85 VITRIFICATION OF IN VITRO-MATURED PORCINE OOCYTES AT THE METAPHASE-II STAGE

2008 ◽  
Vol 20 (1) ◽  
pp. 123
Author(s):  
B. Ogawa ◽  
S. Ueno ◽  
N. Nakayama ◽  
H. Matsunari ◽  
Y. Ikeda ◽  
...  
Keyword(s):  
Survival Rate ◽  
Mitotic Spindle ◽  
Early Stage ◽  
Formation Rate ◽  
Calf Serum ◽  
Slight Modification ◽  
Combination Group ◽  
Chi Square ◽  
The Difference

Cryopreservation of mammalian metaphase-II (M-II) oocytes is still impractical compared to that of early stage embryos. In this study we examined the effects of delipation and mitotic spindle stabilization in order to improve the post-vitrification survival rate of in vitro-matured (IVM) porcine oocytes at the M-II stage. Cumulus–oocyte complexes that had been collected from slaughterhouse ovaries were matured in vitro in NCSU23 supplemented with 0.6 mm cysteine, 10 ng mL–1 epidermal growth factor (EGF), 10% porcine follcular fluid (PFF), and 10 IU mL–1 eCG and hCG. The denuded M-II oocytes were vitrified in the presence of 30% ethylene glycol and 0.5 m sucrose using the minimum volume cooling (MVC) method with a MVC plate (Cryotop�; Kitazato Supply, Tokyo, Japan). Vitrified embryos were rewarmed by immersing the MVC plate directly into rewarming solution containing 1 m sucrose and 20% calf serum at 39�C for 1 min, followed by stepwise dilution of the cryoprotectants. We compared the effects of previtrification treatments, namely, (1) delipation, (2) mitotic spindle stabilization, (3) delipation + mitotic spindle stabilization, and (4) no treatment. For delipation, we used a noninvasive method (Esaki et al. 2004 Biol. Reprod. 71, 432–437) that we had published previously with slight modification. The embryos were treated with 4% trypsin at 38�C for approximately two min to expand the zona pellucida, and then centrifuged (12 000g, 38�C 23 min) with 7.5 µg mL–1 cytochalasin B to polarize cytoplasmic lipid droplets within the perivitelline space. For mitotic spindle stabilization, M-II oocytes were vitrified in the presence of 1 µm paclitaxel. After the oocytes were rewarmed, electrical activation of the oocytes (150 V mm–1, 100 µs, one time) was carried out to induce parthenogenesis. These parthenogenetic embryos were cultured in PZM-5 for 7 days, and the number of vitrified embryos that developed into blastocysts with respect to each treatment was determined. The blastcyst formation rate and mean cell numbers of the blastcysts were compared among the treatment groups (chi-square test, Tukey's test). Of the 50 M-II oocytes that had been vitrified without pretreatment, only one oocyte (2.0%) developed into a blastocyst with 20 cells. By contrast, the number of vitrified embryos that developed into blastocysts was significantly high when they were delipated prior to vitrification (37.8%, 14/37, 64.0 � 9.6; P < 0.01). Mitotic spindle stabilization also improved the survival rate of vitrified oocytes (18.6%, 21/113, 56.7 � 9.6; P < 0.01). The combination of delipation and mitotic spindle stabilization produced the highest number of vitrified oocytes that developed into blastocysts (43.8%, 35/80, 69.4 � 6.4), although the difference between the combination group and the delipation group was not significant. These results indicate that blastocysts can be produced very efficiently from IVM porcine oocytes that have been vitrified at the M-II stage using both noninvasive delipation and mitotic spindle stabilization procedures.

