Biological characteristics of pregnancy in captive Yangtze finless porpoises revealed by urinary metabolomics†.

The Yangtze finless porpoises (Neophocaena asiaeorientalis a.) are an endemic and critically endangered species in China. Intensive captive breeding is essential for understanding the biology of critically endangered species, especially their pregnancy characteristics, knowledge of which is crucial for effective breeding management. Urine metabolomics can reveal metabolic differences, arising from physiological changes across pregnancy stages. Therefore, we used the urinary metabolomic technology, to explore urinary metabolite changes in pregnant Yangtze finless porpoises. A total of 2281 metabolites were identified in all samples, which including organic acids and derivatives (24.45%), organoheterocyclic compounds (20.23%), benzenoids (18.05%), organic oxygen compounds (7.73%), and phenylpropanoids and polyketides (6.48%). There were 164, 387, and 522 metabolites demonstrating differential abundance during early pregnancy, mid pregnancy, and late pregnancy, respectively, from the levels observed in nonpregnancy. The levels of pregnenolone, 17α-hydroxyprogesterone, and tetrahydrocortisone were significantly higher during all pregnancy stages, indicating their important roles in fetal development. The differential metabolites between nonpregnancy and pregnancy were mainly associated with amino acid and carbohydrate metabolism. Moreover, metabolic activity varied across pregnancy stages; steroid hormone biosynthesis was predominant in early pregnancy, and amino acid biosynthesis and carbohydrate metabolism were predominant in mid pregnancy and late pregnancy, respectively. Our results provide new insights into metabolic characteristics in the Yangtze finless porpoises' urine during pregnancy, and indicate that the differential levels of urine metabolites can determine pregnancy in Yangtze finless porpoises, providing valuable information for the husbandry and management of pregnant Yangtze finless porpoises in captivity.

Viral Diversity and Epidemiology in Critically Endangered Yangtze Finless Porpoises (Neophocaena asiaeorientalis asiaeorientalis).

The Yangtze finless porpoise (YFP) (Neophocaena asiaeorientalis asiaeorientalis) is a critically endangered freshwater cetacean, with about 1,249 individuals thought to be left in the wild. However, viral entities and viral diseases of YFPs remain obscure. In this study, anal swabs for virome analysis were collected during the physical examination of YFPs in the Tian-E-Zhou Oxbow (TEO) ex situ reserve. A total of 19 eukaryotic viral species belonging to 9 families, including Papillomaviridae, Herpesviridae, Picornaviridae, Picobirnaviridae, Caliciviridae, Retroviridae, Parvoviridae, Virgaviridae, and Narnaviridae, and other unclassified viruses were identified based on metasequencing. Among these detected viruses, a novel herpesvirus (NaHV), two different kobuviruses (NaKV1-2), and six different papillomaviruses (NaPV1 to -6) were considered potential risks to YFPs and confirmed by PCR or reverse transcription-PCR (RT-PCR). Most YFPs sampled were found to harbor one or more kinds of detected viral genomes (52/58 [89.7%]). Surveillance results demonstrated that kobuvirus and herpesvirus displayed obvious age distribution and PVs showed significant gender difference in YFPs. According to species demarcation criteria in individual genera in Papillomaviridae, two novel species (referred to as Omikronpapillomavirus 2 and 3) and four novel isolates of PV were identified in YFPs. Further evolutionary analysis suggested that NaPVs would occupy the mucosal niche and that virus-host codivergence mixed with duplications and host-switching events drives the evolution of cetacean PVs. Divergence times of PVs in YFP and other cetacean reflect the incipient speciation of YFPs. In summary, our findings revealed the potential viral entities, their prevalence, and their evolutionary history in YFPs, which raises an important issue regarding effects of viral infection on the fitness of YFPs. IMPORTANCE The Yangtze finless porpoise (YFP) is the only cetacean species in freshwater following the functional extinction of the baiji (Lipotes vexillifer). Health management, disease treatment, and other special measures are important for maintaining the existing YFP populations, especially in in situ and ex situ reserves. The discovery of potential viral entities and their prevalence in YFPs raises an important issue regarding the effects of viral infection on the fitness of YFPs and may contribute to the conservation of YFPs. The evolutionary history of papillomaviruses in YFP and other cetaceans reflects the phylogeny of their hosts and supports the status of incipient species, opening a window to investigate the evolutionary adaptation of cetaceans to freshwater as well as their phylogeny to remedy the deficiency of fossil evidence.

