H. G. Kattesh

Effect of menhaden fish oil supplementation and lipopolysaccharide exposure on nursery pigs. I. Effects on the immune axis when fed diets containing spray-dried plasma.

The objective of the present study was to evaluate the potential immunological benefit of adding menhaden fish oil to the diet of weaned pigs. Twenty-four crossbred male pigs were weaned at approximately 18 days of age and placed on a complex nursery diet containing 30% lactose and 7% plasma protein with 6% corn oil as the fat source (Cont, n=12) or with 5% menhaden fish oil and 1% corn oil as the fat source (MFO, n=12) for a period of 15 days. Body weights did not differ (P>0.78) between dietary groups either at the beginning or end of the 15 days feeding period. On day 15, all pigs were non-surgically fitted with an indwelling jugular catheter. On d 16, pigs received an i.v. injection of either saline (n=6/dietary group) or lipopolysaccharide (LPS; 150 microg/kg body weight; n=6/dietary group) and blood samples were collected at 30 min intervals for a period of 5h. Serum was harvested and stored at -80 degrees C for analysis of cortisol (CS), corticosteroid-binding globulin (CBG), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). There was no significant effect of diet on basal concentrations (Time 0) of any of the blood parameters analyzed. A Time x Treatment x Diet interaction (P<0.03) was observed for serum CS such that those pigs which consumed the MFO diet followed by LPS treatment had a reduced CS response as compared to the LPS-treated pigs on the Cont diet. A Time x Treatment interaction (P<0.01) was observed for serum CBG such that LPS treatment reduced circulating CBG as compared to the saline-treated pigs. Time x Treatment x Diet interactions were also observed for serum concentrations of TNF-alpha (P=0.084) and IFN-gamma (P=0.022) such that both the TNF-alpha and IFN-gamma response to the LPS challenge was lower in those pigs receiving the MFO diet as compared to the LPS-treated pigs on the Cont diet. Overall, serum CS was negatively correlated with the CBG response (r=-0.40, P<0.001), however, the strongest negative correlation was observed in the LPS-treated pigs which consumed the MFO diet (r=-0.63, P<0.001). While further studies are needed to evaluate the immunological response of including MFO in the nursery pig diet, the present study demonstrates that supplementation with MFO does indeed alter the immunological response to an LPS challenge.

Plasma Cortisol Distribution in the Pig from Birth to Six Weeks of Age

Plasma levels of cortisol and percent distribution of cortisol among protein-bound (corticosteroid-binding globulin (CBG) and albumin) and unbound forms were measured in naturally born/conventionally reared pigs from birth to 6 weeks of age. Total cortisol and percent unbound cortisol were highest in pigs at birth and decreased (p less than 0.01) in a linear fashion over the sampling period. Percent CBG-bound cortisol was lowest on days 3-21 relative to the peak value seen on day 42. However, actual CBG-bound cortisol was not different after day 1. Percent albumin-bound cortisol was highly correlated with percent CBG-bound cortisol (r = -0.90; p less than 0.001). These results suggest that a rapid shift in cortisol distribution from unbound and albumin-bound forms to that which is bound to CBG occurs by approximately day 28 of age in the neonatal pig.

Prenatal stress effects on pig development and response to weaning

Exposing a pregnant sow to stress has been shown to affect the resulting offspring. Our objective was to determine if rough handling of pregnant sows altered the physiology of her offspring and if these alterations were different from an experimentally induced model of prenatal stress. Sow treatments consisted of i.v. injections of ACTH (1 IU/kg of BW), exposure to rough handling for 10 min (Rough), or no treatment (Control) once a week during d 42 to 77 of gestation. To determine the plasma cortisol response to treatments, blood (5 mL) was collected from 30 sows after treatment administration. To conduct the prenatal stress study, a separate group of 56 sows was used in 1 of 4 replicates. At birth, production data were collected for each litter, including birth weight, number born, anogenital distance, and pig viability. At weaning, pigs were blocked by BW and sex, and placed in a nursery pen of 6 pigs, with 2 pigs from each treatment group. To assess the effect of treatments on cortisol, corticosteroid-binding globulin (CBG), and hematological cell profiles, blood was collected every other day for 10 d after weaning. Application of treatments caused plasma cortisol concentrations to be greatest in ACTH sows compared with Control sows (P < 0.001), with Rough sows having intermediate values (P = 0.07). Treatments did not affect the number of pigs born, number of stillborn, or pig viability (P > 0.40). The ratio of cortisol to CBG did not differ between treatments (P = 0.09). Hematological variables did not differ between treatments (P > 0.19). Pigs born to ACTH sows had a smaller anogenital distance compared with controls (P < 0.03), with pigs from Rough sows being intermediate. Our data indicate that swine exposed to prenatal stress (ACTH injection) can have alterations in sexual morphology without effects on growth or the immune cell populations measured in this study.

