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Unité Mixte de Recherches sur le Veau et le Porc, Institut National de la Recherche Agronomique, St-Gilles, France
1To whom correspondence should be addressed. E-mail: luron{at}roazhon.inra.fr
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • weaning • feed intake • pancreas • gene expression • pig
The capacity of young pigs to rapidly adapt to, and adequately digest, dry feed after weaning has a major influence on their subsequent performance. However, these capabilities are questionable during wk 1 postweaning because the apparent digestibility of nutrients is usually low at this time (1
,2
). Weaning stress is characterized by a sudden decline in most pancreatic enzyme activities (3
–7
). A period of time ranging from 2–3 wks postweaning is required to recover comparable enzyme contents in the pancreas. This period of time is dependent on dietary protein source but mainly on the level of dry feed intake. At d 3 postweaning, "noneaters" apparently accumulated large amounts of enzymes in their pancreas without substantial release into the gut (8
). Therefore, it can be expected that weaning affects gene expression and synthesis of enzymes in the pancreas, which in turn could affect the capacity for adaptation of the pancreas to dry feed intake.
The aim of this study was to examine changes in digestive capacity of the exocrine pancreas during the 2 wk after weaning, by measuring gene expression and activities of digestive enzymes in the pancreas. Moreover, effects of feed intake levels during the immediate postweaning period were analyzed. To identify potential humoral signals linking the weaning process and the pancreatic response, the relationship between circulating concentrations of gut regulatory peptides and exocrine pancreatic function was also determined.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Treatments and experiments were conducted according to the European Community regulations concerning the protection of experimental animals. Saving piglets that are above the rearing capacity of the sow (9
) by weaning at 6–8 d of age is accepted by the European legislation [Report of the Scientific Veterinary Committee 1997 (10
)]. Two trials involving 56 crossbred Large White x Landrace piglets of both sexes were designed to study the effect of weaning at 7 d of age (trial 1) and the effect of postweaning feed intake (trial 2) on the adaptation of pancreatic function. According to a previously described procedure (11
), weaned (W)2 piglets were tube fed to prevent feed intake variability, which is usually observed just after weaning (2
). In addition, a blood catheter was placed in a jugular vein of W and sow-reared (SR) piglets. The catheter of SR piglets was held in a bag placed on the dorsal side of the piglet’s neck and fixed using surgical tape. SR piglets were replaced in their original litter 
3 h after surgery. W piglets were housed individually in stainless steel metabolic cages providing visual contact with each other and allowing separate collection of feces and urine. Room temperature was initially set at 32°C, progressively decreased to 28°C on d 7 postweaning and was constant thereafter. W piglets were fed a complete commercial formula (Porkisec; Celtilait, Ploudaniel, France) (Table 1
) based on dairy products, extruded cereals, fish meal, toasted soybean, soybean and coconut oil and containing levels of vitamins and minerals meeting or exceeding the NRC requirements for piglets (12
). From a 10-d total collection of feces and urine starting on d 4 postweaning, the average (mean of d 4 to d 7 and d 8 to d 14) metabolizable energy (ME) value of the diet was estimated to be 16.64 kJ/g. Fecal digestibility of energy intake was 89.3 ± 0.5% from d 4 to d 7 after weaning and increased to 91.1 ± 0.4% (P < 0.05) during the 2nd wk postweaning (trial 1). Nitrogen digestibility was not significantly changed over these two periods (82.3 ± 0.7 and 83.9 ± 0.4%, respectively). Before each feeding, the powdered diet was vigorously mixed with water to a final concentration 200 g/L from d 1 to d 7 postweaning and 250 g/L during the remaining period. The liquid feed was warmed to 37°C and slowly administered with a syringe via the cannula. The first meal was fed 2 h after surgery. Piglets were fed their calculated feed allocation six times daily (at 0700, 1000, 1300, 1600, 1900 and 2300 h), and had no access to additional water.
