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Cytosine methylation patterns have not yet been thoroughly studied in horses. Here, we profile n  = 333 samples from 42 horse tissue types at loci that are highly conserved between mammalian species using a custom array (HorvathMammalMethylChip40). Using the blood and liver tissues from horses, we develop five epigenetic aging clocks: a multi-tissue clock, a blood clock, a liver clock and two dual-species clocks that apply to both horses and humans. In addition, using blood methylation data from three additional equid species (plains zebra, Grevy’s zebras and Somali asses), we develop another clock that applies across all equid species. Castration does not significantly impact the epigenetic aging rate of blood or liver samples from horses. Methylation and RNA data from the same tissues define the relationship between methylation and RNA expression across horse tissues. We expect that the multi-tissue atlas will become a valuable resource. Methylation levels of specific sites in the genome is correlated with aging. Here the authors develop a human-horse clock which could assist in translating anti-aging interventions from humans to horses and vice versa.

Age‐associated DNA‐methylation profiles have been used successfully to develop highly accurate biomarkers of age (\"epigenetic clocks\") in humans, mice, dogs, and other species. Here we present epigenetic clocks for African and Asian elephants. These clocks were developed using novel DNA methylation profiles of 140 elephant blood samples of known age, at loci that are highly conserved between mammalian species, using a custom Infinium array (HorvathMammalMethylChip40). We present epigenetic clocks for Asian elephants (Elephas maximus), African elephants (Loxodonta africana), and both elephant species combined. Two additional human‐elephant clocks were constructed by combining human and elephant samples. Epigenome‐wide association studies identified elephant age‐related CpGs and their proximal genes. The products of these genes play important roles in cellular differentiation, organismal development, metabolism, and circadian rhythms. Intracellular events observed to change with age included the methylation of bivalent chromatin domains, and targets of polycomb repressive complexes. These readily available epigenetic clocks can be used for elephant conservation efforts where accurate estimates of age are needed to predict demographic trends. DNA methylation aging clocks for African and Asian elephants. Some of these clocks apply to humans as well. Parts of the graphic were created by Hollis Burbank‐Hammerlund, Sandra Oviedo, and BioRender.com.

Perceived loudness is challenging to study in non-human animals. However, reaction time to an acoustic stimulus is a useful behavioral proxy for the assessment of perceived loudness. Understanding the effect of sound frequency and level on perceived loudness would improve prediction and modeling of anthropogenic noise impacts on marine mammals. In this study, behavioral hearing tests conducted with two killer whales were analyzed to capture conditioned vocal response latency, which is the time between the onset of the acoustic signal and the onset of the response (i.e., reaction time). The results showed that vocal reaction times decreased with increasing sensation level (i.e., sound pressure level above the baseline hearing threshold), while the effect of frequency on reaction time varied between the subjects. Reaction time as a function of sound duration is described, and equal-latency contours are presented. The data suggest that vocal reaction time decreases with increasing sensation level, therefore supporting the use of reaction time as a proxy for loudness perception in killer whales.

The study examined how season, age, sex, and pregnancy outcomes influenced serum total thyroxine (TT4) and triiodothyronine (TT3) levels in killer whales (Orcinus orca). Total T4 and TT3 concentrations were quantified in 1513 serum samples collected voluntarily over ~40 years from 14 males and 24 females (ages 1–54) under managed care. Data were analyzed using LMM to determine the effects of age, sex, season, and pregnancy status (normal vs. abnormal outcomes). Age, season, and pregnancy significantly influenced thyroid hormone concentrations, while sex did not. Juveniles exhibited higher concentrations consistent with increased thermoregulatory needs and growth demands. Seasonal analysis showed TT4 peaked in summer and declined in winter suggesting thermoregulatory adaptation. Pregnancies with abnormal outcomes (abortion, dystocia, stillbirth) were associated with atypical thyroid hormone profiles; specifically, dystocia was linked to consistently low TT3/TT4, while stillbirths correlated with elevated late-term TT3. Females experiencing abortion showed decreased TT3 and TT4 during the late gestation. These findings suggest that in pregnancies with adverse outcomes, metabolic imbalances or transient hyperthyroid-like states may negatively impact fetal health. Consequently, in killer whales, variation in thyroid hormone levels may reflect a complex interplay between environmental adaptation, reproductive status, and underlying evolutionary physiology.

