WO1998048273A1

WO1998048273A1 - Method for fertility detection

Info

Publication number: WO1998048273A1
Application number: PCT/US1998/008067
Authority: WO
Inventors: Richard J. Harrison
Original assignee: Harrison Richard J
Priority date: 1997-04-23
Filing date: 1998-04-22
Publication date: 1998-10-29
Also published as: AU7146198A

Abstract

The present invention describes a method for detecting heat, ovulation and fertility periods of a lactating mammal based on a determination of changes in the levels of luteinizing hormone metabolites (LH metabolites) in the milk of lactating mammals.

Description

METHOD FOR FERTILITY DETECTION

Field of the Invention The present invention refers generally to a diagnostic method for detection of the stages of fertility in lactating mammals. More particularly, the present invention concerns the identification of changes in LH metabolite levels in the milk of a lactating mammal as a method for detection and prediction of the fertility period and ovulation. The method can be used to determine fertility in both human and non-human lactating female mammals. The invention can be used by persons interested in breeding animals, such as cows, to provide a simple and convenient method for determining when the animals are fertile and should be artificially inseminated. Alternatively, a human female who recently has given birth and who wishes to avoid another pregnancy while she is nursing her child can use the method to determine when she is fertile and thus should avoid unprotected sexual intercourse .

Background of the Invention

For many reasons artificial insemination is preferred to natural mating in both beef and dairy cattle. Unfortunately, many of the reasons for artificial insemination also have resulted in increased breeding cost due to failed estrus detection and failed inseminations . Some of the reasons for these increased costs include genetic selection which has led to animals which are less active during estrus, artificial insemination which has eliminated the efficient pasture bull and larger herd size which has reduced or eliminated human monitoring of behavioral estrus in individual animals. USDA Dairy Herd Improvement Association (DHIA) records indicate the U.S. dairy industry losses resulting from failed estrus detection and failed inseminations are about 1.5 billion dollars per year.

"Open Days" is a term used to describe the number of days between postpartum and next pregnancy. The desired minimum open days for dairy cows is reported by the USDA as 42-63, while the actual current U.S. average is 126 with a DHIA goal for producers of 100 open days .

The dairy and cattle industries have recognized the need for ovulation detection and have employed a number of methods to reduce open days through increased estrus detection, which currently is less than 50%, and improved artificial insemination conception rates, which currently are in the 40-45% range--a rate significantly lower than the 70% achieved from natural mating. The occurrence of ovulation at the research level can be established with some certainty through application of various prior art methods applicable to humans. While the only irrefutable method of proving ovulation is the occurrence of conception (occasionally the actual recovery of the eggs), several testing techniques, including surgical, clinical and biochemical or histological techniques are available which presumptively confirm the occurrence of ovulation. These techniques, however, are impractical for commercial application to the cattle industry. Visual and Mechanical Detection of Estrus Behavior, but not Ovulation . - Current commercial methods for determining the timing of artificial insemination of lactating bovine in the dairy and pure bred cattle industry are based on the detection of heat. Heat detection has four major drawbacks which reduce the detection of estrus and conception rates . The first drawback is the tendency of most currently available detection methods to give a high number of false positive results, the second is the inability to determine if ovulation occurs with heat, the third is time of ovulation during heat and the fourth is failure to identify heat.

Currently practiced methods for determination of timing for insemination are either visual or mechanical and are based upon the concept of detecting increased movement or mounting by other animals which generally occurs during heat. Years of artificial insemination using selected sires to improve genetic traits and production yields have created more docile animals that often exhibit less activity during estrus and are more likely to have an undetected or silent heat. In addition, heat detection identifies a period of time in the estrus cycle when ovulation can occur, but it does not predict ovulation timing or confirm that ovulation has occurred. Knowledge of both ovulation occurrence and timing are important factors in achieving high conception rates and reducing operating costs.

Visual tests are based on the observation of markings or changes in markings. Commonly used visual tests include chalk balls placed on chains tied to the necks of neutered bulls, tail painting, K-mar ink packs and general observation. Most mechanical tests tend to be of the leg strap battery operated counting device variety. A typical device will have a mercury switch which activates a counter when the animal moves. A rapid increase in the number of counts per day is considered to be an indication of heat. All of these methods are currently used in dairy farming operations and have helped the farmer to reach the current average 126 open day efficiency rates. It is believed that they fail to achieve significantly higher rates for some of the following reasons: a) devices detect onset of heat and not ovulation, b) activated by general herd activity, c) devices are activated by accidental contact, d) washed off markings, and e) no time indicator.

Progesterone Determina tion for the Detection of Ovula tion - In the late 1980 's a number of biotechnology companies introduced cowside progesterone tests which were initially targeted at the market need for improved insemination. Subsequently, these tests were found to have their own drawbacks in that they only confirmed, but did not predict, ovulation. The tests are still available through veterinarians who identified a method for evaluation of reproductive defects in non-conceiving animals.

Other Methods for Detecting Ovula tion - The literature contains a number of proposed approaches to ovulation detection. In the late 1980' s several researchers proposed the detection of ovulation based on vaginal electrical resistance measurements. More recently, one researcher proposed using rheological behavior of the vaginal fluid and a second has proposed purifying and characterizing the estrus signaling pheromone from cow urine. None of these approaches, however, have been demonstrated successfully in commercial practice.

