Non-invasive kit and method for rapid visual pregnancy detection in mammals using milk and reagent

Abstract

ABSTRACT NON-INVASIVE KIT AND METHOD FOR RAPID VISUAL PREGNANCY DETECTION IN MAMMALS USING MILK AND REAGENT The invention relates to a non-invasive, cost-effective, and field-deployable kit (100) and method (200) for detecting pregnancy in mammals using milk samples. The kit comprises a first vial (102) for milk collection, reagent bottle(s) (104) containing bromophenol blue, a color reference chart (106), a second vial or test strip (110) for reaction, and an instruction manual (108). In use, milk is mixed with the reagent in a 1:2 milk-to-reagent ratio, gently swirled for 5–10 seconds, and the resulting color change is visually interpreted. A whitish-blue color indicates a pregnancy-positive result, while a dark blue or purple color indicates pregnancy-negative. The reaction is stable within an observation window of 2 minutes –3 days, requires no specialized training, and operates in ambient conditions of 15–40 °C. The invention enables early-stage pregnancy detection, especially for cattle, buffalo, goats, and sheep, and is suitable for rural settings without requiring laboratory infrastructure or invasive procedures. (FIGURES 1 & 3)

Description

FIELD OF THE INVENTIONThe present disclosure pertains to analytical method and kit thereof. The presentdisclosure relates to a kit and method of analysis of milk sample qualitativelywherein it comprises chemical reagent(s) based analysis, which selectivelyanalyse the milk sample and confirms the pregnancy of mammal from which thesample is obtained.BACKGROUND OF THE INVENTIONReproductive performance is one of the important factors that can affect dairyfarms' professional and economical success. Finding new ways to improvefertility parameters as well as dairy cows' welfare on the farms can lead to aneconomic improvement. Early identification of non-pregnant dairy cows afterArtificial insemination (AI) improves reproductive efficiency and the 21-daypregnancy rate by decreasing the interval between AI services, thereby increasingthe AI service rate. Reducing the insemination interval for non-pregnant cows isexpected to reduce the time to pregnancy and improve pregnancy rate. From 55 to70% of lactating dairy cows fail to become pregnant after AI and need to be reinseminated. However, non-pregnant cows need to be identified before beingsubjected to re-insemination to further reduce the interbreeding interval.The diagnosis of pregnancy in cattle is traditionally performed on around day 40after insemination by transrectal palpation of the reproductive tract foridentification of the amniotic vesicle or approximately 26 days after inseminationwith the aid of ultrasonography. Note that direct palpation of the conceptus earlyin gestation has also been associated with pregnancy loss in some studies.However, it is worth mentioning that recent studies did not find any impact of therectal palpation on pregnancy loss. However, the rectal palpation procedure likelyleads to discomfort, resulting in physiological and behavioral stress reactions.Recently, more and more often, attention is paid to the negative impact of stressaccompanying the rectal palpation and thus violating animal welfare.Also, pregnancy diagnosis can be a problem for dairy farmers in areas whereveterinary support is limited. An alternative approach is to use a pregnancydetection assay. In the 1980s, several companies marketed on-farm, milkprogesterone tests (Nebel, J. Dairy Sci., 71, pp. 1682-1690, 1988). They offeredthe dairy worker the convenience of collecting milk samples and immediateresults. However, progesterone is an indirect indicator of pregnancy status. Cattlesmay have high concentrations of progesterone for reasons other than pregnancy.For that reason, the technology was very effective at identifying nonpregnantcows (>90% accuracy), but less so for pregnant cows (<80% accuracy, Nebel,1988). Adoption of this technology was too low to remain economically viable.In 2002, a pregnancy-detection assay based on the measurement of pregnancyassociated glycoproteins (PAG) became available to dairy farmers (BioPRYN,BioTracking LLC, Moscow, ID). The assay is limited to serum or plasma samplesand must be sent to a laboratory for analysis, which are having turnaround time isusually 2 to 3 days, depending on the laboratory. The PAG are synthesized andsecreted by placental tissue, and therefore are direct indicators of pregnancy.These assays are accurate at identifying both pregnant and nonpregnant cows at27 day post-insemination or later (>90% accuracy). Though successful, theapplication of this technology has been limited by the requirement for collectingblood samples.Thus, there is a need in the art to make and use a kit and / or test strip, and methodof analysis of simple milk samples to confirm the pregnancy in mammals likecattle, cows, etc. in an effective, and non-invasive manner, and this plays a keyrole in management strategies to improve reproductive efficiency, dairy cowwelfare and profitability on dairy farms.The present disclosure satisfies the existing needs, as well as others, and generallyovercomes the deficiencies found in the prior and general art.OBJECTIVES OF THE INVENTIONIt is an objective of the present disclosure to provide a kit for in vitrodetection / analysis of sample by preliminarily confirming a pregnancy in amammal.It is another objective of the present disclosure to provide a kit for in vitrodetection / analysis of sample by qualitatively confirming a pregnancy in amammal.Yet another objective of the present disclosure to provide a method of detection oranalysis of milk samples for checking either presence or absence of pregnancy ina mammal.SUMMARY OF THE INVENTIONThe present invention provides a n, non-invasive, rapid, and cost-effective methodfor detecting pregnancy in cattle through a simple colorimetric reaction using milksamples. Unlike conventional techniques such as rectal palpation orultrasonography-which are invasive, expensive, and require skilled personnel-this method relies on the interaction of specific chemical reagents withcomponents in the milk to produce a distinct color change that indicates thepregnancy status of the animal. The method is optimized to be accessible andapplicable directly in the field, especially by small-scale farmers in rural areas,and has the potential for commercial translation into a portable diagnostic kit.The process involves collecting milk from cows suspected of pregnancy andmixing it with chemical reagents that react with milk constituents to produce avisual change. Three reagents were tested: copper sulfate, acetocaramine red, andBhromophenol Blue. Among these, Bhromophenol Blue emerged as the preferredreagent due to its reliability and clarity in differentiating between pregnant andnon-pregnant samples. At an optimized milk-to-reagent volume ratio of 1:2, adistinct whitish-blue color with slight curdling was observed in milk frompregnant cows, while milk from non-pregnant cows produced a dark blue orpurple hue with minimal curdling. This reaction was consistently visible within 5-10 seconds after gentle swirling, making it a highly practical tool for earlypregnancy detection.The method's key advantages include being completely non-invasive (eliminatinganimal stress), requiring no electricity or laboratory setup, and offering nearinstant results without technical expertise. It supports early-stage detection-potentially within 30 days of conception-allowing for improved herdmanagement and reproductive planning. Furthermore, this technique circumventsthe need for expensive equipment and lab infrastructure, thereby