Development and validation of a sensitive enzyme immunoassay (EIA) for blood plasma cortisol in female cattle,buffaloes, and goats

A highly sensitive enzyme immunoassay (EIA) that used the second antibody coating technique and the cortisol-horseradish peroxidase conjugate as a label for determination of free and total cortisol in blood plasma of dairy animals (cows, buffaloes, and goats) was developed. For biological validation of the EIA, blood samples were collected from the animals at 48 and 24 h before and 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, and 132 h after dexamethasone administration. The EIA was performed directly with 20 μL of fresh plasma (for free cortisol) and also with 20 μL of heat-treated plasma (for total cortisol) after 1:5 dilutions with PBS. Cortisol standards ranging from 0.39 to 200 pg/well/20 μL were used, and the sensitivity of the EIA procedure was found to be 0.39 pg/well/20 μL, which corresponded to 0.02 ng/mL. In comparison with RIA the EIA was at least 4 times more sensitive and required 5 times less cortisol antiserum. In female cattle, buffaloes, and goats, the total, free, and bound plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than the total, free, and bound cortisol after dexamethasone administration. It can be concluded from these studies that the direct, sensitive EIA validated for estimating the free and total cortisol concentrations was sufficiently reliable and quick for studying the dynamics of cortisol distribution in blood plasma of dairy animals.     

Physiological Changes in Mink (Mustela vison) Dams Subjected to Weaning at Different Times during Lactation

The influence of time of weaning on weight changes, and on hormone and electrolyte status in domestic mink (Mustela vison) dams raising at least five kits per litter was examined. The kits were weaned at either day 42 (group 1) or day 49 (group 2) after birth. The dams were weighed and urine and blood were sampled from day 29 to day 56 after delivery. A considerable loss of dams' body weight, constituting 6.3% in group 1 and 8.1% in group 2, was noticed on the day after weaning, indicating a greatly reduced food consumption on the day of weaning. No changes in plasma aldosterone and no significant decrease in urinary sodium concentration were seen in dams after removal from their litters, whereas, irrespective of time of weaning, urinary sodium and chloride concentrations were halved a few days after weaning compared with the other group. The plasma cortisol concentration was high before and during weaning and was nearly halved 1 week after weaning, implying less strain on the dams after weaning. It is concluded that the weaning period is a most vulnerable and stressful period to the dams irrespective of the actual time of weaning.     

Cortisol Response of Nile Tilapia,Oreochromis niloticus (L.), to Temperature Changes

Abstract

Nile tilapia, Oreochromis niloticus (L.), is a commercially-valuable fish species with high nutritional value. As a result of the intensive aquaculture of this species, handling, transport, and environmental changes that causes stress on these fish are unavoidable. The objective of this study was to determine the effects of gradual and acute temperature changes on juvenile tilapia. No significant difference (P > 0.05) was found among serum cortisol levels in juvenile tilapia when the water temperature was gradually increased from 27°C to 32°C, or 40°C, and maintained for 1 hour, although the levels were five times pretreat-ment levels. When tilapia acclimated to 27°C were subjected to 18°, 27°, 30°, 32°, 34°, 36°, 38°, or 40°C water for 10 minutes in a water bath, followed by a recovery period of 10 minutes at 27°C in the original aquaria, no significant difference (P > 0.05) in cortisol levels was observed among treatments except for significantly elevated levels at 38°C and 40°C. When tilapia acclimated to 27°C were subjected to the same temperature exposures but given a recovery period of 60 minutes at 27°C in the original aquaria, there was no significant (P > 0.05) increase in cortisol levels in tilapia among treatments from 18° to 36°C; but there was a significant (P > 0.05) increase between values from those treatments at 38° and 40°C. Acute temperature changes initiated the cortisol response as early as 10 minutes in fish following exposure to 38°C or 40°C and resulted in significant increases in the 38°C and 40°C treatments following 1 hour of recovery at 27°C. These results have implications for the management of tilapia during bacterial challenge, vaccination, and handling and transport during aquacultural activities.     

Stress in a conservation context: A discussion of glucocorticoid actions and how levels change with conservation-relevant variables

Over the past few decades, there has been a steep increase in the number of conservation-related field studies that measure glucocorticoid hormones (corticosterone or cortisol) as a marker for stress. Endocrine tools show great potential for informing conservation, however interpretation of results is often complicated. This paper reviews the role of glucocorticoids for non-physiologists, evaluates select applications of glucocorticoid measures to conservation field studies, and proposes a theoretical model to focus future research. Because levels of glucocorticoids typically increase in response to challenge and sometimes predict mortality or decrease reproduction, it is often assumed that high levels of glucocorticoids indicate stress. However, literature review suggests that glucocorticoid measures fail to change consistently in a predictable manner with adverse conditions and do not always show a linear correlation with survival or reproductive success. Inconsistencies relate in part to methodological problems but also have a physiological basis. We propose that relationships between (1) glucocorticoids and fitness and (2) glucocorticoids and disturbance may be more log quadratic rather than linear. We hope these models will be useful in generating predictions for future studies and resolving the inconsistencies that currently complicate interpretation for conservation endocrinologists.     

Interday variation and effect of transportation on indirect blood pressure measurements,plasma endothelin-1 and serum cortisol in Standardbred and Icelandic horses

Background

Systemic hypertension is a prominent feature in humans with metabolic syndrome (MS) and this is partly caused by an enhanced endothelin-1 (ET-1) mediated vasoconstriction. There are indications that systemic hypertension might be a feature in equine metabolic syndrome (EMS) but if ET-1 is involved in the development of hypertension in horses is not known. Increased levels of cortisol have also been found in humans with MS but there are no reports of this in horses. Before blood pressure, plasma ET-1 and serum cortisol can be evaluated in horses with EMS, it is necessary to investigate the interday variation of these parameters on clinically healthy horses. The aims of the present study were therefore to evaluate the interday variation and influence of transportation on systemic blood pressure, plasma ET-1 and serum cortisol in healthy Standardbred and Icelandic horses, and to detect potential breed differences.

Methods

Nine horses of each breed were included in the study. Blood pressure was measured and blood samples were collected between 6 and 9 am on two separate days. Eight of the horses (four of each breed) were transported to a new stable were they stayed overnight. The next morning, the sampling procedure was repeated.

Results

The interday variation was higher for plasma ET-1 (37%) than for indirect pressure measurements (8-21%) and serum cortisol (18%). There were no differences in systemic blood pressure between the two breeds. The Icelandic horses had significantly lower serum cortisol and significantly higher plasma ET-1 concentrations compared to the Standardbred horses. Plasma ET-1 was significantly elevated after transportation, but systemic blood pressure and serum cortisol did not differ from the values obtained in the home environment.

Conclusions

Indirect blood pressure, plasma ET-1 and serum cortisol are of interest as markers for cardiovascular dysfunction in horses with EMS. The elevated plasma ET-1 concentrations recorded after transportation was likely caused by a stress response. This needs to be considered when evaluating plasma ET-1 in horses after transportation. The differences detected in plasma ET-1 and serum cortisol between the two breeds might be related to differences in genetic setup, training status as well as management conditions.     

Cortisol release,heart rate,and heart rate variability in transport-naive horses during repeated road transport

Domestic animals are often repeatedly exposed to the same anthropogenic stressors. Based on cortisol secretion and heart rate, it has been demonstrated that transport is stressful for horses, but so far, changes in this stress response with repeated road transport have not been reported. We determined salivary cortisol concentrations, fecal cortisol metabolites, cardiac beat-to-beat (RR) interval, and heart rate variability (HRV) in transport-naive horses (N = 8) transported 4 times over a standardized course of 200 km. Immunoreactive salivary cortisol concentrations always increased in response to transport (P < 0.001), but cortisol release decreased stepwise with each transport (P < 0.05). Concentrations of fecal cortisol metabolites increased from 55.1 ± 4.6 ng/g before the first transport to 161 ± 17 ng/g the morning after (P < 0.001). Subsequent transport did not cause further increases in fecal cortisol metabolites. In response to the first transport, mean RR interval decreased with loading of the horses and further with the onset of transport (1551 ± 23, 1304 ± 166, and 1101 ± 123 msec 1 d before, immediately preceeding, and after 60–90 min of transport, respectively; P < 0.05). Decreases in RR interval during subsequent transports became less pronounced (P < 0.001). Transport was associated with a short rise in the HRV variable standard deviation 2 (P < 0.001 except transport 1), indicating sympathetic activation. No consistent changes were found for other HRV variables. In conclusion, a transport-induced stress response in horses decreased with repeated transport, indicating that animals habituated to the situation, but an increased cortisol secretion remained detectable.     

