Yujiang Hao

B-Mode ultrasonographic evaluation of the testis in relation to serum testosterone concentration in male Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) during the breeding season

The use of ultrasonography as a noninvasive tool for assessing the reproductive status of the male Yangtze finless porpoise (YFP; Neophocaena phocaenoides asiaeorientalis) was validated by correlating ultrasonographically determined testicular volume (TV) and testicular parenchyma pixel intensity (PI) with serum testosterone (T) concentration. The testes of 13 free-ranging male YFPs from the Tian-e-Zhou Reserve and three captive animals from the Baiji Dolphinarium (Wuhan, China) were examined ultrasonographically during April 2008. Testis volume was determined using Lamberts formula for an ellipsoid. Testicular parenchyma PI was evaluated by analyzing testicular ultrasonograms using pixel analysis software (Image J). Serum T concentrations were determined using a single-antibody radioimmunoassay. The TV, PI, and serum T concentration were low and similar in animals with body length <133 cm, highest in those with body length >or=142 cm, and highly variable in those with body length from 133 to 141 cm. Both TV and PI were closely correlated with serum T concentration (r=0.91 and r=0.85, respectively; P<0.01), indicating a consistent association between structural and functional development of the testis. In conclusion, we inferred that puberty onset in male YFPs occurred when TV was >150 cm(3) and PI was >60 during the breeding season and that testicular ultrasonography and pixel analysis was an efficient, noninvasive, real-time tool to evaluate testicular function of live male YFPs.

Variation in sexual behaviors in a group of captive male Yangtze finless porpoises (Neophocaena phocaenoides asiaeorientalis): motivated by physiological changes?

Most male mammals in temperate regions demonstrate seasonal sexual behaviors that coincide with seasonal variations in gonadal activities and androgen hormones. The Yangtze finless porpoise is a temperate freshwater cetacean species and an obvious seasonal breeder. To investigate the relationship between sexual behavior and gonadal activity in this animal, testicular size (volume) and structure (ultrasonogram pixel intensity) of two adult male porpoises (AF, AB) and one sub-adult male (TT) were longitudinally monitored from November 2008 to November 2009. Serum testosterone concentration was also monitored during the same period. Variations in the frequency of sexual behavior in AF and AB had similar, but seasonal trends. Their testicular size and pixel intensity also varied seasonally. Testicular size increased in March, peaked from April through June, and decreased gradually from August through September, whereas testicular pixel intensity started to increase in early February. The frequency of sexual behavior was positively correlated with testicular volume and pixel intensity (P = 0.000018 and P = 0.00012, respectively) in AF. Serum testosterone concentrations also varied. The sub-adult male porpoise, TT, was undergoing puberty, as evidenced by its marked increase in testicular volume, testicular pixel intensity, and serum testosterone concentrations from the beginning of 2009. Interestingly, TT exhibited the highest frequency of sexual behavior, most of which was same-sex pairing. However, its oversexed behavior neither quantitatively correlated with its smaller testicular volume (P = 0.61) nor with its testicular pixel intensity (P = 0.69).

Some observations on the sociosexual behavior of a captive male Yangtze finless porpoise calf (Neophocaena phocaenoides asiaeorientalis)

Sociosexual behavior in a captive male Yangtze finless porpoise calf (Neophocaena phocaenoides asiaeorientalis) was studied during its first year postpartum. This behavior was first observed at 1 month postpartum. During the year, (1) each event of the sociosexual behavior typically lasted 6–9 s and the frequency was 2.8 occurrences h−1; (2) ventral–ventral was the predominant sociosexual gesture; (3) prior to sociosexual interactions, the calf generally bonded with the receiver; (4) the mother was his preferred sociosexual partner but other adults of both sexes were also involved; (5) the frequency of sociosexual behavior decreased until the 9th month postpartum and increased thereafter.

Fecal Bacterial Composition of the Endangered Yangtze Finless Porpoises Living Under Captive and Semi-natural Conditions

Intestinal microbiota is essential to the health and physiology of host animals. We undertook the first microbiological study of the fecal bacterial composition from critically endangered (CR) Yangtze finless porpoises (Neophocaena asiaeorientalis asiaeorientalis; YFPs) living under captive and semi-natural conditions using both high-throughput sequencing method and 16S rRNA gene clone library method. As determined by high-throughput sequencing of V3–V4 regions of the 16S rRNA gene, semi-natural samples harbored 30 and 36 operational taxonomic units (OTUs), which was more than the 22 and 27 OTUs detected from YFPs living in captivity. In captive YFPs Firmicutes was the predominant phylum, whereas this was Proteobacteria for YFPs living in semi-nature conditions. This suggests habitat-specific fecal bacterial composition of YFPs. Plesiomonas spp. and Aeromonas spp., which are potentially pathogenic, were identified in all the feces. Bacterial diversity from one porpoise living in captivity was also determined by constructing a 16S rRNA gene clone library and only 1 phylum was identified. High-throughput sequencing was more effective at determining the bacterial diversity compared to the 16S rRNA gene clone library. This study provides important information for the management and conservation of the CR YFPs.

