Intracytoplasmic sperm injection (ICSI) is an assisted fertilization technique and has been widely applied in human medicine to overcome some obstacles of infertility. However, this technology has not yet been used as a mainstream technique for animal production, including the rabbit, due to its limited success. The aim of this study was to improve ICSI techniques and establish an efficient ICSI method for rabbits. Spermatozoa used for ICSI were collected from mature New Zealand white male rabbits. They were washed two to three times with HEPES-buffered TCM199 containing 10% FBS and then mixed with 10% polyvinylpyrrolidone (PVP) prior to microinjection. Oocytes were harvested from superovulated donor rabbits after 14–15 h hCG treatment and were fertilized by microinjection of a single living spermatozoon into the ooplasm of each oocyte without additional activation treatment. A total of 317 injected oocytes resulted in the high survival rate of 86.1%. Among the surviving oocytes, 273 were placed into culture dishes for in vitro development. The fertilization, cleavage and blastocyst rates were 59.0%, 88.2% and 45.3% respectively. Furthermore, ICSI embryos were produced with spermatozoa from an infertile male rabbit, and 21 early-stage embryos (2-cell and 4-cell) were surgically transferred into the oviducts of two adult female rabbits. On day 31 after transfer, one out of the two recipients gave birth to two normal and healthy young rabbits. These results demonstrate that rabbit oocytes can be successfully activated and fertilized by the new ICSI protocol. Spermatozoa derived from infertile rabbits can successful fertilize oocytes and produce offspring by the simple ICSI technique.

To determine the fertilization and embryonic potential of immature metaphase I (MI) oocytes in patients with low oocyte maturity rate in whom the percentage of mature oocytes obtained was less than 75% of the total retrieved ones. In vivo matured metaphase II (MII) oocytes (MII-ICSI, n = 244), and in vitro matured MI oocytes (MI-MII-ICSI, n = 202) underwent an intracytoplasmic sperm injection (ICSI) procedure. Maturation rate, fertilization rate and early embryonic development were compared in both groups. In total, 683 oocytes were collected from 117 ICSI cycles of 117 patients. Among them, 244 (35.7%) were mature MII and 259 (37.9%) were MI after the denudation process. Of those 259 MI oocytes, 202 (77.9%) progressed to MII oocytes after an incubation period of 18–24 h. The maturation rate was 77.9%. Fertilization rate was found to be significantly higher in the rescued in vitro matured MI oocyte group when compared with the in vivo matured MII oocyte group (41.6% vs 25.8%; P = 0.0006). However, no significant difference was observed in terms of cleavage rates on days 2 and 3 between the groups (P = 0.9126 and P = 0.5031, respectively). There may be unidentified in vivo factors on the oocyte maturation causing low developmental capacity in spite of high fertilization rates in the group of patients with low oocyte maturity rate. Furthermore, studies are needed to determine the appropriate culture characteristics as well as culture period and ICSI timing of these oocytes.

During fertilisation in starfish oocytes, the fertilisation cone develops temporarily beneath the penetrating sperm. The role of the fertilisation cone in sperm incorporation in the starfish Asterias amurensis was examined using cytochalasin B (CB). CB (2 μM) allowed sperm acrosomal process–egg plasma membrane fusion and egg activation, but inhibited the development of the fertilisation cone containing actin microfilaments. When sperm were added to intact oocytes (with the jelly coat and vitelline coat) in seawater containing CB, the sperm head did not penetrate the fertilisation membrane. Although the acrosomal process fused with egg plasma membrane, the sperm head remained outside the fertilisation membrane. On the other hand, denuded oocytes without the jelly coat and vitelline coat allowed sperm penetration even in the presence of 2 μM CB. Electron microscopy revealed that sperm organelles, including the acrosomal process, nucleus, mitochondrion and tail, were incorporated into the slightly electron-dense cytoplasm, which was similar to the cytoplasm of the fertilisation cone. These results show that the development of the fertilisation cone/actin filament complex is not essential for incorporation of the sperm, since incorporation can occur in denuded oocytes. However, the cone is required for fertilisation of intact oocytes, suggesting that this actin-filament-containing structure is necessary for getting the sperm through the outer egg coats.

This study examined the event of protein phosphorylation in bovine oocytes in response to sperm penetration and parthenogenetic activation. In vitro matured oocytes were labelled with [32P]orthophosphate at 3 h intervals from 3 h to 18 h or from 0 h to 12 h following in vitro fertilisation and parthenogenetic activation, respectively. The level of protein dephosphorylation, at approximately 43 kDa, was similar in fertilised and parthenogenetically activated bovine oocytes. However, the level of protein phosphorylation at 40 kDa, 23 kDa and 18 kDa was different between these two samples. There were no such changes of protein phosphorylation and dephosphorylation in the control oocytes. Further, by two-dimensional gel electrophoresis there is a difference in the level of protein phosphorylation at 18 kDa between the fertilised and activated oocytes. These results suggest that this protein phosphorylation may be related to the formation of the male pronucleus in bovine oocytes.

