WO2009146043A1

WO2009146043A1 - Assay for semen optimization

Info

Publication number: WO2009146043A1
Application number: PCT/US2009/039022
Authority: WO
Inventors: B. Arial Cohen
Original assignee: Cohen B Arial
Priority date: 2008-03-31
Filing date: 2009-03-31
Publication date: 2009-12-03

Abstract

A method for semen optimization for use in artificial insemination is described. The method involves incubating a semen collection, monitoring changes in protein and/or fluorochrome binding to the sperm contained therein and terminating the incubation prior at use or processing for storage. The method allows increasing fertilization success and/or creation of a gender bias.

Description

Assay For Semen Optimization

BACKGROUND

[001] Treatment of mammalian semen to achieve a higher proportion of fertility and/or a higher proportion of one gender over another can be advantageous. For example, a dairy herd would obtain economic benefit from an increase in numbers of cows pregnant at any given time and/or birthing a higher percentage of heifers relative to bulls. . In such a situation, replacement animals for the herds are produced more efficiently. In addition, especially with low-beef value animals such as Holsteins, the expense of bull calves, and the cruelty these animals face when used in veal production is reduced.

[002] The availability of replacement animals born at the farm eliminates the need to import replacements and the attendant risk of disease introduction into a herd. Additional advantages are found for businesses housing elite sires that produce dairy bull semen. Since these bulls are evaluated, i.e. "sire-proofed," for genetic quality only through their daughters, an elite bull can be brought into semen production more quickly if he produces daughters more quickly and often. This speeds improvement of the sire genotype, with the attendant competitive advantage. This further produces a savings in feed, vet, and other costs associated with bull farming. It also accelerates the improvement of the genetic base of dairy herds using semen from these processors, with the attendant economic savings to dairy farmer and semen processor alike.

[003] In addition, achieving good fertility by increasing the quality of sperm used in artificial insemination is considered to be the single greatest determinant of the success or failure of dairy farms. Since "open" or non-pregnant cows are not productive, they decrease profit. Consequently, any increase in fertility is considered worthwhile. Similar situations exist for other types of animals raised for dairy such as goats, sheep, cattle, buffalo, swine, etc.

[004] In another example, optimized sperm quality can lead to improvement and/or expansion of a particular population of animals. For instance, sperm collected from elite race horses or other champion animals, such as cattle or other livestock and particular breeds of dogs and cats, is commonly used for artificial insemination to maximize the probability of maintaining particular features in the gene pool. Sperm quality is particularly important in the breeding programs directed to exotic and endangered animals where the number of captive individuals is limited. Here, the ability to increase overall birth rates, thereby increasing the potential for rapid expansion of the population, are critical for success.

[005] In another example, the personal suffering and costs associated with human infertility can in many cases be reduced through maximizing sperm quality. Couples whose infertility is caused by low sperm count or poor sperm motility can benefit by increasing the number of intact and viable sperm that result after the washing and preparations steps needed prior to intrauterine artificial insemination (IUI) or intracytoplasmic sperm injection (ICSI).

[006] With respect to gender bias, the suffering and costs of human sex-linked diseases can be reduced through birth of females in affected human families. Female births are the only way to eliminate over 300 X-linked diseases, many of which shorten and impair quality of life and create staggering medical costs. Currently, the costs and suffering associated with these diseases can be minimized through pre-implantation genetic diagnosis. In this process, eggs are harvested by laproscopy following injections of hormones and fertility drugs. Eggs are fertilized in vitro and, after embryos have reached sufficient size, a single cell is microdissected from each embryo for genetic analysis. A suitable unaffected female embryo is chosen for implantation. Alternatively, sperm is collected and treated with mutagenic dye in preparation for fluorescent activated cell sorting (FACS). X-bearing sperm are obtained, however, they are so damaged that the sperm nucleus must be injected into an isolated egg in vitro using intracytoplasmic egg injection. Embryos are then cultured and implanted in recipients. Both of these techniques are expensive and raise unresolved questions about the effect of exposure to DNA-binding dyes with respect to their cytotoxicity and mutagenic potential (Downey et al. (1991) J. Histochem. and Cytochem. 39: 485-489; Durand and Olive (1982) J. Histochem. and Cytochem. 30:111-116).

[007] The scientific literature describes several methods for achieving gender bias through treatment of mammalian semen. They differ in process; some involve physical separation of sperm while others do not. They also differ at point of application; to sperm, to female mammals, to clutches of eggs in egg-laying animals. What they share in common is that they cannot be applied effectively on-site.

