DESC:F O R M 2

THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]

1. TITLE OF THE INVENTION: BIOMOLECULES FOR DETECTION OF FERTILITY BIOMARKERS IN BOS INDICUS BULLS AND METHODS THEREOF

2. APPLICANT (A) NAME: ADIT BIOSCIENCE PVT. LTD.

(B) ADDRESS: ADIT BIOSCIENCE PVT. LTD, RM # 303, KIIT-TBI, CAMPUS 11, KIIT UNIVERSITY, BHUBANESWAR, ODISHA, PIN 751024

3. NATIONALITY (C) INDIA

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED

[001] TECHNICAL FIELD OF THE INVENTION
[002] The present invention is in the technical field of biomolecules for the detection of fertility biomarkers in Bos indicus bulls. More particularly, invention deals with detection of TIMP2 (Tissue Inhibitor of MetalloProteinase-2) in seminal plasma and in sperm of Bos indicus species, as a biomarker associated with semen fertility.
[003] BACKGROUND OF THE INVENTION
[004] Bulls are used either for natural services in dairy herd, or their semen is collected and cryo-preserved for later use for artificial insemination in the cows. Therefore, fertility screening of bulls is of high economic importance to the dairy farming. Bull breeding soundness evaluation (BSE) is performed for each bull and a performance index is assigned respectively. This essentially means reproductive performance of bulls or bull semen in getting the cow pregnant in single service is measured. Several factors such as physical traits, health, and genetics are also considered for the bull evaluation [1].
[005] However, the results are often unreliable despite all these examinations [2]. Thus, new method, which is based on presence of fertility-associated factors/biomarkers in the bull semen, offers a potentially practicable and reliable technique in screening high performing bulls. Bull semen contains various proteins, enzymes etc. that helps in the process of fertilization of spermatozoa with the ova. The liquid fraction of the semen, the seminal plasma contains these factors, which subsequently help sperms to undergo physiological modification, a necessary condition for fertilization [3, 4].
[006] Differential expression of heparin binding proteins in seminal plasma is positively correlated to fertility of the bull semen [5].
[007] During and after ejaculation, these proteins bind to sperms. Heparin capacitates sperm in-vitro by binding to the ligand proteins [6, 7]. Similarly, tissue inhibitor of metalloproteinase-2 (TIMP2), a heparin binding protein, is well-studied fertility-associated biomarker in Bos taurus [8, 9]. However, for Bos indicus there is no report of any such factors associated with sperm fertility.
[008] In this context, we confirmed presence of TIMP2 in seminal plasma and sperm from Bos indicus species, as a biomarker associated with semen fertility. Thus, proceeded with development of screening technology to assess presence of TIMP2 in Bos indicus semen as indicator for bull fertility screening.
[009] Thus, there is an urgent need in the art to develop biomolecules and methods for the detection of fertility biomarkers in Bos indicus bulls.
[010] In summary, there is a need for development of technology for detection of markers in seminal plasma and sperm from Bos indicus species, as a biomarker associated with semen fertility.
[011] SUMMARY OF THE INVENTION
[012] According to an exemplary aspect, the present invention discloses a method for the detection of fertility biomarkers in Bos indicus bulls.
[013] In an embodiment, invention deals with TIMP2 in seminal plasma and in sperm from Bos indicus species, as a biomarker associated with semen fertility.
[014] In further embodiment, invention relates to a screening technology to assess presence of TIMP2 in Bos indicus semen as an indicator for bull fertility screening.
[015] According to a further exemplary aspect of the present invention, exogenous addition of TIMP2 to bull semen could improve the fertile worthiness of sub-fertile bulls.
[016] In further embodiment, invention also mentions testing kits for development of point-of-care device for qualitative estimation of fertile worthiness of bull semen.
[017] In an embodiment, a method for evaluating the fertility of Bos indicus spermatozoa/semen, comprising: assessing in a sample of Bos indicus spermatozoa the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof; wherein said sperm fertility protein is selected from the group consisting of TIMP2 (Tissue inhibitor of metalloproteinase-2), fragment of TIMP2 and combinations thereof; and wherein said amount and/or activity is predicative of said fertility.
[018] In further embodiment, spermatozoa are Bos indicus bulls spermatozoa/semen, wherein the sperm fertility protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1- TIMP2 amino acid sequence (192 aa), SEQ ID NO: 2 -TIMP2 Domain-1 (109 aa) and SEQ ID NO: 3- TIMP-2 Domain-2 (83 aa).
[019] In an embodiment, a method of in vitro fertilization and/or artificial insemination comprising:
[020] (a) screening a sample of Bos indicus bulls spermatozoa/semen for assessing in sperm cells the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof, said sperm fertility protein is TIMP2; and
[021] (b) selecting for in vitro fertilization and/or artificial insemination a batch of sub population of spermatozoa of higher fertility potential based upon said screening.
[022] In an embodiment, a method for assigning a fertility index to a Bos indicus bulls semen sample comprising:
[023] (a) measuring in the semen sample the amount and/or activity of a sperm fertility protein, and/or of a fragment, isoform or homolog thereof, said sperm fertility protein is TIMP-2; and
[024] (b) assigning a fertility index to said semen sample based upon said measurement.
[025] In an embodiment, a kit for evaluating in a Bos indicus bulls sperm sample a fertility-related parameter, the kit comprising a user manual or instructions and kit components for measuring in the Bos indicus bulls sperm sample the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof, said sperm fertility protein is TIMP 2.
[026] The advantages and uses of this invention include but not limited to,
[027] Anti-TIMP2 antibodies can be used for bull fertility assessment in various assays such ELISA for quantitative estimation of TIMP2 in bull semen.
[028] Invention can be used for development of lateral flow assay-based point-of-care fertility testing kit.
[029] The recombinant antigen can be added exogenously to enhance sperm capacitation reaction and likely to improve conception rate in artificial insemination.
[030] This is especially important for liquid nitrogen storage of sperm or sexed sperm, where it is reported that the quality of frozen sperm is reduced compared to the fresh liquid semen.
[031] Exogenous addition of TIMP2 to bull semen could improve the fertile worthiness of sub-fertile bulls.
