1. Enviropigs
Kristen Greer

2. What is an “Enviropig”?
Enviropig™ is a trademark used to designate a genetically modified line of Yorkshire pigs that produces phytase in the salivary glands, and secretes the enzyme in the saliva.

3. Why Phytase?
Approximately 50 to 75% of the phosphorus present in cereal grains is present in an indigestible compound called phytate.
Phytate that passes through the digestive tract is enriched in the manure approx. 4-fold because the protein and carbohydrates in cereals are digested and absorbed.
Phytase is an enzyme that can break down the undigestible phytate releases digestible phosphorus, calcium and other nutrients.

4. How does phytase work?
The promoter of the gene directs constitutive production of the active phytase enzyme in secretory cells of the salivary glands including the parotid, submaxillary and sublingual glands.
The phytase is secreted in the saliva and enters the mouth where it mixes with feed consumed.
Tissue localization of phytase expression. Dark brown staining indicates the presence of phytase protein in acinar cells and in the parotid ducts. Left, phytase positive staining tissues; d, parotid
duct; s, serous cells; m, mucous cells. Right, negative staining in parotid gland from a non-transgenic pig and gluteal muscle from the transgenic pig.

5. How does phytase work? (cont.)
The phytase is most active in the acidic environment of the stomach.
There the enzyme digests the phosphorus rich phytate molecules releasing phosphate molecules that are readily absorbed from the small intestine.
The phytase produced by the Enviropig™ is as active as the enzyme produced in Escherichia coli.
There is only one protein produced from the transgene and that is the phytase enzyme.

6. The problem with Phosphorous
In areas of intense swine production there is a build up of phosphorus in the soil.
High phosphorus content in water is a major factor allowing extensive algal growth .
This eventually leads to a reduction in oxygen content, fish kills, and water that is no longer suitable for drinking.

7. Phytase transgene constructs.
How does it work?
A transgene containing the murine (mouse) parotid secretory protein promoter gene sequence and the Escherichia coli phytase gene was constructed.
The appA gene from E. coli was inserted downstream of the salivary-specific promoters R15-PRP and PSP, to obtain the inducible R15/APPA and the constitutive PSP/APPA constructs. A B
Phytase transgene constructs.
(A) Inducible R15/APPA
transgene.
(B) Constitutive PSP/APPA transgene.

8. How does it work? (cont.)
The transgene was introduced into the pig chromosome by pronuclear microinjection.
This embryo along with other embryos was surgically implanted into the reproductive tract of an estrous synchronized sow.

9. Results
After a 114 day gestation period, the piglets born were checked for the presence of the transgene and for phytase enzyme activity in the saliva.
When the mature genetically modified pig was crossed with a conventional pig, approximately half of the pigs contained the phytase transgene.
This showed that the transgene was stably inserted into one of the chromosomes of the pigs and was inherited in a Mendelian fashion.

10. Results (cont.)
The Enviropig excretes from 30 to 70.7% less phosphorus in manure depending upon the age and diet.

11. Results (cont.) Experiments have shown no impact on growth rate.
No change in numbers of live born or still births compared to controls.
No change in numbers of piglets weaned compared to controls.

12. Benefits of Raising Enviropigs
By raising Enviropigs instead of ordinary pigs a more expensive manure phosphorus application limit could be avoided, and would contribute to an overall phosphorus pollution reduction.
In addition, the feed cost could be reduced by eliminating the need to supplement the diet with either phosphate or phytase.

13. References
Golovon, S. et al. (2001). Pigs expressing salivary phytase produce low-phosphorous manure. nature biotechnology, 19. Retrieved from
Enviropig. (2010). Formally published manuscript, Technology, University of Guelph, Ontario, Canada. Retrieved from
Golovon, S. et al. (2001). Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control. Nature Biotechnology, 19. Retrieved from