Presence of cumulus cells during in vitro fertilization protects the bovine oocyte against oxidative stress and improves first cleavage but does not affect further development

Zygote ◽  
2005 ◽  
Vol 13 (2) ◽  
pp. 177-185 ◽  
Author(s):  
A. Nader Fatehi ◽  
Bernard A.J. Roelen ◽  
Ben Colenbrander ◽  
Eric J. Schoevers ◽  
Bart M. Gadella ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Formation Rate ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Physical Contact ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Vitro Fertilization

The present study was conducted to evaluate the function of cumulus cells during bovine IVF. Oocytes within cumulus–oocyte complexes (COCs) or denuded oocytes (DOs) were inseminated in control medium, or DOs were inseminated in cumulus cell conditioned medium (CCCM). DOs exhibited reduced cleavage and blastocyst formation rates when compared with intact COCs. The reduced blastocyst formation rate of DOs resulted from reduced first cleavage but subsequent embryo development was not changed. Live-dead staining and staining for apoptotic cells revealed no differences in blastocysts from oocytes fertilized as COC or DO. Fertilization of DOs in CCCM partially restored the cleavage rate, suggesting that factors secreted by cumulus cells are important for fertilization but that physical contact between oocytes and cumulus cells is required for optimal fertilization and first cleavage. Exposure of COCs to hydrogen peroxide shortly before fertilization reduced the cleavage rate, but did not lead to enhanced death of cumulus cells or oocyte death. Exposure of DOs to hydrogen peroxide, however, resulted in oocyte death and a complete block of first cleavage, suggesting that cumulus cells protect the oocyte against oxidative stress during fertilization.

294 IN VITRO DEVELOPMENT OF RAT OOCYTES FOLLOWING MICROINJECTION OF A CUMULUS CELL INTO THE OOPLASM AND CHEMICAL ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 262
Author(s):  
W. Fujii ◽  
H. Funahashi
Keyword(s):  
Chemical Activation ◽  
Formation Rate ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Female Rats ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Pronuclear Formation ◽  
Vitro Development

If diploid zygotes constituted with a somatic and a maternal genome could successfully develop to term, a new reproductive method would be developed to produce animals. However, there appears to be little information on this subject. In the present study, in vitro early development of the constituted zygotes was examined. A cumulus cell was microinjected into a rat non-enucleated oocyte, the reconstructed oocyte was chemically activated, and the pronuclear formation and in vitro development of the embryo was observed. Prepubertal Wistar female rats (21–27 days old) were induced to superovulate with an IP injection of 15 IU of eCG, followed by 15 IU of hCG 48 h later. Cumulus cells were removed from oocytes by pipetting with 0.1% hyaluronidase. Experiment 1: The DNA content of cumulus cells for microinjection was evaluated by flow cytometry. Experiment 2: The optimal concentration of SrCl2 for activation of rat oocytes was examined. Experiment 3: Cumulus cells were injected into mature oocytes in BSA-free HEPES-buffered mKRB containing 0.1% polyvinyl alcohol (PVA) and cytochalasin B (5 �g mL-1), and were then chemically activated by treatment in Ca2+-free mKRB containing 5 mM SrCl2 for 20 min at 0 to 0.5 (A), 1 to 1.5 (B), or 3 to 3.5 h (C) after injection. Activated embryos were cultured in droplets of mKRB in an atmosphere of 5% CO2 in air at 37�C for 9 to 12 h. After being observed for pronuclear formation, the embryos were transferred into mR1ECM-PVA, and the cleavage and blastocyst formation rates were examined 24 and 120 h later, respectively. Results from 3 to 7 replicates were analyzed by ANOVA and Duncan's multiple range test. A total of 90.0 and 9.5% of cumulus cells derived from ovulated oocyte–cumulus complexes contained 2C and 4C DNA contents, respectively. Survival rates did not differ among oocytes stimulated with 0 to 5 mM SrCl2 (96.7–100%) but did differ between those stimulated with 1.25 and 10 mM SrCl2 (100 and 72.9%, respectively). Activation rates of oocytes increased at higher SrCl2 concentrations and were higher at 5 and 10 mM (92.6 and 98.5%, respectively) than at other concentrations. When cumulus-injected oocytes were activated after various periods after the injection, the incidences of pronuclear formation and cleavage did not differ among the periods (A: 95.0 and 81.3%; B: 85.6 and 85.0%; and C: 82.7 and 84.6%, respectively). Although a majority of the embryos developed to the 2- to 4-cell stages (78.7%; 152/208), the blastocyst formation rate was very low (0.8%; 2/208). In conclusion, rat non-enucleated oocytes injected with a cumulus cell can form pronuclei and cleave following chemical activation, but blastocyst formation of the embryos is very limited.

