C H A P T E R

4.3

N

Nutrition of theLaboratory Mouse

UTR

ITION

567

Merel Ritskes-Hoitinga

H

Radboud University Nijmegen Medical Centre,The Netherlands

USB

Graham Tobin

AN

Harlan Laboratories UK, Bicester, Oxfordshire, UK

DRY

Tanja Lyholm Jensen

AN

Novo Nordisk A/S, Måløv, Denmark

DM

Lars Friis Mikkelsen

A

MSD, Translational Medicine Research Center, Singapore

INTEN

ANCE

IntroductionLaboratory mice and rats have always made upa large percentage of the total number ofanimals used for biomedical research purposes.This percentage is usually around 80–90% ofthe total number of animals used. For thisreason, these species have been well-character-ized in many ways. The use of the laboratorymouse (Mus musculus) has increased even moredramatically over the last decades, due to thepossibility of studying gene function in vivo bythe use of genetic modification techniques

The Laboratory Mouse� 2012 Elsevier Ltd. All rights reserved.ISBN 978-0-12-382008-2

which have resulted in many newly establishedmouse strains.

The nutrition of the laboratory mouse (andrat) has also been well-studied and well-definedin comparison with that of other species (for anoverview see [1]). This chapter concentrates onimportant aspects of feeding laboratory mice.One needs to be aware of how nutrition andfeeding as an environmental factor can interactwith experimental results and animal welfarewhen nutrition is not the main focus of study.Moreover, when the mouse is used as an animalmodel for human nutritional conditions, specificexperimental conditions need to be taken care of.

DOI: 10.1016/B978-0-12-382008-2.00024-6

HUSB

ANDRY

ANDM

AINTE

NANCE

568

NUTR

ITIO

N

This is important in order to obtain reliableexperimental results and optimal welfare of theanimals simultaneously.

NutritionalrequirementsEnergyUnder ad libitum feeding conditions animalsusually eat an amount of food that is determinedby the their energy requirements [2]. In Table4.3.1 the energy need is given according to thestage of life the mouse (or other animal species)is in. During lactation an animal has a muchhigher energy need than during themaintenancephase where the animal has stopped growing andis not pregnant or lactating. ‘Metabolic kilos’(kg0.75) are used in order to be able to comparespecies of different sizes. One cannot comparespecies on the basis of mass in kilograms, asdepending on the size of the animal, the meta-bolic rate per kilogram of body weight isdifferent. Using metabolic kilos compensatesfor these differences, so that a reliable compar-ison between species can be made.

That part of the energy in the diet capable oftransformation by the body is called metabolizableenergy [3]. Usually the metabolizable energycontent (MEC) of a diet can be obtained fromthe feed manufacturers. In case it is not provided,it can also be estimated. For this purpose one usesthe levels of energy-producing substances in thediets, i.e. fat, carbohydrates and protein. Fatshave an MEC of about 37 kJ/g, and protein andcarbohydrates about 17 kJ/g. As different types

TABLE 4.3.1: Food intake under ad libitum condi-tions is based on the energy requirement, whichis related to the stage of life

Stage of life Energy need (MJ/kg0.75)

Growth 1.20

Maintenance 0.45

Pregnancy 0.60

Lactation 1.30

Source: Beynen and Coates [2].

of carbohydrates have different MECs, metabolicstudies are required in case one needs to knowthe exact MEC for specific studies. The contribu-tion made by fibre to dietary energy content isusually negligible [2].

The need for energy depends not only on thestage of life of the mice, but also on other factorssuch as the environmental temperature and theamount of activity. As laboratory conditions areusually standardized to a certain room tempera-ture and cage size, which prevents unusuallyhigh activity, these general formulae for energyrequirements according to the stage of life canbe used reliably in the current laboratory setting.A mouse showing stereotyped behaviour, such ascircling, will have a higher energy need than thecagemate that does not exhibit this behaviour.This will increase variation in results. In orderto prevent stereotyped behaviour, environ-mental conditions must meet the animals’ essen-tial needs [4].

Food intake according to energy need willonly hold when the rest of the diet lives up tothe minimal needs. In case there is a shortage ofan essential nutrient, the animal may become(sub)clinically affected, which may lead toa reduced food intake. A reduced food intakemay also occur in the case of a test substancewith a bad taste being added to the diet. Thiscan interfere with the reliability of the experi-mental results. On the other hand, where dietshave a very good taste, mice are expected toingest more than their energy need, leading toobesity.

Nutrient requirementsThe National Research Council (NRC) providesscientific documentation on nutritional require-ments for mice and other species [5]. There isa lot of data published on these requirements,and the NRC establishes committees to reviewthese as new scientific data become available.On the basis of this review process, these commit-tees then publish guidelines on the estimatednutrient requirements [5, 6]. The estimatednutrient requirements for the mouse are shownin Table 4.3.2 [4]. The nutrient amounts per kgdiet as well as the nutrient amounts per 100 kJof diet are shown. As mice eat according toenergy need under ad libitum conditions, it is

TABLE 4.3.2: Estimated nutrient requirements of mice

NutrientAmount

Per kg diet Per 100 kJ

Metabolizable energy 16 500 kJ

Lipid 50.0 g 0.30 g

Linoleic acid 6.8 g 0.04 g

Protein (growth) 180.0 g 1.09 gAMINO ACIDS

Arginine 3.0 g 18.18mg

Histidine 2.0 g 12.12mg

Isoleucine 4.0 g 24.24mg

Leucine 7.0 g 42.42mg

Valine 5.0 g 30.30mg

Threonine 4.0 g 24.24mg

Lysine 4.0 g 24.24mg

Methionine 5.0 g 30.30mg

Phenylalanine 7.6 g 46.06mg

Tryptophan 1.0 g 6.06mg

MINERALS

Calcium 5.0 g 0.03 g

Chloride 0.5 g 3.03mg

Magnesium 0.5 g 3.03mg

Phosphorus 3.0 g 18.18mg

Potassium 2.0 g 12.12mg

Sodium 0.5 g 3.03mg

Copper 6.0mg 36.36 mg

Iron 35.0mg 0.21mg

Manganese 10.0mg 60.61 mg

Zinc 10.0mg 60.61 mg

Iodine 150.0 mg 0.91 mg

Molybdenum 150.0 mg 0.91 mg

Selenium 150.0 mg 0.91 mg

VITAMINS

A (retinol) 0.72mg 4.36 mg

D (cholecalciferol) 0.03mg 0.15 mg

E (R,R,R-a-tocopherol) 22.0mg 0.13mg

K (phylloquinone) 1.0mg 6.06 mg

Biotin (D-biotin) 0.2mg 1.21 mg

Choline (bitartrate) 2.0 g 12.12mg

Folic acid 0.5mg 3.03 mg

Niacin (nicotinic acid) 15.0mg 90.91 mg

Panthothenate 16.0mg 96.97 mg

Riboflavin 7.0mg 42.42 mg

Thiamin 5.0mg 30.30 mg

B6 (pyridoxine-HCl) 8.0mg 48.48 mg

B12 10.0 mg 0.06 mg

Source: Council of Europe [4].

NUTR

ITION

569

HUSB

ANDRY

ANDM

AINTEN

ANCE

HUSB

ANDRY

ANDM

AINTE

NANCE

570

NUTR

ITIO

N

advantageous to present the diets in amountsper kJ. This makes it easier to judge whetherexperimental diet compositions live up to allthe essential nutrient needs under ad libitumconditions. The NRC guidelines [5] recommendnutrient allowances that are greater than theminimum requirements, as they are often basedon the criterion for obtaining maximum growth[2]. This is not necessarily the best situation forobtaining optimal (long-term) health. However,as these recommendations are the best scientifi-cally documented requirements of essentialnutrients available at the moment, it is advisableto use them until new scientific proof becomesavailable.

The recommendations do not take intoaccount that there can be differences inminimum requirements between differentstrains [2]. Interactions between the nutritionalrequirements and genetic background of mousestrains can occur. Dystrophic cardiac calcifica-tion is a postmortem finding in various strains,which coincides with calcifications in the tongue,lungs and diaphragms [7]. The inbred strainsDBA/2Ola and C3H/Ola are susceptible to thedevelopment of soft tissue calcifications,whereas C57BL/6Ola and BALB/cOla are resis-tant towards the disorder (see Table 4.3.3; [7]).The region on chromosome 7 containing thegene Hrc (coding for the histidine-rich calcium-binding protein in the sarcoplasmatic reticulum)is likely to be associated with soft tissue calcifica-tions in DBA/2 mice [8]. Nutritional measure-ments can be taken to prevent excessivecalcifications. The diet of susceptible mice,

TABLE 4.3.3: Cardiac calcification in four mouse strain

Strain/parameter DBA/2

Heart histology

Incidencea 7/7

Scoreb 0.3e2.6

Mineral content(mmol/g dry wt)

Calciumc 43.6 � 16.7aThe number of mice that were scored positive in histological sections fbRange of average scores per mouse.cMean � standard deviation.Source: Van den Broek et al. [7].

especially at a young age, should containadequate amounts of magnesium and fluoride[9], whereas excessive phosphorus and vitaminD intake should be avoided [10].

Genetic modification may also alter nutrientrequirements. In order to make sure of satisfyingthe requirements of a particular geneticallymodified strain, it may be necessary to addspecial nutrients to the diets.

The recommendations do not necessarilyhold for germ-free mice either. Vitamins K andB12, for example, are synthesized by the gut floraof conventional mice and will be sufficientlyingested as a result of coprophagy. Grit floors donot prevent coprophagy, as the mice can still eatthe faeces directly from the anus. For germ-free and specified pathogen free (SPF) mice it isadvisable to include higher vitamin B and Klevels in the diets, as the microflora of SPFanimals may not contain all vitamin-synthesizingorganisms [2].

Toxic levelsToxic levels of nutrients have been defined fora range of nutrients for laboratory rodents [5],and specifically for minerals [11] and vitamins[12]. These tend to focus on extreme excessesthat are unlikely to occur in carefully formulatedand appropriately used diets, but even modestexcesses of some nutrients, for example ofprotein [1], calcium and phosphorus [13], may bedeleterious, though not toxic. In Europe, delib-erate experimental use of toxic levels of nutri-ents would be controlled by both institutional

s

BALB/c C3H C57BL/6

0/7 7/7 0/7

0.0e0.0 0.1e2.8 0.0e0.0

17.2 � 1.8 42.9 � 33.6 17.1 � 2.2

or the presence of cardiac calcification out of a group of sevenmice.

NUTR

ITION

571

HUSB

AN

and national bodies tasked with the regulation ofanimal welfare.

Toxic effects of nutrients occur rarely andare usually due to lack of knowledge or accident.The following are examples:

• Hyperhomocysteinaemia, an independentrisk factor for atherothrombosis, was inducedin apoE-deficient mice by adding extramethionine to the diets [14]. As shown inFigure 4.3.1, both the ‘low’ and ‘high’ methio-nine groups did not live until the scheduledend of the study. The total dietarymethioninelevels fed to the mice in these groups were2.2% and 4.4%, respectively, levels that withhindsight were obviously toxic for mice. Theminimum recommended dietary methioninelevel for mice during growth is only 0.3% [5].As a dietary methionine level of 2.3%decreased body weight in Wistar rats [15],higher dietary concentrations must be chosenwith care, in order to avoid the risk of toxiceffects. Decreasing the excess dietary methi-onine level to 1.4% allowed themice to surviveuntil the end of the study without obviousclinical or toxicity problems (Figure 4.3.2;[14]). By carefully screening the literature

Figure 4.3.1 Body weight development in apoE-deficie‘high’ (4.4%, High-Met group) and ‘low’ (2.2%, Low-Metmice caused weight loss and death before the scheduZhou, J. et al. 2001 [14].

before starting animal experiments, the mostoptimal high dietary levels can be calculatedand chosen in accordance with the purpose ofthe study without causing toxicity.

• A change in the optical isomer of cholinebitartrate led to the development of kidneyand bladder stones in rodents, followed byrenal failure. Synthetic DL-tartaric acid wassubstituted for the previously used naturalL-tartaric acid isomer without informing thediet manufacturers. DL-Tartaric acid has beenshown to induce renal damage in rats [16, 17].

ContaminantsContaminants can be defined as undesirablesubstances (usually extraneous in origin) which,when present at a sufficiently high concentrationin the food, may affect the animal and thereforethe outcome of the experiments [18]. Possiblecontaminants include industrial chemicals (e.g.PCBs), pesticides (e.g. DDT), mycotoxins (e.g. afla-toxin), heavy metals, nitrosamines, nitrates andnitrites. The list also typically includes microbio-logical parameters, some of which may be delete-rious to the animal (e.g. salmonella) while others,

nt mice fed five different experimental diets [10]. Agroup) dietary methionine level fed to apoE-deficientled end of the study. Reproduced with permission from

DRY

ANDM

AINTEN

ANCE

Figure 4.3.2 The body weight development af apoE-deficient mice on seven different purified diets. The‘high’ methionine level of 1.4% did not appear to cause adverse effects during the course of the study [39].

HUSB

ANDRY

ANDM

AINTE

NANCE

572

NUTR

ITIO

N

such as total viable count and moulds, are indica-tors of hygiene standards in the feed mill,although at very high levels they could be delete-rious to the animal.

Both the US Environmental ProtectionAgency (EPA) and FDA (Food and Drug Adminis-tration) were central in introducingmonitoring ofcontaminant levels in laboratory animal diet, but itis important to distinguish between their guide-lines for in vivo toxicology and similar studiescarried out in accordance with Good LaboratoryPractice (GLP), and for studies carried out fortheir own requirements or purposes. Those forthe latter were really contractual specificationsto be met by diet suppliers.