Accumulation and risk prioritization of psychoactive substances in the critically endangered Yangtze finless porpoise.

Psychoactive substances have been identified as a kind of emerging contaminants in aquatic environment and pose potential adverse effects on aquatic animals. Yangtze finless porpoise, a critically endangered species in China, is also facing the threat of psychoactive substances. In this study, the accumulation characteristics and risk prioritization of psychoactive substances were investigated in Yangtze finless porpoise collected from Poyang Lake (PYL) and Tian-E-Zhou Oxbow (TZO) in Yangtze River basin. The levels of psychoactive substances were detected in the range of below method detection limits (MDLs) to 98.4 ng/mL in the serum of Yangtze finless porpoise. Codeine (COD) and methamphetamine were identified as the major substances due to the highest residual levels with a median concentration of 0.72 ng/mL and 0.33 ng/mL, respectively. The total concentrations of psychoactive substances in the porpoise collected from TZO was significantly higher than those from PYL. Risk analysis based on effect ratio derived from the ratio of steady-state psychoactive substance serum concentration in the porpoise and human therapeutic plasma concentration revealed that COD was the substance with the highest risk among the psychoactive substances detected, followed by lysergic acid diethylamide (LSD), morphine, alprazolam (ALPZ) and lormetazepam. Location-specific risk prioritization of psychoactive substances found that the top 3 substances are LSD, lorazepam (LORZ) and ALPZ in PYL, and COD, LSD and LORZ in TZO. The results disclose the accumulation of psychoactive substances in Yangtze finless porpoise and suggest that the potential adverse effects should be concerned.

Reliability of caretakers' ratings of captive bottlenose dolphins (Tursiops truncatus) and finless porpoises' (Neophocaena asiaeorientalis sp.) behavioral style for welfare monitoring.

Qualitative ratings are increasingly used to assess animal welfare. We investigated the associations between daily caretakers' ratings of individuals' behavioral style, behavior during free time, participation during training sessions, and environmental variables in captive Yangtze finless porpoises, East-Asian finless porpoises, and bottlenose dolphins. Associations between caretakers' ratings and behavior were found, including social swimming, fast swimming, play, interaction with humans, and agonistic and socio-sexual interactions. Ratings were also associated with participation during training sessions and food intake. Social separation, transport to a new pool, high visitors' density, and holding of a sick conspecific were associated with ratings indicating a deteriorated human-animal relationship, a lower interest/fear of the environment, and/or a higher social cohesion depending on the group. Through principal component analyses, new variables that represented both caretakers' ratings and behavior and that could be used for an easier interpretation of the data for welfare monitoring within each group were extracted. Caretaker ratings and behavior should be used as a complement to previously validated indicators to detect changes in welfare state and understand the potential causes of these changes.

Biofilm formation, antimicrobial assay, and toxin-genotypes of Clostridium perfringens type C isolates cultured from a neonatal Yangtze finless porpoise.

This is a culture-dependent study with the objective of pure culturing and characterizing pathogenic bacteria from the blowhole, lung, stomach and fecal samples of a neonatal crucially endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) that died 27 days after birth. Bacteria were inoculated using a swab onto blood and MacConkey agar plates and representative isolates were identified through 16S rRNA gene sequence analysis. A total of three Clostridium perfringens type C strains from the fecal samples were isolated. Toxin genes, including cpa, cpb and cpb2, were detected by PCR amplification, whereas the etx, iap and cpe genes were not detected. Biofilm formation of the three strains was then examined. Only one strain was capable of biofilm formation. In addition, isolates showed strong resistance against the antibiotics amikacin (3/3), erythromycin (1/3), gentamicin (3/3), streptomycin (3/3), and trimethoprim (3/3), while sensitivity to ampicillin (3/3), bacitracin (3/3), erythromycin (2/3), penicillin G (3/3), and tetracycline (3/3). The results suggested C. perfringens type C could have contributed to the death of this neonatal porpoise.

