41 DEVELOPMENTAL POTENTIAL OF BUFFALO OOCYTES VITRIFIED AT THE GERMINAL VESICLE STAGE: EFFECTS OF DIFFERENT CRYOPROTECTANT COMBINATIONS AND CRYODEVICES

2016 ◽  
Vol 28 (2) ◽  
pp. 150
Author(s):  
A. S. El-Shalofy ◽  
A. R. Moawad ◽  
G. M. Darwish ◽  
S. T. Ismail ◽  
A. B. Badawy
Keyword(s):  
Solid Surface ◽  
Germinal Vesicle ◽  
Subsequent Development ◽  
The Other ◽  
Control Group ◽  
Developmental Potential ◽  
High Frequencies ◽  
Nuclear Maturation ◽  
Maturation Rate

The cryopreservation of immature oocytes would generate a readily available, nonseasonal source of female gametes for both research and reproduction. In domestic animals, the most promising results in the field of oocyte cryopreservation have been reported in cattle, and a few experiments have been conducted on buffalo. The aim of the present study was to compare the effects of different cryoprotectant combinations and different cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle stage. Cumulus-oocyte complexes obtained at slaughter from mature buffalos were vitrified by using either straw or open pulled-straw or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol or 20% EG + 20% dimethylsulfoxide (DMSO). Following vitrification and warming, viable oocytes were matured in vitro for 22 h. Matured oocytes were either evaluated for nuclear maturation or fertilized and cultured in vitro for 7 days. Recovery rate was significantly higher (P < 0.05) in the oocytes vitrified by straw in 20% EG + 20% glycerol (92.6%) as compared with the other groups. Percentages of viable oocytes were significantly higher (P < 0.05) in the oocytes vitrified in 20% EG + 20% DMSO using SSV (95.7%) than those in the other groups (from 80 to 88.0%). Among the vitrified groups, the highest maturation rate was achieved in SSV with 20% EG + 20% DMSO group (56.7%). This value was comparable with those in the control group (62.1%). After IVF and embryo culture, the highest cleavage and blastocyst rates were obtained in SSV with 20% EG + 20% DMSO group (35.7 and 21.4%, respectively), and these values were nearly similar to those in the control group (38.7 and 25.8%, respectively). Vitrification of oocytes by straw or open pulled-straw resulted in significantly lower (P < 0.05) blastocyst rates (2.6 and 11.5%) as compared with the control. In conclusion, buffalo oocytes vitrified at the germinal vesicle stage can be matured, fertilized, and developed in vitro and produce high frequencies of blastocyst embryos. Solid surface vitrification may be superior to straw and open pulled-straw in vitrification of immature buffalo oocytes because this technique results in higher survival and embryo development rates.

scholarly journals Effect of resveratrol on the in vitro maturation of ovine (Ovis aries) oocytes and the subsequent development of handmade cloned embryos

2019 ◽  
Vol 5 (4) ◽  
Author(s):  
José Luis Martínez-Ibarra ◽  
Eugenia Adriana Espinoza-Mendoza ◽  
Raymundo Rangel-Santos ◽  
Demetrio Alonso Ambriz-García ◽  
María Del Carmen Navarro-Maldonado
Keyword(s):  
Embryo Quality ◽  
Developmental Stages ◽  
Subsequent Development ◽  
Poor Quality ◽  
Ovis Aries ◽  
Control Group ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Maturation Rate

