In this paper, with the aim to find new genes involved in mammalian spermatogenesis, we isolated, for the first time in the rat testis, a partial cDNA clone that encoded EH domain binding protein 1-like 1 (Ehbp1l1), a protein that has a single calponin homology domain (CH). Bioinformatic analysis showed that EHBP1l1 contains three domains: the N-terminal C2-like, the CH and the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domains, which are evolutionarily conserved in vertebrates. We found that Ehbp1l1 mRNA was expressed in several rat tissues, including the liver, intestine, kidney and also in the testis during its development, with a higher level in testis from 12-month-old animals. Interestingly, in situ hybridization experiments revealed that Ehbp1l1 is specifically expressed by types I and II spermatocytes, this result was validated by RT-PCR performed on total RNA obtained from enriched fractions of different testicular cell types. As EHBP1l1 has been described as linked to vesicular transport to the actin cytoskeleton and as an effector of the small GTPase Rab8, we hypothesized that it could participate both in cytoskeletal remodelling and in the regulation of vesicle sorting from the trans-Golgi network to the apical plasma membrane. Our findings provide a better understand of the molecular mechanisms of the differentiation process of spermatogenesis; Ehbp1l1 may also be used as a new marker of testicular activity.

Experiments were conducted to study in vitro maturation of prepubertal goat oocytes and their developmental potential after chemical activation. In Experiment 1, cumulus–oocytes complexes collected from the ovaries of prepubertal goats slaughtered at a local abattoir were matured in vitro in TCM-199-based medium supplemented with 10 µg/ml luteinizing hormone (LH) (treatment 1) or 10 µg/ml LH + 0.1 mM l-cysteine (treatment 2). In Experiment 2, mature oocytes were activated with either 5 µM ionomycin or 7% ethanol. After 18 h, some oocytes were randomly fixed and stained to evaluate their chromatin status, while others were cultured in embryo culture medium to study their further development. In Experiment 3, oocytes activated with 5 µM ionomycin were cultured for 7 days in one of the four different culture media [Charles Rosenkrans medium (CR-1), TCM-199, potassium simplex optimization medium (KSOM) and synthetic oviductal fluid (SOF)] to study their developmental potential. The maturation rate in control, treatment 1, and treatment 2 media did not differ from each other (P > 0.05). However, the lowest degeneration of oocytes was observed in treatment 3 (P < 0.05) when compared with the other two groups. The proportion of activated oocytes was higher, while non-activated oocytes were lower in ionomycin group when compared with the group activated with ethanol (P < 0.05). The proportions of oocytes cleaved were 65.7, 56.8, 61.0 and 54.4% in CR-1, TCM-199, KSOM and SOF medium, respectively, with no significant difference. However, further development of cleaved oocytes was better in KSOM followed by SOF.

The seminal characteristics of Moenkhausia oligolepis are described. Three males were induced with a single dose of carp pituitary. Semen was collected 6 h after induction, and diluted in dibasic sodium phosphate extender solution. For motility analysis, 1 µl of diluted semen was added to 10 µl of distilled water to achieve gamete activation. The average duration of total motility was 76.67 s; while the average sperm motility rate at intervals of 15 s was 95.3, 85.3, 59.6, 31.7, 13.0, 4.6 and 1.2%. To determine sperm concentration in samples, 0.5 μl of semen was diluted with 500 μl of glutaraldehyde. An aliquot of 10 μl of this dilution was utilized for cell counting. An average count of 4.97 × 109 ± 3.46 sperm/ml was obtained. Morphological analyses were performed using eosin–nigrosine dye; 20.33% of the sperm were observed to be dead. Live sperm, comprising the other 79.67%, had an average length of approximately 30 µm, with a head diameter of 4.488 ± 0.7 µm; and a flagella plus mid-piece length of 26.071 ± 12.4 µm. Of those sperm, 69% had a normal morphology, while 31% had primary and secondary abnormalities. The observed abnormality rate did not have a detrimental effect on artificial fertilization potential for the species. The description of the seminal characteristics of a species is one of the most important sets of information required for artificial reproduction of fish in captivity. It also contributes significantly to the total biological knowledge of the studied species.

Oxygen concentration influences oocyte quality and subsequent embryo development, but it remains unclear whether oxygen concentrations affect the developmental competence and transcriptomic profile of yak oocytes. In this study, we investigated the effects of different oxygen concentrations (5% versus 20%) on the developmental competence, reactive oxygen species (ROS) levels, glutathione (GSH) content, and transcriptomic profile of yak oocytes. The results showed that a low oxygen concentration significantly increased the maturation rate of yak oocytes (81.2 ± 2.2% vs 75.9 ± 1.3%) and the blastocyst quality of yak in vitro fertilized embryos. Analysis of ROS and GSH showed that a low oxygen concentration reduced ROS levels and increased the content of GSH (75.05 ± 7.1 ng/oocyte vs 50.63 ± 5.6 ng/oocyte). Furthermore, transcriptomic analysis identified 120 differentially expressed genes (DEGs) between the two groups of oocytes. Gene enrichment analysis of the DEGs indicated multiple cellular processes, including oxidative phosphorylation, transcription regulation, mitochondrial regulation, oestrogen signalling pathway, HIF-1 signalling pathway, TNF signalling pathway, were involved in the response to oxygen concentration alterations. Taken together, these results indicated that a low oxygen concentration improved the developmental competence of yak oocytes.

The cause of hybrid sterility and inviability has not been analyzed in the fin-fish hybrid, although large numbers of hybridizations have been carried out. In this study, we produced allo-diploid hybrids by cross-fertilization between female goldfish (Carassius auratus) and male golden venus chub (Hemigrammocypris rasborella). Inviability of these hybrids was due to breakage of the enveloping layer during epiboly or due to malformation with serious cardiac oedema around the hatching stage. Spontaneous allo-triploid hybrids with two sets of the goldfish genome and one set of the golden venus chub genome developed normally and survived beyond the feeding stage. This improved survival was confirmed by generating heat-shock-induced allo-triploid hybrids that possessed an extra goldfish genome. When inviable allo-diploid hybrid cells were transplanted into goldfish host embryos at the blastula stage, these embryos hatched normally, incorporating the allo-diploid cells. These allo-diploid hybrid cells persisted, and were genetically detected in a 6-month-old fish. In contrast, primordial germ cells taken from allo-diploid hybrids and transplanted into goldfish hosts at the blastula stage had disappeared by 10 days post-fertilization, even under chimeric conditions. In allo-triploid hybrid embryos, germ cells proliferated in the gonad, but had disappeared by 10 weeks post-fertilization. These results showed that while hybrid germ cells are inviable even in chimeric conditions, hybrid somatic cells remain viable.

The present study evaluated the effect of the aqueous extract from leaves of E. speciosa on some physiological and biochemical parameters of reproduction and the onset of puberty in pregnant mare serum gonadotropin (PMSG)-primed immature female rats. High pressure liquid chromatography (HPLC) was used to quantify the phenolic compounds in the methanol/methylene chloride (1:1) extract, the ethanolic and ethyl acetate fractions and the aqueous residue of E. speciosa. E. speciosa (0, 8, 32 or 64 mg/kg) were administered for 15 days to 24 non-PMSG-primed and 24 primed rats with 0.01 IU of PMSG. At the end of the treatment period, animal were sacrificed and their body, ovarian, uterine weight, ovarian protein or cholesterol level, as well as data on puberty onset were recorded. Of the 16 polyphenolic compounds quantitatively revealed in the extracts and fractions of E. speciosa after HPLC analysis, quercetin, rutin, apigenin and eugenol were the most abundant. Non-primed rats showed a significant increase (P < 0.05) in the uterine relative weight at the dose of 8 mg/kg when compared with the other treatments. The uterine proteins and the ovarian cholesterol (P < 0.05), respectively, showed a reduction at doses of 64 mg/kg and 32 mg/kg in non-primed rats. However in PMSG-primed rats, a significant decrease (P < 0.05) was observed in ovarian cholesterol at 64 mg/kg. In conclusion, E. speciosa potentializes the PMSG-inducing effect on folliculogenesis in PMSG-primed rats.

The present study evaluated the effect of binder of sperm protein 1 (BSP1) and/or heparin on in vitro bovine capacitation and fertilization rates using epididymal and ejaculated bovine sperm. Frozen–thawed sperm were selected and used in the following treatments. Control group: Fert-TALP medium without heparin; heparin (HEP) group: Fert-TALP with heparin (10 UI/ml); BSP1 group: Fert-TALP medium with BSP1 (10 µg/ml for ejaculated sperm; 40 µg/ml for epididymal sperm); HEP + BSP1 group: Fert-TALP medium with heparin (5 UI/ml) and BSP1 (5 µg/ml for ejaculated sperm; 20 µg/ml for epididymal sperm) and determined in vitro capacitation rates in different interval times (0, 15, 30 and 60 min) using the chlortetracycline fluorescence (CTC) method. Also, we evaluated the development rates of oocytes fertilized with ejaculated or epididymal sperm into the same treatments. Capacitation was greater and faster when ejaculated sperm were treated for 60 min with heparin compared with other treatments. However, developmental rates were similar in all treatments. For epididymal sperm, the treatments with BSP1 presented higher capacitation and fertilization rates compared with heparin (P < 0.05). The effects of heparin + BSP1 on capacitation and developmental rates did not cause any increase in capacitation or blastocyst rates compared with other groups for ejaculated or epididymal sperm. In conclusion, this study confirmed that either BSP1 and heparin can be used as capacitator agents for bovine ejaculated sperm during IVF. However, BSP1 seems to be more efficient compared with heparin for epididymal sperm. Furthermore, BSP1 and heparin have no synergic effects on sperm capacitation.

This study aimed to evaluate the relationship between antral follicular count (AFC) and ovarian volume (OV), preantral follicular population and survival, meiotic progression and ultrastructure of cumulus–oocyte complexes (COCs) after in vitro maturation. In experiment 1, the relationship between AFC and preantral follicle population and survival was evaluated by classical histology. In experiment 2, the relationship among AFC, OV, ability of oocytes to resume meiosis and ultrastructure of in vitro matured bovine COCs was studied. A positive correlation (P < 0.05) between AFC and the numbers of healthy primordial, degenerate and total follicles was observed, as well as with healthy secondary follicles and total follicles. The numbers of grades I and II oocytes in ovaries of high AFC class were higher compared with those with intermediate or lower AFC. After in vitro maturation, COCs from ovaries of high AFC had a higher percentage of oocytes in metaphase II compared with those of intermediate and low AFC (P < 0.0001). Ovaries of intermediate AFC had a higher percentage of oocytes in metaphase II compared with ovaries with low AFC (P < 0.0001). The proportion of oocytes in metaphase I, telophase I and anaphase I in COCs from ovaries of intermediate AFC (26.04%) was higher (P < 0.05) compared with that seen in COCs of ovaries with high (8.55%) and low (14.15%) AFC. No differences in the ultrastructure of oocytes were seen. In conclusion, after in vitro maturation, cow ovaries with high AFC have higher numbers of oocytes that reach in metaphase II (MII), but they also have higher numbers of degenerated primordial and primary follicles.

This study aimed to evaluate the effects of dexamethasone on development, viability, antrum formation and ultrastructural integrity of bovine secondary follicles cultured in vitro for 18 days. Bovine ovaries were obtained from slaughterhouses and secondary follicles of ~150–200 µm diameter were isolated and cultured in the laboratory in TCM-199+ alone or supplemented with different concentrations of dexamethasone (1, 10, 100 and 1000 ng/ml). Follicle viability was evaluated after the culture period, using calcein-AM (viable) and ethidium homodimer (nonviable). Follicle diameters and antrum formation were evaluated at days 0, 6, 12 and 18. Before or after in vitro culture, follicles were fixed for histological and ultrastructural analysis. Follicle diameters were evaluated using analysis of variance and Kruskal–Wallis test, while chi-squared test was used to evaluate the percentage of viable follicles and antrum formation (P < 0.05). Follicles cultured for 6 days with all treatments increased their diameters significantly, but there was no significant difference between treatments at the end of the culture period. In vitro cultured follicles showed antral cavity formation at the end of the culture period, but no influence of dexamethasone was seen. Ultrastructural analysis showed that follicles cultured with dexamethasone (1, 10, 100 and 1000 ng/ml) had well preserved granulosa cells. However, oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone showed signs of degeneration. It can be concluded that follicles cultured in vitro in the presence of dexamethasone demonstrated continuous in vitro growth, but oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone had poor ultrastructure.

Clinical outcomes following frozen–thawed cleavage embryo transfer versus frozen–thawed blastocyst transfer in high responder patients undergoing in vitro fertilisation/intracytoplasmic sperm injection cycles are still debated. In a retrospective study, 106 high responder patients who were candidate for ‘freeze-all embryos’ were recruited. Frozen–thawed embryos were transferred at the cleavage stage (n = 53) or the blastocyst stage (n = 53). Clinical pregnancy was considered as the primary outcome and chemical pregnancy, ongoing pregnancy, implantation rate, and fertilization rate, as well as miscarriage rate, were measured as the secondary outcome. Clinical (47.2% vs. 24.5%), chemical (56.6% vs. 32.1%), and ongoing pregnancy rates (37.7% vs. 17%) as well as implantation rates (33.6% vs. 13.5%) were significantly higher in the blastocyst group compared with the cleavage group respectively (P < 0.05). Miscarriage rate was comparable between groups (P > 0.05). Transfer of frozen–thawed embryos at the blastocyst stage was preferable in the high responder patients to increase implantation, pregnancy and live birth rates compared with cleavage stage embryo transfer.

In this report, we present a case of a couple who obtained a live birth with a single oocyte fertilized by intracytoplasmic sperm injection. The oocyte was collected at 36 h post trigger and was found to be at metaphase II when sperm injection was performed. At 18 h post injection, the oocyte was found to be fertilized with two clear pronuclei. The embryo divided and generated a four-cell embryo on day 2, which was replaced to the uterine cavity. Pregnancy test gave a positive β-human chorionic gonadotropin result, the scan performed at 7 weeks, revealed the presence of one amniotic sac with fetal heartbeat. Healthy live birth was obtained after 39 weeks of gestation.