Antioxidant procyanidin B2 protects oocytes against cryoinjuries via mitochondria regulated cortical tension

Background:Irreversible cryodamage caused by oocyte vitrification limited its wild application in female fertility preservation.Antioxidants were always used to antagonist the oxidative stress caused by vitrification.However,the comprehensive mechanism underlying the protective role of antioxidants has not been studied.Procyanidin B2(PCB2)is a potent natural antioxidant and its functions in response to vitrification are still unknown.In this study,the effects of PCB2 on vitrified-thawed oocytes and subsequent embryo development were explored,and the mechanisms underlying the protective role of PCB2 were systematically elucidated.Results:Vitrification induced a marked decline in oocyte quality,while PCB2 could improve oocyte viability and further development after parthenogenetic activation.A subsequent study indicated that PCB2 effectively attenuated vitrification-induced oxidative stress,rescued mitochondrial dysfunction,and improved cell viability.Moreover,PCB2 also acts as a cortical tension regulator apart from strong antioxidant properties.Increased cortical tension caused by PCB2 would maintain normal spindle morphology and promote migration,ensure correct meiosis progression and finally reduce the aneuploidy rate in vitrified oocytes.Further study reveals that ATP biosynthesis plays a crucial role in cortical tension regulation,and PCB2 effectively increased the cortical tension through the electron transfer chain pathway.Additionally,PCB2 would elevate the cortical tension in embryo cells at morula and blastocyst stages and further improve blastocyst quality.What's more,targeted metabolomics shows that PCB2 has a beneficial effect on blastocyst formation by mediating saccharides and amino acids metabolism.Conclusions:Antioxidant PCB2 exhibits multi-protective roles in response to vitrification stimuli through mitochondria-mediated cortical tension regulation.

Isorhamnetin protects porcine oocytes from zearalenone-induced reproductive toxicity through the PI3K/Akt signaling pathway

Background Zearalenone(ZEA)widely exists in moldy grains,which seriously destroys the fertility of females.Isorhamnetin,a natural flavonoid,has extensive of pharmacological activities.However,the beneficial effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced porcine oocyte damage have not been investigated.Methods Oocytes were treated with different concentrations of ZEA(3,5,8 and 10μmol/L)and isorhamnetin(5,10,20 and 30μmol/L)for 44 h at 39℃.ZEA(5μmol/L)and isorhamnetin(10μmol/L)were selected for subsequent studies.Polar body exclusion rate,apoptosis rate and apoptosis related proteins,ROS levels and SOD2 protein,mitochondrial membrane potential and distribution,endoplasmic reticulum distribution and proteins expression,and PI3K,Akt and p-Akt proteins expression of oocytes were detected.In addition,the effect of PI3K antagonist(LY294002)on oocyte nuclear maturation and apoptosis were used to determine the involvement of PI3K/Akt signaling pathway.Results Our findings showed that ZEA exposure damaged oocytes and isorhamnetin therapy restored the developmental capability of porcine oocytes.Isorhamnetin promoted polar body extrusion rate to rescue ZEA-induced meiotic arrest in porcine oocytes.Isorhamnetin alleviated ZEA-induced oxidative stress by stimulating SOD2 protein expression and inhibiting ROS production.Moreover,isorhamnetin enhanced normal mitochondrial distribution and mitochondrial membrane potential to prevent mitochondrial dysfunction induced by ZEA.Changing the expression of endoplasmic reticulum stress-related marker proteins(CHOP,GRP78)and the distribution rate of normal endoplasmic reticulum showed that isorhamnetin relieved ZEA-caused endoplasmic reticulum stress.Mechanistically,isorhamnetin decreased Bax/Bcl-2 protein expression and inhibited ZEA-induced apoptosis through PI3K/Akt signaling pathway.Conclusions Collectively,these results suggest that isorhamnetin protects oocytes from ZEA-caused damage through PI3K/Akt signaling pathway,which enhances meiotic maturation and mitochondrial function,and inhibits early apoptosis,oxidative stress and endoplasmic reticulum stress in porcine oocytes.Our study provides a new strategy for solving the reproductive toxicity induced by ZEA and treating woman infertility.

