Use of Nanotechnology/nanoparticles in Livestock and Poultry Feeding

1. Pankaj Kumar Singh
Assistant Professor(Animal Nutrition),
Bihar Veterinary College, Patna, Bihar, India-800014
E-mail: vetpank@gmail.com 1/61

2. HISTORY
“There’s plenty of room at the bottom”
“I would like to describe a field, in which little has been done, but
in which an enormous amount can be done in principle. …..What
I want to talk about is the manipulating and controlling thing on
a small scale”
“There’s plenty of room at the bottom”
Richard Feynman, Caltech (1959)
Father of Nanotechnology
1974: Norio Taniguchi coined the term
‘Nanotechnology’ 2/61

3. Nano word is derived from the Greek nanos meaning dwarf.
Nanoparticle: Ultrafine particle of size 1-100 nm, material
with all three external dimension in the nano scale.
Nanoscience: The study of phenomena and manipulation of
material at nano scale, where properties differ significantly from
those at larger scale.
( Laurence, 2010)
Principles of Nanotechnology
3/61

4. 1. Nanoparticles
2. Nano emulsion
3. Nano clays
4. Buckey balls
5. Micelles
6. Quantum dots 4/61

5. INORGANIC NANOPARTICLES
SILVER 5/61

6. ORGANIC NANOPARTICLES
6/61

7. Preparation of Nano-particles
Top down method (Physical method)
Bottom-up method (Chemical method)
Methods:
1. Physical Methods
a) Mechanical method (Ball milling)
b) Physical vapour deposition (PVD)
c) Gas phase synthesis
2. Chemical Methods:
a) Cross linking micro-emulsion
b) Precipitation (Huang et al., 2007)
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8. Physical Methods
1. Mechanical method (Ball milling):
Uses different versions of mechanical dispersion viz Electro explosion,
laser-induced electro-dispersion, supersonic jets etc.
eg. Ball milling.
2. Physical vapour deposition (PVD):
Transferring the substrate to form a film by evaporation and sputtering.
In evaporation: Matters are removed from the source by thermal means.
In sputtering: Atoms or molecules are dislodged from solid target through
impact of gaseous ions.
(Cardenas et al., 2007).
3. Gas phase synthesis:
Involve atmospheric or low pressure evaporation of powders or the co-
evaporation of the two elemental components.
eg. Zinc and sulfur. Gold decorated silica nanoparticles .
(Adam et al., 2011).
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9. BALL MILLING
Principle: Balls rotate with high energy inside a drum and then fall on the
solid with gravity force and crush the solid into nano crystals.
• Equipped with grinding media composed of wolfram carbide or steel.
High Energy Ball Milling (HEBM) is more efficient:
 The impact energy of HEBM is 1000 times higher
 To achieve desired structural changes.
 Controlled milling atmosphere and temperature
 A longer milling time
Use: Preparation of Nano Zinc Oxide
Drawbacks:
1. Not uniform particle size
2. Contamination during milling 9

10. 1. Cross linking micro-emulsion methods
2. Precipitation methods
CHEMICAL METHODS
Advantages of chemical methods:
 Avoid contamination during physical methods
 Uniform sized nano particle Production
 Stabilization of nano particles from agglomeration
 Surface modification and application
 Processing control
 Mass production.
(Lane et al., 2002) 10/61

11. Cross linking Emulsion method
 Micro-emulsions are complex liquids consisting of oil,
water, surfactant (e.g. CTAB) and co-surfactant that
form a clear solution.
 Micro emulsions bring together the metal precursor
(water-soluble) and the reactant (oil-soluble) to enable
the reduction of the metal to occur.
 As the water concentration alters, the system can change
from a w/o to an o/w micro-emulsion.
 Syntheiss of nano particles occur
(Eastoe and Warne, 1996)
11/61

