223 RELATIONSHIP BETWEEN THE LENGTH OF CELL CYCLES, CLEAVAGE PATTERN, AND DEVELOPMENTAL COMPETENCE DURING IN VITRO CULTURE OF IN VITRO-MATURED/IN VITRO-FERTILIZED BOVINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 209 ◽  
Author(s):  
T. Somfai ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
S. Kobayashi ◽  
...  
Keyword(s):  
Cell Division ◽  
In Vitro Culture ◽  
Cell Mass ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Pattern ◽  
Cell Cycles ◽  
Bovine Oocytes

In in vitro embryo production systems, there is a need to select embryos with good developmental competence at the early stages. This study was conducted to determine whether there was any relationship between the duration of the first 3 cell cycles, the cleavage pattern of the first cell division, and the developmental competence of embryos during in vitro culture. A total of 320 in vitro-matured and in vitro-fertilized bovine oocytes were cultured in microdrops of CR1aa medium supplemented with 5% calf serum covered by mineral oil in 5% CO2 and 20% O2 at 38.5°C. The kinetics of embryo development were measured by time-lapse cinematography. Embryos were classified according to their cleavage pattern at the first cell division. Of 285 cleaved embryos, 119 had 2 blastomeres of the same size (normal cleavage: NC), 49 had 2 blastomeres with multiple small fragments (multiple fragments: MF), 34 had 2 blastomeres and a protrusion (protrusion: PT), 45 showed direct cleavage from 1 cell to 3 or 4 blastomeres (3–4BL), and 60 oocytes cleaved to 2 blastomeres of different sizes (unequal blastomeres: UB). (Twenty-two embryos belonged to 2 classes.) After 175 h of culture, blastocysts were either subjected to differential inner cell mass/trophectoderm (ICM/TE) staining or karyotyped. The first and second cell cycles (mean ± SEM) of viable embryos (that could develop to the blastocyst stage) were significantly shorter than those of nonviable embryos (24.9 ± 0.3 h and 8.7 ± 0.1 h v. 26.6 ± 0.7 h and 10.0 ± 0.1 h, respectively); however, the length of the third cell cycle did not differ (P < 0.05, paired t-test). The duration of 1 cell stage in the NC group was significantly shorter than that of MF, PT, 3–4BL, and UB groups (24.7 ± 0.4 h, 26.6 ± 0.5 h, 26.3 ± 0.6 h, 26.0 ± 0.2 h, and 27.7 ± 0.9 h, respectively). The length of the second and third cell cycles did not differ among the groups. The percentage of NC embryos that developed to the blastocyst stage was similar to that of the 3–4BL group (66.9 and 56.7%, respectively) but was significantly higher than those of the MF, PT, and UB groups (40.5, 26.5, and 35.6%, respectively; P < 0.05, ANOVA). The mean cell numbers of NC blastocysts did not differ from those of the MF, 3–4BL, and UB groups but were higher than those of PT embryos (147.1, 155.6, 121.6, 146.4, and 115.1, respectively). There was no difference in ICM/TE rates between the groups. Unlike NC, MF, PT, and UB embryos, most (6 of 8 karyotyped) 3–4BL blastocysts had abnormal ploidy, such as haploid, triploid, mixoploid, or chaotic chromosome numbers, in blastomeres. Our results revealed that not only the length of the first cell cycles, but also the cleavage pattern during first cell division can be a marker of developmental competence and should be considered for the selection of good-quality embryos for embryo transfer. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

236 KINETICS AND PATTERN OF THE FIRST CLEAVAGE OF IN VITRO-FERTILIZED EMBRYOS BY IN VIVO-MATURED OOCYTES AND X-SORTED SPERMATOZOA IN DAIRY CATTLE

2013 ◽  
Vol 25 (1) ◽  
pp. 266
Author(s):  
S. Matoba ◽  
S. Sugimura ◽  
H. Matsuda ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Holstein Cows ◽  
Cleavage Pattern ◽  
Cell Cleavage ◽  
Significant Difference