Plasma levels of cortisol and corticosteroid-binding globulin (CBG) and hepatic CBG mRNA expression in pre- and postnatal pigs.

The relationships among hepatic corticosteroid-binding globulin (CBG) mRNA expression and plasma concentrations of cortisol and CBG was evaluated in fetal pigs (n=7-14 per age) on days 50, 70, 80, 90, and 104 of gestation and postnatal pigs (n=8 per age) on days 1, 3, 10, 20, 30, and 40 following birth. In fetal pigs, hepatic CBG mRNA expression was highest (P<0.01) on day 50 as compared to days 90 and 104, exhibiting an overall negative relationship (r=-0.63; P<0.01) with estimated gestation age. Plasma porcine CBG (pCBG) concentration was correlated (r=0.34; P<0.05) with hepatic CBG mRNA level. Plasma cortisol concentrations were not different over this same period. In postnatal pigs, hepatic CBG mRNA expression increased (P<0.01) from days 3 to 40. The pCBG concentration increased (P<0.01) from days 1 (6.1+/-3.4 microg/ml) to 10 (15.1+/-3.7 microg/ml), while plasma cortisol concentration remained constant. An understanding of the relation between hepatic CBG mRNA and circulating pCBG concentrations may provide insight into the mechanisms determining the bioavailability of cortisol necessary in prenatal development and the conservation of cortisol during postnatal development in the pig.

Distribution of cortisol among its free and protein-bound fractions in rainbow trout (Orhynchus mykiss): Evidence of control by sexual maturation

1. Total cortisol concentrations did not differ in sexually matured female, male, and immature rainbow trout. 2. The per cent cortisol bound to a corticosteroid binding protein was greater in mature female fish (48.2%) than in mature male (16.0%) and immature fish (19.5%). 3. The mature female fish exhibited a lower percentage of free cortisol (21.8%) compared to mature males (44.8%) and immature fish (43.2%). 4. Parallel aspects of the teleostean cortisol binding-protein and the mammalian counterpart are compared and commented upon.

Physiological and immunological responses to weaning and transport in the young pig: Modulation by administration of porcine somatotropin

To examine the effects of exogenous porcine (p) ST on measures of stress and immune function in weaned pigs with or without transport, pigs (20 +/- 1 d of age) received daily injections of pST (0.5 mg/kg; n = 16) or saline (n = 16) for 5 d. On d 5, a blood sample was collected immediately before injection. At 4 h postinjection, pigs were weighed, sampled for blood, injected with di-nitrophenyl-conjugated keyhole limpet hemocyanin, and weaned. One half of the pigs in each group were transported for 3 h before placement in the nursery. Pigs were weighed, and blood was collected on 1, 7, and 14 d postweaning. Statistical significance was set at P < 0.05. Serum IGF-I concentrations were increased by pST and decreased by weaning, but not affected by transport. The free cortisol index was elevated in all pigs 1 d postweaning, although less in transported versus nontransported pigs. By 7 d postweaning, the free cortisol index returned to prewean values. Serum concentrations of immunoglobulin (Ig) G increased in all pigs by 14 d postweaning, but were not affected by pST or transport. Serum IgM concentrations were elevated at 7 and 14 d postweaning. Before weaning and again 1 d postweaning, pigs treated with pST had greater concentrations of IgM than did control animals. Circulating neutrophils increased in pST-treated pigs 4 h after the final pST injection. Improved immune function in weaned pigs by pST may lead to greater health and growth in a commercial setting.

Isolation and identification of porcine embryonic basic protein as a fragment of pregnancy-associated glycoprotein-2☆

Between days 11 and 12 of gestation, the porcine conceptus undergoes a metamorphosis from a spherical blastocyst to an elongate thread-like form. During this process, the conceptus secretes a variety of products. One of these products is protein previously referred to as porcine embryonic basic protein (BP). This protein has been shown to be a major secreted product between days 13 and 18. In this study, we report a simple two-step procedure to isolate BP from day 15 porcine conceptus conditioned medium, utilized ion-exchange chromatography and reverse-phase HPLC. Purified BP was subjected to Edman degradation amino-terminal sequencing and a 25 amino acid residue sequence was obtained. Comparing the N-terminal sequence of BP to sequences in the GenBank database determined that BP shared amino acid homology with porcine pregnancy-associated glycoprotein-2 (PAG-2). The region of identity corresponded to an internal site of PAG-2, suggesting BP was a proteolytic fragment of PAG-2. The purified protein was confirmed to be BP by Western blot using a previously characterized anti-BP antiserum. Also, the BP was immunolocalized with the trophectoderm of day 11 blastocysts. Staining intensity was diminished in spherical blastocysts compared to elongated blastocysts. Although the function of PAG-2 and its cleavage product BP are unknown, the large quantity produced by the porcine conceptus and its sequence conservation across species may indicate a necessary role in early pregnancy.

