Leticia Martínez-Fernández

Physiological Stress Responses, Fecal Marking Behavior, and Reproduction in Wild European Pine Martens (Martes martes)

The relationship among physiological stress responses, fecal marking behavior, and reproduction in male and female European pine martens was investigated. Between July 2004 and June 2007, 145 fresh fecal samples were collected in a protected area of northwest Spain. Fecal DNA was used for specific identification by using the polymerase chain reaction-restriction fragment length polymorphism technique. Glucocorticoids (cortisol) and sex steroid hormones (P, progesterone; E, estradiol; T, testosterone) were quantified by enzyme immunoassays. Sex was assigned according to concentrations of T+P+E and the T/P ratio. Fecal cortisol concentrations were higher in males than in females. Feces with a presumptive marking function (on conspicuous substrates, above ground level, and/or in latrines) had higher mean levels of cortisol than those that were on inconspicuous substrates and/or at ground level, for both males and females. Fecal mark density was highest in spring, when mean levels of fecal cortisol were more elevated. Therefore, the higher physiological stress levels in females could be due to female physiological state (late-term pregnancy and lactation), competition for resources connected to birth, or food resources for offspring rearing. In males, the increase could be due to higher male competition for access to females during pro-estrus and estrus. Our results suggest that scent marking in European pine martens is related to reproduction and is involved in intersexual and intrasexual communication.

Isoflavones and their effects on the onset of puberty in male Wistar rats.

This study was performed to determine how two of the most important isoflavones, genistein and daidzein, affect the gonadal axis in male prepuberal rats. One hundred and seventy‐five prepuberal male Wistar rats were allocated into seven groups: one control group and six experimental groups that were orally administered a high or low dose of genistein, daidzein or a mixture of both. Testosterone determination was assayed by EIA. The testes and body weights were measured, and the histology of the epididymis with the sperm content and epididymal sperm count were evaluated. In the control group, we observed an increase in the serum testosterone levels (>2.5 ng ml−1) at the third week (52 days), which corresponded to the onset of puberty in these rats. The same increase in serum testosterone levels was observed at the fourth week in rats that received low doses of isoflavones; therefore, we concluded that the onset of puberty was delayed. At high doses, there was no significant increase in testosterone levels, which could be related to the fact that these male rats did not reach puberty. These findings were supported by the results obtained from the analysis of the epididymal content as well as the testes/body weight ratio.

Assessment of ovarian cycles in the African elephant (Loxodonta africana) by measurement of salivary progesterone metabolites

Monitoring ovarian cycles through hormonal analysis is important in order to improve breeding management of captive elephants, and non-invasive collection techniques are particularly interesting for this purpose. However, there are some practical difficulties in collecting proper samples, and easier and more practical methods may be an advantage for some institutions and/or some animals. This study describes the development and validation of an enzymeimmunoassay (EIA) for progestins in salivary samples of African elephants, Loxodonta africana. Weekly urinary and salivary samples from five non-pregnant elephant cows aged 7-12 years were obtained for 28 weeks and analyzed using EIA. Both techniques correlated positively (r = 0.799; P < 0.001), and the cycle characteristics obtained were identical. The results clearly show that ovarian cycles can be monitored by measuring progestins from salivary samples in the African elephant. This is a simple and non-invasive method that may be a practical alternative to other sampling methods used in the species.

The Effects of Isoflavones on Androgens and Glucocorticoids During Puberty on Male Wistar Rats

Isoflavones are the most common phytoestrogens found in human diets. However, it is still not clear whether isoflavones have effects on the reproductive and the endocrine systems under normal dietary intake and overdose. The aim of this study was to determine how the most important isoflavones, genistein and daidzein, affect androgen and glucorticoid levels on male prepuberal rats. A hundred and seventy-five 30-day-old male Wistar rats were dosed orally by stomach tube every day for 35 days, with saline solution, low and high doses of genistein, daidzein and a mixture of both. Serum samples were analysed by an enzyme immunoassay for hormone determinations. In control group, there was a peak of testosterone (T) and dihydrotestosterone levels associated to the onset of puberty, at the third week. However, in low-dose groups, the same peak was found at the fourth week (p < 0.05), indicating a delay in the onset of puberty in these groups. Moreover, high doses groups serum androgen levels were significantly lower (p < 0.05) than the control group from the first week until fifth week. This fact was supported by a epididymal histological analysis that indicate in low doses there were several content of spermatozoa at fourth week and in high doses there were few content of spermatozoa. Besides, corticosterone levels followed the same pattern of androgens in all groups. We can conclude that oral administration of isoflavones in male rats decreased the secretion of androgens and glucocorticoids causing a delay in the onset of puberty and may cause physiological and developmental problems.

Faecal sexual steroids in sex typing and endocrine status of great bustards

Faecal sexual steroids have been used in field studies evaluating the relationships between gender and the multiple factors influencing endocrine status of individuals. The determination of faecal steroids has been also proposed as an alternative, non-invasive sexing method when other methods were deemed impractical or risky for the health of birds. In this study, we quantified sexual steroid hormones in faeces of the great bustard (Otis tarda), a large and sexually dimorphic polyginic bird species that it is threatened and subjected to intense wildlife management. We evaluated differences between captivity and wild conditions, flocks and sexes, and used faecal steroids to develop sex determination procedures. We found similar steroid levels in captive and wild bustards, no differences between unisexual wild flocks and clear between-sexes differences in testosterone but not estradiol. Faecal steroids accurately discriminated gender in both captive and wild known-sex great bustards. Total testosterone concentration was always higher than estradiol concentration in faecal samples from males, but estradiol was not always higher than testosterone in females. Faecal steroids failed to reveal the presence of young males in female flocks during winter, despite faecal testosterone levels increased with age in a small sample of captive males. Our results show that faecal steroid measurement for both sexing and characterizing the endocrine status of great bustards is feasible, and therefore it should be valuable in wildlife management, especially in combination with additional information obtained from faeces as diet.

Circadian rhythm of salivary cortisol secretion in female zoo‐kept African elephants (Loxodonta africana)

Salivary samples were collected over a 24-hr period from one group of six juvenile (7-12 years) and one group of three adult (24-25 years) African elephant females, Loxodonta africana, and the cortisol concentration was measured in unextracted samples by EIA. Samples were collected during May, June, and November 2012 (n = 147) using cotton swabs at 4-hr intervals from 20:00 to 20:00 of the next day (seven samples per animal in each trial). The animals are kept under standard zoo management: the herd is maintained in their indoor enclosures until 10:00 and then released into the outdoor enclosures until 21:00-21:30 (May/June) and 18:30-19:00 (November). No adult elephant bull was present at the zoo during this time. The results demonstrate a clear diurnal pattern of cortisol secretion with the lowest concentration observed at 20:00 (2.03 ± 0.08 ng/ml saliva) and the peak concentrations at 08:00 (5.26 ± 0.35 ng/ml saliva). Although the cortisol values were higher in the adult cows compared to the juvenile cows in the May-June period, the differences were not significant. However, the values obtained in November from the juvenile group were significantly higher (P < 0.05) than the concentrations measured in this group in June. In conclusion, salivary cortisol in zoo elephants follows a circadian rhythm (sleep-wake cycle) adapted to daily zoo husbandry routines.

Effects of different fentanyl anaesthetic mixtures on cortico-adrenal function in rabbits

The involvement of the adrenal gland in short- and long-term adaptation of organisms to stress-inducing agents is known. A number of studies have shown that anaesthetics might regulate these systems and, consequently, influence serum glucocorticoid concentrations in rabbits (Gonzalez Gil and others 2001). In order to assess the cortico-adrenal response to different anaesthetics mixtures in rabbits, 10 New Zealand White rabbits maintained under conventional conditions (12/12 light/dark, 20–22°C, 50–55 per cent relative humidity, 10–15 air changes/hour), were assigned to four treatment groups (n = 10/group). The experimental protocols adhered to the Council of EU (European Union) rules, and were approved by the Institutional Animal Care and Use Committee of the Veterinary Faculty of Madrid at Universidad Complutense of Madrid, Spain. Treatment order was randomised, and each animal received all treatments with a minimum interexperiment interval of 10 days. The groups were: control group (C), 1 ml normal saline solution; group K/F, ketamine (25 mg kg−1, Imalgene 1000, Merial, Barcelona, Spain) and fentanyl (0.02 mg kg−1, Fentanest, Kern Pharma, Barcelona, Spain); group F/M, fentanyl (0.02 mg kg−1) and medetomidine (0.2 mg kg−1, Domtor, Pfizer, Madrid, Spain); and group F/M/D, fentanyl (0.02 mg kg−1), medetomidine (0.2 mg kg−1) and diazepam (1 mg kg−1, Valium, Roche Farma, Madrid, Spain). The dosages were based upon preliminary studies and from a review of previous anaesthetics studies using rabbits (Baumgartner and others 2010). In order to minimise …