scholarly journals Effect of storage tube material and resveratrol during liquid storage of matured bovine oocytes on subsequent development

2017 ◽  
Vol 65 (4) ◽  
pp. 546-555
Author(s):  
Tayita Suttirojpattana ◽  
Tamás Somfai ◽  
Satoko Matoba ◽  
Takashi Nagai ◽  
Rangsun Parnpai ◽  
...  
Keyword(s):  
Embryo Development ◽  
Subsequent Development ◽  
Control Group ◽  
Tube Material ◽  
Blastocyst Development ◽  
Liquid Storage ◽  
Glass Tubes ◽  
Bovine Oocytes

This study determined the optimum storage vessel and the effects of resveratrol for the storage of in vitro matured (IVM) bovine oocytes. After IVM, the oocytes were kept in a Hepes-buffered medium at 25 °C for 20 h in different containers including Eppendorf tubes (ET) made of polypropylene (PP) and polystyrene (PS), and tissue culture tubes (TCT) made of PP, PS, and glass. Then oocytes were subjected to IVF and subsequent in vitro embryo development was compared among the groups and to that of a control group without storage. The percentage of blastocyst development in the control group was significantly higher than in the stored groups (P < 0.05). Among oocytes stored in TCT, the percentage of blastocyst development of oocytes stored in glass TCT was significantly higher than that of oocytes stored in PP and PS TCT (P < 0.05); however, it did not differ from that of oocytes stored in ET. The quality of blastocysts did not differ among the control and stored groups. Embryo development was not affected when 0.1, 1 or 10 μM resveratrol was added to the medium during oocyte storage. In conclusion, glass tubes were optimal for oocyte storage and resveratrol did not improve the development of stored oocytes.

329 SUPPRESSION OF BOVINE OOCYTE IVM BY SERUM AND FSH DEPRIVATION OR BY α-AMANITIN: EFFECT ON FERTILIZATION AND EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 272
Author(s):  
K. Kananen-Anttila ◽  
M. Eronen ◽  
J. Matilainen ◽  
M. Kallio ◽  
J. Peippo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Embryo Cleavage ◽  
Bovine Oocytes ◽  
Sperm Aster

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μ­m; serum+FSH-free: 12 ± 0.5 μ­m, P < 0.05; α-amanitin: 10 ± 0.6 μ­m, P < 0.001) and sperm asters (control: 86 ± 4 μ­m; serum+FSH-free: 82 ± 4 μ­m, P < 0.01; α-amanitin: 49 ± 7 μm, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription. Table 1. Average percentages ± (n) for nuclear maturation, fertilization (min two pronuclei), embryo cleavage, and blastocyst development

315 SELECTION OF BOVINE OOCYTES BY BRILLIANT CRESYL BLUE BEFORE IN VITRO MATURATION IMPROVES BLASTOCYST DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 273 ◽  
Author(s):  
A. Sugulle ◽  
S. Katakawa ◽  
S. Yamamoto ◽  
S. Oomori ◽  
I. Itou ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Control Group ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Bovine Oocytes ◽  
Selection Of

The morphological identification of immature oocytes has commonly been used to select the bovine oocytes for IVF. However, &lt;30% of the recovered oocytes reach the blastocyst stage after fertilization, and this is probably due to the quality of the oocytes at the beginning of maturation. The brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. The aim of this study was to evaluate the effect of the BCB stain on the selection of bovine oocytes and on the subsequent embryo development for in vitro production (IVP). Cumulus–oocyte complexes (COCs) were collected by the aspiration of 2- to 6-mm follicles. A total of 559 oocytes were divided into 2 groups: (1) a control group, immediately cultured, and (2) a BCB-incubated group. After 90 min of BCB staining (Pujol et al. 2004 Theriogenology 61, 735–744), the oocytes were divided into oocytes with blue cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB−). The COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg mL−1 FSH at 38.5°C under an atmosphere of 5% CO2 in air. The matured COCs were inseminated with 5 × 106 sperm mL−1. After 18 h of gamete co-culture, the presumed zygotes were cultured in CR1aa supplemented with 5% CS for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (proportion of ≥5-cell stage, the total cleavage rates) and on Days 7 to 9 (blastocyst rate). The experiment was replicated 5 times, and the data were analyzed by a chi-square test and ANOVA. The results are presented in Table 1. The proportion of embryos with ≥5-cell stage was significantly higher (P &lt; 0.01) in the BCB+ group than in the BCB− group, but not in the control group. The total cleavage rate for the BCB+ embryos was significantly higher than that of either the BCB− or the control group (P &lt; 0.01). There were also significant differences (P &lt; 0.01) in the blastocyst development between the BCB+ and BCB− embryos and between the BCB− and the control embryos (P &lt; 0.05). This result showed that the selection of bovine oocytes by BCB staining before in vitro maturation may be useful for selecting oocytes that are developmentally competent up to Day 9 for IVP. Table 1.Effect of selection of oocytes by brilliant cresyl blue (BCB) staining on the subsequent embryo development of in vitro-matured/in vitro-fertilized bovine embryos