137 EFFECT OF THE ADDITION OF FOLIC ACID TO MATURATION AND CULTURE MEDIA ON DEVELOPMENT OF THE BOVINE BLASTOCYST AND ITS SURVIVAL RATE AFTER FREEZE-THAWING

2017 ◽  
Vol 29 (1) ◽  
pp. 177
Author(s):  
S. Sato ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Folic Acid ◽  
Survival Rate ◽  
Culture Media ◽  
Cell Damage ◽  
Calf Serum ◽  
Cleavage Rate ◽  
Chi Square ◽  
Exact Test ◽  
Chi Square Test

Reactive oxygen species (ROS) are the main causes of cell damage in bovine embryos in vitro. Folic acid (FA) is an antioxidant that protects cells from ROS. We studied the effect of the addition of FA to maturation and culture media on development of bovine blastocysts and their survival rate after freeze-thawing. Cell-oocyte complexes (COC) were allowed to mature in HEPES (25 mM)-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS), 0.02 AU mL−1 of FSH, and FA (0, 2.5, 25, and 50 µM) for 20 hours (20–25 COC/100-µL droplet of the medium). After 6 hours of gamete co-culture (5 × 106 sperm/mL), presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS and FA (0, 2.5, 25, and 50 μM) for 9 days (day of fertilization = Day 0). Expanded blastocysts that developed from Day 7 to 9 were frozen for further study. Each embryo was frozen in Dulbecco’s PBS (D-PBS) supplemented with 20% CS, 1.5 M ethylene glycol (EG), and 0.1 M sucrose (SUC). Embryos were equilibrated with their freezing medium for 15 min and loaded individually into a 0.25-mL straw. These straws were put into the cooling chamber of a programmable freezer precooled at −7°C. After 2 min, straws were seeded and held for 13 min at −7°C. Next, straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. Frozen embryos were thawed by allowing straws to stand in air for 7 s and warming them in a 30°C water bath for 20 s. Thawed embryos were washed twice with D-PBS supplemented with 20% fetal calf serum (FCS), which was warmed to 38°C. They were immersed into the same medium at 38°C for 10 min, and each embryo was cultured in a 20-μL droplet of TCM199 supplemented with 10% FCS and 0.1 mM β-mercaptoethanol (TCM-199-βME) for 72 h. Embryo cleavage rate was observed at 55 h post-insemination. Blastocyst rates were analysed at 9 days post-insemination. Rates of embryos developing into reexpanded, hatching, and hatched blastocyst stages were determined after 72 h of thawing. All data were analysed by the chi-square test and Fisher’s exact test. Cleavage and blastocyst rates after insemination at 55 hours and 9 days, respectively, were not significantly different among media containing 0 μM (n = 278; 74.1% and 39.9%), 2.5 μM (n = 260; 74.2% and 45.8%), 25 μM (n = 258; 75.6% and 45.7%), and 50 μM (n = 253; 76.3% and 42.7%) FA. Survival and hatching rates of frozen and thawed expanded blastocysts after 72 h in culture were 62.5% and 56.3%, respectively, in 0 μM FA (n = 16); 85.2% and 74.1% in 2.5 μM FA (n = 27); 66.7% and 62.5% in 25 μM FA (n = 24); and 68.0% and 64.0% in 50 μM FA (n = 25). Blastocysts cultured in media containing 2.5 μM FA tended to have a higher survival rate than those cultured in media containing 0 μM FA, although this difference was not significant (P = 0.09). Inclusion of FA did not appear to influence development or post-thaw survival of bovine blastocysts produced in vitro.

237 THE EFFECTS OF BULLS AND X-SORTING OF SPERM ON THE ACCURACY OF NONINVASIVE CRITERIA TO PREDICT BLASTOCYST FORMATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 208
Author(s):  
S. Matoba ◽  
T. Somfai ◽  
T. Nagai ◽  
M. Geshi
Keyword(s):  
Formation Rate ◽  
Calf Serum ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Chi Square