[008] For example, several methods are reported for generating sex bias by physical separation of sperm, all of which involve complex laboratory manipulations and equipment. Fluorescence activated cell sorting (FACS) resolves sperm into X (female) and Y (male) bearing pools, after cell labeling with mutagenic DNA-binding dyes to reveal chromosome content (Abeydeera et al. (1998) Theriogenology 50: 981-988; Cran and Johnson (1996) Human Reproduction Update 2: 355-363). Methods of artificially biasing the sex of mammalian offspring through physical separation have also included methods based upon density sedimentation of spermatozoa (e.g. Brandriff et al. (1986) Fertil. Steril. 46:678-685) and by separating the population of spermatozoa into fractions that differ by the sex-linked electrical charge resident thereon (U.S. Patent Number 4,083,957). Methods have also been described that rely on mechanical sorting of sperm by sex-type. U.S. Patent Number 5,514,537, for example, uses a column packed with two sizes of beads. The large beads are of a diameter so that the smaller beads will fall between the interstices created between the larger beads. Then the interstices between the smaller beads allow Y-bearing sperm to enter them while the X-bearing sperm are excluded, thereby affecting separation of the two subpopulations. Separation based on immunological methods and cell surface markers have also been proposed (Blecher et al. (1999) Theriogenology 52: 1309-1321). In another example, U.S. Patent Number 3,687,806 discloses an immunological method for controlling the sex of mammalian offspring using antibodies that react with either X-bearing sperm or Y- bearing sperm which uses an agglutination step to separate bound antibodies from unaffected antibodies. U.S. Patent Number 4,191,749 discloses a method for increasing the percentage of mammalian offspring of either sex by using a male-specific antibody coupled to a solid- phase immunoabsorbant material to selectively bind male-determining sperm while female- determining sperm remain unbound in a supernatant. U.S. Patent Number 5,021,244 discloses a method for sorting living cells based upon DNA capacity, particularly sperm populations to produce subpopulations enriched in X-bearing sperm or Y-bearing sperm by means of sex- associated membrane proteins and antibodies specific for such proteins.

[009] Some methods have combined various aspects of the immunological and mechanical separations such as U.S. Patent Numbers 6,153,373 and 6,489,092 which use antibodies coupled to a magnetic particle for separation of sperm.

[0010] Separation based on a miniscule size difference between X- and Y-bearing sperm has also been attempted (Van Munster et al. (1999) Theriogenology 52: 1281-1293; Van Munster (1999) Cytometry 35: 125-128; Van Munster 2002 Cytometry 47: 192-199).

[0011] In addition, sex bias without physical separation of sperm into X and Y bearing classes has been described. For example, stress (Catalano et al. (2006) Human Reproduction 21: 3127-3131), good or poor physical condition (Trivers and Willard (1973) Science 179:90-92), feed composition (Alexenko et al. (2007) Biol. Reprod. 77:599-604), temperature (Crews (1996) Zoological Science 13: 1-13) and other factors (Wedekind (2002) Animal Conservation 5:13-20) have been shown to affect offspring sex ratio. Lechniak has also shown that time-based sexing of semen can occur when semen is held for various times before use in insemination.

[0012] Lechniak et al. (2003, Reprod. Dom. Anim. 38:224-227; incorporated herein by reference in its entirety) describes a study to determine whether or not sperm preincubation prior to fertilization in vitro (IVF) influences the rate of fertilization, embryo development, and/or the sex ratio among blastocysts. In the study, oocyte-cumulus- complexes (OCC) were aspirated from follicles of slaughterhouse ovaries; collected in Hepes-buffered Ham's F- 10; and matured in maturation medium under silicone oil for 24 hours at 39 degrees Centigrade (⁰C). Frozen-thawed sperm cells were used. After swim-up, the motile fraction of sperm was incubated in Sperm-Talp (no heparin included) at 39°C for 0, 6 and 24 hours. Sperm count was carried out and sperm motility was evaluated. The number of motile sperm cells was kept similar in each experimental group. The motile sperm decreased with time, as did fertility The authors reported 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.

SUMMARY

[0013] The high variability of semen quality across ejaculates (collections) used in artificial insemination (AI) is a source of significant problems, the results of which range from the economic losses farms experience due to the inability to consistently control the sex of the animals produced, to the adverse affects experienced by those seeking medical treatment for fertility issues because of sperm quality issues resulting from semen processing in clinical settings. An ability to monitor sperm biological processes as they occur during semen processing would provide a way to tailor semen processing to each individual collection, thereby optimizing sperm quality.

[0014] Clearly, the full advantage of increased fertility and/or sex selection for farm economics or reduction of human disease has been unreachable due to the lack of an effective fertility and/or gender bias technique suitable for on-site use (e.g., on-farm and in clinics). What is needed is a method that preserves fertility and/or generates a moderate sex bias when used with standard on-farm and in-doctor's office methods of artificial insemination, and eliminates exposure of sperm to mutagens and damaging conditions. An ability to monitor sperm biological processes as they occur during semen processing would provide a way to tailor semen processing to each individual collection, thereby optimizing sperm quality and maximizing fertility and/or creating bias. The instant assay provides a solution which makes it possible to increase fertility and/or skew the sex ratio of births in methods that is applicable on-site, while simultaneously eliminating exposure of sperm to deleterious conditions and agents.

[0015] The assay described herein is designed to capture these advantages. In addition, because the method simply imposes process control on biological processes common to all mammalian sperm, it is broadly applicable. This is especially relevant for breeding of exotic species, including primates, where fertility maintenance is a key factor and is related to the number and quality of cells used during insemination. It is likewise relevant for on-farm use with cattle, sheep, goats and swine or with champion livestock and animals such as race horses and pedigreed dogs and cats. The technology also has advantages for human users who suffer from fertility issues or sex-linked diseases.

