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).

208 TIME LAPSE CINEMATOGRAPHIC ANALYSIS OF CLEAVAGE AND BLASTULATION IN BOVINE EMBRYOS OBTAINED BY OVUM PICKUP AND IN VITRO FERTILIZATION

2009 ◽  
Vol 21 (1) ◽  
pp. 202
Author(s):  
K. Imai ◽  
T. Somfai ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Calf Serum ◽  
Development Program ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Cell Divisions

Since the 1980s, several different bovine in vitro embryo production systems have been developed, and more than 291 000 embryos have been transferred throughout the world (Thibier M 2007 IETS Newsletter 25(4), 15–20). However, we have limited knowledge about the cleavage pattern of the first, second, and third cell divisions and the developmental activities of embryos during in vitro culture (IVC). The present study was conducted to determine the developmental activities of bovine embryos obtained by ovum pickup (OPU), in vitro maturation (IVM), and in vitro fertilization (IVF). We analyzed embryonic development by time-lapse cinematography (TLC). A total of 92 cumulus–oocyte complexes were collected by OPU from Japanese Black cows and were subjected to IVM and IVF as reported previously (Imai et al. 2006 J. Reprod. Dev. 52(Suppl.), S19–S29). Inseminated oocytes were cultured in microdrops of CR1aa medium supplemented with 5% calf serum covered by mineral oil in 5% CO2 in air at 38.5°C. Kinetics of embryo development were measured by TLC for 168 h after IVF by using a Cultured Cell Monitoring System (CCM–M1.4ZS, Astec, Fukuoka, Japan). A total of 672 photographs of the embryos were taken (1 photograph every 15 min) during IVC. Image stacks were analyzed by the CCM–M1.4 software. Timing of the first, second, and third cell divisions, blastulation, and embryonic contractions were recorded. The results are reported as time (h) passed after insemination. In total, 75 (81.5%) embryos cleaved and 61 (66.3%) embryos developed to the blastocyst stage. The first, second, and third cell divisions in these viable embryos occurred at 24.0 ± 0.5, 32.1 ± 0.2, and 39.4 ± 0.4 h (mean ± SE) after IVF, respectively. On the other hand, in nonviable embryos (those that failed to develop to the blastocyst stage; n = 14), these cell divisions occurred at 29.5 ± 2.2, 41.3 ± 3.3, and 57.2 ± 7.6 h after IVF, respectively. There tended to be a difference (P = 0.06; paired t-test) in the timing of the first cell division between viable and nonviable embryos. Blastulation of embryos began at 114.4 ± 1.1 h, embryos developed to the blastocyst stage at 127.3 ± 1.4 h, and blastocysts began to expand at 138.4 ± 1.7 h after IVF, respectively. During blastocyst development, embryonic contractions (shrinkage attributable to the rupture of the blastocoele) and tight-shrinkage (shrinking of the embryo to less than 70% of its surface area) were observed in all embryos. The mean numbers of contractions and tight-shrinkages in blastocysts were 5.3 ± 2.7 and 2.1 ± 1.0 times, respectively. The frequency of contractions from the beginning of blastulation to the blastocyst stage was significantly lower (P < 0.01) than after the blastocyst stage. It took 6.9 ± 4.6 h for the embryos to re-expand after the tight-shrinkages. These results indicate that viable in vitro-produced embryos can be selected at early stages by TLC. Further studies are necessary to clarify the importance of the pulsating activity in OPU–IVF embryos. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

scholarly journals 275FOLLICULAR ATRESIA IN SMALL, NON-FSH-DEPENDENT BOVINE FOLLICLES IS ASSOCIATED WITH INCREASED DEVELOPMENTAL POTENTIAL OF OOCYTES FOLLOWING IVP

2004 ◽  
Vol 16 (2) ◽  
pp. 258
Author(s):  
H. Irving-Rodgers ◽  
S. Morris ◽  
R. Collett ◽  
K. Catanzariti ◽  
T. Peura ◽  
...  
Keyword(s):  
Basal Lamina ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Early Cleavage ◽  
Blastocyst Development ◽  
Chi Square ◽  
Developmental Potential ◽  
Chi Square Analysis