Previously, an early first cleavage and a second cleavage after IVF with a normal cleavage pattern defined by even blastomeres without fragments or protrusions was found to be a potent marker for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLoS ONE 7, e36627). The aim of this study was to investigate the effects of bulls and X-sorting of sperm on the ability of these simple noninvasive markers to predict the potency of bovine IVF embryos to develop to the blastocyst stage in vitro. Immature oocytes were matured in TCM199 supplemented with 0.02 armour unit mL–1 FSH and 5% calf serum at 38.5°C in 5% CO2 and 95% air for 22 to 23 h. After maturation, oocytes were inseminated with either of non-sorted frozen-thawed sperm from 3 bulls (A–C) or X-sorted sperm of bull A. Putative zygotes were cultured (IVC) in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually by time-lapse cinematography (CCM-1.3Z; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). First and second cleavage kinetics and pattern were categorized according to Sugimura et al. (2012). For each bull, blastocyst development from embryos possessing the following 3 selection markers was compared: (marker 1) the first cleavage within 28 h after IVF, (marker 2) marker 1 combined with 2 even blastomeres without fragments or protrusions, and (marker 3) marker 2 combined with the second cleavage within 50 h after IVF with ≥6 even blastomeres without fragments or protrusions, respectively. Data were analysed by the Yates' corrected chi-square test. A total of 823 oocytes were used in at least 3 replications. When non-sorted sperm was used for IVF, there was not difference (P > 0.05) in total blastocyst formation rates on Day 8 (Day 0 = IVF) among bulls (ranging between 49.5 and 60.8%); however, blastocyst formation rate of embryos generated from X-sorted sperm of bull A (39.5%) was lower (P < 0.05) compared with other groups despite of similar cleavage rates. Embryos having marker 3 criteria developed to the blastocysts stage at significantly higher rates than those having marker 1 criteria in case of non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A (75.9, 87.0, 90.0, and 75.0% v. 59.5, 62.2, 63.6, and 46.3%, respectively). In groups produced from non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A, blastocyst development rates of embryos with marker 2 criteria (73.7, 75.0, 90.0, and 65.8%, respectively) were higher (P < 0.05) than those of embryos having marker 1 criteria but did not differ significantly from those with marker 3 criteria. Our results reveal that a first cleavage within 28 h after IVF to 2 even blastomeres without fragments or protrusions are potent predictive markers of the developmental competence of bovine embryos to the blastocyst stage regardless of bulls and sperm sorting.Research was partly supported by JSPS KAKENHI (26450388).

scholarly journals Serum CYR61 As A Potential Biomarker for The Diagnosis of Esophagogastric Junction Tumor

2021 ◽  
Author(s):  
Ling-Yu Chu ◽  
Jian-Yuan Zhou ◽  
Yi-Xuan Zhao ◽  
Yan-Ting Ou ◽  
Tian Yang ◽  
...  
Keyword(s):  
Early Stage ◽  
Area Under The Curve ◽  
Serum Levels ◽  
Enzyme Linked Immunosorbent Assay ◽  
Operating Characteristics ◽  
Tumor Patients ◽  
Chi Square ◽  
Potential Biomarker ◽  
The Difference ◽  
Normal Controls

Background：Esophagogastric junction tumor (EGJ) is a rare but fatal disease with a rapid rising incidence worldwide in the late 20 years, and it lacks a convenient and safe method for diagnosis. This study aimed to evaluate the potential of serum CYR61 as a biomarker for the diagnosis of EGJ tumor. Methods: Enzyme-linked immunosorbent assay (ELISA) was used to estimate CYR61 levels in sera of 152 EGJ tumor patients and 137 normal controls. Receiver operating characteristics (ROC) was carried out to evaluate the diagnostic accuracy. The Mann–Whitney’s U test was used to compare the difference of serum levels of CYR61 between groups. And chi-square tests were employed to estimate the correlation of the positive rate of serum CYR61 between/among subgroups. Results: Serum CYR61 levels were statistically lower in EGJ tumor and early-stage EGJ tumor patients than those in normal controls (P&lt;0.0001). The sensitivity, specificity, and the area under the curve (AUC) of this biomarker in EGJ tumor were 88.2%, 43.8% and 0.691, respectively, and those for early stage of EGJ tumor were 80.0%, 66.4% and 0.722, respectively. Analyses showed that there was no correlation between the clinical data and the levels of CYR61 (P&gt;0.05). Conclusion: This study showed that CYR61 might be a potential biomarker to assist the diagnosis of EGJ tumor.