The effect of resveratrol on the in vitro maturation (IVM) of ovine (Ovis aries) oocytes and the development of handmade cloned embryos was evaluated. The nuclear maturation and reactive oxygen species (ROS) levels in the oocytes, as well as the early development and morphological cloned embryo quality, were evaluated under different resveratrol concentrations (0, 0.5, 2 and 5 μM). After IVM, no significant difference was observed in the maturation rate of oocytes treated with 0.5 μM (81.3 %) and 2 μM (72 %) resveratrol compared to that of the control group (0 μM) (74.2 %), but the rate significantly decreased at 5 μM (56 %) (p < 0.05). When the oocyte ROS levels were determined, no significant differences among the groups were observed (p > 0.05). For cloned embryo development, the embryos obtained from the oocytes treated with 0.5 μM resveratrol showed higher (p < 0.05) compacted morula rates (10.7 %) compared to the embryos obtained from the oocytes treated with 0, 2 and 5 μM (6.2, 0 and 0 %, respectively). Regarding embryo morphological quality, the embryos from the oocytes treated with 0.5 μM resveratrol showed a lower rate of poor quality morulae (4.7 %) in comparison to those treated with 0, 2 and 5 μM (23.8, 23.3 and 33.3 %, respectively) (p < 0.05). In conclusion, resveratrol showed no significant improvement on the IVM or ROS levels in domestic ovine oocytes. However, treatment with 0.5 μM resveratrol during IVM improved embryo quality and promoted morulae compaction of Ovis aries handmade cloned embryos.Figure 3. Different developmental stages of the HMC sheep embryos cultured in the WOW system. Cleaved embryos (a-d), 8‒16 blastomere embryos (e-h), morulae (i-l) and compact morulae (m-p) (200X).

scholarly journals Effects of prematuration culture with a phosphodiesterase-3 inhibitor on oocyte morphology and embryo quality in in vitro maturation

2021 ◽  
Vol 48 (4) ◽  
pp. 352-361
Author(s):  
Mohammed Ashraf Cheruveetil ◽  
Prasanna Kumar Shetty ◽  
Kamini A Rao ◽  
Arya Rajendran ◽  
Muhammed Asif
Keyword(s):  
In Vitro Maturation ◽  
Embryo Quality ◽  
Germinal Vesicle ◽  
Control Group ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Phosphodiesterase 3 ◽  
The Impact ◽  
Experimental Group

Objective: The study assessed the developmental potential of germinal vesicle (GV) oocytes subjected to in vitro maturation (IVM) after prematuration culture with cilostamide (a phosphodiesterase-3 inhibitor) and the impact of cilostamide exposure on the morphology of meiosis II (MII) oocytes and subsequent embryo quality. Methods: In total, 994 oocytes were collected from 63 patients. Among 307 GV oocytes, 140 oocytes were selected for the experimental group and 130 oocytes for the control group. The denuded GV-stage oocytes were cultured for 6 hours with cilostamide in the experimental group and without cilostamide in the control group. After 6 hours, the oocytes in the experimental group were washed and transferred to fresh IVM medium. The maturational status of the oocytes in both groups was examined at 26, 36, and 48 hours. Fertilization was assessed at 18 hours post-intracytoplasmic sperm injection. Embryo quality was assessed on days 3 and 5.Results: In total, 92.1% of the oocytes remained in the GV stage, while 6.4% converted to the MI stage (p<0.01) after cilostamide exposure. In both groups, more MII oocytes were observed at 36 hours (25.8% vs. 21.5%) than at 26 hours (10.8% vs. 14.6%) and 48 hours (13% vs. 7.9%) (p>0.05). With the advenet of cilostamide, blastocyst quality was better in the experimental group than in the control group (p<0.05). Conclusions: Cilostamide effectively blocked nuclear maturation and promoted cytoplasmic growth. Prematuration culture with cilostamide enabled synchronization between cytoplasmic and nuclear maturity, resulting in better blastocyst outcomes.

Improvement of the developmental competence of canine oocyte using caffeine supplementation during IVM at different maturation time

Zygote ◽  
2018 ◽  
Vol 26 (2) ◽  
pp. 162-167 ◽  
Author(s):  
Mohamed Fathi ◽  
A. Salama ◽  
Magdy R. Badr
Keyword(s):  
Developmental Competence ◽  
Control Group ◽  
Free Medium ◽  
Maturation Time ◽  
Fluid Medium ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Oviductal Fluid