Chinchilla lanigera is an endangered species therefore the development of cryopreservation protocols for its gametes is a useful tool in the application of assisted reproduction techniques. A study of the functionality of the spermatozoa punctured from the cauda epididymis was performed on fresh or frozen–thawed samples with three cryoprotective media (test-yolk buffer, sucrose and glycerol). The effect that these media had on sperm physiology during the freezing, storage and later thawing process was analysed. A decrease in the percentages of viability, motility, membrane integrity and capacity to undergo the induced acrosome reaction was found with all the media assayed, an increase in the percentages of DNA fragmentation was also observed. The comparative analysis of the effect of the different cryoprotectants assayed showed that the best medium to use to cryopreserve epididymal sperm in this species is test-yolk buffer. This medium had the least effect on the abovementioned physiological parameters, especially at the level of genetic material.

The scope of this article is to answer two questions: first, when were the terms embryo (E), gamete (G) and zygote (Z) coined, and second, who were the scientists who put them in circulation? In order to clarify and document the origin of these three words, a vast amount of biomedical literature, particularly from the late 19th and early 20th centuries, has been reviewed; an undertaking made possible by the free access to the massive repository of available scientific literature on the Internet. Furthermore, as a result of this analysis, I would like to raise some ethical aspects of scientific publication, in particular the obligation of scientists to personally assess the quality and source of data they take from the literature and use in their publications.

Polyploid mouse embryos are important models for understanding the mechanisms of cleavage and preimplantation development in mammals. In this study, hexaploid (6n) mouse embryos were produced by the electrofusion of blastomeres from diploid (2n) and tetraploid (4n) embryos at the 2-cell stage. Furthermore, the developmental pattern of hexaploid embryos was evaluated by blastocyst rate, cell number, karyotype analysis, cytoskeleton staining and Oct-4 immunofluorescence. The results showed that 72.7% of the hexaploid embryos were able to develop to the blastocyst stage, which is a lower number than that found with normal diploid embryos (98.0%, p < 0.05). The cell number in hexaploid blastocyst was 12.3 ± 2.0, which was less than that found in diploid or tetraploid blastocysts (41.2 ± 7.2; 18.4 ± 3.5). Karyotype analysis confirmed that the number of chromosomes in hexaploid embryos was 120. β-Tubulin and Oct-4 immunofluorescence indicated that the hexaploid blastocysts were nearly lacking inner cell mass (ICM), but some blastomeres did show Oct-4-positive expression.

Infertility affects 15% of all couples worldwide and 50% of cases of infertility are solely due to male factors. A decrease in motility in the semen is considered one of the main factors that is directly related to infertility. The use of supplementation to improve the overall sperm quality has become increasingly popular worldwide. The purpose of this study was to evaluate whether sperm motility was affected by the combination of serotonin (5-HT), selenium (Se), zinc (Zn), and vitamins D, and E supplementation. Semen samples were incubated for 75 min at 37°C in medium containing varying concentrations of 5-HT, Se, Zn, vitamin D, and E. 5-HT (200 μM), Se (2 μg/ml), Zn (10 μg/ml), vitamin D (100 nM), and vitamin E (2 mmol) have also been shown to increase progressive sperm motility. Three different mixtures of supplements were also tested for their combined effects on sperm motility and reactive oxygen species (ROS) production. While the total motility in the control group was 71.96%, this was found to increase to 82.85% in the first mixture. In contrast the average ROS level was 8.97% in the control group and decreased to 4.23% in the first mixture. Inclusion of a supplement cocktail (5-HT, Se, Zn, vitamins D and E) in sperm processing and culture medium could create an overall improvement in sperm motility while decreasing ROS levels during the incubation period. These molecules may enhance the success of assisted reproduction techniques when present in sperm preparation medium.

