One of the most important concerns in assisted reproduction (ART), and in particular ICSI, is the quality of sperm DNA. Oxidative stress is one of the major causes of damage to DNA and attempting to reduce generation of DNA damage related to reactive oxygen species (ROS) through consumption of antioxidants is often tempting. However, current antioxidant treatments, given irrespectively of clinically quantified deficiencies, are poorly efficient, potentially detrimental and over-exposure is risky. Here we discuss new treatments in relation to present day concepts on oxidative stress. This discussion includes stimulation of endogenous anti-ROS defense i.e. glutathione synthesis and recycling of homocysteine, the epicentre of multiple ROS-linked pathologies.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive and accurate tool for quantitative estimation of gene transcription levels in preimplantation embryos. To control for possible experimental variations, gene expression data must be normalized using internal control genes commonly known as reference genes. However, the stability of reference genes can vary depending on the state of development and/or experimental conditions; hence the assessment of their stability is essential before initiating a gene expression analysis. In the present study, we used RT-qPCR to measure the transcript levels of 10 commonly used reference genes and analyzed their expression stability in bovine blastocysts produced by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). Using the geNorm program, we found the best combination of genes to normalize gene expression data in bovine embryos at the blastocyst stage produced by IVF (HMBS, SF3A1, and HPRT1), ICSI (H2A, HMBS, and GAPDH), SCNT (ACTB, SF3A1, and SDHA) and/or between blastocysts produced by these methods (GAPDH, HMBS and EEF1A2). We also demonstrated that not only the culture conditions may affect the expression patterns in bovine blastocysts but also the choice of embryo production method may have an important effect.

Cryopreservation is a technique used to preserve cells for long-time storage. It is widely used in agriculture to store male gametes in liquid nitrogen. The aim of this study was to determine the optimum thawing temperature and time for samples subjected to annexin V magnetic-activated cell sorting (AnMACS) as the sperm preparation technique. Pooled semen samples from three ejaculates were divided into two groups. The treatment group was subjected both to AnMACS and to being cryopreserved, whilst the control group was cryopreserved directly without MACS. Post-thaw analysis was carried out for samples thawed at either 20°C for 13 s, 37°C for 30 s, 40°C for 7 s, 60°C for 6 s or 80°C for 5 s. Sperm kinematics, viability and capacitation status were determined for samples subjected to all thawing temperatures described. Results showed that thawing at 37°C for 13 s for MACS-processed samples was a superior option compared with other thawing procedures; there was a significant difference in P < 0.05 values for curvilinear velocity (VCL μm/s) and sperm straightness (STR %) when samples were thawed at 40°C for 7 s, with fewer capacitated spermatozoa (P < 0.05) when samples were thawed at 37°C for 30 s, 40°C for 7 s or 60°C for 6 s. Hence, we can speculate that the use of AnMACS as the sperm preparation technique can somehow enhance sperm cryosurvival rate after cryopreservation, however the fertilization potential of these cells has yet to be determined.

Low-density lipoproteins (LDL) is known to protect boar sperm during freezing–thawing, but little information is known about the effects of LDL extracted from different avian egg yolks on post-thaw boar semen quality. The purpose of this study was to compare and analyze the effects of LDL at various concentrations and different species on boar sperm quality after freezing–thawing. LDL extracted from the yolk of hen egg, duck egg, quail egg, pigeon egg or ostrich egg was added to the extender at the concentrations of 0.06, 0.07, 0.08, 0.09 and 0.1 g/ml, respectively, and their effects on frozen–thawed boar sperm quality were assessed. According to all measured parameters, the results showed that sperm motility, acrosome integrity and plasma membrane integrity were 43.20%, 52.57% and 48.13%, respectively, after being frozen–thawed with 0.09 g/ml LDL extracted from pigeon egg yolk. All these quality parameters were higher than that of other groups (P < 0.05). In conclusion, our results confirmed that LDL extracted from pigeon egg yolk had the best cryoprotective effects on frozen–thawed boar sperm among all of the groups supplemented with LDL from five kinds of avian egg in extender. The optimum concentration of LDL extracted from pigeon egg in boar semen freezing extender was 0.09 g/ml.

