Doctoral Dissertations

Jeonghoon Heo

Date of Award

8-2001

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Animal Science

Major Professor

Henry G. Kattesh

Committee Members

James D. Godkkin, Judith M. Grizzle, Alan G. Matthew, Hugo Eiler

Abstract

Three experiments were conducted to investigate the relationships of plasma cortisol, CBG concentration, and CBG mRNA expression during development and heat and social stress in pigs. The first experiment was conducted to develop and characterize a porcine CBG cDNA probe in order to examine porcine CBG mRNA expression in some major tissues from the postnatal pig. The reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted to develop the porcine CBG cDNA probe from liver total RNA extracted from pigs on 40 days of age. The RT-PCR product was subcloned into the pGEM vector and subjected to the treatment of restriction enzymes and DNA sequencing. Northern blot analysis was conducted using total RNA extracted from samples (~ 200 mg) of liver, lung, kidney and whole adrenal tissue that were collected from neonatal pigs either 3 (n = 2) or 40 (n = 2) days of age. A 500 base pair (bp) partial porcine CBG cDNA encoded 166 amino acids and had 83%, 78%, and 77% homology to a 494 bp nucleotide sequence of sheep, human, and rabbit, respectively. The deduced amino acids sequence of the partial porcine CBG showed 77%, 62%, 60% and 51% homology to sheep, human, and rabbit, and rat CBG sequences, respectively. An approximately 1.53-kilobase CBG mRNA was detected only in the liver tissue The second experiment was conducted to evaluate the relationships among hepatic CBG mRNA expression and plasma concentrations of cortisol and CBG during prenatal and postnatal periods in the pig. Blood and liver tissue were collected from fetal pigs (n = 7-14 per age) on day 50, 70, 80, 90, and 104 of gestation, as estimated by fetal crown-rump length, and from postnatal pigs (n = 8 per age) on day 1,3, 10, 20, 30, and 40 following birth. Plasma cortisol and CBG concentrations were determined by a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA), respectively. CBG mRNA expression was determined from liver total RNA by Northern blot analysis and expressed relative to p-actin mRNA expression level. In fetal pigs, CBG mRNA expression was highest (P < 0.01) on day 50 compared to day 90 exhibiting a negative relationship (r = - 0.63, P < 0.01) with estimated gestation age. Plasma CBG concentrations were correlated (r = 0.34, P < 0.05) to CBG mRNA levels. Plasma cortisol concentrations were not different over this same period. In postnatal pigs, CBG mRNA expression increased (P < 0.01) from day 3 to day 40. Plasma CBG concentration increased (P 7lt; 0.01) from day 1 (6.1 ± 3.4 |j,g/ml) to day 10 (15.1 ± 3.7 ng/ml). Plasma cortisol concentrations remained constant. The third experiment was conducted to examine the relationships among plasma cortisol and CBG levels, and hepatic CBG mRNA expression in pigs subjected to elevated environmental temperature in conjunction with establishing social hierarchy. Twenty-four pigs (three or six pigs per litter) were weaned at 25 days of age and housed by litter for 2 weeks at 23 ± 2°C. On day 0, animals were weighed and placed under general anesthesia for collection of blood (10 ml) and liver tissue ( ~ 100 mg). On day 1, three pigs of similar weight (23 ± 0.9 kg) but from different litters were allotted to eight nursery pens within two environmentally controlled rooms (12 animals/room). From days 1 to 7 treatment period), one room was maintained at 23 ± 2°C (controi, CON) and the other at 33 ± 2°C (heat treatment, HEAT). From days 8 to 14 (recovery period), both rooms were maintained at 23 ± 2°C. Animals were videotaped for 72 hours beginning on days 1 and 8 to document behaviorai changes in response to room temperature and to determine sociai order. Blood and liver tissue were collected again on days 7 and 14. Plasma haptoglobin increased (P < 0.05) from 467 ± 123 µg/ml on day 0 to 763 ± 113 µg/ml on day 7 in HEAT pigs. Plasma cortisol and CBG decreased (P < 0.05) from 99.3 ± 8.3 ng/ml and 11.4 ± 1.1 ug/ml on day 0 to 85.1 ± 8.3 ng/ml and 9.9 ± 1.1 µg/ml on day 7 in HEAT pigs, respectively. Hepatic CBG mRNA level and neutrophil:lymphocyte ratio were not affected (P > 0.1) by treatment. HEAT pigs displayed increased (P < 0.01) drinking but reduced feeding (P < 0.01) and lying in contact with other pigs (P < 0.05) behaviors. Average daily gain of body weight (ADG) tended (P = 0.06) to be lower for HEAT (0.64 ± 0.06 kg/d) compared to CON (0.82 ± 0.06 kg/d) pigs. During the recovery period, HEAT pigs had similar (P > 0.1) ADG, plasma cortisol, CBG, haptoglobin, and drinking and feeding behavior but increased (P < 0.01) lying with contact behaviors compared to CON pigs. Measured physiological and behavioral responses were not related to social status In summary, a 500 bp partial porcine CBG cDNA developed from pig liver total RNA had high homology with CBG cDNA from human, sheep, and rabbit. Liver was the primary source of CBG biosynthesis in the postnatal pig. In fetal pigs, plasma CBG level was mainly determined by hepatic CBG mRNA expression that was inversely related to gestational age. In postnatal pigs, vi hepatic CBG mRNA was directly related to age but plasma CBG levels did not appear to be determined by hepatic CBG mRNA expression alone. Also, reduced circulating levels of cortisol and CBG In pigs following a 7-day exposure to elevated temperature was not be attributed to changes In hepatic CBG mRNA expression.

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