2.1. Species Studied and Collection Location

To obtain the embryos and larvae, five female L. marmoratus and five male adult and mature P. reticulatum were used, belonging to the Piraí fish farm stock (Figure 1), located in the city of Terenos, Mato Grosso do Sul, Brazil. The use of these animals followed the guidelines of the Ethics Committee for the Use of Animals at UNESP, Ilha Solteira Campus (approval no. CEUA-FEIS/UNESP 09/2023). The reproducers were collected with a trawl net in the excavated tank, where they were kept and then transferred to concrete tanks located in the Piscicultura Piraí reproduction laboratory for hormonal induction of reproduction.

The average weight of females was 4.06 ± 0.35 kg, and the average weight of males was 3.28 ± 0.38 kg. The study was conducted during the reproductive period of the parental species starting in November 2022.

2.2. Obtaining Embryos

Hormone induction was carried out by applying a solution of the crude extract of the carp pituitary gland (EBHC) (Cyprinus carpio). Two doses of pituitary extract were applied to the females; the first dose was 5.0 mg/kg body weight, and after 12 h, the second application was carried out at a dosage of 1.0 mg/kg. Males received a dose of 1.0 mg/kg body weight during the second application of the females [15].

Approximately 6 h after applying the last dose of the hormone, oocytes were extruded, and sperm was collected through massaging in the cephalocaudal direction. The oocytes from females and semen from males were then pooled. Fertilization was carried out following the “dry” method, consisting of first mixing the spermatozoa with the oocytes in a dry container, without contact with water, and soon after, adding water for spermatozoa activation, fertilization promotion, and egg hydration. Afterward, the eggs were deposited in four vertical incubators of 200 L each, with ~200 g of eggs placed in each incubator. Incubation took place at an average temperature of 27.98 ± 0.42°C, with constant water renewal. In this study, the hybrid was termed Hybrid LP, a term coming from the initials of the scientific name of the parental species.

2.3. Collection Method

Embryo collection began shortly after fertilization and was carried out at 15 min intervals during the first 2 h of embryogenesis and then at 1-h intervals until the larvae hatched. Subsequently, collections were carried out every 6 h for the first 2 days and at 12 h intervals until the fifth day.

2.4. Processing the Material for Analysis

At each collection, two samples of 50 embryos were collected; one sample was fixed in a fixative solution of 2.5% glutaraldehyde in phosphate buffer (pH 7.2) and the other in a solution of 4% paraformaldehyde and 2% glutaraldehyde in Sorensen’s phosphate buffer (pH at 7.2) (Karnovsky’s fixative). After 24 h, the samples fixed in Karnovsky’s fixative were placed in 70% alcohol for subsequent analysis. The collected material was taken to the Neotropical Ichthyology Laboratory (L.I.NEO.), located at UNESP, Ilha Solteira Campus, São Paulo, Brazil, for further processing and analysis.

2.5. Light Microscopy Analysis

For the histological analysis, 20 prefixed specimens representing each stage of development were individually embedded and oriented in a glycol methacrylate plastic resin for microtomy. Serial transverse and sagittal histological sections of 3 µm thickness were obtained and stained with Harris hematoxylin and eosin, analyzed, and photomicrographed under a Carl Zeiss Scope A1 AX10 microscope equipped with an AxioCam MRc5 ZEISS digital camera and ZEN 3.6 (blue edition) image analysis program. To analyze the material “in toto,” the embryos and larvae were analyzed using a MOTIC SMZ-168 stereomicroscope and photomicrographed using the same image analysis system mentioned above.

2.6. Scanning Electron Microscopy (SEM)

For SEM analysis, the larvae prefixed in 2.5% glutaraldehyde were transferred to a microtube, washed with distilled water, postfixed in 0.5% osmium tetroxide, dehydrated in sequence with increasing ethanol content (7.5%–100%), and dried in critical point equipment (LEICA EM CPD030). Next, the samples were placed on stubs and covered with a 10 µm gold film in a metallizer (BAL-TEC-SCD 050 Sputter Coater), analyzed, and electromicrographed under an SEM (EVO LS15; Carl Zeiss). These procedures were performed at the Electron Microscopy Center of the Biosciences Institute, UNESP, Botucatu Campus, São Paulo, Brazil.

3.1. Embryogenesis

The eggs had a yellowish color, clear chorion, and small perivitelline space after hydration, with no oil drop observed inside the yolk sac during the entire period of embryonic development.

The development period of the hybrid embryos, from the moment of fertilization until the larvae hatched, was 13 h at an average temperature of 27.98°C. After fertilization, the following stages of hybrid embryogenesis were established: zygote formation, cleavage, gastrula formation, organogenesis, and hatching (Table 1).

