Introduction
Attempts at producing transgenic pigs by means of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) or pronuclear injection require considerable amounts of MII/mature oocytes or zygotes at the time of microinjection, therefore the effective use of ovaries from slaughterhouse females by means of in vitro techniques is crucial. Micromanipulation of oocytes is time consuming during ICSI or NT experiments, however the ‘time frame’ to manipulate oocytes without a drop in efficiency is narrow, due to the use of both not completely matured and/or aged MII oocytes. The nuclear status and morphology of porcine MII oocytes and the extent of oocyte aging cannot be visually (without specific staining) distinguished during maturation culture for 36–72 h (Kikuchi et al., Reference Kikuchi, Naito, Noguchi, Kaneko and Tojo2002). However, cytoplasmic changes affecting oocyte quality seem to occur when the arrest period is prolonged. These quality changes, occurring in oocytes arrested at MII during prolonged culture, are called ‘aging’ and those oocytes that have aged but not degenerated are called ‘aged oocytes’ (Kikuchi et al., Reference Kikuchi, Naito, Noguchi, Kaneko and Tojo2002). Zygotes from aged oocytes are reported to show limited developmental success and undergo programmed cell death or apoptosis (reviewed by Fissore et al., Reference Fissore, Kurokawa, Knott, Zhang and Smyth2002). Aged oocytes exhibit abnormal morphological characteristics before fertilization, such as the disappearance of the microfilament-rich area over the meiotic spindle, disruption of and abnormal spindle location and chromatin disorganization (Webb et al., Reference Webb, Howlett and Maro1986). Mitochondrial dysfunction is also likely to play an important role in the predisposition of aged oocytes to spontaneous and fertilization- or activation-induced fragmentation (reviewed by Fissore et al., Reference Fissore, Kurokawa, Knott, Zhang and Smyth2002). Moreover, Holker et al. (Reference Hölker, Petersen, Hassel, Kues, Lemme, Lucas-Hahn and Niemann2005) indicated that only a narrow time window (4 h) is compatible with higher developmental rates in porcine NT.
Roscovitine can be applied to allow prolonged and more flexible maturation schedules in the laboratory (Coy et al., Reference Coy, Romar, Ruiz, Cánovas, Gadea, García Vázquez and Matás2005), and is one of the meiotic inhibitors more commonly used in the in vitro production (IVP) of pig embryos, and its effect on the resumption of meiosis has been shown previously in pigs (Ju et al., Reference Ju, Tsay and Ruan2003; Coy et al., Reference Coy, Romar, Ruiz, Cánovas, Gadea, García Vázquez and Matás2005; Garcia-Rosello et al., Reference Garcia-Rosello, Coy, Vázquez, Ruiz and Matas2006; Romar & Funahashi, Reference Romar and Funahashi2006). Piglets have been obtained derived from oocytes that have been artificially arrested with roscovitine during the 24 h before in vitro maturation and fertilization (Coy et al., Reference Coy, Romar, Ruiz, Cánovas, Gadea, García Vázquez and Matás2005), suggesting that this substance did not impair the viability of treated embryos. In pigs, the times of roscovitine treatments assayed in the literature varied from 22–48 h (Krischek & Meinecke, Reference Krischek and Meinecke2001; Coy et al., Reference Coy, Romar, Ruiz, Cánovas, Gadea, García Vázquez and Matás2005; Garcia-Rosello et al., Reference Garcia-Rosello, Coy, Vázquez, Ruiz and Matas2006; Romar & Funahashi, Reference Romar and Funahashi2006) and in no study was further embryo development impaired by roscovitine treatment, although expectations of improvement were not fulfilled.
On the other hand, large numbers of micromanipulated embryos (from ICSI or NT) are needed for embryo transfer to obtain satisfactory pregnancy and delivery results (reviewed by Garcia-Rosello et al., Reference Garcia-Rosello, García-Mengual, Coy, Alfonso and Silvestre2008). Therefore, a short roscovitine treatment might be an interesting option to extend the temporary window in which MII oocytes are supposed to be ready to be submitted to ICSI or NT per day. Despite the numerous papers published in recent years on IVP of pig embryos, there are no data assessing possible beneficial/detrimental effects of roscovitine treatment shorter than 22 h on pig oocytes and their further embryo development. Only a few studies with butyrolactone I or cycloheximide (CHX) treatment for 10–12 h to synchronize nuclear progression during meiotic maturation in porcine oocytes were found (Ye et al., Reference Ye, Flint, Campbell and Luck2002, Reference Ye, Campbell, Craigon and Luck2005; Marques et al., Reference Marques, Nicacio, de Oliveira, Nascimento, Caetano, Mendes, Mello, Milazzotto, Assumpcao and Visintin2007). Ye et al. (Reference Ye, Campbell, Craigon and Luck2005) observed an improvement in embryo developmental competence with a pretreatment of 12 h with CHX.
