1. October 2005 Saccharomyces cerevisiae bio-active cell European patent (n.0111202) European patent (n.98116181.3)

2. AN INACTIVATED SACCHAROMICES CEREVISAE (SC) PRODUCED BY A PATENTED PROCESS TO PRESERVE NUTRITIONAL CONTENT

3. USE OF YEAST IN RUMINANTS 40S: first research in USA 50s-60s: researches on SC show growth effects of +6% with 8 gr/head/day

4. USE OF YEAST IN RUMINANTS PUBLISHED STUDIES:  2 BEFORE 1980  41 BETWEEN 1980-1990  150 FROM 1990 TO DATE In USA dairy cows using SC move from 19% in 90s from 51% to day

5. DOXAL starts researching and manufacturing SC 1975

6. Dead or alive cells? Two main questions: Why Saccharomyces Cerevisiae?

7. Piva et al (1984) SC increase fiber digestion in rumen Dawson(1990)Williams(1991) Girard(1996):SC stimulates Megasphaera Elsdenii Federique Chacheyras-Durand INRA (1995): SC stimulates Megasphaera Elsdenii MECHANISM OF ACTION:

8. Chacheyras(1995): SC increases cellulosolytic fungi Feedstuffs n.24(10-6-96) SC inactivates mycotoxins in rumen

9. MECHANISM OF ACTION: Erasmus(1992)-Dawson(1990) Megasphaera Elsdenii and Selenomonas Ruminantium are stimulated by peptides of SC Nisbet-Martin(1991):Selenomonas ruminantium is stimulated by malic acid of SC Wallace(1991): SC increase dry matter intake

10. Explanations involving compounds contained INSIDE the cell: Dead or Alive Calleway-Martin(1997) :vitamins, aminoacids,organic acids Nisbet-Martin((1991) : malic acid Dawson(1990)-Girard(1996) short chain peptides INF. ZOOTEC(1995)many authors:increase of Ruminococcus albus by short chain peptides. Cevolani –Pepe(2001)Vit b,glutamic acid,glutathion,nucleotides,nucleosides, Erasmus(2002):short chain peptides

11. Explanations to support live yeast Newbold(1996) Wallace (1998) SC increases O 2 consumption in rumen?

12.  VanSoest - Nutritional Ecology of Ruminants  Fantuz (1999)  Ilkyu Yoon Feed International (2003)  University of Turin:propionic acid at 0,5% toxic for SC (in rumen 4%) WHY THIS LAST EXPLANATION IS FALSE? SC cannot survive in rumen:

13. In 1995 University of Piacenza (PIVA) made a trial in vitro with Yea –sacch (ALL-TECH) where it proved to be effective in stimulating Megasphaera Elsdenii. The in vitro medium was completely anaerobe

14. How should be the ideal yeast for ruminants? BIO-ACTIVE BUT NOT ALIVE

15. It has to keep its content intact until it reaches the rumen. It should not have any activity that delays the release of this substances in rumen. The cell wall must be degradable in the rumen It must have a high number of cells per gram of product BIO-ACTIVE BUT NOT ALIVE

16. Why THEPAX ® is the ideal yeast ? 30 years experience 2 European Patents (n.0111202) Bio-active but not alive It is the best strain for rumen enviroment High number of cells per gr of product (30 x 10 9 )

17. THEPAX ® cells viewed at electron microscope

18. WHY ADMINISTERING LIVE SC WHEN IT DOES NOT SURVIVE IN THE RUMEN? BECAUSE THEY ARE NOT ABLE TO “KILL” THE CELL WITHOUT DESTROYING IT

20. THEPAX ® in ruminants

21. UNIVERSITY OF PIACENZA- ITALY IN VITRO: rumen content from fasting heifers containing 250ppm Thepax ControlThepax VFA119mg/ 100ml150 mg/100 ml

22. UNIVERSITY OF PIACENZA- ITALY IN VIVO:fistulated heifers, fed Thepax 10g/head ControlThepax Acetic Acid (mMol%) 5769 pH 6.696.89 Butyric Acid (mMol%) 9.0514.71 (Piva et All., Microbial.Alim. Nutr.1991)

23. COMPARATIVE TEST ON 40 FIRST-LACTATION COWS (South France, 2003),BETWEEN LIVE YEAST VS THEPAX TEST CONTROL LIVE YEAST (lb) THEPAX (lb) DIFFERENCE (lb) P. VALUE PRE-TEST WEIGHTS 75.676.30.70.583 WEEK 162.169.97.80.001 WEEK 272.774.41.70.560 WEEK 365.670.14.50.115 WEEK 464.371.16.80.020 WEEK 561.769.17.40.005 Avg. test weights65.370.95.60.021 POST TEST WEIGHTS 64.366.72.40.530

24. MEGASPHAERA ELSDENII

25. The pH drop induced by concentrate feeding is somewhat reduced by feeding THEPAX©

26. THEPAX© stabilises the daily variation in rumen pH.

27. Tartari-Benatti -University of Turin Bullocks from 336 to 430 kg ControlThepax3gr Thepax 6gr Avilamycin340mg 839 918 986 958 Daily Gain (gr)

28. Mr Busso Marco Farm Cavour(Torino) Italy Trial : March,27th,1995

29. Breed used in the trial Blonde d’Aquitaine

30. Results of the trial: Control Initial weight:377kg Final weight:462,7kg Adg : 1,948kg Intake: 8,99 kg DM THEPAX ® 10gr Initial weight:395kg Final weight: 484,7kg Adg : 2,040kg Intake:9,50 kg DM (feed and hay given ad libitum)

31. Feed composition: Hay ad libitum Feed composition: Corn 63 % Wheat bran 20 % Soyabean meal 12 % Doxal premix 3 % Calcium soap 2 %

32. Trial on fattening lambs : 54 lambs from 4.5 to 38 kg ADG (gr): Control : 343 Thepax : 359 (400g/ton) Feed Consumption: Control : kg 100.7 Thepax : kg 104.6 Licee’ de Saint Affrique-Guitard Feb. 2005