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Non-invasive study of changes in body composition inrabbits during pregnancy using X-ray computerized

tomographyGábor Milisits, Róbert Romvári, Antonella Dalle Zotte, Zsolt Szendrõ

To cite this version:Gábor Milisits, Róbert Romvári, Antonella Dalle Zotte, Zsolt Szendrõ. Non-invasive study of changesin body composition in rabbits during pregnancy using X-ray computerized tomography. Annales dezootechnie, INRA/EDP Sciences, 1999, 48 (1), pp.25-34. �hal-00889779�

Original article

Non-invasive study of changes in body compositionin rabbits during pregnancy using X-ray

computerized tomography

Gábor Milisitsa Róbert Romvária Antonella Dalle Zotte

Zsolt Szendrõa

a Pannon Agricultural University, Faculty of Animal Science,Guba S. u. 40, 7401 Kaposvar, Hungary

Dtparhmento dí Scienze Zootecniche, Università di Padova, Agripolis,35020 Legnaro (PD), Italy

(Received 20 January 1998; accepted 25 May 1998)

Abstract - The objectives of this study were: 1 ) to follow changes in body composition duringpregnancy using a non-invasive method; 2) to compare the results obtained with X-ray computerizedtomography (CT) and with the slaughter method; and 3) to study the influence of litter size onchanges in body reserves in pregnant does. Seventeen pregnant Pannon White rabbit does wereexamined by means of CT on the day of insemination, on days 14, 21 and 28 of pregnancy, and a fewhours after parturition. At comparable time points, non-pregnant does (n = 26) were also scanned asa control group. During the CT procedure 27 images (scans) were taken from all of the animals in thebody region from the scapular arch to the femoral-tibial articulation. The scans were processed bymeans of the computerized imaging technique, to obtain three-dimensional (3D) histograms thatrepresented the changes in the water content, and in the fat and muscle tissues of the does. On the dayafter parturition all the does were slaughtered and the fat content of their empty bodies was sub-jected to chemical analysis. In the first 14 days of pregnancy little change was detected in bodycomposition. Thereafter, in the abdominal region a marked increase was noted in densities corre-sponding to water and water-rich tissues, in correspondence with the growth of uterine contents.From day 21 of pregnancy a decrease in fat was noted in the pregnant does and an increase in the samereserves in the non-pregnant control group. To compare the fat content of animals in different physio-logical states so-termed fat indices were created by relating pixels corresponding to fat densities tothe total number of pixels. In the same way muscle indices were created to demonstrate the changesin the muscle reserves during the experimental period. To test the accuracy of estimation by fatindices a regression analysis was carried out between the index numbers and the fat content sub-jected to chemical analysis. The result of this analysis showed a high correlation between the para-

* Correspondence and reprintsE-mail: milisits@atk.kaposvar.pate.hu

meters examined. The same fat indices demonstrated a higher mobilisation of body reserves in doeswith higher litter size than in those with smaller litters. © Elsevier / Inra

X-ray tomography / body composition / pregnancy / rabbit / reproduction

Résumé — Étude de l’évolution de la composition corporelle au cours de la gestation chez lalapine, à l’aide d’une méthode non invasive : la tomographie aux rayons X assistée par ordi-nateur. La tomographie assistée par ordinateur (CT) a été utilisée pour : 1) déterminer la variationde la composition corporelle des lapines durant leur gestation ; 2) comparer les résultats obtenus à l’aidede la CT et avec la méthode de l’abattage comparatif ; 3) étudier l’influence de la taille de la portéesur l’évolution des réserves corporelles pendant la gestation. Dix-sept lapines ont été explorées àcinq moments (insémination, 14, 21 et 28 j de gestation et quelques heures après la mise bas). Vingt-six femelles non gestantes (NP) ont été examinées en même temps, comme groupe de référence.Toutes les femelles ont été sacrifiées le lendemain de la mise bas. Pendant les premiers 14 j de ges-tation, les changements de composition corporelle ont été très faibles. Toutefois, au niveau de larégion abdominale une augmentation de densité a été observée, correspondant à la croissance des tis-sus utérins et des foetus. À partir du 21e jour de gestation, les réserves corporelles lipidiques diminuentchez les femelles gestantes tandis que pendant cette même période elles augmentent chez les femellesnon gravides. Pour comparer les quantités des tissus adipeux et musculaire chez les femelles soumisesaux différents états physiologiques, des « Indices du gras » et des « Indices musculaires » ont été créés.L’analyse de régression a mis en évidence une étroite corrélation entre ces indices et les contenus dugras analysé par voie classique. Les mêmes Indices ont démontré que plus les femelles ont une por-tée de grande taille, plus la mobilisation de leurs réserves corporelles est forte. @ Elsevier / Inra

tomographie aux rayons X / composition corporelle / gestation / lapin / reproduction

1. INTRODUCTION

It is widely known that the body compo-sition of rabbit does is markedly affectedby pregnancy and lactation, as described byParigi Bini et al. [4, 5] and Xiccato et al.[ 17]. After the 21 st day of pregnancy, foetaldevelopment is accelerated and maternalreserves become progressively depleted [3].The initially exponential growth of foetusesgradually changes to linearity from day 23[6] and the food intake of the does becomesprogressively restricted due to the increasingsize of the reproductive tract and the uterinecontents. The usual energy content of com-mercial diets (10-12 MJ DE/kg) does notmeet the requirement of a doe carrying morethan four foetuses and results in the mobili-sation of body reserves [1]. ] .

