Comparison of Protein Fermentation Characteristics in Rumen Fluid: Gas Production vs Nylon Bag Technique

1. بسم الله الرحمن الرحيم Comparison of protein fermentation characteristics in rumen fluid determined with the gas production technique and the nylon bag technique By : M.A.Al-badwi Prof. Dr. Ahmed M. El-Waziry

2. Introduction • Protein is one of the most important Components of rommant diets • Protein metabolism in the rumen is the result of metabolic activity of ruminal microorganisms. • Ruminants have protein requirement at two levels:- - firstly, to meet the nitrogen (N) needs of rumen microbes. - secondly, to meet the amino acids requirements of the host animal .

3. Evaluation of Nutritive Value of Ruminant Feeds 1. Chemical analysis:

4. 1.1. Near infrared reflectance spectroscopy (NIRS). • 1.2. Electrophoretic analysis .

5. 2. In vivo methods • 2.1- Digestion trials . • 2.2- In Situ technique(nylon bag technique or in sacco technique)

6. In Situ technique • The principle of in situ method . • Standard technique in many feed evaluation systems . • Determine rate and extent of feed degradation in the rumen. • Divides a feed sample in a washout (W), (U) and (D) fraction. • The advantage : - That degradation characteristics of all chemical components in a feed can be investigated. • Disadvantages: - very laborious and so expensive • a) Sample size to bag surface ratio • b) Bag pore size • c) Diet of the host animal • d) Number and species of animals • f )Sample preparation • g) Washing the bags • h) Interpretation of results

7. 3. In vitro methods: • Techniques that measure feed composition, digestibility of nutrients, feed degradation, ruminal fermentation and passage rate from the rumen as following: • 3.1- The two stages technique (Tilley & Terry, 1963). • 3.2- The rumen simulating technique (Rusitec), 1977. • 3.3- Enzymatic method . • 3.4- In vitro gas production technique .

8. In vitro gas production technique • Anaerobic digestion of carbohydrates by ruminal microbes produces VFA, C02, CH4 ,and traces of H2 . So, measurement of gas production in vitro can be used to study the rate and extent of digestion of feedstuffs. • Developed to estimate protein fermentation in the rumen. • The advantage : • It is quick and cheap, and many data can be collected in one run. • a) Sample preparation and size. • b) Buffer . • c) Incubation time & time of reading.

9. In situ measurements Three mature rumen fistulated cows fed ad libitum a ration containing: 720 g/kg forage (DM basis), including on a DM basis Grass hay (23 g/kg) Grass silage (143 g/kg) Maize silage (437 g/kg) Pea silage (65 g/kg) Maize gluten meal (127 g/kg) Corncob silage (96 g/kg) Solvent extracted soybean meal (52 g/kg) Solvent extracted rapeseed meal (52 g/kg) Minerals (5 g/kg) feeds were incubated at the same time (3, 8, 16, 48 and 336 h) 35 bag/cow Sample 5 g (collected , washed for (40 min) ,dried at 70 ◦C (24 h analysed for DM, ash and CP The washout fraction (W) of N (CP) was determined by washing with cold tap water. The residue of Nafter 336 h incubation was considered to be the undegradable fraction (U). The degradable fraction (D)was calculated as 1 − W − U.

10. In situ measurements Two non-lactating rumen fistulated • fed on a diet composed of 1 kg of standard compound feed containing : • - 150 g/kg starch, offered in the morning, and ad libitum • grass hay in the morning and afternoon. • Animals had free a ccess to water and to mineral–vitamin blocks Rumen fluid samples (after the morning feeding ) Rumen fluid was strained through two layers of cheesecloth Pre-warmed insulated flasks, previously filled with CO2, and kept at 39◦ C under CO2

11. buffer/mineral solution - N-free and contained. - To avoid a too high input of N from the rumen fluid. • Rumen fluid was diluted 1:19 with the buffer/mineral solution. • Added to the buffered rumen fluid rapidly fermentable carbohydrates and incubated at 39 ◦C to be certain that N was the limiting factor to fermentation.

12. pre-incubation was performed in • IN bottle 5 l with continuous flushing of CO2 . • In four small bottles in the gas production equipment, with 60 ml buffered rumen fluid to follow the gas production. • After only 4 h of incubation, gas production had ceased. • All laboratory handlings were carried out under continuous flushing with CO2 . • All samples were incubated in one run in duplicate. • Gas production was recorded for 72 h, using a fully automated system

14. Results:1. Feed samples

15. 2. In situ degradation of CP

17. 3. Gas production incubations :

20. Discussion: • That the excess of rapidly fermentable carbohydrates depleted all obtainable N. • Carbohydrates never became limiting. • Fermentation after the maximum capacity of the buffer did not occur. • N was limiting and stopped fermentation. • The gas production for casein and urea (Fig. 3)longer period than incubations with the other samples (Fig. 4a and b).

21. The N in the feed samples was partly soluble (Fig. 1), which means that the other part is not soluble. • The gas production for casein showed higher gas productions at higher amounts of casein per bottle, was not seen for urea. • Casein and N in the feed samples contain amino acids, whereas urea is a non-protein N source.

22. All urea samples (15–100 mg N), gas production stopped completely after 15–20 h. • This implies that the microorganisms need to synthesize these amino acids themselves, needing energy for this process.

23. Most of the gas production appeared to indicate a linear gas production between 5 and 20 h of incubation. • Gas production at different incubation periods correlated with the results obtained by the nylon bag incubations (Table 2). • Highest correlations between the W-fraction and gas production were obtained after 5 h of incubation (r2 = 0.63), and between the rate of degradation (kd) and gas production after 20–30 h of incubation (r2= 0.49–0.51).

24. There was high correlation between the calculated amount of rumen escape protein (REP, Table 2) and the gas production after 12-25 h (r2= 0.83-0.85). • The relationship between the amount of REP and the gas production after 20 h (r2= 0.85) is shown in Fig. 5

25. Fig. 5. Relationship between the amount of rumen escape protein (g/kg CP), as determined with the in situ nylon bag technique,and the gas production per 15mgN after 20 h of incubation (r2 = 0.85).

26. Conclusions : • The gas production technique was originally developed to determine fermentation kinetics of organic matter in rumen fluid. • The gas production technique can be adapted to study protein fermentation kinetics in rumen fluid, by using a N free buffer and using an excess of rapidly fermentable carbohydrates. • There was a close relationship (r2= 0.85) between the gas production and the amount of rumen escape protein, determined with the nylon bag technique

27. السلام عليكم ورحمة الله وبركاته