Lecture 1 Feeding and Growth

Fish generally more efficient

- Can maintain position in water

- Do not regulate body temperature
- Excrete ammonia directly into water
- nutrient dense feed

Ingestion and post-ingestion processes

1. Increase Ghrelin, stimulate GH increase

2. Food in: Decrease Ghrelin
3. Food digested: GH-R increase
4. Nutrients into blood, Plasma FAA and IGF-I increase

Fish feeding

- Appetite regulated by circadian rhythms

- Fish prepare for growth before meal (if predictable)
- Feeding first meal at same time each day beneficial
- Nutrient dynamics and endocrinology make feeding rhythm better
- Barramundi digest and absorb faster than salmon, minimise starvation overnight

Lecture 2 Experimental Design

Experimental Design

- Treatment vs natural
- Experimental unit: tank
- Standardization and replication

Experimental feeds

1. Same batch
2. Same energy contents (isocaloric)
3. Same protein content (isonitrogenous)
4. Ration -> satiation/ restricted

Measuring feed intake

1. Direct observation
2. Radiographic techniques
3. Stomach content analysis
4. Markers and dyes in feeds

Problems with length only based measures

1. Does not account for tissue growth

 - Shell length may increase, Tissue mass may decrease
- Length increase, weight may decrease (fish)
2. Condition factor (k)
- r/s between body mass and length
- fish: [weight (g)/ length (cm)^3] x 100
- bivalves: [meat weight (g)/ internal shell volume (ml)] x 100
- higher value = better condition

Feed efficiency

1. Feed conversion ratio (FCR)

- Feed consumed/ mass produced
2. Protein efficiency ratio (PER)
- Mass produced/ mass of protein fed
- Growth per unit protein
3. Net protein utilization (NPU%)
- [Protein gain (g)/ protein intake (g)] x 100
- Better than PER

Lecture 3 Ingredient Evaluation

Raw material composition

- protein (aa)
- moisture
- ether extract (fat/ fatty acids)
- ash
- fiber
- nitrogen-free extract (NFE/ energy)
- vitamins
- minerals
- energy

Proximate composition
NFE: soluble carbohydrates estimated by difference after analysis of other components

Protein

1. Kjeldalh nitrogen -> digestion with conc H2SO4 at high temp

- Colorimetric reaction
- Official standard
2. Dumas
- Combustion and nitrogen gas detection

Fat

1. Soxhlet
- Low phospholipid capture, lower fat readings
2. Acid hydrolysis
- More complete analysis
3. Chloroform/methanol

Energy

- Sample ignited in excess of O2 inside sealed container, inside insulated water vessel

Antinutritional factors

1. Digestibility
- Difference between food and faeces
- Apparent vs true digestibility -> endogenous losses, assume marker move through
gut same rate as food and not digested
 2. Species-specific digestibility
3. Palatability/ attractiveness (sapionin)
4. Ingredient effect on feed production
- Expansion of feed during extrusion
- Oil absorption capacity of the final pellet
- Pellet durability: physical integrity
- Pellet stability in water: physical integrity + nutrient leaching
5. Feed and nutrient utilization

Lecture 4 Feed Formulation

Price of raw materials vs protein content

- Protein expensive
- Dense sources of protein important to meet nutrient requirement

Formulation

1. Species-specific nutrient requirements

- Nutrient digestibility
- Fat metabolism
- Protein utilization
- Micro-nutrition
2. Max/minimum raw material inclusion
- Limited by specific antinutrient levels in formulation or max raw material
- Select low-alkaloid lupins (maximum sapionin levels in formulation for soy)
- Phytic acid -> in vege as storage form of inositol, bind to P Zn Mg
3. Requirements for feed production
- Antioxidants: prevent rancidity of lipids
- Oxidation: loss of vitamins, EAA and EFA and production of toxic by-products
- Improve animal antioxidant capacity
- Natural: vit E (tocopherols), vit C
- Artificial: ethoxyquin, Butylated HydroxyToluene (BHT), BHAnisole (BHA)
4. Seasonal requirements
5. Health requirements
- Product or challenge specific
- Support barrier function of fish, immune system, antioxidant, anti-inflammatory
functions
 6. Customer-specific requirements

Formulation limits due to feed production

1. Mash fat -> lubricates and decreases friction

2. Minimum starch level -> binding and expansion, float/sink properties
3. Densification
4. RVA/ Shear cells
5. Raw materials and moisture

Nutritional strategies

1. Appropriate raw materials

2. Feeds tailored to challenges
3. Cost effective feeds and feeding
4. Water treatment for water soluble antinutrients
5. Water + alcohol extraction removes antinutrient in soy and concentrations to form
SPC

Lecture 5: Feed Manufacture

Purchasing – quality criteria and cost

- Minimum and maximum standards

- Antioxidants needed? Rancidity issues
- Traceability
- Certification criteria

Intake

- Pre-cleaners (magnets and sifters)

- Storage (silos) -> oil tanks mild steel, stainless expensive, higher volumes self heat
more readily, antioxidants important

Moving raw materials

- Bucket elevators
- Screw conveyors
- Pneumatic conveyor

Silo storage: bulk vs micro

- How many raw materials used

- How many days of storage available
- Appropriate storage -> use before out of date, safety stock, accuracy of weighing and
batch size (depend on volume of input)
Mixing

- Ribbon mixer
- Single shaft paddle mixer
- Twin shaft paddle mixer

Particle size reduction

- Reduce particles to <1/3 of die open area

- Hammer mills: 200-250 microns, good for 1mm feed
- Screen size dictate particle size

Pulverisers

- Beater plates force particles against wear liners

- Particle reduction on impact
- <100 micron particle size

Extrusion

- Need prescreening to avoid large particles blocking die

- Live bin: one/more batch of meal mix and buffer to continuous system
- Feed system -> preconditioner

Preconditioning

- Mixing, hydration, heat

- Starch hydration
- Gelatination: amylose leak from starch granules, amylopectin deformed. As cool, H
bonds form and expel water. Amylopectin forms stronger bond than amylose

Extrusion

- Energy
1. Mechanical -> kneading elements, motor size, back-pressure
2. Thermal -> hot water, steam, thermoblocks
- Twin screw: more mixing, more homogenous, more cost, higher range of fat moistyre
formulation flexibility
- Die: metal plate with holes through which feed mixes pass, cut by rotating blade

Drying

- Spread pellets to constant bed depth

- Time, temperature, airflow control drying

Oil Coating

- Dry (8-15% moisture) kernel placed into vacuum chamber

- 100-200 mbar
Cooling

- Ensure within 7 degree of ambient temp

- Packing hot pellets cause sweating -> mold
- Speed of cooling affects crystalisation of saturated fats – fat leakage

Buoyancy control: moisture content, degree of starch gelatinization

- Bulk density: <480g/L floating, >640g/L, in between slow sinking

- Starch 7% sinking, 20% floating

Moisture content and water activity

- Excess water: mold

- Mycotoxin risk
- Aw water activity measure of free water, no microbial activity at Aw <60
- Mold inhibitor and salts decrease Aw

Benefits of extruded feeds

1. Water stability: starch binding

2. Improved feed digestibility
3. Deactivation of anti-nutritional factors
4. Buoyancy control
5. Bacteria/fungus free product
6. Up to 30% fat

Lecture 6 Feed Formulation

Raw material limitations

- Fish meal and oil

- Antinutrients
- Mycotoxins: aflatoxins, deoxynivalenol, zeraleonone, fumonisin, ochratoxins

Pearson Square
Protein in feed

- Most expensive
- Formulation program cheapest cost/unit protein up, within constraints
- Feather meal 5% -> soy -> poultry -> ….
- If there is space in recipe, it will be filled with the cheapest raw material (eg wheat)

Lecture 7 Feed storage and transportation

Types of feeds

- Wet feeds
1. Bacterial degradation rapid
2. - 20C
- Formulated wet or moist feed
1. Ground, pasteurized or ensilaged fish
2. Supplemented with vitamins, minerals, oils
- Dry feeds
1. Nutritionally efficient
2. Consistent nutrient profile
3. Convenient handling
4. Better shelf life than wet/ moist

Factors influencing storage life

- Feed management
1. Plan purchases
2. Do not accept moldy, damp, insect infested delivery
3. Know shelf life

Nutrient degradation
 - Oxidation -> rancidity -> palatability decrease, toxic chemicals depress growth. High
lipid more susceptible
- Carbo and AA stable
- Vitamins: heat, pressure, humidity, friction, light redox reaction

Antioxidants

- Ethoxyquin
- BHT
- BHA
- Vitamin E
- Rosemary extract

Self-heating and combustion

- Problematic for blood meal and fish meal

- High volume storage of finished feed problematic
- Antioxidants and check temp

Feed storage

- Bag stacked safely

- Airflow and storage away from wall and floor
- Radiant heat near walls can damage feed
- Cool, dry, pest proof
- No direct sunlight
- Minimise storage time

Lecture 8

Rotifers

- Small suitable for larvae

- Hardy and relatively easy to mass produce at very high densities
- Cultured on a variety of diets
- Fresh, brackish, and marine

Morphology

- 3 distinct parts: head, truck, and foot

- Segmented and externally covered by a transparent cuticle
 - Head cilia draws water into the mouth, sifts for food
- Trunk encloses digestive tract and reproductive organs
- Final region is the foot – it has toes for attachment

Reproduction

- Life cycle 7-10 days

- Favourable: asexual, amictic eggs (females)
- Unfavourable: sexual, smaller haploid mictic eggs. Unfertilised -> males. Fertilised ->
resting eggs with dehydration resistant granulated outer shell. Dormant for years and
hatch into females when conditions favorable again. Suitable for storage.

