Colorimetric urine test for early pregnancy detection in livestocks

Abstract

The present invention provides an optical density based non-invasive, field usable method for the detection of pregnancy in healthy ruminant livestock using naturally micturated urine. The present invention also provides a colorimetric kit for detection of pregnancy in ruminants livestock. The test also helps in differentiating between pregnant and non-pregnant ruminant females using urine samples.

Description

TITLE: COLORIMETRIC URINE TEST FOR EARLY PREGNANCY DETECTION IN LIVESTOCKRELATED APPLICATIONS OF INVENTION

[0001] The present application claims priority to India Provisional Patent Application No. IN202411050036 filed on June 29, 2024.FIELD OF INVENTION

[0002] The present invention relates to the field of animal biochemistry. The present invention provides a method for pregnancy detection in ruminant livestock animal through the detection of a specific metabolites in urine. The invention also provides kits and chemicals useful for the detection of early pregnancy in livestock. More specifically, the present invention relates to reagent composition for pregnancy estimation by colorimetry and a test kit assembly and its method of use.BACKGROUND OF THE INVENTION

[0003] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Livestock play a pivotal role in the socio-economic and cultural fabric of rural and agricultural communities worldwide, particularly in countries like India, where they are integral to the livelihood of millions. Livestock contribute to essential resources such as meat, milk, and draught power, forming the backbone of various agricultural ecosystems. These ecosystems range from traditional grazing systems to semi-intensive (mix of grazing and supplemental feed) and intensive farming setups (controlled and specialised diet for commercial demands), with livestock serving as a vital resource for economic sustenance, food security, and labor efficiency.

[0005] Early detection of pregnancy in livestock is a cornerstone of reproductive management and has far-reaching implications for productivity and profitability in the dairy and agricultural sectors. Timely identification of pregnancy enables farmers to optimize breeding programs, ensuring the early rebreeding of non-pregnant animals, better management of pregnant animals, and increased revenues through enhanced milk production. Furthermore, it facilitates timely decision-making for animals whose ovarian activity may resume late after parturition, thereby improving reproductive efficiency and reducing economic losses associated with reproductive disorders or missed breeding opportunities.

[0006] Traditional methods for pregnancy detection, such as rectal palpation and ultrasonography, have limitations in terms of timing, accessibility, and practicality in field conditions. Rectal palpation, a commonly used method, can only confirm pregnancy after approximately 60 days of gestation. On the other hand, ultrasonography, while more advanced, requires specialized equipment and trained personnel and is typically conducted approx. 30 days post-conception. These limitations underscore the need for alternative solutions that are both accessible and effective in the early stages of gestation. Reproductive management, assisted reproductive technologies (ART) such as artificial insemination are increasingly being used for both livestock. The challenge of distinguishing pregnant from non-pregnant animals shortly after insemination is difficult. This delay in pregnancy detection can lead to significant economic losses, as open (non-pregnant) animals are often not identified and re-inseminated promptly, resulting in extended calving intervals, reduced milk production, and suboptimal use of resources.

[0007] Mohita Rai et al (2018) found in their study that a high Abscisic Acid concentration of 170.62 nanomoles / ml was found in pregnant cows compared to 74.46 nanomoles / ml in non- pregnant cows. The authors also hypothesised that the presence of ABA could be one of the factors reducing seed germination. However, this is a crude method that requires seed germination (4-7 days) and therefore cannot be used for the immediate detection of pregnancy in dairy cows. In addition, seed germination is influenced by temperature, humidity and seasonal variations.

[0008] Bethapudiet al. (2015) published a further extension of the above method using a simple, non-invasive technique developed based on ancient Egyptian knowledge called the Punyakoti test (Veena, 1997) to detect early pregnancy at day 26 post-fertilisation. Significant inhibition of seed germination after 48 hours and shoot length after 5 days was used as a criterion for determining true pregnancy. Pregnancy was confirmed by rectal palpation 45-60 days after fertilisation. However, this is also a crude method that requires seed germination (5 days) and therefore cannot be used for immediate confirmation of pregnancy in dairy cows.

[0009] Mellin et al (1965) have described a method for the chemical detection of oestrogen in bovine urine. Specifically, this method uses internal 14C standards, a combination of enzyme and acid hydrolysis, paper chromatography and fluorimetry for the determination of four oestrogens in bovine pregnancy urine. In comparison to this study, the present invention provides a kit that demonstrates a field test using a colour reaction that does not require any instrumentation such as chromatography and fluorimetry.

