Reproduction in Domestic Animals

334 C Galli, I Lagutina, R Duchi, S Colleoni and G Lazzaricouplets, the cleavage rate was 35–50% and onlyapproximately 2.5% of NT-embryos reached the blastocyststage. Hinrichs (Hinrichs et al. 2006, 2007) usedpiezoelectric micromanipulation both to enucleate themetaphase oocyte and to inject the somatic cell directlyinto the enucleated oocyte, thus overcoming the limitationof cell fusion. The horse oocyte tolerates themicroinjection procedure very well as demonstrated alsoby the success of the ICSI procedure (Galli et al. 2007).For the zona-free method, the zona pellucida ofoocytes with extruded polar bodies is digested with0.5% pronase in PBS. Zona-free oocytes are enucleatedunder UV light with a blunt micropipette (with perpendicularbreak). All manipulations are performed inSOF-Hepes containing 10% FCS. Subsequently, zonafreecytoplasts are individually washed for few secondsin 300 lg ⁄ ml phytohemagglutinin P in TCM 199-Hepesand then quickly dropped over a single donor cell (Vajtaet al. 2003) settled to the bottom of a drop of TCM 199with 0.5% of FCS. Formed cell couplets are washed in0.3 M mannitol (50 lM Ca and 100 lM Mg) solutionand fused one or two times at 15–30 min intervals by asingle DC-pulse of 1.2 kV ⁄ cm applied for 30 ls at 26–27 h of maturation. With such an approach, the fusionrate approximates 100% and because of the lowerelectric field required, both cleavage rate and postcleavagedevelopment to the blastocyst stage are higher(Lagutina et al. 2005, 2007). Activation is usuallyperformed 2–4 h after fusion. In our experience, horseoocytes are difficult to activate and the standardprotocols used in ruminants do not yield a high rate ofcleavage. The authors found that the use of ionomycin(a calcium ionophore) followed by the synergistic effectof 6-DMAP (1 mM) and Cycloheximide (5 lg ⁄ ml)resulted in over 90% of parthenogenetic activation(Lazzari et al. 2002b; Galli et al. 2007) and cleavage ofcloned embryos (Lagutina et al. 2005). Woods (Woodset al. 2003) increased the calcium level during activationof in vivo matured oocytes. Hinrichs (Hinrichs et al.2006, 2007) extensively compared different activationprotocols including the injection of sperm extract.Sperm extract in combination with ionomycin, whilenot statistically different from other treatments, providedthe highest blastocyst development rate althoughthis was not superior to that reported by Lagutina(Lagutina et al. 2005) using the association of ionomycinwith both 6-DMAP and cycloheximide.Embryo CultureThe progress in in vitro maturation and ICSI technologyhas increased efforts to design suitable culture systemsfor early cleavage-stage embryos. Very little is knownabout the specific in vitro culture requirements of equineembryos. The assessment of culture conditions has beencarried out with embryos produced in vitro by ICSI andusing various media. Many different culture conditionshave been reported for pre-implantation development ofICSI fertilized horse oocytes, including defined mediasuch as G1.2 (Choi et al. 2002), DMEM-F12 and CZB(Choi et al. 2004) and modified SOF (Galli et al. 2002a).Earlier work evaluated co-culture with somatic cellsincluding Vero cells (Dell’Aquila et al. 1997), oviductepithelial cells (Battut et al. 1991), cumulus cells (Liet al. 2001), granulosa cells (Rosati et al. 2002) orculture in conditioned media (Choi et al. 2001). In mostof these systems, yet, the blastocyst rates remained low,ranging from 4% to 16% of injected oocytes. Incontrast, the culture of presumptive zygotes followingICSI, in vivo either in the mare oviduct or in thesurrogate sheep oviduct, allowed much higher development,being approximately 30% (Galli et al. 2002b,2007; Lazzari et al. 2002b; Tremoleda et al. 2003; Choiet al. 2004) (Table 6). When cell number counts werecompared among in vivo produced embryos and thoseproduced by in vitro culture in a modified SOF medium,both on day 7 of development, in vitro producedembryos had significantly lower cell numbers, resemblinga day 5 embryo rather than a day 7 (Tremoledaet al. 2003). This difference is now taken into accountwhen embryos are transferred to synchronized recipients(see Section ‘Embryo Transfer and Offspring’).The results reported above where obtained using aTCM 199-based medium for oocyte maturation. Interestingly,the authors found that maturing the oocytes ina DMEM-F12-based medium allows to increase blastocystdevelopment to a much closer rate to in vivo culture,being in the range of 26% when compared with 27–50%for the sheep oviduct system, both for ICSI and forSCNT embryos (Galli et al. 2007). Yet, other laboratorieshave achieved equivalent blastocyst development(25–35%) with oocytes matured in M199, usingDMEM-F12 medium for embryo culture under a mixedgas atmosphere (Hinrichs et al. 2005; Choi et al.2006a,b).In our laboratory, equine embryos are cultured in20 ll microdrops under oil with up to 20 embryos perdrop of mSOF. The culture of zona-free embryos isperformed individually in 5 ll drops or in the WOWsystem (Vajta et al. 2000). Fifty per cent of the media isreplaced on D 4 with mSOF and on D6 withDMEM ⁄ F12 supplemented with 5%FCS and 5% SR(serum replacement).The residual difference in embryo development ratesbetween the in vitro and in vivo culture environmentsindicates the need for further improvement of theTable 6. Oocyte recovery rate by OPU and effect of in vitro or in vivo sheep oviduct culture on embryos development (Galli et al 2007)No. ofOPUsNo. of oocytes(no. per OPU)No. metaphase II(% of oocytes)(no. per OPU)No. of cleaved(% of injected)(no. per OPU)No. of comp.morulae ⁄ blastocysts(% of injected) (no. per OPU)Sheep oviduct culture 20 60 (3.0) 46 (76.7) (2.3) 41 (89.1% a) (2.1) 23 (50.0 a) (1.2)In vitro culture 12 46 (4.1) 36 (73.5) (3.0) 25 (69.4% a) (2.1) 5 (13.9 b) (0.4)Student’s t-test. Numbers within columns with different letters are significantly different (p < 0.05).Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag