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. 2026 Sep 8;55(5):e70175. doi: 10.1111/ahe.70175

Histology of Liver Vascular‐Biliary Tracts and Associated Exocrine Pancreas in Guppies (Poecilia reticulata) at Three Post‐Hatch Ages

Sisandra Sousa 1,2, Maria J Rocha 1,2, Eduardo Rocha 1,2,✉
PMCID: PMC13552196  PMID: 42708559

ABSTRACT

To describe the structural development of the liver in young guppies ( Poecilia reticulata ) and complement the adult characterisation, we conducted a histological study at 7, 17, and 28 days post‐birth. We used serial sections to trace the organisation of blood vessels, bile ducts, and pancreatic tissue within and around the liver. At all ages, the liver was highly cellular and composed mainly of hepatocytes. Pigmented melanomacrophage centres or aggregates were not observed, whereas rodlet cells were rare and restricted to extrahepatic connective tissue. At 7 days, we identified four distinct venous entry points. This multi‐afferent pattern persisted at Days 17 and 28. Arterioles remained restricted to the outer hilar region and were not observed within the liver. The gallbladder and common hepatic duct were recognisable at Day 7. In contrast, intrahepatic bile ducts were not discernible at Day 7, were scarce at Day 17, and were more frequent, although still limited, at Day 28. Exocrine pancreatic tissue was mainly extrahepatic at Day 7; intrahepatic pancreatic acini were detected in one fish at this age and were increasingly observed at Days 17 and 28 in association with afferent venous tracts. No vascular–biliary associations comparable to those described in adult guppies were observed. These findings establish a histological baseline for normal post‐hatching liver development in guppies and provide a reference for future studies in comparative anatomy and of environmentally associated changes in liver structure during early life stages.

Keywords: afferent‐efferent vessels, biliary tree, fish, liver, stromal tracts

1. Introduction

The importance of studying fish liver structure and function has increased over the last few decades. In particular, more detailed knowledge of this organ enables evaluation of fish health and environmental quality (Kroon et al. 2017; Topić Popović et al. 2023). Hundreds of studies have evaluated fish livers as part of biomonitoring (e.g., Könemann and Van Leeuwen 1980; Fanta et al. 2003; Carrola et al. 2009; Orso et al. 2023). For example, when studying the impacts of endocrine disruptors on fish reproduction, the liver is often a key organ of interest, owing to its role in producing the egg‐yolk precursor lipoprotein vitellogenin (e.g., Arukwe and Goksøyr 2003; Hara et al. 2016). In addition, the fish liver can serve as a model in biomedically relevant studies, such as induced hepatocarcinogenesis (e.g., Sato et al. 1973; Nunez et al. 1991). Lastly, liver morphophysiology is also relevant to aquaculture, particularly for studying the effects of diet (e.g., Caballero et al. 1999; Figueiredo‐Silva et al. 2005). In all the settings mentioned, the liver may display an array of structural and functional biomarkers of physiological adaptation or disease that can be translated into microanatomical changes or even gross lesions (van der Oost et al. 2003; Kroon et al. 2017; Topić Popović et al. 2023).

Histological studies are central for diagnosing acute and chronic effects of biological and chemical agents and for evaluating adaptive changes and lesions in fish (Gernhöfer et al. 2001; Hinton and Lauren 1990). To identify liver microanatomical alterations, including subtle early changes and advanced injuries, the histopathologist must know the organ's normal histology in detail. The histopathologist must also distinguish exogenous from endogenous influences, particularly those related to age and sex. To complicate matters further, fish comprise over 36,100 species (FishBase 2025), and their hepatic histology exhibits phylogenetically acquired features that may differ even among closely related species (Akiyoshi and Inoue 2004; Shiojiri et al. 2018). Some divergences are clear, such as species‐specific variation of hepatic exocrine (acinar and ductal) pancreocytes (Rocha et al. 1994; Figueiredo‐Fernandes et al. 2006). Other differential interspecific traits are evident with advanced methods, such as immunohistochemistry and 3D reconstructions (Figueiredo‐Fernandes et al. 2007; Shiojiri et al. 2018; Sousa et al. 2018).

Sex and age may influence fish liver histology, but sex has received the most attention. Qualitative and quantitative sex differences in the liver have been described mainly in salmonids (Hampton et al. 1989; Rocha et al. 2010; de Barros et al. 2026). They have also been reported in other fish, such as the Japanese medaka (Teh and Hinton 1998; Qiao et al. 2016) and the zebrafish (Yao et al. 2012; de Oliveira et al. 2016). Rarely, other species have been studied for sex‐specific variation, for example, the Nile tilapia (Figueiredo‐Fernandes et al. 2006). Much less attention has been given to liver ontogenesis during embryonic development and throughout the growth of fry and young juveniles. In post‐hatched and juvenile fish, the few studies focus mainly on developmental anatomy (Hinton et al. 2004) or specific cells, such as macrophages in brown trout (Resende et al. 2002) and hepatocytes in sea bream (Guyot et al. 1995) or sea trout (Byczkowska‐Smyk 1967a, 1967b). A study in domestic carp covered ages from embryo to 15 years of age and analysed hepatocytes and intrahepatic pancreas (Fishelson and Becker 2001).

