Survival Rate Of Embryos Research Articles

Ovulation of juvenile, mature, and aged female C57BL/6 mice following coadministration of inhibin antiserum and equine chorionic gonadotropin

Superovulation technique is important to improve the efficiency of oocyte and animal production and reduce the number of oocyte donors. Previously, we have reported that the coadministration of inhibin antiserum (IAS) and equine chorionic gonadotropin (eCG) results in the production of >100 oocytes in a 4-week-old female C57BL/6 mice. It is well established that superovulation depends on the age of the female mice. However, detailed data regarding the ovulation of juvenile, mature, and aged female mice following the administration of IAS and eCG as well as the performance of reproductive technologies using oocytes have not yet been investigated. In the present study, we examined the effect of the age of female mice (3–50 weeks old) on the number of ovulated oocytes via the coadministration of IAS and eCG or eCG alone. Treatment with IAS plus eCG produced the maximum number of oocytes at 4 weeks of age. Moreover, IAS plus eCG produced more oocytes than eCG alone in mice aged between 3 and 5 weeks or 7 and 30 weeks. The fertilization and birth rates were similar between the two treatments at any age. Moreover, after vitrifying and warming the embryos, the survival and birth rates of two-cell embryos were similar between the two treatments. Subsequently, we examined the optimal ages of female mice (between 24 and 34 days) to obtain a high and stable number of oocytes. In mice aged between 24 and 32 days, IAS plus eCG induced the production of more eggs than eCG alone. Notably, the coadministration of IAS and eCG in mice aged between 25 and 31 days resulted in stable ovulation and high number of oocytes. Using the tip of the optimal female aged between 25 and 31 days old, we demonstrated an efficient production of embryos and offspring between homozygous knockout males and few females aged 26–28 days via in-vitro fertilization and embryo transfer. In summary, the coadministration of IAS and eCG resulted in a higher number of oocytes in juvenile, mature, and aged female mice. This treatment may be useful for the efficient production of homozygous mutant mice from a limited number of female mice.

Haploid Induction in Multiplier Onion (Allium cepa var aggregatum) through in vitro Gynogenesis

The haploid induction study is an effective method for inbred and hybrid development quickly. The optimization of media composition for specific indigenous genotypes was a critical factor. Two media namely, Gamborg’s medium (B5) and Murashige and Skoog’s medium (MS) with six different combinations of plant growth regulators were used to evaluate the gynogenic potential of six different genotypes of multiplier onion. Days for embryo induction was earlier in Acc. 1 (46-54), whereas Acc. 4 was the best responding genotype with a higher percent of embryo induction (0.76) followed by Acc. 15 (0.48). The embryo induction rate and survival rate of 0.21% and 54.54% respectively were recorded in B5 medium whereas in MS medium the induction and survival rate was 0.34 % and 34.86 % respectively. Embryo induction ranged from 0 to 1.41% in this study. Five haploids and one mixoploid in B5 medium, five haploids in MS medium were obtained. Ploidy status of the plants was confirmed by flow cytometry, cell cytology, and stomatal dimension analysis..

Mitigative effects of natural and model dissolved organic matter with different functionalities on the toxicity of methylmercury in embryonic zebrafish

Dissolved organic matter (DOM) occurs ubiquitously in aquatic environments and plays an intrinsic role in altering the chemical speciation and toxicity of methylmercury (MeHg). However, interactions between MeHg and natural DOM remain poorly understood, especially at the functional group level. We report here the mitigative effects of three natural organic matter (NOM) and five model-DOM under different concentrations (0, 1, 3, 10, 30 and 100 mg-C/L) on the toxicity of MeHg in embryonic zebrafish (<4 h post-fertilization, hpf). NOM are those from the Mississippi River, Yukon River, and Suwannee River, while model-DOM include those containing thiosalicylic acid, L-glutathione, dextran, alginic acid, and humic acid. We selected a MeHg concentration (100 n-mol/L) that reduces the survival rate of embryos at 24 hpf by 18% and increases malformations at 72 and 96 hpf. In the presence of DOM, however, the malformation rates induced by MeHg can be mitigated to a different extent depending on DOM concentrations, specific functional groups, and/or specific components. Model DOM with aromatic thiols was the most effective at mitigating the effects of MeHg, followed by L-glutathione, carbohydrates, and humic acid. NOM also mitigated the toxicity of MeHg dependent on their composition and/or effective DOM components as characterized by fluorescence excitation-emission matrix techniques. Specifically, humic-like DOM components are more effective in reducing the MeHg toxicity in the embryonic zebrafish compared to protein-like components. Further studies are needed to elucidate the interactions between DOM and MeHg and the mitigative mechanisms at the molecular level.

