Presentation during the IV International Symphosium on Nutritional requirements of Poultry and Swine on March 29th - by Gilberto Litta DSM Animal Nutrition. Main Message of the presentation is the importance of vitamin in Poultry Nutrition as well as the multiple benefits achievable.

1. Recommended Vitamin Levels for High
Performance Broilers and Layers
IV Simposio Internacional Sobre Exigencias Nutricionais de Aves e Suinos
Universidade Federal de Vicosa, Vicosa, Brazil March 29th – 30th , 2017
Gilberto Litta
Animal Science & Advocacy Manager
DSM Nutritional Products, Kaiseraugst (Switzerland)

2. The Century of the Vitamins
1900 20171950
1929-1967: 12 Nobel Prices
awarded to 20 Scientists for
Synthesis, Characterization
und Elucidation of Function of
the Vitamins
1912: Term
“Vitamins”
coined by
Casimir Funk
1946: First Large-
scale, industrial
Synthesis of Vitamin A
by Roche
1934-87: Development of industrial
Production Processes for Vitamins by Roche,
starting with Vitamin C
2016: 13th Edition of the
Vitamin Recommendation Folder
DSM Vitamin Supplementation
Guidelines for Domestic
Animals published
1906-41: 13 Vitamins
identified and characterized

3. ▪ Fat soluble vitamins (4):
▪ vitamin A (retinol) vitamin D (calciferols)
▪ vitamin E (tocopherols) vitamin K (phylloquinone)
▪ Water soluble vitamins (9):
▪ vitamin B1 (thiamin) vitamin B2 (riboflavin)
▪ vitamin B6 (pyridoxine) vitamin B12 (cobalamin)
▪ niacin (B3;PP) pantothenic acid (B5)
▪ folic acid (B9; M) biotin (B7; H)
▪ vitamin C (ascorbic acid)
Vitamins
Classification and Nomenclature
▪ Choline is sometimes included in the list;
▪ Inositol is essential for aquatic species only

4. ▪ Vitamins are essential micronutrients, required for optimum
health and normal physiological functions such as growth,
development, maintenance or reproduction of the animal.
▪ Vitamins exercise catalytic functions; they facilitate both
synthesis and degradation of the nutrients, thereby controlling
the metabolism.
▪ Most vitamins cannot be synthesized by the animals and
therefore they must be obtained from the feed.
Vitamins: Definitions and Functions

5. Functions of Vitamins
Vitamin Basic function(s) Deficiency disorders/diseases
Vitamin A photosensitive retinal pigment, regulation of epithelial cell
differentiation, regulation of gene transcription
blindness, xerophthalmia,
keratomalacia, impaired growth
Vitamin D promotion of intestinal Ca absorption, mobilization of Ca from
bone, stimulation of renal Ca resorption, regulation of PTH
secretion, possible function in muscle
Rickets, Osteomalacia
Vitamin E antioxidant protector for membranes nerve, muscle degeneration
Vitamin K co-substrate for γ-carboxylation of glutamyl residues of several
clotting factors and their Ca-binding proteins
impaired blood clotting
Vitamin B1 coenzyme for oxidative decarboxylation of 2-keto acids,
coenzyme for pyruvate decarboxylase and transketolase
Beriberi, polyneuritis, Wernicke-
Korsakoff syndrome
Vitamin B2 coenzyme for numerous flavoproteins that catalyze redox
reactions in fatty acid synthesis/degradation, TCA cycle
dermatitis
Vitamin B6 coenzyme for metabolism of amino acids symptoms vary by species
Vitamin B12 coenzyme for conversion of methylmalonyl-CoA to succinyl-
CoA, methyl group transfer from 5-CH3-FH4 to homocysteine in
methionine synthesis
megaloblastic anemia, impaired
growth
Pantothenic
Acid
co-substrate for activation/transfer of acyl groups to form
esters, amides, citrate, triglycerides, etc.
symptoms vary by species
Niacin co-substrate for hydrogen transfer catalyzed by many
dehydrogenases, e.g. TCA cycle respiratory chain
Pellagra
Folic Acid coenzyme for transfer of single-carbon units megaloblastic anemia
Biotin coenzyme for carboxylations dermatitis, cracked hooves
Choline component of acetylcholine and the membrane structural
component phosphatidylcholine
poor growth, Perosis (deformity of
leg bones in young birds), fatty
liver
Vitamin C co-substrate for hydroxylations in collagen synthesis, steroid
metabolism
Scurvy

6. • Vitamins can almost never be regarded as nutrients in isolation
given that they display a wide range of interactions
▪ Fat soluble vitamins must be fed in correct ratios as they all
compete for intestinal absorption
▪ Water soluble vitamins are regulators of the intermediary
metabolism of protein, fats and carbohydrates (energy): a lack
of one of them increases the need of the others
• It’s very difficult to estimate precisely the optimum
requirements for maximizing performance for each vitamin
separately and mathematical relationships can’t be established
(Whitehead, 1987)
Vitamins
Functions and requirements