124 TAXOL™ COULD PROMOTE EMBRYO DEVELOPMENT OF BOVINE OOCYTES VITRIFIED BY OPS

2007 ◽  
Vol 19 (1) ◽  
pp. 179
Author(s):  
R. Morató ◽  
D. Izquierdo ◽  
M. J. Palomo ◽  
B. Anguita ◽  
A. R. Jiménez-Macedo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Calf Serum ◽  
Subsequent Development ◽  
Control Group ◽  
Control Groups ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Microtubule Stabilizer ◽  
Calf Oocytes

Stabilizing the cytoskeleton system during vitrification could be beneficial for improving post-thawed survival and subsequent development of vitrified oocytes. Taxol™, paclitaxel, is a microtubule stabilizer that has been found to improve development competence of vitrified mouse and human oocytes. The objective of this work was to study the effect of a Taxol pretreatment before OPS vitrification on the post-thaw cow and calf oocyte development. Oocytes were aspirated from slaughterhouse-derived ovaries and matured in TCM-199. Oocytes were randomly assigned to one of 3 experimental groups: (1) control oocytes matured in vitro for 24 h, (2) oocytes matured for 22 h and vitrified by the OPS method (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58), and (3) oocytes matured for 22 h and vitrified by OPS method with 1 µM Taxol. OPS and Taxol–OPS oocytes were transferred back into the maturation dishes and matured for 2 additional h before being subjected to fertilization. Fertilization was performed using frozen–thawed Percoll-selected sperm. At 22 h after insemination, presumptive zygotes were pipetted and then cultured in drops of 25 µL SOF medium and 5% fetal calf serum under paraffin oil at 38.5°C in 5% CO2, 5% O2, 90% N2, and maximum humidity. The Taxol–OPS group provided a significantly higher cleavage rate than the OPS group in cows (41.9% and 34.0%, respectively) or in calves (33.7% and 23.5%, respectively). However, cleavage rate in the experimental groups was significantly lower than in the control group (78.3% and 69.7% for cow and calf control groups, respectively). Blastocyst yield was also higher for the Taxol–OPS group (3.2%) than the OPS group (0%) in cow oocytes. There was no blastocyst development when calf oocytes were vitrified with or without Taxol pretreatment. As expected, cow and calf vitrification groups triggered a significantly lower blastocyst yield when compared with their control (26.7% and 14.9% for cow and calf control groups, respectively). In conclusion, this study showed that supplementation of 1 µM Taxol could promote embryo development after thawing. Further research is indicated to clarify the function of Taxol and its optimal concentration in order to improve the rate of embryo development. Table 1. Effect of Taxol pretreatment on development of cow and calf oocytes vitrified by OPS

The effect of temperature during liquid storage of in vitro–matured bovine oocytes on subsequent embryo development

2016 ◽  
Vol 85 (3) ◽  
pp. 509-518.e1 ◽  
Author(s):  
Tayita Suttirojpattana ◽  
Tamas Somfai ◽  
Satoko Matoba ◽  
Takashi Nagai ◽  
Rangsun Parnpai ◽  
...  
Keyword(s):  
Embryo Development ◽  
Effect Of Temperature ◽  
Liquid Storage ◽  
Bovine Oocytes

PSX-B-13 Effect of epidermal growth factor and prolactin on the quality of bovine oocytes aging in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-327
Author(s):  
Ekaterina Shedova ◽  
Galina Singina ◽  
Irina Y Lebedeva ◽  
Aleksandr Lopukhov
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Control Group ◽  
Tunel Staining ◽  
Apoptosis Resistance ◽  
Prolonged Culture ◽  
Epidermal Growth ◽  
Bovine Oocytes

