Nutritional status, suckling behavior, and prolactin release during lactation

Physiology& Behavior,Vol. 54, pp. 1015-1019, 1993

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Nutritional Status, Suckling Behavior, and Prolactin Release During Lactation K E R R Y J. S C H U L Z E A N D K A T H L E E N M. R A S M U S S E N 1

Division o f Nutritional Sciences, Cornell University, Ithaca, N Y 14853 Received 1 M a y 1992 SCHULZE, K. J. AND K. M. RASMUSSEN.Nutritional status, suckling behavior, and prolact& releaseduring lactation. PHYSIOL BEHAV 54(5) 1015-1019, 1993.--This experiment was conducted to determine whether suckling-inducedprolactin (PRL) values in chronically food-restricted lactating rats resulted from the inability of their litters to suckle vigorously. Rats were randomly assigned to be fed ad lib (AL) or to be chronically restricted (CR) to 50% of AL before and during pregnancy and lactation. Rats nursed their own litter (own) or a litter of the opposite dietary treatment group (other) for 90-min periods at early or peak lactation. Blood was collected regularly through a catheter implanted several days earlier. Suckling contributed to differencesin PRL between AL and CR dams in early lactation as demonstrated by an interaction between dietary treatment and type of litter. However, tests of AL own vs. AL other and CR own vs. CR other were not significant.This is probably because the inattentive behavior of CR dams attenuated the PRL response in this group. At peak lactation, PRL values were related to recovery time from catheter implantation; therefore, determinants of PRL release change over the course of lactation. Lactation

Prolactin

Nutritional status

Suckling

BECAUSE prolactin (PRL) is a major regulator of metabolism (28) and synthesis of milk components (27) during lactation in rats, its role during the metabolically stressed state of chronic undernutrition may be particularly important. For this reason, we chose to examine the relationship of chronic undernutrition to circulating PRL in the lactating rat. In an initial study (16), lower suckling-induced PRL values were observed during early (day 7) and peak (day 14) lactation in rats chronically restricted (CR) to 50% of the consumption ofad lib-fed controls (AL). In that study, a single blood sample was drawn from either the tail vein (day 7) or the heart (day 14) of dams under diethyl ether anesthesia after 30 min of nursing following 2 h of separation from their litters. Chronic undernutrition may have directly affected the ability of the dam to produce, secrete, or catabolize PRL, thereby resuiting in different PRL responses attributable to nutritional status. However, given our observation that pups of CR dams were considerably smaller than pups of AL dams at these times in early and peak lactation, we hypothesized that lower PRL values in CR dams resulted from the inability of their litters to suckle vigorously enough to elicit a typical PRL response. Suckling vigor, represented by acutely underfed pups or ranges in litter size, is known to influence PRL release in well-nourished dams. Acutely underfed pups elicited a greater PRL response from

Rat

dams than normally fed pups at peak lactation (14), and increasing litter size elevated the PRL response of dams at both early and peak lactation (19). If differences in PRL between AL and CR dams are driven by pup suckling capabilities, crossfostering litters between AL and CR dams should result in an increased suckling-induced PRL response in CR dams and a decreased suckling-induced PRL response in AL dams. If these trends were not seen, it could be concluded that the differences in PRL values are attributable to the dams' physiological response to undernutrition. In the present study, therefore, litters were crossfostered between AL and CR dams during a standardized nursing period at early or peak lactation. Dietary treatment groups were replicated from our previous experiment (16). However, to eliminate any influence of anesthesia [ether causes the release of PRL (9)] and to evaluate a more complete plasma PRL response to suckling, catheterized dams were used to permit repeated blood sampling during an extended period of interaction between dams and litters. METHOD

Animal Protocol Female Sprague-Dawley rats were obtained from a commercial supplier (Charles River Laboratories, Inc., Kingston,

Requests for reprints should be addressed to Dr. Kathleen M. Rasmussen, 111 Savage Hall, Cornell University, Ithaca, NY 14853-6301.