Most studies in the fish liver have focused on oviparous species, regarded as the “typical fish”. However, some fish use another reproductive mode: ovoviviparity. Members of the family Poeciliidae are ovoviviparous and include species of ecological and economic importance, such as popular pet mollies, platies, swordtails, and guppies, the latter being widely distributed and common in the pet trade (Manenti et al. 2025). Among them, the guppy is one of the most extensively studied, featuring in numerous publications across diverse topics (e.g., Imai et al. 2022; Searle et al. 2023; Fox et al. 2024), as shown by a Scopus search for “guppy” that retrieved 3485 documents (21 May 2026).

Despite histopathological evaluations of the guppy liver (e.g., Sato et al. 1973; Wester et al. 1985; Wester and Canton 1992) and evidence that gender‐specific responses to stressors may occur (Antunes et al. 2017), the normal microanatomy of the organ has been neglected. To the best of our knowledge, only one detailed histological study of guppy liver has appeared in an internationally indexed journal (Sousa et al. 2018). This study was conducted in adults and examined the types of intrahepatic vascular‐biliary tracts and their three‐dimensional distribution. The work raised new questions about how such tracts are established early in ontogeny. To the best of our knowledge, only one publication addresses changes in the guppy liver anatomy throughout life, reporting not only pigment accumulation but also degenerative cytological changes (Woodhead 1978). The build‐up of lipofuscin in the livers of adult guppies with age has been confirmed, accompanied by an increase in melanomacrophage centre size (Imai et al. 2022). In contrast, no published studies have examined the early development of hepatic structure in the postnatal guppy, specifically the stromal tracts and associated pancreas.

The present study aimed to characterise the postnatal ontogeny of hepatic vascular, biliary, and pancreatic organisation in fry and juvenile guppies using serial histological sections. It complements previous work on guppy adult liver histology (Sousa et al. 2018) and provides new data that clarify the organ's structure and development, both of which are essential for evaluating external‐factor‐induced abnormalities.

2. Materials and Methods

2.1. Animal Handling

The work complied with national animal care and use laws. Adult guppies reared in Israel were purchased from a local authorised importer company. The fish were acclimatised to laboratory conditions for 3 weeks, kept in glass aquaria with dechlorinated, continuously filtered, and aerated tap water at 26°C ± 1°C and under a 12:12 h light–dark photoperiod. The pH was measured with a benchtop meter (WTW, Germany), and water quality was assessed every other day by monitoring NO3, NO2 −, and NH3/NH4 using kits (PRODAC, Italy) to ensure they remained within optimal ranges. In addition, partial water exchanges were made once weekly to maintain quality.

To obtain offspring, pregnant adult females were transferred to individual five‐litre aquaria within floating livebearer breeding boxes, equipped with a bottom grid trap to prevent the mother from eating the offspring. Water quality was maintained as described. Animals were fed once a day with TetraMin Tropical Fish Flakes.

After parturition, the newborns were separated from the mother and left to grow for 7, 17, and 28 days (Figure 1). Fish were maintained under constant water conditions and fed twice daily with TetraMin Tropical Flakes, crushed to a particle size appropriate for each developmental stage. At each age, 5 females and 5 males were studied histologically. Because sex could only be determined by histology, additional animals were initially sacrificed and randomly selected until 5 of each sex were obtained. The initial pool had an approximately 1:1 male‐to‐female ratio.

FIGURE 1.

FIGURE 1

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Photos of guppies taken with a stereo microscope. (A) 7‐day‐old guppy; (B) 17‐day‐old guppy; (C) 28‐day‐old guppy.

2.2. Fixation and Tissue Processing

Each guppy was humanely killed by immersion in a bath of an ice‐cold solution of the anaesthetic ethylene glycol monophenyl ether (Merck, Germany), at a concentration of 1 μL/mL. Before further processing, each fish was weighed and photographed with a binocular microscope (SZ61, Olympus, Japan) equipped with a digital camera (DP21, Olympus, Japan) for later length measurement using the free software ImageJ. Then, the animal was fixed in toto in Bouin's solution at room temperature for 24 h. The fixed individuals were washed in 70% ethanol and dehydrated with an ascending series of alcohols (70%, 95%, and 100%), followed by clearing in xylene. The samples were then impregnated in high‐quality paraffin (Paraplast Plus, Sigma‐Aldrich) and processed using an automatic tissue processor (Leica TP1020, Germany). Each animal was oriented in the paraffin block to produce longitudinal sections. Each block was exhaustively serially sectioned into 25‐μm thick paraffin sections using an automated rotary microtome (Leica RM2155, Germany). The sections were picked up on glass slides coated with 3‐aminopropyl‐triethoxysilane (Sigma, Germany) and stained with haematoxylin‐eosin (H&E). Slides were mounted with DPX (Sigma‐Aldrich, Germany) and examined using a light microscope (Olympus BX50, Japan) equipped with a high‐numerical‐aperture (NA 1.35) 100× oil‐immersion objective, which provided high resolving power and a shallow depth of field. The objective was used when required for detailed assessment of small vascular and biliary structures. All sections containing liver tissue were studied. Photos were taken with a digital camera (DP21, Olympus, Japan).

