Concept of bypass protein

Concept of bypass protein, with results of different articles published and different techniques along with values

1. Concept of By-pass nutrients Bypass Protein Vishnu Vardhan Reddy.P TVM/2015-029 Department of Animal nutrition College of Veterinary Science, Tirupati Sri Venkateswara Veterinary University

2. Definition of Bypass Protein ‘‘Rumen protected’’ has been defined by the Association of American Feed Control Officials (Noel, 2000) as: ‘‘ A nutrient(s ) fed in such a form that provides an increase in the flow of that nutrient(s), unchanged, to the abomasum, yet is available to the animal in the intestine .’’ (NRC 2001 Pg.no:53)

3. Sources of Bypass Protein 1. Naturally Protected Proteins 2. Heat Treatment 3 . Chemical Treatment 4. Esophageal Groove 5 . Post Rumen Infusion (Fistula ) 6. Encapsulation of Proteins 7. Amino Acids Analogs 8. Lowering Ruminal Protease Activity 9. Decreasing Retention Time in Rumen

4. Naturally Protected Proteins Feed UDP % Maize (grain) 65 Barley 21( 11-27) Sorghum 52 Bajra 68 Oat grain 14–20 Wheat grain 20–36 Cotton seed meal 41–50 Linseed meal 11–45 Ground nut meal 30 Rapeseed meal 23 Soybean meal 28 ( 15–45) Sunflower meal 24 Subabul 51 – 70 Feed UDP % Blood meal 76 – 82 Fish meal 71 – 80 Meat meal 53 – 76 Brewers dried 53 Corn gluten 53 Wheat bread 29 Corn silage 27 Rice straw 63 Wheat straw 45 Para grass 52 Cow pea 32 – 45 Berseem 37 – 52 Alfa-Alfa 28 (NRC, 1985; Dutta et. al ., 1997)

5. Heat Treatment Heat processing of feed decreases protein degradation in the rumen by denaturing proteins and the formation of protein–carbohydrate cross-links called as Maillard reactions and protein–protein cross-links. (Animal Nutrition by McDonald seventh edition Pg.no:566)

6. According to the article “ Estimates of protein fractions of various heat-treated feeds in ruminant production” by Ho Thanh Tham , Ngo Van Man and T R Preston. Experiment feeds: Cassava or Tapioca (Manihot esculenta, Crantz) ( CLM ) Sesbania (Sesbania grandiflora) (SG ) Leucaena or S ubabul (Leucaena leucocephala) (LL ) Gliricidia (Gliricidia sepium) (GS ) Water hyacinth (Eichornia crassipes) (WH). Processing methods: Heat treatment 60°C , 100°C , 140°C for 2 hours at each temperature.

7. Effect of heating on Fraction A (g/kg CP) in leaf samples Feed 60°C 100°C 140°C Decrease in % Cassava 114 111 71 37.7 % Sesbania 465 452 411 2.8 % Subabul 390 375 333 14.6 % Gliricidia 186 170 78 58 % Water hyacinth 142 133 115 19 % Effect of heating on Fraction B1 (g/kg CP) in leaf samples Feed 60°C 100°C 140°C Decrease in % Cassava 42 29 3 92.8 % Sesbania 19 10 7 63 % Subabul 126 42 25 14.6 % Gliricidia 138 134 57 80 % Water hyacinth 155 67 62 60 %

8. Effect of heating on Fraction B2 (g/kg CP) in leaf samples Effect of heating on Fraction B3 (g/kg CP) in leaf samples Feed 60°C 100°C 140°C Decrease in % Cassava 663 651 359 92.8 % Sesbania 434 450 309 63 % Subabul 421 497 545 14.6 % Gliricidia 553 503 244 80 % Water hyacinth 213 263 15 60 % Feed 60°C 100°C 140°C Increase in % Cassava 93 109 429 78.3 % Sesbania 45 54 241 81.3 % Subabul 93 96 100 7 % Gliricidia 22 84 494 95.5 % Water hyacinth 437 485 760 42.5 %

