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.

In zebrafish, a punctate band of F-actin is reported to develop in the external yolk syncytial layer (E-YSL) during the latter part of epiboly in zebrafish embryos. Here, electron microscopy (EM) and fluorescence confocal microscopy were conducted to investigate dynamic changes in the E-YSL membrane during epiboly. Using scanning EM, we report that the surface of the E-YSL is highly convoluted, consisting of a complex interwoven network of branching membrane surface microvilli-like protrusions. The region of membrane surface protrusions was relatively wide at 30% epiboly but narrowed as epiboly progressed. This narrowing was coincident with the formation of the punctate actin band. We also demonstrated using immunogold transmission EM that actin clusters were localized at the membrane surface mainly within the protrusions as well as in deeper locations of the E-YSL. Furthermore, during the latter part of epiboly, the punctate actin band was coincident with a region of highly dynamic endocytosis. Treatment with cytochalasin B led to the disruption of the punctate actin band and the membrane surface protrusions, as well as the attenuation of endocytosis. Together, our data suggest that, in the E-YSL, the region encompassing the membrane surface protrusions and its associated punctate actin band are likely to be an integral part of the localized endocytosis, which is important for the progression of epiboly in zebrafish embryos.

Mammalian oocytes not fertilized immediately after ovulation can undergo ageing and a rapid decline in quality. The addition of antioxidants can be an efficient approach to delaying the oocyte ageing process. Onion peel extract (OPE) contains quercetin and other flavonoids with natural antioxidant activities. In this study, we investigated the effect of OPE on mouse oocyte ageing and its mechanism of action. The oocytes were aged in vitro in M16 medium for 16 h after adding OPE at different concentrations (0, 50, 100, 200, and 500 μg/ml). The addition of 100 μg/ml OPE reduced the oocyte fragmentation rate, decreased the reactive oxygen species (ROS) level, increased the glutathione (GSH) level, and improved the mitochondrial membrane potential compared with the control group. The addition of OPE also increased the expression of SOD1, CAT, and GPX3 genes, and the caspase-3 activity in OPE-treated aged oocytes was significantly lower than that in untreated aged oocytes and similar to that in fresh oocytes. These results indicated that OPE delayed mouse oocyte ageing by reducing oxidative stress and apoptosis and enhancing mitochondrial function.

Estradiol and progesterone have been recognized as important mediators of reproductive events in the female mainly via binding to their receptors. This study aimed to characterize the immunolocalization of the estrogen receptor alfa (ERα), estrogen receptor beta (ERβ) and progesterone receptor (PR) in the ovarian follicles of the lizard Sceloporus torquatus. The localization of steroid receptors has a spatio-temporal pattern that depends on the stage of follicular development. The immunostaining intensity of the three receptors was high in the pyriform cells and the cortex of the oocyte of previtellogenic follicles. During the vitellogenic phase, the granulosa and theca immunostaining was intense even with the modification of the follicular layer. In the preovulatory follicles, the receptors were found in yolk and additionally, ERα was also located in the theca. These observations suggest a role for sex steroids in regulating follicular development in lizards, like other vertebrates.

In this study, we built on our previous research that discovered that autophagy activated the metaphase I stage during porcine oocytes in vitro maturation. We investigated the relationship between autophagy and oocyte maturation. First, we confirmed whether autophagy was activated differently by different media (TCM199 and NCSU-23) during maturation. Then, we investigated whether oocyte maturation affected autophagic activation. In addition, we examined whether the inhibition of autophagy affected the nuclear maturation rate of porcine oocytes. As for the main experiment, we measured LC3-II levels using western blotting after inhibition of nuclear maturation via cAMP treatment in an in vitro culture to clarify whether nuclear maturation affected autophagy. After autophagy inhibition, we also counted matured oocytes by treating them with wortmannin or a E64d and pepstatin A mixture. Both groups, which had different treatment times of cAMP, showed the same levels of LC3-II, while the maturation rates were about four times higher after cAMP 22 h treatment than that of the 42 h treatment group. This indicated that neither cAMP nor nuclear status affected autophagy. Autophagy inhibition during in vitro oocyte maturation with wortmannin treatment reduced oocyte maturation rates by about half, while autophagy inhibition by the E64d and pepstatin A mixture treatment did not significantly affect the oocyte maturation. Therefore, wortmannin itself, or the autophagy induction step, but not the degradation step, is involved in the oocyte maturation of porcine oocytes. Overall, we propose that oocyte maturation does not stand upstream of autophagy activation, but autophagy may exist upstream of oocyte maturation.

