5 million, more preferably is less than about 1 million and still more preferably is less than about 0.5 million.

In preferred embodiments of the invention, semen collection, incubation and preparation techniques are efficient and gentle to spermatozoa cells that are easily damaged, and most of the cells processed using the methods of this invention retain their activity as compared to conventionally processed sperm cells. This means that conception rates for animals inseminated with incubated cells can be maintained at levels similar to that using conventionally processed cells. In contrast, prior art methods of other parties for cell treatment often compromise the motility and fertilization ability of spermatozoa due to the use of harsh conditions including exposure to laser light and dye molecules (FACS), shear forces, etc., so that fertilization utilizing such separated spermatozoa requires complicated and expensive techniques and lowers the efficiency of conception. Further, such techniques are not suitable for use on a farm. Thus, in accord with such embodiments of the invention, for incubated spermatozoa that are then used in standard insemination procedures, the conception rate of offspring resulting from the insemination is, in preferred embodiments is at least about 50% of the conception rate obtained using conventionally prepared non-incubated spermatozoa. In more preferred embodiments, the conception rate is higher and approaches that seen using conventionally prepared non-incubated spermatozoa (e.g., at least about 70%, 80%, 90%, or 95% of the conception rate obtained using conventionally prepared non- incubated spermatozoa). These methods, therefore, are useful for creating a sex bias in mammalian offspring without the use of IVF, embryo transfer or other expensive procedures or equipment. By using the preferred methods described herein, spermatozoa of a mammal can be incubated and processed without a substantial loss of quality. Quality includes, but is not limited to: motility, progressive motility, grade of motility, acrosomal integrity, immediate and incubated post-thaw motility and morphology. Thus, the quality of the incubated spermatozoa using these methods is at least about 50% of the unprocessed spermatozoa. Preferably, the functionality of the fractionated spermatozoa is at least about 60% of the unprocessed spermatozoa, at least about 70% of the unprocessed spermatozoa, at least about 80% of the unprocessed spermatozoa, or is at least about 90% of the unprocessed spermatozoa. More preferably, the quality of the fractionated spermatozoa is at least about 95% of the unprocessed spermatozoa, still more preferably is at least about 97% of the unprocessed spermatozoa, yet even more preferably is at least about 98% of the unprocessed spermatozoa, and most preferably is at least about 99% of the unprocessed spermatozoa. Thus, populations of incubated spermatozoa preferentially determinative of one sex having the foregoing levels of quality relative to unprocessed spermatozoa are provided.

The invention will be described further in the following examples.

Example 1 Semen was collected from proven artificial insemination bulls according to the following procedure.

1. Ejaculate was collected into a modified Collection tube comprising of a 15 ml collection tube completely immersed in a container of freezer pack gel and brought to 32 ⁰C prior to use. Immediately before the ejaculate was collected but after the completion of the required false mountings, the collection apparatus was attached to the end of the artificial vagina and the ejaculate collected.

2. Immediately, the insulated collection tube was transfered to a circulating water bath at 12 ⁰C to begin the cooling incubation process.

3. Incubate for 4 hours at 12 ⁰C. 4. Prepare straws according to conventional procedures.

Conveniently, a jacketed collection tube was made by filling a 50 ml conical tube with freezer pack gel, covering the top with a membrane in which a cross-cut opening was made, and inserting into the gel through the opening a 15 ml conical tube into which the semen was collected.

The final dilution volume (with extender) was calculated by dividing the total initial cells by 660 x 10⁶. For example, from a 7ml ejaculate, if the cell concentration is 1 billion/ml, you have 7 billion cells total. 7 billion cells divided by 660 x 10⁶ = 10.6 ml for final volume.

The collected and incubated semen was extended and frozen using conventional freezing procedures. Straws were prepared at about 20 million sperm cells/straw (calculated).

Cows and heifers in working dairy herds were inseminated with semen by artificial insemination (AI) with the incubated semen.

Example 2

Five separate ejaculates were collected from a single bull on different days. The ejaculate in its collection tube was placed in 20OmL of 32⁰C water in a beaker, which was placed into a water bath at 12⁰C to begin slow cooling. The ejaculates were then split so portions could be treated differently. The control portions were processed—extended and frozen into straws— by methods standard at that semen processing site, with 20 million sperm cells/straw (calculated). The 6h hold portions were held (incubated) as neat semen in the water bath for 6h before processing by methods standard at that semen processing site. Cows and heifers in working dairy herds were inseminated with semen by AI. Semen was introduced by methods standard in the field— with semen deposition into the uterus just beyond the cervix.

55 straws prepared as set forth above and 55 control straws were sent into the field for AI. Evaluation of the sex ratio of embryos, as determiined by fetal ultrasound scanning, for the cows and heifers inseminated with control or "6h hold" semen (i.e., held 6h before processing) are tabulated below in Table 1. Conception rate appears to be lower for the "6h hold" semen, although data may not be complete.

Post thaw motilities of the control and incubated (i.e., "6h hold") semen are tabulated in Table 2.

TABLE 1

Treatment Female Feti Male Feti Total Feti % Female

6h Hold 23 10 33 69.7%

Control 24 31 55 43.6%

TABLE 2

Ejaculate Number

Treatment 1 2 3 4 5

6h Hold 50% 50% 30% 25% 60%

Control 75% 70% 45% 50% 70%

The invention has been described in detail including preferred embodiments thereof. However, modifications and improvements within the scope of this invention will occur to those skilled in the art. The above description is intended to be exemplary only. The scope of this invention is defined only by the following claims and their equivalents.

All patent and literature references disclosed hereinabove hereby are incorporated by reference in their entirety.

Claims

What is claimed is:

1. A method for treating a specimen of semen useful for artificial insemination to increase the conception of female offspring in a mammal, the method comprising incubating the specimen at a predetermined temperature for at least a predetermined period of time and, thereafter, using the semen for artificial insemination of said mammal.

2. The method of claim 1 , wherein the predetermined temperature is in the range of about 4°C to about 2O⁰C.

3. The method of claim 1 , wherein the predetermined temperature is in the range of about 6°C to about 17°C.

4. The method of claim 1 , wherein the predetermined temperature about 12°C.

5. The method of claim 1 , wherein the predetermined period of time is from about 2 hours to about 24 hours.

6. The method of claim 1, wherein the predetermined period of time is from about 2 hours to about 12 hours.

7. The method of claim 1 , wherein the predetermined period of time is from about 2 hours to about 8 hours.

8. The method of claim 1 , wherein the predetermined period of time is from about 4 hours to about 6 hours.

9. The method of claim 1 , further comprising collecting the specimen of semen from a corresponding male donor using a jacketed collection tube comprising a material preheated to a temperature in the range of from about 30°C to about 4O⁰C.

10. The method of claim 1 , further comprising collecting the specimen of semen from a corresponding male donor using a jacketed collection tube comprising a material preheated to a temperature in the range of from about 32°C to about 35°C.

11. The method of claim 1 , further comprising collecting the specimen of semen from a corresponding male donor using a jacketed collection tube comprising a material preconditioned to a temperature in the range of from about 4°C to about 20°C.

12. The method of claim 1, further comprising collecting the specimen of semen from a corresponding male donor using a jacketed collection tube comprising a material preconditioned to a temperature in the range of from about 6°C to about 17°C.

13. The method of any one of claims 9-12, further comprising placing the jacketed collection tube into a temperature controlled device, wherein the temperature is controlled in the range of from about 4°C to about 20°C.

14. The method of any one of claims 9-12, further comprising placing the jacketed collection tube into a temperature controlled device, wherein the temperature is controlled in the range of from about 6⁰C to about 17⁰C.

15. The method of any one of claims 9-12, further comprising placing the jacketed collection tube into a temperature controlled device, wherein the temperature is controlled at about 12°C.

16. The method of claim 1, further comprising collecting the specimen of semen from a corresponding male donor using a collection tube and placing the collection tube into a container having sufficient liquid to surround the test tube in a region of the specimen, wherein the temperature of the liquid is in the range of from about 30°C to about 40°C.

17. The method of claim 1, further comprising collecting the specimen of semen from a corresponding male donor using a collection tube and placing the collection tube into a container having sufficient liquid to surround the test tube in a region of the specimen, wherein the temperature of the liquid is in the range of from about 32°C to about 35°C.

18. The method of claim 16 or 17, further comprising placing the collection tube in the container into a temperature controlled device, wherein the temperature is controlled in the range of from about 6°C to about 17°C.

19. The method of claim 16 or 17, further comprising placing the collection tube in the container into a temperature controlled device, wherein the temperature is controlled in the range of from about 6⁰C to about 17°C.

20. The method of claim 16 or 17, further comprising placing the collection tube in the container into a temperature controlled device, wherein the temperature is controlled at about 12°C.

21. The method of any one of claims 1-20 further including artificially inseminating a suitable female recipient with a unit dose of semen.

22. The method of claim 21 , wherein the mammal is selected from the group consisting of cattle, sheep, pigs, goats, horses, dogs and cats.