2.3. Histological Tracking of Serial Sections

All liver‐containing sections from each fish were analysed in detail, regardless of fragment size; no sections were skipped. The mean number of liver‐containing sections examined per fish was 25, 30, and 35 at 7, 17, and 28 days post‐birth, respectively. Observations were confirmed by two observers (Sisandra Sousa and Eduardo Rocha). As a first approach, we searched for the localisation of zone(s) where: (1) afferent blood vessels could be perceived as entering the liver; (2) bile duct(s) could be spotted emerging from the liver; and (3) hepatic vein(s) could be seen leaving the organ with efferent blood towards the heart. To meet these goals, we tracked small venous and arteriolar blood vessels and bile ducts along serial sections from the hilum or other potential vascular afferent points. We also tracked isolated veins and small starting bile ducts in reverse, up to the point where they joined other afferent elements or left the organ (as efferent structures). As such, afferent vessels were all those entering the organ through the hilum or hilar region, recognised in principle by the presence of bile ducts, arterial vessels, and pancreatic tissue. Efferent veins were defined as vessels originating from the capillary network, not linked to hilar vessels and emerging from the liver at a non‐hilar location. Isolated vascular and/or biliary elements and their associations were named according to the nomenclature used in the adult guppy liver (Sousa et al. 2018).

Vessel type was assessed morphologically in serial sections based on lumen size and shape, wall thickness, and the presence or absence of a discernible smooth‐muscle layer. Venous vessels were recognised by their typically larger lumen and thin wall, whereas arterioles were identified by their smaller lumen and comparatively thicker wall. The bile ducts were readily distinguished by their cuboidal epithelium, even when the epithelial cells were low in height. The course of each vessel and duct was followed through consecutive serial sections to further support the distinction between venous vessels, arterioles, and ducts, and to determine their relationship with the hepatic parenchyma and associated structures. Only after analysis of all sections containing a given structure and serial correlation of the tracked elements was the classification of each vascular and/or biliary element recorded.

3. Results

Observations of the serial sections are summarised in six image plates. Both general aspects and details are given for Days 7 (Figures 2 and 3), 17 (Figures 4 and 5), and 28 (Figures 6 and 7). Females and males did not differ in development; thus, the descriptions are unified.

FIGURE 2.

FIGURE 2

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Histological images from 7‐day‐old guppies. (A) General aspect at low magnification, showing the relative position of different organs —brain (B), eye (E), gills (G), intestine (I), liver (L)—within the animal (the ventral side is on the right side); (B) Higher magnification of the liver, displaying a compact aspect, and whose parenchyma consists essentially of hepatocytes (He). The stroma is yet scarce and barely perceivable at low magnification; (C) General aspect showing three of the four entrance points of blood vessels in the liver (ventral 1, ventral 3, and dorsal 4); (D) Closer observation of the most ventral and anterior entrance of a small afferent vein (V), originating from the intestinal wall (corresponds to ventral 1 in image C); (E) Detail of another, more posterior and bigger, afferent vein (V) (that corresponds to ventral 3 in image C), typically surrounded by exocrine pancreatic acini (P); (F) Section that captured the tiny vein (V) entering the liver at position ventral 2. One islet of Langerhans (IL) appears as a roundish cluster of cells near the liver, pancreatic acini (P), and gallbladder (GB). Staining: H&E.

FIGURE 3.

FIGURE 3

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Histological images from 7‐day‐old guppies. (A) Image showing the entry of the exocrine pancreas (P) into the liver (L), coming from the intestine (I), together with an afferent vein (V). (B) One emerging hepatic vein (HV), presenting two intrahepatic branches (limited by dashed lines); the one in the middle of the image runs anteroposteriorly, crossing almost the entire liver. (C) Image of the principal hilar zone of the liver, where one arteriole (AT) is seen outside the organ and surrounded by exocrine pancreas (P). One extrahepatic bile duct (BD) is observed. (D) Image showing exocrine pancreas (P) inside the liver (L), adjacent to a small vein near the organ periphery. (E) Principal hilar liver area, containing exocrine pancreas (P), islets of Langerhans (IL), an arteriole (A), and an afferent vein (V) approaching the liver (L). One bile duct emerges from the liver towards the forming gallbladder (GB). (F) Detail of an emerging larger bile duct (BD) resulting from several smaller ducts. Staining: H&E.