Seasonal Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) movements in the Poyang Lake, China: Implications on flexible management for aquatic animals in fluctuating freshwater ecosystems.

Identifying seasonal high-use habitats and migration corridors is the basis for migratory species conservation. Previous studies have focused on Marine Protected Areas, while freshwater ecosystems, often accompanied by severe hydrological fluctuations, suggest new perspectives for flexible management. Poyang Lake is an essential habitat for the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, YFP), supporting almost half of their natural population. However, studies on the movement patterns and habitat preferences of YFPs still lack there, preventing potential protected areas' effectiveness. In this study, we conducted 10 surveys for the YFP population in the main body and tributaries of Poyang Lake from October 2018 to August 2020. We used habitat modeling to analyze their seasonal habitat use and the environmental predictors. YFPs showed a seasonal movement pattern consistent with water level fluctuations. They entered the tributaries from the main lake body in low and medium water levels and returned to the main lake during high water level periods. The water depth was the most important environmental variable in each hydrological season that affects YFPs' habitat selection. The suitable water depth was about 4-8 m during the low water level, 6-12 m during the medium water level, and 7-20 m during the high water level. YFPs' 50% core habitats distribution showed noticeable seasonal changes. The proportion of their suitable habitats in the whole lake was relatively low, which was highest during high water level (16.89%), and the weakest during low water level (12.11%). Considering the seasonal movements of the YFPs in the lake, we recommend flexible management measures for their core habitats to alleviate human interference and restore their movement rhythm between the river and the lake, which shed light on protected area management for aquatic animals in such seasonal fluctuating habitats.

Blubber fatty acid compositions in different geographic populations of finless porpoise in Chinese waters: implications for thermal adaptation.

The stratification of blubber fatty acids (FAs) is a widely used strategy in marine mammals and can be influenced by many factors, including their diet, environmental temperature, and physiological status. There are 3 distinct finless porpoise species/subspecies in Chinese coastal and inland waters in a wide range from 20°N to 40°N. The biochemical stratification of the blubbers of finless porpoises in different regions may provide valuable information for understanding their environmental adaptations. The FA compositions of 4 geographic populations of finless porpoise (FP) collected from the Bohai Gulf, East China Sea, South China Sea, and Yangtze River were therefore analyzed and compared. The blubber FA compositions of finless porpoises were dominated by ΣMUFAs, followed by ΣSFAs and ΣPUFAs, and were generally consistent with those of other cetaceans. The blubber of finless porpoises was significantly stratified with increasing levels of ΣMUFAs and decreasing levels of ΣSFAs and ΣPUFAs from the inner to the outer layers. The 3 marine populations shared more similarities in their FA compositions and stratifications compared to the freshwater porpoises, particularly regarding the PUFAs in the inner layers, which might have been mainly influenced by the diet compositions of marine and freshwater porpoises. Contrary to what was expected, in the 3 marine populations, the SFA and MUFA levels showed opposite correlations (negative and positive, respectively) with habitat temperatures, possibly indicating an adaptation mechanism in finless porpoises characterized by a trade-off between the insulation and fluidity properties of the blubber through the adjustment of the compositions and gradients of MUFAs and SFAs across the blubber depth in response to environmental temperature/latitude changes.

The Click Production of Captive Yangtze Finless Porpoises (Neophocaena asiaeorientalis asiaorientalis) Is Influenced by Social and Environmental Factors.

