This study was carried out to compare the efficacy of different methods to activate buffalo A + B and C + D quality oocytes parthenogenetically and to study the in vitro developmental competence of oocytes and expression of some important genes at the different developmental stages of parthenotes. The percentage of A + B oocytes (62.16 ± 5.06%, range 53.8–71.3%) was significantly higher (P < 0.001) compared with that of C + D oocytes (37.8 ± 5.00%, range 28.6–46.1%) retrieved from slaughterhouse buffalo ovaries. Among all combinations, ethanol activation followed by culture in research vitro cleave medium gave the highest cleavage and blastocyst yields for both A + B and C + D grade oocytes. Total cell numbers, inner cell mass/trophectoderm ratio and apoptotic index of A + B group blastocysts were significantly different (P < 0.05) from their C + D counterpart. To determine the status of expression patterns of developmentally regulated genes, the expression of cumulus–oocyte complexes, fertilization, developmental competence and apoptotic-related genes were also studied in parthenogenetically produced buffalo embryos at different stages, and indicated that the differential expression patterns of the above genes had a role in early embryonic development.

To study the role of orexin A in the reproductive regulation of Mongolian sheep, ovine ovarian granulosa cells were cultured in vitro. The cells were divided into groups after luteinization, the experimental group was given orexin A and the transcriptome was sequenced together with that of the control group. The different genes related to reproduction were screened out. qRT-PCR, western blot and enzyme-linked immunosorbent assay (ELISA) were used to verify the selected genes and detect the effect on progesterone secretion. In total, 123 differentially expressed genes were obtained by sequencing. Six genes with high expression related to reproduction (PRRT2, ABCG1, SOX4, TBX3, ID1 and ATP8) were screened. The results of qRT-PCR were consistent with those of sequencing; western blot and ELISA were used to verify the protein levels of steroidogenic acute regulatory protein (StAR) and its related PRRT2 and ABCG1, and to detect their effect on progesterone secretion. Validation results were consistent with those of qRT-PCR and sequencing. The experimental group was given orexin A and compared with the control group. Expression of PRRT2 protein was significantly increased (P < 0.05), ABCG1 protein expression was significantly decreased (P < 0.05), StAR expression was significantly increased (P < 0.05), and progesterone secretion was significantly increased (P < 0.05). The results showed that orexin A promoted the expression of StAR by upregulating PRRT2 and downregulating ABCG1, therefore affecting secretion of progesterone. Gene expression characteristics of orexin A affecting progesterone secretion were preliminarily explored; this study provides a theoretical basis for further study on signalling pathways and reproductive regulation in Mongolian sheep.

Although bovine embryo in vitro production (IVP) is a common assisted reproductive technology, critical points warrant further study, including sperm traits and oxidative status of sperm for in vitro fertilization (IVF). Our aim was to evaluate whether the lipid peroxidation index of commercial bull semen is influenced by sperm traits and oxidative status of sperm populations selected using Percoll® gradient. Semen straws from 48 batches from 14 Nelore bulls were thawed individually, analyzed for motility and subjected to Percoll selection. After Percoll, the lipid peroxidation index of the extender was evaluated, whereas selected sperm were analyzed for motility, acrosome and membrane integrity, mitochondrial membrane potential, chromatin resistance and oxidative potential under IVF conditions. Batches were divided retrospectively in four groups according to lipid peroxidation index. Sperm from Group 4 with the lowest index of lipid peroxidation had, after Percoll selection, greater plasma membrane integrity (81.3%; P = 0.004), higher mitochondrial potential (81.1%; P = 0.009) and lower oxidative potential (135.3 ng thiobarbituric acid reactive substances (TBARS)/ml; P = 0.026) compared with Group 1 with highest lipid peroxidation index (74.3%, 73% and 213.1 ng TBARS/ml, respectively). Furthermore, we observed negative correlations for the lipid peroxidation index with motility, membrane integrity and mitochondrial potential, and positive correlations with oxidative potential. In conclusion, oxidative stress in semen straws, as determined using lipid peroxidation in the extender, is associated with sperm traits and their oxidative potential under IVF conditions. These results provided further insights regarding the importance of preventing oxidative stress during semen handling and cryopreservation, as this could affect sperm selected for IVF. Finally, Percoll selection did not completely remove sperm with oxidative markers.

The aim of the present study was to investigate several common conditions that may potentially be correlated with follicular oxidative status during an intracytoplasmic sperm injection (ICSI) cycle and that include the serum oestrogen level on the day of oocyte pick-up, maternal age and pregnancy outcome. Patients that were enrolled in the study were classified randomly into three groups using their numerical order. The first group were classified based on maternal age (<35 and ≥35 years) (n = 398), the second group on the serum oestradiol (E2) level on the day of human chorionic gonadotropin (hCG) administration (levels >90th percentile and ≤ 90th percentile) (n = 491) and the third group on pregnancy outcome (positive/negative) (n = 376). The groups were matched for the other variables (stimulation protocol, dose of gonadotropin, duration of stimulation, antral follicle count, body mass index, basal follicle stimulating hormone (FSH), and E2 levels and day of hCG trigger) to prevent the possible contribution of those parameters to the results. Each group was matched for other variables (stimulation protocol, dose of gonadotrophin, duration of stimulation, antral follicle count, body mass index, basal FSH and E2 levels and day of hCG trigger) that may have affected the outcome, except for the parameter under investigation. Maternal age (P = 0.044,168 r = 0.418), oestrogen level on day of hCG administration (P = 0.001, r = 0.436) and pregnancy outcome (AUC = 0.65, P = 0.071) were found to be correlated with follicular oxidative status. The results obtained will help us to shield patients from possible situations that may cause oxidative stress and therefore adverse outcomes of an ICSI cycle.

The objective of this study was to investigate any effect of cryopreservation of donated eggs on laboratorial and clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles. This retrospective cohort study included 320 oocyte recipients undergoing 307 vitrified and 119 fresh oocyte recipient ICSI cycles, participating in an egg-sharing donation programme, from 2015 to 2018, in a private university-affiliated in vitro fertilization (IVF) centre. A review of donor and recipient ICSI cycles was charted. A general mixed models fit by restricted maximum likelihood, followed by Bonferroni post hoc test was used to compare the means between fresh and warm oocyte donation groups and investigate the effect of cryopreservation on recipient ICSI outcome. The main outcome measure was blastocyst development rates. Fertilization rate, high-quality embryo rates on days 2 and 3, normal cleavage speed rates on days 2 and 3, and blastocyst development rate were significantly higher for the fresh oocyte donation cycles compared with warmed oocyte donation cycles. In the egg-sharing donation programme, fertilization and embryo developmental competence were reduced when vitrified oocytes from infertile couples were used for ICSI compared with fresh oocytes.

The objective of this study was to investigate the effect of oocyte and sperm cryopreservation on donated eggs submitted to intracytoplasmic sperm injection (ICSI) cycles. Medical charts of 122 oocyte recipients undergoing 152 oocyte recipient ICSI cycles, from 2017 to 2018, in a private university-affiliated in vitro fertilization (IVF) centre, were reviewed in this historical cohort study. Cycles were divided into four groups according to the gamete status: the FO/FS Group, recipients in which fresh oocytes were injected with fresh sperm (n = 19); the FO/CrS Group, recipients in which fresh oocytes were injected with cryopreserved sperm (n = 14); the CrO/FS Group, recipients in which cryopreserved oocytes were injected with fresh sperm (n = 85); and the CrO/CrS Group, recipients in which cryopreserved oocytes were injected with cryopreserved sperm (n = 34). Generalized mixed models fit by restricted maximum likelihood, followed by Bonferroni post hoc test for the comparison of means amongst groups were used to investigate the effect of cryopreservation on recipient ICSI outcomes. The results were expressed as mean differences with 95% confidence intervals and P-values. The main outcome measure was the implantation rate. Normal day 3 cleavage speed, blastocyst development and implantation rates were significantly lower in the CrO/CrS Group compared with the FO/FS Group. In conclusion, embryo developmental competence and implantation potential were reduced when vitrified oocytes were injected with frozen sperm in an egg-sharing donation programme.

The present study investigated the effects of c-type natriuretic peptide (CNP) on the development of murine preantral follicles during in vitro growth (IVG). Preantral follicles isolated from ovaries of Kunming mice were cultured in vitro. In the culture system, CNP was supplemented in the experimental groups and omitted in the control groups. In Experiment 1, CNP was only supplemented at the early stage and follicle development was evaluated. In Experiments 2 and 3, CNP was supplemented during the whole period of in vitro culture. In Experiment 2, follicle development and oocyte maturity were evaluated. In Experiment 3, follicle development and embryo cleavage after in vitro fertilization (IVF) were assessed. The results showed that in the control groups in all three experiments, granulosa cells migrated from within the follicle and the follicles could not reach the antral stage. In the experimental groups in all three experiments, no migration of granulosa cells was observed and follicle development was assessed as attaining the antral stage, which was significantly superior to that of the control group (P < 0.0001). Oocyte meiotic arrest was effectively maintained, hence giving good developmental competence. In conclusion, CNP supplementation in the culture system during IVG benefited the development of murine preantral follicles.

This study was conducted to examine whether glucose in maturation medium containing reduced NaCl could improve oocyte maturation and embryonic development in pigs. The base medium was bovine serum albumin-free porcine zygote medium (PZM)-3 containing 10% (v/v) pig follicular fluid (FPZM) or 0.1% (w/v) polyvinyl alcohol (PPZM). Using each medium, the effects of NaCl concentrations (108 and 61.6 mM) and 5.56 mM glucose supplementation (designated as PZM108N, PZM108G, PZM61N, and PZM61G, respectively) were examined using a 2 × 2 factorial arrangement. When oocytes were matured in FPZM, glucose supplementation improved nuclear maturation compared with no supplementation, regardless of the NaCl concentrations. FPZM61G showed a higher blastocyst formation compared with FPZM108N and FPZM108G after parthenogenesis (PA). Blastocyst formations of somatic cell nuclear transfer (SCNT) embryos derived from FPZM61N and FPZM61G were higher compared with those of oocytes from FPZM108N. When oocytes were matured in PPZM, glucose added to PPZM108 and PPZM61 increased nuclear maturation compared with no supplementation. However, glucose added to PPZM108 did not alter embryonic development after PA. Additionally, oocytes matured in PPZM61G showed a higher blastocyst formation compared with those from PPZM61N. In SCNT, blastocyst formation was not influenced by glucose supplementation of PPZM108, but was increased by maturation in glucose-supplemented PPZM61. In embryonic development of in vitro fertilization (IVF), oocytes matured in medium with reduced NaCl and glucose showed significantly higher blastocyst formation compared with those matured in PPZM108G. Our results demonstrated that glucose in maturation medium containing 61.6 mM NaCl increased oocyte maturation and embryonic development after PA, SCNT, and IVF.

Mammalian sperm have to undergo capacitation to be fertilization competent. Capacitated sperm in vitro show hyperpolarization of the membrane potential. It has been reported that in mouse membrane hyperpolarization is necessary for the acrosome reaction. We recently found that the fluid of the hamster oviduct, where fertilization occurs, contained a high potassium (K+) concentration (~20 mEq/l). This high K+ concentration could depolarize the membrane potential and prevent the acrosome reaction/fertilization. Conversely, some beneficial effects on capacitation of high K+ concentration or a high K/Na ratio were also reported. In the present study, we investigated the effects of oviduct high K+ concentration on hamster sperm capacitation-associated events and fertilization. The present study confirmed that, in hamster sperm, membrane potential was hyperpolarized upon in vitro capacitation, indicating that capacitation-associated hyperpolarization is a universal phenomenon among mammalian species. An increase in KCl concentration in the medium to 20 mM significantly depolarized the membrane potential and suppressed hyperpolarization when in the presence of >101 mM NaCl. However, an increase in the KCl concentration to 20 mM did not significantly affect the percentage of motile sperm, hyperactivation or the acrosome reaction. No effect of 20 mM KCl on in vitro fertilization was observed. In addition, no correlative changes in hyperactivation and the acrosome reaction with K/Na ratio were observed. These results suggested that in hamsters the oviduct K+ concentration suppressed hyperpolarization but had no effect on capacitation and in vitro fertilization. Our results raised a question over the physiological significance of capacitation-associated hyperpolarization.

This study aimed to investigate the effects of eugenol on growth, viability, antrum formation and mRNA expression of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1) and peroxiredoxin 6 (PRDX6) in bovine secondary follicles cultured in vitro. To this end, bovine ovaries were collected from a local slaughterhouse and in the laboratory the follicles were isolated from the ovarian cortex. The follicles were then cultured in TCM-199+ alone or supplemented with different concentrations of eugenol (0.5, 5.0 and 50.0 μM). Follicular diameters and antrum formation were evaluated on days 0, 6, 12 and 18. Viability analysis was performed using calcein and ethidium homodimer. Real-time PCR was used to quantify mRNA levels for SOD, CAT, GPX1 and PRDX6 in cultured follicles. Follicular diameters and mRNA levels in follicles cultured in vitro were compared using analysis of variance and Kruskal–Wallis tests, while follicular survival and antrum formation were compared using the chi-squared test (P < 0.05). The results showed that secondary follicles cultured with eugenol maintained similar morphology and viability to follicles cultured in the control group. A progressive increase in follicular diameter was observed between days 0 and 12 for all treatments, except for follicles cultured with 50 µM eugenol. Eugenol (5.0 and 50.0 μM) increased mRNA levels for GPX1 in cultured follicles, but 0.5 μM eugenol reduced mRNA levels for SOD. The addition of eugenol did not influence mRNA expression for CAT and PRDX6. In conclusion, eugenol supplementation reduces mRNA levels for SOD and increases mRNA levels of GPX1 in bovine secondary follicles cultured in vitro.

The process of cytodifferentiation in spermatogenesis is governed by a unique genetic and molecular programme. In this context, accurate ‘tuning’ of the regulatory mechanisms involved in germ cells differentiation is required, as any error could have dramatic consequences on species survival and maintenance. To study the processes that govern the spatial–temporal expression of genes, as well as analyse transmission of epigenetic information to descendants, an integrated approach of genetics, biochemistry and cytology data is necessary. As information in the literature on interplay between DNA methylation and histone H3 lysine 4 trimethylation (H3K4me3) in the advanced stages of murine spermatogenesis is still scarce, we investigated the effect of a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, at the cytological level using immunocytochemistry methodology. Our results revealed a particular distribution of H3K4me3 during sperm cell differentiation and highlighted an important role for regulation of DNA methylation in controlling histone methylation and chromatin remodelling during spermatogenesis.

This research aimed to examine the effects of thyme, celery and salinomycin on ovarian sex hormones, reproductive traits and antioxidant status during the estrous cycle. Seventy-five mature Barki ewes aged 2–3 years with an average weight of 40 ± 1.5 kg were assigned randomly into five groups (15 head/group). Group 1 was kept as the control; groups 2 and 3 received 20 g/head/day thyme (T) and celery (C) as dried herbs, respectively. Group 4 (T×C) received 10 g thyme + 10 g celery/head/day, and group 5 was treated with salinomycin 1 g/head/day. Blood samples were collected during follicular and luteal phases of the estrous cycle. Thyme and celery and the mixture of T×C increased (P < 0.01) estradiol-17β (E2) during the follicular phase of the estrous cycle, while only the celery group showed a marked (P < 0.001) increase in progesterone (P4) during the luteal phase compared with the control. Salinomycin supplementation decreased (P < 0.05) E2 concentrations during the follicular and luteal phases of the estrous cycle. Supplementation with thyme and celery enhanced (P < 0.001) antioxidant capacity in the luteal phase compared with the follicular stage. The salinomycin group showed increased (P < 0.01) levels of reduced glutathione (GSH) and decreased malondialdehyde (MDA) levels compared with the control group throughout luteal phase. For the interaction between estrous phases and treatments, thyme, celery, and T×C supplementation revealed an increase (P < 0.05) in superoxide dismutase (SOD), GSH, and glutathione disulfide (GSSG) levels compared with the control group during the follicular and luteal phases. Thyme and celery supplementation improved the number of services per conception and fertilization from 1st and 2nd inseminations, respectively. In conclusion, the applied treatment had significant effects on reproductive performance and antioxidant status in ewes throughout the estrous cycle.

Oocyte cryopreservation has become an important component of assisted reproductive technology with increasing implication in female fertility preservation and animal reproduction. However, the possible adverse effects of oocyte cryopreservation on epigenetic status of the resulting embryos is still an open question. This study evaluated the effects of MII-oocyte vitrification on gene transcripts linked to epigenetic reprogramming in association with the developmental competence and epigenetic status of the resulting embryos at 2-cell and blastocyst stages in dromedary camel. The cleavage rate of vitrified oocytes following intracytoplasmic sperm injection was significantly increased compared with the control (98.2 ± 2 vs. 72.7 ± 4.1%, respectively), possibly due to the higher susceptibility of vitrified oocytes to spontaneous activation. Nonetheless, the competence of cleaved embryos derived from vitrified oocytes for development to the blastocyst and hatched blastocyst was significantly reduced compared with the control (7.7 ± 1.2 and 11.1 ± 11.1 compared with 28.1 ± 2.6 and 52.4 ± 9.9%, respectively). The relative transcript abundances of epigenetic reprogramming genes DNMT1, DNMT3B, HDAC1, and SUV39H1 were all significantly reduced in vitrified oocytes relative to the control. Evaluation of the epigenetic marks showed significant reductions in the levels of DNA methylation (6.1 ± 0.3 vs. 9.9 ± 0.5, respectively) and H3K9 acetylation (7.8 ± 0.2 vs. 10.7 ± 0.3, respectively) in 2-cell embryos in the vitrification group relative to the control. Development to the blastocyst stage partially adjusted the effects that oocyte vitrification had on the epigenetic status of embryos (DNA methylation: 4.9 ± 0.4 vs. 6.2 ± 0.6; H3K9 acetylation: 5.8 ± 0.3 vs. 8 ± 0.9, respectively). To conclude, oocyte vitrification may interfere with the critical stages of epigenetic reprogramming during preimplantation embryo development.

Studies on the larval development of fish are essential for conservation and improvements in cultivation techniques. Geophagus brasiliensis popularly known as Cará has potential as a fish of interest in ornamental aquaculture. Wild adults of G. brasiliensis were kept in an aquarium for spontaneous reproduction. Newly hatched larvae were transferred to 5-litre aquaria at 22, 26 and 30°C until total yolk sac resorption. Histological slides were used for biometric analysis and monitoring of larval ontogenesis at different temperatures. Histologically, from the first to the fourth days it was possible to identify myomeres, optic vesicle, yolk syncytial layer, brain, heart and differentiation of the eye layers. From the fourth to the seventh days, it was possible to identify mandibular and gill cartilages, swim bladder, liver, prismatic epithelium with striated border in intestine and renal epithelium. All biometric measurements increased over the days, except height and length of the yolk sac, which gradually decreased until the complete resorption of the yolk sac that occurred on the fifth day at a temperature of 30°C, the sixth day at 26°C and the seventh day at 22°C. Morphological events at 30°C such as the reabsorption of the yolk sac, the appearance of cartilage in the branchial arches and differentiation of the layers of the eyes occurred faster compared with the other temperatures tested. Opening of the mouth and digestive tract occurred at a similar time on the fourth day in all temperatures.

This study examined the effects of zinc chloride (ZnCl2) and sodium selenite (Na2SeO3) supplementation in maturation medium on in vitro maturation (IVM) rate, oxidative biomarkers and gene expression in buffalo oocytes. Ovaries from a slaughterhouse were aspirated and good quality cumulus–oocyte complexes (COCs) with at least four layers of compact cumulus cells and evenly granulated dark ooplasm were selected. COCs were randomly allocated during IVM (22 h) to one of four treatment groups: (1) control maturation medium (basic medium), or basic medium supplemented with (2) ZnCl2 (1.5 µg/ml), (3) Na2SeO3 (5 µg/l), or (4) ZnCl2 + Na2SeO3 (1.5 µg/ml + 5 µg/l, respectively). Oocytes were denuded after 22 h of IVM in the first four replicates. Specimens were fixed and stained to evaluate the stage of nuclear maturation. The spent medium was collected for biochemical assays of total antioxidant capacity (TAC), malondialdehyde (MDA) and hydrogen peroxide concentrations. A second four replicates were used for COCs for RNA extraction. The expression levels of antioxidant (SOD1, GPX4, CAT and PRDX1), antiapoptotic (BCL2 and BCL-XL) and proapoptotic (BAX and BID) genes were measured. Supplementation with ZnCl2 and Na2SeO3 during IVM increased the ratio of oocytes reaching metaphase II at 22 h, increased TAC and decreased MDA and H2O2 concentrations in the maturation medium (P < 0.05). Moreover, beneficial effects were associated with complementary changes in expression patterns of antioxidative, antiapoptotic and proapoptotic genes, suggesting lower oxidative stress and apoptosis. Supplementation medium with zinc chloride and sodium selenite improves the maturation rate, reduces oxidative stress and increases expression levels of antioxidative and antiapoptotic genes.

Mutation in CFAP43 leads to severe asthenozoospermia and multiple morphological abnormalities of the sperm flagellum (MMAF) in both human and mouse. Previous studies have shown that disruption of intra-manchette transport (IMT) caused failure of flagellum assembly and sperm head shaping. In a previous study, therefore, we postulated that disruption of IMT may contribute to the failure of sperm flagellum formation and result in MMAF, however the mechanisms underlying these defects are still poorly understood. Cfap43-deficient mice were studied here to reveal the cellular mechanisms of abnormal sperm head morphology and MMAF. Depletion of Cfap43 led to abnormal spermiogenesis and caused MMAF, sperm head abnormality and oligozoospermia. Furthermore, both abnormal manchette and disorganized ectoplasmic specialization (ES) could be observed at the elongated spermatids in Cfap43-deficient mice. Therefore, our findings demonstrated that, in mice, CFAP43-mediated IMT is essential for sperm head shaping and sperm flagellum formation.

Maternal age is a significant factor influencing in vitro fertilization (IVF) outcomes. Oxidative stress (OS) is one of the major causes of age-related cellular and molecular damage. The purpose of this work was to investigate the correlation between maternal age with intrafollicular antioxidants and OS markers in follicular fluid (FF), and also to determine the OS status in patients of advanced age. This study was a prospective study including 201 women undergoing IVF whose age was between 24 and 45 years old. FF samples were obtained from mature follicles at the time of oocyte retrieval. After treatment of FF, lipid peroxidation levels (MDA) and enzyme activities such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione (GSH) level were evaluated using spectrophotometry. The results indicated that the age cutoff point for increasing the MDA level was fixed at 37 years, allowing the study to be differentiated into two age groups. Group I included patients whose age was less than 37 years, and group II included patients whose age was greater than or equal 37 years. Statistical analysis revealed that MDA and GSH levels and GR activity were significantly higher in group II compared with group I. The SOD and CAT activities were significantly less in group II compared with group I. We concluded that from 37 years old a reproductive ageing was accompanied by a change in the antioxidant pattern in FF that impaired reactive oxygen species scavenging efficiency.

In this study, we tried to maintain the vitality of Chinese sturgeon (Acipenser sinensis) ova before fertilization with several treatments in vitro. The ovulated eggs were allocated to groups with different incubation medium (coelomic fluid and artificial media), temperature (4°C and 16°C) and storage duration (2 h and 6 h). The maximum fertilization and hatching rate were observed for the control group in which the ova were fertilized immediately after spawning, with the values of 82.45% and 84.73%, respectively. Compared with the control group, the fertilization and hatching rate of all the treatment groups stored at 4°C or in coelomic fluid decreased significantly (P < 0.05). The fertilization rate of the treatment group stored in artificial medium at 16°C did not change obviously in the first 2 h (P > 0.05), but declined dramatically (P < 0.05) after 6 h. In comparison with the control group, no significant (P > 0.05) reduction was shown in hatching rate of the treatment group stored in artificial medium at 16°C for 6 h. The results showed that the ova of Chinese sturgeon can be stored for at least 6 h at 16°C in artificial medium without weakening; this provides a practical application method for the routine hatchery practice of Chinese sturgeon, as well as certain relevant research.

The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus–oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

Dicliptera verticillata is a medicinal plant traditionally used in western Cameroon to cure female infertility. This experiment was designed to assess the effects of the aqueous extract of Dicliptera verticillata (AEDv) on fertility and gestation in female rats. Oral increasing doses of AEDv were administered to immature female rats over 20 d. After this time, some animals were mated with fertile males and some fertility parameters were assayed; the other animals were euthanized for preliminary toxicity parameters analysis. The effects of AEDv on the different stages of gestation were assayed on selected animals previously controlled for estrous cycle regularity and mated. AEDv led to an increase in serum, uterine and ovarian proteins as well as in ovarian and uterine weights (P < 0.05) in immature female rats. Hepatic proteins significantly decreased (P < 0.01) in high dose-treated animals (50 and 100 mg/kg) compared with controls. The number of implantation sites and the fertility rate were significantly lower (P < 0.05), while the antifertility activity increased significantly (P < 0.05) in treated rats compared with controls. When administered from the 1st to the 5th day of pregnancy, AEDv led to a decrease of more than 60% in the implantation rate in high dose-treated rats (50, 100, and 400 mg/kg). From the 6th to the 9th day, the implantation, gestation rates and the number of fetuses decreased significantly in all treated groups. From the 11th to the 20th day, a 50% resorption and decrease in gestation rate were reported in 50 mg/kg dose-treated animals. AEDv possesses weak contraceptive and abortifacient effects during pregnancy.