Starting and maintaining rotifer cultures (300-1000rotifer/ml)

1. Inoculate microalgae culture with rotifer. Starter culture could be either live culture
or resting eggs
2. Rotifers consume the microalgae and their population rapidly increase
3. constant food availability
4. Some culture water removed from the rearing vessels daily and replaced with similar
volume of live microalgae culture
5. Water removed by siphoning through 60micron sieve to retain rotifer

Food

- Live microalgae or algal pastes (Nannochloropsis, tetraselmis, isochrysis, pavlova)

- Bakers yeast
- Enrichment: fatty acid (microalgae, emulsion oil, microencapsulated diet, homemade
emulsion) protein (protein selco, algamac protein plus)

Physical parameters

- 20-35% salinity
- strong aeration
- 15-35C depend on type
- 7.5-8pH

Culture method

1. Batch culture (3-6day)

- cultured to a certain density when total culture volume harvested
2. semi continuous culture (15-40day)
- proportion of culture harvested regularly, replaced with new water, complete harvest
and restart after certain period
3. continuous culture
- continually harvesting from culture with new water entering culture system at same
rate of harvest
Lecture 9 Artemia

Reproduction and fecundity

- favourable: ovoviviparous -> fertilised eggs develop inside uterus of female into free
swimming nauplii released into water column
- unfavourable: oviparous -> egg develop to gastrula stage, shell gland of female
secrete thick shell surrounding egg, deposit as dormant cyst
- 50-200 cyst/nauplii per reproductive cycle

Artemia life cycle

- Cysts: 200-300micron
- Hatching or umbrella stage: cyst hydrated, metabolism of embryo activated, cyst
outer membrane burst, embryo emerges but still enclosed in egg membrane
- Nauplii: egg membrane rupture and free swimming nauplius released (0.4-0.5mm,
0.002mg) first instar orange (yolk) 3 pair of appendage, red ocellus (eye)
- Metanauplii: series of molts, trunk and abdomen elongate and digestive tract
becomes functional

Hatching cysts

- 25% salinity optimal

- 2g/L
- 25-30C vigorous aeration, DO > 2mg/L
- 2000lux at water surface
- pH 7.5-8.5
- 24hrs hatch, 12 hrs after start feeding (Nauplius II)

Harvesting

1. Stop aeration, egg float

2. Nauplii positively phototactic, shine light at bottom to concentrate
3. Reduce cyst shell if possible, bacteria + digestive disorders
Decapsulation can solve this
- Sodium hypocloride solution, hydration of cysts
- Decapsulation
- Washing to deactivate chlorine solution
- Advantage: limit digestive and disease problem by empty cyst, improve hatch rate,
larvae can feed directly on decapsulated cyst, decapsulated nauplii higher energy
content
- Disadvantages: extra labour and chemical costs, inappropriate handling -> low hatch
rate
Issues

- Reliability of supply
- Nutritional inconsistency (depend on source and nutritional composition of food)
- Nutritional deficiency (inadequate fatty acid for larvae, have to enrich = more costs)

Enrichment

- Fatty acid: microalgae, commercial emulsion oil, microencapsulated diet, homemade

emulsion
- Factors influencing success: duration (longer better, 3-5hr, overnight or 48hr).
Concentration (higher better, shorter time taken). Artemia density (more enrichment
per individual better)

On-growing artemia (5-7kg/m3 production rate)

- Larger artemia required for late larvae or juveniles of some species (eg lobster)
- Nutritional value of on-grown adult Artemia superior than nauplii (protein increase
47% in nauplii to 60% adult, adult rich in ALL amino acid, exoskeleton of adult
artemia extremely thin good for digestion)
- 1000-3000nauplii/L initially
- Feed microalgae
- Cleaned regularly to remove detritus/ faecal matter and maintain water quality
- Good aeration
- Good water quality
- Readily available food supply
- Low light
- 25-30C
- 30-35% salinity

Lecture 10 Copepod

Copepod

- Represent 80% of zooplankton

- Improve larval survival, growth, pigmentation, gut development and metamorphosis
- Readily digested by fish larvae
- superior nutritional value (higher HUFA and DHA contents and essential fatty acids
from copepods more digestible, compared to enriched rotifers and artemia)
- Smalls size nauplii ideal for early larvae with small mouth gape (eg grouper)
- Copepod swimming pattern elicit strong feeding response in fish larvae
- High enzyme activity of copepod important to early fish larvae since they have
insufficient enzymes and need prey enzymes to help digestion
Copepods for aquaculture

1. Calanoid copepods
o Eurytemora
o Acartia
o Bestiolina
o Parvocalanus
2. Harpacticoid copepods
o Tisbe
o Tigriopus
o Euterpina

Problems with Copepod culture

- Species specific algal species (feed requirements)

- Good water quality required for planktonic calanoid copepod culture
- Very low culture density for calanoid copepods (4-5/ml, best 20-30/ml)
- Cannibalism

Other small zooplankton

- Protozoa (ciliates)
- Larvae of bivalve
- Cladoceran

Utilising natural zooplankton

- Copepods, rotifers, cladocerans

- Copepods make up 80%, 500-10,000/m3
- Concentrations higher in lagoons and estuaries

Extensive larval culture in ponds

- Advantages
o Low cost, low input system
o Variety of larval food organisms (more balanced)
o Effective for species that cannot be cultured on traditional hatchery prey
(rotifers and artemia)
- Disadvantages
o Little control over larval feeding -> binge feeding may exhaust zooplankton
o Little control over water quality and disease
o Little control over plankton composition and abundance
o Unreliable, typically low survival while total maturity is also common, results
often unrepeatable
How are live feeds chosen for use in hatcheries? What are the properties of commonly used live

feeds and what are their limitations? Use examples.

