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Progesterone concentrations in female and male gray whales Differences in concentrations were statistically different among reproductive groups for female and male gray whales (ANOVA: F = 31.09, df = 6.00, P< 0.001) of known reproductive status. Boxplots denote median (thick line), upper (75%) and lower (25) quartile (boxes) and largest and smallest value within 1.5 times interquartile range below 25% and above 75% (whiskers).
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Most of our knowledge on reproductive biology of gray whales dates back to scientific research conducted during commercial whaling in the late 1950s and 1960s. The goal of the present study was to provide updated insights on reproductive physiology of gray whales, using progesterone and testosterone as biomarkers. We measured hormone concentrations...
Citations
... A growing body of data indicates that annual cyclicity in male testosterone occurs in some baleen whale species (Vu et al., 2015;Hunt et al., 2018Hunt et al., , 2022Cates et al., 2019; . Melica et al., 2021), including some resident non-migratory populations (e.g. fin whales, Balaenoptera physalus, Carone et al., 2019). ...
... Recent advancements in non-lethal collection and analysis of non-plasma biological samples (e.g. blubber, Melica et al., 2021;and faecal samples, Lemos et al., 2020;Fernandez Ajó et al., 2023) have enabled a greater understanding of reproductive profiles, seasonality and variability of reproductive hormones in gray whales. However, much of this research has focused on progesterone (i.e. for pregnancy diagnosis) and cortisol (i.e. for examination of impacts of stressors (Lemos et al., 2022a(Lemos et al., , 2022bPirotta et al., 2023), while patterns of testosterone in males remain understudied. ...
... blubber) and histological examination from whaling data are consistent with our findings. For example, Melica et al. (2021) analyzed hormones from blubber biopsies and found that testosterone levels in blubber were elevated in adult male gray whales during the fall season compared to the summer months. Rice and Wolman (1971) examined testes from both immature and adult male gray whales collected during scientific whaling efforts, observing spermatogenesis in the seminiferous tubules of adult males. ...
Understanding wildlife reproductive seasonality is crucial for effective management and long-term monitoring of species. This study investigates the seasonal variability of testosterone in male Pacific Coast Feeding Group (PCFG) gray whales, using an eight-year dataset (2016–2023) of individual sightings, drone-based photogrammetry and endocrine analysis of faecal samples. We analyzed the relationship between faecal testosterone levels and total body length (TL), body condition (body area index, BAI), sexual maturity and day of the year using generalized additive mixed models. Our findings reveal a significant increase in faecal testosterone levels in mature males (MM) towards the end of the foraging season. This increase was not observed in JM, highlighting age-dependent development of sexual haracteristics. No significant relationship was found between testosterone levels and TL. Additionally, BAI was not significantly associated with testosterone levels. Our results suggest that the increasing testosterone levels in MM gray whales may indicate preparation for mating before the southbound migration. These findings provide valuable insights into the reproductive biology of PCFG gray whales and underscore the importance of non-invasive faecal sampling for studying reproductive seasonality in large whales. Our approach not only provides further insights into the seasonality of male reproduction for the PCFG gray whales but also offers tools to enhance the understanding of male reproduction in baleen whales broadly with non-invasive approaches.
... Evaluation of hormonal states can provide researchers with critical information about an individual's reproductive physiology and responses to stress. In mammals, the steroid hormones progesterone and testosterone often reflect reproductive states including pregnancy, sexual maturity and reproductive cycles (Lasley and Kirkpatrick, 1991;McCormick and Romero, 2017;Melica et al., 2021), while the glucocorticoids (cortisol, corticosterone) provide insight into exposure to stressors and the resulting impacts on health (Sheriff et al., 2011;Matas et al., 2016). Evaluating hormonal states of in situ and ex situ wildlife populations can be accomplished through a wide variety of sample matrices. ...
