Nuclear Maturation Rates Research Articles

Dose-dependent effects of gonadotropin on oocyte developmental competence and apoptosis

The aim of the present study was to evaluate the effect of gonadotropins (Gn) on oocyte maturation, developmental competence and apoptosis in an animal model. Bovine cumulus-oocyte complexes (COCs) were matured for 24 h in media supplemented with varying concentrations of Bravelle (B), B + Menopur (B+M) or B + Repronex (B + R) (Ferring Pharmaceuticals, Parsiappany, NJ, USA). Then, nuclear maturation, embryo development, and apoptosis in cumulus cells and oocytes were evaluated. Low to moderate Gn concentrations (75-75 00 mIUmL(-1)) effectively improved nuclear maturation and in vitro development. Higher concentrations of Gn (75 000 mIUmL(-1)) did not have any added beneficial effects and nuclear maturation and blastocyst rates in the presence of these concentrations were comparable to control (P>0.05). Most COCs showed slight apoptosis when exposed to 75, 750 and 75 00 mIUmL(-1) Gn; however, when the concentration was increased to 75 000 mIUmL(-1), the proportion of moderately apoptotic COCs increased. In conclusion, extremely high concentrations of Gn have detrimental effects on oocyte nuclear maturation and embryo development and increase apoptosis in cumulus cells, suggesting the importance of judicious use of Gn in assisted reproductive technologies (ART).

Factors affecting nuclear maturation, cleavage and embryo development of vitrified bovine cumulus-oocyte complexes

The objective was to investigate the effects of cryodevice, vitrification solutions, and equilibration time on in vitro maturation, cleavage, and embryo development of vitrified bovine oocytes. In Experiment 1, the nuclear maturation (MII) rate of immature bovine COCs vitrified was compared between two equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P < 0.0001). Equilibration time did not affect MII rate (P = 0.964); however, the MII rate was higher for COCs vitrified on cryotops than in straws (23 vs 9%, P = 0.007). In Experiment 2, bovine COCs were vitrified on cryotops using two equilibration times (0 vs 5 min) in VS1 and two kinds of vitrification solutions (freshly prepared vs frozen). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocysts rate 31 vs 0.4%). Cleavage rate of COCs vitrified using frozen solutions with 5 min equilibration was higher (P = 0.05) than other treatment groups. However, blastocyst rate did not differ (P = 0.993) among treatment groups. In conclusion, cryotop was a better cryodevice than 0.25 mL straw for vitrification of bovine COCs. Furthermore, 5 min equilibration in VS1 improved cleavage. Compared with control, the vitrification procedure per se damaged bovine COCs, resulting in poor nuclear maturation and embryo development. However, vitrification did not immediately kill oocytes, as the cleavage rate was acceptable.

252 EFFECT OF FOLLICLE-STIMULATING HORMONE ADDITION ON IN VITRO MATURATION AND CLEAVAGE OF ALPACA (VICUGNA PACOS) EMBRYOS

We have previously reported that alpaca oocytes require between 38 and 42 h of maturation time (Huanca et al. 2010 Reprod. Fertil Dev. 22(1), 327). The objective of this study was to evaluate the effect of the addition of FSH in the maturation medium on nuclear maturation and cleavage rate. Ovaries were collected from a slaughterhouse and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. Cumulus–oocyte complexes (COC) were obtained by slicing of ovaries with a scalpel and were pooled in a conical tube for sedimentation before evaluation. The 476 COC with homogeneous cytoplasm and 2 or more layers of cumulus cells were transferred to plates with a 40-μmL drop of maturation medium TCM-199 supplemented with 10% FCS (vol:vol), 10 μg mL–1 of hCG, 0.2 mM sodium pyruvate, 50 μg mL–1 of gentamicine, and 1 μg mL–1 of oestradiol plus 0.5 μg mL–1 of FSH (Folltropin, Bioniche Animal Health, Belleville, Ontario, Canada) according the following treatments: T1 (Control): FSH by 42 h, T2: 21 h with FSH + 21 h without FSH, T3: 21 h without FSH + 21 h with FSH. The COC were maintenance under mineral oil with 10 to 12 oocytes per drop and maturated 42 h at 39°C in an atmosphere of 5% CO2 and high humidity. After the maturation time, part of the COC were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mg/mL of hyaluronidase and fixed in ethanol: acetic acid (3:1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5 min. The slides were examined under a phase contrast microscope at ×400 to evaluate the status of nuclear maturation and classify as germinal vesicle (GV), metaphase I (MI), anaphase–telophase, metaphase II (MII), and degenerated. The other part of oocytes was fertilized in vitro using epididymal sperm. Motile spermatozoa were obtained by centrifugation at 600 × g on a percoll discontinuous gradient (22.5:45.0%) for 10 min. After the supernatant was removed by aspiration, the pellet was resuspended in TL HEPES and centrifuged again at 300 × g by 5 m. The pellet was resuspended in TL Stock. Gametes were coincubated for 18 h at 39°C with 5% CO2 and high humidity. Presumptive zygotes were culture in KSOM supplemented with 1 mM of glutamine, 0.3 mM of sodium pyruvate, 50 μg mL–1 of gentamicine, ethylenediaminetetraacetic acid, amino acids essentials and nonessentials, and BSA by 3 days and cultured in SOF medium for 7 days. Cleavage rate was evaluated at 72 h. Proportional data were compared by chi-square test. The proportions of oocytes reaching MII stage were 64.9 ± 8.1, 49.2 ± 9.4, and 53.8 ± 7.5% for T1, T2, and T3, respectively. Cleavage rates were 39.1, 36.3, and 33.1% and blastocysts rates were 13.6, 16.1, and 14.5% for T1, T2, and T3, respectively. The results suggest that the moment of addition of FSH does not have an effect on the maturation and cleavage rate of alpaca oocytes. This work was supported by Grant 064 – FINCyT – PIBAP 2008 and Grant 032 – PROCYT – CONCYTEC.

