Production of Bovine Hand-Made Cloned Embryos by Zygote–Oocyte Cytoplasmic Hemi-complementation

Abstract

The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; hand-made cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote–oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

Introduction

Mammalian cloned embryos have been shown to develop faulty epigenetic configurations at a high and unpredictable level after cloning by somatic cell nuclear transfer (SCNT), usually associated with abnormal phenotypes in the course of development (Beaujean et al., 2004; Bourc'his et al., 2001; Dean et al., 2001; Kang et al., 2001; Reik et al., 1993; Wells, 2010; Xue et al., 2002). Thus, temporal and spatial failures at gene expression level and embryonic development are probably caused by inadequate epigenomic reprogramming during and after the SCNT procedures per se. The recipient cytoplast plays a key role on nuclear reprogramming, but the ooplasmic components responsible for nuclear reprogramming after fertilization appear not to be sufficient to modify the differentiation marks from most somatic cells after SCNT (Bird, 2002; Giraldo et al., 2008, 2009; Wells, 2010), a process also known in cloning as “erasure of somatic cell differentiation status” (Cezar, 2003). Many factors intrinsic to the cytoplast (e.g., activation status, stage of the cell cycle, cytoplasmic maturation, and volume) and to the karyoplast (e.g., donor cell differentiation, stage of the cell cycle, genotype, and epigenotype) are known to play critical roles in the chromatin remodeling and nuclear reprogramming events after cloning (Bordignon and Smith, 2006; Fulka et al., 1996; Wells, 2010). In fact, MII cytoplasts are regarded as the most appropriate cytoplast type for cloning, despite the cell cycle asynchrony with the somatic cell, as they are postulated to contain chromatin remodeling properties that are necessary for reprogramming somatic cell nuclei (Fulka et al., 1996), mainly when cells are arrested at G0/G1 of the cell cycle (Bordignon and Smith, 2006). Recent advances in chromatin therapy (Egli et al., 2007; Giraldo et al., 2009; Wells, 2010) and induced pluripotent stem (iPS) cells (Takahashi and Yamanaka, 2006; Zhou et al., 2009), for instance, are technologies that promise to aid the identification of key cellular factors required for proper epigenetic reprogramming of cells.

The functional and molecular synchrony between cytoplast and karyoplast is an additional important factor on SCNT, because this synchrony between the donor nucleus and the ooplasmic components might lead to an inadequate or proper genomic reprogramming after cloning (Wells, 2010). For instance, the cytoplasmic mosaicism, that is, differences in cytoplasmic components and their intracellular compartmentalization (Wolpert, 1971), and level of mitochondrial heteroplasmy caused by fusion of distinct cytoplasms during cloning, usually originated from enucleated MII oocytes (cytoplast) and somatic cells (karyoplast), may either be detrimental or promote the developmental capacity of reconstructed embryos (Liu and Keefe, 2000; Alberio et al., 2001). Schurmann et al. (2006) demonstrated that the use of one-cell stage IVF-derived embryos as cytoplasts for SCNT cloning, enucleated 4 h after the onset of IVF, resulted in similar in vitro development as embryos produced with enucleated MII oocytes. However, in vivo development of such cloned blastocysts, reconstructed with IVF-derived cytoplasts, was significantly higher, manifested by higher pregnancy and calving rates. As the use of a more physiological strategy to activate oocytes has been reported in cattle (Schurmann et al., 2006) and horses (Hinrichs et al., 2006), with sperm-mediated activation considered beneficial for epigenetic reprogramming, perhaps a better cell cycle synchrony or favorable cytoplasmic mosaicism between cytoplast and karyoplast may have played important roles in improving development. This clearly demonstrates the differences with respect to the epigenetic reprogramming capacity between karyoplasts and cytoplasts, in the process of cloning by SCNT. Collectively, such phenomena are examples of the wide spectrum of biological events intertwining during the cloning procedure, which demonstrate the complexity of the cell and molecular processes that still need to be elucidated in biology, for which experiments in cloning are very useful.

Studies on in vitro development of bovine cloned embryos comparing the effect of embryo reconstruction with somatic cells (usually at the G0/G1 phases of the cell cycle) fused to single cytoplasts or a combination of cytoplasts at distinct cell cycle phases and/or activation status (hemi-oocytes at metaphase, hemi-zygotes at early postfertilization stages, previously activated by the sperm) require further systematic investigation. For that purpose, the hand-made cloning (HMC) procedure is an interesting model for studies in cell and developmental biology. The cytoplasmic mosaicism and heteroplasmy caused by the fusion of cytoplasts from distinct oocytes after cloning by HMC might affect in vitro embryo developmental capacity either positively or negatively (Vajta et al., 2005). When proven positive, complementation of cytoplasmic factors by fusing distinct cytoplasts has been shown to cause an increase in total embryo cell number (Giraldo et al., 2008; Tecirlioglu et al., 2005) or to rescue, in part, pronuclei from oxidative stress when in a healthy cytoplasm (Liu and Keefe, 2000). Alternatively, cloning efficiency may fall due to the negative effect caused by biologically compromised cytoplasts (Vajta et al., 2005) or perhaps due to an incomplete cytoplasmic complementation. Also, the absence of zona pellucida associated to the well-of-the-well (WOW) system provides an excellent tool for the study of embryo aggregation (Boiani et al., 2003; Misica-Turner et al., 2007; Ribeiro et al., 2009), cytoplasmic volume (Peura et al., 1998; Ribeiro et al., 2009; Wells, 2010), cell allocation (Misica-Turner et al., 2007), serial nuclear transfer (Wells, 2010), and also in studies on the effects of cytoplasmic hemi-complementation on development. As the use of IVF cytoplasts (preactivated by the sperm) appears to enhance embryo viability after SCNT (Schurmann et al., 2006), the aim of this study was to determine the effects of the oocyte-zygote hemi-complementation, using distinct types of cytoplasts (nonactivated enucleated MII hemi-oocytes, early pronuclear stage enucleated IVF-derived hemi-zygotes), and/or karyoplasts (somatic cells, nonactivated MII hemi-oocytes containing the metaphase plate, early pronuclear stage IVF-derived hemi-zygotes containing the sperm and egg chromatin) for embryo reconstruction. In addition, the effect of specific embryo activation processes (sperm-mediated or chemically induced) on in vitro development, cell density and cell allocation of bovine embryos cloned by HMC was examined.