The goal of this project was to determine whether the originating strain of mouse embryonic stem (ES) cells affects the maintenance of their pluripotency under uniform culture conditions. ES cells from two strains of mice, E14 and C2J, were tested. Both ES cell lines were cultured in KOSR + 2i medium and then injected into C57BL/6J blastocysts. Our results demonstrate that this medium could support both E14 and C2J ES cells to keep their pluripotency, though E14 ES cells were found to have a higher chimeric rate than C2J ES cells. However, analysis by backcrossing revealed that C2J and E14 ES cells have the same ability for germline transmission. Our results demonstrate that ES cells derived from E14 and C2J cells have the same capacity for germline transmission when injected into C57BL/6J blastocysts; however, due to the limitation of mixed genetic background between E14 cells and host C57BL/6J embryos, C2J ES cells are preferable to E14 ES cells for use in gene-targeting and should become the cell line of choice for the generation of genetically engineered mutant mouse lines.

In a previous study, we have identified a set of conserved spermatogenic genes whose expression is restricted to testis and ovary and that are developmentally regulated. One of these genes, the transcription factor Mael, has been reported to play an essential role in mouse spermatogenesis. Nevertheless, the role of Mael in mouse oogenesis has not been defined. In order to analyse the role of Mael in mouse oogenesis, the expression of this gene was blocked during early oogenesis in mouse in vitro using RNAi technology. In addition, the role of Mael during differentiation of embryonic stem cells (ESC) into germ cells in vitro was analysed. Results show that downregulation of Mael by a specific short interfering RNA disrupted fetal oocyte growth and differentiation in fetal ovary explants in culture and the expression of several germ-cell markers in ESC during their differentiation. These results suggest that there is an important role for Mael in early oogenesis and during germ-cell differentiation from embryonic stem cells in mouse in vitro.

The oocyte meiotic spindle is comprised of microtubules (MT) that bind chromatin and regulate both metaphase plate formation and karyokinesis during meiotic maturation; however, little information is known about their role in meiosis reinitiation. This study was conducted to determine if microtubule integrity is required for meiotic induction and to ascertain how it affects activation of AMP-activated protein kinase (AMPK), an important participant in the meiotic induction process. Treatment with microtubule-disrupting agents nocodazole and vinblastine suppressed meiotic resumption in a dose-dependent manner in both arrested cumulus cell-enclosed oocytes (CEO) stimulated with follicle-stimulating hormone (FSH) and arrested denuded oocytes (DO) stimulated with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR). This effect coincided with suppression of AMPK activation as determined by western blotting and germinal vesicle immunostaining. Treatment with the MT stabilizer paclitaxel also suppressed meiotic induction. Targeting actin filament polymerization had only a marginal effect on meiotic induction. Immunolocalization experiments revealed that active AMPK colocalized with γ-tubulin during metaphase I and II stages, while it localized at the spindle midzone during anaphase. This discrete localization pattern was dependent on MT integrity. Treatment with nocodazole led to disruption of proper spindle pole localization of active AMPK, while paclitaxel induced excessive polymerization of spindle MT and formation of ectopic asters with accentuated AMPK colocalization. Although stimulation of AMPK increased the rate of germinal vesicle breakdown (GVB), spindle formation and polar body (PB) extrusion, the kinase had no effect on peripheral movement of the spindle. These data suggest that the meiosis-inducing action and localization of AMPK are regulated by MT spindle integrity during mouse oocyte maturation.

