After 16 h (for concentrate feeds) and 24 h (for roughage samples) of incubation (The termination of the incubation should be at the time at which the efficiency of microbial production is maximum, which is difficult to ascertain under normal feed evaluation experiments for a large number of feeds. An approach that could be applied is running of 200 mg sample for the kinetic experiment and calculating the time (t/2) at which the gas production is half of the potential gas production. The time ‘t/2’ is near to the time at which the efficiency of microbial production is the maximum. This means that one has to run a kinetic experiment before determination of truly degraded organic matter and microbial protein using purines or 15N approaches. The time 16 h for concentrate feeds and 24 h for roughages might be a good compromise for routine feed evaluation), measure:
a) True organic matter degradability and gas production (weigh air dried 375 + 5 mg sample in 30 ml medium containing double the level of bicarbonate ions than the conventional medium of Menke et al. (1979) used for estimation of metabolizable energy (ME) and degradability of organic matter (DOM)). Record net gas production (ml gas production in syringe containing feed minus ml gas production in blank). After termination of the incubation, transfer the contents of the syringe quantitatively in a beaker and digest it with the neutral digestion solution for 1 h (the purpose of this treatment is to solubilize the microbes and obtain only the undegraded feed). Filter the contents through crucible no. 2, wash the residue on the crucible with hot water till the residue is free of the detergent. Dry the crucibles at 130o C for 2 h or at 100o C for 10 h (overnight). Record weight of the crucibles after transferring them to a desiccator. This weight minus weight of empty crucible gives the weight of undegraded feed in that particular syringe. [Note: this method of measurement of undegraded residue does not work satisfactorily with tannin-containing feeds/samples (presence of tannin-protein complexes as artifacts in the residue) and starch-rich feeds (some starch might not be degraded by microbes up to 16 h of incubation but it would get solubilized in the neutral detergent solution leading to underestimation of truly undegraded residue). It may be noted that this method of measurement of undegraded residue should not be applied at the initial hours of incubation (before 16 h of incubation) since during this period a portion of the feed, which is undegraded by microbes could be solubilised in the neutral detergent solution.]. Let the weight of this residue (undegraded feed) be ‘a’. Now transfer the crucibles containing this residue to Muffle furnace and ash the sample. The organic matter will disappear leaving the ash. After transferring the crucibles to a desiccator, weigh them and subtract from this weight the weight of the empty crucible to obtain the weight of ash (called as ‘b’), i.e. subtract ‘b’ from ‘a’ to obtain truly undegraded organic matter (a-b). Calculate the organic matter degradability in percentage unit. Please note that for calculation of organic matter degradability, one needs to have dry matter and ash contents of the feed sample. This will enable to calculate organic matter in 375 + 5 mg of the air-dried matter weighed in the syringe for the incubation.
Organic matter weighed into the syringe
=(375 x DM in percentage/100)) minus (375 x (DM in percentage/100) x (Ash in percentage/100)).
Or (375 x DM in percentage/100) (1 – Ash in percentage/100)
Let this value be ‘c’.
Percent organic matter degradability is (a-b)100/c.
b) For most conventional feed resources (not the tannin-containing feeds and starch-rich feed ingredients), microbial mass production can be estimated at the time of termination of the incubation. Please note that this approach should not be applied for the initial hours of incubation (earlier than 16 h).
mg microbial mass production = ((a-b) – 2.2 x net gas in ml)); (a-b) is in mg and 2.2 is the stoichiometric factor.
Efficiency of microbial mass production = ((a-b) – 2.2 x net gas in ml)/(a-b)
c) Calculation of Partitioning Factor (PF). PF is a measure of efficiency of microbial mass production or efficiency of microbial protein production. Higher the factor, higher is the efficiency.
PF = (mg truly degraded organic matter)/ml gas
PF = c – (a-b) in mg truly degraded organic matter divided by ml gas produced in that particular syringe for which ‘c – (a-b)’ has been calculated. This value is the PF.