SummaryThe aim of the current study was to investigate the effect of caffeine supplementation during in vitro maturation (IVM) for different maturation times on the developmental potential of canine oocytes recovered from ovariohysterectomized bitches. The recovered cumulus–oocytes complexes were in vitro matured for 72 h. Here, 10 mM caffeine was added to the maturation medium for different incubation times (caffeine from 0–72 h maturation, caffeine for the first 24 h of maturation only, caffeine addition from 24 to 48 h maturation time, caffeine addition from 48 to 72 h maturation or in caffeine-free medium, control group). The matured oocytes were in vitro fertilized using frozen–thawed spermatozoa. The presumptive zygotes were in vitro cultured in synthetic oviductal fluid medium for 5 days. The results showed that both maturation and fertilization rates were significantly higher (P ˂ 0.05) using caffeine-treated medium for the first 24 h of maturation compared with the control and other two groups of caffeine treatment (from 24 to 48 h and from 48 to 72 h), whereas use of caffeine-treated medium for a 0–72 h incubation time did not affect these rates (P > 0.05). Interestingly, the matured oocytes in caffeine-supplemented medium for the first 24 h or from 0–72 h showed a significant (P ˂ 0.05) increase in the total number of cleaved embryos compared with the control group. In conclusion, supplementation of the maturation medium with 10 mM caffeine for the first 24 h of maturation or during the whole maturation time (0–72 h) improved nuclear maturation and subsequent embryo development preimplantation following in vitro fertilization.

scholarly journals Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 851-859 ◽  
Author(s):  
Hsiao Yun Yang ◽  
Shae-Lee Cox ◽  
Graham Jenkin ◽  
Jock Findlay ◽  
Alan Trounson ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Germinal Vesicle ◽  
Fertilization Rate ◽  
Ovarian Tissue Cryopreservation ◽  
Control Group ◽  
Developmental Potential ◽  
Oocyte Yield ◽  
Tissue Grafts

Ovarian tissue cryopreservation and subsequent transplantation can restore fertility in cancer patients. This study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, the kidney capsule or subcutaneous) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts and whether using a standard gonadotrophin stimulation protocol would increase oocyte yield from the grafts. Mouse ovarian tissue was grafted into four week old mice and collected three weeks later. Graft recipients were treated either with or without exogenous gonadotrophin stimulation prior to graft collection. Grafted ovaries yielded oocytes that were either at the germinal vesicle (GV) stage or mature metaphase II (MII) stage at collection. These GV oocytes were matured beforein vitrofertilization (IVF), while the MII oocytes underwent IVF immediately. Oocytes collected from the oviducts of non-grafted superovulated mice of the same age served as controls. Two-cell embryos were transferred to pseudopregnant recipients and recovered at day 15 of gestation or left to go to term. Graft retrieval and the number of oocytes from each graft were lowest from the subcutaneous graft site. The number of two-cell embryos produced was significantly higher for oocytes from the grafts to the bursa as compared with the other sites. All graft sites gave rise to embryos with comparable implantation rates and developmental potential to fetuses and offspring following transfer. However, the oocytes from grafted ovaries had a significantly lower developmental potential when compared with the control group. Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation and development. In conclusion, graft site affects the number and quality of oocytes produced from ovarian grafts.

155 EFFECT OF CO-CULTURE WITH CUMULUS-DERIVED SOMATIC CELLS DURING IN VITRO MATURATION ON PORCINE CUMULUS–OOCYTE COMPLEXES AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

2014 ◽  
Vol 26 (1) ◽  
pp. 191 ◽  
Author(s):  
J. D. Yoon ◽  
L. Cai ◽  
S. U. Hwang ◽  
Y. Jeon ◽  
E. Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mrna Expression ◽  
In Vitro Maturation ◽  
Somatic Cells ◽  
The Other ◽  
Control Group ◽  
Cortical Granules ◽  
Developmental Potential ◽  
Significant Difference

