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.

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

32 Vitrification of in vitro-matured bovine oocytes in triacetate cellulose hollow fibres

2019 ◽  
Vol 31 (1) ◽  
pp. 142
Author(s):  
E. V. Kornienko ◽  
A. B. Romanova ◽  
M. A. Ikonopistseva ◽  
G. P. Malenko
Keyword(s):  
Ethylene Glycol ◽  
Fertilization Rate ◽  
Control Group ◽  
Hatching Rate ◽  
High Survival ◽  
Hollow Fibres ◽  
Group 2 ◽  
Bovine Oocytes ◽  
Group 1

A prospective method of vitrification in triacetate cellulose hollow fibres (HF) introduced by Matsunari et al. (2012 J. Reprod. Dev. 58, 599-608) allowed significant simplification and standardization of vitrification/warming procedures and was successfully used for group cryopreservation of various pre-implantation mammalian embryos. The goal of the current work was to evaluate the effectiveness of the HF vitrification method for cryopreservation of in vitro-matured bovine oocytes. The base medium for all the vitrification and rewarming solutions was calcium-free TBP-like protein-HEPES supplied with 20% of fetal bovine serum. Groups of 15 morphologically normal in vitro-matured bovine oocytes were equilibrated with 3% (vol/vol) ethylene glycol for 15min, loaded into HF, and transferred into vitrification solution containing 30% ethylene glycol and either 0.5M (Group 1) or 1.0M (Group 2) sucrose. Hollow fibres were incubated for either 60s (Group 1) or 30s (Group 2) and immediately plunged into LN. Rewarming was conducted at 39°C. Oocytes within HF were placed in decreasing concentrations of sucrose solutions to remove cryoprotectants. Then, oocytes were subjected to IVF. Non-vitrified denuded oocytes were used as a control. Survival rates were evaluated at 21h post-rewarming. Part of the presumptive zygotes were fixed and stained with acetolacmoid for fertilization rate. The remaining zygotes were cultured for 10 days. Developmental rates were evaluated at 44h and 7 and 10 days post-IVF. All results are presented as mean percentage±standard deviation. Data were analysed using Mann-Whitney U test. Significance was set at P&lt;0.05. Survival rate was significantly lower in Group 1 (79.0±8.0%) and Group 2 (75.0±5.0%) compared with the control group (97.0±4.0%). Fertilization rate in Group 1 differed significantly from the control (80.5±18.3% v. 95.5±9.1%). Cleavage rates in Groups 1 and 2 did not differ significantly from the control (42.5±15.7% v. 60.7±11.1% v. 63.0±15.8%, respectively). Blastocyst yields at 7 days post-IVF were 0.9±2.3 (1/116) and 9.6±5.4% (6/65) in Groups 1 and 2, respectively. The former was significantly lower than in the control group (17.0±10.3%, 23/154). It should be noted that hatching in the control group started at 8 days post-IVF and was delayed in Groups 1 and 2 for at least 24h. Day 10 blastocyst yields were 3.0±3.3 (P&lt;0.05), 20.9±13.8, and 30.4±9.6% in Groups 1 and 2 and the control group, respectively. All obtained Day 10 blastocysts (3/116) in Group 1 hatched. Hatching rate in Group 2 was significantly lower than in the control group. Both Groups 1 and 2 showed relatively high survival and fertilization rates, but embryo development rates in both groups had a tendency to be lower than in the control. However, the obtained results indicate that the modifications of the protocol may increase the effectiveness of HF vitrification. The HF vitrification method remains a prospective option for simultaneous cryopreservation of a group of bovine oocytes.

scholarly journals Developmental competence and expression of the MATER and ZAR1 genes in immature bovine oocytes selected by brilliant cresyl blue

Zygote ◽  
2009 ◽  
Vol 18 (3) ◽  
pp. 209-216 ◽  
Author(s):  
Gustavo Bruno Mota ◽  
Ribrio Ivan Tavares Pereira Batista ◽  
Raquel Varella Serapião ◽  
Mariana Cortes Boité ◽  
João Henrique Moreira Viana ◽  
...  
Keyword(s):  
Developmental Competence ◽  
Control Group ◽  
Blue Dye ◽  
Brilliant Cresyl Blue ◽  
Immature Oocytes ◽  
Relative Expression ◽  
Cresyl Blue ◽  
Blastocyst Rate ◽  
Bovine Oocytes

SummaryThe objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus–oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 °C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB− (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB− group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB− (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB− (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB− groups. Despite the relative expression of MATER in holding control, BCB+ and BCB− were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.

24 Survival rates of vitrified biopsied bovine in vitro-produced blastocysts using the VitTrans device

2019 ◽  
Vol 31 (1) ◽  
pp. 138
Author(s):  
N. González ◽  
J. Scherzer ◽  
M. Reichenbach ◽  
C. Otzdorff ◽  
H. Zerbe
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Embryo Transfer ◽  
Zona Pellucida ◽  
Survival Rates ◽  
Petri Dish ◽  
Blastocyst Stage ◽  
Control Group ◽  
Embryo Survival

In breeding programs, the application of a vitrification method suitable for direct transfer of biopsied embryos can increase the genetic improvement of cattle and help reduce the costs of embryo transfer. The aim of this study was to determine the in vitro survival of biopsied vitrified blastocysts using the new VitTrans device (Morató and Mogas 2014 Cryobiology 68, 288-293), a 1-step in-straw warming system. Immature bovine oocytes were in vitro matured, fertilized, and cultured to the blastocyst stage. A total of 110 grade 1 blastocysts (IETS codes 6 and 7) were randomly allocated to 2 groups: (1) biopsy (n=49) and (2) without biopsy, or control (n=61). Blastocysts were biopsied using a microblade mounted on a micromanipulator. A small portion of the trophoblast, approximately 15%, was cut off and a significant part of the zona pellucida was sliced away. Both groups were then vitrified using the VitTrans device. For vitrification, all blastocysts were exposed to an equilibration medium with 7.5% ethylene glycol+7.5% dimethyl sulfoxide in holding medium (HM) consisting of TCM-199 with 20% FCS, moved into a drop with 16.5% ethylene glycol+16.5% dimethyl sulfoxide+0.5M sucrose in HM, and then placed in a microdroplet on the VitTrans. The VitTrans was plunged into LN and covered with a 0.5-mL straw. For warming, the protective cover was removed from the VitTrans while still submerged in LN. Subsequently, a new 0.5-mL plastic embryo transfer straw was placed on the VitTrans while flushing the warming solution (0.3mL of 0.5M sucrose in HM at 45°C) with a syringe through the lumen of the device. By entering the warming solution into the VitTrans device, the embryo is flushed inside the plastic straw. The straw containing the embryo can then be readily used for transfer after the VitTrans is removed. To recover the embryo in the laboratory, the content of the straw was put into a Petri dish and blastocysts were placed in the culture medium and incubated at 38.5°C in 5% CO2 and 5% O2 in air. Morphology and re-expansion were evaluated 24h post-warming. The embryo survival rate was defined as the ratio of blastocysts that were able to re-expand with regards to the total number of warmed blastocysts. Due to the attachment of embryos inside the straw, a total of 18 embryos were lost during recovery (12 from the biopsied group and 6 from the nonbiopsied group). The ratio of re-expanded blastocysts from the recovered embryos was 40% in the biopsy group and 61% in the control group. In conclusion, vitrification using the VitTrans device showed good results with intact embryos compared with biopsied embryos. In addition, biopsied embryos had a tendency to adhere to the inside of the straw, which is probably due to the damage or loss of the zona pellucida. Additional research is required to minimize the loss of embryos.

40 THE EFFECT OF EXPOSURE TIME ON TOXICITY OF VITRIFICATION SOLUTION ON PORCINE CUMULUS–OOCYTE COMPLEXES BEFORE IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 127 ◽  
Author(s):  
R. Appeltant ◽  
T. Somfai ◽  
E. C. S. Santos ◽  
K. Kikuchi
Keyword(s):  
Ethylene Glycol ◽  
Propylene Glycol ◽  
Exposure Time ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Control Group ◽  
Dibutyryl Camp ◽  
Porcine Oocyte ◽  
Vitrification Solution

Although offspring have been produced from porcine cumulus-oocyte complexes (COC) vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731), embryo development rates have remained low. Numerous vitrification protocols are reported with a wide variation in the applied exposure time to the vitrification solution. Because cryoprotectants in the vitrification solution can be detrimental to the oocytes and their subsequent development, it is important to verify the effect of their exposure time to the COC. In this study, we compared the development of a control group with 3 toxicity control (TC) groups in which COC were exposed to the vitrification solution for 30 s, 1 min, or 1.5 min (TC1, TC2, and TC3, respectively) at 38.5°C. Before exposure, the COC were rinsed and equilibrated in 7 µg mL−1 cytochalasin B. The equilibration solution consisted of 2% (vol/vol) ethylene glycol + 2% (vol/vol) propylene glycol and the vitrification solution contained 17.5% (vol/vol) ethylene glycol + 17.5% (vol/vol) propylene glycol, 50 mg mL−1 polyvinylpyrrolidone and 0.3 M sucrose. The COC were not exposed to liquid nitrogen. After washing in a warming solution of 0.4 M sucrose at 42°C, COC were washed in a sucrose gradient from 0.2 to 0.0 M. Subsequently, the COC were subjected to in vitro maturation in porcine oocyte medium. During the first 20 h of in vitro maturation, the porcine oocyte medium was supplemented with 10 IU mL−1 eCG, 10 IU mL−1 hCG, 1 mM dibutyryl cAMP, and 10 ng mL−1 epidermal growth factor. Then, the medium was replaced with dibutyryl cAMP-free porcine oocyte medium for an additional 28 h. After in vitro maturation, oocytes were parthenogenetically activated (Day 0) and cultured for 7 days in porcine zygote medium. Survival, nuclear maturation, cleavage, and blastocysts rates (Days 6 and 7) were assessed. All parameters were statistically analysed by binary logistic regression. Only the survival rate of TC3 was significantly lower than that of the control group (89.2 v. 95.6%). Exposure to cryoprotectants significantly decreased maturation rates in TC1, TC2, and TC3 compared with the control (72.6%, 75.2%, 76.3% v. 86.1%). Cleavage rates were significantly lower in TC2 and TC3 than that in the control (82.8% and 81.7% v. 92.9%). Concerning blastocyst rates on Day 6 and Day 7 of in vitro culture, only TC1 could reach the same level as the control, expressed on the total number of activated oocytes (54.6% v. 67.7%, and 64.0% v. 72.9%, respectively) as well as expressed on the cleaved oocytes (61.4% v. 72.4% and 72.0% v. 78.0%, respectively). Consequently, despite the reduced maturation rate, TC1 provides the same quantity of blastocysts from matured oocytes as the control. In conclusion, exposure to the vitrification solution for longer than 30 s has toxic effects on COC and therefore is not recommended for vitrification. R. Appeltant is an International Research Fellow of the JSPS Japan (P15402).

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.

247 EFFECTS OF SILDENAFIL OR SNAP SUPPLEMENTATION, OR BOTH, DURING IN VITRO MATURATION ON LIPID AND REACTIVE OXYGEN SPECIES ACCUMULATION IN BOVINE OOCYTES AND EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 213
Author(s):  
M. Del Collado ◽  
R. C. Botigelli ◽  
K. R. L. Schwarz ◽  
C. Elias ◽  
C. L. V. Leal ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Lipid Accumulation ◽  
Reactive Oxygen ◽  
Epifluorescence Microscopy ◽  
Control Group ◽  
Oxygen Species ◽  
Lipid Quantification ◽  
Development Rates ◽  
Bovine Oocytes

Previous studies have demonstrated that a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillmaine, SNAP) and a phosphodiesterase inhibitor (Sildenafil, SILD) delay the meiotic resumption of oocytes removed from the follicular environment, and therefore could be used to improve the quality of in vitro-matured (IVM) oocytes. However, it has been reported that SILD-treated cells have increased lipid metabolism and that NO supplementation can modulate the oxidative stress. This study aims to determine the effects of SNAP or SILD supplementation, or both, during IVM on embryo developmental rates, on lipid accumulation of IVM oocytes and on reactive oxygen species (ROS) and lipid accumulation of embryos derived from IVM oocytes. Bovine oocytes were cultured in TCM199 containing 1.0 μg mL–1 of FSH, 50 μg mL–1 of hCG, 1.0 μg mL–1 of oestradiol, 0.2 mM pyruvate, 83.4 μg mL–1 of amikacin, 10% FBS (control group; GCONT), supplemented with 10 µM SILD (GSILD), 0.1 µM SNAP (GSNAP) or both (GS+S). After 24 h of IVM, matured oocytes were assessed for lipid quantification (approximately 49 per group) or used for in vitro embryo production (IVP; approximately 340 oocytes per group). For lipid quantification, denuded oocytes were fixed with 5% triton in 4% paraformaldehyde (PFA) for 30 min and stained with 1 ng mL–1 of Nile Red for 30 min. Embryo lipid analyses (approximately 55 per group) were performed as described for oocytes. For ROS assessment (approximately 58 per group), IVP embryos were stained with 10 µM of H2DFFDA for 1 h and fixed for 30 min in 4% PFA. Stained oocyte and embryo assessments were performed on epifluorescence microscopy, and captured images were analysed on ImageJ (NIH, Bethesda, MD, USA) to quantify the fluorescence intensity (f.i). Statistical analyses were performed with data from 3 replicates for oocytes and 4 for embryos: statistical differences were assessed for lipid and ROS quantity and development rates by split-plot ANOVA. Variables considered in the model were SNAP (presence/absence) and SILD (presence/absence). Means were compared by Student's t at P < 0.05. Regarding oocyte lipid accumulation, groups with SILD (GSILD and GS+S) presented higher lipid quantity (f.i: 52.11 and 47.24, respectively) compared with GCONT and GSNAP (f.i: 38.86 and 41.86, respectively). Supplementation during IVM did not affect development rates (cleavage of 88.1, 88.2, 88.8, and 89.5% and blastocyst rates of 41.2, 38.6, 40, and 41.2% for GCONT, GSNAP, GSILD, and GS+S, respectively). Regarding embryo lipid quantity, similar to oocyte results, SILD groups (GSILD and GS+S) presented higher lipid accumulation (f.i: 68.9 and 68.5, respectively) compared with GCONT (f.i: 55.8) and GSNAP (f.i: 58.9). Considering embryo ROS quantity, GCONT (f.i: 35.9) and GS+S (f.i: 34.2) had the highest levels; however, GS+S did not differ from GSNAP (f.i: 32.85), which was similar to GSILD (f.i: 30.4). In conclusion, SILD had a negative effect on lipid accumulation, which could be due to increased lipid synthesis without increasing lipid oxidation because no increase of embryo ROS levels was observed.

43 MASS VITRIFICATION OF GERMINAL-VESICLE STAGE EQUINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 151
Author(s):  
H. S. Canesin ◽  
I. Ortiz ◽  
J. G. Brom-de-Luna ◽  
Y. H. Choi ◽  
K. Hinrichs
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Fetal Bovine Serum ◽  
Control Group ◽  
Cleavage Rate ◽  
Fetal Bovine ◽  
Vitrification Solution

Oocyte cryopreservation has the potential to preserve female genetics. In addition, equine oocytes are not readily available in some areas, and vitrification could be used to accumulate oocytes at remote locations to provide material for research. To preserve large numbers of oocytes, a method for rapid vitrification of multiple oocytes is needed. First, we determined whether immature equine oocytes could be held overnight before vitrification, and we tested the use of a mesh+capillary-action media-removal vitrification platform. Oocytes were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to either immediate vitrification or overnight holding (24 to 27 h in 40% M199-Earle’s salts, 40% M199-Hanks’ salts, 20% fetal bovine serum, and 0.3 mM pyruvate) then vitrification. Oocytes were vitrified using different times (1 or 4 min) in vitrification solution and first warming solution: 1v1w, 1v4w, 4v1w, and 4v4w. The base solution was MH (80% M199-Hanks’ salts and 20% fetal bovine serum). Cryoprotectant concentration (vol/vol) was increased in 3 steps until reaching 7.5% dimethyl sulfoxide and 7.5% ethylene glycol. The oocytes were then held in vitrification solution (MH with 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose) for either 1 or 4 min, according to treatment, and 3 to 10 oocytes were transferred to a 75-μm sterile stainless steel mesh. The mesh was placed on sterile paper to absorb excess medium, then plunged in LN. The oocytes were warmed in MH solution with 1.25 M sucrose for either 1 or 4 min, then placed in 0.62 M and 0.31 M sucrose solutions for 5 min each and undetermined time in MH. After warming, oocytes were cultured for maturation (in vitro maturation) in M199-Earle’s salts, 5 mU mL–1 FSH, and 10% fetal bovine serum. After 30 to 36 h, the oocytes were denuded and stained with Hoechst 33258. Data were analysed by Fisher’s exact test. There were no significant differences (P > 0.05) in rates of meiotic resumption among timing treatments (35, 24, 26, and 39% for 1v1w, 1v4w, 4v1w, and 4v4w, respectively), nor between immediately vitrified (17/55, 31%) and overnight held-vitrified groups (18/56, 32%). In the second experiment, all oocytes were held overnight. They were vitrified and warmed using only the 1v1w and 4v4w schedules, then subjected to in vitro maturation, intracytoplasmic sperm injection, and embryo culture. The MII rate of the control group (27/37, 73%) was higher (P < 0.05) than that for 1v1w (12/33, 36%) or 4v4w treatments (10/35, 29%). The cleavage rate for control (25/27, 93%) was higher than that for 1v1w (5/9, 56%) but not than that for 4v4w (6/9, 67%). Blastocyst rates were 19% (5/27), 11% (1/9), and 0% (0/9) for control, 1v1w, and 4v4w, respectively (P > 0.05). These results indicate that blastocysts may be produced from equine immature oocytes vitrified en masse; however, both the maturation and blastocyst production rates were relatively low. Additional studies are required to improve the efficiency of this technique. This work was supported by the Clinical Equine ICSI Program, Texas A&M University.

scholarly journals 104EFFECT OF HATCHING STATUS ON VITRIFICATION OF CLONED BOVINE BLASTOCYSTS

2004 ◽  
Vol 16 (2) ◽  
pp. 174
Author(s):  
C. Laowtammathron ◽  
T. Terao ◽  
C. Lorthongpanich ◽  
S. Muenthaisong ◽  
T. Vetchayan ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Sucrose Solution ◽  
Survival Rates ◽  
Nuclear Transplantation ◽  
Fibroblast Cells ◽  
Anova Test ◽  
University Of Technology ◽  
Vitrification Solution ◽  
Cloned Bovine

Bovine blastocysts produced by nuclear transplantation have mechanical slits in their zonae pellucidae, and therefore initiate hatching earlier than the non-manipulated embryos. The present study was undertaken to examine whether the hatching stage of cloned blastocysts is among the factors influencing their survival after vitrification and warming. Cloned bovine blastocysts were produced by using adult ear fibroblast cells as reported previously (Parnpai et al., 2002, Theriogenology 57, 443), except that fused couplets were co-cultured with bovine oviductal epithelial cells in mSOFaa medium supplemented with 0.1% linoleic acid-albumin (LAA)+0.2% BSA (Hochi et al., 1999, Theriogenology 52, 497–504). Hatching blastocysts harvested on Day 7 were classified into one of three groups according to the ratio of extruding embryonic diameter from zona (D2) to embryonic diameter inside the zona (D1); category-A: D2/D1=0.01–0.70; category-B: D2/D1=0.71–1.00; category-C: D2/D1=1.01–1.70. The blastocysts were first exposed to 10% DMSO+10% ethylene glycol in TCM199+20% FCS for 2min, and then equilibrated in 20% DMSO+20% ethylene glycol+0.5M sucrose with or without 10% Ficoll in TCM199+20% FCS for 30s. One to three blastocysts were placed on a Cryotop sheet (Kitazato Supply Co., Tokyo, Japan) and vitrified in liquid nitrogen. The samples were warmed in 0.5M sucrose solution for 2min and transferred into TCM199+20% FCS in five steps (5min per step). The post-warm survival of the blastocysts was assessed by in vitro culture for 24h. When Ficoll-free vitrification solution was used, post-warm survival rate of the category-A blastocysts (77%, 23/30) was not significantly different (ANOVA test) from those of category-B and category-C blastocysts (74%, 20/27; and 80%, 24/30; respectively). Inclusion of 10% Ficoll in the vitrification solution did not improve (ANOVA test) the post-warm survival rates of cloned blastocysts (category-A: 65%, 22/34; category-B: 54%, 15/28; category-C: 59%, 19/32). Groups of fresh nonsurgical embryos, vitrified with or without Ficoll, yielded 66.7% (4/6), 66.7% (2/3) and 40.0% (2/5), respectively, of recipients pregnant at 48 days of gestation. In conclusion, cloned bovine blastocysts, regardless of their hatching stages, were relatively resistant to cryopreservation by vitrification. (Supported by Thailand Research Fund and R&amp;D Fund of Suranaree University of Technology.)

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