Effects of cholecystokinin on liquid diet intake of early weaned piglets

Physiology & Behavior 68 (1999) 163–168

Effects of cholecystokinin on liquid diet intake of early weaned piglets E. Baranyiová,a,* R.L. Hullingerb a

Department of Biochemistry, University of Veterinary and Pharmaceutical Sciences, 612 42 Brno, and Veterinary Research Institute, 621 32 Brno, Czech Republic b Department of Basic Medical Sciences, PU School of Veterinary Medicine, West Lafayette, IN 47907-1246, USA Received 7 August 1998; received in revised form 19 July 1999; accepted 11 August 1999

Abstract The effect of cholecystokinin octapeptide (CCK-8) on the consumption of a liquid diet was investigated in 10 piglets, weaned on Day 1, housed individually in cages, and offered a commercial diet for suckling from feeding bottles nine times a day at 2-h intervals with an 8-h break during the night. CCK-8 was administered to piglets (n 5 5) on Days 2, 4, 6, 8, 10, 12, 20, and 23 in single intraperitoneal doses of 4 mg·kg21 in saline, 5–7 min before the first morning feeding at 0600 h. The relative diet intake in CCK-treated animals was reduced significantly (p , 0.05) on Days 2, 4, 6, 8, 10, 12, and 23 compared to saline-treated controls, and on Days 2, 4, 6, 8, 10, 12, and 23 when the consumption at 0600 and 0800 h in CCK-treated piglets was compared. Our data support the notion that the short-term mechanisms involved in food intake control of piglets are relatively complex and operative at the time of birth, and that CCK plays a role in their food intake regulatory cascade. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Early weaning; Newborn piglets; High-fat diet; CCK; Food intake; Control

1. Introduction Ample evidence has accumulated of cholecystokinininduced satiety across species from invertebrates to mammals [1,2]. Gibbs et al. [3] first reported that cholecystokinin (CCK) reduced the food intake of rats. Effects of CCK have also been extensively studied in swine. Various aspects of the inhibitory action of CCK following peripheral and central administrations on operant [4,5] and voluntary [6–8] food intake and feeding behaviour have been described in weaned [9] and prepubertal animals whose food intake pattern and controls are typical of adult individuals. The effects of CCK in this species have also been studied using its specific receptor agonists [54] and antagonists [8,11,12]. CCK has also been shown to reduce operant sugar intake in pigs [13]. Experimental superalimentation in growing pigs has been achieved via immunization against CCK [10,14]. However, food intake of piglets and its control mechanisms are different during the early postnatal period [15–23], when: (1) milk is the sole source of energy and fluid, and (2) its distribution is initially fully controlled by the mother. Other regulatory systems come into play during the weaning period [18,24]. Similarly, it has been shown in newborn rats that food intake controls develop gradually [25].

* Corresponding author. Tel.: 420-5-4156-2505; Fax: 420-5-4121-1151 E-mail address: [email protected]

To our knowledge, the role of CCK and its interactions with other factors involved in food intake control during the postnatal period have been studied only in rats. In this altricial species, there is evidence for an ingestive and other roles for CCK postnatally [26–30,55,56]. Indirect evidence of CCK action in precocial animals, piglets, has been demonstrated, when substances known to elicit CCK secretion, suppressed feeding of piglets on the sow, as measured by their body mass changes [22]. Moreover, there is also immunohistochemical evidence of CCK presence in the cerebral cortex of adult pigs [31] and its early presence in brain and gastrointestinal tract of porcine fetuses [32]. The aim of the present experiment was to determine whether exogenous CCK is an effective satiety agent during the early postnatal period in piglets. We employed a rearing system that excludes nutritional dependence on the sow and competition for milk with littermates [15,33–35]. This system allows the piglets to determine only the style of suckling and sizes of their diet intakes but not the feeding frequency or any extra intake of food and fluid. A preliminary report has appeared [36]. 2. Materials and methods Ten (crossbred—Landrace, Yorkshire, Pietrain) littermate piglets (seven females and three males) were kept under conventional farm conditions during Day 1 after birth (with approximately 12 h available to them for colostrum

