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.

The aim of the study was to investigate the continuous changing pattern of H4K12 acetylation, and the expression levels of histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs) in mouse oocytes during meiosis and after parthenogenetic activation (PA). The immunofluorescence results showed hyperacetylation of lysine-12 on histone H4 (H4K12) in the germinal vesicle (GV) oocytes that then decreased during germinal vesicle breakdown (GVBD), and disappeared in metaphase II (MII). However, it reappeared in the early 1-cell embryos derived after 4 h of PA. The expression levels of some selected HATs and HDACs also validated the changing pattern of H4K12 acetylation during meiosis and PA. In conclusion, H4K12 is deacetylated in GVBD and MII, and re-hyperacetylated after PA.

The present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

The oocyte is known from recent studies in the mouse, cow, sheep and human to be a central regulator of follicular cell function. However, in the pig, little information is known about the regulation of cumulus expansion by oocyte-secreted factors and oocyte quality. We investigated the possible effects of oocyte-secreted factors during in vitro maturation on cumulus expansion and on porcine oocytes as judged by subsequent embryonic development after parthenogenetic activation. Cumulus–oocyte complexes (COC) from antral follicles of pig ovaries collected from a local abattoir were divided into control and treatment groups and were cultured in tissue culture medium 199 supplemented with follicle-stimulating hormone. Treatment groups consisted of increasing numbers of denuded oocytes (DO) co-cultured with COC (at ratios of COC to DO of 1:1, 1:2, 1:3, 1:4 and 1:5). After incubation for 44 h, cumulus expansion and maturation rates were assessed and oocytes were activated parthenogenetically. Cumulus expansion in the 1 COC:4 DO and 1 COC:5 DO groups was low and altered because full dispersion of the outer layer did not occur. Cell viability was not affected, as measured by the automated cell counter, but scanning electron microscopy revealed only a scanty extracellular matrix. Blastocyst rate was significantly higher in the 1 COC:4 DO (34.4%) and in the 1 COC:5 DO (34.9%) groups (p < 0.05) when compared with other groups. Maturation rate, cleavage rate and total cell number showed no significant difference between control and treatment groups. Amplification by reverse transcription polymerase chain reaction (RT-PCR) showed up-regulation of growth differentiation factor 9 (GDF9) in the cumulus cells in the 1 COC:4 DO group at 44 h. We conclude that denuded porcine oocytes could improve the maturation of COC as evidenced by increased blastocyst development in the 1 COC:4 DO, even though cumulus expansion was poor. This improvement could be a result of the GDF9 up-regulation.

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 μg/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX– blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

Oocyte activation is an essential step in animal cloning to allow subsequent development of the reconstructed embryos. A special activation protocol is required for different animal species. The present study investigated low temperature, electrical pulses, ethanol, ionomycin and strontium for goat oocyte activation in order to optimize the protocols. We found, as a result, effective activation and parthenogenetic development of goat oocytes that had been derived from ionomycin, strontium and electrical pulse groups. Within each group 79.3–81.6%, 2.2–78.8% and 65.5% of the oocytes cleaved and 16.2–24.8%, 0–15.6% and 11.1% of the cleaved embryos developed into blastocysts when the oocytes were activated by ionomycin combined with 6-dimethylaminopurine, strontium plus cytochalasin B and electrical pulses combined with cytochalasin B, respectively. However, low temperature and ethanol were both unable to activate goat oocytes under our experimental conditions. When ionomycin combined with 6-dimethylaminopurine and strontium plus cytochalasin B was applied to activate somatic cell nuclear transfer embryos derived from cultured cumulus, 51.0% and 72.5% of the embryos cleaved, respectively. After transfer of 4-cell embryos into recipients, one (1/19 and 1/7) of the recipients from each group was found to be pregnant as detected by ultrasound, but both of these recipients lost the embryos between 45 and 60 days of pregnancy.

In nuclear-transferred or round spermatid-injected oocytes, artificial activation is required for further development in mammals. Although strontium chloride is widely used as the reagent for inducing oocyte activation in mice, the optimal method for oocyte activation remains controversial in rats because ovulated rat oocytes are spontaneously activated in vitro before artificial activation is applied. In our previous study, we found that cytostatic factor activity, which is indispensable for arrest at the MII stage, is potentially low in rats and that this activity differs greatly between two outbred rats (Slc: Sprague-Dawley (SD) and Crj: Wistar). Therefore, it is necessary to establish an optimal protocol for oocyte activation independent of strains. Given that comparative studies of the in vitro development of oocytes activated by different activation protocols are very limited, we compared four different protocols for oocyte activation (ethanol, ionomycin, strontium and electrical pulses) in two different SD and Wistar rats. Our results show that oocytes derived from SD rats have significantly higher cleavage and blastocyst formation than those from Wistar rats independent of activation regimes. In both types of rat, ethanol treatment provided significantly higher developmental ability at cleavage and blastocyst formation compared to the other activation protocols. However, the initial culture in a fertilization medium (high osmolarity mR1ECM) for 24 h showed a detrimental effect on the further in vitro development of parthenogenetic rat oocytes. Taken together, our results show that ethanol treatment is the optimal protocol for the activation of rat oocytes in SD and Wistar outbred rats. Our data also suggest that high-osmolarity media are inadequate for the in vitro development of parthenogenetically activated oocytes compared with fertilized oocytes.

The aims of the study were to measure the mRNA expression of brain-derived neurotrophic factor (BDNF) in bovine oocytes and early embryos derived from in vitro fertilization (IVF), parthenogenetic activation (PA) and nuclear transfer (NT), and to investigate the effects of BDNF on the development of IVF and parthenogenetic embryos. Bovine oocytes matured in vitro for 22 h were in vitro fertilized or parthenogenetic activated. By reverse transcription-PCR and quantitative real-time PCR, we found that germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, 4-cell and 8-cell embryos, morulae, and blastocysts were all shown to express mRNA for BDNF. The mRNA levels for BDNF gene were different in bovine oocytes and IVF embryos at different stages (P < 0.01), with the highest expression in MII oocytes and the lowest expression in 8-cell embryos. The mRNA for BDNF was highly expressed in the PA and IVF blastocysts compared to the NT blastocysts (P < 0.01). Supplementation of culture media with BDNF at the concentration of 40 μg/l caused a significant increase in the rates of in vitro-fertilized blastocyst formation (P < 0.05) and parthenogenetic blastocyst formation (P < 0.05). However, the rate of oocyte cleavage in BDNF groups was not significantly different from that in the BDNF-free control (P > 0.05) after IVF or PA. We have also investigated the effects of BDNF on the growth of granulosa cells, which were used for co-culture of bovine early embryos. The results revealed that supplementation of culture media with 20 μg/l BDNF promoted the growth of granulosa cells (P < 0.01). Taken together, these results provided evidence for the role of neurotrophins in promoting early embryonic development as well as in the growth of granulosa cells by the co-culture system, indicating that BDNF can directly or indirectly promote bovine early embryo development.

The viability of SCNT embryos is poor, with an extremely low cloned piglet production rate. In the present work, we studied the effect of three activation protocols based on ionomycin treatment (5 μM ionomycin for 5 min and incubated in 2 mM 6-DMAP for 3.5 h) or electric stimuli (two square wave electrical DC pulses of 1.2 kV/cm for 30 μs) combined or not with 6-DMAP on parthenogenetic embryo development. Oocytes activated by ionomycin plus 6-DMAP showed lower cleavage (47.2 vs. 78.5–81.5; p < 0.05) and blastocyst rates (11.3 vs. 29.2–32.1; p < 0.05) than those activated by electrical and electrical plus 6-DMAP treatments. Also, we studied the effect of addition of serum to maturation medium (0% vs. 10%) on nuclear maturation and further parthenogenetic and SCNT embryo development. We observed in the parthenogenetic embryos that cleavage rates in the serum-free group were significantly higher than in the serum-supplemented group (81.8 vs. 69.6% respectively; p < 0.05), although these differences were not detected in blastocyst rates or blastocyst nuclei numbers. Regarding SCNT embryos, no significant differences were observed in cleavage or blastocyst rates between different experimental groups of SCNT embryos. In conclusion, electrical pulse followed or not by 6-DMAP was found to be an efficient procedure to artificially activate MII porcine oocytes. Moreover, the addition of serum to oocyte maturation media did not seem to improve parthenogenetic or SCNT porcine embryo development.

The present study investigated the effects of the sexual maturity of oocyte donors on in vitro maturation (IVM) and the parthenogenetic developmental capacity of fresh minke whale oocytes. The effects of cytochalasin B (CB) pretreatment and two types of cryoprotectant solutions (ethylene glycol (EG) or ethylene glycol and dimethylsulfoxide (EG + DMSO)) on the in vitro maturation of vitrified immature whale oocytes were compared, and the developmental capacity of vitrified immature whale oocytes following IVM and intracytoplasmic sperm injection examined (ICSI). The maturation rate did not differ significantly with sexual maturity (adult, 60.9%; prepubertal, 53.1%), but the parthenogenetic activation rate of oocytes from adult donors (76.7%) was significantly higher (p < 0.05) than that of oocytes from prepubertal donors (46.4%). The maturation rates after vitrification and warming were not significantly different between the EG (22.2%) and EG + DMSO groups (30.2%), or between the CB-treated (30.4%) and non-CB-treated groups (27.3%). These results indicate that parthenogenetic activation of in vitro matured oocytes from adult minke whales was superior to that from prepubertal whales, but that the developmental capacity of the whale oocytes after parthenogenetic activation or ICSI was still low. The present study also showed that CB treatment before vitrification and two kinds of cryoprotectants did not improve the IVM rate following the vitrification of immature whale oocytes.

We applied intracytoplasmic sperm injection (ICSI) to the rat comparing three different sperm injection techniques: conventional setup with a sharp needle bearing a spike (method 1), combination of partial zona dissection (PZD) needle and blunt pipette (method 2) and piezo-injection using a blunt pipette (method 3). We also investigated the timing of sperm pronuclear formation after injection. Survival rates after injection were 8%, 24% and 71% for the methods 1, 2 and 3, respectively. All surviving oocytes formed pronuclei by about 6 h after injection. Although the survival and activation rates following sperm injection using piezo-injection were high, the incidence of normal fertilisation, as evidenced by second polar body extrusion and formation of two pronuclei, was only 10%. The vast majority of the zygotes were multinucleated, although most of them subsequently underwent cleavage. Fixation and staining of injected oocytes at different times after injection revealed that replacement of sperm nuclear protamines by histones takes place by 15 min after injection, sperm head swelling occurs within 0.5–1 h after injection and pronuclei become fully developed by 7 h after injection. Although the rate of normal fertilisation in the rat following ICSI was low under the present experimental conditions, the results indicated that direct ICSI using a piezo-driven pipette would be a potentially valuable method of producing rat offspring.

Fertilised mouse eggs develop the oolemma block to sperm penetration within 1 h. This block makes zona-free eggs at the pronuclear stage (zygotes) fully resistant to sperm penetration. In this study we investigated whether this block can spread – as a result of cell fusion – to the oolemma of eggs that are competent to be penetrated by spermatozoa. Preovulatory (GV) oocytes, ovulated oocytes in metaphase II (MII) and 1-cell parthenotes were fused with zygotes and the hybrid cells inseminated at various intervals after fusion. Sperm penetration was assessed on the basis of the presence of Giemsa-positive sperm heads in the air-dried preparations. The oolemma block to sperm penetration develops in all types of hybrids although at different speeds: it develops fast (2–3 h) in oolemma derived from MII oocytes and artificially activated eggs, and slowly in oolemma derived from GV oocytes. In the GV oocyte–zygote hybrids the time of formation of the block varied: while 50% of cells lost the ability to fuse with sperm by 2 h after fusion, in the remaining cells the block must have developed some time between 5 and 18 h after fusion. How these sperm-induced modifications of the oolemma of fertilised egg spread in the hybrid cell and render the ‘virgin’ part of oolemma resistant to sperm penetration remains unknown.