Effect of number of oocytes and embryos on in vitro oocyte maturation , fertilization and embryo development in bovine

The aim of this study was to identify the in vitro development stage at which the culture of a single or low number (n = 5 or 10) of oocytes/embryos could impair development in comparison with culture in group (n = 50). In the Experiment 1, it was confirmed that single in vitro embryo production yielded lower cleavage and blastocyst rates than in group (49.4 vs. 83.0%; 0% vs. 37.8%, respectively; p < 0.05). In Experiment 2 and 3, it was observed no effect on embryo development of culturing single or low number of oocytes during maturation and fertilization, respectively. In Experiment 4, it was observed a detrimental effect on blastocyst rate when cultured single or low number of embryos during post-fertilization in vitro culture (2.9; 10.2-10.8; 33.2% in single, low number of embryos (5-10), and controlgrouped, respectively; p < 0.05). In Experiment 5, it was observed that the last part of the culture period (day 3 onwards) seemed to be more affected by the low number of embryos placed in culture. In conclusion, post-fertilization culture, especially on days 3 to 7 after fertilization, seems to be the most important stage for embryo development on single and/or low number (5-10) of embryos culture. Additional key words: single/low embryo culture.


Introduction
The in vitro production (IVP) of bovine embryos using single or low numbers of oocytes and embryos during culture is increasing among researchers linked to the production of embryos from oocytes collected by ovum pick-up (OPU) (see Carolan et al., 1996;Vajta et al., 2000Vajta et al., , 2008;;Ward et al., 2000;Goovaerts et al., 2009).
For experimental purposes in bovine, in vitro embryo production from oocytes collected from abattoir ovaries is in general quite efficient when a high number of oocytes are cultured together, although the quality of these IVP embryos continually lags behind that of embryos produced in vivo (Lonergan et al., 2003).These in vitro systems usually culture approximately 40-50 oocytes/embryos in 400-500 µL of medium, obtaining blastocyst rates around 30-40% (Holm et al., 1999;Gordon, 2003).However, for commercial purposes such as use in OPU, where it is usually necessary just to keep the immature oocytes from one donor together, a small number of oocytes/embryos can be cultured (approximately three to six oocytes recovered per nonstimulated cow; Rizos et al., 2005;Chaubal et al., 2006).It has been widely reported that in vitro embryo development in bovine and other mammalian species tends to be suppressed in cultures with a single or low number of embryos (in mouse: Paria and Dey, 1990;Canseco et al., 1992;Lane and Gardner, 1992;Kato and Tsunoda, 1994;in bovine: Palma et al., 1992;Ferry et al., 1994;Keefer et al., 1994;Blondin and Sirard, 1995;Carolan et al., 1996;Donnay et al., 1997;O'Doherty et al., 1997;Ward et al., 2000;Oyamada and Fukui, 2004;Pereira et al., 2005;Fujita et al., 2006).It seems that the culture systems developed to culture embryos in groups could be unsuitable and/or incomplete for individual embryo culture, and there must be some kind of limiting factor or condition in single embryo in vitro culture systems which remain still without knowing (Nagao et al., 2008).
Even though several reports have addressed single embryo IVP with varying levels of success (Carolan et al., 1996;Hagemann et al., 1998;Oyamada et al., 2004), there is still lack of repeatability and high variability in the results among laboratories.
The aim of this work was to identify the stage of the in vitro process at which single and low number of embryo development could be impaired, in order to be able to optimise and develop an effective bovine oocyte culture system.To this end, it was studied the effect of single embryo and/or small number embryo culture during the whole in vitro process (maturation, fertilization and culture), and also in each process separately, on the bovine embryo development and quality.

