Protective effect of resveratrol against cadmium-induced toxicity on ovine oocyte in vitro maturation and fertilization

Background:Heavy metal cadmium(Cd)is a widespread environmental contaminant with a potential toxicity that might negatively affect female reproduction and fertility.It has been reported that Cd exposure impaired the quality of oocytes and led to a defective maturation and fertilization,through oxidative stress induction.Resveratrol(Res)is a natural polyphenol with strong antioxidant properties that exhibited protective role in preventing oocyte redox homeostasis disruption and quality decline.Here,we explored whether the addition of Res to in vitro maturation(IVM)medium might act as a protection against Cd-induced toxicity on ovine oocyte maturation and fertilization.Firstly,we evaluated the effect of supplementing IVM medium with two different Res concentrations(1and 2μmol/L)on nuclear maturation and fertilization of oocytes matured under CdCl2(2μmol/L)exposure.Therefore,the concentration of 1μmol/L Res was selected to analyse the effects of this compound on intracellular ROS levels,mitochondrial(mt)distribution and activity,chromatin configuration,cytoskeleton morphology,cortical granules(CGs)distribution and mRNA expression of genes associated with cellular response to oxidative stress(i.e.SIRT1,SOD 1,GPX1,GSR,CAT)in Cd-exposed in vitro matured oocytes.Results:We found that 1μmol/L Res restored the reduced oocyte meiotic competence induced by Cd exposure as well as,Res sustained oocyte ability to be normally fertilized and decreased polyspermic fertilization at both tested concentrations.Moreover,we demonstrated that 1μmol/L Res mitigated Cd-induced alterations of oocyte cytoplasmic maturation by reducing reactive oxygen species(ROS)accumulation,preventing mt dysfunction,maintaining the correct meiotic spindle and cortical F-actin assembly and the normal cortical granule distribution as well as up-regulating SIRT1,SOD1 and GPX1 genes.Conclusions:Taken together,our findings highlighted the beneficial influence exerted by Res in preventing Cdinduced disturbance of nuclear and cytoplasmic maturation and subsequent fertilization in ovine oocytes.Res treatment may help to establish defence strategies counteracting Cd-induced toxicity on the female gamete.

Effect of zeaxanthin on porcine embryonic development during in vitro maturation

Zeaxanthin is a common carotenoid, which is a powerful antioxidant that protects against damage caused by reactive oxygen species. The aim of the present study was to investigate the effects of zeaxanthin supplementation on in vitro maturation of porcine embryo development. We investigated nuclear maturation, intracellular glutathione （GSH）, and reactive oxygen species （ROS） levels during in vitro maturation, and subsequent embryonic development following parthenogenetic activation and in vitro fertilization OVF）. The oocytes were maturated and used at the metaphase II stage. After 42 hours of in vitro maturation, the zeaxanthin-treated group （0.5 μmol/L） showed significant increases in nuclear maturation （89.6%） than the control group （83.4%） （P 〈 0.05）. The intracellular GSH levels increased significantly （P 〈 0.05） as zeaxanthin concentrations increased; ROS generation levels decreased with increased zeaxanthin concentrations, but there were no significant differences. There were no significant differences in subsequent embryonic development, cleavage rate, blastocyst stage rate, and total blastocyst cell numbers following parthenogenetic activation and IVF when in vitro maturation media was supplemented with zeaxanthin. These results suggest that treatment with zeaxanthin during in vitro maturation improved the nuclear maturation of porcine oocytes by increasing the intracellular GSH level, thereby slightly decreasing the intracellular ROS level.

The Role of Brain-derived Neurotrophic Factor in Mouse Oocyte Maturation in vitro

