Excretion Of Corticosterone Metabolites Research Articles

Effects of rearing methods on feather-damaging behavior and corticosterone metabolite excretion in the peach-faced lovebird (Agapornis roseicollis Vieillot)

Hand-reared birds are more likely to develop behavioral disorders such as feather-damaging behavior (FDB) than parent-reared birds; this may adversely affect the human–animal bond and lead to poor animal welfare. Therefore, it is important to determine the effects of rearing methods on FDB to improve the welfare of companion birds. We determined the effects of rearing methods on FDB in the peach-faced lovebird (Agapornis roseicollis Vieillot) using a case–control study and by comparing the concentrations of corticosterone metabolites (CM) in bird droppings among the following 3 groups: (1) non-FDB hand-reared (N-HR) birds; (2) FDB hand-reared (FDB-HR) birds; and (3) parent-reared (PR) birds with neonatal handling. The case–control study included sex, age, rearing method, signs of separation anxiety, staple food, fresh food, enrichment, and the presence of conspecifics as potential risk factors for FDB. Droppings were collected once a month over a 3-day period for 2 months, and the CM concentrations in the droppings were determined using a competitive enzyme immunoassay. The prevalence of FDB was higher in the HR birds than in the PR birds. Furthermore, there was no difference in the mean CM concentrations in the droppings between the N-HR and FDB-HR birds; however, the mean CM concentrations in the droppings of these birds were higher than those of the PR birds. The presence of conspecifics resulted in significantly lower CM concentrations in the 3 groups than when conspecifics were absent. These findings suggest that rearing methods influence FDB development and dropping CM concentrations in the peach-faced lovebirds. Therefore, we propose parental rearing with neonatal handling over hand-rearing and maintaining with conspecifics for the welfare of the peach-faced lovebirds.

Softened food reduces weight loss in the streptozotocin-induced male mouse model of diabetic nephropathy.

The streptozotocin (STZ)-induced diabetic mouse is a widely used model of diabetes and diabetic nephropathy (DN). However, it is a well-known issue that this model is challenged by high weight loss, which despite supportive measures often results in high euthanization rates. To overcome these issues, we hypothesized that supplementing STZ-induced diabetic mice with water-softened chow in addition to normal chow would reduce weight loss, lower the need for supportive treatment, and reduce the number of mice reaching the humane endpoint of 20% weight loss. In a 15 week STZ-induced DN study we demonstrated that diabetic male mice receiving softened chow had reduced acute weight loss following STZ treatment ( p = 0.045) and additionally fewer mice were euthanized due to weight loss. By supplementing the diabetic mice with softened chow, no mice reached 20% weight loss whereas 37.5% of the mice without this supplement reached this humane endpoint ( p = 0.0027). Excretion of corticosterone metabolites in faeces was reduced in diabetic mice on softened chow ( p = 0.0007), suggesting lower levels of general stress. Finally, it was demonstrated that the water-softened chow supplement did not significantly affect the induction of key disease parameters, i.e. %HbA1C and albuminuria nor result in abnormal teeth wear. In conclusion, supplementation of softened food is refining the STZ-induced diabetic mouse model significantly by reducing stress, weight loss and the number of animals sacrificed due to humane endpoints, while maintaining the key phenotypes of diabetes and nephropathy.

An association between feather damaging behavior and corticosterone metabolite excretion in captive African grey parrots (Psittacus erithacus).