Materials and Methods

All chemicals were from Sigma Chemical Co. (St. Louis, MO, USA), unless stated otherwise. The entire study was carried out in one laboratory, during a consecutive period of time, and performed by the same team of skilled technicians.

Establishment of primary somatic cell cultures

Bovine primary somatic cell cultures were derived from the ear biopsy of an adult Nellore female, with cultures maintained and preserved according to our standard procedures (Gerger et al., 2010; Ribeiro et al., 2009). Cells were cultured in DMEM culture medium (Dulbecco's modified Eagle's Medium, Gibco-BRL, Grand Island, NY, USA), supplemented with 0.22 mM sodium pyruvate, 26.2 mM sodium bicarbonate, 10,000 IU/mL penicillin G, 10 mg/mL streptomycin sulfate, and 10% fetal calf serum (Nutricell, SP, Brazil), until high confluence and either replated for culture (passage) or cryopreserved for further utilization. Only cells at a high confluence (>95%) and up to the fourth passage were used for cloning.

In vitro maturation (IVM)

IVM was performed as described previously (Gerger et al., 2010; Ribeiro et al., 2009). Briefly, selected cumulus–oocyte complexes (COCs) from bovine ovaries collected at two regional slaughterhouses were in vitro-matured in groups of approximately 50 per well in four-well dishes (Nunclon, Roskilde, Denmark) containing 400 μL IVM medium, composed of manipulation medium (MM; TCM-199 with Earle's salt, 25 mM HEPES) + 10% estrous mare serum (EMS), supplemented with 26.2 mM sodium bicarbonate, 0.2 mM sodium pyruvate, 0.01 UI/mL FSHp (Folltropin, Bioniche, Animal Health, Belleville, Ontario, Canada), 0.5 μg/mL LH (Lutropin, Bioniche, Animal Health, Canada), and incubated at 38.5°C in 5% CO₂ in air and high humidity. Matured COCs were used either for cloning or for in vitro fertilization, as below.

In vitro fertilization (IVF)

Bovine in vitro capacitation and IVF were carried out as previously described (Ribeiro et al., 2009). After 17 h of IVM, matured COCs were coincubated with in vitro-capacitated sperm cells (1 × 10⁶ motile sperm/mL), in four-well dishes containing 400 μL TALP-fert medium per well, at 38.5°C, with 5% CO₂ in air and 95% humidity. Depending on the experimental group, IVF was done for 6 h (experimental groups for embryo reconstruction using zygotes) or for 18–22 h (IVF controls).

Cumulus cells and zona pellucida removal, oocyte and zygote manual bisection, and selection of hemi-cytoplasts and hemi-karyoplasts

After 17–18 h of IVM (for the batch of COCs used for cloning) or 6 h after the onset of IVF (for groups of in vitro-matured COCs used for IVF), COCs or presumptive zygotes were denuded by pipetting. This was followed by screening for the presence of one or two polar bodies, as markers for maturation (MII phase) and fertilization, respectively. For zygotes, both polar bodies were not always visibly close to one another, requiring a careful examination. Only matured or fertilized oocytes were used in the experiment.

Zona pellucida (ZP) digestion was performed in MII oocytes by a brief exposure (<30sec) to a 0.5% protease solution in serum-free MM. Protease was inactivated by rinsing several times in MM with 10% fetal bovine serum (FBS). However, as zygotes were more sensitive to the protease, their exposure time for zona removal was shorter (around 15 sec), which was also followed by a rinse in pure FBS, followed by rinsing in MM + 10% FBS.

Zona-free oocytes were exposed to 5 μg/mL cytochalasin-B (CCB) in MM and distributed in dishes in groups of two to three oocytes per 5 μL microdrop under mineral oil. Oocytes were manually bisected using splitting blades (Ultrasharp Splitting Blade, Bioniche, Pullman, WA, USA), under a stereomicroscope. Resulting hemi-oocytes were segregated as enucleated (MII cytoplasts) or nonenucleated (MII karyoplasts) under UV light and 10 μg/mL bisbenzimide (Hoechst 33342) exposure using an inverted epifluorescence microscope (XDY-1, China). Both types of hemi-oocytes were used for embryo reconstruction according to the experimental groups, as shown below.

Zona-free zygotes were placed individually in dishes containing 5–10 μL of 5 μg/mL CCB + 10 μg/mL bisbenzimide in MM for manual bisection performed under UV light control to assure proper enucleation, that is, to obtain one enucleated half (enucleated hemi-zygote, IVF cytoplasts) and another nucleated half (hemi-zygote with both the sperm and the haploid maternal chromosomes, IVF karyoplasts). Zygote exposure to UV light was not continuous, being done quickly for the initial localization of the sperm and egg chromatins and polar bodies, if present. Then, the splitting blade was manually positioned close to the zygote, when the filter was reopened, with the zygote maintained under UV exposure until the completion of the manual bisection (Fig. 1). From the first UV light exposure to the completion of bisection, the average exposure time was no longer than 10 sec.

FIG. 1.

Manual bisection of zona-free bovine one-cell stage zygote. (A) Zygote prior to manual bisection under UV-light control, with decondensing sperm (narrow arrow) and oocyte (wide arrow) chromatin. (B) IVF-karyoplast (wide arrow) and IVF-cytoplast (narrow arrow) after manual bisection.

Experimental design: reconstruction of cloned embryos, electrofusion, and embryo activation

Figure 2 depicts the experimental design used for embryo reconstruction, describing the type of cytoplast (MII hemi-oocyte, IVF hemi-zygote) and karyoplast (MII hemi-oocyte, IVF hemi-zygote, somatic cell), the electrofusion groups, and the embryo activation process (no activation vs. sperm-mediated activation vs. chemical activation vs. sperm + chemical activation).

FIG. 2.

