99 EFFECT OF CYSTEAMINE ON SURVIVAL OF BOVINE AND OVINE OOCYTES VITRIFIED USING THE MINIMUM VOLUME COOLING (MVC) CRYOTOP METHOD

2006 ◽  
Vol 18 (2) ◽  
pp. 158 ◽  
Author(s):  
J. Kelly ◽  
D. Kleemann ◽  
M. Kuwayama ◽  
S. Walker
Keyword(s):  
Ethylene Glycol ◽  
Sucrose Solution ◽  
Comparable Result ◽  
Individual Treatment ◽  
Minimum Volume ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Blastocyst Rate ◽  
Main Effects ◽  
Bovine Oocytes

The addition of cysteamine to maturation (IVM) media increases glutathione (GSH) synthesis in bovine oocytes and improves embryo development and quality (de Matos et al. 1995 Mol. Reprod. Dev. 42, 432–436). This study assesses the effect of adding cysteamine to IVM media on the survival of bovine and ovine oocytes following vitrification using the MVC cryotop method (Kuwayama and Kato 2000 J. Assist. Reprod. Genet. 17, 477 abst.). Abattoir-sourced bovine and ovine cumulus–oocyte complexes (COC) were matured in IVM media with or without 100 μM cysteamine for 24 h. After maturation, the COC were partially denuded and a proportion vitrified. Oocytes were equilibrated with 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 30 s and then exposed to 20% EG, 20% DMSO, 0.5 M sucrose, and 20% FCS for 15 s. Oocytes were loaded onto a MVC plate (Cryotop, Kitazato Supply, Tokyo), and plunged into liquid nitrogen. After 5 days, oocytes were thawed directly into 1.25 M sucrose solution at 38.5°C, followed by stepwise dilution of the cryoprotectants. Ova were subsequently fertilized (Day 0) and cultured in modified SOF. Oocyte survival was assessed by cleavage and development to Day 8 compared with the development of fresh oocytes. Main effects, interactions and individual treatment differences were tested using procedure CATMOD in SAS. Cleavage rate was higher (P < 0.001) for fresh oocytes than for vitrified oocytes and it increased (P < 0.05) only for fresh ovine oocytes when cysteamine was added to maturation media. Blastocyst development was influenced by a significant (P < 0.001) interaction between species and whether or not oocytes were vitrified. This interaction occurred because cysteamine improved blastocyst rate in fresh ovine and vitrified bovine oocytes but not in other treatments. These results show that bovine oocytes (38.3% blastocyst rate) can be vitrified successfully when maturation occurs in the presence of cysteamine; however, a comparable result did not occur in ovine oocytes (10.6% blastocyst rate) despite >70% cleavage. Table

scholarly journals 100EFFECTS OF USING MICRO-PIPETTE TIPS OF DIFFERENT DIAMETERS AND VOLUME OF VITRIFICATION SOLUTION ON THE VIABILITY OF IVM BOVINE OOCYTES AFTER VITRIFICATION

2004 ◽  
Vol 16 (2) ◽  
pp. 171
Author(s):  
Y. Inaba ◽  
O. Dochi ◽  
H. Koyama
Keyword(s):  
Ethylene Glycol ◽  
Calf Serum ◽  
Cleavage Rate ◽  
Chi Square ◽  
Pipette Tip ◽  
Blastocyst Rate ◽  
Vitrification Solution ◽  
Different Diameters ◽  
And Control ◽  
Bovine Oocytes

