A Test of Bone Mobilization Relative to Reproductive Demand: Skeletal Quality Is Improved inCannibalistic Females with Large LittersAuthor(s): Wendy R. HoodReviewed work(s):Source: Physiological and Biochemical Zoology, Vol. 85, No. 4 (July/August 2012), pp. 385-396Published by: The University of Chicago PressStable URL: http://www.jstor.org/stable/10.1086/666057 .Accessed: 19/06/2012 12:38

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385

A Test of Bone Mobilization Relative to Reproductive Demand:

Skeletal Quality Is Improved in Cannibalistic Females

with Large Litters

* E-mail: wrhood@auburn.edu.

Physiological and Biochemical Zoology 85(4):385–396. 2012. � 2012 by TheUniversity of Chicago. All rights reserved. 1522-2152/2012/8504-1124$15.00.DOI: 10.1086/666057

Wendy R. Hood*Department of Biological Sciences, Auburn University,Auburn, Alabama 36849

Accepted 3/28/2012; Electronically Published 6/1/2012

ABSTRACT

In species with repeated bouts of reproduction, a female’s abilityto retain sufficient tissue for self-maintenance is essential toher survival and capacity for future reproduction. Loss of bonemineral content results in bone fragility and the possibility ofreduced survival, so females should guard against the overuseof their bone mineral during reproduction. Given these con-straints, I predicted that bone mobilization would increase withlitter size in mice but plateau before maximum litter size wasreached. To test this idea, I manipulated the litter sizes of housemice on the day of parturition to 3, 8, 13, and 18 offspring.At weaning, I euthanized the females and calculated whole-body and bone mineral composition. The total mineral contentof females’ femurs dropped as litter size increased to the averagelitter size for this strain of mouse (13) but surprisingly, femoralmineral content was higher for females assigned the largest littersizes (18). Seven of the nine females assigned 18 young can-nibalized some of their offspring. For females assigned to theselarger litters, femoral ash content was not correlated with num-ber of young consumed, suggesting that mineral recycling hadlittle effect on final bone mineral content. However, nursingeffort (accounting for young lost to cannibalism) was correlatedwith maternal femoral ash at weaning. These finding suggestthat the high bone mineral content of females assigned thelargest litters was associated with a reduction in endogenousmineral allocated to the litter.

Introduction

In iteroparous species, the capacity for future reproduction isdependent on an individual’s ability to retain adequate body

condition during previous bouts of reproduction (Clutton-Brock 1991; Stearns 1992). Mammalian females are burdenedwith supplying the building blocks required for offspring de-velopment both in utero and during postnatal dependency. Tomeet this demand, females adopt one or a combination ofstrategies, including increasing food intake and mobilizing so-matic tissues from their own bodies (Drent and Daan 1980;Speakman 2008). Use of somatic tissue can protect females andtheir young from unpredictable or predictably poor food avail-ability during pregnancy and lactation (Drent and Daan 1980),but reliance on stored nutrients may affect a female’s residualreproductive value by influencing her probability of survival,the residual amount of stores she has available for the nextreproductive bout, and the time required to build new tissueto support subsequent reproductive efforts (Clutton-Brock1984).

If the residual reproductive value of a female is greater thanzero, the allocation of somatic resources to offspring should begoverned by priority rules that prevent excessive use of somatictissue (Zera and Harshman 2001). Numerous studies haveshown that mammalian females will forgo reproduction or re-duce allocation when they are in poor condition and when theyare still developing themselves. For example, Ono et al. (1987)showed that California sea lions transferred less milk to theiryoung when they were in poor condition during an El Ninoevent, and Landete-Castillejos et al. (2004) showed that pri-miparous female Iberian red deer prioritize self-maintenanceduring reproduction while they are still growing by bearingmore smaller daughters than larger sons. Negative relationshipsbetween litter size and nutrient allocation can also reflect pro-cesses that protect maternal condition. Rogowitz and McClure(1995) showed that maternal body mass in lactating cotton ratswas comparable between females suckling three offspring andthose suckling six. In these examples, priority rules manage andprotect the catabolism of maternal tissues.

During uterine and postnatal development, females supportthe growth of their offspring’s skeletons by increasing intakeof calcium and by mobilizing mineral from their bone (Brom-mage 1989). The skeletal mass of small mammals is low relativeto that of larger species (Prange et al. 1979), and thus smallspecies have relatively less bone mineral available for mobili-zation (Hood et al. 2006). Small mammals produce large vol-umes of milk relative to their size (Oftedal 1984), while thecalcium and phosphorus content of their milk is comparablewith that of larger species (Studier and Kunz 1995), suggestingthat mineral transfer between females and offspring is relativelyhigh. Yet despite a relatively small somatic resource, bone min-

386 W. R. Hood

eral loss during reproduction is proportionally higher in thefew small mammals that have been examined relative to thatin larger species (Wysolmerski 2002).