Experimental design for control and experimental groups. Control groups: the IVF control group was obtained after IVF for 18–22 h. The parthenogenetic control groups, zona-intact (PG) oocytes, zona-free (ZFPG) oocytes, and HMC-PG were control groups for monitoring the process of manipulation, culture conditions, and chemical and spontaneous activation. The HMC-SCNT Control Group is the conventional hand-made cloning model, reconstructed by pairing two MII cytoplasts and a somatic cell. Experimental groups: group G-I differed from HMC-SCNT control by the use of one cytoplast that had already been preactivated by the sperm (IVF-cytoplast) along with a MII cytoplast and a somatic cell. Group G-II used two IVF cytoplasts and a somatic cell for embryo reconstruction. Both groups G-III and G-IV were reconstructed by pairing one IVF karyoplast with either one MII cytoplast (G-III) or one IVF cytoplast (G-IV). Embryos in PG, ZFPG, and HMC-SCNT groups were chemically activated (CA). Subgroups of HMC-PG were either chemically activated (CA) or nonactivated (spontaneous activation). Experimental subgroups from G-I to G-IV were either spermatically (SA) or spermatically and chemically activated (CA). SA: Sperm-mediated activation; CA: Chemical activation (ionomycin/6-DMAP); Nonactivated oocyte/karyoplast/cytoplast (MII, HMC-PG); Chemically activated Zygotes (PG, ZFPG, HMC-PG CA, HMC-SCNT); Sperm-activated Zygote/Karyoplast/Cytoplast (IVF, G-II SA, G-IV SA); Sperm-activated Zygote reconstructed with an IVF cytoplast/karyoplast and an MII cytoplast (G-I SA, G-III SA); Sperm-activated and chemically activated Zygote reconstructed with an IVF cytoplast/karyoplast and an MII cytoplast (G-I SA + CA, G-III SA + CA); Sperm-activated and chemically activated zygote reconstructed with an IVF cytoplast/karyoplast (G-II SA + CA, G-IV SA + CA); Somatic cell karyoplast or pro-nuclei.

Embryo reconstruction

Depending on the treatment group (Fig. 2), embryo reconstruction was accomplished by a quick exposure of two hemi-structures (MII or IVF hemi-cytoplasts and/or hemi-karyoplasts) and/or a somatic cell to 500 μg/mL phytohaemoagglutinin (PHA) solution in MM (protein-free) + PVA 0.01%. The IVF cytoplasts and IVF karyoplasts were obtained from the same pool of IVF oocytes from the IVF control group, except that IVF lasted for only 6 h. In every replication, groups of zona-intact and zona-free oocytes were separated for use as zona-intact and zona-free parthenogenetically activated control oocytes (PG and ZFPG), respectively, being kept in the incubator up to the chemical activation, as described below. The parthenote group HMC-PG was used as a control for the manipulation process per se.

Electrofusion

According to the scheme in Figure 2, following reconstruction, couplets from groups HMC-PG, G-III, and G-IV, and triplets from groups HMC-SCNT, G-I, and G-II were equilibrated in electrofusion solution (0.3 M mannitol, 0.05 mM CaCl₂•2H₂O, 0.1 mM MgSO₄•7H₂O, 0.5 mM HEPES, and 0.01% polyvinyl alcohol), in pools of up to 20 couplets/triplets, placed in a 3.2-mm fusion chamber (BTX453, BTX Instruments Inc., San Diego, CA, USA) and subjected to electrofusion after a prepulse of 15 V (AC) during 12 to 15 sec, followed by a double pulse of 1.2 kV/cm (DC) for 20 μsec each (BTX Electro Cell Manipulator 200, BTX Instruments Inc.). Structures were rinsed, individually placed in microdrops of MM under oil, and incubated for at least 50 min until fusion assessment. The mean interval between fusion and fusion assessment was 2 h. Only structures with complete fusion were used on the following steps.

Embryo activation

Parthenote groups PG and ZFPG, and the HMC-SCNT control group were always submitted to the chemical activation protocol. For parthenote group HMC-PG, approximately half of the fused structures were chemically activated (CA) and the other half was left without activation to assess spontaneous activation and developmental rates due to the manipulation procedures. Groups G-I to G-IV were equally allocated to one of two subgroups, being one of the subgroups maintained in MM until in vitro culture (IVC) for the assessment of the effect of the sperm-mediated activation (SA), whereas the other subgroups were also subjected to chemical activation (SA + CA), as depicted in Figure 2. The protocol used for chemical activation consisted of a 5-min exposure to 5 μM ionomycin in MM, followed by incubation in 2 mM 6-dimethyl aminopurine (6-DMAP) in MM for 6 h. The mean interval between fusion and chemical activation was 2.0 ± 0.2 h.

IVC

Embryos from all groups were in vitro-cultured in microwells using the WOW system, according to Vajta et al. (2000), and modified by Feltrin et al. (2006), in modified SOFaaci medium, based on Ribeiro et al. (2009). Microwells were manually produced into four-well dishes containing 400 μL SOF medium supplemented with 0.34 mM trisodium citrate, 2.77 mM myo-inositol, 30 μL/mL BME essential amino acids, 10 μL/mL MEM nonessential amino acids, and 5% EMS, under 400 μL of mineral oil. Dishes were cultured at 38.5°C, in humidified gas mixture (5% CO₂, 5% O₂, and 90% N₂), into laminated foil bags (Vajta et al., 2000).

Assessment of in vitro viability, embryo quality, and postfertilization events

Cleavage rate was evaluated 48 h after chemical activation (set as time = 0 h for all groups), with the recording of the number of cleaved blastomeres present in each microwell. Development to the blastocyst stage was evaluated on day 7, with blastocysts classified according to stage of development and morphological quality following the IETS guidelines (Stringfellow and Seidel, 1998). In the case of zona-free embryos, the stages of development were assessed by comparing embryo size and morphology with zona-intact blastocysts.

The estimation of the total cell number (TCN) and the proportion of cells in the inner cell mass (ICM) and in the trophectodermal (TE) cell lineages per individual blastocyst were performed by differential staining, based on Cesari et al. (2006), with a few modifications. In brief, following morphological evaluation, blastocysts from each group were incubated in a Dulbecco's phosphate-buffered saline (DPBS) solution containing 10 μg/mL propidium iodide and 1 mg/mL Triton X-100 for 40 sec. Then, embryos were fixed in absolute ethanol containing 15 μg/mL bisbenzimide for additional 7 min. Fixed embryos were placed on a slide in a 5-μL glycerol droplet, under a coverslip for immediate evaluation in an epifluorescent inverted microscope.