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

The precision of embryo evaluation using stereoscopic microscopy (SM) and inverted phase contrast microscopy (PCM) was compared in 20 Bos indicus cows superovulated at two different times of the year. In total, 118 embryos were collected and classified according to their developmental stage and quality by two independent evaluators using SM and inverted PCM. Cohen's kappa coefficient was used to determine concordance between SM and PCM observations. A good level of agreement (k = 0.616) was found for quality level, and a moderate one (k = 0.464) for developmental stage, particularly at the morula stage. Using the TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) technique, concordance level was deemed to be low with the SM (k = 0.169), and poor with the PCM (k = 0.217). Differences in concordance levels were also found between observations made at the two times of year, 78 embryos were evaluated in the rainy season when concordance level was good (k = 0.68), in contrast to the 40 embryos evaluated in the dry season when agreement was found to be poor (k = 0.24). In conclusion, inverted PCM was somewhat more effective for evaluating embryos, particularly at the morula stage. However, considering the high cost of an inverted PCM, the differences observed do not justify its purchase for routine embryo evaluation.

This study investigated mRNA levels for insulin-like growth factors (IGFs) IGF1 (IGF-I) and IGF2 (IGF-II), IGF receptors (IGF1R and IGF2R), and binding proteins (IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6) in bovine follicles of 0.2, 0.5 or 1.0 mm in diameter. mRNA expression levels in in vitro cultured follicles that reached approximately 0.5 mm were compared with that of in vivo grown follicles. IGF1R and IGF2R expression levels in 0.5 mm in vivo follicles were higher than in 1.0 or 0.2 mm follicles, respectively. IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6 showed variable expression in the follicular size classes analyzed. In vitro grown follicles had significantly reduced expression levels for IGF1, IGF1R, IGFBP-3, IGFBP-5 and IGFBP-6 mRNA when compared with 0.2 mm follicles, but, when compared with in vivo grown follicles (0.5 mm), only IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 showed a reduction in their expression. In conclusion, IGFs, their receptors and IGFBPs showed variable expression of mRNA levels in the follicular size classes analyzed.

Spermatogenesis is a process in adult male mammals supported by spermatogonial stem cells (SSCs). The cultivation of SSCs has potential value, for example for the treatment of male infertility or spermatogonial transplantation. Testicular interstitial fluid was added to culture medium to a final concentration of 5, 10, 20, 30 or 40%, in order to investigate its effects on proliferation of mouse SSCs in vitro, Alkaline phosphatase (AKP) assay, reverse transcription polymerase chain reaction (RT-PCR) analysis and indirect immunofluorescence of cells were performed to identify SSCs, and the proliferation rate and diameters of the SSCs colonies were measured. The results showed that the optimal addition of testicular interstitial fluid to culture medium was 30%. When medium supplemented with 30% testicular interstitial fluid was used to culture mouse SSCs, the optimum proliferation rate and diameter of the cell colonies were 72.53% and 249 μm, respectively, after 8 days in culture, values that were significant higher than those found for other groups (P < 0.05). In conclusion, proliferation of mouse SSCs could be promoted significantly by supplementation of the culture medium with 30% testicular interstitial fluid. More research is needed to evaluate and understand the precise physiological role of testicular interstitial fluid during cultivation of SSCs.

Dwarf rainbowfish (Melanotaenia praecox) are an ornamental fish with a high added value and considerable commercial interest. However, little information is known about the organism's reproductive biology, especially the early stages of embryonic development (initial ontogeny). Melanotaenia praecox embryos were examined throughout development to describe the dwarf rainbowfish initial ontogeny. Eggs were incubated at 28°C, and observations were recorded at pre-determined times. Development stages were identified and classified according to morphophysiological characteristics. The initial size of the eggs ranged between 0.99 and 1.04 mm. Oil droplets were observed in the eggs, and fixing filaments for adhesion were observed in the corium. Embryonic development was similar to that of other species in the genus Melanotaenia, in which hatching begins 119.50 h post-fertilisation or 3405.75 degree-h post-fertilisation. The main features of the newly hatched larvae were excellent swimming activity, a reduced yolk sac, mouth movement and an apparently functional digestive system.

