This study was aimed to establish embryonic stem (ES)-like cells from blastocysts derived from somatic cell nuclear transfer (SCNT) in pig. Somatic cells isolated from both day-30 fetus and neonatal cloned piglet were used for donor cells. A total of 60 blastocysts (46 and 14 derived from fetal and neonatal fibroblast donor cells, respectively) were seeded onto a mitotically inactive mouse embryonic fibroblast (MEF) monolayer and two ES-like cell lines, one from each donor cell type, were established. They remained undifferentiated over more than 52 (fetal fibroblast-derived) and 48 (neonatal fibroblast-derived) passages, while retaining alkaline phosphatase activity and reactivity with ES specific markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-4, TRA-1–60 and TRA-1–81. These ES-like cells maintained normal diploid karyotype throughout subculture and successfully differentiated into embryoid bodies that expressed three germ layer-specific genes (ectoderm: β-III tubulin; endoderm: amylase; and mesoderm: enolase) after culture in leukemia inhibitory factor-free medium. Microsatellite analysis confirmed that they were genetically identical to its donor cells. Combined with gene targeting, our results may contribute to developing an efficient method for producing transgenic pigs for various purposes.

Ocean acidification is now recognized as a threat to marine ecosystems; however, the effect of ocean acidification on fertilization in marine organisms is still largely unknown. In this study, we focused on sperm flagellar motility in broadcast spawning reef invertebrates (a coral and a sea cucumber). Below pH 7.7, the pH predicted to occur within the next 100 years, sperm flagellar motility was seriously impaired in these organisms. Considering that sperm flagellar motility is indispensable for transporting the paternal haploid genome for fertilization, fertilization taking place in seawater may decline in the not too distant future. Urgent surveys are necessary for a better understanding of the physiological consequences of ocean acidification on sperm flagellar motility in a wide range of marine invertebrates.

Fine structure observations were performed by means of electron microscopy on oogenesis and vitellogenesis and the special functions of follicular cells in the Chinese soft-shelled turtle (Pelodiseus sinensis). Histological examination of the ovary showed a well developed lacunae system containing fine granules, fibres or gelatiniform materials with one or two germinal beds dispersed on the dorsal surface of the ovarian cortex. The process of oogenesis comprised 10 consecutive phases according to the morphology of the yolk platelets, follicular cells and zona pellucida (ZP). Electron microscopy of vitellogenesis revealed some of the mitochondria gradually being transformed into yolk granules. In the advanced stage of vitellogenesis, large amounts of rough endoplasmic reticula, Golgiosomes and other cell organelles that are involved in synthesis and secretion were observed in follicular cells. The ZP was formed by microvilli, thus increasing the absorptive surface of the oocyte and facilitating transport of nutrients from the follicular epithelium to the ooplasm. This study demonstrated that the ovaries of members of the Testudinidae share more features with Archosaurs than with Squamates, indicating that these features were phylogenetically conserved in the Archosauria. The present observations suggest that the accumulation of yolk materials was controlled by the intrinsic and extrinsic pathways as well as by the activity of follicular cells. These results might also support a sibling relationship of the Testudinidae with the Archosauria and not with all extant reptiles.

The effect of bovine embryo culture on a flat surface, (without a surface-active compound) on the level of mRNA expression of hyaluronan (HA) synthases (Has1, Has2 and Has3), Ha receptors RHAMM and C44 receptors was evaluated by mitochondrial DNA concentration and in vitro development. Cultures were evaluated up to 96 h post-insemination (hpi) using SOFaa medium. Of the three Has isoforms, Has2 expression only increased in the bovine serum albumin (BSA)-only supplemented groups regardless of time of BSA addition. Expression of RHAMM receptors was highly dependent on the addition of HA, irrespective of the presence of BSA in the medium. In contrast, expression of the CD44 receptor gene was not affected by any treatment. The cleavage rates and number of embryos that developed to ≤8-cell stage by day 4 were not affected by lack of BSA in the medium, but increased numbers of blastocysts developed in medium supplemented with BSA from days 1 or 4 with or without HA than in medium that had HA only. Addition of both HA and BSA at day 4 increased mtDNA copy numbers at the blastocyst stage. Data suggest that the addition of BSA and/or HA at 96 hpi increased expression of RHAMM and Has2 genes, but not CD44, Has1 or Has3 genes. Higher expression levels of Has2 than Has1 and the three isoforms indicate that high- rather than low-molecular-weight HA should be used for preimplantation bovine embryo culture.

The developmental stages for the embryonic and larval periods of the silver catfish (Rhamdia quelen) kept at different temperatures (21, 24, 27 and 30°C) are described. Fish were analysed under light and scanning electron microscopy. For embryonic development, we described 25 stages, which were grouped into seven periods named zygote, cleavage, blastula, gastrula, segmentation, pharyngula and hatching periods. For larval development, we defined three stages (early, mid, and late larvae). Additionally, the main ontogenetic events during the post-larvae and early juvenile periods were also described. This species presents a well developped lateral line and chemosensory systems that grow up during the larval period, maturing in the post-larvae. All tested temperatures are viable to R. quelen development, but a shorter incubation period was necessary to complete the development at lower temperatures. However, some malformations (heart edema) were verified at 30°C.