Brain-derived neurotrophic factor (BDNF) can promote developmental competence in mammalian oocytes during in vitro maturation (IVM),but the role of BDNF in oocyte maturation at cellular level is not still clear.In this study,mouse cumulus-enclosed oocytes subjected to IVM were fertilized and cultured to blastocyst stage.Meiotic spindle configuration and cortical granules distribution during oocyte maturation in vitro were assessed by using immunofluorescence and laser confocal microscopy.The results showed that BDNF contributed to the complete preimplantation development of mouse oocytes compared to the control oocytes (13.78% vs.5.92%;P【0.05).Further,BDNF did not accelerate nuclear maturation of IVM oocytes.For the BDNF-treated oocytes at meiosis Ⅰ,Meiotic spindle areas were significantly smaller and the number of cytoplasmic microtubule organizing centers was greater than that in the control,and the percentages of oocytes showed spindles positioned near the oolemma and a well-formed cortical granule-free domain were significantly higher than that of the control.These morphological characteristics of the BDNF-treated oocytes were much closer to the oocytes matured in vivo than those of the control oocytes.In conclusion,BDNF can promote the developmental competence of mouse IVM oocytes,by improving the meiotic spindle configuration and location and cortical granules distribution at meiosis Ⅰ.

Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection (ICSI) using ejaculated and testicular spermatozoa

Aim: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). Methods: Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. Results: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P < 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. Conclusion: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.

Developmental competence of bovine oocytes with increasing concentrations of nano-copper and nano-zinc particles during in vitro maturation

Objective: To evaluate copper and zinc concentrations in plasma and follicular fluid from cattle ovaries, and estimate the impact of rational concentrations of copper and zinc oxide nanoparticles (CuO-NPs & ZnO-NPs) supplemented during in vitro maturation (IVM) against DNA damage of cumulus cells, glutathione content in oocytes and on consequent embryo development. Methods: Oocytes were obtained from 2 to 8 mm follicles by aspiration method for IVM. Replicates of experiments were performed on different days, with a separate batch of cumulus oocytes complex for each day. Results: The DNA damage of oocytes and cumulus cells significantly decreased with supplemental CuO-NPs or ZnO-NPs concentrations of 0.7 and 1.0 μg/mL in the IVM medium compared to medium without CuO-NPs or ZnO-NPs (P < 0.01). Total glutathione concentrations in oocytes and cumulus cells significantly increased following supplementation with both 0.7 and 1.0 μg/mL CuO-NPs or ZnO-NPs in comparison with 0 and 0.4 μg/mL CuO-NPs or ZnO-NPs supplemented groups (P < 0.01). Supplementation of CuO-NPs or ZnO-NPs during IVM medium at any concentration had no significant effect on cleavge rate. Both CuO-NPs and ZnO-NPs significantly increased blastocyst rates when oocytes were matured with 0.7, 1.0 μg/mL CuO-NPs concentrations (P < 0.01). In contrast, addition of 1.5 μg/mL of CuO-NPs or ZnO-NPs to the maturation media resulted in detrimental effects on the developmental competence of bovine oocytes confirming toxicity induced by CuO-NPs and ZnO-NPs in high concentrations. Conclusions: CuO-NPs or ZnO-NPs-treated bovine oocytes during IVM show low level of DNA fragmentation and increased intracellular glutathione content of cumulus cells. In vitro embryo development is improved by supplementation of rational concentrations of CuO-NPs or ZnO-NPs to culture media. Toxicity induced by CuO-NPs and ZnO-NPs is confirmed in high concentrations.

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining： a study using the mouse model

选择是最可能的发展的卵母细胞是关键的为在 vitrofertilization 并且动物克隆。染色的灿烂的 cresyl 蓝色(BCB ) 在大型家畜被用于卵母细胞选举，但是它的更宽的用途需要推进评估。我们再划分进染色的那些(BCB+) 和那些的老鼠卵母细胞未沾污(BCB-) 根据他们的 ooplasm BCBcoloration。染色质配置，积云房间 apoptosis，细胞质的成熟和发展胜任在 BCB+ 和 BCB- 卵母细胞之间被比较。BCB+ 卵母细胞的胜任上的卵母细胞直径，性成熟和 gonadotropin 刺激的效果也被分析。在thelarge尺寸和中等尺寸的组，BCB+卵母细胞更大并且显示出更多的包围 nucleoli ( SN )染色质配置和早闭锁的更高的频率，并且他们也获得了更好细胞质的成熟(象细胞内部的 GSH 水平和 mitochondrialdistribution 的模式坚定)并且在试管内成熟( IVM )以后的更高发展的潜力比BCB卵母细胞。当没与 PMSG 告知时，成年老鼠比 prepubertal 老鼠与更高的胜任生产了更多的 BCB+ 卵母细胞。PMSG priming 增加了比例和 BCB+oocytes 的发展力量。在大尺寸的组的 BCB+ 卵母细胞在 medium-sizegroup 比他们的对应物显示出更多的 SN 染色质配置，更好细胞质的成熟和更高发展的潜力。染色的 BCB 能为卵母细胞选择被用作一个有效方法，这被结束，但是 BCB+ 卵母细胞的胜任可以与卵母细胞直径，动物性成熟和 gonadotropin 刺激变化。总起来说，这里描述的标准的系列将更好允许在为更好的开发选择卵母细胞的选择。