democratizingaccess to pregnancy diagnostics for economically challenged farmingcommunities.A core of the invention lies in the identification and optimization ofBhromophenol Blue as a pregnancy-indicating reagent in milk, with the 1:2 milkto-reagent ratio delivering the most consistent diagnostic outcome. The processdoes not merely rely on chemical presence but on a defined and reproduciblevisual indicator within seconds-a feature not disclosed in prior art. No existingmethods provide such a rapid, low-cost, and easy-to-interpret solution using milkand a visual reagent.The invention is structured for scalability and practical deployment. A futurediagnostic kit could contain pre-measured reagents, droppers, milk collectiontubes, and a standardized color reference chart for result interpretation. Futuredevelopments may also include digital integrations, such as smartphone-basedcolor sensors or mobile apps, to automate reading and reduce human error.Additionally, research into the biochemical interaction mechanism ofBhromophenol Blue with pregnancy-associated milk components could furtherrefine the test's specificity and sensitivity. Broader validation across differentcattle breeds, environmental conditions, and possibly other livestock species isalso envisaged.In conclusion, this invention introduces a technically advanced method fordetecting pregnancy in cattle. It bridges a critical gap in rural veterinarydiagnostics by offering an affordable, user-friendly, and animal-friendly solution.With clear potential for commercialization and widespread adoption, it representsa significant advancement in livestock reproductive health management.The present disclosure provides a new and improved test strip and kit thereof fordetection and analysis of a milk sample to convey qualitatively and preliminarilythe presence or absence of pregnancy in a mammal.In an aspect, the present disclosure relates to a kit (100) for in vitrodetection / analysis of a sample, the kit comprises:i. 1st vial (102) for collecting the sample,ii. bottle(s) (104) containing chemical reagent(s),iii. colour reference chart (106),iv. an instruction manual (108), andv. a test strip or 2nd vial (110) for mixing or applying the sample withchemical reagent(s);wherein the chemical reagent(s) and the sample is applied or mixed in said teststrip or 2nd vial to obtain a mixture with qualitative change in colour confirmingpositive or negative result of the sample.In an embodiment, the sample is milk sample.In another aspect, the present disclosure relates to a method (200) ofdetection / analysis of a sample, comprising:a) providing (202) the sample in a 1st vial or test strip;b) mixing (204) a chemical reagent from bottle, with said sample from 1stvial in a 2nd vial under stirring at temperature in the range of 25-40°C fortime period in the range of 5-10 seconds to obtain a solution; andc) observing and comparing (206) a colour of the solution obtained in step b)with colour reference chart to analyse and confirm the sample being eitherpositive or negative for pregnancy.In an embodiment, the test strip or 2nd vial comprises mixing or applying of thesample with chemical reagent(s) in weight ratio in the range of 1:1 to 1:3.Various objects, features, aspects and advantages of the inventive subject matterwill become more apparent from the following detailed description of preferredembodiments.BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGSFIGURE 1 shows representative illustration covering the components of the kit, inaccordance with an exemplary embodiment of the present invention.FIGURE 2 shows representative illustration covering the methods steps followedfor the analysis and detection of the sample for pregnancy of mammal like cattle, ,in accordance with an exemplary embodiment of the present invention.FIGURE 3 shows representative drawing showing the test results of positiveand / or negative sample with colour differentiation, in accordance with anexemplary embodiment of the present invention.FIGURES 4A-4I shows representative drawings showing the test results ofpositive and / or negative sample with colour differentiation from pregnancy of 1month to pregnancy of 9 months, in accordance with an exemplary embodimentof the present invention.FIGURE 5A-5H experimental result conducted to see effect of addition of SDS(Sodium dodecyl sulphate) of 0.08gm till 20ml in both pregnant and non-pregnantanimal milk respectively, in accordance with an embodiment of the presentinvention.DETAILED DESCRIPTION OF THE INVENTIONThe present invention provides a, non-invasive, rapid, and cost-effective methodfor detecting pregnancy in cattle through a simple colorimetric reaction using milksamples. Unlike conventional techniques such as rectal palpation orultrasonography-which are invasive, expensive, and require skilled personnel-this method relies on the interaction of specific chemical reagents withcomponents in the milk to produce a distinct color change that indicates thepregnancy status of the animal. The method is optimized to be accessible andapplicable directly in the field, especially by small-scale farmers in rural areas,and has the potential for commercial translation into a portable diagnostic kit.The process involves collecting milk from cows suspected of pregnancy andmixing it with chemical reagents that react with milk constituents to produce avisual change. Three reagents were tested: copper sulfate, acetocaramine red, andBhromophenol Blue. Among these, Bhromophenol Blue emerged as the preferredreagent due to its reliability and clarity in differentiating between pregnant andnon-pregnant samples. At an optimized milk-to-reagent volume ratio of 1:2, adistinct whitish-blue color with slight curdling was observed in milk frompregnant cows, while milk from non-pregnant cows produced a dark blue orpurple hue with minimal curdling. This reaction was consistently visible within 5-10 seconds after gentle swirling, making it a highly practical tool for earlypregnancy detection.The method's key advantages include being completely non-invasive (eliminatinganimal stress), requiring no electricity or laboratory setup, and offering nearinstant results without technical expertise. It supports early-stage detection-potentially within 30 days of conception-allowing for improved herdmanagement and reproductive planning. Furthermore, this technique circumventsthe need for expensive equipment and lab infrastructure, thereby democratizingaccess to pregnancy diagnostics for economically challenged farmingcommunities.A core of the invention lies in the identification and optimization ofBhromophenol Blue as a pregnancy-indicating reagent in milk, with the 1:2 milkto-reagent ratio delivering the most consistent diagnostic outcome. The processdoes not merely rely on chemical presence but on a defined and reproduciblevisual indicator within seconds-a feature not disclosed in prior art. No existingmethods provide such a rapid, low-cost, and easy-to-interpret solution using milkand a visual reagent.The invention is structured for scalability and practical deployment. A futurediagnostic kit could contain pre-measured reagents, droppers, milk collectiontubes, and a standardized color reference chart for result interpretation. Futuredevelopments may also include digital integrations, such as smartphone-basedcolor sensors or mobile apps, to automate reading and reduce human error.Additionally, research into the biochemical interaction mechanism ofBhromophenol Blue with pregnancy-associated milk components could furtherrefine the test's specificity and sensitivity. Broader validation across differentcattle breeds, environmental conditions, and possibly