Effects of weaning and weaning weight on neuroendocrine regulators of feed intake in pigs.

A depression in feed intake and growth often occurs in the weaned pig. Spray-dried plasma is often added to nursery diets in an attempt to stimulate feed intake during this lag. The current study evaluated gene expression of appetite regulators in hypothalamus and adipose tissue 4 d after weaning. Barrows (2 wk of age) were cross-fostered to a sow (SOW, n = 8) or weaned and fed a nursery diet containing either 0 or 7% spray-dried plasma (NP, n = 8, and SDP, n = 8, respectively). Piglets were allocated such that 2 size groups existed within each experimental group: small (3.5 to 4.3 kg of BW piglets) and large (4.6 to 5.7 kg of BW piglets) subsets, based on weaning weight (WW), existed within each experimental group: small (3.5 to 4.3 kg piglets) and large (4.6 to 5.7 kg piglets). Animals were killed 4 d after weaning for tissue collection. There was a weaning group x WW interactive effect (P < 0.05) on hypothalamic neuropeptide Y messenger RNA expression, such that expression was least in the small SDP piglets. No WW or weaning group effects were seen on adipose leptin, hypothalamic leptin receptor, or hypothalamic proopiomelanocortin gene expression. An effect of WW was seen on hypothalamic neuropeptide Y, agouti-related protein, orexin, and type 2 orexin receptor gene expression, such that large pigs expressed greater amounts of these transcripts (P < 0.002). Strong positive correlations in gene expression were found among all of these genes, whose products are known to stimulate appetite. Partial correlation controlling for initial WW revealed that preweaning size explained most if not all of these associations. These data suggest that the postweaning expression of appetite-regulating genes is more dependent on preweaning conditions than on weaning diet.

Orexin-B modulates luteinizing hormone and growth hormone secretion from porcine pituitary cells in culture.

To test the hypothesis that orexin-B acts directly on the anterior pituitary to regulate LH and growth hormone (GH) secretion, anterior pituitary cells from prepuberal gilts were studied in primary culture. On day 4 of culture, 10(5) cells/well were challenged with 0.1, 10 or 1000 nM GnRH; 10, 100 or 1000 nM [Ala15]-hGRF-(1-29)NH2 or 0.1, 1, 10 or 100 nM, orexin-B individually or in combinations with 0.1 and 1000 nM GnRH or 10 and 1000 nM GRF. Secreted LH and GH were measured at 4 h after treatment. Basal LH and GH secretion (control; n = 6 pigs) was 183 +/- 18 and 108 +/- 4.8 ng/well, respectively. Relative to control at 4 h, all doses of GnRH and GRF increased (P < 0.0001) LH and GH secretion, respectively. All doses of orexin-B increased (P < 0.01) LH secretion, except for the 0.1 nM dose. Basal GH secretion was unaffected by orexin-B. Addition of 1, 10 or 100 nM orexin-B in combinations with 0.1 nM GnRH increased (P < 0.001) LH secretion compared to GnRH alone. Only 0.1 nM (P = 0.06) and 100 nM (P < 0.001) orexin-B in combinations with 1000 nM GnRH increased LH secretion compared to GnRH alone. All doses of orexin-B in combination with 1000 nM GRF suppressed (P < 0.0001) GH secretion compare to GRF alone, while only 0.1 nM orexin-B in combination with 10 nM GRF suppressed (P < 0.01) GH secretion compared to GRF. These results indicate that orexin may directly modulate LH and GH secretion at the level of the pituitary gland.

Partial nucleotide sequence of the porcine corticosteroid-binding globulin (CBG) cDNA and specification of CBG expression sites in postnatal pigs.

The purpose of this study was to develop a porcine CBG cDNA probe in order to examine the porcine CBG mRNA expression in major tissues from the postnatal pig. The reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted to develop the porcine CBG cDNA probe using total RNA extracted from liver of 40-day-old pig. The RT-PCR product was subcloned into the pGEM vector (Promega, Madison, WI) and subjected to restriction enzyme treatments and DNA sequencing. Northern blot analysis was conducted using total RNA extracted from samples (approximately 200 mg) of liver, lung, kidney, and whole adrenal tissue that were collected from pigs on day 3 (n = 2) or day 40 (n = 2) postpartum. A 500 bp partial porcine CBG cDNA encoded 166 amino acids and had 83, 78, and 77% homology to a 494-nucleotide sequence of CBG from sheep, human, and rabbit, respectively. The deduced peptide sequence of the partial porcine CBG showed 77, 62, 60, and 51% homology to sheep, human, rabbit, and rat CBG sequences, respectively. An approximately 1.53 kb CBG mRNA was detected only in the liver tissue. In conclusion, the development of a partial CBG cDNA for swine makes it possible to study the ontogeny and the regulation of CBG synthesis at the molecular level and, based on tissues examined in this study, the liver appears to be the primary source of CBG biosynthesis in the postnatal pig.