Fertilization of IVF/ICSI Using Sibling Oocytes from Couples with Subfertile Male or Unexplained Infertility

The significance of the performance of conventional in vitro fertilization and intracytoplasmic sperm injection (IVF/ICSI) using sibling oocytes from couples with subfertile male or unexplained infertility was evaluated. A total of 410 sibling oocyte cumulus-corona complexes (OCCC) from 21 couples with subfertile male (group A) and 11 unexplained infertile couples (group B) were randomly divided, in order of retrieval, into two groups inseminated either by conventional IVF or by ICSI. The treatment outcomes and the influence of infertility factors on fertilization in each group were compared. The results showed that although the two pronuclear (2PN) fertilization rate per injected sibling oocytes was significantly higher after ICSI (group A: 68.2 %±28.8 %; group B: 66.2 %±24.9 %) than after conventional IVF (group A: 41.8 %±32.7 %; group B: 40.1 %±22.1 %), the other variables studied included: the fertilization rates of per allocated sibling oocytes IVF/ICSI, the fertilization rates of sibling oocytes IVF/ICSI after excluding failed IVF fertilization cycles, as well as the cleavage rates of normal fertilization were not statistically significant (P>0.05). Similarly, though the total fertilization failure rate in the IVF group (group A: 42.9 %; group B: 36.4 %) was significantly higher than in the ICSI group (group A: 4.8 %; group B: 0), we did not cancel cycles due to the normal fertilization of sibling oocytes. Embryo transfer was possible in all 32 couples. There were 10 clinical pregnancies in the two groups. We also discovered a possible association between some semen parameters and sperm functions of group A, and women age and duration of infertility of group B and fertilization. It is suggested that adoption of the split IVF/ICSI technology in the above cases may help eliminate fertilization failures. This is also a useful method to investigate the effect of single factor on the employment of assisted reproductive technology.

Effect of Lamb Age and in vitro Culture System of Oocytes on JIVET Technology

[Objectives] This study was conducted to investigate the effects of lamb age and in vitro culture system of oocytes on the results of juvenile in vitro embryo transfer( JIVET). [Methods]Ten Dorper × small-tailed Han lambs aged 5 to 10 weeks were induced to superovulate via i. p. injection of pregnant mare's serum gonadotropin( PMSG). The oocytes were matured in basal maturation solution or modified maturation solution,which was prepared by adding 200 μmol/L cysteine to the basal maturation solution. Then,the oocytes were fertilized in fertilization medium I containing 2% estrus sheep serum( ESS) or fertilization medium II containing 3 mg/ml bull serum albumin( BSA). Finally,the number of oocytes,oocyte maturation rate and cleavage rate of the lambs of different ages were determined. [Results]The average number of oocytes recovered per lamb was( 111. 00 ± 16. 97),( 139. 50 ± 28. 99),( 108. 50 ± 17. 68) and( 42. 00 ± 11. 31) for5-,7-,8-and 10-week-old Dorper × small-tailed Han lambs,respectively. The number of oocytes obtained from 5-,7-and 8-week-old lambs was significantly higher than that from 10-week-old lambs( P < 0. 05),but there was no significant difference among 5-,7-and 8-week-old lambs( P > 0. 05). The maturation rate of oocytes cultured in modified maturation solution was 3. 64% higher than that in basal maturation solution. The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II( P < 0. 01). [Conclusions] The results of JIVET can be improved by harvesting oocytes from lambs aged 5-8 weeks,adding a certain amount of cysteine into oocyte maturation solution,and a certain amount of ESS into fertilization medium.