In addition to bovines, the need for prediction of fertility also is known for other members of the bovidea family. One example of this is the determination of fertility in sheep and goats for breeding purposes or for the creation of transgenic animals. The need for fertility detection also exists in mammals such as swine which are part of the family scientifically classified as suidae and tayassuidae. Much like the dairy industry, commercial swine producers now are using artificial insemination techniques to improve productivity.

Thus, a need continues to exist for a reliable, fast, and inexpensive procedure for predicting ovulation in non-human mammals.

Human females also frequently desire a simple method of knowing when they are ovulating and, therefore, fertile. Determination of fertility can be sought by women who wish to become pregnant and also by women who wish to avoid pregnancy, including women who recently have given birth and wish to avoid becoming pregnant again during the time that they are nursing. Accordingly, there is a broad-based need for a simple and reliable method for determining fertility in human and non-human mammals.

Summary of the Invention

The present invention is based on the discovery that metabolites of LH are present in the milk of a female mammal at significantly increased levels just prior to the ovulatory portion of the mammal's estrus cycle and that the increased protein or peptide level (s) can be detected as a means of predicting ovulation. Milk was selected as the medium of choice because of its convenience to the person making the determination and the impracticality of collecting output of other bodily functions as well as the undesirability of performing invasive procedures. It now has been discovered that luteinizing hormone metabolites exist in milk and they can be used as an indicator of ovulation. LH is a pituitary- secreted hormone which is known to surge and, in concert with follicle stimulating hormone (FSH), causes the initiation of ovulation (Veterinary Endocrinology and Reproduction 2nd Edition; McDonald L.E., Lea & Febiger, Philadelphia, 1976) . LH is a glycoprotein composed of an α-chain and β-chain of amino acids and constituent carbohydrate moieties . The bovine form of the hormone (bLH) has a molecular weight of 29070 with an α- subunit of 10796 (protein) and 3253 (carbohydrate) and a β- subunit of 12980 (protein) and 2041 (carbohydrate) (Pierce, J.G. and Parsons, T. F., "Glycoprotein Hormones: Structure and Function" Ann . Rev. Biochem . 50:465-95 (1981)). Significant sequence homology can exist between the LH sequences of different animal species. For example, rat (rLH) and bovine (bLH) share an 83% sequence homology.

In the case of mammals, LH is the hormone universally responsible for the rupture of the mature ovarian follicle, i.e. ovulation. Its baseline serum levels are fairly stable during the estrous cycle, with the exception of a large (-10 fold) above baseline pituitary-derived pulse or surge preceding ovulation.

The present invention describes a method for detection of the fertility period and ovulation in lactating mammals, preferably bovines . It has been discovered that by monitoring the concentration of luteinizing hormone metabolites (LH metabolites) in milk, a reliable diagnostic indication of fertility and ovulation can be obtained.

In one aspect, the present invention provides a method for determining the condition of fertility in a female lactating mammal which comprises detecting the surge in level of LH metabolite (s) present in the milk of the mammal which occurs immediately preceding ovulation. Shortly after increases in LH blood levels occur during the LH surge, the LH metabolite (s) level in milk increases significantly above its normal basal level and rapidly declines as blood levels decrease, thus mimicking the LH surge in blood. In a particular aspect, the invention involves a method for detecting the surge in concentration of one or more of the LH metabolites present in milk by introducing into a milk sample at least one antibody capable of binding to an LH metabolite of interest and determining whether the antibody has reacted with the LH metabolite (s) . Preferably, the antibody is associated with a label capable of producing a visually detectable signal which can be detected when the level of the metabolite which binds to the antibody is above the basal level.

Another aspect of this invention is a diagnostic kit for determination of the condition of fertility in a female mammal based upon changes in the level of LH metabolites in milk. This kit contains at least one antibody to one or more LH metabolites which can be employed in an assay for the performance of the method of this invention. The antibody in the kit desirably is conjugated to a detectable label. The diagnostic kit further can comprise typical buffering, washing and other diagnostic reagents conventional in such kits. In addition, the kit can contain special media, filters or other separation tools which enable the user to perform the assay using untreated milk samples or milk which has been treated to separate potential blocking agents prior to performance of the desired assay. Other aspects and advantages of the present invention are described further in the following detailed description of preferred embodiments of the present invention.

Brief Description of the Drawing Figure 1 is a graph showing that a portion of radioactive LH injected into the blood of lactating female rats passed through the animals' mammary glands and into the stomachs of the nursing rat pups.

Detailed Description

In the following detailed description of the method of the invention, conditions such as temperature, pressure and the like are generally not critical unless noted otherwise. Unless specified, process steps are carried out at room temperature and atmospheric pressure.

The present invention is based on the discovery that luteinizing hormone metabolites are present in the milk of lactating mammals and that levels of the luteinizing hormone metabolites present in milk have a direct correlation to luteinizing hormone levels present in blood.

This discovery allows the use of assays for the detection of fertility and ovulation. The invention involves identification of a detectable increase (above basal levels) in LH metabolites in collected milk samples from a lactating female mammal. As used herein, a detectable increase above basal levels refers to at least about a 25% increase, preferably at least a 50% increase, more preferably at least about a 100% increase, and most preferably about a 1000% increase, above average basal levels of one or more metabolites of LH. Increased LH metabolite levels in milk are directly correlated to the LH surge in blood and the LH surge has a direct correlation with ovulation.