The purpose of this study was to investigate the effects of co-culture with cumulus-derived somatic cells (CSC) during porcine in vitro maturation (IVM) and subsequent embryonic development after IVF. The CSC were cultured in Dulbecco's modified Eagle medium for 48 h with various numbers of cumulus-derived somatic cells (0.0, 2.5, 5.0, and 10.0 × 104), and then cultured in TCM-199 for 4 h before the oocytes were added. Cumulus-oocytes complexes from 3- to 6-mm follicles were matured in 500 μL of TCM-199, with eCG and hCG, for 22 h, and then cultured in M199 without hormones for 22 h. Each experiment consisted of at least 4 replicates. Statistical analyses were carried out using SPSS 17.0 software (SPSS Inc., Chicago, IL). Percentage data were compared by one-way ANOVA, followed by Duncan's multiple range test. Data were presented as means ± s.e.m. Differences were considered to be significant if the P-value was 0.05. After IVM, no significant difference (P < 0.05) was observed in nuclear maturation rate among the 0.0, 2.5, 5.0, and 10.0 × 104 groups (88.0 ± 2.37, 81.5 ± 2.17, 87.0 ± 1.98 and 86.0 ± 1.93%, respectively). The 2.5 × 104 group showed a significant (P < 0.05) increase in intracellular glutathione (GSH) levels compared with that of the other groups. Intracellular reactive oxygen species (ROS) levels of mature oocyte in all groups showed no significant differences. The developmental competence of matured oocytes in all groups was evaluated after IVF. The 2.5 and 5.0 × 104 groups showed significantly (P < 0.05) high cleavage rates (60.0 ± 4.7 and 64.52 ± 5.9%, respectively) compared with the 0 and 10.0 × 104 groups (43.15 ± 5.0 and 53.8 ± 5.0%, respectively). The 2.5 × 104 group showed a significantly (P < 0.05) higher BL formation rate (35.7 ± 2.9) than control group (21.0 ± 3.8%, respectively), and higher total cell number (127.25 ± 7.7) compared with the 0 and 10 × 104 groups (89.3 ± 4.0 and 92.6 ± 3.7, respectively). In the analysis of gene expression, IVF-BL derived from the 2.5 and 5.0 × 104 groups showed higher (P < 0.05) mRNA expression of PCNA, which is an essential component of the DNA replication and repair machinery and POU5F1 has been used to evaluate developmental potential in embryos. The 10.0 × 104 group showed higher (P < 0.05) mRNA expression of caspase-3 and Bak as known pro-apoptotic factors, compared with the control group IVF-BL. The results of cortical granules distribution which leads digesting sperm receptor proteins ZP2 and ZP3 to block polyspermy, showed that the 2.5 × 104 group was increased significantly (P < 0.05) compared with the other co-culture groups (13.7 ± 6.1, 29.2 ± 9.5, 18.3 ± 0.8 and 19.52 ± 5.3, respectively). In conclusion, co-culture with 2.5 × 104 cumulus-derived somatic cells during IVM improved the developmental potential of porcine IVF embryos by increasing the intracellular GSH level and distribution of cortical granules during oocyte maturation. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

49 Developmental Potential of Dromedary Camel Oocytes Vitrified at the Germinal Vesicle Stage: Effects of Different Cryoprotectant Combinations and Cryo-Carriers

2018 ◽  
Vol 30 (1) ◽  
pp. 164
Author(s):  
M. Fathi ◽  
A. R. Moawad ◽  
M. R. Badr
Keyword(s):  
Survival Rates ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Dromedary Camel ◽  
In Vitro Production ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
And Control ◽  
Vitro Production

Cryopreservation of oocyte would be an alternative to overcome the limited availability of dromedary camel oocytes and allow improvements in in vitro production in this species. Our aim was to develop a protocol for vitrification of dromedary camel oocytes at the germinal vesicle (GV) stage using various cryoprotectant combinations and cryo-carriers. In experiment 1, cumulus–ppcyte complexes (COC) obtained at slaughter were equilibrated in a solution composed of 10% ethylene glycol (EG) and 0.25 M trehalose. The oocytes were then exposed for 60 s to vitrification solutions (VS) composed of 20% EG and 20% dimethyl sulfoxide (DMSO; VS1) or 25% EG plus 25% DMSO (VS2) or 25% EG and 25% glycerol (VS3). The COC were then transferred into decreasing concentration of trehalose solution (toxicity test). In experiment 2, COC were randomly divided into 4 groups and vitrified by using straw or open pulled-straw (OPS) or solid surface vitrification (SSV) or cryotop in VS1 or VS2. Following vitrification and warming viable oocytes were matured in vitro for 30 h at 39°C in 5% CO2 in air. Matured oocytes were fertilized in vitro by epididymal spermatozoa of mature male camels and then cultured in modified KSOMaa medium for 7 days. Oocyte viability, maturation, fertilization, and embryo development were evaluated. Data were analysed using one-way ANOVA and t-test. Viability and nuclear maturation rates were significantly lower (P ≤ 0.05) in oocytes exposed to VS3 (44.8% and 34.0%) than those exposed to VS1 (68.2% and 48.0%) and VS2 (79.3% and 56.9%). Although recovery rates were significantly lower (P ≤ 0.05) in oocytes vitrified using SSV or cryotop in either VS1 or VS2 solutions (66.9% to 71.1%) than those vitrified by straws using VS1 or VS2 solutions (86.3% to 91.0%), survival rates were higher in SSV and cryotop groups (90.7% to 94.8%) than straw and OPS (68.2% to 86.5%) groups. Among vitrified groups, maturation and fertilization rates (51.8% and 39.2%, respectively) were the highest in the cryotop-VS2 group. Those values were comparable to those seen in the controls (59.2% and 44.6%, respectively). Cleavage (22.5% to 27.9%), morula (13.2% to 14.5%), and blastocyst (6.4% to 8.5%) rates were significantly higher (P ≤ 0.05) in SSV and cryotop groups than in straws. No significant differences were observed in these parameters between cryotop and control groups. Together, the results show that both vitrification solution and cryodevice affect viability and developmental competence of vitrified/warmed dromedary camel oocytes. We report for the first time that dromedary camel oocytes vitrified at the GV stage have the ability to be matured, fertilized, and subsequently develop in vitro to produce blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.

