Temperature is a key environmental driver of hepatic physiology in ectotherms, and three-dimensional (3D) fish liver models may serve as an ethically advantageous platform to investigate warming effects under controlled conditions while reducing the need for experimental animals. Despite their increasing use in toxicology, their application to assess climate-relevant temperature effects on liver function remains limited. This study investigated the temporal effects of a warming scenario on primary hepatocyte spheroids from juvenile brown trout (Salmo trutta), a bioindicator species. The study explores how a + 3 °C increase can affect spheroid development and maintenance, as well as its impact on metabolic activity, cell proliferation and death, and morphology over time. Spheroids were maintained at 18 °C and 21 °C for 25 days and analysed at five time points using metabolic, morphometric, immunocytochemical, and ultrastructural approaches. Mitochondrial metabolic activity, assessed by resazurin reduction, showed no significant temperature-related differences. In contrast, warming accelerated spheroid formation and produced larger spheroids. Proliferative activity, assessed by proliferating cell nuclear antigen (PCNA) immunostaining, was significantly reduced at 21 °C, while caspase-3 levels remained unchanged, indicating no increase in apoptosis. The autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3A/B) showed lower immunoreactivity at 21 °C, with no temporal variation. Ultrastructural analysis revealed preserved hepatocyte integrity at both temperatures and abundant cytoplasmic dense bodies consistent with autolysosomal structures, which increased over time. Overall, a realistic warming scenario altered growth dynamics and cellular morphology. Further, this data reinforces that 3D fish liver models are viable alternative systems for assessing climate-driven effects.

The brown trout (Salmo trutta) is a commercially and ecologically significant salmonid fish, yet its hepatic cellular and functional dynamics throughout the reproductive cycle remain poorly characterised, particularly in males. This study investigated seasonal and sex-specific liver plasticity across four reproductive stages: spawning capable (December), regressing (March), regenerating (July), and developing (November). We quantified mRNA and protein abundance of key oestrogen-responsive targets—vitellogenin (VtgA) and zona pellucida (ZP) proteins—alongside cell turnover markers, caspase 3 (Casp3) and proliferating cell nuclear antigen (PCNA). These molecular endpoints were integrated with stereological analyses to estimate hepatocyte, nuclear, and cytoplasmic volumes. Results revealed stage-dependent mobilisation and transient hepatic retention of reproductive proteins; females exhibited stronger vitellogenic signatures and more pronounced seasonal shifts than males. Although VtgA and ZP mRNA levels peaked during the developing and spawning-capable stages, males maintained low but consistent levels throughout the cycle, indicating constitutive hepatic oestrogen sensitivity. Regarding cell turnover, PCNA protein data indicated heightened proliferative activity during the spawning-capable and regressing stages. In contrast, while Casp3 mRNA levels remained stable across all stages, protein detection suggested a post-transcriptional increase in apoptotic signalling during the developing phase, consistent with controlled tissue remodelling rather than extensive cell loss. Stereological data confirmed enlarged hepatocyte and nuclear volumes during periods of high secretory and proliferative demand. Overall, these findings demonstrate significant stage-dependent and sex-specific plasticity in brown trout liver, providing a robust reference framework for ecological monitoring, endocrine disruption assessments, and studies of teleost reproductive physiology.

Polycyclic aromatic hydrocarbons (PAHs) and global warming impact aquatic ecosystems, eventually interacting. Monolayer (2D) cultures of cell lines, such as the rainbow trout liver RTL-W1, are employed for unveiling toxicological effects in fish. Nonetheless, three-dimensional (3D) models constitute an alternate paradigm, better emulating in vivo responses. Here, ultra-low attachment (ULA) plates were used to generate ten-day-old RTL-W1 spheroids for exposure to a control, a solvent control (0.1% DMSO) and the model PAH benzo[k]fluoranthene (BkF) at 10 and 100 nM and at 18 and 23 °C (thermal stress). After a 4-day exposure, spheroids were analyzed for viability (alamarBlue and lactate dehydrogenase), biometry (area, diameter and sphericity), histocytology (optical and electron microscopy), and mRNA levels of the detoxification-related genes cytochrome P450 (CYP)1A, CYP3A27, aryl hydrocarbon receptor (AhR), glutathione S-transferase (GST), uridine diphosphate–glucuronosyltransferase (UGT), catalase (CAT), multidrug resistance-associated protein 2 (MRP2) and bile salt export protein (BSEP). Immunocytochemistry (ICC) was used to assess CYP1A protein expression. Neither temperature nor BkF exposure altered the spheroids’ viability or biometry. BkF modified the cell’s ultrastructure. The expression of CYP1A was augmented with both BkF concentrations, while AhR’s increased at the higher concentration. The CYP1A protein showed a dose-dependent increase. Temperature and BkF concurrently modelled UGT’s expression, which increased in the 100 nM condition at 23 °C. Conversely, CYP3A27, MRP2, and BSEP expressions lowered at 23 °C. CAT and GST mRNA levels were uninfluenced by either stressor. Overall, BkF and temperature impacted independently or interactively in RTL-W1 spheroids. These seem to be useful novel tools for studying the liver-related effects of temperature and PAHs.