Abstract The evaluation of factors responsible for the protection of the oocytes attained the metaphase-II stage from aging is importance for successful in vitro embryo reproduction. The aim of the present research was to study dose-dependent effects of epidermal growth factor (EGF) and prolactin (PRL) on the quality of bovine oocytes after their aging in vitro. Bovine cumulus-enclosed oocytes (CEOs) were matured in vitro for 20 h in TCM 199 containing 0.2 mM sodium pyruvate, 10% fetal calf serum (FCS), 10 μg/ml FSH and LH. At the end of in vitro maturation, oocytes were transferred to TCM 199 supplemented with 10% FCS (aging medium) and cultured for additional 24 h in the absence (Control) and in presence of EGF (10 and 50 ng/ml) and PRL (20 and 50 ng/ml). After prolonged culture oocytes were used for apoptosis detection (TUNEL staining, n=251) and the state of chromosomes evaluation (Tarkowski’s cytogenetic method, n=359). The data from 3–4 replicates were analyzed by ANOVA. At the end of prolonged culture (24 h) the rate of apoptotic oocytes in the Control group was 47.4±8.5%. EGF at concentration of 10 ng/ml and PRL at both doses decreased this rate to 15.0–22.1% (p &lt; 0.05). Furthermore, PRL (not EGF) reduced the frequency of abnormal chromosome modifications (decondensation, adherence, clumping) at concentrations of 20–50 ng/ml from 58.7±2.1% (Control) to 41.2±1.9 and 45.6±2.7% respectively (p &lt; 0.01). Thus, EGF and PRL is able to maintain the apoptosis resistance of bovine oocytes during their prolonged in vitro culture as well as PRL have the decelerating effect on abnormal modifications of M-II chromosomes. The research was supported by RFBR (17-29-08035) and the Ministry of Science and Higher Education of Russia.

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.

242 USE OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN IN VITRO PREMATURATION OF BOVINE OOCYTES SUBJECTED TO PARTHENOGENETIC ACTIVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 200
Author(s):  
T. H. C. De Bem ◽  
R. Rochetti ◽  
P. R. L. Pires ◽  
F. F. Bressan ◽  
P. R. Adona ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Polar Body ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Parthenogenetic Activation ◽  
Bovine Oocytes ◽  
Hatching Rates

Prematuration provides an additional time for oocyte capacitation and maturation in an attempt to improve in vitro embryo production (IVP) rates and allows media supplementation during this period for IVP. The aim of this study was to use brain-derived neurotropic factor (BDNF) in prematuration to improve maturation of bovine oocytes subjected to parthenogenetic activation and cultured with different media. Oocytes were subjected to prematuration in TCM-199 medium supplemented with 10 µm butyrolactone I, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin for 24 h in the absence of BDNF (control) or in the presence of 10 ng mL–1 BDNF (BD). Oocytes were then in vitro-matured (IVM) in TCM-199 medium supplemented with 10% FCS, 0.5 µg mL–1 FSH, 5.0 µg mL–1 LH, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin at 38.5�C under 5% CO2 in air. After 19 h oocytes were denuded using hyaluronidase and vortexing for 3 min for the 1st polar body (1PB) selection. Those which extruded the 1PB were maintained in IVM until 26 h, when parthenogenetic activation was performed (5 min in 5 µm ionomycin, followed by 3 h in 2 mm 6-DMAP). Activated oocytes were then transferred to in vitro culture (IVC) for embryo development evaluation. Embryos from both groups were cultured in SOF medium with 2.5% FCS, 0.05 g mL–1 BSA, 0.2 mm pyruvate, and 10 mg mL–1 gentamicin. Cleavage rates on the second day of in vitro culture (D2), embryo production at Days 7 and 8 (D7 and D8), and hatching rate at Day 8 were evaluated. Data regarding 1PB extrusion, cleavage, blastocyst development on D7 and D8, and blastocyst D8 hatching rates of three replicates were analyzed by chi-square test at 5% significance using the BIOESTATS 4.0 software. Control and BD, respectively, did not show differences (P > 0.05) regarding 1PB extrusion (n = 164, 63.81%, and n = 175, 66.79%) or cleavage (n = 117, 71.34%, and n = 138, 78.86%). However, for control and BD, respectively, blastocyst development on D7 (n = 63, 38.41%, and n = 89, 50.86%), D8 (n = 63, 38.41%, and n = 91, 52.00%), and hatching on D8 (n = 22, 34.92%, and n = 39, 43.82%) were all significantly higher for BD when compared with control (P < 0.05). In conclusion, BDNF during prematuration improved in vitro embryo development by increasing blastocyst and hatching rates of parthenogenetic embryos.