[032] The sequence homology between bovine and homosapien TIMP2 indicates similar applicability in other mammalian species too.
[033] Based on binding of sperms to TIMP-2 antibodies, quick FACS based assays could be developed for quantitative estimation of fertile worthiness of bull semen.
[034] BRIEF DESCRIPTION OF THE DRAWINGS
[035] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.
[036] FIG. 1 illustrates a schematic representation of the steps of the invention, according to the aspects of invention.
[037] FIG. 2 shows PCR amplification product of TIMP2, according to the aspects of invention.
[038] FIG. 3 shows schematic presentation of construct of plasmid, according to the aspects of invention.
[039] FIG. 4 shows expression of recombinant antigen (rTIMP2) confirmation, according to the aspects of invention.
[040] FIG. 5 shows western blot detection of TIMP2 in Bos indicus using polyclonal anti-TIMP2, according to the aspects of invention.
[041] FIG.6 illustrates shows Immunocytochemistry detection of TIMP2 in Red Sindhi sperms, (a) Phase contrast (b) DAPI and (c) anti- TIMP2, according to the aspects of invention.
[042] FIG. 7 shows the anti-P2 binding to the seminal plasma sample (S3) at ~23 kDa, according to the aspects of invention.
[043] FIG. 8 shows the Schematic presentation of fertility test kit, according to the aspects of invention.
[044] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
[045] DETAILED DESCRIPTION OF THE INVENTION
[046] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
[047] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
[048] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.
[049] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.
[050] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.
[051] Example embodiments of the present invention are described with reference to the accompanying figures.
[052] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
[053] Definitions
[054] The following terms are used as defined below throughout this application, unless otherwise indicated.
[055] "Marker" or "biomarker" are used interchangeably, and in the context of the present invention refer to a polypeptide, which is differentially present in a sample collected from Bos indicus bulls as compared to a comparable sample taken from control Murrah buffaloes.
[056] The phrase "differentially present" refers to differences in the quantity of the marker present in a sample taken from bulls as compared to a control subject. A biomarker can be differentially present in terms of frequency, quantity or both.
[057] "Diagnostic" means identifying a certain quantity of marker in a condition.
[058] The terms "detection", "detecting" and the like, may be used in the context of detecting markers or biomarkers.
[059] A "test amount" of a marker refers to an amount of a marker present in a sample being tested. A test amount can be either in absolute amount (e.g., µg/ml) or a relative amount (e.g., relative intensity of signals).
[060] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. "Polypeptide," "peptide" and "protein” can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
[061] "Detectable moiety" or a "label" refers to spectroscopic, photochemical, biochemical, immunochemical, or chemical means of detection of a composition. For example, labels may include 32P, 35S, fluorescent dyes, biotin-streptavidin, dioxigenin, haptens, electron-dense reagents and enzymes. The detectable moiety generates a measurable signal that can quantify the amount of bound detectable moiety in a sample. Quantitation of the signal is done by scintillation counting, densitometry, or flow cytometry.
[062] "Antibody” refers to a polypeptide ligand encoded by an immunoglobulin gene(s), which specifically binds and recognizes an epitope.
[063] The terms "subject", "patient" or "individual" generally refer to a human or mammals.
[064] "Sample" refers to a polynucleotides, antibodies fragments, polypeptides, peptides, genomic DNA, RNA, or cDNA, polypeptides, a cell, a tissue, and derivatives thereof may comprise a bodily fluid or a soluble cell preparation, or culture media, a chromosome, an organelle, or membrane isolated or extracted from a cell.
[065] EMBODIMENTS OF THE INVENTION:
[066] In an embodiment, a method for evaluating the fertility of Bos indicus spermatozoa/semen, comprising: assessing in a sample of Bos indicus spermatozoa the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof; wherein said sperm fertility protein is selected from the group consisting of TIMP2 (Tissue inhibitor of metalloproteinase-2), fragment of TIMP2 and combinations thereof; and wherein said amount and/or activity is predicative of said fertility.
[067] In further embodiment, spermatozoa are Bos indicus bulls spermatozoa/semen, wherein the sperm fertility protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1- TIMP2 amino acid sequence (192 aa), SEQ ID NO: 2 -TIMP2 Domain-1 (109 aa) and SEQ ID NO: 3- TIMP-2 Domain-2 (83 aa).
[068] In an embodiment, the method comprising the steps of: comparing said amount and/or activity of said sperm fertility protein, fragment, isoform or homolog thereof to one or more predetermined threshold values; and evaluating fertility of the Bos indicus bulls spermatozoa/semen based upon said comparison.
[069] In an embodiment, the protein is TIMP2, wherein a higher amount and/or activity in the sample compared to a predetermined threshold value is indicative of a higher fertility potential.
[070] In an embodiment, a method for evaluating the fertility of ejaculated bovine spermatozoa/semen, comprising assessing in a sample of bovine spermatozoa the amount of one or more sperm fertility protein, said sperm fertility protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1- TIMP2 amino acid sequence (P1; 192 aa), SEQ ID NO: 2 -TIMP2 Domain-1 (P2; 109 aa) and SEQ ID NO: 3- TIMP-2 Domain2 (83 aa).
[071] In an embodiment, a method of in vitro fertilization and/or artificial insemination comprising:
[072] (a) screening a sample of Bos indicus bulls spermatozoa/semen for assessing in sperm cells the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof, said sperm fertility protein is TIMP2; and
[073] (b) selecting for in vitro fertilization and/or artificial insemination a batch of sub population of spermatozoa of higher fertility potential based upon said screening.
[074] In an embodiment, a method for assigning a fertility index to a Bos indicus bulls semen sample comprising:
[075] (a) measuring in the semen sample the amount and/or activity of a sperm fertility protein, and/or of a fragment, isoform or homolog thereof, said sperm fertility protein is TIMP-2; and
[076] (b) assigning a fertility index to said semen sample based upon said measurement.
[077] In an embodiment, the said detection comprises LFA or ELISA or RIA or Western blot or any combination thereof.
[078] In an embodiment, the said ELISA is a sandwich ELISA comprising binding of a TIMP 2 to a first antibody preparation fixed to a substrate and a second antibody preparation labeled with an enzyme.
