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

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.

242 USE OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN IN VITRO PREMATURATION OF BOVINE OOCYTES SUBJECTED TO PARTHENOGENETIC ACTIVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 200
Author(s):  
T. H. C. De Bem ◽  
R. Rochetti ◽  
P. R. L. Pires ◽  
F. F. Bressan ◽  
P. R. Adona ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Polar Body ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Parthenogenetic Activation ◽  
Bovine Oocytes ◽  
Hatching Rates

Prematuration provides an additional time for oocyte capacitation and maturation in an attempt to improve in vitro embryo production (IVP) rates and allows media supplementation during this period for IVP. The aim of this study was to use brain-derived neurotropic factor (BDNF) in prematuration to improve maturation of bovine oocytes subjected to parthenogenetic activation and cultured with different media. Oocytes were subjected to prematuration in TCM-199 medium supplemented with 10 µm butyrolactone I, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin for 24 h in the absence of BDNF (control) or in the presence of 10 ng mL–1 BDNF (BD). Oocytes were then in vitro-matured (IVM) in TCM-199 medium supplemented with 10% FCS, 0.5 µg mL–1 FSH, 5.0 µg mL–1 LH, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin at 38.5�C under 5% CO2 in air. After 19 h oocytes were denuded using hyaluronidase and vortexing for 3 min for the 1st polar body (1PB) selection. Those which extruded the 1PB were maintained in IVM until 26 h, when parthenogenetic activation was performed (5 min in 5 µm ionomycin, followed by 3 h in 2 mm 6-DMAP). Activated oocytes were then transferred to in vitro culture (IVC) for embryo development evaluation. Embryos from both groups were cultured in SOF medium with 2.5% FCS, 0.05 g mL–1 BSA, 0.2 mm pyruvate, and 10 mg mL–1 gentamicin. Cleavage rates on the second day of in vitro culture (D2), embryo production at Days 7 and 8 (D7 and D8), and hatching rate at Day 8 were evaluated. Data regarding 1PB extrusion, cleavage, blastocyst development on D7 and D8, and blastocyst D8 hatching rates of three replicates were analyzed by chi-square test at 5% significance using the BIOESTATS 4.0 software. Control and BD, respectively, did not show differences (P > 0.05) regarding 1PB extrusion (n = 164, 63.81%, and n = 175, 66.79%) or cleavage (n = 117, 71.34%, and n = 138, 78.86%). However, for control and BD, respectively, blastocyst development on D7 (n = 63, 38.41%, and n = 89, 50.86%), D8 (n = 63, 38.41%, and n = 91, 52.00%), and hatching on D8 (n = 22, 34.92%, and n = 39, 43.82%) were all significantly higher for BD when compared with control (P < 0.05). In conclusion, BDNF during prematuration improved in vitro embryo development by increasing blastocyst and hatching rates of parthenogenetic embryos.

The effect of steer sera generating low or high concentrations of ammonia in culture on bovine embryo development and cell number in vitro

1999 ◽  
Vol 1999 ◽  
pp. 2-2 ◽  
Author(s):  
M. Kuran ◽  
M.E. Staines ◽  
G.J. McCallum ◽  
A.G. Onal ◽  
T.G. McEvoy
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Cell Monolayers ◽  
High Concentrations ◽  
Oviduct Fluid ◽  
Bovine Oocytes

Ovine embryos produced in synthetic oviduct fluid (SOF) medium or in coculture with granulosa cell monolayers supplemented with low (A; 120 μmol/l) and high (B; 190 μmol/l) ammonia-producing steer sera caused different degrees of fetal oversize (Carolan et al., 1998). The objective of the present study was to determine whether the effects on fetal growth induced by these sera were associated with alterations in early embryo development.A total of 911 bovine oocytes, used in 8 replicates to test the effect of three culture treatments on embryo development, were matured and fertilized in vitro (IVF= Day 0). Presumptive zygotes were allocated on Day 1 to culture in SOF supplemented with 10% v/v steer serum (SOF+A, n=308; SOF+B, n=302) or with amino acids plus 0.4% w/v crystalline BSA (SOFaaBSA, n=301). All cultures were in 20 μl droplets under oil (38.5°C; 5% CO2, 5% O2; 4 zygotes per drop) and droplets were renewed every 48 h. Cleavage rate was recorded on Day 3. On Days 7 and 8, blastocyst yields, grade 1 and 2 blastocysts, their cell numbers (by staining with Hoechst 33342) and their stage and diameter were determined.