Nicotinamide mononucleotide supplementation improves the quality of porcine oocytes under heat stress

Background:Elevated ambient temperature-caused heat stress is a major concern for livestock production due to its negative impact on animal feed intake,growth,reproduction,and health.Particularly,the germ cells are extremely sensitive to the heat stress.However,the effective approach and strategy regarding how to protect mammalian oocytes from heat stress-induced defects have not been determined.Methods:Germinal vesicle(GV)porcine oocytes were cultured at 41.5℃ for 24 h to induce heat stress,and then cultured at 38.5℃ to the specific developmental stage for subsequent analysis.Nicotinamide mononucleotide(NMN)was dissolved in water to 1 mol/L for a stock solution and further diluted with the maturation medium to the final concentrations of 10μmol/L,20μmol/L,50μmol/L or 100μmol/L,respectively,during heat stress.Immunostaining and fluorescence intensity quantification were applied to assess the effects of heat stress and NMN supplementation on the key processes during the oocyte meiotic maturation.Results:Here,we report that NMN supplementation improves the quality of porcine oocytes under heat stress.Specifically,we found that heat stress resulted in oocyte maturation failure by disturbing the dynamics of meiotic organelles,including the cytoskeleton assembly,cortical granule distribution and mitochondrial function.In addition,heat stress induced the production of excessive reactive oxygen species(ROS)and DNA damage,leading to the occurrence of apoptosis in oocytes and subsequent embryonic development arrest.More importantly,we validated that supplementation of NMN during heat stress restored the meiotic defects during porcine oocyte maturation.Conclusions:Taken together,our study documents that NMN supplementation is an effective approach to improve the quality of oocytes under heat stress by promoting both nuclear and cytoplasmic maturation.

Studies on Electrical Activation of Porcine Oocytes Matured in vitro and Embryo Culture Systems

Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical field strength and the pulse duration at 130Vmm-1/80 us, with a blastocyst development rate of (20.12 ± 8.18) % (P > 0.05). No significant difference was found between treatments of multiple pulses and a single pulse (P > 0.05). Parthenogenetic embryos were cultured with different methods and air conditions for 7 days in vitro, blastocyst development rate of embryos with changed culture media [ (26.44 ± 8.35) % ] or changed media with 10% fetal bovine serum (FBS) [ (17.68 ± 5.39)% ] on the fifth day showing no significant difference from that of embryos without change of culture media [ (25.30 ± 7.55) % , P > 0.05 ], while cell numbers of blastocysts from embryos with changed culture media (15.78 + 5.46 and 14.55 ± 4.81) were significantly lower than number of blastocysts from embryos without change of culture media (18.01 ± 6.79, P<0.01). Blastocyst development rate and blastocyst cell number of embryos cultured in lower O2(5%CO2: 7%O2:88%N2) also showed no significant difference from those in high O2(5%CO2 in air) [(20.78 ± 8. 80)% and 17.0016.12 vs. (25.30 ± 7.55)% and 18.0116.79, P>0.05]. It is concluded that change of culture media with the same new one or changing over to media with 10% fetal bovine serum (FBS) on the fifth day and low O2 environment are not necessary for porcine embryos development.

CENP-A Regulates Chromosome Segregation during the First Meiosis of Mouse Oocytes

Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.

Experimental Study on Meiotic Spindles and Chromosomes of Mouse Mature (MⅡ) Stage Oocytes under Laser Scanning Confocal Microscopy

Taking the mouse as a model, the experimental method of observing the morphology of meiotic spindles and chromosomes in mature oocytes were investigated in order to evaluate the effects of various interventions on the quality of oocytes accurately and rapidly. Laser scanning confocal microscope （LSCM） was used to examine the meiotic spindles and chromosomes by the technologies of optical section and three-dimensional （3D） image reconstruction. The results showed that the configurations of meiotic spindles and chromosomes could be observed clearly by LSCM. The normal rate of meiotic spindles and chromosomes was 82% and 86% respectively. It was concluded that the LSCM was a valid instrument to observe the meiotic spindles and chromosomes of mature oocytes and could be used as a valid method to evaluate the quality of MⅡocytes.