Hormone monitoring of at-risk species can be valuable for evaluation of individual physiological status. Traditional non-invasive endocrine monitoring from urine and faeces typically captures only a short window in time, poorly reflecting long-term hormone fluctuations. We examined toenail trimmings collected from African (Loxodonta africana) and Asian (Elephas maximus) elephants during routine foot care, to determine if long-term hormone patterns are preserved in these slow-growing keratinized tissues. We first measured the growth rate of elephant toenails biweekly for one year, to establish the temporal delay between deposition of hormones into nail tissue (at the proximal nail bed) and collection of toenail trimmings months later (at the distal tip of the nail). In African elephants, toenails grew ~0.18 ± 0.015 mm/day (mean ± SEM) and in Asian elephants, toenails grew ~0.24 ± 0.034 mm/day. This slow growth rate, combined with the large toenail size of elephants, may mean that toenails could contain a ‘hormone timeline’ of over a year between the nail bed and nail tip. Progesterone, testosterone and cortisol were readily detectable using commercial enzyme immunoassays, and all assays passed validations, indicating that these hormones can be accurately quantified in elephant toenail extract. In most cases, variations in hormone concentrations reflected expected physiological patterns for adult females and males (e.g. ovarian cycling and musth) and matched individual health records from participating zoos. Progesterone patterns aligned with our calculations of temporal delay, aligning with female ovarian cycling from over six months prior. Unexpectedly, male testosterone patterns aligned with current musth status at the time of sample collection (i.e. rather than prior musth status). Though this sample type will require further study, these results indicate that preserved hormone patterns in elephant toenails could give conservationists a new tool to aid management of elephant populations.
... Blubber testosterone shows annual cyclicity in several large baleen whales where it has been studied, including male blue whales, Balaenoptera musculus (Melica, Atkinson, Gendron, et al., 2021), fin whales, Balaenoptera physalus (Carone et al., 2019), grey whales, Eschrichtius robustus (Melica, Atkinson, Calambokidis, et al., 2021), and humpback whales, Megaptera novaeangliae (Vu et al., 2015;Cates et al., 2019). In all these cases, males experience higher levels of testosterone during the breeding compared to the feeding season, as is expected for seasonal breeders. ...
How the underlying forces of sexual selection impact reproductive tactics including elaborate acoustic displays in cetaceans remains poorly understood. Here, I combined 26 years (1995-2020) of photo-identification, behavioural, (epi)genetic, and endocrine data from an endangered population of humpback whales (New Caledonia), to explore male reproductive success, age, physiology, and population dynamics over almost a third of the lifespan of a humpback whale. First, I conducted a paternity analysis on 177 known mother-offspring pairs and confirmed previous findings of low variation in reproductive success in male humpback whales. Second, epigenetic age estimates of 485 males revealed a left-skewed population age structure in the first half of the study period that became more balanced in the second half. Further, older males (> 23 years) more often engaged in certain reproductive tactics (singing and escorting) and were more successful in siring offspring once the population age structure stabilised, suggesting reproductive tactics and reproductive success in male humpback whales may be age-dependent. Third, using enzyme immunoassays on 457 blubber samples, I observed a seasonal decline in male testosterone in the population over the breeding season. Testosterone levels appeared highest during puberty, then decreased and levelled off at the onset of maturity, yet were highly variable at any point during the breeding season and across males of all ages. Lastly, I investigated the influence of genetic diversity at the major histocompatibility complex (MHC) class I and class IIa (DQB and DRB-a) on patterns of male reproductive success in humpback whales. Mating pairs shared fewer alleles than expected under random mating at MHC class I and IIa, thus, providing evidence of an MHC-mediated female mate choice in humpback whales. This thesis provides novel, critical insights into the evolutionary consequences of commercial whaling on the demography, patterns of reproduction and sexual selection of exploited populations of baleen whales.
... In addition, analysis of reproductive hormones in baleen, while only attainable from dead whales, provides the opportunity to track the recent reproductive histories of individuals, increasing understanding of calving intervals, age of sexual maturity, and timing of the breeding season (Hunt et al. 2016(Hunt et al. , 2022Lowe et al. 2021). In gray whales, analysis of reproductive hormone concentrations in fecal samples and blubber biopsies have focused on evaluating how reproductive hormones vary with age, reproductive status, season, and life history stage (Lemos et al. 2020, Melica et al. 2021. Although analysis thus far has been limited to a single sample, Hunt et al. (2017) demonstrated that reproductive hormones can be detected in gray whale baleen. ...
