Heart rate and salivary cortisol concentrations in foals at birth.

Heart rate (HR), HR variability (HRV) and salivary cortisol concentrations were determined in foals (n = 13) during the perinatal phase and until 5 months of age. In the fetus, HR decreased from 77 ± 3 beats/min at 120 min before birth to 60 ± 1 beats/min at 5 min before birth (P <0.01). Within 30 min of birth, HR increased to 160 ± 9 beats/min (P <0.01). Salivary cortisol concentrations immediately after birth were 11.9 ± 3.6 ng/mL and within 2 h increased to a maximum of 52.5 ± 12.3 ng/mL (P <0.01). In conclusion, increases in HR and salivary cortisol concentrations in foals are not induced during parturition, but occur immediately after birth.

Reduced-size microchips for identification of horses: response to implantation and readability during a six-month period.

In this study, readability of reduced-size microchips in horses and the response to implantation were analysed. It was hypothesised that small microchips can be implanted stress-free but are less readable than larger microchips. Adult mares (n=40) were implanted with a reduced-size microchip (10.9×1.6 mm) at the left side of the neck (size of conventional microchips 11.4×2.2 mm). Microchips were identified with three different scanners (A, B, C) immediately, and at 6, 12 and 28 weeks after implantation. Twelve out of the 40 mares were submitted to microchip implantation and control treatments and cortisol, heart rate and heart rate variability (HRV) were determined. From the chip-bearing side of the neck, microchips were identified with all scanners in all horses at all times. From the contralateral side, correct readings were always 100 per cent with scanner C and with scanners A and B ranged between 60 and 100 per cent. Heart rate and HRV variable sd of beat-to-beat interval increased slightly (P<0.01) at microchip implantation and control treatment, but cortisol concentration did not increase. In conclusion, reduced-size microchips are highly reliable for identification of horses. Compared with conventional microchips, the reduction in size did not impair readability. Microchip implantation is no pronounced stressor for horses.

Physiological and behavioral stress parameters in calves in response to partial scrotal resection, orchidectomy, and Burdizzo castration.

Establishing artificial cryptorchids by partial scrotal resection without removing the testicles is a technique for castration of bull calves that recently has gained new interest. In contrast to orchidectomy and Burdizzo castration, the stress response of calves to shortening of the scrotum is unknown. In this study, partial scrotal resection in bull calves was compared with orchidectomy, Burdizzo castration, and controls without intervention (n=10 per group, ages 56 ± 3 d). Procedures were performed under xylazine sedation and local anesthesia. We hypothesized that partial scrotal resection is least stressful. Salivary cortisol, heart rate, heart rate variability, behavior, and locomotion were analyzed. Cortisol concentration peaked 60 min after start of the procedures. Cortisol release was at least in part xylazine induced and none of the experimental procedures released additional cortisol. Heart rate increased in calves of all groups with initial handling, but immediately after xylazine sedation decreased to 30% below initial values and was not modified by surgical procedures. The heart rate variability variables standard deviation of beat-to-beat interval and root mean square of successive beat-to-beat differences increased when calves were placed on the surgery table but effects were similar in calves submitted to surgeries and control calves. Locomotion increased, whereas lying time decreased in response to all surgeries. Locomotion increase was most pronounced after orchidectomy. Plasma fibrinogen concentrations increased after orchidectomy only. With adequate pain medication, orchidectomy, Burdizzo castration, and partial scrotal resection do not differ with regard to acute stress and, by inference, pain. Partial scrotal resection when carried out under xylazine sedation and local anesthesia thus is an acceptable castration technique in bull calves.

Configuration of antibodies for assay of urinary cortisol in dogs influences analytic specificity.