Lecture 11: Ingestion and Digestion

Basic digestive process

1. Ingestion of food -> Capturing, swallowing

Bivalves: filter feed using ciliary action and food grooves (susceptible to ingesting
microplastics

Gastropods: rasp food using radula -> oesophagus

Crustaceans: prehensile appendages for trituration -> oesophagus. Chewing occurs in

stomach (gastric mill)

Teeth -> restrain object until swallowed &/or scraping and cutting

Buccal cavity -> area where food is first encountered, extend from jaw to oesophageal
sphincter, posterior of buccal cavity known as pharynx, gill rakers enable retention of food
particles, wide variation among fish species

2. Mechanical breakdown of food -> Size reduction and increase surface area

During ingestion and digestion, increase SA of food. Physical fragmentation of food ->
tearing, grinding, peristaltic action of stomach and intestine

3. Movement (peristalsis) -> Propulsion of food through digestive tract

Coordinated contractions and relaxations of smooth muscle in gut wall

Moves (propulsion) and mixes (segmentation) the digesta

Gut motility -> neural and hormonal control, temp, fibre

4. Chemical breakdown of food -> secretion and release of digestive juices in response
to stimuli (require 50% moisture)

Digestion: digestive fluids from stomach, pancreas, liver, intestine

- Break down ingested food into molecules readily absorbed and utilised
 - Digestion ability -> enzyme and fluids and appropriate anatomical apparatus (species
specific

Gastric juice

- Secreted in stomach
- Pepsinogen and hydrochloric acid
- Pepsinogen -> pepsin hydrolyse peptide bond

Bile (pyloric/anterior intestine)

- Produce in liver, store in gall bladder, release in proximal intestinal

- Bile acids: emulsification for lipase digestion
- Bicarbonate: pH increase
- Bilirubin: waste elimination
- Cholesterol and fatty acids

Pancreatic juice

- Diffuse tissue around pyloric intestine

- Water, bicarbonate and digestive enzymes (prote,lip,carbohydr-ases)
- Enzymes act in chyme (semidigested digesta) and also in brush border membrane of
enterocytes
- Fish adjust enzyme secretion according to nutrient composition

Proteases

- Protein -> aa by hydrolysis

- Substrate specific -> endo (trypsin, pepsin, chymotrypsin, cleave non terminal aa)
and exo-peptidase (cleave terminal)

Lipase

- Lipid -> free fatty acid by hydrolysis

- Not substrate specific (hydrolyse most organic esters)

Carbohydrase

- Di,tri,oligo,poly-saccharides -> monosaccharides by enzyme specific steps

Alkaline digestion through mid-gut

hind gut (distal intestine) balances minerals/moisture + enterohepatic recycling (bile salts)
 5. Adsorption & utilisation -> molecules into blood/lymphatic system for metabolism

Proximal and mid intestinal -> simple diffusion (fatty acids), facilitated diffusion (aa,
fructose), active transport (aa, glucose)

Pinocytosis: absorption of larger polypeptides by gut epithelial cells. Surface membrane of

epithelial cells engulf the molecule. Larvae

AA & monosaccharide -> blood capillaries

FAs & monoglycerides -> convert to triglycerides in epithelial cells -> coated with proteins to
form chylomicrons -> lymphatic capillaries

(Arthropods) Hepatopancreas: synthesis and secretion of digestive enzymes, adsorption of

digested dietary products, maintenance of mineral reserves and organic substances,
distribution of stored reserves

6. Elimination -> removal of undigested food and wastes

Digestibility: quantification of the efficiency of digestion and absorption (provides relative

measure of the extent to which ingested food have been digested and absorbed)

Crude vs digestible basis: composition of diet may show nutrient adequacy but dependent
on the processes of digestion and absorption to make nutrients available

Otogenic development
 1. Hatch: tract is straight tube, histologically undifferentiated unchanged from mouth
opening until the completion of yolk absorption. Liver and pancreas formed,
functional at first feeding
2. Segmented into buccopharynx, foregut, midgut, hindgut
3. Larval period ends with development of stomach with gastric glands and pyloric
caeca

Lecture 12 Energy Partitioning

Energy

- Required for all metabolic reactions

- Fat most energy dense macronutrient
- Far more energy efficient energy source

Catabolism: break down large molecule (release energy)

Anabolism: builds up molecules (uses energy)

Adenosine Triphosphate (ATP) delivers energy by dephosphorylations to ADP

- Phosphate groups are high energy molecules and highly negatively charged

Cellular respiration

- Set of metabolic processes to transfer energy from nutrients into ATP

- Mitochondria main site of most energy production reactions

Energy production pathway

Krebs cycle

Oxidative phosphorylation and electron transport chain

- Electrons are passed from one member to another

- Exergonic reactions release free energy
- Energy capture to create a proton gradient
- Resultant electrochemical proton gradient generates chemical energy -> ATP
- Electrons in water are of low energy
Aerobic metabolic scope

- Difference between MR at active (max: sustained swimming) and basal (min)

- Greater the aerobic metabolic scope, the greater potential for growth
- Measuring metabolism: respirometry

- Hypoxia creates conflicts in energy budget -> lower aerobic metabolic scope ->
reduced growth potential
Feed nutrient requirements: requirements for growth (retained nutrients) + requirements
for metabolism (maintenance) + allowance for digestibility = nutrient requirements

Specific dynamic action

- Metabolic rate increases after feeding

- Energy required for digestion, absorption and growth (metabolic cost)
- Increased oxygen consumption
- High protein feeds increase oxygen demand but not necessarily growth (higher
energy cost)

