Nce) to keep does under anaesthesia during laparoscopy. Females were slaughtered 6 days later and parthenote blastocysts were recovered by uterine horns perfusion with 20 mL of Dulbecco Phosphate Buffered Saline (DPBS) supplemented with 0.1 of BSA.Materials and MethodsAll chemicals in this study were purchased from Sigma-Aldrich ?Quimica S.A. (Madrid, Spain) unless stated otherwise.AnimalsMature (adult) rabbit does belonging to the 18325633 New Zealand White ?line from the ICTA (Instituto de Ciencia y Tecnologia Animal) at the Polytechnic University of Valencia (Spain) were used as oocyte and embryo donors 23727046 and recipient does. The Ethics and Animal Welfare Committee of the Universidad Politecnica de Valencia ?approved this study. All animals were handled according to the principles of animal care published by Spanish Royal Decree 1201/2005 (BOE, 2005; BOE = Official Spanish State Gazette).Control embryo recovery at day 6 of developmentSix receptive does were artificially inseminated with pooled sperm from CAL120 fertile males [14] and induced to ovulate as previously described. In vivo fertilised embryos were collected from does slaughtered at 6 days of pregnancy by flushing uterine horns as previously described.Parthenogenetic oocyte activationTo obtain oocytes for parthenogenetic activation, 32 receptive does were induced to ovulate with an intramuscular dose of 1 mg of Buserelin acetate. Does were slaughtered 16?8 h postinduction of ovulation and the reproductive tract was immediately removed. Oocytes were recovered by perfusion of each oviduct with 5 mL of pre-warmed Phosphate Buffered Saline without calcium chloride (PBS) and supplemented with 0.1 of Bovine Serum Albumin (BSA). Recovered oocytes were submitted to two sets 1 h apart of two DC electrical pulses of 3.2 kv/cm for 20 ms at 1 sec apart in an activation medium (0.3 M mannitol supplemented with 100 mM MgSO4 and 100 mM CaCl2), followed by 1 h exposure in TCM199 medium supplemented with 5 mg/mL of cycloheximide and 2 mM of 6-DMAP. A total of 369 oocytes were activated.RNA extraction, amplification and sample labellingAs the amount of RNA present in a single embryo is rather limited [15], for each experimental group (parthenotes and in vivo fertilised embryos) four independent pools consisting of seven embryos were produced. Total RNA was isolated using traditional phenol/chloroform extraction by sonication in the Trizol 1113-59-3 custom synthesis reagent (Invitrogen). Concentration, quality and integrity of RNA were evaluated by Bioanalyzer 2100 (Agilent Technologies). Afterwards, 150 ng of Total RNA were amplified and labelled using QuickAmp Labelling Kit (Agilent Technologies, Madrid, Spain), following the manufacturer’s instructions, which employs a linear amplification method with T7 polimerase. Control embryo samples were labelled with Cyanine 5 dye (Cy5) and parthenoteTable 1. Information on primers used for real-time qPCR.Correlation (R2) 0.Gene IMPACTAccession number ENSOCUTSequence 59R39 GCGTCTTCTTCACCTCATGG TGTTTCTTGGCACAGTTGTTGAFragment size (pb)Efficiency ( ) 104.SMARCAENSOCUTAATCCGCAACCACAAGTAAC GAACACTGACTGTAAGACGAT103.0.EMPENSOCUTAATGTTGGTGTTACTGGCTG GATGCGTTAATAGAGTCTGAA100.0.SCGB1AENSOCUTCCAGTTACGAGACATCCCTGA CATACACAGTGGGCTCTTCACT0.DPYENSOCUTGCAGAGAACCCTCATTCTGAG CGCACAACTGTCTGATCCTGGT98.0.CALCENSOCUTGCTAGAGACTGAGGGCTCCA CACGAAGTTGCTCTTCACCA90.0.H2AFZAFAGAGCCGGCTGCCAGTTCC CAGTCGCGCCCACACGTCC98.GAPDHLGTTCTTCTCGTGCAG ATGGATCATTGATGGCGACAACAT93.H2AFZ: H2A histone family member Z [35]; GAPDH: glycerald.Nce) to keep does under anaesthesia during laparoscopy. Females were slaughtered 6 days later and parthenote blastocysts were recovered by uterine horns perfusion with 20 mL of Dulbecco Phosphate Buffered Saline (DPBS) supplemented with 0.1 of BSA.Materials and MethodsAll chemicals in this study were purchased from Sigma-Aldrich ?Quimica S.A. (Madrid, Spain) unless stated otherwise.AnimalsMature (adult) rabbit does belonging to the 18325633 New Zealand White ?line from the ICTA (Instituto de Ciencia y Tecnologia Animal) at the Polytechnic University of Valencia (Spain) were used as oocyte and embryo donors 23727046 and recipient does. The Ethics and Animal Welfare Committee of the Universidad Politecnica de Valencia ?approved this study. All animals were handled according to the principles of animal care published by Spanish Royal Decree 1201/2005 (BOE, 2005; BOE = Official Spanish State Gazette).Control embryo recovery at day 6 of developmentSix receptive does were artificially inseminated with pooled sperm from fertile males [14] and induced to ovulate as previously described. In vivo fertilised embryos were collected from does slaughtered at 6 days of pregnancy by flushing uterine horns as previously described.Parthenogenetic oocyte activationTo obtain oocytes for parthenogenetic activation, 32 receptive does were induced to ovulate with an intramuscular dose of 1 mg of Buserelin acetate. Does were slaughtered 16?8 h postinduction of ovulation and the reproductive tract was immediately removed. Oocytes were recovered by perfusion of each oviduct with 5 mL of pre-warmed Phosphate Buffered Saline without calcium chloride (PBS) and supplemented with 0.1 of Bovine Serum Albumin (BSA). Recovered oocytes were submitted to two sets 1 h apart of two DC electrical pulses of 3.2 kv/cm for 20 ms at 1 sec apart in an activation medium (0.3 M mannitol supplemented with 100 mM MgSO4 and 100 mM CaCl2), followed by 1 h exposure in TCM199 medium supplemented with 5 mg/mL of cycloheximide and 2 mM of 6-DMAP. A total of 369 oocytes were activated.RNA extraction, amplification and sample labellingAs the amount of RNA present in a single embryo is rather limited [15], for each experimental group (parthenotes and in vivo fertilised embryos) four independent pools consisting of seven embryos were produced. Total RNA was isolated using traditional phenol/chloroform extraction by sonication in the Trizol reagent (Invitrogen). Concentration, quality and integrity of RNA were evaluated by Bioanalyzer 2100 (Agilent Technologies). Afterwards, 150 ng of Total RNA were amplified and labelled using QuickAmp Labelling Kit (Agilent Technologies, Madrid, Spain), following the manufacturer’s instructions, which employs a linear amplification method with T7 polimerase. Control embryo samples were labelled with Cyanine 5 dye (Cy5) and parthenoteTable 1. Information on primers used for real-time qPCR.Correlation (R2) 0.Gene IMPACTAccession number ENSOCUTSequence 59R39 GCGTCTTCTTCACCTCATGG TGTTTCTTGGCACAGTTGTTGAFragment size (pb)Efficiency ( ) 104.SMARCAENSOCUTAATCCGCAACCACAAGTAAC GAACACTGACTGTAAGACGAT103.0.EMPENSOCUTAATGTTGGTGTTACTGGCTG GATGCGTTAATAGAGTCTGAA100.0.SCGB1AENSOCUTCCAGTTACGAGACATCCCTGA CATACACAGTGGGCTCTTCACT0.DPYENSOCUTGCAGAGAACCCTCATTCTGAG CGCACAACTGTCTGATCCTGGT98.0.CALCENSOCUTGCTAGAGACTGAGGGCTCCA CACGAAGTTGCTCTTCACCA90.0.H2AFZAFAGAGCCGGCTGCCAGTTCC CAGTCGCGCCCACACGTCC98.GAPDHLGTTCTTCTCGTGCAG ATGGATCATTGATGGCGACAACAT93.H2AFZ: H2A histone family member Z [35]; GAPDH: glycerald.