294 USE OF FORSKOLIN TO PRODUCE IN VITRO BOVINE EMBRYOS UNDER TWO CONCENTRATIONS OF FETAL CALF SERUM

2010 ◽  
Vol 22 (1) ◽  
pp. 303
Author(s):  
D. M. Paschoal ◽  
M. J. Sudano ◽  
L. C. O. Magalhães ◽  
L. F. Crocomo ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Fetal Calf Serum ◽  
Formation Rate ◽  
Calf Serum ◽  
Basic Medium ◽  
Intracellular Camp ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Viability ◽  
High Concentration

The increased storage of lipid granules in in vitro-produced (IVP) bovine embryos seems to be related to the presence and concentration of fetal calf serum (FCS) during culture. The presence of high concentration of lipids on embryos reduces their viability after cryopreservation, which has been one of the main obstacles for the success of vitrification of IVP bovine embryos (Moore et al. 2007 Theriogenology 68, 1316-1325). The present experiment aimed to induce cytoplasmic lipolysis in IVP bovine embryos using forskolin (Sigma-Aldrich, St. Louis, MO, USA), which raises the levels of intracellular cAMP (Seamon et al. 1981 Proc. Natl. Acad. Sci. USA, 78, 3363-3367). Nelore oocytes were matured in TCM-199 + 10% FCS, FSH, and LH in 5% CO2 in air atmosphere, at 38.5°C. After 24 h of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions. Presumptive zygotes were cultured in 2 concentrations of FCS: Control 0% (SOFaa + 5 mg mL-1 BSA; basic medium, BM), and Control 2.5% (BM supplemented with 2.5% FCS). On Day 6 of culture embryos were divided into 2 additional treatments: Forskolin 0% (BM + 10 μM forskolin; and Forskolin 2.5% (BM supplemented with 2.5% FCS and 10 μM forskolin). All embryos were cultured in a 5% CO2, 5%O2, and 90% N2 atmosphere at 38.5°C for 7 days, when blastocyst formation rate was evaluated. Embryo viability was also checked by staining the embryos with Hoechst 33342 and propidium iodide. Data were analyzed by ANOVA followed by Tukey’s test, using a 5% significance level. No statistical differences were observed among treatments on cleavage rates, evaluated on Day 3 of culture, or on blastocyst formation rates. Although no statistical differences was observed between treatments on percentage of viable cells, embryos cultured with 0% FCS, independently of the presence of forskolin, presented significantly more damaged cells than embryos cultured with 2.5% FCS (P < 0.05). The results indicate that the presence of FCS is important to reduce degeneration of blastomeres during culture. Moreover, the presence of forskolin on Day 6 of culture did not influence embryo development, indicating that this drug could be a good alternative to reduce embryo lipid content in bovine IVP embryos produced in presence of FCS. Table 1.Effect of fetal calf serum and forskolin on embryo culture Acknowledgments: FAPESP 07/53505-1.

242 PROMISING TWO-STEP EVALUATION SYSTEM FOR SELECTING HIGH DEVELOPMENTAL COMPETENCE IN VITRO FERTILIZED EMBRYOS IN CATTLE USING WELL-OF-THE-WELL DISH

2015 ◽  
Vol 27 (1) ◽  
pp. 210
Author(s):  
M. Taniai ◽  
M. Takayama ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Evaluation System ◽  
Evaluation Method ◽  
Calf Serum ◽  
Time Lapse ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test

