Tailing Optical Pulling Force on a Metal-Dielectric Hybrid Dimer with Electromagnetic Coupling.

In this work, we demonstrate that optical pulling forces (OPFs) can be induced by a hybrid dimer consisting of a Si nanoparticle (NP) and a coated nanoparticle with a gain core and Au shell under normal plane wave illumination. Analytical theory reveals that the underlying physical mechanism relies on interactions between the electric dipole (ED) modes excited in the NPs. As compared with the individual NP, it is found that the magnitude of optical force can be enlarged by almost three orders for the Si NP and one order for the coated gain NP in the coupled dimer. In addition, we find that the OPFs exerted on the NPs are heavily dependent on the gain level of the core materials, the incident polarization angle and the sizes of the NPs. More interestingly, we find that the OPF can also be exerted on a trimer system consisting of two identical Si NPs and a coated NP arranged in a line. The related results could be used to propose a versatile platform for manipulating NPs.

Research progress of visual detection in rapid on-site detection of pathogen nucleic acid.

Nucleic acid detection is widely used in pathogen screening and detection due to its high sensitivity and specificity. With the increase of detection requirements and the development of amplification technology, nucleic acid detection methods are gradually developing towards simple, fast and low-cost. Quantitative polymerase chain reaction (qPCR), as the "gold standard" for nucleic acid detection, relies on expensive equipment and professional operators, which is not suitable for rapid on-site detection of pathogens. The visual detection method without relying on excitation light source or complex equipment can present the detection results in a more intuitive and portable way after combining with rapid and efficient amplification technology, which has the potential of point-of-care testing (POCT). This paper focuses on the reported application of amplification technology and CRISPR/Cas technology in visual detection and compares their advantages and disadvantages, so as to provide reference for POCT strategy based on pathogen nucleic acid.

An enriched environment delays the progression from mild cognitive impairment to Alzheimer's disease in senescence-accelerated mouse prone 8 mice.

A previous study demonstrated that middle-aged (5-6 months of age) senescence-accelerated mouse prone 8 (SAMP8) mice can be used as animal models of mild cognitive impairment (MCI). An enriched environment (EE) can mitigate cognitive decline and decrease the pathological changes associated with various neurodegenerative diseases. In the present study, the learning-memory abilities of SAMP8 mice during the MCI phase (5 months of age) was evaluated and neuropathological changes in the hippocampus were examined after the mice were exposed to an EE for 60 days. In the Morris water maze test, EE-exposed mice demonstrated significantly decreased escape latency and increased time spent in the target quadrant and number of platform crossings compared with control mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling and Nissl staining showed that EE-exposed mice had reduced neuronal apoptosis and increased number of surviving neurons compared with control mice. Golgi staining, transmission electron microscopy, and immunohistochemical staining demonstrated that EE-exposed mice exhibited increased dendritic spine densities among secondary and tertiary apical dendrites; increases in synaptic numerical density, synaptic surface density, and expression of synaptophysin; and reduced deposition of amyloid-ß (Aß) and expression of amyloid-precursor protein (APP) in the hippocampal CA1 region compared with control mice. These results demonstrate that EE exposure effectively decreases neuronal loss and regulates neuronal synaptic plasticity by reducing the expression of APP and the deposition of Aß in the hippocampal CA1 region, thereby mitigating cognitive decline in SAMP8 mice during the MCI phase and delaying the progression from MCI to Alzheimer's disease.

An antidiabetic nutraceutical combination of red yeast rice (Monascus purpureus), bitter gourd (Momordica charantia), and chromium alleviates dedifferentiation of pancreatic ß cells in db/db mice.

