genetic and environmental effects on complex traits...

Copyright � 2006 by the Genetics Society of AmericaDOI: 10.1534/genetics.106.060004

Genetic and Environmental Effects on Complex Traits in Mice

William Valdar,*,1 Leah C. Solberg,† Dominique Gauguier,* William O. Cookson,*J. Nicholas P. Rawlins,‡ Richard Mott* and Jonathan Flint*

*Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom, †Medical College of Wisconsin, HMGC,Milwaukee, Wisconsin 53226 and ‡Department of Experimental Psychology, University of Oxford, Oxford, OX1 3UD, United Kingdom

Manuscript received April 26, 2006Accepted for publication July 23, 2006

ABSTRACT

The interaction between genotype and environment is recognized as an important source ofexperimental variation when complex traits are measured in the mouse, but the magnitude of thatinteraction has not often been measured. From a study of 2448 genetically heterogeneous mice, we reportthe heritability of 88 complex traits that include models of human disease (asthma, type 2 diabetesmellitus, obesity, and anxiety) as well as immunological, biochemical, and hematological phenotypes. Weshow that environmental and physiological covariates are involved in an unexpectedly large number ofsignificant interactions with genetic background. The 15 covariates we examined have a significant effecton behavioral and physiological tests, although they rarely explain .10% of the variation. We found thatinteraction effects are more frequent and larger than the main effects: half of the interactions explained.20% of the variance and in nine cases exceeded 50%. Our results indicate that assays of gene functionusing mouse models should take into account interactions between gene and environment.

IT is widely recognized that environmental variables,such as who carries out the experiment and when,

and physiological variables, such as sex and weight, areconfounds that need to be accounted for during thecollection of mouse phenotypes. Many articles attest tothe effect of these variables on phenotypic values (e.g.,Chesler et al. 2002a; Champy et al. 2004) and point outthe need for rigorous standardization of laboratorypractice (Henderson 1970; Crabbe et al. 1999; Brown

et al. 2005). It is also acknowledged that the size andeven direction of environmental effects on a phenotypecan vary with genotype, a phenomenon known as gene-by-environment interaction, and this has been docu-mented in studies of rodents over the past 50 years (e.g.,Cooper and Zubek 1958).

Following a report on the importance of laboratory-by-strain interaction (Crabbe et al. 1999), recent inter-est has focused on the prevalence and size of suchinteractions, as well as their ability to increase power ingenetic mapping experiments (Wang et al. 2006). Table1 summarizes the available data and shows that thepicture of how much genetic and environmental factorsinteract is piecemeal: our knowledge of the relative sizeof interaction and main effects is limited to a handful ofphenotype–covariate combinations.

During an investigation of the genetic basis of com-plex traits in 2448 genetically heterogeneous stock (HS)

mice (1220 female, 1228 male) (Solberg et al. 2006),we collected environmental and physiological covari-ates. The mice we used were descended from eightinbred strains (A/J, AKR/J, BALBc/J, CBA/J, C3H/HeJ, C57BL/6J, DBA/2J, and LP/J) (Demarest et al.2001), incorporating more genetic variation from asingle cross than has hitherto been assessed in mice.The generality of our findings is enhanced by our use ofa battery of tests that includes both behavioral and abroad range of physiological phenotypes (Solberg et al.2006), summarized in Table 2 (the names of all pheno-types are given in Table 3).

METHODS

Animals: Original Northport HS mice were obtainedfrom Robert Hitzemann at the Oregon Health SciencesUnit, Portland, Oregon. At the time the animals arrivedthey had passed 50 generations of pseudorandombreeding (Demarest et al. 2001). A breeding colony inopen cages was established at Oxford University togenerate animals for phenotyping. The animals’ pedi-gree comprising the parents and grandparents of thephenotyped animals was recorded.

Phenotypes and covariates: The phenotypes used inthis study and the protocol used to collect them are fullydescribed in Solberg et al. (2006) and summarized inTable 2. We collected 15 covariates (Table 4). Seven aremouse-specific covariates (short names quoted in brack-ets where needed): sex, age, cage identifier (i.e., a unitof shared environment), weight at 9 weeks (‘‘weight’’),number of animals in a cage (‘‘cage density’’), sibship

1Corresponding author: Wellcome Trust Centre for Human Genetics,Roosevelt Dr., Headington, Oxford OX3 7BN, United Kingdom.E-mail [email protected]

Genetics 174: 959–984 (October 2006)

(‘‘family’’), and which litter the mouse came from(‘‘litter’’; e.g., ‘‘3’’ means the animal came from hisparents’ third litter); three are test-specific covariates:experimenter, test order, and apparatus (if more thanone was used); and five covariates are for the time of theexperiment: year, season (the group of three months),month, hour (time rounded to the nearest hour), and‘‘study day,’’ defined as the number of days from start ofthe study on January 20, 2003.

In the analysis, we fitted statistical models for eachphenotype, first testing the significance of each covari-ate as a main effect and then its interaction with geneticbackground. Covariates were either treated as con-tinuous variables [age, cage density, litter, study day(continuous), weight] or encoded as categorical factorstaking discrete levels (apparatus, cage, experimenter,sex, hour, month, season, year, and family). Note thatalthough hour could have been treated as continuous,that would have allowed detection of only linear trends

between time and phenotype, whereas as a factor it canbe used to detect nonlinear relationships.

Statistical analysis: All analysis was carried out usingthe R statistical package (R Development Core Team

2004), along with the add-on packages lme4 (Pinheiro

and Bates 2000), MASS (Venables and Ripley 2002),and regress (Clifford and McCullagh 2005).

We applied normalizing transformations to each phe-notype, guided by the Box–Cox procedure (Venables

and Ripley 2002), and in most cases this comprised asimple exponentiation or log transform to correct skew-ness (see Table 5). Phenotypes with symmetrical buthighly long-tailed distributions were corrected with asimplified Blom transformation (Blom 1958), in whichthe value is replaced by the probit of its empirical dis-tribution function probability. Asymmetric highly skewedlong-tailed distributions best modeled as exponential orgamma distributions were excluded from the analysis, aswere categorical phenotypes and latency phenotypes

TABLE 1

Recent reports of gene-by-environment interactions in mouse

Covariate Phenotype

QTL (i.e., singlelocus) or

polygenic (e.g.,strain) effect

Main-effectvariancea (%)

Interaction-effectvariancea (%) Reference

Laboratory Elevated plus maze Polygenic 32.7b 21b Crabbe et al. (1999)Body weight Polygenic 20.4b 7.1b

Cocaine-induced activity Polygenic 5.3b 8.6b

Sex Body weight Polygenic 63.7b 7b

Open field test Polygenic — 4.5b

Diet Obesity QTL — — York et al. (1999)

Diet (food shortage) Amphetamine-inducedactivity

— — Cabib et al. (2000)

Maternal lactationalenvironment

Plasma glucose Polygenic — — Reifsnyder et al.(2000)

Experimenter Tail-withdrawal latency Polygenic 42 18 Chesler et al. (2002)SexTesting orderTime of day

Laboratory Locomotion Polygenic 11.9–28.4b 10.9–16.5b Wahlsten et al. (2003)Elevated plus maze Polygenic 25.2–30b 13–14.3b

Diet Agressiveness Polygenic — — Nyberg et al. (2004)

Diet Liver weight QTL — — Ehrich et al. (2005)Serum insulin QTL — —Fat pad QTL — —

Diet Liver weight Polygenic — — Biddinger et al. (2005)Leptin Polygenic — —Glucose tolerance test Polygenic — —

Laboratory Open field test Polygenic 0–20.3 0.1–8.7 Kafkafi et al. (2005)

Sex Gonadal fat mass QTL — — Wang et al. (2006)

a The proportion of variance attributable to the main or interaction effect of the covariate, with ‘‘—’’ representing cases wherethis figure was not reported.

b The proportion of variance is given as the partial v2-statistic.

960 W. Valdar et al.

that require survival analysis. After transformation, eachphenotype was trimmed by removing values more than3 standard deviations from the mean to moderate theeffects of outliers.

Modeling the heritability and the effect of common en-vironment: We used a variance-components approachto model the effect of genetic background. Here thegenetic effect on an animal’s phenotype is a value drawnfrom a normal distribution constrained such that thegenetic effects of different animals correlate with theirrelatedness. First we fitted a standard additive genetic,common environmental error, unique environmentalerror (ACE) model to obtain estimates of the propor-tion of phenotypic variance attributable to additivegenetic effects (i.e., the heritability) and to shared en-vironmental effects. Second, we used an approximationto the ACE model that could be extended to test for theeffect of individual environmental covariates.

We formulated the ACE model as follows. Let n be thetotal number of animals, ncage be the number of cages,m be the grand mean, yij be the phenotype of the ithanimal in the jth cage, aij be that animal’s additivegenetic random effect, xijðcÞ be its value for covariate c,bc be the fixed effect associated with covariate c, C be theset of fixed-effect covariates, dj be the random effect of

cage j, and eij be the random effect of uncorrelatedenvironmental noise. Then

yij ¼ m 1X

c2C

bcxijðcÞ1 aij 1 dj 1 eij ; ð1Þ

where, the n-vector e � N ð0;s2EIÞ, the ncage-vector d �

N ð0;s2cageIÞ, and the n-vector g � N ð0;s2

AAÞ, where A isthe n 3 n additive genetic relationship matrix (e.g., seeLynch and Walsh 1998) computed from the pedigree.We estimated the heritability of each phenotype, i.e., theproportion of variance attributable to additive geneticvariation, as h2 ¼ s2

A=s2y and the size of the common

environmental effect as s2cage=s2

y , where s2y is the phe-

notypic variance. The set of covariates chosen for C wassex, litter, and, for phenotypes not directly related tobody mass, weight. Fitting was done by restricted esti-mate maximum likelihood (REML), using the R pack-age regress.

Testing main effects of covariates: For each pheno-type we tested the significance of individual covariatesusing an approximation to the ACE model above. Weemployed a random family effect as a surrogate for thegenetic effect, replacing the random effect ai , specificto individual i, with a random effect fq , specific to familyq. As explained below, this substitution amounts to a

TABLE 2

Summary of phenotypes analyzed, number of animals, and mean age (in days) at which the animals were analyzed

Phenotype Description No. of animals Mean age (days)

Weight, 6 wk Body weight at the beginning of testing. 2516 42Immunology CD4, CD3, CD8, and B220 antibody staining. 1872 42OFT Open field arena: distance in the perimeter, the center, and total

distance in 5 min.2504 45

EPM Elevated plus maze: distance traveled, time spent, and entries intoclosed and open arms.

2452 46

FN Food hyponeophagia: time taken to sample a novel foodstuff(overnight food deprivation).

2474 47

Burrowing No. of pellets removed from burrow in 1.5 hr. 2455 48Activity Activity measured in a home cage in 30 min. 2445 48Startle Startle to a loud noise. 1948 52Context freezing Freezing to the context in which a tone is associated with a foot shock. 2070 55Cue freezing Freezing to a tone after association with a foot shock. 2110 56Plethysmography Animals sensitized by injection with ovalbumin inhale metacholine and

changes in lung function are measured by plethysmography(a model of asthma). Respiratory rate, tidal volume, minute volume,expiratory time, inspiratory time, and enhanced pause are recordedwith and without exposure to metacholine.

2304 63

IPGTT Glucose and insulin values taken at 0, 15, 30, and 75 min after i.p.glucose injection (a model of type 2 diabetes mellitus).

2334 68

Weight, 10 wk Body weight at the end of testing. 2319 70FBC Full blood count (hematocrit, Hb concentration, mean cellular volume,

mean cellular Hb concentration, white cell count, platelet count).1892 71

Tissue harvest Adrenal weight. 2309 71Wound healing Reduction in size of a 2-mm ear punch hole. 2273 71Biochemistry Albumin, alkaline phosphatase, alanine transaminase, aspartate

transaminase, calcium, chloride, creatinine, high-densitylipoprotein, low-density lipoprotein, phosphorous, sodium, totalcholesterol, total protein, triglycerides.