Age-related changes in porcine corticosteroid-binding globulin (pCBG) as determined by an enzyme-linked immunosorbent assay

The objectives of this study were to develop an assay for the direct measure of porcine corticosteroid-binding globulin (pCBG) and to confirm age-related changes in plasma pCBG concentration. Isolation and purification of pCBG from plasma was performed by affinity chromatography and HPLC-DEAE anion exchange techniques. Analysis by SDS-PAGE revealed two polypeptides (54 and 59 kDa) having similar amino acid homology (>50%) to previously reported sequences of seven mammalian species for the first 33 amino acids. Porcine CBG (20 ng/well) was immobilized to microtiter plates and standards or samples added along with rabbit antiserum developed against the purified pCBG. Goat anti-rabbit IgG-alkaline phosphatase conjugate was added followed by p-NPP substrate. The resultant color development was read at 405 nm. Intra- and interassay coefficients of variation (n=26) of a pooled sample were 10 and 15%, respectively. Age-related changes (P<0.001) in plasma pCBG concentration (n=203) from day 3 through 168 of age confirmed that, in the pig, changes seen in the percent distribution of cortisol among protein bound and free forms around day 28 of age are associated with an increase in CBG concentration.

Effect of prenatal stress on subsequent response to mixing stress and a lipopolysaccharide challenge in pigs

Sows subjected to prenatal stress have been found to produce offspring that have altered responses to stress. Our objective was to determine if exposing a sow to stress would alter the response of the offspring to lipopolysaccharide (LPS) at 2 mo of age or their response to mixing stress at 4 mo of age. Sow treatments consisted of intravenous injections of ACTH (1 IU/kg of BW), exposure to rough handling for a 10-min duration (rough), or no treatment (control) once per week from d 42 to 77 of gestation. At 2 mo of age, pigs from each treatment, 1 per litter (n = 21, 17, and 15 for the ACTH, rough, and control treatments, respectively), were challenged with 2 μg of LPS/kg of BW or saline, or served as a noninjected control. Their behavioral response to a human approach test and salivary cortisol were measured. At 4 mo of age, 1 pig from each treatment (n = 14, 14, and 15 for the ACTH, rough, and control treatments, respectively) was taken from its home pen and placed in a pen of unfamiliar pigs. At this time, a punch biopsy wound (6 × 6 mm) was created to measure the ability of the pig to heal the wound. At this same time, each pig received a 1-mL intramuscular injection of 20% ovine red blood cells (oRBC), and then a second injection of oRBC at 21 d postmixing. Blood samples were collected 3 times per week for 2 wk and then once a week for 4 more weeks. Blood samples were analyzed for cortisol, porcine corticosteroid-binding globulin, antibody response to oRBC, and nitric oxide production by macrophages. Behavior was recorded during the first 5 d after mixing. All pigs in the LPS challenge responded with characteristic sickness behavior; however, pigs in the rough treatment showed less sickness behavior than those in the other 2 treatments (P < 0.05). Maternal stress treatment did not affect (P < 0.43) salivary cortisol. Pigs from all treatments responded similarly to mixing stress with regard to cortisol, porcine corticosteroid-binding globulin, antibody titers, nitric oxide production, and hematology measures, and all pigs experienced the same amount of aggression in response to mixing. Without altering peripheral measures of stress responsivity, prenatal stress enhanced the ability of pigs to cope with a simulated immune challenge, which could prove to be an adaptation to challenging environments.

PLASMA AND UTERINE CORTISOL, PROGESTERONE AND PROTEIN CHANGES IN PSEUDOPREGNANT GILTS TREATED WITH HYDROCORTISONE ACETATE

Abstract To determine the effects of cortisol concentrations during pregnancy, gilts, made pseudopregnant through twice daily administration of 5 mg estradiol benzoate on Days 11 to 15 (Day 0 = first day of estrus), received either 5 mg/kg body weight of hydrocortisone acetate (HA) in sesame oil (n=5) or sesame oil alone (n=6) twice daily on Days 21 to 30. Blood samples (20 ml) were collected on Days 11, 21 and 31. Uterine flushings were obtained surgically on Day 31. The HA-treated gilts had higher (P