FIGURE 4.

FIGURE 4

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Histological images from 17‐day‐old guppies. (A) General aspect at low magnification, showing the relative position of different organs—gills (G), heart (H), intestine (I), kidney (K), liver (L), and thymus (T)—within the animal (the ventral side is on the left side). (B) General aspect at low magnification, showing several entry points of blood vessels into the liver (1, 2, 3, 4). Exocrine pancreatic acini (P) surrounding a small vein coming from the intestinal wall. The parenchyma consists essentially of intensely stained hepatocytes (He). (C) Aspect of a more posterior hepatic zone, showing two small veins (V), with associated pancreatic tissue (P), piercing the organ at two positions (at left, dorsal 4, at right, ventral 3). (D) Higher magnification of a vein (V) associated with exocrine pancreas (P) and surrounded by hepatic parenchyma. (E) Image showing the principal (larger) hilum (H) and another (more posterior) zone of entrance of an afferent vein associated with exocrine pancreatic acini and a small arteriole, together forming a single venous–arteriolar tract with pancreatic acini (VAT‐P). (F) Detail of the VAT‐P seen in E, with an arteriole (A), a vein (V), and exocrine pancreatic acini (P). Staining: H&E.

FIGURE 5.

FIGURE 5

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Histological images from 17‐day‐old guppies. (A) Overall view of the liver (L), showing a long, straight, centrally positioned intrahepatic vein (V) that crosses almost the entire liver (L) in a postero‐anterior direction and connects directly to a hepatic vein (HV). The top of the image corresponds to the anterior end of the fish, as in panel F. (B) Fusion of two (efferent) veins (V) within the liver (L). (C) Detail of an afferent vein (V) with associated pancreatic acini (P), piercing the liver (L) and starting to be encircled by parenchyma. (D) Detail with three sections of small bile ducts (B), one of them in close contact with exocrine pancreocytes (P). (E) Afferent vein (V) associated with exocrine pancreatic acini (P) observed already inside the liver (L). (F) Two efferent intrahepatic veins (V) merging into one hepatic vein (HV). Staining: H&E.

FIGURE 6.

FIGURE 6

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Histological images from 28‐day‐old guppies. (A) General aspect at low magnification that shows four entry points (ventral 1, 2, 3, and dorsal 4) of afferent veins into the liver. Veins in positions 2 and 3 show adventitial exocrine pancreatic acini (P). (B) Image at low magnification showing two small veins (V) coming from the intestinal wall and going towards the liver; both are sheathed by pancreatic acini (P). (C) Image at low magnification with veins (V), attached to the exocrine pancreas (P), piercing into the liver at different locations, including near the hilar zone (H); nearby, there is an islet of Langerhans (IL) and one small arteriole (A), associated with the exocrine pancreas (P). (D) Image detailing the hilum (H), where one vein (V), the main portal vessel associated with pancreatic acini (P), enters the liver (L); one arteriole (A), also surrounded by pancreas, is seen at the hilum. (E) Image showing three small intrahepatic bile ducts (B) and one (afferent) vein (V) partly sheathed with exocrine pancreas (P), running just parallel to the liver surface. (F) Detail of a biliary duct (BD) adjacent to a small intrahepatic bile duct (B), transitioning to the extrahepatic portion directed towards the gallbladder (GB). I—Intestine. Staining: H&E.

FIGURE 7.

FIGURE 7

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Histological images from 28‐day‐old guppies. (A) Image showing two more posteriorly located entry points of afferent veins (V) into the liver (L), each associated with exocrine pancreatic acini (P) and in proximity to an islet of Langerhans (IL). (B) Image showing two long afferent veins (V) that extend deep within the liver (L). (C) Section showing the liver (L) at the level of the main hilum (H), revealing three additional afferent veins: Two associated with pancreatic acini (P) and one without associated pancreatic acini (V). (D) Detail of three small bile ducts (B), one of them side‐by‐side with an afferent vein (V). (E) Two afferent veins (V) associated with pancreatic acini (P), one located deep within the liver (L). (F) Anterior‐most zone of the liver (L) at the level of the efferent single hepatic vein (HV). I—Intestine. Staining: H&E.

From 7 to 28 days, a noticeable feature was the continuous and parallel increase in body and liver size, to the point that in fish aged 28 days, it was no longer feasible to capture in a single micrograph the juvenile body profile (Figure 6A), in contrast to Days 7 (Figure 2A) and 17 (Figure 4A). The initial overall shape of the sectioned liver at Days 7 and 17 provided “triangular” profiles, while at 28 days the organ shape was elongated and somewhat more irregular. Up to Day 28, the liver was highly cellular, with virtually no discernible connective tissue within the organ. Although the endothelial cells lining the capillaries were visible, the liver's cellularity was composed mainly of hepatocytes. The liver lacked pigmented macrophages either in interhepatocytic or stromal locations.