Antidiabetic properties of red yeast rice, bitter gourd, and chromium have gained scientific support. This study aimed to test whether a nutraceutical combination of these 3 materials prevented dedifferentiation of pancreatic ß cells. Male db/db mice (8 weeks of age) were allocated into four groups (DB, DB/L, DB/M, and DB/H; n = 8-10) and fed a high-fat diet containing 0%, 0.2%, 0.4%, or 1% nutraceutical, respectively, whereas wild-type mice receiving a standard diet served as a healthy control (C; n = 10). The nutraceutical contained 10 mg/g monacolin K, 165 µg/g chromium, and 300 mg/g bitter gourd. After 8-weeks dietary treatment, diabetic syndromes (including hyperglycemia, hyperphagia, excessive drinking, polyuria, glucosuria, albuminuria, and glucose intolerance), were improved by the nutraceutical in a dose-dependent fashion. Decreased insulin and increased glucagon in serum and pancreatic islets in db/db mice were abolished in the DB/H group. Furthermore, supplementation curtailed dedifferentiation of ß cells, as evidenced by decreasing the dedifferentiation marker (Aldh1a3) and increasing ß-cell-enriched genes and transcription factors (Ins1, Ins2, FOXO1, and NKX6.1), as well as nuclear localization of NKX6.1 in pancreatic islets when compared to the DB group. We concluded that this nutraceutical, a combination of Monascus purpureus, Momordica charantia, and chromium, could be used as an adjunct for type 2 diabetes treatment and delay disease progression by sustaining ß-cell function.

New molecular diagnostic technologies for clinical detection of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an ongoing pandemic of new coronavirus pneumonia (corona virus disease 2019, COVID-19). The virus has a long incubation period and strong infectivity, which poses a major threat to global health and safety. Detection of SARS-CoV-2 nucleic acid lies at the center of rapid detection of COVID-19, which is instrumental for mitigation of the ongoing pandemic. As of August 17, 2020, The National Medical Products Administration in China has approved 15 new coronavirus nucleic acid detection kits, 10 kits of which are based on reverse transcription-real-time quantitative PCR (RT-qPCR) technology. The remaining kits use five molecular diagnostic technologies different from RT-qPCR. This article reviews the principles, reaction time, advantages and disadvantages of above 15 detection kits, in order to provide references for rapid screening, diagnosis, prevention and control of COVID-19 and similar infectious diseases.

Dietary Exposure to Oxidized Frying Oil from Fetus to Adulthood Suppresses Male Reproductive Development by Altering Testicular Cholesterol and Testosterone Homeostasis in Sprague Dawley Rats.

BACKGROUND: Dietary frying oil may have endocrine-disrupting effects, as a feminization effect was observed in cohorts of C57BL/6J male mice fetuses from dams consuming oxidized frying oil (OFO) during pregnancy. OBJECTIVE: The aim of present study was to test the hypothesis that OFO is an anti-androgen. METHODS: In experiment 1, male progeny of Sprague Dawley female rats fed fresh oil or an OFO diet (10 g fat/100 g, from fresh or 24-h-fried soybean oil; [control diet (C) and OFO groups, respectively] from midgestation through lactation were studied. Pups were weaned at 3 wk of age and then consumed their mothers' diet until 9 wk of age. In addition, a group of dams and pups that consumed a high-fat diet (HF; 10 g fried and 20 g fresh soybean oil/100 g) was included to counteract body-weight loss associated with OFO ingestion. Indices of male reproductive development and testosterone homeostasis were measured. In experiment 2, male rats were allocated to C and OFO groups (treated as above) and indices of male fertility compared at 9-10 wk of age. RESULTS: In experiment 1, final body weights of the HF group were lower (17%) than the C group but higher (14%) than the OFO group (P < 0.0001 for each). In addition to abnormalities in seminiferous tubules, HF and OFO groups did not differ from one another, but, compared with the C group, had delayed preputial separation (4.9 d) and reductions in serum testosterone concentrations (17-74%), anogenital distance (8-20%), weights of androgen-dependent tissues (8-30%), testicular testosterone and cholesterol concentrations (30-40%), and mRNA levels of genes involved in steroidogenesis and cholesterol homeostasis (30-70%). In experiment 2, OFO-exposed males had 20% lower sperm motility (P < 0.05); however, when mated to normal females, pregnancy rates and litter sizes did not differ between OFO and C groups. CONCLUSIONS: The anti-androgenic effect of OFO in Sprague Dawley rats was attributed to decreased testicular concentrations of cholesterol (testosterone precursor) and not body-weight loss.

