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Laboratory Animal NutritionLaboratory Animal Nutrition

Timo NevalainenTimo Nevalainen

University of Eastern Finland

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Nutrient requirementsNutrient requirements

Each species has specific needs for Each species has specific needs for essential nutrientsessential nutrients

e.g.e.g. vitamin C essential for guinea pigs, vitamin C essential for guinea pigs, not for ratsnot for rats

National Research Council has published National Research Council has published detailed documents for each speciesdetailed documents for each species

Requirements are ”minimum needs” under Requirements are ”minimum needs” under ad libad lib / maximum growth conditions / maximum growth conditions

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Nutrient requirements documents for all species freely available:

http://lab.nap.edu/nap-cgi/discover.cgi?term=nutrient+requirements&restric=NAP&mw=&submit.x=6&submit.y=11

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Why to fulfill nutrient requirements?Why to fulfill nutrient requirements?

Essential needs of Essential needs of specific nutrients for specific nutrients for each species and each species and each period in life, for each period in life, for maintaining good maintaining good health, andhealth, and

For obtaining reliable For obtaining reliable resultsresults

Skin lesions in Wistar rats due to dietary Vitamin B 6 and B12 deficiency

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Toxic levels of nutrientsToxic levels of nutrients

Influence of Influence of methionine on methionine on atherosclerosis in atherosclerosis in apoE deficient miceapoE deficient mice

2.2 and 4.4% dietary 2.2 and 4.4% dietary methionine methionine ->toxic effects->toxic effects

Zhou Art.Thromb.Vasc.Biol.2001Zhou Art.Thromb.Vasc.Biol.2001

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Responsible concentrationsResponsible concentrations

Purified AIN dietPurified AIN diet

Control 0.5% methionine Control 0.5% methionine (NRC level)(NRC level)

Highest level: 1.5% Highest level: 1.5% methioninmethionin

No toxic effectsNo toxic effects

Reliable resultsReliable results

Good welfareGood welfare

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Types of eating & digestionTypes of eating & digestion

Rodents practice coprophagiaRodents practice coprophagia

Rabbit also coprophagic; and is a rear end Rabbit also coprophagic; and is a rear end fermentorfermentor

Dog and cat are carnivoresDog and cat are carnivores

Sheep and goat are ruminants; and front Sheep and goat are ruminants; and front end fermentorsend fermentors

Swine is a omnivore like humansSwine is a omnivore like humans

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Types of diets usedTypes of diets used

Natural ingredient Natural ingredient diets = made of diets = made of natural ingredients, natural ingredients, such as maize gluten, such as maize gluten, corn oil, wheatcorn oil, wheatFrequently usedFrequently usedComposition such Composition such that pelleting is that pelleting is possiblepossibleRelatively cheapRelatively cheap

Ritskes-Hoitinga et al. 1991

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Natural-ingredient dietsNatural-ingredient diets”Chow diets””Chow diets”

””Standard” diets Standard” diets commercially commercially available for each available for each speciesspeciesSpecial diets for Special diets for Growth/maintenance Growth/maintenance and Reproduction and Reproduction Composition meets Composition meets the needs for each the needs for each species and condition species and condition more than sufficientlymore than sufficiently

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Diet types Diet types

Natural chowsNatural chows

Chemically definedChemically defined

Purified dietsPurified diets

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Problem with Problem with natural ingredient diets??natural ingredient diets??

Beynen 2001:Beynen 2001: Between brand Between brand

variationvariation Between batch Between batch

variationvariation

Batch analysis Batch analysis certificate strongly certificate strongly advisedadvised

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Between-brand variation in Between-brand variation in 9 natural-ingredient minipig diets9 natural-ingredient minipig diets

Metabolizable energy Metabolizable energy content MJ/kgcontent MJ/kg

9.5 - 11.29.5 - 11.2

Cu mg/kgCu mg/kg 10.0 - 38.010.0 - 38.0

Fe mg/kgFe mg/kg 130 - 770130 - 770

Vitamin C mg/kgVitamin C mg/kg 0.0 - 100.00.0 - 100.0

Ritskes-Hoitinga, Bollen 1999

Catalogue values

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Between-batch variationBetween-batch variation4 batches of minipig diet4 batches of minipig diet