In the following discussion, the focus will be on the usefulness of the invention for determining the onset of ovulation in non-human female mammals, primarily for purposes of determining optimal times for artificially inseminating the animals for desired breeding purposes. It should be recognized, however, that the invention also can be useful for determining when a nursing human mother is fertile, albeit for a very different purpose. Many nursing mothers do not wish to become pregnant again while they are nursing their infants . By determining when her surge in LH occurs through the determination of elevated levels of LH metabolites in her milk, a nursing mother can determine when she is ovulating and avoid unprotected sexual intercourse.

Although in the examples below the animals used in the experiments, for purposes of simplicity, were rats, it should be understood that the methods of the present invention can be applied to ovulation detection in all female mammals which exhibit estrus behavior during lactation. This includes all scientific classification members of the bovidea family, which includes, but is not limited to, bovine, oxen, bison, water buffalo, sheep and goats, and the tayassuidae family, which includes swine. In addition, as noted above, it further includes nursing human females. It also is understood that references to LH metabolites in milk includes any active metabolite of LH which will bind to one or more anti-LH metabolite antibodies.

Turning now to the application of this invention to determining fertility in non-human lactating mammals, this invention responds to an unmet need in the field of animal artificial reproduction and provides a simple test for use by animal owners. The method and products provided by this invention have a number of advantages over present methods for detecting the onset of ovulation in animals.

The detection of a substance in a milk sample as opposed to other body fluids samples, such as urine and blood, is a clear advantage to the bovine stockman, veterinarian or researcher. Urine is obviously difficult to collect, and blood collection is an invasive procedure which may create health risks for the animal. In addition, since no "in the field" tests are currently available for urine or blood, results must be determined in a laboratory. Thus, the method and assays associated with this invention provide the user with direct results which are determined from samples which are easy to obtain.

The method and products of this invention also provide significant advantages in ovulation detection versus the alternative inferential methods currently available for use. Current methods infer ovulation based on detection of heat whereas the present invention provides exact timing regarding the initiation of the fertility period and ovulation.

In the practice of the method of this invention, a variety of assay formats can be employed which use one or more antibodies capable of binding to epitopes on one or more of the LH metabolites. Such metabolites can include, for example, the alpha or beta chain of LH or one or more glycoprotein, protein or peptide components thereof comprising an amino acid sequence which is within or derived from the amino acid sequence of LH.

A preferred embodiment of this invention is a method for detecting fertility in a lactating female dairy cow by detecting the surge in LH metabolite levels in milk which occurs in conjunction with the surge in LH level in the blood which precedes ovulation. The method can take advantage of a number of well-known immunoassay methods which employ antibodies for detection of specific peptide or protein substances. Such assays can include, for example, the use of at least one labeled antibody to detect the presence of a significantly increased level of at least one LH metabolite in milk above the baseline level. Alternatively, as described below, an assay can use two antibodies which bind to different epitopes of the desired metabolite to detect the increased level of LH metabolites .

The antibodies to LH metabolites for use in the assays of this invention can be polyclonal antibodies (Pabs) , monoclonal antibodies (Mabs) , CDR-grafted antibodies, chimeric antibodies, active binding fragments (e.g., FAB, Fab' or F(ab)₂ fragments), anti- idiotype antibodies, synthetic single-chain antibodies or the like which bind to an epitope on an LH metabolite. It may be preferable for purposes of increased target specificity to utilize Mabs, which can be generated using known techniques. It should be understood that other antibodies, fragments and binding proteins also can be employed using procedures well known to those skilled in the art.

In order to carry out any of the antibody-based assays described below, the metabolites of LH present in the milk of the animal species of interest are isolated and identified using procedures known in the art or described in the examples below and these metabolites are used to obtain one or more antibodies which bind to at least one metabolite of interest (desirably, a metabolite present in a relatively large quantity in comparison to other metabolites present) using standard procedures known to persons of skill in the art. Techniques for obtaining polyclonal antibodies, monoclonal antibodies and antibody fragments which bind to an antigen of interest are well known.

As discussed below, in certain assays more than one antibody desirably is used. In such assays, the antibodies selected desirably bind to epitopes on the metabolite of interest at a sufficient distance from one another that steric hindrance can be avoided.

Immunoassays for LH metabolites in milk can be configured in any of the formats well known to those skilled in the art. They can be in competitive or sandwich formats. They can be simultaneous or sequential and can be heterogeneous (involving a separation step) or homogeneous. Moreover, any of the detection methods known to those skilled in the immunoassay art can be employed. For example, the assay for LH metabolites can be a radioimmunoassay, an enzyme immunoassay, a fluorescent immunoassay, a fluorescence polarization immunoassay, a luminescent (chemluminescent or bioluminescent ) immunoassay, a latex particle (e.g. color particle) agglutination assay, an assay employing an immunoelectrode, or the like. To facilitate separation, antibodies to LH metabolites can be immobilized on magnetic particles, beads, walls of microtiter plates wells, walls of tubes, or the like.