9. Effect of heating on Fraction C (g/kg CP) in leaf samples Feed 60°C 100°C 140°C Increase in % Cassava 88 100 138 36.2 % Sesbania 36 34 32 -11 % (decrease) Subabul 37 55 60 38.3 % Gliricidia 101 109 128 21 % Water hyacinth 54 53 48 -11 % (decrease) Heating the leaves to temperatures of 140°C for 2 hours reduced the proportion of the protein in the A and B2 fractions and increased the B3fraction . Conclusions

10. According to the article ”Optimization of roasting conditions for soybean cake evaluated by in situ protein degradability and N-fraction method” by Snjay kumar , t.k.walli , rajani kumari . Feed sample: Soybean cake Treatment method: Roasting at 140, 150, 160, 170°C for 30 min.

11. Different nitrogen (% of total N) fractions in raw and roasted soybean, roasted at different temperatures for 30 min A+B1 fractions are positively related to ECPD B2 fraction negatively related to ECPD Sample A+B1 (PBSN) B2 (PBIN-NDIN) B3 (NDIN-ASIN) C (ADIN) ECPD Raw (Without roasting) 35.22 63.30 0.405 1.08 58.5 140°C/30 min 32.28 66.03 0.780 0.81 50.2 150°C/30 min 29.67 67.90 2.008 0.482 48.3 160°C/30 min 25.88 70.89 2.14 1.09 46.0 170°C/30 min 22.88 71.12 3.36 2.64 45.1

12. ECPD=33.63424026+0.89824611(A+B1)-0.174329925(B2)-0.2541073921(B3)+1.398296608(C) ECPD=46.256225949+0.8253277191(A+B1)-0.320238478(B2)-0.419856296(C) ECPD=42.73418756+0.810303725(A+B1)-0.251985896(B2) ECPD=22.50895891+0.917516113(A+B1) Conclusion Roasting at 160°C for 30 min was optimum time and temperature fro soybean to make it good bypass protein

13. Chemical Treatment Formaldehyde treatment Lignosulfonate treatment Xylose Treatment Tannic acid treatment

14. Formaldehyde treatment Treatment of high quality proteins result in the formation of cross-links with amino group and makes the protein less susceptible to microbial attack ( Czerkawski , 1986). These bonds are highly stable in the near neutral pH of the rumen but are readily hydrolyzed in the acidic pH of the lower digestive tract.

15. Action of Formaldehyde as follows: Formation of methylol groups on terminal amino groups of protein chain and epsilon amino group or lysine Condensation of these groups with primary amide of group of asparagine and glutamine, and guanindyl group of arginine. the condensation results in formation of intermolecular and intramolecular methylene bridges. These bridges are broken down in acidic medium of abomasum with liberation of formaldehyde (Frankel- Convat and Oleott 1948)

16. According to the article “Effect of processing on protection of highly degradable protein sources in steers” by M.Yugandhar K umar and A.Ravi . Experiment feeds: Babul seed cake, Coconut cake, Dried poultry waste, Guar meal, Mustard cake, Rape seed meal, Tobacco seed cake. Processing methods: Heat treatment at 125°C for 3 hours. Extrusion cooking at temperature 100-120°C screw speed 300-320rpm, feeder rate 10-12 rpm, and products were cut into 1.5 cm and sundried and ground. Formaldehyde treatment with 3.5 gm. HCHO/100 gm. of CP. Animals used: Four Ongole X Holstein crossbreed steers.

17. Effect of treatment on protein degradation kinetics of protein supplements

18. Effect of treatment on degradation kinetics of protein fractions and effective protein degradability

19. Heat treatment is better for reducing EDP of Rape seed meal, and Coconut cake Formaldehyde treatment for mustard cake, Tobacco seed cake, Heat or HCHO treatment for Babul seed meal, Rape seed meal, and Mustard cake. Dried poultry waste and guar meal were resistant to different processing methods. Extrusion cooking was least effective of the three methods. Conclusion:-

