Nuclear Maturation Rates Research Articles

The Effect of the Addition and Removal of Various Cryoprotectants on the Nuclear Maturation and ATP Content of Immature Porcine Oocytes

This study was undertaken to investigate the influence of exposure and removal of four different cryoprotectants (CPAs) on the ATP content of cumulus cell-enclosed (COs) and cumulus cell-denuded (DOs) immature porcine oocytes. The in vitro nuclear maturation of the COs, exposed to and removed from the CPAs was also assessed. Both COs and DOs were exposed to 1.5 M concentrations of each CPA (ethylene glycol (EG); propylene glycol (PG); dimethyl-sulfoxide (DMSO); and glycerol (G)) for durations of 5, 15, and 30 minutes at room temperature (23.5±1.5°C), and immersed in physiological saline supplemented with 20% (v/v) fetal bovine serum for 5 minutes (39°C) to remove each CPA. Before, during and after exposure to each CPA, the ATP content of both the COs and the DOs was measured. After removal from each CPA an aliquot of the COs was matured for 44±2 h, and their nuclear maturation rates were measured up to the metaphase stage of the second meiotic division (the M-II stage). The maturation rates up to the M-II stage were not significantly different between all the groups that were exposed to each CPA for 5 minutes. For 15 and 30 minute exposures, the maturation rates of the COs exposed to PG, DMSO and EG were almost the same as those of the control groups; however, the rates of G group exposed for 15 and 30 minutes were significantly lower (p<0.05) than the control group. These groups were also found to have a decrease in the ATP content of COs and DOs during and after exposure for the same periods (p<0.05). In addition, although the ATP contents of the COs after exposure to EG for any period were the same as the controls, the ATP content of the DOs exposed to EG for any period were significantly lower (p<0.05) than those of the controls. When the ATP content of the COs and DOs of each CPA were compared, the DOs exposed to PG were found to have a significantly greater level (p<0.05) than DOs exposed to G for any duration. In addition, the ATP content of DOs exposed to PG for 30 min and removal was also higher (p<0.05) than when exposed to DMSO for the same period. These findings indicate that PG may be a useful CPA for the cryopreservation of immature porcine oocytes.

The use of R-roscovitine to fit the ‘time frame’ on in vitro porcine embryo production by intracytoplasmic sperm injection

Micromanipulation of oocytes is time consuming during ICSI experiments; however the 'time frame' to manipulate oocytes without a drop in efficiency is not very wide due to the use of not completely matured and/or aged MII oocytes. Therefore, the aim of this work was to study the effect of a short roscovitine pretreatment for 5 h and two different IVM periods (5R + 40IVM or 5R + 45IVM) and a prolonged IVM time from 45 h (45IVM) to 50 h (50IVM) on parthenogenetic and ICSI embryo development, in order to fit the time frame to manipulate pig oocytes to the whole labour day session. In the first experiment, oocytes, pretreated with roscovitine and IVM cultured for 5 h, showed a similar nuclear stage as non-cultured oocytes and a significantly higher percentage of GVI-GVII oocytes compared with non-roscovitine treated oocytes cultured for 5 h in IVM conditions. When COC were cultured under the 5R + 40IVM system, nuclear maturation and cleavage rates after electrical activation were significantly lower than when COC were cultured under the 45IVM, 50IVM and 5R + 45IVM culture systems (54.2% vs. 72.6-76.8% and 58.8% vs. 81.4-88.3%, respectively). However, this difference was not statistically significant for parthenogenote blastocyst rate. No differences were observed in MII and in parthenogenote and ICSI embryo development among 45IVM, 50IVM and 5R + 45IVM experimental groups. In conclusion, under our conditions and using parthenogenetic and ICSI embryos, we observed that it is feasible to prolong the pig oocyte manipulation 'time frame' by at least 5 h with no significant drop in blastocyst rate.

Effect of Oocyte Diameter on in vitro Embryo Production in Dogs

Summary In vitro embryo production has not been confidently applied to the dog successfully. Up to date, only one blactocyst have been achieved by in vitro culture. Therefore, the aim of the present study was to examine the effects of different oocyte diameters (≤100 μm and >100 μm) on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of immature dog oocytes. The study was performed in two steps. At the first step (experiment I), effects of two different oocyte diameters on IVM of dog oocytes were investigated. The nuclear maturation rates were evaluated by aceto-orcein staining method at the end of the IVM. At the second step (experiment II), in vitro matured oocytes were fertilized with fresh spermatozoa for 24 h and in vitro cultured for 7 d. At the end of the IVC period, embryonic development was assessed by microscopic observation at 24 h intervals and fixed for staining by aceto-orcein staining method after 7 days. In comparison relating in IVM and IVF rates, larger oocytes have higher maturation (P<0.05) and cleavage (P<0.01) rates than the smaller ones. Unfortunately, none of the oocyte was reached to morula or blactocyst stage in both groups. In conclusion, it is demonstrated that the oocyte diameter may be a helpful selection criteria for dog in vitro embryo production.