EPA and FDA both stipulated that for in vivotoxicology studies carried out to GLP the feedshould be analysed periodically to ensure thatcontaminants known to be capable of inter-fering with the study and reasonably expectedto be present in such feed were not present atlevels above those specified by the tester in theprotocol [19, 20]. These requirements havebeen reaffirmed in updates to the originalpublications. More recently, the OECD [21] hasstated that, for regulatory carcinogenicity

studies, the content of dietary contaminants,such as pesticide residues, persistent organicpollutants, phytooestrogens, heavy metals andmycotoxins, that might influence the outcomeof the test, should be as low as possible. Further-more the diet should be analysed for suchcontaminants at least at the beginning of thestudy and when there is a change in the batchused. The introduction of phytooestrogens tothe list is novel and is discussed below. None ofthese agencies defined either specific contami-nants or limits to their levels in diet but assumedthese would be determined by those conductingthe studies in light of the substances being testedand the potential for interference from possiblecontaminants in the diet. In further guidelines,FDA cautioned against assuming that analysisof a blanket range of analytes would always beappropriate [22]; nevertheless this does happenin practice.

Despite the general nature of contaminantguidelines from the EPA and FDA, they haveprovided specific statements on contaminantsand the maximum allowable concentrations(MAC). However, thesewere in the formof specifi-cations for diet to be used as part of the testing

programme to support the EPA Toxic SubstancesControl Act [23] and for in-house studiesby government agencies such as theNational Insti-tute of Environmental Health Sciencesand National Centre for Toxicological Research[24–26]. These generally became de facto standardsfor US diet manufacturers and their customers.

In Europe, these guidelines have beenextended by BARQA [18] and GV-Solas [27] toinclude guidelines on appropriate ranges forthe nutrient levels claimed by manufacturers.The list of contaminants and MAC (see Table4.3.4) show some commonality but also reflect

TABLE 4.3.4: Comparison of three different guidelinenants for mice (and rats)

Contaminant BARQAa

Fluorine mg/kg 40

Nitrate mg/kg 100

Nitrite mg/kg 5.0

Nitrosamines mg/kg

Lead mg/kg 3.0

Arsenic mg/kg 1.0

Cadmium mg/kg 0.5

Mercury mg/kg 0.1

Selenium mg/kg 0.5

Aflatoxins mg/kg 5.0

PCB mg/kg 50

DDT (total) mg/kg 100

Dieldrin mg/kg 20

Endrin mg/kg

Lindane mg/kg 100

Heptachlor mg/kg

HCB mg/kg

a,b,d-HCH mg/kg

a,g-chlordan mg/kg

a,b-endosulfan andendosulfate mg/kg

Malathion mg/kg 500

Fenitrothion mg/kg

individual historical experiences. Individual dietmanufacturers have also generated lists ofcontaminants, generally based on the lists above,but also reflecting their historical experienceand that of their major customers.

In recent years, phytooestrogens in labora-tory animal diet have been recognized as havingpotentially significant confounding effects onexperimental studies. Although endogenous innature, they may be regarded as contaminants[21]. These substances, mainly in the form of iso-flavones and primarily associated with soybeanmeal, can vary considerably in concentration in

s on maximum allowed dietary levels of contami-

GV SOLASb EPAc

150

15.0

NDEAd 0.01NDMAe 0.01

0.01

1.5f 1.5

1.0 1

0.4 0.15

0.1 0.1

0.1e0.5

B1 10B2 5

5g

G1 5G2 5

50 50

50h 100

10i 20

10

100 20

20

10j

20

20

100

1000 2500

1000

(Continued)

NUTR

ITION

573

HUSB

ANDRY

ANDM

AINTEN

ANCE

TABLE 4.3.4: Comparison of three different guidelines on maximum allowed dietary levels of contami-nants for mice (and rats)dcont'd

Contaminant BARQAa GV SOLASb EPAc

Pirimiphos (-methyl)mg/kg

1000

Chlorpyriphos(-methyl) mg/kg

1000

Other phosphoricacid esters mg/kg

500

Oestrogenic activitymg/kg

1

TVO per gk 20 000 Fibre <7% 100 000

Fibre >7% 500 000

Mesophilic sporesper g

20,000

Salmonellae per g none None

E. coli per g none 10

Fungal units per g 200 Fibre <7% 1000Fibre >7% 5000

Fusarium toxinsmg/kg

Deoxynivalenol 0.50Ochratoxin 0.10Zearalenone 0.10

A/B activity per g None NoneaBritish Association of Research Quality Assurance, 1992 [18].bGerman Association for Laboratory Animal Science, 2002 [27].cEnvironmental Protection Agency, 1979 [19].dNitrosodiethylamine.eNitrosodimethylamine.fWith a dietary protein level of over 20% or with a crude fibre level of over 12%, values of up to 2.5mg/kg feed are possible.gAflatoxin B1, B2, G1, G2.hDDT þ DDE þ DDD.iDieldrin þ aldrin.jHeptachlor and heptachlor epoxy.kTotal viable organisms.

HUSB

ANDRY

ANDM

AINTE

NANCE

574

NUTR

ITIO

N

different batches of feed, and affect the animalthrough a variety of mechanisms. They can influ-ence reproduction, the response to endocrinedisruptor chemicals, tumour studies (e.g.mammary, prostate, lung, colorectal and bladdercancers), the cardiovascular system, immunologyand inflammation, diabetes, bone metabolismand neurobiology studies (e.g. memory, cognition,anxiety and neurodegeneration) [1, 28].

Unfortunately most contaminant lists areoutdated and include contaminants that are nolonger found in diet, but exclude some of morecurrent relevance. For example it is difficult tounderstand the continuing inclusion of oestro-genic activity in some parts of Europe (thoughnot in the USA) which was primarily intendedto detect contamination with highly potent

diethylstilbestrol and which should not be, buthas been, confused with the detection ofphytooestrogens [29]. Likely contaminants indiet have been reviewed by Tobin et al. [1].

The nature and level of contaminants areinfluenced by the ingredients used in the diet:fish meal, a very good quality protein sourceand widely used in laboratory animal diets, isalso potentially a source of nitrosamines andheavy metals. Much of the mercury in fish mealis in the form of organic methyl mercury, whichis highly absorbed, in contrast to the inorganicmercury associated with other ingredients [30].Soybean meal, and alfalfa are rich sources of oes-trogenic isoflavones. Avoidance of certain ingre-dients can be used to minimize the level ofcontaminants thatmight be of concern to the user.

NUTR

ITION

575

HUSB

ANDRY

ANDM

AINTEN

ANCE

Most commercial manufacturers providediets with batch analysis certificates that includeanalysis of key nutrients and contaminants. Thisprovides buyers with the opportunity to judgewhether this specific batch is suitable for thepurpose of their particular study or to buy analternative batch. After the completion of thestudy, the batch analysis certificate is also valu-able for the interpretation of the results.Although small deviations in nutrients may beacceptable, if explained, it should be unusualfor a manufacturer to release a batch thatexceeds an MAC for an inorganic and organiccontaminant since this is likely to require signifi-cant justification to regulatory authorities.

Types of dietsNatural-ingredient dietsThe two most commonly used types of mousediets in the laboratory are natural-ingredientand purified diets. A third category of diets, thechemically defined diets in which amino acidsmay be used instead of a protein source, or fattyacids instead of fat, are of minor interest, rarelyused, and are not discussed in this chapter (butsee Beynen and Coates [2] for information). Thepelleted diets that are often standard in mostlaboratory animal facilities are usually producedfrom natural ingredients.

Natural-ingredient diets can also be dividedinto open- and closed-formula diets [31]. In open-formula diets, all dietary ingredients and theirconcentrations are reported and should not varyfrom batch to batch. In closed-formula diets, thedietary ingredients used are usually reported,but the concentration of each dietary ingredientis not stated by the manufacturing company.

Closed-formula diets can be manufactured toeither a fixed or a variable formula (open-formula diets are always fixed). In a variable-formula diet, the objective is to try to maintainthe nutrient levels as constant as possible, byadjusting the inclusion of ingredients to allowfor any nutrient variability in different batchesof ingredients. In practice it is impossible toachieve such consistency and the focus is usuallyon consistency of protein level. The concentra-tion of dietary ingredients in the diet may vary

from batch to batch or with availability of ingre-dients [31].

In a fixed-formula diet, the same proportionsof raw material ingredients are used each timea batch is produced [18]. As natural ingredientscan differ in nutrient levels, natural-ingredientdiets are subject to variation, though with carefulselection of ingredients and suppliers, and qualitycontrol of incoming materials, variation of nutri-ents can be minimized. It should be possible ina fixed-formula diet to achieve a coefficient ofvariation in a nutrient such as protein as low as2–3%, which compares favourably with variableformula diets. For a fuller discussion see Tobin [1].

While most natural-ingredient diets for miceare pelleted, it is possible to obtain extruded(expanded) rodent diets. These offer severaladvantages particularly for autoclaving, forhigherfat diets, and for weaker strains of mice [1, 32].

Manufacturers provide information on thediets in their catalogues and on their websites.The amount of information provided and theway the catalogue values for nutrient concentra-tions have been established is not standardized,though it is usually possible to contact the indi-vidual firms to find out any additional detailsrequired. There is considerable differencebetween manufacturers in the nutrient levelsfor diets with a similar purpose (i.e. breedingand maintenance diets). In some cases a mainte-nance diet from one manufacturer can havehigher or similar nutrient levels to those ina breeding diet from another. It is important tocompare suitability of diets based on nutrientlevels rather than name.

Researchers often assume that the nutrientlevels in the manufacturers’ data sheets will beaccurately reflected in a particular batch ofdiet. Manufacturers’ data sheets should be treatedas a guide to nutrient levels and usually reflectthe values calculated from typical nutrient levelsin the ingredients, either from the manufac-turers’ own ingredient data or published tables.Only by carrying out analytical measurementscan the values be stated with certainty. Wherehigh nutrient accuracy is required a priori, that isbest achieved by the use of purified diets [33].The deviation between actual and datasheetvalues can be considerable and is brought aboutmainly by variation in nutrients in ingredients,mixing inhomogeneity, sampling errors (typical

HUSB

ANDRY

ANDM

AINTE

NANCE

576

NUTR

ITIO

N

batch sizes can be as great as 20 tonnes), andanalytical errors. Even legislation on nutrientdeclarations and specifications [27] canencourage deliberate under-reporting of averagefor crude protein and over-reporting of crudefibre to minimize the risk of deviating from spec-ifications (the data are normally distributed butthe specifications may not be).

Inevitably between-batch variation canincrease variation within a study and certainlybetween studies: any increase in the standarddeviations increases the number of animalsrequired to find statistically significant findings[34]. This runs counter to the objectives of Russelland Burch’s principle of the 3Rs (reduction,refinement and replacement) in which one ofthe goals is to limit the number of animals used.From the point of view of standardization, it isadvisable to use purified diets in experimentalstudies instead of natural-ingredient diets wher-ever practicable. Where natural-ingredient dietsare used, it is advisable to buy them witha batch-analysis certificate, so that one at leasthas information on the exact levels of selectednutrients and contaminants of the specific batchuse; the user should at least measure the nutrientsof concern for the experiment.

Purified dietsPurified or semi-purified diets (also calledsynthetic or semi-synthetic diets) are defined asbeing formulated with a combination of ingredi-ents, each of which is largely a source of a nutrienttype. This results in diets having a much more

TABLE 4.3.5: Critical nutrients in five purified diets in cing to actual analysis

Type of diet Vitamin B6 Vita

(mg/kg) (mg/

Control 8.9 (8) 6.4

Low folate 7.2 (8) 10 (

Low vitamin B6 3.7 (4) 5.3

Low methionine 6.8 (8) 6.4

High vitamins 18 (24) 31 (aAs low as possible.Source: Zhou [14].

standardized composition than natural-ingredientdiets, consequently leading to more reproducibleresults, bothwithin andbetween laboratories. Puri-fied diets also allow precise and accurate changesin individual nutrients in a way not achievablewith natural-ingredient diets. One commonmistake in many studies is to use a natural-ingredient diet as the control and a purified dietas the ‘experimental’ diet. This may mean thata considerable part of the observed difference isbecause of the control, rather than the variablebeing investigated [35].

Improvements in accuracy and precisionachieved with purified diets contribute toa more responsible use of laboratory animals, asthe number of animals needed for reachingstatistically significant results can be reduced,and experimental results between studies andlaboratories can be compared more directly.Table 4.3.5 illustrates the analysed values versusthe targeted levels in purified diets [14].

The American Institute of Nutrition hasdeveloped general-purpose purified diets,initially AIN-76A (the original AIN-76 was modi-fied because it resulted in vitamin K deficiency)and, in 1993 further improvements were made,resulting in AIN-93G for growth and AIN-93Mfor maintenance [36–38]. It is possible that thediets can be further improved by further supple-mentation with vitamin B12 and selenium [14, 39].Table 4.3.6 gives a typical example of a dietdesigned according to AIN-93 guidelines, withthese modifications. The AIN-76A and AIN-93diets are often the basis for furthermodificationsfor specific experimental purposes.

oncentrations as aimed for (in brackets) and accord-

min B12 Folate Methionine

kg) (mg/kg) (g/kg)

(10) 0.53 (0.5) 4.3 (5)

10) 0.02a 4.5 (5)

(10) 0.86 (0.5) 4.1 (5)

(10) 0.8 (0.5) 13.7 (15)

30) 2.3 (1.5) 4.4 (5)

TABLE 4.3.6: Composition of a purified mousediet, according to AIN-93 guidelines

Ingredient Amount (g)

Casein 14.0

Corn flour 71.5

Solkafloc (cellulose) 5.0

Corn oil 4.0

Choline bitartrate 0.2

L-Cystine 0.2

Vitamin and mineral mix 5.0

Methionine 0.1

Total 100.0

Source: Reeves et al. [38]; Zhou [14]; Zhou et al. [39].

NUTR

ITION

577

HUSB

ANDRY

ANDM

AINTEN

ANCE

Most of the major diet manufacturers havea technical group that can design or recommendpurified diets for customers, and some compa-nies manufacture almost entirely purified diets.In Harlan, about 900–1000 new formulas aredesigned each year.