Yangtze finless porpoises use high-frequency clicks to navigate, forage, and communicate. The way in which click production may vary depending on social or environmental context has never been investigated. A group of five captive Yangtze finless porpoises was monitored for one year, and 107 h of audio recordings was collected under different conditions. Using a MATLAB-generated interface, we extracted click density (i.e., number of clicks per minute) from these recordings and analyzed its variation depending on the context. As expected, click density increased as the number of animals present increased. The click density did not exhibit diurnal variations but did have seasonal variations, with click density being highest in summer and fall. Yangtze finless porpoises produced more clicks when socially separated than when not (136% more), during training/feeding sessions than outside of such sessions (312% more), when enrichment was provided (265% more on average), and when noisy events occurred rather than when no unusual event occurred (22% more). The click density decreased when many visitors were present in the facility (up to 35% less). These results show that Yangtze finless porpoises modulate their click production depending on the context and suggest that their echolocation activity and their emotional state may be linked to these changes. Such context-dependent variations also indicate the potential usefulness of monitoring acoustical activity as part of a welfare assessment tool in this species. Additionally, the click density variation found in captivity could be useful for understanding click rate variations of wild populations that are hardly visible.

Community-based population monitoring for large baleen whales: the case study of Bryde's whale in Beibu Gulf of China.

Citizen science is a popular method for the long-term monitoring of the distribution of wild animals. The application of these methods in different species and environments still poses challenges, especially for aquatic animals. In this study, we investigated the distribution of the Bryde's whale (Balaenoptera edeni) in the Beibu Gulf of China by using scientific boat-based surveys and citizen science approaches using 2 different methods of data collection. First, we built our mobile app (Whale Guard) and installed it on fishermen's phones. Second, we used a popular instant messaging app (WeChat) to create an online fisherman community. We found that the mobile phone app collected far fewer reports (5 reports) than the online community group (42 reports, P < 0.01). By using a variety of incentives, we maintained the fisherman's community's activity without significant user loss (P < 0.01). We also found that the locations collected by social media applications in this study were consistent with observations from scientific boat-based transect surveys. The sightings distribution of Bryde's whales differed from those in previous surveys in that they were present across larger areas. Social media apps reported that 69% of reports was confirmed by more than one person, whereas Whale Guard reports were much lower (0, P <0.001). Community-based citizen science can greatly contribute to the long-term monitoring of Bryde's whales, and it has successfully overcome the challenges of data accessibility, accuracy, and fragmentation. Our study shows how to appropriately use citizen science in different community groups and community-based approaches make them useful for large baleen whale surveys.

Thresholds of population persistence for the Yangtze finless porpoise: implications for conservation managements.

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis), a critically endangered species, is the only cetacean species in the Yangtze River following the functional extinction of baiji (Lipotes vexillifer). To inform conservation actions, two important questions need to be addressed: what is the threshold value of survival rate, and what is the threshold value of population size? We calculate the instantaneous rate of population increase ( r ¯ ) for the Yangtze finless porpoise for various combinations of the calf and the non-calf survival rates. We also test the probability of extinction for different minimum carrying capacities for 100 and 500 years using a stable population model. The threshold value of the non-calf survival rate is never lower than 0.869, but current estimates from field data have been far below this threshold. Our model based on extinction probability and carrying capacity suggests that the threshold for the population size to persist 100 years required 113 animals, and 472 animals are required to persist 500 years. Therefore, we recommend establishing an ex situ reserve network to guarantee the minimum 100-year carrying capacity. To ensure the long-term population viability, we suggest establishing two in situ reserve zones in two lakes and their surrounding reserves to meet a minimum 500-year carrying capacity. In addition, measures to avoid further habitat fragmentation should be priority.

Sexual maturity, seasonal estrus, and gestation in female Indo-Pacific bottlenose dolphins Tursiops aduncus inferred from serum reproductive hormones.