Values for Triglycerides,Insulin, Cortisol,and ACTH in a Herd of Normal Donkeys

Reference ranges for triglycerides, insulin, cortisol, and adrenocorticotropic hormone (ACTH) are used in diagnosing hyperlipemia and pituitary pars intermedia dysfunction (PPID) in the donkey. Values are currently compared to reference ranges of the horse so as to diagnose disease. Previous studies found differences between hematological, serum biochemical, and hormone values of the horse and donkey. We suspected that similar differences existed between horse and donkey triglyceride, insulin, cortisol, and ACTH levels. Blood samples were drawn from 44 healthy mammoth donkeys and 1 miniature donkey, ranging in age from 3 weeks to 21 years, and varying in sex and pregnancy status. All but one donkey scored 3 of 5, “ideal,” body condition scoring. Samples were tested for triglycerides, insulin, cortisol, and ACTH levels. A marked difference was found between horse and donkey normal values for triglycerides, insulin, and ACTH. The mean values and standard deviation in the tested population were 66.4 ± 34.2 mg/dL for triglycerides, 2.1 ± 2.05 μU/mL for insulin, and 66.7 ± 20.7 pg/mL for ACTH. The reference ranges in the horse are 14–77 mg/dL for triglycerides, 4.9–45.5 μU/mL for insulin, and 18.7 ± 6.8 pg/mL for ACTH. Cortisol levels were similar in the two species, a 4.0 ± 1.2 μg/dL for donkeys being within the reference range for the horse, 2.9–6.6 μg/dL. Values were not correlated to age. The sample size prevented us from determining any correlation according to sex or pregnancy status. Differences between horse and donkey triglyceride and ACTH values may be significant for accurately diagnosing and treating hyperlipemia and PPID, respectively, in the donkey.     

Oxidative stress, steroid hormone concentrations and acetylcholinesterase activity in Oreochromis niloticus exposed to chlorpyrifos

We investigated the endocrine disrupting effects of chlorpyrifos-ethyl which is suspected to be originated from oxidative stress. Initially, the 96 h LC₅₀ values of chlorpyrifos in juvenile and adult of Oreochromis niloticus were determined to be 98.67 μg/L and 154.01 μg/L, respectively. Sub-lethal concentrations of chlorpyrifos-ethyl (5 ppb, 10 ppb, 15 ppb) were administrated to adult fish for 15 and 30 days. Fish were then left to depurate for 15 days in pesticide-free water. Gonadal somatic indices, serum sex steroids as indicators of reproductive function and cortisol level as indicator of stress condition were measured to observe the endocrine disruption effects of chlorpyrifos-ethyl. Gonadal glutathione S-transferase and antioxidant enzyme activities and lipid peroxidation as indicators of oxidative stress were also measured. Acetylcholinesterase activity was measured as a marker of chlorpyrifos toxicity. Results showed that serum estradiol, testosteron and cortisol levels in fish exposed to chlorpyrifos were lower than those of the control fish while gonad somatic indices did not change during the experiments. After 30 days, chlorpyrifos exposure decreased GST activity, and increased SOD enzyme activity by up to 215-446% compared with the control, suggesting there was a oxidative stress. No statistically significant differences between GPx and CAT specific activities, protein contents and lipid peroxidation were determined between control and treatment groups in all exposure concentrations and periods. Acetylcholinesterase activity decreased (45.83-77.28%) in gonad tissues. After recovery serum estradiol and testosteron levels were similar to those of the control levels. An increase in the GST and SOD enzyme activities were determined. Cortisol level and AChE activity in all exposure groups decreased after the depuration period, and fish were unable to overcome the stress of chlorpyrifos. Thus, this study revealed that after chlorpyrifos treatments there exists a protective function of antioxidant enzymes against lipid peroxidation in gonad tissue of O. niloticus. There also exist lower testosteron and estradiol levels in exposed fish than those of the control fish without any alterations in oxidative stress, which is attributed to the capability of chlorpyrifos to impair steroid hormone levels.     

Hormone profile of an edible, freshwater teleost, Sarotherodon mossambicus (Peters) under endosulfan toxicity

The hormone profile of Sarotherodon mossambicus (Peters) under endosulfan (an organochlorine pesticide) toxicity was investigated by estimating the serum levels of T₃ (triiodothyronine), T₄ (thyroxine), cortisol, prolactin and insulin in control and sub-lethal (0.001 mg L⁻¹) endosulfan-exposed fish for 1, 6, 12, 24 h and 5 days. Exposure of fish to sub-lethal concentration of endosulfan caused varying changes in the levels of serum hormones studied. Based on the results obtained, it was concluded that (i) the fish registered an increased metabolic activity during the initial period followed by an adaptive lowering of metabolic rate as indicated by changes in the serum T₃ and T₄ levels, (ii) the fish showed an adaptive stress response as indicated by the reduced cortisol level which could be probably due to severe interrenal exhaustion, (iii) the increased serum prolactin levels in the fish under endosulfan exposure is indicative of a possible action of prolactin on gills and kidney towards hydromineral regulation in the fish body under pesticide stress, (iv) the fish adaptively increases its serum insulin level thereby registering a possible adaptive hypoglycemic role of the hormone to favour the transport of glucose to different tissues besides a possible lipogenic activity of the higher insulin level in fish tissue under pesticide toxicity and (v) the fish appears to exhibit a possible adaptive recovery response in its hormone profile following prolonged exposure for 24 h and 5 days.