7. Metabolic functions and interactions of B-
group vitamins
Amino acid
pool
B6 FA B12 Krebs (citric
acid) cycle
B2 PP B1
B1 = Thiamin
B2 = Riboflavin
PP = Nicotinamide
B6 = Pyridoxine
B12 = Cobalamine
H = Biotin
FA = Folic acid
PA = Pantothenic acid
Amines
Purines
Pyruvic
acid
Glucose
B6
FA
B2
B6
Proteins Carbohydrates
Uric acid Urea
B6
B6
Glycogen
Glycerol Fatty acids
Acetyl CoA
B1
B2
PP
Fats Diet
Cell
Cell
Excretion
B2
PP
PA
B2 PP H
PP
B1

8. B Vitamins and Methionine cycle
Methionine
Homocysteine
Vitamin B12
B9, CH3-THF
SAM - SAH
CholineBetaine
CH3 acceptor (nucleic acid, protein & amine synthesis,
phosphatidylcholine, creatine
Cysteine
Vitamin B6

☺

9. Vitamin
Demand
Housing
Conditions
Genetics/
Breed
Feed
Composition
Life Stage
Performance
Stress
Infectious
Pressure
Temperature
/Humidity
Factors influencing Vitamin Requirements

10. Comparison of Ross broiler live weight for age
over time (as hatched)
Source: Ross Performance and Management Guide, 1980, 2007 and 2014

11. Genetic driven improved performance requires each year
an adjustment of vitamin supplementation in the range of 1%
The improved efficiency reduces vitamin intake per unit of productivity
and dictates the need for more dietary vitamin
1985 2005 ∆/year
Layer (1 kg) 2.7 IU/egg 2.1 IU/egg -1.1%
Broiler (2 kg) 40 IU/kg gain 34 IU/kg gain -0.8%
Turkey (14 kg) 55 IU/kg gain 48 IU/kg gain -0.6%
All diets containing 20 IU Vitamin E/kg feed
Impact of Genetic Improvement
on Vitamin Supplementation
Source: S. Leeson, World’s Poultry Sci. Journal, 2007 63 255-266

12. minimum
optimum
adequate
▪ Avoid clinical (and sub-clinical?) deficiency symptoms
▪ Generate maximum performance and feed utilization
▪ Satisfy needs for efficient nutrient metabolism
▪ Maintain adequate vitamin plasma and tissue levels
▪ Enable successful reproduction
▪ Support optimum health and welfare
▪ Develop superior product quality
Criteria of Vitamin Requirements

13. Increased Blood-clotting TimeDepression of Appetite
Resorption DisordersInflamed MouthSusceptibility to Infection
Hepatic NecrosisPododermatitisOsteomalacia
Cervical ParalysisFatty Liver and Kidney Syndrome
Muscular MyopathyNecrosis of Heart FibersPerosis
DiarrhoeaAscitesPoor Absorption of Nutrients
Deformed /Brittle BonesLow Immune Response
Fertility ProblemsParalysis / Lameness
What is the Reason for Production
Problems such as …
Vitamin Deficiencies
Dry / Scaly Skin

14. Encephalomalacia –
Vitamin E (and Selenium)
Rickets - Vitamin D3 Perosis - Biotin
Vitamin B1 Deficiency Vitamin B2 Deficiency Fatty Liver Hemorrhagic Syndrome
(FLHS) Vitamin B12 and E Deficiencies
Vitamin Deficiencies in Poultry

15. ▪ Inadequate vitamin intake with the feed:
▪ low natural vitamin levels in feedstuffs, low availability
▪ presence of vitamin antagonists (avidin [egg white] biotin)
▪ insufficient vitamin supplementation levels / mixing errors.
▪ Poor digestion and absorption of vitamins
▪ gut health issues impair fat soluble vitamins absorption
▪ Increased vitamin requirements:
▪ depending on diet composition (e.g. vitamin E for PUFA’s)
▪ for immune response and during phases of stress or diseases.
Causes of Vitamin Deficiency

16. Vitamins and…
1. Bone development and health
2. Immune system modulation
3. Gut health
4. Stress
5. Performance
6. Meat and egg quality

17. Bone development and health
▪ Vitamina D3
▪ 25OHD3
▪ Vitamina K
▪ Vitamin C

18. 0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
7 14 21 28 35 42
GutLesionScore
(1=mild;2=mod;3=severe
Average Age (days)
Histopathic Evidence  Day 14-21
Gross Evidence  Day 21-28
Legs  Day 28-finish
Malabsorption-induced lameness
Malabsorption reduces efficiency of vitamin D metabolism (intestinal
absorption) with direct negative impact on bone development/health….
….and bone health is also impaired by the immune response…

19. Diseases and immunological aspects antagonize
production functions
Anorexia and Fever (IL-1,
TNF-a)
Increased skeletal muscle protein
degradation (IL-1)
Infective disease
Cytokine production
(TNFα, IL-1b, and IL-6)
Acute phase protein response
Decreased Body Weight Gain and Muscle Growth
Lymphocyte/Macrophage
stimulation
Decreased bone deposition and Increased bone resorption
Increased lipolysis in adipocytes (IL-1,
TNF-a)
Increased release of
corticosterone (IL-1, IL-6)

