Spent culture medium can provide valuable information regarding the physiological state of a bovine preimplantation embryos through non-invasive analysis of the sum/depleted metabolite constituents. Metabolomics has become of great interest as an adjunct technique to morphological and cleavage-rate assessment, but more importantly, in improving our understanding of metabolism. In this study, in vitro produced bovine embryos developing at different rates were evaluated using proton nuclear magnetic resonance (1H NMR). Spent culture medium from individually cultured embryos (2-cell to blastocyst stage) were divided into two groups based on their cleavage rate fast growing (FG) and slow growing (SG; developmentally delayed by 12–24 h), then analyzed by a 600 MHz NMR spectrometer. Sixteen metabolites were detected and investigated for sum/depletion throughout development. Data indicate distinct differences between the 4-cell SG and FG embryos for pyruvate (P < 0.05, n = 9) and at the 16-cell stage for acetate, tryptophan, leucine/isoleucine, valine and histidine. Overall sum/depletion levels of metabolites demonstrated that embryos produced glutamate, but consumed histidine, tyrosine, glycine, methionine, tryptophan, phenylalanine, lysine, arginine, acetate, threonine, alanine, pyruvate, valine, isoleucine/leucine, and lactate with an overall trend of higher consumption of these metabolites by FG groups. Principal component analysis revealed distinct clustering of the plain medium, SG, and FG group, signifying the uniqueness of the metabolomic signatures of each of these groups. This study is the first of its kind to characterize the metabolomic profiles of SG and FG bovine embryos produced in vitro using 1H NMR. Elucidating differences between embryos of varying developmental rates could contribute to a better understanding of embryonic health and physiology.

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

Previous investigations have indicated that c-Jun N-terminal kinase (JNK) regulates the maturation and aging of oocytes produced by deuterostome animals. In order to assess the roles of this kinase in a protostome, oocytes of the marine nemertean worm Cerebratulus were stimulated to mature and subsequently aged before being probed with phospho-specific antibodies against active forms of JNK and maturation-promoting factor (MPF). Based on blots of maturing oocytes, a 40-kD putative JNK is normally activated during germinal vesicle breakdown (GVBD), which begins at 30 min post-stimulation with seawater, whereas treating immature oocytes with JNK inhibitors downregulates both the 40-kD JNK signal and GVBD, collectively suggesting a 40-kD JNK may facilitate oocyte maturation. Along with this JNK activity, mature oocytes also exhibit high levels of MPF at 2 h post-stimulation. However, by ~6–8 h post-GVBD, mature oocytes lose the 40-kD JNK signal, and at ~20–30 h of aging, an ~48-kD phospho-JNK band arises as oocytes deactivate MPF and begin to lyse during a necroptotic-like mode of death. Accordingly, JNK inhibitors reduce the aging-related 48-kD JNK phosphorylation while maintaining MPF activity and retarding oocyte degradation. Such findings suggest that a 48-kD JNK may help deactivate MPF and trigger death. Possible mechanisms by which JNK activation either together with, or independently of, protein neosynthesis might stimulate oocyte degradation are discussed.

Parthenotes are characterized by poor in vitro developmental potential either due to the ploidy status or the absence of paternal factors. In the present study, we demonstrate the beneficial role of sperm-derived factors (SDF) on the in vitro development of mouse parthenotes. Mature (MII) oocytes collected from superovulated Swiss albino mice were activated using strontium chloride (SrCl2) in the presence or absence of various concentrations of SDF in M16 medium. The presence of SDF in activation medium did not have any significant influence on the activation rate. However, a significant increase in the developmental potential of the embryos and increased blastocyst rate (P < 0.01) was observed at 50 µg/ml concentration. Furthermore, the activated oocytes from this group exhibited early cleavage and cortical distribution of cortical granules that was similar to that of normally fertilized zygotes. Culturing 2-cell stage parthenotes in the presence of SDF significantly improved the developmental potential (P < 0.05) indicating that they also play a significant role in embryo development. In conclusion, artificial activation of oocytes with SDF can improve the developmental potential of parthenotes in vitro.