219 APPLICATION OF A MICROFLUIDIC SPERM SORTER TO THE IN VITRO PRODUCTION OF PORCINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 208
Author(s):  
H. Sano ◽  
K. Matsuura ◽  
K. Naruse ◽  
H. Funahashi
Keyword(s):  
Seminal Fluid ◽  
Formation Rate ◽  
Defined Medium ◽  
Laminar Flows ◽  
Efficient Production ◽  
Blastocyst Formation ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Vitro Fertilization

In porcine in vitro fertilization (IVF), polyspermy is a persistent obstacle to the efficient production of normal embryos. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) was developed to isolate motile human spermatozoa (from diluted semen by two laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. We previously reported that the monospermy rate was higher when oocytes were co-cultured with isolated spermatozoa in an MFSS for 5 min than when spermatozoa were co-cultured traditionally in drops for 8 h (P < 0.05; Reprod. Fertil. Dev. 20, 187 abst). The present study was undertaken to compare the effect of oocyte location within the MFSS chamber on early development after IVF in the MFSS. A sperm-rich fraction from Berkshire boars was diluted at 1 × 108 cells mL–1 with modified Modena solution containing 20% seminal fluid. In the first experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine (mm199-caf) for 5 min at room temperature. Before flowing, porcine IVM oocytes were positioned in a part of the MFSS chamber where motile spermatozoa would accumulate with mm199-caf. After flowing for 5 min, those oocytes were cultured in caffeine-free mm199 for 8 h and then in a chemically defined medium (PZM-5) for 7 days. In the second experiment, denuded oocytes were co-cultured with isolated spermatozoa at several locations in the MFSS chamber, and the penetration and monospermy rates were estimated. The concentration of motile spermatozoa was also measured at each place in the MFSS chamber after isolation for 5 min. Statistical analyses of results based on 4 to 5 replicates were carried out by ANOVA and Fisher’s PLSD post hoc test (significance, P < 0.05). When IVM oocytes were co-cultured with spermatozoa gradually accumulated in the chamber of the MFSS for 5 min, the cleavage rate (83.7 ± 6.3% of 121 oocytes) was not different from that of control oocytes co-cultured with spermatozoa (5.7 × 105 cells mL–1) in 100-μL drops for 5 min (84.6 ± 6.6% of 126 oocytes). However, the blastocyst formation rate (38.2 ± 3.3%) was higher than for the controls (20.6 ± 6.8%; P < 0.05). After flowing for 5 min, the distance from the inflow opening of the MFSS chamber to the location of the oocytes did not affect the sperm penetration rate, but did affect the monospermy rate (14.0 ± 4.0% of 48 oocytes at the nearest position to 50.0 ± 5.6% of 43 oocytes at the furthest position; P < 0.05). After flowing for 5 min, the concentration of motile spermatozoa was also different at each location (57.5 ± 5.6 × 104 cells mL–1 at the nearest position to 0.8 ± 0.5 × 104 cells mL–1 at the furthest position; P < 0.05). These observations demonstrate that co-culturing oocytes with spermatozoa that gradually accumulated in the MFSS chamber improved the efficiency of blastocyst formation in the pig, whereas efficiency was affected by the position where oocytes were located in the chamber.

scholarly journals 288EFFECTS OF OVIDUCTAL EPITHELIAL AND CUMULUS CELLS ON FERTILIZATION AND EMBRYO DEVELOPMENT OF PIG OOCYTES IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 264
Author(s):  
B.S. Yang ◽  
B.N. Day
Keyword(s):  
Epithelial Cells ◽  
Embryo Development ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Treatment Groups ◽  
Sperm Penetration

