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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
,4,5
,6
* Department of Animal Science, University of Tennessee, Knoxville 37996;
and
Animal Physiology Research Unit, ARS, USDA, Columbia, MO 65211; and and
Department of Animal Sciences, University of Missouri, Columbia 65211
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: appetite • growth • neuroendocrine • piglet • weaning
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; McCracken et al., 1995). The biological mechanisms underlying the postweaning growth lag are poorly understood. Suppressed intake may result from stressors encountered at weaning, such as maternal separation, relocation to new housing, introduction into new social groups, and changing to a dry diet (Hampson and Kidder, 1986; Ekkel et al., 1995).
Few studies have evaluated metabolic and endocrine responses of the pig during weaning (Carroll et al., 1998; Matteri et al., 2000). Typical of the endocrine profile seen during inadequate feed intake (Buonomo and Baile, 1991), weaning the piglet increases concentrations of GH and decreases serum concentrations of IGF-I and IGF-II (Carroll et al., 1998; Matteri et al., 2000). Importantly, although weaning clearly decreases feed intake, associated changes in the expression of appetite-controlling genes such as leptin, neuropeptide Y (NPY), orexin, type 2 orexin receptor, proopiomelanocortin (POMC), and agouti-related protein remain to be determined. An inadequate understanding of the effects of weaning on these gene products limits our capability to develop targeted strategies for maintaining early postweaning performance.
The objective of the current study was to evaluate the expression of appetite-regulating genes in response to weaning. To produce contrasting states of growth and feed intake, pigs were not weaned (cross-fostered to a lactating sow within the same farrowing room), weaned onto a standard starter diet, or weaned onto a diet containing spray-dried plasma. The use of spray-dried plasma has been reported to significantly improve feed intake, intestinal development, and growth in weaned pigs (Coffey and Cromwell, 1995; de Rodas et al., 1995; Touchette et al., 1997, 1998; Matteri et al., 2000).
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experimental Design
A total of 24 barrows (14 d of age), with an initial BW of 4.69 ± 0.13 kg, were allotted to 1 of 3 treatments groups (8 pigs per group) as follows:
SOW: pigs were cross-fostered to a sow in 1 farrowing crate, such that the sow was not the natural mother to any of the pigs;
NP: pigs were contained in 1 pen and given a standard phase I diet containing 0% spray-dried plasma; and
SDP: pigs were contained in 1 pen and given a standard phase 1 diet containing 7% spray-dried plasma.
Pigs were allocated by size, resulting in groups of small (3.95 ± 0.07 kg of BW, 3.5- to 4.3-kg range) and large (5.10 ± 0.09 kg of BW, 4.6- to 5.7-kg range) animals distributed across weaning groups (4 small and 4 large/group).
Pigs were checked at least once daily for health problems and feed availability. All pigs were allowed access to food and water at all times. Body weights were recorded daily for each pig. The phase I diets were formulated to contain equal amounts of digestible essential AA and also ME and are described in detail by Matteri et al. (2000).
On d 4 of treatment, all pigs were euthanized by electrical stunning followed by exsanguination. Animals were euthanized randomly with regard to treatment group from 0800 to 0930 h. Hypothalamic and s.c. adipose tissues were quickly removed and placed on dry ice until stored at –80°C for subsequent RNA isolation. The collected hypothalamic tissue was bounded by the cranial edge of the optic chiasm, the caudal edge of the mammillary bodies, and dorsally by the hypothalamic sulcus. The adipose tissue was collected from the back of the neck at the site of decapitation. A blood sample was also collected for serum hormone analyses, the results of which were previously published (Matteri et al., 2000) and reviewed briefly in the discussion section of this manuscript.
Hypothalamic gene expression was measured for NPY, orexin, type 2 orexin receptor, long-form leptin receptor, agouti-related protein, and POMC; adipose lep-tin expression was also measured (note that orexin as the term is used in this manuscript refers to the prepro-orexin transcript; the gene products orexin-A and orexin-B are both end products of this transcript).
Riboprobe Generation
Polymerase chain reaction was used to amplify complementary DNA (cDNA) for NPY, orexin, type 2 orexin receptor, long-form leptin receptor, agouti-related protein, POMC, leptin, and 28S ribosomal RNA. The PCR products were cloned into vectors, and the identities of the cDNA clones were identified by sequencing. Transcription with biotinylation (Maxiscript kit, Ambion, Austin, TX) was performed on each vector to produce biotinylated complementary RNA probes for use in chemiluminescence-based hybridization detection. Primer sequences used for PCR amplification of cDNA, references describing the initial probe generation for each target, and Northern blot data are shown in Table 1. The sequence of the porcine type 2 orexin receptor cDNA fragment, which had not been previously characterized, has been submitted to GenBank (accession number AF059740).
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Statistical Analysis
Homogeneity of variance was confirmed throughout the data set by F-test. Body weight data were evaluated by ANOVA [weaning weight (WW) x weaning group with repeated measures over time). Expression data for feed intake regulators were analyzed by 2-way ANOVA (WW x weaning group). Weaning weight was removed from the model for any endpoints for which there was not a significant effect, and the results from the simplified model were presented. Fisher’s LSD test was used to compare means. These analyses were performed with Statview (SAS, Cary, NC). To better understand the apparent interrelationships among the expression of appetite-regulating genes, partial correlations were performed, controlling for initial pig WW. This statistical analysis was performed with SAS.
It can be argued that pen and treatment are confounded, and therefore, the results are due to the effect of pen; however, we have not observed an effect of pen in previous studies in these facilities (Carroll et al., 2002; Touchette et al., 2002; Frank et al., 2003).
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Cross-fostered pigs gained more BW over the 4-d experimental period than did those pigs fed dry feed, regardless of SDP content (P < 0.001, Figure 1B). At d 4, however, the SDP group gained the same amount as the SOW group; the NP group did not experience that gain (P < 0.001, Figure 1C). There was no effect of WW on these gain-related endpoints. Body weight data from this study (along with serum concentrations and gene expression of growth-related gene products) have been previously reported and discussed (Matteri et al., 2000).
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). A weaning group x WW interaction (P < 0.05) was detected for hypothalamic NPY mRNA expression, such that expression was least in the small SDP pigs. Expression of genes encoding adipose leptin, hypothalamic long-form leptin receptor, or hypo-thalamic POMC expression was not affected by weaning group or WW (P > 0.1).
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). The strength of the relationship between the expression levels of any 2 genes was in most cases due in large part to the association of gene expression to initial pig WW. After controlling for pig WW, only the relationships between the expression of type 2 orexin receptor and POMC and between agouti-related protein and POMC remained very strong (P < 0.001). Relationships between expression of orexin and type 2 orexin receptor, orexin and agouti-related protein, type 2 orexin receptor and agouti-related protein, and type 2 orexin receptor and leptin receptor remained (P < 0.05) but were weakened considerably by controlling for WW.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), likely influenced by the well-established effects of depressed feed intake on somatotropic function (Vance et al., 1992; Straus, 1994). Having established this time frame, we evaluated early postweaning changes in gene expression related to appetite control. Matteri et al. (2000) described growth data (serum hormone concentrations and gene expression of known regulators of growth) observed in this study. Briefly, levels of specific mRNA for growth regulatory genes did not differ between nursed and dry-fed piglets 4 d after weaning. Serum IGF-I and IGF-II concentrations were decreased in both NP and SDP compared with SOW. Small pigs had lower levels of liver IGFBP acid labile subunit, muscle IGF-II, and muscle GH receptor mRNA, as well as serum concentrations of IGF-II. In contrast, adipose tissue IGF-I and IGF-II mRNA levels were greatest in the small piglets.
Little information exists concerning the regulatory in-fluences of weaning, nutrition, and development on expression of porcine appetite-regulating genes such as NPY, leptin, orexin, and agouti-related protein. Neuro-peptide Y is a hypothalamic peptide with potent appetite-stimulating activity (Billington et al., 1991). The expression of NPY is elevated in undernourished animals (Brady et al., 1990; McShane et al., 1993) and is regulated in part by leptin (Stephens et al., 1995), an appetite-suppressing hormone secreted by fat cells (Zhang et al., 1994). Leptin production corresponds with body fat content, thus creating a monitor of fatness by which to regulate appetite (Considine et al., 1996). Orexin has been shown to increase feed intake in rats (Sakurai et al., 1998; Muroya et al., 2004), pigs (Dyer et al., 1999), and sheep (Sartin et al., 2001). Like NPY, it is expressed at increased levels in undernourished rats (Sakurai et al., 1998). Recent evidence suggests that orexin directly regulates NPY-containing and other glucose-responsive neurons in various regions of the hypothalamus (Muroya et al., 2004).