The aim of this work was to fit the time frame for manipulating pig oocytes to the whole labour day session in the laboratory, without a drop in efficiency, by using roscovitine treatment and longer IVM culture. Therefore, the objective was to study the effect of a short roscovitine pretreatment (5 h) and a prolonged IVM, to 50 h, on parthenogenetic and ICSI embryo development.
Materials and Methods
Culture media
Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich Química. Handling medium (HM199) was composed of medium 199 supplemented with 25 mM HEPES, 7.5% (v/v) heat-inactivated foetal calf serum (FCS; Invitrogen) and antibiotics. Maturation medium (MM199) consisted of Medium 199 supplemented with 0.1% polyvinyl alcohol, 0.57 mM cysteine and 10 ng/ml epidermal growth factor and antibiotics (Abeydeera et al., Reference Abeydeera, Wang, Cantley, Rieke, Murphy, Prather and Day2000) with hormonal modifications (0.1 IU/ml recombinant human FSH and recombinant LH; Silvestre et al., Reference Silvestre, Alfonso, García-Mengual, Salvador, Duque and Molina2007). The embryo culture medium (CM) was PZM-3 (Yoshioka et al., Reference Yoshioka, Suzuki, Tanaka, Anas and Iwamura2002) containing 75 μg/ml potassium penicillin G (P3032) and 50 μg/ml streptomycin sulphate (S6501). R-roscovitine (R7772) was solubilized in dimethyl sulphoxide (DMSO) before freezing at –20 °C as a 10 mM stock, as previously described, without observing problems in DMSO concentration (Romar & Funahashi, Reference Romar and Funahashi2006). Media with bicarbonate were equilibrated overnight in a humidified atmosphere containing 5% CO2 in air and at 38.5 °C, and manipulated covered with previously equilibrated mineral oil.
Preparation and culture of cumulus–oocyte complexes
Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported within 45 min to the laboratory in 0.9% (w/v) NaCl at 35 °C supplemented with antibiotics. Cumulus–oocyte complexes (COC) were obtained by antral follicle (3–7 mm diameter) aspiration with an 18-gauge needle connected to a 10 ml syringe and prefilled with HM199.
In vitro maturation and culture
Approximately 50–60 COC were cultured in 500 μl medium in a Nunc 4-well multidish (Nunc, Roskilde, Denmark) for 45 h in a static culture system. During the first 22 h of culture, COC were incubated in MM199 supplemented with hormones. Then, COC were washed twice and cultured in hormone-free MM199 for the remaining time, depending on the experimental design. The oocytes pretreated with roscovitine were cultured for 5 h in presence of 50 μM roscovitine in 2.5 ml of hormone-free MM199. After the treatment, COC were washed extensively twice in roscovitine-free MM199 and allocated in a conventional IVM system as described above. In vitro maturation and culture were performed at 38.5 °C in an atmosphere of 5% CO2, 5% O2.
After ICSI or electrical activation (EA), presumptive zygotes were washed twice in embryo CM and groups of approximately 20 zygotes were transferred to a Nunc 4-well multidish containing 500 μl of CM per well and incubated at 38.5 °C, in 5% CO2 and 5% O2 in air during 7 days. On day 5 of culture, each well of CM was supplemented with 2% FCS.
Intracytoplasmic sperm injection and parthenogenetic activation
Sperm was supplied by a commercial artificial insemination nucleus and stored at 17 °C for a maximum of 3 days. Semen sample was submitted to light centrifugation (50 g for 3 min) to eliminate foreign particles and dead cells. After the first washing, the cleanest fraction of semen was gathered and put through a second centrifugation (1250 g for 4 min). The pellet was re-suspended in HM199.
For ICSI, matured oocytes were washed and transferred to HM199 drops. Intracytoplasmic sperm injection was conducted on an inverted microscope with attached micromanipulators as previously described by Silvestre et al. (Reference Silvestre, Alfonso, García-Mengual, Salvador, Duque and Molina2007). Briefly, six to eight microdrops were placed in each lid surrounding central sperm drops (polyvinylpyrrolidone solution 10% (PVP360) + 1 μl of the sperm suspension). Microdrops were covered with mineral oil. One single sperm was immobilized by crushing the midpiece with the tip of the injection pipette. The immobilized sperm was aspirated tail first and microinjected into the ooplasm.