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). Because of the high frequency of feeding (see above) and the low level of dry feed intake per meal, we assumed recovery of digestive proteins in W piglets was complete at the time of killing. Piglets were killed by an intracardiac administration of an overdose of sodium thiopental (30 mg/kg body; Nesdonal, Merial, France) and exsanguinated. Immediately after slaughter, the pancreas was gently removed, weighed and cut in several small pieces. Randomly sampled pieces (1 g in total) were homogenized in deionized cold water, sampled, frozen in liquid nitrogen and stored at -20°C until analysis. Another set of about 1 g of pancreatic pieces was homogenized with a Polytron in a 50 mmol/L Tris-base buffer, pH 7.5, containing 5 mol/L guanidium thyocyanate, 25 mmol/L EDTA and 10 mL/L 2-mercaptoethanol. These samples were frozen and stored at -80°C until extraction of RNA. In trial 1, blood samples were collected both in W piglets at 8, 10, 12, 14, 17 and 21 d of age and in SR piglets at 8, 14, 17 and 21 d of age. In trial 2, blood samples were collected at 7, 9, 10, 11, 12 and 14 d of age. In both trials, blood samples were collected 1 h after suckling in SR piglets and 3 h after the first morning meal in W piglets. In trial 2, an additional blood collection was carried out 3 h after the first meal fed 4 h after surgery at 7 d of age. Trial 1.
Thirty piglets were used in trial 1. Twenty-four piglets were weaned at 7 d of age. On the basis of litter origin and body weight, they were allocated to the four following treatments (six piglets per treatment): killed at weaning (SR7), at 3 (W10), 7 (W14) or 14 d (W21) after weaning. Actual ages of killing were 7, 10, 14 and 21 d, respectively. Six contemporary piglets were sow-reared until killing at 21 d (SR21). Body weights at birth and at 7 d of SR piglets were similar to those of W piglets. No creep feed was provided during the suckling period, although pigs had access to sow feed. However, examination of the stomach contents at killing revealed that they had not consumed solid feed. Daily amounts of ME provided to W piglets (Table 2
) were calculated by mimicking those found in a large number of piglets (9
). In SR piglets, milk ME intake was calculated from the conversion rate of ME into body weight gain (2
). Estimated daily milk ME intake gradually decreased from 2100 kJ/kg body0.75 at 5 d of age to 2050, 1800, 1750 and 1300 kJ/kg body0.75 at 8, 12, 16 and 19 d of age, respectively.
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Twenty-six piglets were weaned at 7 d of age and assigned to five treatment groups (Table 2)
. Ten piglets were fed a high level of feed and killed at 3 (Group H; n = 4) or 7 d (Group HH; n = 6) postweaning. Ten other piglets were fed a low level of feed and killed at 3 (Group L; n = 4) or 7 d postweaning (Group LL; n = 6). A fifth group of piglets (LH; n = 6) was fed the low level of feed during the first 3 postweaning d and switched to the high level of feed during the remaining period and killed at 7 d postweaning.
DNA, RNA, protein and enzyme assays.
Pancreatic DNA contents were assayed according to the Labarca and Paigen (14
) method, whereas RNA contents were determined using the technique of Munro and Fleck (15
). Protein concentration was measured as described by Lowry et al. (16
). Trypsin (EC 3.4.21.4) activity, which is the main pancreatic proteolytic activity after weaning, was measured according to Laine et al. (17
) using N-
-benzoyl-L-Arg-p-nitroanilide as substrate. Lipase (EC 3.1.1.3) and amylase (EC 3.2.1.1) activities were determined as previously described (18
). The resulting enzymatic units are expressed both as nanomoles of hydrolyzed substrate/min (IU) per mg of homogenate protein (IU/mg protein; specific activity) and per kg of body (IU/kg body weight; relative activity).
mRNA quantification by Northern blot.
Samples were prepared according to the extraction procedure of Chirgwin et al. (19
), adapted by Le Huërou et al. (18
). RNA concentration was spectrophotometrically measured at 260 nm and the integrity of RNA was verified by analyzing ribosomal contents by electrophoresis on formaldehyde/agarose gels.