Effects of MⅡ stage oocytes zona pellucida birefringence on pregnancy outcome

Objective:To explore the effects of different MⅡstage oocytes zona pellucida birefringence on pregnancy outcome.Methods:A total of 46 couples with infertile which induced by single cause received in-vitro fertilization treatment were analyzed retrospectively,and randomly divided into the high zona birefringence(HZB)/HZB group.HZB/low zona birefringeuce(LZB) group and LZB/LZB group according to different oocytes zona pellucida birefringence.Intracytoplasmic sperm injection outcome was analyzed and compared.Results:The proportion of HZB oocytes, implantation rate and the pregnancy rate were decreased in three groups(HZB/HZB group】HZB/ LZB group】LZB/LZB group)(P【0.05).But there was no significantly different between the number of oocytes and fertilization rate of these groups(P】0.05).Logistic regression analysis showed that factors allecl MⅡstage oocytes zona pellucida birefringence were age.basal FSH level and the LH level on the day of HCG injection.Age and KSH levels were negatively correlated with the single oocyte zona pellucida birefringence:While the LH level on the day of hCC injection was positively correlated with the single oocylc zona pellucida birefringence.Conclusions:The primary influence factors on MⅡstage oocytes zona pellucida are age.basal FSH level and the LH level on the day of hCG injection.The birefringence value of zona pellucida can affect the pregnancy outcome.

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.

High pregnancy and implantation rates can be obtained with preincubation of oocytes before insemination in IVF and ICSI procedures

Purpose: Evaluate the effect of preincubation of oocytes prior to IVF or ICSI cycles with embryo transfer at blastocyst stage. Methods: Retrospective non randomized study based on secondary analysis of data. Setting: Laboratory of Assisted Reproduction at the Alcivar Hospital. Patients: One hundred-eighteen cycles of IVF and ICSI were analyzed in the present study. The evaluated groups were formed for those patients whose oocytes, after retrieval, were inseminated at 1-3 h (Group I) or 4-6 h (Group II). Results: There was no difference in fertilization rate (83.6% and 78.1%), Day 3 cleavage rate (95.1% and 97.1%), and blastocyst formation (31.1% and 39.1%) for groups I and II respectively. Clinical pregnancy rates (PR: 53.0% vs 22.9%) and implantation rates (IR: 38.1% vs 13.0%) were significantly higher in group II versus group I, respectively (P < 0.05). Conclusions: Preincubation of oocytes before insemination is a factor which raises the PR and IR after the blastocyst transfer.

Collection of superovulated mouse oocytes continuously by surgery and their development after activation

Objective:To establish a new way to collect superovulated oocytes or zygotes repeatedlyfrom an individual mouse.Methods:Superovulations were induced by injection PMSG and hCG in Kunming strainmice.The ampullaes of oviduct of all anaesthetised mouse were put in a specially designed'U'sink and released.The second and third times of PMSG injection were made on the sixth day andeleventh day after the first superovulation injection.The capacity of development was examinedby in vitro culture of parthenogenesis activation oocytes.Results:Development to blastocyst stage was not significantly different between the first andsecond time collection.The percentage of blastocyst stage in CD and Sr^(++) treatment was signifi-cantly higher(P<0.05)than the oocytes treated in CB and Sr^(++).Conclusion:This method enables us to collect oocytes or zygotes repeatedly from one individ-ual mouse in an interval as short as 5 days and without influence on the quality of oocytes.

Effect of vitrification procedure on chromosomal status of embryos achieved from vitrified and fresh oocytes

Background: In order to assess the chromosomal status in embryos obtained from vitrified and fresh donated oocytes, preimplantational genetic diagnostic (PGD) was performed after biopsy of one blastomere at day 3. METHODS: A total of 249 oocytes were obtained from 23 oocyte donors, 80 oocytes were used in the vitrified group and 151 oocytes were used in the fresh group. Nine chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y) were investigated by fluorescence in situ hybridization (FISH) analysis in 56 and 121 embryos from vitrified and fresh group respectively. Fertilization, cleavage rate, embryo quality and chromosomal abnormality rate were compared between groups evaluated. Results: Vitrified oocytes showed a survival rate of 97.5%. There was no significant difference in the fertilization rate (82.7% and 91.4%), Day 2 cleavage rate (90.3% and 87.7%) or blastocyst formation rate (31.1% and 44.6%) for the vitrified and fresh groups respectively. Chromosomal abnormality rate (66.1% versus 71.9%), percentage of abnormal blastocysts (61.1% versus 64.8%) and percentage of abnormalities for each analyzed chromosome were similar for the vitrified group compared with the control group. Conclusions: The rates of chromosomal abnormalities in embryos from vitrified oocytes are similar to those published previously;and comparable to those observed in embryos from fresh oocytes. These results confirm that the developmental competence and chromosomal status of embryos obtained from vitrified oocytes is not affected by the vitrification procedure, and they preserve the potential to be fertilized and to develop in to blastocyst stage similar to embryos from fresh oocytes.

Effects of EGF and IGF-I on in vitro Maturation and Cleavage of Ovine Oocytes

The study investigated the effects of epidermal growth factor（EGF） and insulin-like growth factor 1 （ IGF-I）, alone or together, on the in vitro maturation and cleavage of ovine oocytes, aimed to optimize the in vitro maturation conditions for ovine oocytes. The results showed that the maturation and cleavage rates were 71.2% and 45.5% respectively when the medium was supplemented with 50 ng/mL EGF alone, which was significantly higher than other EGF supplemented groups （0, 10, 20, 30, and 40 ng/mL） （P 〈0.05）. The highest maturation and cleavage rates were 72.9% and 45. 7% when the EGF concentration reached 100 ng/mL. The maturation and cleavage rates were 70. 7% and 58. 5% with 40 ng/mL IGF-I supplemented, which were significantly higher than other treatments （0,10,20,60,80, and 100 ng/mL） （P 〈0.05）. The lowest maturation and cleavage rates were 38.8% and 20.0% when the IGF-I concentration reached 100 ng/mL （ P 〈 0.05 ） When 50 ng/mL EGF and 40 ng/mL IGF-I were used concomitantly,the maturation and cleavage rates were 85. 6% and 61. 0% respectively, which were significantly higher than the treatments with EGF or IGF-I alone （ P 〈 0.05 ）.

Comparative proteomic analysis of mature and immature oocytes in domestic cats

Objective:To evaluate changes of feline(Felis catus)oocytes proteins during in vitro maturation by using the proteomic approach.Methods:Immature oocytes(germinal vesicle)isolated from female cats were cultured and collected at 0 h and 24 h.After collection,oocytes were investigated into immature(germinal vesicle)and mature(metaphaseⅡ)stages.The qualitative profiles of the proteins at the immature and mature stages were determined by one-dimensional electrophoresis and liquid chromatography-mass spectrometry.Results:Our data revealed that following 24 h in vitro maturation the maturation rate(metaphaseⅡstage)was 58.7%.Eighty-one of the 260 proteins analyzed were differentially expressed between the germinal vesicle stage and the metaphaseⅡ-arrest stage.Proteomic analysis of germinal vesicle and metaphaseⅡoocytes showed abundant expression of proteins involved in transportation(10%),indicating that this was a major characteristic of germinal vesicle oocytes.Similarly,analysis of the proteome of metaphaseⅡoocytes indicated that cell cycle proteins were overexpressed.Interestingly,proteins involved in DNA repair and apoptosis were only expressed in germinal vesicle oocytes and proteins involved in fertilization were only expressed in metaphaseⅡoocytes.Conclusions:The overexpression of certain proteins in germinal vesicle and metaphaseⅡis necessary for oocyte development and maturation.Our findings provide a valuable resource for further investigations into protein expression in oocytes at different developmental stages.