Feline infectious peritonitis (FIP) is caused by a feline coronavirus (FCoV) and it is one of the most important viral diseases of cats. Recently, new drugs have been developed and used to face the inexorable fatal prognosis of FIP when untreated. More than ever, a definitive ante-mortem diagnosis is crucial to identify the cats that would benefit from antiviral treatment and to avoid euthanasia of unaffected animals. Identification of FCoV in macrophages using immunohistochemistry (IHC) is the gold standard method for diagnosing FIP. In this pilot study, the role of IHC applied to cell tube blocks (CTBs) of effusion fluids of cats clinically suspected to have FIP was assessed. A prospective cohort of seven cats was enrolled. IHC results in CTBs was positive in six out seven cases. Necropsy, histology and IHC in organs/tissues confirmed FIP infection in all the seven cases. The false negative in a CTB corresponded to a case of bacterial peritonitis secondary to FIP. As proof-of-concept, CTBs are valuable as a non-invasive technique for a definitive ante-mortem/clinical diagnosis of FIP and it is recommended to obtain CTBs from effusion fluids of all suspected FIP cases.

Polycyclic aromatic hydrocarbons (PAHs), prevalent aquatic contaminants, arise from burning fossil fuels, a major source of greenhouse gases driving global warming. PAHs and warmer temperatures individually exert diverse negative effects on aquatic organisms. However, the effects of PAH exposure and/or rising temperature remain largely unknown. Liver in vitro models, like the rainbow trout (Oncorhynchus mykiss) RTL-W1 liver cell line, have been employed to unravel PAH-exposure effects, primarily on cell viability and enzymatic activity. Here, monolayer-cultured (2D) RTL-W1 cells were used to assess the co-exposure effects of temperature (18 and 21 °C) and two PAHs, benzo[a]pyrene (B[a]P) and benzo[k]fluoranthene (B[k]F), at 10 and 100 nM. After a 72 h exposure, the cell density and viability were evaluated using the trypan blue and LDH assays. The mRNA levels of the detoxification-associated genes aryl hydrocarbon receptor (AhR), cytochrome P450 (CYP)1A, CYP3A27, glutathione S-transferase omega 1 (GSTO1), uridine diphosphate-glucuronosyltransferase (UGT), catalase (CAT), and multidrug resistance-associated protein 2 (MRP2) were measured by RT-qPCR. Temperature influenced cell viability and LDH leakage. Both PAHs reduced the cell density and upregulated the mRNA levels of AhR, CYP1A, CYP3A27, and UGT, while GSTO1 and MRP2 were only augmented after the higher B[k]F concentration. Temperature influenced CAT and UGT expression. There was no interaction between temperature and the PAHs. Overall, the results show that B[k]F has more effects on detoxification targets than B[a]P, whereas a temperature increase mildly affects gene expression. The RTL-W1 in 2D seems useful for unravelling not only the liver effects of PAH but also the impact of temperature stress.

Three-dimensional (3D) fish hepatocyte cultures are promising alternative models for replicating in vivo data. Few studies have attempted to characterise the structure and function of fish 3D liver models and illustrate their applicability. This study aimed to further characterise a previously established spheroid model obtained from juvenile brown trout (Salmo trutta) primary hepatocytes under estrogenic stimulation. The spheroids were exposed for six days to environmentally relevant concentrations of 17α-ethinylestradiol—EE2 (1–100 ng/L). The mRNA levels of peroxisome (catalase—Cat and urate oxidase—Uox), lipid metabolism (acyl-CoA long chain synthetase 1—Acsl1, apolipoprotein AI—ApoAI, and fatty acid binding protein 1—Fabp1), and estrogen-related (estrogen receptor α—ERα, estrogen receptor β—ERβ, vitellogenin A—VtgA, zona pellucida glycoprotein 2.5—ZP2.5, and zona pellucida glycoprotein 3a.2—ZP3a.2) target genes were evaluated by quantitative real-time polymerase chain reaction. Immunohistochemistry was used to assess Vtg and ZP protein expressions. At the highest EE2 concentration, VtgA and ZP2.5 genes were significantly upregulated. The remaining target genes were not significantly altered by EE2. Vtg and ZP immunostaining was consistently increased in spheroids exposed to 50 and 100 ng/L of EE2, whereas lower EE2 levels resulted in a weaker signal. EE2 did not induce significant changes in the spheroids’ viability and morphological parameters. This study identified EE2 effects at environmentally relevant doses in trout liver spheroids, indicating its usefulness as a proxy for in vivo impacts of xenoestrogens.