253 OOCYTE PREMATURATION IN THE PRESENCE OF MILRINONE IMPROVES NUCLEAR BUT NOT CYTOPLASMIC MATURATION OF MACAQUE OOCYTES

During oocyte growth chromatin configuration of the germinal vesicle (GV) oocyte undergoes modification in relation to changes in transcriptional activity crucial for conferring meiotic as well as developmental competence on the oocyte. In the macaque oocyte, there are 3 distinct GV states: GV1, noncondensed chromatin; GV2, an intermediate state; and GV3, condensed chromatin. The aim of this study was to test the effects of a prematuration culture (PMC) system, using the phosphodiesterase type 3 inhibitor milrinone (MIL), on the synchronization of GV chromatin to the GV3 stage and assess metaphase II (MII) oocyte reduced glutathione (GSH) content as a measure of cytoplasmic maturation. Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. To assess the effect of PMC on GV chromatin status, immature oocytes retrieved from unstimulated ovaries were either fixed (2% paraformaldehyde+0.1% Triton-X100) immediately after follicular aspiration (t = 0) or after culture in a humidified atmosphere of 6% CO2 in air at 37°C for 24 h in modified Connaught Medical Research Laboratories medium (mCMRL) supplemented with 10% FCS (Hyclone, Logan, UT, USA) and 12.5 μM MIL in the absence (MILNil) or presence of 1.0 IU of FSH (MILFSH). For chromatin assessment, fixed GV oocytes were stained with 5 μg mL–1 of 4′,6-diamidino-2-phenylindole (Molecular Probes, Leiden, the Netherlands) and imaged using confocal microscopy. Following PMC, MILFSH oocytes were transferred to fresh mCMRL+FCS supplemented with 1.0 IU of recombinant human FSH and 1.0 IU of hLH and cultured for a further 30 h. Control and MILFSH oocytes were denuded of cumulus cells and assessed for maturation. The MII oocytes were prepared for GSH analysis, and total GSH content was determined using a commercial 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay kit (North-West Life Science). The MII rates were compared using chi-square. Differences in oocyte GSH content were compared using t-test. Significant differences were determined at P &lt; 0.05. There was no significant difference in the proportion of oocytes remaining at the GV stage following 24 h of PMC in MILNil or MILFSH (42/44, 96% v. 32/35, 91%, respectively). However, there was a significant reduction in GV1 chromatin (15/49, 31% v. 28/54, 52% and 22/58, 38%) and a significant increase in GV3 chromatin (23/49, 47% v. 14/54, 26% and 16/58, 28%) observed in MILFSH oocytes compared with both MILNil and t = 0 oocytes, respectively. The MII rate of MILFSH oocytes following in vitro maturation was significantly higher compared with the MII rate of control in vitro matured oocytes (91/167, 55% v. 83/243, 34%). There was no significant difference in the GSH content of GV oocytes from the time of oocyte collection (t = 0) or GV oocytes following PMC in MILFSH (3.69 ± 0.16 and 4.14 ± 0.28 pmol/oocyte, n = 39–49 oocytes). The GSH content of control in vitro matured MII oocytes was significantly greater than that of MILFSH-treated MII oocytes (3.13 ± 0.16 v. 2.02 ± 0.04 pmol/oocyte, n =53–54 oocytes). The PMC supported high rates of nuclear maturation, but cytoplasmic maturation, assessed by GSH content, was negatively affected. Further assessment following fertilization and development is required to determine the practical utility of PMC in a primate in vitro maturation setting.