In any of the foregoing assays, the assay can be set up as either a qualitative or a quantitative assay. That is, the assay can be designed either to simply indicate if the amount of metabolite (s) being measured is in excess of the basal level or it can actually measure the actual amount (s) of the metabolite (s) . Obviously, the former approach is simpler and more practical for assays used outside of the laboratory. In a qualitative assay, the assay would be set up such that if only the baseline level of the LH metabolite (s) in milk are present the assay would not show a positive indicator, but if the milk sample contains increased levels of metabolite (s) which are present following the surge in LH released by the pituitary to the blood stream preceding ovulation the assay would indicate a positive result. If quantitative results were desired, the same assay could be run in a laboratory using instruments to quantitate the results against predetermined standards. For example, a quantitative assay could be designed much in accordance with that taught by Sarda, A. and M. Nair, J. Clin . Endocrinol . Metab . 59(4): 826 (1981), which uses a radioactive label to determine exact amounts of a desired component in a sample. Alternatively, this assay could be converted to a qualitative assay by using a nonradioactive label, such as a colorimetric label, and designing the assay such that the label showed a positive result only if the amount of the desired metabolite present in the sample was above the basal level. Depending upon the nature of the label, the detection means can be mechanical, electronic, magnetic or visual. Labels detectable visually are preferred for use in diagnostic kits for use in the field and even in clinical or laboratory applications due to the rapidity of the signal and its easy readability. For colorimetric detection, a variety of enzyme systems have been described in the art which will operate appropriately in the homogeneous assay format. As one example of a two-enzyme colorimetric assay, a first enzyme, glucose oxidase, can be employed which uses glucose as a substrate. Interaction between glucose and glucose oxidase releases peroxide as a product. A peroxidase then can be employed as the second enzyme. The peroxidase catalyzes the reaction of peroxide and a hydrogen donor such as tetramethyl Benzedrine (TMB) produces an oxidized product that appears as a blue color .

Other useful assays employ colorimetric enzyme systems, such as horseradish peroxides (HR) or alkaline phosphate (AP) , (using, e.g., indoxyl phosphate as the substrate) . When using such assays, the reaction advantageously is read within 5 to 15 minutes, preferably 10 minutes, to obtain an accurate result. A longer reaction time can lead to color changes induced by trace amounts of enzyme remaining on the reaction surface. In some embodiments, if desired, a stop solution can be employed to disable the enzyme reaction. Examples of such solutions are known to those skilled in the art.

Also useful are label systems which employ commercially available colored latex microparticles in which a dye has been embedded. Such particles can be used instead of enzymes to form conjugates with the antibodies and metabolites in the methods of this invention and provide a visual signal in the assay.

Other conventional label systems that can be used in the methods of this invention include fluorescent compounds, radioactive compounds or elements and immunoelectrodes .

If desired, a two antibody assay system can be used. For example, one Mab which binds to a first selected epitope on an LH metabolite of interest can be attached to a colorimetric indicator, such as colloidal gold. The amount of indicator is selected such that if the milk sample tested contains a level of the desired metabolite above the basal level the assay will indicate a positive result. A second Mab which binds to a different epitope on the same LH metabolite is attached to one half of a natural binding or affinity pair, such as, for example, biotin and streptavidin . Both antibodies are mixed with a milk sample, then the resultant mixture flows down a test strip which has the second half of the natural binding or affinity pair bound to the strip in an area where results are to be read. As the mixture passes over the reading area, the affinity pairs will bind and a color will develop as the antibodies which are bound to the LH metabolite and also the antibody with the colloidal gold collect in the reading area. Parameters such as quantity of sample and the amount of labeled antibody are used to establish result visual reading levels.

In an alternative assay, one Mab which binds to a first selected epitope on an LH metabolite of interest can be conjugated to a conventional solid matrix, such as, for example, latex beads. A second Mab, which binds to a different epitope on the metabolite of interest, is conjugated to colored latex particles. A milk sample taken from the mammal being evaluated is incubated with, and subsequently separated from, the antibody-conjugated matrix. A predetermined amount of the second Mab is added to the carrier and allowed to react with the metabolite attached to the matrix through the first antibody. The carrier then is washed. The washing of the sample will be sufficient to determine whether there are large quantities of the metabolite present, as the washing will fail to remove the second antibody conjugated to the colored particles if all of the conjugated antibody has bound to the metabolite attached to the matrix via the first antibody and the sample will retain the color of the colored particles. If the sample does not contain an elevated level of the metabolite, a majority of the colored particles, conjugated to excess second antibody will be washed away and the wash water will be highly colored from the presence of the colored particles. These assays can be designed to be either qualitative or quantitative in the expression of their results. In a typical competitive assay format, using a single anti-metabolite antibody, a sample of milk containing an unknown quantity of LH metabolite is combined with a known amount of labeled LH metabolite and the mixture is contacted with an antibody to the LH metabolite under immunoreaction conditions. The amount of LH metabolite in the sample is inversely proportional to the amount of labeled LH metabolite that reacts with the antibody and can be determined from a calibration curve. Depending upon the particular assay format, the antibody may be conjugated to a solid surface. The reaction could take place in solution with precipitation after the reaction or it could take place on a tube or microtiter plate wall. If desired, the LH metabolite, LH metabolite conjugates or antibodies used in an assay in accordance with this invention can be synthetically generated. A synthetic compound or material is one which is manufactured or derived to be identical or to mimic its naturally occurring counterpart. Once the sequence of a desired metabolite, metabolite conjugate or antibody is determined using standard techniques, a corresponding compound can be synthesized using standard techniques and procedures.

In a second competitive assay using a synthetic metabolite, a milk sample is contacted with and incubated with predetermined amounts of an unlabeled antibody specific for a desired metabolite and a labeled synthetic version of the same metabolite. The labeled synthetic metabolite competes with the natural metabolite in the milk sample for binding to the antibody. If there is an elevated amount of the natural metabolite in the sample, it will react with a higher percentage of the antibody binding sites and less of the labeled synthetic metabolite will bind to the antibody. By determining how much of the known amount of the labeled synthetic metabolite did not bind to the antibody one can determine whether there was an elevated amount of the natural metabolite in the milk sample .