Quantification of mRNA was carried out by Northern blot analysis of total RNA samples with specific cDNA probes. Denatured RNA samples (20 µg per lane) were separated by electrophoresis through a 1.2% agarose/formaldehyde/ethidium bromide gel at 60 V for 3 h and further treated and transferred to a positively charged nylon membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) by downward capillary action. Finally, they were covalently crosslinked by UV rays (0.6 J/cm2). Bovine amylase, trypsin and lipase cDNA (18
) were labeled with [-32P]dCTP (Amersham, Orsay, France) by random priming extension (Gibco BRL, Cergy Pontoise, France) and purified on G50 Sephadex columns (Amersham Pharmacia Biotech, Orsay, France). Prehybridization of nylon membranes was carried out in 500 mL/L formamide (F7508; Sigma, St. Quentin Fallavier, France), 6x standard sodium citrate (SSC), 0.01 mol/L EDTA (pH 8.0), 5x Denhardt’s solution (20
), 5 g/L sodium dodecyl sulfate (SDS) and 0.1 g/L denatured salmon sperm DNA (O17543; Eurobio, Les Ulis, France) for 6 h at 42°C. Radiolabeled probe was added and hybridization was carried out for 20 h at 42°C. Hybridized nylon membranes were washed twice in 2x SSC with 1 g/L SDS at room temperature for 5 min, twice in the same conditions for 20 min and finally twice in 0.1x SSC with 1 g/L SDS at 55°C for 20 min. The radioactive signal was detected using a phosphor screen (Molecular Dynamics, Sunnyvale, CA) and quantified with a scanning and imaging system [phosphoImager STORM 840 (Molecular Dynamics) and ImageQuant software (Amersham Biosciences, Piscataway, NJ)]. Each blot was rehybridized with a random primed fragment coding for 18s RNA to quantify total RNA fixed on membranes.
Peptide hormone radioimmunoassays.
Blood samples were drawn into tubes containing aprotinin (500 kIU/L blood; Antagosan, Hoechst Marion Roussel, Paris, France) and EDTA (4 g/L blood), were mixed gently and were immediately centrifuged (1000 x g for 5 min) to obtain plasma. The chilled plasma samples were stored at -80°C until analyzed. All samples of a trial assay for a given hormone were tested in one session. Gastrin was measured by radioimmunoassay as previously described (21
). Antibodies, human gastrin-17 standard and 125I-gastrin were obtained from RAS7186, SC102 and Y7186, respectively (Peninsula Laboratories, San Carlos, CA). Plasma samples (100 µL) and human gastrin-17 standards were incubated with rabbit gastrin antiserum raised against human gastrin-17; the antiserum recognizes both gastrin-17 and gastrin-34. The sensitivity of the assay was 1 pmol/L and the intraassay CV was 9.3%. CCK concentrations were measured on ethanol-extracted plasmas by using a commercially available radioimmunoassay kit (Euro-diagnostica, Malmö, Sweden). Synthetic CCK 26-33 was used as a standard. The sensitivity of the assay was 0.3 pmol/L and the intraassay CV was 5.5%.
Statistical analysis.
For data of trials 1 and 2, ANOVA was performed using the general linear procedure of Statistical Analysis System (SAS Institute, Cary, NC). In trial 1, differences between treatments were assessed using the least significant difference (LSD) test. In trial 2, differences between treatments at 3 and 7 d postweaning and paired comparisons (L vs. LL, L vs. LH and H vs. HH) were assessed using the LSD test. The regression procedure of SAS was used to assess relationships between plasma concentrations of CCK and gastrin and 1) pancreatic enzyme (activities and mRNA), 2) postweaning amount of feed intake (both trials); between pancreatic enzyme activities and 1) N and energy apparent digestibility, 2) daily body weight gain and 3) amount of feed intake (trial 2), and between enzyme mRNAs and specific activities. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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As expected, because of the dramatic decrease in feed intake, W piglets experienced a growth check immediately postweaning (Table 3
). W piglets lost 9% of their body weight on d 1 postweaning; however, they recovered their weaning weight by d 4 postweaning. During the overall postweaning period (d 7 to d 21), W piglets grew at a mean rate of 152 ± 7 g/d, which was lower than the 270 ± 26 g/d (P < 0.05) in SR piglets. At 21 d of age, W piglets were 24% (P < 0.05) lighter than SR piglets.