1890 71

Gene–Environment Effects in Mice 961

reparameterization that affects in a predictable fashiononly the estimated variance of random terms. Also,because we wish to examine the effects of individual en-vironmental covariates, we excluded a catch-all randomeffect for cage, which would otherwise be heavily con-founded with any individual environmental covariate.Using notation similar to that above, the model fortesting the significance of covariate c1 was

yiq ¼ m 1X

c2C

bTc xiqðcÞ1 bT

c1xikðc1Þ1 fq 1 eiq ; ð2Þ

where bc are the fixed effects associated with covariate c,xiqðcÞ is the component of the design matrix represent-ing the ith animal’s value for covariate c, bc1

and xiqðc1Þare defined similarly for c1, and fq is such that if thereare nF nuclear families then the nF-vector f � N ð0;s2

FIÞ.We measured the significance of the covariate c1 asthe improvement in fit conferred by covariate c1 aftercertain basic covariates (C) had already been included.The set C usually comprised sex and, for phenotypesnot directly related to body mass, weight. When c1 wasweight, C comprised only sex; when c1 was sex, C wasempty. The significance of the fixed effect c1 was as-sessed using an approximation to the sequential F-test

TABLE 3

Phenotypes assessed in the project

Test Measure

Open field arena Total activityFecal boli

Elevated plus maze Closed-arm distanceOpen-arm distanceClosed-arm timeOpen-arm timeClosed-arm entriesOpen-arm entries

New home-cageactivity

Total beam breaks (30 min)Total beam breaks (first 5 min)Total beam breaks (last 5 min)Fine movement

Context freezing Time freezing to context (sec)Cue conditioning Time freezing during cue (sec)

Time freezing after cue (sec)Fecal boli

Fear-potentiatedstartle

Startle responseChange in startle after training

Plethysmography Enhanced pause (baseline)Enhanced pause (metacholine)Expiratory time (baseline)Expiratory time (metacholine)Inspiratory time (baseline)Inspiratory time (metacholine)PenH differenceRespiratory rate (baseline)Respiratory rate (metacholine)Tidal minute volume (baseline)Tidal minute volume (metacholine)Tidal volume (baseline)Tidal volume (metacholine)

IPGTT AUC-G (mg/dl)AUC-IRI (ng/ml)AUC-IRI/AUC-GDG (mg/dl)DIRI (ng/ml)DIRI/DGGlucose 0 (mg/dl)Glucose 15 (mg/dl)Glucose 30 (mg/dl)Glucose 75 (mg/dl)Insulin 0 (ng/ml)Insulin 15 (ng/ml)Insulin 30 (ng/ml)Insulin 75 (ng/ml)Insulin slopeK (glucose slope)

Immunology %B2201

%CD31

%CD41

%CD41/CD31

%CD81

%CD81/CD31

%NK cellsHematology Hematocrit (%)

Hemoglobin (g/dl)Mean cellular volume (fl)Platelets (n/ml)

(continued)

TABLE 3

(Continued)

Test Measure

Red blood cell count (n/ml)White blood cell count (n/ml)Mean cellular Hb concentration (%)Red cell distribution widthMean corpuscular hemoglobin (pg)LymphocytesPlateletcrit (%)

Biochemistry Alkaline phosphatase (units/liter)Alanine transaminase (units/liter)Aspartate transaminase (units/liter)Albumin (g/liter)Calcium (mmol)Chloride (mmol)High-density lipoproteins (mmol)Low-density lipoproteins (mmol)Phosphorous (mmol)Sodium (mmol)Total cholesterol (mmol)Total protein (g/liter)Triglycerides (mmol)Urea (mmol)

Weight, length,and growth

Body lengthBody mass indexGrowth slopeWeight, 10 wkWeight, 6 wkWeight, 7 wkWeight, 8 wk

Adrenal weight Adrenal weight (g)Wound healing Ear hole area (from ear punch) (mm2)


based on the Wald test (Pinheiro and Bates 2000). Wefit all models by REML using the lmer function from theR package lme4 (Pinheiro and Bates 2000).

Testing interaction effects between covariates andfamily: We define the ‘‘interaction model’’ for thecovariate c1 and family by adding a term to the main-effects model in Equation 3 to allow each family to haveits own effect for that covariate. For factor covariates, theinteraction model included a random intercept nestedwithin family, i.e.,

yiqk ¼ m 1X

c2C

bTc xiqðcÞ1 bT

c1xiqkðc1Þ1 fq 1 uqk 1 eiqk

¼ m 1X

c2C

bTc xiqðcÞ1 bc1k 1 fq 1 uqk 1 eiqk ; ð3Þ

where bc1k is the fixed effect associated with category kof covariate c1, and uqk is the random effect for cate-gory k in family q, such that if there are nU uniquecombinations of category and family then the nU-vectoru � N ð0;s2

UIÞ. For continuous covariates, the interac-tion model included a random slope for c1 conditionedon family, i.e.,

yiq ¼ m 1X

c2C

bTc xiqðcÞ1 ðuq1 1 bc1

ÞTxiqðc1Þ1 fq 1 eiq

¼ m 1X

c2C

bTc xiqðcÞ1 ðuq 1 bc1

Þxiqðc1Þ1 fq 1 eiq ; ð4Þ

where bc1is the fixed coefficient of covariate c1, uq is the

random deviation from that coefficient in family q, andthe correlation between the random intercept f andslope u is unrestricted. We assessed the significance of

the interaction model (Equation 3 or Equation 4) by alikelihood-ratio test (LRT) with the correspondingmain-effects model. Note that by using the change inthe number of degrees of freedom to parameterize thechi-square distribution used for the LRT, our P-valuesfor interaction effects are slightly conservative (Self andLiang 1987). We used the Dunn–Sidak correction, anexact form of the Bonferroni correction (Sahai andAgeel 2000), to take account of the number of testsperformed. For N tests, the corrected 5% threshold islog P ¼ �log10ð1� ð1� 0:05Þ1=N Þ.

The magnitude of a covariate’s effect is defined as thepercentage of phenotypic variance it explains, esti-mated in the model used to test its significance. Forfixed effects, this is the percentage of the total sum ofsquares attributable to the effect in a sequential ANOVAtable after fitting the other covariates (known in someliterature as h2; Olejnik and Algina 2003). For randomeffects, it is the estimated variance of the effect ex-pressed as a percentage of the total phenotypic variance.Where the random effect is based on an interaction withfamily, we report the percentage variance as twice thatof the estimated amount, in accordance with the repa-rameterization formulas described below.

Our use of family as a surrogate for the genetic effectmeans we underestimate the effect size of interactionsby a factor of two. However, this difference is entirelysuperficial. Suppose the n animals are sorted in order oftheir nF nuclear families. When fitting the family ef-fect, the n-vector of random effects is distributed asf � N 0;s2

FF� �

, where the matrix F is block diagonalsuch that Fij is 1 if i and j are in the same sibship and 0

TABLE 4

Covariates used in the study

Covariate Encoding Description Summary

Age Integer Age in days Mean ¼ 61, SD ¼ 4, 31–85Apparatus Categorical Experimental unit used Groups ¼ 4, size ¼ 348–526Cage Categorical Cage in which animal was housed Groups ¼ 435–549, size ¼ 1–7Cage density Integer No. of animals in a cage Mean ¼ 4.7, SD ¼ 1.1, 2–7Experimenter Categorical Who performed the test Groups ¼ 2–12, size ¼ 7–457Family Categorical Sibship of animal Groups ¼ 160–180, size ¼ 1–52Hour Categorical Hour of the day test was performed Groups ¼ 1–11, size ¼ 1–2307Litter Integer No. litter the animal came from Mean ¼ 2.2, SD ¼ 1.3, 1–8Month Categorical Month test was performed Groups ¼ 12, size ¼ 32–314Season Categorical Season test was performed Groups ¼ 4, size ¼ 284–788Sex Categorical Sex of the animal Groups ¼ 2, size ¼ 806–1293Study day Integer Day into study that test was performed

(day 1 is Jan. 20, 2003)Mean ¼ 306, SD ¼ 160, 1–621

Test order Integer Order in which animal was tested that day Mean ¼ 2.8, SD ¼ 1.4, 1–7Weight Real no. Body weight (g) at 9 wk Mean ¼ 23.9, SD ¼ 4.2, 12–39.1Year Categorical Year of test Groups ¼ 2, size ¼ 711–1517

‘‘Encoding’’ refers to how a covariate was modeled statistically. For numerical covariates, the column headed ‘‘Summary’’ givesthe grand mean and standard deviation over all phenotypes, followed by the minimum and maximum values observed for anygiven phenotype. For categorical covariates Summary gives the number and size of categories seen for a typical phenotype. Forexample, for phenotypes in which the experimenter covariate was present, there were between 2 and 12 experimenters who eachrecorded data for between 7 and 457 mice.


TABLE 5

Transformations, heritabilities, and common environment effects for 88 phenotypes listed in order of heritability

Phenotype Transformation Category% variance due to additive genetic

variation (i.e., heritability)% variance due to

common environment

%CD81 x Physiological 88.91 11.09CD41/CD81 x�(1/3) Physiological 80.48 14.59Weight, 7 wk (g) x1/3 Physiological 79.36 20.64Weight, 6 wk (g) x1/3 Physiological 74.48 25.52%CD41/CD31 x2 Physiological 72.73 18.48Weight, 8 wk (g) x1/3 Physiological 71.99 18.29High density lipoproteins (mmol) x Physiological 69.11 17.01Alkaline phosphatase (units/liter)

ffiffiffixp

Physiological 62.83 20.47Weight, 10 wk (g) x1/3 Physiological 62.35 18.02%B2201

ffiffiffixp

Physiological 59.86 24.66Glucose 0 (mg/dl)

ffiffiffixp

Physiological 55.33 32.08Red cell distribution width x�2 Physiological 55.29 12.98Mean cellular Hb conc. (%) x Physiological 52.16 39.97%CD31 x2 Physiological 51.30 22.51Ear hole area (mm2)

ffiffiffixp

Physiological 51.02 14.46Mean cellular volume (fl) x Physiological 50.89 20.60Calcium (mmol) x Physiological 48.89 31.39Lymphocytes

ffiffiffixp

Physiological 48.29 17.85Mean corpuscular hemoglobin (pg) x Physiological 47.94 20.24Inspiratory time (metacholine) x�1 Physiological 44.96 10.81Chloride (mmol) x Physiological 44.78 38.43Open-arm distance x1/3 Behavioral 42.06 6.19%CD41 x Physiological 40.70 26.46Startle response x1/3 Behavioral 40.67 4.20White blood cell count (n/ml) log10(x 1 1) Physiological 40.65 23.15Sodium (mmol) x Physiological 39.34 37.83Closed-arm distance x Behavioral 38.81 7.95Open-arm entries

ffiffiffixp

Behavioral 38.57 5.46Open-arm time

ffiffiffixp

Behavioral 37.92 6.08Enhanced pause (baseline) log10(x) Physiological 37.81 26.70Total cholesterol (mmol) x Physiological 37.50 17.62Total beam breaks (30 min)

ffiffiffixp

Behavioral 37.17 11.14Respiratory rate (metacholine) log10(x) Physiological 36.13 12.81Expiratory time (metacholine) log10(x) Physiological 35.00 13.94Glucose 15 (mg/dl)

ffiffiffixp

Physiological 34.83 29.86Total activity x Behavioral 33.86 5.81Inspiratory time (baseline) x�2 Physiological 32.76 16.55Alanine transaminase (units/liter) log10(x 1 3) Physiological 32.20 29.18Respiratory rate (baseline) x Physiological 31.57 18.95Tidal volume (metacholine) x1/3 Physiological 30.95 21.69Low density lipoproteins (mmol) log10(x) Physiological 30.70 18.25Urea (mmol) log10(x 1 1) Physiological 30.59 21.54Growth slope x Physiological 30.52 37.39Time freezing during cue (sec) x Behavioral 30.51 0.00Expiratory time (baseline) log10(x) Physiological 29.52 21.37Fine movement x2 Behavioral 29.45 10.04Total beam breaks (first 5 min)

ffiffiffixp

Behavioral 29.27 12.26Enhanced pause (metacholine) log10(x) Physiological 27.30 28.26Adrenal weight log10(x) Physiological 27.00 36.09Closed-arm time

ffiffiffixp

Behavioral 26.65 7.47Tidal minute volume (metacholine) x1/3 Physiological 26.59 20.01Albumin (g/liter) x Physiological 26.42 22.42Insulin 30 (ng/ml) x1/4 Physiological 26.34 21.62Glucose 75 (mg/dl)

ffiffiffixp

Physiological 26.28 22.94Insulin 15 (ng/ml) log10(x) Physiological 25.85 22.24Time freezing to context (sec)

ffiffiffixp

Behavioral 25.23 12.09PenH difference x1/3 Physiological 25.20 28.85

(continued)


otherwise (note that parents are not included in theanalysis because phenotypes were collected only on theoffspring). The covariance matrix for all random effectsis therefore

V ¼ s2FF 1 s2

EFI; ð5Þ

where s2EF

is the environmental variance when usingfamily for the genetic effect. This models all animalswithin a sibship as if they were genetically identical andall sibships as nuclear. Treating sibships as nuclear isreasonable in our case since the sparsity of our additivegenetic relationship matrix means that A � S, whereSij ¼ 1 when i ¼ j, 0.5 when i and j are sibs, and 0otherwise, and we found empirically that in this data setthe likelihood ratios using the full pedigree A matrixwere very close to those obtained using the nuclearapproximation S. Using the approximation S for A, ourheritability models a covariance matrix

V ¼ s2AS 1 s2

EAI: ð6Þ

Substituting the equality S ¼ 0:5ðF 1 IÞ and equat-ing the coefficients of F and I, it follows that V ¼s2

A0:5� �

F 1 s2A0:5 1 s2

EA

� �I such that when estimated,

s2A ¼ 2s2

F, which agrees with our observed discrepancybetween family-effect size and heritability. Similarlys2

A0:5 1 s2EA¼ s2

EF. Thus the two models are reparame-

terizations of each other. When fitted, they have iden-tical likelihood ratios, and hence 2s2

F is an estimate ofthe true additive genetic variance.

Our estimates of the variance attributable to gene-by-environment effects also rely on the use of the familysurrogate. Applying a similar argument to that above wecan show that those variance estimates are also half whatthey would be if we used the S matrix. The variance ofthe interaction model for categorical covariates (Equa-tion 4) is

V ¼ s2FF 1 s2

MFMF 1 s2

EAI; ð7Þ

where s2MF

is the variance of the interaction and MF is itscorrelation matrix, which is simply F but with Fij ¼ 0

TABLE 5

(Continued)

Phenotype Transformation Category% variance due to additive genetic

variation (i.e., heritability)% variance due to

common environment

Platelets (n/ml) x Physiological 25.07 19.94DIRI/DG x1/4 Physiological 24.61 23.89Triglycerides (mmol) log10(x) Physiological 22.55 21.94AUC-IRI/AUC-G x1/4 Physiological 22.48 22.86Total beam breaks (last 5 min)

ffiffiffixp

Behavioral 22.39 7.58Glucose 30 (mg/dl)

ffiffiffixp

Physiological 22.18 27.00% NK cells x�(1/2) Physiological 21.88 30.24DG (mg/dl) x Physiological 21.82 24.96Body length (cm) x Physiological 21.34 19.92AUC-G (mg/dl) x Physiological 21.24 24.88DIRI (ng/ml) x1/3 Physiological 21.02 19.04Aspartate transaminase (units/liter) x�(1/2) Physiological 20.96 18.47AUC-IRI (ng/ml) x1/2 Physiological 19.24 18.87Closed-arm entries x Behavioral 19.20 7.00Tidal volume (baseline) x1/3 Physiological 18.56 25.07Insulin 0 (ng/ml) log10(x) Physiological 17.83 26.01Tidal minute volume (baseline) x1/3 Physiological 16.51 22.21Phosphorous (mmol) log10(x 1 1) Physiological 16.10 28.41Insulin slope x1/3 Physiological 15.21 6.49Red blood cell count (n/ml) x3 Physiological 15.14 18.00Hemoglobin (g/dl) x3 Physiological 15.12 17.83Time freezing after cue (sec) x Behavioral 13.81 0.00Change in startle after training Blom(x) Behavioral 13.61 4.48Fecal boli

ffiffiffiffiffiffiffiffiffiffiffix 1 1p

Behavioral 13.38 13.02Body mass index x Physiological 13.21 14.75Insulin 75 (ng/ml) x1/3 Physiological 13.11 26.72Plateletcrit (%)

ffiffiffixp

Physiological 12.91 20.24Hematocrit (%) x3 Physiological 10.86 18.98Fecal boli after cue

ffiffiffixp

Behavioral 9.91 6.97Total protein (g/liter) x2 Physiological 8.51 28.59K (glucose slope) x1/2 Physiological 7.60 10.28

Transformations use the following conventions: x¼ phenotype; log10, log to base 10; Blom, replace each point with the probit ofits relative cumulative frequency.


when animals i and j are in different categories. If wewere to use S (an approximation for A) in place of F wewould have

V ¼ s2AS 1 s2

MAMS 1 s2

EAI; ð8Þ

with s2MA

being the interaction between the categoricalcovariate and the additive genetic effect. However, sinceS ¼ 0:5ðF 1 IÞ and MS ¼ 0:5ðMF 1 IÞ, it follows thatV ¼ s2

A0:5� �

F 1 s2MA

0:5� �

MF 1 s2A0:5 1 s2

MA0:5 1 s2

EA

� �I

and therefore s2MA¼ 2s2

MF. For interactions between a

continuous covariate x and family (Equation 5) thevariance is

V ¼ s2FF 1 s2

MFZFZT 1 s2

EAI; ð9Þ

where Z ¼ diagðxÞ when x is the n-vector of x for the nanimals. If we were to use S-approximation for A thevariance would be

V ¼ s2AS 1 s2

MAZSZT 1 s2

EAI: ð10Þ

Substituting S ¼ 0:5ðF 1 IÞ as before, V ¼ s2A0:5

� �F 1

s2MA

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ZFZT 1 s2MA

0:5ZZT 1 s2A0:5 1 s2

EA

� �� I, which

implies s2MA¼ 2s2

MF. Hence, in all cases the estimated var-

iance of an additive genetic component is simply twicethat of the corresponding family component.

RESULTS

Of the 102 phenotypes available for analysis (Solberg

et al. 2006), 88 could be accommodated in our linearmixed modeling framework (see methods). We ob-tained data for 15 covariates (Table 4): age, apparatus(for those tests where multiple units were used), cage(a variable indicating animals that were housed in thesame cage), cage density (the number of animals in acage), experimenter, family (defined as the offspring oftwo parents), sex, hour, litter (a number represent-ing the birth order of each litter for a given sire anddam), month, season, study day, test order, weight, andyear. An average of 10.3 covariates were recorded perphenotype (since not all phenotype–covariate combi-nations were available), leading to an average of 69.4phenotypes measured per covariate. In total, we per-formed 1804 statistical tests. The significance of resultsis reported as the negative base 10 logarithm of theP-value (log P) of the relevant test. We took account ofmultiple testing by using the Dunn–Sidak correction,which for a ¼ 5% comparisonwise error rate yielded asignificance threshold of log P ¼ 4.55.

We assessed initially the importance of three physio-logical covariates (sex, weight, and age). We fitted thecovariates sequentially in the order sex, then weight,then age, so that, for instance, our reported significancefor weight refers to how much it improved the fit of amodel that already included sex. We included family inall models to ensure tested covariates were significantover and above genetic effects. Family, modeled as a

random effect, is highly correlated with heritability(correlation of 0.89) and so acts a surrogate for theeffect of additive genetic variation (see methods). Wereport estimates of heritability for all phenotypes inTable 5.

The effects of sex, weight, and age were relativelysmall (Figure 1b, ‘‘main effect’’ rows): sex effects ex-plained .10% of the variance for 14 phenotypes; inmore than half of the cases the effect was ,5%; weightaccounted for .10% of the variance for three pheno-types; all age effects were ,2% (see appendix).

We estimated the significances and effects of theremaining covariates by adding each to a model thatalready included family, sex, and weight. Significantmain effects of covariates were more common in phys-iological than behavioral phenotypes (33% of the timevs. 13%; see Table 6). Overall, 21 of the 258 significanteffects explained .10% of the variance; the five cases ofwhen a covariate explained .25% of the variance in-volved sex. Table 6 provides a summary for each covar-iate, splitting results by category of phenotype. Figure 1plots log P-values and the percentage of phenotypicvariance explained by significant covariates. Figure 2summarizes the variance explained by significant covar-iates for the 16 subcategories of phenotype.

We then extended our model to test for gene-by-covariate interactions, taking the main-effects modelsreported above and then assessing how much addinginteraction terms improved the fit. We found 389significant interaction effects. Figure 3 illustrates theinteraction between sex and family on the percentage ofB-cells (%B2201) among white blood cells. It shows thatthe effect of sex is often marked within families but itsdirection can vary between families. Similarly, Figure 4illustrates the interaction between family and season onmean adrenal weight measured at 10 weeks. It shows sea-sonal means (spring in green, summer in red, autumnin brown, and winter in blue) for 28 families. In 11families, adrenal glands are heaviest when harvested inwinter, whereas in 9 families they are heaviest in sum-mer. The seasonal effects are strong within but incon-sistent across families, reflecting the greater importanceof interaction over main effects.

The distribution of the 389 significant interactioneffects differed from that of the main effects (Figure 1and appendix). Remarkably, half of the effects couldexplain .20% of the variance. In nine cases the in-teraction could explain .50% of the variance. Thelargest numbers of interactions were with month (65significant effects), season (55), sex (53), litter (51), andcage density (40). There were only 13 significant in-teractions with experimenter.

Physiological phenotypes showed the largest num-ber of interactions with covariates (56% of interac-tions tested were significant; Table 7). Largest effectswere found on mean cellular hemoglobin concentra-tion, serum sodium and serum chloride concentrations,


and plethysmography measures. There were fewer in-teractions with behavioral phenotypes (5% of interac-tions tested were significant, amounting to 11 in total),although the effect sizes were much the same on average(mean of 18.1% for behavior compared with a mean of18.6% for physiology; see Figure 2).

DISCUSSION

We have carried out the first systematic analysis ofa range of covariates across multiple phenotypes(see appendix). We have estimated the heritability of88 phenotypes, assessed the impact of a number of

Figure 1.—Main effects and interactions. (a)The log P (i.e., the�log10 of the P-value) for mainand interaction effects of 12 covariates. Each boxshows significance scores for one covariate on allapplicable phenotypes. The shaded bar marksthe corrected 5% threshold for significance(log P¼ 4.55). For example, Apparatus has signif-icant main effects for a few phenotypes but signif-icant interactions for none, whereas Hour has fewsignificant main effects but has significant inter-action effect for a number of phenotypes. (b)The estimated percentage of variance significanteffects contributed to the phenotype. Note thatlog P ’s are capped at 20 for display purposes andthat results for test order, which had no significanteffects, are not shown.

TABLE 6

Summary of main effects

Physiological phenotypes Behavioral phenotypes

Covariate Median log PMean %variance SD

No. observed(significant/all)

Medianlog P

Mean %variance SD


Age 0.82 0.98 0.40 6/65 0.73 0.86 0.17 3/18Apparatus — — — — 31.59 7.80 3.47 4/5Cage density 1.01 0.68 0.34 9/70 0.84 — — 0/18Experimenter 2.04 3.30 2.44 7/25 2.50 1.80 0.56 6/20Hour 1.55 1.16 — 1/29 1.51 1.41 — 1/20Litter 0.97 0.90 0.31 9/70 0.88 — — 0/18Month 8.96 3.56 2.24 51/65 2.14 1.75 — 1/18Season 5.47 1.90 1.25 38/65 1.57 — — 0/18Sex 12.41 9.47 11.62 48/70 2.06 2.19 1.79 5/18Study day 1.00 2.03 1.60 15/65 1.27 0.73 — 1/18Test order 0.37 — — 0/25 0.72 — — 0/16Weight 2.92 3.06 3.89 27/65 2.06 0.90 0.23 6/18Year 1.87 1.30 0.71 19/65 1.61 0.85 — 1/18

Variances (means and standard deviations) refer only to effects that were significant at log P . 4.55.


environmental factors, and measured the size of gene-by-environment interactions. Our large data set pro-vides the most robust assessments to date of thesemeasures in both behavioral and physiological domains.

We found large interactions between gene and envi-ronment and report that the effects are not restricted tobehavioral phenotypes (see appendix). We do notbelieve this is an artifact of our analysis. Our calculationsof percentage variance for random interaction effectsand for fixed main effects are only roughly comparablewith each other (see methods) and the interaction ef-fects are subject to a slight upward bias. However, that isnot sufficient to account for the substantially highereffect of significant interactions (18.6%) compared withsignificant main effects (3.7%). Second, inhomogeneityof phenotype variance across families is also unlikelyto account for our findings since in many cases therank order of covariate effects differs between families(Ungerer et al. 2003) as illustrated in Figures 3 and 4.

We report the effects of covariates as the percentageof phenotypic variance they explain and in doing soprovide one assessment of how environmental covari-ates influence a phenotype. But the true nature of this

interaction is more complex. For example, the concen-tration of alanine transaminase is subject to gene-by-environment interactions of month, accounting for48.49% of the phenotypic variance, of season, account-ing for 45.51%, and of litter, accounting for 18.17%. Yetthese effects combine, with further covariates, to pro-duce 100%. How is this possible?

The correlational structure of our data complicatesan assessment of the relative importance of differentcovariates and interactions. The observed phenotypicvariance is the sum of the variances of the covariatesminus twice the covariances between the covariates.This means that two covariates could have individualeffects of 50% but a summed effect of 60% if they arepositively correlated (or one of ,50% if they are nega-tively correlated). An observed covariate effect, just likean observed QTL effect, therefore includes a portion ofthe effect of any element that correlates with it; an actualmonth effect will partly manifest as observed litter andseason effects and vice versa. A more comprehensiveanalysis would build a complete picture of each pheno-type in the context of a path diagram or structuralequation model that enumerated all relationships, both

Figure 2.—Main and interaction effects of co-variates on 88 phenotypes from 16 experimentaltests. The y-axis gives the percentage variance ex-plained by significant covariates; the x-axis liststhe test performed with the number of pheno-types measured from that test in parentheses.Physiological tests are listed first and behavioraltests second. Boxes show the median (centralline) and interquartile range (IQR; box perime-ter), whiskers indicate the furthest data point,1.58 IQRs from the median, and circles showoutliers.

Figure 3.—Interaction between sex and familyfor the immunological phenotype percentage ofB-cells among lymphocytes in 2056 mice. Foreach of 69 families (x-axis) we plot means (solidcircles) and standard errors (bars) of the pheno-type value for males (blue) and females (pink).The y-axis gives the phenotype as the square rootof the percentage of white blood cells presentingB220. The graph shows that sex can have a strongeffect within families but that the direction of theeffect varies between families (interaction log P¼10.7). For example, in families plotted on the left,males are enriched in the B-cell compared with fe-males, whereas for families on the right this sex ef-

fect is reversed. The graph also illustrates the marginal effects on the trait of family (differing overall heights; heritability¼ 59.9%)and sex (females higher overall; main effect log P ¼ 13.0).


raw effects and correlations, between actors (e.g., Lynch

and Walsh 1998).The importance of gene-by-environment interactions

has been emphasized in the analysis of mouse behaviorand largely ignored in studies of mouse physiology. Inthe light of this, we designed our phenotyping protocolto minimize the effects of covariates on behavioralmeasures. All such tests were automated, so that theexperimenter’s intervention was limited to placing ani-mals in the apparatus. This may explain why some co-variates, previously suspected to influence behavioralphenotypes, were found to make a small contribution tothe variance: time of day (hour) was a nonsignificant (orhardly significant with negligible effect) contributor toall measures including those that utilize explorationas a measure of anxiety (elevated plus maze, which hadobservations from 9 different hours of the day, andopen field, which had observations from 10), despitethe fact that exploratory activity has been reported tovary throughout the day (Aschoff 1981). The order in

which animals are tested is also considered to have animportant effect on behavior (Harro 1997), but wefound no evidence for this: its effect was nonsignificanton all phenotypes measured.

Physiological phenotypes were not so controlled.There are no automated ways of administering an intra-peritoneal glucose tolerance test, for example, and weobserved large experimenter effects on these tests. Thisraises the question as to whether some phenotypesare more susceptible to interaction effects than others.Differences in the assessment protocols cannot be theonly factor that accounts for the smaller number ofinteractions in behavioral tests. There are a number ofcovariates common to all phenotypes whose effectswe could not ameliorate: month, season, year, sex, andweight. All of these covariates impinge more on physi-ological than on behavioral phenotypes (Tables 6 and 7).

Importantly, we observed many significant and largegene-by-environment interactions in our analysis ofphysiological phenotypes. Biochemical measures showed

Figure 4.—Interaction between season andfamily for the physiological phenotype mean ad-renal weight in 696 mice. For each of 28 families(x-axis) we plot the seasonal means (solid circles)and standard errors (bars) of the phenotype foranimals phenotyped in winter (blue), spring(green), summer (red), and autumn (brown).The y-axis gives the phenotype as the logarithmto the base 10 of the mean weight in grams of ad-renal glands at 10 weeks old. The graph showsthat the effect of season is consistent within familybut can vary between families. For example, forthe rightmost family adrenal glands are lightestin animals tested in summer and heaviest in au-tumn. Yet the rank order of seasons varies consid-erably through the graph.

TABLE 7

Summary of interaction effects between covariates and family

Physiological phenotypes Behavioral phenotypes

CovariateMedian

log PMean %variance SD


Medianlog P

Mean %variance SD


Age 2.39 1.22 0.59 26/65 0.25 — — 0/18Apparatus — — — — 0.00 — — 0/5Cage density 5.05 10.80 4.45 40/70 0.37 — — 0/18Experimenter 4.28 26.41 6.89 11/25 2.35 16.65 1.62 2/20Hour 6.17 23.69 7.78 21/29 1.96 — — 0/20Litter 9.17 13.58 7.21 51/70 0.53 — — 0/18Month 11.84 29.94 11.63 60/65 3.87 18.49 3.50 5/18Season 8.04 29.24 11.66 52/65 2.25 18.94 3.92 3/18Sex 6.82 22.33 6.66 52/70 2.21 16.38 — 1/18Study day 2.86 0.03 0.02 22/65 0.39 — — 0/18Test order 0.21 — — 0/25 0.00 — — 0/16Weight 3.88 0.59 0.14 28/65 1.39 — — 0/18Year 2.09 39.77 15.72 15/65 0.69 — — 0/18

Variances (means and standard deviations) refer only to effects that were significant at log P . 4.55.


strong (.10% effect) gene-by-environment interactionswith month (in 14 of 16 biochemical phenotypes), sex(12), season (9), and litter (8). We saw a similar patternof strong seasonal and sex effects for hematology, im-munology, plethysmography (which also had a stronghour interaction), and the glucose tolerance test (whichalso had a strong experimenter interaction). This hasprofound implications for QTL studies.

QTL detection experiments suffer when covariatesare not adequately accommodated in the experimentaldesign and subsequent analysis. First, a QTL may owesome, or indeed all, of its significance to an environ-mental effect confounded with the allelic variant. Whena phenotype is strongly affected by who performed theexperiment, any nonfunctional variant that correlateswith the experimenter will manifest as a significant, butspurious, effect. The random nature of recombinationmeans that in any experimental cross a fully balanceddesign is impossible and so confounds of this type areineluctable. While the impact of covariates can be mini-mized by regressing out their effects prior to mapping(e.g., Valdar et al. 2006), this is highly conservative,since in the converse scenario, where experimenter actsas a surrogate variable for an actual QTL effect, the QTLwill be missed.

Second, an interaction between a QTL and an envi-ronmental covariate may conceal the effect of both,even when covariate and QTL are in the model. Forinstance, if mice with allele a fear experimenter Johnmore than experimenter Alice, but mice with allele Afear Alice more than John and all four conditions occurin about equal proportion, then neither experimenternor QTL will have an observed effect. To recover thegenetic effect in this case it is necessary to model theinteraction in the mapping procedure (e.g., Wang et al.2006).

Our analyses are limited by the relatively smallnumber of covariates that we collected. We have noinformation on temperature fluctuation and humiditylevels [shown to be important for behavioral tests ofnociception (Chesler et al. 2002a,b)], which mightexplain month and seasonal effects. We have no in-formation on noise levels that are significantly increasedduring working hours (Milligan et al. 1993). The pre-dominance of significant temporal covariates reflectsthe importance of many other unknown environmentalfactors whose effect is moderated through the animals’genotypes. Thus the dissection of complex phenotypesin the mouse will require far more sophisticated ob-servation and analysis of these interactions than hashitherto been attempted.

W.V. gratefully acknowledges receipt of an Access to ResearchInfrastructures fellowship under Orjan Carlborg, Uppsala University,Sweden, and additionally thanks Mike Neale, Tom Price, and PeterVisscher for helpful discussions. This work was funded by grantsfrom the Wellcome Trust and the European Union Framework 6Programme, contract no. LHSG-CT-2003-503265.

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Communicating editor: G. Gibson


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11.7

723

.24

Bio

chem

istr

yA

lkal

ine

ph

osp

hat

ase

(un

its/

lite

r)St

ud

yd

ay17

01—

—6.

200.

02B

ioch

emis

try

Alk

alin

ep

ho

sph

atas

e(u

nit

s/li

ter)

Year

1701

——

4.95

24.4

5B

ioch

emis

try

Asp

arta

tetr

ansa

min

ase

(un

its/

lite

r)L

itte

r19

33—

—9.

8310

.96

Bio

chem

istr

yA

spar

tate

tran

sam

inas

e(u

nit

s/li

ter)

Mo

nth

1629

9.03

3.02

8.89

25.0

9B

ioch

emis

try

Asp

arta

tetr

ansa

min

ase

(un

its/

lite

r)Se

aso

n16

295.

731.

3510

.02

27.8

7B

ioch

emis

try

Asp

arta

tetr

ansa

min

ase

(un

its/

lite

r)Se

x19

4227

.03

4.91

——

Bio

chem

istr

yA

spar

tate

tran

sam

inas

e(u

nit

s/li

ter)

Stu

dy

day

1629

25.5

25.

27—

—B

ioch

emis

try

Asp

arta

tetr

ansa

min

ase

(un

its/

lite

r)W

eigh

t19

4210

.20

1.72

——

Bio

chem

istr

yA

spar

tate

tran

sam

inas

e(u

nit

s/li

ter)

Year

1629

7.03

1.35

5.49

27.4

2

(con

tin

ued

)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

oty

pe

Co

vari

ate

No

.o

bse

rved

log

P%

vari

ance

exp

lain

edlo

gP

%va

rian

ceex

pla

ined

Bio

chem

istr

yC

alci

um

(mm

ol)

Age

1688

——

4.70

0.77

Bio

chem

istr

yC

alci

um

(mm

ol)

Cag

ed

ensi

ty20

04—

—4.

728.

84B

ioch

emis

try

Cal

ciu

m(m

mo

l)L

itte

r19

94—

—13

.69

15.8

9B

ioch

emis

try

Cal

ciu

m(m

mo

l)M

on

th16

8812

.22

3.66

14.9

535

.51

Bio

chem

istr

yC

alci

um

(mm

ol)

Seas

on

1688

6.95

1.59

13.6

038

.72

Bio

chem

istr

yC

alci

um

(mm

ol)

Sex

2004

11.4

81.

6515

.18

32.1

8B

ioch

emis

try

Cal

ciu

m(m

mo

l)St

ud

yd

ay16

8813

.11

2.55

10.2

80.

02B

ioch

emis

try

Cal

ciu

m(m

mo

l)W

eigh

t20

0410

.36

1.48

11.4

20.

83B

ioch

emis

try

Cal

ciu

m(m

mo

l)Ye

ar16

8811

.79

2.26

——

Bio

chem

istr

yC

hlo

rid

e(m

mo

l)A

ge17

44—

—13

.36

2.21

Bio

chem

istr

yC

hlo

rid

e(m

mo

l)C

age

den

sity

2068

——

7.82

11.1

0B

ioch

emis

try

Ch

lori

de

(mm

ol)

Lit

ter

2058

——

26.6

038

.71

Bio

chem

istr

yC

hlo

rid

e(m

mo

l)M

on

th17

447.

692.

3943

.88

69.0

6B

ioch

emis

try

Ch

lori

de

(mm

ol)

Seas

on

1744

——

30.2

870

.86

Bio

chem

istr

yC

hlo

rid

e(m

mo

l)Se

x20

6823

.74

3.32

20.7

435

.48

Bio

chem

istr

yC

hlo

rid

e(m

mo

l)St

ud

yd

ay17

448.

331.

4214

.12

0.02

Bio

chem

istr

yC

hlo

rid

e(m

mo

l)W

eigh

t20

6810

.63

1.40

14.0

40.

78B

ioch

emis

try

Hig

h-d

ensi

tyli

po

pro

tein

s(m

mo

l)A

ge16

12—

—5.

360.

45B

ioch

emis

try

Hig

h-d

ensi

tyli

po

pro

tein

s(m

mo

l)M

on

th16

12—

—5.

8612

.67

Bio

chem

istr

yH

igh

-den

sity

lip

op

rote

ins

(mm

ol)

Sex

1912

173.

5522

.74

14.9

621

.61

Bio

chem

istr

yH

igh

-den

sity

lip

op

rote

ins

(mm

ol)

Stu

dy

day

1612

4.58

0.49

——

Bio

chem

istr

yH

igh

-den

sity

lip

op

rote

ins

(mm

ol)

Wei

ght

1912

19.4

62.

0114

.86

0.71

Bio

chem

istr

yH

igh

-den

sity

lip

op

rote

ins

(mm

ol)

Year

1612

5.34

0.58

——

Bio

chem

istr

yL

ow

-den

sity

lip

op

rote

ins

(mm

ol)

Cag

ed

ensi

ty19

47—

—4.

778.

53B

ioch

emis

try

Lo

w-d

ensi

tyli

po

pro

tein

s(m

mo

l)M

on

th16

469.

203.

055.

5418

.53

Bio

chem

istr

yL

ow

-den

sity

lip

op

rote

ins

(mm

ol)

Sex

1947

13.4

42.

327.

7319

.19

Bio

chem

istr

yP

ho

sph

oro

us

(mm

ol)

Age

1495

6.27

1.41

——

Bio

chem

istr

yP

ho

sph

oro

us

(mm

ol)

Mo

nth

1495

——

12.7

740

.25

Bio

chem

istr

yP

ho

sph

oro

us

(mm

ol)

Seas

on

1495

——

5.77

26.5

4B

ioch

emis

try

Ph

osp

ho

rou

s(m

mo

l)Se

x17

83—

—5.

2124

.81

Bio

chem

istr

yP

ho

sph

oro

us

(mm

ol)

Stu

dy

day

1495

4.86

1.05

——

Bio

chem

istr

yP

ho

sph

oro

us

(mm

ol)

Year

1495

8.05

1.84

——

Bio

chem

istr

ySo

diu

m(m

mo

l)A

ge17

34—

—9.

721.

85B

ioch

emis

try

Sod

ium

(mm

ol)

Lit

ter

2048

——

28.5

433

.14

Bio

chem

istr

ySo

diu

m(m

mo

l)M

on

th17

346.

632.

1037

.62

62.0

2B

ioch

emis

try

Sod

ium

(mm

ol)

Seas

on

1734

——

21.6

250

.65

Bio

chem

istr

ySo

diu

m(m

mo

l)Se

x20

5834

.14

5.01

17.4

531

.96

Bio

chem

istr

ySo

diu

m(m

mo

l)St

ud

yd

ay17

347.

041.

1711

.85

0.02

Bio

chem

istr

ySo

diu

m(m

mo

l)W

eigh

t20

5814

.14

1.96

12.6

40.

76 (con

tin

ued

)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

oty

pe

Co

vari

ate

No

.o

bse

rved

log

P%

vari

ance

exp

lain

edlo

gP

%va

rian

ceex

pla

ined

Bio

chem

istr

ySo

diu

m(m

mo

l)Ye

ar17

347.

081.

17—

—B

ioch

emis

try

To

tal

cho

lest

ero

l(m

mo

l)M

on

th17

0415

.12

3.48

6.68

16.0

2B

ioch

emis

try

To

tal

cho

lest

ero

l(m

mo

l)Se

aso

n17

0411

.76

2.09

——

Bio

chem

istr

yT

ota

lch

ole

ster

ol

(mm

ol)

Sex

2018

97.5

915

.23

5.81

14.0

8B

ioch

emis

try

To

tal

cho

lest

ero

l(m

mo

l)W

eigh

t20

185.

430.

66—

—B

ioch

emis

try

To

tal

pro

tein

(g/

lite

r)C

age

den

sity

1882

——

5.86

10.4

0B

ioch

emis

try

To

tal

pro

tein

(g/

lite

r)M

on

th15

6516

.74

5.29

12.8

640

.36

Bio

chem

istr

yT

ota

lp

rote

in(g

/li

ter)

Seas

on

1565

12.9

13.

238.

3840

.76

Bio

chem

istr

yT

ota

lp

rote

in(g

/li

ter)

Sex

1882

12.4

92.

345.

7120

.71

Bio

chem

istr

yT

ota

lp

rote

in(g

/li

ter)

Stu

dy

day

1565

——

5.86

0.02

Bio

chem

istr

yT

ota

lp

rote

in(g

/li

ter)

Wei

ght

1882

8.64

1.57

——

Bio

chem

istr

yT

rigl

ycer

ides

(mm

ol)

Cag

ed

ensi

ty17

38—

—7.

3010

.48

Bio

chem

istr

yT

rigl

ycer

ides

(mm

ol)

Mo

nth

1448

——

6.31

20.7

9B

ioch

emis

try

Tri

glyc

erid

es(m

mo

l)Se

x17

3885

.67

16.3

4—

—B

ioch

emis

try

Ure

a(m

mo

l)C

age

den

sity

1992

——

8.79

13.8

8B

ioch

emis

try

Ure

a(m

mo

l)L

itte

r19

82—

—10

.54

11.8

6B

ioch

emis

try

Ure

a(m

mo

l)M

on

th16

736.

802.

448.