Fish are exposed to increased water temperatures and aquatic pollutants, including endocrine-disrupting compounds (EDCs). Although each stressor can disturb fish liver metabolism independently, combined effects may exist. To unveil the molecular mechanisms behind the effects of EDCs and temperature, fish liver cell lines are potential models needing better characterisation. Accordingly, we exposed the rainbow trout RTL-W1 cells (72 h), at 18 °C and 21 °C, to ethynylestradiol (EE2), levonorgestrel (LNG), and a mixture of both hormones (MIX) at 10 µM. The gene expression of a selection of targets related to detoxification (CYP1A, CYP3A27, GST, UGT, CAT, and MRP2), estrogen exposure (ERα, VtgA), lipid metabolism (FAS, FABP1, FATP1), and temperature stress (HSP70b) was analysed by RT-qPCR. GST expression was higher after LNG exposure at 21 °C than at 18 °C. LNG further enhanced the expression of CAT, while both LNG and MIX increased the expressions of CYP3A27 and MRP2. In contrast, FAS expression only increased in MIX, compared to the control. ERα, VtgA, UGT, CYP1A, HSP70b, FABP1, and FATP1 expressions were not influenced by the temperature or the tested EDCs. The RTL-W1 model was unresponsive to EE2 alone, sensitive to LNG (in detoxification pathway genes), and mainly insensitive to the temperature range but had the potential to unveil specific interactions.

This study aimed to provide a comprehensive analysis of the histological structure of intestinal tissues of platyfish (Xiphophorus maculatus) and swordtail fish (Xiphophorus helleri). Specifically, the objectives were: (1) to compare the structural adaptations of their intestines related to their distinct feeding habits, diet, and digestive strategies; and (2) to explore their potential as animal models for intestinal disease research. Through detailed examination of tissue morphology, cell types, and structural features, this study found that both species lack a stomach, with the intestine directly connected to the esophagus. Additionally, this study proposes a new division of the intestine into anterior and posterior segments based on distinct histological characteristics. The anterior segment may be adapted for temporary food storage and digestion and was characterized by elongated epithelial cells and thin intestinal folds. In contrast, the posterior segment displayed shorter villi and higher concentrations of goblet cells. This study is the first to describe in detail the intestinal morphology of platyfish and swordtail fish. These findings contribute significantly to the understanding of the comparative anatomy and physiology of these fish species, highlighting their potential as valuable models for intestinal biology research.

Immunolabeling on Romanowsky-stained cytology (RSC) slides can be used, although there is limited evidence of its suitability for phenotyping canine and feline lymphomas. A comparison with matched cell blocks (CB) is missing. Immunolabeling on RSC and CB was compared for lymphoid markers (CD3 and PAX5) in 53 lymphomas and 4 chylous effusions from dogs and cats. The influence of pre-analytical variables (species, time of archive, type of specimens and coverslipping) and the interobserver agreement among the 2 observers was assessed. Fewer CD3+ lymphocytes were identified in RSC, while the PAX5 positivity by RSC and CB had a substantial agreement. Immunodetection of CD3 and the diagnosis of a T-cell population on RSC was more difficult. Lower intensity and higher background were noted in RSC. Immunophenotyping was inconclusive in 54% RSC and 19% CB. The interobserver reproducibility of immunophenotyping on CB was substantial, being higher than in RSC. The immunolabeling performance on the RSC of effusion and feline samples was unsatisfactory. The detection of lymphoid markers, especially membranous antigens in retrospective RSC, is affected by the pre-analytical variables: species, time of the archive, and type of specimens. CB are a more consistent type of sample for immunophenotyping purposes.