İn Vitro Köpek Embriyosu Üretimi Üzerine İki Farklı Medyumun Etkisi

Summary Embryo production via in vitro fertilization and nuclear transfer has been accomplished in the dog, and the transfer of the cloned embryos has recently resulted in the birth of puppies. However, the efficiency of these technologies is still very limited. Until now, only two morulas and single blactocyst have been achieved in vitro. Therefore, the aim of the present study was to examine the effects of two different media (mSOF and TCM 199) on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of immature dog oocytes. The study was performed in two steps. At the first step, the effects of two different media, on IVM of dog oocytes were investigated. At the end of the IVM period, the nuclear maturation rates were evaluated by aceto-orcein staining method. At the second step, after the IVM period the oocytes inseminated with fresh spermatozoa for 24 h and left for IVC for 7 d. At the end of the IVC period, embryonic development was assessed by microscopic observation at 24 h intervals and then fixed and stained by the same method. Consequently, maturation rates of oocytes in mSOF medium were significantly higher than those of the TCM 199 (P<0.001). After 7 d of the IVC period, only one oocyte was cleaved in the TCM 199, while eight oocytes cleaved and one of them developed to morula stage in mSOF medium group (P=0.037).

Nuclear maturation and structural components of nonhuman primate cumulus–oocyte complexes during in vivo and in vitro maturation

To compare cumulus cell structure and timing of oocyte maturation of in vitro-matured (IVM) and in vivo-matured (VVM) nonhuman primate oocytes. In vivo maturation and in vitro maturation of oocytes. Animal cell culture laboratory. Forty-eight female rhesus macaques. Fifteen animals were administered FSH, and aspirated oocytes were cultured in vitro for 0, 3, 6, 12, or 24 hours (IVM). Thirty-three animals were administered FSH and hCG, and oocytes were collected 3, 6, 12, or 28-30 hours after hCG (VVM). Nuclear maturation and microtubule scores of oocytes and actin and tubulin transzonal processes of cumulus cells. Embryo development was observed for VVM oocytes. The rate of nuclear maturation was faster for IVM oocytes compared with VVM oocytes. Actin transzonal processes decreased 0-12 hours after hCG administration for VVM oocytes. Tubulin transzonal processes of IVM and VVM oocytes decreased from 0 to 24 hours and from 0 to 3 hours, respectively. Embryo development improved as VVM time increased. Nuclear maturation and remodeling of cumulus-oocyte complex structural components associated with in vitro maturation do not parallel those of oocyte maturation in vivo, indicating that in vitro culture conditions continue to be suboptimal.

254 DYNAMICS OF GOLGI APPARATUS DURING BOVINE OOCYTE IN VITRO MATURATION: EFFECTS OF INHIBITORS ON CDC2A AND CYTOPLASMIC-DYNEIN ATPase ACTIVITY

The process of maturation encompasses a complex series of molecular and structural events. Completion of the nuclear changes to produce a metaphase II (MII) oocyte does not reflect the molecular and structural maturity of an oocyte, which is sometimes termed cytoplasmic maturation. The Golgi apparatus phosphorylates, fragments, and changes the localization during oocyte maturation. GM130 and phospho-GM130 are used as markers for the Golgi apparatus and phosphorylated Golgi apparatus, respectively. The goal of this study was to analyze the dynamics of the Golgi apparatus and its association with microtubules in bovine oocytes at different stages of in vitro maturation [IVM; i.e. germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and MII]. The roles of CDC2A kinase (also known as p34cdc2) and cytoplasmic-dynein ATPase on Golgi dynamics were studied by using specific inhibitors. The distribution of the markers was assessed by immunocytochemistry and laser confocal microscopy. To unravel the role of CDC2A and cytoplasmic dynein ATPase on the dynamics of the Golgi apparatus, the inhibitors roscovitine (ROS) and sodium-orthovanadate (SOV), respectively, were used. In the first experiment, the nuclear maturation rate was checked in the presence of the inhibitors at different times and for different incubation times to explore whether oocytes were able to reach the MII stage. At the GV and GVBD stages, the Golgi apparatus is observed as fragments named mini-Golgies and at the MI and MII stages as punctate foci throughout the cytoplasm. Our results showed 2 well-defined movements of the Golgi apparatus toward opposite directions, depending on the maturation stage. The first movement was observed between 5 and 9 h of IVM (i.e. the GVBD stage), when the Golgi apparatus relocalized from the ooplasm to the periphery. The second movement was observed between 9 and 15 h of IVM (i.e. the MI stage), when the Golgi apparatus moved from the cortex to throughout the cytoplasm and remained there up to the MII stage. The use of inhibitors on CDC2A and cytoplasmic-dynein ATPase at selected time points revealed that CDC2A played a crucial role on the distribution of this organelle during the first movement, whereas the final localization at the GVBD stage was dependent on cytoplasmic-dynein transport. The second movement of the Golgi apparatus was disturbed by the SOV treatment, but not by the use of ROS, suggesting a role of cytoplasmic-dynein-dependent transport during the distribution and organization of the punctate foci at the MI stage. The phosphorylation status of Golgi was not affected at the different incubation times with inhibitors, except in those oocytes incubated with ROS for 24 h, suggesting a role of CDC2A. In conclusion, we describe the involvement of CDC2A during the first movement of the Golgi apparatus and the importance of cytoplasmic-dynein ATPase activity in the first and second relocalization of Golgi during bovine oocyte maturation. DAAD.

250 EFFECT OF LOW OXYGEN TENSION ATMOSPHERE AND MATURATION MEDIA ON IN VITRO-MATURED SWINE OOCYTES

The aim of this study was to evaluate the efficiency of a low oxygen tension atmosphere (5% CO2, 5% O2, and 90% N2) on swine oocyte maturation in chemically defined media or when supplemented with porcine follicular fluid (PFF). Briefly, oocytes were in vitro matured for 44 h in TCM-199 with 10% PFF or 0.1% PVA added, under a low oxygen tension atmosphere or a normal oxygen tension atmosphere (5% CO2 in air, approximate 20% O2). At 0 and 44 h of maturation, cumulus oophorus cells were removed. To evaluate the migration of cortical granules, oocytes were fixed, permeabilized, and incubated in 100 μg of FITC-PNA mL–1 for 30 min. Oocytes were then incubated in 10 μg mL–1 of Rnase for 30 min and in 10 μg mL–1 of propidium iodide for 10 min to verify nuclear maturation by confocal microscopy (Zeiss LSM 510 Meta). Heat shock protein 70 (HSP70) content was assessed as described in Kawarsky and King (2001 Zygote 9(3), 39–50) to verify oxidative stress. Data were analyzed by the SAS System for Windows (2000). The nonparametric ANOVA NPAR1WAY procedure was applied to evaluate nuclear maturation rate by comparing groups in pairs. Migration of cortical granules and HSP70 content were analyzed using PROC GLM (LSD test of means). The effects of treatment and manipulation were verified in all analyses. The significance level was 5%, and data were presented as means ± SEM. Results indicated that the percentage of metaphase II oocytes did not differ among groups after 44 h of maturation [PFF 5% O2 (89.16 ± 3.73a), PFF 20% O2 (86.59 ± 6a), PVA 5% O2 (79.62 ± 8.22a), and PVA 20% O2 (93 ± 5.17a)]. However, these groups were different from the 0-h group (0 ± 0b). Results for the percentage of cortical granule migration showed that 0-h oocytes (38.92 ± 2.75a) had lower migration rates compared with other groups. After 44 h of maturation, migration of the cortical granule rate of the PFF-supplemented group under a 5% O2 atmosphere (61.66 ± 3.21b) was different when compared with the PVA 20% O2 group (50.97 ± 3.48c). The other groups showed intermediate results, but without statistical differences [PFF 20% O2 (58.51 ± 2.5bc) and PVA 5% O2 (53.75 ± 3.14bc)] for the migration of cortical granules. Moreover, no difference at pixel quantification of HSP70 was observed among groups after 44 h of maturation [PFF 5% O2 (116.45 ± 40.94a), PFF 20% O2 (44.44 ± 12.66a), PVA 5% O2 (29.95 ± 7.95a), and PVA 20% O2 (58.49 ± 22.2a)], although these groups were different from the 0-h group (247.41 ± 38.59b). Although the HSP content decreased throughout in vitro maturation of swine oocytes under the low and high oxygen tension atmospheres, it can be concluded that a low oxygen tension atmosphere did not affect nuclear maturation and rates of cortical granule migration regardless of maturation media supplementation. Financial support: FAPESP (grant no. 05/01420-7).

Effects of heat stress during in vitro maturation on cytoplasmic versus nuclear components of mouse oocytes

The objectives of this study were to investigate the effect of heat stress during in vitro maturation on the developmental potential of mouse oocytes and to determine whether the deleterious effect was on the nuclear or cytoplasmic component. While rates of oocyte nuclear maturation (development to the metaphase II stage) did not differ from 37 to 40 degrees C, rates for blastocyst formation decreased significantly as maturation temperature increased from 38.5 to 39 degrees C. Chromosome spindle exchange showed that while blastocyst formation did not differ when spindles matured in vivo or in vitro at 37, 40 or 40.7 degrees C were transplanted into in vivo matured cytoplasts, no blastocyst formation was observed when in vivo spindles were transferred into the 40 degrees C cytoplasts. While oocytes reconstructed between 37 degrees C ooplasts and 37 or 40 degrees C karyoplasts developed into 4-cell embryos at a similar rate, no oocytes reconstituted between 40 degrees C ooplasts and 37 degrees C spindles developed to the 4-cell stage. Immunofluorescence microscopy revealed impaired migration of cortical granules and mitochondria in oocytes matured at 40 degrees C compared with oocytes matured at 37 degrees C. A decreased glutathione/GSSG ratio was also observed in oocytes matured at 40 degrees C. While spindle assembling was normal and no MAD2 was activated in oocytes matured at 37 or 40 degrees C, spindle assembling was affected and MAD2 was activated in some of the oocytes matured at 40.7 degrees C. It is concluded that 1) oocyte cytoplasmic maturation is more susceptible to heat stress than nuclear maturation, and 2) cytoplasmic rather than nuclear components determine the pre-implantation developmental capacity of an oocyte.