The time elapsed at each in vitro manipulation step from the onset of IVM through IVC for zygotes used for cloning is shown in Table 1. Parallel to the cloning studies, and to position the biological events during the manipulation (Table 1), the kinetics of cytoplasmic and nuclear chromatin postfertilization and pronuclear formation events were evaluated by fluorescence microscopy in groups of bovine IVF embryos after chromatin staining with 10 μg/mL bisbenzimide from 6 to 19 h after the onset of IVF.

Table 1.

Manipulation Steps During Early Zygote Development from Onset of IVM to IVC for Zygotes Used for Cloning by Hand-Made Cloning

	Time elapsed, h (mean ± SD)
In vitro manipulation process	After the onset of IVF	After the onset of IVM
IVF	0.0 ± 0.0	17.0 ± 0.1
Cumulus cells removal	6.0 ± 0.1	23.0 ± 0.1
Polar body selection	7.2 ± 0.4	24.2 ± 0.4
End of embryo splitting	9.8 ± 1.1	26.8 ± 1.1
Membrane electrofusion^a	11.1 ± 1.0	27.9 ± 1.2
Embryo chemical activation^a	13.0 ± 1.1	29.8 ± 1.1
In vitro culture	19.0 ± 0.4	35.8 ± 1.1

Based on the evaluation of the kinetics of cytoplasmic and nuclear chromatin post-fertilization events, most IVF zygotes should be at the pronuclear stage by 11 to 13 h after IVF, the time for the mean fusion and activation procedures for cloned embryos using IVF cytoplasts and/or karyoplasts.

Statistical analysis

Data analyses were done using the Minitab software (State College, PA). Fusion, cleavage and blastocyst rates, and ICM:TCN, TE:TCN, ICN:TE ratios, were compared using the chi-square test. Data regarding TCN and cell number in the embryonic lineages (ICM, TE), based on morphological quality and stage of development, were analyzed by analysis of variance (ANOVA). A level of significance of 5%, with embryo type (IVF, PG, ZFPG, HMC-PG, HMC-SCNT, and experimental groups G-I to G-IV), stage of development (early blastocyst, blastocyst, expanded blastocyst, or hatching/hatched blastocyst), and embryo quality (good, fair, or poor) was determined. For zona-free blastocysts, the size of zona-intact embryos was used for comparison to assure a more accurate evaluation. However, the size of blastocysts can be highly variable, at least in our experimental conditions. For that reason, zona-free embryos potentially at stages 8 (hatching blastocyst) and 9 (hatched blastocyst) were pooled for the analysis. Pairwise comparisons between treatment groups were performed using the Tukey test. Simple Pearson's correlation and linear regression tests were used for the analyses of relationships and dependence between traits.

Results

Oocyte and zygote manipulation, and embryo reconstruction

A total of 15,222 bovine COCs were in vitro-matured after 20 replications, with 4638 COCs used for production of MII cytoplasts and MII karyoplasts and 9845 for IVF cytoplasts and IVF karyoplasts, from which 9280 (94.3%) were usable for the experiments, and 739 used for the IVF control group. The mean maturation rate for the pool of oocytes used for MII structures, based on polar body selection, was 63.5% (2946/4638), which was similar to the rate observed for the pool of oocytes 6 to 7 h after the onset of IVF (65.7%; 6099/9280), with 37.9% (3731/9280) and 25.5% (2368/9280) having one (MII presumptive unfertilized oocytes) and two (zygotes) polar bodies upon screening, respectively. To note, 3.0% (71/2368) of the zygotes were polyspermic, with the percentage of polyspermy appearing to be higher when oocyte quality was lower, based on their morphology and lower survival through the cloning steps. Due to some degree of variation in egg numbers, egg quality, and zygotes with two polar bodies, approximately 70% (60 to 100%) of all groups were consistently present in each replication, with the alternating exclusion of control parthenote groups when possible.

The splitting of zygotes under controlled UV light exposure resulted in 79.7% survival (1888/2368); the screening of MII hemi-cytoplasts resulted in 54.5% MII cytoplasts (2425/4448) and 44.5% MII karyoplasts (1981/4448). A total of 1756 IVF cytoplasts, 787 IVF karyoplasts, 1748 MII cytoplasts, and 297 MII karyoplasts were used for embryo reconstruction (HMC-PG, HMC-SCNT control group, and G-I to G-IV experimental groups). The mean cell confluence in culture dishes used as somatic cell karyoplasts for cloning (HMC-SCNT and G-I and G-II groups), assessed morphologically prior to cloning procedures, was 90.0 ± 6.4%.

Following electrofusion, fusion rates (Table 2) were higher in the HMC-PG group and lower in the HMC-TNCS control group, and in G-II and G-IV groups, which used IVF hemi-cytoplasts or IVF hemi-karyoplasts for reconstruction (p < 0.05). Such difference was likely due to the biological nature of the structures being fused. Pooled fusion rates for structures composed of a hemi-cytoplast and a hemi-karyoplast (HMC-PG, G-III, G-IV; 906/1084, 83.6%) were higher (p < .05) than for structures composed of two hemi-cytoplasts and a somatic cell (HMC-TNCS, G-I, G-II; 909/1,210, 75.1%).

Table 2.

Fusion Rates (%) Between Cloned Control and Experimental Groups After Embryo Reconstruction

	Reconstructed structures	Fusion rate
Treatment groups^*	n	N	%
HMC-PG	297	283	95.3^a
HMC-TNCS	365	274	75.1^cd
G-I	375	303	80.8^bc
G-II	470	332	70.6^d
G-III	346	291	84.1^b
G-IV	441	332	75.3^cd

a–d

Numbers in columns without common superscripts differ, p < 0.05.

Abbreviations for treatment groups are defined in Figure 2.

Cytoplast/karyoplast type, activation protocol, and in vitro embryo development

Table 3 summarizes data from in vitro embryo development in control groups (IVF, PG, ZFPG, HMC-PG, HMC-SCNT) and experimental groups (G-I to G-IV, and activation subgroups within each group).

Table 3.