The methods described herein can be used efficiently through means of a diagnostic kit. Such a kit comprises the components necessary to enable one to carry out a particular assay. For example, the kit can contain a first antibody associated with a first enzyme, a second antibody conjugated to a second enzyme and a substrate for the first enzyme, wherein the reaction product of the first enzyme and the substrate can interact with the second enzyme to produce a visible reaction or product, and a container, such as a vial for containing the milk sample. Alternatively, the kit can contain an antibody, bound to a solid carrier and associated with a first enzyme, which specifically binds to a desired LH metabolite, a second antibody, associated with a second enzyme, which binds to a different epitope of the same LH metabolite and a sufficient amount of a substrate for the first enzyme wherein, again, the reaction product of the first enzyme and the substrate can interact with the second enzyme to produce a detectable reaction or product.

The kits further can contain other useful components, as needed. If a stop solution is desirable, for example, such solution would be included. If the detectable label is one that is detectable by non-visual means, the components necessary for that detection are provided. The kit further can contain special media, filters or other separation tools which enable the user, if desired, to perform the assay using a milk sample which has been treated to separate potential blocking agents prior to performance of the desired assay. As an example, the kit can contain a commercial membrane filter with controlled pore sizes which would selectively separate milk components with molecular weight greater than those of the LH metabolites from the raw milk sample. In addition, the kit components also can enable the user to accomplish steps commonly used in solid phase separation procedures in the assay.

The following examples illustrate how the existence of LH metabolites in the milk of lactating mammals was proven and demonstrates correlation between levels present in milk and onset of fertility and ovulation which can be measured through the practice of the present invention.

Example 1

Detection of Labeled LH Metabolites in the Stomachs of Rat Pups Nursing from Normally Cycling Lactating

Rat Dams Injected Intraperitoneally with Labeled

LH at Levels Simulating LH Surge Levels and the Correlation of LH Metabolites obtained from Rat Milk to Rat Blood Serum Levels of LH.

This experiment was set up to determine if rat LH or its metabolites is transferred from the blood of normally lactating rat dams through the mammary gland and into the dam's milk. In addition, the experiment was designed to determine transfer rates and correlations should LH or its metabolites pass through the mammary gland. Rat pups were used to collect milk from the dams and a series of pup substitutions were used to create timed milk collection . The experiment was conducted as follows:

Milk Collection Protocol:

• Seven timed pregnant CD rats were bred to deliver within a 24 hour period. Rat litters were designated as A through G.

• On postpartum day (PPD)l two litters (A&B) were reduced to 10 pups and the remaining litters were reduced to 8 pups (C-G) . In some cases pups were switched between litters to achieve a 1:1 sex ratio and the desired number of pups per litter. • On the morning of PPD 4, rat luteinizing hormone

(rLH) obtained from the National Hormone and Pituitary Program, Rockville, MD., was iodinated (lmCi/2μg) by the chloramine-T method, as previously described by Agrawal (Agrawal A. K., Pampori N. A. and Shapiro B. H. "Sex- and dose-dependent effects of neonatally administered aspartate on the ultradian pattern of circulating growth hormone regulating hexobarbital metabolism and action." Toxicol . Appl . Pharma col . , 108: 96-106, 1991). The iodinated hormone was purified over Sephadex G-100 gel filtration columns to separate it from free iodide.

• At approximately 1PM on PPD 4 four pups, one male and one female pup from each of litters A&B were removed as controls. Dams A & B then were injected intraperitoneally with ¹²⁵I-rLH with approximately 1 μg per dam.

• At approximately 3:30PM A & B dams were separated from their pups and a male and female pup was removed from each litter (i.e. four pups) . Replacement pups, one male and one female from each of litters C & D were marked on the head and back with a large swatch of black magic marker, painted with a fecal slurry from the adopted dam, and integrated with other pups in the A & B litters. A & B litters were then returned to their respective dams and observed to insure that all pups in each litter were accepted by their respective dams.

• At approximately 6PM the four marked pups in litters A & B were removed and new pups replaced in a similar fostering procedure to that previously described and replaced with 4 pups from litters E & F.

• The same replacement and fostering procedure was followed at approximately 9AM and 6PM on each of PPD 5, PPD 6 and PPD 7.

• On the evening of PPD 7 all of the remaining pups and dams in litters A - G were euthanized using pentobarbital . The non-radioactive pups and dams (C - G) were disposed of and the remaining animals analyzed.

• Pups removed from A & B litters during the experiment and all A & B litter animals remaining after 6PM PPD

7 were euthanized and checked for radioactivity.

• Those pups which showed signs of radioactivity were then washed to remove the possibility of contamination from maternal feces, urine, or saliva and rechecked for radioactivity.

• If pups remained radioactive, their stomach contents were washed out and collected and both stomach contents and bodies were counted and recorded.

Results

With this experiment we were able to demonstrate that a portion (> 25% of the injected LH) of the radioactive LH injected into the blood of lactating dams passed through the mammary gland and into the stomachs of the dam's nursing pups. The data from this experiment is shown graphically in Figure 1. This figure demonstrates that a direct correlation exists between the time of injection of the radioactive LH into the dam and its appearance in the nursing rat pups' stomach.