Effects of Ages of Donors and Conditions of Preserving Ovaries on Porcine Oocytes Maturation in vitro and Efficiency of Parthenogenetic Activation

Effects of different ages of donors and different conditions of preserving ovaries onporcine oocytes maturation in vitro and efficiency of parthenogenetic activation werestudied. The experiments included: 1) effects of different temperatures (22, 30, 37, 38.5and 40℃) of preserving ovaries on porcine oocytes maturation in vitro and developmentalpotential; 2) effects of periods of preserving ovaries on porcine oocytes maturation invitro and development in vitro; 3) effects of different ages of donors on porcine oocytesmaturation in vitro and developmental potential. The results of the experiment showed:1) There were no statistical differences (p>0.05) of the parthenogenetic cleavage rate(79.64% vs 76.18%) and blastocyst rate (18.11% vs 33.82%) between oocytes from ovariespreserved at 38.5℃ and those preserved at 37℃. When the preserving temperature wasincreased to 40℃, the cleavage rate (21.68%) and the blastocyst rate (0) were greatsignificantly lower than those at 37℃(p<0.01). The cleavage rate (80.79% vs 76.18%) andblastocyst rate (29.61% vs 33.82%) were not different between 30 and 37℃(p>0.05). Whenthe preserving temperature was decreased to 22℃, the rate of cleavage was not different,but the rate of blastocyst was significantly lower, compared with that at 37℃; 2) Thecleavage and blastocyst rates of the porcine oocytes collected after slaughter 2 or 6hwere not different (p>0.05); 3) The cleavage rate of oocytes from gilts and sows aftermaturation was not different, but the blastocyst rate of the sow group was significantlyhigher than that of gilt group (p<0.05). The blastocyst cell number of sows and giltshowed no difference (p>0.05).

Expression and kinetic characteristics of muscle type acetylcholine receptors in Xenopus oocytes

Nicotinic acetylcholine receptors(N2-ChRs)were synthesized in Xenopus oocytes after injection of mRNAs extracted from denervated rat muscle and mRNAs transcribed from Torpedo N2-ChR subunit cDNAs in vitro.Membrane inward current in the injected oocytes wa

Development of Bovine Embryos from Two Different Activated-time Oocytes for Parthenogenesis and Two Kinds of Nucleus Donors for Cloning

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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.

Effect of Specific Nutrients on Ovulation, Oocytes Development, Gene Expression and Coupling Success in Mice

Introduction: The roles of genetic, epigenetic, metabolic and other environmental factors such as nutrition and stress, are becoming evident for a successful and healthy pregnancy. This raises the possibility and question, if and how, we improve the probability of pregnancy and of a healthy fetus? The present study examined the role of metabolic, antioxidant and minerals and the results suggest that these factors may positively influence the oocyte quality and the pregnancy rate. Methods: CD1 female mice aged 15 and 5 weeks were divided into four groups of ten each and treated by intragastric gavage daily for 3 weeks. G1: Vehicle;G2: Carnitines (L-carnitine 0.4 mg and acetyl-L-carnitine 0.12 mg/mouse);G3: Microelements (Zinc 4 ng, Copper 0.8 ng, Iron 7 ng/mouse);G4: G3+G2. At the end of the treatment period superovulation was induced and oocytes were collected to assess their quantity and quality. Further, in vitro fertilization (IVF) experiments were performed to assess the preimplantation embryo development. The birth success rate was also analyzed in old and young female. The mice were in vivo fertilized. qRT-PCR were performed to analyze a possible modulation in key genes of the reproductive process. Results: The number of oocytes was significantly higher in groups 2 and 4 compared to the control group. The oocyte number in group 3 was not affected. The level of degraded oocytes was 29.1% and 19.3% (group 2 and 4) versus 34.3% (control). Concomitantly, the numbers of embryos arriving to successful birth were also increased in G4, both in the old and young group of mice. Preliminary analysis of genes affected evidenced that AMH was up regulated in the ovary and KITL in the uterus in group 2. Conclusion: Results showed that L-carnitine, acetyl-L-carnitine and micronutrients were able to improve both oocytes quality and success rate of pregnancy. Further studies are planned to further examine ways to improve pregnancy and fetal health.