The largest use of purified diets is in the studyof metabolic syndrome (diabetes, obesity). Puri-fied diets are also used to obtain low concentra-tions of particular nutrients that typicallycannot be reached when using natural-ingredientdiets, due to the relatively high concentrationsnaturally present in the raw materials.

Particularly with high fat inclusion, it may beimpossible to pellet purified diets, though thiscan be influenced by the type of fat and carbohy-drate: the inclusion of dextrin can often improvepelletability. The example of the diet given inTable 4.3.6 is of such a composition. When pellet-ing is not possible, the use of special feedingdevices becomes necessary [40].

The use of purified diets in toxicologystudies has been promoted from time to time[41, 42], primarily because of their ability tominimize nutrient variability within andbetween studies, and avoid potentially con-founding non-nutrients and contaminants asso-ciated with natural-ingredient diets. AIN-93Mwas developed for possible use in toxicologystudies. Unfortunately there are some practicaldifficulties in its widespread use in toxicology,including significantly increased cost, avail-ability of sufficient New Zealand white casein,difficulty of manufacture of the large amounts

needed for such studies and limited experienceof their use. Furthermore, studies in the 1980s ofcomparisons between natural-ingredient dietsand AIN-76A were disappointing [43, 44] butthis may have been due in part to the highlevels of sucrose and protein in the diet [45].Later studies carried out with AIN-93M, whichcorrected these issues, have provided someencouragement but mortality on purified dietsseems to exceed that on natural-ingredient diets,even with diet restriction [46–48].

Storage conditionsIn order to ensure that the nutrient contents ofthe natural-ingredient diets remain within thespecifications until the recommended expirydate, diets should be stored in a cool (about18–21 �C) and dry place (less than 65% relativehumidity) and free from pests. In order to avoidcross-contamination, the storage area must bededicated to non-medicated diets. A full discus-sion on diet storage is given in Tobin et al. [1].

Storage of purified diets, particularly high-fatones, poses particular problems. Fullerton et al.[49] reported that vitamin losses and ranciditycould be substantially decreased by reducingstorage temperature from ambient to 4 �C, and inthe case of AIN-76A (which would be typical ofmanypurifieddiets), expirationcouldbeextendedto 6months. Shelf life can be further prolongedby keeping the diets in a freezer at�20 �C insteadof 4 �C. Storage of AIN-93 at �20 �C for 3monthsdid not lead to fat oxidation as measured by theperoxide value, in contrast to storage at þ5 �C(unpublished observations). When storing diet at�20 �C, addition of antioxidants is not considerednecessary. This is important where antioxidantsmay interfere with the purpose of studies, e.g.atherosclerosis induction, and are best avoided.Working with highly unsaturated oils like fish oilneeds particular care since they are very easilyoxidized, altering the nature of the fatty acidsand possibly decreasing vitamin E levels. Whenadding fish oil to diets, the best procedure is tokeep the oil stored at �80 �C, and then mix the oilfreshly through the purified diet each day, justbefore feeding [50].

HUSB

ANDRY

ANDM

AINTE

NANCE

578

NUTR

ITIO

N

Pellet hardnessAlthough mice are less sensitive than rats to diethardness [51], it is still an important factor in theirgrowth and reproduction, particularly for weakeranimals such as many inbred, genetically modi-fied and mutant strains.

Even mice that seem unaffected by diet hard-ness may be affected in periods where increasedfood intake is required. Koopman and colleagues[52, 53] showed that even robust female outbredSwiss mice were restricted in their ability to raiselitters when fed a hard diet because they wereunable to increase their food intake adequately tomeet the increased energy demands of lactation.Subsequently, although the pups had a lower wean-ingweight, theoffspringwereable togrowatalmosta normal rate despite the hardness of the diet.

There are several reviews on themeasurementof pellet hardness, and the influence of processconditions and raw materials on diet hardness[1, 54–56]. Thehardnessmeasurement is influencednot only by the truehardness of the diet but also bythe method of measurement and the size of thepellet, making it difficult to compare measure-ments carriedout indifferent laboratories. Inaddi-tion there is often considerable variation betweenindividual pellets from a single batch of diet[53, 57], and a reliable mean value needs to beobtained from at least 10 pellets, and possiblyup to 30.

Autoclaving/irradiationDiets that are to be used behind microbiologicalbarriers are usually steam autoclaved or gammairradiated [58]. The most common autoclavingconditions are 121–124 �C for 15–30minutes or134–137 �C for 3minutes. Autoclaving has littleeffect on the overall protein level, but damagesindividual amino acids, particularly methionine,cystine and lysine, and causes reactions betweenamino acids, and between amino acids and carbo-hydrates that reduce the availability and biolog-ical quality of the protein [59, 60]. There is alsoa considerable loss of some vitamins, particularly

thiamine (B1), vitamin B12, vitamin K3, vitamin B6and pantothenate. Losses of vitamin A, D3 andfolic acid are modest while riboflavin, biotin,niacin and vitamin E are little affected.

Measurements of crude protein, crude fatand crude fibre, ash [61, 62], and fatty acidpatterns do not appear to be measurably influ-enced by autoclaving. Manufacturers providespecial diets with increased levels of vitamins(and sometimes amino acids such as methionineand lysine) in order to compensate for auto-claving losses.

Autoclaving also has adverse effects on thephysical nature of pelleted diets, often resultingin diet clumping and hardness [1, 57]. Theseeffects can be largely eliminated by the use ofextruded diets.

For irradiation it is advisable to use 21 kGy asa minimal dose (typically equating to an averageof 25 kGy) for diets to be used in SPF units, and40 kGy for germ-free animals (average 50 kGy).Lower doses can be used when an analysis of themicrobial quality prior to irradiation indicatesthat this will be sufficient, for example, with highfat purified diets. Irradiation is less damagingto nutrients than a comparable level of steam ster-ilization, for example, protein and amino acids[61–63]. Vitamin losses are typically less than 20% [1].

The main concern with irradiation of diet isthe potential for the free radicals that areproduced to oxidize fats and produce peroxides[64, 65]. This seems not to be a problem with stan-dard diets but may adversely affect the intake ofirradiated high-fat diets [66].

Quality controlBoth BARQA [18] and GV-Solas [27] give guid-ance on the quality control of laboratory animaldiets. Users should follow the adage ‘Trust butverify’, ideally by auditing the manufacturer (byvisit or questionnaire). The quality of the finishedproduct depends on the quality of the incomingmaterials, care in the manufacturing process(including pest control and cleaning), the qualityand training of staff, and rigorous QC/QAsystems, including documented operating proce-dures. Finally, a series of physical and analyticaltests should confirm that the product is what it

NUTR

IT

purports to be and that it meets specificationbefore it is released for use. Any storage areasfor finished product should be clean, well-orga-nized, pest free and of the desired temperatureand humidity: ideally the store should be temper-ature controlled. It should be possible to obtainevidence of all these factors by examiningretained records. Ideally independent verifica-tion of the quality system should be available,for example ISO 9000 certification. For GLPstudies, the diet is usually analysed for a widerange of nutrients and contaminants beforerelease, though the analytes will vary betweendifferent countries. Such a diet is known byvarious termsdcertified diet, extended analysisdiet, GLP diet. Apart from the analysis, thereshould be no difference in the quality of dietfor GLP and non-GLP studies.

ION

579

HUSB

ANDRY

ANDM

AINTEN

ANCE

Ad libitum feedingversus foodrestrictionAd libitum feeding means that the diet is availableat all times. Restricted feeding refers to restrict-ing the amount of food while still ensuring nutri-tional adequacy [67]. This implies that only theamount of energy has been restricted. It is stillcommon practice to feed laboratory mice (andrats) ad libitum (less than 1% of the total numberof papers on rodents mention the use of foodrestriction; [68]), even though this is undesirablefrom the point of view of animal welfare, aswell as the validity of the experimental results.Keenan et al. [69] have stated that ad libitumfeeding is currently our worst standardizedexperimental factor in laboratory units. In sometoxicology laboratories it has become commonpractice to restrict feed intake of rodents to 75–80% of ad libitum intake, as this gives better stan-dardization and longer survival. This implies,however, that animals are housed individually,as there are no appropriate housing and feedingsystems available for the restricted feeding ofgroup-housed rodents. As rodents are socialspecies, the resolution of the Council ofEurope [4] requires that social species need to

be group-housed whenever possible. For thatreason it is necessary that appropriate feedingsystems and/or methods be developed that bothguarantee restricted feeding as well as socialhousing conditions. Until then considerationshould be givendwhenever possibledto feedingthe animals individually, and then providingsocial housing as soon as the feeding period isover. For rats the use of diet boards has securedworking for food and, as a consequence, loweringof body weight, even though the diet is available24 h/day, and additionally led to a longer survivaltime as compared to normal ad libitumconditions [70].

When comparing ad libitum feeding versusfood restriction, it becomes clear that ad libitumfeeding has a negative impact on the health ofrodents. Ad libitum feeding will lead to moreobesity, a shorter survival time, increased degen-erative kidney and heart diseases, a shorterlatency time and higher incidence of cancer ascompared with restricted feeding [67]. Table 4.3.7shows a comparison between ad libitum and food-restricted B6C3F1 mice that served as controlgroups in long-term toxicity studies [67]. Foodrestriction gave a reduced body weight ascompared with ad libitum feeding, and alsoa decreased incidence of liver tumours in bothsexes, decreased lung tumour incidence in malesand decreased malignant lymphomas in females.Food restriction led to an improved survival time,especially in females. A recent meta-analysisrevealed that mice in control groups in theNational Toxicology Program showed an 11.8%increase in body weight per decade from 1982to 2003 in females. In male mice there wasa 10.5% increase of body weight per decade [71].

A large and elaborate study has been con-ducted on the effects of diet composition andfood consumption in rats. Significant effects ofdiet reduction (20% reduction) were found onlongevity, degenerative disease development andneoplasia in Wistar rats [72]. Similar effects canbe expected for mice.

During the 1980s and 1990s the variation inresults of long-term bioassays in rodents thatwere on ad libitum feeding schemes increased[69]. Therefore the feeding of a standardizedrestricted amount of food to each individual inthis type of experiment is considered a necessityin order to obtain standardized results without

TABLE 4.3.7: Some results of control groups ofB6C3F1 mice on ad libitum or food restrictionschedules

Tumour type andsurvival Males Females

AL FR AL FR

Liver tumours (%) 54 22** 45 8**

Lung tumours (%) 28 17* 2 12

Malignant lymphoma (%) 9 8 16 4*

Haemangio(sarco)ma (%) 5 2 8 2

Survival (%) 80 88 65 94**

AL, ad libitum; FR, food restriction.*(p < 0.05),**(p < 0.01) significantly different from AL-fed controls.Source: Hart et al. [67].

HUSB

ANDRY

ANDM

AINTE

NANCE

580

NUTR

ITIO

N

unnecessarily large variations. This will alsoincrease the reliability of the interpretation oftoxicity of test substances, as the amount of thetest substance ingested through the diet becomesstandardized for each individual. The standardiza-tion of food intake will also contribute to a lowervariation in experimental results, and thusprovide Reduction potential [71]. However,a recent study in rats into the Reduction potentialof food restriction as compared to ad libitumfeeding revealed that the resulting variationdepended on the parameter measured [73]. Forbody weights the Reduction effect was clear andsignificant, but the type of feeding had no consis-tent effect on the variation of several standardblood parameters measured. Besides a better stan-dardization, another advantage of food restrictionis that animals become ‘more robust’, i.e. they arebetter in coping with stress factors [69]. That waythey can be exposed to test substances in higherconcentrations and/or for longer periods withoutcompromising the animal’s health, physiology ormetabolic profile [67]. As food restriction to 75%of ad libitum intake improves longevity (as seenabove), animals can be exposed to test compoundsfor a longer period, thereby improving the sensi-tivity of bioassays to detect compound-specificchronic toxicity and carcinogenicity [67].

The level of nutrients in natural-ingredientdiets is usually far above the levels needed forfulfilling the nutrient requirements [5]. Manufac-turers do this in order to guarantee that no defi-ciencies will occur, even after longer storage

periods. Moreover, the NRC requirementssupport maximum growth. Therefore limitingfood intake to 75% of ad libitum intake will stillmake sure that the diet lives up to the minimumlevels of essential nutrients.

Pair feedingWhere a carcinogenic test substance is mixedthrough the diet and has a bad taste, this willreduce food intake, particularly when feeding isad libitum. The control group will ingest a higheramount of food than the test group, therebydeveloping cancer at a higher frequency and atan earlier age. The outcome could thus be false-negative, i.e. that one does not judge the carcino-genic test substance to be carcinogenic. In orderto avoid these problems, pair-feeding is neces-sary. How much food the test animals ingest ismeasured, and then the same amount of food isgiven to the control group/animals the nextday. This assumes that the food intake of eachindividual is measured [2, 40].