[079] In an embodiment, a kit for evaluating in a Bos indicus bulls sperm sample a fertility-related parameter, the kit comprising a user manual or instructions and kit components for measuring in the Bos indicus bulls sperm sample the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof, said sperm fertility protein is TIMP 2.
[080] In an embodiment, the kit as claimed in claim 10, wherein said kit components comprise:
[081] (a) specific antibodies directed against said sperm fertility protein, capture polyclonal and/or monoclonal anti-P2 and/or P3 antibody;
[082] (b) enzyme conjugated detection monoclonal anti-P3 antibody; and
[083] (c) control Bovine anti-IgG antibody; and/or
[084] (d) TIMP2 antigen standard (P1).
[085] The aim of the invention is to detect fertility biomarker in Bos indicus semen. Inventors chose TIMP2 among a dozen of reported fertility biomarkers in Bos taurus after meticulous analysis and experimentation. After bioinformatics-based data mining, inventors chose the conservative domain, and designed the sequence for gene synthesis. The synthetic gene was used as template to amplify three constructs (P1, P2 and P3). These three constructs were cloned in expression vector and used for recombinant protein production in bacterial/yeast expression host system. The protein was isolated using His-tag column and purified further using affinity column and size exclusion chromatography.
[086] The purified recombinant antigens were used for polyclonal/monoclonal antibody production. These antibodies can be used to detect TIMP2 expression in Bos indicus bull sperm and seminal plasma fluids through Western blot, mass spectroscopy, immunohistochemistry, lateral flow, FACS assay for qualitative detection, and ELISA for quantitative detection. Flow chart A and the FIG 1 illustrate a schematic representation of the steps of the invention for detection and expression of TIMP2, according to the aspects of invention.
[087]
[088] Flowchart A: Describes the method of invention
[089] Flowchart A
[090] The present invention relates to detection of fertility-associated biomarker in the seminal plasma and spermatozoa of Bos indicus using polyclonal antibody against TIMP2. The polyclonal and/or monoclonal antibody are used to develop point-of-care (PoC) diagnostic kits based on lateral flow assay technique to assess for bull fertility worthiness, and a more robust quantitative ELISA technology to qualitatively evaluate bull’s breeding soundness. Based on this invention, fertile worthiness of bull/bull semen could be screened for breeding purpose. This will significantly increase the much needed breeding efficiency of dairy herd, and hence the overall profitability of dairy farming sector. In addition to screening of TIMP2, inventor further document and confirm that this polyclonal antibody could be used to assess other reported fertility-associated factors from seminal plasma such as deoxyribonuclease1 like 3 (DNASE1L3) and osteopontin ?10, 12, 13?. Thus, making this antibody comprehensive for screening multiple fertility associated factors. Further, the recombinant protein/fertility biomarkers can be exogenously added to collected semen to enhance fertility of the cryo-preserved bull semen intended for use in Artificial Insemination (AI) ?8?. This approach could potentially enhance fertility and pregnancy rate of sub-fertile bulls of high production traits.
[091] Cloning of the target gene:
[092] For the purpose of recombinant protein expression in prokaryotic host and subsequent antibody generation inventor constructed three customized plasmid DNA suitable for the purpose.
[093] P1: Inventor first analyzed the amino acid sequences of TIMP-2 of bovine species. Based on the comparative analysis, the conserved domains of TIMP2 protein of Bos indicus was targeted for recombinant protein expression with suitable modification to primary amino acid sequences. Modification was done to get better protein expression levels in E.coli host. The codon optimized DNA sequence (CDS) of TIMP2 was synthesized commercially (GeneArt). In the customized construct, a couple of dozens of amino acid residues signifying a putative signal peptide in the N-terminal of TIMP2 was deleted to prevent accumulation of the de novo protein in bacterial inclusion bodies and further enhance recovery of expressed protein from bacterial cell lysates. The rest of the sequence was amplified by polymerase chain reaction using primers (5’ CGTGGATCCAGTCCCGTCCATCCACAAC3’) and (5’ CGTAAGCTTATGGATCTTCGATGTCCAAGAAC 3’). These primers were engineered with flanking BamHI and HindIII restriction sites respectively to facilitate cloning process in the expression vector. In this process inventor made ~190 aa long construct and the resultant protein was used for antibody production as well as kept as positive control for further use in developing ELISA.
[094] P2. Further truncated version of P1 containing 109 amino acid residues. By bionformatics tool, inventor learned that TIMP-2 structure has two domains. For antibody generation purpose, and subsequent use in ELISA plate development, inventor re-designed the TIMP-2 protein into two separate non-overlaping fragments. Inventor named it as Domain-1 and Domain-2. The TIMP-2 Domain-1 sequence was amplified using (5’ CGTGGATCCAGTCCCGTCCATCCACAAC3’) and 5’ CGTAAGCTTAGGACAACGTATCCCAAGGC3’). Both the sequences were cloned into pST50Trc1-STRHISNDHFR plasmid (Fig 2) under the Lac promoter.
[095] P3. In the third construct, which is also a truncated version of P1, inventor used rest of the protein primarily at C-terminal end to make a construct to be used in protien production as well as in generation of detection antibodies for ELISA.
[096] Recombinant protein production:
[097] The recombinant plasmid was further used for the protein expression. The recombinant protein was expressed in E.coli BL-21 (DE3) PlysS expression host. Briefly, the recombinant plasmid was transformed into “E.coli BL-21 (DE3) PlysS” and recombinant colonies were screening on ampicillin and chloramphenicol supplemented LB plates and later confirmed by DNA analysis and sequencing. Well-isolated colony was inoculated for protein expression at 37°C until the OD600 reached to 0.8. Bacterial culture was then induced with 0.2 mM IPTG at 18°C for 12 hrs.
[098] Following induction, the bacterial cells were harvested by centrifugation at 7,000 rpm for 5 min at room temperature. The bacterial cell pellet was resuspended in 1x P300 (50 mM phosphate buffer, 300 mM NaCl, pH7.0), flash-cooled in liquid nitrogen, and thawed at 30°C for 30 min followed by sonication on ice (70% amplitude for 3sec On/Off for 5 min). The whole sample was spun down at 18,000 rpm for 30 min at 4°C. Inventor confirmed the presence of target protein in the inclusion bodies (pellet) by SDS-PAGE and Western blot analysis. The protein was further purified under denatured conditions (8M urea) using affinity Ni-NTA beads followed by size exclusion chromatography. The isolated protein was extensively dialyzed in 1xPBS at 4°C.