The objective of this study was to investigate the effects of the diameters of micro-pipette tips and the volume of vitrification solution (VS) on viability of IVM bovine oocytes after vitrification. COCs were aspirated from 2–5mm follicles of ovaries obtained at a local abattoir. COCs were matured for 19h in TCM-199 supplemented with 5% calf serum (CS) and 0.02mgmL−1 FSH at 38.5°C in an atmosphere of 5% CO2 in air. The matured oocytes were then vitrified on the basis of Kuwayama and Kato (2000 J. Assist. Reprod. Genet. 17, 477 abst). Matured oocytes were first exposed to 7.5% ethylene glycol (EG) and 7.5% DMSO in holding medium (HM; Dulbecco’s PBS supplemented with 20% CS) for 3min, and then equilibrated for 1min in 15% EG, 15% DMSO, and 0.5M sucrose in HM. Ten oocytes were loaded into each micro-pipette tip (MidAtlantic Diagnostics, Inc., Marlton, NJ, USA), and directly plunged into liquid nitrogen. Warming was performed by placing the narrow end of the micro-pipette tips directly into HM containing 0.5M sucrose; the tips maintained in this medium for 5min. After washing in HM, oocytes underwent an additional 3h of maturation. They were then subjected to IVF (Day 0). After IVF, morphologically intact oocytes were cultured. Oocytes matured for 20–21h were used as a control. The cleavage rate at Day 3 and blastocyst rate at Day 7 to 9 were based on the number of cultured oocytes, and analyzed using the chi-square method. In experiment 1, the oocytes were vitrified with 0.5μL of VS in micro-pipette tips with 150-, 200-, or 275-μm inner diameters (ID) (100 eggs per tip size). The number of morphologically intact oocytes was 64 (150μm), 62 (200μm), and 54 (275μm). The cleavage rates of morphologically intact oocytes at Day 3 of 150μm (45.3%) and 200-μm tips (45.2%) were significantly lower than that of 275-μm tips (53.7%) and the control (63.6%) (P&lt;0.05). The blastocyst rate of morphologically intact oocytes at Day 7 to 9 of 150-μm (9.4%) and 275-μm tips (14.8%) were significantly lower than that of the control (33.0%) (P&lt;0.05), and that of 200-μm tips (19.4%) also showed a tendency of being lower than that of the control (P&lt;0.1). In experiment 2, the oocytes were vitrified with 0.3 (70 eggs), 0.5 (60 eggs), or 1μL (60 eggs) of VS in micro-pipette tips with 200-μm ID. The number of morphologically intact oocytes was 40 (0.3μL), 32 (0.5μL), and 28 (1μL). The cleavage rates of morphologically intact oocytes at Day 3 of the 0.3μL (45.0%), 0.5μL (37.5%), and 1μL solutions (35.7%) were significantly lower than that of the control (67.6%) (P&lt;0.05). However, there were no differences in the blastocyst rate of morphologically intact oocytes at Day 7 to 9 among 0.3μL (15.6%), 0.5μL (28.1%), and 1μL solutions (17.9%), and control (23.9%). These results suggest that the viability of IVM bovine oocytes after vitrification may be improved by using micro-pipette tips with 200-μm ID and containing 0.5μL of VS.

108 NEGLIGIBLE LEVEL OF DNA DAMAGE IN BOVINE OOCYTES VITRIFIED USING MINIMUM VOLUME METHODS

2006 ◽  
Vol 18 (2) ◽  
pp. 162
Author(s):  
K. Papis ◽  
E. Stachowiak ◽  
M. Kruszewski ◽  
T. Iwanenko ◽  
T. Bartlomiejczyk
Keyword(s):  
Comet Assay ◽  
Dna Fragmentation ◽  
Sucrose Solution ◽  
Objective Assessment ◽  
Significant Proportion ◽  
Positive Control ◽  
Negative Control ◽  
Dna Integrity ◽  
Minimum Volume ◽  
Bovine Oocytes