Effect of recombinant porcine somatotropin on fetal and placental growth in gilts with reduced uterine capacity.

Crowded uterine conditions were induced by unilateral hysterectomy-ovariectomy (UHO) in 42 gilts to determine the effect of recombinant porcine somatotropin on fetal and placental growth. Gilts were randomly assigned across three replicates to one of three treatments: Control (C; n = 14), daily injections of 1 mL saline from d 0 to 64 of gestation, Early (E; n = 12), 5 mg of rpST/d from d 0 to 30, followed by 1 mL saline from d 31 to 64, and Late (L; n = 16), 1 mL saline/d from d 0 to 29, followed by 5 mg of rpST/d from d 30 to 64 of gestation. Blood was collected from each gilt via jugular venipuncture at d 0 and every 15 d thereafter. Gilts were hysterectomized on d 65 of gestation. Length of placental attachment and fetal crown-rump length were measured. Placentas and fetuses were weighed. Placental length, wet weight, and dry weight were recorded. Treatment with rpST (either E or L) increased (P < 0.0001) maternal plasma IGF-I concentrations relative to controls. Treatment with rpST did not affect placental wet weight or placental DNA content. However, E and L treatments increased the percentage of placental protein (P = 0.01) and placental dry matter (P = 0.10) and increased contact area of uterine-placental interface (P = 0.01). Despite changes in placental composition and morphology, weights of fetuses collected from L-treated gilts did not differ from controls, whereas weights of fetuses collected from E-treated gilts tended to be less than controls (P < 0.06). Administration of rpST increased maternal IGF-I concentrations and placental surface area but failed to increase fetal growth in the UHO model. Therefore, mechanisms that are independent of maternal IGF-I or placental contact area may control early fetal growth under crowded uterine conditions.

Effect of spray-dried plasma and lipopolysaccharide exposure on weaned pigs: II. Effects on the hypothalamic-pituitary-adrenal axis of weaned pigs.

A study was conducted with 20 weaned barrows (14 d, 4.98 +/- 0.21 kg) to determine the effect of feeding spray-dried plasma (SDP) after weaning on the pig's stress response to a lipopolysaccharide (LPS) challenge. After weaning, pigs were fed a diet containing 0 or 7% SDP for 7 d. On d 6 after weaning, all pigs were nonsurgically fitted with a jugular catheter. On d 7 after weaning, the pigs were given i.p. injections of either saline or LPS (150 microg/kg BW) followed by serial blood collection every 15 min for a 3-h period. Following the 3-h blood collection, all pigs were killed and tissue was collected for mRNA analysis. Pig weight on d 7 after weaning was not affected by dietary treatment (P > 0.21). Pigs fed the diet with SDP had lower (P < 0.05) levels of hypothalamic corticotropin-releasing hormone (CRH) mRNA, pituitary gland CRH receptor mRNA, and adrenal gland adrenocorticotropin-releasing hormone (ACTH) receptor mRNA. Dietary treatment did not affect pituitary gland proopiomelanocortin (POMC) mRNA. No effect of LPS treatment was observed in any of the mRNA levels examined. For both serum ACTH and cortisol, there was a significant diet x LPS treatment interaction (P < 0.01) such that both the ACTH and cortisol responses to the LPS challenge were greater in the pigs fed the diet with SDP than in the pigs fed the diet without SDP. For pigs given the saline injection, diet did not affect basal serum cortisol concentration; however, basal serum ACTH concentration was lower in those pigs fed the diet with SDP (P < 0.0001). A diet x LPS treatment interaction (P < 0.024) was observed for adrenal gland mRNA expression for steroidogenic acute regulatory (StAR) protein such that the LPS-induced increase in StAR mRNA was greater in the pigs fed SDP than in pigs fed the diet without SDP. These results demonstrate that pigs fed a diet with SDP have an increased activation of the pituitary-adrenal axis following an LPS challenge compared to pigs fed a diet without SDP.

Effect of spray-dried plasma and lipopolysaccharide exposure on weaned pigs: I. Effects on the immune axis of weaned pigs.