20. Cytokines promote bone resorption
TNFα, IL-1b and IL-6 are cytokines promoting osteoclast* synthesis and activity
TNFα, IL-1b and IL-6
* Osteoclast are the cells in the bone tissues responsible of bone resorption
(i.e.«destroying» bone tissue) for mobilizing Ca++ from bones to blood

21. Trait
(Ca/P: 8.0/3.5 g/kg)
Vitamin D3
200 IU/kg
Vitamin D3
800 IU/kg
Vitamin D3
5,000 IU/kg
Vitamin D3
10,000 IU/kg
Experiment 1
Liveweight 315a 311a 316a 336b
Tibia breaking
strength
61.0a 78.1b 90.9c 93.5c
TD incidence 88a 51b 6c 8c
Experiment 2
Liveweight 295a 297a 303a 351b
Tibia breaking
strength
36.4a 44.5a 61.4b 76.1c
TD incidence 78a 84a 22b 0b
Source: Whitehead et al., 2004
Effect of Vitamin D3 on Bone Structure and Functionality

22. Skin
Liver
Kidney
hydroxylation
hydroxylation
Cholecalciferol (Vitamin D3)
25-hydroxycholecalciferol
(25-OH-D3, Circulating form)
1,25-dihydroxycholecalciferol
(Active form)
Skin
Liver
Kidney
7-dehydrocholesterol Dietary Vitamin D3
UVB
irradiation
Skin
Liver
Kidney
hydroxylation
hydroxylation
Cholecalciferol (Vitamin D3)
25-hydroxycholecalciferol
(25-OH-D3, Circulating form)
1,25-dihydroxycholecalciferol
(Active form)
Skin
Liver
Kidney
7-dehydrocholesterol 25OHD3 (Hy•D®)
Vitamin D3 and 25OHD3 metabolism
UVB irradiation
(280-315 nm)*
Absorption occurs
virtually independent of
fat digestion

23. Source: Rebel A. And G. Weber, 2009, XVI World vet. Poultry Congress, Marrakesh
14,71
14,27
12,96
29,98
37,67
36,84
9,88
0 0
19,87
14,04
16,17
0
5
10
15
20
25
30
35
40
3 d p.i. 6 d p.i. 8 d p.i.
25OHD3plasma(ng/ml)
Days post-infection
Uninfected Control Uninfected Hy•D®
Infected Control Infected Hy•D®
* Day-old broiler chicks were inoculated either with saline (Uninfected) or with MAS (Infected) on the day of hatch.
Control: vitamin D3 2.760 IU/kg feed; HyD: 69 mg 25-OH-D3/kg feed
Effect of Malabsorption Syndrome (MAS) on 25OHD3
serum level

24. 25OHD3 vs Vitamin D3 effect on bone strength
Single trials results 2003 - 2012
25OHD3 (Hy•D®) improved bone strength 10,8% against vitamin D3
Source: summary of experimental and field trials 2003 - 2012
4,5%
18,5% 18,1%
9,5%
5,7%
1,5%
4,8%
3,5%
29,9%
17,2%
5,6%
10,8%
0,0%
5,0%
10,0%
15,0%
20,0%
25,0%
30,0%
35,0%
Bone Strength % improvement HyD vs Vitamin D3
Page 23

25. Effects of vitamin D3 or 25OHD3 on the incidence of TD,
Head Femur Necrosis and Valgus Various defects
0
10
20
30
40
50
60
70
80
90
Control Hy·D® Control Hy·D® Control Hy·D® Control Hy·D®
21 - 28 29 - 35 36 - 42 43 - 49
Tibial Dyschondroplasia, %
0
20
40
60
80
100
120
Control Hy·D® Control Hy·D® Control Hy·D® Control Hy·D®
21 - 28 29 - 35 36 - 42 43 - 49
Head Femur Necrosis, %
0
5
10
15
20
25
30
35
40
Control Hy·D® Control Hy·D® Control Hy·D® Control Hy·D®
21 - 28 29 - 35 36 - 42 43 - 49
Valgus Various defects, %
25OHD3 in the feed reduced
bone disorders vs vitamin D3
weeks
Source: Naas et al., 2012

26. 25OHD3 vs Vitamin D3 and Tibial Dyschondroplasia
0
2
4
6
8
10
12
TD Severity TD Incidence
%affectedBirds D3: 2’760 IU/kg Hy•D: 68.9 µg/kg
Hy•D: 344 µg/kg
AUBURN UNIVERISTYROSLIN INSTITUTE
Control D3
(75 µg/kg)
Hy•D
(75 µg/kg)
Normal Tibial
Growth Plates:
35.0% 88.0%
Abnormal Tibial
Growth Plates:
Severity 0 25.0% 2.0%
Severity 1 12.5% 2.0%
Severity 2 16.7% 6.0%
Severity 3 10.4% 2.0%
100.0% 100.0%
75 mg/kg = 3,000 IU/kg

27. 0
5
10
15
20
25
30
35
40
45
50
28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
Cumulativelameness(n.cases)
Days
Control HyD
25OHD3 (Hy•D®) reduced late lameness incidence against vitamin D3
Control diet with vitamin D3 5.500 IU/kg; HyD 69 mg/kg in the water on top of control diet
Late Lameness Study
Source: Wideman et al., Poultry Sci., 2015

