Nucleotides and Bioactive peptides. Boosting the ... · 500 ppm of Nucleoforce™ in the feed had a...

AQUAFEED HORIZONS. MARCH 27, 2018, BITEC, BANGKOK, THAILAND

Francisco J. González DVMProject and Area Manager for APAC. Bioiberica, Spain.

Nucleotides and Bioactive peptides.Boosting the functionalities of aquafeeds

Introduction:

• Ocean will not be able to coverworld demand for fish in the future…it needs to come from farming!

• Aquaculture production has grownsteadily in the last five decades

AQUACULTURE: Efficient

Sustainable

Source: FAO 2014. The State of World Fisheries and Aquaculture

Francisco González. Bangkok 27th March 2018

Introduction:


• IMPROVES IN AQUACULTURE industry:improved genetics, increased knowledge on biology and handling, improved facilities and advances in NUTRITION

• Current CHALLENGES: Adverse climate conditions, globalization of pathologies, tendency to ↑seeding densities, prohibition of growth promoters, variations in cost of raw materials for diets

→ Need for new tools and mechanisms:

1-enhance steady productivity and economic growth

2-while avoiding negative impact of these challenges

→ increase adaptability to stressful scenarios (diet, environment, handling and diseases)

Using NUTRITION to improve Immune and Digestive Health

1. What do we do at Bioiberica?

2. SOLUTIONS FOR AQUACULTURE:

• Dietary nucleotides

• Hydrolysed protein (bioactive peptides)3. Scientific Evidences

4. A look to the future


What we do:

Research, development, manufacturing and marketingof BIOMOLECULES designed to improve

HUMAN, ANIMAL and PLANT health

Introduction:


Introduction:

Specialists in BIOMOLECULES

APPLICATIONS IN AQUACULTURE


The CONCEPT:

Use NUTRITION to improveIMMUNE and DIGESTIVE Health

❶DIETARY

NUCLEOTIDES

❷BIOACTIVEPEPTIDES

↑ PRODUCTIVITYIMPROVE INTESTINAL HEALTH,

DIGESTIBILITY AND PALATABILITY

PROMOTE GROWTH,IMPROVE IMMUNE & GI HEALTH

AND SURVIVAL RATE


❶Dietary NUCLEOTIDES

Nucleotides can polymerize to form anucleic acid macromolecule such as DNA or RNA,the basic building blocks of all living organisms



• Low molecular weight intracellular compounds.Key roles in biochemical processes

• Nucleotide patterns are species-specific

Structure: NUCLEOBASE (nitrogenous base) FIVE-CARBON SUGAR (ribose/desoxyribose) 1 or more PHOSPHATE GROUPS

Purine Nucleotides ( ATP, GTP, IMP )Pyrimidine Nucleotides ( UTP, CTP, TMP )

Fontana et al. Role of Dietary Nucleotides in Immunity Functional Food Reviews, Vol 2, No 3 (Fall), 2010: pp 00–00



Semi-essential nutrients – conditionally essential nutrientsEssential during rapid growth, malnutrition, infection, stress or injury: High energetic cost!

Rapidly proliferating tissues (immune system, intestine) not able to fulfillneeds of cell nucleotides exclusively by de novo synthesis→ Salvage pathway: recovering of nucleosides and nucleobases from blood and DIET


Younger animals: higher cell replication rates, lower endogenous synthesis and poorer diet digestion

Stress: nucleotide demand increases to support the immune system and the availability of energy is reduced

Special situations or periods when tissues are developing: Immune system, bone marrow, intestinal mucosa, liver, reproductive system...

OUR APPROACH: Tailor-made products for AQUACULTURE

Nucleotides considered semiessential nutrients

Aquatic species show a faster growthcompared to terrestrial species

Aquatic species have been recently domesticated, so they are

more sensitive to stress

Use of vegetable proteins causesintestinal damage that should be repaired

quickly to avoid production losses



NUCLEOTIDES in pediatric nutrition:

1980s: Epidemiological studies:Breastfed babies less diarrhea episodes than those fed with processed milkThe difference between breast milk (human) and formulated milk (cow): NUCLEOTIDES→Addition of nucleotides to infant formulations

1991: EU Scientific Committee on Food:Nucleotides included in therecommendations of infant formulas



Bioiberica decided to extrapolate to other species...but NUCLEOTIDE PROFILES ARE SPECIES-SPECIFIC!

