Laparoscopic Evaluation of the Reproductive Tract in Two Female Polar Bears (Three Procedures) (Ursus maritimus).

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.

Relationship between red blood cell lifespan and endogenous carbon monoxide in the common bottlenose dolphin and beluga.

Certain deep-diving marine mammals [i.e., northern elephant seal (Mirounga angustirostris), Weddell seal (Leptonychotes weddellii)] have blood carbon monoxide (CO) levels that are comparable with those of chronic cigarette smokers. Most CO produced in humans is a byproduct of heme degradation, which is released when red blood cells (RBCs) are destroyed. Elevated CO can occur in humans when RBC lifespan decreases. The contribution of RBC turnover to CO concentrations in marine mammals is unknown. Here, we report the first RBC lifespans in two healthy marine mammal species with different diving capacities and heme stores, the shallow-diving bottlenose dolphin (Tursiops truncatus) and deep-diving beluga whale (Delphinapterus leucas), and we relate the lifespans to the levels of CO in blood and breath. The belugas, with high blood heme stores, had the longest mean RBC lifespan compared with humans and bottlenose dolphins. Both cetacean species were found to have three times higher blood CO content compared with humans. The estimated CO production rate from heme degradation indicates some marine mammals may have additional mechanisms for CO production, or delay CO removal from the body, potentially from long-duration breath-holds.NEW & NOTEWORTHY This is the first study to determine the red blood cell lifespan in a marine mammal species. High concentrations of carbon monoxide (CO) were found in the blood of bottlenose dolphins and in the blood and breath of belugas compared with healthy humans. Red blood cell turnover accounted for these high levels in bottlenose dolphins, but there may be alternative mechanisms of endogenous CO production that are contributing to the CO concentrations observed in belugas.

DNA methylation networks underlying mammalian traits.

Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

Pan-primate studies of age and sex.

Age and sex have a profound effect on cytosine methylation levels in humans and many other species. Here we analyzed DNA methylation profiles of 2400 tissues derived from 37 primate species including 11 haplorhine species (baboons, marmosets, vervets, rhesus macaque, chimpanzees, gorillas, orangutan, humans) and 26 strepsirrhine species (suborders Lemuriformes and Lorisiformes). From these we present here, pan-primate epigenetic clocks which are highly accurate for all primates including humans (age correlation R = 0.98). We also carried out in-depth analysis of baboon DNA methylation profiles and generated five epigenetic clocks for baboons (Olive-yellow baboon hybrid), one of which, the pan-tissue epigenetic clock, was trained on seven tissue types (fetal cerebral cortex, adult cerebral cortex, cerebellum, adipose, heart, liver, and skeletal muscle) with ages ranging from late fetal life to 22.8 years of age. Using the primate data, we characterize the effect of age and sex on individual cytosines in highly conserved regions. We identify 11 sex-related CpGs on autosomes near genes (POU3F2, CDYL, MYCL, FBXL4, ZC3H10, ZXDC, RRAS, FAM217A, RBM39, GRIA2, UHRF2). Low overlap can be observed between age- and sex-related CpGs. Overall, this study advances our understanding of conserved age- and sex-related epigenetic changes in primates, and provides biomarkers of aging for all primates.

Multi-tissue DNA methylation aging clocks for sea lions, walruses and seals.

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.

Behavior Skills Training with Zoological Staff to Increase Killer Whale Attending Behavior.

Attending is a requisite behavior for interactions between animals in zoological care and their care specialist (trainers). Head-dropping behavior (HDB) is incompatible with attending and was observed with two killer whales during whale-trainer interactions (WTI). Initial observations also noted inconsistencies in the trainers' application of shaping procedures during WTI. A three-part plan was developed to address trainers procedural fidelity, increase whale attending during WTI, and program for the behavioral generalization and maintenance of the whale-trainer dyads (dyad). First, behavior skills training (BST) was used to instruct trainers on discrete trial training (DTT). Second, the trainers' applied their acquired skill of DTT, targeting attending behavior, with their whale during WTI. Third, behavioral generalization was programed by switching the dyad pairs in an additional DTT generalization phase. The findings demonstrated a strong positive correlation between trainer DTT fidelity and whale attending. Finally, the results suggest that future assessments of behavioral management programs with zoological trainers and animals are warranted.

The emergence and early development of socio-sexual behavior in beluga calves (Delphinapterus leucas).