In order to obtain parthenogenetic embryos, MII oocytes were placed between two platinum wire electrodes (0.5 mm apart) and covered with Electropulsing Medium (EM) (pH 7.0; 280–300 mOsm) consisting of 0.3 M mannitol, 1.0 mM CaCl2·2H2O, 0.1 mM MgSO4·6H2O and 0.5 mM HEPES. MII oocytes were equilibrated in EM for 1 min before being placed in the microfusion chamber. The temperature was maintained at 38.5 °C throughout both procedures using a heated microscopic stage. The oocytes were then stimulated with one electrical set of two DC pulses (1-second interval) of 1.2 kV/cm for 30 μs delivered on a cell electroporator (Multiporator® Eppendorf). After activation, oocytes were washed twice and cultured in CM.
Assessment of nuclear status of oocytes and embryo development
For the assessment of nuclear status in Experiment 1 (design described below), before in vitro maturation, COC were gently pipetted through a small-bore pipette to strip ova. Oocytes were fixed and stained for 15 min in ethanol with bisbenzimide (Coy et al., Reference Coy, Romar, Ruiz, Cánovas, Gadea, García Vázquez and Matás2005; Marques et al., Reference Marques, Nicacio, de Oliveira, Nascimento, Caetano, Mendes, Mello, Milazzotto, Assumpcao and Visintin2007), and mounted on glass slides. Oocytes were examined under an epifluorescence microscope at ×200 and ×400 magnification and classified into two groups according to the morphological criteria for characterization of meiotic stages by Ye et al. (Reference Ye, Flint, Campbell and Luck2002) with minor modifications: group A: oocytes at GVI to GVII stage, where chromatin was stained and formed a shape of ring corresponding with the nucleolus; group B: oocytes at GVIII, GVIV and GVBD stages, where an irregular network of filamentous bivalents or a cluster of chromatin and no nucleolus was observed.
For the assessment of embryo development in Experiments 2 and 3 (design described below), after in vitro maturation, the COC were briefly placed in HM199 supplemented with 1 mg/ml bovine testes hyaluronidase and then gently pipetted through a small-bore pipette to strip ova. Oocytes were scored under stereoscopic vision for presence of the first polar body and therefore corresponded to MII oocytes. Regarding fertilization and embryo development, cleavage and blastocyst rate were evaluated respectively at 48 and 168 h after EA or ICSI under a stereomicroscope. In order to observe the cell nuclei, embryos developing to blastocyst stage were fixed and stained in absolute ethanol with bisbenzimide and cell nuclei were scored under an epifluorescence microscope.
Experimental design
Experiment 1: Nuclear status of pig oocytes after 5 h culture in roscovitine
This experiment was carried out to study whether roscovitine pretreatment in MM199 medium without FSH, LH for the first 5 h of maturation (5R group) kept the nuclear stage at the same level as found in oocytes just after recovery (before culture group: 0IVM). COC were divided in three groups:
• 0IVM (before culture group): COC were denuded, fixed and stained to evaluate nuclear status just after aspiration from follicles.
• 5R (roscovitine group): after recovering COC were cultured in presence of 50 μM of roscovitine for 5 h under IVM conditions.
• 5IVM (maturation group): COC were cultured for 5 h under IVM conditions in absence of roscovitine.
Experiment 2: Effect of different IVM systems on pig oocyte nuclear maturation and embryo development after electrical activation
We adopted a parthenogenetic model to determine the developmental potential of oocytes under different maturation conditions. To this end, MII oocytes from all groups were electrically activated.
The experimental design is schematically represented in Fig. 1. COC were divided into four different maturation groups:
• 45IVM (control group): COC were collected and matured for 45 h as previously described.
• 50IVM: COC were matured for 50 h as previously described.
• 5R + 40IVM: COC were cultured in 50 μM of roscovitine during the first 5 h in IVM medium without hormonal supplementation. Following this period, COC were washed twice in fresh MM199 and allowed to mature for 40 h.
• 5R + 45IVM: COC were cultured in 50 μM of roscovitine for the first 5 h in IVM medium without hormonal supplementation. Following this period, COC were washed twice in fresh MM199 and allowed to mature for 45 h under conditions as described above.