NutrientNutrient Catalogue Catalogue valuevalue

LowestLowest

valuevalue

Highest Highest valuevalue

Ca Ca

%%

1.021.02

0.90.9

1.11.1

Protein Protein

%% 13.913.9 14.214.2 16.516.5

Vit EVit E

mg/kgmg/kg 53.053.0 32.032.0 216.0216.0

Ritskes-Hoitinga, Bollen 1999

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Results from feeding10 brands Results from feeding10 brands of rodent chowsof rodent chows

ParameterParameter Lowest-highest Lowest-highest mean valuemean value

Food intake g/dFood intake g/d 12.1 – 15.612.1 – 15.6

Growth g/dGrowth g/d 2.5 - 3.52.5 - 3.5

Water intake ml/dWater intake ml/d 18.2 – 24.818.2 – 24.8

Kidney wt % of BWKidney wt % of BW 0.45 – 0.510.45 – 0.51

Urine prod ml/dUrine prod ml/d 8.6 – 17.08.6 – 17.0

Urine pHUrine pH 6.2 – 7.96.2 – 7.9

Caecal wt. % of BWCaecal wt. % of BW 1.8 – 3.91.8 – 3.9

Ritskes-Hoitinga et al. 1991

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Variable composition gives variable Variable composition gives variable results: 2 yr tox studies in 5 labsresults: 2 yr tox studies in 5 labs

40 studies 40 studies controls controls

Results in Results in F344 malesF344 males

Results in Results in F344 femalesF344 females

% survival% survival 44 - 7844 - 78 50 - 8650 - 86

Liver Liver neoplasias %neoplasias % 0 - 120 - 12 0 - 12 *0 - 12 *

Mammary Mammary tumors %tumors % 0 - 80 - 8 2 - 44 *2 - 44 *

Haseman 1984, Roe 1994

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Standardization requires Standardization requires descriptiondescription

Survey of scientific articlesSurvey of scientific articles Brand name told in 10-15%Brand name told in 10-15% Composition shown in 2%Composition shown in 2%

Conclusion:Conclusion: If diet has no effect, then OKIf diet has no effect, then OK If diet has effect, no way to repeatIf diet has effect, no way to repeat

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Solution for variation?Solution for variation?

Stick to one batchStick to one batch Difficulty: limited durationDifficulty: limited duration

Analysis for critical elements in the Analysis for critical elements in the dietdiet

Detailed description of diet in Detailed description of diet in scientific articlesscientific articles

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Diet formsDiet forms

Full chows in useFull chows in use Only diet and water availableOnly diet and water available

Standard and expanded pelletStandard and expanded pellet

Powdered dietsPowdered diets Test substances can be mixedTest substances can be mixed Diet is wastedDiet is wasted Special equipment for feedingSpecial equipment for feeding

Liquid and colloid dietsLiquid and colloid diets

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Measuring diet consumptionMeasuring diet consumption

Diet used = eating + spillageDiet used = eating + spillage

Metabolism cage – only reliableMetabolism cage – only reliable

In group housing get averagesIn group housing get averages

Values calculated or from reports are Values calculated or from reports are for guidance onlyfor guidance only

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Designing special dietsDesigning special diets

If one wishes to change the fat content of the If one wishes to change the fat content of the diet, isocaloric exchange is needed, because,diet, isocaloric exchange is needed, because, Fat contains more calories per gram than Fat contains more calories per gram than

carbohydrates and proteincarbohydrates and protein When changing the fat concentration in the diet, the When changing the fat concentration in the diet, the

energy concentration changesenergy concentration changes A change in fat concentration must be accompanied A change in fat concentration must be accompanied

by an exchange with by an exchange with e.g.e.g. carbohydrates on the basis carbohydrates on the basis of caloriesof calories

If this is done on the basis of weight, then the animals If this is done on the basis of weight, then the animals will ingest a different amount of ALL nutrientswill ingest a different amount of ALL nutrients

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Diet changesDiet changes

Dilution effectDilution effect All additions have someAll additions have some

Energy rich additions are problematicEnergy rich additions are problematic Energy rich drinking solution are no differentEnergy rich drinking solution are no different

Maximum dilution 10-20 %Maximum dilution 10-20 %

Results in different amounts of feed Results in different amounts of feed eateneaten

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Feeding typesFeeding types

Ad libitum. Ad libitum. Takes place mostly during darkTakes place mostly during dark

MealsMeals Day or night? Usually restricted feeding timeDay or night? Usually restricted feeding time

