Bellwethers of change: population modelling of North Pacific humpback whales from 2002 through 2021 reveals shift from recovery to climate response.

For the 40 years after the end of commercial whaling in 1976, humpback whale populations in the North Pacific Ocean exhibited a prolonged period of recovery. Using mark-recapture methods on the largest individual photo-identification dataset ever assembled for a cetacean, we estimated annual ocean-basin-wide abundance for the species from 2002 through 2021. Trends in annual estimates describe strong post-whaling era population recovery from 16 875 (± 5955) in 2002 to a peak abundance estimate of 33 488 (± 4455) in 2012. An apparent 20% decline from 2012 to 2021, 33 488 (± 4455) to 26 662 (± 4192), suggests the population abruptly reached carrying capacity due to loss of prey resources. This was particularly evident for humpback whales wintering in Hawai'i, where, by 2021, estimated abundance had declined by 34% from a peak in 2013, down to abundance levels previously seen in 2006, and contrasted to an absence of decline in Mainland Mexico breeding humpbacks. The strongest marine heatwave recorded globally to date during the 2014-2016 period appeared to have altered the course of species recovery, with enduring effects. Extending this time series will allow humpback whales to serve as an indicator species for the ecosystem in the face of a changing climate.

A call to implement preclinical study registration in animal ethics review.

Protocol registration is required in clinical trials. Registration of animal studies could improve research transparency and reduce redundancy, yet uptake has been minimal. Integrating study registration into institutional approval of animal use protocols is a promising approach to increase uptake.

Development and characterization of a fecal-induced peritonitis model of murine sepsis: results from a multi-laboratory study and iterative modification of experimental conditions.

BACKGROUND: Preclinical sepsis models have been criticized for their inability to recapitulate human sepsis and suffer from methodological shortcomings that limit external validity and reproducibility. The National Preclinical Sepsis Platform (NPSP) is a consortium of basic science researchers, veterinarians, and stakeholders in Canada undertaking standardized multi-laboratory sepsis research to increase the efficacy and efficiency of bench-to-bedside translation. In this study, we aimed to develop and characterize a 72-h fecal-induced peritonitis (FIP) model of murine sepsis conducted in two independent laboratories. The experimental protocol was optimized by sequentially modifying dose of fecal slurry and timing of antibiotics in an iterative fashion, and then repeating the experimental series at site 1 and site 2. RESULTS: Escalating doses of fecal slurry (0.5-2.5 mg/g) resulted in increased disease severity, as assessed by the modified Murine Sepsis Score (MSS). However, the MSS was poorly associated with progression to death during the experiments, and mice were found dead without elevated MSS scores. Administration of early antibiotics within 4 h of inoculation rescued the animals from sepsis compared with late administration of antibiotics after 12 h, as evidenced by 100% survival and reduced bacterial load in peritoneum and blood in the early antibiotic group. Site 1 and site 2 had statistically significant differences in mortality (60% vs 88%; p < 0.05) for the same dose of fecal slurry (0.75 mg/g) and marked differences in body temperature between groups. CONCLUSIONS: We demonstrate a systematic approach to optimizing a 72-h FIP model of murine sepsis for use in multi-laboratory studies. Alterations to experimental conditions, such as dose of fecal slurry and timing of antibiotics, have clear impact on outcomes. Differences in mortality between sites despite rigorous standardization warrants further investigations to better understand inter-laboratory variation and methodological design in preclinical studies.

A collaborative and near-comprehensive North Pacific humpback whale photo-ID dataset.

We present an ocean-basin-scale dataset that includes tail fluke photographic identification (photo-ID) and encounter data for most living individual humpback whales (Megaptera novaeangliae) in the North Pacific Ocean. The dataset was built through a broad collaboration combining 39 separate curated photo-ID catalogs, supplemented with community science data. Data from throughout the North Pacific were aggregated into 13 regions, including six breeding regions, six feeding regions, and one migratory corridor. All images were compared with minimal pre-processing using a recently developed image recognition algorithm based on machine learning through artificial intelligence; this system is capable of rapidly detecting matches between individuals with an estimated 97-99% accuracy. For the 2001-2021 study period, a total of 27,956 unique individuals were documented in 157,350 encounters. Each individual was encountered, on average, in 5.6 sampling periods (i.e., breeding and feeding seasons), with an annual average of 87% of whales encountered in more than one season. The combined dataset and image recognition tool represents a living and accessible resource for collaborative, basin-wide studies of a keystone marine mammal in a time of rapid ecological change.

mtDNA heteroplasmy gives rise to a new maternal lineage in North Pacific humpback whales (Megaptera novaeangliae).