With the development of embryo technologies, such as in vitro fertilization, cloning and transgenesis, cryopreservation of mammalian gametes and embryos has acquired a particular interest. Despite a certain success, various cryopreservation techniques often cause significant morphological and biochemical alterations, which lead to the disruption of cell organelles, cytoskeleton damages, cell death and loss of embryo viability. Ultrastructural studies confirm high sensitivity of the cell membrane and organelle membrane to freezing and thawing. It was found that many substances with low molecular weights have a protective action against cold-induced damage. In this concern, an anti-freeze protein (AFP) and anti-freeze glycoproteins (AFGPs), which occur at extremely high concentrations in fish that live in Arctic waters and protect them against freezing, may be of potential interest for cryostorage of animal embryos at ultra-low temperatures. This mini-review briefly describes several models of AFP/AFGP action to preserve cells against chilling-induced damages and indicates several ways to improve post-thaw developmental potential of the embryo.

In zebrafish chimaerism experiments, the cell injection can involve intra-embryonic cell lyses by osmolar effects. Moreover, the donor cells can be injured during manipulation due to osmolar changes into the transplant pipette. Therefore, the present study aimed to assess the effects of manipulation medium osmolarity on embryonic survival and donor cell viability.

In Experiment I, 0.1 μl to 0.15 μl approximately of an isosmolar solution (300 mOsm) was injected into recipient embryos, which were kept at 300 (E1) or 30 mOsm (E2). Survival at day 1 was significantly higher in the E2 group than in E1 (E1: 68% vs E2: 81%, p < 0.05), but after 5 days embryo survival in the E1 group was slightly higher. In Experiment II, donor cells from zebrafish embryos were exposed (or not) to a possible osmolarity change (inner pipette medium: 300 mOsm vs external medium: 30 or 300 mOsm) using two different micropipette outer diameters, 40–50 and 60–70 μm. Cell mechanical damage was detected in the 40–50 μm pipette (p < 0.05), but not by the handling medium osmolarity. Results recommend the use of a 300 mOsm manipulation medium and bore-sized pipettes adjusted as closely as possible to the donor cell size.

The black armoured catfish Rhinelepis aspera is an important commercial species found in the São Francisco River (Brazil) that has great potential for pisciculture. This paper presents, for the first time, information on the embryogenesis and larval development of this species. The breeder fish were subjected to spawning induction with crude carp pituitary extract. Eggs were kept in incubators at 24°C and embryogenesis was observed with a stereomicroscope until hatching. Ovary fragments, samples of recently extruded oocytes and larvae were fixed in Bouin's solution and subjected to routine histology techniques. Recently extruded oocytes were also analyzed with a scanning electron microscope (SEM). Females (80%) had a positive response to hypophysation. Oocyte extrusion was performed at 8 h and 30 min after the second hormone injection and the fertilization rate achieved 72.4 ± 8.8%. Recently extruded oocytes had a mean diameter of 1360.80 ± 40.87 μm and presented a conspicuous jelly coat covering the zona pellucida. Histochemical reactions indicated the presence of neutral glycoproteins in the yolk globules and in the zona pellucida. These reactions also indicated the presence of neutral glycoproteins and carboxylated acid glycoconjugates in the follicular cells and the jelly coat. These compounds form mucosubstances that can be related to the adhesiveness of the eggs. Under the SEM the oocytes presented a micropyle disc and a thick jelly coat over their surface. The closure of the blastopore occurred 9 h and 40 min after fertilization and embryogenesis was completed at 45 h and 50 min after fertilization, when the embryos were incubated at 24°C. The total absorption of the yolk sac occurred during the fifth day indicating the need for an exogenous food supply. These results provide essential information for the successful breeding and management of the promising R. aspera.

The embryogenesis of Brycon cephalus was established in seven stages: zygote, cleavage, blastula, gastrula, segmentation, larval and hatching, in an incubation period of 11 h (26°C). The zygote phase was observed directly after fertilization and egg hydration. Cleavage began at 0.5 h of incubation and extended up to the morula phase (1.5 h; +100 blastomeres). Cleavage was meroblastic and underwent the following division pattern: the first five divisions were vertical and perpendicular to each other, following the model 2 × 2, 4 × 2, 4 × 4 and 4 × 8. The sixth division was horizontal and occurred at 1.25 h after fertilization, giving rise to two cell layers (4 × 8 × 2) with 64 blastomeres. At the blastula stage (1.25–1.5 h), an irregular space between the blastomeres, the blastocoele, could be detected and the periblast structure initiated. The gastrula (1.75–6.0 h) was characterized by the morphogenetic movements of epiboly, convergence and cell involution, and formation of the embryonic axis. The segmentation stage (7–9 h) comprised the development of somites, the notochord, optic, otic and Kupffer's vesicles, neural tube, primitive intestine and ended with the release of the tail. The larval stage (up to 10 h) was characterized by the presence of 30 somites and growth and elongation of the larvae. At the hatching stage, the embryos presented more than 30 somites and exhibited swimming movements and a soft chorion. The blastomeres presented euchromatic nuclei, indicating a high mitotic activity and many yolk globules in the cytoplasm. The periblast was constituted of a layer with several nuclei and many vesicles, which grew during the epiboly movement.