20. According to article “ Effect of Varying Levels of Formaldehyde and Heat Treatment on in situ Ruminal Degradation of Different Vegetable Protein Meals” by Faran hameed and Talat naseer pasha Feed samples: Maize gluten meal (60 %), Rapeseed meal, Sunflower meal, Cottonseed meal Treatment method: Formaldehyde treatment 0.50, 1.00 and 1.50% levels Autoclaving for 0, 30, 45 and 60 minutes at 15 pound steam pressure Animals used: Fistulated male buffalo calf

21. % of RUP values of feed samples as treated by formaldehyde and autoclaving at 24 hours incubation Formaldehyde Treatment Maize Gluten Meal, 60% Rapeseed Meal Sunflower Meal Cotton seed Meal Un treated 66.82 ± 1.61 19.62 ± 1.18 7.15 ± 0.58 20.6 ± 1.39 0.5% 93.68 ± 1.06 74.62 ± 0.56 78.67 ± 3.24 46.29 ± 2.33 1% 97.09 ± 3.10 84.75 ± 0.87 68.9 ± 3.61 50.59 ± 1.54 1.5% 89.55 ± 0.85 89.75 ± 1.35 79.25 ± 1.27 49.46 ± 0.68 Autoclaving timing Maize Gluten Meal, 60% Rapeseed Meal Sunflower Meal Cotton seed Meal Un treated 66.82 ± 1.61 19.62 ± 1.18 7.15 ± 0.58 20.6 ± 1.39 30 min 87.27 ± 0.82 47.33 ± 1.43 9.61 ± 0.51 40.92 ± 1.78 45 min 84.38 ± 1.68 41.53 ± 0.60 13.49 ± 0.85 43.0 ± 3.26 60 min 91.09 ± 1.31 50.79 ± 0.61 15.89 ± 1.36 64.05 ± 2.82 autoclaving

22. Conclusion To achieve higher commercial rumen by-pass values maize gluten meal (60 %) and sunflower meal should be treated with 0.5% formaldehyde whereas, for cotton seed meal heat treatment through autoclaving for 60 minutes gave better results. While comparing both treatments formaldehyde treatment is practicable and economical.

23. Lignosulfonate treatment In general , the term "Lignosulfonate" is used to describe any product derived from the spent sulfite liquor that is generated during the sulfite digestion of wood and containing a percentage of lignosulfonic acid or its ash as well as hemicellulose and sugars . ( Windschitl and Stern, 1988)

24. Because lignosulfonates can bind and precipitate protein , it was hypothesized that protein meal treated with lignosulfonates could be rendered less degradable in the rumen. It was concluded that heat and the presence of wood sugars in the lignosulfonate preparation were necessary for a positive response . ( Windschitl and Stern, 1988)

25. According to the article “ Effect of lignosulphonate treatment of groundnut and mustard cake on ruminal protein degradability in cattle” by G Mondal , T K Walli and A K Patra Feed sample: Groundnut cake, M ustard cake Treatment method: Calcium lignosulphonate treatment at the rate of 0, 5, 6 and 7 percent on fresh basis (91.5% DM) with the addition of 10 percent water (weight basis of fresh samples ) Then the treated samples were heated to 95°C for 2 h in a hot air oven (Wright et al 2005 ). Experimental animal used: T hree mature Fistulated male crossbred cattle

26. Feed Level of treatment CP RDP UDP Post ruminal digestibility EPD, % Digestible UDP GNC 0 % 43.2 32.4 10.8 88.3 74.9 9.55 5 % 43.1 24.1 18.9 89.32 56 16.9 6 % 43.1 24.9 18.2 83.5 57.7 15.2 7 % 43.4 24.0 19.4 80.6 55.4 15.6 MC 0 % 33.2 24.5 8.69 85.7 74.1 7.43 5 % 33.5 23.5 9.97 85.4 70.2 8.51 6 % 33.2 22.4 10.8 86.7 67.6 9.36 7 % 33.2 20.1 13.1 83.6 60.6 10.9 Effect of lignosulphonate treatment of GNC and M) on rumen degradable (RDP), undegradable (UDP) protein and digestible UDP, and post ruminal digestibility (percent) of UDP (percent of DM basis)

27. Conclusions The effective ruminal degradability of GNC protein decreased at 5 percent LSO3 treatment and post-ruminal digestibility of GNC protein decreased beyond 5 percent LSO3 treatment. The effective degradability of protein of LSO3 treated MC decreased at 7 percent treatment and post-ruminal protein digestibility was not affected up to 7 percent level. Therefore , from this study it can be concluded that GNC and MC may be treated with LSO3 at 5 and 7 percent levels , respectively, to reduce the ruminal CP degradability without affecting the post ruminal protein digestibility.