12. Cross linking Emulsion
Water in oil/ oil in water emulsion preparation
Vigorous shaking
Separation & Hardening of particle
Type of Surfactant used is critical to
stability of final emulsion
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13. 2.Precipitation method
Soluble form of mineral
Alkaline solution
Filtration & Centrifugation
Rinsing with hot & cold
water
2.2 g of Zn (CH3COO)2.2H2O and
2 g of NaHCO3 are mixed at room
temperature.
Pyrolyzed at 300º C for 3 h.
Zn (CH3COO)2.2H2O is changed into
ZnO nano-particles, while NaHCO3 is
changed into CH3COONa.
Washed with deionized water.
ZnO nano particles obtained by
thermal decomposition process.
(Bagum et al., 2008)
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14. Surface effect
Particle size < 100nm
Lesser stability of atoms
Lesser energy needed to join adjacent atoms
Lower fusion point
Quantum effect
Special arrangement allow to have different properties
than parent element
More surface area than micro particles
Chemical reaction rate increases 1000 times
(Buzea et al., 2007)
14/61

15. Application of Nanotechnology in Animal Nutrition
Feed Biosafety
(Livestock, Environment)
Feed Quality Control
Pathogens/contaminant
Detection & Control
(Nano sensor/Biosensor)
Digestion & Absorption improvement
(Nano-particles)
Packaging/storage/Stability
(Smart packaging
Nanomaterials)
Feed supplements/
Nanocapsulation
Nanotechnolog
y
(Nano-feed)
15/61

16. ZnO-NPs inhibits growth of fungus:
Hydroxyl group of cellulose molecules of fungi
Oxygen atom of ZnO-NPs
H2O2 on the surface of ZnO-NPs
Inhibition of the fungi growth.
(Moraru et al., 2003)
Mycotoxin Binding
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17. Shelf life of Feed
• Silicate nano-particles enriched films (SiO2/TiO2)
Indicate color change in presence of toxins /Microorganisms
• Prevent drying of contents
• Protection from moisture & oxidation
• Antibacterial Nano Ag/ ZnO/ MgO has repellent surfaces
• Enhanced mechanical & thermal stability
• Increases shelf life & protection
(La Coste, 2005) 17/61

18. V
V
V
V
V
V
Low particle size
More particles at
Surface
Large surface area
Higher exposure
per unit mass
Basic concept of Nanoparticles as Feed Additive
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19. Nanoparticle can enter the GIT:
Directly from food & water
As feed additive & Supplements
As nano-drug
Particle uptake in GIT -
Uptake by Passive Diffusion
Through mucus and cells
Smaller particle Faster diffusion
Easily cross GIT barrier
Insoluble NPs are readily taken up across the intestinal barrier
Better absorption than macro equivalents
(Hoet et al., 2004)
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20. GI Uptake and Translocation of NPs
Increases surface area available to interact with
biological support (Arbos et al., 2002)
Penetrate deeply into tissues through fine capillaries.
Efficient uptake by cells
Particles diffusion rate through GI depends on
Size & charge (Szentkuti, 1997)
Surface coating (Lai et al., 2007)
Efficient delivery of active compounds to target sites
Improve the bioavailability of Nutrients (Chen et al., 2006)
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21. Nanoparticles
Absorption of NPs Through the GIT
21/61

22. Additive contain minerals with a nano formulation such as
Nano Zn, Nano Se, Nano Cu , Nano Ag, etc.
Nano-additive can also be in incorporated in micelles or
capsules of protein or natural feed ingredient (Morris,2005)
Chitosan, Liposome etc. are used to protect the potency
and efficacy of oral nano-additive by-
Protecting from undesired enzymatic activity
Protecting from undesired bile salt
Protecting from commensal microorganism
Enhance bioavailability
(Handy, 2007)
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23. 23

24. 24

25. Effects of nano zinc oxide on milk production and immunity in
Holstein Friesian crossbred cows (Rajendran et al., 2013)
48 lactating cows (2-4th
lactation)
Milk yield: 8.58 kg; Period: 75 days
4 group
Healthy Subclinical mastitis affected cows
(tested by California mastitis test)
Control group Zinc oxide
@ 60 ppm
Zinc methionine
@ 60 ppm
Nano Zn
@60 ppm
25