Embryological studies in fish species are useful to the understanding of their biology and systematics. The available biological data in Leiarius marmoratus are scarce and additional information about its reproductive biology is needed, mainly because this species has been commercially exploited and used in production of hybrid lineages. In order to evaluate the temporal–morphological embryonic modifications in L. marmoratus, samples of nearly 200 embryos were collected at random at different stages of development, starting from fecundation (time zero). Embryos were fixed in modified Karnovsk's solution and 2.5% glutaraldehyde, processed and analysed under optic and electron microscopy. The incubation period of L. marmoratus was equal to 14.42 h at a mean temperature of 28.3 ± 0.07°C. The following stages of embryonic development were established: zygote, cleavage, gastrula, organogenesis and hatching. These stages were divided into phases, as follows: cleavage – phases of 2, 4, 8, 16, 32 and 64 cells and morula; gastrula – phases of 25, 50, 75 and 90% of epiboly and blastopore closure; and organogenesis – neurula, segmentation and pre-larval phases. The embryogenesis of L. marmoratus was typical of neotropical teleosteans, with peculiarities in species development.

Domestic cat embryos generated by in vitro fertilization (IVF) and cultured without the zona pellucida have a reduced implantation capacity after embryo transfer at the blastocyst stage. The objective of this study was to evaluate the expression of trophectoderm markers in domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were selected: (1) domestic cat embryos generated by IVF and cultured in vitro normally (zona intact group, ZI); and (2) domestic cat embryos generated by IVF and cultured in vitro without a zona pellucida (zona-free group, ZF). In the ZF group, the zona pellucida of the presumptive zygote was removed and these were cultured using the well of the well (WOW) system. In vitro culture was carried out for 7 days. The cleavage, morula and blastocyst rates were estimated. Finally, the relative expression levels of the trophectoderm markers TEAD4, YAP1, CDX2 and EOMES, the cell adhesion marker E-cadherin and the apoptosis marker CASP3 were evaluated by RT-qPCR in the blastocysts. The Wilcoxon test was used to evaluate differences (P < 0.05). No differences were observed in the cleavage, morula and blastocyst rates between the ZF and ZI groups. No differences were found in the expression of TEAD4, CDX2, E-cadherin and CASP3 between groups. The expression of YAP1 and EOMES was higher in ZF blastocysts than in ZI blastocysts. In conclusion, the in vitro culture without the zona pellucida generates an overexpression of YAP1 and EOMES in the domestic cat blastocysts. More studies are needed to confirm if this overexpression might affect in vivo development.

Regarding the low number of embryos that reach the blastocyst stage when cultured in vitro, this study aimed to evaluate the effects of quercetin on pre-implantation mouse (Mus musculus) embryos obtained using in vitro fertilization, especially during the passage from morula to blastocyst. Furthermore, we studied whether quercetin also affected the expression of hypoxia-inducible factor 1α (HIF-1α). The culture medium for the embryos was supplemented with quercetin, for long or short periods of time, and then the development potential, total cell number, apoptosis rates and expression of HIF-1α were studied to determine the effect of quercetin. Embryos failed to develop when cultured for long periods of time with quercetin, implying the possible toxic effects of this, alternatively antioxidant, compound. However, a short culture from morula to blastocyst significantly improved the development potential of in vitro produced embryos, increasing the final total cell number and reducing the apoptosis rate, observing similar results to those embryos cultured in low-oxygen concentrations or developed in utero. Furthermore, in embryos treated with quercetin for 2 or 4 h we found an increase in HIF-1α compared with untreated embryos. This work could imply a way to use quercetin in fertility clinics to improve the production of healthy blastocysts and, consequently, increase the success rates in assisted reproduction techniques.

To find more morphological characteristics useful for discussion on aralian or non-aralian Panax affinity, pollen morphological diversity was comparatively analysed in P. ginseng, Aralia elata and Oplopanax elatus collected during their pollination periods. In the anthers of both the buds and open flowers, the pollen average diameter varied between some species-specific maximum and minimal measurement. However, the larger pollen grains were typically found in the buds whereas the smaller pollen prevailed in the open flowers, testifying to the pollen size diminution during anther maturation. Based on this finding, the subsequent examination of pollen according to size decrease was put into operation as a method of pollen modification for the study. The structural mechanisms of pollen metamorphosis were identified as not being species specific but rather universal. These mechanisms are suggested to be the shrinkage of the pollen vegetative cytoplasm, the intine enlargement, the deepening of three colporate apertures provided by exine sunken into enlarged intine areas, the aperture accretion as well as the transformation of the exine from thick/sculptured into thin/less sculptured. During ‘size-reducing metamorphosis’, the pollen grains changed dramatically, going through a species-specific set of intermediate morphs to the final species-specific morphotype. In P. ginseng this morphotype is round (diameter is about 16 μm), in A. elata it is round with a single projection (diameter is about 15 μm) and in O. elatus it is ovoid with a single projection (average diameter is about 18 μm). In addition, every species is peculiar in having the unique vegetative cytoplasm inclusions and individual construction of the largest pollen exine. From a phylogenetic perspective, these findings presumably add support to the option of equal remoteness of P. ginseng from A. elata and O. elatus. The characteristics found seem to be suitable for examination of Panax affinity, by the subsequent study of more Araliaceae representatives.