Effects of Ovarian Preservation Time on in vitro Maturation and in vitro Fertilization of Oocytes from Slaughtered Sheep

[Objectives] The aim was to study the effects of ovarian preservation time on in vitro fertilization of oocytes from slaughtered sheep. [Methods] The collected ovaries were randomly and evenly divided into four groups. They were preserved in physiological saline containing penicillin（ 100 IU/ml） and streptomycin（ 100 μg/ml） at 15-20 ℃ for 0（ Control）,6,12 and 18 h,respectively. Then,the oocytes were subjected to in vitro fertilization. [Results]The maturation rates,cleavage rates and blastocyst rates of the oocytes preserved for 6 and 12 h showed no significant differences compared with those of the oocytes preserved for0 h（ 72. 03%,70. 87% vs. 73. 68%; 74. 12%,72. 60% vs. 74. 49%; 22. 22%,20. 75% vs. 23. 29%）（ P 〉 0. 05）. There were also no significant differences in maturation rate,cleavage rate or blastocyst rate between the oocytes preserved for 18 and 0 h（ P 〉 0. 05）. [Conclusions] Within a certain rage（ 0-18 h）,storage time of ovary at 15-20 ℃ does not affect the continued development of oocytes from slaughtered sheep.

In-vitro maturation treatment for infertile women with polycystic ovary syndrome

Polycystic ovary syndrome(PCOS) is the most common cause of anovulatory infertility.If PCOS infertile women fail to conceive after conventional induction of ovulation,the assisted reproductive therapy is an alternative method for pregnancy.In-vitro maturation is an efficient,more economical and simple method without ovarian hyperstimulation syndrome complication。

LH Plays a Role to Enhance the Nuclear and Cytoplasm Synchronization During In vitro Maturation of Ovine Oocytes

This study is aimed to investigate the effect of luteinizing hormone （LH） on maturation and fertilization in vitro of ovine oocytes. The ovine oocytes were cultured in vitro in medium with or without LH, and then checked by confocal laser scanning microscope to observe the distribution of cortical granules stained with FITC-LCA during different maturation periods. Similarly, some in vitro matured oocytes were also fertilized in vitro for analysis of their developmental potentiality further. After being cultured in vitro for 4 h, there were significant differences about the rate of germinal vesicle break down （GVBD） between the treatment （with LH） and the control groups （without any hormones） （36.76% vs 18%, P〈0.05）. Further, there were also significant differences of the cleavage and blastocyst rates between these two groups （67.15% vs 42.37%, 21.9% vs 12.71%, P〈0.05, respectively）. The distribution of cortical granules appeared to spread from the edges to the central site of sheep oocytes following their delaying durations of maturation in vitro. It can be concluded that LH may play a role to delay the occurence of GVBD, prolong the maturation duration of cytoplasm, and enhance the nuclear and cytoplasm synchronization of ovine oocytes matured in vitro and finally improve their in vitro developmental potentiality.

Effects of Chemical Activation on the Parthenogenetic Development of Porcine in vitro Maturation Oocytes