26. Particulars Control Zinc oxide
@ 60 ppm
Zinc methionine
@ 60 ppm
Nano Zn
@60 ppm
Milk Prodn. (kg /cow/day)Milk Prodn. (kg /cow/day)
Before Expt. 10.53a
±0.45 8.63.21b
±0.34 8.57b
±0.15 8.53b
±0.21
1st
fortnight 10.58a
±0.41 9.21b
±0.39 9.04b
±0.18 9.46b
±0.27
5th
fortnight 10.29a
±0.34 9.32b
±0.32 10.46b
±0.25 10.92a
±0.18
Fortnightly milk Somatic Cell Count (1000’S per ml)
Before Expt. 193.33a
±8.82 375.00b
±17.27 375.5b
± 21.90 386.67b
±17.83
1st
fortnight 194.17c
±3.96 355.00a
±16.0 320.83a
±18.73 236.67b
±10.93
5th
fortnight 185.83ab
±4.17 196.67b
±6.54 195.00b
3.65 172.50b
±6.02
Serum Zn level
(μmol/l) , 75th
day
31.83b
±1.05 29.17b
±1.19 30.33b
±2.03 40.67a
±1.54
Effects of nano zinc oxide on milk production and immunity in
Holstein Friesian crossbred cows (Rajendran et al., 2013)
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27. 27/61

28. Effect of Nano-Se and Se–Yeast in Feed Digestibility, Rumen
Fermentation in sheep
18 male sheep (42.5±3.2 kg of BW)
Control
group
3 mg Se/ kg
diet from
Nano-Se
(NS)
3 mg Se/ kg
diet from
Se-yeast
(YS)
(Shi et al., 2011)
Ration: Roughage (Alfalfa Hay+ Maize stalk) : Conc. (Maize, WB, SM, SFM):: 70: 30
Period : 20 days
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29. Effects of NS and SY supplementation on ruminal pH
and fermentation in sheep (She et al., 2011)
Item Control NS YS
pH
Ammonia N (mg/100 mL)
Acetate (A) (mol/100 mol)
Propionate (P) (mol/100 mol)
Butyrate (mol/100 mol)
A/P
Total VFA (mM)
6.79c
11.05c
60.52
18.23a
6.01
3.32c
91.13a
6.34a
8.35a
58.42
21.38c
5.89
2.73b
96.41c
6.57b
9.79b
59.03
19.56b
5.92
3.02b
94.19b
29

30. Effects of NS, SY supplementation on
nutrient digestibility (She et al., 2011)
Nutrient
Digestibility
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31. Effect of Nano-Se and Se-Y on Purine Derivatives in Sheep
(She et al., 2011)
Urinary
excretion
(mmol/day)
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32. 32

33. 40 Male Taihang black goats
(17.6±0.8 kg)
Age of 90±3 days
4 treatments
Period: 90 days
Effect of Nano-Se on semen quality, GPx activity, and testes
ultrastructure in male Boer goats (Shi et al., 2010)
Control
0.3 ppm
Sodium
Selenite (SS)
@0.3 ppm
Yeast- Se
(SY)
@0.3 ppm
Nano-Se
(NS)
@ 0.3 ppm
33/61

34. Effect of Na Selenite (SS) , Yeast Se (SY) & Nano Se (NS) on
growth performance, Se concentration & antioxidant status in
growing male goats (Shi et al., 2011)
Particulars Control
0.3 ppm
SS
0.3 ppm
SY
0.3 ppm
NS
0.3 ppm
Initial Wt Kg (AVR) 17.43 17.22 17.68 17.35
Final Wt Kg (AVR) 21.92a
24.01b
25.39b 24.97b
ADG (g/d) 49.9a
75.3b
85.7c 84.7c
Blood Se(µg/ml) 90d 0.19a
0.29b
0.31b 0.38c
Serum Se (µg/ml) 90d 0.07a
0.15b
0.17b 0.21c
Liver(µg/g) 1.1a
2.5b
2.8bc 3.1c
GSH-Px(U/ml) 90d 150a
233b
291c 367d
SOD(U/ml) 90d 181a
252b
250b 313c
34

35. “Effect of elemental Nano Se on feed digestibility, rumen
fermentation & PD in goat (Shi et al., 2011)
Particulars Control
0.3 ppm
SS
0.3 ppm
SY
0.3 ppm
NS
0.3 ppm
Rumen pH 6.88 6.71 6.68 6.80
NH3N (mg%) 12.49 10.30 9.95 11.22
Propionate
mol/100mol
15.67 17.21 18.10 17.26
Total VFA (mM) 73.63 75.18 77.72 75.42
DMD 0.63 0.67 0.67 0.63
NDF dig. 0.46 0.57 0.58 0.52
CP dig. 0.64 0.71 0.72 0.64
Total PD 15.43 19.26 19.75 16.28
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36. Effect of Nano-Se on semen quality, GPx activity, and testes
ultrastructure in male Boer goats (Shi et al., 2010)
42 Weaning Boer Goat buck
Two experimental treatment
Control (n=20)
@0.3 mg/kg Se
Nano selenium (n=22)
@ 0.3 mg/kg nano Se
Period: 12 weeks (Weaning to Sexual maturity)
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37. Effect of Nano-Se on Semen Characteristic
(Shi et al.,2009)
Particulars Control Nano-Se
Ejaculate volume (ml) 0.87 ± 0.31 0.97 ± 0.58
Sperm motility (%) 75.20 ± 4.69 80.51 ± 3.40
Sperm density (109 ml−1) 49.38 ± 4.10 51.95 ± 3.00
Sperm abnormality rate (%) 16.23a
± 2.68 4.34b
± 2.10
Sperm pH 6.01 ± 0.11 5.75 ± 0.07
Semen GSH-Px concentration
(U/ml)
13.6 a
± 3.15 30.0 b
± 2.87*
ATPase concentration (U/ml) 5.41a
± 1.07 16.01b
± 2.00*
Testicular GSH-Px
concentration (U/mg)
65a
± 5.89 107 b
± 9.56*
(Shi et al., 2010)
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38. Normal nuclear membrane Defective nuclear membrane
Normal mitochondria Abnormal mitochondria
Mitochondria array tightly Extensive vacuolation b/w mitochondria
38

39. 39/61

40. Effect of Nano-Cu on growth performance & serum traits of
piglets (Gonzales Eguia et al., 2009)
36 Piglets, 4 months of age
Two experimental treatment
Period: 47 days
Control
(9.6mg/kg)
CuSO4
(50 mg/ kg)
Nano Cu
(50mg/kg)
40/61

41. Item Control
(9.6mg/kg)
CuSO4
(50 mg/ kg)
Nano Cu
(50mg/kg)
Initial Body Wt. (kg) 9.57 9.68 9.67
Final Body Wt (kg) 39.00 39.97 40.50
ADG (g) 626c
639b 656a
Feed Intake (kg/d) 0.94a
1.07b
1.04c
FCR 1.63a
1.59b 1.50c
Cu availability % 23.6c
34.2b 44.0a
Serum Cu (mg/dl) 65.8 66.1 70.1
IgG, mg/ml 41.02b
46.39a 45.17a
SOD,IU/ml 43.1c
109.0b 173.3a
Effect of Nano-Cu on Cu availability, nutrient digestibility,
growth performance & serum traits of piglets
(Gonzales Eguia et al., 2009)
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42. Effect of 20 or 40 mg/kg of silver nanoparticles on
Productive performance (5 weeks after weaning)
Control 20mg/kg 40mg/kg
Feed Intake (g /d)
0–2 weeks 154 189 148
3–5 weeks 527 670 630
Daily gain (g/d)
0–2 weeks 2.1 1.9 1.7
3–5 weeks 1.6 1.8 1.8
Feed to gain (kg/kg)
0–2 weeks 2.1 1.9 1.7
3–5 weeks 1.6 1.8 1.8
(Fondevila et al., 2009) 42/61