The aim of this study was to describe, for the first time, the embryogenesis and larval growth of the Paraitinga Brycon nattereri Günther, 1864 reared in captivity. After artificial fertilization, eggs were incubated at constant temperature (~19°C) and collected every 15 min during the first 3 h and then every 3 h until hatching. Five larvae were collected daily over 15 days for evaluation of the length, yolk sac volume and specific growth rate. The following stages of embryonic development were identified: zygote, cleavage, gastrula, segmentation and larval. The hatching occurred after 50–54 h, with larvae poorly developed and fully depigmented, devoid of mouth and swimming capacity, presenting 6.32 mm total length and 3.64 mm3 yolk sac volume. The mouth opening was observed between days 3–4 after hatching. The yolk sac absorption was slow during the first 3 days, increasing sharply after this period, being completed on the day 11. During this period there was a decrease in the larval growth rate. After yolk sac absorption, an increase in the growth rate was observed that coincided with the start of exogenous feeding. Cannibalism was not observed during the 15 days of evaluation. The initial development of B. nattereri was slow and poorly developed larvae in relation to other Brycon species, certainly due to the lower temperature required for egg incubation and larval rearing. Other studies are needed in order to develop techniques to improve the methods of incubating eggs and feeding larvae.

We produced a new chemical compound based on methylxanthines and polyphenols (CCMP) present in the chemical matrix of guaraná (Paullinia cupana), a seed extract with antioxidant properties. After supplementation with the standard extract of resveratrol, a well documented antioxidant found in other plant sources, we investigated whether this resveratrol-enriched compound could improve sperm viability and modulate differentially reactive oxygen species (ROS) and nitric oxide (NO) levels in thawed sperm. Sperm samples obtained from healthy young donors were treated with different concentrations of guaraná extract (0.1, 1, 5 or 10 mg/ml) and cells were frozen at −80°C for 24 h. In addition, the potential protective effects of guaraná treatment on sperm treated with pro-oxidant compound (200 µM hydrogen peroxide, H2O2) were assessed. Samples were also exposed to three concentrations of CCMP before being frozen in liquid nitrogen (−196°C) or in an ultrafreezer (−80°C) for 24 h, and both pre-freezing and post-thaw measurements of viability and oxidative stress were performed. Guaraná supplementation at 10 mg/ml significantly increased post-thaw viability and decreased oxidative metabolism of the sperm. Moreover, selected concentrations of CCMP improved viability and oxidative metabolism in sperm samples pre-freezing. Furthermore, CCMP showed cryoprotective activity by increasing viability and decreasing oxidative stress in post-thaw samples. In summary, these findings suggested that CCMP supplementation acts as a cryoprotectant to modulate ROS and NO levels in thawed sperm. CCMP could be used to enhance sperm quality and reproductive success.

In fish with external fertilization, sperm must reach the oocyte through the micropyle to enter the cytoplasm. Fertilization success is then influenced by characteristics of oocytes or sperm. In this study, we evaluated oocyte morphology and sperm motility parameters and their effects on the inseminating dose in a teleost fish Astyanax altiparanae. Interestingly, we found one of the lowest yet described inseminating doses in teleosts (2390 spermatozoa oocyte−1 ml−1). Such a fertilization efficacy may be explained by the long duration of sperm motility (>75 s), the small oocyte diameter (695.119 µm), large micropyle diameter (7.57 µm), and the presence of grooves on the oocyte surface that guides spermatozoon to the fertilization area. Additionally, we have described for the first time a structure that combines grooves on the chorion surface and a ridge in the micropylar area.

