Livestock Science 143 (2012) 35–42

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Zona-free and with-zona parthenogenetic embryo production in goat(Capra hircus) — effect of activation methods, culture systems andculture media

Arun Kumar De⁎, Dhruba Malakar, Manoj Kumar Jena, Rahul Dutta,Shweta Garg, Yogesh S. AksheyAnimal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India

a r t i c l e i n f o

⁎ Corresponding author at: Current address: AniCentral Agricultural Research Institute, Port Blair, AIslands-744101, India.

E-mail address: biotech.cari@gmail.com (A.K. De).

1871-1413/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.livsci.2011.08.012

a b s t r a c t

Article history:Received 6 March 2011Received in revised form 12 May 2011Accepted 20 August 2011

Studies on parthenogenetic activation of oocytes are important to improve the efficiency of nu-clear transfer as artificial activation of oocytes is an essential component of nuclear transferprotocol. The present study was carried out to investigate the effects of different activationmethods, culture systems and culture media on in vitro development of zona-free and with-zona parthenogenetic embryos in goat. In case of zona-free parthenogenesis, there was a sig-nificant (pb0.05) increase in cleavage rate and blastocyst yield when oocytes were activatedby electrical pulse (76.29±0.52% and 19.07±0.39% respectively) than when Ca-ionophorewas used for activation (63.45±0.73% and 14.09±0.65% respectively). The quality of blasto-cysts was evaluated by determination of cell number and by expression profile of pluripotentrelated gene Oct-4. No significant (pb0.05) difference was found in quality of blastocysts pro-duced by different activation methods. In culturing of zona-free parthenogenetic embryos, flatsurface (FS) was proved to be the best system. A significant (pb0.05) decrease in cleavage rateand blastocyst yield was found in Microdrop culture of zona-free embryos (43.67±2.08% and0.72±0.72% respectively) in comparison to WOW of zona-free embryos (73.88±1.70% and15.51±1.34% respectively) and FS of zona-free (75.14±0.81% and 23.93±2.71% respectively)as well as with-zona (72.16±1.55% and 18.16±0.68%) embryos. Zona-free flat culture systemyielded significantly (pb0.05) higher blastocyst rate than zona-free WOW system as well aswith-zona flat culture system. The zona-free and with-zona parthenogenetic embryos werecultured in three different media — Research Vitro Cleave media from Cook® Australia(RVCL), Embryo Development Medium (EDM) and Modified Synthetic Oviductal Fluid(mSOF). In case of zona-free parthenogenesis, significant (pb0.05) increase was found in blas-tocyst development rate in RVCL medium (18.61±1.52%) than EDM (11.29±0.77%) or mSOF(11.53±1.86%). In case of with-zona parthenogenesis, RVCL medium and EDMwere found su-perior to mSOF. The results of the study will be helpful to improve the efficiency of nucleartransfer in goat.

© 2011 Elsevier B.V. All rights reserved.

Keywords:ParthenogenesisGoatNuclear transferBlastocyst

mal Science Division,ndaman and Nicobar

ll rights reserved.

1. Introduction

Parthenogenesis is the biological phenomenon by whichembryonic development is initiated without male contribu-tion. The parthenogenetic activation of oocytes is an impor-tant tool to investigate the comparative roles of paternal andmaternal genomes in controlling early embryo development.As artificial activation of oocytes is an essential component

36 A.K. De et al. / Livestock Science 143 (2012) 35–42

of nuclear transfer protocols, studies on parthenogenetic oo-cyte activation are important to improve the efficiency of nu-clear transfer (Kim et al., 1996). An optimized activationprotocol may enhance nuclear reprogramming of the recon-structed embryo which in turn increases the efficiency of nu-clear transfer.

