The actin filaments on the surface of echinoderm oocytes and eggs readily undergo massive reorganization during meiotic maturation and fertilization. In sea urchin eggs, the actin cytoskeletal response to the fertilizing sperm is fast enough to accompany Ca2+ signals and to guide sperm's entry into the egg. Although recent work using live cell imaging technology confirmed changes in the actin polymerization status in fertilized eggs, as was previously shown using light and electron microscopy, it failed to provide experimental evidence of F-actin depolymerization a few seconds after insemination, which is concurrent with the sperm-induced Ca2+ release. In the present study, we applied Raman microspectroscopy to tackle this issue by examining the spectral profiles of the egg's subplasmalemmal regions before and after treating the eggs with actin drugs or fertilizing sperm. At both early (15 s) and late (15 min) time points after fertilization, specific peak shifts in the Raman spectra revealed change in the actin structure, and Raman imaging detected the cytoskeletal changes corresponding to the F-actin reorganization visualized with LifeAct-GFP in confocal microscopy. Our observation suggests that the application of Raman spectroscopy, which does not require microinjection of fluorescent probes and exogenous gene expression, may serve as an alternative or even advantageous method in disclosing rapid subtle changes in the subplasmalemmal actin cytoskeleton that are difficult to resolve.

Maintaining genomic stability is crucial for normal development. At earlier stages of preimplantation development, as the embryonic genome activation is not fully completed, the embryos may be more prone to abnormalities. Aneuploidies are one of the most common genetic causes of implantation failure or first-trimester miscarriages. Apoptosis is a crucial mechanism to eliminate damaged or abnormal cells from the organism to enable healthy growth. Therefore, this study aimed to determine the relationship between the expression levels of genes involved in apoptosis in human aneuploid and euploid blastocysts. In total, 32 human embryos obtained from 21 patients were used for this study. Trophectoderm biopsies were performed and next-generation screening was carried out for aneuploidy screening. Total RNA was extracted from each blastocyst separately and cDNA was synthesized. Gene expression levels were evaluated using RT-PCR. The statistical analysis was performed to evaluate the gene expression level variations in the euploid and aneuploid embryos, respectively. The expression level of the BAX gene was significantly different between the aneuploid and euploid samples. BAX expression levels were found to be 1.5-fold lower in aneuploid cells. However, the expression levels of BAK and MAD2L1 genes were similar in each group. This study aimed to investigate the possible role of genes involved in apoptosis and aneuploidy mechanisms. The findings of this investigation revealed that the BAX gene was expressed significantly differently between aneuploid and euploid embryos. Therefore, it is possible that the genes involved in the apoptotic pathway have a role in the aneuploidy mechanism.

Commercial application of embryo transfer in pig breeding is dependent on the storage of embryos. The aim of this study was to assess the embryo quality of in vitro-produced blastocysts after 3 h liquid storage at 37°C in CO2-free medium by evaluating morphology, in vitro developmental capacity and apoptosis. Blastocysts at days 5 and 6 post-fertilization were randomly allocated to the storage group (HEPES-buffered NCSU-23 medium including bovine serum albumin in a portable embryo transport incubator at 37°C) or a control group (porcine blastocyst medium in a conventional culture incubator). Thereafter, blastocysts were evaluated for morphology and stained to assess apoptosis straight after the 3 h storage period or after a further 24 h conventional incubation. There was no significant difference between the storage and control group after 3 h storage and the further 24 h conventional incubation for any of the parameters, nor for apoptosis straight after the 3 h storage. Embryos that reached the blastocyst stage at day 5 showed less apoptosis (6.6% vs 10.9%, P = 0.01) and a trend for a higher rate of developmental capacity (70.6% vs 51.5%, P = 0.089) than embryos reaching the blastocyst stage on day 6. In conclusion, in vitro-produced porcine blastocysts can be stored for 3 h at physiological temperature in transportable incubators using a CO2-independent medium without compromising quality.

Induction of puberty in cattle breeds that attain puberty in later stages, such as Gir, allows the earlier beginning of reproductive life and it might increase oocyte quality. Here, the ovulatory capacity of prepuberal Gir heifers was studied and its relationship to follicular growth, luteinizing hormone (LH) secretion and oocyte quality was evaluated. Peripubertal Gir heifers were treated with a progesterone-based protocol and according to ovulatory response were separated into groups: not-ovulated (N-OV) and ovulated (OV). Serial blood samples were taken 24 h after estradiol treatment on day 12 to evaluate LH secretion. Cumulus-oocyte complexes (COCs) were collected using ovum pick-up and assessed for brilliant cresyl blue (BCB) staining rate, IVF-grade oocytes rate, and mean oocyte diameter, in comparison with cow oocytes. Gene expression of developmental competence markers (ZAR1, MATER, and IGF2R) was also analyzed. The largest follicle diameters were similar between N-OV and OV groups on the day of estradiol treatment (d12) and the next day and decreased (P = 0.04) in the N-OV group thereafter. LH pulse secretion was different between groups (N-OV = 3.61 ± 0.34 vs OV = 2.83 ± 0.21 ng/ ml; P = 0.04). COC assessment showed that the number of recovered oocytes, BCB+ rate, IVF-grade oocytes and oocyte size was similar (P > 0.05) among groups, resembling adult cow patterns. ZAR1, MATER and IGF2R gene expression in oocytes were also similar (P > 0.05) in N-OV and OV groups. In conclusion, our results demonstrate a lower LH secretion profile in peripubertal Gir heifers prone to ovulate after induction protocol, and that oocyte quality is not affected on a short-term basis by ovulation itself.

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.

The objective of the current study was to investigate the influence of synergism of the dry powder of Alpinia galanga rhizomes (AGR) and/or zinc sulfate in the diet on semen quality and reproductive traits of California rabbit bucks. The study was conducted in two stages. First stage: appreciation of semen characteristics, 36 California rabbit bucks (aged 5 months) with average body weights of 2980 g were divided randomly into six treatments (six individuals each). The treatment groups were: first group, control fed basal diet (C); second group, fed basal diet plus 1 g AGR/kg dry matter (DM) (AGR1); third group, fed basal diet plus 2 g AGR/kg DM (AGR2); fourth group, fed basal diet plus 200 mg Zn/litre drinking water (Zn); fifth group, fed basal diet plus 1 g AGR/kg DM and 200 mg Zn/litre drinking water (AGR1 + Zn); sixth group, fed basal diet plus 2 g AGR/kg DM and 200 mg Zn/litre drinking water (AGR2 + Zn). Second stage: the previous bucks were used to determine the efficiency of semen on reproductive fertility traits, 48 mature does (aged 6 months, nulliparous) with an average body weight of 3050 ± 20.7 g were divided randomly into six treatments and inseminated with previous groups of treated bucks. The results of the first stage, recorded high activity on gonadotropins hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH), free testosterone (FT), progesterone (P4) and oestrogen (E217β) concentrations for AGR1 + Zn and AGR2 + Zn compared with the control group. Groups AGR1, AGR2, AGR1 + Zn and AGR2 + Zn had significantly lowered concentrations of triglycerides, total cholesterol, low-density lipoprotein, and malondialdehyde (MDA), whereas high-density lipoprotein and total antioxidant capacity (TAC) were increased significantly compared with the control group. The group supplemented with AGR with or without Zn had significantly improved ejaculate volume, advanced motility, sperm concentration, and cell integrity. Fertility rate and litter size were improved in all groups compared with the control. It was concluded that supplementing diets with Alpinia galanga and Zn significantly increased sperm percentage, motility and reproductive hormones (testosterone, FSH, LH, E217β, P4). This suggested that this plant when used may be favourable for improved sperm quality and fertility parameters.

The role of hyaluronic acid (HA) as a 'physiologic selector' is also well recognized in vitro: it has been demonstrated that spermatozoa that bind to immobilized HA in vitro are those having completed their plasma membrane remodelling, and cytoplasmic and meiotic maturation. Sperm selection using HA has been expected to increase the implantation rate in intracytoplasmic sperm injection (ICSI) cycles. This work was designed to evaluate an alternative product for slowing sperm motility that contains HA and measures its outcomes: fertilization rate, embryo quality, and implantation and pregnancy rates. The present study found a positive drift in embryo quality that was statistically significant in the study group (SpermSlow™-ICSI) with teratozoospermia compared with PVP-ICSI in the same group. There were differences in the pregnancy rate (statistically insignificant in normozoospermia, asthenozoospermia, oligozoospermia, and teratozoospermia) in the SpermSlow-ICSI group compared with PVP-ICSI. The HA-ICSI technique in assisted reproduction technology (ART) is an important way to improve fertilization rate, embryo quality, and pregnancy rate.

This study aimed to evaluate the ploidy and survival of larvae resulting from crosses between tetraploid females and diploid males of yellowtail tetra Astyanax altiparanae, both females (three diploids and three tetraploids) and males (n = 3 diploids). Breeders were subjected to hormonal induction with pituitary gland extract from common carp fish (Cyprinus carpio). Females received two doses at concentrations of 0.3 and 3.0 mg/kg -1 body weight and at intervals of 6 h. Males were induced with a single dose of 3.0 mg/kg -1 applied simultaneously with the second dose in females. Oocytes from each diploid and tetraploid female were fertilized with semen from the same male, resulting in two crosses: cross 1 (diploid male and diploid female) and cross 2 (diploid male and tetraploid female). The procedures were performed with separate females (diploid and tetraploid) and diploid males for each repetition (n = 3). For ploidy determination, 60 larvae from each treatment were analyzed using flow cytometry and cytogenetic analyses. As expected, flow cytometry analysis showed that progenies from crosses 1 and 2 presented diploid and triploid individuals, respectively, with a 100% success rate. The same results were confirmed in the cytogenetic analysis, in which the larvae resulting from cross 1 had 50 metaphase chromosomes and those from cross 2 had 75 chromosomes. The oocytes have a slightly ovoid shape at the time of extrusion. Diploid oocytes had a size of 559 ± 20.62 μm and tetraploid of 1025.33 ± 30.91 μm. Statistical differences were observed between eggs from crosses 1 and 2 (P = 0.0130). No significant differences between treatments were observed for survival at the 2-cell stage (P = 0.6174), blastula (P = 0.9717), gastrula (P = 0.5301), somite (P = 0.3811), and hatching (P = 0.0984) stages. In conclusion, our results showed that tetraploid females of the yellowtail tetra A. altiparanae are fertile, present viable gametes after stripping and fertilization using the 'dry method', and may be used for mass production of triploids. This is the first report of these procedures within neotropical characins, and which can be applied in other related species of economic importance.

The aim of this study was to assess the consequences of treatment with pentoxifylline (PTX), an inducer of sperm motility, on sperm DNA fragmentation (SDF) and clinical characteristics in non-obstructive azoospermia (NOA) patients. The pilot study included 15 NOA patients. Half of each sperm sample before and after rapid freezing, was treated with PTX (3.6 mM /l, 30 min) as the PTX group and the remaining samples were considered as the control. SDF and sperm motility were assessed in each group. The clinical study comprised 30 fresh testicular sperm extractions (TESE) and 22 post-thawed TESE intracytoplasmic sperm injection cycles. Half of the mature oocytes from each patient were injected with PTX-treated spermatozoa and the remaining oocytes were injected with non-treated spermatozoa. Fertilization was assessed at 16 h post injection. Embryo transfer was carried out on day 2 after fertilization. Chemical pregnancy was assessed 2 weeks after transfer. PTX was found to significantly increase (P < 0.05) sperm motility. There was an insignificant difference in SDF rates between the groups (P > 0.05). In patient ovaries given fresh TESE, there was not any significant difference in clinical characteristics (P > 0.05). In patient ovaries given post-thawed TESE, there was a significant difference in the number of 2PN and in embryo formation (P < 0.05). Differences in the results of chemical pregnancy were insignificant (P > 0.05) between the groups. In addition, there was not any correlation between DNA fragmentation index and sperm motility and laboratory outcomes. Therefore, obtaining viable spermatozoa using PTX was more effective in post-thawed TESE regime patients in terms of 2PN and in embryo formation, deprived of damaging effects on sperm DNA integrity.

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.