Mammal hepatocyte spheroids have been investigated as alternative experimental models in several contexts, since three-dimensional (3D) systems have shown the potential to mimic in vivo scenarios. The description of fish hepatocyte 3D models is still minimal. This study intends to further characterize brown trout primary hepatocyte spheroids at distinct time points up to 25 days in culture. Viability, biometry, histomorphology, and basal expression of a selection of genes (metabolism and detoxification, efflux transport, and estrogenic signalling) were considered. The gene expression of whole liver samples from the same fish donor were evaluated concurrently. After 12 days in culture, the hepatocyte spheroids exhibited biometric and morphological stability. From the 12th to the 20th day in culture, the basal expression levels for most of the selected genes did not vary. The targeted mRNA levels were higher in brown trout liver samples compared to hepatocyte spheroids. Despite that, data supported that this model resembles some in vivo features. As an experimental alternative model, it showed potential to be used in a stable time window that can be exploited for exposure tests to different xenobiotics, namely, estrogenic compounds.

Veterinary cytology faced a remarkable evolution in the last 15 years, in part due to increase recognition of the advantages of the cytology by veterinary clinicians. Simultaneously, there has been a growing awareness by the owners about the importance of a complete diagnostic workup aimed at defining a proper treatment protocol. With the extended use of cytology, challenging diagnostic cases are more frequent, and more clinically useful answers are requested. In this scenario, the use of cytology specimens to perform ancillary techniques is a valid approach. Rather than being simply archived, cytology slides can be a valuable source and a good platform to carry out cytochemistry, immunocytochemistry, and molecular techniques. Therefore, several diagnostic techniques can be applied in tiny samples, thus following the “doing more with less” principle. The aim of this approach is to refine the cytologic diagnosis and provide additional prognostic and therapeutic information. Herein, we detailed this principle in veterinary cytology and reviewed the use of cytology specimens for ancillary techniques as a single procedure, i.e., using the whole slide, or multiple procedures, i.e., multiple procedures applied in the same slide.

Three-dimensional (3D) fish liver cultures mimic the in vivo cellular microenvironment, which is ideal for ecotoxicological research. Despite that, the application of these cultures to evaluate toxic effects in fish is scarce. A 3D model of brown trout (Salmo trutta f. fario) primary hepatocyte spheroids was optimized in this study by using DMEM/F-12 with 15 mM of HEPES, 10 mL/L of an antibiotic and antimycotic solution and FBS 10% (v/v), at 18 °C with ∼100 rpm. The selection of optimal conditions was based on a multiparametric characterization of the spheroids, including biometry, viability, microanatomy and immunohistochemistry. Biometric and morphologic stabilization of spheroids was reached within 12-16 days of culture. To our knowledge, this study is the first to culture and characterize viable spheroids from brown trout primary hepatocytes for over 30 days. Further, the 3D model was tested to explore the androgenic influences on lipidic target genes after 96 h exposures to control, solvent control, 10 and 100 µM of 5α-dihydrotestosterone (DHT), a non-aromatizable androgen. Spheroids exposed to 100 µM of DHT had decreased sphericity. DHT at 100 µM also significantly down-regulated Acox1-3I, PPARγ and fatty acid synthesis targets (i.e., ACC), and significantly up-regulated Fabp1. Acsl1 was significantly up-regulated after exposure to both 10 and 100 µM of DHT. The results support that DHT modulates distinct lipidic pathways in brown trout and show that this 3D model is a new valuable tool for physiological and toxicological mechanistic studies.

Veterinary Clinical PathologyVolume 51, Issue 2 p. 194-195 YESTERDAY • TODAY • TOMORROW Mesothelial cells: The messed-up cells that make a confusion out of an effusion Carla Marrinhas, Carla Marrinhas Cytology Diagnostic Services, Laboratory of Histology and Embryology, ICBAS-UP, University of Porto, Porto, PortugalSearch for more papers by this authorCélia Lopes, Célia Lopes Cytology Diagnostic Services, Laboratory of Histology and Embryology, ICBAS-UP, University of Porto, Porto, PortugalSearch for more papers by this authorMarta Santos, Marta Santos Cytology Diagnostic Services, Laboratory of Histology and Embryology, ICBAS-UP, University of Porto, Porto, PortugalSearch for more papers by this authorRicardo Marcos, Corresponding Author Ricardo Marcos rmarcos@icbas.up.pt Cytology Diagnostic Services, Laboratory of Histology and Embryology, ICBAS-UP, University of Porto, Porto, PortugalSearch for more papers by this author Carla Marrinhas, Carla Marrinhas Cytology Diagnostic Services, Laboratory of Histology and Embryology, ICBAS-UP, University of Porto, Porto, PortugalSearch for more papers by this authorCélia Lopes, Célia Lopes Cytology Diagnostic Services, Laboratory of Histology and Embryology, ICBAS-UP, University of Porto, Porto, PortugalSearch for more papers by this authorMarta Santos, Marta Santos Cytology Diagnostic Services, Laboratory of Histology and Embryology, ICBAS-UP, University of Porto, Porto, PortugalSearch for more papers by this authorRicardo Marcos, Corresponding Author Ricardo Marcos rmarcos@icbas.up.pt Cytology Diagnostic Services, Laboratory of Histology and Embryology, ICBAS-UP, University of Porto, Porto, PortugalSearch for more papers by this author First published: 02 June 2022 https://doi.org/10.1111/vcp.13146Citations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume51, Issue2June 2022Pages 194-195 RelatedInformation

