Physiological and analytical validations of fecal steroid hormone measures in black howler monkeys

Abstract

The measurement of hormones in fecal samples allows for the noninvasive assessment of the endocrine status of free-ranging primates. However, procedures and techniques for hormone analysis in feces must be validated, both analytically and physiologically. Few studies have addressed the endocrinology of black howler monkeys (Alouatta pigra). Due to its conservation status, direct handling of individuals from this species and invasive sample collection are highly regulated, and therefore traditional methods for the validation of hormone assays, such as pharmacological challenges, are not allowed. As a consequence, sometimes studies of the fecal hormones of free-ranging black howler monkeys do not report physiological validations and therefore the biological reliability of such measurements cannot be assessed. In order to stimulate future research with this species, the present study aimed at providing methodological bases for fecal endocrine monitoring. Specifically, we compared the validity of two immunoassays (radioimmunoassays, RIA; solid-phase chemiluminescent enzyme immunoassay, SPCEI) performed with commercial kits to measure cortisol, testosterone, estradiol, and progesterone; and demonstrate how the physiological functions of these steroid hormones can be determined through non-pharmacological validations. We found no differences between the analytical validity of RIA and SPCEI assays to measure cortisol and testosterone, whereas for estradiol and progesterone RIA showed better results. Concerning the physiological validation of our assays, we demonstrated that: (1) comparisons between pre- and post-stress situations may be used to assess cortisol response, (2) comparisons between females and males may be used to assess variation in testosterone levels, and (3) comparisons between pregnant and non-pregnant females may be used to determine variation in estradiol and progesterone activity. The analytical and physiological validations that we performed demonstrate that there are currently commercial kits that allow for correct endocrine monitoring of this species, and that there are non-pharmacological alternatives to assess the biological validity of hormone measurements.

Maternal glucocorticoid levels affect sex allocation in black howler monkeys

“…Inter‐assay variation, estimated for the five assays from fecal pools with varying levels of cortisol, averaged 19.3% (low), 15.1% (medium) and 7.2% (high). The protocols that we used to extract and quantify glucocorticoid levels in feces had been previously validated to detect the activation of the HPA axis in response to stressful stimuli in mantled howler monkeys (Rangel‐Negrín et al ., ). All samples were run in duplicate, and glucocorticoid values are reported as ng g −1 (dry feces).…”

MEASUREMENT OF SERUM TESTOSTERONE IN KACANG GOAT BYUSING ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TECHNIQUE: THE IMPORTANCE OF KIT VALIDATION (Pengukuran Testosteron Serum Kambing Kacang dengan Teknik Enzyme-Linked Immunosorbent Assay (ELISA): Pentingnya Validasi Kit)

“…To examine the capability and precision of a commercial testosterone ELISA kits (DRG EIA-1559) for the quantifying concentration of testosterone in kacang goat, an analytical validation was performed. The procedure of this validation was adopted from previous studies (Pettitt et al, 2007;Rangel-Negrin et al, 2014). The analytical validation comprises of parallelism test, accuracy, and precision.…”

The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle

“…In order to reliably measure C and T concentrations in cattle using a human commercial ELISA kit, the ELISA kit needs to be validated analytically, physiologically or biologically 4,14 . Analytical validation can be performed by examining the specificity (cross-reactions), sensitivity, precision, and parallelism (linearity) of the ELISA kit 1,4 .…”

“…Analytical validation can be performed by examining the specificity (cross-reactions), sensitivity, precision, and parallelism (linearity) of the ELISA kit 1,4 . In addition, it is crucial to perform the biological validation of the assay to examine the ability of the assay to differentiate the variation of the hormone concentration based on the physiological conditions of the animals 14 . The biological validation of T measurements can be achieved by comparing the concentrations of T from individuals of different age (juvenile versus adult), or sex (male versus female) classes 4 .…”