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Early undernutrition: Subsequent growth of male and female rats
Pergamon Preen
Life Sciences Vol . 22~ pp . 179-186 Printed in the U.S .A .
EARLY UNDERNUTRITION :
SUBSEQUENT GROWTH OF MALS AND FEMALE RATS
T .S . Whatson*, W.J . Mooret, J .L . Smart and J . bobbing Department of Child Health, The Medical School, University of Manchester, Manchester United Kingdom (Received in final form November 21, 1977) Male and female rata were growth retarded by feeding their mothers a restricted quantity of food throughout pregnancy and lactation . They were weaned at 26 days of age and allowed free access to food from that time . All animals were weighed at weekly intervals from 26 to 139 days of age . Both male and female undernourished rats were significantly lighter than their respective well-fed caatrols at birth and weaning. They remained significantly smaller throûghThe weight diffexL out the period of nutritional rehabilitation . sacs was greater in melee both absolutely and proportionately. Analysis of the growth velocity curves revealed that whereas previously undernourished male rata showed no catch-up growth, previously undernourished females grew significantly faster than their controls between the ages of 48-55 and 55-62 days . The peak of the growth velocity curve was significantly delayed by early undernutritioa is female but not male rats . Irrespective of nutrition the peak occurred about 2 weeks earlier in female than in male rats . The trajectory of recovery growth appears to depend partly an how much of the body growth spurt is covered by the undernutrition . Rats which have been undernourished for a short period may or may not grow to be as large as control animals whose growth has not been restricted, depending as the timing of the nutritional insult (1) . That is, they may or may not show complete catch-up growth . Catch-up growth is defined here with respect to body weight as a rate of increase in the absolute body weight of previously undernourished (PU) animals which exceeds that of well nourished control (C) animals of the same age . This results is some convergence of growth trajectory towards that of controls ; and catch-up may be complete or incomplete . It is well established that male rata which have been undernourished throughout much or all of the suckling period show deficits in body weight which are resistant to nutritional rehabilitation from weaning and which persists throughout life (1,2,3,4,5,6,7) . Characteristically the absolute weight deficit caatinues to increase well into adulthood. * East of Scotland College of Agriculture, West Mains Road, Edinburgh . t Department of Community Health, University of Bristol, Canyng Hall, Whiteladies Road, Bristol, B58 2PR. 179
Early Undernutrition and Growth
Vol . 22, No . 2, 1978
Visual The situation with respect to female PU rats is less clear . inspection of published growth curves indicates that they remain on average However, when statistical analysis of the lighter than C females (6,8,9,10) . final body weights has been carried out the difference has sometimes proved Certainly the growth curves ôf C and significant (9) and sometimes not (10) . PU females do not diverge to the same extent after weaning ss those of corresponding males and it is even possible that PU females have some capacity for true catch-up growth . The present experiment was undertaken to investigate in depth whether this is so by recording the individual weight increments of large numbers of C and PU rate at weekly intervals during nutritional rehabilitation This analysis was conducted according to and comparing them statistically . the recent recommendation of Abbey and Howard (11) and is hence more appropriate thaw that in earlier studies (see Statistics below) . Methods Animals Female rata of the black sad white hooded Liater strain which had borne On the day of mating, one or two litters were housed 3 to a cage with 1 male . determined by examination of the vaginal lavages for sperm, females were housed Control females singly and allocated to either the control or underfed group. Underfed females were given were well fed throughout pregnancy and lactation . a daily ration of good quality diet (Mouse cubes, Oakes Millers Ltd ., Congleton, Cheshire)* equal to about one half of the amount eaten by well fed females at These quantities were 10 g/day throughout pregnancy, the respective times . p 15 g/day during the first week post artum , 20 g/day during the second week and All pupa were fed thereafter 25 g/day until the litter was weaned at 26 days . Water was freely available at all times . ad lib after weaning . All litters were culled at birth to 8 pups, comprising as nearly as possSexes were separated at 26 days and housed in ible 3 females and 5 melee . littermate groups of 2 or 3 . The animals were weighed at birth, weaning and weekly thereafter until 139 days of age . Twenty male and 30 female C rate from 10 litters and 32 male and 45 female PU rats from 16 litters were used . Statistics Following the recommendation of Abbey and Howard (11) the mean weight of all males or females in each litter was computed at each age and this figure Thus used in the analyses rather than the weight of each individual animal . the number of independent observations at each age was 10 and 16 for C and PU rats respectively . Students t teat has been used except where stated otherwise and all P values are two-tailed . Results and Discussion The body weight of the PU animals was significantly (p < 0 .001) lower than that of C animals of the same sea, at birth, weaning and all ages thereafter (Fig . 1) . The way in which the weight difference between groups changed At 26 days of age the with age depended on the sea of the animals (Fig . 2) . mean weight difference between C and PU male rate was 48 g and this had increased to 113 g by 139 days of age . The mean absolute weight difference increased each week throughout almost the entire post weaning period . The only
* An analysis of this diet is given in (12) .