Four entry points for presumed afferent blood vessels were observed. From Day 7, on the ventral face of the organ, three points were spotted (Figure 2C,F). In parallel, at the dorsal side of the organ, there was one fourth entrance point. For descriptive purposes, these points were numbered from 1 to 4, starting from the anterior‐ventral side. The presumed afferent vessels were venous; we never found arterial vessels entering the liver. At Day 7, those piercing the liver at points 1 and 2 were thin‐walled, very short, and derived from the intestinal serosa (Figure 2D,F). The main venous afferent vessels pierced the liver at points 3 and 4 and were notably larger, showing exocrine pancreocytes attached to the vascular adventitia. The four venous entrance points continued to be visible on Day 17 (Figure 4B) and Day 28 (Figure 6A).

From Day 7 onward, vascular organisation, particularly the hilar specialisation, increased in size and underwent progressive morphological differentiation. The liver hilus became progressively more evident with time and developed around the initial point 4, corresponding to the dorsal border of the organ. A gallbladder marked the hilar zone. By Day 7, the gallbladder was present, distinguishable by its simple cuboidal, basophilic epithelium (Figure 2C–F) and a tiny circular lumen. On Day 17 (Figure 4D) and Day 28 (Figures 6C,D and 7C), gallbladder growth was evident, with the organ's wall being irregular, showing some folds and delineated by a simple columnar epithelium. The common hepatic bile duct was recognisable from Day 7 (Figures 2E and 3E) and elongated over time, as evident on Day 28 (Figures 6D and 7C). Small and infrequent intrahepatic bile ducts were discernible on Day 17 (Figure 5D) and more frequent on Day 28 (Figures 6E and 7D).

During formation of the hilum, the area circumventing the gallbladder showed afferent veins, the adventitia of which displayed, from Day 7, plenty of exocrine pancreatic acini (Figure 2C–E) and one conspicuous endocrine Langerhans islet (Figures 2F and 6C,D). A small artery was in the area (Figures 3E and 6C,D), but we did not see it entering the liver. The pancreatic structures, together with the gallbladder and the small artery, define the liver hilum. All its natural markers were present on Days 17 (Figure 4E) and 28 (Figure 7A,C). However, some of the described structures were not exclusive to the hilum. In particular, in the above‐cited “entrance point 3”, an afferent vein was associated with pancreatic acini but lacked bile ducts. This arrangement constituted a secondary afferent entry region. The latter was noticed on Days 7 (Figure 2C,E), 17 (Figure 4B,C) and 28 (Figures 6B and 7C). The afferent vein of this secondary or pseudo‐hilum was not only bordered by pancreocytes but was associated with one tiny arterial vessel, forming a venous–arteriolar tract with pancreatic acini (Figure 4E,F); there were no Langerhans islets, and, like in the main hilum, we did not see arterial branches entering the liver.

In addition to the extrahepatic pancreas, exocrine pancreatic acini were also observed within the liver. They were found from Day 7 onward, either close to its periphery (mainly at 7 days) or located deep in the organ (especially at 17 and 28 days). The pancreocytes were distinct as focal and essentially basophilic groups of pancreocytic acini (Figures 3D, 4D, 5E and 7E), partially surrounded by hepatocytes and attached to the walls of small (afferent) veins. Such venous tracts with pancreatic acini originated from either a true hilum or a pseudo‐hilum (Figure 3A). Despite being present, those intrahepatic acini were rarely observed.

As to post‐capillary efferent vascularisation, from Day 7 onwards, two distinct elongated veins were visible. They formed from deep within the organ, after the capillary network, did not contact any hilus, and ran towards the already distinct hepatic vein (Figure 3B). Using the hepatic vein as a reference point, the linear efferent veins radiated through the liver in a radial pattern. The distance between them progressively increased. The length and diameter of the veins increased with age (Figures 5A and 7B). The veins opened at the anterior part of the organ and merged once piercing the liver capsule (Figures 5F and 6F). Those efferent veins were isolated vessels because they were not paired along their entire course with other veins, bile ducts, or pancreocytes.

A final aspect warranting specific mention is the presence of rare, scattered rodlet cells in 90% (27 in 30) of the studied animals. These cells were exclusively located within the connective tissue and were identified across all three age groups. Their distribution was strictly extrahepatic: they were observed either in the vascular adventitia in close proximity to the liver or within the connective tissue separating the pericardial and abdominal cavities. Figure 8 displays representative images of the rodlet cells and their locations at Days 7, 17, and 28.

FIGURE 8.