Recently, we reported that high rates of good-quality blastocysts can be produced by IVF of in vivo-matured oocytes, obtained by ovum pick-up (OPU) after superstimulation in Holstein cows, with X-sorted sperm [Matoba et al. 2012 Reprod. Domest. Anim. 47(Suppl. 4), 515]. However, we have limited knowledge concerning the normality of embryonic cleavages in such embryos. The present study examined their kinetics and pattern of the first cell cycle. In vivo-matured oocytes were collected by OPU from non-lactating Holstein cows just before ovulation after superstimulation and ovulation induction by gonadotropin-releasing hormone. The oocytes were inseminated with 5 × 106 sperm mL–1 of X-sorted sperm and cultured in CR1aa supplemented with 5% newborn calf serum and 0.25 mg mL–1 of linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually using a microwell culture dish and time-lapse cinematography (CCM-1.4MZS, Astec, Fukuoka, Japan) (Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken every 15 min during the in vitro culture period, and images were analysed by CCM-1.4 software (Astec). The cleavage pattern was categorised into normal cleavage (2 even blastomeres without fragment or protrusion) or abnormal cleavage (those with 2 uneven blastomeres, with fragments or protrusions and those dividing into 3 to 5 blastomeres at the first cleavage). Data were analysed by ANOVA, chi-square, and discriminant function. A total of 117 embryos were examined; of this number, 63.2% developed to the blastocyst stage and the rest were degenerated. A high rate of normal cleavage and a low rate of abnormal cleavage, including those with 2 uneven blastomeres and those with fragments or protrusions in the first cleavage pattern, were recorded in embryos that could develop to blastocysts compared with degenerated ones (P < 0.01 or P < 0.05, respectively; Table 1). No significant difference was found in those dividing into 3 to 5 blastomeres between the blastocysts and degenerated embryos (Table 1). Embryos developing to the blastocyst stage had a shorter duration of the first cell cycle [27.2 ± 2.3 h post-insemination (hpi)] compared with those undergoing degeneration (30.6 ± 5.7 hpi; P < 0.001). The threshold of duration of the first cell cycle was calculated by (X – 27.2)/2.3 = (30.6 – X)/5.7, resulting in X = 28.2. Blastocysts with a short duration of the first cell cleavage (≤28.2 hpi) showed a higher frequency of the normal cleavage pattern than those with a duration of the first cell cleavage longer than 28.2 hpi (71.7 and 53.6%, respectively; P < 0.05). Our results revealed that those IVF embryos that finished their first cleavage before 28.2 h of IVF and showed a normal cleavage pattern had superior developmental competence. Table 1.The first cleavege pattern reflects the developmental competence: blastocysts versus degenerated embryos This work was supported by the Research and Development Projects for Application in Promoting New Policy of Agriculture, Forestry and Fisheries (22016).

346 BOVINE EMBRYO DEVELOPMENT AFTER IVM/IVF/IVC OF OOCYTES STORED FOR 22 H IN VARIOUS MEDIA

2007 ◽  
Vol 19 (1) ◽  
pp. 288
Author(s):  
C. Kubota ◽  
T. Kojima ◽  
T. Nagai ◽  
X. Tian ◽  
X. Yang
Keyword(s):  
Subsequent Development ◽  
Industrial Applications ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Bovine Embryo ◽  
Becton Dickinson ◽  
In Vitro Storage ◽  
Bovine Oocytes

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco&apos;s PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus&ndash;oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10&percnt; fetal bovine serum (FBS) or 1 mg mL&minus;1 polyvinyl alcohol (PVA). COCs were collected from follicles (3&ndash;8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5&deg;C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10&percnt; FBS and 20 &micro;g mL&minus;1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281&ndash;286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS&ndash;IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM&ndash;IVF&ndash;IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student&apos;s t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC (bFF (n &equals; 87): 0&percnt;, 0&percnt;; PBS/FBS (n &equals; 72): 84&percnt;, 1&percnt;; and PBS/PVA (n &equals; 81): 89&percnt;, 6&percnt;, respectively) were significantly lower than those of the control group (n &equals; 406; 97&percnt; and 29&percnt;, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS&ndash;IVM&ndash;IVF (M199A/FBS (n &equals; 97): 82&percnt;, 28&percnt;; and M199A/PVA (n &equals; 111): 98&percnt;, 31&percnt;, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.

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.

132 The Developmental Competence of Bovine Oocytes Exposed to Progesterone and Luteotrophic Hormones During the Second Step of Two-Step Maturation

2018 ◽  
Vol 30 (1) ◽  
pp. 206
Author(s):  
G. Singina ◽  
I. Lebedeva ◽  
T. Taradajnic ◽  
E. Shedova ◽  
A. Lopukhov ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Calf Serum ◽  
Developmental Competence ◽  
Second Step ◽  
Russian Science ◽  
Cleavage Rate ◽  
System 1 ◽  
And Control ◽  
Bovine Oocytes