[099] Custom antibody production of recombinant TIMP2
[0100] The purified recombinant TIMP2 (rTIMP2; P1) was used as antigen for polyclonal antibody production (Abgenex). Two healthy New Zealand white rabbits weighing around 1.5-2.0 kg were immunized with the antigen to raise the anti-sera. Before immunization process 5 ml of pre-immune sera were collected from each animal. About 200 µg of antigen was emulsified thoroughly with Complete Freund’s Adjuvant (CFA) (Sigma) at 1:1 ratio. The animals were injected subcutaneously with the emulsion (0.5 ml per rabbit), which contained 100 µg of immunogenic protein. The animals were booster injected subcutaneously on 7th, 14th, 21st and 28th day of immunization with 50 µg of immunogen emulsified with Incomplete Freund’s Adjuvant (Sigma) before collecting the first immune sera. The animals were bled individually on 42nd days. Subsequently the animals were bled two more time in 15 days interval with two boosters in alternative weeks. Necessary approval of Institute’s Animal Ethic Committee was in place for all experiments.
[0101] The collected blood was allowed to clot at room temperature for 2 h and then left at 4ºC over night. The blood samples were centrifuged at 4,500 rpm for 15 min and the individual serum was collected separately. The polyclonal antibodies were purified using protein A column and sodium azide (0.05%) was added as preservative. The immune sera were aliquoted and preserved at -20ºC for further use.
[0102] Custom antibody production: TIMP2 Domain 1
[0103] The purified TIMP2 Domain 1 (P2; 109 aa) was used as antigen for polyclonal antibody production (Abgenex). The rest of the procedure was as described for recombinant TIMP2.
[0104] Detection of TIMP2 expression in Bos Indicus
[0105] Western blot with anti-TIMP2 Domain 1 (anti-P2):
[0106] The purified antibody was tested for binding to seminal plasma samples from Bos Indicus through Western blot analysis. Briefly, 1 µg of recombinant TIMP2 (P1) and TIMP2 Domain-1 (P2) were used as control and seminal samples at 1:50 dilution in 1X PBS were run in SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad Trans-Blot Turbo Transfer). The membranes were incubated at 25oC for 1hr and at 4oC overnight in blocking buffer (5% skim milk in 1x TBS-T) followed by washing in 1X TBS-T (0.1% Tween 20).
[0107] The membranes were then incubated for 1hr at 25oC and 1hr at 4oC in anti-TIMP2 Domain-1 at 1:500 dilution in 1X TBS-T containing 1% skim milk. The membranes were washed, incubated in HRP conjugated anti-rabbit IgG (Bio-Rad) at 1:3000 dilutions as above for 1hr at 25oC. The membranes were then developed in 1ml ECL reagent mix with 1 sec exposure time (Bio-Rad). As a negative control for primary antibody, rabbit pre-bled serum was used at 1:5,000 dilution in PBS (data not shown).
[0108] Experimental design:
[0109] Quantitative Sandwich ELISA Kit:
[0110] Components:
[0111] Anti-TIMP2 microplate (polyclonal or monoclonal as capture antibody)
[0112] Lyophilized TIMP2 standard
[0113] Conjugated TIMP2 detection antibody (Mouse monoclonal)
[0114] Avidin-HRP conjugate (anti-mouse secondary antibody)
[0115] Standard & sample diluent (PBS, pH 7.4)
[0116] Antibody diluent
[0117] Conjugate diluent
[0118] Wash buffer
[0119] Stop solution (2 M H2SO4)
[0120] TMB substrate (3, 3’, 5, 5’-tetramethylbenzidine)
[0121] FACS-staining to detect presence of TIMP-2 on surface of sperms:
[0122] Flow cytometry is being extensively used to study human sperm quality in the areas of reproductive biology. Thus, in instant case the FACS experiments would determine presence of TIMP2 in bound form with sperm cells. Any standard FACS counter with ability to detect green or red flourescence emission would identify the same. For this testing, about 1 million sperm cells will be taken for the experiments. Presence of surface TIMP-2 indicates better fertlie worthy sperms. The fluorescence signals of the sperm samples need to be analyzed by any FACS digital flow cytometer (example BD Biosciences) by counting a minimum of 30,000 events at low acquisition rate (5000 /sec). Prior to the experiment, all alignment and calibration checks must be done and the instrument color optimization should be done with positive color beads supplied by the company.
[0123] Staining protocol:
[0124] Take 100 µl of cell sperm suspension to each tube containing 1 million cells
[0125] The blocking antibody step is required: Add 100 µl of Fc block to each sample (Fc block diluted in PBS buffer at 1:50 ratio).
[0126] Incubate the mixture on ice for 20 min.
[0127] Next, centrifuge at 500 g for 5 min at 4°C. Discard supernatant.
[0128] Add 0.1-10 µg/ml of the primary anti-TIMP-2 antibody.
[0129] Incubate the mixture for 30 min at room temperature or 4°C in the dark.
[0130] Wash the sperm cells 3 times by centrifugation 500 g for 5 min at 4°C
[0131] Add secondary anti-rabbit IgG (Example Rhodamine/ FITC) 1: 5000 ratio
[0132] Incubate the mixture for 30 min at room temperature or 4°C in the dark.
[0133] Wash the sperm cells 3 times by centrifugation 500 g for 5 min at 4°C
[0134] Finally resuspend them in 200 µl to 1ml of ice cold FACS buffer (PBS containing 1 % BSA buffer).
[0135] Fix the cells with 1% paraformaldehyde for 5 minutes in room temperature.
[0136] Keep the cells in the dark on ice or at 4°C in a fridge until the scheduled time for analysis (within 2 weeks, better to do immediately).
[0137] Analysis: for best results, analyze the cells on the flow cytometer as soon as possible. Interpretation: -ve control: No primary antibody
[0138] Compared to negative control, check the presence of TIMP-2 bound antibodies on surface of sperm cells by comparing FACS plot.