other livestock species isalso envisaged.In conclusion, this invention introduces a highly inventive, and technicallyadvanced method for detecting pregnancy in cattle. It bridges a critical gap inrural veterinary diagnostics by offering an affordable, user-friendly, and animalfriendly solution. With clear potential for commercialization and widespreadadoption, it represents a significant advancement in livestock reproductive healthmanagement.In general embodiment, the present disclosure provides new and improved teststrip and kit thereof for detection and analysis of a milk sample to conveyqualitatively and preliminarily the presence or absence of pregnancy in amammal.Accordingly, to address said problems, in one embodiment, the present disclosurerelates to a kit (100) for in vitro detection / analysis of a sample. FIGURE 1 showsrepresentative illustration covering the components of the kit in accordance withan exemplary embodiment of the present invention.The invention provides a novel kit and method for rapid, non-invasive pregnancydetection in mammals, particularly suited for cattle, buffalo, goats, and sheep. Thekit (100) comprises five key components: a first vial (102) for collecting a milksample, reagent bottle(s) (104) containing a chemical reagent-preferablybromophenol blue-a color reference chart (106) for result interpretation, asecond vial or test strip (110) for conducting the reaction, and an instructionmanual (108) detailing usage steps. In implementation, a fresh milk sample iscollected and mixed with the bromophenol blue reagent in a volumetric ratio of1:2 (milk:reagent) in the second vial or on the strip. This mixture is gently swirledfor 5-10 seconds at room temperature (25-40°C). Within an observation windowof 2-60 seconds, a visible color change occurs: whitish-blue with slight curdlingindicates a pregnant animal, while dark blue or purple with minimal curdlingdenotes a non-pregnant state. This reaction is compared with the pre-printed colorreference chart for easy diagnosis. The reagents are formulated to be shelf-stable,do not require electricity or skilled personnel, and are optimized for on-field, ruralapplications where conventional pregnancy detection methods like ultrasound orhormonal assays are impractical or costly. The method thus offers a quick, lowcost, and reliable diagnostic tool that improves herd management and enablesearly pregnancy detection, even within 30 days of conception.The present invention provides a solution to the longstanding problem ofpregnancy detection in livestock, particularly in resource-limited rural settings.Traditional methods such as rectal palpation and ultrasound are either invasive,require expensive equipment, or demand trained personnel-factors that renderthem impractical for widespread use among small-scale farmers. The novelty ofthe present invention lies in the use of bromophenol blue as a specific chemicalreagent to elicit a rapid and visually distinct colorimetric reaction when mixedwith a milk sample, with a defined volumetric ratio (1:2) and a short reaction time(5-10 seconds). The invention is the first to disclose a milk-based color-changereaction that reliably distinguishes between pregnant and non-pregnant animalswithin seconds, using no laboratory equipment or invasive procedures.From an inventive step perspective, the combination of multiple technicalfeatures-such as the specific reagent (bromophenol blue), the optimized milk-toreagent ratio, the defined swirling and observation times, and the inclusion of acolor reference chart shows a synergistic integration. While bromophenol blueand other dyes have been known in laboratory chemistry, their application infield-level pregnancy diagnostics using milk as a biological medium has not beenpreviously disclosed or suggested. Moreover, no prior art teaches the preciseconditions under which these reagents produce color differences specifically tiedto pregnancy biomarkers in milk.The invention demonstrates a clear technical advancement by translating achemical detection principle into a portable, self-contained diagnostic kit that canfunction in ambient rural environments (15-40°C), without electricity,refrigeration, or laboratory infrastructure. It also offers early-stage pregnancydetection, potentially within 30 days of conception-earlier than manyconventional techniques. The reaction is non-invasive, animal-friendly, andcompleted in under a minute, offering immediate feedback to farmers.The technical benefits are significant: it lowers the cost of pregnancy detection,enables timely herd management decisions, reduces stress to the animals, andempowers untrained users in remote areas with a reliable diagnostic tool.Additionally, the shelf-stable nature of the reagents and the inclusion of step-bystep instructions further reinforce the invention's usability and robustness. In sum,the claims as drafted encapsulate an inventive and field-ready solution that isnovel, technically superior, non-obvious, and socially impactful-well alignedwith the requirements of the Indian Patents Act, including inventive step andindustrial applicability.Referring again to FIG. 1 the kit comprises:i. 1st vial (102) for collecting the sample,ii. bottle(s) (104) containing chemical reagent(s),iii. colour reference chart (106),iv. an instruction manual (108), andv. a test strip or 2nd vial (110) for mixing or applying the sample withchemical reagent(s);wherein the chemical reagent(s) and the sample is applied or mixed in said teststrip or 2nd vial to obtain a mixture with qualitative change in colour confirmingpositive or negative result of the sample.In an embodiment, the sample is milk sample.In an embodiment, the milk sample is obtained from a mammal selected fromcattle, buffalo, goat, and sheep.In an embodiment, the colour reference chart comprises at least two colourreferences confirming positive or negative results for pregnancy.In an embodiment, the positive sample means the sample is positive for pregnancyi.e., sample may contain pregnancy biomarkers. In the context of the presentdisclosure, a "positive sample" refers to a sample indicative of pregnancy, i.e., thesample may contain one or more pregnancy-associated biomarkers. Thesebiomarkers may be proteins or other biological molecules that undergomeasurable changes during pregnancy, such as variations in concentration,structural conformation, or binding affinity. In certain embodiments, suchbiomarkers may interact with a dye or reagent, thereby producing a detectablecolor change or signal.In one example, the positive sample indicative of pregnancy may comprisespecific biomarkers that selectively interact with the chemical reagent to yield alight-blue coloration. Such biomarkers may include, but are not limited to, humanchorionic gonadotropin (hCG), pregnancy-associated plasma protein-A (PAPPA), alpha-fetoprotein (AFP), pregnancy-specific glycoproteins (PSGs), or estriolbinding proteins, all of which are known to exhibit altered concentration orconformation during pregnancy. These biomarkers, individually or incombination, may bind to or influence the binding of the chemical reagent (forexample, a dye with protein-binding affinity), thereby producing a characteristiclight-blue signal. The invention contemplates that variations in concentration,conformational changes, or differential binding of these pregnancy-associatedproteins or glycoproteins may underlie the observed selective reactivity andresultant color development.In an embodiment, the negative sample means the sample is negative forpregnancy i.e., sample may contain pregnancy biomarkers.In an embodiment, the colour reference chart comprises light blue colour as onecolour