Previous studies have reported that the first polar body (PB1) morphology reflects embryo development competence, but the effects of PB1 on porcine embryo development remain unknown. This study aims to determine whether the ability of porcine embryo development is related to oocytes’ PB1 in vitro. The distribution of type II cortical granules (CGs) of porcine matured oocytes in grade B PB1 is significantly greater compared with those in grades A and C PB1 (71.43% versus 52.46% and 50%; P < 0.05). The ratio of porcine parthenogenetic blastocysts and the mean cell number in each blastocyst in the group with grade B PB1 is significantly greater than that with grades A and C PB1 (30.81% vs. 19.02% and 15.15%; P < 0.05) and (36.67 versus 24.67, 28.67; P < 0.05), and no significant differences are found in the embryo cleavage for all groups (79.75%, 84.30%, and 78.18% in grades A, B, and C PB1; P > 0.05). The acetylation level of porcine embryos in the group with grade B PB1 is significantly greater compared with those in the other groups (P < 0.05), and is almost 2.5 times higher than that in grade A. Therefore, porcine oocytes with PB1 in grade B are more competitive in cytoplasmic maturation and further embryo development in vitro.

Treatment with 6-dimethylaminopurine (6-DMAP) or demecolcine (DE) for several (at least 2) hours after artificial activation is known to improve in vitro development of porcine embryos. However, several reports have also shown that treatments with these chemicals induce apoptosis. The aim of this study was to find out whether short-term treatment with 6-DMAP and DE combined with electrical or thimerosal/dithiothreitol (Thi/DTT) activation had a beneficial effect on development of parthenogenetically activated porcine oocytes. We additionally treated embryos with 6-DMAP (2 mM) and/or DE (0.4 μg/ml) for a short time (40 min) after an electrical pulse (EP) or Thi/DTT. As a result, short-term treatment with 6-DMAP and DE successfully induced development of electrically or Thi/DTT-activated porcine parthenogenetic embryos with no significant difference in cleavage rate, blastocyst formation rate and total cell number compared with long-term treatment. To find optimal activation protocol, cleavage rate, blastocyst formation rate and total cell number were compared between EP and Thi/DTT treatments. Thi/DTT + 6-DMAP + DE showed significantly higher blastocyst formation rate (36.1 ± 3.5%) and total cell number (46.9 ± 1.0) than other groups (EP + 6-DMAP + DE, EP + Thi/DTT + 6-DMAP + DE: 23.3 ± 3.0%, 42.2 ± 1.1 and 17.2 ± 2.7%, 36.7 ± 1.5, respectively). In conclusion, this study demonstrates that short-term treatment with 6-DMAP and DE is as effective as the standard long-term treatment and Thi/DTT + 6-DMAP + DE exerts a synergistic effect.

The development of an ovarian follicle is a complex process at the cellular and molecular level that is mainly regulated by follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR). To elucidate the contribution of these receptors to ovarian follicle development, it is necessary to determine their expression profiles during this biological process. Therefore, this study aimed to investigate the relationship between ovarian development pattern and the differential ovarian expression pattern of FSHR and LHR genes as well as proteins at different developmental stages. Ovaries were collected from 30 New Zealand rabbits at day 0 (birth), week 2 (neonate), week 4 (cub), week 16 (maturity), and day 18 pregnancy. Ovarian histology, and gene as well as protein expression were determined using light microscopy, real-time PCR and western blotting, respectively. The results showed that the expression levels of FSHR mRNA and protein increased coincidently with age and the growth of ovarian follicles. The levels of LHR mRNA and protein remained low from the day of birth until week 4 and became significantly higher by week 16 coinciding with appearance of growing and antral follicles, which have a defined thecal layer. FSHR gene and protein expression decreased with pregnancy, whereas LHR increased, reaching a peak level during pregnancy. It can be concluded that changes in FSHR and LHR gene and protein expression could be related to the growth and development of follicles, indicating the regulatory role for these receptors in rabbit folliculogenesis.