Reproductive hormones in serum concentrations of progesterone, estradiol, and testosterone in female Indo-Pacific bottlenose dolphins (Tursiops aduncus, n = 12) housed in Ocean Park Hong Kong were investigated in the present study. Results showed that, onset of puberty of captive Indo-Pacific bottlenose dolphins was at 5 years while sexual maturity was at 6. Average serum progesterone concentrations in non-pregnant sexually mature individuals was 0.33 (0.25-0.97) ng/mL (interquartile), significantly higher than in immature ones 0.26 (0.25-0.38) ng/mL. This study found significant difference in serum estradiol concentrations between individuals at the onset of puberty (9.5 ± 1.7 pg/mL, ±SD) and not (below detection limit 9 pg/mL). A slightly seasonal breeding pattern, with progesterone values tend to be higher from February to October (0.38 [0.25-1.07] ng/mL) was inferred. During pregnancy, serum progesterone concentrations range from 10.54 ± 8.74 ng/mL (indexed month post-conception [IMPC] 0) to 25.49 ± 12.06 ng/mL (IMPC 2), and display a bimodal pattern with 2 peaks in early- (25.49 ± 12.06 ng/mL, IMPC 2) and late-pregnancy (21.71 ± 10.25 ng/mL, IMPC 12), respectively. Serum estradiol concentrations can seldom be detected in early-pregnancy and increase constantly in mid- (9.45 ± 1.83 pg/mL) and late-pregnancy (11.88 ± 3.81 pg/mL), with a spike (15.45 ± 6.78 pg/mL) 1 month prior to delivery. Serum testosterone concentrations elevate significantly in IMPC 7 (0.36 ± 0.10 ng/mL) compared to other months (0.16 ± 0.10 ng/mL) of the year. The present study provides normal concentration profiles for some reproductive hormones in female Indo-Pacific bottlenose dolphins and can contribute to the breeding monitoring of this species. Also, our study would shed further light on the reproductive physiology of small cetaceans.

Contextual impacts on individual and synchronous breathing rate variations in three captive odontocete groups.

Breathing rates are often collected both in the wild and in captivity to inform on cetaceans' internal state. However, few studies have investigated the effect of various factors on this breathing rate. We investigated the variations of individual and synchronous breathing rates depending on individual features (species, sex, age), displayed behavior, social parameters (social grouping), and environmental parameters (diurnal variation, presence of enrichment, unusual events, and presence of visitors in three groups of captive odontocetes (Yangtze finless porpoises, Neophocaena asiaeorentalis asiaeorientalis, East-Asian finless porpoises, Neophocaena asiaeorentalis sunameri, bottlenose dolphins, Tursiops truncatus). Both individual and synchronous breathing rates were the highest when animals engaged in energetic or social behaviors. Individual breathing rate decreased but synchronous breathing rate increased with the presence of enrichment. Both rates increased during unusual events (e.g., pool cleaning, presence of a diver in the pool, noise, transport) and when public was present for Yangtze finless porpoises. Finally, synchronous breathing rate increased for Yangtze finless porpoises when experiencing social separation. We suggest that individual and synchronous breathing rates are useful parameters to measure, both in wild and captive animals, to obtain information on their arousal/stress state. However, these rates should be interpreted with caution and should be used together with other parameters to allow more accurate inferences.

Pneumonia in endangered aquatic mammals and the need for developing low-coverage vaccination for their management and conservation.

Anthropogenic activities can lead to several devastating effects on the environment. The pollutants, which include the discharge of effluents, runoffs in the form of different lethal and sub-lethal concentrations of pesticides, heavy metals, and other contaminants, can harm exposed fauna and flora. The aquatic environment is the ultimate destination for many pollutants which negatively affect aquatic biodiversity and even can cause a species to become extinct. A pollutant can directly affect the behavior of an animal, disrupt cellular systems, and impair the immune system. This harm can be reduced and even mitigated by adopting proper approaches for the conservation of the target biota. Among aquatic organisms, cetaceans, such as the Yangtze finless porpoise, Irrawaddy dolphin, Ganges River dolphin, Amazon River dolphin, and Indus River dolphin, are at a higher risk of extinction because of lack of knowledge and research, and thus insufficient information with respect to their conservation status, management, and policies. Pneumonia is one of the leading causes of mass mortalities of cetaceans. This article reviews the limited research reported on stress and pneumonia induced by pollution, stress-induced pneumonia and immunosuppression, pneumonia-caused mass mortalities of aquatic mammals, and vaccination in wildlife with a specific focus on aquatic mammals, the role of genomics in vaccine development and vaccination, and the major challenges in vaccine development for biodiversity conservation.