Sperm preparation, in vitro fertilization and artificial activation
Two straws with 0.25 mL of frozen semen from two bulls were thawed at 37°C in a water bath for 1 min and centrifuged for 20 min at 700 × g through a Percoll gradient of 2 mL 90%, 2 mL 45% Percoll, in 15 mL centrifuge tubes.The Percoll 90 was made according to the protocol described by Parrish et al. (1995).To prepare the Percoll 45, the Percol 90 was mixed 1:1 with HM199.The sperm pellet was isolated and washed in 5 mL of HM199 by centrifugation at 350 × g for 5 min.Approximately 50 µL of semen pellet remained after the f inal centrifugation and was diluted with approximately 100 µL of HM199.Final concentration of 1 × 10 6 sperm mL -1 was used for in vitro fertilization (IVF).After IVM, COCs were washed three times in fertilization Fert-Talp medium (Parrish et al., 1988) with 10 μg mL -1 of heparin (Sigma H9399), and co-Single and low number in vitro embryo culture incubated with the spermatozoa for 18-20 h in 5% CO 2 at 38.5°C under mineral oil (Sigma M8410).
To study a complete effect on cytoplasmic maturation and to separate the sperm interaction, artificial activated oocytes were used.In order to obtain parthenogenetic embryos, in vitro matured oocytes were denuded of cumulus cells and oocytes with one visible polar body were considered as metaphase II (MII) oocytes.Then, the MII oocytes were activated by exposure to 5 mM ionomycin (Sigma I0634) for 5 min, washed twice, and incubated in 2 mM 6-dimethylaminopurine (DMAP; Sigma D2629) for 3 h.After that, 6-DMAP was washed off, and the oocytes were in vitro cultured.
After IVF or parthenogenetic activation, oocytes were washed three times in culture medium, and cultured at 38.5°C in a humidified atmosphere with 5% CO 2 and 5% O 2 , under mineral oil, for the in vitro culture (IVC).After IVF, oocytes were previously denuded from surrounding cumulus cells in HM199 before IVC.In Experiments 1 to 4, at day 5 (D5) of culture, IVC medium was supplemented with FBS to reach 10% concentration.In Experiment 5, this FBS supplementation was done at D3, when changing of volume culture conditions was done (see below in experimental design), in order to reduce handling of embryos.Cleavage and blastocyst formation rates were recorded at D2 (48 h post-fertilization), and at D7-8 of culture, respectively.Blastocysts were f ixed and stained in ethanol with 25 µg mL -1 of bisbenzimide (Hoechst 33342, Sigma B2261), and the total number of cells was counted under an epifluorescence microscope.

Experimental design
Experimental design is summarised in Table 1.Experiment 1 was performed to study the effect of single

Statistical analysis
At least, three to six replicates were performed in each experiment.Results of maturation, cleavage, and blastocyst rates were analysed using the χ2 test.When a single degree of freedom was involved, the Yates' correction for continuity was carried out.Results of blastocyst cell number were analysed using analysis of variance (ANOVA).A probability of p < 0.05 was considered to be statistically different.

Results
In Experiment 1 (Table 2), it was observed that when the whole embryo production process is performed maintaining individually maturation, fertilization and culture (sIVP), a signif icantly lower cleavage and blastocyst rates are yielded compared to grouped culture (gIVP).
In Experiment 3 (Table 4), no significant difference was observed in cleavage, blastocyst rate or number of cells per blastocyst, between low number fertilization (r5IVF) and control-grouped fertilization (gIVF).
In Experiment 4 (Table 4), it was observed that embryos cultured in control-grouped (gIVC) conditions showed signif icant higher cleavage rate than those cultured single (sIVC) or in low number n = 5 (r5IVC) conditions (p < 0.05), although no difference reached statistical significance when compared to low number n = 10 (r10IVC) conditions.Regarding blastocyst yield, sIVC embryos showed the lowest blastocyst rates (2.9%; p < 0.05), whereas gIVC embryos yielded the highest blastocyst rates (33.2%;p < 0.05).No differences on number of cells per blastocyst were observed.
In Experiment 5 (Table 4), it was again observed that culturing embryos in r5IVC resulted in a significant lower cleavage than culturing in gIVC, and gIVC resulted in a higher blastocyst yield than any other group tested (p < 0.05).When variation of the culture conditions took place, embryos cultured under low number (n = 5) conditions from D1 to D3 and changed to control-grouped conditions D4 onwards (r5IVC + gIVC) showed higher blastocyst yield and number of cells per blastocyst, than when variation of the culture