Whether the variation in the reported urinary corticoid-to-creatinine ratio in dogs is affected by the application of 2 commonly applied anticortisol antibodies was investigated. Free-catch morning urine samples of 50 healthy dogs were analyzed in duplicate with the use of 2 different polyclonal antibodies (antibody A and B) raised in different rabbits. Antibody A was raised against cortisol-3-carboxymethyl-oxime and antibody B against cortisol-21-hemisuccinate linked to BSA. Enzyme immunoassays were applied by using corresponding biotinylated labels. To examine possible cross-reactions with conjugated and nonconjugated cortisol metabolites, EIA measurements were performed with urine samples both before (directly assayed) and after diethyl-ether extraction, as well as after reversed-phase HPLC. Although the results correlated (P < 0.001), urinary corticoid concentrations and accordingly the urinary corticoid-to-creatinine ratios were 8 times higher when using antibody A than when using antibody B (mean ± SD corticoid concentrations, 223 ± 131 vs 29 ± 12 nmol/L; P < 0.001). Irrespective of the antibody used, extraction significantly decreased measured corticoid concentrations (antibody A, 158 ± 120 nmol/L; antibody B, 15 ± 8 nmol/L; P < 0.001), but the decrease was conspicuous when antibody A was used. Antibody A cross-reacted significantly with polar (eg, conjugated) metabolites, clearly depicted in the chromatogram by 3 additional peaks in earlier fractions well separated from cortisol. In contrast the assay that used antibody B was specific, showing only 1 major peak in the fractions eluting authentic cortisol. In summary, the study indicates that the configuration of the antibody considerably influences the analytic specificity of cortisol assays and underlines the pivotal importance of assay validation for each species and sample material.

Measurement of fecal glucocorticoid metabolites and evaluation of udder characteristics to estimate stress after sudden dry-off in dairy cows with different milk yields.

Sudden dry-off is an established management practice in the dairy industry. But milk yield has been increasing continuously during the last decades. There is no information whether the dry-off procedure, which often results in swollen and firm udders, causes stress, particularly in high-producing dairy cows. Therefore, we evaluated the effect of a sudden dry-off on extramammary udder pressure and the concentration of fecal glucocorticoid metabolites (i.e., 11,17-dioxoandrostane, 11,17-DOA) as an indirect stress parameter. Measurements were carried out within the last week before dry-off and until 9d after dry-off considering 3 groups of milk yield (i.e., low: <15 kg/d, medium: 15-20 kg/d, and high: >20 kg/d). Udder pressure increased in all yield groups after dry-off, peaked at d 2 after dry-off and decreased afterwards. Pressures were highest in high-yielding cows and lowest in low-yielding cows. But only in high-yielding cows was udder pressure after dry-off higher than before dry-off. Baseline 11,17-DOA concentrations depended on milk yield. They were highest in low-yielding (121.7 ± 33.3 ng/g) and lowest in high-yielding cows (71.1 ± 30.0 ng/g). After dry-off, 11,17-DOA increased in all yield groups and peaked at d 3. Whereas in medium- and high-yielding cows 11,17-DOA levels differed significantly from their respective baseline during the whole 9-d measuring period, low-yielding cows showed elevated 11,17-DOA levels only on d 3 after dry-off. However, especially the increase in 11,17-DOA after dry-off between the 3 yield groups was considerably different. Mean 11,17-DOA increase from baseline to d 3 was highest in high-yielding cows (129.1%) and considerably lower in low-yielding cows (40.1%). The highest fecal 11,17-DOA concentrations were measured on d 3 after dry-off, indicating that the stress was most intense on d 2, which is due to an 18-h time lag; at about the same time, udder pressure peaked. Our results showed a negligible effect of a sudden dry-off on low-yielding cows. High-yielding cows, however, faced high extramammary pressures and increased glucocorticoid production. Considering animal welfare aspects, a review of the current dry-off strategies might be warranted.

Pain management with flunixin meglumine at dehorning of calves.

Dehorning (DH) of calves is a common procedure on commercial dairy farms. Pain management of calves has been investigated in several studies. It is generally accepted that the use of local anesthesia before DH is essential for pain management. Postoperative inflammatory pain should be treated by using a nonsteroidal antiinflammatory drug. The objective of this controlled, randomized, and blinded clinical trial was to determine the effects of the nonsteroidal antiinflammatory drug flunixin meglumine before DH on cortisol concentrations in sera of 5- to 9-wk old calves. Furthermore, selected behavioral characteristics and heart and respiratory rate were examined to assess pain in the hours after dehorning. A total of 80 calves were allocated to 4 groups. In each of 20 replicates, 4 calves were randomly assigned to the following groups: in 3 treatment groups, calves received a local anesthetic (10 mL of procain hydrochloride) and a first treatment (i.v.) with flunixin meglumine or a placebo 20 min before hot-iron dehorning, and a second treatment with flunixin meglumine or a placebo (0.9% saline) 3 h after DH. Calves in the control (CON) group were not dehorned and did not receive any treatment. Groups received 2.2 mg of flunixin meglumine/kg followed by a placebo (FP), 2.2 mg of flunixin meglumine/kg for both treatments (FF), or a placebo for both treatments (PP). Blood samples were collected from all calves, including CON calves, 20 min before restraint in a headlock for DH, 2 min after DH, as well as 30 min and 1, 2, 4, 6, and 8 h after DH. Samples were analyzed for concentration of cortisol by enzyme immunoassay. It was found that concentration of cortisol, calculated as area under the curve, was greater in PP compared with FF and tended to be greater compared with FP. Significant differences between PP and FF were detected at 30 min and 2 h after DH. Throughout the observation period, cortisol concentrations were in both flunixin meglumine-treated groups at a similar level as in the CON group. The heart and respiratory rates showed neither difference between the CON group and the 3 dehorned groups nor between the treatment groups.