28. Pullet Development
Nutrition of pullets is critical for building a strong, healthy and high yielding laying hen,
especially for bones
Source: adapted from Bregendhal K., Nutrition of laying hens, Hy-Line
Immune and digestive
system and bones
Bones, muscles &
feathers (frame)
Oviduct and
medullary bone
85% of the skeleton
develops within 6
weeks and 95%
within 10 weeks

29. CONFIDENTIAL
Vitamin D3 vs 25OHD3 (Hy•D®) on mineral
apposition rate in pullets
Vitamin D3
(2,760 IU/kg)
Vitamin D3
(5,520 IU/kg)
Vitamin D3 (2,760 IU/kg) + Hy•D® (69 µg/kg )
Mineral apposition rate: Calcein labeling technique
Sourec: Kim et al. 2016, University of Georgia (preliminary data, unpublished)

30. 25OHD3 vs Vitamin D3 effect on bone health in layers
• Hy•D® increased bone strength (greater cortical bone density)
• Hy•D® groups used more efficiently the medullary bone instead of destroying the cortical tissue
(structural bone)
Source: Korver & Saunder-Blades, 2005
The greater the cortical bone the lower the risk of bone fractures and cage layer fatigue
(with a decrease in egg production and lower shell quality)

31. 0
50
100
150
200
250
0 160 320 640 1280 2560
Without Vitamin C
100 mg Vitamin C/kg feed
Effect of Vitamin C on Plasma Concentrations of
1,25OH2D3
Vitamin D3 (IU/kg feed)
pg/mlplasma

32. 0
5
10
15
20
25
0 160 320 640 1280 2560
Without Vitamin C
100 mg Vitamin C/kg feed
Effect of Vitamin C on the Intestinal Ca-binding
Capacity
Vitamin D3 (IU/kg feed)
%binding

33. 0
50
100
150
200
250
0 160 320 640 1280
Without Vitamin C
100 mg Vitamin C/kg feed
Newton
Breaking Load of Tibia as Affected by the
Supplementation of Vitamin C
Vitamin D3 (IU/kg feed)

34. Biotin and Pododermatits

35. Biotin Group (2000 mcg/kg) Control Group (200 mcg/kg)
Source: Buda et al., 2000
Surface of Reticulate Scales at Digital Foot Pad
of Turkeys

36. Control Group (200 mcg/kg)Biotin Group (2000 mcg/kg)
Surface of the Stratum Corneum at the
Reticulate Scale of Turkeys
Source: Buda et al., 2000

37. Immune system modulation
▪ Vitamina A
▪ Vitamina E
▪ Vitamina C
▪ Vitamina D3
▪ 25OHD3

38. The immune system

39. Vitamin A and cell-mediated immune response
Flow cytometric analysis of intraepithelial lymphocytes
Source: Dalloul et al., 2002 Poultry Sci.
▪ Pale grey bars: 8.000 IU/kg vitamin A;
Dark grey bars: vitamin A deficient
diet
▪ Lymphocytes expressing the surface
markers CD3,CD4,CD8,αβTCR, and
γδTCR, as well as surface IgA are
reported as the percentage of total
lymphocytes
Vitamin A deficiency, altering the
lymphocytes subpopulations,
reduced local cell-mediated
immunity and lowered the ability
of broilers to resist
Eimeria infection.

40. • Female broiler chicks (day-old)
• 4 dietary treatments:
• Basal diet (20 ppm Vit E)
• Basal diet + 100 ppm Vit E
• Basal diet + 200 ppm Vit E
• Basal diet + 300 ppm Vit E
• Vaccination at 28 days of age with
inactivated and emulsified
Newcastle disease virus
• Blood sample on days 7, 14, 21,
28, 38, 48 and 58
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
5,0
7 14 21 28 38 48 58
HIAntibodyTiters(log2) Days after Vaccination
Basal Diet BD + 100 ppm Vit E
BD + 200 ppm Vit E BD + 300 ppm Vit E
Adapted from Franchini et al., 1986 Clinica Vet.
Vitamin E modulates humoral immune
response in broilers
Vitamin E supplementation induced a higher production of antibodies

41. Group 1 2 3 4 5 6 7 8 9 10
Vitamin E,
mg/kg
0 0 0 0 15 15 50 50 200 200
Vaccine BI No No Yes Yes Yes Yes Yes Yes Yes Yes
+ Virus Yes No No Yes No Yes No Yes No Yes
Group 1 2 3 4 5 6 7 8 9 10
Vitamin E,
mg/kg
0 0 0 0 15 15 50 50 200 200
Vaccine BI No No Yes Yes Yes Yes Yes Yes Yes Yes
+ Virus Yes No No Yes No Yes No Yes No Yes
Bacteria count Nitric oxide (µM) produced by macrophages
Vitamina E and macrophages bactericide
activity against Salmonella enteritidis
Fonte: Almeida et al., 2014

42. Vitamina C and immune response in broilers
vaccinated against Gumboro
Parameters Control
no Vitamina C
+ 1000 ppm
Vitamin C
IgM+ in bursa (N)
Pre-vaccination (7 days) 65.32 76.24*
Post-vaccination (21 days) 82.82 92.00*
Post-adminstration of virus (31 days) 56.68 66.68*
Antibodies IgG production
(x106 cells)
Post-vaccination (21 days) 19.6 47.6*
Post-administration of virus (31 days) 29.6 37.6*
Fonte: Wu et al., 2000
* P<0.05