FIGURE 8

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Histological images showing extrahepatic rodlet cells of guppies at different ages. (A) Rodlet cell from a 7‐day‐old fish, located close to the liver (L). The cell is longitudinally sectioned, revealing the typical peripheral nucleus and intracytoplasmic elongated rodlets. (B) Two rodlet cells from a 17‐day‐old fish, also located close to the liver (L). The rodlet cell on the left is cross‐sectioned, and the one on the right is longitudinally sectioned. (C and D) depict the same rodlet cell, somewhat obliquely sectioned, at low (C) and high (D) magnification—40× and 100× objective lenses, respectively—to better illustrate its size relative to surrounding structures, namely the intestinal epithelium (IE), exocrine pancreatic acini (PA), and a liver (L) fragment. Staining: H&E.

The main developmental features observed at Days 7, 17, and 28 are comparatively summarised in Table 1.

TABLE 1.

Summary of hepatic vascular, biliary, pancreatic, and associated cellular features in guppies at 7, 17, and 28 days post‐birth (n = 10/age).

Feature Day 7 Day 17 Day 28
Body and liver size Small; the whole juvenile body profile could be captured in one micrograph Increased body and liver size; the whole‐body profile could still be captured in one micrograph Further marked increase; whole‐body profile could no longer be captured in a single micrograph
Overall liver profile Triangular Triangular Elongated and somewhat irregular
Hepatic parenchyma Highly cellular, composed mainly of hepatocytes; virtually no discernible intraparenchymal connective tissue Same general pattern Same general pattern
Venous entry points Four entry points: three ventral and one dorsal Four entry points maintained Four entry points maintained
Afferent veins Points 1 and 2: short, thin‐walled veins derived from the intestinal serosa. Points 3 and 4: larger veins associated with exocrine pancreatic acini Same entrance pattern maintained Same entrance pattern maintained
Arterioles Present in the outer hilar region; no intrahepatic arterioles Present in the outer hilar region; no intrahepatic arterioles Present in the outer hilar region; no intrahepatic arterioles
Liver hilus Present around dorsal entry point 4; marked by the gallbladder, afferent veins with pancreatic acini, a Langerhans islet, and a small arteriole Hilar markers maintained and more evident Hilar markers maintained and more evident
Common hepatic bile duct Recognisable Present and elongated Further elongated
Intrahepatic bile ducts Not discernible Small and infrequent More frequent than at Day 17, although still limited
Secondary afferent entry region at entry point 3 Present; afferent vein associated with pancreatic acini but devoid of bile ducts Present Present
Intrahepatic pancreatic acini Absent in 9/10 fish; present in 1/10 fish, where acini were very peripheral Present within the liver, along afferent venous tracts; more frequently located deep within the liver than at Day 7 Present within the liver, along afferent venous tracts; more frequently located deep within the liver
Efferent veins Two elongated veins visible after the capillary network Increased length and diameter Further increased length and diameter
Pigmented macrophages Not observed in 10/10 fish Not observed in 10/10 fish Not observed in 10/10 fish
Rodlet cells Rare and extrahepatic; present in 10/10 fish Rare and extrahepatic; present in 9/10 fish Rare and extrahepatic; present in 8/10 fish
Individual variation One fish showed very peripheral pancreatic acini; otherwise, findings were consistent among fish No relevant variation observed No relevant variation observed
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Note: All liver‐containing serial sections from each specimen were examined. Rodlet cells were confined to extrahepatic connective tissue, particularly the vascular adventitia near the liver and the connective tissue separating the pericardial and abdominal cavities. Findings are presented jointly for females and males (no sex‐related differences).

4. Discussion

This study shows that the early ontogeny of hepatic vascular–biliary organisation in the guppy follows a distinctive developmental pattern. Rather than undergoing fully coordinated maturation, the structural components of the liver develop asynchronously, with vascular architecture becoming established before substantial biliary differentiation and pancreatic integration. Thus, adult hepatic organisation appears to arise through a temporally non‐synchronous sequence of structural maturation.

In adult guppies, the liver contains exocrine pancreatic tissue (hepatopancreas) (Sousa et al. 2018), as also reported in other species such as Nile tilapia (Figueiredo‐Fernandes et al. 2007). In both species, this tissue is often associated with blood vessels and bile ducts. During ontogenesis, exocrine pancreatic tissue develops around blood vessels and may remain extrahepatic or extend into the liver (Bruslé and Anadón 1996; Bombonato et al. 2007). In the present material, no intrahepatic pancreatic tissue was detected in most 7‐day‐old fish; only one individual showed exocrine pancreatic acini, and these were located very peripherally. By contrast, pancreatic tissue was frequently observed near the liver surface, particularly at the ventral (3) and dorsal (4) venous entry points, sometimes accompanying the vessel up to the liver border but not crossing it. In 17‐ and 28‐day‐old fish, exocrine pancreatic acini were present within the liver along afferent blood vessels. This pattern is consistent with the independent development of hepatic parenchyma and pancreatic tissue. It also indicates a progressive increase in intrahepatic pancreatic acini associated with afferent venous tracts, approaching the adult pattern. This recurring spatial association suggests that afferent venous tracts may provide a structural route for the progressive establishment of the hepatopancreatic pattern. However, their role in directing pancreatic extension cannot be determined solely from morphology. The origin of the pancreatic tissue associated with the efferent veins in adult fish also remains unclear (Sousa et al. 2018). Whether the pancreatic tissue associated with the efferent veins results from an internal metaplastic transformation of biliary epithelium (Wolf et al. 1990), or reflects a heterotopic origin (Lawrence et al. 2015) remains unconfirmed.