Bovine IVF embryos are evaluated morphologically using light microscopy just before transfer. However, this evaluation method is subjective, and an objective method with more certainty is needed. Sugimura et al. (PLoS ONE 2012 7, e36627) reported a promising system for selecting healthy IVF bovine embryo by using time-lapse cinematography and 5 prognostic factors. This study was to investigate the efficacy of a 2-step evaluation system of IVF embryos using microscopy for selecting high developmental competence IVF embryos. Cumulus-oocyte complexes (COC) were collected by ovarian follicular aspiration (2 to 5 mm diameter) obtained from a local abattoir. The COC (n = 488) were matured in TCM-199 medium supplemented with 5% calf serum (CS) and 0.02 IU mL–1 of FSH at 38.5°C for 20 h in an atmosphere of 5% CO2 (20 COC 100 µL–1 droplets). After 10 h of gametes co-culture (5.0 × 106 sperm cells mL–1), the presumptive zygotes were cultured in 125 µL of CR1 aa medium supplemented with 5% CS in well of-the-well culture dishes (AS ONE, Japan; 25 zygotes well–1) at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days. Two-step evaluations of embryos were done at 27 and 55 h post-IVF (hpi). In the first step of evaluation, cleavage patterns at 27 hpi were categorized as mono-cell, 2-cell with even blastomeres and without fragments (normal cleavage), 2-cell with uneven blastomeres, and ≥3 blastomeres. During the second step of evaluation, embryos were classified by their number of blastomeres (2 to 5 cells, 6 to 8 cells, and >8 cells) and the absence or presence of multiple fragments (<20 or >20%) at 55 hpi. The data were analysed by chi-square test. The blastocyst rate (BL%) of embryos cleaved before 27 hpi (56.6%, n = 106) was higher (P < 0.01) than those of embryos cleaved after 27 hpi (37.0%, n = 235). A greater percentage (P < 0.05) of 2-cell embryos with normal cleavage (68.0%, n = 50) developed to blastocysts than from with =3 blastomeres at 27 hpi (40.6%, n = 32). Superior BL% (P < 0.01) was obtained from embryos categorized as 6- to 8-cell stage (58.6%, n = 140) and >8 cell stage (70.6%, n = 25) compared with those embryos at the 2- to 5-cell stage at 55 hpi (26.1%, n = 176). Embryos with no fragments (58.0%, n = 467) had higher BL% (P < 0.01) compared with those with <20% fragments (30.7%, n = 127) and having with >20% fragments (17.5%, n = 25) at 55 hpi. The highest of BL% was observed in embryos showing a normal cleavage to 2-cells with at 27 hpi and having >6 cells with no fragments at 55 hpi (95.2%, n = 21, P < 0.01). These results demonstrate that the 2-step evaluation system at 27 and 55 hpi using microscopy is an effective method for selecting IVF embryos with high developmental competence.

232 ADDITION OF VERY LOW AMOUNTS OF SERUM (ESTRUS COW SERUM) IMPROVES IN VITRO EMBRYO PRODUCTION IN DAIRY CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 205 ◽  
Author(s):  
E. Mullaart ◽  
F. Dotinga ◽  
C. Ponsart ◽  
H. Knijn ◽  
J. Schouten
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Calf Serum ◽  
High Serum ◽  
In Vitro Production ◽  
Embryo Production ◽  
Chi Square ◽  
Embryo Yield ◽  
Vitro Production

Improving the efficiency of the in vitro production (IVP) process is very important because it results in more embryos to be used in breeding programs or as commercial service. At CRV, a culture medium consisting of SOF with amino acids and BSA is used. In the past, richer culture media were used with 10% fetal calf serum combined with BRL cell co-culture. Although the efficiency of the IVP process of these media was good, these rather high serum concentrations were quite often related to large offspring syndrome (LOS). The switch to a culture system without serum resulted in a significant reduction in LOS but also in a reduction of embryo yield. The aim of the present study was to investigate the effect of adding low amounts of serum to the culture medium on efficiency of embryo production. Immature cumulus-oocyte complexes (COC) were recovered from ovaries 6 to 8 h upon slaughter. The COC were matured in vitro in TCM199/FCS/LH/FSH supplemented with cysteamine (0.1 mM). Subsequently, matured oocytes were fertilised with frozen-thawed gradient-separated semen and further cultured for 7 days in SOFaaBSA. The SOF medium contained either 0 (control), 0.1, 0.5, or 1.0% oestrus cow serum (ECS). Embryos development was scored at Day 7. Three replicates were performed and results were analysed by chi-square analyses. The results clearly show that adding ECS significantly improved embryo production (Table 1). Interestingly, already very low amounts (0.1%) of serum gave a significant increase in embryo percentage. In conclusion, addition of very low amounts of ECS (0.1%) is beneficial for embryo production, resulting in significantly higher embryo production (from 19 to 27%). In a subsequent field trial with OPU-derived embryos, the effect of addition of 0.1% ECS on birth weight (LOS) of the calves has to be investigated. Table 1.Percentage of blastocysts at Day 7 after culture in SOF medium with different amounts of serum