A relatively high number of bovine cryopreserved oocytes analyzed by the comet assay (Men et al. 2003 Mol. Reprod. Dev. 64, 245) showed compromised DNA integrity. The DNA fragmentation (comet tails) was found in 29% of slow cooled oocytes, in 20% of oocytes vitrified in straws and in 24% of oocytes vitrified in open pulled straws (OPS). Present study used the comet assay to compare the DNA status of 151 in vitro matured bovine oocytes vitrified in straws, in OPS or in droplets. It was assumed that the droplet method (Papis et al. 2000 Theriogenology 54, 651), which has gentle pre-equilibration prior to vitrification, would offer better protection of DNA. OPS vitrification was performed using a solution consisting of 20% DMSO, 20% ethylene glycol (EG), and 0.5 M sucrose. For in-straw and in-droplet vitrification, VS14 (5.5 M EG and 1.0 M sucrose) solution was used. In these two methods pre-equilibration in 3% EG solution for 15 min was applied. Fresh oocytes exposed to 0.5 mM of hydrogen peroxide for 5 min served as the positive control. Fresh M II oocytes served as the negative control. The comet assay was performed according to the procedure of Men et al. (2003) with some modifications aimed at enhancing the sensitivity of the method. The zona pellucida was removed using 0.5% pronase solution, followed by placing of the oocytes in droplets of low-melting agarose on slides. These were subjected to overnight treatment in lysis buffer, followed by 40 min of DNA unfolding and 30 min electrophoresis. Following air drying, the slides were stained with DAPI fluorochrome and photographed. The pictures were saved as anonymous consecutive files to enable objective assessment. Of 119 vitrified oocytes, 112 (94%) were evaluated. The remainder were lost or displayed atypical pictures. The comets could not be analyzed with the Comet v.3.0 software, possibly due to the large size of each oocyte. Six main classes of comet tails were distinguished ranging from no tail (class 0) to heavy and long tail (class 5). Positive control oocytes displayed class 4 (36%) or 5 (64%) tails. Negative control oocytes formed class 0 (18%) to class 3 (47%) comet tails. The oocytes vitrified using minimum volume methods fell within the same range, with 80% and 76% of oocytes vitrified in droplets and OPS, respectively, forming class 1 or 2 tails. One OPS vitrified oocyte (2.2%) expressed a class 5 tail. A small but significant proportion of oocytes vitrified in straws (15.4%, P d 0.05, ANOVA) formed class 4 tails typical of positive control oocytes. In conclusion, in spite of pre-equilibration, a significant proportion of oocytes vitrified in straws contained detectable levels of DNA fragmentation, due probably to the lower cooling rate. The minimum volume protocols (the droplet and OPS methods) caused virtually no damage as assessed by the DNA comet assay. Results presented here differ from those reported previously. Reasons for differences remain to be established.

62 EXPOSURE TO ETHYLENE GLYCOL AND DIMETHYL SULFOXIDE CAUSES ACTIVATION AND SPINDLE ANOMALIES IN BUFFALO (BUBALUS BUBALIS) OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 131 ◽  
Author(s):  
M. De Blasi ◽  
E. Mariotti ◽  
M. Rubessa ◽  
S. Di Francesco ◽  
G. Campanile ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Embryo Development ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Meiotic Spindle ◽  
Blastocyst Development ◽  
Chi Square ◽  
Chi Square Test ◽  
Simple Exposure

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo. Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants

Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws

2000 ◽  
Vol 54 (1) ◽  
pp. 119-128 ◽  
Author(s):  
A.E. Hurtt ◽  
F. Landim-Alvarenga ◽  
G.E. Scidel ◽  
E.L. Squires
Keyword(s):  
Ethylene Glycol ◽  
Sucrose Solution ◽  
Bovine Oocytes

353 IN VITRO FERTILIZATION WITH FLOW-SORTED BUFFALO SPERM

2006 ◽  
Vol 18 (2) ◽  
pp. 283 ◽  
Author(s):  
M. Zhang ◽  
X. W. Liang ◽  
Y. Q. Lu ◽  
K. H. Lu
Keyword(s):  
Mineral Oil ◽  
Motile Sperm ◽  
Percoll Gradient ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Blastocyst Rate ◽  
Maximum Humidity ◽  
Cattle And Sheep

