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.

70 NON-PLATED GRANULOSA AND CUMULUS CELLS AND FIRST PASSAGE FIBROBLASTS AS NUCLEUS DONOR FOR GOAT CLONING

2006 ◽  
Vol 18 (2) ◽  
pp. 143
Author(s):  
D. Salamone ◽  
M. Catala ◽  
A. Gibbons ◽  
F. Pereyra Bonnet ◽  
M. Cueto
Keyword(s):  
Granulosa Cells ◽  
Room Temperature ◽  
Uv Light ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
First Passage ◽  
Chi Square ◽  
Donor Nucleus ◽  
Chi Square Test

Different types of somatic cells have been used as nucleus donors for cloning. Most of them were previously cultured in vitro as a monolayer through several plate passages. The experiment reported here was conducted to study the potential usages of granulosa and cumulus cells for cloning without previous culture as a monolayer. A first-plate-passage fibroblast was also used. Oocytes were aspirated by laparoscopy from Criolla goats and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL HEPES with 1 mg/mL bovine testis hyaluronidase. Metaphases were assessed and oocytes were enucleated by visualization with Hoechst 33342 (5 μg/mL) under UV light (<6 s). Granulosa and cumulus cells were also recovered by laparoscopy and maintained in maturation medium in cryotube for 20 h at room temperature or 39°C, respectively. Goat adult ear fibroblasts were cultured for 1 or 2 weeks and used 2 days after confluence. All types of donor cells were transferred to the perivitlline space of enucleated oocytes and fused by an electrical pulse. After 2 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium and atmosphere of 5% CO2 + 5% O2 + 90% N2. Cleavage (Day 2) and development to blastocysts (Day 6) were recorded and analyzed by chi-square test. The cleavage rate for non-plated granulosa cells was higher than for the other treatment goups; cumulus cells had a lower rate of development to blastocysts (Table 1). These results suggest that granulosa cells collected and maintained for 24 h at room temperature could be used to produce cloned blastocysts. Table 1. Effect of non-plated granulosa and cumulus cells and first passage fibroblasts as donor nucleus oocytes in goat cloning

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

71 EFFECT OF CELL QUANTITY IN MICE CHIMERA PRODUCTION BY DEMI-BLASTOCYST AGGREGATION

2012 ◽  
Vol 24 (1) ◽  
pp. 148
Author(s):  
C. Pontes Godoi ◽  
P. D. Moço ◽  
B. Cazari ◽  
P. T. Mihara ◽  
P. V. Silva ◽  
...  
Keyword(s):  
Cell Mass ◽  
Farm Animals ◽  
Control Group ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Chi Square Test ◽  
Aggregation Technique

Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early stages, synthesise cell adhesion molecules that increase the aggregation chances among them (Vestweber et al. 1987 Develop. Biol. 124, 451–456). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al. 2010 Transgenic Res. 19, 344–345). Based on the evidence of less permissive aggregation in post-compaction-stage embryos and the need to expose the inner surface of those embryos to improve aggregation rate, the aim of this study was to evaluate, in mice, the influence of cell quantity (i.e. the quantity of half-embryos put together to aggregate themselves) in the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/EGFP and Swiss Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008 Transgenic Res. 17, 1015). Eight-cell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona pellucida removed using pronase treatment (2 mg mL–1 for 15 min), whereas blastocysts (recovered 3.5 dpc) were split with a microblade controlled by micromanipulator in an inverted microscope (NK2; Eppendorf, Hamburg, Germany and Eclipse Ti; Nikon, Tokyo, Japan, respectively). The aggregation groups were a control (C) with 2 pre-compaction whole embryos (8C or PCM, or both) and 2 experimental with post-compaction embryos [i.e. 2 (2DB) or 4 (4DB) demi-blastocysts]. The structures (2 or 4) of the groups were stuck to each other with the use of phytohemagglutinin (1 mg mL–1) and cultured in vitro by 24 h (37°C, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified by detection of a single, cohesive cell mass or a structure in an 8 shape with more than one-half of its total diameter aggregated. For the 4DB group, a successful aggregation was considered when, at least 2 of 4 DB had aggregated. The results were analysed using chi-square test, Fisher's exact test and Kruskal-Wallis (to compare among groups, between groups and among medians of group replicates, respectively) and significance was considered when P < 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a; 8.3b and 36.4%c (P < 0.001). The increasing of the aggregation technique efficacy, in post-compaction stages, would be particularly interesting in farm animals (e.g. bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as 8 cells) and when only trophectoderm aggregation is wanted. It was concluded that cell increasing (from 2 to 4 DB) improved the chimerism rate, but not enough to be similar to the control group. Supported by FAPESP of Brazil.