Cortisol release, heart rate and heart rate variability, and superficial body temperature, in horses lunged either with hyperflexion of the neck or with an extended head and neck position.

Bringing the head and neck of ridden horses into a position of hyperflexion is widely used in equestrian sports. In our study, the hypothesis was tested that hyperflexion is an acute stressor for horses. Salivary cortisol concentrations, heart rate, heart rate variability (HRV) and superficial body temperature were determined in horses (n = 16) lunged on two subsequent days. The head and neck of the horse was fixed with side reins in a position allowing forward extension on day A and fixed in hyperflexion on day B. The order of treatments alternated between horses. In response to lunging, cortisol concentration increased (day A from 0.73 ± 0.06 to 1.41 ± 0.13 ng/ml, p < 0.001; day B from 0.68 ± 0.07 to 1.38 ± 0.13 ng/ml, p < 0.001) but did not differ between days A and B. Beat-to-beat (RR) interval decreased in response to lunging on both days. HRV variables standard deviation of RR interval (SDRR) and RMSSD (root mean square of successive RR differences) decreased (p < 0.001) but did not differ between days. In the cranial region of the neck, the difference between maximum and minimum temperature was increased in hyperflexion (p < 0.01). In conclusion, physiological parameters do not indicate an acute stress response to hyperflexion of the head alone in horses lunged at moderate speed and not touched with the whip. However, if hyperflexion is combined with active intervention of a rider, a stressful experience for the horse cannot be excluded.

Cortisol and progestin release, heart rate and heart rate variability in the pregnant and postpartum mare, fetus and newborn foal.

The mechanisms leading to parturition in the horse in many aspects differ from those in other species. Pregnancy is maintained not by progesterone but by 5α-pregnanes and the progestin precursor pregnenolone originates from the fetus. As parturition approaches, the fetal adrenal switches from pregnenolone to cortisol synthesis but it is not known whether cortisol crosses the placenta. We hypothesized that in parallel to fetal cortisol release, cortisol in the maternal circulation increases before foaling and this increase can be determined in both saliva and plasma. In addition, maternal, fetal and neonatal heart rate and heart rate variability were measured. In 25 pregnant mares, saliva for cortisol analysis was collected 4 times daily from 15 days before to 5 days after foaling. In 13 mares, in addition, fetomaternal electrocardiogram (ECG) recordings were made and blood samples for progestin and cortisol analysis were collected once daily. Heart rate (HR) was recorded until 5 days after foaling. The heart rate variability (HRV) variables standard deviation of the beat-to-beat (RR) interval (SDRR) and root mean square of successive RR differences (RMSSD) were calculated. From Days 15 to 4 before parturition, progestin concentration increased (peak 267 ± 42 ng/mL) and decreased thereafter (P < 0.05, day of foaling 113 ± 18 ng/mL). A prepartum increase in maternal cortisol concentrations was evident in blood (P < 0.05) and saliva (P < 0.05) and paralleled the decrease in progestin concentrations. In mares, HR remained constant during the last days of pregnancy but decreased within one day after parturition (P < 0.05) while maternal HRV did not change. In the fetus and neonate, HR increased from before to after birth (P < 0.05) indicating increasing demands on the cardiovascular system with adaptation to extrauterine life.

Physiological and behavioural responses of young horses to hot iron branding and microchip implantation.

