172 ADMINISTRATION OF GLUTATHIONE OR THIOREDOXIN TO MEDIUM REDUCES INTRACELLULAR REDOX STATUS AND IMPROVES EMBRYONIC DEVELOPMENT TO THE BLASTOCYST STAGE OF PORCINE IVM/IVF OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 194
Author(s):  
M. Ozawa ◽  
T. Nagai ◽  
M. Fahrudin ◽  
N. W. K. Karja ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Culture Medium ◽  
Redox Status ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Normal Embryo ◽  
Control Group ◽  
Intracellular Redox Status ◽  
Intracellular Redox

Successful in vitro production of blastocysts from immature oocytes can be carried out using in vitro oocyte maturation (IVM), fertilization (IVF), and embryo culture (IVC) at a high level of repeatability in the porcine. However, the rates of in vitro development of IVM/IVF oocytes to the blastocyst stage remained around 20%. The environment in vitro is so simple and materially limited that there exist several stressors in vitro that disturb normal embryo development. Oxidative stress, which is caused by excess production of reactive oxygen species, is a major disturbing factor for the development of pre-implantation embryos in vitro. The series of present experiments were conducted using culture conditions with enhanced reducing capacity by the addition of glutathione (GSH) or thioredoxin to the culture medium to monitor developmental competence of porcine embryos and to verify their intracellular redox status. Cumulus-oocyte complexes were obtained from ovaries recovered from prepubertal gilts. Putative zygotes were produced by IVM of oocytes, followed by IVF (designated as Day 0). They were then cultured in modified NCSU-37 media containing GSH or thioredoxin as an antioxidant, or without any antioxidant (control), and blastocyst development rates on Day 6 were monitored. In addition, intracellular GSH content as a reducing parameter and intracellular H2O2 level as an oxidative parameter were measured; the intracellular redox status in the embryo was verified by the ratio of the GSH to the H2O2. Measurements in each group were replicated six times. Percentages of the embryos that developed to the blastocyst stage were significantly increased when 0.5 or 1.0 �M GSH (29.6 � 2.7% or 30.4 � 3.5%, and P < 0.05 or 0.01, respectively) or 1.0 mg/mL thioredoxin (30.6 � 2.4%, P < 0.01) was added to the medium compared to the percentage in the control group (20.1 � 2.2%). Intracellular redox status in embryos at the 8- to 12-cell stage or blastocysts was drastically reduced in GSH- or thioredoxin-added groups compared to that in the control group (P < 0.05 to 0.001). Furthermore, GSH or thioredoxin addition to the medium increased total cell numbers (48.3 � 2.1 to 49.2 � 2.1) and lowered ratios of apoptotic cells (6.2 � 0.6% to 7.0 � 0.7%) in blastocyst compared to those values in the control group (P < 0.05; cell number = 39.3 � 2.0, apoptosis rate = 11.1 � 1.1%) (37 to 53 embryos in each group were used for the TUNEL assay). These results suggest that the administration of GSH or thioredoxin to the culture medium improves in vitro embryonic development after IVM/IVF of oocytes, and that these beneficial effects are associated with maintenance of the intracellular redox status in a reduced state in porcine embryos.

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.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

2018 ◽  
Vol 30 (1) ◽  
pp. 189
Author(s):  
L. Landeo ◽  
R. S. Molina ◽  
M. E. Zuñiga ◽  
T. R. Gastelu ◽  
C. Sotacuro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptomyces Griseus ◽  
Petri Dish ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Morula Stage

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

scholarly journals Porcine follicular fluid derived from > 8 mm sized follicles improves oocyte maturation and embryo development during in vitro maturation of pigs

Zygote ◽  
2020 ◽  
pp. 1-6
Author(s):  
Ji-Eun Park ◽  
Sang-Hee Lee ◽  
Yong Hwangbo ◽  
Choon-Keun Park
Keyword(s):  
Embryonic Development ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Group ◽  
Cumulus Expansion ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Summary The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3–6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus–oocyte complexes (COCs) were collected from follicles 3–6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.

133 VITRIFICATION CAUSES DAMAGE OF THE ANTIOXIDANT DEFENSE SYSTEM IN IVM PORCINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 184 ◽  
Author(s):  
T. Somfai ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
K. Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Antioxidant Defense System ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Sperm Penetration ◽  
Second Polar Body ◽  
Polar Body Extrusion

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P &lt; 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P &lt; 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P &lt; 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P &lt; 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P &lt; 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes. This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).