This study was designed to evaluate the quality and viability of bovine embryos produced by in vitro fertilization (IVF), after intracytoplasmic injection of pCX–EGFP–liposome complexes or pBCKIP2.8–liposome complexes (plasmids that codify the human insulin gene). Cleavage, blastocysts and expanded blastocysts rates of these both groups were not different from that of controls (IVF or IVF embryos injected with liposomes alone; IVF-L). The percentage of EGFP-positive (EGFP+) blastocysts was 41.8%. In Experiment 2, the blastocysts obtained after injection of pCX–EGFP–liposome complexes that did or did not express the transgene, were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay at days 6, 7 and 8 of culture in vitro(Bd6, Bd7 and Bd8), in order to evaluate DNA fragmentation. The EGFP+ blastocysts showed different proportions of TUNEL-positive cells (T+) at Bd6, Bd7 and Bd8 (91, 73.7 and 99.5%, respectively) while blastocysts without EGFP expression (EGFP−) showed statistically lower numbers of fragmented nuclei (0, 44.6 and 85%, respectively; P < 0.05). There was no evidence of DNA fragmentation in either Bd6 or Bd7 IVF and IVF-L control blastocysts, but T+ nuclei were detected at Bd8 in both groups (66.4 and 85.8% respectively). Finally, IVF blastocysts (n = 21) injected with insulin–liposome complexes, cultured for 6, 7 and 8 days, were transferred to recipient cows. Pregnancy rates of 18.2% (2/11) and 40% (2/5) resulted from the transfer of Bd6 and Bd7 cells, respectively. Two pregnancies developed to term but they were not transgenic for the insulin gene. In conclusion, EGFP expression affects DNA integrity but not embryo development. Moreover, additional transfers are required in order to overcome the drawbacks generated by in vitro culture length and transgene expression.

Microtubule-associated protein light chain 3 (LC3)-II is a marker of autophagosome. In this study, LC3-II expression was used to identify autophagy, during the in vitro maturation of porcine oocytes. In a time-course experiment, cumulus–oocyte complexes (COCs) were cultured in NCSU23 medium for 0 h, 14 h, 28 h or 42 h. The cumulus cells were removed and denuded oocytes were processed for western blotting or immunostaining. Western blotting showed that the LC3-II levels changed over time, with maximum levels observed at 14 h and minimum levels at 42 h. Immunostaining of LC3 showed the signals with dot shapes and ring shapes in oocytes at every group that probably represent autophagosomes. To ascertain whether autophagic induction and degradation were occurring, we treated the cultures with autophagic inhibitors. Lysosomal protease inhibitor E64d and pepstatin A increased the LC3-II levels and wortmannin, inhibitor of autophagic induction, decreased the LC3-II levels. Western blotting and immunostaining demonstrated that LC3-II is present in porcine oocytes cultured in vitro. The decreased LC3-II levels after wortmannin treatment suggest that it is newly generated in porcine oocytes, a phenomenon that represents autophagic induction. Furthermore, increased LC3-II levels after E64d and pepstatin A addition imply that LC3-II is degraded by lysosomal proteases, an indication of autophagic degradation. Our results suggest that autophagy, which is a dynamic process whereby autophagosomes are newly generated and subsequently degraded, is probably occurring in porcine oocytes during in vitro maturation.

