Reproduction in Domestic Animals

418 P Loi, K Matzukawa, G Ptak, Y Natan, J Fulka Jr and A Aravlyophilized for 24 h under an inlet pressure of 1 mtorr at)50°C, following the protocol used for sperm celllyophilization (Wakayama and Yanagimachi 1998). Atthe end of the process, each ampoule was flame sealed,placed into cardboard and stored at room temperature(18–23°C) until use.On the light of the massive nuclear damage in rehydratedcells lyophilized using the sperm cell protocol,different solutions were tested for lyophilization. Briefly,peripheral blood lymphocytes were isolated fromperipheral blood (from Assaf ewes) through a Ficoll–Paque density gradient; the purity of the cells wasassessed by an automatic cell counter (Pentra 60; ABX,Montpellier, France), more than 80% of the cells werelymphocytes.Granulosa cells were obtained by COCs collectedfrom the ovaries of slaughtered Assaf ewes. CumulusOocyte Complexes were matured for 24 h in the mediumTCM 199 plus 10% FCS, FSH, LH and oestradiol inincubator at 38.5°C with 5% CO 2 . At the end ofmaturation, expanded COCs were shortly incubatedin hyaluronidase (300 USP units ⁄ ml; Sigma-Aldrich,Milano, Italy) and dissociated into single-cell populationby repeated pipetting.The freezing solution (2000 ll) was added to the cells(lymphocytes or granulosa) to be lyophilized. Thefreezing solution was composed of 50% FCS and0.1 M trehalose in Hepes Talp buffer. Samples at avolume of 2 ml were frozen using the MTG freezingapparatus (IMT, Nes Ziona, Israel) at a cooling rate of5.1°C ⁄ min. The concentration of the cells ranged from1–6 · 10 6 cells ⁄ ml. After freezing, the samples werestored in liquid nitrogen until transfer into the lyophilizer(Freezone Plus 6; Labconco). Samples werelyophilized for 72 h, after which each ampoule wasflame sealed, placed into cardboard and stored at roomtemperature (18–23°C) until use. Aliquots of the sampleswere dispatched by air mail to Teramo, Italy, fornuclear transfer studies.RehydrationImmediately before use for nuclear transfer, the ampouleswere opened and 100 ll or 2000 ll of milliQ water(according to the freeze-drying protocol) were added.Rehydrated cells were washed twice with medium 199plus Hepes, antibiotics and BSA before use for nucleartransfer. For each ampoule, viability was assessed onsmall aliquots cells by propidium iodine staining (200cells counted). DNA fragmentation was assessed withcomet essay according the manufacturer instruction(R&D Systems, Milano, Italy) in every replicate.DNA integrityDNA integrity was evaluated using the single cell gelelectrophoresis assay (aka comet assay) (R&D Systems)as described by the manufacturer. Cells were diluted to aconcentration of 105 cells ⁄ ml in PBS. The cells werecombined with molten LM agarose at a ratio of:1 : 10(v ⁄ v), then 75 ll were placed on comet slides.The slides were put in the dark at 4°C for 10 min. Slideswere then immersed in a pre-cooled (4°C) lysis solutionfor 1 h at 4°C. Afterward, the slides were immersed in afreshly prepared alkali solution, which consisted of 0.6 gNaOH pellets, 250 ll of 250 mM EDTA, pH 10.0 and49.75 ml deionized water, for 60 min in the dark atroom temperature. Finally, the slides were washed in aTBE buffer (Tris base, Boric acid and EDTA) for 5 minand then the slides were submerged in TBE buffer in ahorizontal electrophoresis apparatus; and 1 V ⁄ cm wasapplied for 10 min. In addition, each experiment wasconducted with two controls; the first one was of cellsthat were previously treated with 100 lM of hydrogenperoxide for 10 min at 2–8°C as described in themanufacturer kit (this served as a positive control forthe assay showing damaged DNA) and the secondcontrol was of fresh cells, indicating endogenous levelsof damage within the cells. After the samples were dried,they were stained with SYBR green. Scoring was carriedout using a fluorescent microscope (Zeiss, Vertrieb,Germany) connect to a digital camera (Sony, Tokyo,Japan) and analysed using the Image J free software(NIH, Baltimore, MD, USA).SEMFor evaluation of the morphology of dry samples,Scanning Electron Microscopy (Philips Inc., Milano,Italy) was used. The samples were gold plated beforebeing placed in the SEM, and observed with a voltage ofthe electron scatter of 25 kV.Oocyte maturationMethods of in vitro embryo production were as previouslydescribed (Ptak et al. 1999a,b). Briefly, followingcollection of ovaries from adult sheep (Comisana andBergamasca breeds) at slaughter, oocytes were aspiratedand evaluated under a dissecting microscope. Onlyoocytes surrounded by at least two layers of granulosacells and with evenly granulated cytoplasm were selectedfor in vitro maturation (IVM). Oocytes were maturedin vitro in bicarbonate-buffered TCM-199 (Gibco) (275mOsm) containing 2 mM glutamine, 100 lM cysteamine,0.3 mM sodium pyruvate, 10% foetal bovine serum(FBS) (Gibco Milan, Italy), 5 lg ⁄ ml FSH (OvagenAuckland, New Zealand), 5 lg ⁄ ml LH, 1 lg ⁄ ml oestradiolin a humidified atmosphere of 5% CO 2 in air at39°C for 24 h.Oocyte enucleation and nuclear transferMatured oocytes were enucleated generally after 22 h,after the start of the maturation. Cumulus OocyteComplexes were shortly incubated with hyaluronidase,and the granulosa cells removed by repeated pipetting.Oocytes were incubated in Hepes-buffered 199 mediumcontaining 4 mg ⁄ ml of BSA and 7.5 lg ⁄ ml of cytochalasinB and Hoechst 33342, 10 mg ⁄ ml for 15 min inincubator.Enucleation was carried out in Hepes-buffered 199medium plus BSA and cytochalasin B using the routinemanipulation procedures.Enucleated oocytes were allowed to recover fromcytochalasin B treatment for 10 min in the incubator,Ó 2008 Teramo University