De Novo Assembly and Characterization of Narrow-Ridged Finless Porpoise Renal Transcriptome and Identification of Candidate Genes Involved in Osmoregulation

During the evolutionary transition from land to water, cetaceans have undergone numerous critical challenges, with osmoregulation being the major one. Two subspecies of the narrow-ridged finless porpoise (Neophocaena asiaeorientalis), the freshwater Yangtze finless porpoise (N. a. asiaeorientalis, NAA) and the marine East Asian finless porpoise (N. a. sunameri, NAS), provide excellent subjects to understand the genetic basis of osmoregulatory divergence between freshwater and marine mammals. The kidney plays an important and well-established role in osmoregulation in marine mammals and thus, herein, we utilized RNA-seq to characterize the renal transcriptome and preliminarily analyze the divergence between the NAA and the NAS. Approximately 48.98 million clean reads from NAS and 49.40 million clean reads from NAA were obtained by RNA-Seq. And 73,449 (NAS) and 68,073 (NAA) unigenes were assembled. Among these annotations, 22,231 (NAS) and 21,849 (NAA) unigenes were annotated against the NCBI nr protein database. The ion channel complex GO term and four pathways were detected as relevant to osmoregulation by GO and KEGG pathway classification of these annotated unigenes. Although the endangered status of the study species prevented analysis of biological replicates, we identified nine differentially expressed genes (DEGs) that may be vital in the osmoregulation of the narrow-ridged finless porpoise and worthwhile for future studies. Of these DEGs, the differential expression and distribution of the aquaporin-2 (AQP2) in the collecting duct were verified using immunohistochemical experiments. Together, this work is the first report of renal transcriptome sequencing in cetaceans, and it will provide a valuable resource for future molecular genetics studies on cetacean osmoregulation.

Concentrations of osmotically related constituents in plasma and urine of finless porpoise (Neophocaena asiaeorientalis): implications for osmoregulatory strategies for marine mammals living in freshwater

BackgroundMost cetaceans inhabit the hyperosmotic marine environment with only a few species living in freshwater habitats. The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is the only freshwater subspecies of the genus. Our aim was to study whether the osmoregulation mechanism of the Yangtze finless porpoise is different from the marine subspecies, the East Asian finless porpoise (Neophocaena asiaeorientalis sunameri). We assayed and compared the concentrations of the constituents involved in osmoregulation in the blood and urine in the Yangtze finless porpoise and the East Asian finless porpoise. We also compared the corresponding urine constituents of the porpoises with existing data on fin whales (Balaenoptera physalus) and bottlenose dolphins (Tursiops truncatus).ResultsThe mean plasma osmolality of Yangtze finless porpoise was significantly lower than that of the marine subspecies (P < 0.01). Similarly, the urine osmolality of Yangtze finless porpoise was also significantly lower than that of its marine counterpart (P < 0.05). However, the urine sodium concentration of freshwater finless porpoise was significantly lower than that in the marine subspecies (P < 0.01), even though their serum sodium has no significant difference. Moreover, the freshwater porpoise has significantly lower urine urea concentration but much higher serum urea than in the marine finless porpoise (P < 0.05).ConclusionsThese results suggest that the freshwater finless porpoise does have different osmoregulatory mechanism from marine cetaceans. Conserving sodium by excreting urine with low ion levels may be an essential strategy to maintain the serum electrolyte balance for the freshwater subspecies that also appears to be more susceptible to hyponatremia.

Physiological responses to capture and handling of free-ranging male Yangtze finless porpoises (Neophocaena phocaenoides asiaeorientalis)

We captured free-ranging male Yangtze finless porpoises over three seasons and assayed leukocytes and serum biochemistry to investigate physiological responses to the capture and handlings. Serum thyroid hormones (THs) declined sharply in those porpoises compared with hormone variation in a captive male finless porpoise. Hypernatremia and hypokalemia were also significant in the free-ranging animals suggesting that conservation of serum sodium might be acutely vital for this freshwater subspecies. The animals captured in spring showed more significant neutrophilia and eosinopenia than those captured in autumn suggesting that they may be more affected by capture during the breeding season. Furthermore, physical examination of porpoises when out of the water was apparently stressful, particularly when they were kept out of the water for longer periods. However, an increase in circulating THs may be an adaptive response to accommodate these short-term stresses.

Topographical distribution of blubber in finless porpoises (Neophocaena asiaeorientalis sunameri): a result from adapting to living in coastal waters

BackgroundBlubber has many functions, among which energy storage, thermoregulation, buoyancy, and hydrodynamic streamlining are the most frequently cited. Within and between taxa, variations in its structure and distribution likely reflect different adaptations of a species to its life history requirements, environment, health, and function. Here, we use ultrasound to describe the distribution of blubber in the finless porpoise (Neophocaena asiaeorientalis sunameri) based on examinations of 34 fresh cadavers recovered as accidental fisheries bycatch.ResultsMeasurements of blubber depth determined by ultrasound positively correlated with conventional measurements using a scalpel and calipers. Whereas conventional surgical incision and visual examination revealed two layers of blubber, ultrasound revealed up to three layers; thus, ultrasound reveals additional structural detail in blubber while crude necropsy techniques do not. Across life history categories, ultrasound revealed the distribution of inner blubber to be topographically consistent with that of full-depth blubber. Blubber in the dorsal region was stratified into three layers and was significantly thicker than that in the lateral and ventral regions, in which a middle layer was normally absent.ConclusionsUltrasound provides a fast, effective, and accurate means to determine blubber thickness and structure, and thus, assessment of the health of fresh finless porpoise carcasses. Blubber depth is determined largely by the thickness of the inner and middle layers, wherein lipids are concentrated. The thickening of blubber in the dorsal thoracic-abdominal region suggests multiple roles of thermal insulation, lipid storage, and, we speculate, to facilitate vertical stability in the complex shallow and estuarine waters in which this animal absent of a dorsal fin occurs.

Physicochemical Evolution and Molecular Adaptation of the Cetacean Osmoregulation-related Gene UT-A2 and Implications for Functional Studies

Cetaceans have an enigmatic evolutionary history of re-invading aquatic habitats. One of their essential adaptabilities that has enabled this process is their homeostatic strategy adjustment. Here, we investigated the physicochemical evolution and molecular adaptation of the cetacean urea transporter UT-A2, which plays an important role in urine concentration and water homeostasis. First, we cloned UT-A2 from the freshwater Yangtze finless porpoise, after which bioinformatics analyses were conducted based on available datasets (including freshwater baiji and marine toothed and baleen whales) using MEGA, PAML, DataMonkey, TreeSAAP and Consurf. Our findings suggest that the UT-A2 protein shows folding similar to that of dvUT and UT-B, whereas some variations occurred in the functional So and Si regions of the selectivity filter. Additionally, several regions of the cetacean UT-A2 protein have experienced molecular adaptations. We suggest that positive-destabilizing selection could contribute to adaptations by influencing its biochemical and conformational character. The conservation of amino acid residues within the selectivity filter of the urea conduction pore is likely to be necessary for urea conduction, whereas the non-conserved amino acid replacements around the entrance and exit of the conduction pore could potentially affect the activity, which could be interesting target sites for future mutagenesis studies.

Effect of Photoperiod Extension on the Testicular Sonographic Appearance and Sexual Behavior of Captive Yangtze Finless porpoise (Neophocaena asiaeorientalis asiaeorientalis)

Xueying Yu, Yujiang Hao, Brian CW Kot, and Ding Wang (2016) Photoperiod plays an important role in the seasonal regulation of reproduction in mammals. In the present study, we examined the effect of an extended photoperiod (light/dark: 14 h/10 h) during the usual sexually inactive phase (Jan-Mar) on testicular sonographic appearance and sexual behaviors of two captive Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, YFP). During the extended photoperiod treatment, the parenchyma pixel intensity, cross- sectional area of testicular sonographic images, as well as the frequency of sexual behaviors of the two male porpoises increased significantly compared to the control treatment. Parenchyma pixel intensity, the cross- sectional area of testicular sonographic images and the frequency of sexual behaviors of the two captive males all increased in response to extended photoperiod. These results may suggest that photoperiod is a potent factor regulating the reproductive seasonality in YFPs.