Discussion
This work was planned to try to identify the main step of the in vitro process at which single or low number embryo development could be impaired.In Experiment 1, it was observed that when the embryo production process is performed maintaining individually maturation, fertilization and culture, a significant lower cleavage and blastocyst rate was obtained, as it was observed in other works (Keefer et al., 1994;Ward et al., 2000;Fujita et al., 2006).
Oocyte maturation environment has been demonstrated to be essential for high in vitro blastocyst yields (Sirard and Blondin, 1996;Jewgenow et al., 1999;Rizos et al., 2002).To investigate this, it was studied the effect of varying the number of oocytes placed in culture during in vitro maturation, but maintaining the ratio oocytes/volume of IVM medium, on their in vitro development of bovine embryos after IVF or parthenogenetic activation.Blastocyst yield after single, low number (n = 5-10) or control (n = 50) in vitro maturation was no significantly different.Results obtained here are in agreement with those reported by other authors (Carolan et al., 1996;Ward et al., 2000); however, it can be found some contradictory results in the bibliography, where it is also observed that single oocyte maturation could reduce the developmental capacity of oocytes compared with group maturation (Blondin and Sirard, 1995;Jewgenow et al., 1999) even maintaining the same oocyte/volume ratio (1 oocyte in 10 µL vs. 10 oocytes in 100 µL) (Feng et al., 2007).By other hand, it seems that developmental potential of oocytes following IVM, IVF and IVC could greatly depend on the presence and morphology of the surrounding cumulus cells and follicular environment (Araki et al., 1998;Gordon, 2003;Feng et al., 2007).Araki et al. (1998) observed that the tight layer of cumulus cells enhanced the developmental capacity of individual cultured oocytes, achieving higher rates of blastocyst when oocytes with homogeneous ooplasm and surrounding with more than four layers of cumulus cells were individually cultured through IVM to IVC, compared to that with two to three layers.Furthermore, Feng et al. (2007) showed that blastocyst rates of bovine oocytes were highly correlated with the degree of cumulus expansion.In the present work, oocytes with more than three layers of surrounding cumulus cells and homogeneous cytoplasm were used, so it could be thought that the high intrinsic quality of oocytes used could explain the fact that individual IVM did not impair blastocyst yield during in vitro fertilization and culture in group.
Regarding IVF process, it was observed that when five oocytes were fertilized in a drop of 50 µL (r5IVF), no significant effect was observed either in cleavage rate, blastocyst yield or blastocyst cells number com-pared with control-grouped fertilization when in the remaining steps (IVM, IVC) they were cultured in group.Although Carolan et al. (1996) observed that maintaining the control in-group oocyte/volume ratio for individual in vitro fertilization (1 embryo in 10 µL) seriously compromised subsequent embryo development, these authors observed that individual oocytes could be successfully fertilized by increasing the volume of IVF to 50-100 µL, with no differences in terms of embryo development compared to in-group.
In the conditions of the present work, the postfertilization culture conditions seem to be the most important step in the whole process of in vitro embryo production concerning to number of oocytes/embryos.This effect has also been observed by Ward et al. (2000), but furthermore, in the present work it was detected that the last part of embryo culture, D3 to D7, is the stage when is more noticeable the impairment of small number of embryos in culture on blastocyst formation.
It was observed that culture of embryos in controlgrouped conditions resulted in a higher cleavage and blastocyst rate than individual or low embryo number (n < 10) culture conditions.Single embryo culture results obtained here are very low, but this level is also observed in other works culturing embryos individually (sIVC), and even regrouped for the remaining steps (gIVM, gIVF) (Carolan et al., 1996;Ward et al., 2000;Goovaerts et al., 2009).It is suggested in several animal species (mice, bovine, porcine) that suppression of early development of embryos cultured individually might be caused by a deficiency of cooperative interaction among embryos (Paria and Dey, 1990;Keefer et al., 1994;Stokes et al., 2005).It seems that several growth factors could act as possible embryotrophic factors reciprocally stimulating embryo development, in a paracrine/autocrine fashion, when embryos are cultured in vitro in groups (Paria and Dey, 1990;Palma and Brem, 1995;Thibodeaux et al., 1995;Lim and Hansel, 1996;Stokes et al., 2005;Contramaestre et al., 2008).In addition, Nagao et al. (2008) concluded that the cooperative interaction among bovine early embryos during in vitro culture may be also mediated by the reduction of toxic factors, so at low embryo density, reduced oxygen tension or the exclusion of inorganic phosphate from the medium enhances blastocyst development.Moreover, volume of medium also seems to be an important factor for individual embryo culture.Very small volumes (< 10 µL) may accumulate toxic metabolites (Lane and Gardner, 1992;Carolan et al., Single and