5824

.17

Bio

chem

istr

yU

rea

(mm

ol)

Seas

on

1673

5.68

1.35

——

Bio

chem

istr

yU

rea

(mm

ol)

Sex

1992

——

10.6

227

.54

Co

nte

xtfr

eezi

ng

Tim

efr

eezi

ng

toco

nte

xt(s

ec)

Ap

par

atu

s16

7118

.88

4.13

——

Co

nte

xtfr

eezi

ng

Tim

efr

eezi

ng

toco

nte

xt(s

ec)

Exp

erim

ente

r16

715.

221.

70—

—C

on

text

free

zin

gT

ime

free

zin

gto

con

text

(sec

)Se

x16

7124

.20

5.14

——

Co

nte

xtfr

eezi

ng

Tim

efr

eezi

ng

toco

nte

xt(s

ec)

Wei

ght

1671

6.04

1.14

——

Cu

eco

nd

itio

nin

gF

ecal

bo

liaf

ter

cue

Sex

1768

4.80

0.98

——

Cu

eco

nd

itio

nin

gT

ime

free

zin

gaf

ter

cue

(sec

)A

ge17

915.

081.

05—

—C

ue

con

dit

ion

ing

Tim

efr

eezi

ng

afte

rcu

e(s

ec)

Ap

par

atu

s16

6543

.68

10.9

8—

—C

ue

con

dit

ion

ing

Tim

efr

eezi

ng

du

rin

gcu

e(s

ec)

Ap

par

atu

s16

6546

.03

10.5

4—

—E

leva

ted

plu

sm

aze

Clo

sed

-arm

entr

ies

Exp

erim

ente

r22

294.

571.

44—

—E

leva

ted

plu

sm

aze

Clo

sed

-arm

entr

ies

Wei

ght

2229

5.68

0.89

——

Ele

vate

dp

lus

maz

eC

lose

d-a

rmti

me

Mo

nth

2221

——

4.98

15.6

1E

leva

ted

plu

sm

aze

Op

en-a

rmd

ista

nce

Exp

erim

ente

r22

617.

141.

71—

—E

leva

ted

plu

sm

aze

Op

en-a

rmd

ista

nce

Mo

nth

2260

——

7.52

17.0

6E

leva

ted

plu

sm

aze

Op

en-a

rmd

ista

nce

Wei

ght

2261

4.82

0.63

——

Ele

vate

dp

lus

maz

eO

pen

-arm

entr

ies

Wei

ght

2261

5.90

0.80

——

Ele

vate

dp

lus

maz

eO

pen

-arm

tim

eE

xper

imen

ter

2261

6.78

1.69

——

Ele

vate

dp

lus

maz

eO

pen

-arm

tim

eM

on

th22

60—

—7.

1817

.03

Ele

vate

dp

lus

maz

eO

pen

-arm

tim

eW

eigh

t22

615.

300.

72—

—F

ear

po

ten

tiat

edst

artl

eSt

artl

ere

spo

nse

Age

2005

5.40

0.82

——

Fea

rp

ote

nti

ated

star

tle

Star

tle

resp

on

seA

pp

arat

us

2005

31.5

95.

54—

— (con

tin

ued

)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

oty

pe

Co

vari

ate

No

.o

bse

rved

log

P%

vari

ance

exp

lain

edlo

gP

%va

rian

ceex

pla

ined

Fea

rp

ote

nti

ated

star

tle

Star

tle

resp

on

seSe

x20

0515

.69

2.65

——

Fea

rp

ote

nti

ated

star

tle

Star

tle

resp

on

seSt

ud

yd

ay20

054.

860.

73—

—F

ear

po

ten

tiat

edst

artl

eSt

artl

ere

spo

nse

Wei

ght

2005

7.53

1.19

——

Fea

rp

ote

nti

ated

star

tle

Star

tle

resp

on

seYe

ar20

055.

600.

85—

—G

luco

seto

lera

nce

test

AU

C-G

(mg/

dl)

Age

2130

——

5.36

0.94

Glu

cose

tole

ran

cete

stA

UC

-G(m

g/d

l)C

age

den

sity

2130

——

8.35

12.5

1G

luco

seto

lera

nce

test

AU

C-G

(mg/

dl)

Exp

erim

ente

r21

309.

452.

118.

7726

.37

Glu

cose

tole

ran

cete

stA

UC

-G(m

g/d

l)H

ou

r21

30—

—5.

0416

.56

Glu

cose

tole

ran

cete

stA

UC

-G(m

g/d

l)L

itte

r21

17—

—12

.48

11.9

6G

luco

seto

lera

nce

test

AU

C-G

(mg/

dl)

Mo

nth

2130

4.74

1.59

18.7

136

.65

Glu

cose

tole

ran

cete

stA

UC

-G(m

g/d

l)Se

aso

n21

305.

101.

017.

8025

.18

Glu

cose

tole

ran

cete

stA

UC

-G(m

g/d

l)St

ud

yd

ay21

30—

—5.

600.

01G

luco

seto

lera

nce

test

AU

C-G

(mg/

dl)

Wei

ght

2130

——

6.26

0.53

Glu

cose

tole

ran

cete

stA

UC

-IR

I(n

g/m

l)C

age

den

sity

2105

——

5.10

7.54

Glu

cose

tole

ran

cete

stA

UC

-IR

I(n

g/m

l)M

on

th21

05—

—10

.38

24.6

5G

luco

seto

lera

nce

test

AU

C-I

RI

(ng/

ml)

Seas

on

2105

——

4.63

16.3

5G

luco

seto

lera

nce

test

AU

C-I

RI

(ng/

ml)

Sex

2105

——

7.95

21.3

3G

luco

seto

lera

nce

test

AU

C-I

RI

(ng/

ml)

Wei

ght

2105

6.66

1.07

——

Glu

cose

tole

ran

cete

stA

UC

-IR

I/A

UC

-GC

age

den

sity

1982

——

6.51

10.9

2G

luco

seto

lera

nce

test

AU

C-I

RI/

AU

C-G

Lit

ter

1970

——

5.85

8.22

Glu

cose

tole

ran

cete

stA

UC

-IR

I/A

UC

-GM

on

th19

82—

—10

.08

26.8

5G

luco

seto

lera

nce

test

AU

C-I

RI/

AU

C-G

Seas

on

1982

——

5.08

19.6

5G

luco

seto

lera

nce

test

AU

C-I

RI/

AU

C-G

Sex

1982

——

6.70

19.9

6G

luco

seto

lera

nce

test

DG

(mg/

dl)

Age

2131

——

5.88

1.01

Glu

cose

tole

ran

cete

stD

G(m

g/d

l)C

age

den

sity

2131

——

8.53

12.8

2G

luco

seto

lera

nce

test

DG

(mg/

dl)

Exp

erim

ente

r21

3111

.18

2.43

9.80

28.1

5G

luco

seto

lera

nce

test

DG

(mg/

dl)

Ho

ur

2131

——

5.36

17.2

9G

luco

seto

lera

nce

test

DG

(mg/

dl)

Lit

ter

2118

——

12.7

512

.24

Glu

cose

tole

ran

cete

stD

G(m

g/d

l)M

on

th21

315.

151.

6818

.64

36.7

2G

luco

seto

lera

nce

test

DG

(mg/

dl)

Seas

on

2131

5.79

1.13

7.61

25.0

2G

luco

seto

lera

nce

test

DG

(mg/

dl)

Stu

dy

day

2131

——

5.87

0.01

Glu

cose

tole

ran

cete

stD

G(m

g/d

l)W

eigh

t21

31—

—6.

620.

56G

luco

seto

lera

nce

test

DIR

I(n

g/m

l)C

age

den

sity

2107

——

5.14

7.64

Glu

cose

tole

ran

cete

stD

IRI

(ng/

ml)

Lit

ter

2095

——

5.02

6.49

Glu

cose

tole

ran

cete

stD

IRI

(ng/

ml)

Mo

nth

2107

——

9.93

24.0

2G

luco

seto

lera

nce

test

DIR

I(n

g/m

l)Se

aso

n21

07—

—4.

8517

.02

Glu

cose

tole

ran

cete

stD

IRI

(ng/

ml)

Sex

2107

——

7.48

20.0

5G

luco

seto

lera

nce

test

DIR

I(n

g/m

l)W

eigh

t21

076.

661.

06—

—G

luco

seto

lera

nce

test

DIR

I/D

GC

age

den

sity

1984

——

6.53

10.8

5

(con

tin

ued

)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

oty

pe

Co

vari

ate

No

.o

bse

rved

log

P%

vari

ance

exp

lain

edlo

gP

%va

rian

ceex

pla

ined

Glu

cose

tole

ran

cete

stD

IRI/

DG

Lit

ter

1972

——

6.95

9.39

Glu

cose

tole

ran

cete

stD

IRI/

DG

Mo

nth

1984

4.86

1.71

11.1

728

.02

Glu

cose

tole

ran

cete

stD

IRI/

DG

Seas

on

1984

——

6.05

21.7

3G

luco

seto

lera

nce

test

DIR

I/D

GSe

x19

84—

—6.

7419

.65

Glu

cose

tole

ran

cete

stG

luco

se0

(mg/

dl)

Age

2225

4.66

0.43

22.8

11.

50G

luco

seto

lera

nce

test

Glu

cose

0(m

g/d

l)C

age

den

sity

2225

4.71

0.43

18.0

012

.92

Glu

cose

tole

ran

cete

stG

luco

se0

(mg/

dl)

Exp

erim

ente

r22

2523

.96

2.95

24.0

434

.21

Glu

cose

tole

ran

cete

stG

luco

se0

(mg/

dl)

Ho

ur

2225

——

12.9

522

.62

Glu

cose

tole

ran

cete

stG

luco

se0

(mg/

dl)

Lit

ter

2212

13.6

31.

3819

.37

19.7

1G

luco

seto

lera

nce

test

Glu

cose

0(m

g/d

l)M

on

th22

258.

091.

4246

.92

41.4

1G

luco

seto

lera

nce

test

Glu

cose

0(m

g/d

l)Se

aso

n22

255.

890.

7226

.62

39.0

4G

luco

seto

lera

nce

test

Glu

cose

0(m

g/d

l)Se

x22

2515

8.57

20.4

216

.37

24.8

2G

luco

seto

lera

nce

test

Glu

cose

0(m

g/d

l)St

ud

yd

ay22

2514

.64

1.51

24.4

90.

01G

luco

seto

lera

nce

test

Glu

cose

0(m

g/d

l)W

eigh

t22

25—

—15

.79

0.72

Glu

cose

tole

ran

cete

stG

luco

se0

(mg/

dl)

Year

2225

28.2

93.

0910

.67

30.4

0G

luco

seto

lera

nce

test

Glu

cose

15(m

g/d

l)A

ge22

04—

—11

.08

1.30

Glu

cose

tole

ran

cete

stG

luco

se15

(mg/

dl)

Cag

ed

ensi

ty22

04—

—15

.31

18.0

4G

luco

seto

lera

nce

test

Glu

cose

15(m

g/d

l)E

xper

imen

ter

2204

53.7

08.

4316

.69

30.8

2G

luco

seto

lera

nce

test

Glu

cose

15(m

g/d

l)H

ou

r22

045.

291.

166.

0219

.58

Glu

cose

tole

ran

cete

stG

luco

se15

(mg/

dl)

Lit

ter

2192

8.02

1.12

9.18

15.0

6G

luco

seto

lera

nce

test

Glu

cose

15(m

g/d

l)M

on

th22

044.

721.

4031

.77

44.5

5G

luco

seto

lera

nce

test

Glu

cose

15(m

g/d

l)Se

aso

n22

045.

540.

9619

.27

40.9

0G

luco

seto

lera

nce

test

Glu

cose

15(m

g/d

l)Se

x22

0415

.01

2.21

7.35

21.4

0G

luco

seto

lera

nce

test

Glu

cose

15(m

g/d

l)St

ud

yd

ay22

044.

790.

6318

.57

0.01

Glu

cose

tole

ran

cete

stG

luco

se15

(mg/

dl)

Wei

ght

2204

——

5.56

0.55

Glu

cose

tole

ran

cete

stG

luco

se15

(mg/

dl)

Year

2204

12.4

91.

825.

7436

.94

Glu

cose

tole

ran

cete

stG

luco

se30

(mg/

dl)

Age

2187

——

6.94

0.94

Glu

cose

tole

ran

cete

stG

luco

se30

(mg/

dl)

Cag

ed

ensi

ty21

87—

—11

.78

16.1

4G

luco

seto

lera

nce

test

Glu

cose

30(m

g/d

l)E

xper

imen

ter

2187

21.2

34.

0411

.68

29.4

1G

luco

seto

lera

nce

test

Glu

cose

30(m

g/d

l)H

ou

r21

87—

—5.

5920

.08

Glu

cose

tole

ran

cete

stG

luco

se30

(mg/

dl)

Lit

ter

2174

4.68

0.67

8.05

11.0

2G

luco

seto

lera

nce

test

Glu

cose

30(m

g/d

l)M

on

th21

875.

031.

5919

.07

34.3

5G

luco

seto

lera

nce

test

Glu

cose

30(m

g/d

l)Se

aso

n21

875.

721.

0811

.18

31.2

7G

luco

seto

lera

nce

test

Glu

cose

30(m

g/d

l)Se

x21

879.

871.

534.

8418

.00

Glu

cose

tole

ran

cete

stG

luco

se30

(mg/

dl)

Stu

dy

day

2187

——

10.3

30.

01G

luco

seto

lera

nce

test

Glu

cose

30(m

g/d

l)W

eigh

t21

87—

—5.

300.

63G

luco

seto

lera

nce

test

Glu

cose

30(m

g/d

l)Ye

ar21

87—

—4.

7935

.33

Glu

cose

tole

ran

cete

stG

luco

se75

(mg/

dl)

Age

2153

——

7.87

1.02

Glu

cose

tole

ran

cete

stG

luco

se75

(mg/

dl)

Cag

ed

ensi

ty21

53—

—4.

997.

52 (con

tin

ued

)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

oty

pe

Co

vari

ate

No

.o

bse

rved

log

P%

vari

ance

exp

lain

edlo

gP

%va

rian

ceex

pla

ined

Glu

cose

tole

ran

cete

stG

luco

se75

(mg/

dl)

Exp

erim

ente

r21

5310

.63

2.14

17.7

437

.77

Glu

cose

tole

ran

cete

stG

luco

se75

(mg/

dl)

Ho

ur

2153

——

7.83

20.6

7G

luco

seto

lera

nce

test

Glu

cose

75(m

g/d

l)L

itte

r21

40—

—10

.28

10.0

5G

luco

seto

lera

nce

test

Glu

cose

75(m

g/d

l)M

on

th21

535.

901.

7018

.07

32.9

7G

luco

seto

lera

nce

test

Glu

cose

75(m

g/d

l)Se

aso

n21

537.

511.

337.

8323

.33

Glu

cose

tole

ran

cete

stG

luco

se75

(mg/

dl)

Sex

2153

34.8

75.

675.

2415

.93

Glu

cose

tole

ran

cete

stG

luco

se75

(mg/

dl)

Stu

dy

day

2153

——

6.82

0.01

Glu

cose

tole

ran

cete

stG

luco

se75

(mg/

dl)

Wei

ght

2153

——

9.78

0.69

Glu

cose

tole

ran

cete

stIn

suli

n0

(ng/

ml)

Cag

ed

ensi

ty22

06—

—11

.14

14.5

2G

luco

seto

lera

nce

test

Insu

lin

0(n

g/m

l)E

xper

imen

ter

2206

——

12.1

928

.49

Glu

cose

tole

ran

cete

stIn

suli

n0

(ng/

ml)

Ho

ur

2206

——

11.0

232

.11

Glu

cose

tole

ran

cete

stIn

suli

n0

(ng/

ml)

Lit

ter

2193

——

9.26

15.0

6G

luco

seto

lera

nce

test

Insu

lin

0(n

g/m

l)M

on

th22

0611

.67

2.91

26.5

642

.42

Glu

cose

tole

ran

cete

stIn

suli

n0

(ng/

ml)

Seas

on

2206

7.28

1.37

15.2

834

.80

Glu

cose

tole

ran

cete

stIn

suli

n0

(ng/

ml)

Sex

2206

12.7

82.

059.

0624

.42

Glu

cose

tole

ran

cete

stIn

suli

n0

(ng/

ml)

Wei

ght

2206

5.08

0.74

——

Glu

cose

tole

ran

cete

stIn

suli

n15

(ng/

ml)

Cag

ed

ensi

ty21

97—

—6.

4410

.52

Glu

cose

tole

ran

cete

stIn

suli

n15

(ng/

ml)

Exp

erim

ente

r21

97—

—7.

7320

.84

Glu

cose

tole

ran

cete

stIn

suli

n15

(ng/

ml)

Lit

ter

2185

——

9.16

10.5

3G

luco

seto

lera

nce

test

Insu

lin

15(n

g/m

l)M

on

th21

978.

962.

3713

.45

27.7

8G

luco

seto

lera

nce

test

Insu

lin

15(n

g/m

l)Se

aso

n21

975.

951.

126.

0320

.13

Glu

cose

tole

ran

cete

stIn

suli

n15

(ng/

ml)

Sex

2197

——

7.91

20.7

3G

luco

seto

lera

nce

test

Insu

lin

15(n

g/m

l)W

eigh

t21

974.

960.

725.

710.

57G

luco

seto

lera

nce

test

Insu

lin

30(n

g/m

l)C

age

den

sity

2178

——

6.23

9.10

Glu

cose

tole

ran

cete

stIn

suli

n30

(ng/

ml)

Exp

erim

ente

r21

78—

—5.

5116

.19

Glu

cose

tole

ran

cete

stIn

suli

n30

(ng/

ml)

Lit

ter

2166

——

7.56

9.10

Glu

cose

tole

ran

cete

stIn

suli

n30

(ng/

ml)

Mo

nth

2178

7.05

2.03

14.1

829

.40

Glu

cose

tole

ran

cete

stIn

suli

n30

(ng/

ml)

Seas

on

2178

——

8.50

24.7

1G

luco

seto

lera

nce

test

Insu

lin

30(n

g/m

l)Se

x21

78—

—7.

2318

.84

Glu

cose

tole

ran

cete

stIn

suli

n30

(ng/

ml)

Wei

ght

2178

4.62

0.67

——

Glu

cose

tole

ran

cete

stIn

suli

n75

(ng/

ml)

Cag

ed

ensi

ty21

124.

880.

707.

159.

48G

luco

seto

lera

nce

test

Insu

lin

75(n

g/m

l)E

xper

imen

ter

2112

——

5.65

17.0

4G

luco

seto

lera

nce

test

Insu

lin

75(n

g/m

l)H

ou

r21

12—

—8.

5522

.87

Glu

cose

tole

ran

cete

stIn

suli

n75

(ng/

ml)

Lit

ter

2100

——

6.11

12.6

0G

luco

seto

lera

nce

test

Insu

lin

75(n

g/m

l)M

on

th21

126.

861.

9616

.98

30.6

6G

luco

seto

lera

nce

test

Insu

lin

75(n

g/m

l)Se

aso

n21

12—

—6.

9121

.29

Glu

cose

tole

ran

cete

stIn

suli

n75

(ng/

ml)

Sex

2112

30.7

65.

187.

8518

.15

Glu

cose

tole

ran

cete

stIn

suli

n75

(ng/

ml)

Wei

ght

2112

11.8

51.

87—

—G

luco

seto

lera

nce

test

Insu

lin

slo

pe

Sex

1122

5.58

1.83

—— (c

onti

nu

ed)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

oty

pe

Co

vari

ate

No

.o

bse

rved

log

P%

vari

ance

exp

lain

edlo

gP

%va

rian

ceex

pla

ined

Glu

cose

tole

ran

cete

stK

(glu

cose

slo

pe)

Sex

1953

11.1

92.

22—

—G

luco

seto

lera

nce

test

K(g

luco

sesl

op

e)Ye

ar19

534.

830.

87—

—G

row

thG

row

thsl

op

eC

age

den

sity

2418

——

20.0

015

.53

Gro

wth

Gro

wth

slo

pe

Lit

ter

2462

——

26.0

520

.10

Gro

wth

Gro

wth

slo

pe

Sex

2474

135.

7217

.45

19.1

426

.19

Hem

ato

logy

Hem

ogl

ob

in(g

/d

l)M

on

th18

70—

—8.

5923

.95

Hem

ato

logy

Hem

ogl

ob

in(g

/d

l)Se

aso

n18

70—

—5.

0618

.96

Hem

ato

logy

Hem

ogl

ob

in(g

/d

l)Se

x18

709.

071.

73—

—H

emat

olo

gyH

emo

glo

bin

(g/

dl)

Wei

ght

1870

6.22

1.14

4.86

0.40

Hem

ato

logy

Lym

ph

ocy

tes

Age

1833

4.58

0.70

——

Hem

ato

logy

Lym

ph

ocy

tes

Lit

ter

1822

7.18

1.15

7.79

8.82

Hem

ato

logy

Lym

ph

ocy

tes

Mo

nth

1833

——

10.7

225

.40

Hem

ato

logy

Lym

ph

ocy

tes

Seas

on

1833

——

10.2

425

.19

Hem

ato

logy

Lym

ph

ocy

tes

Sex

1833

——

5.90

18.7

1H

emat

olo

gyL

ymp

ho

cyte

sSt

ud

yd

ay18

3314

.16

2.40

——

Hem

ato

logy

Lym

ph

ocy

tes

Year

1833

6.04

0.96

——

Hem

ato

logy

Mea

nce

llu

lar

Hb

con

cen

trat

ion

(%)

Age

1863

9.85

1.40

26.0

41.

83H

emat

olo

gyM

ean

cell

ula

rH

bco

nce

ntr

atio

n(%

)C

age

den

sity

1862

——

33.5

229

.32

Hem

ato

logy

Mea

nce

llu

lar

Hb

con

cen

trat

ion

(%)

Lit

ter

1852

——

39.3

135

.19

Hem

ato

logy

Mea

nce

llu

lar

Hb

con

cen

trat

ion

(%)

Mo

nth

1863

68.9

211

.39

58.1

954

.51

Hem

ato

logy

Mea

nce

llu

lar

Hb

con

cen

trat

ion

(%)

Seas

on

1863

28.1

44.

5440

.00

58.5

8H

emat

olo

gyM

ean

cell

ula

rH

bco

nce

ntr

atio

n(%

)Se

x18

636.

020.

8419

.55

39.1

6H

emat

olo

gyM

ean

cell

ula

rH

bco

nce

ntr

atio

n(%

)St

ud

yd

ay18

635.

960.

8219

.79

0.07

Hem

ato

logy

Mea

nce

llu

lar

Hb

con

cen

trat

ion

(%)

Wei

ght

1863

——

14.6

80.

92H

emat

olo

gyM

ean

cell

ula

rH

bco

nce

ntr

atio

n(%

)Ye

ar18

63—

—31

.10

82.9

4H

emat

olo

gyM

ean

cell

ula

rvo

lum

e(fl

)C

age

den

sity

1875

——

7.63

12.4

7H

emat

olo

gyM

ean

cell

ula

rvo

lum

e(fl

)L

itte

r18

65—

—4.

685.

79H

emat

olo

gyM

ean

cell

ula

rvo

lum

e(fl

)M

on

th18

7613

.39

3.18

15.6

330

.61

Hem

ato

logy

Mea

nce

llu

lar

volu

me

(fl)

Seas

on

1876

——

7.50

21.5

6H

emat

olo

gyM

ean

cell

ula

rvo

lum

e(fl

)Se

x18

76—

—8.

8920

.90

Hem

ato

logy

Mea

nce

llu

lar

volu

me

(fl)

Stu

dy

day

1876

6.15

0.90

——

Hem

ato

logy

Mea

nce

llu

lar

volu

me

(fl)

Wei

ght

1876

——

5.16

0.45

Hem

ato

logy

Mea

nce

llu

lar

volu

me

(fl)

Year

1876

6.67

0.99

——

Hem

ato

logy

Mea

nco

rpu

scu

lar

hem

ogl

ob

in(p

g)A

ge18

71—

—15

.80

1.25

Hem

ato

logy

Mea

nco

rpu

scu

lar

hem

ogl

ob

in(p

g)C

age

den

sity

1870

——

13.3

016

.26

Hem

ato

logy

Mea

nco

rpu

scu

lar

hem

ogl

ob

in(p

g)L

itte

r18

60—

—16

.42

16.5

1H

emat

olo

gyM

ean

corp

usc

ula

rh

emo

glo

bin

(pg)

Mo

nth

1871

32.6

86.

7710

.21

22.9

1H

emat

olo

gyM

ean

corp

usc

ula

rh

emo

glo

bin

(pg)

Seas

on

1871

19.9

43.

628.

8525

.86

Hem

ato

logy

Mea

nco

rpu

scu

lar

hem

ogl

ob

in(p

g)Se

x18

71—

—11

.35

27.4

4

(con

tin

ued

)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

oty

pe

Co

vari

ate

No

.o

bse

rved

log

P%

vari

ance

exp

lain

edlo

gP

%va

rian

ceex

pla

ined

Hem

ato

logy

Mea

nco

rpu

scu

lar

hem

ogl

ob

in(p

g)St

ud

yd

ay18

71—

—9.

470.

04H

emat

olo

gyM

ean

corp

usc

ula

rh

emo

glo

bin

(pg)

Wei

ght

1871

——

5.59

0.55

Hem

ato

logy

Mea

nco

rpu

scu

lar

hem

ogl

ob

in(p

g)Ye

ar18

71—

—8.

5244

.24

Hem

ato

logy

Pla

tele

tcri

t(%

)A

ge18

39—

—5.

100.

73H

emat

olo

gyP

late

letc

rit

(%)

Mo

nth

1839

14.3

94.

066.

2619

.33

Hem

ato

logy

Pla

tele

tcri

t(%

)Se

aso

n18

394.

851.

114.

5919

.65

Hem

ato

logy

Pla

tele

tcri

t(%

)Se

x18

3936

.50

7.50

8.92

27.5

3H

emat

olo

gyP

late

lets

(n/

ml)

Mo

nth

1863

16.4

34.

1810

.19

24.8

1H

emat

olo

gyP

late

lets

(n/

ml)

Seas

on

1863

6.62

1.36

7.75

26.6

4H

emat

olo

gyP

late

lets

(n/

ml)

Sex

1863

30.9

25.

7610

.78

27.6

7H

emat

olo

gyP

late

lets

(n/

ml)

Stu

dy

day

1863

9.44

1.60

——

Hem

ato

logy

Pla

tele

ts(n

/m

l)W

eigh

t18

635.

770.

935.

540.

50H

emat

olo

gyP

late

lets

(n/

ml)

Year

1863

5.56

0.89

——

Hem

ato

logy

Red

blo

od

cell

cou

nt

(n/

ml)

Mo

nth

1870

——

11.8

829

.98

Hem

ato

logy

Red

blo

od

cell

cou

nt

(n/

ml)

Seas

on

1870

——

5.84

21.5

2H

emat

olo

gyR

edb

loo

dce

llco

un

t(n

/m

l)Se

x18

709.

321.

774.