A study was conducted with 20 weaned barrows (14 d, 4.98 +/- .21 kg) to determine the effect of spray-dried plasma (SDP) on the pig's immune response to a lipopolysaccharide (LPS) challenge. After weaning, pigs were fed a diet containing 0 or 7% SDP for 7 d. On d 6 postweaning, all pigs were fitted with a jugular catheter. On d 7 postweaning, the pigs were given an i.p. injection of either saline or LPS (150 microg/kg BW) followed by a 3-h blood collection every 15 min. Following blood collection, all pigs were killed and tissue was collected for mRNA analysis. Additionally, the small intestine was collected for measurement of villus height, crypt depth, and villus height:crypt depth ratio (VCR) at three sites (25, 50, and 75% of the total length). Feeding SDP resulted in reduced (P < 0.05) mRNA expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) mRNA in the adrenal gland, spleen, hypothalamus, pituitary gland, and liver. Additionally, expression of IL-6 mRNA was reduced (P < 0.05) in the spleen and pituitary gland for pigs fed SDP. For pigs fed the diet with SDP, LPS administration did not affect (P > 0.10) cytokine mRNA expression, whereas LPS reduced expression of TNF-alpha mRNA in the spleen and IL-1beta mRNA in the adrenal gland, spleen, and thymus for pigs fed the diet without SDP. For pigs fed the diet with SDP, LPS caused serum TNF-alpha to increase 150-fold compared to a 60-fold increase for pigs fed the diet without SDP. Similarly, interferon-gamma (IFN-gamma) increased 110-fold for pigs fed the diet with SDP compared to a 16-fold increase for pigs fed the diet without SDP. For pigs fed the diet with SDP, LPS caused major villus atrophy, whereas for pigs fed the diet without SDP, LPS had no effect on intestinal morphology. These results demonstrate that the basal activation of the immune system appears to be less for pigs fed the diet with SDP compared to pigs fed the diet without SDP after weaning. Additionally, for pigs fed the diet with SDP, there appeared to be an overresponse of the immune system following LPS administration, which resulted in major damage to the mucosa of the gastrointestinal tract.

Degradation of maternal cdc25c during the maternal to zygotic transition is dependent upon embryonic transcription.

To gain a better understanding of the molecular differences that may contribute to cleavage arrest and the poorer development associated with laboratory produced embryos, a series of experiments were conducted to quantitate the message levels of the cell cycle controller cdc25c, over the maternal to zygotic transition (MZT) in 4-cell in vivo- and in vitro-derived porcine embryos. The experiments were designed to measure both maternal and embryonic derived cdc25c transcripts by quantitative reverse transcription-competitive polymerase chain reaction (RT-cPCR), determine the point of the transition to zygotic genome activation, and study the interaction between initial embryonic transcription and maternal cdc25c degradation. Analysis of in vivo- and in vitro-derived embryos revealed no difference in cdc25c message level for any of the times P4CC (P > 0.05). Comparison of control embryos from 5- to 33-hr P4CC revealed a reduction in transcript quantities in the 10-hr P4CC group that was maintained at later time points (P < 0.05). Embryos cultured in the RNA polymerase inhibitor, alpha-amanitin, from cleavage to 5-, 10-, 18-, 25-, or 33-hr P4CC displayed no difference in cdc25c levels when compared to controls at similar time points (P > 0.05). However, if embryos were first exposed to alpha-amanitin after 18-hr P4CC with this treatment continuing to 33 hr, the levels of cdc25c transcript were reduced (P < 0.04) when compared to those embryos that were first exposed to the inhibitor at either 5- or 10-hr P4CC. This finding and the comparison of these same embryos to the 0-33-hr alpha-amanitin and control groups allowed us to conclude that cdc25c transcription began between 10- and 18-hr P4CC, with the degradation of maternal cdc25c message dependent on transcriptional initiation.

Expression of pregnancy-associated glycoprotein 1 and 2 genes in in vivo, in vitro and parthenogenetically derived preimplantation pig embryos.

The objective of this study was to determine whether porcine PAG (poPAG) genes are expressed in embryos as they develop from the 1-cell stage to expanded blastocysts, and whether expression differed according to how embryos had been derived. Embryos at various preimplantation stages were assayed after in vivo fertilisation, after in vitro fertilisation of in vitro-matured oocytes, or following parthenogenetic activation of in vitro-matured oocytes. The presence of PAG transcripts was determined at the 1-, 2-, and 4-cell, compact morula and blastocyst stages by reverse transcription-PCR procedures with PAG 1- and PAG 2-specific primers, followed by Southern blotting. The mRNAs for poPAG 1 and 2 were detected in in vitro-derived, in vivo-derived and parthenogenetically derived blastocyst stage embryos. In some replications poPAG 1 could be detected as early as the compact morula stage and poPAG 2 could be detected as early as the 4-cell stage. Our study revealed that poPAG 1 and 2 genes are expressed as early as the compact morula stage and 4-cell stage, respectively, in normal embryos and in parthenogenetically derived blastocysts. Thus it appears that the poPAGs are not maternally imprinted and they may be useful as potential candidates for markers of developmental competence.