Oocytes from small, non-FSH-dependent follicles are associated with reduced developmental competence following in vitro embryo production (IVP) compared to oocytes from larger follicles. It has been suggested that, for small follicles, oocytes derived from atretic follicles are more developmentally competent than those from healthy follicles (Blondin P and Sirard MA, 1995 Mol. Reprod. Dev. 41, 54–62). Little is known of the characteristics of small follicles that support developmentally competent oocytes. Here we examine the development to blastocyst stage of oocytes collected from histologically-assessed bovine 2–5mm follicles. Ovaries were obtained at a local abattoir;; 4 follicles were dissected from each ovary and oocytes were recovered. A section of each follicle wall was taken and fixed in 2.5% glutaraldehyde for histological assessment of the follicle and characterization of the morphology of the follicular basal lamina by electron microscopy (Irving-Rodgers HF and Rodgers RJ, 2000 J. Reprod. Fert. 118, 221–228). Oocytes recovered from follicles underwent IVP utilizing a novel single IVP system. Oocytes were matured for 24h (10μL per COC) in TCM199, supplemented with FSH, hCG, FCS, cysteamine and pyruvate. Mature oocytes were inseminated with 1×106 sperm mL−1 for an additional 24h using Bovine Fertilization Medium (10μL per COC;; Cook, Australia). Following insemination, putative zygotes were stripped of remaining cells and placed within individual micro-wells prepared in 1% agar in Bovine Early Cleavage Medium, Cook, Australia. The agar (350μL) was prepared within wells of a 4-well plate and small plugs of agar were removed to form micro-wells. The agar was over-laid with 450μL of Early Cleavage Medium and 250μL mineral oil, and equilibrated overnight before putative zygotes were placed individually within micro-wells. Culture was performed under 7% O2, 6% CO2, and 87% N2 at 39°C. On Day 5 following insemination, fetal calf serum (final concentration 10% v/v) was added to facilitate blastocyst development. Blastocyst formation was assessed on Day 8. A total of 211 oocytes were cultured and 69% were from healthy follicles;; 67 oocytes (32%) had developed to the blastocyst stage by Day 8. Forty-three percent of oocytes recovered from atretic follicles (28/65) had developed to the blastocyst stage by Day 8, as compared to only 27% (39/146) oocytes recovered from healthy follicles, this difference was significant (P&lt;0.05, chi-square analysis). Seventy-eight percent (14/18) of oocytes from healthy follicles with additional follicular basal lamina material (Irging-Rodgers HF and Rodgers RJ, 2000 J. Reprod. Fert. 118, 221–228) failed to develop, whereas only 44% (4/9) of oocytes from healthy follicles with a normal basal lamina failed to develop (P&gt;0.08). The present study finds a direct association between the follicle morphology and oocyte maturational potential within non-FSH dependent follicles, revealing that high levels of development (&gt;40%) can be obtained from atretic follicles. Furthermore, differences between healthy follicles may also contribute to developmental variation.

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.

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.

138 THE RELATIONSHIP BETWEEN THE NORMALITY OF FIRST CLEAVAGE, THE GENE EXPRESSION IN BLASTOMERES, AND THE ABILITY TO DEVELOP TO THE BLASTOCYST STAGE IN IVF-DERIVED BOVINE 2-CELL STAGE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 177
Author(s):  
S. Matoba ◽  
M. Kaneda ◽  
T. Somfai ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Target Genes ◽  
Calf Serum ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Embryonic Cleavage ◽  
The Relationship