Insulin, transferrin and selenium (ITS) supplementation to oocyte maturation medium improves the post-fertilization embryonic development in pigs. ITS is also commonly used as a supplement for the in vitro culture (IVC) of embryos and stem cells in several mammalian species. However, its use during IVC of pig embryos has not been explored. This study investigated the effect of ITS supplementation to IVC medium on the in vitro development ability of pig embryos produced by parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). We observed that ITS had no significant effect on the rate of first cleavage (P > 0.05). However, the rate of blastocyst formation in ITS-treated PA (45.3 ± 1.9 versus 27.1 ± 2.3%), IVF (31.6 ± 0.6 versus 23.5 ± 0.6%) and SCNT (17.6 ± 2.3 versus 10.7 ± 1.4%) embryos was significantly higher (P < 0.05) than those of non-treated controls. Culture of PA embryos in the presence of ITS also enhanced the expansion and hatching ability (29.1 ± 3.0 versus 18.2 ± 3.8%; P < 0.05) of blastocysts and increased the total number of cells per blastocyst (53 ± 2.5 versus 40.9 ± 2.6; P < 0.05). Furthermore, the beneficial effect of ITS on PA embryos was associated with significantly reduced level of intracellular reactive oxygen species (ROS) (20.0 ± 2.6 versus 46.9 ± 3.0). However, in contrast to PA embryos, ITS had no significant effect on the blastocyst quality of IVF and SCNT embryos (P > 0.05). Taken together, these data suggest that supplementation of ITS to the IVC medium exerts a beneficial but differential effect on pig embryos that varies with the method of embryo production in vitro.

Somatic cell nuclear transfer can be used to produce embryonic stem (ES) cells, cloned animals, and can even increase the population size of endangered animals. However, the application of this technique is limited by the low developmental rate of cloned embryos, a situation that may result from abnormal expression of some zygotic genes. In this study, sheep–sheep intra-species cloned embryos, goat–sheep inter-species cloned embryos, or sheep in vitro fertilized embryos were constructed and cultured in vitro and the developmental ability and expression of three pluripotency genes, SSEA-1, Nanog and Oct4, were examined. The results showed firstly that the developmental ability of in vitro fertilized embryos was significantly higher than that of cloned embryos. In addition, the percentage of intra-species cloned embryos that developed to morula or blastocyst stages was also significantly higher than that of the inter-species cloned embryos. Secondly, all three types of embryos expressed SSEA-1 at the 8-cell and morula stages. At the 8-cell stage, a higher percentage of in vitro fertilized embryos expressed SSEA-1 than occurred for cloned embryos. However, at the morula stage, all detected embryos could express SSEA-1. Thirdly, the three types of embryos expressed Oct4 mRNA at the morula and blastocyst stages, and embryos at the blastocyst stage expressed Nanog mRNA. The rate of expression of Oct4 and Nanog mRNA at these developmental stages was higher in in vitro fertilized embryos than in cloned embryos. These results indicated that, during early development, the failure to reactivate some pluripotency genes maybe is a reason for the low cloning efficiency found with cloned embryos.

One of the greatest challenges for reproductive cryobiologists today is to develop an efficient cryopreservation method for human and domestic animal oocytes. The objective of the present study was to optimize a low toxicity solution called VM3 to vitrify porcine oocytes using an open pulled straw (OPS) device and to evaluate the effects on viability, chromosomal organization and cortical granules distribution. Two experiments were conducted in this study. Firstly, we determined the minimum concentration of cryoprotectant present in the VM3 solution required (7.6 M) for vitrification using an OPS device. The appearance of opacity was observed when using a cooling solution at –196°C; no observable opacity was noted as vitrification. In addition, the ultrastructure of oocytes in VM3 or VM3 optimized solution was examined using cryo-scanning electron microscopy. The minimum total cryoprotectant concentration present in VM3 solution necessary for apparent vitrification was 5.6 M when combined with use of an OPS device. Use of both vitrification solutions showed a characteristic plasticized surface. In the second experiment, the relative cytotoxicity of vitrification solutions (VM3 and VM3 optimized) was studied. Oocyte viability, chromosomal organization and the cortical granules distribution were assessed by fluorescent stain. After warming, oocyte survival rate was similar to that of fresh oocytes. The vitrification process significantly reduced correct chromosomal organization and cortical granules distribution rates compared with the fresh oocytes group. However, correct chromosomal organization and cortical granules distribution rates did not differ among oocytes placed in different vitrification solutions. In conclusion, our data demonstrated that the VM3 solution can be optimized and that reduction in concentration to 5.6 M enabled vitrification of oocytes with an OPS device, however use of the VM3 optimised solution had no beneficial effect on vitrification of porcine oocytes.