Vol . 22, No . 2, 1978
181
Early Undernutrition and Growth
sao
aoo 1
30o a F S
w 3 200
100
ZO
10
a0
80
100
1Y0
1A0
AGE Idnnl
FIG . 1 Growth Curves for Male and Female, C and PU Bate from Birth to 139 Days of Age exception to this being between the ages of 84 and 110 days when the mean weight difference remained more or leas constant . The slightly faster growth of the PU males at those times was not statistically significant . ~At all Catchother times the PU males grew more slowly than the C males (Fig . 2) . up grwth was therefore not in evidence . Ia female rats the change in weight difference was not as consistent . At weaning the mean weight of the PU females was 45 g leas than that of the controls and at 40 days the mean weight difference was nearly 50 g . However, at 48 days it had decreased to 47 g and at 55 days it was 42 g, the level at This, albeit fairly small, which it remained into adulthood (Fig . 2) . decrease in the mean absolute weight difference between 48 and 62 days represents true catch-up growth . Thin was principally the consequence of the growth rate is the previously undernourished females being significantly greater than that of control females in the intervals between the ages 48-55 and 55-62 days (p < 0 .02 in each case) . At no other times were the growth rates significantly different (Fig . 3) . These results support the now well documented finding that undernutrition cov®ring most or all of the suckling period, however administered, results in a permanently lower body weight than would normally have been achieved in both male and female rate (3,6,8,9) . This would tend to refute the results of Williams et al (7,10) which indicated that early undernutrition did not perma nently alter the adult body weight of female rata . There are a number of
182
Early Undernutrition and Growth
Vol. 22, No . 2, 1978
W
ci
Z W W W _W
O F W
AGE (dM 1 FIG . 2 Mean Weight Difference Between C and PU Male Rats and C and PU Female Rats at Weekly Intervals from Weaning possible explanations of Chie discrepancy. Firstly, it has been repeatedly pointed out (13,14) that the severity of a particular type of insult imposed during development will to some extent determine its consequences . Amore severe insult will have different conse quences from a mild insult of the same type . It is difficult to assess directly the degree of uadernutrition caused by the various techniques which have been used (e .g . large vs small litters, low vs high protein diet, restricted food intake of the dam etc .) . If weight at a particular age during undernutrition can be assumed to reflect the severity of undernutritioa, then the greater the deficit in comparison with controls of the same age, the more severe will have been the insult . In Table 1 the percentage weight deficit of undernourished ae compared with well fed rata at weaning and in adulthood from 6 studies is presented . Whilst the picture is confounded by the use of different strains of rate, dieted period of undernutrition and ages when weighed it is possible to see a certain order . Those animals with the greatest percentage weight deficit at 21 days (8) also had the greatest percentage weight deficit at 119 days of age . Similarly those animals with the smallest percentage weight deficit at 21 days (7,10) also had the smallest percentage weight deficit at 119 days of age . Thus, the extent of the final deficit in body weight appears to be dependent to some extent on the severity of the insult . Catch-up growth, at least in female rate, certainly occurs but how it is affected by different severities of underautrition, whether it alters in extent or duration, remains to be determined .
