Effect of Repeated Reproductive Cycles on ...

Growth, Development and Aging

Effect of Repeated Reproductive Cycles on Pregnancy Outcome in Ad Libitumâ€”Fedand Chronically FoodRestricted Rats1'2 KATHLEEN M. RASMÃœSSENAND KAREN L. FISCHBECK Division of Nutritional Sciences, Cornell university, Ithaca, /W 14853

INDEXING KEY WORDS: â€¢parity â€¢birth order â€¢pregnancy â€¢rats

â€¢malnutrition

Poor pregnancy outcome is common among women living in deprived circumstances; rates of low birth weight, for example, may exceed 25% (1, 2). In addition to other problems, these women experience the dual nutritional insults of inadequate dietary intake and re peated, closely spaced reproductive cycles. As part of a larger experiment in which the effects of food restric tion and repeated reproductive cycles on lactational performance were examined (3), we investigated the effects of one or both of these factors on maternal weight gain during pregnancy, pregnancy outcome and mater nal weight changes during the interval between peak lactation and the next conception. Data to elucidate some of the important biological interrelationships are available from an analysis of birth certificates filed during 1976 in the United States (4). 0022-3166/87 $3.00 Â©1987 American Institute of Nutrition.

The relationship between parity and birth weight was nonlinear; both first and high parity (fourth and higher) were associated with greater rates of low birth weight. Parity also interacted with maternal age to affect birth weight; women of high parity and young age were more likely to deliver an infant of low birth weight. Com pared to first parity, second parity was associated with a lower weight among teenaged (those under 20 yr old) mothers. An additional interaction existed between maternal age and birth interval; short birth intervals ( 75 > 60 by ScheffÃ©test; and inter action (P < 0.0001 )between reproductive period and diet with PI < P2 for all diet groups (P < 0.0001) by t-test. At d 0 of lactation, the following effects were significant by two-way ANOVA: reproductive period [P < 0.0001) with LI < L2 by ScheffÃ©test, and diet (P < 0.0001) with 100 > 75 > 60 by ScheffÃ©test.

breeding cycle; however, the differences between PI and P2 were much larger in the control group (50 g) than among the restricted groups (26 and 18 g for the rats fed 75 and 60% of the AL intake, respectively). Both reproductive period and diet also affected mater nal weight at the end of pregnancy (d 0 of lactation) (Fig. 1). Dams were consistently heavier in P2 than in PI and there was the expected gradation in weight among the three dietary treatment groups. Both reproductive period and diet affected maternal weight gain during the first half of pregnancy, but the effect of reproductive period was not the same in all diet groups (Fig. 2). Only the restricted dams gained more weight in the first half of P2 than in the first half of PL During the second half of pregnancy there was

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this problem before statistical analyses were carried out. For ease of interpretation, only the untransformed means are reported in the tables, but the statistical results refer to tests carried out on appropriately trans formed data. The one-way analysis of variance (ANOVA) proce dure with the multiple-range ScheffÃ© test available in the SPSS (11) was used to determine if the weights and weight changes of dams and litters differed significantly among the diet groups. The general linear model (GLM), two-way ANOVA procedure with the multiple-range ScheffÃ© test in the SAS statistical package (12) was used to assess the sig nificance of the effects of reproductive period and di etary treatment and their interaction. Differences were termed significant at P < 0.05, and were termed trends at 0.05 < P < 0.10. When the two-way ANOVA re vealed possible (defined as P < 0.20) or significant (P < 0.05) interaction between the effects of reproductive period and diet, a Â£-testwas performed by using the Mini tab statistical package (13) to compare results for each reproductive period within each diet group. For all rats killed in L2, measurements of food intake and of body weights of these same dams and their litters were available from both PI and P2. Therefore, the twoway ANOVA of feed efficiency, weight and weight changes of dams was carried out on these paired data rather than the corresponding unpaired data to take advantage of the greater statistical power available; these data are presented in the tables. For these paired data, the differences between the first and second reproduc tive period were also calculated and the differences among the dietary treatment groups were examined by the one-way ANOVA. The findings from these analyses are included in the text where appropriate, but they are not presented in tabular form.