As in adult guppies, several entry points for afferent blood vessels were observed in the liver, with up to four distinct venous sites identified. This highlights that, even at these early stages, guppies do not fit the classical single‐hilum template or the portal‐triad organisation typical of many other vertebrates (Shiojiri et al. 2018, 2023). Such observations were only possible with serial sections, which allowed us to trace the vessels from the intestinal wall into the liver. Our findings show that venous afferent vessels enter the liver through multiple sites and constitute the predominant morphological component of the hepatic vascular supply. This observation differs from the pattern reported for Nile tilapia, in which the portal vein branches before reaching the hilum–liver border (Figueiredo‐Fernandes et al. 2007). Taken together, these findings emphasise interspecific variation in portal inflow pathways and highlight the importance of species‐specific studies. Serial‐section tracking enabled vessel continuity to be traced. However, the present analysis remains based on two‐dimensional histology and does not provide a complete three‐dimensional reconstruction of the hepatic vascular network. Future studies combining 3D reconstruction with complementary techniques—such as micro‐computed tomography, confocal microscopy, or vascular corrosion casting—could further validate the spatial arrangement and branching of these vessels.

As in adult guppies (Sousa et al. 2018), isolated arterioles were not observed within the liver parenchyma in the present study. Although arterioles were present at all ages, they were restricted to the outer zone near the central hilar region. This finding is consistent with the low degree of hepatic arterialisation previously described in adult guppies (Sousa et al. 2018). Such limited arterialisation is not unique to the guppy; in zebrafish, a formal hepatic artery appears to be lacking, with only occasional hepatic radicles being observed (Lorent et al. 2004; Hrncir and Gracz 2023). Therefore, from the earliest stages of guppy development, the hepatic vascular organisation was morphologically dominated by venous afferent vessels. Guppies can tolerate hypoxic conditions through behavioural and physiological responses and may persist in polluted environments (Kramer and Mehegan 1981; Gomes‐Silva et al. 2020). However, whether the predominantly venous hepatic vascular organisation observed here contributes to hypoxia tolerance or persistence in polluted environments cannot be inferred from the present morphological data.

Melanomacrophage centres or macrophage aggregates (MAs), pigmented aggregates of phagocytic cells involved in host defence (Agius and Roberts 2003; Polinas et al. 2021), were not observed within the hepatic parenchyma, in contrast with adult guppies (Sousa et al. 2018). Their absence in these young fish is consistent with reports that MAs increase in size and frequency with age (Schwindt et al. 2006). Our findings also reinforce that MAs are not reliable markers of afferent vessels, since in adult guppies they may be associated with both afferent and efferent vessels (Sousa et al. 2018), as previously noted in brown trout (Rocha et al. 1994).

A noteworthy observation was the presence of rodlet cells (RCs)—though not in all specimens—within the connective tissue adjacent to extrahepatic blood vessels, near the pancreatic tissue, or associated with the pericardio‐peritoneal septum. The presence of RCs is not universal across all fish species, and there is interindividual variability such that they are undetected in some individuals of the same species (Sayyaf Dezfuli et al. 2022). Although the origin and nature of RCs have long been debated, a growing body of recent evidence supports their endogenous nature (Sayyaf Dezfuli et al. 2022; Manera 2024). Here, RCs were detected in most animals, although generally in low numbers, whereas none were identified in adult guppies despite exhaustive serial sectioning (Sousa et al. 2018). This contrast suggests that their presence and abundance may vary across the guppy life cycle.

Rodlet cells have generally been associated with inflammatory responses and other stressors, including exposure to toxicants (Sayyaf Dezfuli et al. 2022; Manera 2024). In the present study, their low abundance is consistent with the absence of overt pathological changes under the husbandry conditions used. However, their developmental and functional significance, including any relationship with environmental conditions, cannot be inferred from the present material.

