Data from analytical validation tests of parallelism, accuracy, extraction efficiency and within-extraction method variation were compared between extraction protocols. A paired t-test was used to assess differences in measured hormone concentrations of blubber subsamples extracted following each protocol. Hormone values in matched blubber and faecal samples for three individuals, were graphically presented to observe congruence of trends between these alternative matrices. Descriptive statistics (mean ± SEM) were used to summarize the data set. Hormone concentration data were log10-transformed for the following analyses to meet assumptions of normality and homogeneity of variance, which were tested using Shapiro–Wilk test and Levene’s test. Blubber hormone concentrations of live, apparently healthy whales (n = 20 out of 21 biopsy samples; one biopsy (from Eg4510) was excluded due to active entanglement) were examined using a univariate general linear model (GLM). A full factorial model was used to analyse the effect of sex, age class (i.e. juvenile or adult) and their interaction on hormone concentrations (dependent variable) of whale blubber samples, with the following equation: yi = β0 + β1 sexi + β2 age class i + β3 sex × age classi + ϵi where y is the response variable, β is the population slope and fixed effect parameters (including β0 as the population intercept) and ϵ is a random error term associated with the ith observation. To avoid omission of any individual whale due to missing data fields in the GLM, we deliberately classed one female of uncertain age as ‘adult’. This decision was grounded on available data that showed this female was older than 7 years of age (based on sighting records) and successfully calved 17 months after sampling—and therefore, this female was presumed to be nearing reproductive maturity when blubber sampling occurred.

To consider the possible effect of abiotic factors on measured hormone variables in the full set of blubber samples (n = 28; both live and dead whales), we used a multivariate GLM. Key attributes of sample storage time (i.e. number of years from sample collection until hormone analysis), mass of the analysed sample (measured in grams) and whale survival at time of sampling (i.e. live or dead whale, as associated with biopsy or necropsy sampling) were included as explanatory variables into the model designed to analyse all dependent variables (testosterone, progesterone and cortisol concentrations) simultaneously, with the following equation: zik = constant + c1 storage timei + c2 sample massi + c3 whale surivivali where z is the combination of response variables (observation i for the linear combination k) and c is the coefficient measuring the relative contribution of each variable. Univariate between-subjects F-tests that indicated the effect of each factor on each dependent variable were also produced by the GLM framework. All statistical analyses were performed using SPSS (version 25) and significance level was set at 0.05 for all statistical tests.

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