The theoretical range for PF is 2.74 to 4.41. Any value above or below this range should be critically evaluated. For tannin-rich samples, the PF is normally above 4.41. The higher PF (for example 7.2) would mean that 7.2 mg of the truly degraded organic matter produce one ml of the gas. The high values for tannin-rich feeds appear to be due to the solubilization of tannins from the feeds during fermentation, contributing to the dry matter loss but without contributing to the gas, and inhibition of gas production from cell solubles by tannins through inhibition of rumen fermentation (cell solubles will also contribute to dry matter loss). The presence of tannin-protein complexes in the truly undegraded residue and hence underestimation of the truly degraded organic matter also contribute to the errors in the PF, but the presence of these complexes in the truly undegraded will lower the PF. The PF, which is obtained for the tannin-containing sample is the net effect of solubilization of the feed components during fermentation but not contributing to the gas production and the presence of tannin-protein complexes in the truly undegraded residue.
d) For tannin-containing feeds, the syringe contents are not digested after incubation with the neutral detergent solution (see above) to determine truly undegraded organic matter. Instead, the syringe contents are taken to the determination of purines and/or 15N incorporation studies.
Efficiency of microbial mass production can be expressed as: mg net purines in syringe divided by ml of net gas production (both these values need to be corrected for the blanks).
mg (or microgram) of 15N in apparently undegraded residue/ml of net gas production (both these values need to be corrected for the blanks).
If need be, purine amount can be converted to microbial-N by taking rumen fluid sample, centrifuging it at approx. 17,000 g to obtain microbial pellet, washing it once with distilled water and recentrifuging, and lyophilising it. In a weighed lyophilised pellet, purines can be determined by HPLC or using a spectrophotometeric method after precipitation of purine with silver nitrate. Using this purine to microbial mass ratio, the purine can be converted into microbial mass. If there is a need to convert purine to microbial-N, a portion of the same lyophilised pellet can be subjected to the determination of N using micro-Kjeldahl method or by converting N in the pellet to ammonia and determination of ammonia using sodium nitroprusside and hypochlorite reaction (Makkar and Becker, 1999).
Note: Purines are only in the microbes and not in the supernatant, but 15N is present both in the apparently undegraded residue as well as in the supernatant after the incubation. Therefore, there is a need to take apparently undegraded residue for 15N incorporation studies. The apparently undegraded residue is prepared by centrifuging the syringe content using the high speed centrifuge (17,000 g for 20 min at 4o C), discarding supernatant, washing the residue with distilled water followed by again the high speed centrifugation and discarding the supernatant. The residue should be lyophilized or can be dried in a vacuum oven at approx. 50o C. The weight of this residue should be determined (weight of centrifuge tube plus the residue) minus weight of empty centrifuge tube. This weight needs to be taken into account in the calculations at a later stage. The residue should be quantitatively collected and ground to fine powder preferably using ball mill. In addition to determination of 15N, purines could also be determined in this residue. This section addresses the use of this approach for tannin-containing feeds but this could be used for any feed samples.
e) The gas production can be converted to mmol SCFA production using the equations (I) and (II).
f) Estimation of in vivo metabolizable energy (ME) and digestibility of organic matter (DOM) using the net gas value from 375 mg sample and converting the gas value from 375 mg to 200 mg dry sample or directly incubating 200 mg sample for 24 h.
g) Determination of tannin activity by incubating 375 mg of sample in presence and absence of PEG 6000 in 30 ml of the medium containing double the level of bicarbonate ions (see section ‘a’ above); termination of the incubation at 16 and 24 h; and expressing the results at both 16 and 24 h of the incubation, as:
• Difference in the gas production as percent increase on addition of the PEG compared to the syringe without the PEG,
• Difference in purine as percent increase or decrease on addition of the PEG compared to the syringe without the PEG,
• ME and DOM values with and without PEG (after converting gas value for 200 mg sample), and percent change in ME and DOM values on addition of the PEG (taking the value without PEG as 100 %), and
• Change in the efficiency of microbial protein production when expressed as mg purine (or mg 15N incorporation)/ml gas, on addition of the PEG.
h) Determination of the kinetics of gas production (France et al., 2000) by incubating 200 mg of the sample for 96 h for roughages and 72 h for concentrate samples.
i) Microbial N (MN) could also be measured after incubation by following two nitrogen balance approaches (Getachew et al., 2000b). The first approach is:
MN = TN – (NDF-N + Ammonia-N),
where TN is total N i.e., feed N + N in buffered rumen fluid in the syringe before incubation (at 0 time), NDF-N is the N bound to NDF fraction following incubation and Ammonia-N is the ammonia-N in the supernatant following the incubation. In a closed system, the total N present at the start of the incubation can be in microbial mass, NDF-N, ammonia-N and amino acids during any time of the incubation. Negligible amounts of amino acids and peptides are present in the supernatant during fermentation and therefore these can be ignored in calculation of microbial N.