0

500

1000

1500

DairyCow

Sheep Sow Bitch Hen Salmon

Purine N.Pyrimidine N.

μM/Kg


High Quantity and Absorption: absorption

Nucleoproteins

Proteases

Nucleic acids

Nucleases/Phosphoesterases

Nucleotides

NUCLEOSIDES

Alk. phosphatases

Lumen Enterocyte

Nucleotides

NUCLEOSIDES

Hydrolisis grade

❷NUCLEOTIDES: How to choose a good source?


Nucleotide content of some ingredients used in the feed industry

Raw Material Total Nucleotides Mg/Kg ( ppm )

Barley 5

Casein 1

Fish Meal 75

SD Plasma 7

SD Blood Cells 55

Acid Whey 294

Whey Protein Concentrate 282

Common Weaning Feed Diet 71

Shrimps Egg 450-500

Nucleoforce Range 240,000 – 900,000

Concentration




CONCENTRATION

SPECIFICITY AVAILABILITY


Scientific studies with NUCLEOTIDES:Immunity and growth performance of giant tiger prawn (Penaeus Monodon)after administration of dietary nucleotidesVisayas State University. THE PHILIPPINES, 2009

Effects of dietary nucleotides (Nucleoforce Shrimps™) on survival of Litopenaeus vannameiCENAIM RESEARCH CENTER, ESPOL, ECUADOR, 2014

Effects of dietary nucleotides (Nucleoforce Shrimps™) on survival of Litopenaeus vannameiinfected with White spot syndrome virus (WSSV)

CENAIM RESEARCH CENTER, ESPOL, ECUADOR, 2014

Effects of diets supplemented with nucleotides on the performance and health of Tilapia GIFT Foundation in Central Luzon State University (CLSU) in Nueva Ecija. Phillippines 2010


Effects of DIETARY NUCLEOTIDES plus α-glucans on productive parametersof white shrimp Litopenaeus vannameiCENTRO DE INVESTIGACIÓN EN ALIMENTACIÓN Y DESARROLLO, A.C., Mazatlán, MEXICO

Effects of dietary nucleotides plus α-glucans in white shrimp Litopenaeus vannameiaffected by Acute Hepatopancreatic Necrosis Disease (AHPND)CENTRO DE INVESTIGACIÓN EN ALIMENTACIÓN Y DESARROLLO, A.C., Mazatlán, MEXICO

Effects of dietary nucleotides in white shrimp Litopenaeus vannamei performance. Economic analysisFACENDA TECNARÃO, BRASIL, 2016

Effects of dietary nucleotides on the performance in first stages of growth in Litopenaeus vannamei.CTY TNHH TOAN CAU, Bac Lieu, Vietnam 2017

Scientific data: Nucleoforce SHRIMPSImmunity and growth performance of giant tiger prawn (Penaeus Monodon)after administration of dietary nucleotidesVisayas State University. THE PHILIPPINES, 2009

Objectives: Determine effects of NF Shrimps™ administration to giant tiger prawn in pond conditions

Parameters:- Immune system: haemocyte count- Growth- Survival rate

Results:• Significantly increased haemocyte count after 10 weeks• Nucleoforce® inclusion significantly enhanced the proportion of granulocytes,

semigranulocytes and hyalinocytes in the shrimp haemolymph

0

5

10

15

20

25

30

c

b

a

Total Haemocyte Count

Control 250ppm 500ppm


Scientific data: Nucleoforce SHRIMPSImmunity and growth performance of giant tiger prawn (Penaeus Monodon)after administration of dietary nucleotidesVisayas State University. THE PHILIPPINES, 2009

Results:Increased survival rate: 500 ppm and 1000 ppm of Nucleoforce™ significantly improved survival rate during the early stage of the prawns

Approx. Wk4:Heavy continuous rain → freshwater input into the pond

↓salinity to levels detrimental to prawn survival and growth


Scientific data: Nucleoforce SHRIMPSImmunity and growth performance of giant tiger prawn (Penaeus Monodon)after administration of dietary nucleotide supplementationVisayas State University. THE PHILIPPINES, 2009

Results:Increased growth

Conclusion: Dietary supplementation with Nucleoforce® enhances growth performance, survival and immunity of P. monodon, enabling the shrimps to fight infection or disease