OUTCOME

RASMUSSEN AND FISCHBECK

1962

Days 0-10 Days 10-20

90 80

â€¢First pregnancy o Second pregnancy

2 70 e o

en

60

o> O)

50 40 30

75

100

no effect of reproductive period on maternal weight gain (Fig. 2), but dietary restriction resulted in a sig nificantly lower weight gain than AL feeding, and the severely restricted dams gained less than the dams that were moderately restricted. There was no significant difference between PI and P2 in the weight gain of dams during pregnancy that was attributable to the offspring (Fig. 3). This portion of maternal weight gain was significantly lower in the restricted groups, but severe restriction did not produce lower values than moderate restriction. Dietary intake affected net maternal weight gain, but the effect was not the same in the two reproductive periods (Fig. 3). Only the severely restricted dams retained more of the weight gained in pregnancy on d 0 of L2 than they had in LI. Reproductive performance. Conception rate was not affected by either reproductive period or dietary restric tion (data not shown). As expected from the breeding protocol employed, both reproductive period and diet affected age at conception (data not shown). Obviously, dams were older in the second reproductive period and some controls were bred first deliberately to have the

80

70

^

60

40 30 20

Fetal compartment Net maternal gain â€¢ , â€¢First pregnancy o,a Second pregnancy

60 75 Dietary group (% of ad libitum

100 intake)

FIGURE 3 Effect of reproductive period and dietary re striction on the division of pregnancy weight gain between the fetal compartment and maternal gain. Mean Â±SEM;n = 14, 24 and 31 for the controls, 75 and 60% groups, respec tively. For the fetal compartment, only the effect of diet was significant (P < 0.0001) by two-way ANOVA with 100 > 75, 60 by ScheffÃ©test. For net maternal weight gain, the effect of diet was significant (P < 0.0001 ) by two-way ANOVA with 100 > 75 > 60 by ScheffÃ©test as was interaction (P < 0.08) between reproductive period and diet with PI < P2 for 60 (P < 0.001) by Ã®-test.


60

Dietary group (% of ad libitum intake) FIGURE 2 Effect of reproductive period and dietary re striction on weight change of dams during pregnancy. Mean Â±SEM;n = 14, 24 and 32 for the controls, 75 and 60% groups, respectively. During d 0-10 of pregnancy, the following ef fects were significant by two-way ANOVA: reproductive pe riod (P < 0.005) with PI < P2 by ScheffÃ©test; diet (P < 0.0001) with 100 > 75 > 60 by ScheffÃ©test, and interaction (P < 0.001) between reproductive period and diet with PI < P2 for 75 (P < 0.03) and 60 (P < 0.0001) by Â£-test.During d 10-20 of pregnancy, only the effect of diet was significant (P < 0.0001) by two-way ANOVA with 100 > 75 > 60 by ScheffÃ© test.

information necessary to feed the restricted rats. The length of gestation was slightly (0.1 d overall) but sig nificantly shorter in P2 than in PI (data not shown). There was no significant effect of reproductive period on the number of liveborn pups delivered (Table 1). As expected, the number of liveborn pups was lower in the restricted groups. Dietary treatment significantly affected live litter weight at birth (Table 1). There was also possible interaction between reproductive period and diet, but there was no significant difference in litter weight between the two periods in any dietary treat ment group. The birth weight of individual pups was consistently and significantly greater (0.23 g overall) in the second reproductive period (Table 1). Only the dams restricted to 60% of AL intake had lower individual pup weights at birth compared to the AL-fed group; there was no difference between AL-fed dams and the dams that were moderately restricted. Maternal weight between peak lactation and the sec ond conception. During the third week of LI, dietary treatment affected the weight change of dams (Table 2); the restricted dams lost less weight than the con trols, and the severely restricted dams actually gained weight. Dietary treatment also affected the weight

NUTRITIONAL

STATUS, PARITY AND PREGNANCY

1963

OUTCOME

TABLE 1

TABLE 2

Effect of reproductive period and dietary restriction on live litter weight and pup number and weight at d 0 of lactation

Effect of dietary restriction on the weight change and feed efficiency of dams between d 14 of LI and d 0 of P2