28. Xylose Treatment Combination of heat and xylose enhances non- enzymic browning (Maillard reactions) due to the increased availability of sugar aldehydes that react with the protein . Cleale et al. found that treatment of soybean meal with xylose (3 mol xylose/mol lysine) was effective in reducing degradation of soybean protein by rumen microorganisms. (Animal Nutrition by McDonald seventh edition Pg.no:566 )

29. According to the article “ The Effects of Xylose Treatment on Rumen Degradability and Nutrient Digestibility of Soybean and Cottonseed Meals” by P . Sacakli and S. D. Tuncer Feed sample: Soybean and cottonseed meals. Treatment method: water +heat (100°C for 2 hours ) +0.5 % or 1% xylose Experimental animal used : Three ruminally cannulated Merino rams

30. Rumen degradability characteristics and effective degradability values of crude protein of untreated and treated cottonseed meal Feed Treatment a % b % C % /(h) Pe % SBM SBM 12.26 86.70 0.0476 54.60 SBM + WH 12.02 86.50 0.0360 48.20 SBM + 0.5% X 5.90 86.08 0.0247 34.30 SBM +1% X 5.55 74.23 0.0223 28.40 CSM CSM 11.58 81.07 0.0178 32.90 CSM + WH 11.50 77.41 0.0193 33.10 CSM + 0.5% X 11.58 76.57 0.0218 34.80 CSM + 1% X 13.94 86.06 0.0130 31.60 a: the rapidly soluble fraction b: the potentially degradable fraction c: the constant rate of disappearance of b Pe : the effective degradation

31. Conclusion SBM proteins can be effectively protected from degradation in the rumen by xylose treatment through Maillard reaction, without negatively affected in vivo digestibility of protein, whereas xylose treatment appeared to be less efficient on CSM proteins.

32. Tannin treatment The main effect of tannins on proteins is based on their ability to form hydrogen bonds that are stable between pH 3.5 and 8 (approximately). These complexes stable at rumen pH dissociate when the pH falls below 3.5 (such as in the abomasum, pH 2.5-3) or is greater than 8 (for example in the duodenum , pH 8 ). (S . J. Bunglavan and N. Dutta)

33. According to the article “ The Formation of ‘Ruminal Bypass Protein’ ( In Vitro ) by Adding Tannins Isolated from Calliandra calothyrsus Leaves or Formaldehyde” by Elizabeth wina , Dindin abdurohman Experiment material: Crude tannins were isolated from C. calothyrsus leaves protein source is obtained form freeze dried gliricidia leaves, milled soybean meal and casein. Treatment method: Crude tannins at the level of 0, 10, 20, 30, 40 and 50 mg to each 0.5 gm. of protein source in test tubes. Add 10 ml of rumen liquor to each tube and co2 gas id flushed and incubated at 39°C for 48 hours.

34. Dry matter digestibility of casein, soybean meal and gliricidia leaves at different levels of tannin isolate at 48 h of invitro incubation Level of tannin isolate (mg/g protein source) Dry matter digestibility (%) Casein Soybean meal G. sepium leaves 93.27 77.43 49.63 20 88.20 77.70 49.13 40 84.47 74.53 48.23 60 80.67 71.23 43.96 80 80.27 69.68 42.03 100 79.33 69.20 41.10