The live birth rate following embryo transfer is comparable between spontaneous, stimulated and artificial cycles. However, the pregnancy loss rate appears elevated with hormonal therapy, possibly due to luteal insufficiency. This study aimed to determine whether the serum progesterone level on transfer day differed according to the endometrial preparation method for frozen embryo transfer (FET). Twenty spontaneous cycles (SC), 27 ovarian stimulation cycles (OS) and 65 artificial cycles (AC) were retrospectively studied from May to December 2019 in a single French hospital. The primary endpoint was the level of serum progesterone on the day of FET between the three endometrial preparation methods. The mean serum progesterone level on transfer day was 29.47 ng/ml in the OS group versus 20.03 ng/ml in the SC group and 14.32 ng/ml AC group (P < 0.0001). Progesterone levels remained significantly different after logistic regression on age and anti-Müllerian hormone (AMH) level. There was no significant difference in demographic and hormone characteristics (age, body mass index, embryo stage of embryo, type of infertility, basal follicle stimulating hormone, luteinizing hormone, estradiol and AMH levels), endometrial thickness, number and type of embryos transferred, duration of infertility, pregnancy rate, live birth rate and pregnancy loss rate. No difference was found in serum progesterone levels between clinical pregnancy with fetal heartbeat and no clinical pregnancy (no pregnancy or pregnancy loss, 17.49 ng/ml vs 20.83 ng/ml, respectively, P = 0.07). The lower serum progesterone level found on FET day in the AC group should be further investigated to see whether this difference has a clinical effect on the live birth rate.

Oocyte-mediated somatic cell haploidization is a process in which a diploid cell halves its chromosomal content by segregating its homologue within the ooplasm. Replacing the donor oocyte nucleus with a patient’s female diploid somatic nucleus can generate patient-genotyped oocytes. Insemination of these resulting constructs enables their activation and induces a reductive meiotic division, haploidizing the diploid female donor cell that can subsequently support syngamy with the male genome and create a zygote. So far, experimental data for this method have been limited and have not consistently proven the generation of chromosomally normal embryos. Overall, we achieved reconstruction of murine oocytes with a micromanipulation survival rate of 56.5%, and a correct haploidization and fertilization rate of 31.2%, resulting in a 12.7% blastocyst rate. Time-lapse analysis revealed that reconstructed embryos underwent a timely polar body extrusion and pronuclear appearance followed by a satisfactory embryonic cleavage, comparable with the control. Whole genome sequencing of the analyzed embryos indicated that 27.3% (6/22) were properly diploid. Our findings suggest that diploid cell haploidization may be a feasible technique for creating functional gametes in mammals.

Carvacrol (C10H14O), an efficient phenolic antioxidant substance for several cell types, may become a useful antioxidant for female germ cells and embryo culture. This study investigates the effects of carvacrol supplementation on bovine oocytes in in vitro maturation (IVM) and embryo production. In total, 1222 cumulus–oocyte complexes were cultured in TCM-199+ alone (control treatment) or supplemented with carvacrol at the concentrations of 3 µM (Carv-3), 12.5 µM (Carv-12.5), or 25 µM (Carv-25). After IVM, the oocytes were subjected to in vitro fertilization and embryo production, and the spent medium post-IVM was used for evaluating the levels of reactive oxygen species and the antioxidant capacity (2,2-diphenyl-1-picryl-hydrazyl-hydrate and 2,2′-azinobis-3-ethyl-benzothiozoline-6-sulphonic acid quantification). A greater (P < 0.05) antioxidant potential was observed in the spent medium of all carvacrol-treated groups compared with the control medium. Moreover, the addition of carvacrol to the maturation medium did not affect (P > 0.05) blastocyst production on days 7 and 10 of culture; however, the total number of cells per blastocyst was reduced (P < 0.05) in two carvacrol-treated groups (Carv-3 and Carv-25). In conclusion, carvacrol demonstrated a high antioxidant capacity in the spent medium after oocyte maturation; however, although embryo production was not affected, in general, carvacrol addition to IVM medium reduced the total number of cells per blastocyst. Therefore, due to the high antioxidant capacity of carvacrol, new experiments are warranted to investigate the beneficial effects of lower concentrations of carvacrol on embryo production in cattle and other species.