In the present study we set three mainobjectives: 1 ) to follow the changes in bodycomposition of pregnant does using a non-

invasive method; 2) to compare the X-rayCT method with the chemical analysismethod; and 3) to study the influence of lit-ter size on changes in body reserves during gpregnancy.

In previous experiments changes in bodycomposition were only studied using thecomparative slaughter method; therefore theuse of a non-invasive method (CT in thiscase) constitutes the originality of this work.

2. MATERIALS AND METHODS

2.1. Animalsand experimental procedure

A total of 43 nulliparous Pannon White rabbitdoes were kept in individual cages (80 x 50 cm)and fed ad libitum with a commercial pelleteddiet (DE 10.30 MJ/kg, crude protein 17.5 %,ether extract 3.6 %, crude fibre 12.4 %), devel-oped for breeding does. Twenty-seven of these

animals were artificially inseminated at the age of5 months and 3854 (S.D. 198) g mean liveweight. Seventeen of them became pregnant.

The feed consumption and the live weight ofthe does were recorded daily, but due to the12-h fast before CT scanning only one value perweek is represented in figures I and 2.

2.2. CT scanning procedures

CT scanning of pregnant does was performedon the day of insemination, on days 14, 21 and 28of pregnancy, and a few hours after parturition.At the same time 26 non-pregnant does were alsoscanned as control. After a 12-h fast the doeswere anaesthetised with 4 mg/kg b.w. Rompun(Bayer) i.m., and fixed with belts in a stretchedposition in special plexiglass containers [ 10!. Inthis position movements were restricted and thelegs were well separated from the rump. Three

rabbits were scanned simultaneously in the samecontainer. A total of 27 scans were taken fromeach animal by means of a Siemens SomatomDRG Tomograph, with 8 mm thickness and withdifferent distances between the scans, depend-ing on the length of the vertebral column. Thescanning range extended from the scapular archto the end of the femur, and the pictures wereprocessed in accordance with Romvdri et al. [8].The extreme density values (e.g., air and bone)were excluded and those corresponding to fatand muscle were retained, i.e., the range from- 200 to +200 of the Hounsfield scale. This rangewith its 400 values was reduced to 40 so-termedHounsfield variables (HUv) by summarising thenumber of pixels corresponding to 10 consecutivedensity values on the scale. These variables wereused to calculate fat and muscle indices and to

plot three-dimensional histograms to demon-strate changes in body composition. The his-tograms were plotted by negative exponentialinterpolation, using SYSTAT [14] software. To

demonstrate changes in body compositionbetween two time points, so-termed differentialhistograms were plotted by subtracting the formerfrom the latter.

2.3. Slaughterand analytical procedures

At the end of the experimental period, i.e.,on the day after parturition, both experimentaland control animals were slaughtered. Afterremoval of the gut content, the rest of the body,including the empty uterus, was cut into piecesand homogenised by grinding it twice. Fat con-tent was determined by ether extraction in accor-dance with the Soxhlet method.

2.4. Fat indices, muscle indicesand statistical procedures

In order to compare the fat content of the doesin different physiological states, Fat Indices werecreated from CT pictures by relating pixels cor-responding to fat densities to the total numberof pixels according to the formula: (IHUv 7-16/ XHUv 1-40) x 100. In a previous study Romvdii[7] observed that pixels corresponding to HUvariables 7-16 were strongly correlated with thefat content subjected to chemical analysis, andtherefore these variables were used to form fatindices. To test the predictive value of theseindices, regression analysis (SPSS for Windows7.5 [ 13]) was performed between the fat indicesand the fat content of the body determined by achemical method. In this way muscle indiceswere also created, according to the formula:(IHUv 23-40 IHUv 1-40) x 100.

Statistical analysis was carried out using anal-ysis of variance [13], with a factorial modelaccounting for the fixed effect, the physiologicalstate (pregnant or non-pregnant). The effect oflitter size (small or large) was also tested. In thiscase only 13 of the 17 pregnant does were used,because the others had an extremely small (< 4)or an extremely large (> 9) litter size at kindling.The 13 does examined were allotted into two

groups according to litter size at kindling (group 1:n = 7, litter size: 4-6; group 2: n = 6, litter size:7-9).

