This study aims to evaluate the effects of N-acetylcysteine (NAC) on bovine oocyte maturation, mitochondrial activity and transzonal projections (TZP), as well as on the levels of reactive oxygen species (ROS) and messenger RNA (mRNA) for catalase (CAT) superoxide dismutase (SOD), periredoxin-6 (Prdx6), glutathione peroxidase (GPx), growth and differentiation factor-9 (GDF9), histone H1Foo, cyclin B1 (CCNB1) and c-Mos. Bovine cumulus–oocyte complexes (COC) of medium-sized antral follicles (3.0–6.0 mm) were prematured in TCM-199 for 8 h at 38.5°C in 5% CO2. After prematuration in the presence of forskolin and C-type natriuretic peptide, COCs were matured in TCM-199 alone or with 0.1, 0.5 or 2.5 mM NAC. Then, oocytes were classified according to the stage of chromatin. Furthermore, mitochondrial activity and intracellular levels of ROS and TZP were also evaluated. The levels of mRNAs for CAT, SOD, Prdx6, GPx, GDF9, H1Foo, CCNB1 and c-Mos were evaluated using real-time polymerase chain reaction (RT-PCR). The results showed that NAC significantly increased the percentages of oocytes with resumption of meiosis when compared with those oocytes matured in control medium. Oocytes had homogeneous mitochondrial distribution, and those cultured with 0.1 and 0.5 mM NAC had lower levels of ROS when compared with the control. In addition, 0.5 mM NAC reduced TZP and the levels of mRNA for CCNB1. In contrast, NAC did not influence the expression of CAT, GPx, Prdx6, SOD, GDF9, H1Foo, and c-Mos. In conclusion, 0.5 mM NAC reduced the levels of ROS, TZP and mRNA for CCNB1, and improved in vitro resumption of meiosis in oocytes from medium-sized bovine antral follicles.

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.

To investigate the effects of culture media with different lactate concentrations on early embryonic development, data collected from our patients undergoing preimplantation genetic testing (PGT) were assessed using the EmbryoScope™ time-lapse culturing system. After intracytoplasmic sperm injection (ICSI), sibling oocytes were cultured in the same EmbryoScope (Vitrolife) slides including two different commercially available media. The patients with fewer than five mature oocytes were not included in the analyses. All embryos were hatched on day 3, and trophectoderm biopsies (n = 212) were performed accordingly. PGT for aneuploidy (PGT-A) on biopsied materials was carried out using next generation sequencing. Morphokinetic parameters, fertilization, irregular division, degeneration, blastulation, euploidy, and pregnancy rates of embryos cultured in LifeGlobal Global Total medium (LGGT) and Continuous Single Culture-NX Complete medium (CSCM-NXC) were compared. There were no differences observed in time to pronuclear fade, or in time spent as 2-cell (cc2) and 3-cell (s2), to 4-cell, 5-cell, morula and blastocyst stages (P > 0.05). Embryos reached the 2-cell (t2) and 3-cell (t3) stages significantly faster in LGGT (P < 0.05), whereas embryos grown in CSCM-NXC with lower lactate reached starting blastulation significantly sooner (P = 0.026). However, there were no statistical differences observed in fertilization, blastulation, degeneration, irregular division euploidy, and pregnancy rates between the two groups (P > 0.05). Even though pregnancy and fertilization rates did not indicate statistical differences, results are significant to provide better insight on potential roles of lactate in embryo development. These finding will advance the fundamental knowledge of human embryo development and assisted reproductive technologies.

This study evaluated the effect of fibroblast growth factor-2 (FGF-2) on the morphology, primordial follicle activation and growth after in vitro culture of domestic cat ovarian tissue. Ovaries (n = 12) from prepubertal domestic cats were collected and fragmented. One fragment was fixed for histological analysis (fresh control). The remaining fragments were incubated in control medium alone or with 10, 50 or 100 ng/ml FGF-2 for 7 days. After in vitro culture, the following endpoints were analyzed: morphology, activation by counting primordial and developing follicles, and growth (follicle and oocyte diameters). Treatment with 100 ng/ml FGF-2 maintained (P > 0.05) the percentage of normal follicles similar to fresh control. Follicle survival was greater (P < 0.05) after culture in 100 ng/ml FGF-2 than in 50 ng/ml FGF-2. The percentage of primordial follicles decreased (P < 0.05) and the percentage of developing follicles increased (P < 0.05) in all treatments compared with fresh tissue. The proportion of developing follicles increased (P < 0.05) in tissues incubated with 100 ng/ml FGF-2 compared with control medium and other FGF-2 concentrations. Furthermore, culture in 10 or 100 ng/ml FGF-2 resulted in increased (P < 0.05) follicle and oocyte diameters compared with fresh tissues and MEM+. In conclusion, FGF-2 at 100 ng/ml maintains follicle survival and promotes the in vitro activation and growth of cat primordial follicles.