9519

.31

Hem

ato

logy

Red

blo

od

cell

cou

nt

(n/

ml)

Wei

ght

1870

5.90

1.07

4.94

0.48

Hem

ato

logy

Red

cell

dis

trib

uti

on

wid

thL

itte

r18

50—

—4.

648.

40H

emat

olo

gyR

edce

lld

istr

ibu

tio

nw

idth

Mo

nth

1861

8.27

2.18

10.6

823

.56

Hem

ato

logy

Red

cell

dis

trib

uti

on

wid

thSe

aso

n18

61—

—5.

7417

.99

Hem

ato

logy

Red

cell

dis

trib

uti

on

wid

thSe

x18

6112

.88

1.99

——

Hem

ato

logy

Red

cell

dis

trib

uti

on

wid

thW

eigh

t18

61—

—4.

860.

42H

emat

olo

gyW

hit

eb

loo

dce

llco

un

t(n

/m

l)C

age

den

sity

1875

——

5.44

12.8

8H

emat

olo

gyW

hit

eb

loo

dce

llco

un

t(n

/m

l)L

itte

r18

656.

241.

0011

.57

12.5

4H

emat

olo

gyW

hit

eb

loo

dce

llco

un

t(n

/m

l)M

on

th18

76—

—15

.14

33.4

6H

emat

olo

gyW

hit

eb

loo

dce

llco

un

t(n

/m

l)Se

aso

n18

76—

—15

.64

36.7

7H

emat

olo

gyW

hit

eb

loo

dce

llco

un

t(n

/m

l)Se

x18

76—

—6.

8923

.62

Hem

ato

logy

Wh

ite

blo

od

cell

cou

nt

(n/

ml)

Stu

dy

day

1876

10.1

81.

716.

860.

03H

emat

olo

gyW

hit

eb

loo

dce

llco

un

t( n

/m

l)W

eigh

t18

76—

—5.

480.

58H

emat

olo

gyW

hit

eb

loo

dce

llco

un

t(n

/m

l)Ye

ar18

76—

—6.

3342

.86

Hem

ato

logy

Hem

ato

crit

(%)

Mo

nth

1873

——

10.1

327

.26

Hem

ato

logy

Hem

ato

crit

(%)

Seas

on

1873

——

4.92

19.3

1H

emat

olo

gyH

emat

ocr

it(%

)Se

x18

7312

.18

2.39

5.01

18.4

1H

emat

olo

gyH

emat

ocr

it(%

)W

eigh

t18

735.

591.

026.

600.

48Im

mu

no

logy

%B

2201

Age

1723

——

9.73

2.70

Imm

un

olo

gy%

B22

01C

age

den

sity

1677

——

7.72

10.4

9Im

mu

no

logy

%B

2201

Lit

ter

1713

——

9.38

14.2

6Im

mu

no

logy

%B

2201

Mo

nth

1723

11.6

82.

8428

.30

41.2

8Im

mu

no

logy

%B

2201

Seas

on

1723

——

23.9

946

.06

(con

tin

ued

)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

oty

pe

Co

vari

ate

No

.o

bse

rved

log

P%

vari

ance

exp

lain

edlo

gP

%va

rian

ceex

pla

ined

Imm

un

olo

gy%

B22

01

Sex

1723

13.0

22.

0710

.71

28.8

1Im

mu

no

logy

%C

D31

Lit

ter

1723

5.00

0.78

6.68

13.1

9Im

mu

no

logy

%C

D31

Mo

nth

1733

16.6

54.

0719

.89

37.1

7Im

mu

no

logy

%C

D31

Seas

on

1733

5.47

1.12

14.6

935

.98

Imm

un

olo

gy%

CD

31

Sex

1733

——

11.5

730

.57

Imm

un

olo

gy%

CD

41

Cag

ed

ensi

ty16

73—

—4.

928.

94Im

mu

no

logy

%C

D41

Lit

ter

1721

——

11.4

218

.10

Imm

un

olo

gy%

CD

41

Mo

nth

1731

14.9

93.

9423

.00

44.6

4Im

mu

no

logy

%C

D41

Seas

on

1731

——

18.1

644

.24

Imm

un

olo

gy%

CD

41

Sex

1731

——

9.74

28.8

8Im

mu

no

logy

%C

D41

/C

D31

Age

1732

——

6.12

1.24

Imm

un

olo

gy%

CD

41

/C

D31

Cag

ed

ensi

ty16

74—

—5.

406.

88Im

mu

no

logy

%C

D41

/C

D31

Lit

ter

1722

——

15.8

110

.27

Imm

un

olo

gy%

CD

41

/C

D31

Mo

nth

1732

25.7

14.

6513

.23

21.8

2Im

mu

no

logy

%C

D41

/C

D31

Seas

on

1732

8.50

1.35

8.04

17.8

9Im

mu

no

logy

%C

D81

Age

1733

——

5.32

1.08

Imm

un

olo

gy%

CD

81

Mo

nth

1733

14.1

72.

577.

5714

.65

Imm

un

olo

gy%

CD

81

Seas

on

1733

10.5

31.

46—

—Im

mu

no

logy

%C

D81

Sex

1733

——

5.30

12.6

1Im

mu

no

logy

%N

Kce

lls

Cag

ed

ensi

ty16

67—

—5.

2910

.41

Imm

un

olo

gy%

NK

cell

sL

itte

r17

14—

—11

.43

16.7

9Im

mu

no

logy

%N

Kce

lls

Mo

nth

1724

35.2

78.

6816

.15

32.9

1Im

mu

no

logy

%N

Kce

lls

Seas

on

1724

9.04

2.06

16.0

437

.88

Imm

un

olo

gy%

NK

cell

sSe

x17

245.

220.

95—

—Im

mu

no

logy

CD

41/

CD

81A

ge17

29—

—6.

021.

07Im

mu

no

logy

CD

41/

CD

81C

age

den

sity

1671

——

4.85

5.83

Imm

un

olo

gyC

D41

/C

D81

Lit

ter

1719

——

10.6

27.

45Im

mu

no

logy

CD

41/

CD

81M

on

th17

2917

.55

3.26

9.38

17.3

9Im

mu

no

logy

CD

41

/C

D81

Seas

on

1729

8.52

1.29

4.78

12.2

1L

engt

hB

od

yle

ngt

h(c

m)

Age

1942

——

7.56

0.81

Len

gth

Bo

dy

len

gth

(cm

)L

itte

r19

32—

—10

.25

8.30

Len

gth

Bo

dy

len

gth

(cm

)M

on

th19

4216

.46

3.22

11.2

921

.08

Len

gth

Bo

dy

len

gth

(cm

)Se

aso

n19

425.

610.

9113

.42

24.6

9L

engt

hB

od

yle

ngt

h(c

m)

Sex

1942

35.1

25.

17—

—L

engt

hB

od

yle

ngt

h(c

m)

Stu

dy

day

1942

——

4.91

0.02

Len

gth

Bo

dy

len

gth

(cm

)W

eigh

t19

4287

.23

13.9

4—

—L

engt

hB

od

yle

ngt

h(c

m)

Year

1942

5.94

0.76

——

New

ho

me-

cage

acti

vity

Fin

em

ove

men

tA

ge22

945.

140.

72—

—N

ewh

om

e-ca

geac

tivi

tyF

ine

mo

vem

ent

Sex

2294

7.62

1.13

—— (c

onti

nu

ed)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

oty

pe

Co

vari

ate

No

.o

bse

rved

log

P%

vari

ance

exp

lain

edlo

gP

%va

rian

ceex

pla

ined

New

ho

me-

cage

acti

vity

To

tal

bea

mb

reak

s(3

0m

in)

Exp

erim

ente

r22

90—

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4917

.79

New

ho

me-

cage

acti

vity

To

tal

bea

mb

reak

s(3

0m

in)

Mo

nth

2290

——

7.05

18.2

2N

ewh

om

e-ca

geac

tivi

tyT

ota

lb

eam

bre

aks

(30

min

)Se

aso

n22

90—

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8716

.40

New

ho

me-

cage

acti

vity

To

tal

bea

mb

reak

s(fi

rst

5m

in)

Exp

erim

ente

r22

8911

.94

2.90

5.06

15.5

0N

ewh

om

e-ca

geac

tivi

tyT

ota

lb

eam

bre

aks

(firs

t5

min

)M

on

th22

89—

—11

.44

24.5

3N

ewh

om

e-ca

geac

tivi

tyT

ota

lb

eam

bre

aks

(firs

t5

min

)Se

aso

n22

89—

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4923

.45

New

ho

me-

cage

acti

vity

To

tal

bea

mb

reak

s(l

ast

5m

in)

Ho

ur

2275

4.99

1.41

——

Op

enfi

eld

Fec

alb

oli

Mo

nth

2304

5.34

1.75

——

Op

enfi

eld

Fec

alb

oli

Sex

2304

——

5.40

16.3

8O

pen

fiel

dT

ota

lac

tivi

tyE

xper

imen

ter

2302

4.99

1.34

——

Op

enfi

eld

To

tal

acti

vity

Seas

on

2302

——

6.11

16.9

6O

pen

fiel

dT

ota

lac

tivi

tySe

x23

027.

601.

07—

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leth

ysm

ogr

aph

yE

nh

ance

dp

ause

(bas

elin

e)A

ge21

69—

—15

.55

2.18

Ple

thys

mo

grap

hy

En

han

ced

pau

se(b

asel

ine)

Cag

ed

ensi

ty21

69—

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428.

81P

leth

ysm

ogr

aph

yE

nh

ance

dp

ause

(bas

elin

e)H

ou

r21

69—

—18

.39

31.9

6P

leth

ysm

ogr

aph

yE

nh

ance

dp

ause

(bas

elin

e)L

itte

r21

576.

970.

9124

.63

25.4

5P

leth

ysm

ogr

aph

yE

nh

ance

dp

ause

(bas

elin

e)M

on

th21

6977

.44

11.8

724

.29

31.4

3P

leth

ysm

ogr

aph

yE

nh

ance

dp

ause

(bas

elin

e)Se

aso

n21

6948

.57

7.17

13.7

127

.43

Ple

thys

mo

grap

hy

En

han

ced

pau

se(b

asel

ine)

Sex

2169

15.8

02.

2411

.77

25.3

8P

leth

ysm

ogr

aph

yE

nh

ance

dp

ause

(bas

elin

e)St

ud

yd

ay21

69—

—20

.59

0.09

Ple

thys

mo

grap

hy

En

han

ced

pau

se(b

asel

ine)

Year

2169

5.51

0.72

12.0

848

.49

Ple

thys

mo

grap

hy

En

han

ced

pau

se(m

etac

ho

lin

e)A

ge19

43—

—7.

641.

41P

leth

ysm

ogr

aph

yE

nh

ance

dp

ause

(met

ach

oli

ne)

Ho

ur

1943

——

20.3

736

.68

Ple

thys

mo

grap

hy

En

han

ced

pau

se(m

etac

ho

lin

e)L

itte

r19

31—

—10

.94

20.8

6P

leth

ysm

ogr

aph

yE

nh

ance

dp

ause

(met

ach

oli

ne)

Mo

nth

1943

16.7

23.

7619

.83

37.1

3P

leth

ysm

ogr

aph

yE

nh

ance

dp

ause

(met

ach

oli

ne)

Seas

on

1943

11.8

72.

1215

.67

36.3

1P

leth

ysm

ogr

aph

yE

nh

ance

dp

ause

(met

ach

oli

ne)

Sex

1943

21.6

33.

569.

8426

.78

Ple

thys

mo

grap

hy

En

han

ced

pau

se(m

etac

ho

lin

e)W

eigh

t19

436.

510.

97—

—P

leth

ysm

ogr

aph

yE

nh

ance

dp

ause

(met

ach

oli

ne)

Year

1943

——

8.18

50.3

9P

leth

ysm

ogr

aph

yE

xpir

ato

ryti

me

(bas

elin

e)H

ou

r21

65—

—17

.65

34.3

8P

leth

ysm

ogr

aph

yE

xpir

ato

ryti

me

(bas

elin

e)L

itte

r21

53—

—9.

0910

.52

Ple

thys

mo

grap

hy

Exp

irat

ory

tim

e(b

asel

ine)

Mo

nth

2165

17.7

23.

9411

.84

26.4

1P

leth

ysm

ogr

aph

yE

xpir

ato

ryti

me

(bas

elin

e)Se

aso

n21

6513

.74

2.42

10.1

426

.77

Ple

thys

mo

grap

hy

Exp

irat

ory

tim

e(b

asel

ine)

Sex

2165

——

9.43

25.6

0P

leth

ysm

ogr

aph

yE

xpir

ato

ryti

me

(bas

elin

e)St

ud

yd

ay21

65—

—5.

300.

03P

leth

ysm

ogr

aph

yE

xpir

ato

ryti

me

(bas

elin

e)W

eigh

t21

654.

770.

686.

660.

57P

leth

ysm

ogr

aph

yE

xpir

ato

ryti

me

(bas

elin

e)Ye

ar21

659.

981.

53—

—P

leth

ysm

ogr

aph

yE

xpir

ato

ryti

me

(met

ach

oli

ne)

Ho

ur

1935

——

5.56

15.7

5P

leth

ysm

ogr

aph

yE

xpir

ato

ryti

me

(met

ach

oli

ne)

Mo

nth

1935

5.31

1.78

—— (c

onti

nu

ed)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

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pe

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vari

ate

No

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bse

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log

P%

vari

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exp

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ceex

pla

ined

Ple

thys

mo

grap

hy

Exp

irat

ory

tim

e(m

etac

ho

lin

e)Se

x19

35—

—6.

1517

.12

Ple

thys

mo

grap

hy

Insp

irat

ory

tim

e(b

asel

ine)

Cag

ed

ensi

ty21

745.