Toxicity, bioactivity, release of H2S in vivo and pharmaco-kinetics of H2S-donors with thiophosphamide structure

H2S donors are substitutes of H2S with various biological activities like inhibiting the inflammatory response and protecting myocardial cells from injury. In order to confirm whether the H2S donors have drug-like properties, two series thiophosphamide H2S donors were evaluated including toxicity, bioactivity and pharmacokinetic properties in vivo and in vitro. The following results were obtained. Firstly, all the compounds released H2S under measuring condition; with the increase of pH value, the H2S release rate of all the compounds decreased and the amount reduced, but pH value had little effect on the maximum release of H2S. Secondly, in the organs and tissues of rats, the compounds released H2S in the same way as in PBS. In plasma, compound 1 reached the Cmax after administration 55 min, and no compound 1 was detected after 12 h; for compound 18, the Cmax reached only after administration 100 min, and after 6 h, compound 18 was not detected; in organs and tissues, the H2S-release rates were different from those in PBS, but the mechanism of H2S release was the same. Thirdly, in the test of toxicity, all the compounds displayed low toxicities to 5 cancer cells and W138 cell lines; compounds 1 and 18 had slight effect on the physiological tissue and function of rat liver at low concentration; the compounds had almost no effect on the hatching rate, survival rate of zebrafish embryos, and the spontaneous movement of zebrafish embryos at below 0.5 μM, but when they were over 1 μM, the compounds displayed inhibitory effect in the manner of concentration dependence. Fourthly, in the course of anti-inflammatory test, all the tested compounds significantly reduced the level of TNF-α and increased the level of IL-10; when they were 100 μM, the levels of IL-10 were three times as high as those in the control group. Among them, compounds 10 and 18 displayed stronger activities than the others. In addition, the compounds protected H9c2 cells from injure and improved myocardial injury through anti-oxidation pathway. In summary, the compounds have druglike properties due to low toxicity, better activity and good pharmacokinetic property. Therefore, they have potential to be as candidates to investigate further.

Evaluation of the Effect of Aqueous Momordica charantia Linn. Extract on Zebrafish Embryo Model through Acute Toxicity Assay Assessment.

Momordica charantia Linn., commonly known as bitter gourd, has many protective roles due to its medicinal value as it contains bioactive components. However, this extract showed possible toxicity effect on zebrafish embryo. Thus this study was designed to differentiate the toxicity activities in two types of M. charantia sample which are Indian and Chinese M. charantia, as well as to compare between two different aqueous extraction methods, hot and cold aqueous method, using zebrafish embryo assay assessment. It was observed that the survival rate of zebrafish embryo decreased as the concentration of test extract increased for all samples of M. charantia. The LC50 values of hot aqueous Chinese M. charantia, hot aqueous Indian M. charantia, and cold aqueous Chinese M. charantia were 144.54 μg/ml, 199.53 μg/ml, and 251.19 μg/ml, respectively. However, cold aqueous Indian M. charantia has a higher LC50 which was not in the range of the tested concentration. Hatchability of Danio rerio embryo reduced as the concentration of M. charantia extract increased while no hatching was observed in the highest concentration (1000 μg/ml). Scoliosis of zebrafish larvae was only seen in higher concentrations (125-1000 μg/ml) of extract. The heartbeat of zebrafish larvae treated with M. charantia extract was within the normal range, 120-180 bpm, but at higher concentrations (125-1000 μg/ml) the heartbeat differed for all samples of test extract. Hence, although this plant extract was safe to be consumed due to its pharmaceutical effect, it still exhibited mild toxicity effect at higher concentration when it was evaluated on zebrafish embryo.

