STUDY ON A/G RATIO IN COW, BUFFALO AND GOAT.

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STUDY OF A/G RATIO IN COW BUFFALO AND GOAT

• NAME ARUNKUMAR KAVAD
• ROLL NO. 6
• GUIDE DR. S.K.MENON DR. KRUPA NANAVATI

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DECLARATION

• 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.

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• 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.

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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.

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• 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.
• So  
• THE AIM OF THE STUDY IS TO STUDY THE
  ALBUMINGLOBULIN RATIO (A/G RATIO) IN BUFFALO
  COW AND GOAT BLOOD.

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Table showing the values of blood components in
animal studied
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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

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• Biuret Method
• 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.
• Reaction
• 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.
• Reaction
• Albumin BCG Albumin-BCG
  complex
• (yellow) (blue)

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• 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.

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• FOR COLORIMETER
• 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.

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• 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.

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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.
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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.
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• 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.)
• Where
• A AlbuminG GlobulinTo. P Total Protein
• From the formula obtained we can differentiate
  the blood samples between the animals studied
  during work.

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DISCUSSION

• 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.

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•  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.)
• Unknown F1F2/2
• 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. 

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LIMITATION 

• 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.

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CONCLUSION

• 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.

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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.

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BIBILOGRAPHY

• 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).

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Thank you !!!