Impact of environmental temperature on response of neonatal pigs to an endotoxin challenge.

OBJECTIVE: To evaluate the effect of various environmental temperatures (ET) on the ability of neonatal pigs to cope with an endotoxin challenge. ANIMALS: 28 crossbred male pigs that were 24 hours old. PROCEDURE: At 24 hours of age, pigs were placed in environmentally controlled chambers maintained at 18 or 34 C (14 pigs/ET). Rectal temperatures (RT) were recorded at 15-minute intervals for 3 hours following an IP injection of 0.9% NaCl (7 control pigs/ET) or lipopolysaccharide (LPS; 150 microg/kg of body weight; 7 LPS-treated pigs/ET). Tissue specimens and blood samples were collected following the 3-hour challenge period. RESULTS: LPS-treated pigs exposed to 18 C had a period of hypothermia whereas RT for LPS-treated pigs at 34 C did not differ from control pigs. The LPS-treated pigs maintained at 18 C lost the most body weight during the 3-hour period and also had the greatest increase in serum cortisol concentration. Serum prolactin (PRL) concentration was decreased in pigs at 18 C, compared with pigs at 34 C. Challenge with LPS resulted in an increase in serum PRL concentration at 18 C but had no effect on serum PRL at 34 C. Challenge with LPS resulted in an increase in expression of tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 receptor mRNA in the hypothalamus. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to a cold ET can inhibit the ability of neonatal pigs to cope with an exogenous endotoxin challenge. When combined, cold stress and exposure to exogenous endotoxin induces a rapid and potentially dangerous loss of body temperature in neonatal pigs.

Effects of weaning on somatotrophic gene expression and circulating levels of insulin-like growth factor-1 (IGF-1) and IGF-2 in pigs.

The present study evaluated somatotrophic gene expression in liver, muscle and adipose tissue 4 d after weaning, a time point corresponding to greatly reduced serum concentrations of insulin-like growth factor (IGF)-1 and IGF-2 in pigs. Two-week-old barrows were either cross-fostered to a sow (SOW, n = 8) or weaned and fed a phase 1 diet containing either 0 or 7% spray-dried plasma (NP, n = 8 and SDP, n = 8; respectively). Piglets were allocated such that two size groups were equivalently represented in each experimental group (small, 3.5-4.3 kg and large, 4.6-5.7 kg). Animals were weighed daily and sacrificed 4 d after weaning for blood and tissue collection. Daily gains of the SOW piglets were significantly greater than those of the weaned pigs for the first 3 d of the experiment (P < 0.0001). Weight gains in the SOW and SDP pigs between d 3 and 4 were equivalently elevated relative to the NP pigs (P < 0.0001). Serum IGF-1 and IGF-2 concentrations were decreased in both NP and SDP compared to SOW (P < 0.0001). Serum IGF-2 levels were significantly lower in small piglets (P = 0.006). A Weaning Group X Size interaction was noted for liver IGF-2 mRNA (P < 0.03), reflecting a higher level of expression in large SOW piglets relative to small SOW piglets. Weaning did not affect IGF-1, IGF-2, or growth hormone (GH) receptor mRNA levels in liver, muscle, or fat (P > 0.05). Liver IGF-binding protein (IGFBP)-3 and acid-labile subunit (ALS) mRNA levels also were unaffected by weaning. Small pigs had lower levels of liver ALS (P = 0.0003), muscle IGF-2 (P = 0.02), and muscle GH receptor (P = 0.006) mRNAs. In contrast, adipose tissue IGF-1 and IGF-2 mRNA levels were greatest in the small piglets (P = 0.001 and 0.029, respectively).

Administration of ACTH to restrained, pregnant sows alters their pigs' hypothalamic-pituitary-adrenal (HPA) axis.