Normal feedingbehaviour versusfood restrictionRodents are nocturnally active animals andingest most of their food during the dark period.Food restriction (60% of ad libitum intake) ascompared with ad libitum feeding will affect phys-iological variables, circadian rhythms, activityand feeding behaviour [67]. Table 4.3.8 illustratesthe effects of ad libitum feeding and food restric-tion during the dark or the light period, onvarious physiological and behavioural variablesin 28month old male B6C3F1 mice [67, 74]. Totalfood and caloric consumption was decreased infood-restricted groups, as expected. Waterconsumption remained nearly the same in therestricted group fed during the dark period ascompared with the ad libitum fed B6C3F1 mice,so that total water consumption increased in rela-tion to food intake (Table 4.3.8). However, whenfed restrictedly during the light period, water

TABLE 4.3.8: Effects of diet and feeding time on physiological and behavioural variables in old male B6C3F1 mice (aged 28months)

MeasurementAL groupMean � SE

LF restricted groupMean � SE % of AL

DF restricted groupMean � SE % of AL

Significance levels

AL vs LF AL vs DF LF vs DF

Total foodconsumption (g)

5.21 � 0.10 3.35 � 0.02 64.3 3.41 � 0.08 65.5 A*** B*** _****

Caloric consumption(kcal/g)

22.66 � 0.43 14.57 � 0.09 64.3 14.83 � 0.35 65.5 A*** B*** _****

Total waterconsumption (g)

3.64 � 0.38 5.15 � 1.07 141.5 3.72 � 0.07 102.2 A* _**** C*

Number of feedingepisodes

16.51 � 0.69 2.80 � 0.23 17.0 4.32 � 0.37 25.6 A*** B*** C*

Number of drinkingepisodes

11.13 � 1.28 9.03 � 0.44 81.1 8.99 � 0.67 80.8 _*** _*** _***

Average bodytemperature (�C)

36.78 � 0.08 35.54 � 0.15 96.6 35.11 � 0.18 95.5 A*** B*** _***

Maxemin bodytemperature(range; �C)

37.98e35.81 (2.18) 38.15e32.24 (5.91) 271.1 37.52e32.93 (4.59) 210.6 A*** B*** _***

Average activity(pulse/h)

10.54 � 2.30 18.26 � 1.71 173.2 26.50 � 5.57 251.4 A* B** _***

Average O2consumption (g/LBM)(ml ge1 he1)

3.34 � 0.16 3.44 � 0.16 103.0 3.19 � 0.06 95.5 _*** _*** _***

Maxemin respiratoryquotient (range)

0.95e0.86 (0.09) 0.99e0.80 (0.19) 211.0 1.01e0.77 (0.24) 267.0 A*** B*** _***

AL, ad libitum; LF, restricted group fed during light period; DF, restricted group fed during dark period; LBM, lean body mass.Results of Student’s t-test analysis:A[ AL � LF restricted comparison (significant effect)B[ AL � DF restricted comparison (significant effect)C[ LF restricted � DF restricted comparison (significant effect; adapted from Duffy et al., 1990).*p < 0.05,**p < 0.01,***p < 0.001,****p > 0.05.Source: Duffy et al. [74]; Hart et al. [67].

NUTRITION581

HUSBANDRYANDMAINTENANCE

HUSB

ANDRY

ANDM

AINTE

NANCE

582

NUTR

ITIO

N

intake increased to a level of about 141% that ofthe ad libitum fed mice. This may be related tothe fact that the mice do not have food availableduring their normal active period in which theyingest food, thereby possibly using drinkingwater as a substitute. The duration of foodconsumption was compressed by restriction, sothat most food was consumed duringthe first few hours immediately after feedingcommenced. Restricted mice ate fewer meals(feeding episodes) but spent more time feedingper meal and consumed more food per mealthan the ad libitum fed mice. The number ofdrinking episodes was also decreased by foodrestriction. Average body temperature decreased,whereas the range in body temperatureincreased by dietary restriction. Spontaneousactivity was increased by food restriction.Average oxygen consumption increased in therestricted group fed during the light period anddecreased in the restricted group fed during thedark period. This may be explained by the factthat, in the first group, the mice were activelysearching for food during the entire dark period,which is their normal feeding time. The dailyvariations in the respiratory coefficient (RQ)were increased by dietary restriction, indicatingrapid substrate-dependent shifts in metabolicpathways from carbohydrate metabolism (imme-diately after feeding) to fatty acid metabolism(several hours before feeding). This may berelated to an enhanced metabolic efficiency [67].

Becausemice are nocturnally active animals, itis advisable that when animals are fed restrictedly,food is provided during the normal feeding time,i.e. the dark period, in order to fulfil the animals’basic needs. The relationship between circadianrhythms and nutrition is beyond the scope ofthis chapter, but further information can befound in Ritskes-Hoitinga and Strubbe [75].

Individual housingversus grouphousingAfter the 1970s, in mouse studies performed inthe National Toxicology Program, body weights

showed a tendency to increase [67, 76, 77]. Thismay be related to the fact that the protocolchanged from group housing to individualhousing. Individually housed animals had higherbody weights and higher tumour incidence thangroup-housed mice [77]. The variance in bodyweight in group-housed animals (two, four oreight per cage) was smaller than in individuallyhoused mice [78]. According to the NationalToxicology Program, female mice are now onceagain housed in groups, but male mice are stillhoused individually to avoid the problem offighting among group-housed males [67, 77].

Different types of studies based on the routeof administration (e.g. inhalation, feed, corn oilgavage, etc.) are registered in the National Toxi-cology Program database [67]. Each of these typesof study treats control animals differently, result-ing in different results, for example in averagetumour incidence in mice (Table 4.3.9; [67]). Ininhalation studies a clear relationship is seenbetween body weight biomarkers and tumourincidence. Mice are almost always singly housedin inhalation chambers, so that any dietary modu-lation of body weight and spontaneous tumouri-genesis is not confounded by social interactionsamong the animals. Some feed and corn oilgavage studies also use single housing for males,in order to prevent fighting. When comparedwith similar studies that used group housing,group housing increased the percentage ofanimals surviving and decreased the incidenceof lymphomas (Table 4.3.10; [67]). The clear rela-tionship between body weight and survival and(liver) tumour incidences is found in singlyhoused animals only; in group housing this rela-tionship is confounded [67].

Isocaloric exchangeWhen designing experiments in which the die-tary fat content is intended to be altered inthe test groups, it is important to consider thebasic facts in order to reach properly controlledand standardized diet compositions. The MEC offat (37MJ/kg) is about twice as high as that ofcarbohydrates or protein (17MJ/kg). So whenthe dietary fat content is increased, the dietaryenergy concentration will increase. In case of

TABLE 4.3.9: Average body weight at 12months on test (BW12) and tumour incidences for different studytypes for B6C3F1 mice

Type of test i c g f

M F M F M F M F

BW12 (g) 40.1 34.6 44.0 36.2 43.1 38.0 40.9 38.1

Liver tumour incidence (%) 39.6 18.6 41.4 12.2 38.8 10.6 33.2 15.5

Lymphoma incidence (%) 8.7 21.1 16.7 39.1 15.6 43.6 10.0 37.2

Pituitary tumour incidence (%) 0.5 23.2 1.1 33.3 2.6 23.6 0.3 20.0

i, inhalation studies (n¼ 8); c, corn oil gavage studies (n¼ 21); g, water gavage studies (n¼ 5); f, feed studies (n¼ 17); M, males; F, females.Tumour incidences were total incidences after 24months on test.Source: Hart et al. [67].

TABLE 4.3.10: Average survival and lymphomaincidence in single and group-housed maleB6C3F1 mice

Type ofhousing

Single-housed

Group-housed

Survival (%) 65 � 9 84 � 10

Lymphomaincidence (%)

15 � 5 7 � 7

Single-housed combines results of 9 studies, group-housed 12studies.Source: Hart et al. [67].

NUTR

ITION

583

HUSB

ANDRY

ANDM

AINTEN

ANCE

ad libitum food intake, the animals ingest accord-ing to energy need, so the food intake in gramswill decrease. When an exchange with carbohy-drates, for example, is done on the basis ofweight, test and control groups will ingestdifferent amounts of all nutrients (Table 4.3.11;[2]). In order to reach full standardization, theexchange of fats and carbohydrates needs tobe done on the basis of calories (isocaloricexchange; see Table 4.3.9). In this way, the intakein grams of all nutrients will be similar incontrol and test groups; only the intake in fatsand carbohydrates will be different as this isinherent to the design of the diets.

When the intention is to examine the influ-ence of certain dietary fatty acids, it is advisableto keep the total fat content similar and varythe types of fats used, in order to obtain variableconcentrations of fatty acids only. An example ofhow to design two different dietary fat levelswith varying fatty acid concentrations withineach fat level is illustrated by Ritskes-Hoitingaet al. [79].

Mouse models innutrition researchAnimal models can contribute to the under-standing of (parts of) human processes. Russelland Burch [80], in their famous book The Principlesof Humane Experimental Technique, distinguishedbetween two types of animal models: the HighFidelity and the Discrimination models. In theHigh Fidelity model all characteristics resemblethose in humans, whereas in a Discriminationmodel only one characteristic is reproduced.Researchers need to be aware of which type ofmodel they are using, in order to make reliablecomparisons with the human situation [81]. Apart of what is studied is independent of theinterference of scientists, but another part isinfluenced by the choices scientists make [82].The choices that are made regarding the model,the design and environmental conditions are ofmajor importance for the outcome of the study[83]. Results of animal studies should never beextrapolated directly to the human situation,but need discussion and critical evaluation. Liter-ature/systematic reviews of other animal studies,epidemiological studies and clinical trials must bepart of the entire evaluation.

Influence of dietary linoleicacid on mammary cancerdevelopmentIn a study by De Wille et al. [84] the influence ofdietary linoleic acid concentration on mammary

TABLE 4.3.11: Isocaloric exchange

Diet 1 Diet 2 Diet 3 Diet 4

Low-fat High-fat High-fat,adjusted

High-fat,adjusted

DIET INGREDIENT

Protein (g) 20 20 20 20

Carbohydrate (g) 60 40 15 15

Fat (g) 10 30 30 30

Fibre (g) 4 4 4 4

Mineral mix (g) 4 4 4 4

Vitamin mix (g) 1 1 1 1

Test compound (g) 1 1 1 1

‘Inert’compound (g) e e e 25

TOTAL (g) 100 100 75 100

Energy value (kcal/g) 4.10 5.10 5.47 4.10

EXPECTED INTAKE

Energy (kcal/day) 82 82 82 82

Food (g/day) 20 16 15 20

Protein (g/day) 4 3.2 4 4

Carbohydrate (g/day) 12 6.4 3 3

Fat (g/day) 2 4.8 6 6

Fibre (g) 0.8 0.64 0.8 0.8

Mineral mix (g/day) 0.8 0.64 0.8 0.8

Vitamin mix (g/day) 0.2 0.16 0.2 0.2

Test compound (g/day) 0.2 0.16 0.2 0.2

‘Inert’compound (g/day) e e e 5

Source: Beynen and Coates [2].

Figure 4.3.3 Dietary linoleic acid level (LA) andmammary tumour frequency (MT) [84].

HUSB

ANDRY

ANDM

AINTE

NANCE

584

NUTR

ITIO

N

tumour development in transgenic mice(MMTV/v-Ha-ras) was studied. Three levels ofdietary linoleic acid were given, 0%, 1.2% and6.7%. There was a significant reduction ofmammary tumour development on the 0% die-tary level of linoleic acid, as compared with theother two dietary groups (Figure 4.3.3). As linoleicacid is an essential fatty acid necessary for thedevelopment of cell membranes [5], this cannotbe considered a reliable control group. Wherethe diet contains no linoleic acid at all, generalhealth is expected to be compromised. In thestudy by De Wille et al. [84] it was reported thatthere were initially 25 animals in the 0% levellinoleic acid group; however, in the results sectiononly data from 15 animals were presented. Whathappened to the missing 10 animals was not

described. It is essential to mention all details inscientific reports and publications, in order tofully understand the course of events and to beable to reproduce studies. The control group inthe study by De Wille et al. [84] should have con-tained the minimum necessary level of linoleicacid in the diet (0.05%; 5), in order to obtaincontrol animals in good health and reliableresults. By leaving out dietary linoleic acidcompletely, one would not expect that cellmembranes or tumours could develop.

TABLE 4.3.12: Mammary tumour incidence in differen

Reference Strain Type of m

Fischer [85] Sencar mouse DMBA ind

Craig-Schmidt [86] BALB/c mouse DMBA ind

Brown [87] C3H mouse Spontane

De Wille [47] MMTV/v-Ha-ras mouse

MMTV

Ritskes-Hoitinga [45] BALB/c-MTV MMTV

DMBA, dimethylbenz(a)anthracene; MMTV, mouse mammary tumouSource: Ritskes-Hoitinga et al. [79].

In a literature review by Ritskes-Hoitinga et al.[83] the results of studies examining the influenceof dietary linoleic acid in different rodent modelswere compared. The results were dependent onthe type of animal model used. In Table 4.3.12the results from the mouse models aregiven. When using dimethyl-benz(a)anthracene(DMBA) to inducemammary tumours, higher die-tary linoleic acid concentrations were associatedwith increased mammary tumour incidences [85,86]. In spontaneous [87] and the BALB/c-MMTV

t mouse models

odelDietaryfat (%)

Dietarylinoleicacid level (%)

Mammarytumourincidence (%)

uction 15 0.8 23

15 4.5 43

15 8.4 50

uction 20 1.4 36

20 1.5 45

20 11.6 77

ous 5 0.5 13

5 0.9 3

5 1.1 2

5 2.9 3

5 3.0 7

17 1.6 8

17 3.2 15

17 3.6 12

17 9.7 8

17 10.1 5

0 0.0 7

2 1.2 36

11 6.7 52

7 0.9 40

7 1.3 30

7 2.6 44

7 4.3 32

16 0.9 52

16 1.3 30

16 2.6 52

16 4.3 38

r virus.

NUTR

ITION

585

HUSB

ANDRY

ANDM

AINTEN

ANCE

HUSB

ANDRY

ANDM

AINTE

NANCE

586

NUTR

ITIO

N

mouse models [79], no clear association wasdetected between dietary linoleic acid concentra-tions and mammary tumour development. Asmentioned above, the study by De Wille et al.[84] showed a higher mammary tumour incidencein the higher dietary linoleic acid groups. Thismay have been an artefactual finding, as thecontrol group lacked linoleic acid completely.Depending on the model, the latency perioddiffered [83]. It may be that the amount of linoleicacid needed for tumour development is depen-dent on the latency period: by using DMBA, rapidtumour development is induced, which may beassociated with a high linoleic acid ‘requirement’for tumour growth. This illustrates that the choiceof animal models and set of experimental condi-tions, as well as the interpretation of results, needscareful consideration.