35. Instead of tannin 37 % formaldehyde solution was added at the level of 2 g/100 g protein source in another test tube (BARRY , 1976 ).Compare the results between Tannins(60mg/g) and Formaldehyde Binding agent Casein Soybean meal G. sepium leaves DM CP DM CP DM CP g/100 g substrate Tannin isolate 6.7 5.3 24.3 27.9 24.4 34.4 Formaldehyde 77.2 81.4 50.6 54.1 23.5 32.1 Tannin isolated from C. calothyrsus can be used as a protein-binding agent and has a similar activity with formaldehyde to bind forage protein ( Gliricidia sepium) Conclusion

36. Esophageal Groove This is normal function in young one. It is done/ good for liquid proteins . Surgically fitted fistula after the rumen in the lower tract of intestine is an easy method to avoid rumen microbial degradation of proteins, so proteins/ amino acids are available in the intestine. Post Rumen Infusion (Fistula)

37. Encapsulation of Proteins Encapsulation of Proteins is usually done for good Biological value proteins and for individual amino acids . They can be given the form of capsule with a combination of fats or fatty acids sometimes by addition of carbonate , kaolin, lecithin, glucose etc.

38. Amino Acids Analogs Structural manipulation of amino acids to create resistance to ruminal degradation is another potential method for rumen bypass of amino acids . Analogs such as Methionine hydroxy , N-acetyl-DL- Metionine , DLHomocysteine thiolactone-Hcl , DL- Homocysteine , etc. have given satisfactory results.

39. Lowering Ruminal Protease Activity By depressing the proteolysis activity of the rumen microbes we can slow down the protein degradation within the rumen. Bacteria are the mainly responsible for proteolytic degradation. So antibiotics can be used to reduce the protein degradation within the rumen.

40. Decreasing Retention Time in Rumen Less the time in rumen environment causes less degradation. Faster pass of feed in the rumen is the explanation. Factors influencing the rate of passage include food intake, specific gravity, particle size, Concentrate to roughage ratio, rate of rumen degradation etc.

41. Importance of Bypass Protein Required for medium and high l actating and growing animals mainly in early lactation. Increase in Milk production by 10-15 %. Good increase in live weight gain of meat purpose animals. Exposes essential and limiting a mino a cids directly to Intestine. Reduces Milk Production cost.

42. References Nutrient Requirements of Dairy Cattle Seventh Revised Edition, 2001. Animal Nutrition by McDonald seventh edition. Effect of processing on protection of highly degradable protein sources in steers by M.Yugandhar Kumar and A.Ravi . Estimates of protein fractions of various heat-treated feeds in ruminant production by Ho Thanh Tham , Ngo Van Man and T R Preston . Optimization of roasting conditions for soybean cake evaluated by in situ protein degradability and N-fraction method by Snjay kumar , t.k.walli , rajani kumari . Effect of Varying Levels of Formaldehyde and Heat Treatment on in situ Ruminal Degradation of Different Vegetable Protein Meals by Faran hameed and Talat naseer pasha Effect of lignosulphonate treatment of groundnut and mustard cake on ruminal protein degradability in cattle by G Mondal , T K Walli and A K Patra 8 . The Effects of Xylose Treatment on Rumen Degradability and Nutrient Digestibility of Soybean and Cottonseed Meals” by P. Sacakli and S. D. Tuncer

43. 7. Effect of lignosulphonate treatment of groundnut and mustard cake on ruminal protein degradability in cattle by G Mondal , T K Walli and A K Patra 8. The Effects of Xylose Treatment on Rumen Degradability and Nutrient Digestibility of Soybean and Cottonseed Meals” by P. Sacakli and S. D. Tuncer 9. The Formation of ‘Ruminal Bypass Protein’ ( In Vitro ) by Adding Tannins Isolated from Calliandra calothyrsus Leaves or Formaldehyde” by Elizabeth wina , Dindin abdurohman 10. Role of bypass protein in ruminant production by Mayank Tandon , R.A. Siddique and Tanuj Ambwani 11. Evaluation of Calcium Lignosulfonate-Treated Soybean Meal as a Source of Rumen Protected Protein for Dairy Cattle by p.m.windschitl and m.d.stern

44. Thank you Vishnu Vardhan Reddy.P TVM/2015-029