Deficiency or activation of peroxisome proliferator-activated receptor α reduces the tissue concentrations of endogenously synthesized docosahexaenoic acid in C57BL/6J mice.

BACKGROUND/OBJECTIVES: Docosahexaenoic acid (DHA), an n-3 long chain polyunsaturated fatty acid (LCPUFA), is acquired by dietary intake or the in vivo conversion of α-linolenic acid. Many enzymes participating in LCPUFA synthesis are regulated by peroxisome proliferator-activated receptor alpha (PPARα). Therefore, it was hypothesized that the tissue accretion of endogenously synthesized DHA could be modified by PPARα. MATERIALS/METHODS: The tissue DHA concentrations and mRNA levels of genes participating in DHA biosynthesis were compared among PPARα homozygous (KO), heterozygous (HZ), and wild type (WT) mice (Exp I), and between WT mice treated with clofibrate (PPARα agonist) or those not treated (Exp II). In ExpII, the expression levels of the proteins associated with DHA function in the brain cortex and retina were also measured. An n3-PUFA depleted/replenished regimen was applied to mitigate the confounding effects of maternal DHA. RESULTS: PPARα ablation reduced the hepatic Acox, Fads1, and Fads2 mRNA levels, as well as the DHA concentration in the liver, but not in the brain cortex. In contrast, PPARα activation increased hepatic Acox, Fads1, Fads2 and Elovl5 mRNA levels, but reduced the DHA concentrations in the liver, retina, and phospholipid of brain cortex, and decreased mRNA and protein levels of the brain-derived neurotrophic factor in brain cortex. CONCLUSIONS: LCPUFA enzyme expression was altered by PPARα. Either PPARα deficiency or activation-decreased tissue DHA concentration is a stimulus for further studies to determine the functional significance.

Author Correction: Draft genomic and transcriptome resources for marine chelicerate Tachypleus tridentatus.

In the original version of this Data Descriptor the word "Gulf" was incorrectly spelled in the affiliation "Ocean College, Beibu Gulf University, Qinzhou, 535011, Guangxi, China". This has now been corrected in both the HTML and PDF versions.

Draft genomic and transcriptome resources for marine chelicerate Tachypleus tridentatus.

Chinese horseshoe crabs (Tachypleus tridentatus), ancient marine arthropods dating back to the mid-Palaeozoic Era, have provided valuable resources for the detection of bacterial or fungal contamination. However, excessive exploitation for the amoebocyte lysate of Tachypleus has dramatically decreased the population of the Chinese horseshoe crabs. Thus, we present sequencing, assembly and annotation of T. tridentatus, with the hope of understanding the genomic feature of the living fossil and assisting scientists with the protection of this endangered species. The final genome contained a total size of 1.943 Gb, covering 90.23% of the estimated genome size. The transcriptome of three larval stages was constructed to investigate the candidate gene involved in the larval development and validate annotation. The completeness of the genome and gene models was estimated by BUSCO, reaching 96.2% and 95.4%, respectively. The synonymous substitution distribution of paralogues revealed that T. tridentatus had undergone two rounds of whole-genome duplication. All genomic and transcriptome data have been deposited in public databases, ready to be used by researchers working on horseshoe crabs.

Is frying oil a dietary source of an endocrine disruptor? Anti-estrogenic effects of polar compounds from frying oil in rats.