[0016] The assay is directed to a method for semen optimization, thereby increasing fertility and/or creating a gender bias upon artificial insemination, by assessing changes in the sperm cell surface membrane. The method involves (a) obtaining a semen sample, (b) incubating the semen sample, (c) taking at least one sperm sample from the semen sample, (d) contacting the sperm sample with at least a first protein, (e) identifying the number of sperm binding the protein (f) terminating incubation of the semen sample and (g) using or storing the semen sample.

[0017] In one embodiment sperm quality is measured on the basis of sperm motility grade and percent. In another embodiment sperm quality is measured on the basis of the number of intact acrosomes.

[0018] With respect to the treatment, one embodiment envisions a treatment where a sugar polymer, a lipid, an enzyme or combinations thereof is added. Another embodiment uses a treatment that changes the temperature of the semen collection. In yet another embodiment the treatment is mechanical agitation. In some embodiments multiple treatments of the same or different types are performed at the same time or are preformed sequentially.

[0019] The assay assesses how to optimize semen to produce a fertility and/or sex bias by evaluating changes in sperm cell surface markers. In one embodiment the percent of individual sperm in the semen collection displaying a high phosphatidylserine concentration is calculated. In other embodiments the ability to produce a fertility and/or sex bias is assessed using a lectin, an oligosaccharide conjugated to a fluorphore, antibodies, a positively charged protein conjugated to a fluorophore, merocyanine 540, YOPRO-I, or combinations thereof. [0020] In yet another embodiment the ability to produce a fertility and/or gender bias is assessed using a primary antibody in conjunction with a secondary antibody that is conjugated to a fluorophore. In still another embodiment semen optimization, which results in higher fertility and the generation of a gender bias, is assessed using a primary antibody conjugated to an enzyme in conjunction with the enzymatic substrate to produce a colorimetric reaction.

[0021] One embodiment comprises a kit for carrying out the disclosed assay. The kit contains at least a first binding protein and a description of the method. In some embodiments the kit further comprises at least a first binding protein and a second binding protein, a sampling container.

BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1. Comparison of Semen Processing by Standard Method and Method Including Instant Assay.

[0023] Figure 2. Sperm Cell Assays Reveal High Variability. (A) assay of collections on two different days from a Canadian Holstein bull. (B) assay of collections on two different days from an Irish Friesian bull.

[0024] Figure 3. Fertility and Gender Bias Increases In Working Dairy Herds. Percent positive cells = X axis, time = Y axis. (A) bull RDU collection cohorts E (assay-based) and F (fixed time); (B) RDU collection cohorts M (fixed time) and N (assay-based).

DETAILED DESCRIPTION

[0025] The assay is designed for smooth integration into current methods of semen processing (see Figure 1) and creates an increase in fertility and/or a sex bias in a time-based method. This occurs under conditions that maintain fertility in standard on-site techniques of artificial insemination. The method preserves the number of cells processed, as methods that cause a reduction in cell yield are unsuitable in terms of economic and fertility losses. These include all methods requiring a physical separation of sperm.

[0026] The instant assay achieves increased fertility and/or creation of a gender bias by enabling detection of sperm metabolic changes that indicate when collected semen should be processed. Assay results enable users to process semen at a time that will increase fertility and/or cause sex bias upon AI.

[0027] The assay involves obtaining a semen sample, incubating the semen sample, taking at least one sample or aliquot from the semen sample, assessing sperm quality by contacting the sperm sample with at least a first protein, determining the percentage of sperm positive (% positive) for binding the first protein, optionally determining the point at which the % positive begins to decline, terminating the incubation of the semen sample and processing the semen sample for immediate use or for storage. Semen may be stored in straws, or otherwise, for artificial insemination.

[0028] The semen sample is incubated in a suitable collection container which has an overall and uniform temperature equivalent to the ambient temperature or falling within some range with a temperature not lower than 0⁰C and a temperature not higher than the body temperature of the pertinent species or the ambient temperature. To illustrate only, and without further limiting the range endpoints, such temperature ranges can be, for example, 0⁰C - ambient temperature, 0⁰C - 40⁰C, 4°C - 35°C, 10⁰C - 23°C, 15°C - 40⁰C, 18°C - 25°C, or 25°C - 30⁰C. The devices disclosed in application PCT/US09/38134 or in US 2006/0147894 A 1 are examples of suitable collection containers, as is the device disclosed in US 5,895,749. Application PCT US09/38134 is hereby incorporated by reference in its entirety.

[0029] Various reagents and physical conditions to which sperm may be exposed during processing, can affect sperm metabolism. The instant assay provides a means of evaluating treatments most beneficial to sperm and enabling users to improve sperm quality and properties in commercial operations by use of an optional treatment. The treatment minimizes variability in the sperm microenvironment within the semen ejaculate. For example, variability can result from the fact that in some species different pulses of ejaculate have very different biological compositions and consequently there is a gradation within the chemical microenvironment. In other cases the variability is a result of temperature or chemical differences within the semen sample. Therefore, the treatment that homogenizes the semen collection can comprise contact with chemical agents such as, for example, glycosidases (e.g. sialidase, galactosidase, glucosidase, and/or mannosidase), perioxidase, and/or horseradish peroxidase), proteases (e.g. chymortipsin, trypsin, and /or elastase), lipases (e.g. phospholipase C and/or phospholipase A) and kinases (e.g. diglyceride kinase, glycogen synthase kinase and/or inositiol kinase), to name but a few, and/or mono- or oligosaccharides, etc., or incubation at particular temperatures. Suitable temperatures are any temperature between 0⁰C and the ambient and/or the body temperature, or for example, between 0⁰C - 40⁰C, 15°C - 40⁰C, 18°C - 25°C, 25°C - 30⁰C. 4°C - 40⁰C, 15°C - 40⁰C, 18°C - 25°C, or 25°C - 30⁰C, to name but a few. Treatment can also comprise gentle mechanical mixing such as slow rocking of the tube, use of a rotary shaker, or tube inversion Combinations of any of the above treatments that homogenize the semen environment are also envisioned.