Basal metabolism: metabolic expenditure for functions that maintain life

- Temperature major factor (increase with temperature, best within optimal thermal
window)

Voluntary activity

- Fish do not spend energy countering gravity

- Cost of turning and accelerating more expensive than cost of swimming at constant
speed in straight line

Growth of macronutrients
 - Deposition of energy yielding nutrients (protein and lipid) for growth and
reproduction
- Protein content constant across body size
- Lipid replace water as body size increase

Protein sparing of fat

- Non protein energy sources reduce protein use for energy (more efficient)
- Fat retention very efficient, higher fat in feed = higher fat in fish
- Lipids can conserve protein for development than carbohydrates because more
biologically available than any other nutrient

Lecture 13 Protein and AA 1

Dietary protein: one or more chains aa linked together by peptide bonds to form
polypeptide

- Largest fraction of aquaculture diets

- Most expensive
- Understand to -> better FCR, reduce cost
- AA -> amino, carboxyl, R/functional group
- Aa -> peptide. Structure determines function. Defined by DNA/RNA

Protein Digestion

Protein catabolism

- Biosynthesis of proteins requires a continuous supply of aa (constant degradation

and re-synthesis of proteins
- Surplus aa of those needed for protein synthesis cannot be stored -> catabolized as
energy sources (starts w deamination)

Deamination (in liver)

- Amino group removed and remaining carbon component converted to acetyl-CoA or

other krebs cycle intermediates
Dietary Protein: Primary roles

1. Source of aa for protein synthesis

- Structural
- Endocrine
- Immunological
2. Carbon skeleton can be oxidized to provide cellular energy
3. Carbon skeleton can be used for carbohydrate or lipid synthesis
4. Aa are active physiologically
5. Actin and myosin structural proteins for locomotion

Essential nutrient: cannot be synthesised in body/cannot synthesise in sufficient quantity for

optimal body function (indispensable, need to supply in diet)
Limiting aa concept

- 10 essentials aa
- Optimum animal performance (feed, intake, weight gain) is achieved when feed has
o Enough aa
o Right proportion of essential aa
o Supplementation with first limiting aa
o Methionine & lysine

Chirality of aa

- Most aa exist as mirror images of each other

- L-aa most common
- D-met can convert to L-met, DL-Met is most common BUT other aa must be L-isomer

Protein requirements change with size

- Fish and crustaceans decrease requirement with age and size

- Decreased maintenance requirements
- Decreased protein deposition

Estimating requirements

- Whole body composition used as a guide

- Related to Lys as a standard for growth
- Ideal aa concept
 - Several aa active biochemically

Histidine

- Osmoregulation -> forms anserine, carnosine, n-acetyl-histidine osmolytes

- Cataracts -> increasing water temp or other stressors, mitigated by increasing
histidine in feed (osmotic function: 14.4g/kg His)
- Reproduction -> concentrates in gonads and preferentially retained in larval
development 15g/kg His
- Muscle growth and stress effects
- Precursor to haemoglobin

Methionine

- Can provide some of Cys requirement – requires vitamin B6

- 1-carbon reactions (endogenous synthesis of proteins & phospholipids)

Non-amino (non-protein) energy (NPE)

- Energy is required for protein synthesis

- Insufficient NPE forces catabolism of aa (restricts tissue synthesis)
- Non-availability of sufficient NPE thought responsible for high protein requirements
of some aquatic organisms
- Excess energy = fatty fish (less marketable)
- Correct protein to energy ratio (PER) important to optimise growth and product
quality

Sources of NPE

- 2 sources of NPE are lipid and carbohydrate

- Fish (particularly carnivores) are generally unable to effectively utilise dietary
carbohydrate
- Saving protein by increasing NPE is known as protein-sparing
- High lipid diets = high energy diets -> can reduce nitrogen in effluent

Summary

- Fish/crustaceans have high protein requirement compared to terrestrial farm animals

- Fish that utilise NPE better have lower dietary protein requirements
- Vegetable ingredients lack EAA, need to blend ingredients + synthetic aa
Lecture 14 Protein and aa 2

Sources of amino acids

- Most raw material low in lysine and methionine: supplemented as synthetic sources
in most feeds
- Arginine, histidine, methionine may all have conditional requirements – metabolically
active
- Digestibility varies with raw materials, between species and batch

Animal protein – poultry meal

- Bits of birds that humans choose not to eat

- Capturing valuable nutrients in human food chain
- Processing conditions affect digestibility
- Blood meal (whole dried blood 90% protein). Plasma meal (separated by
centrifugation and plasma removed). Haemoglobin meal (separated RBC 97%
protein) -> high digestibility, high histidine, high oxidation

Plant proteins

- Tolerate 100-200mg/kg sparteine (alkaloid)

- Low alkaloid varieties (sweet lupins) 20mg/kg
- Dehulling reduces tannins and increase digestibility
- Max levels 25%
- 49% CP, for lower fat feeds

Increased ingredient use from

- Protein concentration – dehulling, fractionation, reduction of ash

- Plant breeding for improved AA & EFA profiles, reduced antinutrient levels
- Processing to deactivate antinutrients -> heat (extrusion), solvent extraction (protein
concentrates), fermentation

NPE
 - Energy required for protein synthesis, insufficient NPE forces catabolism of aa
(restrict tissue synthesis)
- Lipid and carbohydrate
- Fish generally unable to effectively utilise dietary carbohydrate
- Protein sparing
- High lipid diets (high energy diets) reduce N in effluent