[0010] US 7604950 discloses a method for determining the pregnancy status of a cow by detecting a pregnancy-associated glycoprotein (PAG) comprising proteins present in an acidic protein fraction of bovine placental tissue from day 55 to day 60. This method may involve the use of novel polyclonal and monoclonal antibodies produced using the acidic protein fraction as an immunogen. According to this method, the cow is pregnant if the PAG level is elevated in a sample obtained from the cow. However, this technique requires the isolation of blood samples, which requires experienced veterinarians and therefore cannot be readily applied by dairy farmers who do not have these skills.

[0011] US 10288625 provides compositions and methods for determining the pregnancy status of an animal. The compositions and methods utilise interferon tau (IFNT) and / or antibodies specific for IFNT. This method detects the presence of IFNT in samples obtained from animals as an early indicator of pregnancy. However, the main disadvantage of this method is that it is a laboratory method requiring sophisticated instrumentation and laboratory facilities to analyse the results.

[0012] US 2003 / 0059951 describes a method for producing antibodies against analytes present in body fluids such as blood or blood fractions, saliva, milk, etc. from pregnant animals and for removing antibodies that are reactive with substances in non-pregnant animals. The selected antibodies are then used to prepare tests for the detection of pregnancy in female animals. However, the present method is based on the colour reaction of metabolites in urine and is not an immunoassay requiring sophisticated instrumentation and laboratory facilities for interpretation of the results.

[0013] US.2003 / 0073248 describes three different groups of antigens (antigen groups A, B and C) that are present in whole blood during pregnancy in cattle. Some of these antigens are also present in other fluids such as serum, milk and urine. Group A is a combination of antigens that are most present around days 15-30 after conception. Group B is a combination of antigens that are generally present after day 25 and generally remain at detectable levels throughout pregnancy. Group C antigens are present throughout the pregnancy. This method involves testing a fluid derived from the animal for the presence of at least one Group A or Group B antigen and testing the fluid for the presence of at least one Group C antigen; determining that the animal is pregnant if the test is positive for at least one Group A or Group B antigen and at least one Group C antigen; and determining that the animal is not pregnant if the test is negative for at least one Group C antigen. However, this method is a laboratory-based method requiring sophisticated instruments and laboratory facilities for interpretation of the results, in contrast to the kit and method of the present invention, which is a reHable cow-based method for determiningpregnancy in ruminants based on the colour reaction of urine metabolites and is not an immunoassay.

[0014] US2003 / 0224452 provides a method for detecting pregnancy in a female ungulate or other female ruminant, comprising the following: Obtaining a sample from the animal and detecting the presence of a selected IFN-induced protein, wherein the presence of the selected IFN-induced protein indicates that the animal is pregnant. The tests in this document are useful for analysing cells, blood, plasma, serum, cells, milk, nasal secretions, ocular secretions, vaginal secretions, urine and saliva samples. The tests described in this document are immunoassays such as lateral flow and sandwich assays. However, the method and kit of the present invention is based on the colour reaction of metabolites in urine and is not an immunoassay and does not require sophisticated laboratory equipment. Therefore, the present invention provides a method that is rapid, non-invasive and accurate in detecting pregnancy.

[0015] US2008 / 0026384 discloses compositions and methods for detecting pregnancy in ruminants, preferably cattle. A number of nucleic acid sequences that exhibit differential expression in response to pregnancy, and proteins encoded by them, are used in a variety of tests as pregnancy- specific markers to rapidly and efficiently discriminate between pregnant and nonpregnant animals. These markers include OAS1, LOC33877, bone marrow stromal cell antigen, etc. However, the method of the present invention is completely different as it is simple, fast and straightforward.

[0016] W02004 / 059282 discloses a method for the early detection of pregnancy in farm animals such as ungulates (e.g. laminitis) and in particular in ruminants such as cows, sheep and goats. The method is based on measuring the level of cross -reactive ubiquitin protein (UCRP) [also known as interferon- stimulated gene product- 17 in cattle (ISG17)] and progesterone, whereby an elevated level of ISG17 and progesterone indicates that the cow is pregnant. This method requires extensive laboratory equipment and is therefore not easy to use.