The efficiency of blastocyst production by somatic cell nu-clear transfer (SCNT) is less and is hampered by various bio-logical and technical problems (Vajta et al., 2003); onlyapproximately 1–2% of the embryos reconstructed by nucleartransfer are able to develop to term (Lai and Prather, 2003).Its success rate is dependent upon many factors, includingdonor cell cycle, donor cell type, oocyte quality, enucleation,fusion, oocyte activation procedures and in vitro cultureconditions.

Activation is a crucial parameter affecting blastocyst produc-tion in nuclear transfer. Various chemicals like Ca ionophore, eth-anol and strontium have been used for activation of oocytes(Alberio et al., 2001; Das et al., 2003; Varga et al., 2008). In tradi-tional method for production of cloned goat embryos, electricpulses were applied for activation of reconstructed oocytes(Melican et al., 2005; Shen et al., 2006). Oocyte activation byelectrical pulse is initiated by an elevation of intracellular Ca++.Immediately after electrical stimulation, there is an influx of ex-tracellular Ca++ which in turn triggers an increase of intracellu-lar Ca++ (Cheong et al., 2002). Ionophores such as Calciumionophore and ionomycin induces a great single intracellular cal-cium rise in MII oocytes which originates exclusively from theinternal deposits (Hoth and Penner, 1992) and a likely conse-quence is the activation of several calcium-dependent proteolyt-ic pathways, leading to the destruction of cyclin B, reductionof MPF activity, and resumption of meiosis (Jellerette et al.,2006; Rinaudo et al., 1997; Tomashov-Matar et al., 2005). Ultra-sound was also shown to induce nuclear activation and parthe-nogenetic development of in vitro matured pig oocytes (Satoet al., 2005).

Culture system is another important factor for improvingthe efficiency of blastocyst production by traditional SCNT aswell as hand-made cloning (HMC) method. Several culturesystems like Well-of the wells (WOW) (Vajta et al., 2001),agarose gels (Peura and Vajta, 2003), glass oviduct (Thouaset al., 2003) and microdrops (Oback et al., 2003) have beendeveloped for culture of zona-free cloned embryo with vary-ing in efficiency of blastocyst production. A suitable culturesystem needs to be established for culture of zona-free clonedgoat embryos.

Culture medium is the most determinant factor for im-provement of efficiency of in vitro embryo development. Com-monly used media for in vitro embryo production (IVP), likeM-199 and mSOF, has been shown to be less efficient for invitro development of cloned buffalo embryos producedthrough SCNT (Shah et al., 2008). Commercially available se-quential medium (G1/G2, Vitrolife, Sweden) has been reportedto improve the efficiency to some extent (Simon et al., 2006). Inthe present study, three different types of media — RVCL, EDMand mSOF were evaluated for the efficiency of zona-free andwith-zona blastocyst production.

The aim of the present study was to investigate the effectsof different activation methods, culture systems and culturemedia on yield and quality of zona-free and with-zona par-thenogenetic goat blastocysts. In the first experiment, the

effects of different activation methods (Ca-ionophore andelectrical activation) were evaluated on blastocyst formationand blastocyst quality. Blastocyst quality was evaluated notonly by blastocyst cell number but also by expression of plur-ipotency gene Oct-4 in inner cell mass (ICM) of blastocysts. Inthe second and third experiment, the effect of various culturesystems and culture media were evaluated for production ofzona-free and with-zona parthenogenetic blastocysts. The re-sults of the experiments will help in improving the efficiencyof SCNT and HMC in terms of yield and quality of blastocysts.

2. Materials and methods

All the present experiments comply with all relevant in-stitutional and national animal welfare guidelines, policiesand ethics committee approval.

All chemicals andmediawere purchased fromSigmaChem-ical Co (St. Louis, MO, USA), and disposable plastic wares werefrom Nunc (Roskilde, Denmark) unless specified otherwise.

