The aim of this study was to compare the levels of reactive oxygen species (ROS) in Bos taurus and Bos indicus in vitro embryos cryopreserved using either slow freezing or vitrification. Embryos were divided into four groups based on subspecies and freezing method: Bos indicus slow freezing (BiSF; n = 8), Bos indicus vitrification (BiVT; n = 10), Bos taurus slow freezing (BtSF; n = 9), and Bos taurus vitrification (BtVT; n = 6). After thawing, the embryos were incubated with CellRox Green and images were obtained using a confocal microscope. The fluorescence intensity of each cell was measured and expressed as arbitrary units of fluorescence (auf) and compared using a multiple regression and unpaired t-test with α = 0.05. Results showed that subspecies and the freezing method significantly affected auf (P < 0.001; R2 = 0.1213). Bos indicus embryos had higher auf than Bos taurus embryos, whether frozen by slow freezing (67.05 ± 23.18 vs 51.30 ± 16.84, P < 0.001) or vitrification (64.44 ± 23.32 vs 47.86 ± 17.53, P < 0.001). Slow freezing induced higher auf than vitrification in both Bos taurus (51.30 ± 16.84 vs 47.86 ± 17.53, P < 0.001) and Bos indicus (67.05 ± 23.18 vs 64.44 ± 23.32, P < 0.014). In conclusion, Bos taurus embryos had lower ROS levels when frozen using vitrification, while Bos indicus embryos had consistent ROS patterns regardless of the freezing method. However, Bos indicus embryos frozen by slow freezing tended to have a higher number of cells with elevated ROS levels.

The sequential occurrence of three layers of smooth muscle layers (SML) in human embryos and fetus is not known. Here, we investigated the process of gut SML development in human embryos and fetuses and compared the morphology of SML in fetuses and neonates. The H&E, Masson trichrome staining, and Immunohistochemistry were conducted on 6–12 gestation week human embryos and fetuses and on normal neonatal intestine. We showed that no lumen was seen in 6–7th gestation week embryonic gut, neither gut wall nor SML was developed in this period. In 8–9th gestation week embryonic and fetal gut, primitive inner circular SML (IC-SML) was identified in a narrow and discontinuous gut lumen with some vacuoles. In 10th gestation week fetal gut, the outer longitudinal SML (OL-SML) in gut wall was clearly identifiable, both the inner and outer SML expressed α-SMA. In 11–12th gestation week fetal gut, in addition to the IC-SML and OL-SML, the muscularis mucosae started to develop as revealed by α-SMA immune-reactivity beneath the developing mucosal epithelial layer. Comparing with the gut of fetuses of 11–12th week of gestation, the muscularis mucosae, IC-SML, and OL-SML of neonatal intestine displayed different morphology, including branching into glands of lamina propria in mucosa and increased thickness. In conclusions, in the human developing gut between week-8 to week-12 of gestation, the IC-SML develops and forms at week-8, followed by the formation of OL-SML at week-10, and the muscularis mucosae develops and forms last at week-12.

Over the last decades, extracellular vesicles (EVs) have been found to be implicated in a complex universal mechanism of communication between different cell types. EVs are nanostructures of lipid nature that have an exosomal or ectosomal biogenesis, responsible for the intercellular transport of proteins, lipids, carbohydrates, nucleic acids, ions, among other molecules. The content of EVs can vary due to various factors such as hormonal stimuli, non-physiological conditions, metabolic state, etc. Once EVs reach their target cell, they can modulate processes such as gene expression, metabolism, response to external factors, and can even be associated with the delivery of molecules involved in epigenetic inheritance processes in germ cells. In mammalian reproduction, EVs have been shown to play an important role, either in vivo or in vitro, modulating a variety of processes in sperm, oocytes and embryos, and in their respective environments. Moreover, EVs represent a biodegradable, harmless and specific vehicle, which makes them attractive allies to consider when improving assisted reproductive technologies (ARTs). Therefore, the present review aims to describe the content of the main EVs involved in mammalian reproduction and how they can vary due to different factors, as well as to detail how EVs modulate, directly or indirectly, different molecular processes in gametes and embryos. In addition, we will highlight the mechanisms that remain to be elucidated. We will also propose new perspectives according to the characteristics of each particular EV to improve the different ARTs.