[0030] Mono- or oligosaccharides can be added to the semen collection sample after having been equilibrated to the same temperature as the collection sample. Ideally, this is done at the time of collection, but can be done at any time during the incubation step. Suitable mono- or oligosaccharides are carbohydrates that resemble those present in the isthmus. It is known that the isthmus of the oviduct can serve as a site of sperm binding to create a reservoir of sperm, which are presumed to be in a stabilized state. The mono- or oligosaccharides are chosen to effect membrane stabilization, which stabilize sperm against the manipulations they must undergo. Suitable monosaccharides include sialic acid, mannose, fucose or galactose, while appropriate oligosaccharide polymers are linear or branched chains of any compatible sugar or glycoproteins carrying carbohydrate chains that terminate at their non-reducing end in fucose, galactose, sialic acid or a combination of these sugars. Additional suitable treatments include commercial and/or individually-produced semen diluents and extenders, TALP, glycerol, egg yolk, bicarbonate ions and calcium ions or calcium chelators. Combinations of the above can also be used.

[0031] Aliquots can be taken from the incubated semen sample at regular intervals beginning at the time at which the semen sample is first collected. In one embodiment aliquots are taken every 10 minutes, every 15 minutes, every 20 minutes, every 30 minutes or every 60 minutes. In another embodiment samples are taken at 5 minute intervals. In yet another embodiment aliquots are taken at hour intervals. In still another embodiment only a single aliquot is taken and used. In some embodiments the sampling times are adjusted based on the change detected. Here, sampling times in collections that are rapidly changing are shortened while sampling times in collections that are changing slowly are lengthened. Once the aliquot has been taken, sperm quality as a preliminary assessment of fertility and/or creation of sex bias is evaluated. The evaluation is conducted immediately, within 30 minutes, within an hour or within a day.

[0032] Optimal sperm quality can be defined on the basis of numerous attributes such as number of viable sperm, sperm motility, sperm morphology, fructose level, acrosomal integrity, etc. It is known, for example, that X-bearing sperm are visibly larger than Y- bearing sperm. It is also known that all sperm go through a series of metabolic changes once ejaculation has occurred and the sperm is mixed with plasma from the seminal vesicles and with other fluids. The sperm "maturation" which includes "capacitation" that follows ejaculation is necessary for sperm to achieve fertilizing ability. A number of membrane changes are associated with these processes (Bearer and Friend (1990) J Elecon Micros Tech. 16: 281-297).

[0033] With respect to assessing the ability to produce a sex bias in generated offspring, since the timing of many of the changes that occur during maturation or capacitation may occur at a different rate in X-bearing sperm versus Y-bearing sperm, these sperm cell surface changes can be monitored to assess the ability to produce the highest sex bias in generated offspring. This is done by identifying the time at which the largest group of X-bearing sperm are at their peak with respect to fertilizing performance compared to the Y- bearing sperm. Thus sperm cell surface changes allow one to assess the point at which the highest sex bias can be generated.

[0034] Sperm cell surface changes that have been reported and can be monitored according to the instant assay include increases in net negative surface charge (Bedford (1963) Nature 200: 1178-1180; Yanagimachi et al. (1972) Am J Ant. 135:497-520; Lopez et al. (1989) Gamete Res. 18:319-332), changes in glycoprotein amount or localization (Baker et al. (2004) J Andrology 25:744-751), changes in cholesterol and lipid distribution (Wolfe et al (1998) Biol Reprod 59:1506-1514; Flesch et al (2001) / Cell Sci 114:3543-3555) and phosphatidylserine location (Pena (2007) Asian J Androl 9:731-737), to name but a few.

[0035] Visualization of the sperm cell surface changes can be accomplished in numerous different ways. In some embodiments visualization occurs based on the use of a fluorophore or other light emitting molecule, used alone or in combination with a binding protein or antibody. Suitable fluorophores include carboxifluorescein acetate, phycoerythrin, calcein acetate, alexa fluor 488, YO-PRO-I, SNARF-I, and combinations thereof. In some embodiments combinations of the fluorophores are used where the range of excitation is similar (e.g. 488 nm), but emission occurs in different areas of the spectrum (e.g the green wavelength (515 nm) vs. the red wavelength (610 nm)). Suitable dyes include Annexin-V, Annexin-V-Biotin, biotin, Annexin V-PE, Annexin V-FITC, SAv-FITC, 7-AAD, Hydroethidine, Evans blue, chlorazol Black E, Coomassie Blue and Trypan blue, to name but a few. Combinations of these dyes can also be used. Chromogenic substrates include TMB (3.3',5,5'-tetramethylbenzidine) for peroxidase, ABTS (2.2-Azino-di(3-EthylBenzthiazoline Sulfonic acid) for peroxidase, pNPP (p-nitrophenyl phosphate, disodium salt) for alkaline phosphatase, and 5-bromo-4-chloro-3-indoyl-beta-D-glucuronide (BCIG) for beta galactosidase.

[0036] For example, since sperm develop a net negative surface charge over time, positively charged proteins can be conjugated to an appropriate fluorophore for evaluation. Suitable proteins have a pi greater than or equal to 8.5 so that they will be positively charged at the pH of the binding assay and include histidine-rich proteins such as the late embryogenesis abundant (LEA) proteins (Moons et al. (1995) Plant Physiol 107:177-186). Similarly, lectins conjugated to a fluorophore can be used such as Pisum sativum lectin (PSA), tomato lectin (LEA), peanut lectin (PNA), Aleuria aurantia agglutinin lectin (AAA), Ulex europaeus agglutinin lectin (UEA-I), wheat germ lectin (WGA), Solarium tuberosum (STA) and Tetragonobolus lectin (TPA). Mono- or oligosaccharides suitable for conjugation to a fluorophore include those terminating at the non-reducing end in fucose, galactose, or mannose, or being polymers of lactoseaminoglycans.

[0037] Antibodies to sperm cell surface markers, conjugated to fluorophore or enzymes can also be used. Numerous suitable antibodies are described in the literature. For example, a monoclonal antibody to human germ cells has been described by Naz et al. (1984; Science 225: 342-344), Saxena and Toshimori report a monoclonal antibody to MC31, a cell surface protein that is modified and redistributed during capacitation (2004; Biol Reprod 70:993-1000), Mor et al. describe an anti-heparin-binding sperm membrane protein (HBSM) (2007; Biochem Biophys Res Com 352: 404-409) and Focarelli et al. report that a CD52 antibody presents a different result compared to an anti-gp20 antibody (1999; European Society of Human Reproduction and Embryology 5:46-51). A monoclonal antibody, 4B12, has also been reported that recognizes a surface membrane-associated protein located in the acrosome portion of the spermatozoa that becomes accessible after capacitation (Mollova et al. (2002) Folia Biologica (Praha) 48: 232-236). Other molecules for use as markers during the capacitation process for which antibodies can be made are presented in Cohen-Dayag and Eisenbach (1994; Am J Physiol Cell Physiol 267:C1167- Cl 176).

[0038] In addition, sperm cell surface changes can be monitored with antibodies that are not unique to sperm cells. For example, antibodies to salmonella species (Difco Salmonella H antiserum A-Z product number 224061) can also be used, as can anti- cloxacillin and anti-calponin.

[0039] In some embodiments a second antibody reactive to the first antibody is used to increase ability for detection. For example, if a rabbit anti-salmonella antibody is used as a first antibody, a goat anti-rabbit IgG could be used as a second antibody. In cases where an enzyme reaction is used to visualize binding, the second antibody is conjugated to an enzyme and its substrate is added as a solution. Examples of enzyme-substrate pairs are peroxidase/hydrogen peroxide, glycosidaseM-methyl-umbelliferyl-glycoside, horseradish peroxidase/TMB (3,3',5,5'-tetramethylbenzidine), to name but a few.

[0040] In one embodiment, evaluation of sex bias can be accomplished through use of in vitro fertilization (IVF) to generate embryos. Subsequent evaluation of these embryos for sex by genetic techniques such as PCR of X- and Y-chromosome sequences can be accomplished to reveal sex. It is then possible to correlate observed sex bias with time of incubation and with assay result.

[0041] Once the results of the assessment of fertility and/or the generation of a sex bias are obtained, the semen is washed and diluted (extended) using commercially available products such as Bioxcell® (IMV, L'Aigle, France), and/or individually-produced egg yolk based extenders, or the extenders Triladyl® and Andromed® (Minitube, Tiefenbach, Germany) before packaging into straws.

EXAMPLES

Example 1 - Annexin treatment

[0042] The sperm from a 10 μl sample of ejaculate is washed twice with PBS buffer (8 g NaCl; 0.2 g KCl; 1.44 g Na₂HPO₄ • 7H2O; 0.24 g KH₂PO₄; H₂O to 1 liter. pH 7.2) and the cells resuspended at a concentration of ~1 x 10 cells/ml in IX Binding Buffer (10 X Buffer is 0.1 M HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl₂).

[0043] 100 μl of the solution (~1 x 10 cells) is transferred to a 5 ml culture tube and 2 μl of Annexin V stock solution (Annexin V-PE; BD Biosciences cat. no. 556422, 556421) added. The cells are gently mixed and incubated for 5 min. at room temperature in the dark. 400 μl of IX Binding Buffer is added and the cells analyzed immediately by fluorescence microscopy to determine the number of cells positive for apoptosis (i.e. Annexin V positive).

[0044] Controls are (1) unstained cells, (2) experimental cells stained with Annexin V-FITC for 5 min. and (3) blocked cells stained with 5 mg of unlabeled Annexin V and then Annexin V-FITC.