low number in vitro embryo culture 1996), in contrast, larger volumes of medium or renewal of the media may dilute, or replace, autocrine embryotrophic factors (Paria and Dey, 1990).However, the mechanisms negatively affecting in vitro individual incubation of bovine embryos are still not understood in depth, as there is still high variability in the results among laboratories.The lower limit number of embryos from which reduced efficiency appears is not conclusive, and the comparison with other works is sometimes not easy since, apart from different culture conditions, the ratio oocyte/volume of medium may be changed even in the same work.In the present work, significant differences were observed when 10 embryos in 100 µL (1/10 oocyte µL -1 ) were in vitro cultured.In this sense, Hoelker et al. (2009) detected significant differences in embryo development with 16 embryos (1/32 oocyte µL -1 ) in comparison with the control group (n = 50, 1/10 oocyte µL -1 ).However, other authors (Fujita et al., 2006) detected differences only when a lower number of embryos (n = 3, 1/5 oocyte µL -1 ) were cultured.
Some studies have addressed the use of conditioned medium to investigate the potential beneficial effect of the growth factors secreted in the media, by embryos cultured in groups or cell-coculture, on the promotion of single embryo development (Fujita et al., 2006;Goovaerts et al., 2009).However, to our knowledge, not in the same way as tested in the present work, in which possible depletion renewal or dilution of media was avoided, and only the effect of number of embryos could be studied.Here, it was tried to investigate the effect of changing from low number embryo culture to grouped embryo culture and vice-versa using the same conditioned media in which embryos were previously cultured, and maintaining the control embryo/volume of medium ratio.It was observed that the most deleterious change for embryo development, in terms of blastocyst formation, is when group-cultured embryos are placed in low number embryo culture conditions at D3 and onwards.From the results obtained here, it could be summarised that the last part of embryo culture is likely to be a crucial step when deficiency of cooperative interaction among embryos could arise, impairing blastocyst formation.Although the comparison with porcine embryo development should be cautiously considered, since pig are multiparous while bovine is monoparous, in porcine embryos, it was observed that the beneficial effect of grouped embryo culture, at an optimum distance of 81-160 µm separation among embryos, rose at 48-96 h after insemination and became more evident towards the last part of the culture period, for 96-144 h which corresponded to day 5-6 of culture (Stokes et al., 2005).These latter authors suggested that group culture confers a greater advantage on development post genome activation, and even more so at the morula and blastocyst stage.In bovine embryos, two stages seem to be crucial for embryo development: 8-to 16-cell stage, at days 2-3 of culture after fertilization, in which developmental arrest could occur in the called "8-cell block" (Eyestone and First, 1991); and compaction and cavitation for blastocyst formation from morula, which occurs around the 32cell stage approximately after day 5 of in vitro culture (Van Soom et al., 1997).It was shown in the literature that a serum supplementation to the culture medium at day 5 after fertilization increased the proportion of oocytes that developed to blastocyst (De Moraes and Hansen, 1997;Paula-Lopes et al., 1998;Hagemann et al., 1998), and as it is widely known, growth factors, hormones and other active substances are present in FBS (Gordon, 2003).Taking these data together, it seems likely that some important autocrine/paracrine growth factor(s) and/or other components secreted to the culture media by the embryos could become more important in later stages of culture.
It could be concluded that, in the conditions tested here, post-fertilization culture conditions, regarding number of embryos in culture, seem to be the most important step determining blastocyst yield.Culture of single and/or a low number of embryos (< 10 embryos) drastically impaired the efficiency of blastocyst yield.Moreover, it seems that the final part of the culture (day 3 to day 7) could be crucial for the manifestation of this deleterious effect with a low number of embryos.More research is needed to elucidate the factors negatively affecting individual and low number embryo in vitro culture, and to overcome its lower efficiency.

Table 1 .
Experimental design

Table 2 .
Single and low number in vitro embryo culture 747 Single in vitro bovine embryo production (individual maturation, fertilization and culture) vs. group in vitro embryo production (Experiment 1)

Table 3 .
Effect of different in vitro maturation (IVM) conditions, varying the number of oocytes, on in vitro development of (a) in vitro fertilized bovine embryos (Experiment 2a) and (b) parthenogenetic bovine embryos (Experiment 2b)

Table 4 .
Effect of different culture conditions on in vitro development of bovine embryos i) varying the number of oocytes in fertilization (IVF) (Experiment 3), or ii) varying the number of zygotes in culture (IVC) (Experiment 4), and iii) varying the culture ratio using conditioned medium in IVC(Experiment 5)