Heat stress can have severe deleterious effects on embryo development and survival. The present study evaluated whether CSF2 can protect the developmental competence of the bovine embryo following exposure to a heat shock of 41°C at the zygote and morula stages. In the first experiment, putative zygotes and 2-cell embryos were assigned to receive either 10 ng/ml CSF2 or vehicle, and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Heat shock reduced blastocyst development for embryos treated with vehicle but not for embryos cultured with CSF2. In the second experiment, day 5 embryos (morula) were treated with CSF2 or vehicle and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Temperature treatment did not affect development to the blastocyst stage and there was no effect of CSF2 treatment or the interaction. Results indicate that CSF2 can reduce the deleterious effects of heat shock at the zygote or two-cell stage when the embryo is transcriptionally inactive.

In the treatment of infertile patients with non-obstructive azoospermia (NOA) caused by the deletion of the azoospermia factor c region (AZFc) on the Y chromosome, synchronous and asynchronous surgical strategies are discussed. Clinical data from NOA patients with the AZFc deletion who underwent micro-TESE were analyzed retrospectively. The sperm retrieval rate (SRR) and sperm utilization rate of synchronous and asynchronous operation groups were followed up and compared. The fertilization rate, high-quality embryo rate, clinical pregnancy rate, abortion rate, and cumulative live birth rate of ICSI in patients with successful sperm retrieval were compared between the two groups. The two groups had sperm utilization rates of 98.9% (93/94) and 50.0% (14/28), respectively. The asynchronous group’s sperm consumption rates were much lower than those of the synchronous operation group. Fertilization rate, high-quality embryo rate, clinical pregnancy rate of fresh transfer cycle, abortion rate, and cumulative live birth rate of patients in the synchronous operation group with fresh sperm, and the asynchronous operation group with thawed sperm, respectively, were 30.6% vs 33.8%, 33.8% vs 40.7%, 40.0% vs 12.5%, 30.4% vs 7.1%. Between the two groups, there was no significant difference. This suggests that individuals with NOA caused by the AZFc deletion have a high possibility of successfully acquiring sperm using micro-TESE and ICSI to conceive their own offspring. Synchronous micro-TESE is recommended to improve sperm utilization rate and the cumulative live birth rate.

This study aimed to detect intracellular trehalose in boar sperm that were cryopreserved with liposomes and conduct an analysis of its effects on some characteristics of thawed sperm, including rheological properties. First, soybean lecithin cholesterol-based liposomes were produced and characterized in the presence of 300 mM trehalose. Next, semen samples were frozen in two freezing media: a control medium with 300 mM trehalose and an experimental medium supplemented with 300 mM trehalose and 10% liposomes, both of which were thawed and then studied to ascertain their integrity, motility, rheological response, and trehalose quantities by testing two methods of spermatic lysis via high-performance liquid chromatography with an evaporative light-scattering detector (HPLC-ELSD). The results found spherical liposomes measuring 357 nm that were relatively stable in an aqueous medium and had an entrapment efficiency of 73%. An analysis of the cryopreserved ejaculates showed that their viability and motility did not significantly differ between groups (P > 0.05). The viscous response of the samples was influenced by the extracellular medium rather than by the freezing–thawing process, which resulted in a loss of interaction between the cells and cryoprotectants. Finally, intracellular trehalose levels were determined using HPLC-ELSD, with no differences observed (P > 0.05) when comparing both sperm lysis methods. The use of liposomes with trehalose appears to be a promising option for boar semen cryopreservation, with a marked effect on rheological properties. The proposed HPLC-ELSD method was effective for measuring trehalose in cryopreserved cell samples.

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed ‘FLI’. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

The Mexican tetra Astyanax mexicanus presents two contrasting morphs, a widely distributed surface morph and a cave-adapted morph. These cave-adapted morphs have evolved independently from two different lineages (i.e. ‘old’ and ‘new’ lineages); therefore, this model system gives a unique opportunity to explore parallel adaptive evolution in biological traits. The present study corresponds to the first morphological description of the Astyanax mexicanus maturation process of the spermatozoa and oocytes, using thermal and hormonal stimuli to promote spermatogenesis and oogenesis, considering surface and cave morphs from both lineages. We corroborate the relevance of thermal and hormonal stimuli to promote gamete maturation. The hormone Ovaprim (GnRHa + Domperidone) is an effective promoter of ovarian development, maturation end in oocytes and spawning in Astyanax mexicanus. The sperm morphology of Astyanax mexicanus includes the sperm head, the midpiece, and tail or flagellum. We found differences in the spermatozoan total length between environments (F = 9.929, P = 0.05) and linages (F = 49.86, P = 0.005). The oocytes showed a spherical conformation with a mean diameter of 822.4 ± 194.1 μm for the surface populations, and 604.6 ± 38.3 µm for the cave populations. The oocyte chorion presents ridges and grooves that are arranged radially towards the micropyle. A plug in the micropyle zone was observed after fertilization, confirmed by the outer membrane of the chorion, which provides some weak adhesiveness to the substrate. We observed differences in chorion thickness between the contrasting environmental conditions. This is the first morphological characterization of the Sótanos Vázquez, Escondido and Tigre, which previous to this study were only known from speleological expeditions, with no previous biological information available.