This study examined the effects of meiosis inhibition during bovine oocyte transportation on developmental competence and quality of produced embryos. The transportation medium was supplemented with: 100 μM butyrolactone I (BL), 500 μM IBMX + 100 μM forskolin (mSPOM), 100 μM milrinone (MR) or follicular fluid (bFF), and was carried out in a portable incubator for 6 h. Next, oocytes were in vitro matured (IVM) for 18 h, without the meiotic inhibitors, with the exception of mSPOM group, in which was added 20 μM cilostamide. The three control groups were IVM with 10% fetal calf serum (FCS) (Control Lab FCS) or 0.6% bovine serum albumin (BSA) (Control Lab BSA) in a CO2 in air incubator or in the portable incubator with 0.6% BSA (Control Transp BSA). Higher cleavage rates (P < 0.05) were obtained in the Control Lab FCS group (84.5 ± 5.3%) compared with the other groups (59.6 ± 3.4% to 70.9 ± 2.3%). Embryonic development was higher (P < 0.05) in the Control Lab FCS group (39.8 ± 4.7%) than in the Control Transp BSA (22.7 ± 3.4%) and MR (21.6 ± 2.3%) groups. However, they were similar (P > 0.05) to the other groups (23.6 ± 3.3% to 28.8 ± 2.7%). The total number of blastomeres was higher (P < 0.05) in the Control Lab FCS group (85.2 ± 5.6) than in Control Lab BSA (53.6 ± 2.9), Control Transp BSA (55.5 ± 4.4), BL (58.2 ± 3.0), mSPOM (57.9 ± 4.9) and MR (59.2 ± 3.9), but all these treatments did not differ (P > 0.05) from bFF (67.7 ± 4.2). No differences (P > 0.05) were found in apoptosis by the activity of caspases (139.0 ± 3.2 to 152.4 ± 6.5, expressed in fluorescence intensity) as well as the percentage of TUNEL-positive cells (12.3 ± 2.0% to 15.7 ± 1.7%). In conclusion, the transportation of oocytes over 6 h with BL, mSPOM or bFF enabled the acquisition of developmental competence at similar rates to the Control Lab FCS group.

Superovulatory response is characterized by a high degree of variability and unpredictability. The aim of the present experimental study was to examine whether the amount of maternal body fat can influence the efficiency of ovarian stimulation with gonadotropins. Female mice of two body condition types, normal and obese, produced in a standardized two-generation model, were subjected to ovarian stimulation using eCG and hCG followed by natural mating. Produced ova and embryos were recovered on day 1 and day 4 of pregnancy respectively, and several quantitative, qualitative and developmental parameters were evaluated in them. The overall response of mouse females with normal and elevated amounts of body fat to superovulation was similar: They produced almost the same numbers of ova and embryos on average. Conversely, a higher number of immature oocytes, non-fertilized mature oocytes and lower-stage zygotes were collected from fat females. In both groups, the majority of fertilized oocytes was able to cleave and reach the higher stages of development. However, in the group of fat mice, a lower number of blastocysts was collected, and these blastocysts showed increased incidence of apoptotic cell death. In conclusion, although the response of normal and fat mice to superovulatory treatment was similar, the quality and developmental capacities of produced ova were lower in the group of fat donors.

Prothymosin alpha (PTMA) is a highly acidic, intrinsically disordered protein that was first extracted from rat thymus and characterized as an immunogenic factor but soon detected in a variety of mammalian tissues. The presence of a nuclear localization signal and the adoption of a peculiar random-coil conformation are among the reasons behind its interaction with several molecular partners, hence at this time PTMA is known to be a very conserved and widely expressed molecule, involved in numerous and diverse biological processes. Only few studies have tried to weigh its possible involvement in reproduction, specifically in male gametogenesis: first reports have suggested that PTMA might be associated with the proliferative and early-meiotic phases of mammal spermatogenesis. Some years later, a comparative project on vertebrate spermatogenesis reported the isolation, for the first time, of prothymosin in a non-mammalian species, the amphibian Pelophylax esculentus. PTMA transcript and protein are localized in the germinal compartment, from spermatocytes to spermatozoa. A congruent pattern has been highlighted in studies on the fish Torpedo marmorata and Danio rerio, and in the mammal Rattus norvegicus, in which the expression of PTMA has been found in meiotic and post-meiotic germ cells inside testicular cysts and tubules. Moreover, its presence has been confirmed in rat and human spermatozoa (associated with the acrosome); its retention in the apical region of the head after the acrosome reaction revealed a striking conservation of the pattern during phylogenesis and suggested a possible role for the protein in gametogenesis and in fertilization.