335 EFFECT OF VITAMIN E ON THE DEVELOPMENT OF BOVINE OOCYTES AFTER CHEMICAL ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 283
Author(s):  
J. I. Park ◽  
Y. Jang ◽  
E. S. Lee
Keyword(s):  
Vitamin E ◽  
Apoptotic Cell ◽  
Chemical Activation ◽  
Calf Serum ◽  
Cell Number ◽  
Total Cell ◽  
Chi Square ◽  
Cell Numbers ◽  
Humidified Air

Oxidative stress is known to induce apoptotic cell death by reactive oxygen species (ROS) generated from in vitro culture systems. This study was conducted to evaluate the effect of Vitamin E (VitE), as antioxidant, on development of bovine embryos activated in vitro. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles 3-8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL-1 l-cysteine, 20 mg/mL-1 sodium pyruvate, gonadotropins (250 IU each of eCG and hCG/mL), 10 mg/mL-1 epidermal growth factor, and 100 �M VitE. Oocytes were cultured at 38.9�C in 5% CO2 in humidified air. After 22 hours of culture, oocytes with polar bodies were selected and subjected to activation treatments. Oocytes were exposed to calcium ionomycin (5 �M for 5 min), followed by incubation with 6-DMAP (2 mM) for 3.5 hours in medium supplemented with or without VitE (100 �M). After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9�C in 5% CO2, 5% O2 in humidified air for 7–8 days. Cell numbers were counted by the number of nuclei of blastocysts stained with Hoechst 33342, and apoptosis was detected by TUNEL assay using a MK500 kit (Takara Bio, Inc., Otsu, Shiga, Japan). Total cell and apoptotic cell number were determined under a fluorescence microscope. Data were analyzed using Student&apos;s t-test and chi-square test. The cleavage and blastocyst rates were significantly higher (P &lt; 0.05) after activation with VitE (78.1&percnt; and 16.3&percnt;, n &equals; 80) than without VitE (66.7&percnt; and 11.0&percnt;, n &equals; 60). Total cell numbers were also significantly higher (P &lt; 0.05) in blastocysts after activation with VitE (143.0 &plusmn; 34.02, n &equals; 21) than in those without VitE (127.63 &plusmn; 40.25, n &equals; 20). However, the percentage of TUNEL-positive (apoptotic) cells was similar between blastocysts activated with VitE (5.38 &plusmn; 2.22) and those without VitE (6.76 &plusmn; 1.98). The results of the present study demonstrate that vitamin E added to activation medium promoted further development of activated embryos, although its role in the alleviation of apoptosis remains unclear.

126 EFFECT OF ASSISTED HATCHING ON THE SURVIVAL AND THE DEVELOPMENT OF BOVINE EMBRYOS PRODUCED IN VITRO AFTER CRYOPRESERVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 143
Author(s):  
J. Fukuhara ◽  
T. Takuma ◽  
S. Kasa ◽  
K. Imai
Keyword(s):  
Survival Rates ◽  
Calf Serum ◽  
Assisted Hatching ◽  
Chi Square ◽  
Conventional Procedure ◽  
Custom Made ◽  
Frozen Embryos ◽  
Chi Square Test ◽  
Functional Peptides

The aim of this work is to investigate the effect of assisted hatching (AH) by partial zona pellucida (ZP) dissection on the survival and the development of bovine IVP embryos after ultra-rapid vitrification and slow freezing. COC obtained from abattoir bovine ovaries were matured and fertilized in vitro, and then cultured in IVD101 (Research Institute for the Functional Peptides, Yamagata, Japan) at 38.5�C in 5% CO2, 5% O2, 90% N2. The treatment of AH was done on compacted morulae by partially dissecting ZP with a micromanipulator. As a control, non-treated embryos with intact ZP were used. For vitrification, the blastocysts at days 7 and 8 were placed into a vitrification solution (Dulbecco's PBS (D-PBS) supplemented with 20% glycerol, 20% ethylene glycol (EG), 0.3 m sucrose (SUC), 0.3 m xylose, and 3% polyethylene glycol) for 30 s after two-step equilibration. Then, they were immediately placed on a custom-made vitrification tool made of nylon fishing line with a small piece of iron attached to one end (V-tool), and immersed into liquid nitrogen (LN2). After cooling, the embryos on the V-tool were placed into frozen 0.25 mL straws filled with a diluting solution (D-PBS supplemented with 0.5 m SUC and 20% new born calf serum) using a magnet, and then they were preserved in LN2. For warming, the straws were immersed into 25�C water. The V-tool was then introduced into the column of diluting solution using a magnet. For freezing, the blastocysts at days 7 and 8 were frozen by the conventional procedure with 10% EG. For thawing, the straws were immersed into 30�C water. In this study, 120 embryos were vitrified and 128 embryos were frozen. Warmed and thawed embryos were washed more than two times, and cultured in TCM199 supplemented with 20% fetal bovine serum and 0.1 mm β-mercaptoethanol for 72 h for assessment of survivability and developmental capacity of post-thaw embryos. Data were analyzed with the chi-square test. The survival rates of vitrified embryos were the same with or without AH (81.1 and 82.0%, P > 0.05). The survival rates of frozen embryos were also the same with or without AH (76.3 and 66.7%, P > 0.05). The survival rates of vitrified embryos without AH was significantly higher than that of frozen embryos without AH (82.0 v. 66.7%, P < 0.05). The hatched rates of frozen embryos without AH were significantly lower than that of frozen embryos with AH and those of vitrified embryos with and without AH (43.5 v. 64.4%, 67.9 and 68.9%, P < 0.05). These results indicated that AH enhanced the development of frozen bovine IVP embryos and that our vitrification method using a V-tool did not require AH for development of embryos.

143 EFFECT OF FSH OR EPIDERMAL-GROWTH-FACTOR-LIKE PEPTIDE SUPPLEMENTATION TO MATURATION MEDIUM ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES DERIVED FROM FULLY DEVELOPED FOLLICLES INDUCED BY SUPER-STIMULATION

2017 ◽  
Vol 29 (1) ◽  
pp. 180
Author(s):  
T. Yamanouchi ◽  
S. Sugimura ◽  
H. Matsuda ◽  
M. Ohtake ◽  
Y. Goto ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Formation Rate ◽  
Calf Serum ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Maturation Medium ◽  
Epidermal Growth ◽  
Bovine Oocytes