Chemotherapy, as an important clinical treatment, has greatly enhanced survival in cancer patients, but the side effects and long-term sequelae bother both patients and clinicians. 5-Fluorouracil (5-FU) has been widely used as a chemotherapeutic agent in the clinical treatment of various cancers, but several studies showed its adverse effects on reproduction. Reproductive toxicity of 5-FU often associates with developmental block, malformation and ovarian damage in the females. In males, 5-FU administration alters the morphology of sexual organs, the levels of reproductive endocrine hormones and the progression of spermatogenesis, ultimately reducing sperm numbers. Mechanistically, 5-FU exerts its effect through incorporating the active metabolites into nucleic acids directly, or inhibiting thymidylate synthase to disrupt the function of DNA and RNA, leading to profound effects on cellular metabolism and viability. However, some studies suggested that the toxicity of 5-FU on reproduction is reversible and certain drugs used in combination with 5-FU during chemotherapy could protect reproductive systems from 5-FU damage both in females and males. Herein, we summarise the recent findings and discuss underlying mechanisms of the 5-FU-induced reproductive toxicity, providing a reference for future research and clinical treatments.

The present study aimed to use an in vitro follicle culture (IVFC) biotechnique as a tool to evaluate the influence of whole flaxseed as a feed supplementation in the diet on the in vitro development of caprine early antral follicles (EAFs) and further embryo production. In total, 18 adult goats were homogeneously allocated into two diet groups: Control and Flaxseed. EAFs from both experimental groups (300–400 µm) were isolated and cultured in vitro for 18 days. After IVFC, recovered cumulus–oocyte complexes were submitted to in vitro maturation, and subsequently to IVF and in vitro embryo culture. The endpoints evaluated were follicular growth and morphology, oocyte recovery rate and diameter, sperm penetration, pronuclei formation, embryo development, and estradiol production. The addition of the whole flaxseed in the diet did not affect (P > 0.05) follicular growth and diameter. A higher (P < 0.05) percentage of oocytes ≥ 110 µm was recovered from the flaxseed treatment. However, the sperm penetration rate was higher (P < 0.05) in the control treatment when compared with the flaxseed treatment, but no differences were found regarding the rate of fertilization nor cleaved embryos. In conclusion, dietary flaxseed increased the recovery rate of fully grown oocytes, but it did negatively affect the sperm penetration rate, even though there was no further effect on the cleavage rate.

Intestinal atresia (IA), a common cause of neonatal intestinal obstruction, is a developmental defect, which disrupts the luminal continuity of the intestine. Here, we investigated (i) the process of lumen formation in human embryos; and (ii) how a defective lumen formation led to IA. We performed histological and histochemical study on 6–10 gestation week human embryos and on IA septal regions. To investigate the topology of embryonic intestine development, we conducted 3D reconstruction. We showed that a 6–7th gestation week embryonic gut has no lumen, but filled with mesenchyme cells and vacuoles of a monolayer of epithelial cells. A narrow gut lumen was formed by gestation week-9, the gut was filled with numerous vacuoles of different sizes, some vacuoles were merging with the developing embryonic gut wall. At gestation week-10, a prominent lumen was developed, only few vacuoles were present and were merging with the intestine wall. At IA septal regions, vacuoles were located in the submucous layer, covered by a single layer of epithelium without glandular structure, and surrounded with fibrous tissue. The mucosal epithelium was developed with lamina propria and basement membrane, but the submucosa and the longitudinal smooth muscle layers were not properly developed. Hence, the vacuoles in IA septum could represent a remnant of vacuoles of embryonic gut. In conclusion, the fusion of vacuoles with the developing intestine wall associates with the disappearance of vacuoles and gut lumen formation in human embryos, and perturbation of these developmental events could lead to IA.

