It has been demonstrated that in the zygotes of some mammals a unique checkpoint controls the onset of DNA replication. Thus, DNA replication begins in the maternal pronucleus only after the paternal pronucleus is fully formed. In our experiments we have investigated whether this checkpoint also operates in porcine zygotes produced either by in vitro fertilization (IVF) or by intracytoplasmic sperm injection (ICSI). Our results show that the onset of DNA replication occurs in the maternal pronucleus even in the presence of an intact sperm head in zygotes produced by ICSI, as well as in polyspermic eggs where some sperm heads are intact or male pronuclei are not yet fully developed. We conclude that in porcine zygotes there is an absence of the DNA replication checkpoint that is typical for some other mammals.

This is a retrospective study over a 5-year period. In total, 3139 embryos were individually cryopreserved (Cryotop®) and warmed using the Kitazato vitrification/warming kit. They were classified into three categories based on their expansion degree. Transfer, implantation and pregnancy rates were assessed for each embryo category and compared using SPSS (Statistical Package for the Social Sciences) software. In total, 1139 couples enrolled in infertility treatment programme benefitted from embryo vitrification at day 5. After warming, embryos belonging to the three categories showed similar success rates. Although there was a trend towards better outcomes when grade 3 embryos were transferred, the differences did not reach statistical significance: implantation rates (n fetal sac/n embryo transferred) grade 1: 21.9%, grade 2: 22.7% and grade 3: 30.3% (=0.19). Pregnancy rate (n clinical pregnancy/n transfer) (21.9%, 22.7%, 30.3%, respectively; P=0.11). Miscarriage rate was not statistically different in the three categories (14.5%, 20.4%, 20%, respectively, P=0.51). Our overall results show that it is worth vitrifying slow kinetics embryos as they provide a non-negligible chance to give rise to a pregnancy.

Klotho is a protein that plays different functions in female fertility. We have previously reported that klotho protein supplementation during in vitro maturation improves porcine embryo development, while klotho knockout for somatic cell cloning completely blocks full-term pregnancy in vivo. However, the effects of the microinjection of klotho protein or klotho knockdown dual vector in porcine embryos at different time points and the specific molecular mechanisms remain largely unknown. In this study, we injected the preassembled cas9 + sgRNA dual vector, for klotho knockdown, into the cytoplasm of the germinal vesicle stage of oocytes and into porcine embryos after 6-h parthenogenetic activation. Similarly, the klotho protein was inserted into the cytoplasm of germinal vesicle stage oocytes and porcine embryos after 6-h parthenogenetic activation. Compared with the controls, the microinjection of klotho dual vector markedly decreased the blastocyst formation rates in germinal vesicle stage oocytes and activated embryos. However, the efficiency of blastocyst formation when klotho protein was inserted before in vitro maturation was significantly higher than that after klotho protein insertion into parthenogenetically activated embryos. These results indicated that klotho knockdown may impair embryo development into blastocyst irrespective of injection timing. In addition, klotho protein injection timing in pig embryos may be an important factor for regulating embryo development.

Spermatozoa obtained from hybrid male mice were treated with solubilised zonae pellucidae after a period of capacitation in vitro to induce the acrosome reaction. Single spermatozoa were selected and microinjected into the perivitelline space of mature oocytes. A high proportion of the spermatozoa acrosome-reacted affter treatment with solubilised zonae (63%). However, the fertilisation rate (37%) after subzonal microinjection of oocytes by the treated spermatozoa was not different to the fertilisation rate (40%) of oocytes microinjected with untreated spermatozoa which had a lower rate of acrosome reaction (39%). When spermatozoa were washed by high-speed centrifugation before treatment with solubilised zonae pellucidae and subzonal microinjection, the fertilisation rate (68%) was significantly higher (p < 0.001) than that (29%) for occytes microinjected with untreated spermatozoa and was found to be correlated with a high acrosome reaction rate (74%) (r = 0.8). The washing of spermatozoa by centrifugation itself did not increase the acrosome reaction rate or fertilisation rate of oocytes after microinjection. The results of this study suggests that some modifications other than the acrosome reaction are needed to enable capacitated mouse spermatozoa to fuse with the oocyte plasma membrane. These modifications were achieved by washing spermatozoa by high-speed centrifugation and the replacement of the supernatant with fresh culture medium used for capacitation. Induction of the acrosome reaction by solubilised zonae pellucidae following this treatment leads to a high fertilisation rate of oocytes by subzonal sperm microinjection.