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suckling), after which they were removed from their clinically healthy mother and transferred to the laboratory. They were weighed, placed individually in cages, in a controlled thermoneutral environment [37], with warming pads on the cage floor, and fluorescent light illumination from 0600– 1800 h. Plastic toys were available to each piglet for environmental enrichment. Piglets were offered the Purina Baby Milk Replacer diet reconstituted in water and warmed to 38–408C for suckling ad lib from a feeding bottle nine times a day, at 2-h intervals from 0600–2200 h, followed by an 8-h night pause [24,33,34,38,39]. The diet intake was measured by weighing the bottle before and after feeding of each piglet. All feed weighings were accurate to 60.1 g. Piglets were allowed to nurse until they stopped. Their regular suckling pattern [40] was not observed after CCK administration. Sulphated cholecystokinin octapeptide (Sigma, St. Louis, MO) dissolved in 0.9 NaCl with 7.5% bovine albumin [41] was administered to five piglets on Days 2, 4, 6, 8, 10, 12, 20, and 23, in single i.p. doses of 4 mg·kg21 body mass in 1 mL saline. Except for Day 2, when they were treated at 1200 h, i.e., as soon as their diet consumption from the bottle had stabilized, the treatments were given before the first morning feeding, i.e., before 0600 h and after an 8-h fast. Each piglet was offered the diet 5 min after CCK injection. The control piglets (n 5 5) were given equal volumes of saline (i.p.). The experimental and control groups were alternated on days of CCK administration. All relative diet intakes of CCK-treated and control piglets were compared at the 0600-h feedings; further diet intakes of CCK-treated piglets were compared at the 0600, and the subsequent 0800-h feedings. All diet intakes of CCKtreated piglets at 0600 h were calculated as a percentage of the amounts eaten the day before and the day after the CCK treatment [9]. 2.1. Statistical data analysis This study thus represents an attempt to describe time profiles in daily food intake of pigs in early life phases with

respect to the potential influence of CCK. The food intake data gathered in this way required careful application of parametric statistical methods due to the problems with limited sample size or outliers. Where necessary, selected extreme points (up to one per variable and time unit) were excluded so as to reach more normal distribution. Evaluation of daily food intake time profiles was based on nonparametric Kruskal–Wallis analysis testing the differences among all reasonable experimental variants. The Mann–Whitney test was subsequently applied for detailed inspection of differences among variants [42]. The Mann– Whitney test was also used for testing the differences in Table 1. The experimental procedures had prior approval of the Purdue University Animal Care and Use Committee. 3. Results Postnatal growth and diet consumption of piglets in this experiment did not differ from our previous findings [24,33,34,39]. Their body mass increased from 1327 6 58 g to 5735 6 190 g on Day 23, i.e., more than four times, while their actual whole-day diet intake rose from 308 6 23 to 1912 6 84 g. The relative diet intake per kg21 body mass was lower than that of the saline-treated controls on the respective treatment days except 20. The immediate postinjection relative diet intake in CCK-treated piglets at 0600 h was significantly lower than that at the subsequent feeding at 0800 h, at all days except 20 (Fig. 1). When the CCK-treated piglets served as their own controls, i.e., when comparing their diet intake at 0600 h on the day of treatment with half of sum of that at 0600 h on the days preceding and following the treatment [9], a consistently lower diet intake was found on the day of treatment as a percentage of that eaten on control days (Table 1). This effect occurred after an 8-h night fasting period. Changed feeding behaviour was observed in CCKtreated piglets in that their vocalization and motor activity

Table 1 Relative diet intake at 6 h of CCK-treated and control piglets on days of treatment, compared with half of their relative diet intakes on days preceding and following the treatment (g kg21 6 SEM) Day CCK Day of treatment 1/2(prec. 1 foll.) Significance % diet intake Controls Day of treatment 1/2 (prec. 1 foll.) Significance % Diet intake

4

6

8

10

12

20

23

15.1 6 4.1 19.4 6 1.9 NS 76

15.6 6 3.6 25.7 6 2.5 p , 0.05 60

21.4 6 6.7 33.2 6 2.7 NS 65

20.7 6 2.5 33.2 6 3.2 p , 0.05 59

24.4 6 4.6 35.1 6 2.2 p , 0.05 69

32.3 6 4.6 45.7 6 1.8 p , 0.001 71

27.7 6 2.5 41.0 6 5.0 p , 0.05 68

17.3 6 2.5 22.3 6 3.2 NS 78

33.0 6 1.8 23.8 6 3.1 p , 0.05* 72

41.4 6 5.6 32.8 6 1.9 NS 80

36.1 6 3.4 33.8 6 1.5 NS 94

35.7 6 5.4 33.4 6 3.2 NS 94

34.5 6 4.6 40.3 6 2.1 NS 86

39.0 6 1.2 43.7 6 3.0 NS 89

* Controls ate more on day of experiment. Mann–Whitney test was used to assess the differences between groups.

Fig. 1. Relative diet intake of CCK- and saline-treated piglets at the immediate postinjection feedings, and that of CCK-treated piglets at the next 0800-h feedings. Experimental variants marked by the same capital letter are not significantly different (p , 0.05, Kruskal–Wallis test). The data are presented as means 6 SEM.

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were both decreased within 5 min of CCK administration, and remained low for an interval of about 30 min. They became sleepy, ingested the diet quietly, showed little exploratory or grooming activities, and went to sleep.