250 HUMAN EXHALED AIR CAN EFFECTIVELY SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES AND SUBSEQUENT IN VITRO DEVELOPMENT

The objective of the present study was to examine the feasibility of culturing porcine oocytes and embryos in vitro using the human exhaled lung air atmosphere. In Experiment 1, the effects of lung air atmosphere on nuclear maturation of prepubertal gilt oocytes and subsequent development in vitro of parthenogenetic-activated and somatic-cell-cloned embryos were explored. Abattoir-derived prepubertal gilt cumulus–oocyte complexes (COC) were matured in TCM-199 supplemented with 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, 10 ng mL–1 of epidermal growth factor, and 10% porcine follicular fluid (pFF) for 40 to 44 h at 38.5°C, 100% humidity, and 5% CO2+20% O2 (high oxygen tension) or human exhaled air encapsulated in plastic, airtight bags (lung air) or 5% CO2+7% O2 (low oxygen tension) in the incubator. Nuclear maturation was evaluated by the presence of the 1st polar body. For parthenogenetic activation, denuded oocytes with the 1st polar body were selected and stimulated with a single 1.6-kV/cm, 100-μs direct current pulse followed by culture in porcine zygote medium-3. For NT, denuded metaphase II oocytes were enucleated, and then the donor cell was directly injected into the perivitelline space. After NT, reconstructed couplets were fused and activated electrically followed by treatment in 7.5 μg mL–1 of cytochalasin B and 10 μg mL–1 of cycloheximide for 4 to 6 h before culture in porcine zygote medium-3. We found no significant difference among groups in terms of nuclear maturation rate (66.5% v. 60.2%, 63.2%), cleavage rate (94.8% v. 94.2%, 85.2%), blastocyst formation rate (39.5% v. 40.3%, 32.5%), and total cell number (37 v. 38, 32). Moreover, as for porcine cloned embryo, no significant difference between the lung-air and high-oxygen (20% O2) groups was observed in the cleavage rate (88.3% v. 80.3%), blastocyst formation rate (7.3% v. 10.7%), and total cell number (34 v. 36). The above results indicated that porcine oocytes can be matured in vitro safely and efficiently using the human exhaled lung air atmosphere. In Experiment 2, in vitro developmental competence of porcine zona-free parthenogenetically activated embryos cultured in a lung air, low oxygen (5% O2), or high oxygen (20% O2) tension gas environment was studied. We found no obvious difference among the 3 groups regarding the rates of cleavage (83.0%, 83.6%, 82.8%), but blastocyst formation rate (26.8% v. 48.6%, 48.2%) and total cell number (23 v. 34, 29) in lung air were lower than those in the rest of the groups (P &lt; 0.05). The results show that lung air could be an alternative for preparing a gas environment for in vitro culture of porcine zona-free parthenotes, although not an ideal alternative. Taken together, porcine oocytes and embryos can be cultured in vitro safely and efficiently using the human exhaled lung air atmosphere. Z. B. Cao and L. C. Sui contributed equally to this work. X. R. Zhang and Y. H. Zhang are the corresponding authors. This work was supported by NSFC (30700574), 863 (2008AA101003).

Small Size Hyluronan Promotes Bovine Oocyte Maturation and Embryo Development In Vitro.

The ability of the oocyte to develop to the blastocyst stage following in vitro fertilisation and subsequently a good quality embryo is affected by the microenvironment in which it is cultured. Hyaluronic acid (HA) is an important component of the extracellular matrix which is important for cumulus cells expansion and is present in the follicular oviductal and uterine fluids and is expressed in bovine oocytes and embryos. Hyaluronidase (Hyal) is an enzyme present in the oviduct and uterus and cleaves the HA into biologically active small fragments, facilitating its uptake or degradation. Bovine oocytes or cleaved embryos were matured in vitro in the presence or absence of HA, Hyal-2 or 4-Methylumbelliferone (4-MU), a HA synthesis inhibitor. The rates of oocyte nuclear maturation to the metaphase II stage and development of embryos to blastocyst stage and blastocyst quality were recorded. Large size HA and also Hyal-2 inhibited cumulus cells expansion without affecting oocyte maturation. 4-Mu at 1mM reduced cumulus cells expansion and oocytes maturation rate. Supplementation of embryo culture media with 300 Units/ml Hyal-2 significantly improved the blastocysts yield, and quality by increasing the total cell number and number of cells in the Inner Cell Mass (ICM). Fewer blastocysts were produced when 4-MU was added to the culture medium (P < 0.01). This data suggests that intrinsic HA production is critical for oocyte maturation and blastocyst development and also has an effect on embryo quality. This study therefore provides evidence for critical role of small HA fragments on improving embryo quality and development. (poster)

Effect of the Addition of β-Hydroxybutyrate to Chemically Defined Maturation Medium on the Nuclear Maturation, Sperm Penetration and Embryonic Development of Porcine Oocytes In vitro

We investigated the effects of various concentrations of β-hydroxybutyrate (BHB, 0, 0.1, 1 and 10 mM), a ketone body, added to chemically-defined maturation medium with or without energy substrates (glucose, pyruvate and lactate) on nuclear maturation rates up to the metaphase stage of the second meiotic division (M-II stage). In addition, we also assessed the influence of BHB on glutathione content, sperm penetration rate and embryonic development up to the blastocyst stage of oocytes matured under the presence of these energy substrates. Nuclear maturation rates up to the M-II stage of oocytes matured with BHB in each concentration group did not show a significant increase compared with the control (0 mM) groups in both the presence and absence of energy substrates. Although glutathione contents were not significantly different in each BHB concentration group, the sperm penetration rate in the I mM BHB group was significantly higher (p<0.05) and the embryonic development rate of oocytes up to the blastocyst stage was significantly lower (p<0.05) than the respective values of the control groups. These results suggest that BHB added to a chemically-defined maturation medium may stimulate sperm penetration while inhibiting embryonic development of porcine oocytes.