Bovine oocytes obtained by ovum-pick-up (OPU) following follicle growth treatment (FGT) have improved quality and competence (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). However, the effect of the presence of FSH or epidermal growth factor (EGF) like peptide during in vitro maturation (IVM) on the developmental competence of FGT oocytes has not been well known. This study was undertaken to examine the developmental competence of FGT oocytes following IVM in the presence of FSH (recombinant human FSH) or EGF-like peptide (amphiregulin; Areg) and IVF. Japanese Black cows (n = 17) were used as donors. Five days after arbitrary OPU (opu group), follicles ≥8 mm in diameter were aspirated again, a controlled internal drug release (CIDR) was inserted into the vagina, and then pFSH was injected twice a day from the evening of Day 6 to the morning of Day 10 with decreasing doses (total of 20 AU; 4, 4, 3, 3, 2, 2, 1, 1 AU/day). On the evening of Day 8, PGF2α (0.5 mg of cloprostenol) was administered. On Day 11, oocytes were aspirated from follicles with ≥5 mm in diameter of the treated donors by OPU (fgt group). The cumulus-oocyte complexes (COC) were cultured in the absence (opu-cont and fgt-cont groups) or presence of 0.1 IU mL−1 FSH (opu-fsh and fgt-fsh groups) or 100 ng mL−1 Areg (opu-areg and fgt-areg groups) in IVM medium (mTCM199 containing 5 mg mL−1 BSA) for 20 to 22 h (1 COC/5 µL, total of 162–171 COC per group), and then co-cultured with 3 × 106 sperm/mL for 6 h. The presumptive zygotes were continued to culture in mCR1aa supplemented with 5% newborn calf serum for 216 h (1 zygote/5 µL) using micro-well culture dishes (Dai-Nippon-Print). When repeating this opu-fgt session in the same cow, an interval at least for 50 days was kept, and the session was performed 28 times. Statistical analysis was carried out by Mann-Whitney’s U-test (between opu and fgt groups) or Steel-Dwass test after Kruskal-Wallis test (among all groups). The number of follicles ≥5 mm increased in the fgt than opu group (17.8 v. 2.9; P < 0.01). The number of COC collected was not different between the opu and fgt groups (23.1 v. 19.6; P > 0.05). The blastocyst formation rate was higher in the fgt than opu group (36.9 v. 23.1%; P < 0.01). Within 6 groups, the blastocyst formation rate was higher in the fgt-fsh (43.3%; P < 0.01) and fgt-areg (39.5%; P < 0.05) groups than the opu-cont (16.3%) group. The rate in the fgt-fsh group was also higher than that in the opu-fsh group (43.3 v. 18.7%; P < 0.01). These results suggested that FGT improved the developmental competence of bovine oocytes, probably through improving the ability of the COC to react against FSH/Areg.

Cryopreservation of incomplete compacted morulae and preliminary biopsy of excluded fragments

Zygote ◽  
2019 ◽  
Vol 27 (6) ◽  
pp. 386-391
Author(s):  
Maryna Petrushko ◽  
Taisiia Yurchuk ◽  
Volodymyr Piniaiev ◽  
Natalia Buderatska
Keyword(s):  
Survival Rate ◽  
Formation Rate ◽  
Uterine Cavity ◽  
Embryo Cryopreservation ◽  
Blastocyst Formation ◽  
Significant Difference ◽  
Morula Stage ◽  
Grade 3 ◽  
Further Development

SummaryThe complexity of predicting embryo development potential at the cleavage stages and the emergence of epigenetic risks during prolonged in vitro culture of pre-implantation embryos made it more advantageous to transfer embryos at the morula stage to the uterine cavity. The criteria for estimating embryos at this stage that allow prediction of cryopreservation outcomes have been poorly described. All day 4 embryos (n = 224) were graded 1, 2, 3, 4 or 5 according to blastomere compaction degree (BCD = 100, 75, 50, 25 or 0%, respectively) and the survival and blastocyst formation rate of these morulae were studied after cryopreservation. An inverse dependence was found between survival rate and BCD. Excluded fragments were characterized by low osmotic reaction during exposure to cryoprotective medium and, after freeze-thawing, they were destroyed. As damaged necrotic areas of the embryo can affect their further development rate we proposed blastomeres and biopsy fragments of incomplete compacted morula be removed before embryo cryopreservation. This step led to significant increase in the post-thawing survival rate up to 93.1 ± 4.1%, 75 ± 8.8% and blastocyst formation rate up to 85.2 ± 10.4%, 59.4 ± 5.2% in grade 2 and grade 3 embryos, respectively. There was no significant difference in grade 4 embryos. Therefore the removal of blastomeres and biopsy fragments in incomplete compacted morulae can improve cryopreservation outcomes of grade 2 and grade 3 embryos with BCD.

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