[0045] Blocked Cell Control a. This control includes preincubation of cell samples with recombinant unconjugated Annexin V, which is included as part of the BD Annexin V-FITC Apoptosis Detection Kit II (Cat. No 556570). This serves to block Annexin V-FITC binding sites and thus demonstrates the specificity of Annexin V-FITC staining.

[0046] The protocol is essentially as described above, but 5-15 μg of purified recombinant Annexin V is added instead of Annexin V-PE. The amount of purified recombinant Annexin V required to saturate binding sites may vary according to cell type, and stage of apoptosis. In some cases the cell number is reduced to 0.5 x 10 /100 μl, still adding 5-15 μg of recombinant Annexin V, to obtain optimal results. In addition, the resulting Annexin V mixture is incubated at room temperature for 15 minutes before adding 5 μl of Annexin V-FITC, mixing and incubating again at room temperature for 15 minutes in the dark. 400 μl of IX Binding Buffer is added and the cells analyzed immediately by fluorescence microscopy.

[0047] Optimal sex bias is produced, in the case of annexin V, when 20-40% of sperm show annexin V positivity.

Example 2 - Semen Preparation and Processing

[0048] A device according to PCT/US09/38134 was incubated at 32°C for 60 minutes or more to ensure uniform heating of the device. Just prior to use, the device was removed and within 2 minutes of collection, the sperm was placed in the collection container, inverted once, then immediately placed at 12°C for at least 15 minutes before sampling is begun. During the sampling time the collection of sperm is maintained at 12°C.

[0049] 5 μl of undiluted semen is mixed in a 1.5 ml microfuge tube with 100 μl antibody diluent with Bovine Serum Albumin (Invitrogen SKU # 00-3118). Twenty (20) μl of primary antibody (rabbit anti-salmonellas spp; Salmonella H antiserum A-Z product number 224061; Difco, Detroit, MI; reconstituted according to the manufacturer's instructions) is added, followed by 5 μl of secondary antibody conjugated to Alexa Fluor® 488 (goat anti-rabbit IgG (H + L); Invitrogen, Carlsbad, CA, catalogue no. Al 1008 ) (2 mg/1) and mixed. The solution is then incubated in the dark for 20-30 minutes at ambient temperature. In general, the ambient temperature ranged from 7°C - 27°C.

[0050] After incubation, 1 ml of PBS buffer (8 g NaCl; 0.2 g KCl; 1.44 g Na₂HPO₄ • 7H2O; 0.24 g KH₂PO₄; H₂O to 1 liter. pH 7.2) is added and the solution microfuged for 20 seconds before removing the supernatant. The cell pellet is gently resuspended in 100 μl of PBS buffer and approximately 5 μl transferred to a microscope slide. The number of sperm exhibiting green fluorescence on the head (deemed "positive") is counted as well as the total number of cells. A minimum of 100 cells are counted and the "percent positive" (% positive) is determined by dividing the number of positive cells by the total number of cells and multiplying by 100. [0051] In this case, the processing time is determined based on the % positive. If there are less than 25% positive, incubation is continued for approximately 1 hour before repeating the assay. If more than 25% positive, the assay is repeated at a shorter time interval such as 5, 10 or 15 minutes. Values of the % positive reach a peak and then begin to decline. Two hours after the peak, Bioxcell® extender and/or individually-produced egg yolk-based phosphate buffer extender equilibrated to 12°C is added to the semen, semen is cooled to 4°C - 10⁰C and semen is processed into straws according to standard procedures (e.g. P. Bermejo- Alaverez et al. (2008) Biol of Reproduction 79:594-597) or used immediately.

Example 3 - Variability of Sperm Biology

[0052] Semen was collected from dairy bulls (time = 0) and incubated. At intervals, samples were taken and assayed according to Example 2. (A) assay of collections on two different days from a Canadian Holstein bull named Bacardi. (B) assay of collections on two different days from an Irish Friesian bull named RDU. Results are presented in Figure 2.

[0053] Variability of sperm cell biology between bulls and between collections is revealed by the large differences in assay curve shape and in the time required for attainment of the assay peak point.

Example 4 - Fertility and gender bias increases in working dairy herds correlates with assay peak point

[0054] The first ejaculate of Irish Freisian bull RDU was collected on two different days as shown in Figure 3 (A) and (B). On each day, half of the ejaculate was processed at 6h. The other half of the ejaculate was processed according to Example 2. In Figure 3 (A) bull RDU collection was processed into cohorts RDU-E (assay-based, 5 hour incubation) and RDU-F (fixed time, 6 hours). In Figure 3B, bull RDU collection was processed into cohorts RDU-M (fixed time, 6 hours) and RDU-N (assay-based, 7 hours incubation). A fertility difference of about 10% was observed between the two processing methods, independent of whether the assay indicated processing time should occur before or after the fixed 6 hour time point. Results for gender bias and fertility appear in Examples 4-7 below.

Example 5- Fertility Increase in Working Dairy Herd

[0055] The first ejaculate of elite dairy bulls was collected and sperm cells assayed according to a "fixed time" incubation process or according to Example 2. For fixed time incubation, 12°C extender was added to the 12°C incubated semen and processed into frozen straws 6 hours post-collection. For semen assayed according to Example 2, the incubated semen was extended and processed into straws 2 hours after the assay revealed a peak in the percentage of assay-positive sperm (% positive). The time at which this peak was obtained varied between collections.