This study was designed to examine the physiology and behavior of pigs whose dams were snared and then injected with ACTH during gestation. Administration of ACTH to dams during pregnancy has been shown to replicate the effects of prenatal stress in other species. Control sows (n = 8) were given no treatment, whereas the treatment sows (ACTH, n = 8) were immobilized by snaring the snout and then administered an i.v. injection of ACTH (1 IU/kg BW) weekly from 6 to 12 wk of gestation. A pig was killed from each sow at 1, 30, and 60 d of age. The hypothalamus, pituitary gland, adrenal glands, and liver were immediately obtained to determine the amounts of corticotropin-releasing hormone (CRH), beta-endorphin, and mRNA for pro-opiomelanocorticotropin (POMC), the ACTH receptor (ACTH-R), and insulin-like growth factor I (IGF-I). Pituitary corticotrope and somatotrope cell numbers and adrenal cortex-to-medulla area ratios (CORT:MED) were also determined. Pigs' behaviors were recorded at 6 and 8 wk of age. At 75 d of age, a blood sample was taken and a biopsy puncture was created on one pig from each litter, then pigs were stressed by mixing. Blood was sampled every other day for 10 d to determine plasma cortisol concentrations and differential leukocyte counts. Biopsy damage was evaluated for healing. At 1 d of age, control pigs tended to weigh more (P = .09), have a lower expression of ACTH-R mRNA (P = .01) and IGF-I mRNA (P = .01), and a lower CORT:MED (P = .04) than ACTH pigs. At 30 d of age, control pigs had a greater concentration of beta-endorphin (P = .01) and tended to have a lower concentration of CRH (P = .09) and IGF-I mRNA (P = .10) than ACTH pigs. At 60 d of age, control pigs tended to have lighter pituitary glands (P = .08), a lower expression of POMC mRNA (P = .02), and a CORT:MED (P = .003) than ACTH pigs. At 8 wk of age, control pigs performed a higher frequency of belly nosing (P = .07) and oral vice behaviors (P = .01) than ACTH pigs. In response to mixing stress, control pigs had lesser concentrations of plasma cortisol (P = .03) and healed faster (P = .006) than ACTH pigs. Thus, exogenous ACTH and restraint during gestation alters the HPA axis of the sow's offspring, and during stressful situations later in life health, and therefore welfare, may be compromised.

Postnatal function of the somatotrophic axis in pigs born naturally or by Caesarian section.

This study was designed to determine what effects the birth process would have on development of the somatotrophic axis in neonatal pigs. Eight crossbred sows were selected (n = 4 natural birth and n = 4 Caesarian section) for the present study. Blood and tissue samples from 38 pigs were collected at birth. Twenty pigs were maintained with natural birth sows until sacrificed for blood and tissue collection at 2 wk of age. Gestational age at birth did not differ (P > 0.16) between natural birth and C-section pigs. Average daily gain (ADG) from birth until 2 wk of age was reduced (P < 0.0001) by 39.3% in the C-section pigs as compared to the natural birth pigs. Serum growth hormone (GH) did not differ (P > 0.86) at birth, but was greater (P < 0.024) at 2 wk in C-section pigs. Serum insulin-like growth factor 1 (IGF-1) was greater at birth (P < 0. 0025) and at 2 wk of age (P < 0.035) in the natural birth pigs. Serum concentration of IGF-2 did not differ at birth (P > 0.8) but was greater (P < 0.043) in natural birth pigs at 2 wk of age. Pituitary content of GH mRNA and GH-releasing hormone receptor mRNA did not differ (P > 0.90) between groups regardless of age; however, expression of both mRNAs declined (P < 0.0003) from birth until 2 wk of age. There tended to be a birth type X age interaction (P < 0. 082) for liver IGF-1 mRNA such that C-section pigs had a greater expression at 2 wk of age. Liver IGF-1 mRNA expression increased (P < 0.0001) in both groups from birth to 2 wk of age. Liver expression of GH receptor mRNA was greater in C-section pigs at birth (P < 0. 04) and 2 wk of age (P < 0.03). These data provide evidence that the natural birth process affects postnatal development and/or function of the somatotrophic axis in the neonatal pig.

Long form leptin receptor mRNA expression in the brain, pituitary, and other tissues in the pig.

Much effort has focused recently on understanding the role of leptin, the obese gene product secreted by adipocytes, in regulating growth and reproduction in rodents, humans and domestic animals. We previously demonstrated that leptin inhibited feed intake and stimulated growth hormone (GH) and luteinizing hormone (LH) secretion in the pig. This study was conducted to determine the location of long form leptin receptor (Ob-Rl) mRNA in various tissues of the pig. The leptin receptor has several splice variants in the human and mouse, but Ob-Rl is the major form capable of signal transduction. The Ob-Rl is expressed primarily in the hypothalamus of the human and rodents, but has been located in other tissues as well. In the present study, a partial porcine Ob-Rl cDNA, cloned in our laboratory and specific to the intracellular domain, was used to evaluate the Ob-Rl mRNA expression by RT-PCR in the brain and other tissues in three 105 d-old prepuberal gilts and in a 50 d-old fetus. In 105 d-old gilts, Ob-Rl mRNA was expressed in the hypothalamus, cerebral cortex, amygdala, thalamus, cerebellum, area postrema and anterior pituitary. In addition, Ob-Rl mRNA was expressed in ovary, uterine body, liver, kidney, pancreas, adrenal gland, heart, spleen, lung, intestine, bone marrow, muscle and adipose tissue. However, expression was absent in the thyroid, thymus, superior vena cava, aorta, spinal cord, uterine horn and oviduct. In the 50 d-old fetus, Ob-Rl mRNA was expressed in brain, intestine, muscle, fat, heart, liver and umbilical cord. These results support the idea that leptin might play a role in regulating numerous physiological functions.