3.2. Zygote Stage

With fertilization, the fusion of the male and female pronuclei, cytoplasmic migration, and animal and vegetative poles (yolk bladder) were established. The animal pole comprises active cytoplasm and a nucleus, with a light and translucent yellow color, whereas the vegetative pole is denser and has a darker yellowish color (Figure 2A, 2A ^∗, 2A ^∗∗). The average diameter of the yolk bladder was 703.43 ± 35.43 μm at this stage.

3.3. Cleavage Stage

Cleavage was of the discoidal meroblastic type, also known as partial or incomplete; that is, it occurs only at the animal pole, with the following cleavage pattern being observed: the first five cleavages are vertical in relation to the animal pole and perpendicular to each other, sequentially producing 2, 4, 8, 16, and 32 blastomeres (Figure 2B–F) and the sixth cleavage is parallel to the animal pole, giving rise to two layers of cells, totaling 64 blastomeres (Figure 2G).

The first cleavage was observed at 0.25 h after fertilization (HPF), and the embryos presented two blastomeres (Figure 3A,B). The second cleavage was detected at 0.50 HPF, wherein the embryos had four blastomeres. The third cleavage occurred at 0.75 HPF of development, and many small yolk granules could be identified inside the cells (Figure 3C,D). From this moment of incubation, observation of heterogeneity in the development of embryos began, with embryos with four and eight blastomeres being observed (Figure 3A,C).

The fourth cleavage was observed at 1 HPF, with embryos containing 16 blastomeres. At this phase, a reduction in the size of the blastomeres became evident, owing to the rapidity of the mitotic processes that occur at this stage. The fifth cleavage was observed at 1.25 HPF (32 blastomeres), a phase in which the yolk bladder presented a more rounded appearance than that in the previous phases (Figure 3E,F).

Until the 4th cleavage, the nucleus could not be observed in the cells, and visualization was possible only after the 5th cleavage. Moreover, until this moment, a structure that separated the yolk from the blastomeres was not observed, and blastomeres were formed by incomplete cleavage, remaining connected to each other and to the cytoplasmic layer that surrounds the yolk (Figure 3B,D, and F).

The sixth cleavage was observed at 1.5 HPF, with the mitotic spindle occurring horizontally, and the blastoderm now had cells arranged in two layers, with the lower layer still in incomplete division (Figure 3G,H). At 1.75 HPF, more than 100 cells were observed (Figure 3I,J).

The nuclei in the cells could be identified from the cleavage stage at the 64 blastomere phase (Figure 4A). Until the fifth cleavage, the mitotic spindles are perpendicular to the yolk and promoted incomplete cleavage, as they are unable to cleave the vegetative pole. These cells that were in direct contact with the vegetative pole form the periblast or yolk syncytial layer (Figure 4B), which was better observed in the morula phase.

The cleavage stage ended with the beginning of morphogenetic movements, the most visible being epiboly, which promotes the flattening of peripheral blastomeres by shortening their apico-basal axis. The advanced morula could be observed in the cleavage phase after 3 h of incubation. Because the emergence of a space (blastocele) between the blastoderm and periblast was not observed, rather only irregular spaces (Figures 3A,B) were observed between the blastomeres and the existence of a characteristic blastula stage was not considered.

3.4. Gastrula Stage

At 3 h of embryogenesis, the diameter of the yolk bladder was 695.96 ± 13.34 μm (Figure 3I,J). The beginning of the flattening of the peripheral cells was observed in preparation for epiboly (Figures 3I and 4C). At 4 h of embryogenesis, the blastoderm had already covered half of the yolk sac with the formation of a germ ring or an embryonic shield from the edge of the blastoderm (Figure 3K,L). However, heterogeneity in embryonic development was observed (Table 1).

After 5 h of incubation, most of the embryos had a blastoderm involving 90% of the yolk bladder, with only a small portion of the vegetative pole exposed, the yolk plug (Figure 3M,N). Involution could also be observed, with part of the blastoderm returning, an action that gives rise to the future endoderm (Figure 4C,D).

3.5. Organogenesis Stage

6 h after development, the embryos were in the initial neurula stage (Figure 5A). This stage was marked by cephalocaudal differentiation, the presence of the neural groove, and the segmentation of the mesoderm into more than five somite pairs. After 7 h of development, the following structures were observed: optic vesicles, with a greater distinction between the head and tail (Figure 5B).

At 8 h, a greater segmentation of the mesoderm could be identified (Figure 5C). Histological analysis revealed the structure of the optic vesicle that gives rise to the animal’s eye, which could be observed in the head region (Figure 4E); in the caudal portion, the structure of the Kupfer vesicle was internally lined by cubic to cylindrical ciliated epithelium cells (Figure 4F). At 9 h, the embryos had a longer tail (Figure 5D).

After 10 h, structural differentiation was observed during hindgut formation (Figure 5E). After 12 h, the tail was already very flexible, with many movements observed. The embryo already presents the morphological characteristics of a larva and is ready to hatch. The heartbeat could be observed (Figure 5F).

SEM analysis in the prehatch period with 12 h of development showed that the head was more arched, the opening of the future operculum was deeper (Figure 6A), the nasal pit was developing (Figure 6B), the embryonic fin was clearly visible in the tail region (Figure 6C), and the presence of many somites and a well-defined embryonic fin (Figure 6D).

3.6. Hatching of Larvae

After 13 h, the larvae began to hatch and swim, enabling the observation of heartbeats, straight tails, and clearly visible somites (Figure 6E). At this stage, the larvae were 1127.6 μm in length.

3.7. Hybrid Larvae 12 h After Hatching

After the larvae hatched, their average length was 3070.9 ± 238.6 μm (Figure 7A). Based on the ultrastructure, the operculum could be seen to have already deepened (Figure 7B), and two pairs of developing barbels were also visible (Figure 7C). Additionally, the anus (Figure 7D) and caudal fin (Figure 7E) were being formed.

3.8. Hybrid Larvae 24 h After Hatching

Twenty-four hours after the larvae hatched, the components of the head were further developed, such as a pigmented optic vesicle and a structured mouth. Because of transparency, larval pigmentation could be seen to be more developed owing to the presence of a greater quantity of chromatophores spread across the tail and in the anterior and posterior regions of the yolk sac (Figure 8A). Although the yolk sac remained, the stomach and intestine were already delimited (Figure 8A,B), the latter being already connected to the anus and the notochord extending from the cephalic to the caudal region.

3.9. Hybrid Larvae 30 h After Hatching

At this phase of the organogenesis stage, the larval length has already reached 3754 ± 68 μm (9C). The yolk sac had a very reduced volume with an elongated oval shape. Well-developed and visible wattles were present, and pigmentation was highly evident, with an increased number of chromatophores spread throughout the body. The nervous system was evident with the forebrain, midbrain, and telencephalon visible. The digestive system was well developed, and the oral cavity, esophagus, stomach, and intestine connected to the anal pore could be seen (Figure 8C,D). The eyeball was well developed, with an average diameter of 81.7 ± 10.9 μm, with a well-defined optic cup and a well-pigmented lens (Figure 8D).

The opercular structure was already formed, the nasal pit remained open, but invagination of the tissue towards closure had begun, and a greater number of olfactory sensors were present (Figure 9A,B). The mouth was open, with several teeth in development (Figure 9D), as well as two pairs of mandibular barbels in clear development and one pair lateral to the more developed mouth opening, with sensors arranged in the form of buttons (Figure 9A,D). The tail began to take on a heterocercal shape, characteristic of the species (Figure 9E).

3.10. Hybrid Larvae 60 h After Hatching

At this stage, the stomach was larger (Figure 10D), the yolk was completely absorbed, and the caudal fin had individualized (heterocerca) (Figure 10E) along with the other fins. The two pairs of barbels were larger, and a third pair was visible (Figure 10A). The mouth presented a greater opening, and the teeth were visible under optical microscopy in greater quantities (Figure 10A,B). The anal fin was delimited, and the region where the urogenital pores were positioned was individualized (Figure 10C).

3.11. Hybrid Larvae 108 h After Hatching

At this stage, the region with the nasal pit sensors underwent invagination for the internalization and closure of the nasal pit (Figure 11A). The eyes advanced in development, with an average diameter of 216.3 ± 20.8 μm. The pelvic fin was well-developed (Figure 11B), the operculum was formed (Figure 11C), and gill arches and filaments were observed (Figure 11D).

3.12. Hybrid Larvae 120 h After Hatching

On the fifth day, the barbels and eyes were larger (Figure 12A), with an average eye diameter of 231.8 ± 22.8 μm. An increase in the length of the larva was also observed, which is now measured at 7863 ± 171.2 μm (Figure 12B). Histological analysis revealed that the eyes were formed, allowing visualization of the optic cup, retina, and iris; the myomeres had a V shape (Figure 12C) and had begun transforming into myoblasts for the formation of skeletal muscle fibers. The notochord was still present, and in the gill region, the gill filament supports could be seen to be still cartilaginous (Figure 12D).