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  Section: Plant Lab Protocols
 
 
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Methodology for Amino Acids and Proteins

 
     
 

Determination of net protein utilization, digestibility and biological value
 
The method protein efficiency and net protein ratios was based on the weight gain and feed consumption. For more precise quality of a protein, we need to know the extent of the utilization of protein in the animal system. The amount of protein absorbed and that retained in the body should be determined. Since protein after digestion and absorption gets incorporated into nitrogenous compounds – both proteinaceous and non-proteinaceous of the body – a measure of nitrogen intake, absorption and retention would be an ideal practical approach.
 
Based on nitrogen balanced studies, Net Protein Utilization (NPU), Digestibility (D) and Biological Value (BV) are calculated.
 


 
Principle
The nitrogen content of the test diet is determined. The feed consumption over a five-days-period is measured, leading to a calculation of a total N-intake. The faecal and urinary nitrogens on these five days are also determined. From these values calculation can be made, to know the ‘N retained’ and ‘N absorbed’. NPU, D and BV are then calculated as follows:
NPU =

N retained

N intake

 
 
D =

N retained

N intake

 
BV =

N retained

N intake

 
 
Materials
Wistar male rats (weighing 65 to 68g)
Metabolic cages
Balances for weighing rats
Diet
Plastic container with tight lids
Sieves
N-free mixture
Sucrose                 9.0%
Cellulose               5.2%
Soyabean oil        5.2%
N-free starch        80.6%
 


 
Procedure
Preparation of Diets
Five hundred g diet (dry weight basis) is prepared for each treatment. Initially, determine the protein and dry matter content in the test samples, then calculate the amount of sample required to give 7.5g N on dry weight basis. To this add 20g mineral mixture and 5g vitamin mixture in order to make up 500g diet (dry wt. basis) and rest of the amount from N-free diet after determining the moisture in N-free diet.
1.
Weigh 40.0g diet on dry weight basis (actual weight will vary depending upon the moisture in the diet) into plastic boxes for each rat sufficient for preliminary period for 4days, and tightly close with the lid. Thus each animal receives 150mg N and 10g dry matter per day through the test period.
2.
Put cages on the rack without funnel.
3.
Weigh the rats in the beginning of the experiment, divide the rats into groups of five, such that average weight of the group differs by no more than 0.5g and record the weight (lesser the difference better the standardization).
4.
Transfer diet equivalent to 10g dry weight to the feed box of respective cage. In all the experiments, group 1 is always allotted for casein diet.
5.
Press the feed with suitable flat surface.
6.
Place plastic bowls below each rat cage for the collection of urine and faeces.
7.
Feed every rat once a day in the morning and check for the water in bottles.
8.
On the last day of the preliminary period, when all the diet from the diet box had been transferred to the rat cage feed box, weigh 50g equivalent of dry weight diet and transfer to respective plastic boxes. Apply Vaseline grease to nylon net as well as perspex funnel.
9.
At the end of preliminary period of four days, again weigh the rats and clean the rat cage feed box as well. Clean inside of the rat cage with lukewarm water.
10.
Put both the grease perspex funnel and nylon net in proper position.
11.
Transfer 10.0g dry weight equivalent diet from respective boxes to cage feed box and follow as in preliminary period for the five days of N-balances period.
12.
Put flask containing 35ml 5% H2SO4 and funnel with glass wool below the perspex funnel to collect the urine and beaker containing 50mL 5% H2SO4 below nylon net to collect faeces.
13.
During five days of the N-balance period daily weighing of the feed from diet box and its transfer to cage feed boxes is followed. Any of the faeces remaining, at the neck of the nylon netting is also transferred to beaker with the help of forcep. If by chance faeces have fallen on the funnel, these are also removed to respective beaker.
14.
Every morning spray the net in situ with small quantity of  20% citric acid from a plastic wash bottle  to prevent N losses and then finally wash the glass wool with small quantity of 5% H2SO­4 for the same reason.
15.
Thus following the procedure, during the N-balance period of five days, urine and faeces are collected respectively in flasks and beakers.
16.
At the end of N-balance period, remove all the water bottles and prevent the access of rats to feed box three hours before the termination of experiment.
17.
Weigh the rats and transfer to bigger cages.
18.
Transfer and remaining feed in the feed boxes and spill try to the respective diet boxes.
19.
Wash bottom lids and lower portion of the case, funnel and nylon net with approx. 75mL of lukewarm water, using a soft brush through a large glass funnel down the urine flask with funnel and glass wool. Further, wash funnel with glass wool three times with water to ensure that all N has been washed out.
20.
Transfer urine plus washings quantitatively to a graduated 500mL flask and make up the volume. Mix well.
21.
To beaker containing faeces, add four times 25ml conc. H2SO4 at hourly intervals. After each addition stir and mix thoroughly with spatula and allow it to cool.
22.
This process is followed four times, the resultant faeces solution would become homogeneous. Transfer this mixture to 500mL volumetric flask and make up the volume.
23.
Weigh the remaining feed in diet boxes and record the note book.
24.
Determine N in urine and faeces by taking out 25mL sample of urine and 50mL sample of faeces by macro-kjeldahl method. Samples are analyzed in duplicate.
25.
Calculate the total amount of N excreted in urine and in faeces by each rat during balance period.
 
 


Procedure to Determine Metabolic and Endogenous Nitrogen
Feed a separate group with four per cent freeze-dried, ether-extracted egg protein as the protein source. Since egg protein at this level is completely utilized by rats, nitrogen in the urine and faeces must be of endogenous origin.
Collect the determined nitrogen in the urine and faeces as described, and incorporate in the calculation.


Calculation
NPU =

N retained

=

I – (F – Fk) – (U – Uk)

N intake

I


D =

N retained

=

I – (F – Fk)

N intake

I


and
BV =

N retained

=

I – (F – Fk) – (U – Uk)

N intake

I – (F – Fk)

where I is the intake nitrogen (nitrogen in the diet), F = faecal nitrogen, Fk = endogenous faecal nitrogen, U = urinary nitrogen and Uk = endogenous urinary nitrogen.




Notes
1. The salt mixture and vitamin mixture compositions are given in the preceding experiments.
2. Casein fortified with one per cent DL-methionine is used as internal standard. This diet has an NPU of 88.


References
1. Pellet, P L and Young, V R (1980) In: Nutritional Evaluation of Protein Foods United Nations Univ Food Nutr Bull Supp 4.
 
     
 
 
     




     
 
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