The enzymatic activities of commercially prepared glycosidases were verified by direct chemical assays using defined substrates and fixed and live sea urchin (Lytechinus pictus) embryos to determine if a model cellular interaction of interest to developmental biologists for over a century (interaction of archenteron tip and roof of the blastocoel) was mediated by glycans. Glycosidases (active and denatured) were incubated with microdissected archenterons and blastocoel roofs in a direct assay to learn if their enzymatic activities could prevent the normal adhesive interaction. Of the five glycosidases tested only β-amylase (an exoglycosidase) immediately inhibited the interaction at relatively low unit activity. α-Amylase (an endoglycosidase) had no measurable effect, while other glycosidases (α-glucosidase, β-glucosidase, β-galactosidase) only substantially inhibited adhesion after a 12-h incubation. We demonstrated that the five glycosidases were active (not inhibited) in the presence of embryo materials, and that cleaved sugars could be detected directly after incubation of some enzymes with the embryos. The biochemical purity of the enzymes was examined using gel electrophoresis under denaturing conditions, and the absence of contaminating proteases was confirmed using Azocoll™ substrate. As we cannot entirely rule out the presence of minor contaminating enzymatic activities, only inhibitions of adhesion after very short incubations with enzyme were considered significant and biologically relevant. Although glycans in indirect experiments have been implicated in mediating the interaction of the tip of the archenteron and roof of the blastocoel, to our knowledge, this is the first study that directly implicates polyglucans with terminal 1,4-linked glucose residues in this adhesive event.

Based on the economic and ecological relevance of Brycon amazonicus, the goal of this work was to describe the diameter of oocytes and eggs of this species, as well as the chronological embryonic development. The material was provided by Buriti fish farm, Nova Mutum – MT, Brazil. Samples of both oocytes and eggs were obtained from extrusion to hatching. The material was fixed and measured under stereomicroscope, and the samples were divided for light microscopy or scanning electron microscopy (SEM) analyses. At extrusion, the oocytes were bluish green. The frequency distribution of oocytes revealed that 87.7% of them ranged from 1.11–1.30 mm in diameter. During incubation, the total diameter of the eggs increased from 1.22 ± 0.04 mm to 3.06 ± 0.46 mm in the first 60 min post fertilization (PF), and growth ceased at 180 min PF. Between 10–30 s PF, most eggs were fertilized and fertilization cones were observed from 10 s onwards after gamete activation. The main fertilization events took place asynchronically and spermatozoa were visualized in the micropyle vestibule up to 90 s PF. The first cell was formed in the centre of the blastodisc 20 min PF. The morula stage was identified 2 h PF and, 3 h later, 70% of the yolk was covered by the blastoderm; the blastopore was almost entirely closed at 6 h PF. The cephalic and caudal regions of the embryo could be defined 8 h PF and hatching occurred after 13 h of embryonic development. The larvae hatched with undifferentiated organic systems and with a large yolk sac, free from swimming abilities or visual acuity.

The beneficial effect of supplementing culture medium with melatonin has been reported during in vitro embryo development of species such as mouse, bovine and porcine. However, the effect of melatonin on mouse somatic cell nuclear transfer remains unknown. In this study, we assessed the effects of various concentrations of melatonin (10−6 to 10−12 M) on the in vitro development of mouse somatic cell nuclear transfer embryos for 96 h. Embryos cultured without melatonin were used as control. There was no significant difference in cleavage rates between the groups supplemented with melatonin, dimethyl sulphoxide (DMSO) and the control. The rate of development to blastocyst stage was significantly higher in the group supplemented with 10−12 M melatonin compared with the control group (P < 0.05). Thus, our data demonstrated that adding melatonin to pre-implantation mouse nuclear-transferred embryos can accelerate blastocyst formation.