Title: STUDY ON A/G RATIO IN COW, BUFFALO AND GOAT.
1 STUDY OF A/G RATIO IN COW BUFFALO AND GOAT
NAME ARUNKUMAR KAVAD
ROLL NO. 6
GUIDE DR. S.K.MENON DR. KRUPA NANAVATI
The work done here belongs to Arun Kavad only and any person found to be copying the work or presenting it anywhere will be considered as unlawful and that person will have to face legal actions.
According to Gujarat Pashu-shuraksha Act killing of Cow is punishable.
Animal cruelty is the act of abusing a helpless victim who has no recourse and no voice. It is duty of forensic scientist to be their voice and seek justice for their suffering.
There is paucity of research in field of veterinary forensics however to full fill the keen thirst this study pioneering and completely dedicated to identification and differentiation of domestic animals.
In this study we develop the method for the differentiate the cow blood from the buffalo which is not possible by the present immunological method.
4 AIM OF THE STUDY
Blood is considered very important evidence in any criminal act.
In some cases it becomes necessary to determine the origin of blood sample once it is known that the stain found is of blood.
Test like Precipitin test Haemagglutination-Inhib ition test and Latex test are some test which are been used to determine the origin of blood samples.
But yet work is not been done to determine that to which animal found blood sample belong.
The proteins present in animal blood can give idea to some extend to determine the species origin within animal blood. Albumin and Globulin content in the blood plasma can help to determine the species origin within animal blood.
Albumin and Globulin content in plasma is found to be in different range in different animals.
The AlbuminGlobulin ratio (A/G ratio) can give some specific results which can be helpful to know to which animal the blood sample belong.
THE AIM OF THE STUDY IS TO STUDY THE ALBUMINGLOBULIN RATIO (A/G RATIO) IN BUFFALO COW AND GOAT BLOOD.
6 Table showing the values of blood components in animal studied 7 METHODOLOGY
For study purpose blood samples of animals such as Cow Buffalo and Goat were taken.
Twenty samples of each were taken for analysis. Method use for blood collection in case of animals is different. In case of animals blood can be collected from jugular vein or tail vein.
For collecting samples vacutainers bleeding tubes syringe and needle should be used. The two methods used for collecting blood sample from animals are Jugular bleeding and Tail bleeding.
Here method used for sample collection was Jugular bleeding method.
Mainly two methods are used for analysis
UV Visible Spectroscopy
Colorimeter analysis using Biuret method and Bromocresol green test
Biuret method is based on the principle that Cupric ions (Cu2) in the biuret reagent complex with the groups involved in the peptide bond. In the presence of alkaline media i.e. 3 NaOH (Sodium Hydroxide) and at least two peptide bonds a violet colored chelate is formed.
Proteins Buiret reagent Colored complex.
Bromocresol Green Method
In the presence of Bromocresol Green (BCG) at a slightly acid pH 4.2 serum albumin produces a colour change of the indicator from yellow-green to green-blue. The intensity of the blue colour measured at 630 nm is proportional to the amount of albumin in the sample.
Albumin BCG Albumin-BCG complex
FOR U.V-VISIBLE SPECTROSCOPY
The samples were first centrifuged at 4000 rpm for 5 minutes. As a result of centrifugation all the cells (formed elements) get settle down and plasma is present in the supernatant.
From these supernatant (plasma) is then transferred from centrifuged tube into the test tube and from it 20 microliter is taken out. Into that 20 microliter plasma 5 ml of double distilled water is added to make it dilute. Then that mixture was used to analyze in Ultra-Visible spectroscopy.
Biuret and Bromocresol green test method the samples of blood were centrifuged at 4000 rpm for 5 minutes. Then the supernatant (plasma part) was used for analysis.
In Biuret method supernatant is taken and into it 500µL biuret reagent is added and then it is allowed to get mix for few seconds.After that it is transfer in the cuvette of colorimeter and its analysis is done.
In Bromocresol green method supernatant is taken and into it 500µL bromocresol green reagent is added and then it is allowed to get mix for few seconds. After that it is transfer in the cuvette of colorimeter and its analysis is done.