Cleavage and Blastocyst Rates for Embryos Produced by IVF, Parthenogenetic Activation (PG, ZFPG, HMC-PG), and Cloning (HMC-SCNT, G-I to G-IV) Using Distinct Cytoplasts and Karyoplasts and Activation Protocols

		IVC	Cleavage rate		Blastocyst rate^†
Treatment groups^*	Activation protocol	N	n	%	n	%
IVF	SA	739	544	73.6^d	147	27.0^b
PG	CA	176	166	94.3^ab	74	44.6^a
ZFPG	CA	173	162	93.6^ab	78	48.1^a
HMC-PG	CA	141	133	94.3^a	16	12.0^cd
	NA	149	85	57.0^e	0	0.0^e
HMC-SCNT	CA	260	239	91.9^bc	75	31.4^b
G-I	SA + CA	143	139	97.2^a	19	13.7^c
	SA	143	117	81.8^c	33	28.2^b
G-II	SA + CA	162	140	86.4^c	9	6.4^de
	SA	163	150	92.0^bc	13	8.7^cd
G-III	SA + CA	141	116	82.3^cd	2	1.7^e
	SA	141	102	72.3^c	0	0.0^e
G-IV	SA + CA	150	123	82.0^c	0	0.0^e
	SA	155	125	80.6^c	0	0.0^e

a–e

Numbers in columns without common superscripts differ, p < 0.05.

Abbreviations for treatment groups are defined in Figure 2.

†

Based on cleavage.

SA, sperm-mediated activation; CA, chemical activation; SA + CA, sperm-mediated followed by chemical activation; NA, nonactivated (spontaneous activation).

Control groups

Overall, the parthenote control groups were reliable controls for oocyte quality, early manipulation processes, and IVC conditions, whereas the HMC-TNCS control group and subgroups HMC-PG were additional effective controls for any possible experimental error and bias during the cloning procedure. Between the control groups (IVF, PG, ZFPG, HMC-PG, HMC-SCNT), cleavage was higher in chemically activated structures and lower in the IVF group (which did not undergo oocyte selection by polar body screening). Blastocyst rates also differed between control groups, being higher for PG and ZFPG parthenotes than IVF and HMC-SCNT controls; the lack of difference between both parthenote groups indicates the existence of no apparent effect of zona digestion on subsequent development. In addition, despite being lower than parthenotes, development in the IVF and HMC-SCNT groups was satisfactory and within the expected range, demonstrating a good overall developmental potential of the COCs used in the experiments. In the HMC-PG group, despite the high cleavage rate seen in the CA subgroup, development to the blastocyst stage was lower than anticipated, demonstrating a possible negative manipulation effect on development. Also, even though a fair amount of structures cleaved in the nonactivated HMC-PG subgroup (by spontaneous activation), no blastocyst development was attained after IVC. The lower blastocyst rates observed in the chemically activated HMC-PG subgroup were likely due to the negative effect imposed by the manipulation process per se or even due to the lack of cytoplasmic complementation when this group was compared with the parthenote controls.

Experimental groups

Cleavage rates in G-I to G-IV, irrespective of the activation protocol, fell well within the range observed in control groups. However, development to the blastocyst stage was significantly affected by the type of cytoplast/karyoplast and/or activation protocol used for reconstruction. Surprisingly, blastocyst rate in the G-I SA subgroup was as high as controls, being lower for G-I SA + CA subgroup. The use of IVF hemi-karyoplasts (G-III and G-IV) for embryo reconstruction was proven deleterious for development to the blastocyst stage. Moreover, under most experimental conditions tested in this study, IVF hemi-cytoplasts were not as effective in supporting blastocyst development when used for embryo reconstruction with somatic cells as karyoplasts (G-I SA + CA and both G-II subgroups).

Total cell number and cell allocation in blastocysts from control and experimental groups

In general, the TCN in blastocysts and the cell density in the embryonic lineages (trophectoderm, ICM) varied more according to the stage of development and embryo quality than to embryo type or group. Not surprisingly, embryos of better morphological quality and/or in more advanced stages of development contained more cells, and vice versa. In turn, TCN correlated better with the stage of development (r = 0.696, p < 0.0001) than with embryo quality (r = 0.459, p < 0.0001).

Table 4 shows data regarding TCN in embryos from control and experimental groups, according to the stage of development. No differences existed between groups except for the group of zona-free parthenotes (ZFPG), which had fewer TCN than both the IVF and HMC-SCNT control groups. Also, no differences were seen between groups in terms of stage of development, with differences occurring within groups, by stage, mostly between early stages (5 and/or 6) and more advanced ones (8/9), but not in all groups. Such lack of difference was probably due to a large variation in TCN within each group. However, the combined overall TCN significantly differed between stages. Nevertheless, embryo quality and stage of development were good predictors for ICM number in blastocysts (p < 0.0001). The TCN was strongly correlated with the number of cells in the trophectoderm (r = 0.945, p < 0.0001), with such association not being as strong when with the ICM (r = 0.757, p < 0.0001).

Table 4.

Total Cell Number (LSM ± SEM) in Blastocysts Produced by IVF, Parthenogenetic Activation, and Cloning Using Distinct Cytoplasts and Karyoplasts and Activation Protocols by Stage of Development

				Stage of development
Treatment groups^*	Activation protocol	Embryos n	Total cell number (TCN)	5	6	7	8/9
IVF	SA	94	112.2 ± 11.5^a	84.0 ± 20.6^a^A	76.2 ± 20.6^a^A	111.2 ± 13.3^a^A	177.4 ± 11.9^a^B
PG	CA	66	108.8 ± 7.4^ab	68.8 ± 18.8^a^A	82.0 ± 15.4^a^A	125.3 ± 10.1^a^AB	158.9 ± 13.3^a^B
ZFPG	CA	66	83.0 ± 8.4^b	62.5 ± 23.0^a^A	59.4 ± 16.3^a^A	82.4 ± 12.3^a^A	127.8 ± 13.3^a^A
HMC-SCNT	CA	69	112.3 ± 8.0^a	49.5 ± 23.0^a^A	106.0 ± 13.3^a^AB	130.4 ± 15.4^a^AB	163.3 ± 9.0^a^B
HMC-PG	CA	16	83.6 ± 15.0^ab	38.5 ± 32.6^a^A	62.0 ± 32.6^a^A	72.0 ± 32.6^a^A	161.8 ± 20.6^a^A
	NA	—	—	—	—	—	—
G-I	SA + CA	19	102.5 ± 15.5^ab	47.7 ± 26.6^a^A	87.3 ± 26.6^a^AB	94.0 ± 46.1^a^AB	181.0 ± 17.4^a^B
	SA	31	106.3 ± 12.4^ab	33.3 ± 26.6^a^A	73.5 ± 23.0^a^A	146.0 ± 32.6^a^AB	172.3 ± 12.8^a^B
G-II	SA + CA	13	153.8 ± 26.1^ab	74.0	-	83.0	150.3 ± 24.8
	SA	9	117.1 ± 17.3^ab	64.5 ± 32.6^a^A	82.0 ± 32.6^a^A	131.0 ± 46.1^a^A	191.0 ± 23.0^a^A
G-III	SA + CA	2	173.0 ± 41.3^ab	—	—	83.0	263.0
	SA	—	—			—	—
G-IV	SA + CA	—	—	—	—	—	—
	SA	—	—	—	—	—	—
Overall mean		385	109.1 ± 3.0	60.2 ± 6.5^A	87.7 ± 6.7^B	112.8 ± 6.4^C	155.9 ± 5.5^D

a,b

Numbers in columns without common superscripts differ, p < 0.05.

