Pankaj Kumar SinghAssistant Professor(Animal Nutrition),

Bihar Veterinary College, Patna, Bihar, India-800014

E-mail: vetpank@gmail.com 1/61

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’

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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

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1. Nanoparticles

2. Nano emulsion

3. Nano clays

4. Buckey balls

5. Micelles

6. Quantum dots4/61

INORGANIC NANOPARTICLES

SILVER 5/61

ORGANIC NANOPARTICLES

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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-emulsionb) Precipitation (Huang et al., 2007)

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Physical Methods1. 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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BALL MILLINGPrinciple: 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 (Bakker et al., 1995). 9

1. Cross linking micro-emulsion methods2. 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

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)

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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 12/61

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) 13/61

Surface effectParticle size < 100nm Lesser stability of atomsLesser energy needed to join adjacent atomsLower fusion point

Quantum effectSpecial arrangement allow to have different properties

than parent elementMore surface area than micro particlesChemical reaction rate increases 1000 times

(Buzea et al., 2007)

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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)

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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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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

V

VVV

VV

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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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 barrierBetter absorption than macro equivalents (Hoet et al., 2004)

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GI Uptake and Translocation of NPsIncreases 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) 20/61

Nanoparticles

Absorption of NPs Through the GIT

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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 activityProtecting from undesired bile salt Protecting from commensal microorganismEnhance bioavailability

(Handy, 2007)22/61

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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

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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 194.17 c±3.96 355.00a±16.0 320.83a±18.73 236.67b±10.93

5th fortnight 185.83ab±4.17 196.67b±6.54 195.00b3.65 172.50b±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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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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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

94.19b29

Effects of NS, SY supplementation on nutrient digestibility (She et al., 2011)

Nutrient Digestibility

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Effect of Nano-Se and Se-Y on Purine Derivatives in Sheep

(She et al., 2011)

Urinary excretion (mmol/day)

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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)

Control0.3 ppm

Sodium Selenite

(SS) @0.3 ppm

Yeast- Se (SY)

@0.3 ppm

Nano-Se (NS) @ 0.3 ppm

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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 Control0.3 ppm

SS 0.3 ppm

SY0.3 ppm

NS 0.3 ppm

Initial Wt Kg (AVR) 17.43 17.22 17.68 17.35Final 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

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“Effect of elemental Nano Se on feed digestibility, rumen fermentation & PD in goat (Shi et al., 2011)

Particulars Control0.3 ppm

SS 0.3 ppm

SY0.3 ppm

NS 0.3 ppm

Rumen pH 6.88 6.71 6.68 6.80

NH3N (mg%) 12.49 10.30 9.95 11.22Propionate mol/100mol

15.67 17.21 18.10 17.26

Total VFA (mM) 73.63 75.18 77.72 75.42DMD 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 16.2835/61

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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Effect of Nano-Se on Semen Characteristic

(Shi et al.,2009)

Particulars Control Nano-SeEjaculate volume (ml) 0.87 ± 0.31 0.97 ± 0.58Sperm motility (%) 75.20 ± 4.69 80.51 ± 3.40Sperm density (109 ml−1) 49.38 ± 4.10 51.95 ± 3.00Sperm abnormality rate (%) 16.23a ± 2.68 4.34b ± 2.10Sperm pH 6.01 ± 0.11 5.75 ± 0.07Semen 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-Pxconcentration (U/mg)

65a ± 5.89 107 b ± 9.56*

(Shi et al., 2010) 37/61

Normal nuclear membrane Defective nuclear membrane

Normal mitochondria Abnormal mitochondria Mitochondria array tightly Extensive vacuolation b/w mitochondria

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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)

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Item Control(9.6mg/kg)

CuSO4(50 mg/ kg)

Nano Cu (50mg/kg)

Initial Body Wt. (kg) 9.57 9.68 9.67Final Body Wt (kg) 39.00 39.97 40.50ADG (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 70.1IgG, 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) 41/61

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 1.8Feed 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

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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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 Premix

ME (MJ/kg)

CP (%)

0-21 53 8 3.5 11.15 1.7 2.7 12.41 23.5722-42 60 8 2.4 12.60 1.35 2.9 12.70 20.88

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42 Days

Growth performance (0-42 days) of broilers as influenced by the levels of NP-Cu

Particulars ControlO mg/kg

50 mg/kgNP-Cu

100 mg/kgNP-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)

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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

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Effects of NP-Cu on immune organindexes

IMI(mg/kg)

( Wang et al.,2011) 48/61

Effects of NP-Cu on serum Ig, complements

CONC.(g/L)

( Wang et al., 2011)

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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 supplemented with 0 (control group)

Sodium Selenite @0.15 ppm

Sodium Selenite @0.30 ppm

Nano-Se@0.15 ppm

Nano-Se@0. 30 ppm

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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-Se0.15 ppm

Nano-Se0. 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

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Effect of Supplementation of Different Sources of Selenium onHumoral 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: 25Wheat bran : 24Soya bean meal: 18Mineral mixture: 2Common salt : 0.5Ascorbic 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.

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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 onHumoral Immunity in Guinea Pigs

(Bunglavan and Garg , 2013) 54/61

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

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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)

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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.

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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

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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

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Thank

You

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