55 STUDY ON THE INTERSPECIFIC NUCLEAR TRANSFER OF PRZEWALSKI'S GAZELLES AND BOVINES

2013 ◽  
Vol 25 (1) ◽  
pp. 175
Author(s):  
Y. Gao ◽  
L. Cheng ◽  
G. Su ◽  
Z. Wei ◽  
G. Li
Keyword(s):  
Embryo Development ◽  
Somatic Cells ◽  
Oocyte Nucleus ◽  
Control Group ◽  
Blastocyst Development ◽  
Affymetrix Microarray ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Deacetylase Inhibitor ◽  
Bovine Oocytes

Przewalski’s gazelle (Procapra przewalskii), also known as Platts antelope, is an endangered species only found in China. It belongs to the Artiodactyla order, Bovidae family, antelope subfamily, and Gazella genus. In this study, 5 experiments were designed to examine the developmental potential of Przewalski’s gazelle somatic cells transplanted into bovine enucleated oocytes. Enucleation was conducted by Hoechst 33342 staining of the oocytes and guided by a fluorescent microscope to ensure the removal of the nuclei. The gazelle cells were then transferred to the enucleated oocytes and electrically fused to reconstructed embryos. The study resulted in 5 major findings. (1) When gazelle-bovine reconstructed embryos were treated with the deacetylase inhibitor valproic acid (VPA), at different concentrations and for different times, treatment of the cloned embryos with VPA at 0.5 mM for 24 h significantly increased the 8- to 16-cell-stage embryo development [61.9% (96/155) v. 33.8% (46/136) control]. However, the morula [1.3% (2/155) v. 1.5% (2/155); P > 0.05] and blastocyst (0.7% v. 1.5%; P > 0.05) development were similar to that of the control. In the intraspecific (bovine-bovine) control group, the cleavage, morula and blastocyst development of 3 cloned embryos were 72.6% (127/175), 28.0% (49/175), and 23.4% (41/175). (2) Octamer-binding transcription factor 4 (Oct-4), as a developmental potential and expression marker, was transfected to gazelle cells. When Oct-4-eGFP-confected cells were transferred, the cloned embryo development did not improve either with or without VPA treatment. (3) When the gazelle-bovine embryos were treated with the deacetylase inhibitor trichostatin A (TSA) for 24 h at 10 ng mL–1, blastocyst development was significantly higher than in the control group [3.6% (6/168) v. 0.8% (1/125); P < 0.05]. (4) When a reverse NT protocol, in which the oocyte nucleus was removed after the cell nucleus was fused to the oocyte, was used for NT, the cloned embryo development did not improve. (5) The gazelle-bovine and bovine-bovine cloned embryos at 8- to 16-cell stages, gazelle cells, bovine cells, and bovine oocytes transcriptomes were analyzed by Affymetrix microarray (Affymetrix Microarray Inc., Santa Clara, CA, USA) and repeated twice. A total of 643 genes were activated in gazelle-cattle embryos compared with oocytes, whereas 1527 genes were activated in bovine-bovine clones. A total of 1010 genes that were exclusively expressed in gazelle somatic cells were still expressed in the interspecies cloned embryos. In conclusion, TSA treatment of Przewalski’s gazelle somatic cells transferred into enucleated bovine oocytes improved development of cloned embryos to the blastocyst stage, although still with low efficiency. Data from microarray analyses of the gazelle-cattle embryos showed that over 1000 gazelle-specific genes were still expressed in the interspecific cloned embryos. This work was supported by the National Basic Research Program of China (no. 2012CB22306).

scholarly journals 296 LEUKEMIA INHIBITORY FACTOR INFLUENCES SHEEP OOCYTE PARTHENOGENETIC DEVELOPMENT DURING THE TRANSITION FROM GERMINAL VESICLE TO EARLY PRONUCLEAR STAGE

2005 ◽  
Vol 17 (2) ◽  
pp. 298
Author(s):  
G. Ptak ◽  
F. Lopes ◽  
P. Loi
Keyword(s):  
Embryo Development ◽  
Leukemia Inhibitory Factor ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Inhibitory Factor ◽  
Control Group ◽  
Blastocyst Development ◽  
Human Follicular Fluid ◽  
Oocyte Competence

Leukemia inhibitory factor (LIF) is an indispensable cytokine for female fertility. The influence of LIF on embryo development and particularly implantation has been recently confirmed; however, the effect of this cytokine on the oocyte has not been studied. The presence of LIF in human follicular fluid implies its possible role in the acquisition of oocyte competence. Furthermore, the up-regulation of LIF by steroid hormones in sheep makes entirely feasible the hypothesis that ovulatory estradiol peak plays a role in the preparation of female gamete for fertilization. With this in mind, we studied the effect of LIF during in vitro development of sheep oocytes mimicking the physiological expression of LIF induced by the ovulatory peak of estradiol in mice. GV stage oocytes matured and chemically activated in the presence of LIF and anti-LIF antibody were cultured to the blastocyst stage in our standard media. To eliminate the effect of the putative presence of LIF in heat inactivated fetal calf serum used for oocyte maturation, aliquots of LIF were treated at 56°C for 30 min and added to the maturation medium. The proportion of embryos that reached the blastocyst stage in vitro was significantly higher (P < 0.001) for oocytes matured and activated with LIF (36/93; 39%) than for the group incubated with antibody against LIF (6/68; 9%). The significant effect of anti-LIF antibody (P < 0.001) was also observed when compared with blastocysts developed from the control group of oocytes matured without LIF addition (31/106; 29%). Although the beneficial influence of LIF treatment on embryo development demonstrated with those preliminary data was not confirmed statistically, due to low number of oocytes involved, the proportion of embryos reaching the blastocyst stage in vitro was about 10% higher for those incubated with LIF than for either those cultured without the cytokine or those, matured in the presence of heat-treated LIF (15/55; 27%); however, the rate of blastocyst development appeared very similar to that of the control group. This study revealed for the first time a role of LIF in determining oocyte competence. Further investigation to determine how LIF achieves its effects on the oocyte are ongoing in our laboratory. This work was supported by FIRB RBNE01HPMX, COFIN 2002074357, COFIN 2003073943 002, and British Council 2004.

220 SYNERGISTIC EFFECT ON EMBRYO DEVELOPMENT BY INCLUSION OF SUPPLEMENTAL EMBRYOS IN AGAR CHIPS

2009 ◽  
Vol 21 (1) ◽  
pp. 208 ◽  
Author(s):  
E. M. Senatore ◽  
M. E. Mannino ◽  
M. V. Suarez Novoa ◽  
J. Xu ◽  
S. Chaubal ◽  
...  
Keyword(s):  
Treatment Group ◽  
Embryo Development ◽  
Control Group ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Stage Of Development ◽  
Stage Embryo ◽  
Similar Stage

The main scope of this study was to evaluate the likelihood of a helper effect of agar-embedded cleaved embryos on a low number of free embryos at a similar stage of development within the same culture droplet. Such an improved system could be beneficial within ovum pickup/in vitro embryo production (OPU/IVEP) combined protocols whenever a low number of OPU-derived cleaved embryos are produced per donor. Oocytes were recovered from abattoir ovaries, and after in vitro maturation (IVM) and in vitro fertilization (IVF), presumptive zygotes were deprived of cumulus investment and allocated into culture droplets for 24 h. At 48 h from IVF, 4- to 8-cell cleaved embryos were randomly allocated into a control and a treatment group. Control groups consisted of 1, 3, 5, and 10 embryos, respectively, in 50-μL droplets. Treatment groups consisted of 1, 3, and 5 free embryos with the addition of 9, 7, and 5 embryos, respectively, at a similar stage of development embedded in agar chips, so as to reach a total number of 10 cleaved embryos in each culture droplet. Culture was performed for both the control and treatment groups in SOF medium droplets covered with mineral oil, with the supplementation of essential and nonessential amino acids in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2 at 39°C. Final embryo output was checked at Day 7 from IVF. When considering only free embryos, the difference in progression to blastocyst development was highly significant between the control and treatment groups: 1) group 1 v. 1 + 9: 6.6 v. 84.3% (P = 0.00000); 2) group 3 v. 3 + 7: 11.1 v. 41.3% (P = 0.00001); 3) group 5 v. 5 + 5: 24.4 v. 42.2% (P = 0.00001). Rate of blastocyst development in the control group containing 10 cleaved embryos was not significantly different from free cleaved embryos in the 3 + 7 (39.2 v. 41.3%, P = 0.71) and 5 + 5 treatment groups (39.2 v. 42.2%, P = 0.54), but was significantly lower when compared with the 1 + 9 treatment group (39.2 v. 84.3%, P = 0.000). For 1, 3, 5, and 10 control group embryos, the numbers of replicates and total cleaved embryos used (n) were 30 (n = 30), 27, (n = 81), 27 (n = 135), and 39 (n = 390), respectively. For the 1 + 9, 3 + 7, and 5 + 5 treatment group embryos, the numbers of replicates and total cleaved embryos used were 32 (n = 32), 29 (n = 87), and 27 (n = 135), respectively. In conclusion, a beneficial effect of agar-embedded embryos on the development of free embryos within the same culture droplet was shown. A striking improvement in late-stage embryo development was particularly evident when considering the 1 v. 1 + 9 control and treatment groups. These results may foster a different strategic approach in in vitro culture to enhance embryo development from highly valuable donors.

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