Body Contacts and Social Interactions in Captive Odontocetes Are Influenced by the Context: An Implication for Welfare Assessment.

Research on the welfare of captive odontocetes has increased in recent years, but has been mostly focused on bottlenose dolphins (Tursiops truncatus). Few studies investigated potential welfare indicators using quantitative data linked to a range of conditions or stimuli that are thought to impact the animals' emotional state. Since odontocetes are social animals that engage in various social interactions, these interactions might inform us on their welfare state. We investigated pectoral contact laterality and the effect of the context on several social behaviors in three groups of captive odontocetes (Yangtze finless porpoises, YFPs: Neophocaena asiaeorientalis asiaeorientalis; East-Asian finless porpoises, EAFPs: N. a. sunameri, and bottlenose dolphins, BDs). Animals exhibited patterns depending on the time of the day for most of the social behaviors we analyzed; social separation was associated with lower rates of social behaviors for the two analyzed groups (YFPs and BDs), the accessibility to several pools was associated with higher rates of social behaviors for BDs. The effect of enrichment, disturbances and public presence was less clear and strongly depended on the group, the type of enrichment and disturbance. Our results confirm that captive odontocetes' social behaviors are influenced by the context, and that, depending on the group, some of them, such as pectoral contacts, other body contacts, agonistic interactions or social play exhibit consistent patterns across contexts. Monitoring these behaviors might be useful to adapt the captive management to each species and group. The different responses among the three studied groups confirm that species and groups react differently to a stimulus and therefore, management decisions should be species/group specific. We recommend that more studies should be conducted to validate our findings in other groups of odontocetes under human care.

First evaluation of legacy persistent organic pollutant contamination status of stranded Yangtze finless porpoises along the Yangtze River Basin, China.

Yangtze finless porpoises (Neophocaena asiaeorientalis asiaeorientalis), inhabiting the Yangtze River, are an endangered species in China. They are threatened by various kinds of pollutants, among which persistent organic pollutants (POPs) are of special concern due to their toxicities, high persistency and bioaccumulation potential. To better understand the POP contamination status of Yangtze finless porpoises, an investigation of stranded porpoises along the Yangtze River and adjacent two major lakes in the Yangtze River basin was conducted; the concentrations of four groups of legacy POPs, i.e., hexachlorocyclohexanes (HCHs), dichlorodiphenyltrichloroethanes (DDTs), polybrominated diphenyl ethers (PBDEs), and polychlorinated biphenyl (PCBs), were determined in the blubber samples. The mean concentrations of ΣHCHs (the sum of all congeners/isomers), ΣDDTs, ΣPBDEs and ΣPCBs, were 1670 ± 4210, 28,800 ± 52,300, 141 ± 174, and 1020 ± 1070 ng/g lipid weight, respectively; the high DDTs/PCBs ratio reflected a strong influence of agricultural pollution in the Yangtze River basin, and the high α/γ ratio of HCH isomers indicated the usage of lindane in the corresponding areas; the predominance of low-brominated congeners of PBDEs may be related to congruent patterns in the related environmental matrices. A hazard quotient risk assessment revealed that DDTs could pose a relatively high risk to Yangtze finless porpoises compared with the risks posed by the other POPs.

Swimming features in captive odontocetes: Indicative of animals' emotional state?