180.

74—

—P

leth

ysm

ogr

aph

yIn

spir

ato

ryti

me

(bas

elin

e)H

ou

r21

74—

—17

.02

32.0

9P

leth

ysm

ogr

aph

yIn

spir

ato

ryti

me

(bas

elin

e)L

itte

r21

62—

—12

.07

12.8

7P

leth

ysm

ogr

aph

yIn

spir

ato

ryti

me

(bas

elin

e)M

on

th21

7415

.47

3.57

9.87

24.2

8P

leth

ysm

ogr

aph

yIn

spir

ato

ryti

me

(bas

elin

e)Se

aso

n21

7410

.83

1.95

10.0

927

.17

Ple

thys

mo

grap

hy

Insp

irat

ory

tim

e(b

asel

ine)

Sex

2174

12.3

21.

9512

.50

29.6

1P

leth

ysm

ogr

aph

yIn

spir

ato

ryti

me

(bas

elin

e)St

ud

yd

ay21

74—

—4.

990.

03P

leth

ysm

ogr

aph

yIn

spir

ato

ryti

me

(bas

elin

e)W

eigh

t21

74—

—9.

220.

71P

leth

ysm

ogr

aph

yIn

spir

ato

ryti

me

(met

ach

oli

ne)

Ho

ur

1946

——

7.07

18.9

8P

leth

ysm

ogr

aph

yIn

spir

ato

ryti

me

(met

ach

oli

ne)

Mo

nth

1946

5.41

1.64

——

Ple

thys

mo

grap

hy

Insp

irat

ory

tim

e(m

etac

ho

lin

e)Se

aso

n19

464.

580.

86—

—P

leth

ysm

ogr

aph

yIn

spir

ato

ryti

me

(met

ach

oli

ne)

Sex

1946

20.6

43.

33—

—P

leth

ysm

ogr

aph

yIn

spir

ato

ryti

me

(met

ach

oli

ne)

Wei

ght

1946

6.35

0.93

——

Ple

thys

mo

grap

hy

Pen

Hd

iffe

ren

ceA

ge19

34—

—7.

341.

63P

leth

ysm

ogr

aph

yP

enH

dif

fere

nce

Cag

ed

ensi

ty19

34—

—5.

679.

31P

leth

ysm

ogr

aph

yP

enH

dif

fere

nce

Ho

ur

1934

——

19.0

835

.57

Ple

thys

mo

grap

hy

Pen

Hd

iffe

ren

ceL

itte

r19

22—

—9.

5318

.03

Ple

thys

mo

grap

hy

Pen

Hd

iffe

ren

ceM

on

th19

348.

202.

3221

.79

42.7

5P

leth

ysm

ogr

aph

yP

enH

dif

fere

nce

Seas

on

1934

5.00

1.00

17.3

542

.74

Ple

thys

mo

grap

hy

Pen

Hd

iffe

ren

ceSe

x19

3414

.92

2.52

12.0

229

.38

Ple

thys

mo

grap

hy

Pen

Hd

iffe

ren

ceW

eigh

t19

34—

—6.

270.

60P

leth

ysm

ogr

aph

yP

enH

dif

fere

nce

Year

1934

——

10.7

258

.13

Ple

thys

mo

grap

hy

Res

pir

ato

ryra

te(b

asel

ine)

Cag

ed

ensi

ty21

634.

820.

69—

—P

leth

ysm

ogr

aph

yR

esp

irat

ory

rate

(bas

elin

e)H

ou

r21

63—

—18

.19

33.5

0P

leth

ysm

ogr

aph

yR

esp

irat

ory

rate

(bas

elin

e)L

itte

r21

51—

—11

.93

12.5

3P

leth

ysm

ogr

aph

yR

esp

irat

ory

rate

(bas

elin

e)M

on

th21

6321

.39

4.60

10.4

024

.62

Ple

thys

mo

grap

hy

Res

pir

ato

ryra

te(b

asel

ine)

Seas

on

2163

16.1

42.

849.

6526

.40

Ple

thys

mo

grap

hy

Res

pir

ato

ryra

te(b

asel

ine)

Sex

2163

6.22

0.92

11.0

127

.80

Ple

thys

mo

grap

hy

Res

pir

ato

ryra

te(b

asel

ine)

Stu

dy

day

2163

——

6.20

0.04

Ple

thys

mo

grap

hy

Res

pir

ato

ryra

te(b

asel

ine)

Wei

ght

2163

——

6.07

0.57

Ple

thys

mo

grap

hy

Res

pir

ato

ryra

te(b

asel

ine)

Year

2163

6.34

0.94

——

Ple

thys

mo

grap

hy

Res

pir

ato

ryra

te(m

etac

ho

lin

e)H

ou

r19

28—

—5.

8717

.38

Ple

thys

mo

grap

hy

Res

pir

ato

ryra

te(m

etac

ho

lin

e)Se

aso

n19

285.

211.

08—

—P

leth

ysm

ogr

aph

yT

idal

min

ute

volu

me

(bas

elin

e)A

ge21

58—

—5.

040.

68P

leth

ysm

ogr

aph

yT

idal

min

ute

volu

me

(bas

elin

e)C

age

den

sity

2158

7.51

0.89

——

Ple

thys

mo

grap

hy

Tid

alm

inu

tevo

lum

e(b

asel

ine)

Ho

ur

2158

——

11.5

419

.48

Ple

thys

mo

grap

hy

Tid

alm

inu

tevo

lum

e(b

asel

ine)

Lit

ter

2146

——

6.21

7.69

Ple

thys

mo

grap

hy

Tid

alm

inu

tevo

lum

e(b

asel

ine)

Mo

nth

2158

13.4

92.

5412

.97

20.2

8

(con

tin

ued

)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

oty

pe

Co

vari

ate

No

.o

bse

rved

log

P%

vari

ance

exp

lain

edlo

gP

%va

rian

ceex

pla

ined

Ple

thys

mo

grap

hy

Tid

alm

inu

tevo

lum

e(b

asel

ine)

Seas

on

2158

——

8.70

19.4

1P

leth

ysm

ogr

aph

yT

idal

min

ute

volu

me

(bas

elin

e)Se

x21

5864

.43

9.12

——

Ple

thys

mo

grap

hy

Tid

alm

inu

tevo

lum

e(b

asel

ine)

Wei

ght

2158

68.9

09.

81—

—P

leth

ysm

ogr

aph

yT

idal

min

ute

volu

me

(met

ach

oli

ne)

Ho

ur

1930

——

6.17

11.8

1P

leth

ysm

ogr

aph

yT

idal

min

ute

volu

me

(met

ach

oli

ne)

Lit

ter

1918

——

5.61

5.33

Ple

thys

mo

grap

hy

Tid

alm

inu

tevo

lum

e(m

etac

ho

lin

e)M

on

th19

3018

.51

3.03

4.64

10.6

0P

leth

ysm

ogr

aph

yT

idal

min

ute

volu

me

(met

ach

oli

ne)

Seas

on

1930

10.8

11.

45—

—P

leth

ysm

ogr

aph

yT

idal

min

ute

volu

me

(met

ach

oli

ne)

Sex

1930

105.

0514

.76

5.95

12.0

0P

leth

ysm

ogr

aph

yT

idal

min

ute

volu

me

(met

ach

oli

ne)

Wei

ght

1930

71.2

59.

555.

820.

35P

leth

ysm

ogr

aph

yT

idal

volu

me

(bas

elin

e)A

ge21

49—

—16

.84

1.43

Ple

thys

mo

grap

hy

Tid

alvo

lum

e(b

asel

ine)

Cag

ed

ensi

ty21

49—

—6.

014.

08P

leth

ysm

ogr

aph

yT

idal

volu

me

(bas

elin

e)H

ou

r21

49—

—20

.24

22.3

2P

leth

ysm

ogr

aph

yT

idal

volu

me

(bas

elin

e)L

itte

r21

37—

—16

.96

16.0

6P

leth

ysm

ogr

aph

yT

idal

volu

me

(bas

elin

e)M

on

th21

4939

.79

5.07

20.3

222

.38

Ple

thys

mo

grap

hy

Tid

alvo

lum

e(b

asel

ine)

Seas

on

2149

20.9

02.

4110

.96

20.8

7P

leth

ysm

ogr

aph

yT

idal

volu

me

(bas

elin

e)Se

x21

4913

1.89

16.9

87.

7513

.06

Ple

thys

mo

grap

hy

Tid

alvo

lum

e(b

asel

ine)

Stu

dy

day

2149

——

7.27

0.03

Ple

thys

mo

grap

hy

Tid

alvo

lum

e(b

asel

ine)

Wei

ght

2149

87.9

410

.73

6.12

0.32

Ple

thys

mo

grap

hy

Tid

alvo

lum

e(m

etac

ho

lin

e)A

ge19

32—

—9.

740.

80P

leth

ysm

ogr

aph

yT

idal

volu

me

(met

ach

oli

ne)

Ho

ur

1932

——

10.7

215

.83

Ple

thys

mo

grap

hy

Tid

alvo

lum

e(m

etac

ho

lin

e)L

itte

r19

20—

—8.

867.

08P

leth

ysm

ogr

aph

yT

idal

volu

me

(met

ach

oli

ne)

Mo

nth

1932

26.8

13.

645.

2210

.32

Ple

thys

mo

grap

hy

Tid

alvo

lum

e(m

etac

ho

lin

e)Se

aso

n19

3218

.02

2.07

——

Ple

thys

mo

grap

hy

Tid

alvo

lum

e(m

etac

ho

lin

e)Se

x19

3214

1.45

18.4

36.

0311

.35

Ple

thys

mo

grap

hy

Tid

alvo

lum

e(m

etac

ho

lin

e)W

eigh

t19

3285

.28

10.3

0—

—P

leth

ysm

ogr

aph

yT

idal

volu

me

(met

ach

oli

ne)

Year

1932

6.15

0.59

——

Wei

ght

Bo

dy

mas

sin

dex

Age

1925

5.87

0.79

——

Wei

ght

Bo

dy

mas

sin

dex

Mo

nth

1925

8.83

2.16

7.41

16.7

5W

eigh

tB

od

ym

ass

ind

exSe

aso

n19

256.

211.

079.

9321

.20

Wei

ght

Bo

dy

mas

sin

dex

Sex

1925

113.

9320

.32

——

Wei

ght

Bo

dy

mas

sin

dex

Wei

ght

1925

19.9

13.

03—

—W

eigh

tW

eigh

t,10

wk

(g)

Cag

ed

ensi

ty23

19—

—5.

373.

29W

eigh

tW

eigh

t,10

wk

(g)

Lit

ter

2307

——

10.2

76.

10W

eigh

tW

eigh

t,10

wk

(g)

Sex

2320

Inf

41.3

7—

—W

eigh

tW

eigh

t,6

wk

(g)

Cag

ed

ensi

ty24

325.

570.

3220

.89

9.30

Wei

ght

Wei

ght,

6w

k(g

)L

itte

r24

98—

—39

.27

16.6

9W

eigh

tW

eigh

t,6

wk

(g)

Sex

2511

Inf

30.6

312

.63

12.8

7W

eigh

tW

eigh

t,7

wk

(g)

Cag

ed

ensi

ty24

055.

040.

268.

224.

40W

eigh

tW

eigh

t,7

wk

(g)

Lit

ter

2457

6.16

0.32

21.1

17.

79 (con

tin

ued

)


AP

PE

ND

IX

(Co

nti

nu

ed)

Mai

nef

fect

sIn

tera

ctio

ns

Tes

tP

hen

oty

pe

Co

vari

ate

No

.o

bse

rved

log

P%

vari

ance

exp

lain

edlo

gP

%va

rian

ceex

pla

ined

Wei

ght

Wei

ght,

7w

k(g

)Se

x24

70In

f35

.40

7.13

8.27

Wei

ght

Wei

ght,

8w

k(g

)L

itte

r22

90—

—10

.46

2.53

Wei

ght

Wei

ght,

8w

k(g

)Se

x23

02In

f44

.52

——

Wo

un

dh

eali

ng

Ear

ho

lear

ea(m

m2)

Cag

ed

ensi

ty21

8510

.45

1.37

9.10

9.56

Wo

un

dh

eali

ng

Ear

ho

lear

ea(m

m2)

Lit

ter

2172

6.00

0.76

9.72

10.5

9W

ou

nd

hea

lin

gE

arh

ole

area

(mm

2)

Mo

nth

2185

24.1

74.

349.

9418

.33

Wo

un

dh

eali

ng

Ear

ho

lear

ea(m

m2)

Seas

on

2185

17.8

12.

676.

0814

.33

Wo

un

dh

eali

ng

Ear

ho

lear

ea(m

m2)

Sex

2185

13.9

31.

914.

8712

.85

Wo

un

dh

eali

ng

Ear

ho

lear

ea(m

m2)

Stu

dy

day

2185

——

9.67

0.04

Wo

un

dh

eali

ng

Ear

ho

lear

ea(m

m2)

Year

2185

——

6.42

32.6

4

No

teth

atn

ot

allc

om

bin

atio

ns

of

ph

eno

typ

ean

dco

vari

ate

wer

eav

aila

ble

inth

est

ud

y.L

og

Pd

eno

tes

the

loga

rith

mto

the

bas

e10

of

the

P-v

alu

e.In

fd

eno

tes

aP

-val

ue

that

was

com

pu

tati

on

ally

ind

isti

ngu

ish

able

fro

mze

ro.

Fo

rb

revi

ty,

resu

lts

are

om

itte

dfo

ref

fect

sw

ith

log

P’s

of

,4.

55(i

.e.,

the

corr

ecte

d5%

sign

ifica

nce

leve

l).


genetic and environmental effects on complex traits...

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