4. Discussion The limited number of studies of prepubertal or adult swine have shown that repeated administration of CCK suppresses food intake, elicits an instant response, and this response is of short duration [10,14]. Results obtained in our study are in agreement with these findings, and extend the period of study into the early postnatal period life. They add further evidence to the notion that the mechanisms of food intake control in this species are relatively complex and functional as soon as after birth, and include, for example, the serotoninergic system [43,44]. The major site of CCK action for decreasing meal size in pigs is the vascular bed of the cranial mesenteric or coeliac arteries [7]. In this study, a different approach, namely a 10min infusion of 67 ng/min (i.e., a six times lower total dose than in our study) onto the serosal surface of the duodenum and ileum, yielded inconclusive results. The different responses obtained in these two studies may be due to the dose, length of application but may also be age dependent. On the other hand, CCK-8 infused into jugular vein, carotid artery, and cranial aorta resulted in significant reduction of meal size to 65, 67, and 71% of control meal size in older pigs, similar to meal size reductions in our piglets. The milk ingestion of suckling piglets under natural conditions is short: 8 to 40 s [45]. Its duration was not different in bottle-fed piglets during their suckling period, i.e., until Days 6–10 [40]. In this period, their meal termination is typically abrupt, despite the fact that the diet continues to be available. In piglets in the weaning period [46] with increasingly larger actual amounts of the diet consumed the suckling time tended to exceed 1 min [40]. These suckling times may be too short to allow for a postabsorptive satiety signal to be operative; they are more likely to be governed by rapidly acting preabsorptive signals, presumably also endogenous CCK [9,47]. In this experiment, the suppressing effect of exogenous CCK on diet intake was not even prevented by an 8-h lasting period without access to food, at an age when damnursed piglets may have as many as 20–30 meals during a 24-h day with short intermeal intervals. As in other species studied, the food intake-suppressing effect of CCK was of short duration. However, the diet intake at the next feeding, 2 h later, tended to increase, and more so in piglets approaching the weaning period. At all ages, the piglets did compensate for a smaller diet intake in the morning, as there was no difference in relative whole-day diet consumption compared to control animals (not shown). Intensive vocalization, high motor activity, nosing against the cage walls, salivation, “vacuum” sucking regu-

larly occur in individually housed and diet-fed piglets in anticipation of feeding [40]. Most of these behaviors disappeared in CCK-treated piglets within 5 min, for a period of about 30 min. They suckled the diet quietly, sometimes even reluctantly, and went to sleep with no postingestive rubbing the snout, rooting, or other activities. No signs of discomfort were observed. Our observations are in good agreement with those of Weller and Blass [29,30], who describe CCK as a positive quieting stimulus reducing levels of distress vocalizations in neonatal rats. CCK is known to also elicit postprandial sleep in human babies [48]; in breast-fed infants plasma CCK concentration rise after a meal [49] as they pass into sleep. The sow’s milk is high in fat, especially between Weeks 1 and 4 (7—8%) of lactation, and later, the fat amount decreases to about 6% in Week 8 [50]. The piglets thus consume some 53% of their energy needs in fat in the first week [15] after birth. This amount decreases to 40% in Week 8. Also, the piglets in this experiment were offered a high-fat diet, similar to sow’s milk. CCK release has been shown to response to dietary fat and induce satiety in other species [48], and also in older pigs [6]. It is, therefore, conceivable that considering the high fat amount both in milk and diet, the regulatory mechanisms responding to it, other than just gastric distension signals, are functional after birth in piglets. One of such short-term control mechanisms is, no doubt, the CCK system, involved in suppression of food intake. On the other hand, if the fat content of the diet is decreased by its dilution, piglets younger than 14 days are not able to compensate for this change [18,25]. Some of the food intake control mechanisms, namely the long-term ones, develop gradually in piglets as they enter the weaning period [18,20,22–24]. On the other hand, factors that are involved in the short-term regulation such as the serotoninergic system, are developed and fully functional as soon as on postpartum Day 1 [44]. There is evidence that CCK and serotonin systems may mutually interact to induce a state of satiety [51,52], because the CCK system is also operative soon after birth, as shown in the present experiment, and it can be hypothesized that such a link between these two and other systems in the food intake regulating cascade is operative in newborn piglets. Morphological evidence of CCK in early porcine embryos and fetuses [32] seems to be indicative of other possible roles for CCK in the course of prenatal development, but also in occurrence of neonatal behaviors, similar to findings in newborn rats and possibly involved in establishing the mother–litter bond and its dissolution over time [30,55,56]. Our data indicate that its role in food intake control and feeding behavior comes into play with the onset of milk nutrition. However, the magnitude of the response to CCK administration in the period under study changed, suggesting age-dependent changes during the first month of postnatal life. The results of our study indicate that CCK is an important component of food intake regulation in swine in the early postnatal period.

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Acknowledgments This study was made possible by a J.W. Fulbright Scholarship to E.B., and the generous support of the D.R. Gore Trust. Thanks are due to Professor J. and E. Modrey, and to Professor A. Holub, who have contributed to different aspects of this work, and to Dr. L. Dusek for statistical analysis.

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