While the ontogeny of beluga (Delphinapterus leucas) socio-sexual behavior has been documented in animals between 37- and 108-months-old, the first 36 months of life are yet to be examined. This study investigated how socio-sexual behaviors emerge over the first three years of life in a group of belugas in managed care. The emergence of socio-sexual behaviors was relatively consistent among immature animals. More complex behaviors, like s-posture presentations, developed in a piecemeal fashion (i.e., simple components of the behavior sequence emerged before complex components). The presence of an adult male significantly predicted if immature belugas would initiate and participate in socio-sexual behavior. However, partner preferences changed with age. In the first year of life, belugas engaged in sexual behavior with their mother most frequently but preferred to play with older males if given a choice. By the third year of life, belugas engaged in socio-sexual behavior most frequently with other immature animals. These findings enhance our understanding of how belugas develop sexually and socially and have implications for social housing practices of immature belugas.

Epigenetic clock and methylation studies in marsupials: opossums, Tasmanian devils, kangaroos, and wallabies.

The opossum (Monodelphis domestica), with its sequenced genome, ease of laboratory care and experimental manipulation, and unique biology, is the most used laboratory marsupial. Using the mammalian methylation array, we generated DNA methylation data from n = 100 opossum samples from the ear, liver, and tail. We contrasted postnatal development and later aging effects in the opossum methylome with those in mouse (Mus musculus, C57BL/6 J strain) and other marsupial species such as Tasmanian devil, kangaroos, and wallabies. While the opossum methylome is similar to that of mouse during postnatal development, it is distinct from that shared by other mammals when it comes to the age-related gain of methylation at target sites of polycomb repressive complex 2. Our immunohistochemical staining results provide additional support for the hypothesis that PRC2 activity increases with later aging in mouse tissues but remains constant in opossum tissues. We present several epigenetic clocks for opossums that are distinguished by their compatibility with tissue type (pan-tissue and blood clock) and species (opossum and human). Two dual-species human-opossum pan-tissue clocks accurately measure chronological age and relative age, respectively. The human-opossum epigenetic clocks are expected to provide a significant boost to the attractiveness of opossum as a biological model. Additional epigenetic clocks for Tasmanian devil, red kangaroos and other species of the genus Macropus may aid species conservation efforts.

DNA methylation aging and transcriptomic studies in horses.

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.

Epigenetic models developed for plains zebras predict age in domestic horses and endangered equids.

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.

Epigenetic clock and methylation studies in cats.

Human DNA methylation profiles have been used successfully to develop highly accurate biomarkers of aging ("epigenetic clocks"). Although these human epigenetic clocks are not immediately applicable to all species of the animal kingdom, the principles underpinning them appear to be conserved even in animals that are evolutionarily far removed from humans. This is exemplified by recent development of epigenetic clocks for mice and other mammalian species. Here, we describe epigenetic clocks for the domestic cat (Felis catus), based on methylation profiles of CpGs with flanking DNA sequences that are highly conserved between multiple mammalian species. Methylation levels of these CpGs are measured using a custom-designed Infinium array (HorvathMammalMethylChip40). From these, we present 3 epigenetic clocks for cats; of which, one applies only to blood samples from cats, while the remaining two dual-species human-cat clocks apply both to cats and humans. We demonstrate that these domestic cat clocks also lead to high age correlations in cheetahs, tigers, and lions. It is expected that these epigenetic clocks for cats possess the potential to be further developed for monitoring feline health as well as being used for identifying and validating anti-aging interventions.

Scaling of heart rate with breathing frequency and body mass in cetaceans.

Plasticity in the cardiac function of a marine mammal facilitates rapid adjustments to the contrasting metabolic demands of breathing at the surface and diving during an extended apnea. By matching their heart rate (fH) to their immediate physiological needs, a marine mammal can improve its metabolic efficiency and maximize the proportion of time spent underwater. Respiratory sinus arrhythmia (RSA) is a known modulation of fH that is driven by respiration and has been suggested to increase cardiorespiratory efficiency. To investigate the presence of RSA in cetaceans and the relationship between fH, breathing rate (fR) and body mass (Mb), we measured simultaneous fH and fR in five cetacean species in human care. We found that a higher fR was associated with a higher mean instantaneous fH (ifH) and minimum ifH of the RSA. By contrast, fH scaled inversely with Mb such that larger animals had lower mean and minimum ifHs of the RSA. There was a significant allometric relationship between maximum ifH of the RSA and Mb, but not fR, which may indicate that this parameter is set by physical laws and not adjusted dynamically with physiological needs. RSA was significantly affected by fR and was greatly reduced with small increases in fR. Ultimately, these data show that surface fHs of cetaceans are complex and the fH patterns we observed are controlled by several factors. We suggest the importance of considering RSA when interpreting fH measurements and particularly how fR may drive fH changes that are important for efficient gas exchange. This article is part of the theme issue 'Measuring physiology in free-living animals (Part I)'.