Selection of Rattus norvegicus oocytes for in vitro maturation by brilliant cresyl blue staining

Zygote ◽  
2011 ◽  
Vol 21 (3) ◽  
pp. 238-245 ◽  
Author(s):  
Diego Duarte Alcoba ◽  
Bianca Letícia da Rosa Braga ◽  
Nathallie Louise Sandi-Monroy ◽  
Letícia Auler Proença ◽  
Rui Fernando Felix Lopes ◽  
...  
Keyword(s):  
Rattus Norvegicus ◽  
Blue Staining ◽  
Control Group ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Nuclear Maturation ◽  
Effective Evaluation ◽  
Maturation Rate ◽  
Selection Of

SummaryThe objective of this work was to evaluate the rate of meiosis resumption and nuclear maturation of rat (Rattus norvegicus) oocytes selected for in vitro maturation (IVM) after staining of cumulus–oocyte complexes (COCs) with blue cresyl brilliant (BCB) using different protocols: exposure for 30, 60 or 90 min at 26 μM BCB (Experiment 1), and exposure for 60 min at 13, 20 or 26 μM BCB (Experiment 2). In Experiment 1, the selection of oocytes exposed to BCB for 60 min was found to be the most suitable, as meiosis resumption rates in the BCB+ group (n = 35/61; 57.37%) were the closest to the observed in the control (not exposed) group (n = 70/90; 77.77%) and statistically higher than the values observed for the BCB− group (n = 3/41; 7.32%). Additionally, the more effective evaluation of diagnostic tests (sensitivity and negative predictive value 100%) was observed in COCs exposed for 60 min. In Experiment 2, the 13 μM BCB+ group presented rates of meiosis resumption (n = 57/72; 72.22%) similar to the control group (n = 87/105; 82.86%) and higher than other concentration groups. However, this results of the analysis between BCB− oocytes was also higher in the 13 μM BCB group (n = 28/91; 30.78%) when compared with BCB− COCs exposed to 20 μM (n = 3/62; 4.84%) or 26 μM (n = 3/61; 4.92%) BCB. The nuclear maturation rate in the 13 μM BCB group was similar between BCB+ or BCB− oocytes. The 20 μM BCB group had a lower rate of nuclear maturation of BCB− oocytes than other groups. Thus, our best results in the selection of Rattus norvegicus oocytes by staining with BCB were obtained using the concentration of 13 μM and 20 μM, and an incubation period of 60 min.

scholarly journals Effect of Interleukin-7 on In Vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Developmental Potential after Parthenogenetic Activation

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 741
Author(s):  
Dongjin Oh ◽  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Junchul-David Yoon ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Group ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation ◽  
Interleukin 7