This study was carried out to investigate the effect of adding porcine oviductal epithelial cells (POEC) and also the presence of cumulus cells during in vitro fertilization on fertilization rate and subsequent embryo development of pig oocytes matured in vitro. Cumulus-oocyte complexes (COCs) aspirated from 2- to 6-mm follicles were matured in TCM 199 supplemented with cysteine, EGF, eCG, hCG, and PVA for 20–22h, and cultured in the same medium without hormone for an additional 20–22h. Oviducts attached to ovaries without CL were used to obtain epithelial cells. After removal of the fimbria and utero-tubal junction, oviducts were flushed with MEM supplemented with 10% FBS, and POEC clumps were cultured in the same medium for 48h. After the completion of maturation, COCs were randomly divided into four groups;; cumulus-denuded (D), cumulus-denuded with POEC (DP), cumulus-enclosed (E), and cumulus-enclosed with POEC (EP). Eight to 10 POEC clumps were co-cultured with sperm and oocytes in a 100-μL fertilization drop. Oocytes were fertilized with frozen-thawed spermatozoa for 6h in modified Tris-buffered medium containing caffeine and BSA. Presumptive zygotes were cultured in NCSU23. Oocytes were fixed and stained for the evaluation of penetration at 12h after IVF (n=549 oocytes), and cleavage rate and blastocyst formation were evaluated at 48 and 144h after IVF (n=1531 oocytes), respectively. Results were analyzed by Duncan’s multiple range test using GLM procedure in SAS. Although the sperm penetration rate in group E was lowest among all groups (P&lt;0.05), the monospermic fertilization rate was not significantly different among treatment groups (68.6–81.9%). Although the cleavage rate and percentage blastocyst in group E were significantly lower than other groups (38.1 v. 53.6, 52.0 and 44.6%, and 15.0 v. 21.2, 23.4 and 18.5% in group D, DP, and EP, respectively), blastocyst cell number was not significantly different among treatment groups (24.9–27.3) These results suggested that the presence of cumulus cells alone during fertilization interferes with sperm penetration, cleavage, and blastocyst formation and that POEC may improve both cleavage and blastocyst formation rate.

Antioxidant β-cryptoxanthin enhances porcine oocyte maturation and subsequent embryo development in vitro

2018 ◽  
Vol 30 (9) ◽  
pp. 1204 ◽  
Author(s):  
Yun-Gwi Park ◽  
Seung-Eun Lee ◽  
Yeo-Jin Son ◽  
Sang-Gi Jeong ◽  
Min-Young Shin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Polar Body ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Control Group ◽  
Mrna Levels ◽  
Cleavage Rate ◽  
Antioxidant Genes ◽  
Developmental Potential

Oxidative stress is partly responsible for the poor quality of IVM oocytes. The present study investigated the effects of the antioxidant β-cryptoxanthin on the IVM of porcine oocytes and the in vitro development of the ensuing embryos. Oocytes were matured in IVM medium containing different concentrations of β-cryptoxanthin (0, 0.1, 1, 10 or 100 μM). Treatment with 1 µM β-cryptoxanthin (Group 1B) improved polar body extrusion and the expression of maturation-related genes in cumulus cells and oocytes compared with control. In addition, levels of reactive oxygen species decreased significantly in Group 1B, whereas there were significant increases in glutathione levels and expression of the antioxidant genes superoxide dismutase 1 and peroxiredoxin 5 in this group. After parthenogenetic activation, although the cleavage rate did not differ between the control and 1B groups, the blastocyst formation rate was higher in the latter. Moreover, the total number of cells per blastocyst and relative mRNA levels of pluripotency marker and antioxidant genes were significantly higher in the 1B compared with control group. These results demonstrate that β-cryptoxanthin decreases oxidative stress in porcine oocytes and improves their quality and developmental potential.

10-gingerol induces oxidative stress through HTR1A in cumulus cells: in-vitro and in-silico studies

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Kiptiyah Kiptiyah ◽  
Widodo Widodo ◽  
Gatot Ciptadi ◽  
Aulanni’am Aulanni’Am ◽  
Mohammad A. Widodo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Culture ◽  
Superoxide Dismutase ◽  
In Silico ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Sod Activity ◽  
In Silico Studies ◽  
Incubation Periods

AbstractBackgroundWe investigated whether 10-gingerol is able to induce oxidative stress in cumulus cells.MethodsFor the in-vitro research, we used a cumulus cell culture in M199, containing 10-gingerol in various concentrations (0, 12, 16, and 20 µM), and detected oxidative stress through superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentrations, with incubation periods of 24, 48, 72, and 96 h. The obtained results were confirmed by in-silico studies.ResultsThe in-vitro data revealed that SOD activity and MDA concentration increased with increasing incubation periods: SOD activity at 0 µM (1.39 ± 0.24i), 12 µM (16.42 ± 0.35ab), 16 µM (17.28 ± 0.55ab), 20 µM (17.81 ± 0.12a), with a contribution of 71.1%. MDA concentration at 0 µM (17.82 ± 1.39 l), 12 µM (72.99 ± 0.31c), 16 µM (79.77 ± 4.19b), 20 µM (85.07 ± 2.57a), with a contribution of 73.1%. Based on this, the in-silico data uncovered that 10˗gingerol induces oxidative stress in cumulus cells by inhibiting HTR1A functions and inactivating GSK3B and AKT˗1.Conclusions10-gingerol induces oxidative stress in cumulus cells through enhancing SOD activity and MDA concentration by inhibiting HTR1A functions and inactivating GSK3B and AKT˗1.