Captive welfare studies in odontocete species have been mostly conducted on bottlenose dolphins (Tursiops truncatus) while the welfare of many other species' -including endangered species- remains poorly studied. More research is needed to find and validate potential indicators of welfare for each species and even for each group. Since captive odontocetes spend most of their time swimming, their swimming features are interesting to study in relation to their welfare state. We first analysed the circular swimming direction bias in three groups of captive odontocetes (Yangtze finless porpoises: Neophocaena asiaeorientalis asiaeorientalis; East-Asian finless porpoises: N. a. sunameri; and bottlenose dolphins, Tursiops truncatus). Second, we studied the effect of environmental and social factors (i.e., time of the day, delay to training, enrichment, potential perturbation, social grouping, public presence and housing pool) on circular swimming, fast swimming, group swimming, synchronous swimming and contact swimming in the three groups. Yangtze finless porpoises exhibited a clockwise swimming bias while East-Asian finless porpoises and bottlenose dolphins swam significantly more in the counter-clockwise direction. Each studied factor significantly impacted the animals' swimming behaviour slightly differently depending on the group. However, some patterns were common for the three groups: animals seemed to be more active in the morning than at noon and in the afternoon, and enrichment seemed to decrease circular swimming, fast swimming and social swimming (i.e., synchronous, contact and group swimming), while potential perturbations (e.g., pool cleaning, noise) seemed to increase it. In addition, behaviour differed for Yangtze finless porpoises and bottlenose dolphins right before the training or when other animals were being trained, suggesting an anticipation of this event or an excited/frustrated state in this context. Social separation also impacted these animals' swimming behaviour with less group swimming but more circular swimming, synchronous swimming and fast swimming when separated. The housing pool had an impact on bottlenose dolphins' behaviour with more circular swimming, more fast swimming and less group swimming when having access to a larger space. The effect of the presence of public was unclear and requires further investigation. From our results, we propose that circular swimming, synchronous swimming and contact swimming could be useful to monitor animals' emotional state, but that additional parameters should be added (e.g., swimming speed) since these behaviours can be expressed both in quiet and relaxed contexts and in stressful ones. In addition, fast swimming can be a useful indicator of stress for porpoises but might be more ambiguous for bottlenose dolphins that engage in intense social play bouts for instance. Finally, group swimming might be a good behaviour to monitor when wanting to investigate reactions to various conditions or events that can potentially be stressful. We suggest that further research should be conducted on other groups of odontocetes to validate our findings.

Non-invasive Fecal Steroid Measurements for Monitoring the Reproductive Status of a Critically Endangered Yangtze Finless Porpoises (Neophocaena asiaeorientalis asiaeorientalis).

Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is a critically endangered freshwater cetacean dwelling in the Yangtze River and its adjoining lakes. Affected mainly by the various anthropogenic activities in this region, its population decreased dramatically in the past two decades. To protect this animal from extinction, captive breeding program is an important way to provide basic knowledge for wild population conservation. Non-invasive fecal steroid radioimmunoassay technique was validated in three captive Yangtze finless porpoises for the first time in this study. The seasonality of one captive male and the reproductive status of two females were investigated by longitudinal monitoring their fecal reproductive steroid hormone metabolites. Pregnancy could be diagnosed by an abrupt increase in fecal progesterone metabolites. In late pregnancy (4 months before birth), a significant decrease of fecal progesterone metabolites was observed, which might be referenced for the expectation of parturition date. Seven estrous cycles were recognized in one breeding season of a non-pregnant female judged by the variation of fecal progesterone metabolite levels. The fecal progesterone metabolite level was proved a reliable and precise indicator for estrus and pregnancy diagnosis.

Putative virulence factors of Plesiomonas shigelloides.

Plesiomonas shigelloides is a Gram-negative rod-shaped bacterium which has been isolated from humans, animals and the environment. It has been associated with diarrhoeal disease in humans and various epizootic diseases in animals. In this study P. shigelloides strains were isolated from the faecal material of a captive Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis; YFP) living in semi-natural conditions in China. Plesiomonas shigelloides strain EE2 was subjected to whole genome sequencing. The draft genome was then compared to the genome sequences of ten other P. shigelloides isolates using the Pathosystems Resource Integration Center pipeline. In addition to several virulence factors which have been previously reported, we are proposing new candidate virulence factors such as a repeats-in-toxin protein, lysophospholipase, a twin-arginine translocation system and the type VI secretion effector Phospholipase A1.

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.