Interleukin-7 (IL-7) is a cytokine essential for cell development, proliferation and survival. However, its role in oocyte maturation is largely unknown. To investigate the effects of IL-7 on the in vitro maturation (IVM) of porcine oocytes, we analyzed nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic developmental competence after parthenogenetic activation (PA) under several concentrations of IL-7. After IVM, IL-7 treated groups showed significantly higher nuclear maturation and significantly decreased intracellular ROS levels compared with the control group. All IL-7 treatment groups exhibited significantly increased intracellular GSH levels compared with the control group. All oocytes matured with IL-7 treatment during IVM exhibited significantly higher cleavage and blastocyst formation rates after PA than the non-treatment group. Furthermore, significantly higher mRNA expression levels of developmental-related genes (PCNA, Filia, and NPM2) and antioxidant-related genes (GSR and PRDX1) were observed in the IL-7-supplemented oocytes than in the control group. IL-7-supplemented cumulus cells showed significantly higher mRNA expression of the anti-apoptotic gene BCL2L1 and mitochondria-related genes (TFAM and NOX4), and lower transcript levels of the apoptosis related-gene, Caspase3, than the control group. Collectively, the present study suggests that IL-7 supplementation during porcine IVM improves oocyte maturation and the developmental potential of porcine embryos after PA.

scholarly journals 303 EFFECT OF SERUM SUPPLEMENTATION AND ESTRUS CYCLE STAGE ON IN VITRO NUCLEAR MATURATION OF CANINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 302
Author(s):  
F.Y. Heru ◽  
H.J. Oh ◽  
M.K. Kim ◽  
J. Goo ◽  
M.S. Hossein ◽  
...  
Keyword(s):  
Luteal Phase ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Follicular Phase ◽  
Estrus Cycle ◽  
Immature Oocytes ◽  
Nuclear Maturation ◽  
Serum Supplementation ◽  
Maturation Rate

The present study investigated the effects of the estrus cycle stage and serum supplementation on nuclear maturation of canine oocytes. Ovaries were collected from a private clinic after ovariohysterectomy and classified into follicular, luteal, or anestrus stages through a combination of ovarian morphology and vaginal cytology. A total of 2214 oocytes from 196 ovaries (903 oocytes from 96 anestrus ovaries, 609 oocytes from 36 follicular ovaries, and 702 oocytes from 64 luteal ovaries) were used for experiments. The oocyte retrieval per ovary was 10, 19, and 12 for anestrus, follicular and luteal-phase ovaries, respectively. In Exp. 1, immature oocytes were cultured for 72 h in TCM-199 alone or TCM-199 supplemented with 10% canine anestrus (CAS), estrus (CES), or diestrus (CDS) serum or fetal bovine serum (FBS). In Exp. 2, immature oocytes were cultured for 72 h in TCM-199 supplemented with 0, 5, 10, or 20% CES. After staining with Hoechst 33342, chromatin state and position as well as spindle formation were evaluated to determine the stage of meiosis: germinal vesicle (GV) stage, germinal vesicle breakdown (GVBD), metaphase I (MI) stage, metaphase II (MII) stage. The experiments with anestrus and luteal-phase oocytes were repeated eight times and follicular-phase oocytes were repeated six times. Data were subjected to analysis of variance (ANOVA) and protected least significant difference (LSD) test to determine differences among experimental groups by using the Statistical Analysis System (SAS, SAS Institute, Inc., Cary, NC, USA) program. Statistical significance was determined where P value was less than 0.05. In Exp. 1, the in vitro maturation of oocytes up to MII stage was higher when oocytes were collected from ovaries in follicular phase. The maturation rate up to MII stage was 0.0 to 1.7%, 1.3 to 10.2%, and 1.0 to 3.2% for the oocytes collected from the anestrus, follicular, and luteal-phase ovaries, respectively, depending on the culture media used. In basic TCM media only, 0.0, 1.3, and 2.3% oocytes reached the MII stage for anestrus, follicular, and luteal-phase oocytes, respectively. A significantly higher rate of maturation was obtained when oocytes collected from follicular phase were cultured in TCM-199 supplemented with 10% CES (10.2%), compared to 10% CAS (4.0%), CDS (2.7%), FBS (1.3%), or the control (1.3%). In Exp. 2, supplementing with 10% CES induced the highest (P < 0.05) maturation rate to the MII stage in oocytes collected from follicular-stage ovaries (11.5%) compared to supplementing with 0% (1.0%), 5% (1.3%), or 20% CES (5.1%). Supplementing with CES (5, 10, or 20%) did not have a significant effect on nuclear maturation of canine oocytes collected from anestrus or luteal-stage ovaries. In conclusion, supplementing in vitro maturation medium with 10% CES increased nuclear maturation of canine oocytes, and canine oocytes collected from follicular-stage ovaries are the most suitable to complete nuclear maturation in vitro. This study was supported by grants from the Biogreen 21-1000520030100000.