2.1. In vitro maturation of oocytes

Goat ovaries were collected from local abattoir and trans-ported to the laboratory in a thermo flask containing warm(35 °C) normal saline containing antibiotics (400 IU/ml penicillinand 500 μg/ml streptomycin) within 3 h. Ovaries were trimmed,washed and then oocytes were aspirated by puncturing the visi-ble follicles with a 18-gage needle in the oocyte collection medi-um (OCM) containing TCM 199, 100 μg/ml L-glutamine, 10% FBS(Hyclone, Logan, UT, Cat no. CH30160.02), 50 μg/ml gentamicinand 3 mg/ml BSA (Fraction-V). Oocytes were picked up gentlyunder stereo zoom microscope and kept in a 35 mm Petri dishcontaining OCM and washed 5 times in this medium. Thecumulus-oocyte-complexes (COCs) having≥3 layers of compactcumulus cells were selected for in vitro maturation. COCs werewashed 2 times with the maturation medium containing TCM-199, 10 μg/ml LH, 5 μg/ml FSH, 1 μg/ml estradiol-17β, 50 μg/mlsodium pyruvate, 5.5 mg/ml glucose, 3.5 μg/ml L-glutamine,50 μg/ml gentamicin, 3 mg/ml BSA, and 10% FBS. Four drops of100 μl maturation medium were made in 35 mm Petri dishesand coveredwithmineral oil. These disheswere placed in the in-cubator with 5% CO2 in air at 38.5 °C, 1 h prior to use for equili-bration. Then 15–16 oocytes were placed in each drop ofmaturation medium. The Petri dishes were incubated at 38.5 °Cunder 5% CO2 in air with maximum humidity for 24 h (Malakaret al., 2008).

2.2. Parthenogenetic activation

Matured oocyteswith expanded cumulus cells were trans-ferred into micro centrifuge tube containing 0.5 mg/ml hyal-uronidase in T2 (TCM-199 supplemented with 2.0 mML-glutamine, 0.2 mM sodium pyruvate, 50 μg/ml gentamicinand 2% FBS) and incubated for 1 min at 38.5 °C under 5% CO2

in air. Then vortexing was done for 2–3 min. The contents ofthe tube were transferred to a 35 mm Petri dish containingT2 and completely denuded oocytes were selected andwashed twice in fresh T2 for removal of cumulus cells. Forzona-free parthenogenesis the oocytes were made zona freeby incubating them to T10 (TCM-199 supplemented with2.0 mM L-glutamine, 0.2 mM sodium pyruvate, 50 μg/ml

37A.K. De et al. / Livestock Science 143 (2012) 35–42

gentamicin and 10% FBS) containing 2 mg/ml pronase for 5–8 min.

2.3. Experiment 1: effect of chemical or electrical activation onblastocyst development and quality

Zona-free and with-zona oocytes were randomly dividedinto two groups each and activated by two differentmethods — in one half, oocytes were activated using 2 μMCa ionophore (ionophore A23187) in T20 medium (TCM-199 supplemented with 2.0 mM L-glutamine, 0.2 mM sodiumpyruvate, 50 μg/ml gentamicin and 20% FBS) for 5 min at38.5 °C. Then they were washed thrice in T20 medium andincubated individually in 5 μl droplets of T20 medium con-taining 2 mM 6-Dimethylaminopurine (6-DMAP) in 5% CO2

in air at 38.5 °C for 3 h. In the other group, the oocytes weretransferred to positive and negative electrodes, respectively,of the fusion chamber (BTX microslide 0.5 mm gap, model450; BTX, San Diego, CA) containing the fusion medium con-sisting of 0.3 M Mannitol, 0.1 mMMgSO4, 0.05 mM CaCl2 and3 mg/ml BSA. Then they are activated with a double D.C pulseof 2.31 kV/cm for 15 μs. Following the electrical activation theoocytes were incubated with 2 mM 6-Dimethylaminopurine(6-DMAP) in T20 medium under 5% CO2 in air at 38.5 °C for3 h.