Oocytes eligible for intracytoplasmic sperm injection (ICSI) are those that have progressed through meiosis to metaphase 2 (MII). The remaining delayed mature oocytes can be injected, aiming to achieve more embryos and a better chance to conceive. We aimed to assess the outcome of delayed matured oocytes, derived from either germinal vesicles or metaphase 1 (MI), that reached maturity (MII) 24 h following retrieval. The study population consisted of 362 women who underwent 476 IVF cycles. While fertilization rates were comparable between the sibling delayed mature oocyte group compared with injection on day 0 group (58.4% vs 62%, respectively, P = 0.07), the top-quality embryo rate per injected MII day 0 oocyte was significantly higher compared with day 1 injected oocyte (57.5% vs 43.9% respectively, P < 0.001). Moreover, following fresh transfer of embryos derived from delayed mature oocytes, implantation rate and the clinical pregnancy (CPR) and live-birth rates (LBR) per transfer were 3.9%, 3.3% and 1.6% respectively. When considering the following thawed embryo transfer cycles, implantation, pregnancy and LBR were non-significantly higher (10%, 8.3% and 8.3%, respectively). Although clinical outcomes are significantly lower when using embryos derived from delayed mature oocyte to mature day 0 oocytes, the additional embryos derived from delayed mature oocytes might contribute to the embryo cohort and increase the cumulative live-birth rate per retrieval. Moreover, the embryos derived from delayed mature oocyte favour a transfer in a frozen–thawed cycle rather than in a fresh cycle.

The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) links the folate cycle that produces one-carbon units with the methionine cycle that converts these into S-adenosylmethionine (SAM), the universal methyl donor for almost all methyltransferases. Previously, MTHFR has been shown to be regulated by phosphorylation, which suppresses its activity. SAM levels have been shown to increase substantially soon after initiation of meiotic maturation of the mouse germinal vesicle (GV) stage oocyte and then decrease back to their original low level in mature second meiotic metaphase (MII) eggs. As MTHFR controls the entry of one-carbon units into the methionine cycle, it is a candidate regulator of the SAM levels in oocytes and eggs. Mthfr transcripts are expressed in mouse oocytes and preimplantation embryos and MTHFR protein is present at each stage. In mature MII eggs, the apparent molecular weight of MTHFR was increased compared with GV oocytes, which we hypothesized was due to increased phosphorylation. The increase in apparent molecular weight was reversed by treatment with lambda protein phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. In contrast, LPP had no effect on MTHFR from GV oocytes, 2-cell embryos, or blastocysts. MTHFR was progressively phosphorylated after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing again after egg activation. As phosphorylation suppresses MTHFR activity, it is predicted that MTHFR becomes inactive during meiotic maturation and is minimally active in MII eggs, which is consistent with the reported changes in SAM levels during mouse oocyte maturation.

Sperm motility and viability of cryopreserved semen vary between boars and straws, which influences the outcomes of in vitro embryo production (IVEP). However, progressive motility is usually not considered during IVEP. The aim of this study was to assess fertilization with a 500:1 and 250:1 ‘progressively motile sperm to oocyte’ ratio on IVEP outcomes using semen from three Duroc and three Landrace boars. Frozen–thawed sperm was centrifuged through a 45/90% Percoll® density gradient and sperm quality parameters were assessed. In vitro matured oocytes were fertilized at the two ratios, a portion was stained 10–12 h after start of fertilization to analyze fertilization and polyspermy, while the remaining zygotes were cultured up to day 7. The 500:1 ratio resulted in a higher fertilization and blastocyst yield on day 6 compared with the 250:1 ratio, but no effect of ratio was observed for polyspermy, cleavage rate or blastocyst cell number. Individual differences between boars were observed for fertilization, cleavage and blastocyst rates, but not for the other IVEP outcomes. In conclusion, a higher fertilization and blastocyst yield was obtained with the 500:1 ratio compared with the 250:1 ratio, while polyspermy level was consistent across ratios. Differences in IVEP outcomes were still observed between the individual boars although adjusted for progressive motility. Promising blastocyst yields and high total blastocyst cell counts were obtained with sperm from both breeds.