43. Silver nanoparticles as a potential antimicrobial additive
for weaned pigs (Fondevila et al., 2009)
Experiment 2: Effect on digestive microbiota
in vitro
Experiment 3 : Digestive microbiota
and gut morphology
(μm)
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44. 44/61

45. Effects of copper-Nanoparticles (NP-Cu) and on growth and
immunity in Broiler chicken (Wang et al., 2011)
200 broiler chicks
4 group
Basal diet with 0
(control group)
50 mg/kg of
NP-Cu
100 mg/kg of
NP-Cu
150 mg/kg of
NP-Cu
Days Maize Soybean
meal
Fish
meal
Corn gluten
meal
DCP Premi
x
ME
(MJ/kg)
CP
(%)
0-21 53 8 3.5 11.15 1.7 2.7 12.41 23.57
22-42 60 8 2.4 12.60 1.35 2.9 12.70 20.88
42 Days

46. Growth performance (0-42 days) of broilers as influenced
by the levels of NP-Cu
Particulars Control
O mg/kg
50 mg/kg
NP-Cu
100 mg/kg
NP-Cu
150 mg/kg
NP-Cu
FI (g) 92.49b
96.75a
96.71a
96.59a
ADG (g) 45.81b
48.75a
49.38a
48.73a
FCR 2.02a
1.98a
1.96a
1.98a
( Wang et al., 2011)
46/61

47. Effects of NP-Cu on haematological and micro -biota in ceacal digesta of
broiler chicken
( Wang et al.,
2011)
NP-Cu supplementation
Particulars Control 50 mg/kg 100 mg/kg 150 mg/kg
TP (g/L) 37.86b
40.91a
42.22 a
42.09a
ALB (g/L) 12.97b
14.41a
14.77a
14.34a
UN (mg/dL) 2.24a
1.99b
1.93b
1.98b
Lactobacillus
(cfu/gm)
8.16b
8.32ab
8.43a
8.34ab
Bifidobacterium
(cfu/gm)
8.31b
8.44ab
8.63a
8.52ab
Coliforms
(cfu/gm)
7.36a
7.11b
6.94bc
6.90bc
47/61

48. Effects of NP-Cu on immune organ
indexes
IMI
(mg/kg)
( Wang et al.,2011)
48/61

49. Effects of NP-Cu on serum Ig, complements
CONC.
(g/L)
( Wang et al., 2011)
49

50. Effects of dietary Se source and level on growth
performance and Se concentration in serum and tissue of
broilers ( Hu et al.,
2012)
450 broiler chicks
5 group
Basal diet
supplement
ed with 0
(control
group)
Sodium
Selenite
@0.15
ppm
Sodium
Selenite
@0.30
ppm
Nano-Se
@0.15 ppm
Nano-Se
@0. 30 ppm
50/61

51. Effects of dietary Se source and level on growth performance and
Se concentration in serum and tissue of broilers ( Hu et al., 2012)
Particulars Control
group
Sodium
Selenite
0.15 ppm
Sodium
Selenite
0.30 ppm
Nano-Se
0.15 ppm
Nano-Se
0. 30 ppm
ADG (g/d) 44.3 50.2 49.8 50.4 51.4
Feed Intake, g/d 101.4 103 101.6 103.9 105.4
Feed Efficiency 0.44 0.49 0.49 0.49 0.49
Survial rate 85.6 96.7 98.9 96.7 96.7
Serum GSH-PX
(U/ml)
0.61 1.18 1.19 1.17 1.21
Serum Se (mg/kg) 0.05 0.09 0.14 0.11 0.18
Liver Se (mg/kg) 0.16 0.34 0.47 0.41 0.58
Kidney Se (mg/kg) 1.09 1.57 1.95 1.62 1.92
Muscle Se (mg/kg) 0.07 0.13 0.17 0.20 0.33
51/61