[0056] Two bulls were collected on different days to obtain semen treated by incubation. The control treatment involved the same two bulls with the collections of semen being processed into frozen straws according to standard methods. Success was measured by the non-return rate (NRR) of cows and heifers. NRR is a measure of fertility and is the percentage of animals in the dairy herd that do not return for repeat insemination due to pregnancy or death. A high NRR correlates with high herd fertility and is desirable.

[0057] Table 1 presents the results obtained and reports the number of animals serviced through AI, the number of animals returned after 30 days due to failure to become pregnant and the percent of non-return rate (%NRR). The normal heat cycle of a cow is 21 days.

Table 1 - Increased Fertility in Working Dairy Herd

Incubation

Treatment #Serves 30 Day %NR] method

Control n/a 1010 158 84.36

Fixed time 164 38 76.83 Incubation

Assay-based 139 18 87.05

[0058] From the results presented it is clear that sperm obtained from the assay and used for insemination produced a greater number of pregnancies; that is, as a population had increased fertility. For example, in the fixed time group, 164 animals were serviced by artificial insemination. By 30 days after initial service, 38 of these were returned for repeat insemination, as they were not pregnant. About 77% of the animals were therefore NOT returned for repeat insemination, giving a 76.8% non-return rate. In contrast, for the assay group, 139 animals were inseminated and only 18 were returned for repeat insemination by day 30, meaning 87% of the animals were not returned for repeat insemination.

[0059] In the control group, the non-return rate was 84.36% while in the assay group the NRR was 87.05, which indicates a fertility improvement of 2.69%. The fixed time group showed damage to fertility. Using logistic regression with pregnancy as the outcome, the improvement obtained by the assay method is statistically significantly more likely to increase fertility than the fixed time method (log odds ratio=0.71, p=0.047; a log odds ratio of 0 implies no association).

Example 6 - Fertility Increase Requires Incubation Time Variability

[0060] In order to determine the effect of incubation on fertility, the first ejaculate of two elite dairy bulls, RDU and QUR was collected. One half of the collection was processed according to a "fixed time" incubation process and one half according to Example 2. For the fixed time incubation, 12°C extender was added to the 12°C incubated semen and processed into frozen straws 6 hours post-collection. For semen assayed according to Example 2, the incubated semen was extended and processed into straws 2 hours after the assay revealed a peak in the percentage of assay-positive sperm (% positive).

[0061] Table 2 presents the data obtained and reports the number of animals serviced through AI, the number of animals returned after 30 days due to failure to become pregnant (NNR) and the percent of non-return rate (%NRR). The normal heat cycle of a cow is 21 days.

Table 2 - Fertility Increase Is Correlated With Assay

Collection

Pair and Time, h Method #Serves 30 Day %NI

Bull

RDU 6 Fixed 51 11 78

E&F

5 Assay 74 8 89

QUR 6 Fixed 8 2 75

I&J

7 Assay 20 3 85

RDU 6 Fixed 100 25 75

M&N

7 Assay 39 6 85

[0062] The data in Table 2 indicates that the quality of the sperm obtained from the assay method results in more pregnancies and a lower %NRR.

Example 7 - Gender Bias in Working Dairy Herd

[0063] In order to determine the effect of incubation on gender bias, the first ejaculate of elite dairy bull RDU was collected as described in Example 3. One half of the collection was processed according to a "fixed time" incubation process and one half according to Example 2. For the fixed time incubation, 12°C extender was added to the 12°C incubated semen and processed into frozen straws 6 hours post-collection. For semen assayed according to Example 2, the incubated semen was extended and processed into straws at the time shown in Table 3 (e.g. 2 hours after the assay revealed a peak in the percentage of assay-positive sperm (% positive).

[0064] Table 3 presents the data obtained and reports the numbers of each gender produced as determined by fetal scanning.

Table 3 - Gender bias according to fetal scanning

Collection

Cohorts and Time, h Method # Females # Males % Female

Bull

RDU- 5 Assay-based 15 9 63

E&F

6 Fixed 10 8 56

RDU- 6 Fixed 24 17 59

M&N

7 Assay-based 10 5 67

[0065] As can be seen from the results, the assay-based treated semen produced more females, as desired, than the fixed time treatment.

[0066] Table 4 summarizes the results obtained when the data from Table 3 is grouped according to fixed-time or assay-based treatment.

Table 4 - Summary of gender bias based on semen treatment

Incubation

Treatment # Females # Males % Female* method Control n/a n/a n/a 50

Fixed time 34 25 58

Incubation

Assay-based 25 14 64 *for control, historical percentage of females is reported

Example 8 - Semen Preparation and Processing - Enzymatic

[0067] A device according to PCT/US09/38134 is incubated at 32°C for 60 minutes or more to ensure uniform heating of the device. Just prior to use, the device is removed and within 2 minutes of collection, the sperm is placed in the collection container, inverted once, then immediately placed at 12°C for at least 15 minutes before sampling is begun. During the sampling time the collection of sperm is maintained at 12°C.