Gray whale sexual behavior and copulation are observed throughout their range. The most prominent period for reproductive behavior is during the southward migration from summer feeding areas to wintering areas where some breeding occurs and calves of the year are reared. The seasonal migrations of gray whales are believed to function, in part, to bring together individuals that are otherwise widely distributed during the period of estrus to facilitate mating and reproduction. Sexual behaviors and sexual strategies for this species appear to align closely with those of balaenid (not rorqual) whales, although such comparisons need further investigation. Gray whales are polygynandrous (multi-mate) breeders. There does not appear to be female choice of mates, as groups of numerous females and males aggregate, and multiple copulations occur. Female estrus begins in mid-November and continues to early December; females may undergo a second estrus, extending into February, if they fail to conceive during their first cycle. Male gray whales have large testes and concomitantly produce large volumes of sperm, so they are believed to be sperm competitors; that is, they rely on multiple copulations (and sperm volume) to produce offspring. Multiple copulations with different males during the female estrus period may increase the likelihood that the timing of conception results in the birth of a calf approximately 13 months later near or in the wintering area(s). Mating bouts can last for minutes to hours, interspersed with surface-active-social-sexual behavior. Some all-male groups have been observed with erect penises engaged in social-sexual behavior in the absence of any females. Instances of male aggression toward postpartum females with calves of the year, sometimes resulting in injury or death, have been reported. As a result of dedicated long-term research in the past several decades, the state of knowledge on gray whale reproduction has greatly expanded and updated information on this topic is summarized in this chapter.
... Such epigenetic clocks need to be calibrated using individuals of known age, thus highlighting the crucial role of long-term data collection for the assessment and ground-truthing of such methods Hunt et al. 2017). Hormone concentrations can be measured using multiple matrices: blubber, respiratory vapor ("blow"), and fecal samples (Rolland et al. 2005;Hunt et al. 2013), and for the retrospective and longitudinal assessment of reproductive hormones: baleen plates (Hunt et al. 2014(Hunt et al. , 2016 Progesterone: inference of pregnancy status and rates (e.g., Kellar et al. 2013;Hunt et al. 2016;Pallin et al. 2018a, b;Kershaw et al. 2021) Testosterone: can be used to infer reproductive maturity and status of individuals or seasonal changes in reproductive state (e.g., Kellar et al. 2009;Vu et al. 2015;Cates et al. 2019;Mingramm et al. 2020;Melica et al. 2021) Estradiol: can provide information on female reproductive maturity and receptivity (e.g., Mingramm et al. 2020;Lowe et al. 2022) Bioacoustics ...
While a variety of reproductive tactics are readily witnessed in odontocetes, such behaviors can be far more elusive in baleen whales and in some cases are yet to be observed. This leads researchers to study the reproductive behaviors in mysticetes using a variety of research methods which have improved greatly in recent years. Genetics and genomics tools can provide valuable information on maternity, paternity, age, diversity, and kinship, while acoustic tools can provide new insights into the function of sexual displays such as song. In this chapter, we explore what is known about reproductive strategies and tactics of baleen whales, with a particular focus on the comparatively well-studied right whales ( Eubalaena spp.) and humpback whale ( Megaptera novaeangliae ). Finally, we showcase that by integrating multiple data types, we can explore the interactions between anatomy, physiology, reproductive success, age, population dynamics, and acoustic displays to better understand the mating systems of baleen whales.
... These models estimate the likelihood of an individual belonging to a specific cluster using a latent grouping variable, along with the means and standard deviations of normal probability density functions for each cluster. Mixture models have been successfully applied in similar studies of blue (Balaenoptera musculus) and gray (Eschrichtius robustus) whales (Melica et al., 2021a(Melica et al., , 2021b. ...