Branding is the traditional and well-established method used to mark horses, but recently microchip transponders for implantation have become available. In this study, behaviour, physiological stress variables and skin temperature in foals were determined in response to hot-iron branding (n=7) and microchip implantation (n=7). Salivary cortisol concentrations increased in response to branding (1.8 ± 0.2 ng/mL) and microchip implantation (1.4 ± 0.1ng/mL), but cortisol release over time did not differ. In response to both manipulations there was a transient increase in heart rate (P<0.001) and heart rate variability (P<0.01). Branding and microchip implantation induced a comparable aversive behaviour (branding, score 3.86 ± 0.85; microchip, score 4.00 ± 0.82). Both techniques thus caused similar physiological and behavioural changes indicative of stress. Acutely, implantation of a microchip was as stressful as branding in foals. Branding caused a necrotising skin burn lasting at least 7 days. Moreover branding, but not microchip implantation (P<0.001), was accompanied by a generalized increase in skin temperature which was comparable to low degree post-burn hypermetabolism in humans.

The influence of trilostane on steroid hormone metabolism in canine adrenal glands and corpora lutea-an in vitro study.

Trilostane is widely used to treat hyperadrenocorticism in dogs. Trilostane competitively inhibits the enzyme 3-beta hydroxysteroid dehydrogenase (3ß-HSD), which converts pregnenolone (P5) to progesterone (P4) and dehydroepiandrosterone (DHEA) to androstendione (A4). Although trilostane is frequently used in dogs, the molecular mechanism underlying its effect on canine steroid hormone biosynthesis is still an enigma. Multiple enzymes of 3ß-HSD have been found in humans, rats and mice and their presence might explain the contradictory results of studies on the effectiveness of trilostane. We therefore investigated the influence of trilostane on steroid hormone metabolism in dogs by means of an in vitro model. Canine adrenal glands from freshly euthanized dogs and corpora lutea (CL) were incubated with increasing doses of trilostane. Tritiated P5 or DHEA were used as substrates. The resulting radioactive metabolites were extracted, separated by thin layer chromatography and visualized by autoradiography. A wide variety of radioactive metabolites were formed in the adrenal glands and in the CL, indicating high metabolic activity in both tissues. In the adrenal cortex, trilostane influences the P5 metabolism in a dose- and time-dependent manner, while DHEA metabolism and metabolism of both hormones in the CL were unaffected. The results indicate for the first time that there might be more than one enzyme of 3ß-HSD present in dogs and that trilostane selectively inhibits P5 conversion to P4 only in the adrenal gland.

Environmental influences on Adelie penguin breeding schedules, endocrinology, and chick survival.

To understand how the social and physical environment influences behaviour, reproduction and survival, studies of underlying hormonal processes are crucial; in particular, interactions between stress and reproductive responses may have critical influences on breeding schedules. Several authors have examined the timing of breeding in relation to environmental stimuli, while others have independently described endocrine profiles. However, few studies have simultaneously measured endocrine profiles, breeding behaviour, and offspring survival across seasons. We measured sex and stress hormone concentrations (oestrogens, testosterone, and corticosterone), timing of breeding, and chick survival, in Adelie penguins (Pygoscelis adeliae) at two colonies in two different years. Clutch initiation at Cape Bird South (CBS; year 1, ~14,000 pairs) occurred later than at Cape Crozier East (CCE; year 2, ~ 25,000 pairs); however, breeding was more synchronous at CBS. This pattern was probably generated by the persistence of extensive sea ice at CBS (year 1). Higher corticosterone metabolite and lower sex hormone concentrations at CBS correlated with later breeding and lower chick survival compared to at CCE - again, a likely consequence of sea ice conditions. Within colonies, sub-colony size (S, 50-100; M, 200-300; L, 500-600; XL, >1000 pairs) did not influence the onset or synchrony of breeding, chick survival, or hormone concentrations. We showed that the endocrine profiles of breeding Adelie penguins can differ markedly between years and/or colonies, and that combining measures of endocrinology, behaviour, and offspring survival can reveal the mechanisms and consequences that different environmental conditions can have on breeding ecology.

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 preceding, 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.

Changes in cortisol release and heart rate variability in sport horses during long-distance road transport.