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. In W piglets, relative pancreas weight declined (P < 0.05) during the first 3 d postweaning. However, at 21 d of age, it was higher (P < 0.01) in W than in SR piglets. Pancreatic protein concentration in SR piglets decreased by 14% between 7 and 21 d of age (P < 0.05). This age effect was more marked in W piglets. Therefore, protein concentration was 32% lower (P < 0.05) in W21 than in SR7 piglets and 21% lower (P < 0.01) in W21 than in SR21 piglets. In contrast, no variations in DNA and RNA concentrations were observed (data not shown).
In SR piglets, the specific activity (IU/mg protein) of lipase decreased (P < 0.05) between 7 and 21 d of age (Fig. 1
, upper panels). Compared to SR, specific activities of lipase and trypsin in W piglets were reduced during wk 1 postweaning, but the decrease in amylase was not significant (data not shown). Trypsin- and lipase-specific activities were, respectively, 44 and 79% lower (P < 0.05) in W14 than in SR7 piglets. Thereafter, the specific activity of trypsin returned to its preweaning value, whereas that of lipase remained at a low level. At d 21, lipase-specific activity was 71% (P < 0.001) lower in W21 than in SR21 piglets.
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. In W piglets, the relative activity of all enzymes decreased during wk 1 postweaning. Trypsin, lipase and amylase relative activities were 69, 58 and 88% lower (P < 0.05) in W14 than in SR7 piglets. There were no correlations between relative enzyme activities and N digestibility or BW gain.
The levels of trypsin, lipase and amylase mRNA were not affected by age in SR piglets (Fig. 1
, lower panels). In W piglets trypsin mRNA levels transiently decreased immediately after weaning. Trypsin mRNA was 43% (P < 0.05) lower in W10 than in SR7 piglets. Thereafter, trypsin and lipase mRNA linearly increased (P < 0.05). At d 21, levels of lipase mRNA were 3.1-fold higher in W than in SR piglets.
In SR piglets, plasma CCK concentration increased by 59% (P < 0.05) between 14 and 21 d of age (Fig. 2
). In W piglets, plasma CCK concentration was 78% (P < 0.05) lower at d 1 postweaning than in 8-d-old SR piglets, increased up to d 3 postweaning and remained unchanged afterward. At 14 d of age, values in W piglets were not different from those of their SR counterparts, but were 56% lower (P < 0.05) at 21 d of age. In SR piglets, plasma gastrin concentration increased (P < 0.05) from 8 to 17 d of age. Weaning decreased gastrin concentration, which was 60–67% lower (P < 0.05) in W piglets during the first 2 d after weaning than in 8-d-old SR piglets. Gastrin concentration increased (P < 0.05) up to 5 d postweaning and did not change thereafter. In W piglets, of the two gut regulatory peptides, only plasma gastrin concentrations were correlated with mRNA levels of trypsin and amylase (r = +0.58 and r = +0.68, respectively; P < 0.05). When both SR and W piglets were included in the analysis, there was no correlation between plasma CCK and gastrin concentrations and pancreatic variables.
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Initial body weight at 7 d of age was 2.60 ± 0.02 kg and was not different from those of piglets of trial 1. After weaning, the reduction of body weight was dependent on the level of feed intake (mean of 130 ± 22 g on d 1 postweaning in piglets fed a high level of feed and 241 ± 20 g on the first 3 d postweaning in piglets fed a low level of feed). H and HH piglets recovered their preweaned body weight by 3 d after weaning, whereas LL piglets had not recovered it at the time of killing. By 6 d after weaning, the body weight of LH piglets was similar to their preweaned weight, but that of HH and LH piglets did not differ at 7 d after weaning.