Beluga (Delphinapterus leucas) from the St. Lawrence Estuary, Canada, have been declining since the early 2000s, suggesting recruitment issues as a result of low fecundity, abnormal abortion rates or poor calf or juvenile survival. Pregnancy is difficult to observe in cetaceans, making the ground truthing of pregnancy estimates in wild individuals challenging. Blubber progesterone concentrations were contrasted among 62 SLE beluga with a known reproductive state (i.e. pregnant, resting, parturient and lactating females), that were found dead in 1997 to 2019. The suitability of a threshold obtained from decaying carcasses to assess reproductive state and pregnancy rate of freshly-dead or free-ranging and blindly-sampled beluga was examined using three statistical approaches and two data sets (135 freshly harvested carcasses in Nunavik, and 65 biopsy-sampled SLE beluga). Progesterone concentrations in decaying carcasses were considerably higher in known-pregnant (mean ± sd: 365 ± 244 ng g−1 of tissue) than resting (3.1 ± 4.5 ng g−1 of tissue) or lactating (38.4 ± 100 ng g−1 of tissue) females. An approach based on statistical mixtures of distributions and a logistic regression were compared to the commonly-used, fixed threshold approach (here, 100 ng g−1) for discriminating pregnant from non-pregnant females. The error rate for classifying individuals of known reproductive status was the lowest for the fixed threshold and logistic regression approaches, but the mixture approach required limited a priori knowledge for clustering individuals of unknown pregnancy status. Mismatches in assignations occurred at lipid content < 10% of sample weight. Our results emphasize the importance of reporting lipid contents and progesterone concentrations in both units (ng g−1 of tissue and ng g−1 of lipid) when sample mass is low. By highlighting ways to circumvent potential biases in field sampling associated with capturability of different segments of a population, this study also enhances the usefulness of the technique for estimating pregnancy rate of free-ranging population.
... Biopsy punches are an effective sample collection method of skeletal muscle, with proven utility in transcriptional profiling in fishes (Jeffries et al. 2021), isolation of amino acids and hormones in gray whales (Eschrichtius robustus) (Melicai et al. 2021), and in breast cancer screening in humans (Sharma and Ives 2022). They have also been used as an effective technique for obtaining samples for THg and selenium (Se) quantification (Baker et al. 2004;Peterson et al. 2005;Stahl et al. 2021). ...
Rapid and effective quantification of total mercury concentrations ([THg]) in fish muscle is an important part of ongoing monitoring to provide reliable and near real-time public health guidance. Methods for quantifying THg in fish muscle frequently require the use of large sample mass and numerous preparation steps. Wet (aka fresh weight) biopsy punch samples of fish muscle have been used to quantify THg directly, without drying and homogenization. Both methods have advantages and disadvantages. We compare the use of fresh weight biopsy punches for quantifying THg to using larger, dried homogenized samples. The [THg] determination for the two sampling methods was EPA method 7473. Three separate biopsy punch samples and a large muscle sample were taken from each fish and analyzed on a Direct Mercury Analyzer. There were no statistical differences between mean log transformed wet weight [THg] from biopsy punches and homogenized muscle across all samples or within individual species. Similarly, across the range of [THg] (7.5–612.7 ng/g ww), linear regression of [THg] from biopsy punch and homogenized muscle samples was not different from a 1:1 linear relationship. Linear regression statistics of [THg] with fish fork length produced similar results for both biopsy punch and homogenized muscle samples. However, the coefficient of variation among biopsy punch replicates for individual fish was frequently above the acceptable threshold of 15%. We recommend biopsy punches be used as an effective tool for broad-scale rapid monitoring of fish resources for Hg, while homogenized muscle samples be used for fine-scale ecological and health questions.
... Steroid hormones have been measured in a variety of tissues in multiple marine mammal species, including baleen whales [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. When available, blood serum, blubber, and feces are the matrices of choice for investigating endocrine response to relatively recent events (minutes to days) in living marine mammals [10][11][12][17][18][19][20][21][22][23][24][25]. ...
... Progesterone is a steroid hormone secreted by the corpora lutea in the ovaries during the estrous cycle, and is the predominant compound responsible for sustaining pregnancy in whales and many other mammals [7,31,32]. In general, progesterone concentrations are low in sexually immature whales and elevated in sexually mature females [1,2,4,7,25]. Progesterone concentrations in blubber have been applied as a tool to detect pregnancy in multiple species of baleen whales [1,2,[33][34][35][36][37] and to estimate pregnancy and reproductive rates [4,7,38]. ...