Example 2

Detection of Labeled LH Metabolites in Milk Collected

Directly from Normally Cycling Lactating Rat Dams

Injected Intraperitoneally with LH Surge Simulated

Levels of Radioactivie Labeled LH and Identification of LH Metabolites in Rat Milk

Previously reported work by Johnson and Reeves (Johnson H.E. and Reeves J.J. A luteinizing hormone- releasing hormone-induced serum luteinizing hormone surge is not detectable in the milk of cows. J. Anim . Sci . , 66:442-446,1988) using immunoassays for luteinizing hormone indicated luteinizing hormone was not detectable in the milk of cows, yet we were able to demonstrate in the experiment detailed in Example 1 that some form of radioactive LH was being transferred through the blood into the mammary gland and then into the milk which was being suckled by rat pups. We concluded that the radioactive LH entering the milk must be metabolites of LH and designed our second experiment to demonstrate that the radioactivity entering the milk consisted of radioactive metabolites of the original material. The confirming experiment which was conducted without the aid of rat pups was as follows :

• Six timed pregnant CD rats were bred to deliver within a 24 hour period. Postpartum dams were then kept with their litters until PPD 5. • On PPD 4, rat luteinizing hormone (rLH) obtained from the National Hormone and Pituitary Program, Rockville, MD., was iodinated (lmCi/2μg) by the chloramine-T method, as previously described by Agrawal et.al. (Agrawal A. K., Pampori N. A. and Shapiro B. H. "Sex- and dose-dependent effects of neonatally administered aspartate on the ultradian patterns of circulating growth hormone regulating hexobarbital metabolism and action." Toxicol . Appl . Pharmacol . , 108: 96-106, 1991). The iodinated hormone was purified over Sephadex G-100 gel filtration columns to separate it from free iodide. Purified iodinated hormone was stored at -70°C until the next morning. We previously have observed that iodinated rLH is quite stable for more than a week when stored at -70°C (unpublished) .

• On the morning of PPD 5, each of six lactating rats was intraperitoneally injected with ~1.3xl0⁹ cpm of

¹²⁵I-rLH. Six hours later, milk was collected by a modification of a rodent milking apparatus (Nagasawa H., "A device for milk collection from mice." Labora tory Animal Science 26: 633- 635 , 1979). A sample of the radioactive milk from all six dams as well as control samples, which included ¹²⁵I-rLH and rat milk which was incubated with ¹²⁵I-rLH for 6 hours, were electrophoresed at 15 amps/gel on sodium dodecyl sulfate-polyacrylamide gels containing 12% polyacrylamide according to the general methods of Luemmli (Luemmli U,K., "Cleavage of structured proteins during the assembly of the lead group of bacteriaphage T₄. " Na ture 227:680-685, 1970). Radioautographs of the gels then were prepared.

Results

Radioautographs of the gels revealed 6 to 7 putative metabolites of rLH in the milk collected from the six lactating dams treated with ¹²⁵I-rLH. These identified metabolites had molecular masses from 3 to 20 kilodaltons. Since rat milk incubated with ¹²⁵I-rLH did not generate similar metabolites, we can conclude that the metabolites were produced within the rat and subsequently passed through the mammary gland into the milk. Finally, based on the results of Example 1 and Example 2, we conclude that a correlation can be made between blood levels of luteinizing hormone and the level of luteinizing hormone metabolites in milk, and thus we can identify the luteinizing hormone surge and onset of fertility through measurement of the change in luteinizing hormone metabolite levels in milk.

Example 3

Detection of LH metabolites in the Milk of a Normally Cycling Cow

Twice Daily milk samples are collected from a normally cycling cow using the following protocol. Teats are sprayed with a wash solution and wiped dry while expelling a few milliliters of milk from each quarter. Using a 50 ml graduated polypropylene sample bottle, roughly 45 ml of "first" milk is collected with approximately equal quantities of milk from each mammary quarter. After collection, samples are refrigerated at 40°F prior to the first stage of laboratory sample preparation.

The following laboratory sample preparation procedure is used for each serially collected milk sample. The sample is vortexed and a 12 ml aliquot is placed in a 16 mm x 100 mm test tube. The test tube is centrifuged in a refrigerated swinging bucket centrifuge at 2000 rpm for 10 minutes at 2°C. The cream layer is separated from the skim milk and discarded. Duplicate 16 mm x 100 mm sample tubes are prepared for each sample by pipetting 4 ml of the skim milk sample into 2 tubes. These tubes then are frozen at -70°C until all of the milk samples are collected. The following laboratory RIA sample preparation is performed after all milk samples have been collected. Milk samples are removed from the -70°C freezer and thawed in an ice bath. Trichloroacetic acid (TCA) is used to remove higher molecular weight proteins from each sample as follows: 6 ml of 5% cold aqueous TCA is added to each sample tube and each tube is vortexed for 1 minute and then centrifuged in a refrigerated floor model centrifuge in a swinging buck rotor spinning at 2200 rpm for 25 minutes at 5°C. The supernatant is then decanted into a 50 ml stoppered test tube and washed three times with approximately 30 ml of ethyl ether wash. After the final wash, any remaining ethyl ether is removed under a gentle stream of nitrogen gas until no ethyl ether odor is detectable and then placed under vacuum for 10 minutes. The washed samples then are centrifuged in a refrigerated centrifuge using a swinging buck rotor spinning at 2200 rpm for 20 minutes at 5°C. An 8 ml sample of each supernatant is pipetted into separate 20 ml glass vials and frozen at -70°C. All samples are freeze dried and stored at -70°C for RIA analysis.