[0068] 20 μl of undiluted semen is mixed in a 1.5 ml microfuge tube with 1000 μl antibody diluent with Bovine Serum Albumin (Invitrogen SKU # 00-3118). Five (5) μl of primary antibody (e.g. rabbit anti-salmonellas spp; Salmonella H antiserum A-Z product number 224061; Difco, Detroit, MI; reconstituted according to the manufacturer's instructions) is added, mixed and incubated in the dark for 15 minutes at ambient temperature. 10 μl as supplied by the manufacturer of a peroxidase-conjugated goat anti- rabbit IgG (H+L) (catalogue no. Invitrogen SKU # G-21234) secondary antibody is added.

[0069] After incubation, 1 ml of PBS buffer (8 g NaCl; 0.2 g KCl; 1.44 g Na₂HPO₄ • 7H2O; 0.24 g KH₂PO₄; H₂O to 1 liter. pH 7.2) is added, the resulting solution mixed gently and centrifuged. The supernatant decanted. This wash step is repeated five times.

[0070] To produce signal, 1 ml of peroxidase soluble substrate (TMB/E Ultra Sensitive, Blue, Horseradish Peroxidase Subtrate (soluble); Millipore) is added, mixed gently and incubated at ambient temperature for 10 minutes.

[0071] The tube is placed in a calibrated colorimeter and the absorbance reading taken.

[0072] Proportionally smaller volumes can be used to accommodate performing the assay in a 96 well format or other configurations.

[0073] The processing time is determined based on the optical density (OD) reading. Incubation is continued for approximately 1 hour before repeating the assay. Once the OD reading has increased by about 20% above background, the assay may be repeated at a shorter time interval such as 5, 10 or 15 minutes. With time, the OD reading will again decline. Two hours after the peak OD reading, Bioxcell® extender and/or individually- produced egg yolk-based phosphate buffer extender equilibrated to 12°C is added to the semen, semen is cooled to 4°C - 10⁰C and semen is processed into straws or used immediately.

Example 9 - Gender bias from In Vitro Fertilization Studies

[0074] Semen was collected from an elite diary bull, KSY, and was either placed into a jacketed collection tube pre-warmed to 32°C, which was then placed and held at 12°C (i.e. control), or processed as described in Example 2. [0075] In vivo analysis of fixed time and assay-based artificial insemination of working herds was conducted. Based on fetal screening, the assay-based method gave higher gender bias than the fixed-time method. In contrast, during in vitro analysis of single fixed time and assay-based incubations, the fixed incubations gave higher gender bias than assay- based. In both cases, however, the percent female embryos is elevated above baseline by in vitro analysis. Table 5 presents the results of in vitro analysis.

Table 5 - Gender bias detected by in vitro fertilization of bovine eggs, followed by PCR- based gender analysis.

Claims

CLAIMSWhat is claimed is:

1. A method for optimizing semen comprising: a) obtaining a semen sample containing sperm; b) incubating the semen sample; c) taking at least one sperm sample from the semen sample; d) contacting the sperm sample with at least a first protein; e) identifying the number of sperm binding the protein; f) terminating incubation of the semen sample; and g) using or storing the semen sample.

2. The method of claim 1, wherein the incubation temperature is 0⁰C - body temperature.

3. The method of claim 1, wherein the incubation temperature is 0⁰C - ambient temperature.

4. The method of any one of claims 1-3, wherein the first protein is a polyclonal antibody.

5. The method of any one of claims 1-3, wherein the first protein is a monoclonal antibody.

6. The method of any one of claims 1-5, wherein the first protein is conjugated to a fluorophore and/or a monosaccharide or an oligosaccharide.

7. The method of claim 6, wherein the fluorophore is selected from the group consisting of carboxifluorescein acetate, phycoerythrin, calcein acetate, YO-PRO-I, SNARF-I, and combinations thereof.

8. The method of any one of claims 1-5, wherein the first protein is conjugated to an enzyme.

9. The method of any one of claims 1-8, wherein the first protein is selected from the group consisting of a late embryogenesis abundant (LEA) protein or a lectin.

10. The method of any one of claims 1-9, further comprising a second protein.

11. The method of claim 10, wherein the second antibody binds the first protein.

12. The method of claim 10 or 11, wherein the second protein is conjugated to a fluor.

13. The method of claim 10 or 11, wherein the second antibody is conjugated to an enzymatic substrate.

14. .The method according to any one of claims 10-14, wherein the second protein is a polyclonal antibody.

15. The method according to any one of claims 10-14, wherein the second protein is a monoclonal antibody.

16. The method according to any one of claims 1-15, wherein a sperm sample is taken every 5, 10, 15, 20, 30 or 60 minutes.

17. The method according to any one of claims 1-16, wherein the percent of sperm binding the first protein is determined for each sperm sample.

18. The method according to claim 17, wherein the incubation is terminated at a point after the percent of sperm binding the first protein in a subsequent sample is lower than the previous sample.

19. The method according to any one of claims 1-18, wherein the first protein is an anti- salmonella polyclonal antibody and the second protein is an antibody that recognizes IgG.

20. Use of the method of any one of claims 1-17 for increasing fertility in livestock.

1. Use of the method of any one of claims 1-17 for increasing the sex bias in livestock.