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1016 NY) at 35 days of age. Rats were housed in stainless steel, wirebottom cages at constant temperature (21 °C) and humidity with a 12-h light-dark cycle (light: 0800-2000 h). Animal care and housing were in compliance with applicable National Institutes of Health (NIH) and institutional guidelines. Rats were acclimated to purified diet AIN-76A (Dyers, Inc., Bethlehem. PA) (1,2) over a l-week period. At 42 days of age, rats were randomly assigned to dietary treatment groups either to be fed AIN-76A ad lib (AL) or to be chronically restricted (CR) to 50% of the intake of the ad libfed controls. For each of the two study replicates, randomization was successful in producing dietary treatment groups of similar initial body weights (30). CR rats were fed a modification of the AIN-76A diet, an isocaloric diet with twice the usual proportions of vitamins and minerals. This ensured that CR rats were lacking only in energy and protein. Food intake was monitored daily, and food was distributed as early during the light cycle as possible. A representative group of AL rats was bred first so that their food intake could be monitored throughout pregnancy and lactation to serve as a daily basis for feeding the CR rats. Sufficient intake surrounding parturition and catheter implantation was crucial to enable CR dams to maintain their litters, but food intake was substantially reduced in AL rats at these times. Therefore, CR rats were maintained at a constant level of intake during the days surrounding parturition. Following catheter implantation, they were fed 50% of the amount of food consumed by a group of AL animals not receiving catheters. Rats were weighed twice weekly throughout the study. Rats were bred, as described by Warman and Rasmussen (29), over a 4-week period beginning when they reached 64 days of age. At conception, rats were assigned to nurse their own litters or the litter of a dam of the other dietary treatment group at either early (days 7 and 8) or peak (days 13 and 14) lactation. Animals were paired to exchange litters only if they became pregnant on the same day. At day 20 of pregnancy, rats were moved to solid-bottomed cages with pine-chip bedding and weighed daily until parturition. Day 0 of lactation was defined as the day of parturition or the day following parturition if parturition occurred after 1700 h. Litters were culled to eight pups by day 3 of lactation. Rats were required to maintain litters of at least six pups to remain in the study. Litters of paired dams were maintained at the same number of pups.

Blood Collection A catheter was implanted in the carotid artery 3 or 4 days before blood sampling began. This technique was modified from a previous surgical technique used in this laboratory (24). Heparin-treated polyethylene catheters (PE-50 Intramedic; i.d. 0.58 mm, o.d. 0.965 mm; Becton-Dickinson, Parsippany, NJ) with 3-4 cm silastic tips (i.d. 0.5 mm, o.d. 0.94 mm; Dow-Corning Co., Midland, MI) were inserted into the carotid artery of rats previously anesthetized intramuscularly with xylazine (Rompun, Mobey Co., Shawnee, KS) and ketamine hydrochloride (Ketaset, Fort Dodge Laboratories, Fort Dodge, IA) (AL: 1.8 mg/kg xylazine, 66 mg/kg ketamine; CR: 2.6 mg/kg xylazine, 87 mg/kg ketamine). Catheters were exteriorized on the dorsal side of the rat and drawn through a tether (Alice King Chatham Medical Arts, Los Angeles, CA) attached to a jacket made of Elasticon tape (Johnson and Johnson, New Brunswick, NJ), with the adhesive sides folded together. Holes were cut for the forelegs and teats, and the jacket was stretched around the rat's abdomen and secured with tag cement (Kamar Adhesive, Kamar Inc., Steamboat Springs, CO). Tethers were attached by a swivel hook

SCHULZE AND RASMUSSEN to a line above the cage that ran the length of the cage, permitting the animals free movement. Blood sampling began at 1350 h. Litters were removed from cages for 2 h before blood sampling, and a baseline sample was collected immediately before litters were returned to the dam. Blood was collected in heparinized (one drop of 100 units/ml heparinized saline) 250-ul centrifuge tubes every 10 rain for 90 rain. Between samples, the catheter was flushed with 0.4 ml of sterile heparinized saline (100 units/ml). Blood samples were promptly centrifuged for 4 min ( 13,000 × g, Fisher Model 235B Micro-Centrifuge, Pittsburgh, PA), and the plasma was stored at - 2 0 ° C until analyzed for PRL. Observations of nursing activity were noted during the 90rain study period. Dams were kept with their assigned litters overnight, and the blood collection process was repeated on the following day. Therefore, two PRL response profiles were obtained for each dam.

Prolactin Analysis PRL was measured by radioimmunoassay using the kit provided by the Pituitary Hormones and Antisera Center of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Torrance, CA). Iodinations were performed using the chloramine-T method (8). Nine assays were required to test all the samples. Intraassay variation was within 10%, and interassay variation was within 20%. Samples were randomly assigned to an assay according to animal identification number such that each subtreatment group and day of study were represented in each assay. Because PRL release was sporadic and unrelated to behavioral observations, and because blood samples were taken at equal intervals of time, the PRL data were expressed as the average PRL concentration (ng/ml) for the 90-min blood sampling period. This value is equivalent to the area under the PRL response curve divided by the length of time during which blood was sampled. The erratic nature of PRL release contributed to variability in the PRL data.