Heteroplasmy in the mitochondrial genome offers a rare opportunity to track the evolution of a newly arising maternal lineage in populations of non-model species. Here, we identified a previously unreported mitochondrial DNA haplotype while assembling an integrated database of DNA profiles and photo-identification records from humpback whales in southeastern Alaska (SEAK). The haplotype, referred to as A8, was shared by only 2 individuals, a mature female with her female calf, and differed by only a single base pair from a common haplotype in the North Pacific, referred to as A-. To investigate the origins of the A8 haplotype, we reviewed n = 1,089 electropherograms (including replicate samples) of n = 710 individuals with A- haplotypes from an existing collection. From this review, we found 20 individuals with clear evidence of heteroplasmy for A-/A8 (parental/derived) haplotypes. Of these, 15 were encountered in SEAK, 4 were encountered on the Hawaiian breeding ground (the primary migratory destination for whales in SEAK), and 1 was encountered in the northern Gulf of Alaska. We used genotype exclusion and likelihood to identify one of the heteroplasmic females as the likely mother of the A8 cow and grandmother of the A8 calf, establishing the inheritance and germ-line fixation of the new haplotype from the parental heteroplasmy. The mutation leading to this heteroplasmy and the fixation of the A8 haplotype provide an opportunity to document the population dynamics and regional fidelity of a newly arising maternal lineage in a population recovering from exploitation.

Reproductive Steroid Hormone Patterns in Baleen of Two Pregnant Humpback Whales (Megaptera novaeangliae).

Understanding reproductive physiology in mysticetes has been slowed by the lack of repeated samples from individuals. Analysis of humpback whale baleen enables retrospective hormone analysis within individuals dating back 3-5 years before death. Using this method, we investigated differences in four steroid hormones involved in reproduction and mating during confirmed pregnant and non-pregnant periods in two female humpback whales (Megaptera novaeangliae) with known reproductive histories based on sightings and necropsy data. Cortisol, corticosterone, testosterone, and estradiol concentrations were determined via enzyme immunoassay using subsamples of each baleen plate at 2 cm intervals. There were no significant differences in cortisol or corticosterone during pregnancy when compared to non-pregnancy (inter-calving interval), but there were significant differences between the two whales in average glucocorticoid concentrations, with the younger whale showing higher values overall. For testosterone, levels for the younger female peaked at parturition in one pregnancy, but also had spikes during non-pregnancy. The older female had three large spikes in testosterone, one of which was associated with parturition. Estradiol had large fluctuations in both whales but had generally lower concentrations during non-pregnancy than during pregnancy. There were peaks in estradiol before each pregnancy, possibly coinciding with ovulation, and peaks coinciding with the month of parturition. Both estradiol and testosterone could be useful for determining ovulation or impending birth. Using baleen to investigate retrospective steroid hormone profiles can be used for elucidating long-term patterns of physiological change during gestation. LAY SUMMARY: Case studies of two pregnant humpback whales whose hormones were analyzed in baleen may illuminate when humpback whales ovulate, gestate, and give birth. These physiological metrics could assist in accurate population growth assessments and conservation of the species. This study shows that baleen hormone analysis can be a useful tool for understanding whale reproductive physiology.

Case studies on longitudinal mercury content in humpback whale (Megaptera novaeangliae) baleen.

Quantification of contaminant concentrations in baleen whales is important for individual and population level health assessments but is difficult due to large migrations and infrequent resighings. The use of baleen allows for a multiyear retrospective analysis of contaminant concentrations without having to collect repeated samples from the same individual. Here we provide case studies of mercury analysis using cold vapor atomic absorption spectroscopy in three individual humpback whales (Megaptera novaeangliae), a 44.5-year-old female and two males aged ≥35 and 66 years, over approximately three years of baleen growth. Mercury concentrations in the female's baleen were consistently 2-3 times higher than in either male. Age did not affect mercury concentrations in baleen; the younger male had comparable levels to the older male. In the female, mercury concentrations in the baleen did not change markedly during pregnancy but mercury did spike during the first half of lactation. Stable isotope profiles suggest that diet likely drove the female's high mercury concentrations. In conclusion, variations in baleen mercury content can be highly individualistic. Future studies should compare sexes as well as different populations and species to determine how the concentrations of mercury and other contaminants vary by life history parameters and geography.

Multi-year progesterone profiles during pregnancy in baleen of humpback whales (Megaptera novaeangliae).