Scientific data: Nucleoforce SHRIMPS


Objective: evaluate effects of 2 diets on growth performance and health status of L. vannamei

Treatments: 1,000 animals initially PL27• Treatment 1: supplementation 500 ppm NF SHRIMPS™• Treatment 2: no supplementation (Control)

(5 replicates per treatment)

Methods:Growth Stage: 0.02 g - 4 weeks of life5 tanks of 500 L with 500 Shrimpsper treatment

Parameters:growth, survival rate, biomass




Conclusion:Inclusion of 500 ppm of Nucleforce Shrimps™ had a positive effect on survival rate of L. vannamei

Weight (gr) Biomass (gr) Survival (%)

Control 0.116 a 44.6 b 76.96 b

NF SHRIMPS 0.118 a 58.52 a 99.2 a

0

10

20

30

40

50

60

70

80

90

100

SURVIVAL %

control

NF 500 PPM

0

10

20

30

40

50

60

BIOMASS gr

Results after 4 weeks:


Scientific data: Nucleoforce SHRIMPSEffects of dietary nucleotides (Nucleoforce Shrimps™) on survival of Litopenaeus vannameiinfected with White spot syndrome virus (WSSV)CENAIM RESEARCH CENTER, ESPOL, ECUADOR, 2014

Objective: Determine the effect of Nucleoforce Shrimps™ inclusion on the diet of shrimps (Litopenaeusvannamei) on its performance and survival rate when exposed to White Spot Disease

Treatments: • Group 1: supplementation 500 ppm NF SHRIMPS™• Group 2: no supplementation (Control)

Methods:• Growing Period: 1000 animals of 0.02 g until 3.5 gr

500 animals per treatment, 5 replicatesper treatment of 100 animals in 500L tanks

• Infection Period: Selection of 200 animals, 100 animals per treatment, 10 replicates of 10 animalsper treatment in 40L tanks.

Data Collection: Mortality at 9 days after the infection



Results:Survival after challenge / days after infection

Conclusion: 500 ppm of Nucleoforce™ in the feed had a positive effect on the survival rate of L. Vannamei during the growing period and reduced the mortality when shrimps were infected with WSSV

Effects of dietary nucleotides (Nucleoforce Shrimps™) on survival of Litopenaeus vannameiinfected with White spot syndrome virus (WSSV)CENAIM RESEARCH CENTER, ESPOL, ECUADOR, 2014



OBJECTIVE:To evaluate the effects of NUCLEOFORCE SRHIMPS PLUS and α-glucanson different production parameters of white shrimp Litopenaeus vannamei

MATERIAL AND METHODS:

- Control group: regular diet with no supplementation- NUCLEOTIDES Group: 500ppm Nucleoforce Shrimps Plus (Bioiberica SAU)- NUCLEOTIDES+ α-glucans group: 500ppm Nucleoforce Shrimps Plus + α-glucans

• 50 juveniles of white shrimp (Litopenaeus vannamei) / tank4 replicates

• Inicial mean weight 0.37g

• Intervention during 28 days and four replicates for each group• Controlled conditions of temperature,

oxygen and salinity of water



RESULTS:

ZOOTECNICAL PARAMETERS:

Values with the same superscript do not show significant differences (P> 0.05)

CONTROL NUCLEOTIDES NUCLEOTIDES+ α-glucans

Initial weight (g) 0.37±0.00 0.37±0.00 0.37±0.00

Final weight (g) 2.74±0.60 2.91±0.67 2.98±0.68

Weight gain (g) 2.37±0.13 2.54 ±0.14 2.61±0.21

Individual Food Intake (g/day) 3.85±0.11 3.82±0.22 3.98±0.17

Growth Rate (%) 637.64±34.46 684.81±36.56 702.31±55.80

Food Conversion Ratio 1.63±0.10 1.50±0.08 1.53±0.18

Specific Growth Rate (%/day) 7.13±0.16 7.36±0.16 7.43±0.25SURVIVAL 99.43±0.98 100.00±0.00 98.67±2.31



RESULTS:

2. ZOOTECHNICAL PARAMETERSWEIGHT (g):

• Significantly HIGHER FINAL WEIGHTvs CONTROL with NUCLEOTIDESand with NUCLEOTIDES + α-glucans