[0139] Note: The three recombinant gene constructs (P1, P2 and P3) might be further optimized to include MBP (maltose binding protein) tag in the N or C-terminal for higher yield and enhanced solubility.
[0140] Detection of TIMP2 expression in Bos Indicus:
[0141] Western blot analysis:
[0142] The purified antibody was tested for binding to seminal plasma samples from Bos Indicus through Western blot analysis. Briefly, 1 µg of recombinant TIMP2 (P1) as control and seminal samples at 1:50 dilution in 1X PBS were run in SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad Trans-Blot Turbo Transfer). The membranes were incubated at 25oC for 1hr and at 4oC overnight in blocking buffer (5% skim milk in 1x TBS-T) followed by washing in 1X TBS-T (0.1% Tween 20).
[0143] The membranes were then incubated for 1hr at 25oC and 1hr at 4oC in anti-TIMP2 at 1:10,000 dilution in 1X TBS-T containing 1% skim milk. The membranes were washed, incubated in HRP conjugated anti-rabbit IgG (Bio-Rad) at 1:3000 dilutions as above for 1hr at 25oC. The membranes were then developed in 1ml ECL reagent mix with 1 sec exposure time (Bio-Rad). As a negative control for primary antibody, rabbit pre-bled serum was used at 1:5,000 dilution in PBS.
[0144] Antibody pull down assay:
[0145] Inventor performed antibody-protein pull down assay to isolate TIMP2 from seminal plasma of Sahiwal bull samples. Briefly, 100µl of protein-A beads (Sigma) washed twice in PBS was incubated along with anti-TIMP2 antibody (78 µg) was added and constantly mixed in a rotatory mixer at 25oC overnight.
[0146] The bound protein-A-anti-TIMP2 antibody complex was washed thrice as above. Seminal plasma sample (30 µl) was added and incubated with constant mixing at 25oC for 5 hr. The mixture was washed as above and antibody-antigen bound fraction was eluted in glycine buffer (pH 3.0). The eluted fraction was run in SDS-PAGE, and corresponding gel bands were tested for presence of TIMP2 by mass spec analysis (Qexactive nano-ESI LC MS/MS) by comparing with the bovine gene database.
[0147] Immunocytochemistry of frozen spermatozoa:
[0148] The antibody was further tested using immunohistochemistry method for binding to the Red Sindhi sperm cells retrieved from commercially available artificial insemination straws. The cells were washed twice in PBS, centrifuged at 300g for 5min each at 25oC. One million cells were pipetted onto pre-defined contour on positively charged slide (PathnSitu), and kept at 37oC for 1hr for adsorption and immobilization to the glass slide. The slides were washed in PBS, and fixed using 1% PFA for 10 min at 25oC. After washing, 100 ul of blocking solution (5% BSA) was added, and kept at 4oC overnight. The slides were incubated in anti-TIMP2 at 1:100 for 1 hour in 1% BSA in 1X PBS-Tween-20 (PBS-T, 0.1%) buffer. The slides were then washed in PBS-T and incubated in rhodamine-conjugated anti-rabbit IgG (Life Technologies) at 1:500 for 1hr at 25oC. After washing, the slides were incubated with DAPI (Life Technologies) at 30 nM for 5 min at 25oC. The slides were washed, air dried, mounting media was added and sealed with coverslips. The slides were observed under fluorescent microscope (Olympus).
[0149] Recombinant protein production:
[0150] The custom synthesized TIMP2 gene was PCR amplified (Fig 2). Prior to this, the coding sequences were codon optimized to suit bacterial host and cloned in pST50STRHISNDHFR vector using BamHI and HindIII restriction sites (Fig 3). The recombinant protein was expressed in bacteria and isolated using His-tag column (Fig 4). Protein was further purified using dialysis.
[0151] Western blot analysis of seminal plasma samples:
[0152] Polyclonal antibody was raised in female rabbit against the recombinant TIMP2 (P1) and antibodies were purified by protein-A affinity column. Seminal plasma samples from Sahiwal (Sw) bulls (n= 7) and Murrah (Mu) buffalo (n =10) were tested in Western blot for presence of TIMP2 against rabbit polyclonal anti-Timp2. All the Sahiwal samples showed binding at 32 kDa and ~23 kDa (Fig 5 a). The rTIMP2 has band ~27 kDa. However, buffalo semen samples did not show any binding (Fig 5 b).
[0153] Antibody pull-down assay:
[0154] Further, by using these antibodies inventor did a pull-down assay where Protein-A agarose beads were used as bait to isolate bound antigens. The antibody bound fractions were separated by SDS-PAGE and respective bands of expected protein were cut from the gel in the form of gel bands. The gel bands were run in Qexactive nano-ESI LC MS/MS and pulled down proteins were analyzed and matched agianst bovine gene database. The sequence data confirmed the presence of TIMP2 (24.4 kda) in the bound fraction along with other reported fertility factors such as deoxyribonuclease 1 Like 3 (DNASE1L3; MW 35.1 kDa) and osteopontin (30.9 kda).
[0155] Immunocytochemistry:
[0156] Inventor further tested the eifficacy of antibody in immunocytochemistry for its ability to bind to the spermatozoa. We used frozen Red Sindhi sperm cells for the analysis. The antibody bound majorly in the post acrosomal region of sperm. In some cases it bound to centriol, midpiece and tail (Fig 6). This shows that heparin binding fertility markers, which are abundant in seminal plasma, bind to spermatozoa after ejaculation.
[0157] Result and analysis:
[0158] Figure 2 shows PCR amplification product of TIMP2. The customized TIMP2 CDS was PCR amplified using a 50 ?l reaction mix. Briefly, 10 ng of template, and 0.5 ?M of primers (5’ CGTGGATCCAGTCCCGTCCATCCACAAC3’) and (5’ CGTAAGCTTATGGATCTTCGATGTCCAAGAAC 3’) respectively along with 200 ?M dNTP and 0.05 units/µL Taq DNA polymerase. The reaction condition setup: denature at 95oC for 1 min, annealing at 55oC for 2 min and extension at 72oC for 3 min respectively. The amplified DNA was loaded onto 0.8% agarose gel electrophoresis prestained with ethidium bromide stain. Star denotes the amplification of right size TIMP2 product. Figure 3 shows Schematic presentation of construct of plasmid. Figure 4 Expression of recombinant antigen (rTIMP2) confirmation.