Flow cytometrically sorted X and Y sperm have been successfully used for IVF and the production of offspring in cattle and sheep (Maxwell et al. 2004 Anim. Reprod. Sci. 82–83, 79–95).The objective of this study was to test the feasibility of flow sorted buffalo sperm used in IVF systems and to establish a suitable IVF system for sorted buffalo sperm. Oocytes were aspirated from 2–6 mm follicles on the buffalo ovaries from a slaughterhouse and matured for 22–24 h in a 1-mL dish containing TCM199 + 10% OCS + 3% BFF (bovine folliciular fluid) + hormones at 38.5°C, 5% CO2 in air with maximum humidity. Semen was sorted by a flow-sorter (Clontech, Mountain View, CA, USA) into X- and Y-chromosome bearing sperm at 90% accuracy and stored at 4°C for later use with IVF. Sorted sperm were prepared for IVF by centrifugation (700g, 20 min) through a Percoll gradient (90%:45%), and washed (centrifugation at 700g, 5 min) in mTALP-BSA. For IVF, groups of 10–15 oocytes were transferred to 40-μL drops of mTALP-BSA and incubated with motile sperm at a concentration of 2 × 106 sperm mL−1 in each fertilization drop for 8–10 h under mineral oil. Presumptive zygotes were cultured until Day 8 in 25-µL drops of TCM–199 supplemented with 0.33 mM pyruvate and 10% NCS with granulosa cells at 38.5°C under 5% CO2 in air. Cleavage and blastocyst rates per oocyte insemination were recorded on Day 2 and Days 6–8 after insemination, respectively. Data were analyzed by ANOVA procedures with replicates and treatments in the model. There were significant differences in cleavage rate (42.23% vs. 52.28%) and blastocyst rate (20.62% vs. 27.67%) between sorted and unsorted sperm, respectively (Table 1). There were no significant differences in the proportions of blastocyst development rates on Days 6, 7, or 8 after insemination with sorted and unsorted sperm. The results indicate that sorted buffalo sperm from two bulls have been successfully used in an IVF system to produce sex-controlled embryos. Table 1. Cleavage and blastocyst rates with different sperm types This research was supported by grants from the National Natural Science Foundation of China (30360073) and the Guangxi Department of Science and Technology (0330004–13). The authors (M. Z. and X.W. L.) contributed equally to this work.

123 BOVINE OOCYTE VITRIFICATION USING THE CRYOTOP METHOD: EFFECT OF CUMULUS CELLS AND VITRIFICATION PROTOCOL ON SURVIVAL AND SUBSEQUENT DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 220
Author(s):  
X. L. Zhou ◽  
A. Al Naib ◽  
D. W. Sun ◽  
P. Lonergan
Keyword(s):  
Subsequent Development ◽  
Cumulus Cells ◽  
Theoretical Modelling ◽  
Blastocyst Development ◽  
Oocyte Vitrification ◽  
Permeability Characteristics ◽  
Survival And Development ◽  
Blastocyst Rate ◽  
Improve Rate ◽  
Bovine Oocytes

The ability to successfully cryopreserve mammalian oocytes has numerous practical and economic ethical benefits that may positively affect animal breeding programs and assisted conception in humans. However, oocyte survival and development following cryopreservation remain poor. The aim of the present study was (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) oocytes, (2) to compare empirical and theoretical vitrification protocol, and (3) to assess the effect of adding ice blockers to vitrification media on survival and development competence of bovine oocytes following vitrification using the Cryotop method. Bovine oocytes were collected from the ovaries of slaughtered cross-bred beef heifers. In Experiment 1, cumulus-enclosed and partially denuded GV and MII oocytes were vitrified in 15% EG + 15% DMSO + 0.5 M sucrose in 2 steps. In Experiment 2, GV oocytes were vitrified as above or using theoretical modelling based on permeability and osmotic tolerance characteristics (Wang et al. Reprod. Fertil. Dev. 21 141) in 30% EG +11.4% trehalose in 3 steps or 40% EG + 11.4% trehalose in 4 steps. In Experiment 3, GV oocytes were vitrified in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana, CA, USA) 1% X-1000, 1% Z-1000, or both in 3 steps. The survival, cleavage, and blastocyst rate of cumulus-enclosed oocytes was significantly higher (ANOVA) than those of partially denuded oocytes when vitrified at GV (93.8% v. 81.3%, 65.8% v. 47.3%, 11.3% v. 4.0%, respectively, P < 0.05). However, no significant effect of cumulus cover was detected between the two groups when vitrified at MII (93.0% v. 91.8%, 35.2% v. 36.8%, 5.0% v. 4.4%, respectively). Furthermore, cumulus-enclosed oocytes vitrified at the GV stage exhibited a significantly higher development competence than those vitrified at MII (P < 0.05). In Experiment 2, there were no significant differences in the survival, cleavage, and blastocyst rates among 3 protocols (86.0% v. 92.8% v. 91.2%, 44.8% v. 54.4% v. 45.6%, 5.0% v. 5.4% v. 4.0%, respectively). However, cleavage and blastocyst rate were significantly lower (P < 0.05) than nonvitrified control oocytes. In Experiment 3, the presence of ice-blockers did not improve rate or blastocyst development (P < 0.05). In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes. Theoretical analysis of permeability characteristics and tolerance limits alone may not be sufficient to improve vitrification protocols.