3. RESULTS

3.1. Feed intake and body weight

During the first 16 days of the experi-mental period the daily food intake of preg-nant and control does did not differ signifi-cantly (figure 1); however, the pregnantgroup had a slight tendency to increaseintake with advancing time. After the accel-eration of the development of foetal tissues,the feed consumption of the pregnant doeslagged behind that of the control group !g-ure 1) and the difference reached the level ofsignificance (P < 0.05) on the 24th day ofpregnancy. From the 25th day the differ-ences between the two groups were highlysignificant (P < 0.001).

The body weight of both pregnant andcontrol does increased up to day 29 with-out significant difference. After kindling,the body weight of the experimental doesreturned almost to the initial level and, there-fore, it differed significantly (P < 0.001)from that of the control (figure 2).

3.2. Body composition

The body composition of the rabbit doeson the day of insemination is shown in fcg-ure 3. In the region of muscle tissue (HUv23-40) three peaks could be observed, rep-resenting three groups of skeletal muscles(hind leg, back and foreleg muscles). In theregion of fat tissue (HUv 1-18) the threesmaller peaks show the fat depositions inthe pelvic, perirenal and scapular regions.

Changes in body composition related topregnancy are shown in differential his-

tograms on figures 4 to 7. Little differencewas seen during the first 14 days of preg-nancy (figure 4), but a distinct peak appearedin the abdominal region between days 21 1and 28 (figure 5).

During the last third of pregnancy lossof fat tissue could be seen in the differen-tial histogram of figure 6. During the com-

parable experimental period the non-preg-nant does (figure 7) accumulated fat in thepelvic, perirenal and scapular regions.

Like the histograms, the calculated fatindices also showed the growth of fat tis-sue in the control group during the experi-mental period and its decrease in the preg-nant does during the last 10 days of preg-

nancy (figure 8). Inter-group differenceswere significant (P < 0.001) on days 28 and30 of pregnancy.

To test the predictive value of estimationwith these fat indices, regression analysiswas carried out between the index numbersand the fat contents determined by a chemi-cal method. The correlation was 0.93 and

0.91 for the pregnant and control does,respectively (figures 9, 10).

Unlike the fat indices, the muscle indicesshowed a decrease in muscle in the control

group during the whole experimental periodand in the experimental group during thefirst three weeks of pregnancy (figure 1 I).The ratio of pixels corresponding to mus-cle density values increased during the 4th

week in the pregnant group and decreased

again after kindling.As was mentioned in the Introduction,

the energy content of commercial diets usu-

ally allows only provision for four foetuseswithout major mobilisation of maternalreserves. It was expected, therefore, thathigher litter sizes would result in highermobilisation of body reserves. To test this

hypothesis, the does were allotted to twogroups according to litter size, and their fatcontent was estimated with the fat indices

(figure 12). In the larger (7 to 9) and smaller(4 to 6) litter size groups the fat content ofthe does decreased by 17.9 and 9.3 %,respectively, during the last 10 days of preg-nancy (P < 0.001 ).

4. DISCUSSION

The results obtained in the present studyare in agreement with previous research per-formed with classical slaughter techniques

[2, 3, 17]. During the first 2 weeks of preg-nancy, deposition of fat differed slightlybetween pregnant and control does, whereasduring the last third an intensive mobilisa-tion of fat took place in the maternal body tosupport the accelerated growth of the foe-tuses. A similar tendency was found bySt6rensen [12], who scanned goats duringpregnancy and lactation, and estimatedchanges in the volume of adipose tissue byCT scans, net energy balance and weightgain. Although each of the three methodshad errors and assumptions, CT scanningwas considered the most reliable one.

The decrease observed in the muscle tis-sue correlates to the increasing fat depositionin the control and pregnant does during thefirst three weeks of the experiment. The out-standing muscle index at day 28 of preg-nancy is explained partly by the growinguterus and partly by the finding that the den-sity of foetal tissues is close to that of mus-cle. After kindling, due to the removal ofthe uterine contents, the fat and muscleindices moved towards the original values.

An additional finding of the present studywas the demonstration of the development of

uterine, foetal and placental tissues. Thepeak observed in figure S is located closeto the line of water density (on the rim ofthe region considered to be muscle in thehost animal), which shows, that the foetal-placental unit is rich in water and protein.

The fat indices calculated could be usedto follow the changes in the fat content of thedoes during pregnancy. The accuracy of pre-diction by these indices of the fat contentsubjected to chemical analysis was very sim-ilar to that achieved by other authors [9, 11,15, 16] using other prediction equations.

The difficulty in obtaining higher correla-tions is due to the variability of animal bodycomposition, the limited number of test ani-mals and the error of chemical analysis.

In the present experiment we demon-strated that CT scanning can be used to fol-low in a non-invasive way the changes inbody composition of rabbits in differentphysiological states and to demonstrate theappearance of the foetal-placental unit.

ACKNOWLEDGMENT

This research project was supported by theHungarian grant OTKA (F 025281 ).

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