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.

188 EFFECT OF TRANSFER OF FROZEN-THAWED IVF EMBRYOS ON PREGNANCY IN REPEAT-BREEDER INSEMINATED OR NON-INSEMINATED HOLSTEIN CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 202 ◽  
Author(s):  
O. Dochi ◽  
M. Tanisawa ◽  
S. Goda ◽  
H. Koyama
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Calf Serum ◽  
Pregnancy Rates ◽  
Holstein Cattle ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Chi Square Test ◽  
Repeat Breeder

Repeat-breeding is one of the important factors that affect dairy management. The objective of this study was to investigate the effect of transfer of frozen–thawed IVF embryos on pregnancy in repeat-breeder Holstein cattle. Cumulus–oocyte complexes (COCs) were collected by aspiration of 2–1-mm follicles from ovaries obtained at a local abattoir. COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg/mL of FSH at 38.5°C under a 5% CO2 atmosphere in air. Matured oocytes were inseminated with spermatozoa of 5 × 106/mL in BO solution (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) containing 10 mM hypotaurine and 4 units/mL heparin. After 18 h of gamete co-culture, presumptive zygotes were cultured in CR1aa (Rosenkrans et al. 1991 Theriogenology 35, 266) supplemented with 5% CS for 8 days at 38.5°C under 5% CO2, 5% O2, 90% N2 atmosphere in air. After in vitro fertilization, Day 7 and Day 8 blastocysts were frozen in 1.5 M ethylene glycol (EG) in Dulbecco's PBS (DPBS) supplemented with 0.1 M sucrose and 20% CS. Embryos were transferred into a freezing medium, loaded into 0.25-mL straws, and allowed to stand for 15–20 min for equilibration. The straws were then plunged into a −7°C methanol bath of a programmable freezer for 1 min, seeded at −7°C, maintained at −7°C for 15 min, cooled to −30°C at the rate of −0.3°C/min, and then plunged into liquid nitrogen. Recipient animals (43 heifers, 131 cows) included those that did not conceive after being artificially inseminated (AI) 3 to 15 times. The frozen–thawed IVF embryos were directly transferred to the recipient animals 7 days after estrus or AI. Pregnancy rates were analyzed by chi-square test. The results are presented in Table 1. There were no significant differences in the pregnancy rates between treatments. However, a slightly higher pregnancy rate was achieved by embryo transfer after AI. These results suggest that embryo transfer may increase the pregnancy rate in repeat-breeder Holstein cattle. Table 1. Pregnancy rates after transfer of IVF frozen–thawed embryos in repeat-breeder Holstein cattle

179 EFFECT OF SERUM DURING CULTURE ON DAY 14 ELONGATED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 191 ◽  
Author(s):  
J. Angulo ◽  
G. T. Gentry ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Expression Levels ◽  
The Mean