Transcriptional regulation of pituitary synthesis and secretion of growth hormone in growing wethers and the influence of zeranol on these mechanisms.

This experiment evaluated relationships between pituitary messenger RNA levels of the transcription factor Pit-1, the growth hormone releasing-hormone receptor (GHRHR), and synthesis and secretion of GH in growing wethers. The experiment also evaluated the influence of the estrogenic compound, zeranol, on these relationships. Seventy wethers that were 9.5 +/- 1 day of age were randomly assigned to a control group or to one of three zeranol treatment groups that were implanted (12 mg, Ralgro) at 0, 45, and (or) 90 days of age. Twenty-eight days after implantation (i.e., Days 28, 73, 118) and on Day 135, sera were collected serially from wethers (n > or = 5) from each group and then their pituitary was collected. As wethers gained weight with age, the pituitary increased in size and so did the relative message levels of Pit-1 and GH (effect of time, P < 0.01). However, as wethers reached 135 days of age, serum concentrations of GH had declined while concentrations of IGF-I had increased (linear contrast, P < 0.01). Additionally, zeranol increased serum concentrations of GH and IGF-I and this effect on GH appeared to be a consequence of increased pulse amplitude, particularly at 73 and 118 days of age (treatment x time, P

Expression of two variants of growth hormone receptor messenger ribonucleic acid in porcine liver.

Transcription of GH receptor (GHR) mRNA is initiated from multiple promoters. Most GHR mRNA arise from GHR Promoter 1 (GHR P1) and GHR P2, which transcribe GHR 1A and GHR 1B mRNA, respectively. Our objective was to characterize the expression of GHR 1A and GHR 1B mRNA in liver of neonatal intact (1 d of age) and castrated (14, 28, and 42 d of age) male pigs (Exp. 1; n = 6 per age group), intact male pigs treated with recombinant porcine ST (rpST) or control (Exp. 2; 21, 42, and 77 d of age; n = 4 pigs per treatment per age), and pregnant gilts treated with rpST (n = 6) or control (n = 7) (Exp. 3). Tissue samples were collected at slaughter for mRNA analyses. The porcine GHR 1A and GHR 1B cDNA were cloned and were homologous to GHR cDNA isolated from other species. Ribonuclease protection assays were used to measure GHR 1A and GHR 1B mRNA. Liver expressed GHR 1A and GHR 1B mRNA, whereas muscle, uterus, and ovary expressed GHR 1B mRNA. The GHR 1A mRNA in the liver of neonatal intact and castrated male pigs (Exp. 1) was expressed at very low levels on d 1, 14, and 28, and two of six pigs expressed a high level of GHR 1A on d 42. The GHR 1B mRNA, however, was detected at all ages (d 1 through 42), and the amount of GHR 1B increased (P<.05) on d 42. The liver of intact male pigs (Exp. 2) expressed GHR 1B mRNA by 21 d, whereas a high level of GHR 1A mRNA was not detected until d 42 (P<.10). Administration of rpST had no effect on expression of GHR 1A or GHR 1B mRNA in pigs younger than 77 d (Exp. 2), but it tended to decrease (P<.10) GHR 1A mRNA but not GHR 1B mRNA in pregnant gilts (Exp. 3). In conclusion, GHR mRNA in porcine liver was composed of at least two variants (GHR 1A and GHR 1B). The GHR 1B mRNA was the major GHR mRNA in pig liver before 77 d of age. The GHR 1A mRNA increased after 42 d of age and tended to undergo specific down-regulation in response to rpST in pregnant gilts.

Few differences found between early- and late-weaned pigs raised in the same environment.