The gallbladder and common hepatic duct were recognisable as early as Day 7. By Days 17 and 28, the gallbladder had grown and developed folds, whereas the common hepatic duct had elongated. In contrast, intrahepatic bile ducts are rare at Day 17 and only modestly more frequent at Day 28. Thus, up to the age examined, no mature, dense biliary network was present. These observations indicate that intrahepatic biliary branching lags behind vascular development in the early postnatal guppy liver. This delay may explain the absence, in the present material, of vascular‐biliary associations comparable to the venous‐biliary‐arterial tracts (VBAT) and venous‐biliary tracts (VBT) described in adult guppies (Sousa et al. 2018), brown trout (Rocha et al. 1994), Nile tilapia (Figueiredo‐Fernandes et al. 2007), and grass carp (Mokhtar 2018). Studies on fish biliary development in embryos and juveniles are scarce. Recent post‐hatching data from grass carp provide a useful comparative perspective (Fiala et al. 2026). In this oviparous teleost species, small intrahepatic bile ducts were observed at 20 days post‐hatching, and pancreatic acini began to extend into the liver around portal‐vein branches at the same age. The intrahepatic pancreas was clearly established by 90 days post‐hatching. Direct timing comparisons should nevertheless be made with caution, as grass carp and guppies differ substantially in reproductive mode, growth rate, and developmental schedule. Still, both species show progressive maturation of hepatopancreatic and vascular–biliary organisation rather than its complete establishment immediately after hatching. In Nile tilapia, exocrine pancreatic tissue has likewise been reported to extend into the liver alongside blood vessels during early post‐hatching development (Morrison et al. 2004), indicating that progressive hepatopancreatic integration also occurs in other teleosts. At the mechanistic level, observations in wild‐type and selected zebrafish mutants provide a relevant developmental context for our findings, showing that the emergence of cholangiocytes and the initial stages of bile duct development occur spatially away from blood vessels and independently of the vascular endothelium (Lorent et al. 2004). In zebrafish, this early biliary developmental programme depends on Jagged‐mediated Notch signalling, which is required for biliary cell specification and bile duct formation (Lorent et al. 2004). Whether comparable molecular mechanisms underlie the delayed intrahepatic biliary branching observed in guppy remains to be determined.

5. Conclusions

The present study characterises the early post‐hatching development of the guppy liver and complements existing descriptions of the adult organ. The hepatic vascular organisation was established early, with a multi‐afferent venous supply and no isolated arterioles within the liver parenchyma. In contrast, the intrahepatic biliary system remained poorly developed throughout the period examined, and no vascular–biliary associations comparable to those of adult guppies were observed. Intrahepatic pancreatic acini became more frequent during development and were associated with afferent venous tracts. Rodlet cells were sparse and confined to extrahepatic connective tissue, while pigmented melanomacrophage aggregates were absent. Together, these features distinguish the young liver from the adult condition and provide a developmental reference for comparative anatomical and histopathological studies. The findings also serve as a basis for future controlled studies examining environmentally associated changes in liver structure during early life stages.

Author Contributions

Conceptualisation: S.S., M.J.R., E.R.; Methodology: S.S., M.J.R., E.R.; Formal analysis: S.S., E.R.; Investigation: S.S., E.R.; Resources: M.J.R., E.R.; Writing – Original Draft: S.S., E.R.; Writing – Review and Editing: S.S., M.J.R., E.R.; Visualisation: S.S., E.R.; Supervision: M.J.R., E.R.; Project administration: M.J.R., E.R.; Funding acquisition: M.J.R., E.R.

Funding

This work was supported by FCT—Fundação para a Ciência e a Tecnologia, UIDB/04423/2025, UID/PRR/04423/2025, LA/P/0101/2020.

Ethics Statement

The study complied with the ethical guidelines described in the Directive 2010/63/EU and Decree‐Law no. 113/2013 and was conducted under approval from the Portuguese authority (DGAV, ref. 0421/000/000, 27/02/2018). Further permissions were not required as euthanasia performed by accredited personnel solely for organ or tissue use is not considered a “procedure” under that Directive.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgements

This research benefited from national funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., and by the European Commission's Recovery and Resilience Facility, within the scope of UIDB/04423/2025 (https://doi.org/10.54499/UIDB/04423/2025), UID/PRR/04423/2025 (https://doi.org/10.54499/UID/PRR/04423/2025), and LA/P/0101/2020 (https://doi.org/10.54499/LA/P/0101/2020).

AI Statement: AI‐assisted language tools were used solely for proofreading and language editing during manuscript preparation. Specifically, we used the Superhuman GO assistant (formerly Grammarly) to identify typographical errors and suggest improvements to grammar, clarity, and sentence flow. We did not use the tool to generate, analyse, interpret, manipulate, or fabricate research data, figures, results, references, or scientific conclusions. All suggested edits were reviewed, selected where appropriate, and approved by the authors, who retain full responsibility for the content, accuracy, and integrity of the manuscript.

Data Availability Statement

Data will be made available on reasonable request.

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