Period12Dietary group100(n

intervalEnd Time

pups'12.610.810.412.510.79.9Â± of

LI|d of 14-21)'Between

group100|n changeg-11.4

2.6"Â±

11.5Â± Â±

Â±5.7

2.4Â±

12.0Â±

Â±5.5

24)60(n =

1.6Â±

7.3Â±

Â±6.0

32)100|n =

2.2Â±

12.1Â±

Â±5.9

13)75(n = 24)60(n =

1.8Â±

10.1Â±

Â±5.6

24)60(n =

LIand |d21 P2 Ll-dOP2)3Dietary

2.1WeightLitter270.260.556.575.362.654.8S 9.2Pup35.6 Â±0.50.40.50.40.40.5

4.82.3 Â±

0.020.01 Â±

14)75(n =

0.02-0.22 Â±

2.60.84 Â±

0.180.05 Â±

1.30.36 Â±

0.080.03 Â±

14)75(n =

Â±1.3Feed

Â±0.10

= 32)Weight 'Significant (P < 0.0001) effect of diet on weight change in one way ANOVA; 100 < 75 < 60 by ScheffÃ© test. Significant |P < 0.0001 ) effect of diet on feed efficiency in one-way ANOVA; 100 < 75 < 60 by ScheffÃ©test. 2Mean Â±SD. 3Significant [P < 0.0001) effect of diet on daily weight change in one-way ANOVA; 100 < 75, 60 by ScheffÃ©test. Significant [P < 0.001) effect of diet on feed efficiency in one-way ANOVA; 100 < 75, 60 by ScheffÃ©test.

DISCUSSION Effects of dietary treatment. Although various forms of dietary restriction during pregnancy have been stud ied extensively, the effects of chronic dietary restriction have been examined only in two recent studies from our laboratory (14,15). In those studies as in the present experiment, restricted rats gained less weight during

TABLE 3 Effect of reproductive period and dietary restriction on feed efficiency of dams during pregnancy

Period

Dietary group

Number of dams

Feed efficiency dO-101

d 10-202

eaten0.29gained/g

0.380.250.240.220.250.03 Â±0.033 1210075601007560112432142432g 0.370.03 0.400.05 0.360.04 0.38O.O40.070.040.060.040.28 0.03 0.38 0.06 'Significant (P < 0.0001) interaction between reproductive period and diet in two-way ANOVA; PI > P2 for 100 {P = 0.001) and PI < P2 for 60 |P < 0.0001) by t-test. Significant |P < 0.05) effect of reproductive period in two-way ANOVA; PI > P2 by ScheffÃ©test. 3Mean Â±so. "n = 14.


change between weaning on d 21 of LI and d 0 of P2 (Table 2). These data are expressed as a rate because there were significant (P < 0.0001) differences among the groups in the length of this period (3.8 Â±1.6, 5.0 Â±3.0 and 6.8 Â±2.8 d for the control rats and those rats fed 75 and 60% of the AL intake, respectively). Both groups of restricted dams gained weight during this time, although daily weight gain was not signifi cantly different between the two levels of restricted feeding. In contrast, the AL-fed dams continued to lose weight. Thus, between d 14 of LI and d 0 of P2, control rats lost 23.7 g, whereas the dams fed 75% of the AL intake gained 2.3 g and those dams fed 60% of the AL intake gained 7.6 g. Feed efficiency. The effect of reproductive period on the feed efficiency of dams during the first half of preg nancy was different in each diet group (Table 3). Com pared to PI, the feed efficiency in P2 was lower in the AL-fed dams, unchanged in the moderately restricted dams and higher in the severely restricted dams. During the second half of pregnancy, the feed efficiency of dams was significantly less in P2 than in PI. During the third week of LI and between weaning of d 21 of LI and d 0 of P2, restricted dams had signif icantly greater feed efficiency values than the controls (Table 2). Only during the third week of LI was severe restriction associated with a more positive feed effi ciency than moderate restriction; there was no signif icant difference between restriction levels during the time between weaning and the second conception.

gained/g eaten-0.03 0.02-0.01 Â±

24)60|n =

= 30)Number 'Significant (P = 0.0003] effect of diet in two-way ANOVA; 100 > 75, 60 by ScheffÃ©test. Significant (P < 0.0001) effect of diet in two-way ANOVA; 100 > 75 > 60 by ScheffÃ©test. Possible (P < 0.19) interaction between diet and reproductive period in two-way ANOVA. 3Significant [P = 0.0002) effect of reproductive period in two-way ANOVA; Ll< L2 by ScheffÃ© test. Significant \P < 0.02) effect of diet in two-way ANOVA; 100, 75 > 60 by ScheffÃ©test. "Mean Â±so.