[0159] The amplified PCR product (TIMP2) was cloned and protein expression was done in respective bacterial host. The resultant was loaded to 12% SDS gel and run under 80V for 4 hours at room temperature. Following this, the gel was fixed with acetic acid and stained with commassie blue to vizualize product. The left lane represents marker lane while the right lane represents the expected TIMP2 product.
[0160] Figure 5 Western blot detection of TIMP2 in Bos Indicus using polyclonal anti-TIMP2.
[0161] The polyclonal antibody against rTIMP2 was tested in Western blot analysis: a) Sahiwal seminal plasma samples, and b) Murrah seminal plasma. Briefly, 1 µg of rTIMP2 (positive control) and seminal plasma samples at 1:50 dilution in 1X PBS were run in SDS-PAGE and transferred to nitrocellulose. After blocking overnight in 5% skim milk at 4oC, the membrane was incubated in primary (anti-TIMP2) at 1:10,000 dilution for 1 hour at 25oC followed by secondary antibody incubation (HRP conjugated anti-rabbit IgG) at 1:3000 dilution as above. In panel a, lane 1 represent pre-stained protein ladder. Lane 2,3,4,6,7,8,9 represent samples collected from Sahiwal bulls. While lane 5 represent recombinant TIMP-2 (P1). In panel b, lane 1 represent protein maker, lane 2 represent rTIMP-2 while rest lanes show activity of seminal plasma collected from Murrah buffalo. Figure 6 shows Immunocytochemistry detection of TIMP2 in Red Sindhi sperms.
[0162] The anti-TIMP2 antibody was further tested in immunocytochemistry for binding to the Red Sindhi sperms retrieved from commercially available artificial insemination straw. After thawing and washing, one million cells were pipetted onto predefined contour on positively charged slide for binding at 37oC for 1 hour. The cells were then fixed in 1% PFA for 10 min and treated with 5% BSA as blocking solution overnight at 4oC. Primary antibody (anti-TIMP2) at 1:100 dilutions for 1 hour followed by rhodamine-conjugated anti-rabbit IgG (1:500) for 1 hour, and DAPI (30 nM) for 5 min at 25oC was added to the slides. In panel C red color represent antibody bound to acrosomal region, centriol, midpiece and tail of sperms showing presence of TIMP-2 in Red Sindhi spermatozoa.
[0163] Result of Anti TIMP2 Domain I expression:
[0164] Polyclonal antibody was raised in female rabbit against the recombinant TIMP2 Domain-1 (P2) and antibodies were purified by protein-A affinity column. Seminal plasma sample from Sahiwal (Sw) bull (n= 1) was tested in Western blot for presence of TIMP2 against rabbit polyclonal anti-TIMP2 Domain-1. The Sahiwal sample, which was tested in antibody pull down assay and mass spec analysis, was used in this experiment. The anti-P2 showed binding to the seminal plasma sample (S3) at ~23 kDa. The antigens have bands at ~ 27 kDa and ~17 kDa for P1 and P2 respectively (Fig 7).
[0165] Kit development procedure:
[0166] The flat and clear bottom 96 well immunosorbent microtiter plates (polypropylene or polystyrene surface) will be used for coating the capture antibody (anti-P2 or P3 polyclonal or monoclonal). The concentration of capture antibody will be optimized between 1-10 ug/ml in 0.2 M carbonate/bicarbonate buffer (pH 9.6). 100 ul of capture antibody will be added to the wells and incubated 2 hours at room temperature followed by overnight at 4oC. The plates will be washed 3 times for 5 mins each in 200 ?l of 1XPBS with 0.05% Tween-20 (pH 7.4) using a shaking platform. The plates will be patted dry, sealed and kept at 4oC until use.
[0167] The unbound sites on the plate will be blocked with 300 ?l of 5% BSA per well. The plates will be incubated for 1-2 hours at room temperature or overnight at 4oC followed by washing as before.
[0168] Serial dilution of TIMP2 standards and seminal plasma samples will be prepared in appropriate diluent respectively. Another set of standard dilutions will be made in diluent spiked with equivalent matrix as seminal plasma, which could be either heparin binding protein depleted bovine seminal plasma or bovine serum albumin equal to the total protein content of seminal plasma samples or blocking buffer. This is required for any background correction that might be generated due to other components in the seminal plasma. Physiological concentration of TIMP2 in bovine seminal plasma ranges between 0.9 – 2 mg/ml [14]. So, the standard curve will be generated from 0 – 1000 ng/ml of TIMP2. The samples will be diluted in PBS buffer only. All the standards and blank will be made in triplicate, whereas samples will be made in duplicate. 100 ?l of standard and samples will be added to each well for incubation at 4 to 37oC for 1-2 hours. The wells will be washed 4 times as before.
[0169] Detection antibody (P2 or P3) will be prepared at 0.5 – 5 ug/ml with appropriate buffer (PBS). 100 ul of diluted antibody will be added to each well and incubated for 1-2 hours at 4 to 37oC. The wells will be washed 4 times as before. Diluted secondary antibody (20-200 ng/ml) containing 1% BSA will be added to all wells. It will be incubated for 1-2 hours at room temperature followed by washing as before. 100 ul of TMB will be added to all the wells and incubated for 15-30 min. Equal volume of stop solution will be added and read the optical density at 450 nm.
[0170] The data generated will be analysed using appropriate curve fitting software. Generally, either a linear or semi-log plot is used where concentrations are plotted on X-axis versus the absorbance on the Y-axis. The concentration of TIMP2 in samples can be interpolated using straight-line equation.
[0171] The kit will be optimized and validated for analysing seminal samples from Sahiwal and Red Sindhi (inclusive but not limited to).
[0172] Lateral flow assay kit:
[0173] Fig 8 shows the schematic presentation of fertility test kit. The Zone I is Reaction zone with Detachable Ab and Capture Ab, Zone II is Test zone with Ab firmly attached Nano particle conjugated and The Zone III is Control
[0174] Zone with Detachable Ab and Capture Ab, Ab firmly attached Anti-IgG (bovine).