2.4. Experiment 2: effect of culture systems on blastocystdevelopment and quality

After activation, the with zona oocytes were cultured in flatsystem and zona-free oocytes were cultured in three differentculture systems — (1) WOW: hand-made microwells of300 μm wide and 300 μm deep prepared with the help of asmooth, “V”-shaped darning needles (Booth et al., 2001a) in awell of a four-well dish (4WD) (Nunc; Roskilde, Denmark),otherwise known as the well of wells (WOW) system(Vajta et al., 2001) containing 400 μl of medium. 20–25microwells (WOWs) were prepared in each well of 4WD.The WOWs were prepared a day before in vitro culture(IVC) of embryos, rinsed by pipetting and replaced withfresh medium just before IVC and covered with 400 μl ofmineral oil. A single embryo was cultured in each micro-well. (2) Microdrops (MD): 5 μl droplets of medium wereprepared in a 4WD (10 microdrops in each well) and cov-ered with 400 μl of mineral oil. A single embryo was cul-tured in each microdrop. (3) Flat surface (FS): 400 μl ofmedium added to each well of a 4WD and covered with400 μl of mineral oil. 10–15 embryos were cultured ineach well of a 4WD (FS).

2.5. Experiment 3: effect of culture media on blastocystdevelopment and quality

After activation, the oocytes (bothwith-zona and zona-free)were cultured in three different types ofmedia suchas ResearchVitro Cleavemedium (RVCL, Cook®, Australia; containing NaCl,KCl, KH2PO4, MgSO4, NaHCO3, CaCl2, sodium pyruvate, sodiumlactate, D-glucose, gentamicin, L-glutamine, and EDTA in puri-fied water) supplemented with 1% BSA, Embryo DevelopmentMedia (EDM, containing TCM 199, 30 μg/ml sodium pyruvate,100 μg/ml L-glutamine, 50 μg/ml gentamicin, 10 μl/ml essential

amino acids, 5 μl/ml non-essential amino acids and 10% FCS)supplemented with 1% BSA and modified synthetic oviductalfluid (mSOF)with essential and nonessential amino acids, sodi-um citrate and myoinositol (Holm et al., 1999) with 1% BSA.

2.6. Quality assessment of embryos and determination ofblastocyst cell number

The health of the blastocysts was determined by countingthe cell numbers of blastocysts by differential staining as de-scribed by Thouas et al. (2001). The blastocysts were incubat-ed in sol I (DPBS with 1% Triton X-100 and 100 μg/mlpropidine iodine) for 15 s. the blastocysts were then immedi-ately transferred to 500 μl of solution II (fixative solution of100% ethanol with 25 μg/ml Hoechst 33342) and incubatedat 4 °C overnight. The cell number was counted using aninverted microscope (Nikon) fitted with an UV lamp and ex-citation filters.

2.7. Analysis of Oct-4 gene expression in ICM ofparthenogenetically activated blastocysts

Total RNA was isolated from inner cell mass of blastocystsdeveloped from chemically and electrically activated parthe-nogenetic oocytes for expression study of Oct-4 using “Cells-to-cDNA kit-II” (Ambion, Austin, TX, USA) according to man-ufacturer's instructions. Briefly, the cells were washed withice cold PBS after which 20–50 μl of chilled cell lysis bufferwas added and the mixture was incubated at 75 °C for10 min in a thermal cycler (BIO-RAD, USA). Genomic DNAwas degraded by incubating the cell lysate in DNase-I at37 °C for 30 min and the remaining activity of DNase-I wasinactivated by heating at 75 °C for 5 min. cDNA was preparedusing 10 μl of the cell lysate and random primers. The PCRcycle included denaturation (94 °C for 2 min) followed by re-peated cycles of denaturation (94 °C for 30 s), annealing (for30 s at temperature 58 °C) and extension (72 °C for 45 s) fol-lowed by a final extension (at 72 °C for 10 min). A negativeRT reaction (i.e., RT reaction but without MMLV enzyme)was set up. A house keeping marker gene β actin was alsoamplified at each stage of PCR.

