Enviropig Richard Wolfe

Introduction - Increase food production without degrading the environment. - Manure is used as a fertilizer. - Monogastric (single stomach) animals, pigs and chickens produce high phosphorus manure. - Not able to digest plant phytate

Introduction - Can not remove the phosphorus from phytate. - Up to 80% of the P in the plant based feed is found in phytate. - Phosphorus is supplemented to diet to achieve optimal growth.

Introduction - Required dietary supplements cause excess P in manure. - Run off from using manure as fertilizer adds excess P into streams and rivers. - Algal blooms, oxygen depletion, death of fish and aquatic animals - Phytase is an enzyme that releases P from phytate - Used transgenic mice to deliver E.coli phytase into digestive tract through salivary glands. - Shown E.coli phytase added to poultry diets was effective. - Pigs that produce salivary phytase require almost no supplement and excrete 75% less P.

- Pig eats food - As pig chews phytase released - Phytase and food swallowed - Phytase enzyme activated - Phosphorus absorbed - Low P manure excreted

Results - 4,147 embryos were microinjected. - PSP/APPA transgene (parotid secretary protein promoter linked to the E.coli appA phytase gene) - DNA extracted from blood and tail used to id with PCR - 33 transgenic founder (G 0 ) pigs produced produced 5 to 6000 U/ml of phytase in their saliva at 7-11 days - 15 produced < 5 U/ml. - 4 produced an undetectable amount. - 1U phytase = 1 micro mol phosphate released from phytate per min - Transgene copy number was 35 for WA line, 2 for JA

- Tested to see if salivary phytase promotes digestion of P - Selected transgenic G 1 WA with median salivary phytase 2,420 U/ml - Fed soybean meal containing 53% phytate P - Only source of P

- P in manure reduced 75% in weanling transgenic pigs - P reduced 56% in growing- finishing transgenic pigs - Almost all of the dietary P was digested and absorbed

- Second test to see if salivary phytase promotes digestion of P - G 1 pigs fro JA line fed standard diet - No supplement of inorganic P - Fecal P reduced by 64 – 67% compared to non-transgenic pigs - Average salivary phytase ~190 U/ml - Growth rate transgenic pigs was days to reach 100kg - Non-transgenic was 147 days and they received P supplement

- High phytase activity detected in parotid, sublingual, and submaxillary salivary glands - Low but substantial activity in stomach and duodenal contents - Comparable tissues of non-transgenic pigs contain no detectable amount

- Images correspond to Table 2 - Parotid and submaxillary glands show staining of phytase protein - No staining in transgenic muscle - No staining of non-transgenic parotid

- 55 kDa phytase protein found in B,C,E,F,G - Levels of F,G lower - Mass of D higher

Discussion - Phytase activity present in saliva varied from 5 – 6000 U/ml due to position of transgene insertion - A transgenic pig producing 2-5 U/ml of phytase will satisfy the dietary requirement of P - The transgene copy number was only determine for two lines of transgenic pigs, can not determine if related to phytase expression - Shown that salivary phytase enables digestion of phytate P and no P supplement is required - Also, P in manure is reduced up to 75% - Have not detected any deleterious effect on health or performance

References 1. Golovan SP,et al; Pigs expressing salivary phytase produce low-phosphorus manure. Nature Biotechnology. August 2001, 19, Phytate picture Enviropig image.