Buffalo has many excellent economic traits and it is one of the greatest potential livestock. Compared with cattle, buffalo has poorer reproductivity, it is of great significance to improve the development potential of oocytes. Buffalo oocyte in vitro maturation (IVM) has been widely used in production, but the poor development ability of bovine oocytes IVM limits the development of buffalo reproductivity. Milrinone as a phosphodiesterase inhibitor could affect the maturation of oocytes in goat and mice, but there have been few reported studies in water buffalo. To optimize buffalo oocyte in vitro maturation systems, the effects of phosphodiesterase inhibitor (milrinone) on pre-maturation culture of buffalo oocytes were investigated in this study. Buffalo cumulus–oocyte complexes (COCs) were cultured in medium with different concentrations (0, 12, 25, 50 and 100 mol/l) of milrinone for different times (0, 4, 8, 12, 16, 22 and 24 h). The results showed that the buffalo COCs nuclear maturation process could be inhibited by milrinone (25–100 mol/l) in a dose-dependent manner. The inhibitory effect of milrinone on in vitro maturation of buffalo oocytes did not decrease with the extension of time. This indicated that milrinone can be used as a nuclear maturation inhibitor during the maturation process in buffalo oocytes. In addition, milrinone can inhibit the effect of follicle stimulating hormone (FSH)-induced IVM of buffalo oocytes, but with time FSH partially eliminated the inhibition. Therefore, inhibition of milrinone on the nuclear maturation of buffalo oocytes was reversible, and buffalo oocytes can mature normally after the inhibition is lessened.

Oxidative stress is an undesirable effect of in vitro culture, which requires antioxidant supplementation. This study investigated the analogue of resveratrol (RA33) as an alternative to resveratrol, an antioxidant molecule, for the in vitro culture of in vitro-fertilized bovine embryos. The effect of different concentrations of RA33 on embryo development was evaluated and a comparison between RA33 and resveratrol was performed. The cleavage rate was higher (P < 0.05) with 2.5 μM (69.0 ± 4.4%) than at 0, 0.1 or 0.5 μM RA33 (62.1 ± 2.0%, 60.7 ± 5.9% and 56.7 ± 5.8%, respectively). The blastocyst rates on days 7 and 8 post-fertilization with 2.5 μM RA33 (19.4 ± 3.3% and 24.6 ± 3.3%, respectively) were higher (P < 0.05) than for 0 μM (12.4 ± 2.5% and 15.2±2.5%, respectively). When 2.5 μM RA33 was compared with 0.5 μM resveratrol, similar (P > 0.05) cleavage and blastocyst rates were found between them, but the cleavage rate was higher (P < 0.05) in the control (80.8 ± 3.4%) than for the resveratrol treatment (76.4 ± 3.6%). The numbers of apoptotic cells and the apoptotic index were lower (P < 0.05) with RA33 (6.5 ± 0.6 cells and 6.4 ± 0.7%, respectively) and resveratrol (5 ± 0.8 cells and 5.5 ± 1.0%, respectively) than in the control group (9.8 ± 1.2 cells and 8.9 ± 1.1%, respectively). In conclusion, RA33 can enhance the preimplantation development of in vitro-fertilized bovine embryos and be an alternative to resveratrol in embryo culture medium.

The generation of germ cells from embryonic stem cells in vitro has current historical significance. Western blot, qPCR, immunofluorescence and flow cytometry assays were used to investigate the differences in expression levels of totipotency and specific markers for Wnt regulation and the related signalling pathways during primordial germ cell-like cell (PGCLC) induction and differentiation. During PGCLC induction, activation of WNT3a increased the expression of NANOG, SOX2 and OCT4, but Mvh, DAZL, Blimp1, TFAP2C, Gata4, SOX17, EOMES, Brachyury and PRDM1 expression levels were significantly reduced. Inhibition of the WNT signal demonstrated the opposite effect. Similarly, inhibitors of BMP and the Nodal/Activin signal were used to determine the effect of signal pathways on differentiation. CER1 affected the Wnt signal and differentiation, but the inhibitor SB only regulated differentiation. BMP–WNT–NODAL were mainly responsible for regulating differentiation. Our results provide a reliable theoretical basis and feasibility for further clinical medical research.

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.