Data on effects of progesterone (P4) during in vitro maturation of bovine oocytes on their capacity for embryonic development are contradictory. Our study was aimed at characterising effects of P4 and 2 luteotropic hormones, prolactin (PRL) and LH, on bovine oocyte developmental competence during the second step of two-step maturation (from metaphase (M)I to MII). Slaughterhouse-derived cumulus-enclosed oocytes (CEO) were matured for 12 or 24 h [one-step (OS) Control] in TCM-199 containing 10% fetal calf serum (FCS), 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH at 38.5°C and 5% CO2. The CEO cultured for 12 h were transferred to the following culture systems: (1) TCM-199 containing 10% FCS (Control 1) or (2) a monolayer of granulosa cells (GC) precultured for 12 h in TCM-199 containing 10% FCS (Control 2); then, the oocytes were matured for next 12 h. In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (25 and 50 ng mL−1) or ovine LH (5 μg mL−1). All treatments were repeated 5 to 6 times using 138 to 196 oocytes per group. Following IVM, all oocytes underwent IVF as described previously (Singina et al. 2014 Reprod. Fertil. Dev. 26, 154). Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured to Day 7. Embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst formation. Apoptosis was detected by the TUNEL method using 26 to 47 blastocysts per group (from 4 to 5 separate experiments). For each system, arcsine-transformed data were analysed by one-way ANOVA. In OS Control, the cleavage and blastocyst rates were 68.9 ± 4.4% and 22.0 ± 2.4%, respectively. Regardless of the system or medium of two-step culture, the cleavage rate did not differ from that for OS Control, varying between 57.6 and 68.4%. In the absence of GC (System 1), the blastocyst yield in the P4 group (30.4 ± 0.8%) was greater (P < 0.05) than in OS Control and Control 1 (20.2 ± 2.7%) as well as in the groups treated with LH (19.1 ± 3.0%) and 25 ng mL−1 PRL (20.1 ± 2.7%). In the presence of GC, P4 raised the yield from 16.7 ± 2.3% (Control 2) to 27.7 ± 2.4% (P < 0.05). Furthermore, in System 2, the blastocyst rate in groups treated with P4 and 50 ng mL−1 PRL (25.0 ± 2.8%) was higher (P < 0.05) than in the LH group (13.9 ± 2.6%). Meanwhile, the proportion of apoptotic nuclei (2.3-6.9%) was not associated with the system of oocyte maturation or effects of hormones studied. Our data indicate that P4 (50 ng mL−1) can enhance the developmental competence of bovine oocytes during the second step of two-step maturation regardless of the presence of granulosa cells, whereas the similar effect of PRL (50 ng mL−1) is less pronounced and depends on the granulosa-conditioned environment. This research was supported by the Russian Science Foundation (project 16-16-10069).

Effect of bovine oviductal fluid on development and quality of bovine embryos produced in vitro

2017 ◽  
Vol 29 (3) ◽  
pp. 621 ◽  
Author(s):  
Ricaurte Lopera-Vasquez ◽  
Meriem Hamdi ◽  
Veronica Maillo ◽  
Valeriano Lloreda ◽  
Pilar Coy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Cell Mass ◽  
Water Channel ◽  
Calf Serum ◽  
Cell Counting ◽  
Bovine Embryos ◽  
Oviductal Fluid

To evaluate the effect of bovine oviductal fluid (OF) supplementation during in vitro culture of bovine embryos on their development and quality, in vitro-produced zygotes were cultured in synthetic oviductal fluid (SOF; negative control; C–) supplemented with OF or 5% fetal calf serum (positive control; C+). Embryo development was recorded on Days 7–9 after insemination and blastocyst quality was assessed through cryotolerance, differential cell counting of the inner cell mass and trophectoderm, and gene expression. OF was added to the culture medium at concentrations ranging from 0.625% to 25%. The higher OF concentrations (5%, 10% and 25%) had a detrimental effect on embryo development. Lower OF concentrations (1.25% and 0.625%) supported embryo development until Day 9 (27.5%) and produced higher-quality blastocysts, as reflected by their cryotolerance (53.6% and 57.7% survival at 72 h, respectively, vs 25.9% in C+) and total cell number (mean (± s.e.m.) 165.1 ± 4.7 and 156.2 ± 4.2, respectively, vs 127.7 ± 4.9 in C– and 143.1 ± 4.9 in C+). Consistent with these data, upregulation of the water channel aquaporin 3 (AQP3) mRNA was observed in blastocysts supplemented with 1.25% OF compared with C– and C+. Serum supplementation resulted in a reduction in the expression of glucose and lipid metabolism-related genes and downregulation of the epigenetic-related genes DNA methyltransferase 3A (DNMT3A) and insulin-like growth factor 2 receptor (IGF2R). In conclusion, in vitro culture with low concentrations of OF has a positive effect on the development and quality of bovine embryos.