337 EFFECTS OF CANINE SYNTHETIC OVIDUCT FLUID MEDIUM SUPPLEMENTED WITH THE VARIOUS ENERGY SUBSTRATES ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 276
Author(s):  
H. J. Oh ◽  
M. K. Kim ◽  
Y. H . Fibrianto ◽  
G. Jang ◽  
H. J. Kim ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
The Other ◽  
Germinal Vesicle Breakdown ◽  
Energy Substrates ◽  
Fluid Medium ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Oviduct Fluid

In most mammals, maturation occurs within the ovarian follicle, and preovulatory oocytes are ovulated and ready for fertilization within the oviduct. In contrast, bitch ovulate primary oocytes, over a three day period, undergo both maturation and fertilization within the oviduct. The present study was conducted to evaluate the effects of canine synthetic oviduct fluid (cSOF) supplemented with the various energy substrates on in vitro maturation of canine oocytes. Oocytes were recovered by mincing ovaries collected after ovariohysterectomy in bitches at the follicular stage. Only oocytes with more than two layers of cumulus cells and with homogeneous cytoplasm >100 mm in diameter were selected. Then, oocytes cultured in tissue culture medium (TCM)-199 (control) or cSOF supplemented with various concentrations of glucose (0, 1.11, 3.89, or 5.56 mM, Exp. 1) or fructose (0, 1.11, 3.89, or 5.56 mM, Exp. 1), pyruvate (0, 0.1, 0.25, or 0.5 mM, Exp. 2) or lactate (0, 0.5, 1.0, or 5.0 mM, Exp. 3). In Exp. 4, the combined effects of glucose (1.11 mM), pyruvate (0.5 mM) and lactate (5.0 mM) on nuclear maturation of canine oocytes were investigated. A total of 2990 canine oocytes from 205 ovaries were used for experiments with replication at least three times. The oocytes were cultured for 72 h at 38.5�C in a humidified atmosphere of 5% CO2 in air. After 72 h, the oocytes were stained with 1.9 �g/mL Hoechst 33342 in glycerol and then evaluated under UV light to determine the stage of meiosis as follows: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII) with first polar body. The results of Exp. 1 showed that maturation of canine oocytes to MII was significantly higher (P < 0.05) in medium supplemented with 1.11 mM glucose (4.8%) than for the control (1.8%) and the other glucose-supplemented groups (0 to 1.8%). In Exp. 2, oocytes cultured in cSOF supplemented with 0.5 mM pyruvate showed a significantly higher (P < 0.05) maturation rate to MII (6.3%) than did the other pyruvate-supplemented (0, 0.8, or 2.5%) groups or the control (2.4%). In Exp. 3, more oocytes were matured to the MII stage in cSOF supplemented with 5.0 mM lactate (7.3%) than were the other lactate-supplemented groups (0 to 2.4%) or the control (2.5%). Results of Exp. 4 showed more oocytes progressed to MII in cSOF supplemented with 0.5 mM pyruvate (8.2%), 1.11 mM glucose + 0.5 mM pyruvate (7.4%), or 1.11 mM glucose + 0.5 mM pyruvate 0.5 + 5.0 mM lactate (7.3%) than did the other combination groups (2.2 to 5.2%). In conclusion, the present study demonstrated that supplementing cSOF with 1.11 mM glucose, 0.5 mM pyruvate, or 5.0 mM lactate significantly increased the maturation of canine oocytes to MII, and the combined supplementation of 1.11 mM glucose, 0.5 mM pyruvate, and 5.0 mM lactate further promoted oocyte nuclear maturation compared to 1.11 mM glucose alone and the control. This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).

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