... In general, progesterone concentrations are low in sexually immature whales and elevated in sexually mature females [1,2,4,7,25]. Progesterone concentrations in blubber have been applied as a tool to detect pregnancy in multiple species of baleen whales [1,2,[33][34][35][36][37] and to estimate pregnancy and reproductive rates [4,7,38]. ...
Humpback whales (Megaptera novaeangliae) in Southeast Alaska have been studied for over 50 years, and are largely considered a recovery success since the cessation of commercial whaling. Reproductive physiology is an important factor to consider in studying population health and can provide important insights into the drivers contributing to population abundance fluctuations. Validated assays for progesterone and testosterone were used on blubber biopsies from humpback whales (N = 33 whales, 71 samples) near Juneau, Alaska, in 2020 and 2021. Long-term sighting histories were used to confirm detected pregnancies with calf sightings the following year. Blubber samples were divided into two seasonal bins (early and late summer). Pregnant females sampled in both early and late summer of both 2020 and 2021 showed elevated progesterone concentrations compared to other reproductive states (p
... Reproductive knowledge of most populations comes from gross anatomical and physiological investigations of specimens collected from commercial or indigenous whaling [2][3][4]. Recent studies have used endocrine markers of various tissues to study health, stress and reproductive rates of baleen whales ( [1,[5][6][7][8][9][10], reviewed in [11]). Endocrine systems regulate body processes via negative feedback loops involving steroid hormones, which ultimately control reproductive processes such as maturation, oestrous and ovulation, pregnancy and seasonal mating behaviour [12]. ...
Serial measurements of hormone concentrations along baleen plates allow for reconstructions of mysticete whale reproductive histories. We assessed gestation and calving interval in bowhead whales (Balaena mysticetus) by measuring progesterone, oestradiol, corticosterone and nitrogen stable isotope ratios (δ¹⁵N) along baleen of 10 females from the eastern Canada-west Greenland population. Three immature females (body size < 14.32 m) had uniformly low progesterone concentrations across their baleen, while seven mature females (body size ≥ 14.35 m) had repeated, sustained elevations of progesterone indicative of pregnancies. The mean duration of progesterone elevations (23.6 ± 1.50 months) was considerably longer than the approximately 14 month gestation previously estimated for this species. We consider several possible explanations for this observation, including delayed implantation or sequential ovulations prior to gestation, strategies that would allow females to maximize their fitness in variable Arctic conditions, as well as suggest modified criteria defining gestation as a shorter component of the entire progesterone peak. Calving intervals varied within and among individuals (mean = 3.7 years; range = range 2.8–5.7 years), providing population-specific reproductive estimates for growth models used in bowhead whale management and conservation.
... Therefore, the posterior probability of pregnancy for each combination of fP4m and W50 values in the data was then calculated as the ratio of the probability density for the component with higher means for the two variables to the sum of the two probability densities. We use a non-parametric bootstrap approach, similar to the one applied in Melica et al. [8], to quantify the uncertainty around each probability estimate. Specifically, we resampled the variables with replacement 10 000 times, fitted the mixture model to the bootstrapped dataset, and estimated the probabilities of pregnancy for all combinations of values in the data. ...
... Specifically, we resampled the variables with replacement 10 000 times, fitted the mixture model to the bootstrapped dataset, and estimated the probabilities of pregnancy for all combinations of values in the data. Owing to the small number of data points from pregnant females, those records were included in all bootstrap samples [8]. We calculate the 95% confidence intervals using the 2.5th and 97.5th percentiles of the estimated probabilities of pregnancy. ...
... 10: 230452 6 For each model, we only retained bootstrap samples if the mixture model identified both variables as having a greater mean for the component assumed to correspond to pregnant females (e.g. high fP4m or high W50); and we discarded non-convergent models [8]. Under these conditions, only 65% of the bootstrap samples were retained for the Model 1 and greater than 95% of the bootstrap samples were retained for the two univariate models (Models 2 and 3). ...
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 grey whales to evaluate the utility of faecal progesterone immunoassays and drone-based photogrammetry for regnancy 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 nonpregnant. 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 grey 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.