scholarly journals 94MEIOSIS INHIBITION OF BOVINE OOCYTES: EFFECTS ON SURVIVAL AFTER VITRIFICATION BY OPEN PULLED-STRAW METHOD

2004 ◽  
Vol 16 (2) ◽  
pp. 168
Author(s):  
C. Diez ◽  
M. Carbajo ◽  
L. Fernandez ◽  
C.O. Hidalgo ◽  
S. de la Varga ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
17Β Estradiol ◽  
Bovine Oocytes ◽  
Range Test

Mammalian oocytes remain one of the most difficult cell types to successfully cryopreserve. The in vitro-maturation protocols (IVM) have a large impact on the oocyte maturation. Consequently, inhibition of meiosis has been used to improve developmental competence of oocytes without reducing blastocyst rates. Moreover, the meiotic stage influences the ability of oocytes to survive cryopreservation. This work analyzes the effect of the inhibition of meiosis (prematuration) on the freezability of the bovine oocyte. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries. Inmature oocytes (I) with compact cumulus and evenly granulated cytoplasm were selected. Prematuration (PM) was performed by incubating COCs for 22h in TCM199 NaHCO3 and roscovitine 25μM. IVM was accomplished in TCM199 NaHCO3, 10% FCS, FSH-LH and 17β-estradiol. Oocytes were subjected to 5 treatments prior the vitrification (see table). COCs were partially denuded from cumulus cells and vitrified/warmed using the OPS system (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). Warmed oocytes were fertilized (Day 0) and presumptive zygotes having a normal morphological appearance were cultured in SOFaa+5% of FCS (Day 3); elements with degenerated appearance were discarded and recorded. Fresh oocytes submitted to IVM (c-M) or prematured and matured (c-PM+M) were fertilized and cultured as controls. Data were analyzed by ANOVA and Duncan’s multiple range test and expressed as LSM±SE. Developmental data are referred to the zygotes cultured. Only oocytes vitrified after IVM reached the blastocyst stage, but at lower rates than fresh controls. However, no differences were found between treatments at any developmental stage. Oocytes vitrified both as prematured+matured and immature oocytes showed increased proportions (P&lt;0.01) of degenerated oocytes (37.3±5.9 and 49.9±5.9, respectively), as compared with oocytes matured before vitrification (17.6±5.9). These results show that effects induced by incubation with roscovitine (Lonergan et al. 2003 Mol. Reprod. Dev. 64, 369–378) in combination with cryodamage compromise the oocyte developmental ability. Supported by CICYT, AGL2001-379.

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).

235 PROGESTERONE AFFECTS THE MESSENGER RNA EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 265
Author(s):  
K. Knauer ◽  
H. Stinshoff ◽  
S. Wilkening ◽  
C. Wrenzycki
Keyword(s):  
In Vitro Culture ◽  
Messenger Rna ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Morphological Development ◽  
Bovine Embryo ◽  
Development Rates ◽  
Vitro Model

It is known that the progesterone (P4) provided by the corpus luteum is essential for the maintenance of pregnancy. It has been suggested that supplying external P4 in vivo is beneficial to the establishment and upkeep of pregnancy. The aim of the present study was to assess the effects of supplementation with different concentrations of P4 on either of 2 days of in vitro culture (IVC) on early bovine embryo development in an in vitro model. A total of 5073 cumulus–oocyte complexes were matured and fertilized in vitro. Before culture, they were collected in groups of 30 and allocated to 1 of 9 groups. The groups were supplemented with 10, 20, or 100 ng of P4 on Days 4 or 5 of IVC (IVF = Day 0). Alcohol (ETOH) was used as the solvent, so 8 µL of ETOH was used per supplementation. Therefore, two additional groups were supplemented with only ETOH on Day 4 or 5 of IVC. The presumptive zygotes allocated to group 9 were not supplemented. A culture system without oil overlay was used to prevent the lipophilic P4 from moving into the oil. Embryo cleavage and development rates were determined solely on Day 8 of IVC. Single expanded blastocysts were stored at –80°C for RT-qPCR. Subsequently, the relative amounts of six developmentally important gene transcripts (IGF1R, SLC2A1, HSD3B1, IFNT, PGRMC1, and PGRMC2) were analysed in single embryos of all groups. Statistical analysis was performed using one-way and two-way ANOVA, and the level of significance was set at P ≤ 0.05. Cleavage and development rates did not differ among groups (see Table 1). The relative abundance of IGF1R, SLC2A1, PGRMC1, and PGRMC2 was not affected by either the concentration or the timing of P4 supplementation. Nevertheless, there was a statistically significant interaction between the day of treatment and the concentration used for the expression of HSD3B1 mRNA. When 20 ng of P4 was added on Day 5 of IVC, significantly more HSD3B1 transcripts were detected than if 10 ng, 100 ng, or ETOH alone was added. The expression of IFNT was not affected by the day of supplementation, only by the concentration used. Thus, supplementation with 20 ng of P4 resulted in a significantly higher level of transcripts than when 10 ng or ETOH was supplemented. The results indicate that the amount of P4 present during early embryonic development and the timing of its presence had an impact on molecular developmental competence. However, no effects concerning morphological development up to the blastocyst stage could be detected. Table 1.Cleavage and development rates (± SEM) of embryos supplemented with 10, 20, or 100 ng on Day 4 or 5 of in vitro culture (P ≥ 0.05) The financial support of the FBF e.V. is acknowledged.