7.22-3.1 Â±

4.8g/day-3.6 Â±

30)100|n = 13)75(n =

efficiencyg

1964


examined in control rats. The increased ability of adi pose tissue to synthesize lipid at this time results from an increase in lipoprotein lipase activity and the rate of fatty acid synthesis (18), as well as a decrease in the rate of lipolysis (19). Factors thought to be involved in these changes in net lipid biosynthesis in adipose tissue are not fully understood at present, but the decrease in prolactin values and the increase in insulin values that accompany litter removal are thought to be important. However, they are insufficient to account for the de creases in the lipolytic capacity of adipose tissue (19). It is not known if similar changes occur in underfed dams, but the results of the present investigation sug gest that they do and that changes in lipid biosynthesis in adipose tissue may begin to occur even before wean ing. In the present experiment, we limited the recovery period to the time necessary to rebreed the dams after they weaned their first litter because we wanted to maximize the stress of repeated, closely spaced repro ductive cycles. The continued weight loss among the control rats during this period was unexpected and was unlike the weight gain experienced by both restricted groups. In this study, rats were not killed for carcass com position analysis at the time of weaning their litters or at the second conception, so that the composition of these weight changes remains unknown. From the re sults of Moore and Brasel (17), however, one can spec ulate that the weight gain of the restricted rats con sisted of fat, protein and water, with the gain in fat likely to have been a particularly substantial proportion of the gain. The results of Moore and Brasel (17) also suggest that the weight loss in the AL-f ed animals prob ably was largely water as these animals readjusted the tissue overhydration characteristic of lactation. It is likely that control rats in the present experiment also would have exhibited a net gain in body weight if al lowed a sufficiently long recovery period. Taken together, the findings of the present experi ment and those of Moore and Brasel (17) emphasize the importance of the entire period from peak lactation through whatever recovery is possible for repletion of maternal nutrient stores, particularly fat, in the re stricted rats. These findings suggest the importance of examining events during this period in undernourished human subjects. If similar changes occur, this period represents an important time for nutritional and other interventions designed to improve nutritional status. Effects of reproductive period. From the classic study of Bogart (20), it was expected that the control rats would have heavier litters in P2, primarily as a function of a greater number of pups in each. The control rats in the present experiment did have heavier litters in P2, but as a result of having heavier individual pups, not a greater number of pups. The reason for this dis crepancy between the two studies is not known, but it is not a function of age at breeding because Bogart's


pregnancy and had a lower net maternal weight gain at d 20 of pregnancy than the controls. In contrast, the effect of dietary restriction on the conception rate and the length of gestation has been less consistent. There was much greater breeding success and less variability in the length of gestation in the present experiment than in our previous work. The outcome of pregnancy in the present experiment was most similar to that of Young and Rasmussen (15), in which rats were obtained from the same supplier and a single reproductive cycle was examined. In that experiment and the present study, dietary restriction during the first reproductive period compromised litter number and litter weight at birth but not pup weight at birth. In contrast, there was a strong effect of dietary treatment on pup weight at birth in the second repro ductive period. To our knowledge, the present experiment is the first in which the effects of dietary restriction imposed be fore pregnancy have been examined in the period be tween peak lactation (d 14) and the next conception. The pattern of maternal weight change during both parts of this interval was unexpected. During the third and last week of LI, weight loss was much less in the moderately restricted rats than in the controls, and the severely restricted rats actually gained weight. This in dicates that maternal investment in lactation was much less in the restricted rats than in the controls during this period. This may be the result of less stimulation to produce milk by the smaller, less vigorous litters suckled by the restricted dams. That such pups suckle less vigorously can be inferred from the results of Grosvenor and Mena (16). They compared the amount and proportion of available milk removed during a timed suckling bout by pups of control and underfed dams. Underfed pups were significantly less effective than control pups in removing milk from underfed dams. Weight and carcass composition changes among con trol and food-restricted (to 70% of the AL intake) rats have been studied by Moore and Brasel (17) during the recovery period after lactation. In contrast to the present experiment, food restriction began at conception; rats were killed when their litters were weaned at d 21 postpartum or after a 21-d recovery period. Both control and food-restricted rats gained weight (9 and 21 g, re spectively) during the recovery period. In absolute amounts, the food-restricted rats gained more carcass fat (13.5 vs. 11.8 g) and less carcass protein (as fat-free dry mass) (5.1 vs. 6.8 g) than the controls during this period; the restricted rats gained (8.1 g) and the controls lost (3.8 g) water over this same interval. Moore and Brasel (17) noted that the particularly re markable recovery in carcass fat experienced by their food-restricted rats resembled that of previously starved rats studied by others and suggested that the postweaning recovery period is an active one metabolically. The nature of the metabolic changes that occur immedi ately (within the first 48 h) after litter removal has been