reference.In an embodiment, the colour reference chart comprises dark blue colour as onecolour reference.In an embodiment, the colour reference chart is designed to correlate the extent ofbiomarker-reagent interaction with a visual indicator, wherein the chart compriseslight blue colour as one reference, indicative of a lower level or thresholdpresence of the pregnancy-associated biomarker(s). In another embodiment, thecolour reference chart comprises dark blue colour as a reference, corresponding toa higher concentration or stronger interaction of the said biomarker(s) with thechemical reagent. The gradation from light blue to dark blue thereby provides asemi-quantitative or qualitative indication of the biomarker level in the sample,enabling a clear and user-friendly interpretation of pregnancy status.In an embodiment, the colour reference chart comprises dark purple colour as onecolour reference.In an embodiment, the positive sample shows light blue colour confirming thesample is positive for pregnancy.In an embodiment, the negative sample shows the dark blue or purple colourconfirming the sample is negative for pregnancy.In an embodiment, the 1st vial is made of material selected from plastic, woodenand steel.In an embodiment, the bottle(s) containing chemical reagent(s) comprisesbottle(s), and chemical reagent(s) in solution or in dry powder.In an embodiment, the bottle(s) is / are made of material selected from transparentplastic, ambered coloured plastic, wooden and steel.In an embodiment, the chemical reagent solution(s) is / are selected frombromophenol blue, acetocaramine red dye, and copper sulphate or any ofcombination thereof.In an embodiment, the kit additionally comprises a glove, and a mixing tool formixing the sample with chemical reagent(s).In an embodiment, the instruction manual comprises a set of paper covering stepsfor the testing and analysis of the sample, and use of the kit for said analysis.In an embodiment, the test strip or 2nd vial comprises mixing or applying of thesample with chemical reagent(s) in weight ratio in the range of 1:1 to 1:3.In an embodiment, the kit provides early detection and analysis of the sample forpregnancy in said mammals i.e., within 5-15 seconds considering the specificcolour change and confirmation of the presence or absence of positive or negativesample as disclosed above.In an embodiment, the amount or volume of the sample is in the range of 0.5-5mL per test / analysis.In an embodiment, the amount or volume of chemical reagents in said kit is in therange of 0.25-10 mL per test / analysis.In an embodiment, the volume by volume ratio of the sample: chemical reagent(s)is in the range of 1:0.5 to 1: 3.In an embodiment, the mixing and reaction time of the sample and chemicalreagent(s) under gentle swirling is done for time period in the range of 5-15 seconds.In an embodiment, the kit and / or testing is / are used / done at temperature in therange of 15-40°C.In an embodiment, the reagent bottle(s) containing said chemical reagent(s) is / arestored at temperature in the range of 4-40°C, and at pH in the range of 6.5-8.5 asaqueous solution or powder.In another embodiment, the present disclosure relates to a method (200) ofdetection / analysis of a sample. FIGURE 2 shows representative illustrationcovering the methods steps followed for the analysis and detection of the samplefor pregnancy of mammal like cattle, in accordance with an exemplaryembodiment of the present invention. The method includes the steps of:a) providing (202) the sample in a 1st vial or test strip;b) mixing (204) a chemical reagent from bottle, with said sample from 1stvial in a 2nd vial under stirring at temperature in the range of 25-40°C fortime period in the range of 5-10 seconds to obtain a solution, andc) observing and comparing (206) a colour of the solution obtained in step b)with colour reference chart to analyse and confirm the sample being eitherpositive or negative for pregnancy.In an embodiment, the sample is milk sample.In an embodiment, the milk sample is obtained from a mammal selected fromcattle, buffalo, goat, and sheep.In an embodiment, the colour reference chart comprises at least two-colourreferences confirming positive or negative results for pregnancy.In an embodiment, the positive sample means the sample is positive for pregnancyi.e., sample may contain pregnancy biomarkers.In an embodiment, the negative sample means the sample is negative forpregnancy i.e., sample may contain pregnancy biomarkers.In an embodiment, the colour reference chart comprises light blue colour (208) asone colour reference.In an embodiment, the colour reference chart comprises dark blue colour (210) asone colour reference.In an embodiment, the colour reference chart comprises dark purple colour (210)as one colour reference.In an embodiment, the positive sample (212) shows light blue colour confirmingthe sample is positive for pregnancy.In an embodiment, the negative sample (214) shows the dark blue or purple colourconfirming the sample is negative for pregnancy.In an embodiment, the 1st vial is made of material selected from plastic, woodenand steel.In an embodiment, the bottle(s) containing chemical reagent(s) comprisesbottle(s), and chemical reagent(s) in solution or in dry powder.In an embodiment, the bottle(s) is / are made of material selected from transparentplastic, ambered coloured plastic, wooden and steel.In an embodiment, the chemical reagent solution(s) is / are selected frombromophenol blue, acetocaramine red dye, and copper sulphate or any ofcombination thereof.In an embodiment, the test strip or 2nd vial comprises mixing or applying of thesample with chemical reagent(s) in weight ratio in the range of 1:1 to 1:3.In an embodiment, the amount or volume of the sample used in said method is inthe range of 0.5-5 mL per test / analysis.In an embodiment, the amount or volume of chemical reagents used in saidmethod is in the range of 0.25-10 mL per test / analysis.In an embodiment, the volume by volume ratio of the sample: chemical reagent(s)is in the range of 1:0.5 to 1: 3.In an embodiment, the mixing and reaction time of the sample and chemicalreagent(s) under gentle swirling is done for time period in the range of 5-15 seconds.In an embodiment, the kit and / or testing is / are used / done at temperature in therange of 15-40°C.In an embodiment, the reagent bottle(s) containing said chemical reagent(s) is / arestored at temperature in the range of 4-40°C, and at pH in the range of 6.5-8.5 asaqueous solution or powder.While the foregoing description discloses various embodiments of the disclosure,other and further embodiments of the invention may be devised without departingfrom the basic scope of the disclosure. The invention is not limited to thedescribed embodiments, versions or examples, which are included to enable aperson having ordinary skill in the art to make and use the invention whencombined with information and knowledge available to the person havingordinary skill in the art.EXAMPLESThe present disclosure is further explained in the form of following examples.However, it is to be understood that the foregoing examples are merely illustrativeand are not to be taken as limitations upon the scope of the invention. Variouschanges and modifications to the disclosed embodiments will be apparent to thoseskilled in the art. Such changes and modifications may be made without departingfrom the scope of the invention.Example 1: Analysis of Sample using the kit disclosed in the present disclosure:1. Sample Collection:- Fresh milk samples are collected from cows, both pregnant and nonpregnant.