Early first and second cleaved embryos after IVF associated with even blastomeres without fragments or protrusions were found to be a potent criterion for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLOS ONE 7, e36627). The aim of this study was to examine the relationship between an early normal first cleavage pattern, the transcript abundance, and their development to the blastocyst stage in each blastomere in 2-cell stage bovine embryos. The IVF-derived bovine embryos were cultured individually in well-of-the-well culture dishes 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. The first embryonic cleavage was categorized as being either normal (occurring within 28 h after IVF with 2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with/without fragment/protrusion and/or later than 28 h after IVF). Then, cleaved embryos were placed in 0.5% actinase-E in Ca- and Mg-free PBS and blastomeres were separated by pipetting (n = 85; 4 replicates). In each embryo, one blastomere was subjected to quantitative RT-PCR to analyse the expression of developmentally important genes. The remaining blastomere was subsequently cultured in an individually identifiable manner to verify their ability to develop to the blastocyst stage. Primers were designed for 12 target genes related to pluripotency, cell cycle, metabolism, pregnancy reorganization, placentation, and fetal growth (OCT4, ATP1A1, CCNB1, CDH1, COX1, CTNNB1, GLUT8, MNSOD-3, SOX2, DYNLL1, IGFBP3, and PMSB1) and a reference gene (PPIA). Transcript abundance of target genes in individual blastomeres was compared between embryos showing normal and abnormal cleavage. Values were normalized to the average values of the reference genes and all the means were compared by the Student t-test. Blastomeres resulted from normal cleavage developed to the blastocyst stage on Day 7 to 8 (Day 0 = IVF) at significantly higher rates than those resulted from abnormal cleavage (65.7% v. 37.5%, respectively, P < 0.05). Transcript abundance of OCT4 was significantly higher in blastomeres associated with all abnormal cleavage than in those associated with normal cleavage (P < 0.05). The expression of CCNB1, COX1, ATP1A1, GLUT8, and PMSB1 in blastomeres associated with normal cleavage and blastocyst development was higher than that in those of abnormal cleavage (P < 0.05). However, the level of OCT4, CCNB1, COX1, ATP1A1, and PMSB1 was lower in blastomeres associated with normal cleavage but failure of blastocyst development than those in blastomeres showing abnormal cleavage (P < 0.05). Our results reveal that significantly higher expression of CCNB1, COX1, ATP1A1, and PMSB1 in blastomeres at the 2-cell stage in bovine embryos with superior developmental competence compared with those showing abnormal cleavage and low competence. Research was supported by JSPS KAKENHI (26450388).

288 KINETICS OF FERTILIZATION, DEVELOPMENT, AND SEX RATIO OF IN VITRO-PRODUCED BOVINE EMBRYOS OBTAINED WITH FOUR DIFFERENT BULLS

2007 ◽  
Vol 19 (1) ◽  
pp. 259 ◽  
Author(s):  
M. Alomar ◽  
H. Tasiaux ◽  
S. Remacle ◽  
F. George ◽  
D. Paul ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test ◽  
Kinetics Of

The between-bulls variation in in vitro fertility and the shift of sex ratio toward male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the possible correlation between the kinetics of fertilization, embryo development, and the sex ratio of the resulting embryos. In a first experiment, and using frozen-thawed semen of 4 different AI bulls, the kinetics of pronucleus (PN) formation was evaluated at 8, 12, and 18 h post-in vitro insemination (hpi) after fixation and staining with Hoechst 33342. Fertilized oocytes were classified in 3 PN stages: PN1: showing the first signs of sperm head decondensation; PN2: with two pronuclei of different sizes, the two being far from each other; and PN3: showing two symmetric pronuclei of equal size, close to each other. Differences between bulls were observed at each time point, but were greater at 12 hpi than at 8 or 18 hpi. At 8 hpi and 12 hpi, bull C showed a significantly faster PN formation by comparison with the 3 other bulls (chi-square test: P &lt; 0.05), whereas at 18 hpi, the proportion at each of the PN stages was similar to that of bulls A and D, with bull B showing delayed PN development. In a second experiment, a standard IVP procedure was conducted with the 4 bulls to determine cleavage and blastocyst rates. The timing of first cleavage was measured using time-lapse cinematography. Compared with those of bull B, the embryos generated with bull C led to significantly higher Day 7 blastocyst yields (31.3 � 9.5% vs. 21.9 � 6.7%; ANOVA: P &lt; 0.05). Moreover, the embryos from bull C reaching the blastocyst stage cleaved faster (first cleavage at 23.1 � 2.1 hpi vs. 25.4 � 2.7 hpi for bull B; ANOVA: P &lt; 0.05). In a third experiment, 65 to 76 Day 8 blastocysts were sexed per bull. Embryo sexing was performed by PCR using the co-amplification of a Y-specific bovine SRY sequence and an autosomal btRep-137 sequence. Only blastocysts obtained with bull C showed a shift in sex ratio toward male embryos (76.0% male embryos vs. 53.8% for bull B; chi-square test: P &lt; 0.05), whatever the size of the blastocyst. The shift in sex ratio was already present at the 2-cell stage (64.2% male embryos; n = 53; chi-square test: P &lt; 0.05). In conclusion, for 2 out of 4 bulls, a correlation was observed between the kinetics of PN formation, the timing of first cleavage, and the sex ratio of the resulting embryos.