NUTRITIONAL

STATUS, PARITY AND PREGNANCY

findings were consistent across the three different breeding ages that he investigated. Bogart did not spec ify what strain of rats was used in his experiment; it was presumably less vigorous than the Sprague-Dawley strain used here because the mean litter number of 67 across all parities was much lower than the 12-13 pups seen in this and our previous work with animals from the same supplier (15). Thus, Bogart's rats may have had more room for improvement in litter number than in the present experiment; the litter number at first parity already exceeded the number of nipples available for feeding the litter. In contrast, there was much less room for improvement in pup weight at birth in Bogart's experiment; pups weighed 5.7-6.0 g across

1965

In conclusion, the findings of the present investiga tion serve to focus our attention on both pregnancy itself and the period between reproductive cycles as intervals during which net gains in fat occur, even among food-restricted animals. The implications for lactating women are clear: Both periods represent suitable points for interventions designed to improve birth outcome and lactational performance, and the regulation of changes in body composition during both periods mer its further investigation.

ACKNOWLEDGMENT The authors are indebted to William Fellows for tech nical assistance in this experiment.

LITERATURE CITED 1. DIVISION OF FAMILY HEALTH, WORLD HEALTH ORGANIZA TION (1980] The incidence of low birth weight: a critical re view of available information. World Health Stat. Quart. 33: 197-224. 2. ANONYMOUS (1984| The incidence of low birth weight: an up date. Wkly. Epidem. Ree. 59: 205-212. 3. FISCHBECK, K. L. & RASMUSSEN, K. M. (1987) Effect of repeated reproductive cycles on maternal nutritional status, lactational performance and litter growth in ad libitum-fed and chronically food-restricted rats. /. Nutr. 1967-1975. 4. TAFFEL,S. (1980) Factors associated with low birth weight. Vital and Health Statistics, ser. 21, no. 37, DHEW Publication No. IPHS) 80-1915, Department of Health, Education and Wel fare, Washington, DC. 5. HYTTEN,F. E. (1980) Weight gain in pregnancy. In: Clinical Physiology in Obstetrics (Hytten, F. & Chamberlain, G., eds.|, Blackwell Scientific Publications, Oxford. 6. MORRIS,M. L. & ADAMS,C. E. (1979) Mating post partum, concurrent lactation and reproduction in the laboratory rat. Lab. Anim. 13: 167-170. 7. COLE,H. H. & HART,G. H. (1938) The effect of pregnancy and lactation on growth in the rat. Am. Â¡.Physiol. 123: 589-597. 8. JOHNSON,C. L. (1973) Some aspects of changing body com position of mice during successive pregnancies and lactations. /. Endocrinol. 56: 37-46. 9. AMERICAN INSTITUTE OFNUTRITION (1977) Report of the Amer ican Institute of Nutrition Ad Hoc Committee on standards for nutritional studies. /. Nutr. 107: 1340-1348. 10. AMERICAN INSTITUTE OFNUTRITION (1980) Second report of the American Institute of Nutrition Ad Hoc Committee on standards for nutritional studies. /. Nutr. 110: 1726. 11. NIE, N. H., HULL,C. H., IENKINS,J. G., STEINBRENNER, K. & BENT, D.H. (1975) SPSS: Statistical Package for the Social Sciences, 2nd ed., McGraw-Hill, New York. 12. SAS INSTITUTE (1982) SAS User's Guide: Statistics, SAS (Sta tistical Analysis System), Cary, NC. 13. RYAN,T. A., IOINER,B. L. & RYAN,B. F. (1976) Minitab Student Handbook, Duxbury, North Scituate, MA. 14. WARMAN,N. L. &. RASMUSSEN, K. M. (1983) Effects of mal nutrition during the reproductive cycle on nutritional status and lactational performance of rat dams. Nutr. Res. 3: 527-545. 15. YOUNG,C. M. & RASMUSSEN, K. M. (1985) Effects of varying degrees of chronic dietary restriction in rat dams on reproductive and lactational performance and body composition in dams and their pups. Am. /. Clin. Nutr. 41: 979-987.