Influence of environmental parameters on gametogenesis, spawning and embryo survival in the holothuroid Cucumaria frondosa

The environmental control of reproductive processes is central to both fundamental and applied aspects of animal ecology. The present study experimentally assessed how environmental factors modulate gametogenesis and spawning and impact embryo development and survival in the holothuroid Cucumaria frondosa, a cold-water echinoderm with lecithotrophic development. Adults were exposed to three seawater temperatures, four photoperiod regimes, and two food concentrations during 120 days overlapping the natural gametogenic and spawning periods. Among all the treatments tested, exposure to ambient conditions of water temperature (−1 to 3 °C), photoperiod (8 to 13 h of light) and food concentration (3 × 103 ± 896 rotifers g−1 of sea cucumbers d−1) yielded the greatest reproductive output and embryo survival. At pre-spawning, males and females had the highest gonad index (~33%) and where filled with mature gametes. At spawning, females in ambient conditions released the highest number of oocytes (1.2 × 103 oocytes female−1) and yielded the highest embryo survival rates (21% at 20 days post fertilization). Individuals exposed to warmer water temperatures (6 and 12 °C) experienced delayed gametogenesis and did not spawn. Individuals under 24-h light, 24-h dark, and advanced (4 months) photoperiods showed delayed gametogenesis and low numbers of oocytes released; moreover, embryos died within 10 days post fertilization. Individuals fed with high food concentration (6 × 103 ± 982 rotifers g−1 of sea cucumbers d−1) showed similar results to ambient conditions, suggesting that increased food supply neither decreased nor enhanced reproductive output over the experimental period. This study sheds new light on the environmental drivers of reproductive success in C. frondosa, which is of value for developing conservation strategies and mariculture protocols in cold-water holothuroids with lecithotrophic development.

Boat noise affects the early life history of two damselfishes

Anthropogenic noise can have a negative effect on the physiology and survival of marine fishes. Most research has focused on later life-stages, and few studies have investigated the effects of human-induced noise on embryogenesis. The current study investigated whether playback of motorboat noise affected the embryogenesis of the coral reef damselfishes, Amphiprion melanopus and Acanthochromis polyacanthus. Embryos reared under the playback of boat noise had faster heart rates compared to the ambient reef controls. The effects of noise on morphological development differed between species and the fundamental interrelationships between early life history characteristics changed dramatically under boat noise for Ac. polyacanthus. Noise treatments did not alter the survival rates of embryos under laboratory conditions. Although species specific, our findings suggest that anthropogenic noise causes physiological responses in fishes during embryogenesis and these changes have direct impacts on their development and these alterations may have carry-over effects to later life stages.

Reproduction of endangered river lamprey (Lampetra fluviatilis) in controlled conditions

The research reported focuses on reproduction of the river lamprey Lampetra fluviatilis,(Linnaeus, 1758) in controlled conditions. There was specific emphasis on fish harvesting dates (autumn and spring), holding conditions and reproduction in a controlled environment. Attempts were also made to synchronize the time of ovulation among river lampreys, egg and sperm collections. Hormonal stimulation was conducted using carp pituitary homogenate (CPH) at a total dose of 4 mg/kg which allowed for shortening of the egg-laying period from 2 to 3 weeks to a few days while sustaining embryo survival rates and larvae quality. River lamprey males were found to not require hormonal treatment to yield good-quality sperm, as measured using the CASA system. River lamprey broodstocks adapted well to different manipulations in hatchery conditions when harvested in the autumn and spring. The results of the present study may be used to restore endangered natural populations of the river lamprey (egg and sperm collection, fertilization or gamete preservation) because ovulation and spermiation synchronization is very difficult to achieve without hormonal treatment in controlled conditions.

