Ex situ amphibian populations can experience reproductive dysfunction due to the absence of environmental cues that trigger reproductive events. Assisted reproductive technologies (ART) for amphibians, specifically exogenous hormone regimens, can circumvent these external signals to induce gametogenesis and gamete release. Currently, the use of the mammalian reproductive hormones gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) are used in a species-specific manner to stimulate amphibian breeding. Hormones or hormone mixtures that are effective in all breeding scenarios would provide the best option for conservation practitioners and some commercial products are already in use for breeding other ectotherms. Ovaprim®, which contains salmon GnRH analogue (sGnRHa) and the dopamine antagonist domperidone (DOM), is effective in fish aquaculture and may be effective for amphibians. To test this hypothesis, we treated Fowler's toads (Anaxyrus fowleri) with either sGnRHa alone, a high or low dose of Ovaprim® or hCG. We then compared spermiation response, sperm quantity and quality parameters, and changes in animal mass over time within each treatment. We found administration of Ovaprim® resulted in more males producing sperm with better motility compared to administration of sGnRHa alone. In addition, the Ovaprim® and sGnRHa treatments resulted in lower response rates, lower sperm motilities, more abnormal sperm, and higher aggregations of sperm compared to the hCG treatment. Furthermore, Ovaprim®-treated males gained significant mass, suggesting an anti-diuretic effect of DOM. Together, these results show that neither Ovaprim® nor sGnRHa, at the concentrations tested, are likely suitable replacements for hCG in ex situ bufonid breeding programmes and that hormone mixtures developed for fish may have limited transferability to new world toad species.
Over 25% of cetacean species worldwide are listed as critically endangered, endangered or vulnerable by the International Union for Conservation of Nature. Objective and widely applicable tools to assess cetacean health are therefore vital for population monitoring and to inform conservation initiatives. Novel blubber biomarkers of physiological state are examples of such tools that could be used to assess overall health. Proteins extracted from blubber likely originate from both the circulation and various cell types within the tissue itself, and their expression is responsive to signals originating from other organs and the nervous system. Blubber proteins can therefore capture information on physiological stressors experienced by individuals at the time of sampling. For the first time, we assess the feasibility of applying shotgun proteomics to blubber biopsy samples collected from free-ranging baleen whales. Samples were collected from minke whales (Balaenoptera acutorostrata) (n = 10) in the Gulf of St Lawrence, Canada. Total protein was extracted using a RIPA cell lysis and extraction buffer-based protocol. Extracted proteins were separated and identified using nanoflow Liquid Chromatography Electrospray Ionization in tandem with Mass Spectrometry. We mapped proteins to known biological pathways and determined whether they were significantly enriched based on the proteome profile. A pathway enrichment map was created to visualize overlap in tissue-level biological processes. Amongst the most significantly enriched biological pathways were those involved in immune system function: inflammatory responses, leukocyte-mediated immunity and the humoral immune response. Pathways associated with responses to oxidative stress were also enriched. Using a suite of such protein biomarkers has the potential to better assess the overall health and physiological state of live individuals through remote biopsy sampling. This information is vital for population health assessments to predict population trajectories, and ultimately guide and monitor conservation priorities and initiatives.
Normal reference intervals (RI) of hematologic and biochemical parameters are important for assessing and monitoring the health status of captive and free-living chelonians; however, such information is not available for most wildlife species. Giant Galapagos tortoises are one of the most iconic animals on earth and health information can make an important contribution to their conservation and management. This study provides formal RI of haematology and plasma biochemistry parameters and describes cell morphology along with morphometrics of free-living Eastern Santa Cruz (Chelonoidis donfaustoi), Española (Chelonoidis hoodensis) and San Cristóbal tortoises (Chelonoidis chathamensis). We explored differences in blood parameters between sexes, across the tortoise species in this study and with previously published RI of the Western Santa Cruz tortoise (Chelonoidis porteri). Biochemistry parameters of both Santa Cruz species were overall more similar to each other than to Española and San Cristobal tortoises. This research constitutes the first RI for these three Galapagos tortoise species and may be of value for advising captive-breeding and conservation plans. We recommend further research to establish RI in additional tortoise species so we may better understand and interpret haematology and biochemistry parameters as a valuable conservation tool for species of this critically endangered taxon.
High turbidity and elevated water temperature are environmental stressors that can co-occur in freshwater ecosystems such as when deforestation increases solar radiation and sedimentary runoff. However, we have limited knowledge about their combined impacts on fish behaviour and physiology. We explored independent and interactive effects of sedimentary turbidity and temperature on the swimming activity and both thermal and hypoxia tolerance of the Pugnose Shiner (Miniellus anogenus, formerly Notropis anogenus), a small leuciscid fish listed as Threatened under Canada's Species at Risk Act (SARA). Fish underwent a 15-week acclimation to two temperatures (16°C or 25°C) crossed with two turbidities (~0 NTU or 8.5 NTU). Swimming activity was measured during the first 8 weeks of acclimation. Fish in warm water were more active compared to those in cold water, but turbidity had no effect on activity. Behavioural response to hypoxia was measured after 12 weeks of acclimation, as the oxygen level at which fish used aquatic surface respiration (ASR). Fish in warm water engaged in ASR behaviour at higher oxygen thresholds, indicating less tolerance to hypoxia. Turbidity had no effect on ASR thresholds. Finally, thermal tolerance was measured as the critical thermal maximum (CTmax) after 13-15 weeks of acclimation. Acclimation to warm water increased fish CTmax and Tag (agitation temperature) but reduced the agitation window (°C difference between Tag and CTmax) and thermal safety margin (°C difference between the acclimation temperature and CTmax). Furthermore, fish in warm, turbid water had a lower CTmax and smaller thermal safety margin than fish in warm, clear water, indicating an interaction between turbidity and temperature. This reduced thermal tolerance observed in Pugnose Shiner in warm, turbid water highlights the importance of quantifying independent and interactive effects of multiple stressors when evaluating habitat suitability and conservation strategies for imperilled species.