Segregation and medicated early weaning are technologies used to optimize the productivity and health of pigs, but these practices may also cause aberrant behaviors indicative of stress. Thus, differences in early- (=10 d of age) and late- (=30 d of age) weaned pigs were investigated. At weaning, pigs were housed in groups of four in 16 pens (eight pens per treatment) in the same facility, and, thus, they were not segregated. Body weights were recorded at birth, weaning, and at approximately 42, 65, 102, 137, and 165 d of age (at slaughter). One-minute, instantaneous scan samples during a 10-min period (at 0600, 1000, 1400, and 1800) were used to record the frequency of lying, standing, and sitting, total number of drinks, feeder investigations, and time spent playing/fighting on 2, 3, and 4 d after weaning. Five-minute, direct observations of each pig were conducted at approximately 40, 60, 80, and 150 d of age. Direct observations were also made of the entire pen for 10 min at approximately 50, 95, 123, and 160 d of age to record aberrant behaviors. At 62 d of age, a handling and blood collection stress was imposed. At 165 d of age, a second stress test was conducted in response to rough handling and transport. Early-weaned pigs spent more time playing/ fighting (P < .006) than late-weaned pigs during the 4 d after weaning, manipulated conspecifics more often at 40 d of age (P < .002), had greater percentage of hemoglobin (P < .03) during Stress Test 1, had greater ADG at 42 d of age (P < .03), and had greater hypothalamic growth hormone-releasing hormone receptor mRNA at slaughter (P < .06). Late-weaned pigs had greater ADG between 137 and 165 d of age (P < .03) and greater pro-opiomelanocortin at slaughter (P < .04). Overall, most differences found between early-weaned and late-weaned pigs were evident soon after weaning, but they disappeared before slaughter.

Cyclin B1 transcript quantitation over the maternal to zygotic transition in both in vivo- and in vitro-derived 4-cell porcine embryos.

Using reverse transcription-competitive polymerase chain reaction (RT-cPCR), the quantity of cyclin B1 transcript present over the maternal to zygotic transition was determined for both in vivo- and in vitro-derived 4-cell porcine embryos. After poly(A) RNA isolation, RT-cPCR was performed on single embryos using an introduced, truncated cyclin B1 DNA competitor. Visualization of embryonic cyclin B1 cDNA and competitor for each reaction allowed a ratio to be formed for use in transcript quantity calculations when compared to cPCR standards. Analysis of in vivo- and in vitro-derived control embryos revealed a decline in cyclin B1 transcripts from 5 to 33 h post-4-cell cleavage (P4CC). The quantity of cyclin B1 for the in vivo-derived embryos at 5 and 33 h P4CC was 11.26 and 4.54 attomol/embryo, respectively (P < 0.03), while the in vitro-derived embryos had 20.18 and 7.52 attomol/embryo, respectively (P < 0.03). Treatment with alpha-amanitin from 5, 10, 18, or 25 h P4CC to 33 h P4CC resulted in cyclin B1 quantities that did not differ from those in the 33-h control embryos, irrespective of time spent in the inhibitor. These findings suggest that maternal cyclin B1 transcript degradation occurred over the 4-cell stage with no detectable embryonic cyclin B1 transcripts produced.

Cloning of porcine prepro-orexin cDNA and effects of an intramuscular injection of synthetic porcine orexin-B on feed intake in young pigs.

Early growth is an important determinant of gain and efficiency in growing pigs. A major limiting factor of piglet growth is feed intake. Orexins, newly discovered neuropeptides, may be important regulators of appetite. The orexin gene, which encodes orexin-A and -B, was recently identified in rodents and man. The objectives of this study were to clone the cDNA for porcine orexin, utilize the cDNA sequence information to produce synthetic hormone, and evaluate the effect of orexin administration on feed intake in weanling pigs. Oligonucleotide primers were designed for reverse transcription polymerase chain reaction production of porcine orexin cDNA. The polymerase-chain-reaction products were cloned, sequenced, and found to be 88.5% homologous to the human orexin sequence. Predicted translation of porcine orexin cDNA revealed orexin-A and -B amino acid sequences that were 100% and 96% homologous to the known human peptides, respectively. Porcine orexin-B was synthesized according to the predicted sequence. Twenty-six cross-bred piglets were utilized in three replicates (n = 8-10/replicate). Piglets were weaned between 2-3 wk of age. One week after weaning, equal numbers of animals in each replicate received intramuscular (i.m.) injections of orexin-B (3 mg/kg body weight) or vehicle (sterile water). Feed intake was monitored from -24 to 24 h relative to injection (time 0). The orexin-injected pigs ingested an additional meal at 12 h when compared with the control animals (P = 0.02). Cumulative feed intake was increased by orexin-B administration from 12 to 24 h postinjection (P < or = 0.05). Total feed intake at 24 h was improved by 18% in orexin-treated pigs (P = 0.05). The ability to stimulate appetite during critical periods of early growth, particularly following weaning, could result in significant improvements in swine-production efficiency.