The present study was undertaken to analyze the relative abundance (RA) of pluripotency-associated genes (NANOG, OCT4, SOX2, c-MYC, and FOXD3) in different grades of immature oocytes and various stages of in vitro-produced buffalo embryos using RT-qPCR. Results showed that the RA of NANOG, OCT4, and FOXD3 transcripts was significantly higher (P < 0.05) in A grade oocytes compared with the other grades of oocytes. The RA of the c-MYC transcript was significantly higher (P < 0.05) in A grade compared with the C and D grades of oocytes, but the values did not differ significantly from the B grade of oocytes. The RA of the SOX2 transcript was almost similar in all grades of the oocytes. The expression levels of NANOG (P > 0.05), OCT4 (P > 0.05), c-MYC (P > 0.05) and SOX2 (P < 0.05) were higher in the blastocysts compared with the other stages of the embryos. Markedly, FOXD3 expression was significantly higher (P < 0.05) in 8–16-cell embryos compared with the 2-cell and 4-cell embryos and blastocyst, but did not differ significantly from the morula stage of the embryos. In the study, the majority of pluripotency-associated genes showed higher expression in A grade immature oocytes. Therefore, it is concluded that the A grade oocytes appeared to be more developmental competent and are suitable candidates for nuclear cloning research in buffalo. In buffalo, NANOG, OCT4, SOX2, and c-MYC are highly expressed in blastocysts compared with the other stages of embryos.

The flora of Mediterranean ecosystems contains families with species having fully and under-developed embryos in their seeds. After-ripening for physiological dormancy release and smoke influence germination in many species. We investigated how after-ripening and embryo growth interact with smoke to influence the temporal dynamics of seedling emergence among fire ephemerals. Seeds were placed in the field and under standardized (50% relative humidity, 30°C) laboratory conditions to test the effects of summer conditions on physiological dormancy loss. Germination was tested with water or smoke compounds (smoke water, KAR1) at a simulated autumn/winter temperature (18/7°C). The timing and amount of seedling emergence with smoke was observed for seeds exposed to near-natural conditions. During summer, physiological dormancy was broken in all species, enabling germination at autumn/winter but not summer temperatures; no embryo growth occurred in seeds with under-developed embryos. At the start of the wet season, seedling emergence from seeds with fully developed embryos occurred earlier than from seeds with under-developed embryos. In a non-consistent manner among our study species, smoke and smoke compounds influenced the rate of embryo growth and amount of germination. Effects of smoke were noticeable in terms of number of emergents in the first emergence season. Among ecologically similar species, we have shown (1) that both thermal and embryo traits exclude germination in the summer, (2) how embryo size influences the timing of seedling emergence in autumn–winter, and (3) a reduced requirement for smoke in the second emergence season after a fire with a shift to reliance on seasonal cues for emergence.

Sperm-mediated gene transfer (SMGT) is a simple, fast, and economical biotechnological tool for producing transgenic animals. However, transgene expression with this technique in bovine embryos is still inefficient due to low uptake and binding of exogenous DNA in spermatozoa. The present study evaluated the effects of sperm membrane destabilization on the binding capacity, location and quantity of bound exogenous DNA in cryopreserved bovine spermatozoa using Triton X-100 (TX-100), lysolecithin (LL) and sodium hydroxide (NaOH). Effects of these treatments were also evaluated by intracytoplasmic sperm injection (ICSI)-SMGT. Results showed that all treatments bound exogenous DNA to spermatozoa including the control. Spermatozoa treated with different membrane destabilizing agents bound the exogenous DNA throughout the head and tail of spermatozoa, compared with the control, in which binding occurred mainly in the post-acrosomal region and tail. The amount of exogenous DNA bound to spermatozoa was much higher for the different sperm treatments than the control (P < 0.05), most likely due to the damage induced by these treatments to the plasma and acrosomal membranes. Exogenous gene expression in embryos was also improved by these treatments. These results demonstrated that sperm membrane destabilization could be a novel strategy in bovine SMGT protocols for the generation of transgenic embryos by ICSI.

Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

Autophagy is a major intracellular pathway for the degradation and recycling of cytosolic components. Emerging evidence has demonstrated its crucial role during the embryo development of invertebrates and vertebrates. We recently demonstrated a massive activation of autophagy in Paracentrotus lividus embryos under cadmium stress conditions, and the existence of a temporal relationship between induced autophagy and apoptosis. Although there have been numerous studies on the role of autophagy in the development of different organisms, information on the autophagic process during oogenesis or at the start of development in marine invertebrates is very limited. Here we report our recent data on the occurrence of autophagy at these key phases of development. In order to investigate autophagy trends we performed in vivo assays to detect autophagolysomes, as well as in situ analysis with anti-LC3 antibody to detect autophagosomes before the fusion with lysosomes. From data generated through confocal laser scanning microscopy and quantification of autophagic signals we have drawn several unequivocal conclusions. The results showed a copious and rising number of autophagic organelles that had specific localization. Interestingly the increase in autophagy that occurred just after fertilization has been proved to be crucial for correct initiation of the developmental programme: irreversible developmental delays and morphologic anomalies were induced by short autophagic inhibition. This work focused on the sea urchin model system and corroborates evidence on the need for self-digestion during development, enriching the knowledge on autophagy, a biological mechanism belonging to evolutionarily different organisms.

The Prochilodontidae prepare for reproduction in captivity, but neither ovulation nor spawning occurs, thus requiring induced reproduction for breeding and restocking. This study analyzed for the first time the embryogenesis and larval ontogeny of P. hartii submitted to induced reproduction by hypophysation with crude common carp pituitary extract. The extrusion of oocytes and sperm was performed manually and fertilization was done using the dry method. After fertilization, the eggs were kept in incubators at 23°C. A stereomicroscope was used to measure egg diameter and to monitor embryo development. Samples of larvae were collected daily for 7 days for histological and biometric analyses. The recently extruded oocytes, non-hydrated, are spherical, grey, and non-adhesive with a diameter of 1480 ± 39 μm and after hydration, have a diameter of 2860 ± 120 μm. The positive response to hypophysation was 100% for females and 80% for males. Spawning occurred 7 h after the third hormonal dosage. The fertilization rate was 77% at 23°C. Blastopore closure occurred at 6 h 45 min and embryonic development was completed 36 h 10 min after fertilization. After 204 h post fertilization (hpf) the larvae reached a standard length of 6.56 ± 0.14 mm with the yolk sac completely resorbed. In P. hartii, the oral cavity opening occurred 132 hpf. The results of this study provide knowledge to better understand induced reproduction, breeding, and management of P. hartii, a species with a high potential for pisciculture, and which is commercially important in the Jequitinhonha River basin.

Octamer-binding transcription factor 4 (Oct4) is a critical molecule for the self-renewal and pluripotency of embryonic stem cells. Recent reports have shown that Oct4 also controls cell-cycle progression and enhances the proliferation of various types of cells. As the high proliferation of donor fibroblasts is critical to the production of transgenic pigs, using the somatic cell nuclear transfer technique, we analysed the effect of Oct4 overexpression on the proliferation of porcine fibroblasts and embryos. Porcine endogenous Oct4 cDNA was cloned, sequenced and inserted into an expression vector. The vector was transfected into porcine fibroblasts, and a stable Oct4-overexpressed cell line was established by antibiotic selection. Oct4 expression was validated by the immunostaining of Oct4. Cell morphology was changed to sharp, and both proliferation and migration abilities were enhanced in Oct4-overexpressed cells. Real-time RT-PCR results showed that p16, Bcl2 and Myc were upregulated in Oct4-overexpressed cells. Somatic cell nuclear transfer was performed using Oct4-overexpressed cells, and the development of Oct4 embryos was compared with that of wild-type cloned embryos. The cleavage and blastocyst formation rates were improved in the Oct4 embryos. Interestingly, blastocyst formation of the Oct4 embryos was observed as early as day 5 in culture, while blastocysts were observed from day 6 in wild-type cloned embryos. In conclusion, the overexpression of Oct4 enhanced the proliferation of both porcine fibroblasts and embryos.