When is protein not protein

- Protein = N * 6.25
- L-arginine (C6H14N4O2, mol mass 174.2) contain 32% N (N*6.25=200% protein)
- Nucleotides: nitrogen dense, contain amino group but not carboxyl, tied up in
protein requirement, supplements for specific (appetite, early growth, health)
- Taurine: pdt of methionine-cysteine metabolism. Not an aa (lack carboxyl), sulphonic
acid, conditionally required for some fish (bile acid conjugation, antioxidant and anti-
inflammatory)
Lecture 15 Lipid and FA

Lipids: organic compound, soluble in non-polar organic solvents (alcohol & ether), insoluble
in water (hydrophobic)

FA and Triglycerides

- Carboxyl end (COOH)

- Methyl end (CH3)
- Saturated (C-C only), unsaturated (C=C) can be mono or poly (2-4) or highly (>4)
- Triglyceride (TAG): neutral, ester of glycerol and fatty acid
- FA rarely occur free in nature, incorporated into other molecules
- Different TAG determined by FA composition
- TAG digestion: lipase cleave off FA: TAG -> DAG + FA -> MAG + 2FA

Fatty Synthesis

- Animals lack the desaturase enzymes to convert oleic acid into linoleic acid and
alpha-linolenic acid
- Animals vary with how fast they elongate n-3 fatty acids
- EPA and DHA are essential for all animals
- Some EPA and DHA can be produced in some animals from Alpha Linolenic Acid (ALA)
- Majority of EPA and DHA formed by marine algae

Phospholipid

- Polar lipid, amphiphilic: have polar and non-polar end

- Contain FA, structure similar to TAG but one FA replaced by phosphate molecule
 - CSM: long chain polyunsaturated FA increase fluidity
o change sat:mono FA ratio to change fluidity, response to temperature)
o cholesterol create rigidity
- Change in membrane fluidity lead to acclimation to fluctuating environmental
condition
o fish and shrimp have specific requirements for DHA, EPA and cholesterol

Cholesterol

- Rigidity of CSM
- Cholesterol -> bile acids -> conjugated bile salts
o taurine conjugated cholesterol residues
o emulsify fats In digestion, allow digestion
- Precursor of steroid hormones
o Sterols (4C rings, 17C total, side chains at C3 and C17)
o Steroid hormones
 Corticosteroids (made in adrenal cortex)
 Sex steroids (made in gonads and placenta)
- Cholesterol synthesis
o Vertebrates synthesise cholesterol de novo
o Crustaceans unable to synthesise cholesterol (ecdysone: steroid hormone
control molting and skeleton formation)
o Bivalves limited capacity for sterol synthesis
- Vitamins and carotenoids
o Lipid and fat soluble vitamins
 Vit A D E K
 Absorbed in fat globules (chylomicrons)
 Greater risk for toxicity when consumed in excess than water soluble
vitamins
o Carotenoid
 Lutein and zeaxanthin (yellow pigments in plants)
 Skin colour in yellowish fish
 Astaxanthin
 Essential nutrient for salmon, immune system, provitamin A
 Antioxidant
 Flesh/skin/shell colour for salmonids, red fish and crustaceans

Lecture 16 Lipid and Fatty Acid 2

Fat digestion

- Longer chain FA less digestible

 - Saturated FA less digestible
- Trends in FA content of salmon
o Fish fatty acid profile reflects feed
o Decreased fish oil use over time
o Dilution of EPA and DHA
o Higher fat content than wild salmon
o Higher n-6 from increased use of plant oil

Partitioning of FA

- All FA grp increase in muscle with increasing dietary inclusion

- Cell membranes regulate MUFA and SFA levels but n-3 and n-6 reflect the diet
o RBC most of fat is in phospholipid form from cell membranes
o Heart and liver phospholipids are phospholipids extracted from those tissues

Metabolism of arachidonic acid (20:4n-6) and EPA (20:5n-3)

- Prostaglandins and leukotrienes from ARA are pro-inflammatory

- Those from EPA are anti-inflammatory
- Both use the same enzymatic pathways and so compete

Increasing DHA and EPA in aquafeeds

- GM seeds and microalgae (FO-like levels of DHA and EPA)

Whole body lipid distribution

- Higher fat fish = higher levels of EPA+DHA, expressed as % (g/100g tissue)

Feed formulation

- To meet requirements for proper growth

- To promote optimal health
- To satisfy the consumer demand

EFA deficiencies in finfish

- Poor growth
- High liver and intestinal lipid content
- Poor feed efficiency
- Fin erosion
- Bleeding from gills
- Reduced reproductive performance
- Shock syndrome

EFA deficiency in fish larvae

- Dermal signs
- Reduced growth rate
- Reduced feed efficiency
- Increased mortality
- Reduce tolerance to stress

Provision of energy

- Lipid: 37.5MJ/kg
- Protein: 23.6
- Carbohydrate: 17.2

Feed formulation

- Increase energy (lipid) -> increase growth, decrease feed intake

o Improve FCR
o Sometimes decrease feed can decrease growth
- Up to 42% lipid in high energy diet (salmon)
o Minimise protein catabolism for energy
 o Minimise N excretion
- Crustaceans
o Do not tolerate high levels of dietary fat
o 5-6% optimal, >10% lipid fall in growth rate
o Quality and quantity of dietary lipids (cholesterol) play important role in
growth and health of shrimp

DHA and EPA fate

- Structural role in cell membranes

o Lipid peroxidation may occur -> loss of membrane integrity -> increased
membrane permeability -> structural damage of DNA and cell death
- As source of energy
o Krebs cycle
o EPA selectively oxidized over DHA
 Higher dietary DHA:EPA ratio better for improved n-3 LC-PUFA
deposition in fillet
o Better than other FA as energy source

Oils used In aquaculture

- Canola: high in monounsaturated, digestible energy source

- Poultry: high in saturated fats, cheap energy source
- Fish: expensive, good supply of essential fatty acids
- Algae (Schizochytrium): heterotrophic, produced by fermentation
o 50% oil, up to 60% EPA + DHA
o Cost still higher than fish oil

Lecture 17-18 Carbohydrate nutrients

Carbohydrates: biomolecules that contain carbon, hydrogen and oxygen atoms C(H2O)n

- Sugar group (sweet)

o Mono and disaccharides: 1-2 molecules
o Oligosaccharides: 3-10 monosaccharides
o Polysaccharides: >10 monosaccharide
- Non-sugars group (no sweet taste)
o > 10 monosaccharide (starch, cellulose, chitin, glycogen)
o Homopolysaccharides: identical units
o Heteropolysaccharides: mixture of diff units

Monosaccharides
 - Major building units of complex carbohydrates
- One sugar unit CnH2nOn
- Triose (3C): hyceraldehyde
- Pentose (5C): ribose, xylose, arabinose
- Hexose (6C): glucose, fructose, galactose, mannose
- Hexose most common C6H12O6
- Most abundance monosaccharide in nature is D-glucose

Disaccharides

- 2 monosaccharides linked by glycosidic bonds

- Sucrose (glu and fruc) sugar cane
- Maltose (glu and glu) breakdown product of starch
- Lactose (glu and galac) found in mammal milk

Glycosidic bonds

- 1-6 naming clockwise from anomeric C (w hydroxyl group)

- Alpha and beta bonds – specific enzymes for each
- Animals commonly able to hydrolyse 1-4 and 1-6 alpha linkage but beta (1-4) linkages
(cellulose or fibre) broken by carbohydrases only present in bacteria

Polysaccharides

- >10 monosaccharide units (sugars or non sugars)

- Almost all dietary CHO in fish feed are polysaccharides
- 5 nutritionally important
o Cellulose (fibre): plant cell membrane
o Beta-glucans: plant cell membranes
o Starch: storage molecules in plants
o Chitin: arthropod exoskeletons
o Glycogen: storage molecule in animal tissues

Carbohydrates as functional ingredients

- Oligosaccharides
o Immune stimulation: MOS, FOS
 Immunoglobulin M (IgM) antibody response to disease
 Lysozyme: antibacterial enzyme
o Gut microflora modification
o Skin mucous

Carnivorous fish can benefit from carbohydrase enzyme-treated raw materials

Lecture 20 Vitamins

Vitamins

- Essential micro nutrient for all species (small amt)

- Vitamin-mineral premix
- Cofactors in metabolic reactions
- Coenzymes: non protein molecule bind with enzyme to catalyse reaction
- Divided into 2 types
o Fat soluble A D E K
 Absorbed and transported with lipids (conditions for fat absorption
also enhance absorption of vit) -> dietary intake > metabolic needs.
Accumulate too much fat soluble vitamins, toxic hypervitaminosis
(osteoclast formation, weaken bone, bone pain, blurry vision)
 Can be stored often in liver
 Not easily dissolved when cooking
 Only contain C,H,O
o Water soluble B complex, choline, inositol, C
 Important in nutrient metabolism
 Function of neural and cardiac tissue
 Essential for maintenance, growth, reproduction
 Unstable, dissolve easily when cooking, degradation
 Eliminated in urine, not stored in tissue (constant supply required)
 Vitamin B complex
 8 required in small amt
 Primarily coenzyme functions

Fat Soluble

Water Soluble
Choline

- Component of phosphatidylcholine -> structural function in biological membranes,

fat digestion, absorption, and transport
- Precursor of acetylcholine -> neurotransmitter
- Precursor of betaine -> methylation and osmolyte
- High in vegetable protein source but often unavailable
- Supplementation required

Inositol

- Constituent of phospholipid and important for integrity of lipid membrane

(phosphatidylinositol)
- PI important -> lipid signaling, cell signaling, membrane tracking
- Increased lipid accumulation in inositol deficient diets
- Rich inositol sources: cereal with high bran, bean, leafy green, lecithin
o From animal generally available
o From plant (phytic acid) not digestible and reduce mineral availability

- Ascorbic acid -> not stable in feed so not used

 - Ascorbyl polyphosphate used
- Antioxidant
o Protect cell from free radical
o Protect iron from oxidation (increase iron absorption)
o Recycle oxidized Vit E
- Required for hydroxylation reaction
o Proline to hydroxyproline, lysine to hydroxylysine -> collagen synthesis
(formation of connective tissue, wound repair, bone matrix)
- Deficiency: poor appetite, reduced growth, convulsions, malformed cornea, gill
problem, skin lesion, colouration issue, anaemia, fragile erythrocytes, kidney
disorder, scoliosis, lordosis, black death disease, mortality