Atherosclerosis-inducing dietsMice are historically resistant to the developmentof atherosclerosis [88]. On a normal chow diet(about 4.5% fat and 0.02% cholesterol) most ofthe plasma cholesterol is in the form of highdensity lipid (HDL), the anti-atherogenic fractionof cholesterol [88]. High fat diets will result in thedevelopment of atherosclerotic lesions over timein susceptible strains such as C57BL/6. After7 weeks on the high fat diet, these mice developfatty streaks and progress to more complicatedlesions by 14 weeks. Other strains, such as theBALB/c and C3H strains never develop athero-sclerotic lesions [88]. Special diets used to induceatherosclerosis are a ‘Western-type’ diet, whichcontains about 21% fat and 0.15% cholesterol,and an ‘atherogenic’ diet, which contains 15%fat, 1.25% cholesterol and 0.5% cholic acid [88].The latter is also referred to as the ‘Paigen’sdiet.’Historically, this diet was used to induce gall-stones. It is known to be hepatotoxic and toinduce a proinflammatory state [88]. When usingthe Paigen’s diet, atherosclerotic plaques can beinduced; however, hepatotoxicity and gallstonesare induced simultaneously. Hepatotoxicity mayinterfere with the development of atheroscle-rotic lesions, as was seen in rabbits [50]. It is there-fore considered necessary to at least evaluate thecondition of the liver, and publish this, whenatherosclerosis studies are performed. As

gallstones are painful in humans, it may beexpected to be the same for mice as well.

The use of transgenicmousemodels instead ofwild-type mice models may be a good alternativesolution, as it then becomes possible to omit thecholate from the diet. On a normal chow diet,apoE-deficient mice develop plasma cholesterollevels that are at least 10 times as high as in wild-type mice and most of the cholesterol is inthe highly atherogenic VLDL form [88]. apoE-deficient mice develop atherosclerotic lesions onnormal chow and on a Western-type diet, butlesions develop more rapidly and at an earlierage on the Western-type diet. The atheroscleroticlesions in apoE-deficient mice have strikinglysimilar pathological characteristics and anatomicaldistributions to those of humans [88]. The lowdensity lipid (LDL) receptor-deficient mouse doesnot develop atherosclerosis on a normal chowdiet, but this can be induced by feeding a high-fatdiet. Lesion characteristics are the same as in theapoE-deficient mouse, but lesion formation ismore controllable by dietary changes [88]. Plasmacholesterol levels are lower than in the apoE-deficient mice and are thus more human-like.

If an atherosclerosis-inducing test diet is usednext to a control diet, care must be taken thata proper isocaloric exchange is made between thecontrol and test diet.With isocalorically exchangeddiets, only the fat and carbohydrate intake (ingrams) will be different between the two groups.The intake of all other nutrients will be similar,thereby allowing a more reliable interpretationof results due to optimal standardization.

Diabetes and obesity modelsDiabetes mellitus, often simply referred to as dia-betes, is a group of metabolic diseases character-ized by high blood glucose levels, either becausethe body does not produce enough insulin inthe beta cells of the islets of Langerhans in thepancreas, or because cells in all parts of thebody, except the brain, do not respond ina normal way to the insulin that is produced.This normally means that the cell membranebecomes more permeable to glucose and muscleand liver cells increase conversion of glucose toglycogen. These high blood glucose levelsproduce the classical symptoms of polyuria, poly-dipsia and polyphagia.

NUTR

ITION

587

HUSB

ANDRY

ANDM

AINTEN

ANCE

There are three main types of diabetes: type 1diabetes, type 2 diabetes and gestational diabetes.Type 1 diabetes results from the body’s failure toproduce insulin, and at present requires theperson to inject insulin. It is therefore referredto as insulin-dependent diabetes mellitus(IDDM) or juvenile diabetes. Type 2 diabetesresults from insulin resistance, a condition inwhich the cells fail to respond to insulin properly,sometimes combined with an absolute insulindeficiency and thus formerly referred to asnon-insulin-dependent diabetes mellitus(NIDDM) or adult-onset diabetes. Gestational dia-betes is seen in pregnant women, who have neverhad diabetes before, as a high blood glucose levelduring pregnancy; it may precede developmentof type 2 diabetes.

Other forms of diabetes mellitus include con-genital diabetes, which is due to genetic defectsof insulin secretion, cystic fibrosis-related diabetes,steroid diabetes inducedbyhighdoses ofglucocor-ticoids, and several forms of monogenic diabetes.

Both type 1 and 2 diabetes are chronic diseasesthat usually cannot be cured and need lifelongtreatment with insulin or insulin analogues,although type 2 diabetes at an early stage canbe controlled by rigorous changes in lifestyle,e.g. increased exercise and weight loss. Pancreastransplants have been tried with limited successin type 1 diabetes and gastric bypass surgery hassuccessfully cured type 2 diabetes, but requirea complete change in lifestyle and might havestill unknown long-term side effects. Gestationaldiabetes usually resolves after delivery.

An estimated 285million people, about 6.4% ofthe world’s adult population, were living with dia-betes in 2010. The number is expected to grow to438million by 2030, about 7.8% of the adult popu-lation. Diabetes is one of the major causes ofpremature illness anddeathworldwide.Type2dia-betes is responsible for 85–95% of all diabetes inhigh-income countries and may account for anevenhigherpercentage in low-andmiddle-incomecountries. Non-communicable diseases, includingdiabetes, account for 60% of all deaths worldwide.Almost 80% of type 2 diabetes is preventable bychanging diet, increasing physical activity andimproving the living environment. Yet, withouteffective prevention and control programmes,the incidence ofdiabetes is likely to continue risingglobally.

Insulin is vital for the survival of people withtype 1 diabetes and often ultimately required bypeople with type 2 diabetes. Even though its indis-pensible nature is recognized by its inclusion inthe WHO’s Essential Medicines List, uninter-rupted supplies of insulin are still not availablein many parts of the developing world.

Diabeteswithout adequate treatment can causemany complications. Acute complications are dueto either low blood glucose levels causing hypogly-caemia or high blood glucose levels causing hyper-glycaemia or diabetes ketoacidosis. Chronic andsevere late complications include high blood pres-sure and heart problems leading to heart attacksand heart failure, difficulty in vision and eye prob-lems leading toblindness, kidneyproblems leadingto kidney failure, nerve damage primarily leadingtoproblems of the feet, but also problems like diar-rhoea, constipation, nausea and vomiting.

Adequate research into the pathogenesis,prevention and treatment, and hopefully cure,of diabetes is important and hence the knowl-edge and use of appropriate diabetes and obesityanimal models is extremely important for futureresearch into diabetes [88a, 88b].

The non-obese diabetic (NOD) mouse

The non-obese diabetic (NOD) mouse is a wellknown and important model of type 1 diabetesand has become the model of choice. The NODmouse was originally developed by Makino andcolleagues [89, 90] in Japan during the selectionof a cataract-prone strain derived from theoutbred Jcl:ICR line of mice. During the selectionof this cataract-prone strain, the NOD strain wasestablished, through repetitive brother–sistermating, as a subline that spontaneously developeddiabetes. The incidence of spontaneous diabetes isvery gender dependent, as being 60–80% infemales and only 20–30% in males [90, 91], butalso dependent on various environmental factors.The highest incidence occurs when mice arehoused in a relatively germ-free environmentrather than more conventional ‘dirty’ housingenvironments [92], whereas housing under envi-ronmentally enriched conditions versus non-enriched condition did not show any differencein diabetes onset or incidence [93]. The diabetesonset typically occurs at 12–14 weeks of age infemale mice and slightly later in male mice.

HUSB

ANDRY

ANDM

AINTE

NANCE

588

NUTR

ITIO

N

The histological changes due to the autoimmuneprocess in the pancreas with infiltration ofimmune cells can be noted from the ageof 3–4weeks, when both male and female micebegin to demonstrate mononuclear infiltratessurrounding the islet and later invade the isletover the next few weeks, finally giving rise tosevere insulitis by the age of 10weeks [94]. Studieshave shown that T cells are intimately involved inthe pathogenesis of diabetes in NOD mice withCD4þ T cells being very much involved in thepathogenesis ofdisease anddirectlymediating isletcell destruction. However, the CD8þ T cellspromote the disease as well, and it has been sug-gested that the CD8þ T cells might be causinga sufficient islet cell destruction to prime themore robust CD4þ T cell response [95]. However,the genetically linked autoimmunity in the NODmice is a complex process that likely results fromthe summation of multiple defective tolerancemechanisms, and which shares many similaritiesto human type 1 diabetes. On the other hand,intrinsic differences in the biology of the respec-tive species such as the pathophysiological changesassociated with diabetes in the mouse may not beidentical to those in humans. For example, someof the secondary pathological changes associatedwith the morbidity of chronic type 1 diabetes inhumans, such as kidney lesions, neuropathies, andretinopathy, have not yet been reported in chroni-cally diabetic NODmice [96]. Still the NODmousehas helped investigators in recent development ofa number of potential therapies that may alter thedevelopment of the disease and its progression inpatients with type 1 diabetes.

The obese diabetic mouse (Lepob/Lepob

or ob/ob)

The obese diabetic mouse (ob/ob) is an excellentmodel for the study of obesity and diabetes as ithas a longer lifespan and less severe clinical symp-toms than the diabetic mouse (db/db). The obesespontaneous model was originally developed bythe Jackson Laboratory in 1949 by a spontaneousmutation with only homozygotes being obese,reaching a maximum body weight of 60–70 g atan age of 7–8months, and heterozygotes andwild types being lean, reaching their normalweight of 30–40 g at the age of 3–4months. Infer-tility due to atrophic ovaries and uterus is

characteristic for the ob/ob females [97], whereasthe ob/ob males will occasionally breed.

The obese diabetic (ob/ob) mice are character-ized by marked obesity due to a greatly increasedfood intake 2–3 weeks after weaning. The hyper-phagia is accompanied by an increased efficiencyof energy utilization with the rate of lipogenesisin the liver and the adipose tissue being morethan doubled and both intraperitoneal andsubcutaneous deposit of fat being increased[98]. Further, a transient hyperglycaemia andmarkedly elevated plasma insulin concentrationassociated with an increase in number and sizeof the beta cells of the islets of Langerhans isseen [99]. The secretion of glucagon is alsoelevated and this hyperfunction of the alphacells in the pancreas of the obese mice mightbe involved in the pathogenesis of the obese-hyperglycaemic syndrome [100].

The diabetic mouse (Leprdb/Leprdb or db/db)

The diabetic mouse (db/db) develops severe andpotentially fatal diabetes due to amutational inac-tivation of the leptin receptor gene [101, 102]. Themouse strain was initially developed by Hummelet al. in 1959 by discovering a (Leprdb) mutation inmice with a C57BLKS background. The pheno-type of the homozygous mice includes obesity,insulin resistance and diabetes, with secondaryderangement of renal function and morpholog-ical changes. Thepathogenesis of the renal failuremay be due to renal sensitivity to insulin in thehyperinsulinaemic state with early signs ofproteinuria before evidence of renal lesions,which include extracellular matrix expansion.

Mice homozygous for the diabetes sponta-neous mutation (Leprdb) are sterile and becomeobese at approximately 3–4 weeks of age. Eleva-tions of plasma insulin begin at 10–14 days andelevations of blood sugar at 4–8 weeks. Homozy-gous mutant mice are polyphagic, polydipsicand polyuric. The severity of disease on thisgenetic background leads to an uncontrolledrise in blood sugar, severe depletion of theinsulin-producing beta cells of the pancreaticislets, and death by 10months of age. Exogenousinsulin fails to control blood glucose levels andgluconeogenic enzyme activity increases. Periph-eral neuropathy and myocardial disease are seenin C57BLKS-Leprdb homozygotes as well as

delayed wound healing and increased metabolicefficiency. Mice heterozygous for the diabetesspontaneous mutation (Leprdb) have a normalbody weight, blood glucose and plasma insulinlevels, but increased metabolic efficiency; theywill survive longer fasting periods than controls.

Despite the severe and fatal development ofdiabetes, this model is widely used in diabetesand obesity research, but must be closely moni-tored with well-established humane end-pointsbecause of the severe changes in phenotype.

NUTR

ITION

589

HUSB

ANDRY

ANDM

AINTEN

ANCE

FastingFasting is often used in pharmacokinetic andtoxicological studies and in metabolic tests ofglucose homeostasis.

For metabolic studies, overnight fast (14–18 h)or morning fast (5–6 h) procedures are typicallyused [103]. Since blood glucose varies with foodintake, the mice are fasted in order to obtaina basal blood glucose value [104]. Mice arenocturnal animals and consume about two-thirdsof their total food intake during the night [105–107].Becausemicearenocturnal andhaveahighermetabolic rate than humans [108], overnight fast-ing cannot be considered to cause similar levelsof physiological distress in mice and in humans.

Several changes in physiological andbiochemical processes are seen with fasting, asthe mice readily enter a catabolic state, due totheir metabolic rate (Table 4.3.13). Due to thenumerous physiological changes caused by fast-ing, it can have confounding effects if the appro-priate length of fasting is not considered, therebyinfluencing the scientific results.

The fasting of mice for 18 h causes a 14–17%weight loss [105, 134, 135]. Further, fasting canprovoke mice to enter torpor, a state where themetabolic rate can be lowered to around 30% ofthe basal metabolic rate and the body tempera-ture can decrease by up to 15 �C [108, 137]. Thethermoneutral ambient temperature for miceis approximately 30 �C [138], hence housingmice at about 23 �C will increase the metabolicstress caused by fasting. Fasting causes signifi-cantly increased corticosterone levels [139–141],increasing gradually with increasing duration ofthe fasting period [140].