Influence of different reproductive rhythms on serum estradiol and testosterone levels, features of follicular population and atresia rate, and oocyte maturation in controlled suckling rabbits

The aim of the current work was to analyze the features of ovarian follicular population and their quality in New Zealand white rabbit does synchronized by 24-h controlled doe-litter separation before artificial insemination (AI) during all their reproductive cycles. Synchronized animals were allocated systematically in two groups. A total of 73 rabbit does (group A) were submitted to a 35-day intensive rhythm (AI on day 4 post-partum [pp] and weaning at 25 days of lactation), and 108 rabbit does (group B) were submitted to a 42-day semi-intensive rhythm (AI on day 11 pp and weaning at 35 days of lactation) during 9 months. At the mid-end of their reproductive life, a total of 26 does (5.4 parturitions), under intensive ( n = 15) or semi-intensive rhythm ( n = 11) were either treated in each group with 25 IU eCG 48 h before laparotomy to recover their ovaries ( n = 7 for group A and n = 6 for group B) (according to the Bioethics Committee of the University) or not synchronized with the hormonal treatment ( n = 8 for group A and n = 5 for group B). Blood samples were collected at the moment of ovary recovery; morphometrical parameters, number of total follicles and number of follicles ≥1 mm in size in the ovarian surface were recorded. Oocytes from follicles of one ovary were recovered and matured in TCM 199 supplemented with 10 ng/ml EGF, 100 ng/ml IGF-I and 10% FCS. The counterpart ovaries were fixed in paraformaldehyde solution for histological studies. Detection of cell apoptosis was determined using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) technique. Reproductive performance was affected by the rhythm used, with lower reproductive parameters in the intensive group. The average ovary height and width, the mean number of ≥1 mm follicles and the number of total follicles were similar between groups. Serum concentrations of estradiol (E 2) and testosterone (T) were significantly lower in group A vs. B (E 2: 232.4 ± 56.1 vs. 399.7 ± 53.0 pg/ml; P < 0.05 and T: 1.07 ± 0.10 vs. 1.68 ± 0.23 ng/ml; P < 0.05). No significant differences were found in follicular population or in the mean follicular apoptosis index between groups. Metaphase II rate was significantly lower in group A vs. B (48.5 ± 3.3 vs. 67.6 ± 3.7%; P < 0.01), as well as the migration rate of cortical granules (12.7 ± 2.7 vs. 38.2 ± 6.6%; P < 0.001). On the other hand, neither follicular population, nuclear maturation rate nor apoptosis rate were affected by the eCG treatment, but cytoplasmic maturation was higher in animals treated with eCG in group A (29.2% vs. 5.5%; P < 0.01). In conclusion, rabbit does under transient litter separation during their reproductive life have both their serum estradiol and testosterone concentrations and oocyte quality influenced by the intensive rhythm, leading to a decrease in reproductive parameters. Also, both intensive and semi-intensive rhythms seem to be less receptive to eCG treatment than expected.

Effect of Adding Glucose to Maturation Medium on the Nuclear Maturation and ATP Content of Porcine Oocytes

In this study, we investigated the effect of adding glucose to in vitro maturation (IVM) medium on the nuclear maturation and ATP metabolism of cumulus cell-enclosed porcine oocytes (COs). When various concentrations of glucose (0.00, 2.78, 5.55, 11.10 and 16.65 mM) were added to IVM medium, the nuclear maturation rate up to the metaphase stage of the second meiotic division (M-II stage) in the 2.78 mM group (67.90%) was significantly higher than that of the non-glucose (0.00 mM, 31.66%) group (p<0.05). In addition, when cumulus cell-denuded oocytes (DOs) were matured in medium with or without 2.78 mM glucose, the rate of DOs maturing to the M-II stage with glucose (53.17%) was significantly higher (p<0.05) than that in the group in which glucose was not added (23.53%). However, these values for DOs were significantly lower (p<0.05) than those for COs (with glucose:71.85% and without glucose: 38.10%). Conversely, the ATP content of COs and DOs matured with glucose were almost the same as those matured without glucose. These results indicate that glucose may stimulate nuclear maturation without changing ATP metabolism.

Improving oocyte competence with FSH at time of hCG trigger: a randomized controlled trial

OBJECTIVE: We hypothesize the induction of final oocyte maturation via modification of ovulation trigger with the addition of FSH at the time of hCG will improve developmental competence of the oocyte.DESIGN: Double-blind randomized controlled trial, interim analysis.MATERIALS AND METHODS: Subjects undergoing a long agonist protocol were randomized by sealed envelope allocation to FSH (6 amps) vs placebo at the time of hCG. For each subject, two dominant follicles were aspirated individually and follicular fluid (FF) was collected. Oocytes recovered were handled and cultured individually throughout ICSI and subsequent laboratory procedures. Clinical and laboratory characteristics were recorded, and serum and FF hormone levels were measured with duplicates. Follicular size was extrapolated by follicular volume. The rate of nuclear maturation was assessed by metaphase II oocyte per follicle >12mm. Data were analyzed both at cycle and follicle level using Chi square and Student's t test as appropriate, and analysis was done on an intention to treat basis.Table 1Oocyte ParametersFSHPlaceboMII/follicle >12mm75%74%Mean Follicular Volume (mL)4.10 ±1.94.19 ±1.5ICSI Fertilization (#2PN/MII)81.3%68.0%Day 3 Cleavage >5cells70%75%Fragmentation (1-5, 1 is best)2.3 ±1.22.6 ±1.1Embryos Cryopreserved5.5 ±2.54.5 ±2.6 Open table in a new tab Table 2Hormone ParametersFSHPlaceboFF FSH (mIU/mL)13.3 ±7.69.2 ±5.1FF Estradiol (ng/mL)614.6 ±340548.6 ±301FF Progesterone (mcg/mL)10.3 ± 4.610.3 ±4.3FF hCG (mIU/mL)157.7 ±76.4159.9 ±91.8FF Testosterone (ng/dL)817.1 ±288969.2 ±123 Open table in a new tab CONCLUSIONS: This interim analysis suggests an improvement in oocyte parameters with the addition of FSH at the time of hCG trigger. We hypothesize the increased FF FSH may be enhancing oocyte cytoplasmic maturation and developmental competence. Data collection is ongoing and final data analysis will be presented. OBJECTIVE: We hypothesize the induction of final oocyte maturation via modification of ovulation trigger with the addition of FSH at the time of hCG will improve developmental competence of the oocyte. DESIGN: Double-blind randomized controlled trial, interim analysis. MATERIALS AND METHODS: Subjects undergoing a long agonist protocol were randomized by sealed envelope allocation to FSH (6 amps) vs placebo at the time of hCG. For each subject, two dominant follicles were aspirated individually and follicular fluid (FF) was collected. Oocytes recovered were handled and cultured individually throughout ICSI and subsequent laboratory procedures. Clinical and laboratory characteristics were recorded, and serum and FF hormone levels were measured with duplicates. Follicular size was extrapolated by follicular volume. The rate of nuclear maturation was assessed by metaphase II oocyte per follicle >12mm. Data were analyzed both at cycle and follicle level using Chi square and Student's t test as appropriate, and analysis was done on an intention to treat basis. CONCLUSIONS: This interim analysis suggests an improvement in oocyte parameters with the addition of FSH at the time of hCG trigger. We hypothesize the increased FF FSH may be enhancing oocyte cytoplasmic maturation and developmental competence. Data collection is ongoing and final data analysis will be presented.

Vitrification of in vitro matured oocytes of Mangalica and Large White pigs

The breeding of Mangalica, a native pig breed in Hungary, had been started in 1833, but this pig breed almost became extinct in Hungary in the past decades. In 1991, the number of sows was only 200. Although in these days the existing Mangalica population consists of more than 6000 animals representing different colour variations, the preservation of this traditional pig breed is still very important. Vitrification is a potential tool for the preservation of gametes and embryos of these animals. The aim of this study was to investigate the effects of vitrification on the developmental competence of Mangalica (M) and Large White (LW) oocytes following fertilisation. The oocytes were vitrified by the Open Pulled Straw (OPS) method using different concentrations of ethylene glycol and dimethyl sulphoxide as cryoprotectants. After rehydration the oocytes underwent in vitro fertilisation; the resultant zygotes were then cultured in vitro for four days to assess embryonic development. In the first experiment, in vitro maturation of M and LW oocytes was compared. No significant difference was observed in the nuclear maturation rate of LW and M oocytes. In the second experiment, the sensitivity of oocytes to vitrification was examined by evaluating oocyte morphology after thawing. A higher percentage of LW oocytes showed normal morphology compared to M oocytes, indicating that Mangalica oocytes are more sensitive to cryoprotectants than Large White oocytes. After warming and in vitro fertilisation, more than 50% of the oocytes started embryonic development and by the end of the incubation period morula stage embryos had developed in both groups. The results show that the OPS vitrification technique is well suited to preserve Mangalica oocytes and from these oocytes morula embryos can be produced.

Evaluation of benefits of oocyte denudation timing at ICSI

OBJECTIVE: This study was undertaken to evaluate the results of denudation timing effect of cumulus and corona cells after oocyte retrieval on oocyte nuclear maturation rates, ICSI fertilization rates, cleavage rates and pregnancy rates. DESIGN: Retrospective study. MATERIALS AND METHODS: We analyzed 878 ICSI cycles between January 2006 to March 2008. Cycles were prepared using standart Long, Short Antagonist and Microdose Flare-up protocols where appropriate in each patient. The outcomes were compared in two different age groups [female age < 37 (group 1) and ≥ 37 (group 2)] in order to manage subgroups comparable. Cumulus and corona cells were totally denuded randomly in three subgroups as; immediately denuded after pick-up(H0), after pick-up one hour incubation up to dedudation(H1) and after pick-up 2 hours incubation up to denudation(H2) arranged as timely. Eighteen hours and thirtysix hours after ICSI, fertilization rates and cleavage rates were evaluated in turn. Embryo transfers were done on day 3. Fetal Heart Beat positive patients consented pregnant. Oneway ANOVA test and Chi-square test were used for statistical analysis where appropriate. P< 0.05 was accepted as significant. RESULTS: The table summarizes our results. Mean age, BMI (body mass index), total Gnrh usage, E2 levels at HCG timing, retrieved oocytes, endometrial thickness at transfer time, and transfered embryo numbers were comparable between subgroups (H0,H1,H3) in each group (Group 1 and 2).Table 1Under age 37(Group 1)H0H1H2P ValueNo.Patients68155424Maturation Rates%81%82,3%82,2NSFertilization Rates%71%71,1%75,3(H0,H2 P=0,006) (H1,H2 P=0,0001)Cleavage Rates%99,3%98,9%98,9NSOngoing pregnancy rates per transfer%55,8%61,2%58NSNS=Not Significant. Open table in a new tab Table 2Age 37 and over (group 2)H0H1H2P ValueNo.Patients2146164Maturation Rates%76,3%81,4%84(H0,H2 P=0,003)Fertilization Rates%59,6%69,2%73,1(H0,H1 P=0,03) (H0,H2 P=0,0002)Cleavage Rates%97%98%96,6NSOngoing pregnancy rates per transfer%23,8%26%31NSNS=Not Significant. Open table in a new tab NS=Not Significant. NS=Not Significant. CONCLUSIONS: As conclusion when we evaluate groups overall(group 1 and group2) together, it seems at least 2 hours of incubation is beneficial on fertilization rates for each group and on maturation rates just for older patients. Clevage rates and pregnancy rates didn't differ significantly overall. However more than 2 hours of incubation timing is needed to be assesed for further study.