Restricted feedingRestricted feeding amount given is limitedamount given is limited

Pair feedingPair feeding Same amount for study and control groupsSame amount for study and control groups

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Standardization in relation to Standardization in relation to biological rhythmsbiological rhythms

All processes show All processes show circadian rhythmscircadian rhythmsFeeding should be in Feeding should be in accordance with their accordance with their natural rhythmsnatural rhythmsOtherwise disturbed Otherwise disturbed health, well-being and health, well-being and resultsresultsMore research More research neededneeded

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Advantages of restricted feeding Advantages of restricted feeding

Animals live longerAnimals live longer

Fewer spontaneous tumorsFewer spontaneous tumors

In carcinogenicity studyIn carcinogenicity study test compound may decrease appetite -> test compound may decrease appetite ->

carcinogenicity of compound may be carcinogenicity of compound may be underestimated underestimated

some assessment of diet consumption is some assessment of diet consumption is necessary to find out which is the casenecessary to find out which is the case

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’’Work for food and enrichment’Work for food and enrichment’

Hypothesis: Hypothesis: Rats will work ONLY for food they necessarily Rats will work ONLY for food they necessarily

need, provided work intensity is properly setneed, provided work intensity is properly set

StudyStudy pellets placed in drilled holes of aspen boardpellets placed in drilled holes of aspen board 3 rats (250 g- ) in a cage, 20 week study3 rats (250 g- ) in a cage, 20 week study 3 groups3 groups

A & B = diet enrichment combination (2 ‘doses’)A & B = diet enrichment combination (2 ‘doses’)

C = C = ad libitumad libitum

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Growth of rats

0

100

200

300

400

500

0 2 4 6 8 10 12 14 16 18 20

Week

We

igth

(g

)

Group A

Group B

ad libitum

p < 0.001

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Diet problemsDiet problems

Excessive proteinExcessive protein

Many diets contain 20-28 % proteinMany diets contain 20-28 % protein

Maximum growth with 17% proteinMaximum growth with 17% protein

Suitable protein content for breeding is Suitable protein content for breeding is on 17-20 %on 17-20 %

In long-term studies excessive protein In long-term studies excessive protein may lead to kidney failure may lead to kidney failure

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Rodent chows influence on nephrocalcinosis

•Kidney calcification in female rats

•Severity increases with higher dietary P levels

•Variance in nephro-calcinosis 0/6 – 6/6

•Histological scores 0.0 – 1.3

Ritskes-Hoitinga et al. 1991

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Diet problemsDiet problems

Pesticide & heavy metal residuesPesticide & heavy metal residues

Most common cause for rejection of a Most common cause for rejection of a batch (GLP)batch (GLP)

Ref: Food and Drug Administration (FDA), Ref: Food and Drug Administration (FDA), 1978, Nonclinical Laboratory Studies. Good 1978, Nonclinical Laboratory Studies. Good Laboratory Practice Regulations. Fed Laboratory Practice Regulations. Fed Regis, Part 2, 59986-60025.Regis, Part 2, 59986-60025.

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Why animal breeders use high Why animal breeders use high energy diets?energy diets?

Scientists buy animals by weightScientists buy animals by weight

Ad libitum Ad libitum feeding with high energy feeding with high energy diet ->rats with certain weight fastdiet ->rats with certain weight fast

No change visibleNo change visible

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ReferencesReferences

Quick reference guide on nutrition, Quick reference guide on nutrition, www.felasa.eu, 2001, 2001Beynen, Principles of LAS, 2003Beynen, Principles of LAS, 2003Chwalibog & Ritskes, Handbook of LAS, Chwalibog & Ritskes, Handbook of LAS, 20042004Ritskes and Strubbe, The welfare of lab. Ritskes and Strubbe, The welfare of lab. animals, 2004animals, 2004Ritskes, The laboratory mouse, 2004Ritskes, The laboratory mouse, 2004Dietary Restriction, ILSI Press, 1995Dietary Restriction, ILSI Press, 1995

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Pop quizPop quiz

Study on effects of alcohol on longevityStudy on effects of alcohol on longevity

Rats drink 10 % ethanol in waterRats drink 10 % ethanol in water

What is being studied:What is being studied: Effect of ethanol?Effect of ethanol? Effect of decreased eating?Effect of decreased eating? Effect of decreased drinking?Effect of decreased drinking? All of the above?All of the above?