In general, fasting induces change fromanabolic to catabolic metabolism. The glycogenin the liver provides an energy reserve that canbe utilized immediately to maintain glucosehomeostasis. The liver glycogen content is signif-icantly lowered after 18 h of fasting compared to5 h of fasting [105]. As the liver glycogen reserveis depleted, the breakdown of adipose tissue isinitiated, providing triglycerides utilized forproduction of ketone bodies such as b-hydroxy-butyrate. Plasma b-hydroxybutyrate is signifi-cantly increased in mice fasted for 14 h [142].Hepatic steatosis can be seen after 16 h of fasting[143], but strain differences exist regardinghepatic triglyceride levels during fasting [143].

Haemoglobin A1c (HbA1c) is a long termmeasure of the daily blood glucose average. Ithas been shown in three strains of mice that theglucose level measured after 6 h of fasting corre-lates more closely with HbA1c than the glucoselevel measured after an overnight fast, althoughboth values did correlate [104]. A morning fastof 5–6 h is preferred to an overnight fast in orderto reduce physiological distress and therebyincrease animal welfare and yet result in a validblood glucose measurement [144, 145].

Different responses to fasting have beenreported between male and female mice [140]and between different strains [146], but overall,fasting for more than 5–6 h will result in physio-logical changes that should be considered forconfounding effects and negative animalwelfare impact in all studies [147], and should beused only if considered imperative [78].

When reporting experiments that includefasting procedures, it is important to describethe duration of the fast and the time of initiationof the fast in relation to the light–dark cycle.

Welfareconsiderationsand enrichmentThe use of genetically modified mice hasincreased dramatically. When using thesemodels, the possible health and welfare problemsof the specific models must be evaluated in order

TABLE 4.3.13: Effects of fasting

References Strain Duration of fasting (h)Parametersmeasured

Directionof effect

105, 109 C57BL/6J, CF-1 5e18, 28 Adipose tissue Y

104, 110e113, 131, 139, C57BL, FVB/N, C57BL/6J, SV129,3 strains, A-ZIP/F, SV129/OlaHsd;C57BL/6J

48, 24, 48e72, 24, 6e14, 24,9e24

Blood-b-hydroxybutyrate [

114, 130 C57BL/6J, NMRI 2e16, 24 Blood alanine transaminase 4

114, 130 C57BL/6J, NMRI 2e16, 24 Blood aspartate transaminase 4

130, 131, 139 C57BL, C57BL/6J, FVB/N 48, 2e16, 24 Blood corticosterone [

110e113 C57BL/6J, SV129, 3 strains, A-ZIP/F,SV129/OlaHsd; C57BL/6J

48e72, 24, 6e14, 24, 9e24 Blood free fatty acids [

111, 112 e e Blood glucagon [

110, 112, 113, 124, 130,139

C57BL, C57BL/6J, C57BL/6J, A-ZIP/F,SV129/OlaHsd;C57BL/6J, C3H-S

48, 2e16, 48e72, 24, 9e24,29

Blood glucose Y

116, 130, 132e134 C57BL/6J, 2 strains, Dbh, Aston, WT 2e16, 24, 24, 24, 24 Blood leptin Y

114 Albino 24e120 Blood phospholipids Y

111, 115e118, 127, 131 3 strains, albino, C57BL/6J, SV129,WT, C57BL/6, C57BL/6J

0e72, 5e46, 0e72, 24, 24,16e48, 24e48

Body temperature Y

130, 131, 139 C57BL, C57BL/6J, FVB/N 48, 2e16, 24 Blood testosterone Y

131 FVB/N 24 Blood thyroid stimulatinghormone

4

130, 131, 139 C57BL, C57BL/6J, FVB/N 48, 2e16, 24 Blood thyroxine Y

130, 131 C57BL/6J, FVB/N 2e16, 24 Blood triiodothyronine Y

119 Albino 24e120 Blood volume Y

112, 114, 120, 121, 122,135, 139

C57BL, C57BL/6J, NA, A-ZIP/F, CD1,FVB, NMRI, C57BL/6J

48, 5e18, 24e72, 24, 3e18,72, 24, 24e48

Body weight Y

123e125, 135 CD1, C3H-S, CD1, Bantin and KingmanWhite mice

18, 29, 3e18, 24 Gastric content Y

126 DS 24 Gastric emptying rate constant [

127 Ddy 48 Ghrelin mRNA [

122 C57BL/6J 4e48 Heart rate Y

110 C57BL/6J 48e72 Heart triglyceride 4

HUSBANDRY AND MAINTENANCE

590 NUTRITION

130 C57BL/6J 2e16 Haematocrit Y

130 C57BL/6J 2e16 Haemoglobin 4

114 NMRI 24 Hepatic aminopyrineN-demethylase activity

[

114 NMRI 24 Hepatic aniline hydroxylaseactivity

[

128 Swiss white mice 12 Hepatic aryl hydrocarbonhydroxylase activity

4

110 C57BL/6J 48e72 Hepatic carnitine [

114, 128 NMRI, Swiss white mice 24, 12 Hepatic cytochrome P-450 Y, 4

110, 114, 121, 131 C57BL/6J, FVB/N, C57BL/6J, NMRI 5e18, 24, 48e72, 24 Hepatic glycogen Y

128 Swiss white mice 12 Hepatic glucuronyl transferaseactivity

[

114, 128 NMRI, Swiss white mice 24, 12 Hepatic microsomal protein 4

114 NMRI 24 Hepatic NADPH-cytochromec-reductase activity

4

114 NMRI 24 Hepatic sorbitol dehydrogenase 4

110, 114, 117, 142 C57BL/6J, C57BL/6, 3 strains, NMRI 48e72, 4e16, 6e14, 24 Hepatic triglyceride [

135 CD1 3e18 Intestinal transit time of charcoal Y

110 C57BL/6J 48e72 Kidney triglyceride 4

114 NMRI 24 Liver/body weight 4

114 NMRI 24 Liver water content 4

114, 120 NA, NMRI 24e72, 24 Liver weight Y

120 NA 24e72 Liver weight/100 g body weightratio

Y

122 C57BL/6J 24e48 Mean arterial pressure Y

130 C57BL/6J 2e16 Mean cell haemoglobinconcentration

4

130 C57BL/6J 2e16 Mean cell volume h

130 C57BL/6J 2e16 Mean corpuscular haemoglobin 4

111 SV129 24 Metabolic rate Y

131 FVB/N 24 Muscle glycogen Y

117 C57BL/6 4e16 Muscle-specific glucose uptake [

117 C57BL/6 4e16 Skeletal muscle triglyceride 4

(Continued)NUTRITION591

HUSBANDRYANDMAINTENANCE

139 C57BL 48 Neuropeptide Y mRNA [

139 C57BL 48 Plasma adrenocorticotropichormone

[

130 C57BL/6J 2e16 Plasma amylase [

113 SV129/OlaHsd;C57BL/6J 9e24 Plasma cholesterol [

113, 130, 139, 144 C57BL, C57BL/6J, C57BL/6J, SV129/OlaHsd;C57BL/6J

48, 3e24, 2e16, 9e24 Plasma insulin Y

117 C57BL/6 4e16 Plasma ketone bodies [

110, 111 C57BL/6J, SV129 48e72, 24 Plasma lactate Y

110, 113, 130 C57BL/6J, C57BL/6J, SV129/olaHsd;C57BL/6J

2e16, 48e72, 9e24 Plasma triglyceride h

130, 131 C57BL/6J, FVB/N 2e16, 24 Plasma urea 4

130 C57BL/6J 2e16 Platelets [

130 C57BL/6J 2e16 Red blood cells [

139 C57BL 48 Serum luteinizing hormone 4

129 C57BL/6J 48 Serum resistin Y

131 FVB/N 24 Testis weight 4

117 C57BL/6 4e16 Total hepatic glucose production 4

102, 122, 130 MIL/ha/ICR, 7 strains, C57BL/6J 24e120, NA, 24e48 Water intake h

130 C57BL/6J 2e16 White blood cells h

117 C57BL/6 4e16 Whole body insulin sensitivityindex

[

The arrows indicate whether the parameters increase, decrease, do both or stay level during fasting: Y, decrease; [, increase; h, decrease and increase; 4, no change.Modified from [147].

TABLE 4.3.13: Effects of fastingdcont’d

References Strain Duration of fasting (h)Parametersmeasured

Directionof effect

HUSBANDRY AND MAINTENANCE

592 NUTRITION

NUTR

ITION

593

HUSB

ANDRY

ANDM

AINTEN

ANCE

to prevent unnecessary suffering. Mouse modelsof muscular dystrophy died at a very young age,as these mice were physically unable to reach forthe food in the feeding trough (unpublishedobservations). By providing food pellets at thebottom of the cages and a longer drinking nipple,the animals could survive until a much older agewithout any obvious clinical problems.

After surgery the mice are sometimes unableto stretch their bodies and/or heads towards thefeeding trough and drinking nipple. In this casea small feeding device with feeding pelletsmixed with water must be provided on thebottom of the cage, until the animals arecompletely recovered.

Currently, the enrichment of housing condi-tions receives a lot of attention. Much attentionis given to environmental factors, e.g. space, cagefurniture and nesting material. As animals spenda large proportion of their active time searchingfor food, modifying the way we feed our labora-tory animals has a large potential for enrichment.This has not yet been investigated to a largeextent, and it is the challenge of the coming yearsto investigate and use this potential. For rabbits itwas found that when feeding a restricted amountof food at a time point approaching the ‘normal’feeding hours, stereotyped behaviour was regis-tered as significantly decreased as comparedwith ad libitum or feeding restrictedly at anothertime point [148]. This may imply that whena restricted amount of food is fed at the ‘propertime’, better welfare, better health and more stan-dardized results can be obtained [75].

Mice are social species, so if single housing isa necessity for the experimental design, welfareconsiderations become necessary. According tothe Resolution of the Council of Europe [4] socialspecies must be housed socially, unless there aregood reasons to do otherwise. In certain casesmice may be housed individually at the time offeeding, and housed socially outside the feedingperiod. Another possibility is to divide the cagewith bars, so that the mice can be in close contactwithout being able to reach each other’s food.Human contact may also compensate to a certaindegree for a shortage in social contactwith conspe-cifics. During procedures, e.g. weighing theanimals, individually housed animals may betemporarily housed in a group. It goes withoutsaying that this needs effective individual

identification. The animals may regard the socialcontact provided during procedures as a positivereward, thereby making the handling and proce-dures easier.

References

[1] Tobin G, Stevens KA, Russell RJ. Nutrition.In: Fox JG, Barthold SW, Davisson MT,Newcomer CE, Quimby FW, Smith AL,editors. The Mouse in Biomedical Research.2nd ed., vol. III. London: Academic Press;2007. pp. 321–83.

[2] BeynenAC,CoatesME. In: vanZutphenLFM,BaumansV,BeynenAC, editors. Principles ofLaboratory Animal Science. Amsterdam:Elsevier; 2001. pp. 111–27.

[3] Keenan KP, Ballam GC, Haught DG,Laroque P. The Laboratory Rat. London:Academic Press; 2000. pp. 57–75.

[4] Council of Europe. Resolution on theAccommodation and Care of LaboratoryAnimals; 1997, 30 May.

[5] National Research Council. NutrientRequirements of Laboratory Animals. 4threvised ed. Washington, DC: NationalAcademy Press; 1995.

[6] National Research Council. NutrientRequirements of Laboratory Animals. 3rdrevised ed. Washington, DC: NationalAcademy Press; 1978.

[7] Van den Broek FAR, Beems RB, vanTintelen G, Lemmens AG, Fielmich-Bouwman AXM. Co-variance of chemicallyand histologically analyzed severity ofdystrophic cardiac calcification in mice.Lab Anim 1997;31:74–80.

[8] Van den Broek FAR, Bakker R, denBieman M, Fielmich-Bouwman AX,Lemmens AG, van Lith HA, et al. Geneticanalysis of dystrophic cardiac calcificationin DBA/2 mice. Biochem Biophys Res Co1998;253:204–8.

[9] Van den Broek FAR, Ritskes-Hoitinga J,Beynen AC. Influence of excessive fluorideconsumption on the severity of dystrophiccardiac calcification in DBA/2 mice. BiolTrace Elem Res 2000;78:191–203.

[10] Van den Broek FAR. Dystrophic CarciacCalcification in Laboratory Mice. Thesis.The Netherlands: Utrecht University;1998.

HUSB

ANDRY

ANDM

AINTE

NANCE

594

NUTR

ITIO

N

[11] National Research Council. Mineral Toler-ance of Domestic Animals. Washington, DC:National Academy of Sciences; 1980.

[12] National Research Council. Vitamin Toler-ance of Animals. Washington, DC: NationalAcademy Press; 1987.

[13] Ritskes-Hoitinga J, Beynen AC. Neph-rocalcinosis in the rat: a literature review.Prog Food Nutr Sci 1992;16:85–124.

[14] Zhou J, Moller J, Danielsen CC, Bentzon J,Ravn HB, Austin RC, et al. Dietary supple-mentation with methionine and homo-cysteine promotes early atherosclerosis butnot plaque rupture in ApoE-deficient mice.Arterioscler Thromb Vasc Biol 2001;21:1470–6.

[15] Fau D, Peret J, Hadjiisky P. Effects ofingestion of high protein or excess methi-onine diets by rats for two years. J Nutr1988;118:128–33.

[16] Klurfeld DM. Kidney and bladder stones inrodents fed purified diets. J Nutr 2002;132:3784.

[17] Newland MC, Reile PA, Sartin EA, Hart M,Craig-Schmidt MC, Mandel I, et al.Urolithiasis in rats consuming a dl bitar-trate form of choline in a purified diet.Comp Med 2005;55:354–67.

[18] British Association for Research QualityAssurance. Guidelines for the Manufactureand Supply of GLP Animal Diets. BARQA,3 Wherry Lane, Ipswich, UK: http://www.barqa.com; 1992.

[19] Environmental Protection Agency. ToxicSubstance Control Act (TSCA): Good Labo-ratory Practice Standards. Federal Register1983;48:53922–44.