Birth by caesarian section alters postnatal function of the hypothalamic-pituitary-adrenal axis in young pigs.

Eight crossbred sows were selected for the present study (n = 4 vaginal delivery and n = 4 Caesarian section [C-section]). Gestation length did not differ between vaginal delivery and C-section pigs (113.6 +/- .1 and 113.2 +/- .3 d, respectively; P > .16). Blood and tissue samples from 38 pigs were collected at birth. All remaining pigs were sustained with vaginal-delivery sows until 2 wk of age (n = 39). At 2 wk of age, remaining pigs were catheterized for blood sample collection to assess pituitary-adrenal responsiveness to an injection of corticotropin-releasing hormone (CRH; 10 microg/kg). Blood samples were collected at -30, -15, 0, 5, 10, 20, 40, 60, and 90 min; pigs received CRH or saline at time 0. Pigs were killed and tissue samples were collected immediately following the last blood sample. Serum concentrations of ACTH and cortisol (CS) were measured. Total RNA was isolated from the pituitary and adrenal glands to evaluate gene expression for mRNA specific for pro-opiomelanocortin (POMC) and for the ACTH receptor. Centrifuged clot:blood ratio was reduced in the C-section pigs at birth (P < .001) and at 2 wk of age (P < .043), compared with the vaginally delivered pigs. Basal serum concentration of ACTH was greater in C-section than in vaginally delivered pigs at birth (P = .01) but did not differ at 2 wk of age (P = .42). Basal serum concentration of CS was not different at birth (P = .86) but was greater in C-section pigs than in vaginally delivered pigs at 2 wk of age (P < .04). Serum concentration of ACTH was not different (P > .99) between the two groups of pigs following the CRH challenge. However, serum concentration of CS was greater (P < .05) in C-section pigs following the CRH challenge. Expression of ACTH receptor mRNA tended to be greater in C-section pigs at birth (P < .063) and at 2 wk of age (P < .016). There was no difference in POMC mRNA between treatments (P > .73); however, there was a developmental increase (P < .001) from birth to 2 wk of age. These data indicate that the birth process plays an important role in postnatal function of the hypothalamic-pituitary-adrenal axis in young pigs.

Interactions between environmental temperature and porcine growth hormone (pGH) treatment in neonatal pigs.

Age-dependent interactions between environmental temperature and porcine growth hormone (pGH) treatment on the function of the somatotrophic axis were evaluated in the neonatal pig. At 3 d of age, 40 Landrace x Yorkshire x Duroc piglets received intraperitoneal implants containing either recombinant pGH (0.5 mg/d; n = 20) or vehicle (control; n = 20). Piglets were maintained at either a low (21 degrees C, 50% relative humidity; n = 20) or high (32 degrees C, 50% relative humidity; n = 20) temperature. At 4 and 6 wk of age, 5 pGH-treated and 5 control piglets from each thermal group were sacrificed for tissue collection. Blood samples were collected at the time of sacrifice and analyzed for serum concentrations of GH, insulin-like growth factor 1 (IGF-1), and IGF-2. Liver RNA was analyzed for mRNAs specific for the GH receptor, IGF-1, IGF-2, and IGF binding protein 3. There was no effect of pGH treatment (P = 0.4) on average daily gain; however, both age (P = 0.002) and temperature (P = 0.001) had an effect on average daily gain such that those animals maintained at a low temperature and those sacrificed at 6 wk had greater average daily gains. Serum concentration of GH was elevated (P = 0.003) by pGH treatment and was lowest in the 6-wk-old group (P = 0.008). Serum concentration of IGF-1 was elevated (P = 0.007) by pGH treatment and increased with age (P = 0.01). Liver GH receptor mRNA was unaffected (P > 0.5) by pGH treatment, but was greater in the 6-wk-old group (P < 0.0001) and in piglets maintained at the high temperature (P = 0.04). IGF-1 mRNA was enhanced by pGH treatment (P = 0.0003) and by exposure to the high temperature (P = 0.04), but did not differ (P > 0.5) between age groups. IGF-2 mRNA was greater (P = 0.0009) in the 4-wk-old group and in piglets maintained at the high temperature (P = 0.007), but was unaffected (P = 0.5) by pGH treatment. IGF binding protein 3 mRNA increased with age (P = 0.0004) and was stimulated by pGH treatment in the 6-wk-old group (P = 0.034). The relatively lower level of GH receptor and IGF mRNAs in conjunction with greater growth in the cold environment suggests that somatotrophic gene expression in the liver is not rate limiting for growth in the neonatal pig.