We compared the pregnancy and live birth rates following transfer of early-stage embryos or blastocysts produced by somatic cell nuclear transfer using in vitro-matured oocytes. In total 102 ovaries were collected from dromedary camels at a local abattoir; from these 1048 cumulus–oocytes complexes (COCs) were aspirated and cultured for 42 h in a commercial maturation medium. Metaphase II oocytes were subjected to nuclear transfer. Somatic cell nuclear transfer-derived embryos were cultured in a commercial embryo medium for 2 or 7 days. Next, 71 early-stage embryos were surgically transferred to the left fallopian tube of 28 recipients and 47 blastocysts were transferred to the left uterine horn of 26 recipients. Early pregnancy was detected by serum progesterone (P4), and pregnancy was confirmed using ultrasonography on days 30 and 90 after embryo transfer. Pregnancy rate based on P4 level was 17.86% (5/28) and 11.54% (3/26) for early-stage embryo and blastocyst transfer, respectively. In the early-stage embryo group, out of five recipients, one recipient had lost the pregnancy by the first ultrasonography on day 30; two other recipients aborted at 14 and 24 weeks, and two recipients gave live births. In the blastocyst group, out of three recipients, one lost the pregnancy at an early stage and two recipients gave live births. Therefore, for dromedary camels, we recommend transvaginal blastocyst transfer from the standpoint of the pregnancy and live birth rate, ease of the transfer procedure, and comfort and safety of the recipients.

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.

Male infertility is responsible for 50% of men’s health problems and has always been a concern for personal and social issues. A survey of global statistics suggests an increase in infertility rate as one of the critical issues documented in studies. There are different ways of maintaining fertility in men, depending on their age. In this paper, we review the preservation methods used for fertility treatment in Iran and other countries. Available data were reviewed from Google Scholar, PubMed, Scopus, Web of Science, IranMedex, MEDLIB, IranDoc and Scientific Information Database and searched for articles published up to 2018, using the medical subject heading (MeSH) terms for cryopreservation, sperm, testicular, spermatogonia stem cell, male infertility and/or Iranian and in the world, to provide evidence from evaluation of fertility preservation the methods. Based the search strategy, 274 manuscripts were found. After reviewing the titles, abstracts and manuscripts in their entirety, 119 articles were obtained and selected according to the eligibility criteria. The 85 studies mentioned above were divided into three categories (sperm, testis, and spermatogonia stem cells (SSCs)), and methods of fertility preservation were investigated. Ways to maintain male fertility were different depending on age, and included sperm, testicular, and SSC freezing. The number of studies on testicular tissue and SSCs was low for human samples, and more studies are still needed. Sperm freezing at infertility centres is the top for male fertility preservation.

It has previously been reported that in ex vivo planar explants prepared from Xenopus laevis embryos, the intracellular pH (pHi) increases in cells of the dorsal ectoderm from stage 10.5 to 11.5 (i.e. 11–12.5 hpf). It was proposed that such increases (potentially due to H+ being extruded, sequestered, or buffered in some manner), play a role in regulating neural induction. Here, we used an extracellular ion-selective electrode to non-invasively measure H+ fluxes at eight locations around the equatorial circumference of intact X. laevis embryos between stages 9–12 (˜7–13.25 hpf). We showed that at stages 9–11, there was a small H+ efflux recorded from all the measuring positions. At stage 12 there was a small, but significant, increase in the efflux of H+ from most locations, but the efflux from the dorsal side of the embryo was significantly greater than from the other positions. Embryos were also treated from stages 9–12 with bafilomycin A1, to block the activity of the ATP-driven H+ pump. By stage 22 (24 hpf), these embryos displayed retarded development, arresting before the end of gastrulation and therefore did not display the usual anterior and neural structures, which were observed in the solvent-control embryos. In addition, expression of the early neural gene, Zic3, was absent in treated embryos compared with the solvent controls. Together, our new in vivo data corroborated and extended the earlier explant-derived report describing changes in pHi that were suggested to play a role during neural induction in X. laevis embryos.