Optimizing hormone extraction protocols for whale baleen: Tackling questions of solvent:sample ratio and variation

Ajó, Alejandro Fernández; Hunt, Kathleen E.; Dillon, Danielle; Uhart, Marcela; Sironi, Mariano; Rowntree, Victoria J.; Buck, C. Loren

doi:10.1016/j.ygcen.2021.113828

Cited by 10 publications

(7 citation statements)

References 47 publications

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“…Diluted sample progesterone metabolite concentrations for both kits were parallel to the standard curve (Ajó et al 2022). The intra-assay %CV was 3.1% ( n =19) for the Progesterone EIA kit and 5.1% ( n =22) for the Progesterone Metabolite kit.…”

Section: Resultsmentioning

confidence: 99%

“…We conducted a test of parallelism for both kits by serially diluting fecal extract in assay buffer to produce six dilutions (1:50-1:150 range for Progesterone EIA kit, 1:20-1:640 range for Progesterone Metabolite kit). We compared the linear portions of the different diluted extracts of unknown samples with the progesterone standard binding curve for equality of slope (Aj ó et al 2022).…”

Section: Fecal Sample Enzyme Immunoassay (Eia)mentioning

confidence: 99%

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Evaluating the Efficacy of Noninvasive Fecal Sampling for Pregnancy Detection in Elk (Cervus Canadensis)

Watson

Kurth

Metts

et al. 2023

Journal of Wildlife Diseases

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Elk (Cervus canadensis) were reintroduced to Tennessee, USA in the early 2000s, with limited reproductive monitoring since initial release. We assessed the efficacy of noninvasive sampling for determining pregnancy using invasive (capture) and noninvasive (fecal collection in the field) techniques at the North Cumberland Wildlife Management Area (NCWMA), Tennessee. We captured 20 female elk 2019–2020, used pregnancy-specific protein B (PSPB) in blood to determine pregnancy and compared results to fecal progesterone metabolite (FPM) concentrations using two commercially available enzyme immunoassay (EIA) kits. Based on PSPB concentrations, 8/11 and 3/4 of captured adult elk (≥2.5 yr of age) were pregnant in 2019 and 2020, respectively; no 1.5-yr-old elk were pregnant (n=5). Using the progesterone EIA kit, FPM concentrations were x̄=192.84±38.63 ng/g (95% CI, 96.48–289.20) for nonpregnant and x̄=536.17±74.98 ng/g (95% CI, 375.97–696.36) for pregnant captured females. For the progesterone metabolite kit, FPM concentrations were x̄=188.16±43.39 ng/g (95% confidence interval [CI], 76.63–299.69) for nonpregnant and x̄=693.52±126.52 ng/g (95% CI, 407.31–979.72) for pregnant captured females. From February to May 2019, we collected 357 fecal samples in 65 areas across 489.62 km2 of the NCWMA. Using extracted DNA and analysis of 15 microsatellites, we identified 62 unique individuals from 128 female fecal samples collected on the landscape. We categorized females from landscape-collected feces as nonpregnant (35.5–40.3%; Metabolite-EIA kits), undetermined (1.6–6.5%; Metabolite-EIA kits), or pregnant (62.9–53.2%; Metabolite-EIA kits) based on a 95% CI of captured female FPM concentrations, giving an overall pregnancy rate of 53.2% using the recommended EIA kit. The pregnancy rate in sexually mature females may be higher, as it was not possible to distinguish age classes of landscape-collected fecal samples; therefore, some may have been from younger age classes not expected to be pregnant. Analysis of FPM may be useful at a population level to detect pregnancy.

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Section: Resultsmentioning

confidence: 99%

Section: Fecal Sample Enzyme Immunoassay (Eia)mentioning

confidence: 99%

Evaluating the Efficacy of Noninvasive Fecal Sampling for Pregnancy Detection in Elk (Cervus Canadensis)

Watson

Kurth

Metts

et al. 2023

Journal of Wildlife Diseases

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“…Briefly, we filtered, desalted (to remove spurious inflation of dried faecal mass by salt crystals) and freeze-dried the faecal samples. We weighed the dried and homogenized samples to the nearest 0.1 mg, and excluded samples below 0.02 g from the analysis to avoid inflated values (‘small sample effect’; see [ 4 , 62 ]). Faecal samples contain the metabolized breakdown products of progesterone; we used a progesterone assay kit whose antibody was originally raised against progesterone, but that also cross-reacts to 5α-reduced progestin metabolites (#ADI-900-011), and thus our metric quantifies a subset of faecal progestin metabolites (fP4m).…”

