Reproduction in Domestic Animals

Cloning in Horses 333Table 3. Effect of maturation media on maturation, cleavage rate andICSI embryo development (Galli et al. 2007)MaturationmediumNo. of No. ofoocytes degeneratedNo. ofmetaphaseII (%)No. ofinjectedNo. ofcleaved(% ofinjected)No. ofblastocysts(% ofinjected)TCM 199 434 105 205 (47.2 a) 191 111 (58.1 a) 23 (12.0 a)DMEM ⁄ F12 338 71 159 (45.6 a) 140 108 (77.1 b) 37 (26.4 b)Replicates = 6.Chi square test. Numbers within columns with different letters differ (p < 0.05).Table 4. Effect of maturation media on maturation, cleavage rate andNT embryo development (Galli et al. 2007)MaturationmediumNo. ofoocytesNo. oflysedNo. ofmetaphaseII (%)No. ofNTembryosNo. ofcleaved(% of NT)No. ofblastocysts(% of NT)TCM 199 164 35 64 (39.0 a) 41 38 (92.7 a) 4 (9.77 a)DMEM ⁄ F12 166 35 67 (40.4 a) 47 46 (97.9 a) 13 (27.7 b)Chi square test. Numbers within columns with different letters differ (p < 0.05).(Hinrichs et al. 2005). In that study, high rates ofdegeneration in culture were seen only in oocytes with acompact cumulus; evaluation of oocyte chromatinconfigurations led to the conclusion that these oocyteswere juvenile and that their chromatin was damagedduring storage within the ovary.It is known that in vitro maturation conditionsdramatically influence the development of fertilizedoocytes to the blastocyst stage. Different media are usedfor maturation of equine oocytes, although the mostcommon remains medium TCM199, probably becauseof its widespread use for bovine oocyte maturation. Inthe last 2 years, the authors have tested other mediaincluding DMEM-F12 supplemented with 15% serumreplacement (Invitrogen sri, San Giuliano Milanese,Italy) instead of TCM 199 supplemented with 10%foetal calf serum (FCS). Hormones and growth factorswere added to both media as reported earlier (Lagutinaet al. 2005). We found a clear advantage in the use of aDMEM-F12 based medium not only on cleavage ratebut also on embryo development to the blastocyst stage(Table 3). Preliminary experiments have confirmed similarfindings in horse SCNT (Table 4). This positiveeffect is evident also on blastocyst development, which issignificantly higher in the DMEM-F12 group than in theTCM199 group. This remarkable improvement increasesthe efficiency of equine embryo production andmakes it possible to use a totally in vitro systemfollowing OPU in the horse. The time required forcompleting IVM ranges from 22 to 24 h for expandedoocytes and 26–36 h for compacted onesSource of Donor CellsA variety of donor cells from different origin (foetal andadult) and various tissues have been used for SCNTwithout observing significant differences in the overallefficiency (Oback and Wells 2002). In the horse, foetalfibroblasts (Li et al. 2003; Lagutina et al. 2005; WoodsTable 5. Cleavage and blastocyst development after nuclear transferwith confluent or roscovitine-treated fibroblasts (Hinrichs et al. 2006)TreatmentNo. ofinjected(MII)No. of Cleavage No. of D +cleaved rate 7 blastocystsNo. of D +8 blastocystsTotalblastocyst(%)Confluent 55 40 73 1 1 2 (3.6)Roscovitine 56 44 79 0 2 2 (3.6)et al. 2003) as well as adult fibroblasts and granulosacells have been used (Vanderwall et al. 2004; Lagutinaet al. 2005). For recovering adult fibroblasts, skinbiopsies are taken under local anaesthesia on the neckor the chest of the donor horse. The recovered tissue isthen minced into small pieces that are plated inTCM199 ⁄ DMEM (1:1) culture medium supplementedwith 10% FCS. In 2–3 weeks, the fibroblasts outgrowthe tissue and form a confluent monolayer. Uponsubsequent sub-culture and expansion, the cells arefrozen for later use or prepared for the SCNT procedureat early passages (Li et al. 2003). There is a considerablevariation in the success rate of horse SCNT because ofthe specific cell line source of donor nuclei (Galli et al.2003a; Lagutina et al. 2005) as reported for other species(Oback and Wells 2002). The cell cycle of the donor cellis crucial for success and G0 or G1 stages have beenused in horses. This stages can be achieved throughserum starvation (reduction of FCS to 0.5%), growth toconfluency (contact inhibition) or the use of kinaseinhibitors like Roscovitine (Table 5) (Oback and Wells2002; Hinrichs et al. 2006, 2007). Given the relativelylow number of experiments performed, it cannot beascertained if any of these approaches is better than theother.Embryo Reconstruction and ActivationIn our laboratory, the oocytes are stripped of theircumulus after 22–24 h of maturation. First, by pipettingduring exposure to hyaluronidase; second, in 0.25%trypsin for 1.5 min and finally, in SOF-Hepes containing10% FCS. For reconstruction of NT-embryos, theauthors use both zona-enclosed and zona-free methods(Oback et al. 2003). For the zona-enclosed method,oocytes with an extruded polar body are stained withHoechst 33342 in the presence of cytochalasin B(5 lg ⁄ ml). Enucleation is performed by the aspirationof the polar body and metaphase II plate in a minimalvolume of ooplasm under UV light. All manipulationsare carried out in SOF-Hepes with 6 mg ⁄ ml BSA(Gardner et al. 1994), except fusion. Individual nucleardonor cells are then transferred into the perivitellinespace of zona-enclosed cytoplasts using the enucleationpipette. The cytoplast–karyoplast couplets are transferredinto 0.3 M mannitol (50 lM Ca and 100 lM Mg)solution and fused one or two times within 15–30 mininterval by a double DC-pulse of 1.8–2.4 kV ⁄ cm appliedfor 30 ls at 26–27 h of maturation. Because of theheterogeneous characteristics of the horse zona pellucida,the fusion rate is often inconsistent or low. Li et al.(2002) have found that even high voltage DC pulses of2.2–2.5 kV ⁄ cm were able to fuse less than 60% ofÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag