73 VITRIFICATION OF BOVINE OOCYTES: EFFECT OF PACKAGING AND EQUILIBRATION TIME ON NUCLEAR MATURATION

2009 ◽  
Vol 21 (1) ◽  
pp. 136
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
O. Dochi ◽  
M. Anzar
Keyword(s):  
Complex Structure ◽  
Calf Serum ◽  
Control Group ◽  
Equilibration Time ◽  
Treatment Groups ◽  
Body Tissues ◽  
Vitrification Solution ◽  
Chi Square Analysis ◽  
Bovine Oocytes

The conservation of female animal genetics is challenging because of the scarcity of oocytes and their sensitivity to cryopreservation techniques. During slow, controlled freezing procedures, intracellular ice crystallization often leads to cell damage. Vitrification as an alternate method of cryopreservation exposes cells to a higher concentration of cryoprotectants with an ultra-rapid cooling rate, leading them to an ice-crystal-free, solid glasslike structure. The vitrification procedure has been used successfully for the cryopreservation of embryos and other body tissues, but very few reports of successful oocyte cryopreservation exist because of their complex structure. The present study was designed to compare two packaging methods (Cryotop v. 0.25-mL straw) and two equilibration times (10 v. 0 min) for vitrification of bovine oocytes. COC were aspirated from follicles <8 mm in diameter on bovine ovaries collected from a slaughterhouse. COC with ≥3 layers of cumulus cells and a uniform cytoplasm were selected, washed in Dulbecco’s phosphate-buffered saline (DPBS) + 5% calf serum (CS), and divided into five equal groups. In the control group, the COC were washed in TCM-199 + 5% CS and matured in vitro in TCM-199 containing 5% CS, 5 μg mL–1 of LH, 0.5 μg mL–1 of FSH, and 0.05 μg mL–1 of gentamicin at 38.5°C, 5% CO2, and high humidity for 22 h. In the treatment groups, half the COC were equilibrated with vitrification solution 1 [VS1: TCM-199, 7.5% ethylene glycol (EG), 7.5% DMSO, and 20% CS] for 10 min. After equilibration, COC were exposed to vitrification solution 2 (VS2: TCM-199, 15% EG, 15% DMSO, 20% CS, and 17.1% sucrose) for 30 s. The remaining half of the COC were directly exposed to VS2 without equilibration in VS1. Groups of five equilibrated or nonequilibrated COC were either loaded in a 0.25-mL straw or placed on Cryotop and plunged in liquid nitrogen. The COC were thawed by immersing straws and Cryotops into 37°C thawing solution (TCM-199, 20% CS, and 17.1% sucrose) for 1 min, and were washed and matured in vitro, as described above. After maturation, the COC were denuded using 0.3% hyaluronidase in Ca-Mg free DPBS and mounted on slides. The oocytes were fixed in ethanol:acetic acid (3:1) for 24 h, stained with aceto-orcein for 20 min, and evaluated for stage of maturation. The data (maturation rates) were analyzed using chi-square analysis. In the control group, 61% (n = 54) of the oocytes reached the metaphase-II (M-II) stage. In the treatment groups, more (P < 0.001) oocytes vitrified on Cryotops reached the M-II stage than those vitrified in straws (23.4%; n = 107 v. 9.4%; n = 116). The effect of equilibration time was not significant (P > 0.05) in either packaging method. In conclusion, vitrification of bovine oocytes using the Cryotop method provides an alternative for the cryopreservation of bovine oocytes. Moreover, bovine oocytes can be successfully vitrified without equilibration. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

scholarly journals LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

2004 ◽  
Vol 9 (1) ◽  
Author(s):  
M.G.L. PINTO ◽  
M.I.B. RUBIN ◽  
C.A.M. SILVA ◽  
T.F. HILGERT ◽  
M.F. SÁ FILHO ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mineral Oil ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Bovine Oocytes

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

294 EFFECTS OF FOLLICULAR FLUID OBTAINED FROM REPEAT BREEDER DAIRY HEIFER ON MATURATION OF BOVINE OOCYTES IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 236
Author(s):  
M. Kafi ◽  
M. R. Divar ◽  
S. Gharib-Zadeh
Keyword(s):  
Follicular Fluid ◽  
Present Experiment ◽  
Calf Serum ◽  
Low Fertility ◽  
Control Group ◽  
Normal Fertility ◽  
Maturation Rate ◽  
Bovine Oocytes ◽  
Repeat Breeder

The cause of repeat breeding syndrome is often difficult to explain in dairy heifers with no clinical abnormalities. The aim of the present experiment was to determine the effect of follicular fluid obtained from the preovulatory follicle of repeat breeder heifers on maturation of bovine oocytes in vitro. Holstein virgin heifers either with normal fertility (VH, n = 5) or repeat breeder syndrome (RB, n = 5) were used in the present experiment. The RB heifers had a history of at least 5 unsuccessful consequent artificial breeding. The reason for using such RB heifers was to exclude the possibility of the presence of usual causes of infertility in heifers. Oestrus cycles of all heifers were synchronized using 2 injections of PGF2a 11 days apart. Six to 12 h after oestrus detection, clear follicular fluid samples from the ovulatory follicles were collected transrectally using a long fine-needle covered by a hard plastic tube. Follicular fluid samples were pooled, centrifuged, and frozen until used in the maturation medium. A total of 483 good or excellent quality bovine cumulus-oocytes complexes (COC) were obtained from 2 to 6 mm follicles in diameter from slaughterhouse ovaries and randomly allocated in 3 groups; in group 1 (control, n = 180), oocytes were cultured in TCM-199 supplemented with 10% heat-treated fetal calf serum and hormones (5 IU mL–1 of hCG plus 0.1 IU mL–1 of rFSH); in group 2 (n = 126), oocytes were cultured in TCM-199 supplemented with 10% filtered follicular fluid of VH without hormones; in group 3 (n = 177), oocytes were cultured in TCM-199 supplemented with 10% filtered follicular fluid of RB heifers without hormones. All oocytes were cultured for 24 h at 39°C in an atmosphere of 5% CO2 under 90% humidity. At the end of maturation, the degree of cumulus expansion was evaluated and scored under a stereomicroscope. Then, oocytes were mechanically denuded using 3% sodium citrate and repeated pipetting and were fixed in ethanol/acetic acid (3 : 1) for 24 h. The oocytes were subsequently stained with 1% aceto-orcein and evaluated for meiotic resumption. Proportions were statistically analysed using a Chi-squared test (significant at P < 0.05; SPSS program, 11.5). The percentages of fully expanded COC differed among groups (P < 0.001). The maturation rate (MII stage) was 83% (150/180) in oocytes that were cultured in the presence of FCS as the control group. However, a reduction in the maturation rate was observed when oocytes were cultured either in VH follicular fluid (71.4%, 90/126; P < 0.01) or RB follicular fluid (59.3%, 105/177; P < 0.001) compared to the control group. The percentages of matured oocytes were also different between VH and RB follicular fluid (71.4 v. 59.3%; P < 0.01, respectively). In conclusion, the quality of follicular fluid of the preovulatory follicles of repeat breeder heifers is lower than that of the virgin heifers with normal fertility. This may explain the cause of the low fertility in some repeat breeder Holstein heifers.

165 FETAL CALF SERUM INFLUENCES CYCLIC GMP PATHWAY, WHICH IN TURN AFFECTS THE LIPID CONTENT IN IN VITRO-MATURED BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 196
Author(s):  
K. R. L. Schwarz ◽  
R. C. Botigelli ◽  
F. C. Castro ◽  
M. R. Chiaratti ◽  
C. L. V. Leal
Keyword(s):  
Lipid Content ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Maturation Medium ◽  
Cgmp Pathway ◽  
Bovine Oocytes ◽  
Synthesis And Degradation

The sensitivity of IVP embryos to cryopreservation is often associated with lipid accumulation in the cytoplasm induced by the presence of fetal calf serum (FCS) during culture. Intracellular levels of cyclic (c)AMP and cGMP are involved in the regulation of lipolysis in adipocytes; high levels stimulate lipolysis whereas low levels lead to lipogenesis. Both nucleotides are present in bovine oocytes, together with the enzymes for their synthesis and degradation. The aim of this study was to analysis the effect of FCS on the cGMP pathway and the influence of cGMP on cytoplasmic lipids in bovine oocytes. In experiments 1 and 2, cumulus–oocyte complexes (COC) were cultured for 24 h in maturation medium with different proportions of FCS (2 and 10%) and a control group was matured with 0.4% BSA. After this period, transcripts for cGMP pathway were assessed by real-time PCR (GUCY1B3 and PDE5, cGMP synthesis and degradation enzymes, respectively; experiment 1) in oocytes and cumulus cells, and cGMP levels were measured in COC using commercial enzyme immunoassay kits (EIA; experiment 2). In experiments 3 and 4, COC were matured for 24 h with 0.4% BSA and different concentrations of the phosphodiesterase (PDE)5 inhibitor (0, 10–7, and 10–5 M sildenafil) to inhibit cGMP degradation and a control group was matured with 0.4% BSA. The nucleotide levels were measured in COC (experiment 3) and the oocytes were stained with Nile Red (1 μg mL–1) for evaluation of lipid content (experiment 4). Statistical analyses were performed by ANOVA followed by Tukey post hoc test using SAS software (SAS Institute Inc., Cary, NC, USA). Data for gene expression from 5 replicates and for cGMP measurements and lipid content from 3 replicates were log10-transformed into before analyses. The level of significance was 5%. The presence of FCS reduced GUCY1B3 expression in both cells and increased PDE5A in cumulus cells (P < 0.05). In experiment 2, the groups treated with 2 (0.64 fmol/COC) and 10% FCS (1.04 fmol/COC) showed decreased cGMP levels compared with control (9.46 fmol/COC; P < 0.05). In experiment 3, inhibition of PDE5A increased cGMP levels in the treated groups (36 and 56 fmol/COC for 10–7 and 10–5 M sildenafil, respectively) compared with control (9.5 fmol/COC; P < 0.05). Therefore, sildenafil showed inverse effects compared with FCS (experiment 2). In experiment 4, oocytes treated with 10–7 and 10–5 M sildenafil showed a reduced lipid content compared with controls (11.6 ± 9.4 v. 13.9 μm2 fluorescence intensity, respectively; P < 0.05). The results suggest that FCS in maturation medium affects the cGMP pathway, interfering with the transcription of genes that control its levels, which in turn results in nucleotide reduction. Inhibition of PDE5 increases cGMP levels and reduces the lipid content of oocytes, indicating that changes in this pathway caused by FCS may affect lipid metabolism of oocytes. More studies are underway to better understand this mechanism. The authors acknowledge FAPESP 2012/00170-0 for financial support.

116 EFFECTS OF l-GLUTAMINE ON CRYOPRESERVATION OF IMMATURE BOVINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 167
Author(s):  
C. Yamada ◽  
M. D. Goissis ◽  
H. V. A. Caetano ◽  
A. R. S. Coutinho ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Maturation ◽  
Complex Structure ◽  
Cumulus Cell ◽  
Epifluorescence Microscopy ◽  
Control Group ◽  
Maturation Medium ◽  
Cell Layers ◽  
Bovine Oocytes

The cryopreservation of bovine oocytes remains a challenge despite significant reported progress. Immature bovine oocytes have a complex structure and the conventional cryoprotectants (penetrating cryoprotectants, sugars, and macromolecules) appear to be not sufficient to preserve them efficiently during freezing. Studies on semen and fibroblast cryopreservation indicate that amino acids, particularly l-glutamine, protect enzymes during freezing and increase the post-thaw viability. Therefore, the amino acids may optimize oocyte cryopreservation when associated with conventional cryoprotectants. This work evaluated the effect of l-glutamine on cryopreservation of immature bovine oocytes after in vitro maturation. Oocytes with homogeneous cytoplasm and several cumulus cell layers from slaughterhouse ovaries were distributed randomly in three groups: non-vitrified control, vitrified control, and vitrified with l-glutamine. Oocytes from vitrified groups were exposed for 10 min to PBS + 10% FCS + 10% ethylene glycol (EG) + 0.25 m trehalose (T), and for 30 s to PBS + 10% FCS + 25% EG + 25% dimethylsulfoxide + 0.5 m T at room temperature, adding 80 mm l-glutamine for the third group. Oocytes were loaded into OPS and plunged in liquid nitrogen. For thawing, OPS were immersed in PBS + 10% FCS + 10% EG + 1 m T for three min. Oocytes werethen placed in PBS + 10% FCS + 0.5 m T and in PBS + 10% FCS, remaining three min in each solution. For in vitro maturation, oocytes were washed three times on holding medium (TCM-HEPES + FCS + pyruvate + gentamycin), washed three times in maturation medium (TCM-bicarbonate + FCS + pyruvate + gentamycin + hCG + FSH + estradiol), and cultured in microdrops (90 μL) of maturation medium covered with mineral oil at 38.5°C under 5% CO2 in air and high humidity for 24 h. Oocytes were denuded, fixed in paraformaldehyde and triton, stained with Hoechst 33342, and evaluated under epifluorescence microscopy. Oocytes at metaphase II were considered matured. The group vitrified with l-glutamine had a significantly higher maturation rate than the group vitrified without l-glutamine; however, both had significantly lower maturation rates than the non-vitrified control group. In conclusion, l-glutamine improved the viability of vitrified oocytes. Table 1. Oocyte maturation rates of non-vitrified control, vitrified control, and vitrified with glutamine groups This work was supported by FAPESP 03/08543-1.

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.

scholarly journals 92 BLASTOCYST FORMATION FROM VITRIFIED BOVINE OOCYTES, ZYGOTES, AND TWO-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 196
Author(s):  
A. Moisan ◽  
E. Chamberlain ◽  
S. Leibo ◽  
B. Dresser ◽  
K. Bondioli ◽  
...  
Keyword(s):  
Volume Ratio ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Group Iii ◽  
Treatment Groups ◽  
Group I ◽  
Vitrification Solution ◽  
Group Ii ◽  
Bovine Oocytes

The objective of this study was to devise a protocol to preserve bovine oocytes and early cleavage-stage embryos by vitrification and to compare their subsequent embryonic development after in vitro fertilization (IVF). Mature bovine oocytes from a commercial source (BoMed; Madison, WI, USA) were randomly allocated (in four replicates) to four treatment groups. Group I: control oocytes were subjected to IVF and cultured in CR1aa medium in a humidified atmosphere of 5% O2/5% CO2/90% N2 at 38°C. Group II: MII-stage oocytes were subjected to vitrification and then fertilized by IVF. Group III: presumptive zygotes were vitrified after IVF. Group IV: two-cell embryos resulting from IVF that were cultured for ∼28 h before vitrification. The vitrification solution consisted of TCM199 medium supplemented with 10% fetal bovine serum (mTCM) and containing 20% ethylene glycol (EG)/20% dimethyl sulfoxide (DMSO)/0.65 M trehalose. The oocytes/embryos to be vitrified were rinsed in mTCM, then in 5% EG/5% DMSO, then in 10% EG/10% DMSO, and finally for 45 s in the vitrification solution. For vitrification, groups of 6 to 12 oocytes/embryos were pipetted in <1-μL volume of vitrification medium onto the tip of a CryoTop (Katayama et al. 2003 Fertil. Steril. 80, 223); plunged directly into liquid nitrogen (LN2), and stored for ∼2 h. Vitrified samples were warmed and liquefied by rapidly transferring the Cryotops from LN2 into 0.25 M trehalose in mTCM at 37°C and then sequentially at 1-min intervals into 0.188 M and 0.125 M trehalose. Cleavage was evaluated on Day 3 post-insemination, and blastocyst development was assessed on Days 7 and 9 post-insemination. Of the 251 oocytes in Group I, 71% cleaved by Day 3, 21% formed blastocysts by Day 7, and 29% did so by Day 9; 3% of the total hatched. Of the 116 oocytes in Group II, fewer cleaved (P > 0.05) by Day 3 (54%) and developed into blastocysts by Day 7 (4%) and by Day 9 (8%); none hatched. Group III zygotes (n = 131) responded like Group II oocytes, 53% cleaved, and 5% formed blastocysts on Day 7 and 7% on Day 9; none hatched. In contrast, 19% of the 122 two-cell embryos formed blastocysts by Day 7 and 28% by Day 9, and 3% hatched. Although significantly fewer oocytes/embryos in Groups II and III cleaved compared with Group I, more than 50% of them did so after vitrification. After fertilization and cleavage, the two-cell embryos were much more resistant to the deleterious effects of cryoprotectants and vitrification. Higher survival of two-cell embryos may result from their increased permeability to cryoprotectants, and to water due to their higher surface area to volume ratio.

86 EFFECT OF CRYOPROTECTANT EXPOSURE, VITRIFICATION, AND WARMING TIME OF BOVINE CUMULUS OOCYTE COMPLEXES ON IN VITRO FERTILIZATION AND EMBRYONIC DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 148
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
R. J. Mapletoft ◽  
M. Anzar
Keyword(s):  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Time Interval ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Vitro Fertilization ◽  
Vitrification Solution ◽  
High Concentrations

Despite the importance of cryoprotectants for avoiding ice crystal formation, the high concentrations required for vitrification may be toxic to bovine oocytes. During warming (thawing), the removal of permeating cryoprotectants from cells can lead to osmotic injury, and the most appropriate time interval for warming and cryoprotectant removal from vitrified oocytes is currently uncertain. The present study aimed to evaluate the effect of cryoprotectant exposure, vitrification, and warming time of bovine cumulus oocyte complexes (COC) on fertilization and ability to develop as embryos in vitro. Follicles <8 mm in diameter were aspirated from slaughterhouse-derived bovine ovaries. Cumulus oocyte complexes with ≥3 layers of cumulus cells and a uniform cytoplasm were selected, washed 3 times in Dulbecco’s PBS + 5% newborn calf serum (CS), and randomly divided into 4 groups: 1) control group: no treatment; 2) VS1 group: COC were exposed to vitrification solution 1 [VS1: 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) in TCM-199 + 20% CS] for 5 min; 3) VS1+VS2 group: COC were exposed to VS1 for 5 min followed by vitrification solution 2 (VS2: 15% EG, 15% DMSO, and 0.5 M sucrose in TCM-199 + 20% CS) for 30 s; and 4) vitrified group: COC were exposed to VS1 and VS2, and then vitrified in liquid nitrogen using cryotops. The COC in VS1, VS1+VS2, and vitrified groups were exposed to a warming solution (0.5 M sucrose in TCM-199) for 1 or 5 min. The COC from all groups were in vitro matured (IVM) for 22 h in TCM-199 containing 5% CS, 5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 0.05 μg mL–1 gentamicin at 38.5°C, 5% CO2, and high humidity, incubated with frozen–thawed sperm in Brackett-Oliphant capacitating medium for 18 h, and the presumptive zygotes were cultured in Charles Rosenkrans 1 amino acids (CR1aa) + 5% CS for 9 days. Data were analysed using Proc Glimmix in SAS® 9.2 (SAS Institute Inc., Cary, NC, USA). Cleavage and blastocyst rates in the vitrified group (25 and 2%, respectively) were significantly lower (P < 0.0001) than in control (75 and 27%), VS1 (68 and 19%), or VS1+VS2 (63 and 22%) groups. Cleavage and blastocyst rates did not differ among non-vitrified groups (P > 0.05). In VS1, VS1+VS2, and vitrified groups, warming time had no effect on cleavage or blastocyst rates (P > 0.05). In conclusion, although cryoprotectant exposure and warming times had no apparent adverse effect, vitrification of bovine COC drastically reduced cleavage and blastocyst rates. Further studies are required to understand how vitrification of bovine COC affects subsequent fertilization and embryo development. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

scholarly journals FERTILIZATION OF BOVINE OOCYTES VITRIFIED PRE- AND POST IN VITRO MATURATION

2017 ◽  
Vol 52 (2) ◽  
pp. 104
Author(s):  
Zakiyatul Faizah ◽  
Ninik Darsini ◽  
Aucky Hinting
Keyword(s):  
Ethylene Glycol ◽  
Volume Ratio ◽  
Petri Dish ◽  
Lower Surface ◽  
Control Group ◽  
Oocyte Vitrification ◽  
Treatment Groups ◽  
The Media ◽  
Bovine Oocytes

The success rate of fertilization post save frozen oocytes is still very low, because the oocyte has distinctive features, namely the volume ratio and a lower surface to the limited penetration of water and cryoprotectants penetrate cells. Beside mature oocytes have a thread spindles are particularly vulnerable to the drop in temperature. Keep frozen oocytes is needed, especially in women who needed rescue fertility so their oosit can be fertilized. Maturation is done in TC 100 mL medium covered with mineral oil in a petri dish with a diameter of 36 mm. Oocyte vitrification begins with washing in PBS supplemented medium serum 20% for 1-2 minutes, followed by serum in the medium PBS + 20% + 10% ethylene glycol for 10-14 minutes. Then oocyte vitrification medium is transported in PBS + serum 20% + sucrose 0.5M ethylene glycol + 15% + 15% PROH for 25-30 seconds. Thawing oocytes is done by successive immersed in the media: 1). PBS + 20% serum + 0.5M sucrose, 2). PBS + 20% serum + 0.25M sucrose, and 3). PBS + 20% serum + 0.1 M sucrose. Insemination is done in rosset, and the number of fertilization was observed after 48 hours. Fertilization in the control group amounted to 42.97%, while the K1 and K2 there are no fertilization at all. The analysis showed that fertilization in the control and treatment groups significantly different at p <0.05 in both treatment groups K1 or K2 there are no fertilization at all. The conclusions of this study is there is no difference between the amount of fertilization of bovine oocytes were vitrified pre and post-maturation in vitro.

scholarly journals 292 MATURATION IN A STRAW IS EFFECTIVE ON THE DEVELOPMENT OF BOVINE OOCYTES IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 296
Author(s):  
Y.M. Park ◽  
S.S. Kim ◽  
J.H. Lee ◽  
Y.S. Park ◽  
H.D. Park
Keyword(s):  
Embryo Development ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Development Rate ◽  
Culture Dish ◽  
Chi Square ◽  
Treatment Groups ◽  
Chi Square Test ◽  
Bovine Oocytes

In vitro embryo development is strongly influenced by oocyte maturation environments. Maturation of bovine oocytes is processed in a culture dish. However, the development rate to the transferable blastocyst stage was 10 to 30%. This experiment was to examine the effect of the size of straw and the medium exchange on the development of Korean Native Cow (KNC) oocytes. Ovaries of KNC were obtained from a local slaughterhouse and cumulus oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles. Groups of 15 COCs were matured in TCM-199 supplemented with 10% fetal calf serum (FCS), 1 μg/mL FSH, 10 μg/mL LH, and 1 μg/mL estradiol-17β for 18 h. In vitro-matured oocytes were fertilized using frozen-thawed percoll-separated spermatozoa (Day 0) in fer-TALP medium for 20 h and cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10% FBS (after Day 3). All cultures were maintained in an incubator at 39°C, 5% CO2 in air with maximum humidity. Data from three replicates were analyzed by chi-square test. In Experiment 1, we examined the effect of the instrument of maturation (dish or 0.25-mL and 0.5-mL straws) on embryo development. There were no difference in the cleavage (2-cell) among treatment groups. However, the development rate to the 8-cell and blastocyst stage was significantly higher in the 0.5-mL straw (38.5 and 17.0%) than in the 0.25 mL-straw (26.6 and 7.4%, all respectively). In Experiment 2, the KNC oocytes were matured in 0.5-mL straws based on the results of Experiment 1, and we examined the effect of the conditions such as circulation and exchange of maturation medium at 9 h after the start of IVM on embryo development. The development rates to the 2-cell, 8-cell, and blastocyst stage were significantly higher in the circulation group (83.3, 58.0 and 31.3%) than in the control (72.0, 44.7 and 19.3%) and exchange groups (71.3, 40.0, and 18.0%, all respectively). The results of this study suggest that the maturation of KNC oocytes in 0.5-mL straws accompanied by circulation of medium at 9 h is effective in the development to the blastocyst stage.

166 THE EFFECT OF TEMPERATURE DURING STORAGE OF IN VITRO-MATURED BOVINE OOCYTES IN A HEPES-BUFFERED MEDIUM ON DEVELOPMENTAL COMPETENCE

2016 ◽  
Vol 28 (2) ◽  
pp. 213
Author(s):  
T. Suttirojpattana ◽  
T. Somfai ◽  
S. Matoba ◽  
T. Nagai ◽  
R. Parnpai ◽  
...  
Keyword(s):  
Polar Body ◽  
Calf Serum ◽  
Cell Number ◽  
Developmental Competence ◽  
Effect Of Temperature ◽  
Control Group ◽  
Atp Content ◽  
Developmental Rates ◽  
Bovine Oocytes

The objective of this study was to clarify the effect of the temperature during liquid storage in in vitro matured (IVM) bovine oocytes. IVM bovine oocytes were stored in Eppendorf tube containing 1 mL HEPES TCM-199 supplemented with 10% (v/v) new born calf serum at different temperatures (4°C, 15°C, 25°C, and 38.5°C) for 20 h. The developmental rates of stored and not stored (control) oocytes to the blastocyst stage, cell numbers in resultant blastocysts, and fertilization normality were evaluated after in vitro fertilization and in vitro culture. The ATP content, reduced glutathione (GSH) content, and apoptosis rates in oocytes were also determined in stored and control groups. At least 3 replicates were conducted for each experiment. The data were analysed by 1-way ANOVA followed by post hoc Fisher’s protected least significantly difference test. Percentage data were transformed to arc-sine before analysis. All of the storage groups (4, 15, 25, and 38.5°C groups, respectively) showed significantly lower blastocyst developmental rates (8.5, 14.9, 19.3, and 24.5%, respectively) compared with the control group (39.8%; P < 0.05). Within the storage groups, the 25°C and the 38.5°C groups exhibited the greatest rate of blastocyst formation. In contrast, the total cell number of the 38.5°C group was significantly lower than that of control group (P < 0.05), whereas that of the 25°C group was similar with the control group. The frequency of normal emission of the second polar body (2PB) was significantly greater in the control group compared with the storage groups (P < 0.05). The 2PB emission rate was significantly lower in the 38.5°C group compared with the 4°C group (P < 0.05) but not different from those of the 15°C and 25°C storage groups. The percentage of male pronuclear formation in the control group was significantly higher than those in the stored groups (P < 0.05) except for the 25°C group. During storage at 4°C, the ATP content was significantly decreased compared with the control group (1.3 v. 1.7 pmol; P < 0.05); however, in the 25°C and 38.5°C groups, the ATP content (2.0 and 1.9 pmol, respectively) was significantly higher than that in the control group (1.7 pmol; P < 0.05); whereas the 15°C group showed the same ATP level compared with the control group. Storage of oocytes for 20 h reduced the GSH content compared with the control group without storage (P < 0.05); however, there were no significant differences among storage groups. Annexin-V staining revealed increased incidences of early apoptotic oocytes in the 4°C and 15°C groups (P < 0.05) compared with other groups. In conclusion, based on the embryo developmental competence of stored oocytes and quality of resultant blastocysts, 25°C was determined as the most suitable temperature for temporal storage of matured bovine oocytes. The study was supported by the NARO Institute of Livestock and Grassland Science, Japan (N32G4126), and the Royal Golden Jubilee-PhD scholarship (2.B.TS/53/F.2).

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