217 EFFECT OF PRE-EQUILIBRATION TIME ON THE SURVIVAL RATE OF MATURED BOVINE OOCYTES AFTER VITRIFICATION AND ON SUBSEQUENT EMBRYO DEVELOPMENT

2008 ◽  
Vol 20 (1) ◽  
pp. 188 ◽  
Author(s):  
A. H. Sugulle ◽  
O. Dochi ◽  
H. Koyama
Keyword(s):  
Survival Rate ◽  
Embryo Development ◽  
Exposure Time ◽  
Short Exposure ◽  
Equilibration Time ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Development Rates ◽  
Vitrification Solution ◽  
Bovine Oocytes

Prolonged exposure of oocytes to cryoprotectants causes cell injury, whereas a short exposure time results in insufficient permeation because of ice formation. This study was designed to determine the optimal pre-equilibration time and its effect on the survival rate of matured bovine oocytes after warming and on subsequent embryo development. Bovine COC were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH at 38.5°C in 5% CO2 in air. Then, the COC were partially denuded. The oocytes were pre-equilibrated in 100 μL of vitrification solution 1 (VS1) containing 7.5% ethylene glycol (EG), 7.5% DMSO, and 20% CS in TCM-199 for 0, 1, 3, and 5 min. Then, the oocytes were moved through 100-μL drops of vitrification solution 2 (VS2) containing 30% EG, 30% DMSO, 0.5 m sucrose (Suc), and 20% CS in TCM-199 for 30 s, loaded into cryotops, and immersed into liquid nitrogen. Oocytes were warmed by plunging the cryotops into 1 m Suc in TCM-199 supplemented with 20% CS for 1 min, placed in 0.5 m Suc in TCM-199 supplemented with 20% CS for 3 min, and finally in TCM-199 supplemented with 20% CS alone for 5 min. Frozen–thawed semen from a single bull (5 × 106 spermatozoa mL–1) was used for fertilization. Zygotes were vortexed to remove the cumulus cells 18 h after fertilization and cultured in CR1aa for 9 days. Data were analyzed by the chi-square test. Results are presented in Table 1. There were no differences in the survival rates of the control and vitrified oocytes. The cleavage rate of controls at both 24 and 48 h was greater (P < 0.01) than that of vitrified oocytes. Among pre-equilibration times, the cleavage rate of 0, 1, and 3 min pre-equilibrations at both 24 and 48 h was greater than with the pre-equilibration time of 5 min (P < 0.01). With respect to blastocyst development, the control oocytes showed greater development rates than the vitrified oocytes (P < 0.01), whereas the development rates were lower with the pre-equilibration time of 5 min (P < 0.01) than with the other pre-equilibration times. In conclusion, the results indicated that matured bovine oocytes could survive after vitrification and subsequently develop into blastocysts after IVF. However, the pre-equilibration time before vitrification affects cleavage and blastocyst development; a longer exposure time resulted in lower blastocyst development. Table 1. Effect of pre-equilibration time on matured bovine oocytes survival and on subsequent embryo development

Insulin influences developmental competence of bovine oocytes cultured in α-MEM plus follicle-simulating hormone