all parities and breeding times. The diet fed by Bogart, a stock nonpurified diet developed in 1930 before many of the nutrient requirements of the rat were defined, might also play a role as indicated by the much lower weights of his rats at key ages. For example, at con ception of PI, control rats in the present experiment weighed 228 g, whereas Bogart's rats of the same age weighed only 140-145 g. The most striking effect of reproductive period in this experiment was the unexpectedly larger size of the pups born to the restricted dams in P2. No other studies of repeated reproductive cycles in food-restricted rats are available for comparison. As discussed elsewhere (3), the differences in weight that were present at birth became larger during the nursing period. These two results point to pregnancy and the period between re productive cycles as important for renewal of maternal resources and they suggest, furthermore, that the ac quisition of resources during this time interval is suf ficient not only to produce larger young at birth but also to support improved lactational performance at least through the first third of lactation. What factors could have contributed to the improve ment in pup weight at birth and maternal nutrient stores available to meet the needs of lactation? Evidence from this experiment indicates that all dams were heavier at the second conception because of net maternal weight gain during PI in all groups. It was not unexpected that dams in all groups would experience this net gain. Preg nancy is known to be characterized by a net increase in maternal carcass fat (21, 22), even among food-re stricted rats (23)â€”albeit in greatly reduced amounts. Weight gain in the period between peak lactation and the second conception only among the restricted rats also contributed to the extra weight present at d 0 of P2. In addition, during the first half of P2 restricted dams had a greater feed efficiency and weight gain than during PI. Dams fed 60% of the AL intake also retained more of this extra weight in P2. These findings provide clues to the ability of the restricted rats to improve reproduction and lactation under the stress of repeated, closely spaced reproductive cycles, but they leave open the mechanisms by which these changes in body weight and, presumably, body composition, are controlled.

OUTCOME

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16. GROSVENOR,C. E. & MENA, F. (1983) Effect of underfeeding upon the rate of milk ejection in the lactating rat. /. Endrocrinol. 96: 215-222. 17. MOORE,B. J. & BRASEL,J. A. (1984) One cycle of reproduction consisting of pregnancy, lactation or no lactation, and recovery: effects on carcass composition in ad libitum-fed and food-re stricted rats. /. Ã‘utÃ-.114: 1548-1559. 18. FLINT,D. f., CLEGG,R. A. & VERNON,R. G. (1981) Prolactin and the regulation of adipose-tissue metabolism during lactation in rats. Mol. Cell. Endociinol. 22: 265-275. 19. VERNON,R. G. &. FINLEY,E. (1986) Lipolysis in rat adipocytes during recovery from lactation: response to noradrenaline and adenosine. Biochem. /. 234: 229-231.

Il

20. BOGART, R. (1940) The influence of reproductive state upon growth and the ability to reproduce and lactate in the female rat. Ph.D. thesis, Cornell University, Ithaca, NY. 21. SPRAY,C. (1950) A study of some aspects of reproduction means of chemical analysis. Br. }. Nutr. 4: 354-360.

by

22. NAISMTTH, D. f., RICHARDSON, D. P. & PRTTCHARD, A. E. (1982) The utilization of protein and energy during lactation in the rat, with particular regard to the use of fat accumulated in pregnancy. Br. /. Nutr. 48: 433-441. 23. FELLOWS, W. D. (1985) The partition of nutrients between dam and fetuses in rats underfed acutely and chronically. M.S. thesis, Cornell University, Ithaca, NY.