Duplicate samples are prepared for each of the milk samples and triplicate samples are prepared for a nine point standard displacement curve. All samples are processed using an LH metabolite standard double antibody procedure. Radioactive LH metabolites for use in the assay are prepared using the chloramine T oxidation procedure. Radioactively labeled LH metabolites from the reaction mixture then are separated using a Sephadex column and characterized using gamma counter cpm data.

LH metabolite Assay Reagents Used:

1. Standards: a stock standard solution of 1 mg/ml LH metabolite in a dilution buffer is prepared. A portion of the stock is diluted to 20 ng/ml in dilution buffer, then further diluted to give standard solutions of the following LH metabolite concentrations: 10, 5, 2.5, 0.625, 0.312, 0.156, 0.078 and 0.039 ng/ml. 2. Dilution buffer: 0.01M phosphate buffered saline, pH 7.4 containing 0.1% sodium azide

3. Normal rabbit serum (NRS, Sigma Product No. R- 9133) 2% in dilution buffer

4. Ethylenediamine tetraacetic acid (EDTA) disodium salt solution (Sigma Stock No. ED2SS) 0.1 M, pH 7.8 in distilled water, pH adjusted with ION NaOH.

5. Second antibody: goat anti-rabbit IgG (Sigma Product No. R-0881) is reconstituted in dilution buffer. Reconstituted antiserum is diluted 1:5 in dilution buffer. RIA Protocol

In polypropylene test tubes, 0.5 ml sample or standard, 0.2 ml diluted LH metabolite rabbit antiserum and 0.1 ml radioactive tracer diluted in dilution buffer are added. Tubes are vortexed for 20 seconds, then incubated for 4 hours at 37°C. 0.2 ml 2% NRS is added to each tube, then the tubes are vortexed for 20 seconds. 0.1 ml EDTA is added to each tube and the tubes are vortexed again for 20 seconds. 0.1 ml of second antibody is added to each tube and the tubes again are vortexed for 20 seconds. The tubes then are incubated for 18-20 hours at 4°C then centrifuged for 5 minutes at 4°C in a high speed 90° fixed angle microfuge. Supernatant is removed from each tube and radioactivity present in each precipitate is counted with a gamma spectrometer. A standard curve of the control samples is plotted and sample values are interpolated from their positions on the standard curves . Initiation of fertility occurs when LH metabolite levels increase significantly above normal basal levels .

Example 4 ELISA for Determining LH Metabolites in Milk

A colorimetric enzyme-linked immunosorbent assay (ELISA) can be used as follows to determine whether basal or elevated amounts of LH metabolites are present in milk.

Microtiter plate wells are coated with an antibody which recognizes and specifically binds to a preselected epitope on an LH metabolite antigen of interest and the microtiter plates are incubated for 16-20 hours. The wells then are washed four times and a preselected amount of milk and buffer is added to each well. The plates are incubated for an hour. An antibody which recognizes and specifically binds to a second preselected epitope site on the LH metabolite and has been conjugated to horseradish peroxidase is added and the plates are incubated for an hour. The wells of the plate are washed and peroxide is added to each well. The plates are incubated for 10 minutes, then a stop solution is added to each well. The plate then is read on a microtiter plate reader.

Claims

What is claimed is:

1. A method for detecting the onset of fertility in a female mammal which exhibits estrus during lactation which comprises determining the level of one or more luteinizing hormone metabolites (LH-M(sj) in the milk of the mammal and correlating the level of LH-M(s> with the fertility condition of the mammal, whereby a detectable increase in LH-M.s) above basal levels in the milk indicates the initiation of fertility and onset of ovulation.

2. The method of claim 1, wherein said female mammal is by scientific classification a member of the bovidea family.

3. The method of claim 2, wherein said female mammal is a cow, bison, buffalo, oxen, goat or sheep.

4. The method of claim 1, wherein said female mammal is by scientific classification a member of the tayassuidae family.

5. The method of claim 1, wherein said female mammal is a human.

6. The method in claim 1, wherein each of said luteinizing hormone metabolites comprises a glycoprotein, protein or peptide comprising an amino acid sequence within or derived from the amino acid sequence of LH .

7. The method according to claim 1, wherein said determination comprises contacting a sample of milk from the female mammal with an antibody specific for a desired LH-M, said antibody associated with a detectable label, incubating the sample with the antibody and determining the level of antibody-antigen binding.

8. The method according to claim 7, wherein said antibody is a monoclonal antibody.

9. The method according to claim 7, wherein said antibody is a polyclonal antibody.

10. The method according to claim 7, wherein said label is selected from the group consisting of an enzyme system capable of generating colorimetric signals, colored latex microparticles, fluorescent compounds, luminescent compounds, radioactive compounds or elements, electrochemical mediators, chromophores/dyes, charcoal and immunoelectrodes .

11. The method according to claim 7, wherein said label is capable of mechanical, electronic, radioactive, magnetic or visual detection.

12. The method according to claim 11, wherein said label is capable of visual detection.

13. The method according to claim 11, wherein said label is radioactive.

14. The method according to claim 12, wherein said label comprises horseradish peroxidase and tetramethyl benzidine (TMB) or alkaline phosphatase

(AP) and indoxyl phosphate.