Section: Methodsmentioning

confidence: 99%

Assessment of a non-invasive approach to pregnancy diagnosis in gray whales through drone-based photogrammetry and faecal hormone analysis

et al. 2023

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Knowledge of baleen whales' reproductive physiology is limited and requires long-term individual-based studies and innovative tools. We used 6 years of individual-level data on the Pacific Coast Feeding Group gray whales to evaluate the utility of faecal progesterone immunoassays and drone-based photogrammetry for pregnancy diagnosis. We explored the variability in faecal progesterone metabolites and body morphology relative to observed reproductive status and estimated the pregnancy probability for mature females of unknown reproductive status using normal mixture models. Individual females had higher faecal progesterone concentrations when pregnant than when presumed non-pregnant. Yet, at the population level, high overlap and variability in progesterone metabolite concentrations occurred between pregnant and non-pregnant groups, limiting this metric for accurate pregnancy diagnosis in gray whales. Alternatively, body width at 50% of the total body length (W50) correctly discriminated pregnant from non-pregnant females at individual and population levels, with high accuracy. Application of the model using W50 metric to mature females of unknown pregnancy status identified eight additional pregnancies with high confidence. Our findings highlight the utility of drone-based photogrammetry to non-invasively diagnose pregnancy in this group of gray whales, and the potential for improved data on reproductive rates for population management of baleen whales generally.

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“…Hormones were extracted from powdered baleen samples with modifications of prior studies ( Hunt et al. 2014 , 2016 , 2017a , 2017b , 2018 ; Fernández et al. 2021 ), as follows: 6.00 mL of 100% HPLC-grade methanol was added to 100 mg of baleen powder in 16×100 mm borosilicate glass tubes, vortexed 2 h, and centrifuged for 15 min at 3000 g. Methanol supernatant (containing hormones) was transferred to a 13×100 mm borosilicate glass tube for dry-down, with results corrected for percentage of supernatant recovered.…”

Section: Methodsmentioning

confidence: 99%

Male Bowhead Whale Reproductive Histories Inferred from Baleen Testosterone and Stable Isotopes

Hunt

Buck

Ferguson

et al. 2022

Integrative Organismal Biology

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Synopsis Male mammals of seasonally reproducing species typically have annual testosterone (T) cycles, with T usually peaking during the breeding season, but occurrence of such cycles in male mysticete whales has been difficult to confirm. Baleen, a keratinized filter-feeding apparatus of mysticetes, incorporates hormones as it grows, such that a single baleen plate can record years of endocrine history with sufficient temporal resolution to discern seasonal patterns. We analyzed patterns of T every 2 cm across the full length of baleen plates from nine male bowhead whales (Balaena mysticetus) to investigate occurrence and regularity of T cycles and potential inferences about timing of breeding season, sexual maturation, and reproductive senescence. Baleen specimens ranged from 181–330 cm in length, representing an estimated 11 years (smallest whale) to 22 years (largest whale) of continuous baleen growth, as indicated by annual cycles in stable isotopes. All baleen specimens contained regularly spaced areas of high T content (T peaks) confirmed by time series analysis to be cyclic, with periods matching annual stable isotope cycles of the same individuals. In 8 of the 9 whales, T peaks preceded putative summer isotope peaks by a mean of 2.8 months, suggesting a mating season in late winter / early spring. The only exception to this pattern was the smallest and youngest male, which had T peaks synchronous with isotope peaks. This smallest, youngest whale also did not have T peaks in the first half of the plate, suggesting initiation of T cycling during the period of baleen growth. Linear mixed effect models suggest that whale age influences T concentrations, with the two largest and oldest males exhibiting a dramatic decline in T peak concentration across the period of baleen growth. Overall, these patterns are consistent with onset of sexual maturity in younger males and possible reproductive senescence in older males. We conclude that adult male bowheads undergo annual T cycles, and that analyses of T in baleen may enable investigation of reproductive seasonality, timing of the breeding season, and life history of male whales.

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Optimizing hormone extraction protocols for whale baleen: Tackling questions of solvent:sample ratio and variation

Cited by 10 publications

References 47 publications

Evaluating the Efficacy of Noninvasive Fecal Sampling for Pregnancy Detection in Elk (Cervus Canadensis)

Evaluating the Efficacy of Noninvasive Fecal Sampling for Pregnancy Detection in Elk (Cervus Canadensis)

Assessment of a non-invasive approach to pregnancy diagnosis in gray whales through drone-based photogrammetry and faecal hormone analysis

Male Bowhead Whale Reproductive Histories Inferred from Baleen Testosterone and Stable Isotopes

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