For determination of NDF fraction for N bound to NDF after incubation, the syringe contents are transferred into a 600 ml beaker and the syringes are washed twice with a total of 50 ml neutral detergent solution (NDS) and emptied into the beaker. The contents are refluxed for 1 h, and then filtered through pre-tarred filter crucibles (no. 2). The crucibles are dried overnight at 100o C and weighed. True degradability can be calculated as the weight of substrate incubated minus the weight of the residue after NDS treatment. The residue after NDS treatment (neutral detergent residue, NDF) is subjected to micro-Kjeldahl digestion for determination of NDF-N.
The second approach is:
MN = APUR-N – NDF-N,
where APUR-N is N bound to apparent undegraded residue after incubation. The preparation of apparent undegraded residue is given above (section ‘d’). N bound to this fraction is determined in a manner similar to NDF-N determination.
From the weight of apparent undegraded residue, apparent digestibility can also be calculated.
• Soon after getting the syringes from the firm, mark the plunger and the corresponding graduated outer portion (barrel) with a diamond pencil (just after opening the case containing the syringe comprising of the plunger and the barrel). Give the same number to both the parts of the syringe.
• The plunger should be properly lubricated using white Vaseline (apply less amount of Vaseline for incubations up to 24 h and more for incubations up to 96 h).
• Collect rumen liquor from both the liquid and the solid phase and handle it properly (use of warm containers, flushing the containers with carbon dioxide, always keeping the rumen liquor under carbon dioxide).
• Reducing solution should be prepared fresh on the same day of conducting the experiment.
• Start flushing the medium with carbon dioxide well before (approx. 10 min) adding the reducing solution. Also flush the medium for at least 10 min after adding the rumen liquor and before starting filling the syringes. Keep flushing the medium with carbon dioxide while filling the syringes (the flow could be reduced at this stage).
• While filling the syringes with the medium, keep an eye on the medium (carbon dioxide gas should be flushing into the medium and the medium should be stirring).
• After dispensing 30 ml of the medium into the syringe, create a light vacuum by pushing back the plunger and then open the clip. This procedure will bring the medium lying in the nozzle back into the syringe. Otherwise there could be a loss of the medium and/or sample.
• After filling of the syringes has been completed (might take 30-40 min), shake the syringes. Shake them again after every 30 min till first 2 h of the incubation, and then after every two hours till the first 10 or 12 h of the incubation. Thereafter, shake the syringes after taking the gas volume readings (24, 30, 36, 48, 60, 72, 96 h, as the case may be). Make sure that all feed particles are taken into the medium while stirring (swirling shaking action might help).
• Wash the dispenser with distilled water immediately after finishing filling the syringes, otherwise the dispenser could get stuck up and might not then be usable.
• Check temperature and level of water in the water bath at least twice a day.
• In the evening before going home, if the plunger is above 80 ml level, push it back, record the readings (before and after) pushing back the plunger.
• When you push back the syringe in the evening, give a shake after approx. 30 min in order to prevent taking up the sample along with the bottom portion of the plunger and out from the incubation medium.
• Use the carbon dioxide gas cylinder with caution. Ask someone if you do not know its operation. Misuse could cause an accident.
• While taking the gas volume readings, use the brown ring marked on the plunger and not the bottom end of the plunger. Keep the syringe in inverted position and in parallel with eye while recording the gas reading. Immediately transfer the syringe into the water bath after taking the reading.
• For cleaning the syringes, the syringe should be emptied (preferably pulling back the plunger and removing contents from the back and not from the nozzle). Clips should be removed. The plunger and the outer graduated part of the syringe (barrel) should be separated. Excess Vaseline on the plunger should be cleared with a tissue paper or a piece of soft cloth, and then transfer both the parts in hot water containing detergent (soap) solution. Rub the plunger with hand and inside of the barrel with a soft brush to clean these. Wash thoroughly both the portions with hot water and finally rinse them with distilled water. Dry them well before weighing sample into the syringe.
• Fix the clip in such a manner (by keeping the portion, where pressure is applied to open or close it, facing the syringe) that it does not open by striking on the edges of the lid of the water bath while taking out the syringe for taking reading.
• Mark the crucibles well (preferably with a diamond pencil). Keep them in increasing or decreasing order; this might help you in identifying the crucibles, which have not been marked well, especially after these have been placed in the Muffle furnace.
• For tannin bioassay use only PEG-4000 or PEG-6000 (preferably the latter).
For a slide show on the gas method, refer to: http://www.iaea.org/programmes/nafa/d3/mtc/invitro-slideshowapr01.pdf