Gonadotropin-mediated dynamic changes in cytoplasmic and nuclear maturation in vitro: an animal model

OBJECTIVE: A high level of Gn in follicular fluids may contribute to ovarian hyper-stimulation in ART. The goal of this study was to determine whether there is a dose response effect on oocyte maturation in an animal model.DESIGN: Prospective experimental study.MATERIALS AND METHODS: Experiments were designed according to current popular stimulation protocols in ART, i.e. Bravelle only (B), combination of B and Menopur (B+M) or Repronex (B+R). Bovine oocytes were matured in media supplemented with 75, 750, 7500 or 75 000 mIU/ml Gn or combination of Gn in vitro for 24 h. The control group (NL) had no Gn in the media. Nuclear maturation status, spindle and chromosomal configurations, dynamic changes of the cortical granules (CGs) and mitochondria were evaluated by immunostaining and confocal laser scanning microscope.RESULTS: The nuclear maturation rates were 59.48%, 52.60%, 58.33% and 41.01% in B group, 61.11%, 58.52%, 50.00% and 33.57% in B+M group, 55.26%, 53.97%, 51.47% and 30.83% in B+R group, respectively. During in vitro maturation (IVM) of bovine oocytes, mitochondria formed larger clusters and migrated more centrally and CGs dispersed in cytoplasmic. Except 75000 mIU/ml B subgroup, supplement of different dose of Gn did not influence the migrations of mitochondria and CGs. There were no significant differences between 75, 750, 7500 mIU/ml groups and NL in normal spindle and chromosomal configurations. However, in 75000 mIU/ml groups, only 19.05% (B), 11.76% (B+M) and 0 (B+R) of the MII oocytes had normal spindles.CONCLUSION: Results demonstrated that low to moderate dose of Gn could improve nuclear maturation. However, extremely high dose of Gn had no beneficial effects on nuclear maturation. Although the redistributions of mitochondria and cortical granules (CG) were not altered in a dose-response manner, high dose of Gn did induce spindle and chromosomal abnormalities. Our study highlights the importance of judicious use of Gn and can be applied to clinical stimulation protocols to reduce the potential risks. OBJECTIVE: A high level of Gn in follicular fluids may contribute to ovarian hyper-stimulation in ART. The goal of this study was to determine whether there is a dose response effect on oocyte maturation in an animal model. DESIGN: Prospective experimental study. MATERIALS AND METHODS: Experiments were designed according to current popular stimulation protocols in ART, i.e. Bravelle only (B), combination of B and Menopur (B+M) or Repronex (B+R). Bovine oocytes were matured in media supplemented with 75, 750, 7500 or 75 000 mIU/ml Gn or combination of Gn in vitro for 24 h. The control group (NL) had no Gn in the media. Nuclear maturation status, spindle and chromosomal configurations, dynamic changes of the cortical granules (CGs) and mitochondria were evaluated by immunostaining and confocal laser scanning microscope. RESULTS: The nuclear maturation rates were 59.48%, 52.60%, 58.33% and 41.01% in B group, 61.11%, 58.52%, 50.00% and 33.57% in B+M group, 55.26%, 53.97%, 51.47% and 30.83% in B+R group, respectively. During in vitro maturation (IVM) of bovine oocytes, mitochondria formed larger clusters and migrated more centrally and CGs dispersed in cytoplasmic. Except 75000 mIU/ml B subgroup, supplement of different dose of Gn did not influence the migrations of mitochondria and CGs. There were no significant differences between 75, 750, 7500 mIU/ml groups and NL in normal spindle and chromosomal configurations. However, in 75000 mIU/ml groups, only 19.05% (B), 11.76% (B+M) and 0 (B+R) of the MII oocytes had normal spindles. CONCLUSION: Results demonstrated that low to moderate dose of Gn could improve nuclear maturation. However, extremely high dose of Gn had no beneficial effects on nuclear maturation. Although the redistributions of mitochondria and cortical granules (CG) were not altered in a dose-response manner, high dose of Gn did induce spindle and chromosomal abnormalities. Our study highlights the importance of judicious use of Gn and can be applied to clinical stimulation protocols to reduce the potential risks.

Mechanism of regulation of the EGF-like growth factor amphiregulin in human granulosa cells

OBJECTIVE: Recent evidence supports that LH-induced transactivation of the epidermal growth factor (EGF) signaling network is required for normal fertility. We have previously shown the EGF-like growth factor amphiregulin (AREG) in human follicular fluid is a mediator of cumulus cell expansion and promotes normal rates of oocyte nuclear maturation. We sought to further characterize the molecular pathways in human granulosa cells (GC) that are responsible for production of this critically important growth factor.

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Melatonin (MEL) acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM) on bovine embryos. This study tested the addition of MEL to maturation medium (MM) with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum - FCS) at 39ºC and 5% CO2 in air. After 24 hours of culture in MM with 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH; 10-9 M MEL) or 10-9 M MEL, 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10-9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM.