Vol . 22, No . 2, 1978
Early Uadernutrition and Growth
183
CONTROLS
60 ~
MALE FEMALE PREVIOUSLY UNDERNOURISHED
40
o
MALE
a
FEMALE
30
0~
34
41
48
56
82
~
78
83
90
97
119 132 104 126 139 111
AGE INTERVALS ( d~yt 1 FIG. 3
Mean Increments in Body Weight (g) of C and PU Male and Female Rats for each of 16 Seven day Intervals The timing of the peak of the weight velocity curve may have some bearing as this . In the present study the peak velocity was delayed by aboût ane week In female, but not male rate, this delay was statistically signifi(Fig . 2) . The absence of a significant shift in PU males cant (p < 0.01, Fisher teat) . may have been due to the reported leas well defined peak in male rate (15), No which would tend to obscure any small changes in the timing of the peak . delay appears to have been caused by the undernutrition imposed by Williams et al (10, nee their Fig . 1) and may reflect a lean severe insult in that case . It is noteworthy that the body weight velocity curve for normal females peaks at 4 to 5 weeks, about 2 weeks before that for melee (present Fig. 3 ; 6) . Hence the growth spurt for female rata occurs earlier than that for males and is considerably less in extent . Growth restriction during the suckling period therefore includes a much higher proportion of the female growth spurt thaw of the male growth spurt . This may be the source of the marked sax difference in response to growth restriction during the suckling period in rats . As a general rule, growth restriction which precedes the bodily growth spurt results
184
Early Undernutrition and Growth
Vol . 22, No . 2, 1978
TABLE 1 Percentage Weight Differences between Well Fed and Previously Underfed Male and Female Rats at Weaning and in Adulthood X Weight difference males females
Period of undernutrition
Study
Age (d)
Blackwell et al (1972) (17)
21 112
45 19
48 18
pre- and post natal
Present
26 119
69 25
69 16
pre- and post natal
Slob et al (1973)* (6)
21 119
48 24
48 14
postnatal only
Widdowaon et al (1962)* (8)
21 119
57 30
57 19
postnatal only
Williams et al (1974) (7,10)
21 119
40 10
37 6
postnatal only
Smart et al (1974)* (9)
25 105
56 17
53 14
postnatal from 5 days
* Percentage weight difference obtained by interpolation from published growth curves in divergent growth, and growth restriction during the growth spurt in some degree of convergent (catch-up) growth (16) . Acknowledgements We are indebted to the Medical Research Council, U .K ., and the National Fund for Research into Crippling Diseases, U .K . for financial support . References 1. 2. 3. 4. 5. 6. 7. B. 9. 10 . 11 . 12 . 13 . 14 .
E .M . WIDDUWSON and R.A . McCANCE, Proc . Roy . Soc . ser . B 158, 329-342 (1%3~ E .M . WIDDOWSON and R .A . McCANCE, Proc . Ro . Soc . ser .B _152, 188-206 (1960 B .F . CHOW and C . LEE, J . Nutr . 82, 10-18 (1964 . J . DUBBING and J . SANDS, Biol . Negate 19, 363-378 (1971) B .P .F . ADLARD, J . DUBBING and J .L . SMART, Biol Neonate 23, 95-108 (1973) . A .K . SLOB, C .E . SNOW and E . de NATRIS MATHOT, Developmental Psychobiol . 6, 177-186 (1973) . J.P .G . WILLIAMS, J .M . TANNER and P .C .R . HUGHES, Pediat . Res . _8, 149-156 (1974) . E .M . WIDDUWSON and G.C . KENNEDY, Proc . Roy . Soc . aer .B 156, 96-108 (1962) . J .L . SMART, B .P .F . ADLARD and J . DUBBING, Biol . Neonate25, 135-150 (1974 J .P .G . WILLIAMS, J .M . TANNER and P .C .R . HUGHES, Pediat . Res . _8, 157-169 (1974) H . ABBEY and E . HOWARD, Develo mental Ps chobiol . _6, 329-335 (1973) . J .L . SMART and J . DUBBING, Br . J . Nutr . 3 , 21-430 (1977) . J . DUBBING, in Applied Neurochemistry , (Davison and J . bobbing, eds .) p . 287, Blackwell Scientific Publications, London, 1968 . J . DUBBING, in Scientific Foundations of Paediatrics, (J .A . Davia and J . bobbing, ede . p. 65 William Heinman Medical Books, London, 1974 .
Vol . 22, No . 2, 1478 15 . 16 . 17 .
Early IIndernutrition and Growth
185
A .K . SLOB, aad J .J . VAN DER WERFF TEN BOSCH, Phyaiol . Behau . 14, 353-361 (1975) . J .L . SMART, E .M . BYRNE and J . DOBBING, Proc . Nutr . Soc . 36, 99a, (1977) . B . BLAC&WELL, R .Q . BLACRWELL, T . YU, Y .üENG an B .F . CHOW, J . Nutr . 97 79-84 (1969) .
