Go to the line

Determination of the in vitro gas production.

The in vitro gas production (IVDP) technique measures the evolution of gases (methane and carbon dioxyde) which are produced as end products of fermentation in the rumen. Production of carbon dioxyde is partly from the fermentation and partly as result of formation of short chain fatty acids which expels carbon dioxyde from the bicarbonate buffer solution.

The method is important for feed evaluation, particularly to predict animal performance such as feed digestibility and intake in the ruminant. It has the advantage of the in vitro technique a great number of samples can be evaluate at the same time, it is time saving and cheaper than those methods requiring the use of animals, thus becoming expensives by feed cost.

The IVGP technique provides a great advantage in that the fermentation take place in a glass syringe which allow for several measurements to be made in the same by measuring the gas volume at different intervals of time. This means that not only the possible extent of fermentation can be measured.

The procedure herein described is that of Menke and Steingass (1988)

Instroduction

1/ All substrates should e milled using a 1mm screen. Weigh 200 mg substrate into each   (numbered) syringe and record actual weight. Include a blank (for example. Rumen fluid/ buffer mixture on its own) at the beginning, in the middle of the set, and at the end. A

    sample of hay can be milled and used as a control by including a syringe with the hay at   the beginning and the end of each rum. Samples should be done in duplicate or triplicate.

    After the weighings are completed, grease the plungers with vaseline, and place in   incubator at 38oC. This is normally done the day before the run.

  2/ Measure distilled water, buffer solution, macromineral solution, micromineral solution   and resazurin solution into a round, flat bottomed, flask - warm to 38oC. Then add   reducing solution of sodium sulphide. Place it in a small water bath on magnetic stirrer, put magnet into flask and gently bubble carbon dioxyde through the solution until the blue   color turn to pink, then clear. This means the buffer solution is now reduced. Raise the  carbon dioxyde tube so that it will be above the level of the buffer/ rumen fluid mixture,   but providing a stream of carbon dioxyde into the flask throughout the dispensing   procedure. The pH of buffer should be 7 - 7.3.

  3/ Collect rumen fluid from the donnor animal, strain through gauze into a warm beaker, the   final ratio of rumen buffer fluid should be 1:2 Mix rumen fluid in beaker and transfer to   flask with buffer solution. Make sure the magnet is mixing properly during the whole   process of dispensing the buffer/ rumen fluid mixture into the syringes. Add 30 ml to each  syring using the dispenser (do 2 - 3 times 30 ml amounts into a beaker at the beginning to   be sure the dispenser is properly charger). Fill the syringe, then open the clip and gently   push the plunger of the syringe so that all the gas is removed. Record the level in each   syringe and place it in the water bath.

Times of reading can be chosen to suit the type of substrate in the syringes. For forages 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours and 96 hours are suitable but for concentrate type substrate it may be necessary to take more reading in the first 24 hours. It is advisable to gently mix each syringe     2 - 3 the lectures made at 24 hours and 48 hours.

  Gas production is estimated in ml/ 0.2 g of sample per 48 hours.

Preparation of solution

  1/ Macromineral solution.

        Na2HPO4            5.7 g

        KH2PO4              6.2 g

        MgSO4. 7H2O     0.6 g

    Make up to one liter with distilled water

  2/ Buffer solution

        NaHCO3         35 g

       (NH4)HCO3      4 g

      Make up to one liter with distilled water

  3/ Micromineral solution

        CaCL2. 2H2O         13.2 g

        MnCL2. 4H2O        10.0 g

        CoCL2. 6H2O           1.0 g

        FeCL2. 6H2O           0.8 g

      Make up to 100 ml with distilled water

  4/ Reducing solution

        NaOH 1N                2   ml

        Distilled water      47.5 ml

        Na2.7H2O           0.285 g

  5/ Resazurin solution

        Resazurin 0.100 g

   Make up to 100 ml with distilled water

  6/ Preparation of the buffer solution

      Distilled water                                      474 ml

    Macromineral solution (number 1)      237 ml

    Buffer solution   (number 2)                 237 ml

    Micromineral   (number 3 )                 0.12 ml

    Resazurin solution (number 5)             1.22 ml

  Warm to 38oC, then add reducing solution (number 4), prepared fresh for each run.

  Go to the line