Heat shock protein 70 gene expression in equine blastocysts after exposure of oocytes to high temperatures in vitro or in vivo after exercise of donor mares

Heat above homeothermy can be detrimental to embryonic development, and cells may produce heat shock proteins to try to mitigate these effects. The authors examined the developmental competence of equine oocytes after a single heat exposure (42 °C, 2 or 4 h) during early or late stages of in vitro maturation. Rates of nuclear maturation, cleavage after intracytoplasmic sperm injection, and advanced embryonic development (morula or blastocyst) were compared to those for unexposed controls. Concentrations of heat shock protein 70 ( HSPA1A) mRNA were determined by real-time RT-PCR in resulting blastocysts, and were compared to those for embryos derived in vivo from control or exercised mares. Exposure of oocytes to heat at the onset of in vitro maturation did not affect any measured end point. However, exposure to 42 °C late in maturation culture reduced rates of oocyte nuclear maturation for both the 2 h (43/105 (43%) compared to control 70/103 (68%); P < 0.01), and 4 h (47/106 (44%) compared to control 60/103 (59%); P < 0.05) groups. Additionally, late heat exposure reduced development to morulae and blastocyst stages after intracytoplasmic sperm injection (ICSI; 18/89 (20%) compared to control 43/128 (34%); P < 0.05). Seven days after oocyte heat exposure, resultant blastocysts had a higher abundance of HSPA1A gene transcripts, relative to those for 18S rRNA. In vitro-produced embryos and lower-quality in vivo-produced embryos had significantly higher relative HSPA1A mRNA (lower 18S rRNA) concentrations than did higher-quality in vivo-produced embryos. The authors concluded that equine oocytes were sensitive to heat during late in vitro maturation, and responded to thermal shock with an increased ratio of HSPA1A:18S gene expression that was measurable in the resulting blastocyst. Embryos produced in vitro (including controls) had increased levels of HSPA1A mRNA relative to 18S rRNA compared to in vivo-produced embryos, suggesting a response to environmental insult.

Determination of optimal dose of EGF for bovine oocyte maturation and subsequent in vitro fertilization and culture in two media

The aim of this study was to determine the optimal dose of EGF on in vitro maturation of bovine oocytes and to investigate the effect of different IVF and IVC media on development of bovine embryos matured with EGF. Oocytes collected from a slaughterhouse were randomly divided into 4 groups. Each group of oocytes was incubated with different doses of EGF (Control: 0 ng/mL, Group 1: 1 ng/mL, Group 2: 10 ng/mL, and Group 3: 100 ng/mL) for 24 h to determine the optimal dose. Matured oocytes were fertilized with frozen-thawed bull sperm by using the swim-up method. The oocytes in each group were divided into 2 subgroups and cultured in CR1aa or G1.3/G2.3 media. Embryos were evaluated for cleavage (48th h), and 5-8 cell embryo (day 3) and blastocyst development (day 8) after fertilization. The rates of cumulus expansion and nuclear maturation were higher (P < 0.01) in GI, GII, and GIII compared to the control group. However, no significant difference was observed on oocyte maturation rates between different doses of EGF. Cleavage rates were higher (P < 0.01) in EGF groups than in the control group. No significant differences were obtained in blastocyst development between CR1aa and G1.3/G2.3 media (P > 0.1).

356 MODULATION OF PORCINE NUCLEAR MATURATION STATUS USING A SPECIFIC PHOSPHODIESTERASE INHIBITOR

Compared to other livestock species, IVM porcine COCs have poor developmental competence.Thisismost likely due to poor cytoplasmic maturation and asynchronous nuclear maturation. The resumption of nuclear maturation is largely regulated by cyclic adenosine monophosphate(cAMP), with increasing intra-oocyte levels prolonging cumulus-oocyte gap junction communication and delaying meiotic resumption. Modulation of cAMP levels using phosphodiesterase (PDE) inhibitors during bovine IVM significantly improves developmental competence (Thomas et al. 2004 Biol. Reprod. 71, 1142-1149). Hence, the aim of this study was to determine the effect of a type 3 PDE inhibitor (cilostamide, CIL) supplementation during porcine IVM on nuclear maturation, using a defined culture system. COCs derived from follicles of prepubertal gilts were cultured in groups of 10 in 100μL IVM medium (VitroMat, IVF Vet Solutions, Adelaide, Australia) +100 m IU mL-1 rhFSH +4 mg mL-1 BSA and nuclear maturation was assessed using orcein dye. Exp. 1: IVM medium was supplemented with 0, 5, 10, and 20 μM CIL or 0, 0.01, 0.1, 1, and 10 μM CIL and nuclear maturation was determined at 24 h and 44 h. Exp. 2: COCs were cultured in ± 0.1 μM CIL and nuclear maturation was assessed at 24 h, 44 h, 48 h, 52 h, and 56 h. Proportions of COCs at each stage of nuclear maturation were arcsine transformed and differences determined using a general linear model and Bonferroni post hoc test. Four replicates per experiment were performed with 20 COCs used for each treatment and/or time point. Results for Exp. 1 revealed no differences in the rate of nuclear maturation after 24 h of culture. After 44 h, 77% of COCs incubated in the absence of CIL were at metaphase II (MII) compared to 35-45% MII when cultured in the presence of 1, 5, 10, or 20 μM CIL (P &lt; 0.001). Furthermore, CIL supplementation resulted in approximately half the COCs arresting at germinal vesicle stage (GV) after 44 h of culture. While there were no significant differences in the MII rates of COCs cultured in 0, 0.01, and 0.1 μM CIL, significantly more COCs were at metaphase I (MI) at 44 h compared to control COCs (0 μM CIL, P &lt; 0.05). The time course experiment (Exp 2) demonstrated that nuclear maturation was delayed by 12 h with 0.1 μM CIL, compared to the absence of CIL, with comparable MII rates being achieved at 56 h for 0.1 μM CIL (72%) and 44 h for controls (0 μM CIL = 73%). These results demonstrate that porcine oocyte maturation can be induced in vitro by FSH in the presence of a low dose of type 3 PDE inhibitor resulting in a delay in the resumption and completion of nuclear maturation. Further investigations are underway to determine if CIL treatment prolongs gap junction communication and improves the developmental competence of porcine oocytes. This work was supported by the National Institutes of Health (USA) and National Health and Medical Research Council (Australia).