Several factors may contribute to the total amount of mineralmobilized from the maternal skeleton during reproduction, in-cluding offspring demand and mineral ingestion by the female.Loss of mineral from the femur and lumbar vertebrae is pos-itively correlated with litter size in rats (Peng et al. 1988; Tojoet al. 1998). In addition, the total mass of food consumed byhouse mice, and presumably other rodents, can be limited (Nel-son and Evans 1961; Speakman 2008). Specifically, mice suck-ling large litters are unable to consume more food to com-pensate for the high nutrient demand of their young(Hammond and Diamond 1994). If litter size crosses a thresh-old at which food consumption cannot equal the nutrient de-mand of offspring, then the added mineral demand from moreyoung must come entirely from the female’s skeleton if thesame level of allocation per offspring is to be maintained.

Allocation of minerals from the body of the female comesat a cost. There is a negative correlation between the mineralcontent of a bone and its fragility (Currey 1969, 1988; Boskeyand Coleman 2010). Without an intrinsic rule limiting theamount of bone that can be mobilized during reproduction,females with large litters might allocate so much mineral fromtheir own bones that they suffer catastrophic failure.

Lactation is associated with increased bone turnover andremodeling in long bones, such as the femur and tibia, andaxial bones, such as the lumbar vertebrae, pelvis, and ribs (Mil-ler et al. 1989; Kovacs and Kronenberg 1997; Vajda et al. 1999b,2001). With high amounts of calcium partitioned to milk pro-duction, an increase in bone turnover leads to a reduction inbone mineral content (Bowman and Miller 2001). Corticalbone can display a reduction in cortical thickness and an in-creased number of resorptive spaces, and the mineral contentof new osteons is reduced (Vajda et al. 1999b). Cancellous bonecan display a decrease in number and thickness of trabecularstruts (Shahtaheri et al. 1999). Increases in the porosity ofcortical bone and reductions in the number and thickness oftrabeculae have been shown to negatively affect bone strength(Davison et al. 2006), and as a result, lactating animals havebeen shown to display reductions in bone strength, stiffness,toughness, and ductility (Peng et al. 1988). There is little evi-dence of increased fracture risk during reproduction in women(Kovacs and Kronenberg 1997), lending support to the notionthat there are physiological mechanisms in place to limit theserisks. However, no previous studies have assessed intrinsic limitson the mobilization of bone.

The goal of this investigation is to characterize the patternof bone mineral loss by females during reproduction relativeto litter size, to determine whether there is an intrinsic limitto the amount of bone that females mobilize. Because of itssmall body size and large litters and a strong body of previouswork on food intake and reproductive performance, the ICRlaboratory mouse (Mus musculus, Hsd:ICR(CD-1)) was selectedfor this investigation. I predicted that with increasing litter sizeand thus increasing cumulative demand, the amount of bone

that is mobilized from the maternal skeleton would increase.As a result, females with larger litters would be expected tohave less residual bone and less bone strength at the end ofthe reproduction bout than females with smaller litters. How-ever, I further predicted a physiological cutoff should exist be-yond which females will stop mobilizing bone.

Because house mice are so well characterized, I could makeexplicit predictions. Litter sizes of 11–14 are typical for ICRmice, but litters of 18 and 19 are occasionally observed. Thefood-intake limit of outbred laboratory lactating mice isreached at 8–9 young, according to studies of MF1 and Swiss-Webster mice (Crl:MF1, Crl:CFW(SW); Hammond and Dia-mond 1994; Johnson et al. 2001). The experiment was designedto include two groups with litter sizes below those of the food-intake limit (nonreproductive females and females with threepups) and three groups of females at or above the litter size atwhich food intake can no longer be increased (8, 13, or 18pups). One of these groups was at the average litter size forthis strain of mouse (13 pups), and one was near maximumnatural litter size for this strain (18 pups). The inclusion of thelatter group was essential for examining the response of femalesto especially high demand. I focused on bone loss in the femurbecause the femur is a well-characterized location of bone mo-bilization during reproduction (Peng et al. 1988; Shahtaheri etal. 1999; Vajda et al. 1999a); in addition, I examined whole-body mineral content to determine whether the body as a wholemimicked these bones. I predicted that the amount of mineralin the specified bones and body would decrease as litter sizeincreased from 0 to 8 pups but then be similar to that for 8-pup litters among other treatment groups. Following the con-clusions of previous work, total body fat was also measured forcomparison.

Because a reduction in the mineral content of bone in adultanimals is usually related to increased risk of fraction (Currey1988; Boskey and Coleman 2010); Landete-Castillejos et al.2012, I predicted that the resistance of the femur to fracture(i.e., breaking load) would decrease with litter size up to eightpups and then plateau, the predicted mineral resorption limit.

Material and Methods

Animal Care

All live-animal methods described here were approved by Au-burn University’s Institutional Animal Care and Use Com-mittee (2008-1471). Fifty-five 8–9-wk-old ICR mice were ob-tained from Harlan Laboratories (Prattville, AL; 43 females, 11males). Mice were maintained on a 14L : 10D cycle at 24� �

C. Females were paired in -cm boxes maintained on1� 30 # 30a ventilated rack. All animals were fed Teklad rodent breederdiet 8626 ad lib. (Teklad Diets, Madison, WI; 3.5 kcal/g me-tabolizable energy, 21.3% protein, 10.5% fat, 3.2% crude fiber,1.1% calcium, 0.98% phosphorus). Food intake of all animalswas quantified weekly by recording the mass of food offeredand residual mass of food 1 wk later. Weekly intake was con-verted to daily intake by dividing by 7. Females were randomlyassigned to one of four treatment groups before breeding. The

Bone Mobilization and Cannibalism during Reproduction 387

Table 1: Sample size, litter size at birth and at weaning, and incidence of cannibalism bytreatment group (assigned litter size)

Assigned litter size NLitter sizeat birth

Litter sizeat weaninga

Proportion offemales

cannibalistic

Mean number of pupscannibalized � SEM

(range)

3 9 13.1 � .6 3.0 0/9 08 10 12.2 � .6 8.0 0/10 013 8 13.0 � .4 12.8 � .3 1/8 .3 � .3 (0–2)18 9 11.8 � .9 15.4 � .7 7/9 2.0 � .7 (0–6)

aAccounts for cannibalized pups and pups that died but were not consumed.

treatment name was based on the number of young each femalewas assigned on the day of parturition. Sample sizes per groupare described in table 1. Females were initially paired in a boxwith one other female assigned to a different treatment group;a single male was added to each box for approximately 5 d formating. Five males were introduced to 10 females on any givenday; introductions were staggered by 3–5 d. Females were sep-arated into individual boxes approximately 7 d before partu-rition. Natural litter size ranged from 8 to 16 young, with anaverage of (SEM); litter size at birth did not differ12.5 � 0.3among groups (general linear model: , ; ta-F p 1.02 P 1 0.393, 32

ble 1).To achieve the assigned litter sizes, pups were added or re-

moved from each litter on the day of parturition. Each pupwas assigned a number, and then a random-number generatorwas used to select pups for removal. Cross fostering was in-evitable to achieve the target litter sizes. The effects of crossfostering on females was controlled for by including cross-fostered pups in all treatment groups. Following the manipu-lation, one-third of each litter was born to a different female.On three occasions, only one female gave birth within a 24-hperiod. The size of these litters was adjusted without crossfostering. Pups that were not assigned to a litter on the day ofparturition were euthanized with carbon dioxide.

Six randomly selected pups from each litter (all pups forlitter sizes of three) were marked for identification, retrievedbriefly to measure body mass and tail length on days 1, 7, 14,and 21 postpartum, and analyzed for nutrient composition atweaning (hereafter “select pups”). The number of offspring ineach litter was recorded daily to account for deaths and inci-dents of cannibalism. Cannibalism is common among captiveand free-ranging mice (Labov et al. 1985), and thus this naturalprocess was allowed to occur by leaving dead pups in the boxfor up to 24 h after they were first observed. Pup carcasses thatshowed signs of discoloration indicative of rotting were re-moved. The disappearance of pups is described as an incidenceof cannibalism because it is not known whether the femalekilled the pup or whether the pup died of natural causes beforethe female consumed it. On day 21 postpartum, the typical dayof weaning in lab mice, all females and their litters were eu-thanized with carbon dioxide. This occurred approximately 3h after food was removed from the box in an effort to reducethe effect of intestinal fill on the final body mass of the animals.

After euthanasia, the animals were weighed, the carcasses weredesanguinated, and dissections were completed within 30 min.The major internal organs and both femurs were removed fromthe adults shortly after euthanasia. The femurs were removedby pulling the skin from the lower half of the body, carefullybisecting the muscle and connective tissues surrounding thehead of the femur, dislocating the head of the femur from theacetabulum, removing the bulk of the muscle tissue from thefemur, and finally bisecting the ligaments surrounding the kneeuntil the lower half of the limb could be freed from the femur.The pups were frozen immediately at �20�C and saved for laterdissection. The six select pups from each litter were dissectedand prepared for compositional analyses at a later date. Residualdigesta in the stomach and intestine of adults and pups wascleared by manual expression at the time of dissection.

Laboratory Analyses

All tissues, excluding the removed bones, were dried to a con-stant mass in a forced-convection oven (Binder drying ovenFED 115-UL, Binder, Great River, NY) at 60�C. All dried tissuesfrom the same individual were combined and homogenized.The homogenate was subsampled (0.5 g), and fat extractionswere completed in duplicate. Neutral lipids were extracted withpetroleum ether in a Soxhlet apparatus until samples reachedconstant mass (12 h). The total fat content of each replicatewas determined on the basis of the difference in mass betweenthe pre- and postextracted samples.

The right femur of each mouse was cleaned of residual mus-cle tissue after being soaked in nanopure water in an ultrasonicbath for 32 min. Femoral data for two individuals were basedon the left femur because the right femur was damaged or lost.Fat was extracted from cleaned bones in the Soxhlet as describedabove. Although many investigators use a combination of polarand nonpolar solvents and break bones to remove fat from themarrow, only ( ) more fat was extracted1.05% � 0.37% n p 10with a 2 : 1 petroleum ether–acetone solution than with petro-leum ether alone. There was no difference in the amount offat extracted between bones that were broken and those thatwere left intact (one-tailed t-test: , ). Thus,t p 0.45 P p 0.344

for consistency with other tissues, all extractions were com-pleted with 100% petroleum ether while keeping the bonesintact to avoid inadvertent loss of mineralized tissue. Any re-

388 W. R. Hood

sidual tissue remaining on the bone after ether extraction wasremoved before ashing.