Lecture 21 Minerals

Macro: Ca, P, K, Mg, Na

Micro: Cu, Fe, I, Mn, Se, Zn

Important for structural or metabolic reasons for growth or maintenance of bodily processes

Major structural minerals (bone)

- Formed by hydroxyapatite (Ca5(PO4)3OH

- Ca
o Supplied in feed as CaCO3/ CaSO4
o Mostly supplied by water over gills
o Soft water = Ca deficient (1-15g/kg)

Magnesium MgSO4

- 0.01-0.8g/kg
- Alkaline phosphatase
o Ubiquitous enzyme containing Mg and Zn
o Important for homeostasis
o Liver function and bone formation

Zinc ZnSO4

- 15-240mg/kg
- Trace component of bone (vertebral Zn as response criterion)
- Skin healing -> skin accumulation as criteria for skin health
- Alkaline phosphatase
- Zn-Cu superoxide dismutase (SOD)

Selenium
 - 0.02-1.8mg/kg
- Required for glutathione peroxidase activity (antioxidant system)
- Requirement interacts with other antioxidants
- Se toxicity
o Replaces sulfur in sulfur containing aa
o Proteins become non-functional
o Teratogenic
o Risk decreased organic forms Se-Met or Se-yeast

Mineral-dependent antioxidant systems

- 2O + 2H -> O2 + H2O2 (Zn Cu Mn)

- 2(H2O2) -> 2H2O + O2 (Fe and Histidine)
- H2O2 + 2GSH -> 2H2O + GSSG (selenium)

Iodine

- Thyroid hormone formation

- Triiodothyronine
- Deficiency: goiter
- Rich in seawater (supplement in freshwater feed)

Manganese

- 2-25mg/kg
- Trace mineral in bone formation
- Diverse roles in aa metabolism and vit utilisation
- Mn-SOD

Iron FeSO4.7H2O

- 30-330mg/kg
- Haemoglobin formation
- Measured by haematocrit, blood haemaglobin content
- Catalse requires haem

Trace minerals can form undesirable complexes and interactions with other trace minerals –
reduced bioavailability -> not dissociated in stomach -> reduced interactions
Lecture 22 Functional ingredients

Functional ingredient: ingredient that provides a physiological and/or health benefit to

organisms beyond that achieved by basic nutrition. Can be raw material/ feed additives

Feed formulation

1. Meet requirement for proper growth

2. Promote optimal health
3. Satisfy consumer demand

Novaq

- Decrease FCR
- Increase biomass gain rate
- Gill associated virus resistance

Probiotic

- Bactocell: reduce deformities

Non-nutritional feed additives

- Enhance physical property of feed for manufacture

o Binders
o Antioxidants
o Antimicrobials
o Mycotoxin sequesters
- Cheaper than functional feed additives

Functional feed additives

- Acidifiers
- Phytogenics: plant derived, antioxidant, antimicrobial, antiparasitic, growth
promoter, appetite enhancement
- Prebiotics: compounds (carbs) modify microflora population by promoting growth of
beneficial bacteria. Fiber fermented by good bacteria
- Probiotics
- Symbiotics: mix of pre and pro
- Antibiotics
- Organic acids

Drivers of feed additive use

1. Disease caused by pathogen (economic loss)

2. Excessive antibiotic use lead to bacterial resistance (risk to consumer)
 3. Economical availability of marine ingredients (intestinal health in carnivorous
species)

Restrains of feed additive use

1. Increasing cost of raw materials

2. Regulatory issues

Organic acids

o Antimicrobial in feed (propionic, acetic)

o Intestinal barrier integrity (sodium salt of butyric acid)
o Improve fcr, per, growth rate
o Enhance mineral absorption and mineral availability
- Sodium butyrate
o Trophic effect on intestinal epithelium
o Anti-inflammatory
o Improve intestinal integrity and development -> enhance nutrient absorption
o Intestinal barrier integrity, increase tight junction expression, epithelium
development

Probiotics: any microbial cell provided via the diet or rearing water, optimise microbial
balance of host and ambient environment

- Disease resistance
- Health status
- Growth performance
- Feed utilisation
- Stress response

- Mixed with other ingredient, mix with water and spray on feed, add directly into
water
- Compete for nutrients with pathogen
- Produce antimicrobial compound (bacteriocines, antibiotics, enzymes, organic acid)
 - Antagonize pathogenic bacteria improve absorptive surface area
- Produce extracellular enzyme enhance mineral absorption
- Increase phagocytic and lysozyme activity enhance disease resistance

Lecture 23 Sustainable feeds

BAP

- Fish meal and fish oil conservation

ASC

Strategies for reducing fish meal

1. Use where it counts

a. How much epa dha do fish need? Nutrient requirement, CSM fluidity, neural
development
b. Anti-inflammatory
2. Understand value and limitations of other proteins
3. Explore the next generations of alternatives

a.
b. Algae, GM canola
4. Further increases in trimmings fish meal

Other considerations

- Supply chain
o Land use in terrestrial raw materials
o Carbon footprint
o Modern slavery and other crime
- Outputs
o Nitrogen- total and digestible
o Phosphorous
o Benthic impacts