206 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON IN VITRO EMBRYO PRODUCTION

2008 ◽  
Vol 20 (1) ◽  
pp. 182 ◽  
Author(s):  
K. Imai ◽  
Y. Inaba ◽  
H. Yoshioka ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cumulus Cell ◽  
Oocyte Quality ◽  
Annual Conference ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Follicular Wave ◽  
The Mean ◽  
In Vitro Embryo Production

We previously reported that follicular wave synchronization, by removal of the dominant follicle on Day 5 after ovum pickup (OPU), was effective in increasing oocyte quality in the developing follicles (Imai et al. 2006 32th Annual Conference of the IETS, poster presentation no. 277). The current study was designed to examine the effect of superstimulatory treatment to induce subsequent follicular wave synchronization on embryo production by OPU and IVM-IVF-IVC in Holstein dry cows. Cows were reared under the same feeding and environmental conditions, and 2 OPU sessions were conducted in each cow. In the first session, OPU was performed in 8 cows on arbitrary days of the estrous cycle by using a 7.5-MHz linear transducer with needle (Cova needle, Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200, Aloka, Tokyo, Japan). Follicles larger than 8 mm in diameter were then aspirated and a CIDR was inserted on Day 5 (the day of first OPU session = Day 0). Cows then received 30 mg of FSH (Antrin-R10; Kawasaki Mitaka Pharmaceutical Co., Tokyo, Japan) twice a day from Days 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 mg) by i.m. injection. Cloprostenol (PGF; Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan; 0.75 mg) was administered in the morning of Day 9 (third day of superstimulation). The second OPU session was performed 48 h after PGF administration (Day 11), and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Collected oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. Grades 1 and 2 COC were matured, fertilized, and cultured as described by Imai et al. [2006 J. Reprod. Dev. 52(Suppl.), S19–S29]. Embryo development was assessed by the cleavage rate on Day 2 and by the blastocyst formation rate on Days 7 to 8 (the day of insemination = Day 0). Data were analyzed by Student's t-test. There were no differences in the mean (� SD) number of aspirated follicles or collected oocytes between the first (32.5 � 6.8 and 26.0 � 12.7, respectively) and second (29.3 � 10.4 and 19.0 � 9.4, respectively) OPU sessions (P > 0.1). The percentage of Grade 1 and 2 oocytes for the second OPU session (90.5 � 13.8%) was significantly higher (P < 0.01) than for the first OPU session (63.1 � 6.3%), and significant differences were found for cleavage (79.4 � 14.1, 61.8 � 25.1, P < 0.01) and blastocyst rates (68.1 � 16.7, 24.2 � 22.3, P < 0.001) between sessions. The mean numbers of blastocysts obtained per session were 4.3 � 2.9 and 12.8 � 8.7 in the first and second sessions, respectively (P < 0.01). These results indicate that superstimulatory treatment and subsequent follicular wave synchronization were effective on in vitro embryo production by increasing the oocyte quality.

256 EFFECT OF SPERM SELECTION ON THE RATE OF IN VITRO FERTILIZATION IN ALPACA (VICUGNA PACOS)

2015 ◽  
Vol 27 (1) ◽  
pp. 217
Author(s):  
E. Mellisho ◽  
V. Rivas ◽  
J. Ruiz ◽  
G. Mamani
Keyword(s):  
Extended Period ◽  
Cumulus Cells ◽  
Sperm Selection ◽  
Cleavage Rate ◽  
Slow Cooling ◽  
Vitro Fertilization ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Washing Method

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P < 0.05) using SAS® version 8.0 for Windows. The rate of cleavage (cell division) did not show statistical differences (P = 0.67) for the swim-up method (37%; 155/421) v. washing method (35%; 147/418). The methods of sperm selection (swim-up and washing) did not affect the rate of IVF.