Effects of cumulus cells on rabbit oocyte in vitro maturation

Cumulus cells (CCs) are of great importance in oocyte development and maturation in many species, but detailed influence of CCs has not been extensively examined, especially on rabbit. The present study was designed to investigate the effects of CCs and the elongation of in vitro maturation (IVM) time on rabbit oocyte nuclear and ooplasmic maturation and survival. Cumulus oocyte complexes (COCs) and naked oocytes (NOs) were recovered directly from rabbits super-ovulated with eCG. Corona-enclosed oocytes (COs) and denuded oocytes (DOs) were obtained from COCs after removing a part or whole of CCs. The oocytes were cultured in the following seven groups. (i) Cumulus cell enclosed oocytes (CEOs) were cultured alone (CEOs); (ii) COs were cultured alone (COs); (iii) DOs were cultured alone (DOs); (iv) NOs were cultured alone; (v) DOs were co-cultured with COCs [DOs(COCs)]; (vi) DOs were co-cultured with CCs [DOs(CCs)]; (vii) NOs were co-cultured with CCs [NOs(CCs)]. After the oocytes were cultured for 24 and 30 h, the nuclear maturation was evaluated by first polar body (PB1) extrusion while the ooplasmic maturation was evaluated by the cleavage rate after parthenogenetic activation. The results showed that the nuclear maturation rate of CEOs, COs, DOs(COCs) and DOs(CCs) after 24 h incubation were significantly different from each other (p < or = 0.05), the rate of DOs(CCs) was similar to that of DOs (p > or = 0.05). The cleavage rates in the first two groups were significantly higher than those of the others (p < 0.05). For oocytes cultured for 30 h, the nuclear maturation rates were significantly different for each culture model (p < 0.05). The cleavage rates in first two groups were significantly higher than those of others (p < 0.05). Both the nuclear and cleavage rates significantly increased when the culture time of DOs(COCs) was prolonged from 24 to 30 h. DOs(CCs) nuclear maturation was significantly improved when the culture time was prolonged from 24 to 30 h, but the ooplasmic maturation was not. Few NOs incubated with or without CCs accomplished nuclear maturation (approximately 2% both), even when the culture time was prolonged from 24 to 30 h. The oocyte degeneration rates were significantly different for each culture model after both 24 and 30 h incubation (p < or = 0.05). There was no significant difference in oocyte degeneration in the same groups between 24 and 30 h incubation (p > 0.05). The results suggest that rabbit CCs affect oocyte nuclear and ooplasmic maturation, and their survival. The prolongation of the culture time of rabbit oocyte from 24 to 30 h improves the nuclear and ooplasmic maturation differently in the present system. Rabbit oocytes free of CCs, especially NOs, show weak meiotic resumption potential and compromised viability, which cannot be improved by co-culture with dispersed CCs. The degeneration mostly happens at early time of IVM.

Effect of inhibition of synthesis of inducible nitric oxide synthase-derived nitric oxide by aminoguanidine on the in vitro maturation of oocyte–cumulus complexes of cattle