Relative pancreas weight was not affected by the level of feed intake during the 1st wk postweaning (Table 4
). At d 3 postweaning, the pancreatic protein concentration in H piglets was 25% lower (P < 0.05) than in L piglets. By 7 d after weaning, the relative activity of amylase was 2.3- and 1.1-fold higher (P < 0.05) in HH than in LL and LH groups, respectively (Table 4)
. In contrast, that of lipase was 1.3-fold higher (P < 0.05) in LL than in HH and LH groups. Similar differences were observed for specific enzyme activities (data not shown). Relative trypsin, amylase and lipase activities measured on d 7 postweaning were correlated with daily BW gain measured between d 3 and d 7 postweaning [r = +0.53 (P = 0.06), r = +0.59 and r = -0.70 (P < 0.05), respectively]. Relative amylase and lipase activities were also correlated with feed intake over the 7-d experimental period (r = +0.71 and r = -0.69, P < 0.05, respectively). By 3 d after weaning, mRNA levels of trypsin, amylase and lipase were 2.0-, 3.3- and 1.5-fold higher in H than in L piglets, but only the variation of trypsin mRNA was significant (P < 0.05) (data not shown). At d 7 after weaning, the enzyme mRNA levels paralleled their corresponding enzyme activities, although differences between groups were not significant.
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). Between 3 and 7 d postweaning, plasma CCK concentrations increased 2.4- and 3.1-fold in HH (P < 0.05) and LH (P = 0.06) groups, respectively. At 7 d postweaning, plasma CCK concentration was 5.2-fold higher (P < 0.05) in HH than in LL piglets. By 7 d after weaning, plasma CCK concentration was correlated with the amount of feed (r = +0.56, P < 0.05), amylase-specific activity (r = +0.69; P < 0.05) and trypsin mRNA level (r = +0.61; P < 0.05). Plasma gastrin concentration increased in HH piglets relative to the postweaning age, from d 4 to d 7 (P < 0.01). In the LH group, the increase was delayed by 2 d and started 5 d after weaning. During the 4 to 7 d postweaning period, plasma gastrin concentrations in the HH group were 1.9- to 3.9-fold higher (P < 0.01) than in the LL group. At d 7 postweaning, plasma gastrin concentration in the LH group was intermediate. Therefore, at 7 d after weaning, plasma gastrin concentration was positively correlated with the amount of feed (r = +0.68, P < 0.05). Plasma gastrin concentration was also correlated with lipase-specific activity (r = -0.62; P < 0.05) and with trypsin mRNA level (r = +0.64; P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) in piglets weaned at 21 d of age. In SR piglets, the decrease in the relative pancreas weight between 7 and 21 d of age agreed with data in the literature (3
,5
,6
,22
,23
). In trial 2, the intake achieved in the HH group, which was 4.6-fold greater over the full experimental period than in the LL group, resulted in a difference in body weight between groups at d 7 postweaning. However, by the time of killing on d 7 after weaning, the relative weight of the pancreas was not affected by the amount of feed, in agreement with Kelly et al. (24
).
Protein content and enzyme activities measured in the pancreas are the result of the balance between pancreatic protein synthesis and release via the pancreatic juice. A modification in the level of both pancreatic protein synthesis and secretion may partly explain the 3-d postweaning changes in pancreatic protein contents relative to the level of feed. By 3 d after weaning, the higher protein contents in the pancreas of L piglets compared to H piglets may result from a decreased release of pancreatic protein into the intestinal lumen of piglets fed the lower level of feed (8
). The lack of substrates in the intestinal lumen may explain the low stimulation of pancreatic secretion (12
,25
). Simultaneously, during the first 3-d postweaning period (trial 1), reduced gene expression, as indicated by the lowest levels of specific mRNA, decreased specific enzyme synthesis, illustrated by low specific activities as previously reported in piglets weaned at 21–28 d of age (6
,7
,26
). The transient postweaning decrease in pancreatic gene expression, which correlated with the level of feed intake, was enhanced at low feed intake.