Multi-species and multi-tissue methylation clocks for age estimation in toothed whales and dolphins.

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.

Epigenetic clock and methylation studies in elephants.

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.

Circulating concentrations of thyroid hormones and cortisol in wild and semi-natural Yangtze finless porpoise (Neophocaena asiaeorientalis).

Our understanding about how environmental and biological variables may influence circulating thyroid and adrenal hormones in free-ranging cetaceans is limited. As such, we used liquid chromatography-mass spectrometry to determine concentrations of circulating cortisol and thyroid hormones (THs; tT3, tT4) in 132 Yangtze finless porpoises (YFPs) located in Poyang Lake, (PL, n = 92) and Tian-E-Zhou Oxbow reserve (TZO, n = 40). For overall hormone comparisons, animals were partitioned by age [juvenile and adult (male and non-pregnant, non-lactating female)], sex, season (winter or spring) and geographical location. Geographically, during winter, circulating THs were significantly higher in the PL versus TZO population. Seasonally, within PL, THs were significantly higher in the winter versus spring season. Animals were further binned into groups as follows: juvenile male (JM) and juvenile female (JF), adult male (AM), non-pregnant adult female, pregnant female and non-pregnant lactating female. Intra-group comparisons between locations showed a significant increase in JM THs at PL. Significant increases in THs during winter compared to spring were detected between JM and JF groups. Mean comparisons of cortisol within and between locations for each group identified a significant increase for TZO AM versus TZO pregnant female and JM and JF. Seasonally, in PL, only JF has significantly higher cortisol in winter versus spring. Finally, we established reference values of THs and cortisol for YFPs in different geographical locations. These references are important baselines from which the effects of environmental and biological variables on THs and cortisol may be evaluated.

Effects of sex, age, and season on the variation of blood analytes in a clinically healthy ex situ population of bottlenose dolphins (Tursiops spp.).

BACKGROUND: 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. AIM: To define the variation in hematological and biochemical analytes of dolphins due to sex, age, and season. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Comparative analysis of steroids in cyclic and pregnant killer whales, beluga whales and bottlenose dolphins by liquid chromatography tandem mass spectrometry.

There exists a surprising diversity in the physiology and endocrinology of pregnancy among mammals in both the source (luteal/placental) and metabolism of progesterone. To evaluate the possible diversity of steroid metabolism within toothed cetaceans, we investigated 5α-reduced progesterone metabolites and androgens in cyclic (luteal phase) and pregnant captive killer whales, belugas and bottlenose dolphins (n = 5/species) bled longitudinally in early, mid- and late pregnancy (0.16, 0.50 and 0.85 fractions of 535, 464 and 380 gestation days, respectively). Mid-luteal samples were also collected. Serum was analyzed by liquid chromatography tandem-mass spectrometry as previously validated for (among others) progesterone, 20αOH-progesterone (20αOHP), 5α-dihydroprogesterone (DHP), several additional 5α-reduced metabolites and androgens (dehydroepiandrosterone, androstenedione and testosterone). The predominant mid-luteal pregnanes were: progesterone, belugas; progesterone and 20αOHP, dolphins; allopregnanolone (3α-DHP) and progesterone, killer whales. Progesterone was 2-4-fold higher in early pregnancy than mid-luteal samples but decreased thereafter. The predominant metabolite, 3ß,20α-dihydroprogesterone (3ß,20α-DHP; 40-80 ng/ml) was higher in mid- and late-than early gestation in all 3 species. Concentrations of 20αOHP and 3ß,20α-DHP were similar at mid-gestation but 20αOHP declined in late-gestation in killer whales, and 20αOHP was lower than 3ß,20α-DHP in belugas and dolphins throughout gestation. Other 5α-reduced metabolites, DHP, 3α-DHP and 20α-DHP, were far lower throughout pregnancy (<10 ng/ml). DHP and 3α-DHP decreased from early to mid-gestation in belugas, but changed little in killer whales and dolphins. These data suggest that progesterone metabolism is relatively conserved among these cetacean species. As in equine pregnancies, 3ß,20α-DHP is the major metabolite, increasing at the expense of progesterone as pregnancy progresses. Androstenedione and testosterone also increased detectably in mid- to late-gestation in these species. The tissue source remains unknown, but progesterone metabolism during gestation in these cetaceans is similar to horses and, together with androgens, may be reliable biomarkers of pregnancy.