There has been no report on the outcome of vitrified blastocyst transfer from a vitrified oocyte injected with immotile testicular spermatozoa with only multiple morphological abnormalities of the sperm flagella (MMAF). A couple diagnosed with MMAF returned to the clinic to attempt pregnancy using their vitrified oocytes. Testicular spermatozoa were injected intracytoplasmically, and the following intracytoplasmic sperm injection results were observed. In the second cycle, surplus vitrified oocytes and testicular retrieved sperm were used, but no pregnancy ensued. In the third cycle, a surplus vitrified blastocyst was transferred, and a healthy female child was delivered, with a birth weight of 3050 g and a birth length of 53 cm. In this report we describe a successful pregnancy achieved in a patient presenting MMAF. The successful pregnancy was obtained from vitrified oocytes microinjected with testicular retrieved sperm in a vitrified blastocyst transfer.

Cooling techniques have several applications for reproduction in aquaculture. However, few studies have sought to create protocols for cooling and cryopreservation of Macrobrachium amazonicum embryos. Thus, the objective of this work was to verify the survival of M. amazonicum embryos and the correlation between embryonic volume and mortality of M. amazonicum embryos after cooling. Embryo pools were collected from three females and divided into two treatment groups: dimethyl sulfoxide (DMSO) 3% and ethylene glycol (EG) 0.5%, both of them associated with 2 M sucrose. Positive and negative control groups consisted of seawater 10%. Aliquots of 10 µg of embryos were placed in Falcon® tubes containing a cryoprotectant solution and submitted directly to the test temperature of 2°C for 2 and 6 h of cooling. Further analysis of survival and embryonic volume were performed under a stereoscopic microscope. Data were subjected to analysis of variance (ANOVA), and means were compared using the Tukey test at 5%. The highest embryonic survival rate was observed after the shortest storage time for both the DMSO 3% and the 0.5% EG groups, with survival rates of 84.8 ± 3.9 and 79.7 ± 2.8%, respectively. There was a reduction in survival after 24 h, with the DMSO 3% group presenting a survival rate of 71.7 ± 6.6%, and the EG 0.5% group, 66 ± 6.9%. Survival showed a statistically significant difference when compared with the positive controls after 2 h and 24 h of cooling, with 99 ± 0.5% and 95.8 ± 1.5% survival rates, respectively. There was no significant statistical difference in the embryonic volume, but it was possible to observe a change in the appearance of the embryos, from a translucent coloration to an opaque white or brownish coloration, after 24 h in incubators. Thus, it can be concluded that survival is inversely proportional to storage time and that, although there was no change in the embryonic volume after cooling, a change in the appearance of embryos could be observed.

Heat shock protein 90 (Hsp90) is critical for cell homeostasis but its role on bovine oocyte maturation is not well known. We investigated the importance of Hsp90 for competence of bovine oocyte using 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an inhibitor of Hsp90, during in vitro maturation (IVM). Three experiments evaluated the effect of 17AAG on developmental competence of oocytes matured in vitro under thermoneutral (38.5ºC) or heat shock (HS; 41.5ºC) temperatures. The first experiment found that the blastocyst rates were lower (P < 0.05) with 2 µM 17AAG compared with the untreated control (0 µM). The abundance of HSF1 transcripts was higher in oocytes matured with 2 µM than with 0 and 1 µM 17AAG, whereas the abundance of HSP90AA1 and HSPA1A transcripts was lower (P < 0.05) with 1 and 2 µM than with 0 µM. The second experiment found that 2 µM 17AAG for 12 or 24 h during IVM decreased (P < 0.05) the blastocysts rates. In the third experiment, the association of 2 μM 17AAG with HS for 12 h during IVM resulted in lower (P < 0.05) blastocysts rates than 17AAG, HS or untreated control. In conclusion, inhibition of Hsp90 during in vitro maturation compromises further embryo development; the association of Hsp90 inhibition with HS aggravates the deleterious effect of both on oocyte developmental competence.

To enhance the developmental competency of murine ovarian follicles cultured in vitro, C-type natriuretic peptide (CNP) was supplemented in the culture system. Although the mechanism is not fully elucidated, it was reported that the effect of CNP supplementation was mediated by increased cyclic guanosine monophosphate (cGMP). In the present study, cGMP levels in media for murine preantral follicle culture were compared both between a control group without CNP supplementation and an experimental group with CNP supplementation and between days in each group. In addition, follicle growth patterns and oocyte maturity were assessed and compared between the two groups. Results demonstrated that along with in vitro culture, cGMP levels increased (P < 0.05) both in the control group and the experimental group, whereas cGMP levels were not significantly different between the two groups on the same day of in vitro culture (P > 0.05). The oocyte’s maturity was superior in the experimental group compared with the control group (P < 0.05). As ovarian follicles grew three-dimensionally in the experimental group but were flattened in the control group, CNP might improve oocyte maturity through maintaining the three-dimensional architecture of the ovarian follicle because of increased transzonal projections (TZP) and functional gap junctions between oocyte and surrounding granulosa cells.