337 MITOCHONDRIA AND REACTIVE OXYGEN SPECIES IN PREBUPERTAL LAMB OOCYTES BEFORE AND AFTER IN VITRO MATURATION

Juvenile in vitro embryo transfer (JIVET) reduces the generation interval and increases the rate of genetic gain. The developmental competence of in vitro-produced embryos is strictly related to oocyte quality. Oxidative stress in the oocyte is an emerging problem in reproductive in vitro technologies, due to the gas atmosphere used to incubate oocytes and the lack of physiological defense mechanisms available in the female reproductive tract. The major source of reactive oxygen species (ROS) is represented by mitochondria where ROS are produced during oxidative phosphorylation. The aim of the present study was to analyze mitochondria and ROS in ovine prepubertal oocytes before and after IVM in order to clarify their suitability in JIVET programs. Cumulus-oocyte complexes from the ovaries of 38 slaughtered prepubertal (less than 8 months of age) lambs of the Comisana breed were analyzed at retrieval (group A) or after IVM (group B; Ambruosi et al. 2009 Theriogenology 71, 1093-1104). After cumulus cell removal, all oocytes underwent nuclear chromatin, mitochondria and ROS evaluation by confocal analysis of fluorescence distribution and intensity. Hoechst 33258 and Mitotracker Orange CMTM Ros (Molecular Probes Inc., Eugene, OR) were used to label nuclear chromatin and mitochondria (Ambruosi et al. 2009) and 2′,7′-dichloro-dihydro-fluorescein diacetate was used for ROS labelling (Hashimoto et al. 2000 Mol. Reprod. Dev. 57, 353-360). Out of 65 oocytes from group A, 38 oocytes with regular size (&gt;130 μm in diameter), morphology and nuclear chromatin at the GV stage were selected for analysis. One-hundred-thirty-eight oocytes underwent IVM (group B). Nuclear maturation rate (metaphase II with 1st polar body extruded) was 54%, 75/138. All MII oocytes were used for analysis. Significantly higher rate of oocytes from group B showed heterogeneous (large aggregates, clusters, pericortical, perinuclear) mitochondrial (mt) distribution pattern than oocytes from group A (55%, 41/75 v. 29%, 11/38, respectively; P &lt; 0.05) which showed uniform distribution of small mt aggregates. Fluorescent intensity of mt labeling did not differ between groups (43.05 ± 16.15 v. 45.89 ± 10.36, for group A and B respectively; NS). In most of the oocytes from both groups, intracellular ROS were distributed in small or large aggregates (35/38, 92% and 62/75, 83%). No statistical difference was observed for intracellular ROS levels between oocytes from group A (66.36 ± 13.2) and group B (72.84 ± 20.63; NS). The culture conditions used in this study provided normal mt distribution and intracellular ROS levels. Qualitative and quantitative evaluation of mitochondria and intracellular ROS could be useful to improve in vitro culture methods in ovine prepubertal oocytes.

155 EFFECTS OF CULTURE WITH OVALBUMIN IN ABSENCE OF FETAL BOVINE SERUM AND BOVINE SERUM ALBUMIN ON IN VITRO PRODUCTION OF CATTLE EMBRYOS