Differentially sensitive neuronal subpopulations in the central nervous system and the formation of hindbrain heterotopias in ethanol-exposed zebrafish.

A cardinal feature of prenatal ethanol exposure is CNS damage, resulting in a continuum of neurological and behavioral impairments that are described by the term fetal alcohol spectrum disorders (FASD). FASDs are variable and depend on several factors, including the amount, timing, and duration of prenatal ethanol exposure. To enhance interventions for CNS dysfunction, it is necessary to identify ethanol-sensitive neuronal populations and expand the understanding of factors that modify ethanol teratogenesis. To investigate the susceptibility of different neuronal subtypes, we exposed transgenic zebrafish (Danio rerio) to several ethanol concentrations (0.25, 0.5, 1.0, 1.5, or 2.0%), at different hours post fertilization (hpf; 0, 6, or 24 hpf), for various durations (0-24, 0-48, 4-24, 6-24, 6-48,or 24-48 hpf). Following exposure, embryo survival rates were determined, and CNS neurogenesis, differentiation, and patterning were assessed. Embryo survival rates decrease as ethanol concentrations increase and drastically decline when exposed from 0-24 hpf compared to 4-24 hpf. Abnormal tangential migration of facial motor neurons is observed in isl1:gfp embryos exposed to ethanol concentrations as low as 0.25%, and the formation of IVth ventricle heterotopias are revealed by embryos exposed to ≥1.0% ethanol. Whereas, expression of olig2:dsred and ptf1a:gfp in the cerebellum and spinal cord are largely unaffected. While levels of etv4 mRNA are overtly resistant to ethanol, we observe significant reductions in ptch2 mRNA levels. These data show differentially sensitive CNS neuron subpopulations with susceptibility to low levels of ethanol. In addition, these data reveal the formation of ethanol-induced hindbrain heterotopias.

Green Synthesis of Fluorescent Carbon Dots from Gynostemma for Bioimaging and Antioxidant in Zebrafish.

Fluorescent carbon dots (CDs) have been synthesized via the calcination method using natural gynostemma as the precursor, without any toxic ingredients or surface passivation chemicals. CDs have a narrow size distribution, and the mean particle size is about 2.5 nm. CDs exhibit good water dispersibility and can emit intense blue fluorescence under 365 nm UV light in an aqueous solution, which can be stable in different conditions. The biotoxicity of CDs on the embryonic development of zebrafish is evaluated, the hatch rate and survival rate of embryos are around 90%, and the malformation rate is less than 10%. Because of the excellent fluorescence stability and biocompatibility, CDs can be used in zebrafish for bioimaging. In addition, the antioxidative stress property of CDs is investigated both in vitro and in vivo, and the presence of CDs can promote the mRNA expression of related genes to encode more antioxidant proteins in zebrafish. Therefore, fluorescent CDs would be a potential candidate for bioimaging and treating diseases caused by excessive oxidation damage, such as cancer, senility, and other diseases associated with aging.

Long-term outcomes of freeze-all strategy: A retrospective analysis from a single ART center in Japan.

PurposeTo demonstrate the benefits of the freeze‐all strategy for in vitro fertilization treatment based on retrospective analyses.MethodsPost‐thaw embryo survival rates of slow‐frozen embryos in 294 cycles and vitrified embryos in 12 195 cycles were assessed. Progesterone (P4) and estradiol (E2) levels per mature oocyte by age category were assessed in 9081 cycles and pregnancy rates with fresh embryo transfer and frozen‐thawed embryo transfer by P4 level were assessed in 1535 cycles.ResultsThe survival rates of frozen‐thawed embryos were 92.5% with slow freezing and 99.1% with vitrification. P4 levels on the day of human chorionic gonadotropin (hCG) injection showed a trend toward an increase with age. The pregnancy rate per mature oocyte with fresh embryo transfer decreased dependently upon P4 level, while that with frozen‐thawed embryo transfer was not affected by P4 level. The pregnancy rates with frozen‐thawed embryo transfer were higher than those with fresh embryo transfer in patients aged 42 years or younger.ConclusionsThe freeze‐all strategy is a valuable treatment option which allows the separation of an embryo transfer cycle from an oocyte retrieval cycle, especially for patients with high P4 levels at oocyte retrieval and patients of advanced maternal age.