Hematologic and Biochemical Reference Interval Development and the Effect of Age, Sex, Season, and Location on Hematologic Analyte Concentrations in Critically Endangered Yangtze Finless Porpoise (Neophocaena asiaeorientalis ssp. asiaeorientalis).

In this study, references intervals for 49 clinicopathological parameters were established for the critically endangered Yangtze finless porpoise (YFP) (Neophocaena phocaenoides asiaorientalis). Both from the wild (Poyang Lake) and seminatural (Tian-E-Zhou Oxbow) populations, individual blood samples from 188 animals were collected from 2009 to 2017 and from 2002 to 2015, respectively. For reference interval determination, we used a non-parametric bootstrap-based procedure to determine the 95th percentiles and the associated 90% confidence interval for each analyte. Our results indicated a need to partition the analyte concentrations by sex, age group, or pregnancy; however, we did not find a need to partition results by location. We then used a linear mixed model to determine if evidence existed for mean differences between location with sex and season as covariates and age group as the clustered random variable on mean hematological parameters in the YFP. We found that 88% of the analytes were significantly different between locations. Within the covariates, sex and season showed 31 and 69% significant difference in mean distributions, respectively. Additionally, age group provided a significant source of variation in 25% of the analytes. In summary, our finding suggests that analytes should be grouped according to sex, age, and reproductive status (non-pregnant and non-lactating, pregnant and lactating). Furthermore, we have provided the first set of reference intervals for 49 clinicopathological parameters that could provide guidelines for the initial evaluation of individuals during health assessments.

Sperm handling in aquatic animals for artificial reproduction.

Artificial reproduction involves collection and handling of gametes in a way that secures their quality and maximizes the fertilization outcome. In addition to initial sperm quality, numerous steps can affect the final result of fertilization, from the sperm collection process until gamete mixing (or co-incubation) when the spermatozoon enters or fuses with the oocyte. In this review, we summarize the whole process of sperm handling, from collection until fertilization for fish, penaeid shrimp, bivalve mollusks and marine mammals. To obtain sperm from captive animals, techniques vary widely across taxa, and include stripping by abdominal massage or testis surgical removal in fish, spermatophore collection in penaeid shrimps, gonadal scarification or temperature shock in bivalve mollusks, and voluntary collection via positive reinforcement in mammals. In most cases, special care is needed to avoid contamination by mucus, seawater, urine, or feces that can either activate sperm motility and/or decrease its quality. We also review techniques and extender solutions used for refrigerated storage of sperm across the aforementioned taxa. Finally, we give an overview of the different protocols for in vivo and in vitro fertilization including activation of sperm motility and methods for gamete co-incubation. The present study provides valuable information regarding breeder management either for animal production or species conservation.

Effect of age, sex, and season on the variation in blood analytes of a clinically normal ex situ population of killer whales (Orcinus orca).

BACKGROUND: The effects of sex, age, and season on blood analyte concentrations have not been investigated for the killer whale (Orcinus orca). Defining these changes provides background data for improving the care of managed populations and defines normal changes that could occur in wild counterparts. OBJECTIVES: We aimed to define hematologic and serum biochemical variation by age, sex, and season for an ex situ killer whale population. METHODS: Blood samples collected from killer whales during normal wellness exams were retrospectively identified. Killer whales were categorized by age; calf (0-2.9 years), juvenile (3-10.9 years), early adult (11-20.9 years), adult (21-30.9 years), and aged (>30.9 years); sex; and season. Standard CBC and biochemistry were collated, and only samples without evidence of disease were used. A mixed effects maximum likelihood regression with animal identification (ID) as the random effects variable was used to compare groups with a significance set at P ≤ 0.01. RESULTS: All analytes differed by age, while only four differed by sex. Red blood cell parameters and associated renal analytes increased with age, while liver-associated analytes and glucose decreased. Season affected 59% of the blood analytes. CONCLUSIONS: Aged killer whales showed strong evidence of altered physiology as compared with younger animals. Anemia did not develop with age as was observed in one bottlenose dolphin population. Observed decreases in renal function could be caused by chronic disease or dehydration. Decreases in immune function parameters suggest immune senescence. These results provide background data for evaluating the health of managed and free-ranging killer whales.