Life Sciences Vol . 22~ pp . 179-186 Printed in the U.S .A .
EARLY UNDERNUTRITION :
SUBSEQUENT GROWTH OF MALS AND FEMALE RATS
T .S . Whatson*, W.J . Mooret, J .L . Smart and J . bobbing Department of Child Health, The Medical School, University of Manchester, Manchester United Kingdom (Received in final form November 21, 1977) Male and female rata were growth retarded by feeding their mothers a restricted quantity of food throughout pregnancy and lactation . They were weaned at 26 days of age and allowed free access to food from that time . All animals were weighed at weekly intervals from 26 to 139 days of age . Both male and female undernourished rats were significantly lighter than their respective well-fed caatrols at birth and weaning. They remained significantly smaller throûghThe weight diffexL out the period of nutritional rehabilitation . sacs was greater in melee both absolutely and proportionately. Analysis of the growth velocity curves revealed that whereas previously undernourished male rata showed no catch-up growth, previously undernourished females grew significantly faster than their controls between the ages of 48-55 and 55-62 days . The peak of the growth velocity curve was significantly delayed by early undernutritioa is female but not male rats . Irrespective of nutrition the peak occurred about 2 weeks earlier in female than in male rats . The trajectory of recovery growth appears to depend partly an how much of the body growth spurt is covered by the undernutrition . Rats which have been undernourished for a short period may or may not grow to be as large as control animals whose growth has not been restricted, depending as the timing of the nutritional insult (1) . That is, they may or may not show complete catch-up growth . Catch-up growth is defined here with respect to body weight as a rate of increase in the absolute body weight of previously undernourished (PU) animals which exceeds that of well nourished control (C) animals of the same age . This results is some convergence of growth trajectory towards that of controls ; and catch-up may be complete or incomplete . It is well established that male rata which have been undernourished throughout much or all of the suckling period show deficits in body weight which are resistant to nutritional rehabilitation from weaning and which persists throughout life (1,2,3,4,5,6,7) . Characteristically the absolute weight deficit caatinues to increase well into adulthood. * East of Scotland College of Agriculture, West Mains Road, Edinburgh . t Department of Community Health, University of Bristol, Canyng Hall, Whiteladies Road, Bristol, B58 2PR. 179
Early Undernutrition and Growth
Vol . 22, No . 2, 1978
Visual The situation with respect to female PU rats is less clear . inspection of published growth curves indicates that they remain on average However, when statistical analysis of the lighter than C females (6,8,9,10) . final body weights has been carried out the difference has sometimes proved Certainly the growth curves ôf C and significant (9) and sometimes not (10) . PU females do not diverge to the same extent after weaning ss those of corresponding males and it is even possible that PU females have some capacity for true catch-up growth . The present experiment was undertaken to investigate in depth whether this is so by recording the individual weight increments of large numbers of C and PU rate at weekly intervals during nutritional rehabilitation This analysis was conducted according to and comparing them statistically . the recent recommendation of Abbey and Howard (11) and is hence more appropriate thaw that in earlier studies (see Statistics below) . Methods Animals Female rata of the black sad white hooded Liater strain which had borne On the day of mating, one or two litters were housed 3 to a cage with 1 male . determined by examination of the vaginal lavages for sperm, females were housed Control females singly and allocated to either the control or underfed group. Underfed females were given were well fed throughout pregnancy and lactation . a daily ration of good quality diet (Mouse cubes, Oakes Millers Ltd ., Congleton, Cheshire)* equal to about one half of the amount eaten by well fed females at These quantities were 10 g/day throughout pregnancy, the respective times . p 15 g/day during the first week post artum , 20 g/day during the second week and All pupa were fed thereafter 25 g/day until the litter was weaned at 26 days . Water was freely available at all times . ad lib after weaning . All litters were culled at birth to 8 pups, comprising as nearly as possSexes were separated at 26 days and housed in ible 3 females and 5 melee . littermate groups of 2 or 3 . The animals were weighed at birth, weaning and weekly thereafter until 139 days of age . Twenty male and 30 female C rate from 10 litters and 32 male and 45 female PU rats from 16 litters were used . Statistics Following the recommendation of Abbey and Howard (11) the mean weight of all males or females in each litter was computed at each age and this figure Thus used in the analyses rather than the weight of each individual animal . the number of independent observations at each age was 10 and 16 for C and PU rats respectively . Students t teat has been used except where stated otherwise and all P values are two-tailed . Results and Discussion The body weight of the PU animals was significantly (p < 0 .001) lower than that of C animals of the same sea, at birth, weaning and all ages thereafter (Fig . 1) . The way in which the weight difference between groups changed At 26 days of age the with age depended on the sea of the animals (Fig . 2) . mean weight difference between C and PU male rate was 48 g and this had increased to 113 g by 139 days of age . The mean absolute weight difference increased each week throughout almost the entire post weaning period . The only
* An analysis of this diet is given in (12) .