The agouti-related protein is a melanocortin receptor antagonist that blocks the appetite-suppressing effects of 
-melanocyte-stimulating hormone, a product of the POMC transcript (Kalra et al., 1999). Other products of the POMC transcript, the endorphins, stimulate feed intake (Morley et al., 1983; Levine and Billington, 1989). Expression of POMC in the paraventricular nucleus is increased by NPY (Kalra et al., 1995), and there is evidence that agouti-related protein is co-released with NPY in the paraventricular nucleus (Broberger et al., 1998; Hahn et al., 1998). A potential role for agouti-related protein could be to alleviate the appetite-suppressing effects of 1 POMC product (-melanocyte-stimulating hormone) while allowing another product (the endorphins) to stimulate appetite. In the arcuate nucleus, POMC expression is decreased by NPY (Garcia de Yebenes et al., 1995). We did not see WW- or treatment-specific changes in POMC expression, perhaps because we used complete hypothalamic samples containing both the paraventricular and arcuate areas. Another consideration is that most research in this area (including those publications just discussed) has been performed using rodent models; species differences may certainly apply.
The lack of associations between BW and adipose lep-tin or hypothalamic leptin receptor expression supports the concept that leptin expression may be regulated independently of adiposity or feed intake during early development (Hassink et al., 1996; Nakavisut et al., 1997; Ahima et al., 1998). The positive relationships between WW and hypothalamic NPY, orexin, type 2 orexin receptor, and agouti-related protein expression are suggestive of individual predispositions (either genetically or environmentally induced) to eat and grow. Although it is tempting to speculate that bigger piglets are larger because they eat more due to hypothalamic stimulation of appetite, this relationship must be better characterized by further investigation before that claim may be made.
Increases in both feed intake (Coffey and Cromwell, 1995; de Rodas et al., 1995; Touchette et al., 1998) and intestinal health (Touchette et al., 1997) have been reported in weanling pigs fed spray-dried plasma. Consistent with these previous studies, the present data revealed an increase in BW gain over the last day of the study (from 3 to 4 d after weaning). This increase in growth was not associated with significant changes in most of the appetite regulators examined in this study; changes in appetite regulator gene expression might not be detectable when evaluated at a single time point if the neural stimulation of appetite preceded the increase in intake in the SDP group. Although the SOW group was not weaned, those pigs did experience such stresses as social mixing and having to establish a new teat order. These events may have disrupted feed intake in this group as well, contributing to the failure to observe differences in gene expression due to treatment. The weaning group WW interactive effect on NPY expression observed in the current study, in which NPY mRNA levels were least in small SDP pigs, may be explained by compensatory feedback regulation of NPY following a satiating meal. The increased BW gain seen in these pigs on d 4 indicates that they had ingested a substantial amount of feed before euthanasia. A similar study in which pigs are euthanized at times before d 4 postwean-ing would allow for the observation of changes in neuro-endocrine expression as these animals undergo the weaning process.
The positive relationships observed between pre-WW and postweaning hypothalamic agouti-related protein, orexin, type 2 orexin receptor, and NPY gene expression may reflect physiological roles of these factors in the regulation of growth in the young pig (acknowledging that changes in gene expression at the level of the transcript do not necessarily imply changes at the level of protein function). We have determined that levels of spe-cific mRNA-encoding proteins that regulate appetite do not differ between nursed and dry-fed pigs 4 d after weaning. The increase in growth produced by dietary SDP over the final day of this study was not accompanied by significant changes in appetite-related gene expression. These data raise important questions relating to the temporal relationships between postweaning changes in gene expression and function. Continued studies of the effects of weaning on the physiology of the pig will provide a better understanding of the regulation of appetite, growth, and adaptation to stressors encountered within the production environment.
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Footnotes |
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2 Mention of a trade name or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may be suitable.
4 Current address: Livestock Issues Research Unit, ARS, USDA, Lubbock, TX 79403.
5 Current address: ARS, USDA, Albany CA 94710.
6 Current address: Cargill Inc., Elk River, MN 55330.
3 Corresponding author: ckojima{at}utk.edu
Received for publication November 9, 2006. Accepted for publication May 9, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This article has been cited by other articles:
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  T. A. Cooper, M. P. Roberts, H. G. Kattesh, and C. J. Kojima Effects of Transport Stress, Sex, and Weaning Weight on Postweaning Performance in Pigs Professional Animal Scientist, April 1, 2009; 25(2): 189 - 194. [Abstract] [PDF]
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 C. J. Kojima, H. G. Kattesh, M. P. Roberts, and T. Sun Physiological and immunological responses to weaning and transport in the young pig: Modulation by administration of porcine somatotropin J Anim Sci, November 1, 2008; 86(11): 2913 - 2919. [Abstract] [Full Text] [PDF]
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