Metabolic trial its importance, how to conduct, sample collection, equipment require, analysis and its significance

1. Metabolic Trial
By – Dr Sarang Vajpeyee

2. What actually it is
• Metabolic trials are conducted in animals for determining the balance
of nitrogen, energy, minerals etc. inside the animal body
• In Metabolic trial collection of feces, urine and feeds residue is done
and used for analysis of nutrients intake, digestibility, nitrogen and
mineral balance
• It is generally conducted in confined shed/shelter with underground
place for urine collection

3. Importance
• In animal nutrition the digestibility and metabolism trial aids in
assessing the nutritive value of any feed stuff
• To know about how much a given nutrient is retained in the body for
tissue building and maintenance
• It also helps in judging the suitability of feed for feeding to large
number of animals

4. Why Confinement is Required
• The metabolic cages are used to study the metabolism of feeds in the
animal
• The confinement of animal to enable the total collection of faeces
and urine for determining the digestibility and nutrient (N, minerals)
balance of a feedstuff fed to ruminants

5. Metabolic cage for cattle

6. Cages For Small Ruminant

7. Why it is conducted
• The amount of feed or nutrient intake by an animal which is not excreted in
the faeces is considered to be digested by the animals. The depiction of
digested feed/nutrient as percentage of intake is known as digestibility
coefficient
• However this doesn’t represent the true value of digestibility of a feed or
nutrient because during the period of recording some part of digested
material is returned to the digestive tract after absorption in the body and
excreted in the faeces. In addition to this some secretions of the digestive
system and debris of epithelial cells are also eliminated in the faeces
• These errors are quite large and highly variable for the nitrogenous
constituents and minerals in the feeds

8. Purpose of doing
• The availability of nitrogenous nutrients and minerals is more
accurately determined by their retention (balance) in the body.
Therefore for the determination of the retention of nitrogen and
minerals, metabolism trials are conducted
• The routes of elimination of these constituents from the body are
faeces, urine, expired air, skin secretions, milk ( in case of lactating
mammalian species) and eggs ( in case of laying birds)
• In a conventional balance study the loss of nutrient through skin
being quantitatively little is ignored

9. Procedure
• The feed in question is fed to the animal in known quantity. Precise
recording of feed intake, refusals and faeces voided by the animal are
made, and a sub sample of each is retained for chemical analysis
• When estimates of nitrogen or energy or mineral balance are desired,
urine output also is measured
• Generally a collection period of 5-7 days period is practiced which is
preceded by 7-14 days adaptation for animal to the metabolism
cages, experimental condition and diets

11. Stages
• Pre – collection feeding period
• Collection Period

12. Stages
Pre – collection feeding period
• In order to remove the effect of previous feeding and also to adapt
the animals on the feed to be evaluated, the animals are fed the test
feed for a period of 2-3 weeks
Collection period
• Collection period of 6 or 8 days for ruminant and other herbivorous
animals and 4 days for simple stomached animals

13. Devices used for conducting metabolism trial
• Trial in stall
• Trial in cages

14. Preparation of Feeds
• The roughage to be fed is procured and chaffed (about 2 cm length)
before feeding. Chaffing of roughage is essential for thorough mixing
and drawing of representative sample for chemical analysis
• Total concentrate mixture required for feeding during the pre-
collection and collection period of trial should preferably be prepared
in one lot by mixing all the ingredients thoroughly to ensure uniform
composition
• Diets should be formulated in a such a manner that the animal should
not suffer from the laminitis, rumenitis, acidosis and other
gastrointestinal upsets; however, the primary purpose of metabolic
studies is feed evaluation

15. Preparation of Feeds
• The feeds of pig and poultry are the composite mass of different feed
ingredients prepared by thorough mixing to obtain uniform physio-
chemical composition
• Clean, fresh water should be available to the animal at all times. Food
and water troughs in cages should be located to reduce their
contamination by faeces and/or urine
• Animals should not be without feed for longer than 24 hours, unless
otherwise justified to meet the experimental design protocol, ethical
and welfare issue

16. How to Offer Feed
• The time of feeding is normally fixed between 8 to 10 a.m. the feed is
offered daily at a previously fixed time in the same sequence of
animals
• When diet is made up of concentrate and roughage, the concentrate
mixture is offered first
• Animals normally require 30 min to 1 hour for eating the concentrate
mixture unless the quantity is too large
• Roughage portion of the diet is offered half an hour after eating of
the concentrate mixtures

17. Collection of Feed Sample
• Weighed quantity of the feed is offerd to the animal at a fixed time.
At the same time a small representative sample is taken in a
numbered polythene bag. The sample of feed is brought to analytical
laboratory immediately
• Exactly weighed quantity (50 gm for dry feeds and 100 gm for green
fodders) of feed is dried in hot air oven at 100±1℃ at least for 12 hr
to determine dry matter percent in feed
• Another sample (10 gm for dry feeds and 20gm for green fodders) is
dried in another hot air oven at 60±1℃ for the estimation of gross
energy

18. Collection of Feed Residue
• At the end of 24h the feed left in the trough (feed box) is
quantitatively collected, weighed and mixed thoroughly. After this
sample are drawn and dried for estimation of DM% and gross energy

19. Collection and Aliquoting of Faeces
• Total faeces voided during 24h after offering feed is collected in a
glazed or metallic container and its weight is recorded
• From the thoroughly mixed faeces 3 aliquots are drawn after fixing a
suitable ratio
• The quantity of aliquot may vary from 1/10 to 1/1000 depending on
the total quantity of faecs voided daily.
• One aliquot kept in hot air oven at 100±1℃ for DM estimation and
another aliquot is dried at 60±1℃ for GE estimation
• Aliquot is collected in a numbered wide mouth and stoppered glass
bottle or plastic container in to which 5ml of 25% sulphuric acid is
mixed thoroughly