All fat-extracted bone samples and whole-body homogenates(0.5 g) were ashed in a muffle furnace (Fisher Scientific IsotempMuffle Furnace, Dubuque, IA) at 550�C, with the bones ashedfor 24 h and whole-body samples ashed for 12 h. The percentageof ash in each sample was calculated as the ash content of thesample/fat-free dry mass of the sample before ashing. Ashedsamples were digested in 70% trace metal–grade nitric acidwith one of two methods because of an instrument malfunc-tion. The adult femur samples were digested in a microwavedigestion unit (Speedwave MWS-2, Berghof, Eningen, Ger-many). The microwave temperature was increased gradually to200�C over 15 min, held at 200�C for 15 min, and then de-creased to room temperature over the final 15 min. The re-maining samples were also digested in 70% nitric acid but wereheated on a dry-block heater at 100�C for 1 h. Finally, digestswere diluted for final mineral analysis. All concentrations weredetermined by mass. The calcium concentration of the whole-body and bone samples was measured as an indication of thedegree of bone calcification (Currey 2002), and the sodiumcontent of the whole-body and bone samples was also mea-sured. Sodium can also be stored in bone (Widdowson andDickerson 1964), and thus we evaluated this element becauseit has been shown to be limiting in herbivorous species (Be-lovsky and Jordan 1981; Christian et al. 1993; Grasman andHellgren 1993). Calcium and sodium concentrations were de-termined by inductively coupled plasma optical emission spec-trometry (Perkin Elmer Optima 7300DV, Waltham, MA; wave-lengths: nm; nm). The mineral2� �Ca p 317.93 Na p 589.52contents of whole-body samples run in duplicate ( ) weren p 5compared among digestion methods; the results of these anal-yses were statistically equivalent (two-way ANOVA, partialFmethod: , ). Nevertheless, within any given sta-F p 0.00 P p 0.99tistical test, the method of digestion was similar. All total-body-composition data were corrected for the composition of theremoved femurs (2 # the mineral content of one femur).

Bone strength was measured for the left femurs. The strengthof a bone is largely dependent on two effects: structural vari-ables, such as cortical thickness and number of trabeculae, andmechanical quality of the material (Currey 2002; Davison etal. 2006). In small bones, such as those of mice, it is impossibleto test intrinsic mechanical properties. Thus, as a compositemeasure of mechanical performance of the bone, a whole-femurbending test was employed (Turner and Burr 1993) using aMini Bionix Mechanical Testing System (model 858, MTS Sys-tems, Minneapolis, MN). All bones were wrapped in phos-phate-buffered saline-soaked gauze for at least 3 d before testingthe load required to break each bone (hereafter “breaking load”;Turner and Burr 1993). The gauze and overlying muscle tissuewere removed before testing. Three-point bending tests werecompleted with a 100-N load cell. Each bone was centered ontwo points 9.5 mm apart (gauge length). Force was applied tothe midshaft of the bone at 0.05 mm/s. Minimum force re-quired to break the bone was recorded.

Calculations and Statistics

All statistical analyses were completed in SAS (ver. 9.1.3, SASInstitute, Cary, NC). Food intake was compared among treat-ment groups on day 8 of lactation with ANOVA. Day 8 wasselected for comparison because it was near peak lactation butstill before pups consumed solid food, ensuring that food intakereflected only intake by the adult female. Because pups firstconsume solid food on day 15 of lactation (Hammond andDiamond 1992) and food intake was based on a 7-d average,it was necessary to compare groups 7 d before day 15.

All nutritional analyses were completed in duplicate; the av-erage of these replicates was used for all statistical comparisons.Data were excluded when the coefficient of variation amongreplicates exceeded 10%, resulting in a small variation in samplesize among analyses. All comparisons of maternal, pup, andlitter characteristics among treatment groups were initiallymade with ANCOVA, with litter size at birth included as acovariate. When litter size at birth was not significant, it wasremoved from the model and results based on an ANOVA werepresented. All tests distinguished groups with a Tukey’s test formultiple comparisons. Maternal characteristics included per-centage of body fat, percentage of total body ash, whole-bodyconcentration of Ca2� and Na�, mass and ash content of theright femur, and the Ca2� and Na� concentrations of the fe-murs. Quantifying mineral allocated to the litter was compli-cated by cannibalism. Disappearance of the cannibalized bodiesmade it impossible to directly measure the mass and bodycomposition of these young. Thus, both minimum and max-imum mineral allocation are given for each litter. Maximummineral allocation was estimated on the basis of assigned littersize (an overestimate where cannibalism occurred), and min-imum mineral allocation was estimated on the basis of the littersize at weaning (an underestimate for litters reduced by can-nibalism). To determine litter mass at weaning, the sum of thebody masses of all pups within each litter was determined atthe termination of the study (21 d postpartum). When can-nibalism occurred, total litter body mass was estimated on thebasis of the assigned litter size by adding the estimated massof the consumed young to the total litter mass; the estimatedmass of the consumed young was based on the average of allother young in the litter. The total ash content of the litter wasbased on the average of the six select pups multiplied by theassigned litter size. Independently, the same calculations wereperformed on the basis of litter size at weaning. Proportionaldata do not display a normal distribution; thus, proportionaldata were normalized with an arcsine transformation (Zar2010).

In nearly all instances of cannibalism, most (less a foot orshort length of the tail) or all of the offspring was consumed(W. R. Hood, personal observation). To determine whethercannibalism improved maternal skeletal condition, the rela-tionship between the number of pups consumed and the fem-oral ash content and femoral breaking load of females at wean-ing was assessed with linear correlation. This analysis and allsubsequent analyses were limited to females assigned 18 young

Bone Mobilization and Cannibalism during Reproduction 389

Figure 1. Food intake of females per day relative to assigned litter sizeon day 8 of lactation. Treatment groups that were statistically similarare labeled with the same letter within each bar. Adult females thatdid not reproduce are included as NR. SE bars are given.

because cannibalism was common only in this group. Differ-ences in offspring skeletal size and degree of ossification at thetime each pup was consumed were also expected to affect howmuch mineral females could potentially recover. To examinethis relationship, offspring size at the time of cannibalism wasestimated, and maternal femoral ash content was correlatedwith the sum of the skeletal sizes of the young consumed.Because tail length displayed a strong linear relationship withage before weaning, tail length could be used as a proxy forskeletal size (W. R. Hood, personal observation). Six pups perlitter were measured on days 1, 7, 14, and 21 postpartum. Pupgrowth rates differed among litters but not between male andfemale pups (repeated-measures ANCOVA with pup ID nestedwithin maternal ID: overall , ; maternalF p 60.2 P ! 0.00151, 137

ID: partial , ; sex: partial ,F p 11.5 P ! 0.001 F p 2.79 P 18 1

); therefore, linear-regression equations for tail length were0.09determined independently for each litter (all regressions 2r ≥

). To determine whether cannibalism enhanced femoral0.90mineral by reducing the cumulative mineral demand of thelitter, femoral ash content of females at weaning was correlatedwith nursing effort, the sum of the number of pups suckledfor each day of lactation (e.g., if 18 pups were suckled for 21d, nursing effort p , but if three pups were18 # 21 p 378consumed on day 11, nursing effort p � [(18 # 10) � (15 #

). And finally, to determine whether organic material11)] p 345(primarily collagen in the fat-extracted bone; Campo and Tour-tellotte 1967; Miller and Martin 1968) was being mobilizedfrom bone, the mass of organic material (fat-free dry mass ofthe femur � mass of the ash in the femur) was comparedamong groups by means of ANOVA.

Results

Food intake of females on day 8 of lactation increased withassigned litter size to eight offspring ( , ).F p 45.9 P ! 0.0014, 38

However, there was no difference in intake among groups as-signed 8, 13, or 18 young (fig. 1).

Litter size at birth had a significant effect on litter size atweaning (partial , ) but had no effect onF p 9.7 P p 0.0041

maternal body composition or bone strength ( , all ma-P 1 0.22ternal comparisons). The results of ANOVA suggest that ma-ternal body fat ( , ) but not total body ashF p 6.03 P p 0.0024, 38

of females ( , ) varied with assigned litterF p 1.09 P p 0.3744, 38

size (fig. 2A, 2B). Total body fat was statistically similar betweennonreproductive females and females assigned three and eightyoung (fig. 2A), although means suggest a decrease in residualfat with litter size of 13 young. Body fat did not differ betweenfemales assigned 13 and 18 offspring (fig. 2A). Total-body con-centration of calcium was higher in nonreproductive femalesthan in females assigned 18 young, but all other pairwise com-parisons were similar ( , ). Total body so-F p 8.24 P p 0.0014, 38

dium did not differ among groups ( , ; fig.F p 0.46 P p 0.7614, 38

2C).Treatment had several significant effects on bone character-

istics. The mass of the femurs decreased with increasing littersize ( , ; fig. 3A), whereas the concentra-F p 5.31 P p 0.0024, 38

tion of ash in the femurs decreased with increasing assignedlitter size to 13, but females assigned 18 pups displayed ashconcentrations greater than those of females with 13 pups andsimilar to those of all other groups, including females who didnot reproduce ( , ; fig. 3B). Changes in theF p 5.63 P ! 0.0014, 38

concentration of ash in the femurs reflect changes in the min-eral and not the organic content of the bone because the massof ash in the femur followed a pattern similar to ash concen-tration ( , ), while the organic content ofF p 8.94 P ! 0.0014, 38

bone did not differ among groups ( , ).F p 1.94 P p 0.124, 38

Following femoral ash, concentrations of bone Ca2� declinedwith increasing litter size to 13 and then increased again infemales assigned 18 young ( , ; fig. 3C).F p 4.66 P p 0.0044, 38

There was no difference among groups in the Na� content ofthe bone ( , ; fig. 3C). Bone breaking loadF p 0.27 P p 0.8974, 38

differed significantly among treatments, with the load requiredto break the femur decreasing with increasing litter size up to13 young, but there was no difference between females assigned13 and 18 young (ANOVA, , ; fig. 4).F p 5.30 P p 0.0023, 35