Vol . 22, No . 2, 1978
181
Early Undernutrition and Growth
sao
aoo 1
30o a F S
w 3 200
100
ZO
10
a0
80
100
1Y0
1A0
AGE Idnnl
FIG . 1 Growth Curves for Male and Female, C and PU Bate from Birth to 139 Days of Age exception to this being between the ages of 84 and 110 days when the mean weight difference remained more or leas constant . The slightly faster growth of the PU males at those times was not statistically significant . ~At all Catchother times the PU males grew more slowly than the C males (Fig . 2) . up grwth was therefore not in evidence . Ia female rats the change in weight difference was not as consistent . At weaning the mean weight of the PU females was 45 g leas than that of the controls and at 40 days the mean weight difference was nearly 50 g . However, at 48 days it had decreased to 47 g and at 55 days it was 42 g, the level at This, albeit fairly small, which it remained into adulthood (Fig . 2) . decrease in the mean absolute weight difference between 48 and 62 days represents true catch-up growth . Thin was principally the consequence of the growth rate is the previously undernourished females being significantly greater than that of control females in the intervals between the ages 48-55 and 55-62 days (p < 0 .02 in each case) . At no other times were the growth rates significantly different (Fig . 3) . These results support the now well documented finding that undernutrition cov®ring most or all of the suckling period, however administered, results in a permanently lower body weight than would normally have been achieved in both male and female rate (3,6,8,9) . This would tend to refute the results of Williams et al (7,10) which indicated that early undernutrition did not perma nently alter the adult body weight of female rata . There are a number of
182
Early Undernutrition and Growth
Vol. 22, No . 2, 1978
W
ci
Z W W W _W
O F W
AGE (dM 1 FIG . 2 Mean Weight Difference Between C and PU Male Rats and C and PU Female Rats at Weekly Intervals from Weaning possible explanations of Chie discrepancy. Firstly, it has been repeatedly pointed out (13,14) that the severity of a particular type of insult imposed during development will to some extent determine its consequences . Amore severe insult will have different conse quences from a mild insult of the same type . It is difficult to assess directly the degree of uadernutrition caused by the various techniques which have been used (e .g . large vs small litters, low vs high protein diet, restricted food intake of the dam etc .) . If weight at a particular age during undernutrition can be assumed to reflect the severity of undernutritioa, then the greater the deficit in comparison with controls of the same age, the more severe will have been the insult . In Table 1 the percentage weight deficit of undernourished ae compared with well fed rata at weaning and in adulthood from 6 studies is presented . Whilst the picture is confounded by the use of different strains of rate, dieted period of undernutrition and ages when weighed it is possible to see a certain order . Those animals with the greatest percentage weight deficit at 21 days (8) also had the greatest percentage weight deficit at 119 days of age . Similarly those animals with the smallest percentage weight deficit at 21 days (7,10) also had the smallest percentage weight deficit at 119 days of age . Thus, the extent of the final deficit in body weight appears to be dependent to some extent on the severity of the insult . Catch-up growth, at least in female rate, certainly occurs but how it is affected by different severities of underautrition, whether it alters in extent or duration, remains to be determined .