BackgroundAfrican grey parrots (Psittacus erithacus) are kept as pets and are frequently hand-reared. It has been observed that hand-reared African grey parrots may develop behavioral disorders such as feather damaging behavior (FDB). It is well known that chronic stress is involved in behavioral disorders in captive parrots. The main glucocorticoid in birds is corticosterone; its quantification provides information about adrenocortical activity and is considered to be a reliable indicator of stress levels in birds. We analyzed the differences in the excretion of corticosterone metabolites (CM) in the droppings of African grey parrots characterized by: 1. different rearing histories (parent rearing vs. hand rearing); and 2. the presence or absence of FDB in hand-reared parrots.MethodsA total of 82 African grey parrots that were kept in captivity were considered. According to breeding methods, three groups of birds were defined: 1. The parent-reared (PR) parrots included birds kept in pairs (n = 30 pairs) with a conspecific partner of the opposite sex. All of these birds were healthy and never showed FDB signs; 2. The healthy hand-reared parrots (H-HR) included pet parrots individually kept, that were hand-reared and did not display any sign of FDB (n = 11, 7 males and 4 females); 3. The FDB hand-reared parrot (FDB-HR) included pet parrots individually kept, that were hand-reared and displayed FDB (n = 11, 7 males and 4 females). Droppings were collected in the morning over three alternating days in autumn 2014 and spring 2015. The CM were determined using a multi-species corticosterone enzyme immunoassay kit. Split-plot repeated-measure ANOVA was used to examine any differences using group, season and group × season as the main factors.ResultsDifferent quantities of CM in droppings were found for the three groups. The mean CM value was 587 ng/g in the PR parrots, 494 ng/g in the H-HR parrots and 1,744 ng/g in the FDB-HR parrots, irrespective of the season. The excretion of CM in FDB-HR was significantly higher than in PR or H-HR parrots. CM in droppings were not influenced by the season (autumn vs. spring); furthermore, the interaction between group and sampling season was not significant. Limited to the H-HR and FDB-HR groups, a trend in the significance of the difference in the mean CM excreted by male and female birds was observed, with the levels excreted by males being higher than those excreted by females. When the effect of age was considered (in the two separate groups), there was a statistically significant positive correlation only for H-HR.ConclusionsThe highest amount of CM excretion was found in FDB-HR parrots, and a positive correlation between age and CM excretion was found in H-HR. Given that the CM excretion of both PR and H-HR parrots was similar in our study, future research is recommended to investigate the specific aspects of hand-rearing to improve parrot welfare.

The distorting effect of varying diets on fecal glucocorticoid measurements as indicators of stress: A cautionary demonstration using laboratory mice

The physiological stress response is frequently gauged in animals, non-invasively, through measuring glucocorticoids in excreta. A concern with this method is, however, the unknown effect of variations in diets on the measurements. With an energy dense diet, leading to reduced defecation, will low concentrations of glucocorticoids be artificially inflated? Can this effect be overcome by measuring the total output of glucocorticoids in excreta? In a controlled laboratory setting we explored the effect in mice. When standard mouse chow – high in dietary fiber – was replaced with a 17% more energy-dense diet, fecal mass was significantly reduced. As circulating levels of corticosterone and the total output of corticosterone metabolites over time remained unaffected, the result was an overestimation – more than a doubling – of the corticosterone metabolite excretion if expressed as concentrations. Similar results were obtained for testosterone metabolites. Although measuring the total output is not feasible in, for example, wildlife studies, the present findings highlight the perilousness of relying on concentrations of hormones in excreta with no associated information of the dietary intake as even moderate changes can exert a great influence.

Two novel mutations of the CYP11B2 gene in a Japanese patient with aldosterone deficiency type 1

Isolated hypoaldosteronism is a rare and occasionally life-threatening cause of salt wasting in infancy. A 2-month-old Japanese boy of unrelated parents was examined for failure to thrive and poor weight gain. Laboratory findings were hyponatremia, hyperkalemia, high plasma renin and low aldosterone levels. Spot urine analysis by gas chromatography-mass spectrometry (GC-MS) showed that urinary excretion of corticosterone metabolites was elevated. Whereas excretion of 18-hydroxycortricosterone metabolites was within the normal range, excretion of aldosterone metabolites was undetectable. The patient was therefore suspected to have aldosterone synthase deficiency type 1. Sequence analysis of CYP11B2, the gene encoding aldosterone synthase (CYP11B2), showed that the patient was a compound heterozygote for c.168G>A, p.W56X in exon 1 and c.1149C>T, p.R384X in exon 7. p.W56X was inherited from his mother and p.R384X was from his father. Since both alleles contain nonsense mutations, a lack of CYP11B2 activity was speculated to cause his condition. To our knowledge, this is the first Japanese patient in which the molecular basis of aldosterone synthase deficiency type 1 has been clarified. This case also indicates that spot urinary steroid analysis is useful for diagnosis.