[0175] Components:
[0176] Capture antibody (polyclonal or monoclonal anti-P2 or P3)
[0177] Enzyme conjugated detection antibody (monoclonal anti-P3)
[0178] Control antibody (Bovine anti-IgG)
[0179] Development procedure sketch:
[0180] Sandwich enzyme immunoassay method will be used to develop the LFA kit. The design outlay of the strip is according to the proven concept. Briefly, the sample will be applied to the sample pad. Since the aim is to use this kit at the farm site, the sample after ejaculation is highly viscous, and thus prior processing might be needed. The collected sample must be allowed to stand for 1 hour on ice. The supernatant (seminal plasma) will be collected and diluted further (10 to 100 fold) with a buffer (PBS pH 7.4). Once the sample is applied, it will travel through three zones of reaction. The zone I contain the capture antibody, which will detach after binding to the target analyte (TIMP2) as the sample flows through. Once the sample reaches the zone II, the TIMP2-capture antibody complex will bind to the nano particle conjugated detection antibody. This will release a colour band. The sample will further flow to the zone III, which is the assay control. Nano particle conjugated or bovine anti-IgG will bind to the IgG in the sample and release a different colour.
[0181] The assay optimization parameters will include material for sample pad, conjugate, membrane, absorbent pad, buffering agents, detergents, blocking agents, stabilizers, type of nano particle etc [15].
[0182] SEQUENCE OF TIMP2
[0183] TIMP2 CDS (579 bp)
[0184] ATGAGTCCCGTCCATCCACAACAAGCCTTTTGTAACGCAGACATAGTCATCAGAGCCAAGGCTGTGAACAAGAAGGAAGTTGACTCCGGTAATGATATTTACGGTAATCCTATTAAGAGAATCCAGTACGAAATAAAGCAAATTAAAATGTTCAAAGGACCAGACCAAGATATCGAACTTATATACACTGCTCCATCCTCCGCTGTGTGTGGCGTGAGTTTAGACATCGGTGGCAAAAAGGAGTATTTGATAGCTGGTAAAGCAGAGGGCAACGGCAACATGCATATTACATTGTGTGATTTCATTGTGCCTTGGGATACGTTGTCCGCTACTCAAAAAAAATCATTAAACCATAGATATCAGATGGGCTGCGAATGCAAAATAACGAGGTGCCCAATGATCCCTTGCTACATCTCTAGTCCTGATGAATGTCTATGGATGGATTGGGTTACTGAAAAGAACATAAACGGACACCAAGCAAAATTCTTTGCCTGTATAAAAAGATCCGACGGTTCATGTGCTTGGTACAGAGGTGCAGCTCCTCCTAAGCAGGAGTTCTTGGACATCGAAGATCCATAA
[0185] Primers
[0186] For construct P1
[0187] (5’CGTGGATCCAGTCCCGTCCATCCACAAC3’)
[0188] (5’ CGTAAGCTTATGGATCTTCGATGTCCAAGAAC3’)
[0189] For Construct P2
[0190] (5’CGTGGATCCAGTCCCGTCCATCCACAAC3’)
[0191] (5’CGTAAGCTTAGGACAACGTATCCCAAGGC3’)
[0192] SEQ ID NO: 1 - TIMP2 amino acid sequence (192 aa)
[0193] MSPVHPQQAFCNADIVIRAKAVNKKEVDSGNDIYGNPIKRIQYEIKQIKM
[0194] FKGPDQDIELIYTAPSSAVCGVSLDIGGKKEYLIAGKAEGNGNMHITLCD
[0195] FIVPWDTLSATQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWV
[0196] TEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP
[0197] SEQ ID NO: 2 - TIMP2 Domain-1 (109 aa)
MSPVHPQQAFCNADIVIRAKAVNKKEVDSGNDIYGNPIKRIQYEIKQIKM
FKGPDQDIELIYTAPSSAVCGVSLDIGGKKEYLIAGKAEGNGNMHITLCD
FIVPWDTLS
[0198] SEQ ID NO: 3- TIMP-2 DOMAIN1 (83 AA)
[0199] ATQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWV
[0200] TEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP
[0201] Advantages of the invention:
[0202] TIMP2 is detected for the first time in Bos indicus.
[0203] Based on putative structural information, we designed truncated protein (P1) suitable for accurate detection of TIMP-2 in immunohistochemistry, Western blot and ELISA assays.
[0204] The polyclonal antibody against TIMP2 also detected other reported fertility-associated proteins such as DNASE1L3 and osteopontin in Bos indicus.
[0205] The recombinant construct is designed to enhance protein expression and prevent the accumulation of the protein in the inclusion bodies.
[0206] Best mode to use the invention:
[0207] Lateral flow assay: for point-of-care bull fertility screening: Qualitative.
[0208] ELISA: For quantitative determination of the fertility marker.
[0209] Fertility enhancement: Exogenously TIMP2 supplementation can enhance the fertility of frozen semen straw for AI.
[0210] FACS staining could be developed using these antibodies
[0211] The possible uses of this invention include but not limited to:
[0212] Anti-TIMP2 antibodies can be used for bull fertility assessment in various assays such ELISA for quantitative estimation of TIMP2 in bull semen.
[0213] Can be used for development of lateral flow assay-based point-of-care fertility testing kit.
[0214] The recombinant antigen can be added exogenously to enhance sperm capacitation reaction and likely to improve conception rate in AI.
[0215] This is especially important for liquid nitrogen storage of sperm or sexed sperm, where it is reported that the quality of frozen sperm is reduced compared to the fresh liquid semen.
[0216] Exogenous addition of TIMP2 to bull semen could improve the fertile worthiness of sub-fertile bulls.
[0217] The sequence homology between bovine and Homo sapien TIMP2 indicates similar applicability in other mammalian species too.
[0218] Based on binding of sperms to TIMP-2 antibodies, quick FACS based assays could be developed for quantitative estimation of fertile worthiness of bull semen.