The aim of the present study was to investigate the effects of inhibition of the enzyme inducible nitric oxide synthase (iNOS) by aminoguanidine (AG) on the in vitro maturation of oocyte–cumulus cell complex(es) (COC) of cattle. COC were cultured with different concentrations of AG (0, 1, 10, and 100 mM) for 24 h. In Experiment 1, the extent of cumulus complex expansion, nuclear maturation status and plasma membrane integrity of oocytes and cumulus cells from each treatment were assessed. Nitrate/nitrite (NO 3 −/NO 2 −) concentrations were determined in culture medium by the Griess method. Addition of different concentrations of AG to maturation medium promoted a dose–response inhibitory effect on cumulus expansion ( P < 0.05). Addition of 1 and 10 mM AG to IVM medium did not affect plasma membrane integrity of oocytes or nuclear maturation rates ( P > 0.05), but it did reduce plasma membrane integrity in cumulus cells. One hundred millimolar inhibited pre-metaphase I (pre-MI) to metaphase II (MII) transition, promoted plasma membrane damage in oocytes ( P < 0.05), and increased NO 3 −/NO 2 − concentration when compared to controls ( P < 0.05). To evaluate if this effect was reversible, 10 −5 M sodium nitroprusside (SNP, NO donor) was added, only in the treatment with 100 mM AG that inhibited the nuclear maturation. However, association of 10 −5 M SNP to 100 mM AG did not reverse the effects of AG, but increased NO 3 −/NO 2 −concentration ( P < 0.05). In Experiment 2, the effect of different AG concentrations on cytoplasmic maturation in vitro was assessed based on cortical granule migration, and embryonic development. There was a dose effect on cortical granule migration rate, in which 1 mM AG (83.9 ± 6.2%) did not differ from control oocytes (83.6 ± 8.2%; P > 0.05), but 10 mM partially inhibited migration (3.8 ± 6.4%) and 100 mM totally inhibited migration ( P < 0.05). SNP (10 −5 M) did not revert this inhibitory effect on cortical granules migration in oocytes treated with 100 mM AG. Only those concentrations that did not inhibit IVM were used to assess cleavage and blastocyst development. Addition of 10 mM AG to IVM medium reduced (73.0 ± 8.1%, 15.0 ± 8.9%; P < 0.05) cleavage and blastocyst development, respectively when compared with controls (89.1 ± 3.4%, 37.6 ± 7.3%, respectively), but did not differ, ( P > 0.05), from the group treated with 1 mM AG (80.9 ± 8.4%, 41.5 ± 10.5%, respectively). The results from the present study demonstrate that NO derived from iNOS affects the in vitro maturation of bovine COC, modulating the viability of cumulus cells and of oocyte, the progression of meiosis after GVBD, the migration of cortical granules, and cleavage and blastocyst development.

Immunolocalization of the High‐Mobility Group N2 protein and acetylated histone H3K14 in early developing parthenogenetic bovine embryos derived from oocytes of high and low developmental competence

This study investigated differences in the distribution of acetylated histone H3 at Lysine 14 (H3K14ac) and the High-Mobility Group N2 (HMGN2) protein in the chromatin of early- (before 24 hr) and late-cleaved (after 24 hr) bovine embryos derived from small- (1-2 mm) and large-follicles (4-8 mm). The presence of HMGN2 and H3K14ac has been associated with different nuclear functions including chromatin condensation, transcription, DNA replication and repair. In vitro matured oocytes were parthenogenetically activated (PA) and cultured in synthetic oviduct fluid medium. Early- and late-cleaved embryos were fixed at 36, 50, 60, 70 and 80 hr after PA to detect the presence of H3K14ac and HMGN2. The rates of nuclear maturation (81.1% vs. 58.7%), early cleavage (46.9% vs. 38.9%), and development to blastocyst stage (34.3% vs. 18.9%) were higher (P < 0.05) in oocytes derived from large- compared to small follicles. The proportion of positively stained nuclei at 50 and 60 hr after PA was higher for both H3K14ac (27.2% vs. 4.8% and 64.3% vs. 30%) and HMGN2 (47% vs. 21.3% and 60.6% vs. 46%) in early versus late cleaved embryos derived from small- versus large-follicles, respectively. However, the rate of positive nuclei in early-cleaved embryos from small-versus large-follicles was similar for HMGN2 (87% vs. 93%) but lower for H3K14ac (51% vs. 64.4%) at 80 hr after PA. These data suggest that less developmentally competent embryos derived from small follicles had an altered chromatin remodeling process at the early stages of development compared to those derived from large follicles that are more competent to support development to blastocyst stage.

In vitro maturation, in vitro fertilization and embryonic development of canine oocytes

In this study we have investigated the efficiency of in vitro maturation (IVM) as a basic way to study the development of canine oocytes after in vitro fertilization (IVF). We decided, therefore, to perform two-part experiments. Firstly, experiment I compared the effects of TCM199 without fetal bovine serum (FBS) with TCM199 supplemented with 5% FBS on the in vitro nuclear maturation rate of canine oocytes. For the efficiency of meiotic development to the metaphase II (MII) stage, we found that 4.7% (4/64) of all oocytes grown in TCM199 without FBS developed to the MII stage compared with only 1.7% (1/59) of those grown in TCM199 with 5% FBS for 48 h. Therefore, FBS did not increase in vitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes used for IVF was investigated following heparin treatment. Canine oocytes were fertilized in four groups: Fert-TALP medium without heparin (Fert I) or Fert-TALP medium supplemented with 10, 20 or 30 microg/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes that were grown for 24 h in Fert I following fertilization showed the highest rate of all of the groups, 6.5% (5/77) and developed to the early morula stage. Markedly, the oocytes cultured in Fert I for 24 h following insemination had a higher rate of embryonic development than other groups. We can assert that, unlike findings in other mammals, heparin treatment in canine IVF does not increase the efficiency of the fertilization rate and is therefore not an important factor.

