Foodstuff Total P(%) Phytic acid. (% of Total P). Barley. 0.34. 56. Corn. 0.26. 66. Wheat. 0.30. 67. Soybean .... Reported good germination and no yield redn,.
Genetic Opportunities to Enhance Sustainability of Pork Production in Developing Countries: A Model for Food Animals Cecil Forsberg, S. Golovan, A. Ajakaiye, J.P. Phillips, R.G. Meidinger, M.Z. Fan, J.M. Kelly and R.R. Hacker
The University of Guelph Guelph, Ontario CANADA
Trends in Pig Production in Developed and Developing Countries (FAO Stats, 2003) Live Pigs (Millions)
700
Developed Countries
600
Developing Countries
500
Linear (Developing Countries) Linear (Developed Countries)
400 300 200 1970
1980
1990 Year
2000
Reported Transgenic Pig Projects Gene
Tissue
Effect
Growth hormone…………. All…………. Enhanced growth IGF1………………………...Muscle……. Muscle growth Plant oleate desaturase…. All tissues… Improved nutritional value (for human) Lactoferrin………………… Mammary… Resistance of piglets gland to infection α-Lactalbumin……………. Mammary… Improved piglet gland growth Phytase……………………. Salivary….. Efficient P Utilization gland Reduced P in manure
Eutrophication & Fish Mortalities Caused by Manure Contamination PO43Run-off, tiles
N2
N2O
Anoxia Algal Toxins
Phytate Phosphorus Content of Major Cereal Grains and Protein Supplements Foodstuff
Total P(%) Phytic acid (% of Total P) Barley 0.34 56 OOCorn 0.26 66 O=P- O--O-P=O O O- Wheat O 0.30 67 O=P- OO Soybean meal 0.61 61 H O- - H O=P-O 70 H Cottonseed meal 1.07 H O O -O - =O P Sesame meal 1.27 81 H O O -O - =O Wheat bran 1.37 70 P O-
Phytic acid
Nelson et al. Poultry Sci. 47:1372 ‘68
Action of Phytase + +
=
O O Ca O +Ca - O-C-CH -Protein O=P-O O-P=O 2 Starch O O+ O OH O=P-O Protein -+Mg H O H H O CH H + 2 O=P-O NH+ H + 6 H2 O H O OH 3O - O-P=O O H O H O-P=O OH
Phytase
Phytate
CH2 0H Starch
P. C. M. Simons et al. Brit.J.Nutr. 64:525-540, 1990
OH H H OH
Inositol
OH H
+6
+
H3PO4
Protein Starch Minerals
Pigs 300-1000U Phytase/kg feed 24% ↑ available P 35% ↓ fecal P
How the EnviropigTM Works Phytase produced in the salivary glands and secreted in the saliva
Pancreas
Liver
Indigestible Phytate phosphorus in the diet
Cecum
Stomach Phytase
um n e d Small o u D Intestine
Phytate Phosphate
PHOSPHATE Colon ABSORPTION
Phytate Passes through the non-Transgenic pig Unchanged Phytate is digested in the Enviropig stomach
Manure low in Phosphorus
Important Regions of the pLama2/APPA Plasmid Introduced into Embryos to Create the EnviropigTM
Constitutive Promoter
Mouse-PSP E. coli APPA Promoter coding region
Phytase gene
Segment deleted prior to injection of transgene
Not I
ori pLama2/APPA with pBluescript II plasmid backbone 20.6 kb
β-lactamase coding region
Xho I
Sampling A Pig For Phytase in the Saliva
A 50 kg grower pig produces 0.5 L of saliva when consuming 0.5 kg dry feed
Founder and First Generation EnviropigTM
Founder
Founder First Generation Transgenic Piglets
Different EnviropigTM Lines JA 401-01 40.5 Wks 2nd Gen 160 Kg, June 11/02
Le 571-09 26.5 Wks 1st Gen. 140 Kg
GO Litter 177 3.5 Wks 3rd Gen June 11/02
Conventional Diets Fed to Phytase Pigs Lack Supplemental Phosphorus Ingredient Corn Barley Wheat Soybean meal Fish meal Whey Dical-PO4 CaCO3
Weaner 33.74 8.00 20.00 23.00 5.00 8.00 Nil 0.70
Grower 52.63 8.00 20.00 16.00 Nil 0.88
Finisher 40.98 23.03 20.00 13.00 Nil 0.77
Fecal Phosphate (% of Dry Matter)
Relationship Between Salivary Phytase Activity and Fecal-P Among Non-Tg and Tg Pigs 2.0
Control (Non-transgenic) n=18
1.5 ManonTg
1.0 0.5 0.0
n=10
GordieTg JacquesTg WayneTg n=10
(45%↓)
(57%↓)
n=11
n=10
(64%↓)
(64%↓)
0 50 100 150 200 250 300 Phytase Activity (U/ml of saliva)
Phytase Activity in Saliva of Different Generations of the Cassie Line of Phytase Pigs Males
Activity (U/ml)
600
Females n=10
500 400
n=1
300
n=1 n=1
n=17
n=20
n=7
200 100 0 F F -Founder
G1
G2
G3
Generation
(Assayed morning and evening of day 11)
Number / Litter
Reproductive Viability of The Cassie Line as Compared to Non-Transgenic Yorkshire Pigs
12 10 8 6 4 2 0
Yorkshire Cassie Tg
Live Still- Early Mummies Births borns Deaths
Category
Offspring From a Homozygous Outcross to Non-Tg BoarL JA173, 5 wk,≅6 kg, 3rd Generation (Documents the generation of a homozygous line of pigs suited for breeding)
Nigeria 6 million pigs 97% pigs free range Free Range Pigs Plant materials, scraps and soil Diets Managed Pigs
Energy -Corn & cassava Protein supplement -Soybeans meal/cake or peanut meal Phosphorus -Bone meal (Primarily from cattle) No suppl. Phytase!