The objective of this study was to determine the effects of ionomycin combined with cytochalasin B （CB）, cycloheximide （CHX）, or 6-dimethylaminopurine （6-DMAP） on the activation of porcine oocytes. In Experiment 1, in vitro matured oocytes were activated with 15, 20, 25 or 30 mmol L -1 ionomycin separately. Activation rates of 20, 25 mmol L-1 and 30 mmol L treatments were higher （P〈0.05） than that of 15 mmol L-1 treatment. In Experiment 2, in vitro matured oocytes were activated with 20 mmol L-1 ionomycin for 10, 20, 30, 40 or 50 rain and then incubated with 2 mmol L-1 6-DMAP for 6 h. Cleavage and blastocyst rates [（72.40 ± 13.02）%, （25.37 ± 11.43）%] after treatments for 40 min were higher （P〉0.05） than those of the other treatments. In Experiment 3, matured oocytes were activated with ionomycin and then incubated with 7.5 mg mL-1 CB, 10 mg mL-1 CHX, 2 mmol L-1 6-DMAP, 7.5 mg mL-1 CB ＋ 10 mg mL-1 CHX or 7.5 mg mL-1 CB ＋ 2 mmol L-1 6-DMAP for 6 h. The rates of activation, cleavage and blastocyst of 2 mmol L -1 6-DMAP treatment [（86.05 ± 4.29）%, （61.77 ±8.10）% and （21.62± 3.31）%] were higher （P〈0.05） than those of 7.5 mg mL-1 CB treatment. In Experiment 4, matured oocytes were activated with ionomycin and then incubated with 2 mmol L-1 6-DMAP for 3.5, 5.5 or 7.5 h. Cleavage rates and blastocyst rates of 5.5 h treatment [（66.59 ± 14.36）% and （25.40 ± 10.16）%] were higher （P〉0.05） than those of other treatments. In conclusion, activation of porcine oocytes appears to be most successful using the combination of ionomycin （20 mmol L-1, 40 min） followed by 6-DMAP （2 mmol L-1, 5.5 h）.

Effect of Dynein Inhibitor on Mouse Oocyte in vitro Maturation and Its Cyclin B1 mRNA Level

Objective To evaluate the effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 transcription level. Methods Immature mouse oocytes were cultured in vitro with a known dynein ATPase activity inhibitor-sodium orthovanadate (SOV) to detect the changes of maturation rate, and semi-quantitative RT-PCR and single cell RT-PCR were performed to detect the changes of cyclin B1 mRNA level. Results In dose-dependent experiments, the maturation rates of oocytes were significantly different between 5 mmol/L SOV and control groups (P<0.05), and decreased with SOV increasing doses. However, the elevation of cyclin B1 mRNA level of immatured oocytes cultured for 12 h depended on SOV concentrations ranging from 50 to 500 mmol/L. In incon- tinuity exposed SOV experiments, the maturation rates of oocytes markedly reduced after the first incubation with 400 mmol/L SOV at least for 1 h and were first cultured in SOV-free medium for 4 h or 8 h before exposure to SOV (P<0.05). In time-course experiment, the opposite changes of cyclin B1 mRNA level in oocytes between SOV and control groups were observed. Conclusion Dynein inhibitor might delay oocytes meiosis process, and cause ectopic expression of cyclin B1 in oocytes. Most Oocytes incubated with SOV blocked at germinal vesicles (GV) stage or MⅠto anaphase transition due to dynein dysfunction and ectopic transcription level of cyclin B1.

Expression of Interleukin-6 and Interleukin-6 Receptor in Ovine Oocytes During In vitro Maturation

To study the effects of interleukin-6 （IL-6） and its receptor （IL-6R） during in vitro maturation of ovine oocytes, the mRNA and protein expression levels of IL-6 and IL-6R, along with their localization, were examined during ovine oocytes maturation in vitro through real-time PCR, Western blotting, and immunohistochemistry. Specific patterns of expression of IL-6 and IL-6R were observed at both mRNA and protein levels at each stage of ovine oocytes maturation. IL-6 and IL-6R were distributed primarily on the surface of the cell membrane, with little expression in the cytoplasm or nucleus. IL-6 and IL-6R were expressed significantly at higher levels in the maturation around 4 h, and then decreased dramatically. However the level slightly elevated at 20-24 h. The role of IL-6 and IL-6R on oocytes maturation was studied through in vitro addition of recombinant human IL-6 in different concentrations. The addition of 10 ng mL-1 IL-6 significantly increased the rates of oocytes maturation （P〈0.05）, but did not affect the rates of development of the subsequence IVF ovine embryos. In summary, IL-6 is likely to play an important role in the early ovine oocytes maturation. The expression patterns of the IL-6 and IL-6R on the ovine oocytes maturation open up the possibility of regulatory role of the cytokine in ovine oocytes maturation.