scholarly journals Effect of storage tube material and resveratrol during liquid storage of matured bovine oocytes on subsequent development

2017 ◽  
Vol 65 (4) ◽  
pp. 546-555
Author(s):  
Tayita Suttirojpattana ◽  
Tamás Somfai ◽  
Satoko Matoba ◽  
Takashi Nagai ◽  
Rangsun Parnpai ◽  
...  
Keyword(s):  
Embryo Development ◽  
Subsequent Development ◽  
Control Group ◽  
Tube Material ◽  
Blastocyst Development ◽  
Liquid Storage ◽  
Glass Tubes ◽  
Bovine Oocytes

This study determined the optimum storage vessel and the effects of resveratrol for the storage of in vitro matured (IVM) bovine oocytes. After IVM, the oocytes were kept in a Hepes-buffered medium at 25 °C for 20 h in different containers including Eppendorf tubes (ET) made of polypropylene (PP) and polystyrene (PS), and tissue culture tubes (TCT) made of PP, PS, and glass. Then oocytes were subjected to IVF and subsequent in vitro embryo development was compared among the groups and to that of a control group without storage. The percentage of blastocyst development in the control group was significantly higher than in the stored groups (P < 0.05). Among oocytes stored in TCT, the percentage of blastocyst development of oocytes stored in glass TCT was significantly higher than that of oocytes stored in PP and PS TCT (P < 0.05); however, it did not differ from that of oocytes stored in ET. The quality of blastocysts did not differ among the control and stored groups. Embryo development was not affected when 0.1, 1 or 10 μM resveratrol was added to the medium during oocyte storage. In conclusion, glass tubes were optimal for oocyte storage and resveratrol did not improve the development of stored oocytes.

329 SUPPRESSION OF BOVINE OOCYTE IVM BY SERUM AND FSH DEPRIVATION OR BY α-AMANITIN: EFFECT ON FERTILIZATION AND EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 272
Author(s):  
K. Kananen-Anttila ◽  
M. Eronen ◽  
J. Matilainen ◽  
M. Kallio ◽  
J. Peippo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Embryo Cleavage ◽  
Bovine Oocytes ◽  
Sperm Aster

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μ­m; serum+FSH-free: 12 ± 0.5 μ­m, P < 0.05; α-amanitin: 10 ± 0.6 μ­m, P < 0.001) and sperm asters (control: 86 ± 4 μ­m; serum+FSH-free: 82 ± 4 μ­m, P < 0.01; α-amanitin: 49 ± 7 μm, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription. Table 1. Average percentages ± (n) for nuclear maturation, fertilization (min two pronuclei), embryo cleavage, and blastocyst development

75 ADDITION OF HYALURONAN TO A VITRIFICATION SOLUTION FOR IMMATURE BOVINE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE

2013 ◽  
Vol 25 (1) ◽  
pp. 185
Author(s):  
P. Rodriguez Villamil ◽  
F. Ongaratto ◽  
M. Fernandez Taranco ◽  
G. A. Bó
Keyword(s):  
Ethylene Glycol ◽  
Solid Phase ◽  
Animal Health ◽  
Survival Rates ◽  
Control Group ◽  
Cresyl Blue ◽  
Development Rates ◽  
Vitrification Solution ◽  
Bovine Oocytes