After the 3-d period of alteration of pancreatic function, the expressions of trypsin and lipase genes were increased, whereas that of the amylase gene was unchanged. The increase in trypsin mRNA that occurred 7 d after weaning preceded that of the corresponding enzyme-specific activity during wk 2 postweaning. Between d 3 and d 14 postweaning, lipase-specific activity was diminished when the lipase mRNA level was increased. All these results on pancreatic gene expression indicate that the transcription was probably not the limiting step for enzyme synthesis after weaning. The translation of messengers and the half-life of enzymes might represent important regulatory steps leading to modulation of enzyme synthesis, as suggested by Gestin et al. (27
) for pancreatic elastase genes. In addition, quantitative and qualitative changes in pancreatic protein secretion reported in piglets after weaning (28
,29
) may also participate in the postweaning variations of pancreatic protein and enzyme contents (30
).
Variations among the relative enzyme activities, dietary composition and level of feed intake were not obvious. The postweaning decrease in the relative activity of pancreatic enzymes was previously observed (5
,7
,26
) in piglets weaned between 14 and 28 d of age. The reduction in lipase activity may be related to the abrupt decrease in lipid content in the weaner diet compared to maternal milk (5
), whereas the involvement of the fat source (sow’s milk vs. weaning diet) has been discounted (7
). In agreement with Owsley et al. (4
), our results did not emphasize an increase in amylase relative activity in W piglets compared to SR piglets, although the weaner diet contained an appreciable amount of starch. In contrast, 7 d after weaning, amylase-specific activity was sharply increased in piglets that consumed more feed compared to those that consumed less feed. Therefore, the etiology of postweaning adaptation of pancreatic enzymes is complex and seems to be specific for each enzyme. During the immediate postweaning period, low relative enzyme activities may induce a reduction in the digestive capacity of W piglets. Thereafter, the high energy and nitrogen digestibilities measured in our study indicated that the digestive potential of weaned piglets and their capacity for adaptation were already high as early as the first postweaning days. However, the diet fed to the 7-d-old weaned piglets contained highly digestible milk products. The effects on digestibility may be different with a vegetable-based diet. On the basis of these data, weaning age (7 d in our study vs. 14–28 d in the literature) does not seem to affect the capacity for adaptation of the pancreas to the weaning procedure. The pancreas can react to weaning feed as early as wk 2 of life. Moreover, similar relative enzyme activities in LH and HH groups indicated that, after a 3-d underfeeding period, the adaptive response of the pancreas to an increasing level of feed was not modified.
Control of the development of the pancreatic function could be exerted by a number of hormones. Among gastrointestinal peptides known to play a role in the regulation of the pancreatic function, CCK and gastrin may modulate pancreatic growth and pancreatic protein synthesis and secretion (31
–34
). In the present study, basal concentrations of CCK and gastrin were higher in SR than in W piglets as previously reported (35
,36
). It is worth stressing here that circulating CCK and gastrin were markedly depressed at weaning (trial 1) and were positively correlated with feed intake in W piglets and some pancreatic enzyme activities and mRNA, suggesting that they are potential pancreatic exocrine function regulators. The decrease in plasma CCK and gastrin levels on d 1 after weaning may be early signals of altered digestive function. However, the physiological relevance of these peptides awaits further studies.
Taken together, our results indicated that the expression of pancreatic enzymes was highly modulated by weaning, leading to a biphasic gene expression pattern. During the first 3 d postweaning, enzyme activity and mRNA levels were sharply depressed and the extent of the mRNA level decrease was dependent on the feed intake during this period. Afterward, the expression of specific genes coding digestive enzymes and the enzyme activities measured in the pancreas were restored or stimulated, excepted for lipase-specific activity. Therefore, the pancreas can react to weaning feed as early as the 2nd wk of life.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 9 August 2002. Initial review completed 9 September 2002. Revision accepted 22 October 2002.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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