168 INHIBITION OF BOVINE OOCYTE CUMULUS CELL PROGESTERONE SYNTHESIS DURING IN VITRO MATURATION AFFECTS CHROMOSOME ALIGNMENT AND SPINDLE INTEGRITY IN FERTILIZED EGGS AND EARLY EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 198
Author(s):  
E. Daly ◽  
A. G. Fahey ◽  
M. M. Herlihy ◽  
T. Fair
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Spindle Formation ◽  
Vitro Fertilization ◽  
Time Treatment

We have previously demonstrated the importance of progesterone (P4) synthesis by cumulus cells during oocyte maturation in vitro (IVM) for bovine oocyte acquisition of developmental competence and subsequent embryo development (Aparicio et al. 2011 Biol. Reprod. 84). The aim of this study was to identify key processes that may be deregulated by the inhibition of P4 signalling in the cumulus–oocyte complex (COC) during IVM. To this end, good quality immature COC were placed in IVM medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL–1 epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO2, in the presence or absence of 10 μM trilostane (which blocks P4 synthesis by inhibiting 3 β-hydroxysteroid dehydrogenase; Stegram Pharmaceuticals Ltd., Surrey, UK). Matured COC were washed and placed in 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL–1 fatty acid-free BSA, and 10 mg mL–1 heparin). In vitro fertilization (IVF) was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 106 spermatozoa mL–1 at 39°C under 5% CO2 during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO2, 90% of N2, and 5% O2 atmosphere with maximum humidity. Subsets of presumptive fertilized eggs and developing embryos were recovered at 6, 72, 120, and 192 h postinsemination (hpi) and processed for confocal whole-mount immunocytochemistry. The meiotic and mitotic spindles and chromosomes were visualised by immunofluorescent labelling of α-tubulin and 4′,6-diamindino-2-phenylindole (DAPI), respectively, and classified as normal if the chromosomes were correctly aligned or appropriately segregated, or abnormal if lagging chromosomes or abnormal chromosome segregation were observed. Samples were collected from 5 replicates (n = 50 zygotes/embryos per treatment, per timepoint) and a total of 157 spindles were observed. Logistic regression analysis was conducted to determine the probability of abnormal spindle formation. The incidence of spindle abnormality was regressed on time, treatment, and treatment by time. For all time points, there was significant reduction in the odds of abnormal spindle formation in control samples versus trilostane-treated samples (P < 0.001). In conclusion, our data imply a role for P4 signalling in maintaining spindle integrity during oocyte meiotic maturation and progression through the initial mitotic divisions of early embryo development in cattle.

Technical Report: Effect of commercially available PMSG on maturation, fertilization and embryo development of buffalo oocytes in vitro

2001 ◽  
Vol 13 (6) ◽  
pp. 355 ◽  
Author(s):  
P. S. P. Gupta ◽  
S. Nandi ◽  
B. M. Ravindranatha ◽  
P. V. Sarma
Keyword(s):  
Embryonic Development ◽  
Genetic Manipulation ◽  
Culture Media ◽  
Cell Monolayer ◽  
Basic Research ◽  
Cost Effective ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
Vitro Fertilization

In vitro fertilization (IVF) technology provides an opportunity to produce embryos for genetic manipulation, embryo transfer and basic research in developmental physiology, and can be exploited for emerging biotechnologies such as transgenesis and cloning. In the present study, the effects of different concentrations of commercially available pregnant mare serum gonadotrophin (PMSG) (Folligon; Intervet, International B.V., Boxmeer, Holland) in oocyte culture media, on maturation, fertilization and embryonic development of buffalo oocytes in vitro were investigated. Oocytes aspirated from abattoir-derived ovaries were cultured in media containing TCM-199 + PMSG at 0, 2.5, 20, 30, 40 and 50 IU mL–1 in presence or absence of steer serum (10%) for 24 h in a CO2 incubator. The maturation rate was assessed on the basis of degree of expansion of cumulus cells. The matured oocytes were inseminated with 9–10 x 106 spermatozoa mL–1 in Brackett and Oliphant medium and the cleavage rate was recorded 40–42 h after insemination. Uncleaved oocytes were stained with aceto-orcein for evaluation of fertilization rates. The cleaved embryos were further cultured in TCM-199 + 10% steer serum on buffalo oviducal cell monolayer for 7 days. Maturation, fertilization, cleavage and embryonic development were significantly higher (P<0.05) in oocytes cultured in TCM-199 + 10% steer serum supplemented with 40 and 50 IU PMSG mL–1. It is concluded that commercially available PMSG can effectively be used in place of pure follicle-stimulating hormone for in vitro maturation of buffalo oocytes, making it cost effective for IVF studies.

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