43. CONFIDENTIAL
Hy•D® benefits (Non-classical)
▪ Independent of calcium
metabolism
▪ 1α-hydroxylase enzyme
activity in immune cells
▪ Locally converts circulating 25-
(OH)D3 to 1,25-(OH)2D3
▪ Activity depends on adequate
circulating 25-(OH)
▪ VDR is present in immune
system cells
▪ 1,25-(OH)2D3 binds to VDR and
initiates responses
Sources: Norman, 2008; Shanmugasundaram & Selvaraj, 2012;
Morris et al., 2014, Shojadoost et al., 2015
Vitamin D3 and 25OHD3 and immune function
1a-Hydroxylase mRNA amounts in
different organs in chicks at hatch
0
10
20
30
40
50
60
70
80
RelativemRNAamounts(AU)

44. CONFIDENTIAL
Saunders-Blades and Korver, Pre-Symposium XXIII World’s Poultry Congress, Brisbane, 2008
Early (~32 weeks)
25OHD3 fed to breeders significantly increased
chick’s mature innate immune function*
Mid (~47 weeks) Late (~62 weeks)
* Measured through white blood cell phagocytosis
Effect of Vitamin D3 and 25OHD3 on immune
modulation of breeders and chicks

45. 3. Five poultry studies between 2012 & 2015
4 published and 1 in press

46. CONFIDENTIAL
Morris et al., 2014
Immune modulation▪ Challenge model
▪ Injection of LPS (lipopolysaccharides)
to cause inflammatory responses in broiler chickens
▪ Objective
▪ To evaluate Hy•D® effects in suppressing these responses
▪ Results
▪ Hy•D® improved weight gain compared to D3 post LPS challenge
▪ Exerted anti-inflammatory effects
▪ Beneficial during immune challenges
▪ Beyond starter supplementation maintained the effects
▪ Starter only supplementation did not
Vitamin D3 and 25OHD3 effects on immune modulation
in chickens challenged with LPS injection

47. -2
-1
0
1
2
3
4
5
6
Foldchangefromthe
cholecalciferolsupplemented
group
Cholecalciferol Cholecalciferol + LPS
25-OH Cholecalciferol 25-OH Cholecalciferol + LPS
**
***
*
P values : Diet, P = 0.01; LPS, P = 0.87; Diet*LPS, P < 0.01.
25OHD3 supplementation decreases IL-1β mRNA
amounts in the liver post-LPS injection
Source: Morris A. et al. Poultry science, 2014
Treatment
**
Lipopolysaccharide is a potent immunogen in stimulating an inflammatory response in both in vivo
and in vitro experiments.
Lipopolysaccharide stimulation leads to release of inflammatory cytokines IL-1β and tumor necrosis
factor alpha (TNFa)
IL-1b: pro- inflammatory marker

48. 12 h 48 h
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
Foldchangefromthecontrolgroup
*
* ** **
**
*
*
*
P values: 12 h: 25-hydroxycholecalciferol, P = 0.17; LPS, P < 0.01; 25-hydroxycholecalciferol *LPS, P = 0.25; 48 h: 25-
hydroxycholecalciferol, P < 0.01; LPS, P < 0.01; 25-hydroxycholecalciferol *LPS, P < 0.01.
25OHD3 supplementation increased IL-10 mRNA in
LPS stimulated macrophages
Source: Morris and Selvaraj, 2015
IL-10: anti- inflammatory marker

49. *
0
1
2
3
4
5
6
7
8
21 d 35 d
24hbodyweightgainexpressedas%of
pre-LPSbodyweight
Cholecalciferol 25-OH Cholecalciferol 25-OH Cholecalciferol-14 d
Effects of 25OHD3 supplementation on broiler body
weight gain at 24 h post-LPS challenge
Source: Morris A . et al. Poultry science, 2014
P = 0.03
P = 0.01
**
**
*
***
*
+2,5% more BW gain
+3,8% more BW gain

50. P values: P < 0.01(linear), P = 0.20 (Quadratic)
50,36
130,1
107,05
215,03
312,46
Nitrite(µM)
25OHD3 treatment increased nitric oxide (NO) production
in LPS stimulated macrophage cells
Macrophages secrete a variety of inflammatory mediators and cytokines like TNFa-IL-1, ROS (Reactive Oxygen Species)
and NO (Nitric Oxide)
ROS and NO act as antimicrobicidal compounds responsible for killing pathogenic bacteria
Nitric oxide: immune response aspect
Source: Morris and Selvaraj, 2015
*
* *
**
**

51. CONFIDENTIAL
Morris et al., 2014
Immune modulation▪ 25OHD3 supplementation
▪ Decreased expression of IL-1β (marker for
inflammation)
▪ Increased expression of IL-10 (anti-inflammatory
properties
▪ Increased production of nitric oxide (antimicrobial)
▪ Modulating immune response 25OHD3 limited
weight loss during immune challenge
Conclusions