Embryo quality is influenced by culture conditions, which affect IVM, IVF, and IVC rates. One of the most efficient ways to evaluate the embryonic quality of IVP blastocysts is by differential staining of inner cell mass (ICM) and trophoblast (TF). Bovine embryos of superior quality should present the total number of cells close to the number of cell cycles (Neuber et al. 2002 Theriogenology 57, 2193-2202). In this study, we analyzed the effects of fetal bovine serum (F) and bovine serum albumin (B) replacement for ovalbumin (O) on nuclear maturation, cortical granule migration, pronuclear development, blastocyst rates, and differential staining of ICM and TF in Day 7 blastocysts. The treatment groups were named as follows: the first letter is the protein source used for IVM, the second for IVF, and the third for IVC. When 2 protein sources were used in the same step, the plus symbol (+) was used. The oocytes were IVM in TCM-199, supplemented with the following: 10% F, or 4 mg mL-1 B, or 4 mg mL-1 O, and 1.0 Âμg mL-1 of FSH, 50 Âμg mL-1 of hCG, 1.0 Âμg mL-1 of estradiol, 0.2 mM sodium pyruvate, and 83.4 Âμg mL-1 of amikacin. IVF was accomplished in TALP-IVF medium, with 0.2 mM pyruvate, 83.4 Âμg mL-1 of amikacin, and 6 mg mL-1 B or O. IVC was in SOF, with F, B, or O. The control group (CONT) consisted of the treatment FBF + B. Pronuclear development was compared by the chi-square test, whereas the other results were analyzed by ANOVA followed by the Tukey test, using SAS at 5% significance level (SAS Institute Inc., Cary, NC, USA). For IVM, the treatments F, B, O, and B + O did not affect (P &gt; 0.05) nuclear maturation (73.92 to 78.78%) and cortical granule migration rates (58.89 to 66.76%). Regarding pronuclear development, the treatment FO (76.67%) was similar (P &gt; 0.05) to the control group (82.95%), which was superior (P &lt; 0.05) to the treatments BB (56.98%), BO (39.02%), OB (37.36%), and OO (39.24%). Blastocyst rates in FBF (42.8%) and control (45.0%) groups were superior (P &lt; 0.05) to treatment OOO (26.0%) but similar (P &gt; 0.05) to FOF, BBB, BOB, and OBO (32.0 to 35.8%). The average of blastocyst ICM cells of the group OOO (16.79) was inferior (P &lt; 0.05) to the other groups. However, the average of TF cells on blastocysts of the group OOO (38.25) was similar (P &gt; 0.05) to the groups BBB (45.74) and BOB (45.60) and inferior (P &lt; 0.05) to the groups CONT (57.59), FBF (54.41), FOF (56.74), and OBO (47.35). The total average cells in the blastocysts of the group OOO (56.04) was inferior (P &lt; 0.05) to the groups CONT (84.86), FBF (78.96), FOF (81.32), BBB (68.11), BOB (69.55), and OBO (69.82). The total cell number in the treatments, with several sources of protein supplementation, varied from 56.04 to 84.86. Considering the evaluation interval, this average cell number was discreetly inferior to that expected for the chronological age of the blastocysts. We concluded that it is possible to produce bovine embryos in the absence of F and/or B, with the protein source O, although it reduced blastocyst rates when used in all 3 steps of embryo in vitro production and resulted in blastocysts of inferior quality. Financial support: FAPESP 05/60389-2 and CNPq.

Early metaphase II oocytes treated with dibutyryl cyclic adenosine monophosphate provide suitable recipient cytoplasm for the production of miniature pig somatic cell nuclear transfer embryos

We investigated the effects of in vitro maturation duration and treatment with dibutyryl cyclic adenosine monophosphate (dbcAMP) on the blind enucleation efficiency and developmental competence of miniature pig somatic cell nuclear transfer (SCNT) embryos. Oocytes were cultured for 22 h in NCSU-23 medium with or without 1 mM dbcAMP and then additionally cultured in dbcAMP-free NCSU-23 for 14, 18, or 22 h. Regardless of dbcAMP treatment, the rate of nuclear maturation reached a plateau at 36 and 40 h. However, mitochondrial distribution, a marker for cytoplasmic maturation, differed between the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h. The metaphase II chromosomes were adjacent to the first polar body in 68.8% and 63.5% of the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h, respectively. Furthermore, the blind enucleation efficiency by removing a small volume of cytoplasm was significantly higher in the dbcAMP-untreated oocytes at 36 h (82.9%) and dbcAMP-treated oocytes at 40 h (89.9%) than other groups. The rate of blastocyst formation was highest in the dbcAMP-treated oocytes at 40 h. Hence, this study demonstrated that dbcAMP-treated early metaphase II oocytes are suitable for the production of miniature pig SCNT embryos.

Effect of β-hydroxybutyrate Added to Maturation Medium on Nuclear Maturation of Pig Oocytes

Abstract: The effects of β-hydroxybutyrate (BHB) added to a modified NCSU37 medium during in vitro maturation (IVM) on nuclear maturation, and ATP and glutathione (GSH) contents of porcine oocytes were investigated. Nuclear maturation up to the metaphase stage of the second meiotic division (the M-II stage) of cumulus cell-enclosed oocytes (COs) matured with various concentrations (0.0, 2.3, 3.1, 3.9 and 4.7 mM) of BHB was observed. The nuclear maturation rate of the oocytes of the 3.1 mM group was significantly higher (P < 0.05) than that of the control (0.0 mM) group. Both the ATP and GSH contents of the oocytes matured with or without 3.1 mM BHB and removed from cumulus cells after IVM were not significantly different. These results indicate that 3.1 mM BHB added to the maturation medium may stimulate the nuclear maturation of porcine oocytes without changing their ATP and GSH contents.