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 563-572 ◽  
Author(s):  
Gustavo Bruno Mota ◽  
Ingrid Oliveira e Silva ◽  
Danielle Kaiser de Souza ◽  
Flavia Tuany ◽  
Michele Munk Pereira ◽  
...  
Keyword(s):  
Basal Medium ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Serum Free ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Blastocyst Rate ◽  
Defined Culture ◽  
Bovine Oocytes

SummaryThe aim of this study was to evaluate the dose–response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus–oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus–oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P < 0.05) than the DCM control. Insulin concentrations of 1, 10, or 100 ng/ml decreased the relative levels of GDF9 and HSP70-1 transcripts in oocytes at the end of IVM (P < 0.05). The transcripts levels of PRDX1 decreased (P < 0.05) only when 10 or 100 ng/ml insulin was added to the DCM medium. No difference in levels of GLUT1 transcripts (P > 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.

152 DIELECTROPHORETIC BEHAVIOR OF BOVINE OOCYTES AND ZYGOTES IN RELATION TO DEVELOPMENT AND TRANSCRIPTIONAL ABUNDANCE

2007 ◽  
Vol 19 (1) ◽  
pp. 193
Author(s):  
D. Salilew-Wondim ◽  
F. Rings ◽  
M. Hoelker ◽  
D. Jennen ◽  
E. Tholen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Binding ◽  
Protein Biosynthesis ◽  
Control Group ◽  
Ion Binding ◽  
Transcriptional Level ◽  
Blastocyst Development ◽  
Blastocyst Rate ◽  
Bovine Oocytes

Selection of developmentally competent oocytes and zygotes based on external morphology has been employed to increase in vitro embryo production. However, this method is more often influenced by personal judgments and lacks universal standards. Hence, this study was aimed at investigating the dielectrophoretic behavior of oocytes and zygotes in relation to their development and mRNA abundance. To achieve the objective, 2 experiments were conducted. In the first experiment, matured bovine oocytes (PB+) and zygotes were subjected to a dielectrophoresis (DEP) procedure designed as follows: 4 MHz AC, 450-�m electrode distance, 5 V, and 80-�s cm-1 medium conductivity. The time elapsed for each of the oocytes and zygotes to reach one of the electrodes from the center was recorded. To accomplish this, 457 PB+ oocytes were subjected to the DEP procedure and 152, 121, 90, and 94 were classified as very fast, fast, slow, and very slow, respectively. Moreover, 152 oocytes were used as controls. Similarly, a total of 940 zygotes were subjected to the DEP procedure and classified as very fast (n = 329), fast (n = 329), slow (n = 97), and very slow (n = 245). In addition, 323 zygotes were used as the control group. The oocyte DEP groups were parthenogenetically activated, and the zygote DEP groups were further in vitro-cultured in a CR1 culture medium supplemented with 10% estrous cow serum, 20 mL mL-1 �-mercaptoethanol, and 10 mL mL-1 minimal essential medium. In PB+ DEP groups, the results show that the blastocyst rate (mean � SEM) at 7 days post-parthenogenetic activation was significantly (P &lt; 0.05) lower in the very slow moving group (7.2 � 4.9) compared to the very fast (21.8 � 3.2), fast (21.5 � 4.6), slow (23.3 � 4.5), and the control (19.7 � 3.7) groups. In zygotes, the blastocyst rate at 7 days post-insemination was significantly (P &lt; 0.05) higher in the very fast (16.1 � 2.7) compared to the slow (9.1 � 2.7) and very slow (10.6 � 2.7) groups, but it was not significantly higher than the fast (12.2 � 2.7) and control (12.3 � 2.7) groups. To investigate whether the transcriptional level of DEP separated very fast and very slow oocytes and zygotes, mRNA expression level was analyzed using a bovine cDNA microarray from a pool of 30 zygotes and oocytes in 6 replications including the dyeswap. The data analyzed using statistical analysis for microarray revealed that 31 and 5 genes were up- and down-regulated, respectively, in very fast compared to very slow PB+ DEP groups. The up-regulated genes are known to be involved in RNA binding and protein biosynthesis (RPL2, RPL8, and RPLPO), ion binding (PTGS2 and ANXA2), and cell cycle regulation (CDC91L1, NUSAP1, and CKS1B). Similarly, 25 and 17 genes were up- and down-regulated, respectively, in the very fast compared to the very slow zygote DEP groups. Some of these genes enriching the very fast zygotes are involved in ion binding (ZNF85, ZNF519, and NANOS1), regulation of cell cycle (NASP, SMARCA5, and AURKA), and signal transduction (RALA). Therefore, from this experiment we can conclude that dielectrophoretically separated oocytes and zygotes show differences in the rate of blastocyst development accompanied by differences in transcriptional abundances.