- The milk is separated into distinct samples for testing with differentchemicals.2. Reagent Addition and Reaction:- Chemical Reagents are added to the milk samples in different ratios totrigger chemical reactions that help identify pregnancy.- Reagents Tested:- Chemical copper sulphate:- Result: Effervescence and curdling were observed inpregnant cow milk, but inconsistent results were foundacross samples.- Chemical Acetocaramine red dye:- Result: When mixed at a 1:2 ratio (1 mL of milk to 2 dropsof chemical), a whitish-red curdling color was seen inpregnant milk, and a pinkish-red (dark) color appeared innon-pregnant milk after swirling for 5-10 seconds. Thisreaction was reliable but not the most prominent.- Chemical Bromophenol Blue:- Result: At varying milk-to-reagent ratios (1:1, 1:2, 1:3),whitish-blue color with slight curdling appeared in pregnantmilk, and dark blue / purple color appeared in non-pregnantmilk. The 1:2 ratio showed the most consistent andsignificant results.3. Observations and Results:- Among the chemicals tested, Chemical Bhromophenol Blue provided themost reliable and consistent results, especially when used at a 1:2 milk-toreagent ratio.- The method involves observing the color change and the degree ofcurdling after the reagent is added and swirled for 5-10 seconds.Working Example of the InventionChemical Bromophenol Blue (Preferred Reagent):- Procedure:- Milk samples are collected from both pregnant and non-pregnantcows.- The chemical Bromophenol Blue is added to the milk samples atratios of 1:1, 1:2, and 1:3.- After swirling for 5-10 seconds, the color change is observed.- Results:- Pregnant Cow Sample: Shows a whitish-blue color with slightcurdling.o Non-Pregnant Cow Sample: Shows a dark blue / purple color withminimal curdling.- 1:2 Ratio: Most significant and consistent results across allsamples, making it the optimal ratio for this assay.Key Findings and Discussion- Consistency of Results:- The chemical Bromophenol Blue at a 1:2 ratio demonstrated themost reliable and consistent colour changes, which can be used as adiagnostic tool for pregnancy detection in cattle.- Potential for Commercial Use:- The method is simple, cost-effective, and non-invasive, making itideal for small-scale and rural farmers who cannot access costlyequipment or specialized personnel.Example 21. Non-Invasive Milk-Based Method- Collecting a milk sample from a cow and testing it-no rectalpalpation or ultrasound required.2. Specific Colorimetric Reagent (Chemical Bhromophenol Blue)- The composition and use of chemical Bhromophenol Blue thatreacts with milk components to yield a pregnancy-dependent colorchange.3. Defined Milk-to-Reagent Ratio and Protocol- Volumetric ratio of 1 mL milk : 2 mL reagent- Gentle swirling for 5-10 seconds- Observation window of ≤ 10 seconds4. Distinct Visual Indicators- Whitish-blue curdling for pregnant samples- Dark blue / purple with minimal curdling for non-pregnant samples5. Portable Diagnostic Kit- Kit components (reagent bottles, milk-collection tubes, droppers)- Integrated color reference chart and instructions6. Early-Stage Detection Capability- Reliable detection at stages earlier than conventional methods (e.g.≤ 30 days post-conception)7. Field-Deployable, Farmer-Friendly Format- Shelf-stable reagents, no need for electricity or lab infrastructure- Minimal training requiredFIGURE 3 shows representative drawing showing the test results of positiveand / or negative sample with colour differentiation, in accordance with anexemplary embodiment of the present invention.FIGURES 4A-4I shows representative drawings showing the test results ofpositive and / or negative sample with colour differentiation from pregnancy of 1month to pregnancy of 9 months, in accordance with an exemplary embodimentof the present invention.FIGURE 5A-5H experimental result conducted to see effect of addition of SDS(Sodium dodecyl sulphate) of 0.08gm till 20ml in both pregnant and non-pregnantanimal milk respectively, in accordance with an embodiment of the presentinvention.The addition of SDS (Sodium dodecyl sulphate) of 0.08gm till 20ml afterwards itcan be increased to 0.1gm will lead to enhanced differentiation of colour changein both pregnant and non-pregnant animal milk respectively. This will aid inbetter differentiation of pregnant and non-pregnant animal classification frommilk testing. Also, incubation for just 5 minutes will lead to colour change. Theenhanced colour differentiation: Detection level from 25 days onwards can bedone by comparison of colour chart and adding of the SDS improves the detectionlevels.Table 1: The bromophenol blue addedFigure 5A shows top view of the on spot colour differentiation within 5 minutes. Ifigure P indicates pregnant cow milk sample, and NO indicate non-pregnant cowmilk sample.Figure 5B shows color change in within 10 minutes of stirring.Figure 5C shows color change in 12 hours of incubation.Figure 5D shows color change in 24 hours of incubation.Figure 5F shows color change in 12 hours and 24 hours of incubation.The present invention discloses a non-invasive, colorimetric method for earlypregnancy detection in cattle using milk samples. The process involves theaddition of a specific reagent (Bhromophenol Blue) that reacts with the milkcomponents differently in pregnant and non-pregnant samples, producing a quick,visible color change. The method is cost-effective, easy to use, and suitable forfield application by farmers and veterinarians alike.TableField Accuracy: The method as per the present invention has been tested invarious dairy units with consistent results, especially when using chemicalBhromophenol Blue at the 1:2 milk-to-reagent ratio. However, variations mayoccur depending on individual herd characteristics, lactation stages, and milkcomposition.Potential Interferences: While the current method has been optimized for generaluse, certain environmental factors like temperature or pH may alter the results.Testing across different environmental conditions can provide insights into howthese factors affect the reaction.Reagent Stability: The reagent (chemical Bhromophenol Blue) is stable underambient conditions for a period of several months, provided it is stored in a cool,dry place. Proper storage instructions will be included in the diagnostic kit tomaintain accuracy and consistency.Digital Standardization: Future iterations of this diagnostic method could includea smartphone-based app for image comparison and result interpretation,enhancing the ease of use for farmers. The app could leverage a color-matchingalgorithm to provide instant feedback and even suggest follow-up actions.Regulatory Compliance: This invention will undergo necessary regulatoryapprovals, including veterinary and food safety guidelines, before massdistribution. It is essential that the diagnostic kit complies with local standards forveterinary diagnostics and animal health in different markets.Robustness and Scalability: The kit's design emphasizes portability andsimplicity, ensuring that it can be used in remote or rural areas with minimalinfrastructure. This aligns with the goal of empowering farmers with affordable,reliable pregnancy detection tools, even in resource-limited settings.To summarize, the present invention discloses a field-deployable kit (100) and anassociated method (200) for non-invasive, rapid, and visually interpretabledetection of pregnancy in mammals, particularly in livestock such as cattle,buffalo, goat, and sheep. The kit (100) comprises a series of modular componentsthat are physically arranged and functionally interconnected to facilitate stepwisedetection and interpretation by untrained personnel in rural, resource-constrainedenvironments.Component 1: First Vial for Sample Collection