Understanding calving rates of wild whale populations is critically important for management and conservation. Reproduction of humpback whales (Megaptera novaeangliae) is difficult to monitor and, even with long-term sighting studies, basic physiological information such as pregnancy rates and calving intervals remain poorly understood in many populations. We hypothesized that pregnant whales have sustained elevations in baleen progesterone that temporally correlate with gestation. To test this hypothesis, baleen progesterone profiles from two adult female North Pacific humpbacks, both with extensive sighting records and documented pregnancies, were compared to those of a nulliparous female (adult female never seen with a calf) and a juvenile male. Baleen specimens recovered during necropsy were subsampled every 2 cm from the base to the tip of the plate, with each interval representing 30-45 days of growth. Homogenized baleen powder was assayed for progesterone using enzyme immunoassays. The date of growth of each sampling location on the baleen plate was estimated based on stable isotope analysis of annual δ15N cycles. Progesterone profiles from both pregnant whales showed sustained high progesterone content (>350 ng/g) in areas corresponding to known pregnancies, inferred from calf sightings and post-mortem data. The younger female, estimated to be 13 years old, had higher progesterone during pregnancy than the 44.5 year old, but levels during non-pregnancy were similar. The nulliparous female and the male had low progesterone throughout their baleen plates. Baleen hormone analysis can determine how progesterone concentrations change throughout gestation and has potential for estimating age at first reproduction, pregnancy intervals, failed pregnancies and early calf mortality. Understanding rates of calving and current and historic reproductive patterns in humpbacks is vital to continuing conservation measures in this species.

National Preclinical Sepsis Platform: developing a framework for accelerating innovation in Canadian sepsis research.

Despite decades of preclinical research, no experimentally derived therapies for sepsis have been successfully adopted into routine clinical practice. Factors that contribute to this crisis of translation include poor representation by preclinical models of the complex human condition of sepsis, bias in preclinical studies, as well as limitations of single-laboratory methodology. To overcome some of these shortcomings, multicentre preclinical studies-defined as a research experiment conducted in two or more research laboratories with a common protocol and analysis-are expected to maximize transparency, improve reproducibility, and enhance generalizability. The ultimate objective is to increase the efficiency and efficacy of bench-to-bedside translation for preclinical sepsis research and improve outcomes for patients with life-threatening infection. To this end, we organized the first meeting of the National Preclinical Sepsis Platform (NPSP). This multicentre preclinical  research collaboration of Canadian sepsis researchers and stakeholders was established to study the pathophysiology of sepsis and accelerate movement of promising therapeutics into early phase clinical trials. Integrated knowledge translation and shared decision-making were emphasized to ensure the goals of the platform align with clinical researchers and patient partners. 29 participants from 10 independent labs attended and discussed four main topics: (1) objectives of the platform; (2) animal models of sepsis; (3) multicentre methodology and (4) outcomes for evaluation. A PIRO model (predisposition, insult, response, organ dysfunction) for experimental design was proposed to strengthen linkages with interdisciplinary researchers and key stakeholders. This platform represents an important resource for maximizing translational impact of preclinical sepsis research.

Patterns of cortisol and corticosterone concentrations in humpback whale (Megaptera novaeangliae) baleen are associated with different causes of death.

Baleen whales are subject to a myriad of natural and anthropogenic stressors, but understanding how these stressors affect physiology is difficult. Measurement of adrenal glucocorticoid (GC) hormones involved in the vertebrate stress response (cortisol and corticosterone) in baleen could help fill this data gap. Baleen analysis is a powerful tool, allowing for a retrospective re-creation of multiple years of GC hormone concentrations at approximately a monthly resolution. We hypothesized that whales that died from acute causes (e.g. ship strike) would have lower levels of baleen GCs than whales that died from extended illness or injury (e.g. long-term entanglement in fishing gear). To test this hypothesis, we extracted hormones from baleen plates of four humpback whales (Megaptera novaeangliae) with well-documented deaths including multiple and chronic entanglements (n = 1, female), ship strike (n = 2, male and female) and chronic illness with nutritional stress (n = 1, male). Over ~3 years of baleen growth and during multiple entanglements, the entangled whale had average corticosterone levels of 80-187% higher than the other three whales but cortisol levels were similar to two of the other three whales. The nutritionally stressed and chronically ill whale showed a slow increase in both cortisol and corticosterone spanning ~3 years, followed by a sharp decline in both hormones before death, possibly indicative of adrenal failure in this moribund individual. This whale's correlation between cortisol and corticosterone was significant but there were no correlations in the other three whales. Our results show that cortisol and corticosterone concentrations vary according to the type and duration of illness or injury. Single-point GC concentrations should be interpreted with caution as low values can occur in whales experiencing pronounced stress and individual baselines can be highly variable. Baleen analysis is a promising tissue type for retrospective analyses of physiological responses to various stressors affecting baleen whales.