An experiment was designed to evaluate the effect of brilliant cresyl blue (BCB) selection of immature oocytes and the addition of sodium hyaluronate (HA) to the vitrification solution on survival rates of bovine oocytes vitrified using solid-phase vitrification. Bovine cumulus–oocyte complexes (COC; n = 716) obtained from slaughterhouse ovaries were used in 6 replicates. Cumulus–oocyte complexes were washed in tissue culture medium 199 (TCM-199) and randomly allocated to 2 groups to be exposed to BCB stain (Sigma Chemical Company, St. Louis, MO, USA) for 90 min as described by Alm et al. (2005 Theriogenology 63, 2194–2205) or (control) maintained in Vigro holding medium (Bioniche Animal Health, Belleville, Canada) for 90 min (n = 220). Cumulus–oocyte complexes in the BCB group were selected based on their response to BCB as BCB+ (colored, n = 248) or BCB– (colorless, n = 248), whereas those in the control group were selected morphologically as described by Rodríguez-González et al. (2002 Theriogenology 57, 1397–1409). Oocytes from both BCB groups and 100 oocytes in the control group were vitrified by solid-phase vitrification as previously described by Rodriguez et al. (2012 Reprod. Fertil. Dev. 24, 132). The remaining 120 oocytes in the control group were not vitrified and were matured, fertilized, and cultured in vitro (in SOFaa in a controlled atmosphere) for 7 days. Vitrified oocytes were exposed to 10% ethylene glycol for 10 min, and 20% ethylene glycol + 0.2-M trehalose for 30 s, and then were subdivided to be exposed to 30% ethylene glycol + 0.5-M trehalose with or without 0.1 mg mL–1 HA (MAP 5, Bioniche Animal Health). Vitrified oocytes were stored in liquid nitrogen for at least one week and then placed directly into a 0.5-M sucrose solution (in TCM 199) at 37°C for 5 min, 0.25 M of sucrose for another 5 min, and finally TCM-199 and matured, fertilized, and cultured. Development rates (i.e. proportion of blastocysts) were examined on Day 7 after fertilization. Proportional data were first transformed by square root and then analyzed by ANOVA to detect the effect of replicate, type of oocyte (BCB+, BCB–, controls), and vitrified with or without HA or not vitrified as main effects, using the software Infostat (UNC, Argentina, 2010). There was a significant effect of oocyte type on blastocyst rate (P < 0.01) following vitrification (BCB+, 6.4 ± 0.4%. v. BCB–, 1.6 ± 0.6%). Control oocytes (not exposed to BCB) resulted in 3.0 ± 2.0% blastocysts following vitrification, which was lower to that obtained with the BCB+ oocytes. Vitrification also influenced development rates (3.0 ± 2.0 v. 32.0 ± 1.3%) for blastocysts produced from vitrified v. nonvitrified oocytes, respectively (P < 0.01). Furthermore, the use of HA in the vitrification solutions did not have a significant effect on development rates (4.7 ± 0.9 v. 3.3 ± 0.9%, for blastocysts obtained from vitrified oocytes with or without HA, respectively). In conclusion, the selection of oocytes by BCB increased the in vitro development rates of vitrified immature oocytes, whereas the use of HA in the vitrification solution did not improve the survival rates of vitrified oocytes.

63 QUALITY OF BOVINE EMBRYOS PRODUCED IN VITRO FROM IMMATURE OOCYTES TREATED WITH A SUBLETHAL HYDROSTATIC PRESSURE

2013 ◽  
Vol 25 (1) ◽  
pp. 179
Author(s):  
C. Díez ◽  
B. Trigal ◽  
J. N. Caamaño ◽  
M. Muñoz ◽  
E. Correia ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Pressure Treatment ◽  
Cell Counts ◽  
Development Rates ◽  
Bovine Oocytes