Statistical Analysis PRL data were analyzed as a two-way repeated measures analysis of variance with dietary treatment (AL or CR) and type of litter nursed (own or other) as main effects. The interaction of dietary treatment and the type of litter nursed, therefore, tested the hypothesis that the PRL response is greater in dams nursing litters of AL dams (AL own and CR other) than in dams nursing litters of CR dams (AL other and CR own). Where interaction (p < 0.20) (3) between the main effects occurred (early lactation), the types of litters nursed were compared by Student's t-test within dietary treatment groups. The statistical model was also designed to control for effects of recovery from catheter implantation and consecutive days of blood sampling. Analyses were modified to explore these influences more closely at peak lactation. At both times, square root transformation was required to equalize variance in the PRL data. Data were backtransformed for presentation. A secondary data analysis also was performed because an influence of maternal behavior on PRL response was suspected. Observations of maternal behavior during the suckling periods were used to categorize dams as attentive, having spent the entire period with their assigned litter, or less than attentive. The number of dams from each dietary treatment and nursing group exhibiting each type of behavior was tabulated. Logistic regression analysis was used to estimate odds ratios (ee of~erm ofinteresl)

NUTRITION, SUCKLING, AND PROLACTIN

1017

TABLE 1 EFFECT OF DIETARY TERATMENT ON CHARACTERISTICSOF DAMS AND PUPS AT THE ONSET OF LACTATION (DAY 0) AND AT THE TIME OF STUDY (DAY 7 OR DAY 14) Day 0 AL* Dam weight (g) Litter weight (g) Number of pups Weight/pup(g)

Day 7 CR

355.0 _+ 47.2t 206.9 + 19.5 n=36 n=29 76.9___14.21" 54.6_+ 9.6 n=36 n=29 12.8-+ 2.7t 10.0-+ 2.0 6.1_+ 0.61" 5.5_+ 0.4

Day 14

AL

CR

AL

CR

305.7 _+ 37.41" n=20:~ 89.4_+ 14.7t n = 16§'** 7.8_+ 0.7 11.6+ 2.0

184.5 _+ 10.3 n = 14§ 65.4+ 11.3 n = 18 7.6+ 0.7 8.6-+ 1.1

289.5 + 32.81" n = 11¶ 189.0_+36.7"~ n = 10¶ 7.7_+ 0.5 24.6-+ 4.1

188.5 _ 15.1 n = 11¶ 113.6+ 18.2 n = 12# 7.3_+ 0.9 15.6-+ 2.6

* Abbreviations used: AL, ad lib fed; CR, chronically food restricted. Mean +_ SD, n = number of observations. i" Significantly (p < 0.00 l) different than CR animals. Two missing observations. ¶ Three missing observations. # Four missing observations. ** Litters at day 7 and day 14 represent those used during the study. They are not necessarily of the same dams who completed the study because crossfostering occurred between dams that completed and dams that did not complete this study.

to express the relative effects of dietary treatment and type of litter nursed on maternal behavior. Because dietary treatment and litter type influenced maternal behavior, path analysis (7) was used to estimate the effect of behavioral influences on P R L release in early lactation between three combinations of the four treatment groups (AL own-AL other; AL own-CR other, C R own-CR other). Main effects could not be examined because of the significant interaction between dietary treatment and type of litter on the PRL response. The following regression equations were modelled: Behavior =/3o + flj (groups of interest)

there was no effect of consecutive days of blood sampling in early lactation, baseline data from days 7 and 8 are presented separately because at day 7 rats had not yet been exposed to other litters.

TABLE 2 EFFECT OF DIETARY TREATMENT AND TYPE OF LITTER NURSED ON PLASMA PRL CONCENTRATIONS(ng/ml) AT EARLY (DAYS 7/8) AND PEAK (DAY 13) LACTATION Treatment Group

PRL =/32 + t3 (groups of interest) +/34 (behavior) The coefficient f13 represented the direct effect of the treatmerits of the groups of interest (AL own-AL other, etc.) on P R L release. The product/3| ×/34 represented the indirect effect of these treatments on P R L through their effect on maternal behavior. The total effect of treatments on the P R L response, through both direct and indirect pathways, is estimated by/33 +

/31X/34. All statisticalanalyses were performed with SAS (22,23). RESULTS Dietary treatments resulted in substantial differences in dams and pups by the onset of lactation and throughout the periods of interest at early and peak lactation (Table 1). Weights differed significantly between dietary treatment groups by the end o f the first week of the study. Exchanging litters between A L and C R dams at the time of study was successful in exposing dams to litters whose total mass was very different than their own litters', despite similar numbers of pups per litter (Table 1). Although it is apparent that two distinct dietary treatment groups existed at the time o f study, food intake of AL dams was sharply reduced following catheter implantation in both early and late lactation. At no time following catheterization were AL dams consuming twice as much as C R dams, who were fed 50% of the ad lib consumption of a group o f uncatheterized controls during the postsurgical period. Baseline P R L concentrations were not different among treatment groups at either early or peak lactation (Table 2). Although