The objective was to investigate endocrine-disrupting effects of polar compounds from oxidized frying oil. Estrogenicity of polar compounds was tested with a rat uterotrophic bioassay. Dietary oxidized frying oil (containing 51% polar compounds) or polar compounds isolated from it were incorporated into feed (in lieu of fresh soybean oil) and fed to ovariectomized rats, with or without treatment with exogenous ethynyl estradiol. Exogenous estrogen restored uterine weight, and caused histological abnormalities (stratified epithelia and conglomerate glands) as well as proliferation of uterine epithelial cells. However, tamoxifen or polar compounds reduced these effects. Furthermore, tamoxifen or polar compounds down-regulated uterine mRNA expression of estrogen receptor (ER)-target genes, implicating reduced ER activity in this hypo-uterotrophic effect. Inhibition of ER signaling and mitosis by polar compounds were attributed to reduced MAPK and AKT activation, as well as a reduced ligand binding domain-transactivity of ERα/ß. We concluded polar compounds from frying oil are potential endocrine-disrupting chemicals, with implications for food and environmental safety.

Controllable extension of hairpin-structured flaps to allow low-background cascade invasive reaction for a sensitive DNA logic sensor for mutation detection.

A DNA logic sensor was constructed for gene mutation analysis based on a novel signal amplification cascade by controllably extending a hairpin-structured flap to bridge two invasive reactions. The detection limit was as low as 0.07 fM, and the analytical specificity is high enough to unambiguously pick up 0.02% mutants from a large amount of wild-type DNA. Gene mutations related to the personalized medicine of gefitinib, a typical tyrosine kinase inhibitor, were analyzed by the DNA logic sensor with only a 15 minute response time. Successful assay of tissue samples and cell-free plasma DNA indicates that the new concept we proposed here could benefit clinicians for straightforward prescription of a mutation-targeted drug.

[Progress in molecular identification of snake drugs].

Snake drugs have high values in clinical medication for anti-inflammatory, analgesia activities and dredging collaterals. However, owing to their deficient resource and substantial profit, many counterfeits for snake drugs have appeared on the market. Traditional methods for Chinese medicine authentication include identification of origin, morphology identification, microscopy and physiochemical identification. But these methods are restricted in application because of their high morphological requirement for specimens, complex process for assays and indeterminate results guided by subjective. With the development of molecular biology and molecular genetic techniques, new theories and technologies for molecular detection have been introduced to the authentication of Chinese medicine, such as RAPD, specific PCR amplification, DNA barcoding analysis and so on, improved the authentication system of Chinese medicine. Here, we will give a brief review of molecular detection methods for snake drugs authentication.

Apparent source levels and active communication space of whistles of free-ranging Indo-Pacific humpback dolphins (Sousa chinensis) in the Pearl River Estuary and Beibu Gulf, China.

Background. Knowledge of species-specific vocalization characteristics and their associated active communication space, the effective range over which a communication signal can be detected by a conspecific, is critical for understanding the impacts of underwater acoustic pollution, as well as other threats. Methods. We used a two-dimensional cross-shaped hydrophone array system to record the whistles of free-ranging Indo-Pacific humpback dolphins (Sousa chinensis) in shallow-water environments of the Pearl River Estuary (PRE) and Beibu Gulf (BG), China. Using hyperbolic position fixing, which exploits time differences of arrival of a signal between pairs of hydrophone receivers, we obtained source location estimates for whistles with good signal-to-noise ratio (SNR ≥10 dB) and not polluted by other sounds and back-calculated their apparent source levels (ASL). Combining with the masking levels (including simultaneous noise levels, masking tonal threshold, and the Sousa auditory threshold) and the custom made site-specific sound propagation models, we further estimated their active communication space (ACS). Results. Humpback dolphins produced whistles with average root-mean-square ASL of 138.5 ± 6.8 (mean ± standard deviation) and 137.2 ± 7.0 dB re 1 µPa in PRE (N = 33) and BG (N = 209), respectively. We found statistically significant differences in ASLs among different whistle contour types. The mean and maximum ACS of whistles were estimated to be 14.7 ± 2.6 (median ± quartile deviation) and 17.1± 3.5 m in PRE, and 34.2 ± 9.5 and 43.5 ± 12.2 m in BG. Using just the auditory threshold as the masking level produced the mean and maximum ACSat of 24.3 ± 4.8 and 35.7 ± 4.6 m for PRE, and 60.7 ± 18.1 and 74.3 ± 25.3 m for BG. The small ACSs were due to the high ambient noise level. Significant differences in ACSs were also observed among different whistle contour types. Discussion. Besides shedding some light for evaluating appropriate noise exposure levels and information for the regulation of underwater acoustic pollution, these baseline data can also be used for aiding the passive acoustic monitoring of dolphin populations, defining the boundaries of separate groups in a more biologically meaningful way during field surveys, and guiding the appropriate approach distance for local dolphin-watching boats and research boat during focal group following.

Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming.

Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion.

Dysregulation of galectin-3. Implications for Hermansky-Pudlak syndrome pulmonary fibrosis.

The etiology of Hermansky-Pudlak syndrome (HPS) pulmonary fibrosis (HPSPF), a progressive interstitial lung disease with high mortality, is unknown. Galectin-3 is a ß-galactoside-binding lectin with profibrotic effects. The objective of this study was to investigate the involvement of galectin-3 in HPSPF. Galectin-3 was measured by ELISA, immunohistochemistry, and immunoblotting in human specimens from subjects with HPS and control subjects. Mechanisms of galectin-3 accumulation were studied by quantitative RT-PCR, Northern blot analysis, membrane biotinylation assays, and rescue of HPS1-deficient cells by transfection. Bronchoalveolar lavage galectin-3 concentrations were significantly higher in HPSPF compared with idiopathic pulmonary fibrosis or that from normal volunteers, and correlated with disease severity. Galectin-3 immunostaining was increased in HPSPF compared with idiopathic pulmonary fibrosis or normal lung tissue. Fibroblasts from subjects with HPS subtypes associated with pulmonary fibrosis had increased galectin-3 protein expression compared with cells from nonfibrotic HPS subtypes. Galectin-3 protein accumulation was associated with reduced Galectin-3 mRNA, normal Mucin 1 levels, and up-regulated microRNA-322 in HPSPF cells. Membrane biotinylation assays showed reduced galectin-3 and normal Mucin 1 expression at the plasma membrane in HPSPF cells compared with control cells, which suggests that galectin-3 is mistrafficked in these cells. Reconstitution of HPS1 cDNA into HPS1-deficient cells normalized galectin-3 protein and mRNA levels, as well as corrected galectin-3 trafficking to the membrane. Intracellular galectin-3 levels are regulated by HPS1 protein. Abnormal accumulation of galectin-3 may contribute to the pathogenesis of HPSPF.

Sirolimus decreases circulating lymphangioleiomyomatosis cells in patients with lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis (LAM), sporadic or in women with tuberous sclerosis complex (TSC), is characterized by cystic lung destruction, lymphatic involvement (eg, chylous pleural effusions, lymphangioleiomyomas), and renal angiomyolipomas (AMLs). The multisystem manifestations of LAM appear to result from metastatic dissemination of LAM cells bearing inactivating mutations or having loss of heterozygosity (LOH) of the tumor suppressor genes TSC1 or TSC2, which leads to hyperactivation of the mammalian target of rapamycin. Sirolimus slows the decline of lung function, reduces chylous effusions, and shrinks the size of AMLs. The purpose of this study was to determine the effect of sirolimus on circulating LAM cells. METHODS: Cells from blood were isolated by a density-gradient fractionation system and from urine and chylous effusions by centrifugation. Blood cells were incubated with anti-CD45-fluorescein isothiocyanate (FITC) and anti-CD235a-R-phycoerythrin (PE) antibodies, and urine and chylous effusion cells were incubated with anti-CD44v6-FITC and anti-CD9-R-PE antibodies. Cells were sorted and analyzed for TSC2 LOH. RESULTS: LAM cells with TSC2 LOH were identified in 100% of blood specimens and 75% of urine samples from patients before therapy. Over a mean duration of 2.2 ± 0.4 years of sirolimus therapy, detection rates of LAM cells were significantly decreased to 25% in blood (P < .001) and 8% in urine (P = .003). Following therapy, a greater loss of circulating LAM cells was seen in postmenopausal patients (P = .025). CONCLUSIONS: Patients receiving sirolimus had a progressive loss of circulating LAM cells that depended on time of treatment and menopausal status.