[20] US Food and Drug Administration.Nonclinical laboratory studies. Good labo-ratory practice regulations. Federal Register1978;43:59985–60025.

[21] Organisation for Economic Co-operationand Development. Guideline for the testingof chemicals: Test Guideline 451, Carcino-genicity Studies. Paris: OECD; 2009.

[22] Peterson WA. FDA/GLP Regulations. In:Weinberg S, editor. Good laboratory prac-tice regulations. 3rd ed. New York: MarcelDekker; 2003. pp. 25–113.

[23] Environmental Protection Agency.Proposed health effects test standards forToxic Substances Control Act test rules.Good Laboratory Practice standards forhealth effects. Federal Register 44.1979;Part 2:27334–273375.

[24] Pal BC, Ross RH, Milman HA. Nutri-tional Requirements and ContaminantAnalysis of Laboratory Animal Feeds.Springfield, VA: National TechnicalInformation Service, PB No. EPA-560/6–83–005; 1984.

[25] Rao GN, Knapka JJ. Contaminant andnutrient concentrations of natural ingre-dient rat and mouse diet used in chemicaltoxicology studies. Fundam Appl Toxicol1987;9:329–38.

[26] US Food and Drug Administration (1981).Policies and Procedures for Murine Rodentdiets (NCTR), Arkansas: Jefferson; p. 12.

[27] Society of Laboratory Animal Science GV-SOLAS. Guidelines for the Nutrition ofLaboratory Animals. Berlin: German Societyfor Laboratory Animal Science; 2002. p. 17.

[28] Jensen MN, Ritskes-Hoitinga M. How iso-flavone levels in common rodent diets caninterfere with the value of animal modelsand with experimental results. Lab Anim2007;41:1–18.

[29] Ciana P, Brena A, Sparaciari P, Bonetti E, DiLorenzo D, Maggi A. Estrogenic activities inrodent estrogen-free diets. Endocrinology2005;146:5144–50.

[30] Weiss B, Stern S, Cernichiari E, Gelein R.Methylmercury contamination of labora-tory animal diets. Environ Health Perspect2005;113:1120–2.

[31] Thigpen JE, Setchell KDR, Alhmark KB,Locklear J, Spahr T, Caviness GF, et al.Phytoestrogen content of purified, open-and closed-formula laboratory animal diets.Lab Anim Sci 1999;49:530–6.

[32] Gent N. A study in refining husbandrytechniques for the in-house breeding ofrats and mice. Anim Tech Welfare April2006:3–8.

[33] Ritskes-Hoitinga J, Mathot JNJJ,Danse LHJC, Beynen AC. Commercialrodent diets and nephrocalcinosis inweanling female rats. Lab Anim1991;25:126–32.

[34] Tobin G. The role of diet in the three Rs.In: Cragg P, Stafford K, Love D,Sutherland G, editors. Lifting the Veil:Finding Common Ground. Christchurch,NZ: ANZCAART/Royal SocietyNew Zealand; 2003. pp. 145–62.

[35] Woods SC, Seeley RJ, Rushing PA,D’Alessio D, Tso P. A controlled high-fatdiet induces an obese syndrome in rats.J Nutr 2003;133:1081–7.

NUTR

ITION

595

HUSB

ANDRY

ANDM

AINTEN

ANCE

[36] Reeves PG. Components of the AIN-93diets as improvements in the AIN-76A diet.J Nutr 1997;127:838S–41S.

[37] Reeves Ph G, Nielsen FH, Fahey Jr GC.AIN-93 purified diets for laboratoryrodents: final report of the American Insti-tute of Nutrition ad hoc writing committeeon the reformulation of the AIN-76Arodent diet. J Nutr 1993;123:1939–51.

[38] Reeves PG, Rossow KL, Lindlauf J.Development and testing of the AIN-93purified diets for rodents: results ongrowth, kidney calcification and bonemineralization in rats and mice. J Nutr1993;123:1923–31.

[39] Zhou J, Moller J, Ritskes-Hoitinga M,Larsen ML, Austin RC, Falk E. Effects ofvitamin supplementation and hyper-homocysteinemia on atherosclerosis inApoE-deficient mice. Atherosclerosis 2003;168:255–62.

[40] Curfs J, Chwalibog A, Savenije B, Ristskes-Hoitinga M. Nutrient requirements,experimental design, and feeding sched-ules in animal experimentation. In: Hau J,Shapiro S, editors. Handbook of Labora-tory Animal Science. 3rd ed. Chapter 12.Boca Raton, FL: CRC Press; 2010.

[41] Wise A. Interaction of diet andtoxicitydthe future role of purified diet intoxicological research. Arch Toxicol1982;50:287–99.

[42] Rao GN. Rodent diets for carcinogenesisstudies. J Nutr 1988;118:929–31.

[43] Medinsky MA, Popp JA, Hamm TE,Dent JG. Development of hepatic lesions inmale Fischer-344 rats fed AIN-76A puri-fied diet. Toxicol Appl Pharmacol1982;62:111–20.

[44] Fullerton FR, Greenman DL, Bucci TJ.Effects of diet type on incidence of spon-taneous and 2-acetylaminofluorene-induced liver and bladder tumors inBALB/c mice fed AIN-76A diet versusNIH-07 diet. Fundam Appl Toxicol1992;18:193–9.

[45] Hamm TE, Raynor T, Caviston T. Unsuit-ability of the AIN-76 diet for male F344and CD rats and improvement bysubstituting starch for sucrose. Lab AnimSci 1982;32:414–5.

[46] Duffy PH, Lewis SM, Mayhugh MA,McCracken A, Thorn BT, Reeves PG,et al. Effect of the AIN-93M purifieddiet and dietary restriction on survival in

Sprague-Dawley rats: implications forchronic studies. J Nutr 2002;132:101–7.

[47] Duffy PH, Lewis SM, Mayhugh MA,Trotter RW, Hass BS, Thorn BT, et al.Nonneoplastic pathology in male Sprague-Dawley rats fed the American Institute ofNutrition-93M purified diet at ad libitumand dietary-restricted intakes. Nutr Res2008;28:179–89.

[48] Duffy PH, Lewis SM, Mayhugh MA,Trotter RW, Hass BS, Latendresse JR, et al.Neoplastic pathology in male Sprague-Dawley rats fed AIN-93M diet ad libitum orat restricted intakes. Nutr Res 2008;28:36–42.

[49] Fullerton FR, Greenman DL, Kendall DC.Effects of storage conditions on nutritionalqualities of semipurified (AIN-76) andnatural ingredient (NIH-07) diets. J Nutr1982;112:567–73.

[50] Ritskes-Hoitinga J, Verschuren PM,Meijer GW, Wiersma A, van de Kooij AJ,Timmer WG, et al. The association ofincreasing dietary concentrations of fish oilwith hepatotoxic effects and a higher degreeof aorta atherosclerosis in the ad lib.-fedrabbit. Food Chem Toxicol 1998;36:663–72.

[51] Lane-Petter W. The formulation of labo-ratory animal diets - some practicalconsiderations. In: Tavernor WD, editor.Nutrition, and Disease in ExperimentalAnimals. London: Bailliere, Tindall andCassell; 1970. pp. 64–71.

[52] Koopman JP, Scholten PM, Beynen AC.Hardness of diet pellets and growth of pre-weaned mice: separation of direct effectson the young and indirect effects mediatedby the lactating females. Z Versuchstierkd1989;32:257–60.

[53] Koopman JP, Scholten PM, Roeleveld PC,Velthuizen YW, Beynen AC. Hardness ofdiet pellets and its influence on growth ofpre-weaned and weaned mice. Z Versuch-stierkd 1989;32:71–5.

[54] Thomas M, van der Poel AFB. Physicalquality of pelleted animal feed. 1. Criteriafor pellet quality. Anim Feed Sci Tech1996;61:89–112.

[55] Thomas M, van Vliet T, van der Poel AFB.Physical quality of pelleted animal feed. 3.Contribution of feedstuff components.Anim Feed Sci Tech 1998;70:59–78.

[56] Thomas M, van Zuilichem DJ, van derPoel AFB. Physical quality of pelletedanimal feed. 2. Contribution of processes

HUSB

ANDRY

ANDM

AINTE

NANCE

596

NUTR

ITIO

N

and its conditions. Anim Feed Sci Tech1997;64:173–92.

[57] Thigpen JE, Locklear J, Romines C,Taylor KA, Yearby W, Stokes WS. A stan-dard procedure for measuring pellethardness of rodent diets. Lab Anim Sci1993;43:488–91.

[58] Elias PS, Cohen AJ. Recent Advances inFood Irradiation. Amsterdam: ElsevierBiomedical Press; 1983.

[59] Divakaran S, Decamp O, Cody JJ,Dominy WG. Studies on quality change inshrimp feeds exposed to gamma, E-beamand X-ray irradiation. J Aquacul Trop2002;17:155–65.

[60] Yamanaka M, Saito M, Yamauchi C,Nomura T. Influence of autoclaving ofdiets on digestibility of nutrients andnitrogen retention in conventional mice.Jikken Dobutsu 1973;22:5–14.

[61] Eggum BO. Effect of irradiation on proteinand amino acids in laboratory rodent diet.In: Decontamination of Animal Feeds byIrradiation. Vienna: International AtomicEnergy Agency; 1979. pp. 55–67.

[62] Ford DJ. The effect of methods of sterili-zation on the nutritive value of protein ina commercial rat diet. Br J Nutr1976;35:267–76.

[63] Ericksen WH, Hjarde W, Lieck H, Juul A,Emborg C. Comparison of the BiologicalEffects in Rats of Radiation Sterilized andAutoclave Sterilized Food. Copenhagen:Danish Atomic Energy CommissionResearch Establishment Riso; 1973. p. 16.

[64] Ford DJ. Observations on the influence ofirradiation on fat, and vitamin A in drylaboratory cat diets. In: Decontamination ofAnimal Feeds by Irradiation. Vienna:International Atomic Energy Agency; 1979.pp. 77–81.

[65] Takigawa A, Danbarra H, Ohyama Y.Gamma ray irradiation to semi-purifieddietdperoxide formation, and its effectson chicks. Jpn J Zootech Sci 1976;47:292–302.

[66] Greenberg SM, Frazer AC, Roberts B. Somefactors affecting the growth and develop-ment of rats fed rancid fat. J Nutr1953;50:421–40.

[67] Hart RW, Neumann DA, Robertson RT.Dietary Restriction: Implications for theDesign and Interpretation of Toxicity andCarcinogenicity Studies. Washington, DC:ILSI Press; 1995.

[68] Kasanen I. The Diet BoarddA NovelMethod of Dietary Restriction for Labora-tory Rats. PhD Thesis. University ofHelsinki, Finland. Helsinki, Finland:University Printing House, Helsinki, 2009.

[69] Keenan KP, Ballam GC, Soper KA,Laroque P, Coleman JB, Dixit R. Diet,caloric restriction, and the rodent bioassay.Toxicol Sci 1999;52(Suppl 2):24–34.

[70] Kasanen IH, Inhila KJ, Nevalainen JI,Vaisanen SB, Mertanen AM, Mering SM,et al. A novel dietary restriction method forgroup-housed rats: weight gain and clinicalchemistry characterization. Lab Anim2009;43:138–48.

[71] Nevalainen T, Leenaars M, Crljen V, Friis-Mikkelsen L, Dontas I, Savenije B, et al.Impact of handling, radiotelemetry, andfood restriction, In: The COST Manual ofLaboratory Animal Care, andUsedRefinement, Reduction, andResearch. Boca Raton, FL: CRC Press;2011.

[72] Roe FJC, Lee PN, Conybeare G, Kelly D,Matter B, Prentice D, et al. The BiosureStudy: influence of composition of diet,and food consumption on longevity,degenerative diseases, and neoplasia inWistar rats studied for up to 30 monthspost weaning. Fd Chem Toxic1995;33(S1):S1–100.

[73] Moraal M, Leenaars PPAM, Arnts H,Smeets K, Savenije BS, Curfs JHAJ, et al.The influence of food restriction versus adlibitum feeding of chow and purified dietson variation in body weight, growth andphysiology of female Wistar rats. LabAnim 2011. doi: 10.1258/la.2011.011011.

[74] Duffy PH, Feuers RJ, Hart RW. Effect ofchronic caloric restriction on the circadianregulation of physiological and behavioralvariables in old male B6C3 F1 mice. Chro-nobiol Int 1990;7:113–24.

[75] Ritskes-Hoitinga J, Strubbe J. Nutrition andanimal welfare. In: Kaliste E, editor. TheWelfare of Laboratory Animals. Dor-drecht: Kluwer Academic; 2004. pp. 51–80.

[76] Haseman JK. Value of historical controls inthe interpretation of rodent tumor data.Drug Info J 1992;26:191–200.

[77] Haseman JK, Bourbina J, Eustis SL. Effectof individual housing and other experi-mental design factors on tumor incidencein B6C3F1 mice. Fund Appl Toxicol1994;23:44–52.

NUTR

ITION

597

HUSB

ANDRY

ANDM

AINTEN

ANCE

[78] Claassen V. Neglected Factors in Pharma-cology, and Neuroscience Research.Amsterdam: Elsevier; 1994.

[79] Ritskes-Hoitinga J, Meijers M, Timmer WG,Wiersma A, Meijer GW, Weststrate JA.Effects of two dietary fat levels and fourdietary linoleic acid levels on mammarytumor development in Balb/C-MMTVmice under ad libitum feeding conditions.Nutr Cancer 1996;25:161–72.

[80] Russell WMS, Burch RL. The Principles ofHumane Experimental Technique.London: Methuen; 1959.

[81] Ritskes-Hoitinga J. Professorial Seminar.Animals and Models: Who Cares. Odense:University of Southern Denmark; 1998.

[82] Jacobsen B. Hvad er god forskning?.(in Danish). Copenhagen: Hans ReitzelsForlag; 2001.