15. The method according to claim 1, wherein said determination comprises (a) incubating a sample of milk with a first antibody which binds to a first epitope on the LH-M of interest and is conjugated to a solid carrier, (b) separating said sample from said first antibody-carrier conjugate, (c) adding to said first antibody-carrier conjugate a predetermined amount of a second antibody which binds to a second epitope on the LH-M of interest and is conjugated to a detectable label under conditions wherein said second antibody can react with said LH-M attached to said carrier through said first antibody, (d) washing said carrier (e) and determining the amount of LH-M of interest conjugated to said carrier.

16. The method according to claim 15, wherein said antibodies are monoclonal antibodies .

17. The method according to claim 15, wherein said antibodies are polyclonal antibodies.

18. The method according to claim 15, wherein said label is capable of mechanical, electronic, radioactive, magnetic or visual detection.

19. The method according to claim 1, wherein said determination step comprises (a) combining under antibody-antigen binding conditions (1) a sample of milk containing an LH-M of interest with (2) a known amount of said LH-M to which a label has been attached and an antibody which can specifically bind to an antigen present on both the LH-M of interest and the labeled LH-M and (b) determining the amount of LH-M present in the sample from the amount of labeled LH-M which reacts with said antibody.

20. The method according to claim 19, wherein said antibody is a monoclonal antibody.

21. The method according to claim 19, wherein said antibody is a polyclonal antibody.

22. The method according to claim 19, wherein said label is capable of mechanical, electronic, radioactive, magnetic or visual detection.

23. The method according to claim 1 wherein said determination comprises contacting a sample of milk from the mammal with a predetermined amount of an unlabeled antibody in the sample and a predetermined amount of a labeled LH-M, wherein said antibody specifically recognizes an antigen present on both an LH-M of interest present in the sample and the labeled LH-M, (b) incubating the sample with the unlabeled antibody and the labeled LH-M under antigen-antibody binding conditions, and (c) determining how much of the labeled LH-M has bound to the antibody, wherein the presence of an elevated level of native LH-M in said milk is indicated by a reduction in the relative amount of labeled LH-M bound to the antibody in comparison to the amount of labeled LH-M which binds to the antibody in the presence of a basal level of the LH-M in the milk sample.

24. The method according to claim 23, wherein said antibody is a monoclonal antibody.

25. The method according to claim 23, wherein said antibody is a polyclonal antibody.

26. The method according to claim 23, wherein said label is capable of mechanical, electronic, radioactive, magnetic or visual detection.

27. The method according to claim 23, wherein said labeled LH-M is synthetic LH-M.

28. The method according to claim 1, wherein said determination comprises (a) incubating a sample of milk with a first antibody which binds to a first epitope on the LH-M of interest, is associated with a first enzyme, and is conjugated to a solid carrier, (b) separating said sample from said carrier and said first antibody and first enzyme, (c) adding to said carrier and said first antibody and first enzyme (1) a second antibody which is associated with a second enzyme and which binds to a second epitope on an LH-M of interest under conditions wherein said second antibody can react with the LH-M attached to said carrier through said first antibody and (2) a substrate for the first enzyme, wherein the reaction product of said first enzyme and said substrate will react with said second enzyme to produce a reaction product and (d) determining from the reaction product produced whether the milk sample contained more than a basal level of LH-M.

29. The method according to claim 28, wherein said antibodies are monoclonal antibodies.

30. The method according to claim 28, wherein said first enzyme comprises glucose oxidase, said second enzyme comprises horseradish peroxidase and said substrate comprises glucose.

31. The method according to claim 1, wherein said determination comprises (a) incubating a sample^' of milk with a first antibody which binds to a first epitope on an LH-M of interest and is associated with a first enzyme, (b) adding (1) a second antibody which binds to a second epitope on the LH-M of interest and is associated with a second enzyme and (2) a substrate for said first enzyme, wherein said second enzyme and the reaction product of said first enzyme and said substrate react to produce a reaction product, and (c) determining from said reaction product whether the milk sample contains more than a basal level of LH-M.

32. The method according to claim 31, wherein said antibodies are monoclonal antibodies .

33. The method according to claim 31, wherein said first enzyme comprises glucose oxidase, said second enzyme comprises horseradish peroxidase and said substrate comprises dextrose.

34. The method according to claim 1, wherein said determination comprises (a) incubating a sample of milk with a first antibody which binds to a first epitope on an LH-M of interest and is attached to a detectable indicator and with a second antibody which binds to a second epitope on said LH-M and is attached to one half of a binding or affinity pair, (b) applying the milk sample containing said first and second antibodies to a solid support to which is bound the second half of the binding or affinity pair such that the sample contacts said second half of the binding or affinity pair, and (c) determining from the detectable indicator attached to the solid support whether the milk sample contains more than a basal level of LH-M.

35. The method according to claim 34, wherein said antibodies are monoclonal antibodies .

36. The method according to claim 34, wherein the binding or affinity pair comprises streptavidin and biotin .

37. A diagnostic kit comprising an antibody to an LH metabolite conjugated to a detectable label.

38. A kit in accordance with claim 37, wherein said antibody is conjugated to a solid carrier.

39. A kit in accordance with claim 37, which further comprises a second antibody which specifically binds to a different epitope on said LH metabolite then said first antibody.

40. A kit in accordance with claim 37, which further comprises a synthetic LH metabolite which comprises an antigen specifically recognized by said antibody.

41. A diagnostic kit comprising an antibody to an LH metabolite and a synthetic LH metabolite which comprises an antigen specifically recognized by said antibody, said synthetic LH-metabolite bound to a detectable label.