[102] : At the core of the system isthe first vial (102), a sterile, graduated plastic or borosilicate glass containerdesigned for manual milk collection from the subject animal. This vial mayinclude a snap-on or screw cap and graduation marks in mL (e.g., 1 mL to 10 mL)to ensure accurate volumetric measurement of the milk sample. This container isergonomically designed to be held with one hand and positioned under the udderto aseptically collect 1-2 mL of milk during routine milking operations.Component 2: Reagent Bottle(s)

[104] : Adjacent to the first vial in the kitcontainer is a reagent bottle (104). This is a sealed, light-protective polypropyleneor amber-glass bottle that contains the chemical reagent in liquid or lyophilizedpowder form. The preferred reagent is bromophenol blue, a pH-sensitive dye withspecific binding characteristics toward milk proteins and other pregnancyassociated markers. Alternative or auxiliary reagents such as acetocaramine reddye or copper sulfate may be included for comparative trials or multi-stepvalidation. The reagent bottle is labeled with expiry date, optimal temperaturerange (4-40°C), and pH range (6.5-8.5). The bottle is pre-calibrated to dispense 2mL per squeeze / dropper, ensuring a 1:2 volumetric milk-to-reagent mixing ratio,which has been experimentally determined to yield the most distinct and reliablediagnostic output.Component 3: Second Vial or Test Strip for Mixing