Age determination of wild animals, including pinnipeds, is critical for accurate population assessment and management. For most pinnipeds, current age estimation methodologies utilize tooth or bone sectioning which makes antemortem estimations problematic. We leveraged recent advances in the development of epigenetic age estimators (epigenetic clocks) to develop highly accurate pinniped epigenetic clocks. For clock development, we applied the mammalian methylation array to profile 37,492 cytosine-guanine sites (CpGs) across highly conserved stretches of DNA in blood and skin samples ( n  = 171) from primarily three pinniped species representing the three phylogenetic families: Otariidae, Phocidae and Odobenidae. We built an elastic net model with Leave-One-Out-Cross Validation (LOOCV) and one with a Leave-One-Species-Out-Cross-Validation (LOSOCV). After identifying the top 30 CpGs, the LOOCV produced a highly correlated (r = 0.95) and accurate (median absolute error = 1.7 years) age estimation clock. The LOSOCV elastic net results indicated that blood and skin clock ( r  = 0.84) and blood ( r  = 0.88) pinniped clocks could predict age of animals from pinniped species not used for clock development to within 3.6 and 4.4 years, respectively. These epigenetic clocks provide an improved and relatively non-invasive tool to determine age in skin or blood samples from all pinniped species. DNA methylation aging clocks are generated for several species of pinnipeds enabling the estimation of pinniped age from blood or skin samples.

The development of a precise blood or skin tissue DNA Epigenetic Aging Clock for Odontocete (OEAC) would solve current age estimation inaccuracies for wild odontocetes. Therefore, we determined genome-wide DNA methylation profiles using a custom array (HorvathMammalMethyl40) across skin and blood samples (n = 446) from known age animals representing nine odontocete species within 4 phylogenetic families to identify age associated CG dinucleotides (CpGs). The top CpGs were used to create a cross-validated OEAC clock which was highly correlated for individuals (r = 0.94) and for unique species (median r = 0.93). Finally, we applied the OEAC for estimating the age and sex of 22 wild Norwegian killer whales. DNA methylation patterns of age associated CpGs are highly conserved across odontocetes. These similarities allowed us to develop an odontocete epigenetic aging clock (OEAC) which can be used for species conservation efforts by provide a mechanism for estimating the age of free ranging odontocetes from either blood or skin samples.Todd Robeck and Zhe Fei et al. use DNA methylation profiling to develop an epigenetic clock to measure age in odontocete species, including toothed whales and dolphins. The clocks were highly accurate and represent a valuable tool to help in wildlife conservation efforts.

A comprehensive evaluation of the effects of sex, age, and season on blood analytes in a robust population size of ex situ bottlenose dolphins (Tursiops spp.) has not been investigated to date. To define the variation in hematological and biochemical analytes of dolphins due to sex, age, and season. 1,426 blood samples collected from 156 clinically normal dolphins consisting of 59 males and 97 females in which 37 analytes were measured were retrospectively identified. The dolphins were categorized by age, sex, and season, and categories were compared. About 23 (64%) analytes differed by age. The number of differences between adjacent age groups decreased with advancing age. MPV, glucose, BUN, globulins, GGT and Cl progressively increased with age, whereas Abs lymphs, total protein, ALP, CK and Ca progressively decreased with age. Three (8%) of analytes differed between sex, whereas 16 (44%) analytes differed by season. Female dolphins had higher median iron (33 µmol/L) than male dolphins (25 µmol/L). Female dolphins also had higher Abs lymphs and MCHC, but Abs lymphs and MCHC also differed between age and season, respectively. Sex inconsistently and relatively infrequently influences analytes. Delphinids of advancing age experience immune senescence and decreasing renal perfusion or clearance. These results demonstrate the importance of considering the influences of sex, age, and season on blood data, provide a baseline for accurate interpretation of clinicopathological analytes of delphinids in managed care, and will be useful for investigations into health, disease, and stressors of wild delphinids.