Novel Murine Model of Immune Thrombocytopaenia through Immunized CD41 Knockout Mice.

Immune thrombocytopaenia (ITP) is the most common autoimmune bleeding disorder, where platelets are destroyed by auto-antibodies and/or cell-mediated mechanisms. To understand the pathogenesis of ITP and explore novel therapeutics, three types of animal models have been used: passive ITP, secondary ITP and platelet-induced ITP. However, the first two are not ideal for chronic ITP pathophysiology where both T cell and B cell play important roles in platelet destruction. The most efficient model to mimic chronic ITP is developed by Chow et al through transferring splenocytes from platelet-immune CD61-knockout (KO) mice into mice with severe combined immunodeficiency (SCID). However, placental defects are evident in 25% of CD61-KO females and post-natal haemorrhage does occur, reducing the survival rate of embryos and pups. Compared with CD61-KO mice, CD41-KO ones do not present such problems. In our study, we employ CD41-KO mice as another source of immunized spleen cells. We evaluated our model with existing standards. Transferred SCID mice presented typical features of ITP, such as reduced platelet counts in the peripheral blood, increased anti-platelet antibody levels in the serum and reduced mature megakaryocytes in the bone marrow. What is more, lymphocyte-depletion experiments showed the role of CD8+ T cells in mature megakaryocyte decrease and thrombocytopaenia. And we confirmed the antibody-mediated platelet destruction by phagocytosis in the spleen. Our study develops another efficient murine ITP model through immunized CD41-KO mice.

Direct transfer of frozen-thawed bovine embryos and its application in cattle reproduction management.

Embryo transfer entails many procedures and techniques, of which embryo freezing is an important component in bovine embryo transfer. Embryo freezing techniques have been developed over the last 40 years, allowing practical availability, and have become essential for cattle reproduction management under field conditions. The direct transfer methods of frozen-thawed, in vivo-derived, and in vitro-produced (IVF) bovine embryos using 1.5 M ethylene glycol (EG) with or without sucrose (SUC) are used widely under on-farm conditions, not only in Japan but also globally. The direct transfer method using 1.5 M glycerol (GLY) and 0.25 M SUC (GLY-SUC) is used mainly in Japan. The pregnancy rate with direct transfer of frozen-thawed bovine embryos in either EG or GLY-SUC has been found to not differ from conventional freezing with GLY and traditional dilution techniques. Pregnancy rates following direct transfer of frozen-thawed bovine embryos were affected by the developmental stage of the embryos and the parity of the recipients. The use of ultrasound-guided on-farm ovum pickup is ushering in a new revolution for the commercial application of IVF embryos. Globally, for the first time more IVF bovine embryos were transferred in 2017 than produced in vivo. More than 60% of IVF embryos were transferred fresh due to a low pregnancy rate of frozen-thawed IVF embryos. Many factors seemed to be involved in improving the survival rate of frozen-thawed IVF embryos. Therefore, further research is needed to improve the freezing tolerance of IVF embryos to develop efficient direct transfer methods analogous to those used for in vivo embryos.

