Water intake and confinement effects on estrogen and cortisol production in pregnant mares (continued)

Keywords

Introduction

Materials and
Methods

Results and
Discussion

Conclusion/
Implications

Future Research
Needs

References

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Background

Various biological responses can eliminate or reduce the potential effects of a stressor by changing either the animal's relationship to the stress or its perception of the stressor (Friend and Dellmeier, 1986). Young horses are most affected by psychological stresses such as weaning and separation, as compared to older horses, which are more sensitive to physical stressors (McCall et al., 1987). In general, horses are perceived to be animals of tremendous stamina that possess a remarkable ability to adapt to new environments, adverse or otherwise (McCall et al., 1987).

One of the most common responses of an animal to a stressor is the activation of the hypothalamic-pituitary-adrenal axis (Christison and Johnson, 1972). Adrenocorticotropin (ACTH) itself may have an effect on the pituitary and cause partial inhibition of endogenous cortisol release (James et al., 1970). In a study of pony mares exposed to short-term stressors (twitching), there was an initial rise in plasma cortisol concentrations followed by a return to subnormal values 90 minutes later, indicating the stress was transient (Thompson et al., 1988). When large doses of ACTH were administered, the initial rise in cortisol concentrations was often followed by a period in which the corticosteroids fell below pretreatment concentrations, which probably resulted from suppression of endogenous corticosteroid secretion by the adrenal (Thompson et al., 1988). Normal plasma cortisol values in horses range from 3 to 13 ug/100 ml, with no significant differences due to sex and diurnal pattern similar to that of humans but not as pronounced (James et al., 1970). Exercise has been shown to cause a slight increase (30%) in plasma cortisol concentrations after horses have been subjected to a normal training schedule (James et al., 1970).

During pregnancy, urinary excretion of estrogens increases 300-fold over concentrations in non-pregnant mares, reaching peak values by week 24 (mid-gestation) (Monfort et al., 1991). After mid-pregnancy, estrogen excretion declines sharply, with the decrease becoming more gradual as parturition approaches. Terqui and Palmer (1979) measured total estrogens (conjugated and unconjugated) in blood plasma from day 1 to day 100 in pregnant mares. From day 0 to 35 concentrations were similar to those found during diestrus. An increase in total plasma estrogens occurred from day 35 to 40, followed by a plateau from day 40 to 60 that was greater than the preovulatory surge concentrations. Radioimmunoassay (RIA) techniques revealed low concentrations of estrone, equilin, and equilenin in plasma until day 78 of gestation (Nett et al., 1973). Thereafter, concentrations increased to a peak at day 208 and then declined. The estrogen concentration declined sharply after day 280.

The present study was conducted to determine cortisol and estrogen concentrations in pregnant mare urine taken from mares in long-term confinement (5 months) with restricted or ad libitum access to water, and with or without daily exercise.



Materials and Methods
Experimental Animals
Pregnant mares (n=65) ranging in age from 3 to 11 years, with a mean weight of 1330 lbs ±61.78 lbs, were used in three experimental groups. These mares were either Quarter Horse or draft mares. All animals were housed at the same premise in confinement tie stalls and were accustomed to being handled on a daily basis. Confinement was continuous from 18 weeks to approximately 40 weeks of pregnancy. Each stall had rubber mats on the cement floors. All mares received the same diet, which was automatically delivered at the same time each day. Quantity of feed delivered depended on the size of the individual mare. Vitamin and trace supplements were topdressed on the individual feed aliquot.

Experimental Procedure
Of the 65 mares, 50 were offered water at metered intervals throughout the day. The mares were randomly assigned to three groups. Group I (n=25) mares remained confined at all times with restricted water intake (7 gal/mare /day). Group II (n=25) mares were allowed free exercise for one hour each day with restricted water intake (7 gal/mare/day). Group III (n=15)mares were confined with ad libitum access to water. Restricted water intake was calibrated to provide 7 gal/mare/day. Suggested water intake for mid gestation mares is 7.9 gal/mare/day (Cymbaluk, 1989; Caljuk, 1961). Exercise consisted of freedom to move about a 6 square meter paddock unharnessed for one hour per day. All mares were harnessed with a collection apparatus that collected and dispensed the urine into a 1.3 gallon container.

Sample Preparation
Urine samples (8 ml) were collected daily ai 1000 h for 14 days from each mare and maintained at 32^oF until processed at the laboratory. The 8 ml sample was an aliquot of a 12 hour total collection volume. Crude samples were centrifuged at 3500 rpm ( 38^oF) for 10 minutes and the clear supernatant was decanted and divided into triplicate samples to be used for subsequent analysis.

Hormone analysis
Urinary cortisol concentrations were determined by solid phase radioimmunoassay (Coat-a-Count Cortisol, Diagnostic Products Corporation, Los Angeles, CA). Centrifuged urine samples (8 ml) were extracted with dichloromethane, vortexed, and centrifuged for 10 minutes at 3500 rpm (38^oF). The top layer was aspirated all the way to the interface, and 50 ul of the lower (dichloromethane) phase was transferred to each of two coated tubes. Samples were evaporated to dryness under a nitrogen stream at room temperature. One ml of [¹²⁵I] cortisol was added to each sample tube and allowed to incubate for 45 minutes at 100^oF. Tubes were decanted and counted for one minute in a gamma counter. Cortisol standards ranged from 5 ug/100ml to 50 ug/100 ml and sensitivity was to 0.2 ug/100 ml. Intra- and interassay coefficients of variation were 7.04% and 3.4%, respectively.

Urinary estradiol concentrations were also determined by solid phase radioimmunoassay (Diagnostic Products Corporation, Los Angeles, CA). Cross-reactivity of estradiol antibody to other estrogens was: estriol, 0.235%; d-equilenin, 4.2%; equilin, non-detectable; 17B estradiol-3B-D-glucuronide, 6.0%. Centrifuged urine samples were diluted 1:200 with phosphate buffered saline, extracted with diethyl ether, and flash frozen. A 50 ul sample of the other layer was withdrawn and dried under a nitrogen stream. The sample was reconstituted in gelding serum and incubated for 1 hour with the [¹²⁵I] estradiol tracer. Tubes were decanted and placed in the gamma counter. Estradiol standards ranged from 5 pg/ml to 500 pg/ml, and sensitivity was 1.4 pg/ml. Intra- and interassay coefficients of variation were 2.2 % and 12.0 %, respectively.

Mean creatinine concentrations (Sigma Chemical Co.) were determined from Day 1,7, and 13 samples from each mare to compensate for variations in urine dilution among individual mares. Reaction volume was 3.4 ml in a 10 mm cuvet requiring a 10 to 15 fold urine dilution among individual mares. An alkaline picrate solution (3 ml) was added to 0.3 ml of sample and allowed to stand at room temperature for 10 minutes. Absorbance was read at 500 nm. The colorimetric reaction was stopped by adding 0.1 ml of a sulfuric acid and acetic acid mixture. The sample was vortexed immediately, and the absorbance was read again at 500 nm.

The statistical model included more than one dependent variable; therefore, multivariate analysis of variance (Manova) was used to test the variables water intake and exercise within each experimental unit (mares). Urinary output data was analyzed using a nonparametric Mann-Whitney test.

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Table of Contents – Fall 1997