Figure 1 Experimental design of Experiment 2. Experimental groups: 45IVM and 50IVM treatments correspond to 45 and 50 h of in vitro maturation respectively; 5R + 40IVM and 5R + 45IVM correspond to 5 h of culture in MM199 with roscovitine before 40 and 45 h of maturation, respectively. MM199: maturation medium; EA: electrical activation.
Figure 2 Experimental design of Experiment 3. Experimental groups: 45IVM and 50IVM treatments correspond to 45 and 50 h of in vitro maturation respectively; 5R + 45IVM correspond to 5 h of culture in MM199 with roscovitine before 45 h of maturation. MM199: maturation medium; ICSI: intracytoplasmic sperm injection.
Experiment 3: Effect of different IVM systems on pig oocyte nuclear maturation and embryo development after ICSI
From the results obtained in Experiment 2, the better conditions were used to test the effect of roscovitine treatment on nuclear maturation and in vitro development of embryos obtained by ICSI. The experimental design is schematically represented in Fig. 2. COC were divided into three different maturation groups:
• 45IVM (control group)
• 5R + 45IVM
• 50IVM.
Matured COC from each group were submitted to ICSI as described above.
Statistical analysis
At least four replicates were performed per treatment. Nuclear stage (groups A and B) in Experiment 1, and nuclear maturation, cleavage and blastocyst rates in Experiments 2 and 3, were analysed by a chi-squared test analysis. When a single degree of freedom was involved, the Yates' correction for continuity was carried out. Results of nuclei cells were analysed by the ANOVA test.
Table 1 Nuclear status of pig oocytes after 0 h (0IVM) and 5 h of culture in maturation medium (5IVM) and 5 h in medium with 50 μM roscovitine (5R)
Group A (GVI–GVII), group B (GVIII–GVBD).
COC: cumulus–oocyte complexes; GV: germinal vesicle; GVBD: germinal vesicle breakdown.
Different superscripts in the same column show significant differences (chi-squared test, p < 0.05).
Table 2 Effect of different IVM systems on pig oocyte nuclear maturation and embryo development after electrical activation
1 45IVM and 50IVM treatments correspond to 45 and 50 h of in vitro maturation in MM199, respectively; 5R + 40IVM and 5R + 45IVM correspond to 5 h of culture in MM199 with 50 μmol/l roscovitine before 40 and 45 h of maturation, respectively.
2 Blastocyst rate from cleaved embryos.
Different superscripts in the same column show significant differences (chi-squared test, p < 0.05; ANOVA, p < 0.05).
Results
Nuclear status of oocytes after 5 h culture in roscovitine
Oocytes pretreated with roscovitine (5R) showed similar nuclear stage to non-roscovitine treated oocytes (0IVM), and significantly higher percentage of oocytes in group A (GVI-GVII) than oocytes cultured for 5 h in permissive IVM conditions (5IVM; Table 1). 5IVM group showed higher rate of oocytes in group B (GVIII-GVBD stage) than 0IVM and 5R groups (p < 0.05).
Effect of different IVM systems on pig oocyte nuclear maturation and embryo development after electrical activation
Results of the effect of the different IVM systems tested on embryo development after EA are shown in Table 2. When COC were cultured under the 5R + 40IVM system, nuclear maturation and cleavage rates were significantly lower than when COC were cultured under the 45IVM, 50IVM and 5R + 45IVM culture systems (54.2% vs. 72.6–76.8%, p < 0.05 and 58.8%; 81.4–88.3%, p < 0.05, respectively). However, this difference did not reach statistical significance in blastocyst rate. Regarding embryo quality, 5R + 40IVM group presented the lowest number of cells per blastocyst (p < 0.05). No differences were observed in MII, cleavage, blastocyst rates or in blastocyst cell number among 45IVM, 50IVM and 5R + 45IVM experimental groups.
Effect of different IVM systems on pig oocyte nuclear maturation and embryo development after ICSI
Results of the effect of the different in vitro maturation treatments tested on embryo development after ICSI are shown in Table 3. No significant differences were observed in nuclear maturation, cleavage and blastocyst rates among experimental groups (blastocyst rates ranged from 5.9% to 12.0%). Regarding embryo quality, no differences were observed in blastocyst cell number among groups (ranging from 21.5 to 25.0).
Table 3 Effect of different IVM systems on pig oocyte nuclear maturation and embryo development after ICSI
1 45IVM and 50IVM treatments correspond to 45 and 50 h of in vitro maturation, respectively; 5R + 45IVM corresponds to 5 h of culture in MM199 with 50 μmol/l roscovitine before 45 h of maturation.