[0017] InstavetBiopharm Pvt Ltd. discloses a “visual kit for visual diagnosis of pregnancy in livestock and method for its detection” which detects the level of abscisic acid (ABA) in the urine samples. A red colour in the presence of abscisic acid of about 10 pg / ml and more is an indication of pregnancy and a yellowish- orange colour with a lower amount of abscisic acid is an indication of non-pregnancy, which is then matched with the standard colour bands provided. In the above method, an acid is used as the acidifying agent, which can be selected from HC1, H2SO4, HNO3, phosphoric acid, acetic acid, trichloroacetic acid, etc. in the standard range of 0.1 to 36.8 N and p-hydroxybenzaldehyde (PHB) sulphuric acid (H2SO4) (30 to 36.8N). The above method uses concentrated sulphuric acid, which is not user-friendly.

[0018] The UbioquickVET Bovine Pregnancy Rapid Test is a semi-quantitative immunochromatographic test for the detection of pregnancy-associated bovine glycoprotein in bovine serum, plasma or whole blood. UbioquickVET Bovine Pregnancy is intended for initial screening only and reactive samples should be confirmed by an additional test such as ELISA. In addition, pregnancy can be confirmed using a pregnancy-associated glycoprotein- 1 (PAG-1) test with 94% accuracy between 30- and 35-days post insemination.

[0019] Biolreprod0083 is a highly sensitive and accurate double antibody RIA method for the detection of a pregnancy- associated bovine glycoprotein (bPAG). The detection limit of the assay was set at 0.2 ng / ml. It was confirmed to be specific for bPAG as it does not cross-react with pituitary and placental gonadotropic hormones or other placental or serum proteins. These results suggest that the identification of this placenta- specific antigen in serum could serve as an accurate serological approach for the early detection of pregnancy in cattle from 28 days postbreeding.

[0020] The IDEXX Milk Pregnancy Test is an enzyme-linked immunosorbent assay for the detection of pregnancy-associated glycoproteins (PAGs) in milk samples from various animal species, including cattle, goats, sheep and water buffaloes, and serves as a pregnancy marker. It allows confirmation of pregnancy status from specific time points after breeding or calving: 28 days in cows and goats, 60 days in sheep, 29 days in water buffaloes after breeding and 60 days in cows after calving. This capability allows producers to identify open animals for timely rebreeding.

[0021] BioPRYN Early Cattle Pregnancy Detection Kit measures the presence of PSPB in the bloodstream of cattle with over 99% accuracy. Results are usually available the next day, depending on which BioPRYN laboratory facility is used. However, the method used in this kit is not quick and easy to use by cattle owners themselves.

[0022] The P-TESTTM Livestock Pregnancy Test Kit can be used to monitor pregnancy in cattle, sheep, pigs, goats and alpacas. This kit detects estrone sulphate. This test requires the addition of 1.5 mL of urine and in 10 minutes the P-TEST determines the pregnancy status. However, this test is only reliable from the 100th day of gestation and can therefore only detect late pregnancy (Hamon et al., 1981). Reports indicate that estrone sulphate is not an ideal biomarker for pregnancy as plasma and milk profiles are influenced by many other factors such as genetic make-up, weight, parity status and environment (Lobago et al., 2009).

[0023] ICAR (Indian Council of Agricultural Research) institute Central institute for research on buffalos and DBT (Department of Biotechnology) present the prototype 'Preg-D': A Colourimetric Urine-Based Pregnancy Detection Method for Ruminant Livestock". This versionis intended to succinctly outline the key elements and essential details of the technology (a colorimetric, urine-based pregnancy detection method specifically for ruminant livestock).

[0024] Currently, there is no reliable (i.e. field applicable) method for early pregnancy detection in buffaloes / cattle that enables rapid pregnancy detection and can be used by nontechnical persons such as animal owners or livestock farmers themselves. While urine-based kits for early pregnancy detection in humans are readily available, such methods do not exist for ruminants. This is mainly due to the difference in placentation: humans have a ‘disc-shaped' placenta (disc-shaped area on the chorionic sac), whereas ruminants have a ‘cotyledonary’ placenta (Leiser and Kaufmann, 1994). In ruminants, pregnancy is usually determined by rectal palpation and ultrasound. Recently, some laboratory methods have also become available, mostly based on blood (Balhara et al., 2013). Apart from rectal palpation methods, most other methods for detecting pregnancy in ruminants have not proved successful. A specific, sensitive and rapid method to detect pregnancy at an early stage needs to be available at low cost to be widely accepted and used to improve reproductive performance in livestock.