Telomere shortening during oocyte growth and development is related to reproductive ageing and infertility. The main mechanism involved in the maintenance of telomeres is based on telomerase activity, a specialized enzyme complex, which is capable of adding TTAGGG repeats at the ends of the chromosomes. Mitochondrial dysfunction may cause progressive shortening of telomeres by promoting the generation of reactive oxygen species. Mitofusin-1 is a protein required for mitochondrial fusion. Mice with the mitofusin-1 (Mfn1) deletion in the oocyte are characterized by accelerated follicular depletion and infertility, associated with defective oocyte maturation and follicular development. We hypothesized whether mitochondrial dysfunction in oocytes with targeted deletion of Mfn1 causes telomere shortening. We analyzed telomere length in oocyte and somatic cells in 3-, 6- and 9-month-old Mfn1−/− and wild-type mice. Immunofluorescence in oocyte mice of TRF1 and H2A.X was assessed to evaluate the interplay between the end-protection functions and the response to DNA damage occurring inside the telomeric repeats. Mitochondrial dysfunction due to the deletion of Mfn1 does not seem to affect telomere length in mouse oocytes.

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

Ovine ovarian fragments (3 × 3 × 1 mm) were fixed in neutral buffered formalin (NBF), Carnoy’s solution (CAR), Davidson’s solution (DAV), or paraformaldehyde (PFA) for 12 h or 24 h. After this fixation time, each fragment was prepared for histological analysis. Although fixative and fixation period did not affect follicular and stromal cells density, the percentages of morphologically normal primordial and primary follicles was affected by the fixative type and period of fixation. Paraformaldehyde was not indicated as a fixative for ovarian fragments. Formalin was a suitable fixative only when the period of fixation was 12 h, while Carnoy was efficient after a fixation period of 24 h. In conclusion, the most indicated fixative for the morphological evaluation of ovarian preantral follicles was DAV, regardless of the fixation period, that is 12 or 24 h.

The evaluation of early reproduction traits in Beetal goat was performed for possible effects of genetic and non-genetic factors on litter size at birth (LSB), litter size at weaning (LSW), litter weight at birth (LWB), litter weight at weaning (LWW) and age at first kidding (AFK). The data records consisted of information of pedigree and targeted traits pertained to 223 does born to 25 sires and 122 dams between the years 2004 to 2019. A general linear model was used for assessment of non-genetic factors such as period of birth, type of birth and dam’s weight at kidding on studied traits. Genetic evaluation of targeted traits was done to estimate variance components and genetic parameters under dyadic mixed modelling. The estimates of least-square means for LSB, LSW, LWB, LWW and AFK were observed as 1.27 ± 0.03, 1.25 ± 0.03, 3.24 ± 0.07 kg, 13.08 ± 0.30 kg and 27.56 ± 0.58 months, respectively. Only the period of birth showed significant (P < 0.05) effects for targeted traits in this study. The estimates of direct heritability for LSB, LSW, LWB, LWW and AFK were low in magnitude as 0.08, 0.03, 0.10, 0.03 and 0.06, respectively. The moderate to high genetic and phenotypic correlations among litter traits indicate simultaneous improvement for these traits. It was concluded that low ranged direct heritability estimates for targeted traits indicated modest scope for genetic improvement of reproductive efficiency in Beetal goat through selection and, therefore, adoption of improved managerial practices is necessary to improve reproductive efficiency of Beetal goat.

The cryopreservation of murine ovarian tissue and its transplantation can be a promising technique for the preservation of fertility and an alternative for the future reconstitution of scientific valuable strains of mice. Accordingly, the aim of this study was to describe the entire surgical procedure for ovariectomy and dorsal subcutaneous autotransplantation in mice, and also some data about the efficiency of this procedure. Female C57Bl/6J mice (n = 18) were anaesthetised and bilaterally ovariectomized. After surgery, ovaries were autotransplanted in small subcutaneous pouches in the dorsal region of the forelimbs. The animals were inspected daily and, 23 days after transplantation, euthanasia and recovery of ovarian tissues were performed. Postoperative recovery, oestrous cyclicity, and folliculogenesis progression were evaluated. At 23 days after transplantation, the recovery of the ovaries was feasible, all classes (primordial to antral) of follicles were observed. Additionally, satisfactory efficiency rates were obtained, with 100% of anaesthesia survival rate, survival, graft recovery, folliculogenesis progression and oestrous cyclicity. In general, this short article describes ovarian ectopic autologous transplantation as an effective technique for maintaining rodent oogenesis and endocrine ovarian function. Even more broadly, we can still assume that the application of this technique may reduce the number of breeding matrices and experimental animals in the near future.

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.