Maturation, fertilisation and culture of bovine oocytes and embryos in an individually identifiable manner: a tool for studying oocyte developmental competence

2010 ◽  
Vol 22 (5) ◽  
pp. 839 ◽  
Author(s):  
Satoko Matoba ◽  
Trudee Fair ◽  
Patrick Lonergan
Keyword(s):  
Blastocyst Stage ◽  
Developmental Competence ◽  
Standard Group ◽  
Success Rates ◽  
Group Culture ◽  
Polyester Mesh ◽  
Oocyte Developmental Competence ◽  
Development In Vitro ◽  
Bovine Oocytes

The ability to successfully culture oocytes and embryos individually would facilitate the study of the relationship between follicle parameters and oocyte developmental competence, in order to identify markers of competent oocytes, as well as the ability to use small numbers of oocytes from an individual donor such as when ovum pick-up is carried out. Using a total of 3118 oocytes, the aim of the present study was to develop a system capable of supporting the development of immature bovine oocytes to the blastocyst stage in an individually identifiable manner. Initially, post-fertilisation embryo culture in the Well-of-the-Well (WOW) system, on the cell adhesive Cell-Tak or in polyester mesh was tested and shown to result in similar development to embryos cultured in standard group culture. The results demonstrate that it is possible to culture bovine oocytes to the blastocyst stage in an individually identifiable manner in all three culture systems with comparable success rates. This permits the localisation and identification of individual embryos throughout preimplantation development in vitro while retaining the developmental benefits of group culture. In terms of ease of preparation and use, culture in isolation within the strands of a polyester mesh is preferable.

The effect of vitrification of immature bovine oocytes to the subsequent in vitro development and gene expression

Zygote ◽  
2014 ◽  
Vol 23 (6) ◽  
pp. 933-942 ◽  
Author(s):  
Marwa S. Faheem ◽  
E. Baron ◽  
I. Carvalhais ◽  
A. Chaveiro ◽  
K. Pavani ◽  
...  
Keyword(s):  
Molecular Level ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cell Stage ◽  
Maturation Rate ◽  
Developmental Rates ◽  
Polymerase Chain ◽  
Vitro Development ◽  
Bovine Oocytes

SummaryImmature bovine oocytes were vitrified using the cryotop method and their post-warming survivability and capability to undergo in vitro maturation, fertilization and subsequent embryonic development were evaluated. In addition throughout the embryonic 2-cell, 4-cell, morula and blastocyst stages, the expression of four developmentally important genes (Cx43, CDH1, DNMT1 and HSPA14) was analysed using the real-time polymerase chain reaction (PCR). Immature oocytes (n = 550) were randomly assigned to non-vitrified (fresh) or cryotop vitrification groups using ethylene glycol (EG) with 1,2 propanediol (PROH) or dimethylsulphoxide (DMSO). After warming, oocytes survivability, embryo cleavage and embryonic developmental rates were not statistically different between the two cryoprotectants groups. However, the DMSO group had a lower (P < 0.05) oocyte maturation rate compared with the fresh and PROH groups. For morula and blastocyst rates, the DMSO group achieved a lower (P < 0.05) morula rate compared with the fresh group, while at the blastocyst stage, there were no differences between fresh and both cryoprotectants groups. For molecular analysis, at the 4-cell stage, most studied genes showed an inconsistent pattern of expression either from the PROH or DMSO groups. Noteworthily, these differences were limited at the morula and blastocyst stages. In conclusion, the cryotop method is sufficient for vitrification of immature bovine oocytes, both for embryonic developmental competence and at the molecular level. Moreover, PROH showed some advantage over DMSO as a cryoprotectant.

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