52. Stress
▪ Vitamin A
▪ Vitamin E
▪ Vitamin C

53. 1,72
1,74
1,76
1,78
1,80
1,82
1,84
1,86
1,88
1.560
1.580
1.600
1.620
1.640
1.660
1.680
1.700
1.720
1.740
0 100 200 300 400
FeedConversion
WeightGain
Vitamin C, ppm
Weight Gain, g Feed Conversion
Broilers performance (1 to 42 days ) under heat
stress and with grading levels of vitamin C

54. Effect of grading dosages of Vitamin E on egg yield of
hens under heat stress
25 mg vit E/kg
125 mg vit E/kg
250 mg vit E/kg
a,b P<0,05
Fonte: Panda A. K. et al., 2008
84
84,2
84,4
84,6
84,8
85
85,2
85,4
85,6
85,8
86
86,2
Egg production (%)
b
aa

55. Broilers performance (1 to 42 days ) under heat
stress and with grading levels of vitamin E
1,64
1,66
1,68
1,70
1,72
1,74
1,76
1,78
1,80
1.350
1.400
1.450
1.500
1.550
1.600
1.650
1.700
1.750
1.800
0 75 150 225 300
FeedConversion
WeightGain
Vitamin E, ppm
Weight Gain, g Feed Conversion

56. a,b,c P≤0.001; (vitamin E x temperature 0.001)
3,56
4,89
5,82 5,58
4,02
Totalantibody(log2)
Vitamin E (mg/kg) Temperature
92,4
93,05
94,21
95,12
93,28
Vitamin E (mg/kg) Temperature
a,b P<0.05; (vitamin E x temperature 0.01)
c b a a b
b ab a ba
Antibody response (IgM and IgG) Macrophages (%)
Vitamin E effect on antibody response and macrophages
(%) of broilers under heat stress (Arbor Acres, HS 23.9-
38° C vs 23.9° C)
Higher Vitamin E dosage can improve antibody response (IgM and IgG) and macrophage %
Fonte: Niu Z.Y. et al., 2009

57. Vitamina A effect on immune response in heat-stressed
(HS) layers (Hy-Line, 25 wks, 31.5°C)
Fonte: Lin H. et al., 2002
Humoral response
Vitamin A (IU/Kg)
anti-NewCastleDiseaseVirusAb(log2)
b
a
ab ab
35
40
45
50
55
3,000 6,000 9,000 12,000
T-CellProliferation(%)
Vitamin A (IU/kg)
b
a
bc
c
a
bc
bc
c
No Heat Stress
Heat Stress
Heat-stressed layers
after vaccination
require more vitamin A
for producing more
antiobodies
Measured at 7 and
14 dd post-
vaccination
a,b P<0.05
a,b,c P<0.05

58. Vitamina A and antibody response in heat-stressed
broilers
Source: Niu Z.Y. et al., 2009
a,b,c P≤0.001; (vitamin A x temperature 0.001)
Vitamin A (IU/kg) Temperature
c
b
a
a
b
Higher Vitamina A dosages can improve antibody
response (IgM e IgG) in heat-stressed broilers

59. Zinc and Vitamin A alleviate heat stress (34°C)
effects in broilers
Source: Kucuk et al., 2003 Biological Trace Element Research
Control: diet containing 4,600 and 5,000 IU/kg vitamin A and 45 mg/kg Zn in grower and finisher phases respectively;
Zn: control diet + 30 mg Zn/kg diet;
Vit A: control diet + 4.5 mg (15,000 IU) retinol/kg diet;
Zn + Vit A: control diet + 30 mg Zn/kg diet + 4.5 mg retinol/kg diet.
Vitamin A and Zinc improved
performance in heat stressed
broilers alone and in combination
Blood parameters – emphasis MDA
and glucose – reflect the synergy
in absorption, metabolism and
effects of Vitamin A and Zimc

60. Gut Health
▪ Vitamin A
▪ Vitamin E
▪ Vitamin C
▪ Vitamin D3
▪ 25OHD3
▪ Vitamin B6
▪ Vitamin B2

61. UNSTIRRED WATER LAYER
Vitamin B6
Glutammine-Aminoglycans
Thr-Cyst-Gly-Ser-Pro
Mucin
Vitamin E
Vitamin A
ENTEROCYTE MEMBRANE
INTESTINAL PERMEABILITY
Reduced FAs peroxidation
VitaminD3/
25OHD3

62. Impact of vitamin D3 and 25OHD3 on intestinal
morphology
Source: Chou S. H. et al., 2009
A, B, C and D are measurements at 14, 21, 28 and 35 days of age
Control: 3.000 IU/kg vitamin D3
25OHD3: 3.000 IU/kg vitamin D3 +
69 mg/kg (starter) and 34,5 mg/kg
(grower) of 25OHD3
25OHD3 significantly incresed villi lenght (duodenum and jejunum) and reduced crypt depth
The increased ratio villi lenght/crypt depth may suggest an enhanced rate of nutrient absorption