It has been reported that the addition of serum to embryo culture media alters gene expression and triggers the development of large offspring syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and in vivo-derived (IVD) embryos and to determine the effects of serum on the length of elongated embryos. Abattoir-derived oocytes were obtained from a commercial provider and fertilized at 24 h of maturation with semen from a bull previously used for IVF. At 18 h post-insemination (hpi), embryos were denuded and groups of 15 presumptive zygotes were cultured in 30-μL drops of modified SOF medium with amino acids and 6 mg mL–1 of BSA (mSOFaa). At 72 hpi, cleavage rate was assessed and embryos were randomly allocated into 2 treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed and recorded. Blastocysts (n = 5 to 10) from each treatment were transferred into synchronized recipients, and Day 14 embryos were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at –80°C in a minimal volume of PBS + 0.1% polyvinyl alcohol. Messenger RNA was isolated using a Dynabeads mRNA Direct Kit™ (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA). Quantitative PCR was performed to determine the transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R, and GAPDH for each sample. The GAPDH was used as a reference gene, and gene expression was calculated as a ratio of expression levels between each gene of interest and GAPDH. Expression levels for each gene were determined from standard curves generated by serial dilutions of PCR amplicons starting with 0.4 pg/reaction. Blastocyst development rates were higher in embryos cultured with serum compared with the nonserum treatment (14.9 and 7.4% respectively; chi-square, P < 0.001). Lengths of elongated embryos from the serum (3395.3 ± 414.7 μm) and nonserum (2784 ± 741.8 μm) culture treatments differed from the IVD (6297.7 ± 677.2 μm) treatment (mean ± SE; ANOVA, P < 0.0052). There were no differences in the mean expression levels for COX6A, IFNT1a, PLAC8, and IGF2R across treatment groups, but in the serum treatment, 3 out 11 overexpressed IFNT1a, 4 out of 11 overexpressed IGF2R, and 2 out of 11 overexpressed PLAC8, defined as being 2 standard deviations above the mean of the IVD treatment for each respective gene. In the in vitro-produced nonserum and IVD treatments, overexpression by this definition was not observed. Although mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R, and PLAC8 was observed in some embryos cultured with serum, but not in embryos cultured without serum or IVD embryos.

284 FERTILIZING CAPACITY OF BUFFALO (BUBALUS BUBALIS) SPERM CO-CULTURED WITH OVIDUCT EPITHELIAL CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 299 ◽  
Author(s):  
E. Mariotti ◽  
S. Di Francesco ◽  
M. De Blasi ◽  
C. Siniscalchi ◽  
M. V. Suárez ◽  
...  
Keyword(s):  
Production Efficiency ◽  
Penetration Rate ◽  
Bubalus Bubalis ◽  
Cleavage Rate ◽  
Chi Square ◽  
Chi Square Test ◽  
Group A ◽  
Group B ◽  
Bovine Oocytes

The overall in vitro embryo production efficiency in buffalo is hampered by the poor IVF efficiency. The aim of this work was to evaluate whether the fertilizing ability of buffalo sperm is improved by the presence of bovine oviductal cells (BOEC) during IVF. Because of limited availability of buffalo oocytes, this was assessed by heterologous IVF. Bovine oviducts were obtained at a local abattoir from cows that were in the preovulatory phase of a normal estrous cycle. BOEC recovered from 5 oviducts as previously described (Gualtieri and Talevi 2000 Biol. Reprod. 62, 1754-1762) were pooled and plated in 100 μL drops of TCM-199 + 10% FCS, 100 U mL-1 penicillin, 100 μg mL-1 streptomycin and 0.25 μg mL-1 amphotericin B under mineral oil. Medium was changed every 48 h up to Day 6, when cell confluence and cilia activity were optimal. On day of IVF the medium was removed from the drops and replaced with TALP supplemented with 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin (IVF medium). Frozen-thawed sperm from an IVF-tested buffalo bull, treated by Percoll gradients, were used for all IVF groups (2 × 106 sperm mL-1). In vitro-matured bovine oocytes (n = 409), over 3 replicates, were distributed in 4 fertilization groups: (A) IVF medium alone (control); (B) BOEC monolayer + IVF medium; (C) sperm preincubated for 6 h in IVF medium; and (D) sperm preincubated for 6 h with BOEC + IVF medium. After 20 h of coincubation at 38.5°C and 5% CO2 in air, putative zygotes were denuded, washed, and cultured in SOF medium. Forty-eight hours after IVF, cleavage rate was evaluated, and cleaved and uncleaved oocytes were fixed in 60% methanol and stained with DAPI for nuclei examination under fluorescence microscope. Data were analyzed by chi-square test. Although cleavage rate was not different among groups (46.2, 55.8, 50.0, and 50.0% for A, B, C, and D, respectively), the monospermic penetration rate increased (P < 0.01) in group B (79.3%) compared with group A (69.6%), with intermediate values in groups C (75.2%) and D (76.0%). Interestingly, the percentage of advanced embryos (>4 cells) was higher (P < 0.01) in groups C and D (47.9 and 37.1%, respectively) than in group A (12.1%), whereas group B (21.0%) was only different from group C. We demonstrated that the fertilizing capacity of buffalo sperm, evaluated as oocyte penetration rate after heterologous IVF, is enhanced by the presence of BOEC. This suggests that IVF of buffalo oocytes on BOEC monolayer may improve the IVF efficiency in buffalo. The higher incidence of advanced embryos in both groups with preincubated sperm may be accounted for by an earlier accomplishment of capacitation, leading to anticipated oocyte penetration. However, because the penetration rate in these groups was not improved compared with the control, we hypothesize that sperm viability may have decreased and hence that shorter incubation times should be tested in further studies.

68 EFFECT OF CO-CULTURE DURING FERTILIZATION OF BUFFALO (BUBALUS BUBALIS) IN VITRO-MATURED DENUDED OOCYTES VITRIFIED BY CRYOTOP

2008 ◽  
Vol 20 (1) ◽  
pp. 115
Author(s):  
L. Attanasio ◽  
A. De Rosa ◽  
L. Boccia ◽  
R. Di Palo ◽  
G. Campanile ◽  
...  
Keyword(s):  
Survival Rate ◽  
In Vitro Fertilization ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Group B

Although removal of cumulus cells improves the efficiency of vitrification of buffalo (Bubalus bubalus) in vitro-matured (IVM) oocytes (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342), the lack of cells impairs the fertilization process. Therefore, the aim of the present work was to evaluate the influence of a somatic support during in vitro fertilization (IVF) of buffalo vitrified denuded matured oocytes. Since IVF on a cumulus cells monolayer was inefficient, we verified the effects of co-culture with cumulus-enclosed oocytes (COCs). IVM buffalo oocytes (n = 316) were vitrified by the Cryotop� method (Kuwayama and Kato 2000, J. Assist. Reprod. Genet. 17, 477 abst) that was recently proven suitable for buffalo oocyte cryopreservation (Attanasio et al. 2006 Reprod. Domest. Anim. 41, 302–310). Denuded buffalo oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 3 min, transferred into 20% EG and 20% of DMSO in TCM199 with 20% fetal calf serum (FCS) + 0.5 m sucrose, loaded on Cryotops, and plunged into liquid nitrogen within 25 s. For warming, oocytes were exposed for 1 min to 1.2 m sucrose and then to decreasing concentrations of the sugar (0.6, 0.4, 0.3 m for 30 s) in TCM199 + 20% FCS. Oocytes were rinsed and allocated to IVM drops for 1.5 h. Survival rate was evaluated at this point and the oocytes that had survived (292/316 = 92.4%) were split into 2 fertilization groups: (A) approximately 5 buffalo oocytes per 50-µL drop of IVF medium, and (B) approximately 3 buffalo oocytes + 3 bovine fresh COCs per 50-µL drop of IVF medium. Since buffalo COCs easily lose their cells following IVF, for better identification we used bovine COCs that have a brighter and more compact cumulus mass. In vitro fertilization and culture were carried out as previously described (Gasparrini et al. 2007). As control, buffalo oocytes (n = 104) were in vitro-matured, fertilized, and cultured up to the blastocyst stage. On Day 1, survival rate was evaluated in the two vitrification groups; cleavage and blastocyst rates were recorded on Days 5 and 7, respectively, in all groups. The experiment was repeated 4 times. Differences in the percentages of survival, cleavage, and blastocyst formation among treatments were analyzed by chi-square test. Within vitrification groups, despite similar survival rates on Day 1 (90.6% v. 93.3%, respectively, in Groups A and B), cleavage rate was significantly improved in Group B compared to Group A (59.2% v. 45.4%, respectively; P < 0.01). Interestingly, the cleavage rate in Group B was not significantly different from that recorded in the control group (71.0%). Although blastocysts were produced in both vitrification groups (3.6% v. 4.1%, respectively, in Groups A and B), the yield was significantly lower than that of the control group (29.0%, P < 0.01). In conclusion, co-culture with bovine COC during fertilization improves the capability of buffalo denuded vitrified oocytes to cleave.

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.

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