2 Blastocyst rate from cleaved embryos.
Discussion
In the present work, we approached the ‘time frame’ to manipulate oocytes, mainly for ICSI, in order to optimize the laboratory resources (i.e. to obtain a sufficient number of manipulated embryos to be transferred to recipient female during a single day). One approach to extend the ‘time frame’ to manipulate oocytes is to make a longer culture period, by means of the reversible inhibition of oocyte meiosis using roscovitine. The effect of roscovitine on the maintenance of oocyte meiotic arrest has been assessed previously in pigs using different concentrations, inhibitory periods and sources of oocytes (Krischek & Meinecke, Reference Krischek and Meinecke2001; Marchal et al., Reference Marchal, Tomanek, Terqui and Mermillod2001; Ju et al., Reference Ju, Tsay and Ruan2003; Schoevers et al., Reference Schoevers, Bevers, Roelen and Colenbrander2005). Roscovitine is a selective cdc2 inhibitor, which has been reported to arrest cells in late G1 and at G2/M cell cycle transition (Krischek & Meinecke, Reference Krischek and Meinecke2001; Marchal et al., Reference Marchal, Tomanek, Terqui and Mermillod2001), specifically interferes with MPF kinase activity by blocking the ATP binding pocket of CDK1 (De Azevedo et al., Reference De Azevedo, Leclerc, Meijer, Havlicek, Strnad and Kim1997) and as a consequence prevents chromosome condensation (Krischek & Meinecke, Reference Krischek and Meinecke2001) and cell division (Meijer & Kim, Reference Meijer and Kim1997).
In our first experiment, we studied the stage of the nucleus at the onset of culture and further progression of the nuclear stage during the maturation process. When nuclear stage of pig oocytes was analysed just after COC recovery, we observed a lower rate of GVI–GVII oocytes (group A: approx. 70%) than expected compared to other studies where this percentage ranged from 95 to 100% (Ye et al., Reference Ye, Flint, Campbell and Luck2002; Schoevers et al., Reference Schoevers, Bevers, Roelen and Colenbrander2005). However, in agreement with our results, Marques et al. (Reference Marques, Nicacio, de Oliveira, Nascimento, Caetano, Mendes, Mello, Milazzotto, Assumpcao and Visintin2007) observed that 54.3% of pig oocytes at 0 h of maturation remained at the GV stage. Moreover, Coy et al. (Reference Coy, Romar, Ruiz, Cánovas, Gadea, García Vázquez and Matás2005) obtained 86% of GV oocytes just after oocyte recovery. This divergence in the results could be because in the previous studies (Ye et al., Reference Ye, Flint, Campbell and Luck2002; Schoevers et al., Reference Schoevers, Bevers, Roelen and Colenbrander2005), no differentiation among the different porcine GV stages was made, and it is also possible that environmental conditions might affect the premature breakdown of the germinal vesicle, as suggested by Marques et al. (Reference Marques, Nicacio, de Oliveira, Nascimento, Caetano, Mendes, Mello, Milazzotto, Assumpcao and Visintin2007). In our study, approximately 20% of GVI–GVII oocytes at 0 h progressed to GVIII–GVBD after 5 h of culture, whereas in other studies, the progression of porcine oocytes to GVIV occurred after at least 14 h of culture (Wehrend & Meinecke, Reference Wehrend and Meinecke2001; Sasseville et al., Reference Sasseville, Côté, Guillemette and Richard2006), while other authors observed oocyte progression from the GV to GVBD stages (~30 %) in the first 10 h of in vitro culture (Marques et al., Reference Marques, Nicacio, de Oliveira, Nascimento, Caetano, Mendes, Mello, Milazzotto, Assumpcao and Visintin2007). Ye et al. (Reference Ye, Flint, Campbell and Luck2002) observed that upon removal of cycloheximide synchronization treatment (CHX), the first population of oocytes underwent GVBD at 6 h (28.6%). These latter authors indicated that meiotic progression in oocytes cultured in vitro commonly varies not only among laboratories but also among batches of oocytes. The oocyte nuclear progression observed in the present study was arrested with the applied roscovitine treatment. This reversible arrest with a short treatment (5–12 h) has also been observed using other meiotic inhibitors (Ye et al., Reference Ye, Flint, Campbell and Luck2002; Marques et al., Reference Marques, Nicacio, de Oliveira, Nascimento, Caetano, Mendes, Mello, Milazzotto, Assumpcao and Visintin2007).