63. Faecalibacterium prausnitzii
(prec. Fusobacterium prausnitzii)
▪ G(+), non-spore, anaerobic, difficult to cultivate even in anaerobiosis
▪ SCFAs producer, mostly butyric acid
▪ Reduces pro-inflammatory cytokines production (IFN-g e IL-12)
▪ Promotes production of anti-inflammatory (IL-10)
▪ In humans present in GI tract ≈5% of microbiota in feces
▪ ≈ 15%+: obesity
▪ <5% : Crohn disease; IBD
▪ Isolated in poultry, swine calves (and insects)

64. Riboflavine (Vitamin B2) acts as redox
mediator allowinf the strict anaerobe
F.prausnitzii to grow in intestinal epithelium
despite aerobic condition

65. Performance
▪ OVN
▪ Vitamin D3
▪ 25OHD3

66. Total Vitamin Intake
AverageAnimalResponse
Deficient
Sub-optimum
Optimum
Special Applications
NRC
• Below NRC levels
• Animals at risks of
developing clinical
deficiency signs and
disorders
• Above NRC levels
• Preventing clinical
deficiency signs and
disorders
• Inadequate to permit
optimum health and
productivity
• Offsetting factors
influencing vitamin
requirement
• Permitting optimum
health, productivity
and food quality and
nutritional value
• Above optimum levels
• Optimizing certain
attributes such as
immunity, meat quality,
bone health, etc.
Optimum Vitamin Nutrition Graph

67. Optimum Vitamin Nutrition (OVNTM) is about feeding
animals high quality vitamins in the right amounts and
ratios appropriate to their life stage and growing
conditions.
Optimum Vitamin Nutrition is a cost-effective range of
vitamin supplementation optimizing
• Animal Health and Welfare
• Performance
• Quality and Nutritional Value of Animal-origin Foods
The OVN™ Concept

68. Authors Year Country ROI
1. Coelho 2000 USA n.a.
2. Perez-Vendrell et al. (IRTA) 2002 Spain n.a.
3. Perez-Vendrell and Weber (IRTA) 2007 Spain n.a.
4. Zhang et al.* 2011 China 13:1
5. Araujo et al. 2012 Brazil 5:1
6. Iglesias et al. (Granja Tres Arroyos, Cobb) 2012 Argentina 2:1
7. Aviagen Product Dev. Center 2012 USA 3:1
8. NKP FARM 2012 Thailand n.a.
9. Aviforum 2013 Switzerland 2,8:1
10. INRA & ITAVI 2013 France 5,2:1
* Trial on layers
OVN Trials in Poultry: 2000 - 2013

69. 0
1000
2000
0-21 days 0-40 days
Body Weight (g)
Industry OVN
0
0,5
1
1,5
0-21 days 0-40 days
FCR (g/g)
Industry OVN
a b a b
300
320
340
360
380
Breast weight (g)
Industry OVN
13,5
14
14,5
15
15,5
Breast yield (%)
Industry OVN
a b a b
Perez-Vendrell and Weber, 2007
Effect of Optimum Vitamin Nutrition on
Performance and Meat Yield of Broilers

70. 25OHD3 vs Vitamin D3 effect on breast meat yield:
Single trials results 2004 - 2012
25OHD3 improved 1% breast meat yield against vitamin D3
Source: Summary of experimental and field trials, 2004 - 2012
* Vitamin D3 vs HyD starter & grower; ** Vitamin D3 vs HyD starter; HyD starter vs HyD all feeds
1,0%
2,0% 2,0%
1,7%
0,6% 0,7%
1,3%
0,3%
0,7%
0,6%
0,8%
0,5% 0,4% 0,4%
0,8%
0,3%
0,6%
0,4% 0,3% 0,3% 0,4%
2,0%
3,9%
0,5%
0,6%
1,2%
0,2%
1,0%
0,0%
0,5%
1,0%
1,5%
2,0%
2,5%
3,0%
3,5%
4,0%
4,5%
APSI,2007
APSI,2008
APSI,2009
APSI,Q12010
AgriStats,2010(small…
AgriStats,2010(medium…
AgriStats,2010(largebirds)*
Saunders-Blades&Korver,…
Bray,SFAustin…
Bray,SFAustin…
Bray,SFAustinPenstudy,…
Arkansas,2012
SPR,2012
Poland,2006
IRTA,Spain,2007
ILVO,Belgium,2007
ILVO,Belgium,2008
IRTA,Spain,2009
Poland,2010
Spain,2005
Hungary,2006
Italy,2008(female)
Italy,2008(malemedium)
Italy,2008(malelarge)
Hungary,2010
France,2011
Italy,2012(malelarge)
Average
BreastMeatYield%improvementHyDvsVit.D3
Page 71

71. 72
Vitamin D3 and 25OHD3 studies on meat yield

72. ▪ Birds:
▪ 1440 Cobb broilers ; 0-42days
▪ Treatments:
▪ 4 treatment groups
▪ 12 replications per treatment
▪ 30 broilers per pen
▪ Parameters measured:
▪ Serum 25OHD3 at 21d and 42d
▪ BW, FCR at 21d and 42d
▪ Breast meat yield at 42d
▪ 50 birds/treatment
▪ Expression of protein synthesis-related genes
(mTOR, S6K-1 and IGF-1 ) at 42d
Vignale et al., 2015
Trial Details
Source: Vignale,K., J. of Nutrition, 2015