2.8. Statistical analysis

The data was analyzed using SYSTAT 7.0 (SPSS Inc. Chicago,IL) after arcsin transformation. Differences between meanswere analyzed by one way ANOVA followed by Fisher's LSDtest. Significance was determined at Pb0.05.

3. Results

3.1. Experiment 1: effect of chemical or electrical activation onblastocyst development and quality

The results of Experiment 1 are shown in Table 1. In case ofzona-free parthenogenesis, a significant increase in cleavagerate (Fig. 1A) and blastocyst yield (Fig. 1B, D) (76.29±0.52%and 19.07±0.39% respectively) was found when electricalpulse was used for activation of the oocytes than when calci-um ionophore was used (63.45±0.73% and 14.09±0.65% re-spectively) (Table 1). In case of with-zona parthenogenesis,

Table 1Effect of different activation methods on development of zona-free and with-zona parthenogenetic embryos (data from 4 trials).

Zona-free or with-zonaparthenogenetic embryoproduction

Activation methods Oocytes Cleavage % (n) Blastocyst % (n) Number of nuclei in blastocystsas mean±S.D (n blastocysts)

Zona-free Ca ionophore activation 156 63.45±0.73(99)c 14.09±0.65 (22)c 101.9±4.54 (10)Zona-free Electrical pulse activation 152 76.29±0.52(116)a 19.07±0.39 (29)b 101.4±5.23 (10)With-zona Ca ionophore activation 142 70.44±0.41(100)b 21.09±1.11 (30)ab 103.2±3.03 (10)With-zona Electrical pulse activation 147 72.10±1.32 (106)b 23.11±0.53 (34)a 102.8±5.07 (10)

a,b,cValues having different superscripts in the same column differ significantly (Pb0.05).Results are expressed as mean±s.e.m.

38 A.K. De et al. / Livestock Science 143 (2012) 35–42

no significant difference was found between the activationmethods in terms of cleavage and blastocyst yield (Table 1).When with-zona oocytes were activated by electric pulse ac-tivation, the blastocyst production rate (23.11±0.53%) wassignificantly higher in comparison to zona-free blastocystproduction rates activated either chemically (14.09±0.65%)or electrically (19.07±0.39%). The quality of blastocysts pro-duced by two different activation methods was evaluated bydetermination of number of nuclei/blastocyst and by usingthe gene expression profile of a pluripotent-related geneOct-4 (Fig. 2). No significant difference was found in terms

Fig. 1. (A–D) Stages of zona-free parthenogenetic goat embryo production: A. Zona-free parthenogenetic 2 cell stage goat embryo in WOW system (200×)B. Zona-free parthenogenetic blastocyst stage goat embryo in WOW system (400×). C. Zona-free parthenogenetic 32 cell stage goat embryo in flat system(200×). D. Zona-free parthenogenetic blastocyst stage goat embryo in flat system (400×).

of number of nuclei/blastocyst and expression of Oct-4(Table 1 and Fig. 2).

3.2. Experiment 2: effect of culture systems on in vitroparthenogenetic embryo development and quality

The results of Experiment 2 are shown in Table 2. With-zona parthenogenetic embryos were cultured in flat systemand cleavage rate and blastocyst yield was 72.16±1.55% and18.16±0.68% respectively (Table 2). The zona-free partheno-genetic embryos were cultured in three different systems-

.

Fig. 2. Expression of Oct-4 in parthenogenetic blastocysts. Lane 1: 100 bpladder markers. Lane 2: Negative control. Lane 3: Expression of β-actin.Lane 4: Expression of Oct-4 in Ca-ionophore activated parthenogenetic blas-tocyst. Lane 5: Expression of Oct-4 in electrical pulse activated parthenoge-netic blastocyst.