The in vitro culture of domestic cat embryos without the zona pellucida affects their implantation capacity. MicroRNAs (miRNAs) have an important role in embryo–maternal communication and implantation. The objective of this study was to evaluate the expression of specific miRNAs in domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were done: (1) domestic cat embryos cultured with the zona pellucida (zona intact control group, ZI); and (2) cultured without the zona pellucida (zona free group, ZF). The cleavage, morula and blastocyst rates were evaluated. The blastocysts and their spent medium were used for miRNA expression analysis using RT-qPCR (miR-21, miR-24, mi25, miR-29, miR-96, miR-98, miR-103, miR-191, miR-196, miR-199, miR-130, miR-155 and miR-302). The pre-mature microRNAs (pre-miRNAs) and miRNAs were evaluated in the blastocysts and only miRNAs were evaluated in the spent medium. No differences were observed in the cleavage, morula and blastocyst rates between the ZF and ZI groups (P > 0.05). For miRNAs analysis, miR-103 and miR-191 had the most stable expression and were selected as internal controls. ZF blastocysts had a higher expression of miR-21, miR-25, miR-29 and miR-199 and a lower expression of miR-96 than their ZI counterparts (P < 0.05). Furthermore, higher levels of miR-21, miR-25 and miR-98 were detected in the spent medium of ZF blastocysts (P < 0.05). In conclusion, in vitro culture of domestic cat embryos without the zona pellucida modifies the expression of miR-21, miR-25, miR-29, miR-199 and miR-96 at the blastocyst stage and the release of miR-21, miR-25 and miR-98.

For individual cultures, findings on regulating embryo density by changing the microdrop volume are contradictory. The aim of this study was to investigate the relationship between embryo density and the developmental outcome of day 3 embryos after adjusting covariates. In total, 1196 embryos from 206 couples who had undergone in vitro fertilization treatment were analyzed retrospectively. Three embryo densities were used routinely, i.e. one embryo in a drop (30 μl/embryo), two embryos in a drop (15 μl/embryo) and three embryos in a drop (10 μl/embryo). Embryo quality on day 3 was evaluated, both the cell number of day 3 embryos and the proportion of successful implantations served as endpoints. Maternal age, paternal age, antral follicles and level of anti-Müllerian hormone, type of infertility, controlled ovarian stimulation protocol, length of stimulation, number of retrieved oocytes, number of zygotes (two pronuclei) and insemination type were covariates and adjusted. After adjusting fully for all covariates, the cell number of day 3 embryos was significantly increased by 0.40 (95% CI 0.00, 0.79; P = 0.048) and 0.78 (95% CI 0.02, 1.54; P = 0.044) in the 15 μl/embryo and 10 μl/embryo group separately, compared with the 30 μl/embryo group. The proportions of implanted embryos were 42.1%, 48.7% and 0.0% in the 30 μl/embryo, 15 μl/embryo and 10 μl/embryo groups respectively. There was no statistical significance (P = 0.22) between the 30 μl/embryo group and the 15 μl/embryo group. After adjusting for confounders that were significant in univariate analysis, embryo density was still not associated with day 3 embryo implantation potential (P > 0.05). In a 30-μl microdrop, culturing embryos with an embryo density of both 15 and 10 μl/embryo increased the cell number of day 3 embryos, which did not benefit embryo implanting potential, compared with individual culture of 30 μl/embryo.

Identifying embryos with a high potential for implementation remains a challenge in in vitro fertilization (IVF) cycles. Despite progress in IVF treatment, only a minority of generated embryos has the ability to implant. Another drawback of this practice is the high frequency of multiple pregnancies. This problem leads to economic and health problems. Therefore, the transfer of a single embryo with high implantation potential is the ideal strategy. Morphometric evaluation of two-pronucleus zygote images is a helpful technique when aiming to transfer a single embryo with a high implantation potential. In this study, an automated zygote morphometric evaluation algorithm, called the zygote morphology evaluation (ZME) algorithm, was created to analyze the zygote and provide morphological measurements. The first and most crucial step of the ZME algorithm is the noise reduction step, which was first applied to zygote images. After that, the proposed algorithm detects different parts of the zygote that are indicators of embryo viability and normality, that is the oolemma, perivitelline space, zona pellucida, and nucleolar precursor bodies (NPBs). In addition, a novel dataset was prepared for this task. This dataset consisted of 703 human zygote images, and called the human zygote morphometric evaluation dataset (HZME-DS). Our experimental results in the HZME-DS showed that the ZME algorithm was able to achieve 79.58% average accuracy in identifying the oolemma region, 79.40% average accuracy in determining the perivitelline space, and 79.72% accuracy in identifying the zona pellucida. To calculate the accuracy of identifying NPBs, the proposed algorithm uses Recall and Precision measures, and their harmonic average (F1 measure) reached values of 81.14% and 79.53%, respectively. These encouraging results for our proposed method, which is an automatic and very fast method, showed that the ZME algorithm could help embryologists to evaluate the best zygotes in real time and the best embryos subsequently.