12 RESULTS DISCUSSION
UV VISIBLE SPECTROSCOPY RESULTS
The graph shows the absorbance (ABS) of the blood plasma obtain after running the sample in UV Visible spectroscopy. The wavelength was same for all the samples. 13 The Absorbance ratio obtained after doing the Colorimetric analysis of both the tests Buiret method and Bromocresol green are mention below Test tube showing reaction between bromocresol green reagent and blood plasma. Test tube showing reaction between biuret reagent and blood plasma. 14
The normal value of total protein in cow is 6-8 g/dL albumin is 2.8-3.9 g/dL and of globulin is 2.9-4.9 g/dL.
The normal value of total protein in goat is 6-7.5 g/dL albumin is 2.3-3.6 g/dL and of globulin is 2.7-4.4 g/dL.
The normal value of total protein in buffalo is 6-8 g/dL albumin is 2.8-3.9 g/dL and of globulin is 2.9-4.9 g/dL.
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The obtained data is then statistically analyzed and two formulas are introduced which are as following
FUNCTIONS AT GROUP CENTROIDS
Unstandardized canonical discriminant functions evaluated at group means
F1 21.6137.033(A) 5.8934(G) -9.023(To. P.)
F2 19.0882.569(A) 6.829(G) 7.502(To. P.)
A AlbuminG GlobulinTo. P Total Protein
From the formula obtained we can differentiate the blood samples between the animals studied during work.
Plasma proteins form three major groups and have various functions albumin (60 of total plasma protein) fibrinogen (4) and globulins (36).
In order to study the qualitative difference in blood plasma proteins of near genera goat cow and buffalo blood plasma UV visible spectra analysis is done. The analysis of blood plasma done by using UV visible spectroscopic does not gave conclusive results as the absorbance value does not show much big difference in the absorbance value.
The other methods opted namely Biuret and Bromocresol green method for determination of blood plasma total protein and albumin absorbance shows high variation. In goat it is 7.9cow-12.9 and buffalo-16.8. Such a difference can help in differentiation. Average value of A/G ratio is 1.5 goats 1.9 cows and 1.0 buffalo it also shows a high variation.
Thus it can be said that the results obtain here after doing the analysis will not show various according to the change in the physiological conditions.
The formula formed here has a very less error so it can be use to determine the origin of blood sample of animals which are studied here.
The formula formed will be useful to determine the origin of blood sample as it has only 2 of error.
F1 21.6137.033(A) 5.8934(G) -9.023(To. P.)
F2 19.0882.569(A) 6.829(G) 7.502(To. P.)
From the results it can be concluded that Biuret and Bromocresol green test give conclusive results to determine origin of animal blood samples by estimating A/G ratio.
The method used here is only applicable to animals (studied) with normal health that is not suffering from any kind of disease.
Age can be considered as a factor but during these work it s not taken into consideration.
Diet can be a factor which can affect the results.
The results obtain from UV Visible spectroscopy show very less variation in the absorbance of proteins of goat cow and buffalo blood plasma.
So it can be said that UV Visible spectroscopy method is not so useful in determination of difference among the blood plasma of goat cow and buffalo.
From the Biuret and Bromocresol green tests A/G ratio is help full to trace the animal blood sample (animal studied).
The absorbance value of total protein can be useful to determine the origin of blood sample of animal (animal studied) as the average absorbance value of goat is 7.9 cow is 12.9 and buffalo is 16.8.
The A/G ratio can also be helpful as its average value is 1.5 in goat 1.9 in cow and 1.0 in buffalo.
21 FORENSIC SIGNIFICANCE
There is a paucity of research related to forensic veterinary to determine the origin of Blood samples as killing the animal is crime but there are some animals which is not cover under the law due to their use as human diet.
So it becomes necessary to differentiate between those domestic animals which come under legislation and to kill them is punishable crime like Cow and those which are free from legislation like Buffalo Goat Ox etc.
For the determination of animal blood origin there are different Immunological methods are available in forensic veterinary which require preciseness and need expertise in performing the test.
To overcome from these draw back there is need of simple sensitive and cost effective methods for determine the origin of blood.
From this study the formula formed can help to determine the blood sample origin.
Human Physiology by Dr. C.C. Chatterjee (1st edition).
Criminalistic by Richard Saferstein Ph. D (7th edition).
A Textbook of Animal Husbandry by G.C Banerjee (8th edition).
Introduction to Veterinary Science by James B. Lawhead and Meecee Baker (2nd edition).
Parikhs Textbook of Medical Jurisprudence Forensic Medicine and Toxicology by Dr. C.K. Parikh (6th edition).
23 Thank you !!!
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