Betta splendens is a very important ornamental species. The current paper describes the embryonic and larval development of B. splendens under stereomicroscopy and scanning electron microscopy. Eggs and larvae from natural spawning were collected at different developmental stages at previously established intervals and analysed. The eggs of B. splendens are yellowish, clear, spherical, demersal, translucent and telolecithal with a large amount of yolk. Between 0–2 h post-initial collection (hpIC), the eggs were at the egg cell, first cleavage and morula stages. The blastula stage was identified at 2–3 hpIC and the early gastrula phase was observed at 3–4 hpIC with 20% epiboly, which was finalized after 13–18 hpIC. When the pre-larvae were ready to hatch, the appearance of somites and the free tail were observed, at 23–25 hpIC. At 29 hpIC, the majority of larvae had already hatched at an average temperature of 28.4 ± 0.2°C. The newly hatched larvae measured 2.47 ± 0.044 mm total length. The mouth opened at 23 h post-hatching (hPH) and the yolk sac was totally absorbed at 73 hPH. After 156 hPH, the heart was pumping blood throughout the entire larval body. The caudal fin, operculum and eyes were well developed at 264 hPH. When metamorphosis was complete at 768 hPH, the larvae became juveniles. The current study presents the first results about early development of B. splendens and provides relevant information for its reproduction, rearing and biology.

This study aimed to investigate the effect of melatonin supplementation at different levels in culture medium on embryo development in rabbits. Embryos of 2–4 cells, 8–16 cells and morula stages were recovered from nulliparous Red Baladi rabbit does by laparotomy technique 24, 48 and 72 h post-insemination, respectively. Normal embryos from each stage were cultured to hatched blastocyst stages in either control culture medium (TCM-199 + 20% fetal bovine serum) or control supplemented with melatonin at 10−3 M, 10−6 M or 10−9 M. No effect of melatonin was found on development of embryos recovered at 24 h post-insemination. The high level of melatonin at 10−3 M adversely affected the in vitro development rates of embryos recovered at 48 h post-insemination (52 versus 86, 87 and 80% blastocyst rate; 28 versus 66, 78 and 59% hatchability rate for 10−3 M versus 10−9 M, 10−6 M and control, respectively, P< 0.05). At the morula stage, melatonin at 10−3 M significantly increased the in vitro development of embryos (92% for 10−3 M versus 76% for control, P < 0.05), while the hatchability rate of these embryos was not improved by melatonin (16–30% versus 52% for melatonin groups versus control, P < 0.05). Results show that a moderate level of melatonin (10−6 M) may improve the development and hatchability rates of preimplantation rabbit embryos. The addition of melatonin at a 10−3 M concentration enhances the development of rabbit morulae but may negatively affect the development of earlier embryos. More studies are needed to optimize the use of melatonin in in vitro embryo culture in rabbits.

For Salmo salar, there is a lack of information on the morphology of the first blastomeres formed during embryonic development and which could be used as a diagnostic tool for the first stages of development. The purpose of this investigation, therefore, was to characterize morphometrically the first blastomeres of S. salar. From a pool of eggs incubated at 7.5°C, 100 microphotographs of blastodiscs were extracted and analyzed at different incubation periods: 12, 14, 16, 20 or 24 h. Blastodiscs were characterized morphologically after 16, 20 or 24 h incubation, and classified into symmetric or asymmetric groups according to their morphology. The ratio of length (L) versus width (W) of each blastomere was determined, to establish its symmetry. In addition, 20 microphotographs of blastodiscs of normal appearance were analysed morphologically (control blastodisc: CB) for comparison (20 or 24 h). Results show that the first cleavage ends after 16 h of development. Seven categories were established during blastomere characterization: 47% normal (G1); 27% with dispersed margins (G2); 10% unequal (G3); 9% ‘pie-shaped’ (G4); 3% amorphous (G5); 2% three equal blastomeres and one different one (G6); and 2% with eccentric cleavage (G7). Although the incidence of abnormal cleavage in S. salar is uncertain, there is a potential for some asymmetries to be corrected during embryogenesis to generate viable individuals. More studies are necessary to correlate these abnormal cleavage patterns with indicators of quality in the later stages of embryogenesis in this species, to establish a quality assessment tool for gametes and/or embryos in salmonid species.