MicroRNAs (miRNAs) are small non-encoding RNAs that actively regulate biological and physiological processes, and play an important role in regulating gene expression in all cells, especially in most animal cells, including oocytes and embryos. The expression of miRNAs at the right time and place is crucial for the oocyte’s maturation and the embryo’s subsequent development. Although assisted reproductive techniques (ART) have helped to solve many infertility problems, they cause changes in the expression of miRNA and genes in oocytes and preimplantation embryos, and the effect of these changes on the future of offspring is unknown, and has caused concerns. The relevant genomic alterations commonly imposed on embryos during cryopreservation may have potential epigenetic risks. Understanding the biological functions of miRNAs in frozen maturated oocytes may provide a better understanding of embryonic development and a comparison of fertility conservation in female mammals. With the development of new techniques for genomic evaluation of preimplantation embryos, it has been possible to better understand the effects of ART. The results of various articles have shown that freezing of oocytes and the cryopreservation method are effective for the expression of miRNAs and, in some cases, cause changes in the expression of miRNAs and epigenetic changes in the resulting embryo. This literature review study aimed to investigate the effects of oocyte cryopreservation in both pre-maturation and post-maturation stages, the cryopreservation method and the type of cryoprotectants (CPA) used on the expression of some epigenetic-related genes and miRNAs.

Although ethanol treatment is widely used to activate oocytes, the underlying mechanisms are largely unclear. Roles of intracellular calcium stores and extracellular calcium in ethanol-induced activation (EIA) of oocytes remain to be verified, and whether calcium-sensing receptor (CaSR) is involved in EIA is unknown. This study showed that calcium-free ageing (CFA) in vitro significantly decreased intracellular stored calcium (sCa) and CaSR expression, and impaired EIA, spindle/chromosome morphology and developmental potential of mouse oocytes. Although EIA in oocytes with full sCa after ageing with calcium does not require calcium influx, calcium influx is essential for EIA of oocytes with reduced sCa after CFA. Furthermore, the extremely low EIA rate in oocytes with CFA-downregulated CaSR expression and the fact that inhibiting CaSR significantly decreased the EIA of oocytes with a full complement of CaSR suggest that CaSR played a significant role in the EIA of ageing oocytes. In conclusion, CFA impaired EIA and the developmental potential of mouse oocytes by decreasing sCa and downregulating CaSR expression. Because mouse oocytes routinely treated for activation (18 h post hCG) are equipped with a full sCa complement and CaSR, the present results suggest that, while calcium influx is not essential, CaSR is required for the EIA of oocytes.

Larval testes of four Lepidoptera species were examined using electron microscopy. The testes of one species, the Mediterranean mealmoth Ephestia kuehniella (Pyralidae), were devoid of intracellular pathogens and serve as a control. In this species, metaphase spindles of primary spermatocytes showed a thick layer of perispindle membranes. The membranes were structurally very similar to the agranular endoplasmic reticulum. Membranes of this type occurred also at high frequency throughout the spindle matrix. The analysis of larval testes of Pieris brassicae (Pieridae) revealed virus-like particles within spermatocytes. In another species, Philudoria potatoria (Lasiocampidae), the spermatocytes possessed intracellular bacteria. Whereas the pathogens were found within the germ cells in these species, a fourth species, Plutella xylosteUa (Plutellide), showed microsporidia within somatic cells of the testis sheath. In all the infected animals, the mass of perispindle membranes was reduced in comparison with spermatocytes of E. kuehniella. However, spindle structure appeared regular in the infected animals. This indicates that a thick layer of perispindle membranes is not decisive for spindle assembly and function in male meiosis of Lepidopera

This study aimed to evaluate the effect of the cryopreservation duration (up to 160 months) on the clinical and neonatal outcomes of slow-frozen early-cleavage human embryos. Clinical data collected between February 2013 and August 2017 were included in this retrospective study. Cases were classified into five groups by the duration of cryopreservation: Group 1, 6–12 months; Group 2, 13–36 months; Group 3, 37–60 months; Group 4, 61–84 months; and Group 5, >84 months. The embryo survival rate, implantation rate, clinical pregnancy rate, live-birth rate, newborn sex ratio, singleton gestational age, singleton birth weight and malformation rate were compared between the groups. The cryopreservation duration did not significantly affect the rates of clinical pregnancy (P = 0.119) and live birth (P = 0.354), the newborn sex ratio (P = 0.614) or the singleton gestational age (P = 0.212) and birthweight (P = 0.212). Although decreases in the embryo survival and implantation rates were observed in groups 4 and 5 compared with those in groups 1–3, these differences were not statistically significant (P = 0.329, P = 0.279, respectively). Long-term cryopreservation does not appear to adversely affect the clinical and neonatal outcomes of slow-frozen early-cleavage human embryos.

Adult canine fibroblasts stably transfected with either cytomegalovirus (CMV) or POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) were used to investigate if pre-treatment of these donor cells with two epigenetic drugs [trichostatin A (TSA), or S-adenosylhomocysteine (SAH)] can improve the efficiency of interspecies somatic cell nuclear transfer (iSCNT). Fluorescence-activated cell sorting (FACS), analyses revealed that TSA, but not SAH, treatment of both transgenic and non-transgenic fibroblasts significantly increased acetylation levels compared with untreated relatives. The expression levels of Bcl2 and P53 were significantly affected in TSA-treated cells compared with untreated cells, whereas SAH treatment had no significant effect on cell apoptosis. Irrespective of epigenetic modification, dog/bovine iSCNT embryos had overall similar rates of cleavage and development to 8–16-cell and morula stages in non-transgenic groups. For transgenic reconstructed embryos, however, TSA and SAH could significantly improve development to 8–16-cell and morula stages compared with control. Even though, irrespective of cell transgenesis and epigenetic modification, none of the iSCNT embryos developed to the blastocyst stage. The iSCNT embryos carrying CMV–EGFP expressed EGFP at all developmental stages (2-cell, 4-cell, 8–16-cell, and morula) without mosaicism, while no POU5F1–EGFP signal was observed in any stage of developing iSCNT embryos irrespective of TSA/SAH epigenetic modifications. These results indicated that bovine oocytes partially remodel canine fibroblasts and that TSA and SAH have marginal beneficial effects on this process.

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.

Rivulidae comprises a family of fish largely distributed in Brazil that includes 201 species, of which 125 are considered endangered. This fact emphasizes the need for development of conservation strategies including studies on genetics and reproduction. In this paper, we describe aspects of biology and reproduction of the rivuliid species Hypsolebias sertanejo. We outline the reproductive behaviour of this species under laboratory conditions, analyze ploidy status by flow cytometry, describe reproductive behaviour and performance and test dry and wet incubation of eggs. Although H. sertanejo showed well known patterns of reproductive behaviour, we verified many peculiarities inherent to its reproductive biology. As expected, most individuals were diploid (87.71%), however 14.29% were considered mosaics. Although no sterility was observed within mosaics, infertility of these fish was not fully evaluated. Hatching rate of the eggs collected was very low following both dry and wet incubation (5.04 and 3.79%, respectively). These results provide interesting information regarding the reproductive success of this species, and suggest that chromosomal abnormalities described may reduce the survival of H. sertanejo under natural conditions, limiting the perpetuation of this species, and emphasizing the need for more preservation efforts, including artificial propagation and gene banking.

Recovery of more than one oocyte from a single follicle during laparoscopic egg collection has been reported sporadically and accepted as confirmation of the presence of polyovular or binovular follicles in the human ovary at reproductive age. Most of these reports include conjoined oocytes that share common or fused zona pellucida, and are generally accepted as evidence for true polyovularity due to its certain characteristics. In this study, we report one case of a conjoined oocyte and another case of the recovery of two separate oocytes in a cumulus cell complex and details of their early embryonic development. To our knowledge, this report of the recovery of two separate oocytes without zonal contact is the first in the literature. We reviewed the relevant literature to evaluate information regarding the origin, incidence and significance of polyovularity in reproductive health.