52. 52/61

53. Effect of Supplementation of Different Sources of Selenium on
Humoral Immunity in Guinea Pigs
(Bunglavan and Garg , 2013)
40 male guinea pigs
(462.0 ± 9.3 g BW)
4 Groups
Control group
@ 0 Se
Nano Se
@ 150 ppb
(35 to 50 nm)
Sodium Selenite
@ 150 ppm
Organic Se
@150 ppm
Ration Composition (%)
Maize grain: 30.5
Bengal gram: 25
Wheat bran : 24
Soya bean meal: 18
Mineral mixture: 2
Common salt : 0.5
Ascorbic acid : 0.05
Period: 70 days
4 animals injected with 0.5 ml of Pasteurella multocida vaccine I/M.
Serum antibody titre determined on days 7, 14, 21 and 28 after vaccination.
53/61

54. Particular
(Days)
Control group
@ 0 Se
Nano Se
@ 150 ppb
Sodium Selenite
@ 150 ppb
Organic Se
@150 ppby
Antibody titre (Log10)
7th
day 1.ooa
1.83c
1.08a
1.45b
14th
day 1.45a
2.13b
1.90c
1.98d
21st
day 1.75a
2.66c
2.28b
2.35b
28th
day 1.68a
2.58c
2.20b
2.28b
Mean 1.47a 2.30d
1.87b
2.02c
Effect of Supplementation of Different Sources of Selenium on
Humoral Immunity in Guinea Pigs
(Bunglavan and Garg , 2013) 54/61

55. DST will invest $20 million over the five years for their
Nanomaterials Science and Technology Initiative
IVRI- Zinc & Selenium Nanoparticle as Feed additive
NAINP, Bangalore: Zn nano particle in dairy animals
AIIMS (Delhi) : Targeting and imaging of cancer
IISc (Bangalore), IIT (Mumbai) : Liposomes
NBRC (Gurgaon) : Brain tumor
Panacea Biotec (New Delhi) , Yashnanotech (Mumbai)
Dabur Research Foundation (Ghaziabad) :
Phase-1 Clinical trials of nanoparticle delivery of the
anti- cancer drug paclitaxel, mucosal drug delivery
55/61

56. Safety problem & Potential
Risks • Change in physicochemical
properties
• Change in toxico- kinetic profile
• Can cross Blood Brain Barrier
• Strong anti microbial activity
affects gut natural microflora
• Effects on cellular biochemistry &
homeostasis
• Potential for novel toxicity in GIT
• Inflammatory digestive diseases
(Zhong et al.,2008)
56/61

57. Regulations
Existing laws are inadequate to assess risks posed
by nano based foods and packaging because:
Toxicity risks remain very poorly understood
- because of their unique properties
Not assessed as new chemicals according to
many regulations
Current exposure and safety methods are not
suitable for nanomaterials.
Up to now, there is no international regulation
of nanotechnology or nano-products. 57/61

58. NANOTECHNOLOGY & ANIMAL NUTRITION: FUTURE
CONSIDERATIONS
Establishment of publicly accessible & cost
effective nano tech based feeds.
Risk Assessment & safety (Smart et al., 2006)
Legal framework governing application of
nanotechnology in feed
Legal provision to ensure safety of nano feed
(Food safety Authority of Ireland,2008)
Feed surveillance programmes
Control on disposal/recycling of nanofeed
58/61

59. Conclusion
Nanotechnology can be used in Animal
nutrition sector to improve feed quality,
bioavailability of nutrients, growth, production
performance & immune status in livestock.
Proper legal framework & provisions to be
employed for biologically safe & cost effective
production and utilization of nano-particles for
livestock feeding . 59

60. 60
Than
k
There’s STILL plenty of room at the bottom….