75 Culture of isolated blastomeres supplemented with L-ascorbic acid 2-phosphate in a well-of-the-well culture dish

2019 ◽  
Vol 31 (1) ◽  
pp. 163
Author(s):  
Y. Hasiyada ◽  
H. Matsuda ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
T. Yamanouchi
Keyword(s):  
Formation Rate ◽  
Calf Serum ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Culture Dish ◽  
Cell Stage ◽  
Quality Grade

We have reported monozygotic twin calves that can be produced efficiently by blastomere separation of 2-cell stage embryos and by the use of a commercially provided well-of-the-well culture dish (Hashiyada 2017 J. Reprod. Dev.). The present study was conducted to evaluate the effect of a culture supplement, l-ascorbic acid 2-phosphate (AA-2P), a sustained antioxidant substance that reduces reactive oxygen species. Embryos were produced using oocytes from ovaries collected at an abattoir by in vitro maturation, IVF, and in vitro culture (IVC). TCM199 supplemented with 5% calf serum, Brackett-Oliphant solution supplemented with 10mg mL−1 BSA, and CR1aa containing 5% calf serum were used for each culture step. Two-cell stage embryos were obtained 24 to 27h post-insemination (hpi). Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into each blastomere by gentle pipetting in IVC medium. Each blastomere was introduced into a single conical micro-well of 25 wells, each having a diameter and depth of ~287 and 168µm (Dai Nippon Printing, Tokyo, Japan). Culture of blastomeres in wells was performed covered with a droplet of 2.5 µL/well IVC medium supplemented with 0 (n=212), 250 (n=214), 500 (n=206), and 750 µM (n=204) of AA-2P. The blastocyst formation rate at Day 8 after IVF, the quality of blastocysts assessed by morphological observation, and the cell numbers were compared among each concentration of AA-2P. In addition, the developmental speed to the blastocyst stage was analysed using time-lapse cinematography for 0 and 500 µM of AA-2P (n=40, respectively). Statistical analysis was performed using Fisher’s exact test and ANOVA. The blastocyst formation rate (32-40%), the total cell number (108-114), and inner cell mass cell number (26-28) did not differ among groups. The time to reach the 4-cell stage was significantly shorter in media supplemented with 0 µM (43 hpi) than with 500 µM (52 hpi); however, the time to reach the blastocyst stage did not differ (150 and 155 hpi, respectively). Regarding the proportion of quality grade 1 to 3 blastocysts and the developmental speed to the blastocyst stage, high-quality grade 1 embryos were significantly faster than those of middle and low-quality grade 2 and 3 ones in 0 (145 v. 154 hpi; P&lt;0.05) and 500 µM (150v. 158 hpi; P&lt;0.05) supplemented medium. In this experiment, no effect of AA-2P was observed for the culture of isolated blastomeres from 2-cell stage embryos, although it was suggested that blastomeres with high developmental competence reach the blastocyst stage faster, which might reflect the quality of the embryos.

350 EFFECT OF PROGESTERONE SUPPLEMENTATION OF MATURATION MEDIUM ON DEVELOPMENT OF IVM-IVF-IVC BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 331
Author(s):  
K. Miyata ◽  
H. Koyama ◽  
C. Lessard ◽  
J. Singh ◽  
O. Dochi
Keyword(s):  
In Vitro Maturation ◽  
Formation Rate ◽  
Calf Serum ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Oocyte Competence ◽  
Maturation Medium ◽  
Progesterone Supplementation

Follicular fluid from small and large bovine follicles contains large amounts of progesterone, and during preovulatory period progesterone concen- tration increase markedly by 18 h after LH surge. Furthermore, cumulus cells express membrane progestin receptor beta (Liu et al. 2008 Steroids 73, 1416-1423). For these reasons, we hypothesized that progesterone supports maturation of preovulatory bovine oocytes to MII stage. The object of this study was to investigate the effect of progesterone supplementation of in vitro maturation medium on competence of bovine oocyte to develop into blas- tocysts in vitro. COCs were collected by the aspiration of 2-6 mm follicles from ovaries within 6 h of slaughter. The COCs were divided into 5 groups: (1) a control group, TCM-199 supplemented with 5% calf serum (CS) as IVM medium, and (2 to 5) progesterone (P4) supplementation groups, TCM- 199 supplemented with 5% CS and 1, 3, 5, and 10 μg mL-1 of P4. Groups of 10 COCs were incubated in 50-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. The matured COCs were inseminated with 3 × 106 sperm mL-1. After 18 h of gamete co-culture, the pre- sumptive zygotes were cultured in CR1aa media 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 (total cleavage rates) and on Days 7 to 9 (blastocyst rate). Data was analyzedby chi-square test. The results are presented in Table 1. There were no significant differences in the cleavage rates between treatments. However, the blastocyst formation rate of 5 μg mL-1 P4 supplementation group was significantly higher than that of 10 μg mL-1 P4 supplementation group (P < 0.05). In addition, the blastocyst formation rates of 10 μg mL-1 P4 supplementation group was lower than the other groups. These results suggest that progesterone supple- mentation of in vitro, maturation medium affects the competence of the oocytes to develop into blastocysts in vitro, and 5 μg mL-1 P4 supplementation may be effective in increasing embryo production. Furthermore, 10 μg mL-1 P4 supplementation has negative effect on the oocyte competence. Table 1.Effect of progesterone supplementation on development of IVF bovine embryos