Nine of 36 (25%) reproductive females cannibalized off-spring. The number of offspring cannibalized was significantlygreater among females with an adjusted litter size of 18 thanin all other groups (ANOVA, , ). No fe-F p 8.60 P p 0.0023, 32

males assigned 3 or 8 pups consumed offspring, and only onefemale in the group with 13 pups consumed pups (table 1).The number of young cannibalized per female ranged from 1to 6. Four dead pups were found in the boxes of females with18 young. One of these was missing the next day, and the otherthree dead pups were removed from the box the day after theywere observed. Despite cannibalism, litter sizes remained dif-ferent among groups (ANCOVA, , ; tableF p 337 P ! 0.0014, 38

1), and litter size at birth had a significant effect on final littersize (partial , ).F p 9.7 P p 0.004

The timing of cannibalism varied among individuals. All butone cannibalistic individual consumed pups during early lac-tation (days 1–8), including the one female with an adjusted

390 W. R. Hood

Figure 2. Body composition of females at weaning relative to assignedlitter size. Data include total body fat (% dry mass basis [DMB]; A),total body ash (% fat-free dry mass [FFDM] basis; B), and total calcium(black bars) and sodium content (gray bars; mg/g, FFDM; C). Treat-ment groups that were statistically similar are labeled with the sameletter within each bar. Adult females that did not reproduce are in-cluded as NR. SE bars are given.

Figure 3. Femoral mass (A), percentage of femoral ash (free-fat drymass [FFDM]; B), and percentage (FFDM) of femoral calcium (blackbars) and sodium (gray bars; C) for females at weaning relative toassigned litter size. Treatment groups that were statistically similar arelabeled with the same letter within each bar. Adult females that didnot reproduce are included as NR. SE bars are given.

litter size of 13 that cannibalized offspring. For half of thesefemales, the only incidence of cannibalism occurred duringearly lactation, while half of the females also consumed youngat peak lactation (days 9–17, as defined by Konig et al. 1988;Hammond and Diamond 1992; Speakman and McQueenie1996); only one pup was consumed during late lactation. Three

pups assigned to litters of 18 died but were not cannibalized;these pups died during peak ( ) and late ( ) lactation.n p 2 n p 1

Pup mass decreased with assigned litter size (ANCOVA: over-all: , ; partial: , ; fig. 5A),F p 76.9 P ! 0.001 F p 89.4 P ! 0.0014, 31 3

whereas litter mass increased with assigned litter size up to 13young, but no difference was found in these variables betweenadjusted litters of 13 and 18 (ANOVA: , ;F p 77.9 P ! 0.0013, 31

Bone Mobilization and Cannibalism during Reproduction 391

Figure 4. Femoral bone breaking load (N) relative to assigned littersize. Treatment groups that were statistically similar are labeled withthe same letter within each bar. Adult females that did not reproduceare included as NR. SE bars are given.

fig. 5D). The individual ash, Ca2�, and Na� content of pupsdid not differ among groups (ANOVA: ; fig. 5B, 5C).P 1 0.4The cumulative ash content of the litter followed the samepattern as body mass (ANOVA: , ; fig. 5B).F p 61.0 P ! 0.0013, 29

Cumulative litter Ca2� (ANOVA: , ; fig. 5C)F p 81.8 P ! 0.0013, 26

and cumulative litter Na� (ANCOVA: overall: ,F p 53.44, 24

; partial: , ) increased with assignedP ! 0.001 F p 68.0 P ! 0.0013

litter size, with the total mineral allocated to the litter differingamong all four treatment groups. Because allocation to can-nibalized young could not be determined, actual allocation laysomewhere between allocation based on assigned litter size andallocation based on litter size at weaning. Although allocationwas slightly lower in females with 13 and 18 young when es-timated from litter size at weaning, total mineral allocation byfemales with 18 young did not drop below that of females with13 offspring: litter mass (ANCOVA: overall: ,F p 38.6 P !4, 30

; partial: , ; fig. 5C), ash (ANCOVA:0.001 F p 51.3 P ! 0.0013

overall: , ; partial: , ; fig.F p 32.9 P ! 0.001 F p 73.6 P ! 0.0014, 28 3

5E), Ca2� (ANCOVA: overall: , ; partial:F p 50.0 P ! 0.0014, 25

, ; fig. 5F), and Na� (ANCOVA: overall:F p 64.8 P ! 0.0013

, ; partial: , ) values wereF p 34.4 P ! 0.001 F p 41.0 P ! 0.0014, 24 3

statistically similar among assigned litter sizes of 13 and 18 forall comparisons.

There was no correlation between femoral ash content andnumber of pups consumed for females assigned 18 young( , ; fig. 6A). Findings for bone breaking loadF p 1.87 P p 0.2147

were comparable ( , ). The results of thisF p 1.82 P p 0.2197

regression were not enhanced when I accounted for offspringskeletal size at the time of cannibalism based on the estimatedtail length ( , ; fig. 6B). Finally, the relation-F p 0.43 P p 0.5347

ship between femoral ash and number of pup nursing days wassignificant ( , , ; fig. 6C), suggesting2F p 5.52 P p 0.051 r p 0.447

that cannibalism likely reduced maternal femoral bone deple-tion by reducing the burden of supplying milk to the offspring.