High hydrostatic pressure (HHP) treatment of immature porcine oocytes improves embryo development rates and cell numbers (Pribenszky et al. 2008 Anim. Reprod. Sci. 106, 200–207). However, it is unknown if similar effects can be obtained with bovine oocytes and how HHP affects cryopreservation of the developed blastocysts. In this work, we analyzed the effect of an HHP treatment (Cryo-Innovation Ltd., Budapest, Hungary) on bovine cumulus–oocyte complex (COC) as determined by their developmental ability and embryo quality. Immature COC were submitted to a pressure treatment (200 bar, 1 h at 37°C; HHP group; n = 643) in HEPES-buffered TCM199. Simultaneously, a group of COC was held at 37°C for 1 h (T group; n = 304) in HEPES-buffered TCM199, while other COC were untreated (n = 1182). After in vitro maturation, COC were fertilized in vitro (IVF) and cultured in modified SOF + 6 g L–1 BSA (Holm et al. 1999 Theriogenology 52, 683–700), and embryo development was recorded (5 replicates). Day 7 and 8 excellent- and good-quality embryos were selected for vitrification (cryologic vitrification method; Trigal et al. 2012 Theriogenology 10.1016/j.theriogenology.2012.06.018). After warming, vitrified blastocysts were cultured in modified SOF + 6 g L–1 BSA + 10% FCS for 48 h (3 replicates). Those blastocysts hatching after warming (at 24 and 48 h) were fixed and stained for differential cell counts. Data were analyzed by ANOVA and REGWQ test and are presented as least squares means ± standard error. The HHP-treated oocytes showed increased development rates on Day 3 (Day 3 ≥5-cell embryos: 64.5 ± 2.9a, 53.4 ± 3.9b, 56.7 ± 2.2b for HHP, T, and untreated groups, respectively; a v. b: P < 0.05); however, D8 blastocyst rates were not affected by the pressure treatment (28.5 ± 1.6, 26.4 ± 2.2, and 27.8 ± 1.3 for HHP, T, and untreated groups, respectively). Treatment did not affect survival rates to vitrification (2-h re-expansion rates: 100 ± 6.7, 100 ± 6.7, and 95.4 ± 6.7; 48-h hatching rates: 58.1 ± 9.4, 71.2 ± 9.4, and 62.3 ± 9.4, for HHP, T, and untreated, respectively). Embryos that hatched after warming did not differ in inner cell mass and trophectoderm cell counts (inner cell mass: 15.0 ± 1.9, 12.7 ± 3.0, and 13.0 ± 2.0; trophectoderm: 133.6 ± 8.4, 137.3 ± 12.8, and 138.4 ± 8.6 for HHP, T, and untreated groups, respectively; P > 0.05). Complementary studies are needed to analyze the effects of a sublethal stress in bovine oocytes on the subsequent embryo production and quality. Species-specific mechanisms could underlie the differences in results obtained in bovine and porcine. RTA2011-00090 (FEDER-INIA). Muñoz, Trigal, and Correia are sponsored by RYC08-03454, Cajastur, and FPU2009-5265, respectively.

55 STUDY ON THE INTERSPECIFIC NUCLEAR TRANSFER OF PRZEWALSKI'S GAZELLES AND BOVINES

2013 ◽  
Vol 25 (1) ◽  
pp. 175
Author(s):  
Y. Gao ◽  
L. Cheng ◽  
G. Su ◽  
Z. Wei ◽  
G. Li
Keyword(s):  
Embryo Development ◽  
Somatic Cells ◽  
Oocyte Nucleus ◽  
Control Group ◽  
Blastocyst Development ◽  
Affymetrix Microarray ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Deacetylase Inhibitor ◽  
Bovine Oocytes