[0025] Although there are different companies working on pregnancy detection methods, there is still need to explore the novel ways and compositions that could be more economical, convenient, easily manufactured and scalable and can overcome deficiencies associated with the known arts. The present invention provides a novel, specific and easy to perform, advantageous kit and non-invasive method that is capable of detecting pregnancy in livestock animal. This composition and kit shall be fulfilling the unmet medical need for detection of pregnancy in livestock animal.OBJECT OF THE INVENTION

[0026] An object of the present invention to provide a non-invasive method for detecting pregnancy in livestock animal using their urine sample.

[0027] Another objective of the present invention is to provide a method of pregnancy detection in ruminant livestock animal urine samples by detecting a specific metabolite in urine.

[0028] Another objective of the present invention is to provide a non-invasive method for detecting pregnancy in livestock by detecting anthranilic acid in urine sample as indication of livestock pregnancy.

[0029] Still another object of the present invention to provide a reagent composition for the detection of pregnancy in livestock animal using their urine.

[0030] Yet another object of the present invention to provide a test kit for the detection of pregnancy in livestock animal using their urine

[0031] Still another object of the present invention is to provide a method for detecting pregnancy in livestock animal through the biosensor based on anthranilic acid.SUMMARY OF THE INVENTION

[0032] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0033] The present invention relates to a non-invasive method for detecting pregnancy in livestock using their urine sample. The present invention provides a rapid, non-invasive method for detecting pregnancy in ruminant livestock, such as cows and buffaloes, using a normal urine test. The method involves the use of two reagents: a saturated specific organic acid (Reagent A) and a diluted specific protonating agent (Reagent B). When these reagents are added to a urine sample, they react with a specific pregnancy-associated metabolite, resulting in a color change. The intensity of the color change is indicative of the pregnancy status. A darker color indicates a positive pregnancy result. The present invention also includes a corresponding test kit that contains the necessary reagents and instructions for use. This kit enables rapid and accurate pregnancy detection on-site, without the need for specialized laboratory equipment.

[0034] In one of the aspects of the present invention is to provide a method of pregnancy detection in ruminant livestock animal urine samples by detecting a specific metabolite in urine.

[0035] In another aspect of the present invention is to provide a method of pregnancy detection in livestock animal urine samples by detecting anthranilic acid.

[0036] In still another aspect, the present invention provides a reagent composition for the detection of pregnancy in livestock animal using their urine.

[0037] In yet another aspect, the present invention provides a test kit for the detection of pregnancy in livestock animal using their urine.

[0038] In still another aspect, the present invention provides a method for detecting pregnancy in livestock animal through the biosensor based on anthranilic acid.

[0039] In another aspect, the present invention provides a reagent composition for detecting pregnancy in livestock animal comprising of: a) Reagent A; b) Reagent B;wherein, the Reagent A is a saturated organic acid which is selected from acetic acid, formic acid, concentrated propanoic acid and similar acid thereof; and the Reagent B is a diluted protonating agent of 5-12 N acid and which is selected from hydrochloric acid, sulphuric acid, phosphoric acid and similar acid thereof with 0.1 to 5M of p-dimethylaminocinnamaldehyde.

[0040] In another aspect of the present invention is to provide a non-invasive method for detecting pregnancy in livestock animal, which comprises the steps of: a) collecting urine sample from livestock animal in a test tube; b) adding a Reagent A into the urine sample of step (a); c) adding a Reagent B into the urine sample of step (b); and d) shake the mixture of step (c) vigorously for about 10-20 seconds for detecting pregnancy by development of intense magenta pink colour.

[0041] In another aspect of the present invention, the optical density (OD) of the sample of step (d) is measured at 400 - 800 nm.BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0042] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein:

[0043] Figure 1 shows different colour of the standard anthranilic acid at different concentration.

[0044] Figure 2 shows the intensity of colour development in positive pregnancy in comparison to negative pregnancy.

[0045] Figure 3 shows the colorimeter chart for detection of pregnant and non-pregnant ruminant livestock.DETAILED DESCRIPTION OF THE INVENTION

[0046] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on thecontrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0047] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0048] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0049] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0050] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise.

[0051] Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

[0052] Unless the context requires otherwise, throughout the specification which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to."

[0053] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any unclaimed element essential to the practice of the invention.

[0054] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0055] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0056] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0057] The present invention relates to a non-invasive method for detecting pregnancy in ruminant livestock using their urine sample.

[0058] In one of the embodiments, the present invention provides a method of pregnancy detection in livestock animal urine samples by detecting a specific metabolite in urine.

[0059] In another embodiment, the present invention provides a method of pregnancy detection in livestock animal urine samples by detecting anthranilic acid.

[0060] In yet another embodiment, the present invention provides a test kit for the detection of pregnancy in ruminant livestock using their urine.

[0061] In still another embodiment, the present invention provides a method for detecting pregnancy in livestock through the biosensor based on anthranilic acid.

[0062] In one of the embodiments, the present invention provides biosensor that can be adapted to various diagnostic platforms, including manual, loT-based, lateral flow, strip-based, or any other suitable diagnostic formats.

[0063] In an embodiment, the present invention provides a reagent composition for detecting pregnancy from a urine sample of ruminant livestock comprising of:a) Reagent A; b) Reagent B; wherein, the Reagent A is a saturated organic acid which is selected from acetic acid, formic acid, concentrated propanoic acid and similar acid thereof and; the Reagent B is diluted protonating agent like p-dimethylaminocinnamaldehyde in 5-12 N and which is selected from hydrochloric acid, sulphuric acid, phosphoric acid and similar acid thereof.

[0064] In another embodiment, the present invention provides a reagent composition for detecting anthranilic acid in urine sample as indication of ruminant livestock pregnancy in livestock animal comprising of: a) Reagent A; b) Reagent B; wherein, the Reagent A is a saturated organic acid which is selected from acetic acid, formic acid, concentrated propanoic acid and similar acid thereof and; the Reagent B is diluted protonating agent like p-dimethylaminocinnamaldehyde in 5-12 N acid which is selected from hydrochloric acid, sulphuric acid, phosphoric acid and similar acid thereof.

[0065] In one of the embodiments of the present invention the Reagent A is used in an amount of 150-300pL and the Reagent B is used in an amount of 400-900 pL.

[0066] In one of the embodiments, the present invention provides a non-invasive method for detecting pregnancy in livestock animal which comprises steps of: a) collecting urine sample in a test tube; b) adding a Reagent A into the urine sample of step (a); c) adding a Reagent B into the urine sample of step (b); d) shake the mixture of step (c) vigorously for about 10-20 seconds for detecting pregnancy by development of intense magenta pink colour.

[0067] In some embodiments, the optical density (OD) of the sample of step (d) is measured at 400-800 nm using standardized colorimeter.

[0068] In one of the embodiments, the present invention provides a non-invasive method for detecting pregnancy in livestock animal which comprises steps of: a) collecting urine sample in a test tube; b) adding a Reagent A into the urine sample of step (a); c) adding a Reagent B into the urine sample of step (b); d) shake the mixture of step (c) vigorously for about 10-20 seconds for detecting pregnancy by development of intense magenta pink colour. e) Measure the OD at 400 - 800 nm using standardized colorimeter.

[0069] In some embodiments, the protonating agent is p-dimethylaminocinnamaldehyde in 5- 12 N acid selected from hydrochloric acid, sulphuric acid, phosphoric acid and similar acid thereof. In some embodiments, the p-dimethylaminocinnamaldehyde in the range of 0.1-0.5 M is added to 5-12 N hydrochloric acid. In some embodiments, the urine sample is tested for presence of anthranilic acid, preferably in the concentration range of 1500-2000 pMol / mL.

[0070] The present non-invasive method for detecting pregnancy of animals starts with collecting debris free urine sample in a clean plastic container and taking some amount of it in a test tube for testing.

[0071] The urine sample present in test tube is further added with 150-300pl of Reagent A, and which is further followed by addition of 400-900 pl of Reagent B.

[0072] Formation of intense darkish magenta pink colour differentiates urine sample of pregnant animal with non-pregnant. Intense magenta pink colour is obtained when urine sample of pregnant livestock such as cattle / buffalo is tested, while light pink colour is obtained in case of non-pregnant animal.

[0073] The present method for detection of pregnancy from the urine sample of animal is based on colorimetric reaction between upregulated metabolite present in the urine sample with Reagents A and B, resulting in formation of colour, where light colour is obtained when such metabolite concentration is 200-250 pMol / mL of urine, while dark magenta pink colour is obtained when the metabolite concentration approaches to concentration of 1500-2000 pMol / mL.