Ascidians (Urochordate) are hermaphroditic marine invertebrates that release sperm and eggs to the surrounding seawater. However, several ascidians, including Ciona intestinalis and Halocynthia roretzi, show strict self-sterility due to a self/nonself-recognition mechanism in the interaction between sperm and the vitelline coat (VC) of the eggs. We have previously reported that sperm intracellular Ca2+ level drastically increased immediately after sperm binding to the VC of self eggs but not nonself eggs in C. intestinalis type A, which was potently inhibited by lowering the external Ca2+ concentration, suggesting that sperm Ca2+ influx occurs after sperm self-recognition on the VC. Here, we investigated whether self-sterility was abolished by lowering the external Ca2+ concentration in C. intestinalis. The results showed that the block to self-fertilization was removed by low-Ca2+ (∼1 mM) seawater without decreasing the fertilization rate. Such an effect was not observed with Mg2+ or K+. These results led us to conclude that a low-Ca2+ environment is sufficient to block the self-recognition signal upon fertilization. As low-Ca2+ seawater showed no effect on H. roretzi self-sterility, we propose that the mechanism of self-sterility in Ciona must be distinctive from that in Halocynthia.

Lipopolysaccharide (LPS) endotoxemia has been negatively associated with fertility. This study aimed to investigate the effect of LPS-induced inflammation on gene expression associated with bovine fertility in the uterus and oviduct. Sixteen healthy heifers were divided into two groups. The LPS group (n = 8) received two intravenous (i.v.) injections of 0.5 µg/kg of body weight of LPS with a 24-h interval, and the control group (n = 8) received two i.v. injections of saline solution with the same interval of time. All the animals had the follicular wave synchronized. Three days after the second injection of LPS, all animals were slaughtered and uterine and oviduct samples were collected. Gene expression associated with inflammatory response, thermal and oxidative stresses, oviduct environment quality, and uterine environment quality was evaluated. Body temperature and leucogram demonstrated that LPS induced an acute systemic inflammatory response. In the uterus, the expression of PTGS2 and NANOG genes was downregulated by the LPS challenge. However, no change in expression was observed in the other evaluated genes in the uterus, nor those evaluated in the oviduct. In conclusion, the inflammatory process triggered by LPS did not persist in the uterus and oviduct 3 days after challenge with LPS. Nonetheless, reduction in PTGS2 and NANOG expression in the uterus suggested that, indirectly, LPS may have a prolonged effect, which may affect corpus luteum and endometrial functions.

The aim of this study was to investigate mitochondrial ND5 gene polymorphisms and their relationship with in vitro maturation (IVM) and in vitro culture (IVC) of Sanjabi sheep. Blood and ovarian samples of adult ewes were obtained from a local slaughterhouse. For each ovarian sample, cumulus–oocyte complexes larger than 3 mm in diameter were aspirated from follicles, and their IVM and IVC rates were recorded. A 666-bp fragment of the ND5 gene was amplified using the polymerase chain reaction. The samples were genotyped using a modified single-stranded conformation polymorphism (SSCP) method, and an association study was conducted with IVM and IVC rates. Six different SSCP patterns, designated A, B, C, D, E and F with respective frequencies of 8, 47, 4, 4, 32 and 5%, respectively, were observed. According to the results of association analysis, there was no significant association between the ND1 gene polymorphisms and the IVM and IVC rates (P > 0.05).

Somatic cell nuclear transfer (NT) is associated with aberrant changes in epigenetic reprogramming that impede the development of embryos, particularly during zygotic genome activation. Here, we characterized epigenetic patterns of H3K4me3, H3K9me3, and H3K27me3 in mouse NT embryos up to the second cell cycle (i.e. four-celled stage) during zygotic genome activation. In vivo fertilized and parthenogenetically activated (PA) embryos served as controls. In fertilized embryos, maternal and paternal pronuclei exhibited asymmetric H3K4me3, H3K9me3, and H3K27me3 modifications, with the paternal pronucleus showing delayed epigenetic modifications. Higher levels of H3K4me3 and H3K9me3 were observed in NT and PA embryos than in fertilized embryos. However, NT embryos exhibited a lower level of H3K27me3 than PA and fertilized embryos from pronuclear stage 3 to the four-celled stage. Our finding that NT embryos exhibited aberrant H3K4me3, H3K9me3, and H3K27me3 modifications in comparison with fertilized embryos during early zygotic genome activation help to unravel the epigenetic mechanisms of methylation changes in early NT reprogramming and provide an insight into the role of histone H3 in the regulation of cell plasticity during natural reproduction and somatic cell NT.

The aim of this study was to evaluate the effect of 1 µmol/l zearalenone (ZEN) and 1 µmol/l matairesinol (MAT), alone or in combination, on the morphology of in vitro-cultured ovarian preantral follicles. Ovaries from four adult sheep were collected at a local slaughterhouse and fragmented, and the ovarian pieces were submitted to in vitro culture for 3 days in the presence or absence of the test compounds. The morphology of primordial and primary follicles was impaired by ZEN. The plant lignan MAT alone did not maintain the morphology of the ovarian follicles; its combination with ZEN counteracted the negative effects observed when follicles were cultured in the presence of the mycotoxin alone. However, MAT was not able to promote the in vitro development of the ovarian follicles.