A–D

Numbers in rows without common superscripts differ, p < 0.05.

Abbreviations for treatment groups are defined in Figure 2.

SA, sperm-mediated activation; CA, chemical activation; SA + CA, sperm-mediated followed by chemical activation; NA, nonactivated (spontaneous activation).

The association between both embryonic lineages also existed (r = 0.501, p < 0.0001). However, differences in ICM cell number increased as a function of development, with early blastocysts (stage 5, 14.1 ± 3.1) having similar number of cells in the ICM as blastocysts (stage 6, 19.6 ± 3.2) but fewer than expanded blastocysts (stage 7, 22.7 ± 3.0) or hatching/hatched blastocysts (stages 8/9, 35.7 ± 2.6), for p < 0.05; blastocysts and expanded blastocysts had similar ICM cell number, but lower numbers than stages 8/9. No differences in the proportion of cells within the ICM existed between any groups, regardless of the stage of development, with embryo quality being a good predictor for the ICM proportion than any other factor (p < 0.0001).

Embryo quality did affect TCN and the ICM proportion, with differences being pronounced between embryos of excellent morphological quality and embryos of lower quality (Table 5). Also, the number of blastomeres per embryo, recorded in each microwell, assessed along with cleavage rate on day 2 of development, was a good predictor for stage of development, and consequently, TCN, in blastocysts on day 7 (p < 0.0001). In general, embryos with more blastomeres on day 2 reached more advanced stages of development on day 7, whereas less developed blastocysts on day 7 were usually originated from embryos with fewer blastomeres on day 2. Moreover, the higher the number of blastomeres on day 2, the better the blastocyst quality on day 7 (Table 5). Interestingly, after pooling data by embryo type, reconstructed cloned embryos were kinetically more advanced on day 2 than IVF and parthenote (PG, ZFPG) control groups (6.5 ± 0.5 vs. 5.6 ± 0.3 blastomeres; p < 0.05). Finally, embryos from the HMC-SCNT control group (7.0 ± 0.3) were more advanced on day 2 than embryos from the IVF (5.6 ± 0.3), PG (5.4 ± 0.3) and ZFPG (5.7 ± 0.3) control counterparts. However, number of blastomeres per embryo on day 2 did not have a significant effect on blastocyst rate on day 7 of development.

Table 5.

Relationship Between Embryo Quality and Total Cell Number (LSM ± SEM), Proportion of Cells in the ICM (% of Total Cell Number, TCN) in Blastocysts on Day 7 and Number of Blastomeres at the Cleavage Assessment on Day 2 of Development

Embryo quality	Total cell number	ICM:TCN %	Blastomere number at cleavage
1	124.1 ± 5.5^a	24.9 ± 1.3^a	6.5 ± 0.2^a
2	94.4 ± 5.1^b	20.5 ± 1.2^b	5.9 ± 0.2^b
3	94.0 ± 6.7^b	19.5 ± 1.6^b	5.7 ± 0.3^b

a,b

Numbers in columns without common superscripts differ, p < 0.05.

The evaluation of the postfertilization and pronuclear events in bovine IVF embryos from 6 to 19 h after the onset of IVF showed that the proportion of zygotes with condensed sperm and MII-like oocyte chromatin decreased with time, reaching markedly lower levels (<20%) after 10 to 12 h. Concomitantly, the proportion of zygotes with noncondensed chromatin or showing visible pronuclei increased significantly after 8 to 10 h (>60%), with zygotes showing a mitotic spindle or embryos that had undergone cleavage (two-cell stage embryos) starting to appear after 13 h following the onset of IVF. Most zygotes (>70%) had pronuclei by 11 to 13 h after IVF, which is coincident to the mean time for electrofusion and for embryo chemical activation in groups G-I through G-IV (Table 1).

Discussion

The use of preactivated cytoplasts prior to cloning has been controversial with reports demonstrating successful (Bordignon and Smith, 1998) or faulty (Campbell et al., 1994) results after nuclear transfer, depending on the cell cycle of the donor cell (Oback and Wells, 2002) and on the activation process (Collas and Robl, 1991), among other factors. The donor cell cycle was not determined in our study, but as our strategy to synchronize the cell cycle of cell populations in culture used the cell contact inhibition approach by high cell culture confluence, in association with the selection of small cells for cloning (Batchelder et al., 2005; Boquest et al., 1999; Gerger et al., 2010), it is likely that somatic cell karyoplasts were predominantly in G0/G1. Previous reports have demonstrated that cells in high confluence (putative G1/G0) are better reprogrammed into MII cytoplasts, whereas the use of late telophase cytoplasts does not require high cell confluence, that is, the cytoplast is permissive to receive cells at any stage of the cell cycle (Bordignon and Smith, 2006).