There is growing concern about the potential adverse health effects of phthalates (PAEs) on human health and the environment due to their extensive use as plasticizers and additives in commercial and consumer products. In this study, we assessed PAE concentrations in serum samples from aquarium-based delphinids (Tursiops truncatus, n = 36; Orcinus orca, n = 42) from California, Florida, and Texas, USA. To better understand the physiological effects of phthalates on delphinids, we also explored potential correlations between phthalates and the biomarkers aldosterone, cortisol, corticosterone, hydrogen peroxide, and malondialdehyde while accounting for sex, age, and reproductive stage. All PAEs were detected in at least one of the individuals. ΣPAE ranges were 5.995–2743 ng·mL−1 in bottlenose dolphins and 5.372–88,675 ng·mL−1 in killer whales. Both species displayed higher mean concentrations of DEP and DEHP. PAEs were detected in newborn delphinids, indicating transference via placenta and/or lactation. Linear mixed model results indicated significant correlations between aldosterone, month, location, status, and ΣPAEs in killer whales, suggesting that aldosterone concentrations are likely affected by the cumulative effects of these variables. This study expands on the knowledge of delphinid physiological responses to PAEs and may influence management and conservation decisions on contamination discharge regulations near these species.

Polar bears (Ursus maritimus) face a number of challenges that threaten the survival of the species. Captive breeding represents one essential facet of species conservation, but aspects of the polar bear’s reproductive physiology, such as follicle maturation, coitus-induced ovulation, and pseudopregnancy, are poorly characterized and present challenges for enhancing natural reproductive success and the application of advanced reproductive techniques. Due to the absence of a reliable transrectal or transabdominal ultrasound method for ovarian examination in the species, the ovaries of two adult female polar bears were examined laparoscopically to evaluate the feasibility of surgical access to the ovaries, oviduct, and uterus. The minimally invasive procedure was easily and rapidly performed in both bears and all procedures. Direct visual assessment of the ovary was possible after dissection of a fatty bursal sac, which completely enclosed the ovaries. In the second bear, laparoscopic manipulation of the ovary to draw it closer to the body wall enabled transcutaneous ultrasound. Laparoscopy may be a valuable tool to aid in the application of advanced reproductive technologies in polar bears.

Effective conservation and management of threatened wildlife populations require an accurate assessment of age structure to estimate demographic trends and population viability. Epigenetic aging models are promising developments because they estimate individual age with high accuracy, accurately predict age in related species, and do not require invasive sampling or intensive long-term studies. Using blood and biopsy samples from known age plains zebras (Equus quagga), we model epigenetic aging using two approaches: the epigenetic clock (EC) and the epigenetic pacemaker (EPM). The plains zebra EC has the potential for broad application within the genus Equus given that five of the seven extant wild species of the genus are threatened. We test the EC’s ability to predict age in sister taxa, including two endangered species and the more distantly related domestic horse, demonstrating high accuracy in all cases. By comparing chronological and estimated age in plains zebras, we investigate age acceleration as a proxy of health status. An interaction between chronological age and inbreeding is associated with age acceleration estimated by the EPM, suggesting a cumulative effect of inbreeding on biological aging throughout life.Larison et al. report epigenetic aging models in plains zebras (Equus quagga) using the epigenetic clock and epigenetic pacemaker approaches. Their epigenetic clock allows age to be accurately estimated in endangered sister species, and the pacemaker model identifies an association between inbreeding and accelerating aging.