Effect of Follicle Stimulation Hormone and Luteinizing Hormone on Cumulus Cell Expansion and In Vitro Nuclear Maturation of Canine Oocytes

In general, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in the regulation of cumulus cell expansion and oocyte maturation. We investigated the effects of supplementation of FSH or LH in in vitro maturation (IVM) medium on the incidence of cumulus cell expansion and nuclear maturation in canine oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM-199 supplemented with 10% foetal bovine serum (FBS), 1 mg/ml cysteine, 0.2 mm pyruvic acid and different concentrations of FSH or LH (control, 0.5, 5 or 50 microg/ml) at 38.5 degrees C, 5% CO(2) in air for 72 h. The cumulus cell expansion was measured by microscopic visualization, and nuclear maturation of denuded oocytes was determined by staining with 10 microg/ml Hoechst33342 for 30 min. The cumulus cell expansion in the 5 microg/ml FSH group (397.2 +/- 64.3 microm) was significantly higher than those in the control, 0.5, and 50 microg/ml FSH groups (168.3 +/- 19.1, 286.0 +/- 69.7 and 300.0 +/- 84.3 microm, respectively; p < 0.05). However, there was no difference in cumulus cell expansion among the control, 0.5, 5 and 50 microg/ml LH groups (165.6 +/- 20.2, 160 +/- 26.5, 172 +/- 20.5 and 168 +/- 23.1 microm, respectively; p > 0.05). After 72 h of IVM, the proportion of nuclear development to the MI-MII stage in the 0.5 microg/ml FSH group (15.1%) was higher than those in the control, 0.5 and 50 microg/ml FSH groups (0.9%, 6.5% and 8.0%, respectively; p < 0.05). However, there was no significant difference in nuclear maturation to the MI-MII stage among control, 0.5, 5 and 50 microg/ml LH groups (4.6%, 2.3%, 5.4% and 8.6%, respectively; p > 0.05). This study indicated that a FSH supplement in IVM medium can increase cumulus cell expansion and nuclear maturation, while the nuclear maturation rate remained low. Further studies are required to improve the nuclear development to the MI-MII stages in canine oocytes.

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 degrees C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB-, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 microM BCB. There were fewer BCB+ oocytes after exposure to 13 microM BCB (10%) than after exposure to 26 or 39 microM BCB (57.2 and 61.8%; P<0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4+/-4.2 microm; mean+/-S.E.M.) was higher (P<0.05) than that of BCB- oocytes (136.8+/-4.6 microm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 microM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P<0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB- (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P<0.05); blastocyst development was lowest in BCB- oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.

EFFECT OF SEASON AND OVARIAN STATUS ON IN VITRO NUCLEAR MATURATION OF DOMESTIC CAT OOCYTES

The aim of this experiment was to determine the percentage of oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - and their in vitro nuclear maturation (IVM) rate during two different reproductive seasons experienced by cats in southeast of Brazil – non breeding season (NBS), comprehending January, February and March; and breeding season (BS), which includes the months of August, September and October. Thirty queens were anesthetized and neutering was performed. Ovaries were classified according to their status and were sliced in PBS supplemented with streptomycin, gentamicin and amphotericin B for cumulus oocyte complex (COC) releasing. Only COC presenting dark and uniform ooplasm surrounded by at least four layers of cumulus cells were selected. Grade I COC were washed three times in H-MEM supplemented with 3 mg/mL BSA, 2.0 mM glutamine, 1.0 mM sodium pyruvate, 0.13 mM cystein, 100 mg/mL streptomycin and 100 UI/mL penicillin. Oocytes were incubated in groups of 20–30 in 400 μL of DMEM supplemented with 10 μg/mL FSH, 1 μg/mL LH, 1 μg/mL estradiol, 20 ng/mL IGF-I and 10 ng/mL bFGF (basic fibroblast growth factor) under mineral oil for 30 or 36 hours at 38°C in humidified environment of 5% de O2, 5% CO2 and 90% N2. After 30 to 36 hours, cumulus cells were removed mechanically. After being denuded, COC were stained with Hoechst 33342 and examined under fluorescent microscope (Leica, Wetzlar, Germany). According to nuclear configuration, oocytes were classified as GV (germinal vesicle), GVBD (germinal vesicle breakdown), MI (metaphase I), MII (metaphase II) and D/NI (degenerated/non identifiable). Statistic differences between groups were analysed through ANOVA. During NBS, twenty pairs of ovaries were used. However, during BS 10 pairs were enough to obtain a similar number of oocytes (473 x 445 respectively for NBS and BS). Ten inactive, 08 luteal and 02 follicular ovaries were used during NBS. During BS, 07 inactive, 03 luteal and 01 follicular ovaries were used. The oocyte mean number/female during NBS was 23.65, while during BS, this number increased to 48.20/female. Queens presenting follicular ovarian status provided a low number of oocyte/ovary, however, most of them were classified as in grade I. In the BS 2.8% of oocytes from developed to a morula-like structure through parthenogenesis independently of the ovarian status. During NBS the following rates of GV, VGBD, MI, MII and degenerated oocytes were detected: inactive; 1.46%; 14.08%; 26.7%; 45.14% and 12.62%; luteal; 2.35%; 26.47%; 17.06%; 42.36% and 11.76%, and finally in follicular condition; 2.9%; 18.84%; 20.29%; 46.38% and 11.59%. During BS it was possible to verify the following values: inactive; 2.6%; 17.11%; 24.53%; 45.72% and 10.04%; luteal; 3.45%; 24.14; 14.48%; 46.21 and 11.72% and follicular; 2.27%; 15.92%; 22.73; 50% and 9.09%. The results of this experiment demonstrate that queens neutered in the BS provide a larger number of oocytes than those neutered in the NBS. There is also an indicative that the NBS oocytes have a poor quality compared to those from BS. No statistic difference was verified in the proportion of oocytes in MII between the two reproductive seasons. Financial support: FAPESP (platform)