0

0.5

1

1.5

2

2.5

3

3.5

0 days 14 days 28 days

CONTROL NUCLEOTIDES NUCLEOTIDES + α

P < 0.05



AHPND:

• Acute hepatopancreatic necrosis disease (AHPND) /Early Mortality Syndrome” (EMS)

• Affecting major shrimp producing countries China, Thailand,Vietnam and Mexico. Mortality rate can be beyond 70%

• Caused by specific strains of Vibrio parahaemolyticus that are difficult to eradicate• Transferred through oral and then localizes in the shrimp gastrointestinal tract

affecting hepatopancreas

• Use of antibiotics not desirable: risk of resistance + rejection by legislators and consumersSustainable approaches based on functional feeds are needed

Beneficial effect of NUCLEOTIDES /

NUCLEOTIDES + α-glucans?

Early Mortality Syndrome (EMS) as new Emerging Threat in Shrimp Industry- Mohammad Jalil Zorriehzahra



OBJECTIVE:To evaluate the effectiveness of NUCLEOFORCE SHRIMPS PLUS and α-glucanson the survival of white shrimp affected by AHPND.

MATERIAL AND METHODS:• 50 juveniles of white shrimp (Litopenaeus vannamei) / tank• Controlled conditions of temperature, oxygen and salinity of water

• CHALLENGE: Inoculation of pathogenic strain Vp AHPND+ (confirmed by histopathology)

- Negative Control group (C-) – no challenge: regular diet with no supplementation- Positive Control group (C+) – challenge: regular diet with no supplementation- NUCLEOTIDES: 500ppm Nucleoforce Shrimps Plus- NUCLEOTIDES+α-glucans low dose- NUCLEOTIDES+α-glucans medium dose



RESULTS: MORTALITY 170h post-infectionSignificantly reduced mortality with NUCLEOTIDES + α medium dose vs infected control

0

10

20

30

40

50

60

70

Negative Control Positive Control Nucleotides alone Nucleotides + α low dose

Nucleotides + α medium dose

P < 0.05

0%

62%

49%

39%

45%

Nucleotides + α medium dose



RESULTS: HISTOPATHOLOGY

With infection,• Pale hepatopancreas• Empty midgut

NEGATIVE CONTROL (C-)

INFECTED CONROL (C+)

NUCLEOTIDES + α low dose

NUCLEOTIDES + α medium dose

NUCLEOTIDES

Early Mortality Syndrome (EMS) as new Emerging Threat in Shrimp Industry-Mohammad Jalil Zorriehzahra 2015



RESULTS: HISTOPATHOLOGYTubular necrosis + infiltration

-VE CONTROL +VE CONTROL NUCLEOTIDES + α-glucans medium



CONCLUSIONS:

1. Dietary supplementation with NUCLEOTIDES and NUCLEOTIDES + α-glucansleads to improvements in survival in white shrimp affected byAcute Hepatopancreatic Necrosis Disease (AHPND)


Performance Testing and Disease ControlResearcher: Giovanni ChasinObjetives:

1. Zootecnical results. 2. Evaluation criteria:(1) Survival ratio; (2) Prevalence of clinical signs of the most importante diseases recorded in the farm;(3) Final size.

PRODUCT DOSEA. NUCLEOFORCE SHRIMPS 1g/kg feed (1kg/ton or 1.000ppm)B. PROBIOTIC* 1g/kg feed (1kg/ton or 1.000ppm)C. MOLASSES 2g/kg rfeed (2kg/ton or 2.000ppm)D. CONTROL

*Bacillus cereus var. toyoi 4,0 x 1011 e Bacillus subtilis 4,0 x 1011 UFC

Effects of dietary nucleotides in white shrimp Litopenaeus vannamei performance. FACENDA TECNARÃO, BRASIL, 2016


Experimental design - 3 replicates for each

treatment,including control group.- Average weight: 4,38 gr, same size

and health status- Floating cages 1 m³;- Density 20/m². - Period of testing: 30 days.