21. Collection and Aliquoting of Urine
• Total urine excreted during 24 hr collected in narrow mouth glass or
plastic recepticles containing either 10 ml toluene or 25% H2SO4 at
the rate of 5 ml per litre of urine voided
• These chemicals are put in the urine collection recepticles for
preventing the loss of volatile nitrogen. The urine is measured with
the help of graduated measuring cylinder
• One aliquot used for estimation of nitrogen and another for GE and
mineral content

22. Precautions
• For each diet to be evaluated the number of animals should vary between
5-8 and should be of same species, sex and age
• Male animals are usually preferred due to easy collection of faeces and
urine separately. A large number of animals per experiment also helps in
elimination of experimental errors and variations from one animal to the
other
• The animals selected for the metabolism trials should be fed the same diet
for 3-4 weeks prior to experimental collection of so that residual effect, if
any of the previous diet is removed and the animals are adapted to the
new diet
• The individual intake by animal should exactly be measured so as to
determine the accurate intake of nutrients by the animals

23. Precautions
• After a pre-experimental feeding period of 3-4 weeks, faeces and
urine are collected from individual animals for a period of 6 or 8 days
after every 24 hr
• For determination of digestibility coefficients of various nutrients,
collection of urine is not recorded. But for estimation of balance of
various components like energy, nitrogen, calcium, phosphorus etc. It
is essential to measure the quantity of nutrients excreted in urine
• The feed should be offered to the animals at the same time every day
and almost equal amount should be offered daily. If irregular intake
by the animals is reported, the metabolism trial should either be
delayed or the animal replaced with another healthy animal

24. Criteria for the Metabolic Cages
• Metabolism cages may vary in their size and design depending on the type of
animal, purpose, duration of experiment
• All animals should able to stand up, lie down comfortably and stretch within the
metabolism cages/crates.
• Animals may required to be tethered in cages for the length of the collection
process (generally 10 days) to allow complete collection and maintain separation
of the faeces and urine
• Tethering may also be required to prevent the animals turning around during the
collection process
• Any stresses caused by uncomfortable stalls and rough handling can bias
experimental results therefore, it is necessary to give careful consideration to
facilities used in metabolism studies.
• Cages should be designed to be well-drained and minimise the risk of injury or
disease to the animal

25. Criteria for the Metabolic Cages
• Flooring should permit excreta to pass through readily without
imposing risks of hoof or leg injury
• The building housing metabolism crates and cages should provide
adequate ventilation for respiration, removal of excess heat and
waste gases and have appropriate lighting
• The building should be well insulated and properly sited to reduce
high summer temperatures on animals and maintain adequate
temperatures during winter
• Faeces and urine should not be allowed to accumulate and present a
health risk to the animals. Cages/crates should be cleaned each day

26. Length of confinement
• Animals typically have an introductory phase for adjustment to the
metabolism cage/crate, which may include several days for the animal
to become accustomed to being tethered
• The collection period requires a minimum of 7 days faecal and urine
collection, however, this may need to be varied for specific purposes
but should not exceed 14 days for cattle over 400 kg.
• Feed intake is generally determined 24 hours prior to the collection
period given the lag between feed eaten, urine and faeces excreted
• Animals are not exercised during this period, since faeces and urine
would be lost upon removal of the animal from the crates, negating
the purpose of the experiment

27. Care of animals during/after the procedure
• Careful observation by an expert to ensure the animals’ well-being is
necessary
• Any animals that refuse to eat or become ill, indicated by high rectal
temperatures (> 39° C) or by ‘hot’ or inflamed shins, or by persistent
lying, should be shifted to pasture and be treated appropriately to
ensure recovery

28. Analysis of Biological Samples
• Analysis of feed and biological samples should be done according to
approved accepted procedures. It is imperative to become familiar
with the procedures before analysing the experimental samples
Urine
• Before analysis of urine sample, poured it into a clean beaker and
checked for sediment in the bottom of the collected bottle
• If sediments are present then transfer quantitatively and place clean
magnetic stirring bar for magnetic stirring so as to get a uniform
sample before further analysis

29. Faeces
• In general fresh faecal samples are employed for determination of total
nitrogen. Alternatively, samples for fresh faeces may be frozen and freeze
dried. The dried samples are grounded in a wiley mill using 2 mm screen
for further analysis
• For determination of gross energy of faeces the representative sample is
dried at 60℃ in a forced draft oven and such samples are suitable for routine
determination of both nitrogen as well as faecal gross energy. If the samples
are dried at higher temperature (100 ℃) appreciable amount of nitrogen and
energy will be lost
• Always preserve/retain the biological sub-samples (feeds, faeces and urine)
in fresh state under refrigerated condition until all the analysis are
completed and data have been evaluated and interpreted

30. References
• Briggs, H. M. and W. D. Gallup. 1949. Metabolism stalls for wethers
and steers. J. Anim. Sci. 8:479.
• Erwin, E. S., I. A. Dyer, M. E. Ensminger and Wayne Moore. 1956. A
portable metabolism stall for steers. J. Anim. Sci. 15:435.
• Nelson, A. B., A. D. Tillman, W. D. Gallup and Robert MacVicar. 1954.
A modified metabolism stall for steers. J. Anim. Sci. 13:504.
• Rumsey, T. S. 1969. Collection system for cattle and its effect on EKG
and respiratory patterns. J. Anim. Sci. 28:38.

32. THANKS