[0219] According to a non limiting exemplary aspect of the present invention, the markers can be used for the development of kits that enable sample collection, processing and marker detection. These diagnostic kits developed can then be utilized by breeders/veterinary hospitals/private clinics/ veterinary doctors.
[0220] Merely for illustration, only representative number/type of graph, chart, block and sub- block diagrams were shown. Many environments often contain many more block and sub- block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.
[0221] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.
[0222] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[0223] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.

[000] References
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2) Larson, J. L., & Miller, D. J. (2000). Can relative spermatozoal galactosyltransferase activity be predictive of dairy bull fertility? Journal of Dairy Science, 83(11), 2473–2479. https://doi.org/10.3168/jds.S0022-0302(00)75139-3
3) Karunakaran, M., & Devanathan, T. G. (2017). Evaluation of bull semen for fertility-associated protein, in vitro characters and fertility. Journal of Applied Animal Research, 45(1), 136–144. https://doi.org/10.1080/09712119.2015.1129343
4) Killian, G. J., Chapman, D. A., & Rogowski, L. A. (1993). Fertility-Associated Proteins in Holstein Bull Seminal Plasma1. Biology of Reproduction, 49(6), 1202–1207. https://doi.org/10.1095/biolreprod49.6.1202
5) Singh, A. K., Brar, P. S., & Cheema, R. S. (2016). Heparin binding proteins and their relationship with vital sperm function tests vis-à-vis fertility of buffalo bull semen. Asian Pacific Journal of Reproduction, 5(5), 419–427. https://doi.org/10.1016/j.apjr.2016.07.003
6) Miller, D. J., Winer, M. A., & Ax, R. L. (1990). Heparin-Binding Proteins from Seminal Plasma Bind to Bovine Spermatozoa and Modulate Capacitation by Heparin1. Biology of Reproduction, 42(5–6), 899–915. https://doi.org/10.1095/biolreprod42.6.899
7) Nass, S. J., Miller, D. J., Winer, M. A., & Ax, R. L. (1990). Male accessory sex glands produce heparin-binding proteins that bind to cauda epididymal spermatozoa and are testosterone dependent. Molecular Reproduction and Development, 25(3), 237–246. https://doi.org/10.1002/mrd.1080250305
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9) McCauley, T. C., Zhang, H. M., Bellin, M. E., & Ax, R. L. (2001). Identification of a heparin-binding protein in bovine seminal fluid as tissue inhibitor of metalloproteinases-2. Molecular Reproduction and Development, 58(3), 336–341. https://doi.org/10.1002/1098-2795(200103)58:3<336::AID-MRD12>3.0.CO;2-Z
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13) Erikson, D. W., Way, A. L., Chapman, D. A., & Killian, G. J. (2007). Detection of osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis homogenates, and its potential role in bovine fertilization. Reproduction, 133, 909-917. DOI: 10.1530/REP-06-0228.
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,CLAIMS:I/We Claim,
1) A method for evaluating the fertility of Bos indicus spermatozoa/semen, comprising: assessing in a sample of Bos indicus spermatozoa the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof; wherein said sperm fertility protein is selected from the group consisting of TIMP2 (Tissue inhibitor of metalloproteinase-2), fragment of TIMP2 and combinations thereof; and wherein said amount and/or activity is predicative of said fertility.
2) The method as claimed in claim 1, wherein said spermatozoa are Bos indicus bulls spermatozoa/semen, wherein the sperm fertility protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1- TIMP2 amino acid sequence (192 aa), SEQ ID NO: 2 -TIMP2 Domain-1 (109 aa) and SEQ ID NO: 3- TIMP-2 Domain-2 (83 aa).
3) The method as claimed in claims 1 to 2, wherein the method comprising the steps of: comparing said amount and/or activity of said sperm fertility protein, fragment, isoform or homolog thereof to one or more predetermined threshold values; and evaluating fertility of the Bos indicus bulls spermatozoa/semen based upon said comparison.
4) The method as claimed in claim 3, wherein the protein is TIMP2, wherein a higher amount and/or activity in the sample compared to a predetermined threshold value is indicative of a higher fertility potential.
5) A method for evaluating the fertility of ejaculated bovine spermatozoa/semen, comprising assessing in a sample of bovine spermatozoa the amount of one or more sperm fertility protein, said sperm fertility protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1- TIMP2 amino acid sequence (P1; 192 aa), SEQ ID NO: 2 -TIMP2 Domain-1 (P2; 109 aa) and SEQ ID NO: 3- TIMP-2 Domain2 (P3; 83 aa).
6) A method of in vitro fertilization and/or artificial insemination comprising:
(a) screening a sample of Bos indicus bulls spermatozoa/semen for assessing in sperm cells the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof, said sperm fertility protein is TIMP2; and
(b) selecting for in vitro fertilization and/or artificial insemination a batch of sub population of spermatozoa of higher fertility potential based upon said screening.
7) A method for assigning a fertility index to a Bos indicus bulls semen sample comprising:
(a) measuring in the semen sample the amount and/or activity of a sperm fertility protein, and/or of a fragment, isoform or homolog thereof, said sperm fertility protein is TIMP-2; and
(b) assigning a fertility index to said semen sample based upon said measurement.
8) The method as claimed in claim 7, wherein said detection comprises LFA or ELISA or RIA or Western blot or any combination thereof.
9) The method as claimed in claim 8, wherein said ELISA is a sandwich ELISA comprising binding of a TIMP 2 to a first antibody preparation fixed to a substrate and a second antibody preparation labeled with an enzyme.
10) A kit for evaluating in a Bos indicus bulls sperm sample a fertility-related parameter, the kit comprising a user manual or instructions and kit components for measuring in the Bos indicus bulls sperm sample the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof, said sperm fertility protein is TIMP 2.
11) The kit as claimed in claim 10, wherein said kit components comprise:
(a) specific antibodies directed against said sperm fertility protein, capture polyclonal and/or monoclonal anti-P2 and/or P3 antibody;
(b) enzyme conjugated detection monoclonal anti-P3 antibody; and
(c) control Bovine anti-IgG antibody; and/or
(d) TIMP2 antigen standard (P1).

Dated this 14th day of April 2021

Signature:
(Lipika Sahoo)
Registration Number: IN/PA 2467