Echinoid development progresses through embryonic and larval stages to metamorphosis and the adult form. Despite vast differences in embryos and larval forms, including bilaterally symmetric echinopluteus larvae, ovoid non-feeding larvae and brooded embryos, all metamorphose into juvenile sea urchins with pentaradial symmetry. The adult sea urchin body plan is initiated as the juvenile rudiment. The rudiment has been called the phylotypic stage for the class Echinoidea, a designation that implies little variation at this midpoint in development (e.g. Raff et al., 1991; Richardson, 1995; Raff, 1996). However, right at metamorphosis (upon eversion of the juvenile rudiment), variations in test symmetry, shape and number of spines, and number of skeletal plates, podia and pedicellariae are present in juveniles. This variation suggests either that there is no phylotypic stage or that such a stage occurs earlier in rudiment formation. To distinguish between these possibilities, I explored the patterns by which the juvenile rudiment is formed as well as the variation among juveniles approximately 1 day after metamorphosis in 19 echinoid taxa covering a broad taxonomic range including cidaroids, diadematids, irregular echinoids (spatangoids and clypeasteroids), arbaciids, temnopleurids, echinometrids and strongylocentrotids. Most of the material for analysis of juveniles was obtained by the author. Additional information was gathered from classical studies of metamorphosis. Data were collected on the number and shape of dorsal pedicellariae, juvenile and adult spines, primary and secondary podia, and juvenile test shape. When possible multiple individuals within a species were examined, revealing no or only minor trait variation. These data were mapped on a well-resolved phylogeny established from adult characters.

Cryopreservation of domestic cat semen is mainly performed as a model for the establishment of endangered wild feline protocols. The supplementation of antifreeze protein type I (AFP I) to cryopreservation medium has shown improvement in frozen–thawed sperm quality in other species, but its effect on cat semen has not yet been tested. This study aimed to assess the addition of AFP I to cryopreservation medium in domestic cats. Sperm was obtained from the cauda epididymis of orchiectomized cats; sperm was then pooled in Tris buffer and allocated into three treatments, according to AFP I final concentration: 0 (control), 0.1, and 0.5 µg/ml. Nine replicates were cryopreserved in a two-step protocol and subsequently thawed at 37°C for 30 s. There was no difference (P > 0.05) among the control, 0.1 and 0.5 µg/ml groups for parameters such as motility, vitality, functional membrane integrity, mature chromatin, normal morphology, and sperm binding to egg perivitelline membrane. In the 0.5 μg/ml group only, percentages of live sperm with intact acrosome and of sperm with most inactive mitochondria (DAB III) showed a significant reduction, along with a tendency (P = 0.053) to an increase in the percentage of sperm with most active mitochondria (DAB II). In conclusion, the supplementation of 0.1 and 0.5 µg/ml of AFP I did not promote consistent beneficial effects on the overall sperm cryotolerance in domestic cats.

The effect of hydrostatic pressure treatment on the induction of mitogynogenesis in the eggs of Japanese flounder Paralichthys olivaceus (Temminck et Schlegel) by using heterospecific sperm were studied. Before treatment, the eggs were at metaphase of the first mitosis. The spindle was disassembled by the treatment and then resembled in its pretreatment position, and the chromosomes were rearranged, i.e., the first mitosis was not blocked. During the second mitotic cycle, only a monopolar spindle was assembled in each blastomere and the chromosomes doubled, but cell cleavage was blocked. In the third cycle, mitosis proceeded normally with a bipolar spindle in each blastomere. Flow cytometric analysis of ploidy demonstrated that mitogynogenetic larvae were all diploid. The ultraviolet light-irradiated sperm of the red sea bream (Pagrus major) was condensed, formed a dense chromatin body, and randomly entered one blastomere.

Undifferentiated stem cells may support a greater development of cloned embryos compared with differentiated cell types due to their ease of reprogramming during the nuclear transfer (NT) process. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than are somatic cells. Embryonic germ (EG) cells are undifferentiated stem cells that are isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, the in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated, thus eliminating any variation in genetic differences. The rates of fusion did not differ between NT embryos from EG and NP cells; however, the rate of cleavage in NT embryos derived from EG cells was significantly higher (p < 0.05) than that from NP cells (141/247 [57.1%] vs. 105/228 [46.1%]). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than the rate using NP cells (43/247 [17.4%] vs. 18/228 [7.9%]). The results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. Undifferentiated EG cells may be more amenable to reprogramming after reconstruction compared with differentiated somatic cells.