[83] Ritskes-Hoitinga J, Meijers M, Meijer GW,Weststrate JA. The influence of dietarylinoleic acid on mammary tumour devel-opment in various animal models. ScandJ Lab Anim Sci 1996;23:463–9.

[84] De Wille JW, Waddell K, Steinmeyer C,Farmer ST. Dietary fat promotes mammarytumorigenesis in transgenic mice. CancerLett 1993;69:59–66.

[85] Fischer SM, Conti CJ, Locniskar M,Belury MA, Maldve RE, Lee ML, et al.The effect of dietary fat on the rapiddevelopment of mammary tumorsinduced by 7,12-dimethylbenz(a)anthra-cene in SENCAR mice. Cancer Res 1992;52:662–6.

[86] Craig-Schmidt M, White MT, Teer P,Johnson J, Lane HW. Menhaden, coconut,and corn oils and mammary tumor inci-dence in BALB/c virgin female micetreated with DMBA. Nutr Cancer1993;20:99–106.

[87] Brown RR. Effects of dietary fat on inci-dence of spontaneous and induced cancerin mice. Cancer Res 1981;41:3741–2.

[88] Moazed TC. Continuing EducationSeminar. The American Society of Labo-ratory Animal Practitioners; Cincinnati;USA. October 1998.

[88a] World Diabetes Foundation, www.worlddiabetesfoundation.org; 2009.

[88b] International Diabetes Federation. Dia-betes Atlas, 4th ed. http://www.idf.org/diabetesatlas/; 2009.

[89] Makino S, Kunimoto K, Muraoka Y,Mizushima Y, Katagiri K. Breeding of

a non-obese, diabetic strain of mice. JikkenDobutsu 1980;29:1–13.

[90] Kikutani H, Makino S. The murine auto-immune diabetes model: NOD and relatedstrains. Adv Immunol 1992;51:285–322.

[91] Bach JF. Insulin-dependent diabetes melli-tus as an autoimmune disease. Endocr Rev1994;15:516–42.

[92] Singh B, Rabinovitch A. Influence ofmicrobial agents on the development andprevention of autoimmune diabetes.Autoimmunity 1993;15:209–13.

[93] Mikkelsen LF, Hansen HN, Wulff EM,Haase C, Ottesen JL. The effect of envi-ronmental enrichment on the developmentof diabetes in NOD mice. CALAR MiniSymposium, Copenhagen; 2008.

[94] Anderson MS, Bluestone JA. The NODmouse: a model of immune dysregulation.Annu Rev Immunol 2005;23:447–85.

[95] Wang B, Gonzales A, Benoist C, Mathis D.The role of CD8þ T cells in the initiation ofinsulin-dependent diabetes mellitus. Eur JImmunol 1996;26:1762–9.

[96] Leiter EH, Prochazka M, Coleman DL. Thenon-obese diabetic (NOD) mouse. Am JPathol 1987;128:380–3.

[97] Batt RAL, Harrison GA. The reproductivesystem of the adipose mouse. J Hered1963;54:135–8.

[98] Bray GA, York DA. Hypothalamic andgenetic obesity in experimental animals: anautonomic and endocrine hypothesis. Phys-iol Rev 1979;59:719–809.

[99] Coleman DL. Obese and diabetes: twomutantgenes causing diabetes-obesity syndromes inmice. Diabetologia 1978;14:141–8.

[100] Herberg L, Coleman DL. Laboratoryanimals exhibiting obesity and diabetessyndromes. Metabolism 1977;26:59–98.

[101] Feliers D, Duraisamy S, Faulkner JL, Duch J,Lee AV, Abboud H, et al. Activation ofrenal signalling pathways in db/db micewith type 2 diabetes. Kidney Int2001;60:495–504.

[102] Chen H, Chalet O, Tartaglia LA, Woolf EA,Weng X, Ellis SJ, et al. Evidence that thediabetes gene encodes the leptin receptor:identification of a mutation in the leptinreceptor gene in db/db mice. Cell1996;84:491–5.

[103] Ayala JE, Samuel VT, Morton GJ, Obici S,Croniger CM, Shulman GI, et al. Standardoperating procedures for describing andperforming metabolic tests of glucose

HUSB

ANDRY

ANDM

AINTE

NANCE

598

NUTR

ITIO

N

homeostasis in mice. Dis Model Mech2010;3:525–34.

[104] Han BG, Hao CM, Tchekneva EE,Wang YY, Lee CA, Ebrahim B, et al.Markers of glycemic control in the mouse:comparisons of 6-h- and overnight-fastedblood glucoses to Hb A(1c). Am J PhysiolEndocrinol Metabol 2008;295:E981–6.

[105] Ayala JE, Bracy DP, McGuinness OP,Wasserman DH. Considerations in thedesign of hyperinsulinemic-euglycemicclamps in the conscious mouse. Diabetes2006;55:390–7.

[106] Gannon KS, Smith JC, Henderson R,Hendrick P. A system for studying themicrostructure of ingestive behavior inmice. Physiol Behav 1992;51:515–21.

[107] Kurokawa M, Akino K, Kanda K. A newapparatus for studying feeding anddrinking in the mouse. Physiol Behav2000;70:105–12.

[108] Geiser F. Metabolic rate and body temper-ature reduction during hibernation anddaily torpor. Ann Rev Physiol 2004;66:239–74.

[109] Bronson FH. Susceptibility of the fatreserves of mice to natural challenges.J Comp Physiol BdBiochem Syst EnvironPhysiol 1987;157:551–4.

[110] Hashimoto T, Cook WS, Qi C,Yeldandi AV, Reddy JK, Rao MS. Defect inperoxisome proliferator-activatedreceptor alpha-inducible fatty acid oxida-tion determines the severity of hepaticsteatosis in response to fasting. J Biol Chem2000;275:28918–28.

[111] Kersten S, Seydoux J, Peters JM,Gonzalez FJ, Desvergne B, Wrahli W.Peroxisome proliferator-activated receptoralpha mediates the adaptive response tofasting. J Clin Invest 1999;103:1489–98.

[112] Moitra J, Mason MM, Olive M, Krylov D,Gavrilova O, Marcus-Samuels B, et al. Lifewithout white fat: a transgenic mouse.Genes Devel 1998;12:3168–81.

[113] Oosterveer MH, Van Dijk TH, Grefhorst A,Bloks VW, Havinga R, Kuipers F, et al. Lxralpha deficiency hampers the hepaticadaptive response to fasting in mice. J BiolChem 2008;283:25437–45.

[114] Strubelt O, Dost-Kempf E, Siegers CP,Younes M, Völpel M, Preuss U, et al. Theinfluence of fasting on the susceptibility ofmice to hepatotoxic injury. Toxicol ApplPharmacol 1981;60:66–77.

[115] Hudson JW, Scott IM. Daily torpor in thelaboratory mouse, Mus Musculus var Albino.Physiol Zool 1979;52:205–18.

[116] Wankhade UD, Vella KR, Fox DL, Good DJ.Deletion of Nhlh2 results in a defectivetorpor response and reduced beta adren-ergic receptor expression in adipose tissue.PLoS ONE 2010;5:e12324.

[117] Webb GP, Jagot SA, Jakobson ME. Fasting-induced torpor in Mus musculus and itsimplications in the use of murine modelsfor human obesity studies. Comp BiochemPhysiol AdPhysiol 1982;72:211–9.

[118] Webb GP, Jagot SA, Rogers PD,Jakobson ME. Effects of fasting on ther-moregulation in normal and obese mice.IRCS Med ScidBiochem 1980;8:163–4.

[119] Hodge HC, Maclachlan PL, Bloor WR,Welch EA, Kornberg SL, Falkenheim M.Lipides of the fasting mouse. 3. Phospholi-pide to neutral fat relationship in the bloodlipids. J Biol Chem 1947;169:707–11.

[120] Furner RL, Feller DD. Influence of starva-tion upon hepatic drug metabolism in rats,mice, and guinea pigs. Proc Soc Exp BiolMed 1971;137:816–9.

[121] Sokolovic M, Wehkamp D, Sokolovic A,Vermeulen J, Gilhuijs-Pederson LA, vanHaaften RIM, et al. Fasting inducesa biphasic adaptive metabolic response inmurine small intestine. BMC Genomics2007;8:361.

[122] Williams TD, Chambers JB, Henderson RP,Rashotte ME, Overton JM. Cardiovascularresponses to caloric restriction and ther-moneutrality in C57BL/6J mice. Am JPhysiol – Regul Integr Comp Physiol2002;282:R1459–67.

[123] Osinski MA, Seifert TR, Cox BF,Gintant GA. An improved method ofevaluation of drug-evoked changes ingastric emptying in mice. J PharmacolToxicol Meth 2002;47:115–20.

[124] Pessacq MT, Mercer RG, Rebolledo OR,Gagliardino JJ. Effect of fasting on circa-dian rhythm of serum-insulin levels.Chronobiology 1976;3:20–6.

[125] Yeung CK, McCurrie JR, Wood D. A simplemethod to investigate the inhibitory effectsof drugs on gastric emptying in the mousein vivo. J Pharmacol Toxicol Meth 2001;45:235–40.

[126] Watanabe J,OkabeH, IchihashiT,MizojiriK,Yamada H, Yamamoto R. Gastric-emptyingrate constants after oral-administration of

NUTR

ITION

599

HUSB

ANDRY

ANDM

AINTEN

ANCE

drug solution tomice, rats, and rabbits. ChemPharmacol Bull 1977;25:2147–55.

[127] Asakawa A, Inui A, Kaga T, Yuzuriha H,Nagata T, Ueno N, et al. Ghrelin is anappetite-stimulatory signal from stomachwith structural resemblance to motilin.Gastroenterol 2001;120:337–45.

[128] Walker RM, Massey TE, McElligott TF,Racz WJ. Acetaminophen toxicity in fedand fasted mice. Can J Physiol Pharmacol1982;60:399–404.

[129] Steppan CM, Bailey ST, Bhat S, Brown EJ,Banerjee RR, Wright CM, et al. Thehormone resistin links obesity to diabetes.Nature 2001;409:307–12.

[130] Kutscher CL. Incidence of food-depriva-tion polydipsia in white Swiss mouse.Physiol Behav 1971;7:395–9.

[131] Kanizsai P, Garami A, Solymar M,Szolcsanyi J, Szelenyi Z. Energetics of fast-ing heterothermia in TRPV1-KO and wildtype mice. Physiol Behav 2009;96:149–54.

[132] RaynerDV, SimonE,Duncan JS, TrayhurnP.Hyperleptinaemia in mice induced byadministration of the tyrosine hydroxylaseinhibitor alpha-methyl-p-tyrosine. FEBS Lett1998;429:395–8.

[133] Swoap SJ, Gutilla MJ, Liles LC, Smith RO,Weinshenker D. The full expression offasting-induced torpor requires beta3-adrenergic receptor signaling. J Neurosci2006;26:241–5.

[134] Trayhurn P, Duncan JS, Hoggard N,Rayner DV. Regulation of leptin produc-tion: a dominant role for the sympatheticnervous system? Proc Nutr Soc1998;57:413–9.

[135] Prior H, Ewart L, Fantham E, Pimlott P,Cowell F, Valentin JP. Refinement of thecharcoal meal GI study: Reduction in thefasting period required. J Pharmacol Tox-icol Meth 2009;58:174.

[136] Warwick RO, Smith MP, Graffy MA,Lage GL. Altered distribution and toxicityof digitoxgenin in fasted mice. Life Sci1985;37:775–82.

[137] Brown JCL, Staples JF. Mitochondrialmetabolism during fasting-induced dailytorpor in mice. Biochim BiophysActadBioenerg 2010;1797:476–86.

[138] Williams TD, Chambers JB, Gagnon SP,Roberts LM, Henderson RP, Overton JM.Cardiovascular and metabolic responses tofasting and thermoneutrality in A(y) mice.Physiol Behav 2003;78:615–23.

[139] Ahima RS, Prabakaran D, Mantzoros C,Qu DQ, Lowell B, Maratos Flier E, et al.Role of leptin in the neuroendocrineresponse to fasting. Nature 1996;382:250–2.

[140] Champy MF, Selloum M, Piard L, Zeitler V,Caradec C, Chambon P, et al. Mouse func-tional genomics requires standardization ofmouse handling and housing conditions.Mamm Genome 2004;15:768–83.

[141] Gavrilova O, Leon LR, Marcus-Samuels B,Mason MM, Castle AL, Refetoff S,Vinson C, Reitman ML. Torpor in mice isinduced by both leptin-dependentand -independent mechanisms. Proc NatlAcad Sci U S A 1999;96:14623–8.

[142] Lin XB, Yue P, Chen ZJ, Schonfeld G.Hepatic triglyceride contents are geneti-cally determined in mice: results of a strainsurvey. Am J Physiol Gastroint LiverPhysiol 2005;288:G1179–89.

[143] Heijboer AC, Donga E, Voshol PJ, Dang ZC,Havekes LM, Romijn JA, et al. Sixteenhours of fasting differentially affectshepatic and muscle insulin sensitivity inmice. J Lipid Res 2005;46:582–8.

[144] Andrikopoulos S, Blair AR, Deluca N,Fam BC, Proietto J. Evaluating the glucosetolerance test in mice. Am J PhysiolEndocrinol Metab 2008;295:E1323–32.

[145] Breyer MD, Bottinger E, Brosius FC,Coffman TM, Harris RC, Heilig CW, et al.Mouse models of diabetic nephropathy. JAm Soc Nephrol. 2005;16:27–45.

[146] Kutscher CL. Strain differences indrinking in inbred mice during ad libitumfeeding and food deprivation. PhysiolBehav 1974;13:63–70.

[147] Jensen TL. Fasting of Mice. VeterinaryMaster’s Thesis. Faculty of Life Sciences;University of Copenhagen, Denmark: 2011.

[148] Krohn TC, Ritskes-Hoitinga J, Svendsen P.The effects of feeding and housing on thebehaviour of the laboratory rabbit. LabAnim 1999;33:101–7.