TREATMENT REPLICATES

NUCLEOFORCE SHRIMPS 3

PROBIOTIC* 3

MOLASSES 3

CONTROL 3

Total 12



GROUP SURVIVAL (%) WEIGHT WEIGHT PER

CAGE (Gr/M2)

CONTROL 61,55 9,93 122,24

NF SHRIMPS 73,30 9,65 141,47

PROBIOTIC* 68,30 9,79 133,73

MOLASSES 35,00 10,86 76,02*Bacilus cereus var. Tayoi y Bacillus subtilis




Objective: Evaluate the overall effect of nucleotides on the performance of white shrimp.Treatments:1. Basal diet (Control)2. Basal diet +1g /cubic meter of water in the pond. (Nucleoforce Shrimps Plus™)

ANIMAL, MATERIAL AND METHODS1.4 millions of Zoea divided into two groups of 700,000 and allocated in 3 metric tons of water ponds.

Growing Period: 17 days

Larvae fed with diatom algae and artemia.

Nucleoforce group additionally receives 3 g of NucleoforceShirmps Plus per day for 17 days.


Results: .Inclusion of Nucleforce Shrimps™ had a positive effect on survival rate of L. vannamei.Larger and more uniform size of PL in Nucleoforce gorup


CONTROL NUCLEOFORCE

Survival (%) 48 51

Visual examination Diferent sizes Larger and more uniform size


Results:Inclusion of Nucleforce Shrimps™ had a positive effect on survival rate of L. vannamei.Larger and more uniform size of PL in Nucleoforce gorup.


Nucleoforce Control

Visual examination:


❷ Bioactive peptides

Source of high quality protein (hydrolysed porcine intestinal mucosa)

High% in free amino acids and high digestibility

High palatability, which favors food intake

Directly nurtures the intestinal mucosa, promotingbetter development of villi, improving nutrient absorption

Promotes intestinal recovery in weaned piglets

It translates into improvements inproductivity and also animal health


❷ Bioactive peptides

BIOLOGICAL AND TOXICOLOGICAL SAFETY

Average molecular weight: Palbio 62: 600 Da

(prions generally have molecular weight of 40,000 Da!)

100% Porcine origin 100% controlled by PCR+ comprehensive system of traceability

No heavy metals

Certified process: prionic and viral activity inactivated


Scientific data: PALBIO

Evaluation of Palbio® 62SP (porcine hydrolysed mucosa) supplementation on the production yield of young gilt-head bream fed on 100% plant-based diets with complete elimination of fish mealBiodiversity and Aquaculture Group, Universidad Politécnica de Valencia. Spain, 2014

Objective: evaluate effects of supplementing diet of gilthead bream fed exclusively with plant-based proteins in the complete absence of fish meal with high-quality hydrolysed protein (Palbio® 62 SP)

Animals, materials and methods:Gilt-head bream initial weight of 11grOver 134 days using three experimental fish meal-free feeds

Feeds manufactured by extrusion andfeeding was performed manually twice daily

Fish were kept in 15 cages with a size of 1m3

in 4000-litre tanks, with aeration and including a seawater recirculation system

Temperature 22-24 ºC during the trial

The growth (final weight and growth rate) and nutritional efficiency were evaluated

Ingredients CONTROL PB 1% PB 2%

g/kg g/kg g/kg g/kg

Fish meal - - -Wheat - - -Wheat gluten 290 290 290Beans 44 44 44Soy 182 182 182Pea 44 44 44Sunflower 181 181 181Palbio 62 SP 10 20


Scientific data: PALBIOEvaluation of Palbio® 62SP supplementation on the production yield of young gilt-head bream fed on 100% plant-based diets with complete elimination of fish mealBiodiversity and Aquaculture Group, Universidad Politécnica de Valencia. Spain, 2014

Results:

Conclusion: these results are highly promising as regards feeding gilt-head bream with fish meal-free feed and opens up other possibilities in the search for new sources of protein

TREATMENTS CONTROL PB 1 % PB 2%

Initial weight (g) 12.7 a 9.8 b 10.3 ab

Final weight (g) 87 c 121 a 99 b

EGR – Especific Growth Ratio (%/day) 1.59 c 1.81 a 1.65 b

FCR 1.93 b 1.50 a 1.70 ab0

20

40

60

80

100

120

140

CONTROL PB 1% PB 2%

Initial weight (gr) Final weight (gr)

c

a b


UNIVERSIDAD POLITECNICA DE VALENCIA, ESPAÑA, 2014

% Apparent digestibilitycoefficient

CONTROL 64

PALBIO 62 1% 73

PALBIO 62 2 % 66

Apparent digestibility coefficient

Evaluation of Palbio® 62SP supplementation on the production yield of young gilt-head bream fed on 100% plant-based diets with complete elimination of fish mealBiodiversity and Aquaculture Group, Universidad Politécnica de Valencia. Spain, 2014



group FM 35% Soja 35% Soja + 1% P62 55% Soja 55% Soja +

1% P62% % % % %

Fishmeal Super Prime 25,000 12,500 12,500 0,000 0,000Poultry meal 65 20,000 10,000 9,000 10,000 9,100