165 THE EFFECTS OF L-CARNITINE AND LINOLEIC ACID ALBUMIN SUPPLEMENTATION ON THE DEVELOPMENT AND CRYOSURVIVAL OF BOVINE IN VITRO-MATURED/IN VITRO-FERTILIZED EMBRYOS IN IN VITRO CULTURE MEDIUM

2012 ◽  
Vol 24 (1) ◽  
pp. 194
Author(s):  
S. Miyashita ◽  
Y. Inaba ◽  
T. Somfai ◽  
M. Geshi ◽  
T. Nagai ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
In Vitro Culture ◽  
Culture Medium ◽  
Formation Rate ◽  
Calf Serum ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Formation ◽  
Cleavage Rate

The objective of this study was to investigate the effects of the supplementation of a lipid metabolism inducer, L-carnitine (LC) and a membrane stabilizer, linoleic acid albumin (LAA), on the developmental competence and cryosurvival of bovine in vitro-matured/in vitro-fertilized embryos in in vitro culture medium. Cumulus–oocyte complexes collected from the ovaries of slaughtered cattle were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH at 38.5°C in an atmosphere of 5% CO2 in air. After IVF (Day 0), presumptive zygotes were cultured in CRlaa containing 5% CS at 38.5°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days. The culture medium was supplemented with 0.6 mg mL–1 of LC (LC group; n = 180) or with 0.25 mg mL–1 of LAA (LAA group; n = 180) or with both LC and LAA (LC + LAA group; n = 180) or without LC and LAA (control; n = 178). The cleavage rates were recorded on Day 2 and the blastocyst formation rates were recorded on Day 7 to 9. Expanded blastocysts harvested on Day 7 and 8 (LAA group: n = 31; LC group: n = 29; LC + LAA group: n = 25; control group: n = 33) were used for freezing in modified PBS supplemented with 1.5 M ethylene glycol, 0.1 M sucrose and 20% CS. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1 mM β-mercaptoethanol at 38.5°C under 5% CO2 in air for 72 h. The rates of re-expansion, hatching and formation of hatched blastocysts were determined at 24, 48 and 72 h after thawing, respectively. The rates of cleavage and blastocyst formation were expressed as mean ± s.e.m. and analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by chi-square test. The cleavage rate in the control group (69.1 ± 2.5%) was significantly lower than that in the LAA (81.8 ± 3.8%) and LC + LAA groups (77.9 ± 1.4%) but did not differ from that in the LC group (73.8 ± 2.4%). The blastocyst formation rate in the control group (21.7 ± 2.8%) was significantly lower (P < 0.05) than those in the LAA and LC + LAA groups (33.5 ± 2.8% and 31.4 ± 2.4%, respectively), but it did not differ significantly from that of the LC group (32.1 ± 3.3%) despite a strong tendency (P = 0.06). There were no significant differences among the control, LC, LAA and the LC + LAA groups in post-thaw re-expansion rates (66.7, 75.9, 67.7 and 76.0%, respectively), hatching rates (48.5, 69.0, 58.1 and 64.0%, respectively) and rates of formation of hatched blastocysts (51.5, 62.1, 61.3 and 64.0%, respectively). These results indicate that the addition of LC and LAA to the medium for in vitro culture of in vitro-matured/in vitro-fertilized bovine embryos improved their ability to develop to the blastocyst stage; however, the effects on the freezing tolerance were not verified.

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