Brazil
30 million pigs 15 million in other SA countries Pigs usually on large farms Diet – Corn (60%) & Soybeans (40%) Supplemental P-either dicalcium P04 or phytase Santa Catarina State – 85% of water sources contaminated with fecal organisms due to spreading manure and runoff into streams, ponds and rivers etc.
Brazilian legislation forbids GM livestock.
China 465 million pigs 68% meat production Up to 80% pigs raised in back yards Diet – corn & soybean meal Other protein supplements – peanut meal, rapeseed meal, cottonseed meal, linseed meal, wheat shorts, rice bran (all high phytate). Phosphorus – low quality mineral PO4 or phytase - Bone meal has been banned Serious P & N pollution in some locations (eg. Lake Taihu)
% Reduction in P Excretion
Predicted Relation Between Increased Supplemental Phytase and Reduction in Phosphorus Excretion 60
20%
Reduction attained by Enviropigs
40 20 $1.33 $2.66 $3.99 $5.33 $6.65
0 0
500
1000
1500
2000
2500
Supplemental Phytase (U kg -1)
Prediction developed from data by Kornegay, E.T. 2001. P247 Enzymes in Farm Animal Nutrition, M.R. Bedford and G.G. Partridge
Effect of Reducing the Crude Protein Content of the Diet on Reduction of Phosphorus in the Fecal Matter From Enviropigs % Reduction in Acreage for Spreading Manure
Assumptions: 60% redn in fecal phosphorus 65 60 55
P-limit
50 45 40 35 30
One % redn in diet CP
10% redn fecal N
0 1 2 3 4 5 6 7 % Reduction of Crude Protein in the Diet Note: PO43- is the primary nutrient causing eutrophication followed by nitrogen
Competing Technologies Commercial Phytase Enzymes Requires continuous addition to all diets with feed mixing and preferably pelleted. All phytase to date are partly heat labile and subject to destruction during pelletting. Corn Kernels smaller, 15% yield reduction. Reduced germination. Soybean meal 25% lower yield. Barley Reported good germination and no yield redn, but agronomic characteristics not tested yet.
Canada:
Regulatory Hurdles
Environment Canada – Canadian Environmental Protection (Act Schedule 19) -Document safety to environment including wild animals, flora and fauna Health Canada – Novel Foods Act -Safety of food for human consumption Canadian Food Inspection Agency -Animal well-being, feed and fertilizer acts.
United States:
Food and Drug Admin. (Center for Veterinary Med.) -Animal drug EPA USDA
Pre-Market Safety Assessment of Food Derived from “Genetically Modified Plants” (a) Safety of the inserted DNA (potential for transfer to other organisms) (b) Safety of DNA ingested (potential toxicity) Food safety issues of the newly produced protein: (a) Potential for toxicity of the novel protein (b) Potential for allergenicity of the novel protein (c) Safety of any unintended effects Equivalence of composition to the original food Retention of nutritional value The human dietary exposure No well worn path for food animals!
Conclusions – The EnviropigTM
Documented healthy Enviropigs that digest phytate phosphorus efficiently. Major pig breeding companies and other potential investors are concerned by the uncertain time line for societal acceptance of pork from genetically modified animals. However, there is growing global societal acceptance of GM products.
Environment Issues Remain
¾ World mineral phosphate sources are being depleted. ¾ Bone meal is no longer used in pig diets in a growing number of countries due to the concern of disease transmission (BSE). ¾ Phytase enzyme issues – availability, mixing, pelleting, storage and heat stability? ¾Low phytate crops – germination & yield not fully resolved. None adapted to tropical or subtropical climates?
Thank you Research Support ¾ Ontario Pork Producers Marketing Board ¾ Natural Sciences & Engineering Research Council of Canada ¾ Agriculture & Agri-Food Canada ¾ Ontario Ministry of Agriculture & Food ¾ Food Systems Biotechnology Centre U of G ¾ Mars Landing Program
http://enviropig.uoguelph.ca/#research http://www.uoguelph.ca/microbiology/cf.htm