AL Own*

AL Other

CR Own

CR Other

Day 7/8 Baseline day 7 Baseline day 8 Average for 90 mint

9(2,23) n=9

4(1, !1) n--- 10

5(1, 13) n=7

4(1, 10) n=7

18 (1, 60) n=9

4 (1, 10)

4 (0, 10)

9 (1, 25)

n=10

n=5

n=5

69 (20, 145) n = 18

32 (5, 83) n=20

15 (0, 61) n = 12

37 (2, 108) n = 12

Day 13 Baseline Average for 90 min

3 (1, 8)

3 (1, 6)

47 (21, 83) n=8

18 (6, 37) n=7

5 (0, 15)

6 (0, 25)

24 (2, 49) n=9

21 (3, 56) n=5

* Abbreviations used: AL, ad lib fed; CR, chronically food restricted; Own, nursing its own litter; Other, nursing the litter of a dam of the opposite dietary treatment group. Backtransformed mean (_+1 SD) are indicated, as the distribution of the deviation about the mean is not symmetric; n = number of observations. t Significant (p < 0.05) interaction between dietary treatment and type of litter nursed. Contrasts for AL own-AL other (p = 0.16) and CR own-CR other (p = 0.25) not statistically significant.

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SCHULZE AND RASMUSSEN

During early lactation (days 7 and 8), there was a significant interaction between dietary treatment and the type of litter nursed (Table 2). AL dams nursing the pups of CR dams (AL other) responded with half the average PRL released over the 90-min period as AL dams nursing their own pups. Conversely, CR dams doubled their PRL response on average when they nursed the pups of AL dams (CR other). However, the differences in PRL concentration within each dietary treatment group attributable to type of litter were not statistically significant. At peak lactation, a significant (p < 0.05) elevation in PRL concentration was consistently seen on the second day of blood sampling (days 13, 27 (7.60) ng/ml; days 14. 49 (12, 110) ng/ ml). When observations from day 14 were removed to eliminate the effect of consecutive days of sampling, recovery time from surgery was the most evident effector of PRL release. PRL values were lower in both dietary treatment groups in animals having more time to recover from catheter implantation [3-day recovery: AL, 50 (21,90) ng/ml, and CR, 40 ( 13, 81 ) ng/ml: 4-day recovery: AL, 22 (7.45) ng/ml, and CR, 18 (3, 46) ng/ml, p - 0.07]. Differential behavior of AL and CR dams toward their assigned litters was unexpected. The presence of less attentive behavior was significantly dependent on dietary treatment. Expressed as odds ratios, CR animals were over four times as likely to be less attentive toward their assigned litters (p < 0.01 ). Dams were also over twice as likely to be less attentive toward other litters than their own (p = 0.11 ). In early lactation, maternal behavior was not an important influence on differences in the PRL response between AL own and AL other dams (Table 3). However, the less attentive behavior of CR dams toward other litters explained the majority of the difference in PRL values between AL own and CR other dams. This comparison was chosen to evaluate the role of nutritional status on maternal behavior when suckling vigor was similar between groups (both groups nursing AL litters) and relatively vigorous. Finally, less attentive maternal behavior of CR dams toward other litters attenuated the PRL-elevating effect of nursing the more vigorous pups of AL dams.

DISCUSSION

Our aim was to determine whether the effect of chronic undernutrition on circulating PRL was mediated through an influence on the suckling capabilities of the pups. However, it is apparent that suckling is more accurately characterized as the sum of behaviors of both dams and litters, rather than as an exclusive characteristic of the litter. PRL response profiles reflect the realm of suckling behaviors affected by food restriction and litter exchange, rather than reflecting pup suckling vigor alone. The significant interaction of dietary treatment and the type of litter nursed early in lactation indicates that suckling was, in fact, an important determinant of the differences in circulating PRL attributable to dietary treatment. Although the differences in the PRL response between own and other litters were not significant for either AL or CR dams in early lactation, they were in the hypothesized direction. Differences in PRL due to dietary treatment and litter exchange would have been more easily interpretable had these factors not differentially affected maternal attentiveness. In early lactation, less attentive behavior of CR dams toward other litters contributed substantially to differences between PRL release in AL own and CR other dams. This test indicates that the differences in PRL values between AL and CR dams exposed to litters of the same suckling intensity are primarily attributable to maternal behavior rather than directly to nutritional status