39A.K. De et al. / Livestock Science 143 (2012) 35–42

WOW (Fig. 1A, B), Flat system (Fig. 1C, D) and microdrops. Asignificant decrease in cleavage rate and blastocyst yield wasfound in microdrop of zona-free embryos (43.67±2.08% and0.72±0.72% respectively) in comparison to WOW of zona-free embryos (73.88±1.70% and 15.51±1.34% respectively)and FS of zona-free (75.14±0.81% and 23.93±2.71% respec-tively) as well as with-zona (72.16±1.55% and 18.16±0.68%) embryos (Table 2). Zona-free flat culture system yieldedsignificantly higher blastocyst rate than zona-free WOW sys-tem aswell as with-zona flat culture system. No significant dif-ference was found in terms of number of nuclei/blastocystamong different groups.

3.3. Experiment 3: effect of culture media on in vitroparthenogenetic embryo development and quality

The results of Experiment 3 are shown in Table 3. No sig-nificant difference was found among the three media interms of cleavage percentage both in zona-free and with-zona parthenogenesis. In zona-free parthenogenesis, a signif-icant increase in blastocyst development rate in RVCL medi-um (18.61±1.52%) was observed in comparison to EDM(11.29±0.77%) or mSOF (11.53±1.86%) (Table 3).

In case of with-zona parthenogenetic embryos, blastocystdevelopment rates in RVCL medium (21.6±1.46%) and EDMmedium (20.36±0.36%) were significantly higher thanmSOF medium (10.91±2.494%) but did not differ signifi-cantly with zona-free blastocyst production rate in RVCL me-dium (18.61±1.52%). No significant difference was found interms of number of nuclei/blastocyst among different groups.

Table 2Effect of culture systems on development of zona-free and with-zona parthenogen

Zona-free or with-zonaparthenogenetic embryoproduction

Culture system Oocyte Cleavage %

Zona-free Flat surface 137 75.14±0.8Zona-free WOW 141 73.88±1.7Zona-free Microdrop 128 43.67±2.0With-zona Flat culture 154 72.16±1.5

a,b,cValues having different superscripts in the same column differ significantly (PbResults are expressed as mean±s.e.m.

4. Discussion

For activation of oocytes, maturation-promoting factor(MPF) and mitogen activation promoter factor (MAPK) mustbe suppressed. Multiple and periodic oscillations in intracellu-lar calcium concentrations are responsible for these suppres-sion (White and Yue, 1996). In mammals, the initial calciumincrease is followed by a series of calcium transients that lastfor several hours (Miyazaki et al., 1992). Calcium signal downregulates the activity of the cell cycle regulatory protein M-phase promoting factor (MPF) that leads to exit from themeta-phase arrest (Lorca et al., 1993). During fertilization, thesechanges are induced by interactions between the oocyte andsperm, or by release of a sperm factor into the oocyte cytoplasm(Küpker et al., 1998). The activation of oocyte is induced by anincrease in the oocyte's intracellular free calcium concentra-tion. In normal fertilization, sperms triggers release of calciumfrom the endoplasmic reticulum and this act as a signal for oo-cyte activation (Whitaker, 2006). Intracellular calcium concen-trations can be raised artificially by a combination of severalchemical activators in the absence of sperm (Grupen et al.,2002). Although severalmethods are available for the transientelevation of the intracellular free calcium in oocytes, inducingthe low frequency calcium oscillation is very difficult by artifi-cial means (Machaty and Prather, 1998).