The effects of water and bottom sediments from Lake Balkash (Central Asia) on early development of fish and amphibians

The effects of water and bottom sediments from Lake Balkash (Central Asia) on early development of fish and amphibians

24 Survival rates of vitrified biopsied bovine in vitro-produced blastocysts using the VitTrans device

In breeding programs, the application of a vitrification method suitable for direct transfer of biopsied embryos can increase the genetic improvement of cattle and help reduce the costs of embryo transfer. The aim of this study was to determine the in vitro survival of biopsied vitrified blastocysts using the new VitTrans device (Morató and Mogas 2014 Cryobiology 68, 288-293), a 1-step in-straw warming system. Immature bovine oocytes were in vitro matured, fertilized, and cultured to the blastocyst stage. A total of 110 grade 1 blastocysts (IETS codes 6 and 7) were randomly allocated to 2 groups: (1) biopsy (n=49) and (2) without biopsy, or control (n=61). Blastocysts were biopsied using a microblade mounted on a micromanipulator. A small portion of the trophoblast, approximately 15%, was cut off and a significant part of the zona pellucida was sliced away. Both groups were then vitrified using the VitTrans device. For vitrification, all blastocysts were exposed to an equilibration medium with 7.5% ethylene glycol+7.5% dimethyl sulfoxide in holding medium (HM) consisting of TCM-199 with 20% FCS, moved into a drop with 16.5% ethylene glycol+16.5% dimethyl sulfoxide+0.5M sucrose in HM, and then placed in a microdroplet on the VitTrans. The VitTrans was plunged into LN and covered with a 0.5-mL straw. For warming, the protective cover was removed from the VitTrans while still submerged in LN. Subsequently, a new 0.5-mL plastic embryo transfer straw was placed on the VitTrans while flushing the warming solution (0.3mL of 0.5M sucrose in HM at 45°C) with a syringe through the lumen of the device. By entering the warming solution into the VitTrans device, the embryo is flushed inside the plastic straw. The straw containing the embryo can then be readily used for transfer after the VitTrans is removed. To recover the embryo in the laboratory, the content of the straw was put into a Petri dish and blastocysts were placed in the culture medium and incubated at 38.5°C in 5% CO2 and 5% O2 in air. Morphology and re-expansion were evaluated 24h post-warming. The embryo survival rate was defined as the ratio of blastocysts that were able to re-expand with regards to the total number of warmed blastocysts. Due to the attachment of embryos inside the straw, a total of 18 embryos were lost during recovery (12 from the biopsied group and 6 from the nonbiopsied group). The ratio of re-expanded blastocysts from the recovered embryos was 40% in the biopsy group and 61% in the control group. In conclusion, vitrification using the VitTrans device showed good results with intact embryos compared with biopsied embryos. In addition, biopsied embryos had a tendency to adhere to the inside of the straw, which is probably due to the damage or loss of the zona pellucida. Additional research is required to minimize the loss of embryos.

59 Effect of amniotic progenitor cell microvesicles on freezing of in vitro-produced bovine embryos and on pregnancy rate after embryo transfer

