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.

Microbial communities in different regions of the gastrointestinal tract in East Asian finless porpoises (Neophocaena asiaeorientalis sunameri).

Mammalian gastrointestinal (GI) tract microbial communities are critical for host health. However, the microbiota along the GI tract in cetaceans has not been well characterized compared to other animals. In this study, the bacteria and fungi present in the stomach, foregut, hindgut and feces, of East Asian finless porpoises (Neophocaena asiaeorientalis sunameri, EAFPs) were characterized using high-throughput sequencing analysis. The bacterial and fungal diversity and richness in the stomach, hindgut and fecal samples tended to be higher than those in the foregut. Bacterial taxonomic compositions found in the hindgut and feces were different from those seen in the stomach and foregut. A greater proportion of strict anaerobic bacteria including Clostridia, Fusobacteria, and Ruminococcaceae were found in the hindgut and fecal samples. The fungal communities present in stomach samples differed from those detected in other regions to some extent. Zygomycota and Neocallimastigomycota were more predominant in the stomach. Some potential pathogens, such as Helicobacter spp. and Vibrio spp., were commonly present along the GI tract. Our study confirms that the fecal microbiota can represent the whole GI tract to some extent because of their relatively higher microbial diversity and presence of potential pathogens. Our study provides the first comprehensive characterization of the EAFPs GI microbiota, expanding on the current knowledge about the bacterial diversity in the GI tract of cetaceans. In addition, this is the first study characterizing the fungal diversity of any species of porpoise.

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.

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).

Serum concentrations of gonadotropins and steroid hormones of Neophocaena phocaenoides asiaeorientalis in middle and lower regions of the Yangtze River.

As a relatively isolated and unique freshwater population, the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) is the most endangered subpopulation of this species. The objective of this study was to improve our understanding of their reproductive endocrinology by measuring (radioimmunoassay) serum concentrations of FSH, LH, estradiol (E(2)), progesterone (P(4)) and testosterone (T(2)) in free-ranging animals. Blood samples were collected from 66 Yangtze finless porpoises (41 males and 25 females) captured in the middle and lower reaches of the Yangtze River. Based on significant variation of serum T(2) concentrations in males with body length (BL) >138 cm, we inferred that they were mature; in these animals, there were significant seasonal variations in serum T(2) concentrations, with the highest concentrations in March and April (502.0+/-319.8 ng/dL, mean+/-S.D.) and the lowest in December (79.4+/-83.2 ng/dL). Serum T(2) concentrations positively correlated with serum concentrations of LH and weakly correlated with serum concentrations of E(2), whereas there was a significant negative correlation between serum LH and FSH concentrations in males >138 cm. The smallest apparently pregnant female porpoise had a BL of 130 cm. Serum P(4) concentrations ranged from 13.2 to 112.4 ng/mL (43.9+/-28.3 ng/mL) in pregnant females, and fluctuated under 1.0 ng/mL in non-pregnant females with BL>130 cm. Serum LH concentrations were significantly higher in non-pregnant females >130 cm versus those females <130 cm. To our knowledge, this is the first study of endocrine-related maturity and seasonal breeding characteristics of the Yangtze finless porpoise.