[110] : The second vial or teststrip (110) functions as the reaction chamber. It may be: a small, transparentdisposable vial with a conical base for observing sedimentation or curdling; or astrip made of absorbent cellulose or polymer matrix, coated with dry-formbromophenol blue, where milk is directly dropped.In both formats, the collected milk (from vial 102) is transferred to this mixingzone using a dropper or pipette (either integrated or separately provided). Then,the reagent from bottle (104) is added. The components are mixed by gentleswirling (5-10 seconds). The user observes for a visual colorimetric change that istypically completed within 2 to 60 seconds under ambient temperature conditions(15-40°C).Component 4: Color Reference Chart

[106] : The color reference chart (106) is alaminated, waterproof card affixed to the inside lid of the kit box or attached as aseparate foldable panel. It comprises visual color indicators mapped to diagnosticinterpretations:- Whitish-blue -> Pregnancy-Positive (presence of associated milk-boundbiochemical changes).- Dark blue or purple -> Pregnancy-Negative.The chart may include reference images, RGB hex codes, and pictogramsto assist even illiterate users. It serves as a non-electronic interpretationtool, ensuring no digital infrastructure is required.Component 5: Instruction Manual

[108] : The instruction manual (108) is printedin local languages and comprises stepwise diagrams and QR codes linking tovideo demonstrations for usage. It specifies:- The recommended sample volume (1-2 mL),- The reagent volume (2 mL),- The swirling time (5-10 seconds),- The reaction observation window (2-60 seconds), and- The interpretation protocol using the color chart (106).The manual may also include troubleshooting guidelines, reagent storagerecommendations, and warnings to avoid contamination.Communication and Interaction Among ComponentsThe components are physically arranged within a compact, portable kit box withpartitioned compartments. The communication or operational flow amongcomponents proceeds sequentially as follows:1. Milk sample (collected in 102) is visually assessed for freshness.2. The reagent (104) is then dispensed in a measured dose into reactionchamber (110) already containing milk.3. After mixing, the color change is directly observed, and the result ismatched with chart (106).4. The manual (108) assists in guiding the timing, technique, and diagnosis.In an optional digital implementation, a mobile app or smartphone camera can beused to scan the reaction color and auto-compare it with internal color datasets,increasing objectivity in results. This upgrade may utilize Bluetooth or imageprocessing-based modules for digital color interpretation.Examples of Techniques, Algorithms, and Best Mode- Colorimetric Chemistry Technique: Use of bromophenol blue, a pH- andprotein-binding indicator dye, exploits changes in milk composition duringearly pregnancy (e.g., variations in casein and lactoferrin content).- Best Mode Implementation: The 1:2 milk-to-reagent volumetric ratio, withbromophenol blue at concentration of 0.04% w / v in an aqueous bufferedsolution (pH 7.0-7.2), yields fast and reproducible color responses in pilotfield trials.- Storage Mode: Reagent (104) is stored in a phthalate-free plastic bottlewith silica desiccant sachets in the kit box, maintaining optimal reagentstability across climate zones.- User Interface Mode: The kit is designed for single-person field operation,requiring no power, no refrigeration, and usable during routine milking.Field Implementation of the Invention: To detect pregnancy in a cow using thediagnostic kit (100) comprising components

[102] ,

[104] ,

[106] ,

[108] , and

[110] .Step 1: Sample Collection - First Vial

[102] A 2-year-old Holstein Friesian cow, 28 days post-artificial insemination, issuspected to be pregnant. A fresh milk sample is manually collected duringmorning milking.- Material Used: First vial (102) - a 10 mL sterile plastic sample vial withscrew cap and 1 mL graduation.- Procedure: Using gloved hands and a clean collection cup, 2 mL of milk isaseptically collected directly into vial (102) from one quarter of the udder.Step 2: Preparation of Reagent - Bottle