73. Effect Vitamin D3 and 25OHD3 on Breast Meat Yield (%)
P value= 0.02, SEM: 0.20
19.71 b 19.72 b
20.40 a
20.30 a
19,2
19,4
19,6
19,8
20
20,2
20,4
20,6
Control High D3 HyD 0-42 HyD 0-21
%
25OHD3significantly improved breast meat yield but not additional D3
Source: Vignale, K., J. of Nutrition, 2015

74. Effect Vitamin D3 and 25OHD3 Fractional Synthesis Rate
(FSR) at 42 d
P value= 0.0406, SEM: 3.06
4.44 b 4.38 b
16.27 a
14.77 a
0,00
2,00
4,00
6,00
8,00
10,00
12,00
14,00
16,00
18,00
Control High D3 HyD 0-42 HyD 0-21
Fractionalsynthesisrateat42d%/d
25OHD3significantly improved protein synthesis rate but not additional D3
Source: Vignale, K., J. of Nutrition, 2015

75. 0,00
0,50
1,00
1,50
2,00
2,50
Control High D3 HyD 0-42 HyD 0-21
IGF-1
*
Effect of vitamin D3 and 25OHD3 on the expression of
protein synthesis-related genes at 42 d
0,00
0,50
1,00
1,50
2,00
2,50
3,00
Control High D3 HyD 0-42 HyD 0-21
mTOR
0,00
0,50
1,00
1,50
2,00
Control High D3 HyD 0-42 HyD 0-21
S6K-1
25OHD3 from 0 to 42 d significantly improved the
expression of protein synthesis related genes
* *
*
Source: Vignale, K., J. of Nutrition, 2015
• mTOR (mammalian Target of Rapamycin) is a protein-
kinase important for cell growth and proliferation.
• Its activation is dependent on the effect of hormones or
specific nutrients (Aas)
• Both mTOR and S6K are genes connected with protein
synthesis

76. Meat and Egg Quality
▪ Vitamin A
▪ Vitamin E
▪ Vitamin C

77. Lipid Oxidation and Meat Quality
Lipid Oxidation = Deterioration of Meat Quality
Hydroperoxides,
Cholesterol oxides
Potentially
harmful
substances
Drip loss
Destruction
of Membranes
Formation of
Metmyoglobin
Colour
changes
Aldehydes,
Ketones
Oxidative rancidity,
bad odour / flavour

78. 25 35
100
250
5 25 65 180
a-Tocopherol(mg/gprotein)
Vitamin E (mg/kg feed)
Source: Sheehy et al., 1991
Deposition of alpha-Tocopherol in Thigh Muscle

79. Effect of Optimum Vitamin Nutrition on
Vitamin E in breast meat (mg/100g)
Page 80
P vitamin level 0,0002; P density 0,3645; P interaction 0,5607
Vitamin E level: T1 and T3 18,9 mg/kg; T2 and T4 225 mg/kg
0,00
0,20
0,40
0,60
0,80
1,00
1,20
1,40
Control OVN Normal High
VitaminEinbreastmeat(mg/100g)
Group
Control OVN Normal High
Source: A.M. Perez-Vendrell et al., 2002

80. Effect of Optimum Vitamin Nutrition on
breast lipid oxidation (TBARS, nmol/g)
Page 81
P vitamin level 0,0001; P density 0,0211; P interaction 0,0254
Vitamin E level: T1 and T3 18,9 mg/kg; T2 and T4 225 mg/kg
0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
0,40
0,45
Control OVN Normal High
TBARS(nmol/g)
Group
Control OVN Normal High
Source: A.M. Perez-Vendrell et al., 2002

81. Vitamin fortification of eggs
Vitamin Potential increase & comments
Vitamin A 2-3 fold
Vitamin D3 6 to 10 fold attainable in 2-3 weeks
25OHD3 Naturally present in egg yolk of hens receiving vitamin D3
(0,5 to 8,1 mcg/100 g whole egg)
3-4 fold increase
Vitamin E 4 fold or more increase in 2 to 3 weeks (e.g. 200 IU/kg
feed from 119 to 606 IU/kg egg yolk)
Vitamin K Nearly 5-fold increase in hens fed 7,5 mg/kg feed
Folic acid 2-3 fold increase in 2 to 3 weeks
Biotin 3-5 fold; excess largely goes into albumen
Vitamin B12 3-4 fold; good response but high feed levels needed
Vitamin B2 2-3 fold but fairly refractory to accretion at high feed levels
Pantothenic acid 2-3 fold; plateaus quickly
Niacin 2-3 fold; limited data base
Vitamin B1 ≈2 fold; limited data base
Vitamin B6 ≈2 fold; very limited data base

82. ▪ Vitamins are essential nutrients, required in very small amounts,
playing a pivotal role in all metabolic and physiological functions
Take Home Message
▪ Vitamins have a pretty limited feed cost impact but a great
responsibility in animal’s health and performance as well as on
quality of animal origin foods
▪ Vitamins, besides a direct impact on DWG and FCR, can modulate
several physiological aspects like immunity and stress and hence
providing indirect performance benefits e.g. reduced medications

83. Thank you for your attention
gilberto.litta@dsm.com