Przewalski’s gazelle (Procapra przewalskii), also known as Platts antelope, is an endangered species only found in China. It belongs to the Artiodactyla order, Bovidae family, antelope subfamily, and Gazella genus. In this study, 5 experiments were designed to examine the developmental potential of Przewalski’s gazelle somatic cells transplanted into bovine enucleated oocytes. Enucleation was conducted by Hoechst 33342 staining of the oocytes and guided by a fluorescent microscope to ensure the removal of the nuclei. The gazelle cells were then transferred to the enucleated oocytes and electrically fused to reconstructed embryos. The study resulted in 5 major findings. (1) When gazelle-bovine reconstructed embryos were treated with the deacetylase inhibitor valproic acid (VPA), at different concentrations and for different times, treatment of the cloned embryos with VPA at 0.5 mM for 24 h significantly increased the 8- to 16-cell-stage embryo development [61.9% (96/155) v. 33.8% (46/136) control]. However, the morula [1.3% (2/155) v. 1.5% (2/155); P > 0.05] and blastocyst (0.7% v. 1.5%; P > 0.05) development were similar to that of the control. In the intraspecific (bovine-bovine) control group, the cleavage, morula and blastocyst development of 3 cloned embryos were 72.6% (127/175), 28.0% (49/175), and 23.4% (41/175). (2) Octamer-binding transcription factor 4 (Oct-4), as a developmental potential and expression marker, was transfected to gazelle cells. When Oct-4-eGFP-confected cells were transferred, the cloned embryo development did not improve either with or without VPA treatment. (3) When the gazelle-bovine embryos were treated with the deacetylase inhibitor trichostatin A (TSA) for 24 h at 10 ng mL–1, blastocyst development was significantly higher than in the control group [3.6% (6/168) v. 0.8% (1/125); P < 0.05]. (4) When a reverse NT protocol, in which the oocyte nucleus was removed after the cell nucleus was fused to the oocyte, was used for NT, the cloned embryo development did not improve. (5) The gazelle-bovine and bovine-bovine cloned embryos at 8- to 16-cell stages, gazelle cells, bovine cells, and bovine oocytes transcriptomes were analyzed by Affymetrix microarray (Affymetrix Microarray Inc., Santa Clara, CA, USA) and repeated twice. A total of 643 genes were activated in gazelle-cattle embryos compared with oocytes, whereas 1527 genes were activated in bovine-bovine clones. A total of 1010 genes that were exclusively expressed in gazelle somatic cells were still expressed in the interspecies cloned embryos. In conclusion, TSA treatment of Przewalski’s gazelle somatic cells transferred into enucleated bovine oocytes improved development of cloned embryos to the blastocyst stage, although still with low efficiency. Data from microarray analyses of the gazelle-cattle embryos showed that over 1000 gazelle-specific genes were still expressed in the interspecific cloned embryos. This work was supported by the National Basic Research Program of China (no. 2012CB22306).

315 SELECTION OF BOVINE OOCYTES BY BRILLIANT CRESYL BLUE BEFORE IN VITRO MATURATION IMPROVES BLASTOCYST DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 273 ◽  
Author(s):  
A. Sugulle ◽  
S. Katakawa ◽  
S. Yamamoto ◽  
S. Oomori ◽  
I. Itou ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Control Group ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Bovine Oocytes ◽  
Selection Of

The morphological identification of immature oocytes has commonly been used to select the bovine oocytes for IVF. However, &lt;30% of the recovered oocytes reach the blastocyst stage after fertilization, and this is probably due to the quality of the oocytes at the beginning of maturation. The brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. The aim of this study was to evaluate the effect of the BCB stain on the selection of bovine oocytes and on the subsequent embryo development for in vitro production (IVP). Cumulus–oocyte complexes (COCs) were collected by the aspiration of 2- to 6-mm follicles. A total of 559 oocytes were divided into 2 groups: (1) a control group, immediately cultured, and (2) a BCB-incubated group. After 90 min of BCB staining (Pujol et al. 2004 Theriogenology 61, 735–744), the oocytes were divided into oocytes with blue cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB−). The COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg mL−1 FSH at 38.5°C under an atmosphere of 5% CO2 in air. The matured COCs were inseminated with 5 × 106 sperm mL−1. After 18 h of gamete co-culture, the presumed zygotes were cultured in CR1aa supplemented with 5% CS for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (proportion of ≥5-cell stage, the total cleavage rates) and on Days 7 to 9 (blastocyst rate). The experiment was replicated 5 times, and the data were analyzed by a chi-square test and ANOVA. The results are presented in Table 1. The proportion of embryos with ≥5-cell stage was significantly higher (P &lt; 0.01) in the BCB+ group than in the BCB− group, but not in the control group. The total cleavage rate for the BCB+ embryos was significantly higher than that of either the BCB− or the control group (P &lt; 0.01). There were also significant differences (P &lt; 0.01) in the blastocyst development between the BCB+ and BCB− embryos and between the BCB− and the control embryos (P &lt; 0.05). This result showed that the selection of bovine oocytes by BCB staining before in vitro maturation may be useful for selecting oocytes that are developmentally competent up to Day 9 for IVP. Table 1.Effect of selection of oocytes by brilliant cresyl blue (BCB) staining on the subsequent embryo development of in vitro-matured/in vitro-fertilized bovine embryos

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