Effects of a fire alarm strobe light on fecal corticosterone metabolite concentrations in mice

The type and location of fire alarms are important considerations in animal facility design. The Guide for the Care and Use of Laboratory Animals recommends minimizing animal exposure to such alarms. Nevertheless, it is often necessary to maintain fire alarms within animal housing or procedural areas. The authors exposed male mice to the flashing strobe light component of a standard fire alarm and evaluated mouse fecal corticosterone concentration, which is known to be an indicator of stress. Mice were exposed to the strobe light for 5 min during either the light or the dark phase of the light:dark cycle. The authors collected fecal samples every 6 h for 24 h before exposing mice to the alarm and every 6 h for 24 h after exposure. Fecal samples taken before exposure (baseline samples) showed a normal circadian pattern of corticosterone metabolite excretion. In fecal samples taken after mice were exposed to the fire alarm, metabolite concentrations did not significantly differ from baseline concentrations over time.

Active and passive social support in families of greylag geese (Anser anser).

In general, support by social allies may reduce stress, increase success in agonistic encounters and ease access to resources. Social support was mainly known from mammals, particularly primates, and has been studied in birds only recently. Basically two types are known: (i) 'active social support', which describes the participation of a social ally in agonistic encounters, and (ii) 'passive social support' in which the mere presence of a social ally reduces behavioural and physiological stress responses. In greylag geese (Anser anser) offspring stay with their parents for an entire year or even longer and therefore are a candidate avian model to study support by social allies. We investigated the effects of active and passive social support in ten families (ten males, ten females, 33 juveniles) in a free-roaming, semi-tame flock of greylag geese. Focal individuals were observed during three time periods: (i) re-establishment of the flock in the fall, (ii) stable winter flock, and (iii) disintegration of the flock and break-up of family bonds. We recorded all agonistic interactions of the members of one focal family during morning feedings for two consecutive days: a control day, in which food was distributed widely, and a social density stress situation, in which the same amount of food was spread over a much smaller area. In addition, we collected faeces of all individuals within this family for three hours from the beginning of the feeding situation for determining excreted corticosterone immuno-reactive metabolites by enzyme immuno assay. We found that the small families, i.e. pairs with one or two accompanying young, were involved in more agonistic interactions, mainly through the lack of active social support, as compared to large families in the same situation. Members of greylag goose families lost agonistic encounters significantly less often when actively supported. In addition, the excretion of corticosterone metabolites was significantly decreased in large families during a social density stress situation, probably as an effect of passive social support. Via such a socially induced decrease in hormonal stress response during challenging situations, an individual's long term energy management may benefit.

Furosemide Inhibits 11 -Hydroxysteroid Dehydrogenase Type 2

11β-Hydroxsteroid dehydrogenase 2 (11β-OHSD2) protects the nonselective renal mineralocorticoid receptor from the endogenous glucocorticoid cortisol. Thus, drugs inhibiting 11β-OHSD2 might enhance urinary loss of potassium. As diuretics influence the renal handling of potassium, we analyzed the impact of 13 commonly used diuretics on 11β-OHSD2. Furosemide was the only inhibitor. Its inhibition constant (Ki) was 30 μmol when extracts from COS-1 cells transfected with human 11β-OHSD2 were used as an enzyme source. The type of inhibition was competitive. To establish whether furosemide inhibits 11β-OHSD2 and 11β-OHSD1 in the renal target tissue, isolated tubular segments from rats were analyzed. Furosemide decreased the oxidative activity of 11β-OHSD2 in intact distal tubules and 11β-OHSD1 in proximal convoluted tubules. For the assessment of furosemide on the excretion of corticosterone metabolites in vivo, rats were given furosemide ip, and the ratio of tetrahydrocorticosterone plus 5α-tetrahydrocorticosterone to 11-dehydrotetrahydrocorticosterone was determined in urine. This ratio increased after the administration of furosemide in all animals, indicating inhibition of the oxidative activity of 11β-OHSD. Thus, furosemide inhibits the 11β-OHSD2 enzyme in the target tissue and might by that mechanism enhance the mineralocorticoid effect of 11β-hydroxyglucocorticoids.

Furosemide inhibits 11beta-hydroxysteroid dehydrogenase type 2.

11Beta-hydroxsteroid dehydrogenase 2 (11beta-OHSD2) protects the nonselective renal mineralocorticoid receptor from the endogenous glucocorticoid cortisol. Thus, drugs inhibiting 11beta-OHSD2 might enhance urinary loss of potassium. As diuretics influence the renal handling of potassium, we analyzed the impact of 13 commonly used diuretics on 11beta-OHSD2. Furosemide was the only inhibitor. Its inhibition constant (Ki) was 30 micromol when extracts from COS-1 cells transfected with human 11beta-OHSD2 were used as an enzyme source. The type of inhibition was competitive. To establish whether furosemide inhibits 11beta-OHSD2 and 11beta-OHSD1 in the renal target tissue, isolated tubular segments from rats were analyzed. Furosemide decreased the oxidative activity of 11beta-OHSD2 in intact distal tubules and 11beta-OHSD1 in proximal convoluted tubules. For the assessment of furosemide on the excretion of corticosterone metabolites in vivo, rats were given furosemide i.p., and the ratio of tetrahydrocorticosterone plus 5alpha-tetrahydrocorticosterone to 11-dehydrotetrahydrocorticosterone was determined in urine. This ratio increased after the administration of furosemide in all animals, indicating inhibition of the oxidative activity of 11beta-OHSD. Thus, furosemide inhibits the 11beta-OHSD2 enzyme in the target tissue and might by that mechanism enhance the mineralocorticoid effect of 11beta-hydroxyglucocorticoids.

Evidence of coexisting changes in 11 beta-hydroxysteroid dehydrogenase and 5 beta-reductase activity in subjects with untreated essential hypertension.

We compared corticosteroid metabolite excretion rates and patterns in a group of 68 subjects with untreated essential hypertension and a matched group of 48 normotensive control subjects. The ratio of tetrahydrocortisol plus allotetrahydrocortisol to tetrahydrocortisone and the ratio of allotetrahydrocortisol to tetrahydrocortisol were significantly higher in the hypertensive group. This is qualitatively similar to the situation found in patients with the syndrome of apparent mineralocorticoid excess or subjects treated with licorice or carbenoxolone where hypertension is known to arise from deficiencies of 11 beta-hydroxysteroid dehydrogenase and 5 beta-reductase activities. The equivalent ratios for corticosterone metabolites were not different between groups, but total corticosterone metabolite excretion was higher in the hypertensive group. Plasma cortisol levels were lower in hypertensive than in control subjects, but corticosterone levels were higher. This evidence supports a previous suggestion that the activities of these two enzymes may be reduced in essential hypertension, but the contribution of these changes to hypertension is not known.

Combined virilizing 17-21 hydroxylase deficiency in siblings

Female pseudohermaphroditism with skeletal abnormalities (cranial and antebrachial) was observed in 3 siblings. Marked hirsutism was observed during pregnancies in the mother. Ambiguous genitalia were present at birth but further virilization did not occur. Saltwasting, hypokalemia or hypertension was not present during long-term observation. Adrenal function studies were performed in 2 of the patients. Basal values of urinary cortisol-metabolites and 17-ketosteroids were normal but the excretion of corticosterone-metabolites and pregnanetriol was increased (ratio cortisol-/corticosterone-metabolites 1:1). After ACTH stimulation additional excretion of pregnanetriol and corticosterone-metabolites was found. Also urinary cortisol-metabolites were increased as were 17-ketosteroids, allthough only moderate. Basal ACTH and 17-OH progesterone values were only slightly increased. These observations suggest the presence of a combined partial 17-21 hydroxylase deficiency with skeletal defects. This clinical entity has not previously been described.

Delayed expression of neonatal sexual differentiation of corticosteroid patterns in rat bile.

The excretion of corticosterone metabolites in bile from male and female rats 21 to 56 days of age was studied. No sexual differences were observed in rats 21 and 28 days of age; in these animals 3β, 11β,21‐trihydroxy‐5α‐pregnan‐20‐one disulfate was the major corticosteroid but significant amounts of monosulfurylated 3α,11β,21‐trihydroxy‐5α‐pregnan‐20‐one and 3α,11β,15α,21‐tetra‐hydroxy‐5α‐pregnan‐20‐one were also excreted. From 35 days of age, sexual differences became successively more apparent in the biliary steroid excretion patterns and at 56 days of age female rats had developed a characteristic pattern of mono‐ and disulfurylated 3α,11β,15α,21‐tetra‐hydroxy‐5α‐pregnan‐20‐one whereas the male biliary steroid pattern was characterized by the complete absence of 15‐hydroxylated corticosteroids, by relatively large amounts of 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one disulfate and by the sex‐specific metabolite 5α‐pregnane‐3β,11β, 20β,21‐tetrol disulfate. It is concluded that the “programming” or “imprinting” of hepatic corticosteroid metabolism that occurs in neonatal male rats and results in the absence of 15‐hydroxy corticosterone metabolites and the presence of 5α‐pregnane‐3β, 11β,20β,21‐tetrol in bile of adult male rats is not expressed until after puberty.