TABLE 3 ESTIMATES OF THE DIRECT AND INDIRECT {THROUGH EFFEC'IS ON MATERNAL BEHAVIOR) CONTRIBUTIONS OF TREATMENTS OF INTEREST TO DIFFERENCES IN THE PRL RESPONSE Estimates

Treatment Groups

Direct (6'3)

Indirect (31)c /~4)

I'otal ti~3 ÷ ~31 X fi4)

AL own-AL other* AL own-CR other CR own-CR other

2.595 -0.580 3.156

0.249 1.736 -0.945

-2.346 -2.316 2.211

* Table is read across. For example, the decrease in PRL valuesbetween AL own and AL other is estimated by the value -2.346. The greatest contribution to this differenceis made directlyby the treatments imposed on the animals (-2.595). Behavior was not greatly influenced by these treatments and, therefore, had little impact (0.249).

of the dam. PRL values of CR OTHER dams underestimated the potential PRL response of CR dams, as less attentive maternal behavior attenuated the suckling stimulus to the dam. Similarly, a greater difference in PRL values between CR own and CR other dams would have been seen, reflecting differences in litter suckling vigor, had maternal attentiveness toward other litters not been influenced. Inattentiveness may have been a result of food-seeking behavior. Dams may have expected to be fed when experimenters were present. Such behavior would not be expected and, indeed, was not observed in the AL dams. Because maternal behavior had little influence on the PRL response in AL dams, the differences in PRL values between AL own and AL other could be primarily attributed to the influence of litter suckling vigor. Suckling was not an important determinant of PRL release at peak lactation. Changes in pup behavior and in the responsiveness of the dam to PRL-releasing stimuli over the duration of lactation may explain this finding. Although suckling intensity increases as pups mature (5), rat pups suckle less frequently as lactation progresses (4). Although these long-term changes were not apparent during our 90-min periods of observation, they may have affected the responsiveness of the dams toward PRLreleasing stimuli over the course of lactation. Dams seem to be particularly responsive to the suckling stimulus in early lactation (11,19): the magnitude of the suckling-induced PRL response declines after reaching its peak within the first week of lactation, even when dams are provided with more vigorous foster litters (19). A decreased suckling-induced PRL response may have allowed stress-related release of PRL, typically suppressed during active lactation (6,13), to prevail. The stress of catheter implantation also resulted in the reduced food intake of AL rats. Others who have utilized chronic catheterization have not reported dietary intake data (10-12,14,1921), so it is not known whether this effect is common. A decreased appetite is a concern because it may have caused a period of acute nutritional deprivation in AL animals when adequate nutriture was desired. However, others have not found an effect of short-term food deprivation on plasma PRL concentrations (24). This experiment has underscored the importance of accounting for behavior as an intermediary through which undernutrition works to alter PRL concentrations. Observations of suckling activity during the 90-min periods were not directly related to the PRL response, which, not surprisingly, was sporadic. PRL is secreted in episodic bursts when dams and pups are allow to nurse naturally ( 12,15,20,21). A 2-h period of sep-

NUTRITION, SUCKLING, AND PROLACTIN

1019

aration, designed to motivate interaction between dams and pups, was probably not enough time to replete the pituitary with enough PRL to allow a steady release of P R L over the course of the suckling bout (10). In fact, data collected in a preliminary study (26) suggested that response profiles for dams after a 2-h period of separation were similar to those of dams allowed to nurse naturally. However, the use of a single index of behavior and the statistical technique of path analysis did provide insight into the contribution of behavioral factors to measures of the PRL response. We have since used our results to develop an instrument to characterize the effect of nutritional status on behavioral measures of dams and litters more accurately. With this, we hope to distinguish more accurately how nutritional interventions affect behavior of dams and pups independently, and how these behaviors relate to hormone release in lactating dams.

We expect behavior also to be an important variable in affecting circulating P R L values in undernourished lactating women, the study of which initially motivated the development of this model for chronic undernutrition. In h u m a n populations the relationship of nutritional status to circulating P R L is in the direction opposite to that in rats, with elevated P R L seen in undernourished w o m e n (17,18). Our findings lend credence to the hypothesis that this trend in P R L concentrations is determined by infant suckling and breastfeeding behaviors. ACKNOWLEDGEMENTS This work was supported by a grant from NIH (HD-14953). The gift of the prolactin assay kit from the NIDDK is gratefully acknowledged. The authors would like to thank Michelle McGuire, Debbie Dwyer, and Linda Bennett for technical assistance, and Dr. Edward A. Frongillo, Jr. for statistical consultation.

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