بسم الله الرحمن الرحيم

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

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