It is widely accepted that transport is stressful for horses, but only a few studies are available involving horses that are transported regularly and are accustomed to transport. We determined salivary cortisol immunoreactivity (IR), fecal cortisol metabolites, beat-to-beat (RR) interval, and heart rate variability (HRV) in transport-experienced horses (N=7) in response to a 2-d outbound road transport over 1370 km and 2-d return transport 8 d later. Salivary cortisol IR was low until 60 min before transport but had increased (P<0.05) 30 min before loading. Transport caused a further marked increase (P<0.001), but the response tended to decrease with each day of transport. Concentrations of fecal cortisol metabolites increased on the second day of both outbound and return transports and reached a maximum the following day (P<0.001). During the first 90 min on Day 1 of outbound transport, mean RR interval decreased (P<0.001). Standard deviations of RR interval (SDRR) decreased transiently (P<0.01). The root mean square of successive RR differences (RMSSD) decreased at the beginning of the outbound and return transports (P<0.01), reflecting reduced parasympathetic tone. On the first day of both outbound and return transports, a transient rise in geometric HRV variable standard deviation 2 (SD2) occurred (P<0.01), indicating increased sympathetic activity. In conclusion, transport of experienced horses leads to increased cortisol release and changes in heart rate and HRV, which is indicative of stress. The degree of these changes tended to be most pronounced on the first day of both outbound and return transport.

Comparing plasma and faecal measures of steroid hormones in Adelie penguins Pygoscelis adeliae.

Physiological measurements of both stress and sex hormones are often used to estimate the consequences of natural or human-induced change in ecological studies of various animals. Different methods of hormone measurement exist, potentially explaining variation in results across studies; methods should be cross-validated to ensure that they correlate. We directly compared faecal and plasma hormone measurements for the first time in a wild free-living species, the Adelie penguin (Pygoscelis adeliae). Blood and faecal samples were simultaneously collected from individual penguins for comparison and assayed for testosterone and corticosterone (or their metabolites). Sex differences and variability within each measure, and correlation of values across measures were compared. For both hormones, plasma samples showed greater variation than faecal samples. Males had higher mean corticosterone concentrations than females, but the difference was only statistically significant in faecal samples. Plasma testosterone, but not faecal testosterone, was significantly higher in males than females. Correlation between sample types was poor overall, and weaker in females than in males, perhaps because measures from plasma represent hormones that are both free and bound to globulins, whereas measures from faeces represent only the free portion. Faecal samples also represent a cumulative measure of hormones over time, as opposed to a plasma 'snapshot' concentration. Our data indicate that faecal sampling appears more suitable for assessing baseline hormone concentrations, whilst plasma sampling may best define immediate responses to environmental events. Consequently, future studies should ensure that they select the most appropriate matrix and method of hormone measurement to answer their research questions.

Female androgen patterns and within-pair testosterone compatibility in domestic geese (Anser domesticus).

For successfully raising offspring, long-term monogamous pair partners need to be behaviorally and hormonally coordinated. In the monogamous, biparental greylag geese (Anser anser) a dyadic pairbond-specific measure, 'within-pair testosterone compatibility' (TC) indicated how closely synchronized are seasonal androgen levels, which co-varied with reproductive output. Males, in particular, were assumed to respond to their females' hormonal and fecundity phases. We now present experiments with biparental domestic geese (Anser domesticus) kept as pairs to ask whether TC occurs also in these generally polygynous animals. We further ask how different conditions of mate choice affect TC and whether established TC is maintained during a polygynous flock situation. We measured androgen metabolites (AM) non-invasively from individual droppings. In females, AM was related with gonadal activity as it increased after GnRH but not ACTH challenge. Females with preferred partners had higher maximum AM during egg laying and higher rates of initiating incubation than randomly paired females. Domestic ganders had seasonal AM patterns typical for polygynous males. Within-pair TC ranged from almost perfectly positive to non-correlated in domestic geese but mate choice did not explain TC variation. TC of previous pairs was generally reduced in the flock situation, probably confounded by factors of the social environment, i.e. mating opportunity and availability of multiple partners. On top of the underlying reproductive physiology our results suggest two episodic components of TC: a female androgen responsiveness to the preferred partner at least during egg formation, and the male's facultative potential to respond to her readiness to breed.

Effect of altrenogest-treatment of mares in late gestation on adrenocortical function, blood count and plasma electrolytes in their foals.