Microvesicles (MV) are involved in communication mechanisms based on microRNAs active in the regulation of gene decisive in biological processes such as gametogenesis, fertilization, implantation and embryo development. In this context, in our previous study (Perrini et al. 2018 Reprod. Fertil. Dev. 30, 658-671), amniotic derived MV, and to a lesser extent MV derived from endometrial cells, were effective on stimulating embryo development, demonstrating that MV-exposed embryos show a larger number of cells constituting the inner cell mass, greater viability, higher expression of GPX1 gene (protective against lipid peroxidation) and lower expression of BAX gene (involved in apoptosis) compared with the control. In this study, further effects of amniotic-derived MV on bovine embryo were evaluated. Bovine embryo survival rate after cryopreservation and on pregnancy rate after embryo transfer of fresh or cryopreserved MV-exposed embryos were studied. Embryos were produced from 3782 oocytes. Basing on our previously published protocol (Perrini et al. 2018 Reprod. Fertil. Dev. 30, 658-671), presumptive zygotes were randomly transferred in SOFaa (control, CTR) or cultured by adding 50×106 of amniotic-derived MV mL−1 in the SOFaa on Day 5 post-fertilization (Perrini et al. 2018 Reprod. Fertil. Dev. 30, 658-671). The embryo developmental rate was evaluated at Day 7 (blastocyst stage), and after that, part of the embryos were fresh transferred, whereas part of them were cryopreserved in 1.5 ethylene glycol using a standard slow freezing curve. One fresh or cryopreserved embryo was transferred in each recipient. Statistical analyses were performed by ANOVA with nonparametric Kruskal-Wallis test and for pregnancy rate by chi-square test. Differences were considered statistically significant at P &amp;lt; 0.05. Results showed that blastocyst rate was 34.59±1.32% (709/2050) in CTR and 34.24±1.71% (593/1732) with MV. After cryopreservation, embryo survival was statistically different (P&amp;lt;0.05) between CTR (32.71±6.26%; 87/266) and MV (43.09±5.73%; 78/181), respectively. The pregnancy rate was statistically different (P&amp;lt;0.05) after embryo transfer of fresh embryos, with 66.67% (20/30 cows) of pregnancies for MV and 36.67% (11/30) for CTR. The transfer of cryopreserved embryos again provided a statistically (P&amp;lt;0.05) different result: 36.67% (11/30) for MV embryos versus 10% (3/30) for CTR. These data show that the pregnancy rate of fresh or cryopreserved MV embryos is higher compared with CTR. Based on our previous data demonstrating higher expression of GPX1 gene and lower expression of BAX gene in presence of MV, these results indicate that the higher expression of the GPX1 gene probably limits the damage of lipid peroxidation by improving the survival rate following cryopreservation, whereas the less expressed BAX gene limits damages due to apoptosis. In conclusion, amniotic-derived MV probably play a biological role in the interaction between the embryo and the endometrium, providing a more resource-rich environment.

Generation of CRISPR-Edited Rodents Using a Piezo-Driven Zygote Injection Technique.

Direct modification of the genome of the zygotes (i.e., one-cell embryos) by the CRISPR/Cas9-editing reagents, followed by embryo transfer to pseudopregnant females for live birth, has been the most effective method to generate laboratory rodent models for research. The method relies on proper delivery of the editing reagents into zygotes, which is commonly achieved by a standard or slightly modified pronuclear microinjection technique. In this chapter, we describe in detail an alternative delivery method, named piezo-driven cytoplasmic microinjection, which offers a superior embryo survival and birth rate. Because this method uses a much wider injection needle than that in pronuclear injection, it allows a larger volume of the editing materials to be transported into the zygotes, leading to an increase in the targeting efficiency. This also eliminates the clogging issues seen regularly in pronuclear injection. Moreover, Cytochalasin B that is used to soften zygotes during piezo-driven microinjection has been suggested a role in improving the knockin efficiency, which provides an additional benefit to use this injection method.

Phenology and the physiological niche are co‐adapted in a desert‐dwelling lizard

Abstract A major goal of seasonal biology is to understand how selection on phenology and the physiological niche interact. In oviparous species, fitness variation across the growing season suggests that phenological shifts will alter selective environments experienced by embryos. We hypothesize that physiology could become co‐adapted with phenology; such that embryos perform better in the environmental conditions they are adapted to compared to embryos adapted to other environments (temporal matching). Here, we tested for temporal matching to seasonal changes in the environmental temperatures with toad‐headed lizard, Phrynocephalus przewalskii, which inhabits the temperate desert steppe of China. We used a split‐clutch reciprocal experiment, by incubating eggs from early‐ and late‐breeding females at rising and falling temperature regimes, respectively, to separate the influence of intrinsic (genetic and parental) vs. extrinsic factors (developmental plasticity or acclimatization) on the performance and fitness of offspring. Eggs from early‐breeding females were with higher quality than those from late‐breeding females, likely due to better maternal provisioning. Offspring from early‐breeding females had higher selected body temperatures and metabolic rates than those from late‐breeding females. Falling temperatures that may indicate the end of the growing season, reduced incubation duration and increased metabolic rates for both early and late eggs, compared to rising temperatures. Late hatchlings had higher growth rates when incubated at falling compared to rising temperatures, while growth rates of early hatchlings were not sensitive to incubation temperature. Thus, growth and survival rates of late embryos were similar to early embryos under falling temperatures, despite early embryos being of generally higher quality. Overall, our study confirms that “early is higher quality.” Intrinsic factors dominate offspring performance and fitness, with a general advantage for early embryos throughout the season. We found some support for temporal matching, demonstrating that late embryos with lower quality have physiological strategies that are specialized to late‐season environments, allowing them to attain similar fitness in late‐season environments to that of early embryos. A plain language summary is available for this article.