Despite of physiological and toxicological relevance, the potential of androgens to influence fish lipid metabolism remains poorly explored. Here, brown trout primary hepatocytes were exposed to six concentrations (1 nM to 100 mu M) of dihydrotestosterone (DHT) and testosterone (T), to assess changes in the mRNA levels of genes covering diverse lipid metabolic pathways. Acsl1, essential for fatty acid activation, was up-regulated by T and DHT, whereas the lipogenic enzymes FAS and ACC were up-regulated by the highest (100 mu M) concentration of T and DHT, respectively. ApoA1, the major component of high-density lipoprotein (HDL), was down-regulated by both androgens. PPAR gamma, linked to adipogenesis and peroxisomal beta-oxidation, was down-regulated by T and DHT, while Acox1-3I, rate-limiting in peroxisomal beta-oxidation, was down-regulated by T. Fabp1, StAR and LPL were not altered. Our findings suggest that androgens may impact on lipid transport, adipogenesis and fatty acid beta-oxidation and promote lipogenesis in fish liver.

Brown trout is an environmental freshwater sentinel species and is economically important for recreational fishing and aquaculture. Despite that, there is limited knowledge regarding morpho-physiological variations in adults throughout the reproductive cycle. Thus, this study aimed to analyze the fitness and gonadal maturation of cultured adult brown trout in four reproductive phases (spawning capable—December, regressing—March, regenerating—July, and developing—November). The systematic evaluation of males and females was based on biometric, biochemical, and hormonal parameters, along with a histomorphological grading of gonads and the immunophenotype location of key steroidogenic enzymes. The total weight and lengths reached the lowest levels in December. Gonad weights were higher in December and November, while the opposite pattern was found for liver weights. The lowest levels of cholesterol and total protein were also noted during those stages. The 11-ketotestosterone (11-KT) and testosterone (T) for males, and estradiol (E2) and T for females, mostly explained the hormonal variations. The immunohistochemistry of cytochrome P450c17 (CYP17-I), aromatase (CYP19), and 17β-hydroxysteroid dehydrogenase (17β-HSD) showed sex and site-specific patterns in the distinct reproductive phases. The sex- and season-specific changes generated discriminative multi-parameter profiles, serving as a tool for environmental and aquaculture surveys.

Estrogenic effects triggered by androgens have been previously shown in a few studies. Aromatization and direct binding to estrogen receptors (ERs) are the most proposed mechanisms. For example, previously, a modulation of vitellogenin A (VtgA) by testosterone (T), an aromatizable androgen, was reported in brown trout primary hepatocytes. The effect was reversed by an ER antagonist. In this study, using the same model the disruption caused by T and by the non-aromatizable androgen - dihydrotestosterone (DHT), was assessed in selected estrogenic targets. Hepatocytes were exposed (96 h) to six concentrations of each androgen. The estrogenic targets were VtgA, ERα, ERβ1 and two zona pellucida genes, ZP2.5 and ZP3a.2. The aromatase CYP19a1 gene and the androgen receptor (AR) were also included. Modulation of estrogenic targets was studied by quantitative real-time PCR and immunohistochemistry, using an HScore system. VtgA and ERα were up-regulated by DHT (1, 10, 100 μM) and T (10, 100 μM). In contrast, ERβ1 was down-regulated by DHT (10, 100 μM), and T (100 μM). ZP2.5 mRNA levels were increased by DHT and T (1, 10, 100 μM), while ZP3a.2 was up-regulated by DHT (100 μM) and T (10, 100 μM). Positive correlations were found between VtgA and ERα mRNA levels and ZPs and ERα, after exposure to both androgens. The mRNA levels of CYP19a1 were not changed, while AR expression tended to increase after micromolar DHT exposures. HScores for Vtg and ZPs corroborated the molecular findings. Both androgens triggered estrogen signaling through direct binding to ERs, most probably ERα.