Activation of recipient cytoplast by electric or a combina-tion of electric and chemical stimuli was a critical element ofthe nuclear transfer procedure. Ca ionophore followed by 6-DMAPwas used to obtain cloned embryos in cattle (Bhojwaniet al., 2005; Tecirlioglu et al., 2005). Ca ionophore A 2187 in-creases intracellular CA+2 which in turn destroys the existingCa-sensitive cytostatic factor (CSF), resulting in the reductionof maturation promoting factor (MPF) activity (Swann andOzil, 1994). The combination of calcium ionophores with 6-DMAP induces high rates of activation, pronucleus formationand development to blastocyst stage in sheep (Loi et al.,1998) and cattle (Liu et al., 1998). One disadvantage of acti-vation of oocytes with ionomycin and 6-DMAP was that itcaused DNA damage that was reflected in abnormal patternof karyokinesis at the first cell cycle (De la Fuente and King,1998). Another very effective activating agent is strontiumwhich was used for parthenogenetic activation of mouse,rat, bovine and porcine (Meo et al., 2004; Roh and Hwang,2002; Varga et al., 2008). In traditional method of productionof cloned goat embryos of SCNT, electric pulse has been usedfor activation of reconstructed oocytes (Melican et al., 2005;Shen et al., 2006). Electrical stimulation of oocytes results information of pores in plasma membrane which facilitate

etic embryos (data from 4 trials).

(n) Blastocyst % (n) Number of nuclei in blastocystsas mean±S.D (n blastocysts)

1 (103)a 23.93±2.71 (33)a 99.17±5.91 (6)0 (104)a 15.51±1.34 (22)b 100.00±8.16 (5)8 (54)b 0.72±0.72 (1)c 101 (1)5 (111)a 18.16±0.68 (28)b 98.80±9.73 (5)

0.05).

Table 3Effect of media on development of zona-free or with-zona parthenogenetic embryos (data from 4 trials).

Zona-free or with-zonaparthenogenetic embryoproduction

Media Oocyte Cleavage % (n) Blastocyst % (n) Number of nuclei in blastocystsas mean±S.D (n blastocysts)

Zona-free RVCL 128 70.41±1.11 (90) 18.61±1.52(24)a 99.4±8.45 (5)Zona-free EDM 124 70.21±2.16 (87) 11.29±0.77(14)b 101.8±7.15 (5)Zona-free mSOF 120 69.94±0.82 (84) 11.53±1.86(14)b 98.5±8.71 (6)With-zona RVCL 125 70.39±1.56 (88) 21.6±1.46 (27)a 97.5±10.21 (4)With-zona EDM 113 70.69±1.13(80) 20.36±0.36 (23)a 100.25±1.71 (4)With-zona mSOF 110 70.12±1.20 (77) 10.91±2.49(12)b 100.8±7.30 (5)

a,bValues having different superscripts in the same column differ significantly (Pb0.05).Results are expressed as mean±s.e.m.

40 A.K. De et al. / Livestock Science 143 (2012) 35–42

uptake of extracellular Ca2+ by oocytes (Collas et al., 1993;Onodera and Tsunoda, 1989; Ozil, 1990). This influx of Ca2+

leads to an increase in intracellular Ca2+ concentration andoocyte activation (Sun et al., 1992). In the present study ofzona-free parthenogenesis, a significant increase in cleavagerate and blastocyst yield (76.29±0.52% and 19.07±0.39%respectively) was found when electrical pulse was used foractivation of the oocytes than when calcium ionophorewas used (63.45±0.73% and 14.09±0.65% respectively)(Table 1). The possible reason may be that Ca ionophorehas some toxic effect on zona-free parthenogenetic oocytes.Otherwise, it may be that double electrical pulses followedby 6-DMAP treatment induce a higher and wider calciumtransient ensuring the complete activation of goat oocytes.Similar postulations were reported in porcine (Cerveraet al., 2010; Im et al., 2007). In case of with-zona partheno-genesis no significant difference was found between the acti-vation methods in terms of cleavage and blastocyst yield(Table 1). No significant difference was found in terms ofnumber of nuclei/blastocyst and expression of Oct-4 in theICM of blastocysts produced by two different activationmethods. (Table 1 and Fig. 2). In porcine, over expression ofOct-4 in blastocysts was reported when electrical treatmentswith 6-DMAP was used for activation (Cervera et al., 2010),but we did not find any difference which indicates the spe-cies specific difference in expression of Oct-4.