Next-generation and further transgenerational effects of bisphenol A on zebrafish reproductive tissues

Next-generation effects and further transgenerational effects of an endocrine disruptor, bisphenol A (BPA), were investigated in zebrafish. The effects of BPA treatment through dietary administration in male and female zebrafish on reproductive factors, such as gonadal activity, fertility, hatching rate and malformation in subsequent generations, were examined through the third filial (F3) generation. BPA treatment of initial generation (F0) not only caused retraction of the ovaries and testes but also lowered the survival rate and increased the rate of malformation of the offspring. Although the overall phenotypes of the surviving first filial (F1) generation offspring of treated fish initially appeared to be normal, we found abnormalities in their reproductive tissues after they matured to adulthood. Although the juveniles were fed a normal diet, the ovaries of 40% of the F1 generation fish remained small and did not develop vitellogenic oocytes. Moreover, sterile male fish appeared at a higher percentage (48%) than control (10%). Adverse transgenerational effects on the fecundity of the second filial (F2) and F3 generation fish were also observed. In each generation, survival rate of embryos were significantly low and abnormal embryos were appeared in offspring from BPA treated ancestral. These results demonstrate that the effects of BPA are transferred to subsequent generations not only through oocytes but also through sperm.

Outcomes of effective cryopreservation. A two-year assessment of survival rates of vitrified and warmed blastocysts. The experience of an IVF clinic in a developing country

Cryopreservation of oocytes and embryos by vitrification is currently the method of choice employed by most facilities that practice assisted reproductive technology (ART) and there are several studies reporting impressive outcomes. This specialized technique, however, is operator-dependent, so results do vary amongst embryologists and clinics. In Nigeria, vitrification is rapidly gaining widespread popularity as many hospitals are gradually incorporating this technique into their ART practice. This study aims to show the success of an IVF clinic in a developing country at implementing conventional vitrification and warming protocols with survival rates of vitrified-warmed blastocysts reassessed. A retrospective study of 171 FET cycles with a total of 485 embryos vitrified and subsequently warmed within a period of two years and three months (October 2015-December 2017). This study was conducted at Medical Art Center (MART), a private fertility clinic in Lagos, Nigeria. Participants included infertile couples going through frozen embryo transfers after prior failed in-vitro fertilization (IVF) cycles and in some cases, patients susceptible to OHSS with previous freeze all cycles. Vitrification and subsequent warming was performed with Medicult vitrification and warming media (Origio/Medicult, Målov, Denmark) using the McGill cryoleaf protocol as described by the manufacturer (Medicult, Denmark). A total of 146 vitrification cycles were carried out for patients who went through frozen embryo transfers within the study period (Oct 2015-Dec 2017). In 171 FET cycles, 485 vitrified blastocysts were warmed with 358 blastocysts surviving the procedure. The freeze-thaw survival rate was 73.8%, and the average survival rate per freeze-thaw method was 80.5%. The total number of embryos transferred was 343 (average of 2.1 embryos per patient). 166 embryo transfers were performed. 2 FET cycles required re-vitrification of embryos without a transfer taking place, and only 3 patients had no surviving embryos. Our study supports existing reports from publications highlighting excellent outcomes with vitrification. We were able to achieve high embryo survival rates; therefore, we have adopted Vitrification as the only method of choice for cryopreserving embryos. It may be recommended to other IVF clinics looking to improve their freeze-thaw survival rates.