REASONS FOR PERFORMING STUDY: Mares with compromised pregnancies are often treated with altrenogest to prevent abortion. However, there is only limited information about effects on the foal when altrenogest treatment is continued during final maturation of the fetus. OBJECTIVES: To determine effects of altrenogest treatment during late gestation in mares on maturity, haematology changes, adrenocortical function and serum electrolytes in their newborn foals. METHODS: Six mares were treated with altrenogest (0.088 mg/kg bwt) once daily from Day 280 of pregnancy until foaling and 7 mares served as controls. RESULTS: Foals born to altrenogest-treated mares had a significantly lower neutrophil/lymphocyte ratio on the first day after birth than control foals (P<0.05). Basal plasma cortisol concentrations immediately after birth were higher in foals of altrenogest-treated mares than in control foals (P<0.05). Cortisol release in response to exogenous adrenocorticotropic hormone (ACTH)--except for higher values 15 min after ACTH injection in foals of altrenogest-treated mares on Day 1--revealed no differences in adrenocortical function between the groups of foals. Plasma potassium concentration in foals from altrenogest-treated mares compared to control foals was significantly lower immediately after birth (P<0.05) and plasma ionised calcium concentration was significantly lower 3 h after birth (P = 0.01). CONCLUSIONS AND POTENTIAL RELEVANCE: Altrenogest treatment of pregnant mares prolonged labour had no major effects on adrenocortical function in foals. A reduced neutrophil/lymphocyte ratio in these foals may suggest either immunomodulatory effects of altrenogest or dysmaturity of the foals.

Gestagens and glucocorticoids in chicken eggs.

Avian eggs contain a variety of steroid hormones, which have been attributed as a tool for maternal phenotypic engineering. The majority of studies focuses on androgens, but also significant amounts of progesterone as well as other steroid hormones have been measured. The question if corticosterone is also present in eggs of chickens is currently under debate. The only analytical validation performed so far has failed to demonstrate corticosterone in the yolk of chickens, suggesting that antibodies for corticosterone measurement cross-react with other steroids present in the yolk. In order to investigate this assumption and to characterise potential cross-reacting hormones in more detail, we performed high-performance liquid chromatographic (HPLC) analyses of chicken yolk extracts and determined the concentration of immunoreactive corticosterone, progesterone and cortisol. The progesterone antibody revealed several immunoreactive substances, including progesterone, pregnenolone and two substances with lower polarity. The corticosterone enzyme immunoassay detected immunoreactive substances at exactly the same elution positions as the progesterone assay and a very small peak at the elution position of corticosterone. Immunoreactive cortisol was not found. In addition, inner and outer regions of the yolk sphere were analysed separately via HPLC. We found different concentrations of immunoreactive substances between the inner and outer yolk regions, probably reflecting the steroidogenic activity of the follicle cells during oocyte growth. We conclude that in homogenised yolk extracts without previous clean-up, the measured corticosterone concentrations may actually reflect those of progesterone and its precursors, most probably being 5 alpha- and 5 beta-pregnanes and pregnenolone.

Effect of hen's egg yolk on capacitation and acrosome reaction of diluted canine spermatozoa.