Irreversible neonatal differentiation of corticosterone metabolism in rats in vivo.

The excretion of corticosterone metabolites in bile from gonadectomized male and female rats was studied; the effects of testosterone propionate treatment on steroid excretion in intact and castrated female rats were also studied. Postpubertal ovariectomy did not change the female biliary corticosteroid pattern (mainly characterized by excretion of mono‐ and disulphurylated 3α,11β,15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one); likewise postpubertal testectomy or even testectomy of rats 14 days of age did not change the male biliary corticosteroid pattern (mainly characterized by disulphurylated 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one and 5α‐pregnane‐3β,11β,20β,21‐tetrol). On the other hand neonatal testectomy led to a feminized biliary corticosteroid pattern in the adult rats. The results indicate an irreversible suppression of the hepatic 15α‐hydroxylase activity by testicular secretion products, presumably androgen(s), in the neonatal period. When adult intact or gonadectomized female rats were treated with testosterone propionate the female biliary corticosteroid pattern changed to a male pattern but reverted to a female pattern some time after the therapy was stopped. These results indicate that the 15α‐hydroxylase activity in female rats may be reversibly suppressed by androgen treatment postpubertally.

Steroid metabolism in rats given (1- 2 H 2 )ethanol. Biliary metabolites of corticosterone and administered 4-androstene-3,17-dione.

The rate of excretion of different corticosterone metabolites in rat bile after administration of [1‐2H2]ethanol, and the transfer of deuterium to these metabolites have been studied by gas chromatography‐mass spectrometry. The excretion of corticosterone metabolites increased after injection of ethanol, but the relative amounts of different steroids remained essentially constant. The deuterium excess in 3α,11β, 15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one and 3β(and 3α),11β,21‐trihydroxy‐5α‐pregnan‐20‐one in female rats and of 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one, 5α‐pregnane‐3β‐(and 3α),11β,20β,21‐tetrol and 5β‐pregnane‐3α,11β,20β,21‐tetrol in male rats varied between 7 and 20 atoms per cent. Only monodeuterated molecules were found. All the deuterium in 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one was present in the 3α‐position, and little or no deuterium was found in the 20α‐position of the corresponding 20β‐hydroxysteroid.When 4‐androstene‐3,17‐dione was given to male bile‐fistula rats, 3α‐hydroxy‐5α‐androstan‐17‐one, 3β‐hydroxy‐5α‐androstan‐17‐one, and 3α‐hydroxy‐5β‐androstan‐17‐one, and various hydroxylated metabolites were excreted in bile. Of the former compounds, the 3α,5β‐epimer was predominant in the glucuronide fraction, whereas the 3β,5α‐epimer was the major epimer in the sulphate fraction. During metabolism of [1‐2H2]ethanol the 3β‐ and 3α‐positions of these compounds contained 5–20 atoms per cent of deuterium. No deuterium was found in the 5α‐ or 5β‐positions.The results indicate that the coenzyme pool(s) used in different reductions at C‐3 is metabolically related to NADH formed in the alcohol dehydrogenase reaction. Since little or no deuterium was found at C‐5 and C‐20 other coenzyme pools are probably used for the reductions of these positions.

Steroids in germfree and conventional rats. Distribution and excretion of labelled pregnenolone and corticosterone in male and female rats.

The distribution of intraperitoneally administered [4-14C] pregnenolone and [1,2-3H] corticosterone were studied in male and female conventional rats at different times after injection of the isotopes. The administered steroids were rapidly excreted into the intestinal tract; 2 h after administration 60—70 % of the given dose was localized in the small bowel, mainly in mono sulphurylated form. The elimination in urine and faeces of intraperitoneally administered [4-14C]pregnenolone and [4-14C]corticosterone was studied in germfree and conventional, male and female rats. The steroids were excreted as free and conjugated metabolites. The latter were mainly monosulphurylated, and to a smaller extent disulphurylated. No steroid glucuronides were found. As a probable result of the intestinal microbial sulphohydrolase activities conventional rats excreted more free steroids than germfree both in faeces and urine. Male rats excreted more unconjugated and more polar steroids than female. Corticosterone metabolites were monosulphurylated to a larger extent than pregnenolone metabolites; faecal excretion of corticosterone metabolites was quantitatively more important than of pregnenolone metabolites.