[0074] In a preferred embodiment, the present invention discloses Reagents A and B for detection of metabolites in the urine sample wherein the Reagent A is a saturated organic acid which is selected from acetic acid, formic acid, concentrated propanoic acid and similar acid thereof; and the Reagent B is a diluted protonating agent which is selected from hydrochloricacid, sulphuric acid, phosphoric acid and similar acid thereof with p- dimethylaminocinnamaldehyde.

[0075] The present non-invasive method detects pregnancy of an animal within 2-5 minutes.

[0076] In another embodiment of the present invention there is provided a non-invasive kit for detecting pregnancy in livestock.

[0077] The non-invasive kit for detecting pregnancy of livestock comprises: a) Reagent A; b) Reagent B; c) Test tubes and droppers d) Colorimeter;

[0078] The instructions manual of the non-invasive kit is provided to match the colour generated in the samples.

[0079] The kit optionally comprises test tubes, a colour chart and cotton plugs.

[0080] The kit of the present invention results in immediate colorimetric detection of pregnancy of an animal within 2-5 minutes.

[0081] In an exemplary embodiment, the non-invasive kit for detecting pregnancy of livestock is capable of providing a qualitative and rapid biochemical assay to detect pregnancy non- invasively through urine samples of livestock such as buffaloes. The kit includes two reagents, Reagent A and Reagent B, which react with specific pregnancy- associated metabolites in the urine to form a magenta pink compound. The reaction's outcome is then measured using a digital colorimeter at a wavelength of 400 - 800 nm to determine pregnancy status. Importantly, the kit does not contain any materials of bacterial, viral, parasitic, animal, or human origin, minimizing the risk of infection for lab technicians or during transport. Moreover, the likelihood of reagent leakage is low, ensuring safety for users without significant risk of human infectivity.

[0082] In some embodiments, the Reagent A is prepared by placing 100 ml of 1-10 N formic acid in a glass container and allowing it to stand overnight. In other embodiments, Reagent B is prepared by combining 5-12 N hydrochloric acid with 0.1-5 M p-dimethyl amino cinnamaldehyde in a clean, dry flask. When combined, the solution changes to an orange -red color, indicating the cinnamaldehyde has fully dissolved. This mixture is kept overnight, during which the color transitions from orange-red to yellow. The initial pH is recorded using a calibrated pH meter to ensure consistency.

[0083] In some embodiments, the non-invasive kit for detecting pregnancy of livestock consists Reagent A, a saturated formic acid solution acting as an organic acid reagent, and Reagent B, a protonating agent formed by diluting hydrochloric acid (5-12 N) with 0.1-0.5 Mdimethylaminocinnamaldehyde. The kit includes two types of droppers for applying reagents with precision: a Reagent A dropper with al00-250 pl capacity and a Reagent B dropper with a 500-1000 pl capacity. Fresh urine samples from cattle are collected in clean, dry containers as specimens for testing. If immediate testing is not possible, samples may be stored at 2-8°C for up to 24 hours.

[0084] In a working example, to conduct the non-invasive test for detecting pregnancy, approximately 10 ml of urine is collected in a plastic cup, vessel, or airtight container, ensuring the sample is uncontaminated. 2 ml of the urine is added to a glass test tube provided in the kit, followed by the addition of 4-6 drops of Reagent A, and subsequently 10-15 drops of Reagent B. The test tube is gently tapped to mix the solution, then placed in the digital colorimeter for optical density (OD) measurement. Interpretation of the results is as follows: OD readings of > 0.59 is positive indicating pregnancy, readings of < 0.45 is negative indicating non-pregnancy and readings between 0.46 -0.58 suggest indeterminant samples hence repeating the test after 15 days.

[0085] In an embodiment, the analytical sensitivity and specificity of the kit was evaluated with urine samples from livestock between days 30 and 50 post-artificial insemination (Al) ,and where cross-verified with ultrasonography. Results showed that the kit could reliably detect pregnancy on that particular day, confirmatory test shall be done 15 days post preliminary testing.

[0086] In an embodiment, accuracy of the non-invasive kit for detecting pregnancy of livestock with urine samples from livestock between days 30 and 50 post-artificial insemination (Al) is validated through ultrasonography, serving as a reference method,

[0087] In another embodiment, the sensitivity and specificity for the pregnancy detection kit was identified using urine samples > 30 days post-artificial insemination (Al) from livestock to be greater than 90% when compared with ultrasonography.