Expression of the μ Opioid Receptor and Effects of the Opioid Antagonist Naloxone on In Vitro Maturation of Oocytes Recovered from Anoestrous Bitches

The mu-opioid receptor (MOR) is expressed in bovine, human, equine and canine oocytes, and in seasonal breeders, it is expressed with higher intensity during the anoestrous phase. Supplementation of in vitro maturation (IVM) medium with opioid agents, agonists or antagonists, was shown to affect oocyte maturation in several species such as rat, bovine and equine. This study reports the effects of supplementing IVM medium with naloxone (Nx), an opioid antagonist, on nuclear and cytoplasmic maturation rate of oocytes recovered from anoestrous bitches. Cytoplasmic maturation was examined in terms of mitochondrial (mt) distribution. In order to confirm the receptor-mediated action of Nx, in oocytes of anoestrous bitches, MOR expression was analyzed by Western blot. Cumulus-oocyte complexes, recovered from the ovaries of bitches in anoestrous, were cultured in vitro and Nx was added at the concentrations of 1 x 10(-6), 1 x 10(-8) and 1 x 10(-10) M. The rate of oocytes resuming meiosis after culture in presence of 1 x 10(-6) M Nx (29%) was significantly higher than that of oocytes of control group (12%; p < 0.05). However, treatment with Nx did not affect mt distribution pattern. In denuded oocytes and in corresponding cumulus cells, a doublet of 65 and 50 kDa was observed. We conclude that, in oocytes of anoestrous bitches, MOR is expressed and Nx significantly improves nuclear maturation rate. Further studies should be performed to elucidate the expression of other opioid receptors, such as delta and kappa, and possible interactive effects of their antagonists on canine oocyte maturation.

Influence of metabolic status on oocyte quality and follicular characteristics at different postpartum periods in primiparous rabbit does

Low reproductive performance of high-yield primiparous animals is closely associated with the metabolic stress caused by a simultaneous gestation and lactation. The aim of this work was (1) to analyze body composition and metabolic environment at three time points along lactation (at parturition time; in the lactation period [Day 11 postpartum]; and in the postweaning period [Day 32 postpartum]) of primiparous rabbit does (Oryctolagus cuniculus) and (2) to investigate the ovarian status at insemination time and the possible link with metabolic environment and with their reproductive performance. To this end, does were either submitted to a semi-intensive reproductive rhythm (Group S, inseminated on Day 11 postpartum) or an extensive rhythm (Group E, inseminated on Day 32 postpartum). Body energy (P < 0.05) and protein content (P < 0.001) as well as serum leptin (P < 0.05) and protein concentrations (P < 0.001) increased significantly along the postpartum period. At parturition, body lipid content was significantly lower and serum nonesterified fatty acids concentrations were significantly higher than that on Days 11 postpartum and 32 postpartum. Concerning ovarian status at insemination time, no significant differences were found in mean follicular stages, serum estradiol, progesterone, and prolactin (PRL) concentrations or in prolactin receptor (PRL-R) immunostaining. However, follicles in Group S showed a significantly higher apoptosis index than that of Group E (P < 0.001). The nuclear and cytoplasmic oocyte maturation rates of Group S were also significantly lower than that in Group E. In addition, conception rate and prolificacy were improved in Group E (P < 0.001 and P < 0.05, respectively). In conclusion, in the early postpartum period, metabolic status seems to impact negatively on ovarian follicle and oocyte quality leading to a poor reproductive outcome in primiparous rabbit does.

Effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The present study investigated the effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The donor bitches were categorized into three groups based on stage of estrus cycle: follicular (proestrus or estrous), luteal (diestrus) and anestrus. One ovary of each pair collected from 39 mature bitches was transported in Phosphate Buffer Saline (PBS) at 4 °C while the other was transported at 37 °C. A total of 1138 Grade I COCs obtained from all ovaries were grouped and matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), luteinizing hormone (LH), essential and non-essential amino acids at 38.5 °C in a humidified 5% CO 2, 5% O 2, and 90% N 2 atmosphere for 72 h. The nuclear maturation rates were evaluated by aceto–orcein staining. Oocytes harvested from follicular and luteal ovaries have a significantly higher maturation rates (MI + MII) than the oocytes from anestrual ovaries in the 37 °C group ( p < 0.05). However, oocytes harvested from anestrual ovaries transported at 4 °C had the highest maturation (MI + MII) rate, and the difference between anestrual and luteal ovary groups was significant ( p < 0.05). The oocytes from anestrual ovaries transported at 4 °C have significantly higher maturation rates than those transported at 37 °C ( p < 0.0001). However, the transport temperature (37 or 4 °C) did not significantly affect the maturation (MI + MII) rates of oocytes harvested from the luteal ( p = 0.61) and follicular ( p = 0.48) stage ovaries. It can be concluded from this study that (1) both transport temperature and transport temperature × estrus cycle stage interaction effected the maturation rates, while estrus cycle stage alone did not, and (2) transporting canine ovaries at 4 °C can improve in vitro maturation rates in oocytes harvested from anestrous ovaries.