Effects of Medium on in Vitro Maturation of Pig Oocytes and in Vitro Early Development of Parthenogenetic Embryos

[ Objective] To improve quality of oocytes maturing in vitro and to optimize in vitro culture system of porcine early embryos. [ Method ] Oocytes were cultured in the improved TCM199 and NCSU-23 basic media, which were added pig follicular fluid （PFF） or high-quality fetal bovine serum （FBS） both at a proportion of 10% （V/V）. After in vitro maturation and development, effects of medium on maturation of pig oocytes and development of eady parthenogenetic embryos were investigated using maturing rate of pig oocytes and development rate of parthenogenetic embryos as indicators. [ Result] After 42 h culture, the maturing rates of pig oocytes respectively cultured in the TCM199, TCM199 added FBS, TCM199 added PFF, NCSU-23, NCSU-23 added FBS and NCSU-23 added PFF were （54.2 ±3.5）%, （68.5 ±3.2）%, （69.3 ±3.7）%, （51.6 ±3.3）%, （63.2 ±3.1 ）% and （65.5 ±3.5）%, respectively. The pig oocytes cultured in the TCM199 or NCSU-23 that was added FBS or PFF had significantly higher maturing rate （P 〈 0.05）. The development rates of parthenogenetic embryos were not significantly different between these six experimental media. However, the parthenogenetic embryos which developed in the TCM199 added PFF （36.5 ±4.8） had significantly more blastomeres than those developed in the TCM199 or NCSU-23 （ 18.7 ± 3.2 and 15.5 ± 2.4, respectively） （ P 〈 0.05）. [ Conclusion ] The improved TCM199 and NCSU-23 added PFF or FBS can largely promote in vitro maturation of pig oocytes and in vitro development of early parthenogenetic embryos.

In-vitro Maturation of Immature Oocytes from Preantral Follicles in Prepuberal Mice

Objective To observe the morphological changes in in vitro growth of preantral follicle isolated from prepuberal mice and to assess impacts of gonadotropin （Gn）, insulin transferrin selenium （ITS） and epidermal growth factor （EGF） on their development. Methods Early preantral mice follicles （90-130μm diameter） were mechanical isolated and selected from 2 weeks＇ old mice and then cultured in alpha-minimal essential medium （α-MEM） with or without Gn, ITS and EGF. The preantral follicles were cultured singly in 20 miovliters droplets for up to 14 d. The medium was replaced and the .follicles were observed everyday. Granulosa cells （GC） prolification, antrum formation and oocyte maturation were recorded. Results The medium with Gn supported preantral follicle culture in vitro, during which they retained a three-dimensional structure, maintained oocytes viability and increased in diameter and number of somatic cells＇. Preantral follicles cultured in Gn medium grew obviously, while those without Gn grew slowly and after 6 d＇s culture began to shrink and blacken. Significant increase in survival rate and maturation rate of oocytes was observed in Gn group （P〈0.01）, with 92.9% survived and 28.7%formed an antrum. Further supplementation of the Gn medium with ITS and rLH, resulted in the significant increase in survival and maturation of preantral follicle （P〈0.05） Conclusions α-MEM can be the medium for in vitro culture （IVC） of preantral follicles, but need to be added with rLH/rFSH, rHCG/rEGF to facilitate thecal cell attachment, GC proliferation and oocyte maturation.

A modified protocol for <i>in vitro</i>maturation of mouse oocytes from secondary preantral follicles

A 2-step culture system was designed and tested for the in vitro maturation efficiency of oocytes from pre-puberty preantral follicles of FVB/N inbred mice. The following modifications were made: 1) The concentration of ITS was reduced by half in the basal MIF medium to minimize uncoordinated growth between oocyte and GC cells;2) Heterogeneous preantral follicles were cultured in groups of 3 - 5 follicles in hanging drops of medium with reduced concentration of ITS for six days to induction follicular aggregation. This hanging drop method mimics a 3-D IVM culture system at the early stage of cultivation in which the sphere structure of each follicle is well maintained. It also enables follicles in each aggregate to communicate with each other, synchronize their growth, and thus prevent immature follicular rupture. 3) Medium was further supplemented with retinoic acid to enhance developmental capacity of meiotically arrested oocytes. After a 14-day culture in vitro, ~37% of the collected inbred preantral follicles completed nuclear maturation. Approximately 94% of the mature oocytes tested were able to be fertilized;and 77% of them developed into healthy embryos. These results demonstrate that our IVM system is reliable to produce a satisfactory number of high quality oocytes. In addition, multiple cytoplasmic parameters, including gene expression of key regulators, chromosome/spindle organization, mitochondrial proliferation and distribution, and total ATP content were explored to characterize the supportive and limiting components of our IVM system so that the culture system can be further optimized.

The application of in vitro maturation of oocytes in the infertile patients with polycystic ovarian syndrome

Objective:To evaluate the effects of in vitro maturation(IVM)of oocytes in the infertile pa-tients with polyeystic ovarian syndrome(PCOS).Methods:The infertile patients with PCOS who underwent IVM or IVF/ICSI from January2004 to August 2005 were studied retrospectively.68 unstimulated cycles(48 cases)underwentIVM as IVM group,42 cycles(39 cases)underwent IVF/ICSI as control group.Main outcomesincluding the number of oocytes retrival,the rates of fertilization,embryo cleavage,implanta-tion,pregnancy,miscarriage,ovarian hyperstimulation syndrome(OHSS)and multiple pregnan-cy were assessed.Results:No FSH was administered in IVM group and the mean number of FSH used was(25±6.2)ampoules in control group.When compared with control group,women in IVM grouphad significant increase in fertilization rate(70.7% versus 63.9%)and decrease in cleavage rate(87.9% versus 99.4%)and ovarian hyperstimulation syndrome(0 versus 7.1%).No significantdifferences between IVM group and control group were found in the number of oocytes obtained,implantation rate,clinical pregnancy rate,miscarriage rate and multiple pregnancy rate.Conclusion:Our results suggested that for infertile PCOS women who required assisted con-ception treatment,IVM is a more economical method with less OHSS complication than that ofconventional IVF treatment.

In vitro maturation of human oocytes maintaining good development potential for rescue intracytoplasmic sperm injection with fresh sperm

BACKGROUND The outcomes of the use of commercial in vitro maturation(IVM)medium to culture immature oocytes obtained from conventional ovulation induction,followed by rescue intracytoplasmic sperm injection(RICSI),are not ideal.It is thus difficult to widely adopt this approach in clinical practice.Therefore,it is necessary to explore methods for improving the clinical outcome of IVM.AIM To study the effect of sperm on the developmental potential of in vitro-matured oocytes in conventional culture.METHODS This was a retrospective study of patients whose immature oocytes were harvested from conventional oocyte stimulation cycles and underwent ICSI at our hospital between June 2018 and August 2020.RICSI was performed using sperm collected on the day of oocyte harvest(old)and sperm collected on the day of RICSI(fresh)and oocytes matured in vitro after 24 h of culture in conventional medium.The rates of in vitro oocyte maturation,normal fertilization,normal cleavage,day-3 top-quality embryos,and useful blastocyst formation were compared between the two groups.RESULTS In total,102 germinal vesicle(GV)-stage immature oocytes were cultured in the old sperm group.In the fresh sperm group,122 GV-stage immature oocytes were collected and cultured in vitro for 24 h.There were no significant differences in the general conditions of males and females between the two groups(P>0.05).The oocyte maturation,normal fertilization,and normal cleavage rates of the old and fresh groups were 51.0%vs 55.7%,61.5%vs 64.7%,and 93.8%vs 93.2%,respectively.None of the rates differed significantly(P>0.05)between the two groups.However,the day-3 top-quality embryo and useful blastocyst rates of the old and fresh sperm groups were 16.6%vs 63.4%;6.67%vs 34.6%,respectively.The day-3 top-quality embryos and useful blastocyst rates of the old sperm group were significantly lower than those of the fresh group(P<0.05).CONCLUSION In vitro maturation with conventional culture medium combined with the use of fresh sperm collected on the day of RICSI is an easy-to-implement strategy for patients whose oocytes are completely or mostly immature.