162 EFFECTS OF PROLACTIN AND DITHIOTHREITOL ON THE QUALITY AND DEVELOPMENTAL CAPACITY OF IN VITRO MATURED BOVINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 211
Author(s):  
G. Singina ◽  
I. Lebedeva ◽  
E. Shedova ◽  
N. Zinovieva
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Inhibitory Influence ◽  
Cleavage Rate ◽  
Subsequent Aging ◽  
Vitro Fertilization ◽  
Blastocyst Rate ◽  
System 1 ◽  
Bovine Oocytes

In vitro maturation (IVM) and IVF of domestic animal oocytes is widely used for commercial and research purposes. The oocyte quality and capacity for further development acquired during in vitro maturation and reduced during the subsequent aging are the main limitative factors affecting the embryo production (Miao et al. 2009 Hum. Reprod. Update 15, 573–585). Our objective was to evaluate effects of prolactin (PRL) and dithiothreitol (DTT) on apoptosis and the embryo development of bovine oocytes matured in vitro using 2 different systems. A total of 1437 slaughterhouse-derived cumulus-oocyte complexes (COC) were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. In system 1, 251 COC from a total of IVM oocytes were transferred to the aging medium (TCM-199 supplemented with 10% FCS) and cultured for 24 h in the absence (control) and the presence of either PRL (20 and 50 ng mL–1) or DTT (2.5, 5, and 10 μM). At the end of culture, oocyte apoptosis was detected using the TUNEL method. In system 2, another part of IVM oocytes (1186 COC) was co-incubated for 18 h with sperm in Fert-TALP medium modified by addition of 10 μg mL–1 heparin, PHE (20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine), and 0.1% modified Eagle’s medium (MEM) nonessential amino acids. In this case, PRL and DTT (at the above listed concentrations) were added directly to the fertilization medium. After IVF, oocytes were cultured in CR1aa medium for assessment of the cleavage and blastocyst rates on Days 2 and 8, respectively. The nuclear status of blastocysts was evaluated by the cytogenetic method. The data from 3–7 replicates were analysed by ANOVA. Culture of matured COC in the aging medium (system 1) increased the rate of apoptotic oocytes from 8.1 ± 4.7% (0 h) to 48.6 ± 5.8% (24 h) (P < 0.01). This rate was reduced (P < 0.05) up to 22.5 ± 3.1% and 17.8 ± 5.1% in the presence of PRL (20 and 50 ng mL–1) and up to 15.0 ± 6.9% and 19.5 ± 3.7% in the presence of DTT (2.5 and 5 μM). The direct addition of PRL at a concentration of 20 ng mL–1 to the IVF medium raised the blastocyst rate from 21.6 ± 2.2% to 29.8 ± 2.4% (P < 0.05) but did not affect the cleavage rate (72.1 ± 2.2% v. 74.3 ± 2.1%). By contrast, 50 ng mL–1 PRL did not increase the yield of blastocysts and decreased the cleavage rate (from 74.3 ± 2.1% to 62.9 ± 2.4%, P < 0.05). When added to the IVF medium, DTT raised the blastocyst rate only at a concentration of 2.5 μM (P < 0.05). No effects of PRL and DTT on the number of cells in embryos at the blastocyst stage were found. Our findings indicated that PRL and DTT supplements during in vitro fertilization of bovine oocytes may improve their capacity for the subsequent embryo development. This effect was probably due to the inhibitory influence of PRL and DTT on apoptosis of matured oocytes. The study was supported by the Federal Agency for Scientific Organizations and RFBR (project No. 14–48–03681).

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