Palbio 62 SP 1,000 1,000Corn gluten 2,000 2,550 2,550 2,550 2,550

Soybean meal 48 0,000 35,000 35,000 55,000 55,000Wheat meal 43,900 27,220 27,100 17,380 17,180

Fish oil - SAVINOR 1,300 3,400 3,520 4,300 4,400Vit & Min Premix PV01 1,500 1,500 1,500 1,500 1,500

Soy lecithin - Liquid 2,000 2,000 2,000 2,000 2,000Cholesterol SF 0,100 0,100 0,100 0,100 0,100

Binder (Kieselghur) 0,800 0,800 0,800 0,800 0,800Antioxidant powder 0,200 0,200 0,200 0,200 0,200Sodium propionate 0,100 0,100 0,100 0,100 0,100

MCP 3,100 4,050 4,050 5,000 5,000L-Lysine 0,000 0,050 0,050 0,100 0,100

L-Threonine 0,000 0,120 0,120 0,270 0,270L-Valine 0,000 0,250 0,250 0,400 0,400

DL-Methionine 0,000 0,160 0,160 0,300 0,300Total 100,000 100,000 100,000 100,000 100,000

Effect of the gradual replacement of fishmeal by PALBIO 62and with increasing levels of soybean meal on growth of Litopenaeus vannameiCENAIM RESEARCH CENTER, ESPOL, ECUADOR, 2016


8

8.2

8.4

8.6

8.8

9

9.2

9.4

HP 25 % HP 12,5% + SOJA35%

HP 12,5% + SOJA35% + P62 1%

HP 0% + SOJA 55% HP 0% + SOJA 55%+ P62 1%

12 weeks

HP 25 %

HP 12,5% + SOJA 35%

HP 12,5% + SOJA 35% + P62 1%

HP 0% + SOJA 55%

HP 0% + SOJA 55% + P62 1%


Effect of the gradual replacement of fishmeal by PALBIO 62and with increasing levels of soybean meal on growth of Litopenaeus vannameiCENAIM RESEARCH CENTER, ESPOL, ECUADOR, 2016



NEW Scientific data from a recent study

In vitro study evaluating the ability of porcine hydrolyzed mucosa PALBIO62 to enhance cellular proliferation

LEITAT Technological Center,BARCELONA


Scientific data: PALBIOIn vitro study evaluating the ability of porcine hydrolyzed mucosa PALBIO62to enhance cellular proliferationLEITAT Technological Center, BARCELONA

Objective: Evaluate effects of different fractions ofPALBIO62 on cell proliferation

Results inHUMAN DERMAL FIBROBLASTS

Using low molecular weightfraction of Palbio62

FCS = fetal calf serumUntreated = only Dulbecco’s modified

Eagle basal medium

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

Untreated 10% FCS Dil 1/4 Dil 1/8 Dil 1/16 Dil 1/32

Fold

-in

crea

se o

ver

un

trea

ted

cel

ls

Fraction 9 dilution

HDF primary cultures

*

*

**

*


Summary AQUACULTURE – R&D Bioiberica Animal Health:

Nucleotides for aquaculture: Promote growth and improve immune & intestinal health Good scientific evidence in humans and other species Building up more scientific support for fish and shrimps

Bioactive peptides for aquaculture: Increase productivity, digestibility and palatability Improve intestinal health

Bioiberica R&D: investments in AQUACULTURE

Need for new tools and mechanisms to enhance growth of the aquaculture industry

Design diets and NUTRITIONAL STRATEGIES with positive effects on animal healthwhile avoiding negative impact of current challenges


A look to the future… AQUACULTURE is the future

“We must plant the sea and herd its animals...

using the sea as farmers instead of hunters.

That is what civilization is all about –

farming replacing hunting”

Jacques-Yves Cousteau 1971


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