[104] - Material Used: Reagent bottle (104) containing bromophenol blue solution(0.04% w / v), buffered to pH 7.0, pre-calibrated to dispense 2 mL persqueeze.- Storage & Handling: Bottle stored at room temperature (28°C) in a coolbox. Label checked to confirm expiry and pH range.Step 3: Mixing - Second Vial / Reaction Chamber

[110] - Material Used: Second vial (110) - a 5 mL transparent conical-bottompolypropylene tube with snap cap.- Procedure:- 1 mL of milk from vial (102) is transferred into vial (110) using asterile dropper.- 2 mL of bromophenol blue reagent from bottle (104) is added tothe same vial, achieving the preferred 1:2 milk-to-reagent ratio.- The vial is gently swirled by hand in a circular motion for 7seconds.Step 4: Observation - Color Development (2-60 Seconds)- Ambient Conditions: Room temperature of 31°C, open shed environment,indirect sunlight.- Reaction Result:- Within 5 seconds, the solution begins to curdle slightly.- By 10 seconds, a distinct whitish-blue color forms with visiblethickening.Step 5: Interpretation - Color Chart

[106] - Material Used: Color reference chart (106), laminated card with:- Whitish-blue = Pregnancy-Positive- Dark blue or Purple = Pregnancy-Negative- Procedure: The test result is visually compared to the chart under naturallight.- Result: The mixture matches the whitish-blue zone, indicating that the cowis likely pregnant.Step 6: Instruction Follow-up - Manual

[108] Material Used:Instruction manual (108) printed in English and Marathi, containing:- Illustrated procedure steps- Troubleshooting (e.g., reagent expired, color too faint)- Disposal instructions for used reagents and vialso QR code linking to a demonstration video- User Action: The farmer confirms the interpretation and enters the resultinto a record-keeping notebook for herd tracking.Expected Outcome and Confirmatory Testing:Initial Interpretation: Pregnancy-positive, based on visible whitish-blue reactionwithin 10 seconds.Follow-up: The cow is monitored and later confirmed pregnant through veterinaryultrasound at 45 days post-insemination, validating the test result.Advantages Demonstrated in This Example- Non-invasive: No rectal palpation or ultrasound required.- Low-cost: Reagents and kit are reusable or low-cost per test.- Fast: Result in <10 seconds, actionable in the field.- User-friendly: No technical skills required.- Scalable: Same procedure replicable for buffalo, goat, sheep.ADVANTAGES OF THE INVENTION:- The invention provides a non-invasive, colorimetric, and field-readyanalysis method using milk and simple reagents, for early detection andbroader accessibility of pregnancy in mammals like cattle.- Simple and economic.- A user-friendly kit for detection of pregnancy using milk samples for earlydetection.- Easy accessibility and affordability due to commercial availability.- User-friendliness for point-of-care testing, providing faster and reliableresults.

Claims

1. A kit (100) for non-invasive, in-vitro detection of pregnancy in mammals using milk samples, the kit comprising: a first vial (102) for collecting a milk sample from a mammal; at least one container (104) comprising a chemical reagent comprising bromophenol blue in solution or powder form; a color reference chart (106) comprising at least two color indicators corresponding to pregnancy-positive and pregnancy-negative results; a second vial or test strip (110) for mixing the milk sample with the chemical reagent; an instruction manual (108) describing the procedure for mixing the milk sample and the reagent in a defined volumetric ratio in the range of 1:1 to 1:3 and observing the color change within 5-10 seconds to determine pregnancy status; wherein, upon mixing said milk sample with bromophenol blue at a milkto-reagent ratio of approximately 1:2, milk from a pregnant mammal yields a whitish-blue curdled solution, and milk from a non-pregnant mammal yields a dark blue or purple solution with minimal curdling, thereby enabling early-stage pregnancy detection based on observable colorimetric change.

2. The kit as claimed in claim 1, wherein the mammal is selected from cattle, buffalo, goat, or sheep.

3. The kit as claimed in claim 1, wherein the color reference chart comprises: a) a whitish-blue color indicating a pregnancy-positive sample; and b) a dark blue or purple color indicating a pregnancy-negative sample.

4. The kit as claimed in claim 1, wherein the chemical reagent is bromophenol blue and optionally includes additional dyes selected from acetocaramine red or copper sulfate for comparative testing.

5. The kit as claimed in claim 1, wherein the reagent is shelf-stable at a temperature range of 4-40°C and has a pH in the range of 6.5-8.5.

6. The kit as claimed in claim 1, wherein the reaction is performed under gentle swirling or agitation for a period of 5-15 seconds, and color change is observable within 2 minutes - 3 Days.

7. The kit as claimed in claim 1, wherein the mixing is conducted in ambient temperature conditions ranging from 15-40°C.

8. A method (200) for in-vitro detection of pregnancy in a mammal, the method comprising: collecting a fresh milk sample in a first vial (102); mixing the milk sample with a chemical reagent comprising bromophenol blue from a second vial (104) in a volumetric ratio in the range of 1:1 to 1:3, preferably 1:2, under gentle swirling for 5-10 seconds at ambient temperature (25-40°C); and observing the resultant color and comparing it with a pre-defined color reference chart (106), wherein a whitish-blue color indicates a pregnancy-positive sample, and a dark blue or purple color indicates a pregnancy-negative sample.

9. The method as claimed in claim 8, wherein the milk sample is obtained from cattle, buffalo, goat, or sheep.

10. The method as claimed in claim 8, wherein said visual observation is completed within an observation window of 2 to 60 seconds after reagent mixing.