Pacific spiny dogfish, Squalus suckleyi, move to shallow coastal waters during critical reproductive life stages and are thus at risk of encountering hypoxic events which occur more frequently in these areas. For effective conservation management, we need to fully understand the consequences of hypoxia on marine key species such as elasmobranchs. Because of their benthic life style, we hypothesized that S. suckleyi are hypoxia tolerant and able to efficiently regulate oxygen consumption, and that anaerobic metabolism is supported by a broad range of metabolites including ketones, fatty acids and amino acids. Therefore, we studied oxygen consumption rates, ventilation frequency and amplitude, blood gasses, acid-base regulation, and changes in plasma and tissue metabolites during progressive hypoxia. Our results show that critical oxygen levels (P crit) where oxyregulation is lost were indeed low (18.1% air saturation or 28.5 Torr at 13°C). However, many dogfish behaved as oxyconformers rather than oxyregulators. Arterial blood PO2 levels mostly decreased linearly with decreasing environmental PO2. Blood gases and acid-base status were dependent on open versus closed respirometry but in both set-ups ventilation frequency increased. Hypoxia below Pcrit resulted in an up-regulation of anaerobic glycolysis, as evidenced by increased lactate levels in all tissues except brain. Elasmobranchs typically rely on ketone bodies as oxidative substrates, and decreased concentrations of acetoacetate and β-hydroxybutyrate were observed in white muscle of hypoxic and/or recovering fish. Furthermore, reductions in isoleucine, glutamate, glutamine and other amino acids were observed. After 6 hours of normoxic recovery, changes persisted and only lactate returned to normal in most tissues. This emphasizes the importance of using suitable bioindicators adjusted to preferred metabolic pathways of the target species in conservation physiology. We conclude that Pacific spiny dogfish can tolerate severe transient hypoxic events, but recovery is slow and negative impacts can be expected when hypoxia persists.
How species respond to climate change will depend on the collective response of populations. Intraspecific variation in traits, evolved through genetic adaptation and phenotypic plasticity, can cause thermal performance curves to vary over species' distributions. Intraspecific variation within marine species has received relatively little attention due to the belief that marine systems lack dispersal barriers strong enough to promote locally adapted traits. Here we show that intraspecific variation is present between low- and high-latitude populations of a coral reef damselfish (Acanthochromis polyacanthus). Co-gradient variation was observed when examining aerobic physiology across a thermal gradient that reflected mean summer temperatures of high- and low-latitude regions, as well as projected future ocean temperatures (i.e. 27, 28.5, 30, 31.5°C). Whilst thermally sensitive, no significant differences were observed between high- and low-latitude regions when measuring immunocompetence, haematocrit and anaerobic enzyme activity. The presence of co-gradient variation suggests that dispersal limitations in marine systems can promote local adaptive responses; however, intraspecific variation may not be ubiquitous amongst traits. Identifying locally adapted traits amongst populations remains necessary to accurately project species responses to climate change and identify differences in adaptive potential.
Greater sage-grouse (Centrocercus urophasianus) are a critically endangered species in Canada with fewer than 140 individuals remaining on native habitats in southern Alberta and Saskatchewan. In 2014, the Wilder Institute/Calgary Zoo initiated North America's only zoo-based conservation breeding program for this species to bolster declining wild populations through conservation reintroductions. Within the managed population of sage-grouse, morbidity and mortality have primarily been associated with intestinal bacterial infections. As a preliminary study to assess the gastrointestinal health of this species in managed care, the fecal bacterial microbiome of adult and juvenile captive sage-grouse was characterized with 16S rRNA sequencing. The composition of the microbiome at the phylum level in greater sage-grouse is consistent with previous studies of the avian microbiome, with Bacillota as the most abundant phyla, and Actinomycetota, Bacteroidota and Pseudomonadota also being highly abundant. Antibiotic use and sex did not have a significant impact on the diversity or composition of the microbiome, but the management of juvenile sage-grouse did influence the development of the microbiome. Juveniles that were raised outdoors under maternal care developed a microbiome much more similar to adults when compared to chicks that were incubated and hand-raised. The local environment and parental care appear to be important factors influencing the diversity and composition of the gastrointestinal microbiome in this species.
A key driver of the African savannah elephant population decline is the loss of habitat and associated human-elephant conflict. Elephant physiological responses to these pressures, however, are largely unknown. To address this knowledge gap, we evaluated faecal glucocorticoid metabolite (fGCM) concentrations as an indicator of adrenal activity and faecal thyroid metabolite (fT3) concentrations as an indicator of metabolic activity in relation to land use, livestock density, and human landscape modification, while controlling for the effects of seasonality and primary productivity (measured using the normalized difference vegetation index). Our best-fit model found that fGCM concentrations to be elevated during the dry season, in areas with higher human modification index values, and those with more agropastoral activities and livestock. There was also a negative relationship between primary productivity and fGCM concentrations. We found fT3 concentrations to be higher during the wet season, in agropastoral landscapes, in locations with higher human activity, and in areas with no livestock. This study highlights how elephants balance nutritional rewards and risks in foraging decisions when using human-dominated landscapes, results that can serve to better interpret elephant behaviour at the human-wildlife interface and contribute to more insightful conservation strategies.
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.
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