UNLABELLED: The aim of this study was to investigate the influence of progesterone, cholesterol and calcium (Ca(2+)) in an egg-yolk-containing extender on capacitation and acrosome reactions (AR) of diluted canine spermatozoa during 4 days of cooled-storage. For this purpose, we first investigated the effect of supplementation of a Tris-citrate-fructose buffer (TCF) with progesterone in a final concentration of 0.1, 0.2 and 1.0 microg progesterone/ml TCF-diluted semen. We then compared the effects of TCF and the same buffer-containing 20% egg yolk (TCF-EY). In egg yolks and the TCF-EY, progesterone was measured by enzyme immunoassay, cholesterol by enzymatic colorimetry and Ca(2+) by flame atomic absorption spectrophotometry. For both experiments, ejaculates from eight dogs were used. For the comparison of diluents, one ejaculate was divided and one half diluted with TCF, the other with TCF-EY. One half of each TCF- and TCF-EY-diluted sample was evaluated immediately (D1), the other after storage for 4 days at +4 degrees C (D4). In diluted semen, motility and viability were measured by a computer assisted sperm analyzer (CASA; Sperm Vision, Minitüb, Germany), capacitation and AR were evaluated with a modified chlortetracycline assay (CTC) and the AR additionally by flow cytometry. RESULTS: Supplementation of progesterone revealed, that between D1 and D4, total and progressive motility decreased with all progesterone concentrations, while viability as well as percentage of capacitated and acrosome reacted spermatozoa stayed constant. Progesterone-, cholesterol- and Ca(2+) concentrations in egg yolks were 524.8+/-131.4 ng/g, 13.9+/-2.03 mg/g and 1.27+/-0.17 mg/g, respectively. In the TCF-EY-diluent, the respective values were 210.9 ng/g, 2.52 mg/g and 1.1mg/g. In TCF-semen, at D1, motility and viability were significantly higher than in TCF-EY-samples (p<0.05), however at D4, no significant differences were detectable. Further, in TCF-semen, percentages of spermatozoa with intact membranes decreased significantly (p<0.05) and capacitated spermatozoa increased (p<0.05), which was not seen in TCF-EY-samples. In all samples, low percentages of AR were detected and after 4 days, the highest value of AR in TCF-EY-samples was 5.3% on average, as detected by flow cytometry. We therefore conclude that progesterone from egg yolk in routine extenders does not substantially influence semen longevity or AR of canine semen during cold-storage for 4 days. In contrary, egg yolk seems to prevent a significant increase in capacitated spermatozoa.

Social context rather than behavioral output or winning modulates post-conflict testosterone responses in Japanese quail (Coturnix japonica).

Testosterone regulates the expression of sexual and aggressive behavior in male vertebrates and treatments with testosterone may promote territorial aggression and winning in dyadic contests. Conversely, individual testosterone levels respond to sexual or aggressive interactions and the social environment. Post-conflict testosterone in winner males though appears to be more complex than simply reflecting conflict outcome. Expression and degree of post-conflict testosterone responses may adapt to additional modulators such as repeated winning experience, audience presence, opponent's fighting ability, and self-assessment. We present simulated intrusion experiments with male Japanese quail using mirror-elicited aggression and fights with real opponents ('direct challenge'). We recorded agonistic behavior and measured immunoreactive testosterone metabolites (TM) non-invasively from individual droppings. Frequencies of initiated agonistic behavior were similar whether towards the mirror or in direct challenge tests, although some of the males were behaviorally non-responsive to the mirror ('mirror submissives'). However, there was no TM response to the mirror test in both, mirror fighters and mirror submissives, thus independently of behavioral output. After direct challenges TM levels were elevated in all males (focal males winning or conflict unresolved after 30 min), hence independently of conflict outcome. Thus, in male quail a combination of physical stimuli and the individual perception of own and opponent's fighting ability explained the expression of post-conflict TM responses rather than behavioral performance, conflict outcome, or any of these factors alone. In sum, our results emphasize that the degree of androgen responsiveness to agonistic behavior is fine-tuned by components related with social context and environment.

Corticosterone in chicken eggs.

Birds are discussed as models for prenatal stress. In this study, several experiments were conducted to gain basic knowledge of if, how, and when maternal adrenocortical activity is reflected by corticosterone concentrations in the egg. Radiolabeled corticosterone was administered to 10 laying hens to investigate the uptake into as well as the distribution within the eggs. The yolk was dissected in concentric layers and analyzed. Less than 1% of the administered radioactivity entered the egg but was, however, not evenly distributed. On the day after injection, highest radioactivity (Bq/g) was detected in the albumen and the outmost layer, whereas concentration peaked 4-7 days later in the inner layers. In two other experiments, increased plasma levels of corticosterone were induced by injection of adrenocorticotropic hormone (ACTH) or feeding of corticosterone. Again, yolk disks were cut in layers and analyzed with a corticosterone enzyme immunoassay. No effect of the ACTH administration was detected, whereas feeding of corticosterone resulted in increased immunoreactive corticosterone concentrations in the yolk. Straight-phase high-performance liquid chromatographic (HPLC) separations were also performed to characterize immunoreactive steroids in the yolk. Two close-eluting peaks at the approximate elution position of corticosterone could be observed after the feeding experiment, whereas in untreated control eggs they were absent. It was concluded that transfer from plasma to egg is low for corticosterone and that further investigations concerning the transport mechanisms and the exact nature of yolk steroids are necessary.