Discussion

When female mice were challenged with larger litter sizes, theyincreased the amount of fat and minerals that they mobilizedfrom reserves. This trend in resource allocation occurred onlyup to a litter size of 13, above which females were faced withdemands that may have exceeded their ability to further in-crease allocation. Female mice with litters of 18 young adopteda pattern of nutrient allocation that limited the loss of theirown fat and bone mineral reserves. Cannibalism of young wascommon among females with these large litters; this behaviorimproved the femoral mineral content and thus the skeletalcondition of these females that had faced an especially highdemand.

Somatic Tissue Mobilization

The mineral mobilization imposed by the average litter size forthis strain of mouse (13) approximated a limit to the amountof mineral that females would mobilize for their reproductiveeffort, at least under the conditions of this experiment. In lab-oratory mice, peak lactation occurs before weaning (9–17 dpostpartum; Konig et al. 1988; Hammond and Diamond 1992;Speakman and McQueenie 1996). Because the nutritional de-mand of milk production declines when peak lactation precedesweaning (Oftedal and Iverson 1995), recovery of some somatictissue before weaning is possible. Thus, femoral ash concen-tration displayed by females with 13 young may have been lowerat peak lactation. Factors such as relative maternal age, numberof previous reproductive bouts, availability of dietary calcium,and disease may contribute to differences in the relative amountof bone loss that is tolerated (Peng et al. 1988; Bowman andMiller 1999; Odiere et al. 2010). Because young virgin micewere used for this study and mineral loss from bone duringreproduction decreases with parity in rats (Bowman and Miller1999), it is likely that the patterns described here were morepronounced than would be observed in older, experiencedfemales.

Femoral breaking load decreased with increasing litter sizebut was not statistically different among females with the largestlitter sizes. Thus, the greater bone mineral in females with 18young may not translate to a functional increase in bonestrength. Reduced bone mineral contents and densities andreduced bone strength in females with large litters have beendescribed previously (Peng et al. 1988; Tojo et al. 1998).

Unlike the femur, the whole-body mineral (ash) content offemales was similar among groups. Total-body and femoralsodium contents were also similar, suggesting that sodium avail-ability was not limiting for females in this study. Total-bodycalcium content differed only between females that did notreproduce and those with 18 young, with the latter havingsignificantly less calcium. Error alone could obscure reproduc-tive losses when mineral mobilization is localized. For example,with only 2%–3% of the total calcium in a mouse’s body storedin the femur (Ca2� mass in femur/total body Ca2�), changesin femoral mineral could be hidden with !2% error in whole-

392 W. R. Hood

Figure 5. Allocation of nutrients by females to individual young and the full litter. Data include the body mass, ash content, and calcium andsodium content of individual pups (A, B, and C, respectively) and of the full litter (D, E, and F, respectively). The amount of nutrients thatfemales allocated to dead young and allocated to and recovered from cannibalized young could not be determined. Thus, allocation to thelitter was estimated two ways: (1) as if all pups had survived (black bars; i.e., assigned litter size # average values for pups in litter) and (2)as if the female did not invest in dead or consumed pups (gray bars; i.e., litter size at weaning # average values for pups in litter). Treatmentgroups that were statistically similar are labeled with the same letter within each bar. Adult females that did not reproduce are included as NR.SE bars are given.

body analyses. Losses of mineral during lactation are commonin several appendicular bones and the vertebrae (Rasmussen1977; Zeni et al. 1999); comparable losses are also likely forthe mice in this investigation. Losses of bone mineral fromother regions have also been described during lactation, in-cluding the ribs, skull, mandible, hyoid, and pelvis (Benzie etal. 1955). Cumulative changes in whole-body mineral contenthave been described during periods of high demand (Widdow-son and Dickerson 1964; Hood et al. 2006).

The total-body fat content of females declined with increas-ing litter size up to the average litter size for this strain ofmouse, corresponding to the changes in bone, but that of fe-males assigned the largest litters was similar to that of femaleswith average-sized litters. Allocation to the litter reflected thispattern, with body mass and nutrient accretion increasing toa litter size of 13. Because the nutrient content of cannibalizedpups could not be quantified, nutrient allocation by females

bearing the largest litters could be characterized only as a rangeand not as an exact value. Nevertheless, it is probable thatallocation remained similar for litter sizes greater than 13. Incontrast, allocation to individual pups declined linearly withincreasing litter size, as has been described in several othermammals (Mattingly and McClure 1982; Konig et al. 1988;Sikes 1995; Kunkele 2000; but see Kenagy et al. 1990). It isclear that the food-intake limit alone does not limit allocationto the litter. Females compensate for limited intake by increas-ing body fat and mineral mobilization from the femur betweenlitter sizes of 8 and 13, but the limit to fat mobilization beyondthe average litter size suggests that females may not mobilizetheir own resources beyond the point at which they will likelyjeopardize their own survival (Clutton-Brock 1991).