[0088] In an embodiment, reproducibility of said kit is established using three distinct tests: the inter-run assay, inter-lot assay, and inter-operator assay. In the inter-run assay, identical samples were tested on a single lot by the same analyst at different times of day. The inter-lot assay involved testing the same samples across three different production lots on the same day, while the inter-operator assay was conducted by different analysts on the same lot. The kit achieved 100% reproducibility across these tests, validating consistent performance in varying conditions. In another embodiment of the present invention the pregnancy detection kit for livestock and the reagents associated with the kit were identified to have a shelf life of 12 months when stored at 15 - 25°C.

[0089] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.Examples

[0090] The present invention is based on surprising observation of the inventors about presence of pregnancy upregulated metabolite having active amine and carboxylic group functional moieties in urine of ruminant livestock. It was identified by the present inventors that concentration of such metabolites in urine crosses a threshold level during pregnancy of animals. And at such increased concentration, the metabolite reacts with specific reagents in specific reaction condition to develop colour change.

[0091] It was identified by the present inventors that metabolites having active amine and carboxylic acid functional moieties are present in urine of non-pregnant livestock animal from 200-250 pMol / mL, while it crosses a level of 1500-2000 pMol / mL during pregnancy (Table 1).

[0092] Table 1 : Metabolite Concentration in Urine (6 samples from each day and each group)

[0093] The present inventors developed a method of detecting pregnancy in livestock animal by determining the change in concentration of anthranilic acid having functional moieties. The inventors of the present invention performed NMR, FTIR and Mass spectral studies of urine of livestock animal and observed presence of colour causing moiety in urine which is found upregulated during pregnancy and proposed a chemical reaction mechanism. In the presence of hydrochloric acid (HC1), the aldehyde group of p-dimethylaminocinnamaldehyde is protonated and a Schiff Base is formed: The protonated p-dimethylaminocinnamaldehyde reacts withanthranilic acid to form a Schiff base intermediate and a colour complex is formed (shown inScheme 1)Scheme 1: Proposed chemical reaction mechanism

[0094] Example 1: Urine Sample Collection

[0095] Normal micturated urine (5-10 mL) is collected from the test animal in a clean, airtight glass container or urine cup / container made up of inert plastic material. The urine should be free from any dung or any other contamination. For reference urine sample preparation, urine is collected as described above from a confirmed non-pregnant animal. The test is performed at room temperature within 2-3 hours of the collection of urine samples in the shade. Avoid samples from direct contact with sunlight or heat.

[0096] Example 2: Method of performing the testRaw Materials: Formic acid as Reagent A, 0.1 M p-dimethylaminocinnamaldehyde in 5 N hydrochloric acid as Reagent B, Urine sample, Test Tubes, colorimeterThe test was performed on a sample obtained from animal and the pregnancy status was confirmed by transrectal ultrasonography. The following steps were performed:(a) A 2 mL urine sample was collected in a glass tube;(b) To the sample, 150 pL of formic acid (reagent A) was added;(c) Further, 400 pL of 0.1 M p-dimethylaminocinnamaldehyde in 5 N hydrochloric acid (reagent was added.Development of colour was observed after 10-20 seconds and the colour appeared as darkish magenta pink colour. The development of the colour was due to formation of Schiff base intermediate.The result is shown in Figure 1 and 2. Figure 1 indicates the colour of anthranilic acid in presence of mentioned reagents A and B, Figure 2 indicates the colour intensity in different stages of pregnancy, which can be read from the colour chart as given in Figure 3.

[0097] Example 3: Identification of Agents producing colorimetric reactionThe present team of inventors have identified that there is a change in the concentration of secreted metabolite during pregnancy (NMR studies of Urinary metabolites,1HNMR 400Hz DMSO); high intensity 8 values of coloured compound lies in the range of 0.80 to 8.88 ppm.Also, the FTIR (KBr) cm"1data supports the study that the coloured precipitates showed functional group peak at 1680-1700 (C=O, stretching), 1630-1650 (C=N, stretching, imine), 1315-1340 (C-N, stretching, 3°Amines), 1972-2512 (C=C, C-H stretching), 3290-3476 (N-H, O-H strong stretching), 3700-4356 (O-H, weak stretching). The FTIR data, shows the presence of (C=O), (C=N), (C-N), (N-H), (C-CH2), (O-H), (C=C), (C-O-C) and NH2functional groups in the obtained compound which characterize the functional group data of colour complex.