Effects of single compared with pair housing on hypothalamic-pituitary-adrenal axis activity and low-dose heroin place conditioning in adult male Sprague-Dawley rats.

Whether social isolation of adult rats under standard laboratory conditions produces significant long-term alterations in behavior and physiology is unclear. In the present study, male Sprague-Dawley rats were singly or paired-housed for 10 wk. During this period, they were tested for acquisition, extinction, and reacquisition of heroin (0.3 mg/kg)-conditioned place preference. Fecal corticoid metabolite levels were analyzed several times throughout the period of housing, and food consumption and body weight were monitored. During place conditioning, heroin induced a significant increase in locomotor activity in both singly and pair housed rats, and the resulting place preference was similar in both groups. However, singly housed rats showed increased motor reactivity to heroin on reconditioning after extinction and displayed significant reacquisition of conditioned place preference, compared with pair-housed animals. Over the 10-wk period of the study, there were no differences in body weight or food consumption between groups. Mild significant increases in relative adrenal gland weight and decreases in relative brain weight were noted in singly housed animals compared with those paired. Significant decreases in nocturnal fecal corticoid metabolite output were noted in both groups, with loss of circadian variation in fecal corticoid levels over the course of the study. These data suggest that male Sprague-Dawley rats, irrespective of single or pair housing, develop reduced hypothalamic-pituitary-adrenal axis activity over time under standard laboratory housing conditions. Single housing can enhance both this effect and sensitivity to the stimulatory and rewarding actions of heroin after withdrawal.

Experimental infection of New Zealand white rabbits (Oryctolagus cuniculi) with Leporid herpesvirus 4.

Leporid herpesvirus 4 (LHV4) is a novel alphaherpesvirus recently identified in domestic rabbits (Oryctolagus cuniculi). Little is known about the pathogenesis or time course of disease induced by this virus. We therefore intranasally inoculated 22 female New Zealand white rabbits with 8.4 × 10(4) CCID50 of a clinical viral isolate. Rabbits were monitored for clinical signs, viral shedding in oculonasal secretions, and development and persistence of serum antibodies. Rabbits were euthanized at 3, 5, 7, 14, and 22 d postinfection (dpi) to evaluate gross and microscopic changes. Clinical signs were apparent between 3 to 8 dpi, and included oculonasal discharge, respiratory distress, and reduced appetite, and viral shedding occurred between 2 and 8 dpi. Seroconversion was seen at 11 dpi and persisted to the end of the study (day 22). Severe necrohemorrhagic bronchopneumonia and marked pulmonary edema were noted by 5 dpi and were most severe at 7 dpi. Pulmonary changes largely resolved by 22 dpi. In addition, multifocal splenic necrosis was present at 5 dpi and progressed to submassive necrosis by 7 dpi. Eosinophilic herpesviral intranuclear inclusion bodies were detected in the nasal mucosa, skin, spleen, and lung between 3 to 14 dpi. LHV4 is a pathogen that should be considered for rabbits that present with acute respiratory disease. LHV4 infection can be diagnosed based on characteristic microscopic changes in the lungs and spleen and by virus isolation. Serum antibody levels may be used to monitor viral prevalence in colonies.

Oral gavage in rats: animal welfare evaluation.

The effect of chronic daily orogastric gavage with water (5 mL/kg) on behavior and physiology was evaluated in male Sprague-Dawley rats. Treatment groups included: unmanipulated control, restraint control, dry gavage, and gavage, with all rats singly housed (n = 9 or 10 per group). In addition, a group of pair-housed rats (n = 18) was included to determine whether social housing affected response to gavage. Weekly body weights and food consumption were recorded as well as use of a nylon chew toy for enrichment. Feces were collected biweekly at the end of the light and dark phases for fecal corticoid metabolite determinations. After 28 d of treatment, animals underwent conditioned place preference testing to evaluate sensitivity to motivational properties of the anxiolytic drug chlordiazepoxide (5.6 mg/kg SC). Brain and paired adrenal gland weights were collected at necropsy. Week 2 total fecal corticosterone levels were elevated in all groups and attributed to a fire alarm accidentally tripped during building renovations. No differences occurred in body weight or food consumption between any groups. All groups used a nylon chew toy given for enrichment and demonstrated mild preference for the drug-associated chamber. Fecal weights and corticoid metabolite levels were similar between all groups at week 4 and showed normal diurnal variation. No biologically significant variations were noted in brain or paired adrenal gland to body weight ratios. We conclude that orogastric gavage of aqueous solutions at 5 mL/kg does not negatively affect the welfare of laboratory rats acclimated to handling.