The type of cytoplast and the activation process in our study had a significant role on the results. Prepronuclear stage zygotes used in this study as hemi-karyoplasts or hemi-cytoplasts were fused with the other related structures approximately 11 h after the onset of IVF. At this point, cyclinB/Cdk1 complex (M-Cdk or MPF) activity is supposedly low or absent in the zygotes (Collas et al., 1993; Jones, 2005; Wells, 2010), mimicking a G1 phase of the cell cycle, which would be more synchronous to the donor cell. Interestingly, the subgroup G-I SA in this study was the only experimental group to attain developmental rates to the blastocyst stage similar to the IVF and HMC-SCNT control groups (27.0, 31.4, and 28.2%, respectively), with chemically activated counterpart (G-I SA + CA) halving the blastocyst yield (13.7%). Also, data from the G-I SA subgroup indicated that the IVF hemi-cytoplast was not only able to entrain the nonactivated MII hemi-cytoplast to its ongoing sperm-activated cytoplasm, providing sufficient support for embryo development and sufficient genome reprogramming as much as cloned controls, but also that the additional chemical activation protocol imposed on the subgroup of reconstructed embryos (G-I SA + CA) compromised blastocyst yield. This agrees with previous reports by Campbell et al. (1993a, 1993b), where it was postulated that a cytoplast with low M-Cdk activity should maintain a proper ploidy, as a nonsynchronized karyoplast could fail to develop due to rereplication. It is possible that the association of both cell cycle phases for the cytoplasts and the absence of chemical activation promoted a better synchronicity of the physiological events that favored development.

It is important to mention that the somatic cell in this study was always fused to the IVF hemi-cytoplast during reconstruction of the G-I group, with cell donor and cytoplast cell cycles being potentially more synchronous. The MII cytoplast is known to cause nuclear envelop breakdown (NEBD), premature chromatin condensation (PCC), and all events related to the M phase of the cell cycle to the donor nucleus (Campbell et al., 1993a, 1993b; Heyman et al., 2002; Wakayama and Yanagimachi, 2001; Wakayama et al., 2000; Wells, 2010), and such events are believed to be important for genome reprogramming or embryo development after cloning, providing the basis for the use of MII cytoplasts with delayed artificial activation (2 to 4 h) after membrane fusion (Alberio et al., 2001; Wells, 2010). Perhaps the introduction of the cell genome into the IVF cytoplast, presumably more synchronous to the donor cell, delayed NEBD, PCC, and M-phase-related events for some short period of time, or even prevented it. In such phenomenon, the presence of a nonactivated MII cytoplast appeared to be key for development compared with the use of two IVF cytoplasts (G-II group), even if the nonactivated state was eventually overridden by the activated IVF cytoplast. The cytoplast association in G-I may exert a cytoplasmic complementation effect instead of asynchronic (IVF-MII) or divergent effects. Interestingly, the cytoplasmic complementation by the microinjection of cytoplasmic fractions from viable oocytes into incompetent oocytes has been shown to restore subsequent embryo viability in mice and humans (Barrit et al., 2001; Cohen et al., 1997; Smith et al., 2005; Tesarik and Mendoza, 1996). Under normal development, most intact zygotes at the time used for membrane fusion of reconstructed cloned embryos (11.1 h after the onset of IVF) were at the pronuclear stage (data not shown). Cytoplasms from enucleated zygotes at that developmental stage are postulated to lack critical factors for nuclear reprogramming, which appear to be entrapped into the pronuclei (Egli et al., 2007). Perhaps enucleated IVF-cytoplasts in our study remained at a prepronuclear stage up to membrane fusion. Nonetheless, by preventing such structures from sequestering existing cytoplasmic factors required for proper reprogramming or even embryonic development into pronuclei, by the absence of chromatin, the use of two IVF-cytoplasts for embryo reconstruction (G-II SA group) supported development to the blastocyst stage, even if at lower rates than controls and the G-I SA group. Likely, insufficient moieties or lack of key components may have declined development in this group, with the additional chemical activation reducing development even further, as observed in the G-I SA + CA subgroup. Both the MII and IVF hemi-cytoplast components, along with the sperm-mediated activation, had indeed an important role in development, as seen in the G-I SA subgroup.

When both cytoplasts used for reconstruction were IVF hemi-cytoplasts, as in G-II, the lower developmental potential was observed perhaps due to a less efficient chromatin remodeling process, as the use of cytoplasts 6 h after activation significantly impaired embryo development (Tani et al., 2003), likely due to the loss of key cytoplasmic reprogramming factors in the cytoplasts. Alternatively, the decline in developmental potential may have been caused more due to a low M-Cdk activity in IVF hemi-cytoplasts, which were at a G1-like stage of the cell cycle by the time of embryo fusion and/or activation, which abolishes NEBD and PCC (Tani et al., 2003; Wells, 2010). Consequently, as zygotes were enucleated prior to the pronuclear stage, postulated to sequester key reprogramming factors into the pronuclei of zygotes (Egli et al., 2007), it is likely that the donor nucleus was never exposed to cytoplasmic remodeling factors because the nuclear envelope was not broken down, rather than due to the lack of MII-specific factors in the cytoplast by the time of fusion. Both events can possibly and independently lead to faulty reprogramming and development, as proper genome reprogramming, and adequate cleavage and development are rather dissociated events that are initiated by the embryo activation and the subsequent processes dependent on such pathway (Alberio et al., 2006; Campbell, 2002; Gao et al., 2007). This concept is supported by the fact that the additional chemical activation generally enhanced cleavage rates in this study, with a biological tendency for cleavage to be higher in the chemically activated subgroups. Nonetheless, such advantage was not reflected in further embryo development, as the additional activation of structures reconstructed with IVF hemi-cytoplasts reduced blastocyst yields in the experimental groups. Then, the lower blastocyst rate in chemically activated groups might be explained by the interference with the activation processes set in motion by the sperm. Physiologically, the egg penetration by the sperm causes an increase in calcium concentrations (Cuthbertson et al., 1981), sustained in peaks for a while only in fertilized, but not in parthenogenetically activated eggs (Cuthbertson and Cobbold, 1985; Yoshida and Plant, 1992). By suppressing calcium peaks, the same mechanism by which cortical granule migration is prevented (Kline and Kline, 1992) can be used to evidence that the absence or disruption of the events that play a role in fertilization does not prevent the SCNT embryo development, but may impair the viability of animals originated from such embryos. In the G-I SA + CA subgroup, the additional chemical activation process did not seem to affect the activation events that lead to cleavage, but did compromise further development, with nondeveloped embryos halted at or prior to the stage of embryo blockage, that is, 8- to 16-cell stage, about the time of the embryo genome activation, or EGA (Ma et al., 2001; Meirelles et al., 2004).

Contrary to what was observed in this study, as discussed above, it was anticipated that the fusion of two IVF hemi-cytoplasts to a donor cell (group G-II) would have better developmental potential than the the cytoplastic association in group G-I, yet lower than controls, as seen by others (Shurmann et al., 2006). The blastocyst rate for the HMC-SCNT group was higher (28.8%) than for G-II, irrespective of the additional chemical activation (G2 SA + CA, 5.6%) or not (G-II SA, 8.0%), which is in agreement with a few previous studies regarding MII cytoplasts as recipients of choice for nuclear transfer (Tani et al., 2001), although the opposite was also reported by others (Bordignon and Smith, 1998, 2006; Campbell et al., 1994; Wakayama et al., 2000). However, the lower rate of development may have also been caused by the manipulation of the early zygotes per se, because such structures were more sensitive to certain procedures than MII oocytes (e.g., higher sensitivity to the protease) and were more intensively manipulated (i.e., longer exposure to UV light). In addition, timing for zygote splitting and fusion has also been seen to affect development after cloning. Schurmann et al. (2006) demonstrated that SCNT cloning using IVF cytoplasts attained the same developmental competence as MII oocytes when enucleation was performed 4 h after the onset of IVF, for higher pregnancy and delivery rates. A significant distinction between our study and the work by Shurmann et al. (2006) is that the time for sperm-mediated putative activation (time = 0), and the fusion and chemical activation interval for our IVF cytoplasts were larger than that previous study, which was not longer than 4.5 h. When an interval more similar to ours was used (up to 7 h, being ours between 11 and 13 h), blastocyst yield was lower than 10%, similar to our findings. The authors argued that such positive or negative contributions were likely due to the sperm-mediated activation events, promoting a more physiological pattern of epigenetic reprogramming than conventional chemical activation. We suggest that the events, at such extended period of time after the onset of IVF, are mostly due to the indirect effect of the activation process per se, with less impact of spermatic factors than of zygotic events. Such proposition still needs to be further evaluated.

The experimental groups containing IVF karyoplasts (Groups G-III and G-IV) behaved in a completely distinct pattern when compared with all the other groups, including groups using IVF cytoplasts. Despite cleavage rates being not so different than other experimental groups, embryo development beyond the 8- to 16-cell stage was impaired. It is highly possible that the intense manipulation of zygotes was very crucial for development, as many important biological events are taking place at the time, with most structures likely to be at the chromatin decondensation or pronuclear formation stages during the interval after the onset of IVF (6 to 19 h). The lack of in vitro development in a group of remaining IVF and MII hemi-karyoplasts that were not used in the study to compose the experimental groups above (data not shown) reinforce that concept, as cleavage rates were lower in IVF hemi-embryos and/or in chemically activated IVF embryos (SA + CA), with embryo development beyond embryo blockage being impaired, exactly as seen in groups G-III and G-IV. This fact might also be explained by the deterimental effect of the UV light at a very critical cell cycle phase of the zygote (Smith, 1993; Tsunoda et al., 1988), being this effect avoided by McGrath and Solter (1983) with mouse zygotes without UV light exposure. In an attempt to elucidate the causes of such poor embryo development in groups G-III and G-IV, a pilot study was carried out to determine the effects of the manipulations procedures used for zygote splitting on subsequent development (data not shown). The manipulation steps did not affect cleavage and blastocyst rates. Nevertheless, the UV-light exposure for only 10 sec was detrimental for development up to the blastocyst stage, despite the lack of negative effect on cleavage, in a similar pattern as observed in the experimental groups G-III and G-IV. As the experimental conditions were the same, developmental failure for embryos reconstructed with IVF karyoplasts was likely due to UV-induced DNA breaks and fragmentation during zygote splitting. The detrimental effects on IVF cytoplasts cannot be minimized, as it may have also played a part on the lower blastocysts rates in some related experimental groups in this study.

The kinetics of early cleavage stage embryos is crucial to the success of embryo development, as a close positive relationship exists between the onset of the first cleavage and the embryo developmental competence (Gutiérrez-Adan et al., 2001, 2004; Langendonckt et al., 1997; Lonergan et al., 1999, 2000), that is, embryos with faster rates of development have greater developmental potential than slower developing cocultured embryos (Meirelles et al., 2004). The assessment of embryo quality was also shown to be a good predictor of postimplantation development (Misica-Turner et al., 2007) and for the correct pattern of expression for some genes (Kurosaka et al., 2002). These features are probably due to the quality of the inherited cytoplasm after cloning and may correlate to a more functional maternal-zygotic transition. In fact, the number of blastomeres on day 2 in this study was a good predictor of the stages of development on day 7, but not the blastocyst rate, with cloned embryos tending to express such behavior in a more obvious pattern. In fact, a faster kinetics of development had already been reported for SCNT embryos (Bhak et al., 2006).

In summary, the use of a zygote–oocyte cytoplasmic hemi-complementation for embryo cloning was very effective to support development to the blastocyst stage, with the sperm-mediated activation from the hemi-zygote itself being sufficient to entrain an MII hemi-oocyte to a normal pattern of development without further activation. In fact, additional chemical activation in sperm-activated structures dirsupted development to the blastocyst stage. The use of two IVF sperm-activated hemi-cytoplasts for cloning did not provide any improvement in development, supporting the view that cytoplasmic factors from both MII and sperm-activated zygotes prior to the pronuclear stage may be complementary to support further development. Also, the activation process is mandatory for embryo development when MII hemi-oocytes are used for cloning. Then, it appears that a NEBD induced by a high M-Cdk activity and the presence of cytoplasmic factors provided by MII hemi-oocytes, both events being key for development or chromatin remodeling (which may be lacking or lower in zygotes), along with the more physiologic sperm activation process provided by hemi-zygotes, were necessary requirements for adequate in vitro development. Thus, a logical way to enhance embryo viability after in vitro embryo manipulations, such as cloning by SCNT, is to try to mimic physiological events or to minimize suboptimal conditions, which would favor development. Further studies are still needed to better understand the effect of the interaction between ooplasmic and sperm components on development, also evaluating the in vivo developmental potential of cloned embryos reconstructed by distinct patterns of hemi-complementation and activation protocols.

Footnotes

Acknowledgments

The authors thank the staff and directors of the regional slaughterhouses Frigo Fox Ltda., El Golli Ltda., Verdi Ltda, and Pamplona Ltda, for providing bovine ovaries for this study. This study was supported by CNPq/Brazil (Universal Grant no. 477603/2008-4), CAPES/Brazil (Procad II no. 0192058), and PAP/CAV-UDESC 2008–2009.

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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