We also studied the effect of different IVM systems with or without roscovitine pretreatment on parthenogenetic embryo development. The parthenogenote model has the advantage that a higher amount of electrically activated embryos can be obtained in a shorter period compared to ICSI models, and avoids the confusing variation due to male sperm factor influence (Gupta et al., Reference Gupta, Uhm and Lee2008). The meiotic inhibition induced by roscovitine was fully reversible, since the most of the oocytes reached the MII stage after removal of inhibitor and an additional culture period of 45 h in permissive conditions. On the other hand, with our IVM system (MM199-based media); the 5R + 40IVM experimental group presented significantly lower MII oocyte rates than the other groups after EA, which may be due to a lower time exposure to IVM. Kikuchi et al. (Reference Kikuchi, Naito, Noguchi, Shimada, Kaneko, Yamashita, Aoki, Tojo and Toyoda2000) observed that MII oocytes were first seen after 30 h of culture, and the maturation rate increased to 55% at 36 h; thereafter, the rate was almost constant, at about 65%, until 72 h of culture (Kikuchi et al., Reference Kikuchi, Naito, Noguchi, Shimada, Kaneko, Yamashita, Aoki, Tojo and Toyoda2000). In contrast with our results, other groups observed that less than 45 h in maturation medium was sufficient to obtain a high rate of nuclear maturation in pig oocytes (Grupen et al., Reference Grupen, Nagashima and Nottle1997; Sasseville et al., Reference Sasseville, Côté, Vigneault, Guillemette and Richard2007).
On the other hand, under our conditions it does not seem that oocytes submitted to 50IVM treatment were too aged, since embryo development rates and quality were similar to control group (45IVM). Our data disagreed with other studies, in which porcine oocytes cultured in NCSU based maturation medium for 48 h showed lower parthenogenote blastocyst rates after EA than those cultured for 42 h (Ikeda & Takahashi, Reference Ikeda and Takahashi2001). These authors suggested that oocytes in NCSU based maturation medium reached MII stage slightly earlier than in MM199-based maturation medium. However, different maturation media, hormonal and other maturation supplements, or even the origin of oocytes could be among the reasons for these differences, as shown by Abeydeera et al. (Reference Abeydeera, Wang, Cantley, Prather and Day1998).
Since our main objective is ICSI embryo production, we proceeded to test the IVM systems previously studied with parthenogenote models on ICSI embryos. In the present study, oocytes treated with roscovitine for 5 h (5R–45IVM) showed non-statistical significantly different developmental abilities than the other groups (45IVM, 50IVM). In our conditions, pig oocytes can be matured for 50 h without observing a significant drop in efficiency, although the number of blastocysts obtained was slightly low. Until now, only one reference has been found in the literature about pig oocytes that had been prematured 22 h with roscovitine and fertilized by ICSI (Garcia-Rosello et al., Reference Garcia-Rosello, Coy, Vázquez, Ruiz and Matas2006) without detecting any detrimental effect on fertilization rates. In this study, oocytes matured during 36 h (after 22 h roscovitine pretreatment) achieved a higher proportion of 2 pronuclei stage embryos than those matured with 44 h (Garcia-Rosello et al., Reference Garcia-Rosello, Coy, Vázquez, Ruiz and Matas2006), although no embryo development data was available. In conventional in vitro fertilization systems, a reduction of the oocyte maturation interval from 44 h to 36 h decreased the rate of polyspermic fertilization. However, a significant decrease in the blastocyst rate was also observed, suggesting that maturation for 44 h gives rise to a population of ‘aged’ oocytes that is susceptible to polyspermic fertilization (Grupen et al., Reference Grupen, Nagashima and Nottle1997). However, this problem is bypassed with ICSI.
In conclusion, under our experimental conditions and using parthenogenetic and ICSI embryos, we observed that it is feasible to prolong the ‘time frame’ to manipulate pig oocytes by at least 5 h without apparently loss of efficiency. As implication, these results allow us to fit the time frame to the whole labour day-session, and so optimize the laboratory resources for the production of porcine ICSI embryos.
Acknowledgements
This work was partially supported by Conselleria de Empresa, Universidad y Ciencia (GV05/212) and INIA (RTA2007–0110–00–00) FEDER and Fondo Social Europeo. E. García-Mengual was supported by a research grant from CAPA (GV). The authors would like to thank R. Romar for her critical reading and comments on the manuscript, N. Macowan for revising the English version of this manuscript and the Matadero Mercavalencia for their assistance in obtaining porcine ovaries.