Vol . 22, No . 2, 1978
Early Uadernutrition and Growth
183
CONTROLS
60 ~
MALE FEMALE PREVIOUSLY UNDERNOURISHED
40
o
MALE
a
FEMALE
30
0~
34
41
48
56
82
~
78
83
90
97
119 132 104 126 139 111
AGE INTERVALS ( d~yt 1 FIG. 3
Mean Increments in Body Weight (g) of C and PU Male and Female Rats for each of 16 Seven day Intervals The timing of the peak of the weight velocity curve may have some bearing as this . In the present study the peak velocity was delayed by aboût ane week In female, but not male rate, this delay was statistically signifi(Fig . 2) . The absence of a significant shift in PU males cant (p < 0.01, Fisher teat) . may have been due to the reported leas well defined peak in male rate (15), No which would tend to obscure any small changes in the timing of the peak . delay appears to have been caused by the undernutrition imposed by Williams et al (10, nee their Fig . 1) and may reflect a lean severe insult in that case . It is noteworthy that the body weight velocity curve for normal females peaks at 4 to 5 weeks, about 2 weeks before that for melee (present Fig. 3 ; 6) . Hence the growth spurt for female rata occurs earlier than that for males and is considerably less in extent . Growth restriction during the suckling period therefore includes a much higher proportion of the female growth spurt thaw of the male growth spurt . This may be the source of the marked sax difference in response to growth restriction during the suckling period in rats . As a general rule, growth restriction which precedes the bodily growth spurt results
184
Early Undernutrition and Growth
Vol . 22, No . 2, 1978
TABLE 1 Percentage Weight Differences between Well Fed and Previously Underfed Male and Female Rats at Weaning and in Adulthood X Weight difference males females
Period of undernutrition
Study
Age (d)
Blackwell et al (1972) (17)
21 112
45 19
48 18
pre- and post natal
Present
26 119
69 25
69 16
pre- and post natal
Slob et al (1973)* (6)
21 119
48 24
48 14
postnatal only
Widdowaon et al (1962)* (8)
21 119
57 30
57 19
postnatal only
Williams et al (1974) (7,10)
21 119
40 10
37 6
postnatal only
Smart et al (1974)* (9)
25 105
56 17
53 14
postnatal from 5 days
* Percentage weight difference obtained by interpolation from published growth curves in divergent growth, and growth restriction during the growth spurt in some degree of convergent (catch-up) growth (16) . Acknowledgements We are indebted to the Medical Research Council, U .K ., and the National Fund for Research into Crippling Diseases, U .K . for financial support . References 1. 2. 3. 4. 5. 6. 7. B. 9. 10 . 11 . 12 . 13 . 14 .
E .M . WIDDUWSON and R.A . McCANCE, Proc . Roy . Soc . ser . B 158, 329-342 (1%3~ E .M . WIDDOWSON and R .A . McCANCE, Proc . Ro . Soc . ser .B _152, 188-206 (1960 B .F . CHOW and C . LEE, J . Nutr . 82, 10-18 (1964 . J . DUBBING and J . SANDS, Biol . Negate 19, 363-378 (1971) B .P .F . ADLARD, J . DUBBING and J .L . SMART, Biol Neonate 23, 95-108 (1973) . A .K . SLOB, C .E . SNOW and E . de NATRIS MATHOT, Developmental Psychobiol . 6, 177-186 (1973) . J.P .G . WILLIAMS, J .M . TANNER and P .C .R . HUGHES, Pediat . Res . _8, 149-156 (1974) . E .M . WIDDUWSON and G.C . KENNEDY, Proc . Roy . Soc . aer .B 156, 96-108 (1962) . J .L . SMART, B .P .F . ADLARD and J . DUBBING, Biol . Neonate25, 135-150 (1974 J .P .G . WILLIAMS, J .M . TANNER and P .C .R . HUGHES, Pediat . Res . _8, 157-169 (1974) H . ABBEY and E . HOWARD, Develo mental Ps chobiol . _6, 329-335 (1973) . J .L . SMART and J . DUBBING, Br . J . Nutr . 3 , 21-430 (1977) . J . DUBBING, in Applied Neurochemistry , (Davison and J . bobbing, eds .) p . 287, Blackwell Scientific Publications, London, 1968 . J . DUBBING, in Scientific Foundations of Paediatrics, (J .A . Davia and J . bobbing, ede . p. 65 William Heinman Medical Books, London, 1974 .
Vol . 22, No . 2, 1478 15 . 16 . 17 .
Early IIndernutrition and Growth
185
A .K . SLOB, aad J .J . VAN DER WERFF TEN BOSCH, Phyaiol . Behau . 14, 353-361 (1975) . J .L . SMART, E .M . BYRNE and J . DOBBING, Proc . Nutr . Soc . 36, 99a, (1977) . B . BLAC&WELL, R .Q . BLACRWELL, T . YU, Y .üENG an B .F . CHOW, J . Nutr . 97 79-84 (1969) .