93 PRODUCTION OF LIVE PIGLETS BY CRYOPRESERVATION OF IN VITRO-PRODUCED PORCINE ZYGOTES

2008 ◽  
Vol 20 (1) ◽  
pp. 127
Author(s):  
T. Somfai ◽  
N. Kashiwazaki ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Culture Medium ◽  
Aluminum Foil ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
High Rate ◽  
Cell Numbers ◽  
Embryo Culture Medium ◽  
Vitrification Solution ◽  
And Control

Successful cryopreservation of in vitro-produced porcine zygotes is reported in the present study. Follicular oocytes were collected from prepubertal gilts. They were matured (IVM), fertilized (IVF), and cultured (IVC) in vitro (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Ten or 23 h after IVF, the oocytes were centrifuged at 10 000g at 37�C for 20 min to permit visualization of pronuclei. Zygotes with two or three pronuclei were selected under stereomicroscope and used for solid surface vitrification (SSV). Briefly, after equilibration in 4% ethylene glycol (EG) for 15 min, zygotes were washed in vitrification solution (35% EG, 5% polyvinyl pyrrolidone, and 0.3 m trehalose), and then dropped with about 2 µL vitrification solution onto the dry surface of aluminum foil floating on the surface of liquid nitrogen (LN2). Microdroplets were transferred into cryotubes and stored in LN2. During warming, vitrified droplets were transferred in warming solution (0.4 m trehalose) at 37�C for 1 min, and then consecutively transferred for 1-min periods into 0.2 m, 0.1 m, or 0.05 m trehalose solutions. Survival of vitrified/warmed zygotes was determined by their morphology. To assess their developmental competence, vitrified (SSV), cryoprotectant-treated (CT), and untreated (control) zygotes were cultured in vitro for 6 days. There was no difference in developmental competence between control and CT zygotes in terms of cleavage rates (88.1% and 86.1%, respectively), blastocyst rates (23.2% and 20.8%, respectively), and blastocyst cell numbers (38.0 � 2.0 and 41.2 � 1.7, respectively). The rate of live zygotes after SSV and warming was similar to that of the control (93.4% and 100%, respectively). Cleavage rates (71.7% and 86.3%, respectively) and blastocyst rates (15.8% and 24.5%, respectively) of SSV were significantly reduced after vitrification compared to control (P < 0.05, ANOVA). Blastocyst cell numbers of SSV and control embryos were similar (41.2 � 3.4 and 41.6 � 3.3, respectively). There was no difference in developmental ability between zygotes cryopreserved at an early (10 h after IVF) or late (23 h after IVF) pronuclear stage. When embryo culture medium was supplemented with 1 µm of the antioxidant glutathione, development of cryopreserved zygotes to the blastocyst stage did not differ significantly from that of the control zygotes (18.6% and 22.1%, respectively). To test their ability to develop to term, 150 vitrified zygotes were transferred into a recipient, resulting in pregnancy and the production of five live piglets. These data demonstrate that a high rate of porcine zygotes could be successfully cryopreserved at the pronuclear stage, preserving their full developmental competence.

133 EFFECTS OF ACTIVINA ON mRNA EXPRESSION IN IN VITRO FERTILIZED BOVINE EMBRYOS CULTURED FROM CHEMICALLY DEFINED TWO-STEP CULTURE MEDIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 147
Author(s):  
J. E. Park ◽  
G. Jang ◽  
H. J. Oh ◽  
S. G. Hong ◽  
I. S. Yang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Early Stage ◽  
Activin A ◽  
Developmental Competence ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Defined Culture

During the preimplantation stage, embryo development occurs in a maternal environment within the oviducts and uterine horns. It has been speculated that both the embryo itself and the maternal reproductive tract provide paracrine factors that influence embryo development (Jones et al. 2006 Reproduction 132(5), 799–810). Activins are known for FSH releasers, and several previous studies have reported that activin subunits and activin receptors mRNA were expressed in oocytes, zygotes, and oviduct (Yoshioka et al. 1998 Reprod. Fertil. Dev. 10(3), 293–298; Gandolfi et al. 1995 Mol. Reprod. Dev. 40(3), 286–291). The purposes of the present study were Experiment 1) to evaluate the effects of activin A on developmental competence of bovine embryos derived from two-step defined culture medium (Lim et al. 2007 Theriogenology 67(2), 293–302) and Experiment 2) to analyze the effects of activin A on transcriptional level of the genes in IVF embryos. Cumulus–oocyte complexs were harvested from ovaries obtained from a local slaughter house, matured, and fertilized in vitro. In vitro fertilized zygotes cultured in media supplemented with activin A in the early stage at the concentrations of 0, 10, or 100 ng mL–1 or in the later stage medium at the concentrations of 0, 10, or 100 ng mL–1. Data were analyzed using the Statistical Analysis System (SAS) program. In Exp. 1, although the development competence of embryos that cultured with activin A in the early stage medium was not significantly different, development to blastocysts on day 8 in the later stage medium with 100 ng mL–1 activin A was significantly higher than the control group [22.4% (54/264) v. 34.7% (76/233); P < 0.05]. Hatching rate of blastocyst on day 8 was significantly higher in the presence of 100 ng mL–1 activin A in the later stage culture medium compared with the control group [9.3% (5/54) v. 22.4% (17/76); P < 0.05]. In Exp. 2, the relative expression of 3 genes (Na/KATPase, E-cad, Glut-1) related to blastocyst hatching and implantation was analyzed. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitative reverse transcription-polymerase chain reaction. The expression level of the Na/K ATPase, E-cad, and Glut-1 gene were higher in the presence of activin A in the culture medium compared with the control group. In conclusion, this study suggests that activin A during the later stage of in vitro bovine embryo development can enhance the developmental competence of preimplantation embryos, increase the hatching rate, and affect expression level of genes related to hatching and implantation in defined culture medium. This study was financially supported by KOSEF (grant ? M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.