Evaluation of pig oocyte in vitro maturation and fertilization using three gonadotropin-based hormonal compounds

Objective:To evaluate the effect of human chorionic gonadotropins(hCG)and equine chorionic gonadotropins(eCG)on in vitro gilt oocyte maturation and embryonic development,using frozen semen for fertilization.Methods:Two independent experiments(6 replicates each)were carried out to evaluate gilt oocyte maturation,and fertilization and embryonic development by using ovaries from a local abattoir.Totally,712 oocytes were randomly distributed in four-well dishes to receive Novormon(eCG 5.0 IU),PG600(eCG 5.0 IU and hCG 2.5 IU),Chorulon(hCG 5.0 IU),or no hormones.Oocytes were incubated with 5%CO2,95%air and saturation humidity at 39℃for 44 h.Maturation of the oocytes to metaphaseⅡwas assessed by using the aceto-orcein technique.In addition,741 oocytes were used and randomly distributed in four-well dishes,and then oocyte maturation was carried out as mentioned,but matured oocytes were washed and placed in fertilization medium with frozen-thawed sperm.Gametes were co-incubated for 7 h,and then washed and placed in development medium,and incubated for further 7 days,at which time embryonic development was evaluated.Fertilization and embryo development media were not supplemented with the studied hormones.Results:Novormon(eCG)and PG600(eCG+hCG)treatments significantly improved the percentages of metaphaseⅡoocytes compared to the control group(P<0.05).Furthermore,a significant increase was also observed in the young blastocyst stage between the control group and the PG600 treatment group(P<0.05).Conclusions:Hormonal products Novormon(eCG)and PG600(eCG+hCG)can obtain the highest percentages of in vitro maturation in gilt oocytes;however,this effect is not transferred to fertilization rates.

In Vitro Propagation Potential via Somatic Embryogenesis of the Two Maturing Early Cultivars of European Chestnut (Castanea sativa Mill.)

Turkey ranks the third in the production of chestnuts in the world having an important place both in domestic and global markets. However, the chestnut production and the number of trees have been diminishing in recent years. Therefore, in vitro propagation of the chestnut, in addition to the classical propagation techniques, should be applied. Especially the propogation of the early maturing cultivars and production of the quality chestnuts will provide a better income to the producer. Here, somatic embryo production and regeneration from the immature cotyledons of the early maturing cultivars of the European chestnut (Castanea sativa Mill), Haciibis and Karamehmet, were studied using the somatic embryogenesis, one of the in vitro propagation techniques. To induce the somatic embryogenesis, 168 different combinations were applied to both cultivars. The somatic embryogenesis rate in Haciibis cultivar, in which the interactions were observed among the applications, was found to be 9.9% while it was 11.1% for the Karamehmet cultivar. Dessication, cold treatment, gibberellic acid (GA3) and benzyladenine (BA) + naphthaleneacetic acid (NAA) applications were performed on the regeneration of the somatic embryos, and 40% conversion to plant was obtained with desiccation together with BA + NAA supplementation to the medium.

Effect of Different Reproductive Stages and Culture Times on Domestic Cat <i>in Vitro </i>Oocyte Maturation

The domestic cat has been used as a model to carry out comparative research in assisted reproduction, to be applied in wild cats. The efficiency in domestic cat IVM concerning the reproductive status and/or cultivation times has previously been investigated;however, the studies were carried out separately. The objective of this research was to evaluate the maturation of oocytes of domestic cats of different reproductive stages using two different in vitro culture times. The ovaries were obtained by Ooforo-Salpingo-Hysterectomy of cats that were of the following groups: 1) prepubertal, 2) follicular, 3) pregnant or 4) in anestrus. Maturation was carried out with TCM199 medium supplemented with BSA for 24 h and 48 h. On average, 29 ± 25, 20 ± 15, 17 ± 9 and 17 ± 13 oocytes/cat were recovered from the prepubertal follicular, pregnant, and anestrus stages, respectively, but did not show a significant difference (P > 0.05). Also, meiotic maturation did not show a significant difference between the different reproductive stages at 24 h and 48 h, respectively (P > 0.05). However, in the prepubertal and follicular stages, greater oocyte maturation numbers were observed at 48 h compared to 24 h (P < 0.05). In contrast, the aforementioned result was not observed in the pregnant and anestrus stages (P > 0.05), indicating that the in vitro culture duration is an important factor during in vitro maturation of domestic cat oocytes.