Allocation of minerals to the full litter followed a patternsimilar to that for body mass, but mineral allocation to indi-vidual young was comparable across litter sizes rather than

Bone Mobilization and Cannibalism during Reproduction 393

Figure 6. Relationship between the ash content of females’ femurs atweaning and the number of pups that each female cannibalized (A),the cumulative skeletal size of pups consumed, calculated by the sumof their tail lengths at the time of death (B), and the nursing effort ofthe female, based on the number of pups suckled for all 21 d of lactation(C). Lines show significant regressions at .a p 0.05

declining as litter size increased. It is possible that total energytransfer to the litter was more limiting to offspring developmentthan the minerals necessary to build bone. In general, changein body mass during growth is more sensitive than the skeletonto variation in resource availability (Hoying and Kunz 1998).

Nutrient Recycling, Nutritive Gain by Young, andSavings in Females

There are three possible explanations for higher levels of bonemineral in females suckling the largest litters than in those withaverage-sized litters in this study: (1) reduced maternal mineralallocation to the litter, (2) mineral recycling, and/or (3) in-creased mineral availability. Offspring cannibalism was consid-ered as a method of mineral recycling. The number of youngcannibalized was not significantly correlated with femoral ashcontent or bone-breaking load for females assigned 18 young.However, there was a significant relationship between femoralash content and nursing effort for these females; this relation-ship explained 44% of the variation in femoral mineral contentat weaning. These results suggest that reduced bone mineralloss by females assigned the largest litters was largely associatedwith a reduction in maternal mineral allocation. Owing to thesmall sample size, the possibility that mineral recovered fromconsumed young contributes to maternal bone mineralizationshould not be discounted. Knight et al. (1986) described therelationship between litter size and milk yield in mice. In earlylactation, females suckling 18 young produced significantlymore milk than females with 10 young. At peak lactation, how-ever, these differences diminished. Because milk compositionis not expected to vary for females suckling large litters (Fior-otto et al. 1991), it can be implied that nutrient transfer at peaklactation was likely similar among females assigned 13 and 18offspring regardless of the incidence of cannibalism. Thus, can-nibalistic females most likely incurred the greatest mineral sav-ing but reduced their litter size during early lactation. Increasedmineral absorption by the intestine and decreased mineral ex-cretion by the kidney are common adaptations to the increasedmineral demand of reproduction (Kovacs and Kronenberg1997). These variables were not examined here, but the effectof litter size on these variables is worthy of investigation.

Bone Mobilization and Reproductive Performance

The question of what limits the reproductive performance ofan individual has puzzled biologists for decades. Many ecolog-ical and evolutionary physiologists have approached this prob-lem by looking for intrinsic constraints on the total amountof energy that females transfer to their young. For example,Hammond and Diamond (1992) and others (Drent and Daan1980; Scantlebury et al. 2000) have considered the assimilationcapacity as a possible constraint on energy absorption (central-limitation hypothesis), Brown et al. (2004) suggested that thearchitecture of the circulatory system responsible for nutrientdelivery to the tissues could limit energy expenditure (the met-abolic theory of ecology), and Hammond and colleagues (Ham-

mond and Diamond 1994; Hammond et al. 1994, 1996) andSpeakman and colleagues (Johnson et al. 2001; Krol and Speak-man 2003) have also considered that the synthetic capacity oftissues (peripheral-limitations hypothesis) could also limit en-ergy transfer during lactation. More recently, Krol et al. (2007)have proposed that an animal’s ability to dissipate heat maybe an important constraint on maximum performance during

394 W. R. Hood

reproduction. My observations suggest that risk of detrimentalmineral mobilization is yet another possible constraint onreproduction.

It is particularly fascinating that cannibalism played a rolein improved skeletal condition for females at weaning. Thepossibility that cannibalism could have been a response to theincrease in demand placed on females associated with the ex-perimental manipulation cannot be ruled out, nor is it possibleto rule out the possibility that cannibalistic females targetedalien pups (although limited data suggest otherwise). Never-theless, the changes in maternal condition in cannibalistic fe-males remain intriguing, particularly given that cannibalism iscommon in rodents (Hrdy 1979; Labov et al. 1985; Tuomi etal. 1997; Ebensperger 1998). It is generally assumed that can-nibalistic individuals gain a nutritional reward (Orians and Jan-zen 1974), but tests of the effect of cannibalism on individualcondition were lacking before this investigation (Hrdy 1979;Labov et al. 1985). Here I showed that cannibalistic femalesimproved the mineral content of their femurs by reducing theoverall demand of suckling. After weaning, bone goes througha period of rapid anabolism (Bowman and Miller 1999), andthus cannibalism likely improves a female’s immediate prob-ability of survival during lactation rather than having a sus-tained effect between reproductive events.

Acknowledgments

I thank the tremendous team of undergraduates that workedon this project—particularly Michael Drake, Kasey Gardner,Carolyn Kuhn, and Morgan Luger—for assistance with animaland lab work. I would also like to acknowledge C. Pinkert foruse of the ventilated rack; Auburn University Lab AnimalHealth; and the Hood and Hill laboratories for comments onthe manuscript. This manuscript was improved by the valuablecomments of three anonymous reviewers. This project wasfunded by Auburn University’s College of Sciences andMathematics.

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