scholarly journals Effect of serum-containing and serum-free culture medium-mediated activation of matrix metalloproteinases on embryonic developmental competence

2019 ◽  
Vol 64 (No. 12) ◽  
pp. 473-482
Author(s):  
Sang Hwan Kim ◽  
Jong Taek Yoon
Keyword(s):  
Embryonic Development ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Developmental Competence ◽  
Serum Free Medium ◽  
Vitro Fertilization ◽  
Serum Free ◽  
Foetal Bovine Serum

In this study, we examined whether serum-free and serum-containing media affect matrix metalloproteinase (MMP) activity with respect to embryonic development, and whether MMP expression is correlated with the development of in vitro fertilized eggs. When oocytes were cultured in serum-free medium (containing polyvinylpyrrolidone) and serum (foetal bovine serum)-containing medium, the generation of meiosis 2 (MII) oocytes was 76% and 87.5%, respectively (P &lt; 0.05). After in vitro fertilization using mature oocytes, we observed 39.72% and 64.05% of cleaved oocytes in serum-free and serum-containing groups, respectively (P &lt; 0.05). Our analysis revealed differential expression and activity of MMPs. The serum-containing group showed high MMP-9 activity during oocyte maturation and development of in vitro produced embryos, with particularly high activity in the inner cell mass zone of the embryos. Therefore, this study suggests that the presence or the absence of serum will affect the activity of MMPs, which can be used to measure the rate of embryonic development.

scholarly journals 101 OVARIAN RESPONSE AND DEVELOPMENTAL COMPETENCE OF OOCYTES COLLECTED BY OPU IN SHEEP TREATED WITH GnRH ANTAGONIST

2005 ◽  
Vol 17 (2) ◽  
pp. 201
Author(s):  
F. Berlinguer ◽  
A. Gonzalez-Bulnes ◽  
S. Succu ◽  
G. Leoni ◽  
I. Rosati ◽  
...  
Keyword(s):  
Single Dose ◽  
Gnrh Antagonist ◽  
Ovarian Response ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Gnrh Antagonists ◽  
Group A ◽  
Group B

The use of a single dose of GnRH antagonists during the progestagen treatment prior to superovulatory treatment protocols in sheep increases the number of smaller follicles able to grow and ovulate in response to the exogenous FSH treatment (Lopez-Alonso C et al. 2004 Reprod. Fertil. Dev. 16, 233). The aim of our study was to test if such treatment affects the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from GnRH-antagonist treated sheep during an ovarian by perstimulation protocol. Adult Sarda sheep (n = 18) were synchronized by the insertion of intravaginal sponges (Day 0) which were left in situ for 12 days; on Day 7, group A (n = 10) received a single dose of 3 mg of Antarelix (Teverelix, Europeptides, France) s.c., while group B (n = 8) served as control. All animals received 96 IU of FSH (Ovagen, ICP, New Zealand) administered in 4 equal doses given i.m. every 12 h starting on Day 10. Twelve hours after the last FSH administration oocytes were collected by OPU technique. Follicular growth was monitored by transrectal ultrasonography from Day 7 to Day 11. Collected oocytes were matured, fertilized, and cultured in vitro up to blastocyst stage under standard conditions used in our laboratory (Berlinguer F et al. 2004 Theriogenology 61, 1477–1486). After IVF, uncleaved oocytes were stained with acetolacmoid to evaluate chromatin configuration, while the cleaved ones were cultured in SOF + 0.4% BSA up to the blastocyst stage. Data were analyzed by ANOVA statistical analysis after arcsine transformation of the value percentages. Ultrasonographic monitoring showed a significant increase in the number of follicles (mean ± SEM) present in the ovaries from Day 8 to Day 11 of treatment in group A compared to group B (Day 8: 19 ± 5.1 vs. 13 ± 3.4, P > 0.05; Day 9: 20.1 ± 4.6 vs. 14.1 ± 2.4, P > 0.001; Day 10: 22.5 ± 6.1 vs. 14.7 ± 2.7, P > 0.001; Day 11: 25.3 ± 5.1 vs. 20.5 ± 4.1, P > 0.05), thus confirming that GnRH antagonist administration enhances ovarian response to exogenous FSH stimulation. On the other hand, oocytes collected from untreated sheep lead to a higher blastocyst output (P = 0.014), as illustrated in the table. These results indicated that although GnRH antagonist administration caused a significant increase in the ovarian response to the hormonal treatment, the final blastocyst output was significantly lower compared to that of the control group. This finding seems to suggest an impairment in the developmental competence of treated sheep oocytes. Table 1. In vitro maturation, fertilization, and developmental capacity of oocytes collected from follicles of GnRH antagonist-treated (group A) and untreated (group B) sheep This work was supported by funds from the Spanish MEC (projects SC 00-051-C3.1 and HI2002-0004) and the Italian MIUR (cofin).

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