The embryo culture system plays a significant role in deter-mining the proportion of cloned embryos develops to blasto-cysts. A number of culture systems have been reported forculture of cloned embryos. Das et al. (2003) reported cultureof SCNT goat embryos in feeder free provided with 30–100 μldroplets of medium whereas coculture with cumulus cellswas reported by Keefer et al. (2002). This microdrop systemwas found to be inefficient in case of HMC embryos (Vajtaet al., 2000, 2001). In the present study a single zona-free em-bryo was cultured in 5 μl microdrop of medium and significantdecrease in cleavage rate (43.67±2.08%) and blastocyst pro-duction rate (0.72±0.72) was found in comparison to WOWof zona-free embryos (73.88±1.70% and 15.51±1.34% respec-tively) and FS of zona-free (75.14±0.81% and 23.93±2.71%respectively) as well as with-zona (72.16±1.55% and 18.16±0.68%) embryos (Table 2). WOWwas reported to be most effi-cient culture system for culturing zona-free cloned embryos(Booth et al., 2001a; Vajta et al., 2001) with blastocyst yield ofabout 50% in cattle (Vajta, 2007). WOW system helps to

prevent the disaggregation of blastomeres in the beginning ofembryo development, since in these early stages no cellularjunctions were present. But one disadvantage of WOW systemis the removal of protective surface coating of culture dish dur-ing the preparation of WOW. In our study, FS was found to besuperior culture system than WOW for culture of zona-freeparthenogenetic embryos. There was a significant (pb0.05) in-crease in zona-free blastocyst production rate in FS (23.93±2.71%) compared to WOW system (15.51±1.34%) (Table 2).The possible reason for this may be due to lesser trophoblastadhesion of blastocyst to the plastic surface in flat systemthan WOW system. Similar findings were reported by Vajtaet al. (2001). Another reason may be that in FS, a group of em-bryos (10–15 nos) were cultured, so this group culture hadsome beneficial effect on production of blastocysts.

Culturemedium is very important for in vitrodevelopment ofembryos. Several types of media were reported to be used forculture of cloned zona-free embryos. mSOF supplemented withessential and non-essential amino acids, sodiumcitrate,myoino-sitol and serumwasused in cattle (Booth et al., 2001b) andhorse(Lagutina et al., 2005, 2007) for culture of HMC embryos. In pigs,several media have been developed including Whitten's medi-um (Wright, 1977), North Carolina State University (NCSU)-23and NCSU-37 medium (Petters and Wells, 1993), Beltsville Em-bryoCultureMedium(BECM)-3 (Dobrinsky et al., 1996), porcinezygote medium (PZM)-3 and PZM-4 (Du et al., 2007; Yoshiokaet al., 2002). NCSU-23 has been widely used and is one of themost successful media for culture of porcine embryos after invitro fertilization or nuclear transfer. In case of buffalo, cleavageand morulae formation were better in RVCL medium for clonedembryo production (Shah et al., 2008). In the present study forzona-free parthenogenesis, a significant increase in blastocystdevelopment rate was found in RVCL medium (18.61±1.52%)than EDM (11.29±0.77%) or mSOF (11.53±1.86%) (Table 3).In case of with-zona parthenogenetic embryos also RVCL wasfound better than EDM and mSOF (Table 3).

In conclusion, electrical activation method was found bet-ter for activation of zona-free goat oocytes in terms of blasto-cyst yield without compromising their quality. Flat surfacesystem was superior in supporting the growth of zona-freeparthenogenetic embryos in comparison to WOW or MDand RVCL medium was better than EDM and mSOF in sup-porting growth of zona free parthenogenetic goat embryos.In case of with-zona parthenogenesis, RVCL medium andEDM were found superior to mSOF.

41A.K. De et al. / Livestock Science 143 (2012) 35–42

Acknowledgments

The authors are very thankful to the Indian Council of Ag-ricultural research (ICAR) and Council of scientific and Indus-trial Research (CSIR), India for supporting the work.

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