[0098] Example 4: Demonstrative results of the kitI. Test in lab conditionsThe test was performed on different urine samples obtained from livestock and the pregnancy status was confirmed by transrectal ultrasonography. To perform the test, 2 mL of urine from the test and reference animals were taken in clean test tubes, 150pL of formic acid (reagent A) and 400 pL of 0. IM p-dimethylcinnamaldehyde in 5 N hydrochloric acid (reagent B) were added to each tube. Development of colour was observed after 10-20 seconds.II. Demonstrative results of transrectal ultrasonography:The same animals from which the urine sample was collected were examined by transrectal ultrasonography for pregnancy detection and results were recorded. For this, ultrasound examinations were performed post artificial insemination. Urine from all inseminated animalswas collected and retrogressively classified as pregnant / non-pregnant after performing ultrasonography.Result - The sensitivity of the test done on the animals is greater than 90% and specificity is greater than 80% when compared to ultrasonography.Example 5: Stability Data for KitAccelerated Stability studies for Urine pregnancy detection kit for livestock were evaluated to determine the shelf life when stored at 45°C for 90 days. Testing was conducted at 0,7th,15th,21st, 30th, 41st, 64thand 90thday. At each time point kits stored at 45°C were used to test the stability panel (2 pregnant, 2 non -pregnant samples in duplicate). Urine pregnancy detection kit for livestock showed 100 % sensitivity and specificity when stored at 45°C for 90 days. Though Color change observed in reagent B from yellow to deep yellow, it does not affect the performance of the kit.Based on observations, the shelf life of Urine pregnancy detection kit for livestock is 12 months when stored at 15-25°C.Real time Stability studies for Urine pregnancy detection kit for livestock were evaluated to determine the shelf life when stored at 15 -25°C for 12 months. Testing was conducted at 0,3rd,6th,9th, 12thand 18thmonth. At each time point kits stored at 15-25°C were used to test the stability panel (2 pregnant, 2 non -pregnant samples in duplicate). Urine pregnancy detection kit for livestock showed 100 % sensitivity and specificity when stored at 15 - 25°C for 6 months. The real time stability study will be continued through 18 months to support the shelf life claim.

[0099] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.ADVANTAGES OF THE INVENTION[000100] The present invention to provide a non-invasive method for detecting pregnancy in ruminant livestock animal.[000101] The method of the present invention is pen side-based which is quick, simple and accurate. The method of the invention consumes less time as compared to other pregnancy detection methods i.e. it provides for rapid detection of pregnancy.[000102] The method and kit of the present invention are cost-effective and easy-to use, and highly sensitive and specific for detecting pregnancy early.

Claims

We Claim:

1. A reagent composition for detecting pregnancy from a urine sample of ruminant livestock animal, composition comprising: a. Reagent A; b. Reagent B;Wherein, the Reagent A is a saturated organic acid selected from acetic acid, formic acid, concentrated propanoic acid and similar acid thereof; and the Reagent B is a diluted protonating agent.

2. The reagent composition as claimed in claim 1, wherein the protonating agent is p- dimethylaminocinnamaldehyde in 5-12 N acid selected from hydrochloric acid, sulphuric acid, phosphoric acid and similar acid thereof.

3. The reagent composition as claimed in claim 1, wherein the Reagent A is in an amount ranging from 150-300pL.

4. The reagent composition as claimed in claim 1, wherein the Reagent B is in an amount ranging from 400-900 pL.

5. A non-invasive method for detecting pregnancy in livestock animal, method comprising the steps of: a. collecting a urine sample in a test tube; b. adding a Reagent A into the urine sample of step (a), wherein the Reagent A is a saturated organic acid selected from acetic acid, formic acid, concentrated propanoic acid and similar acid thereof; c. adding a Reagent B into the urine sample of step (b), wherein the Reagent B is a diluted protonating agent; and d. shake the mixture of step (c) vigorously for about 10-20 seconds for detecting pregnancy by development of intense magenta pink colour.

6. The method as claimed in claim 6, wherein optical density (OD) of the sample of step (d) is measured at 400-800 nm using standardized colorimeter.

7. The method as claimed in claim 6, wherein pregnancy is detected through detection of metabolites in the urine sample.

8. A non-invasive kit for detecting pregnancy in livestock animal comprises: a. Reagent A, wherein the Reagent A is a saturated organic acid selected from acetic acid, formic acid, concentrated propanoic acid and similar acid thereof; b. Reagent B, wherein the Reagent B is a diluted protonating agent; c. Test tubes and droppers; and d. Colorimeter

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