Tet1 regulates adult hippocampal neurogenesis and cognition.

DNA hydroxylation catalyzed by Tet dioxygenases occurs abundantly in embryonic stem cells and neurons in mammals. However, its biological function in vivo is largely unknown. Here, we demonstrate that Tet1 plays an important role in regulating neural progenitor cell proliferation in adult mouse brain. Mice lacking Tet1 exhibit impaired hippocampal neurogenesis accompanied by poor learning and memory. In adult neural progenitor cells deficient in Tet1, a cohort of genes involved in progenitor proliferation were hypermethylated and downregulated. Our results indicate that Tet1 is positively involved in the epigenetic regulation of neural progenitor cell proliferation in the adult brain.

The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes.

Sperm and eggs carry distinctive epigenetic modifications that are adjusted by reprogramming after fertilization. The paternal genome in a zygote undergoes active DNA demethylation before the first mitosis. The biological significance and mechanisms of this paternal epigenome remodelling have remained unclear. Here we report that, within mouse zygotes, oxidation of 5-methylcytosine (5mC) occurs on the paternal genome, changing 5mC into 5-hydroxymethylcytosine (5hmC). Furthermore, we demonstrate that the dioxygenase Tet3 (ref. 5) is enriched specifically in the male pronucleus. In Tet3-deficient zygotes from conditional knockout mice, paternal-genome conversion of 5mC into 5hmC fails to occur and the level of 5mC remains constant. Deficiency of Tet3 also impedes the demethylation process of the paternal Oct4 and Nanog genes and delays the subsequent activation of a paternally derived Oct4 transgene in early embryos. Female mice depleted of Tet3 in the germ line show severely reduced fecundity and their heterozygous mutant offspring lacking maternal Tet3 suffer an increased incidence of developmental failure. Oocytes lacking Tet3 also seem to have a reduced ability to reprogram the injected nuclei from somatic cells. Therefore, Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA following natural fertilization and may also contribute to somatic cell nuclear reprogramming during animal cloning.

Natural history of pulmonary fibrosis in two subjects with the same telomerase mutation.

UNLABELLED: Previous studies have identified subclinical lung disease in family members of probands with familial pulmonary fibrosis, but the natural history of preclinical pulmonary fibrosis is uncertain. The purpose of this study was to determine whether individuals with preclinical lung disease will develop pulmonary fibrosis. After a 27-year interval, two subjects with manifestations of preclinical familial pulmonary fibrosis, including asymptomatic alveolar inflammation and alveolar macrophage activation, were reevaluated for lung disease. CT scans of the chest, pulmonary function tests, and BAL were performed, and genomic DNA was analyzed for mutations in candidate genes associated with familial pulmonary fibrosis. One subject developed symptomatic familial pulmonary fibrosis and was treated with oxygen; her sister remained asymptomatic but had findings of pulmonary fibrosis on high-resolution CT scan of the chest. High concentrations of lymphocytes were found in BAL fluid from both subjects. Genetic sequencing and analyses identified a novel heterozygous mutation in telomerase reverse transcriptase (TERT, R1084P), resulting in telomerase dysfunction and short telomeres in both subjects. In familial pulmonary fibrosis, asymptomatic preclinical alveolar inflammation associated with mutation in TERT and telomerase insufficiency can progress to fibrotic lung disease over 2 to 3 decades. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT00071045; URL: www.clinicaltrials.gov.

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).