Reproduction in Domestic Animals

332 C Galli, I Lagutina, R Duchi, S Colleoni and G Lazzaricloning, and not only for beginners is to master all thelengthy and complicated procedures involved in SCNTto a high level of efficiency and reproducibility in orderto obtain scientifically meaningful results (Galli et al.2003b; Ritchie 2006).In principle, all techniques used today in mammalsare based on those described by Willadsen (Willadsen1986). Basically, there is a requirement for maturedoocytes, perhaps in large numbers and of good quality,the oocytes then need to be enucleated before the donorcell is fused or injected in the enucleated oocyte andfinally the reconstructed embryo must be activated. Atthis point, embryos can be transferred to the oviduct ofrecipients for further development. Yet, in large animals,it is preferable to culture in vitro the clonedembryos up to the blastocyst stage, so that a nonsurgicaluterine transfer can be performed (Galli et al.2003b).Source of OocytesOocytes can be harvested from the ovaries of live donorsby ovum pick up (OPU) or from the ovaries ofslaughtered mares (Zhang et al. 1989), the latter beingthe only economically sustainable source of oocytes forSCNT. On average, it is possible to recover three to fouroocytes per abattoir ovary when compared with oocyteretrieval from live donors by OPU, in which, therecovery is slightly more variable and can be from threeto six oocytes per session of OPU (Galli et al. 2007). Thematuration rate of horse oocytes is also quite variable,averaging between 25% and 70% in published studies(Carneiro et al. 2001; Dell’Aquila et al. 2001, 2003;Bogh et al. 2002; Choi et al. 2002; Lorenzo et al. 2002).The recovery of oocytes from abattoir horse ovariesrequires incision and scraping of the follicle wall with acurette and extensive washing to detach the cumulusoocytecomplexes (COCs). Another interesting aspect,peculiar to the horse, is the frequent collection ofoocytes with expanded cumulus; in our laboratory thisaccounts for approximately a third of the recoveredCOCs. In other species such as ruminants and pigs, anexpanded cumulus is linked to collection from atreticfollicles and these oocytes are generally discardedimmediately because of their extremely low developmentalcapacity (de Loos et al. 1989). In the horse, yet,oocytes with an expanded cumulus mature normally andhave normal developmental competence. Table 1 presentsthe data from a large study conducted in ourlaboratory to measure the efficiency of oocyte collectionfrom abattoir ovaries and the expected maturation ratefrom compact and expanded COCs. The data indicatethat each ovary contains 5.3 follicles on an average witha diameter above 5 mm that are suitable for oocytecollection. If these follicles are scraped and washed, therecovery is 3.8 oocytes corresponding to approximately70% efficiency. The need to scrape the follicles derivesfrom the tight connections between the cumulus and themembrana granulosa and between the latter and thefollicle wall. Once the oocytes are collected and selectedon the basis of cumulus morphology, they are transferredto maturation medium and allowed to mature for24 h. In our culture conditions, as shown in Table 1(Galli et al. 2007), the maturation rate ranges from51.1% to 60% for compact and expanded COCs,respectively. This difference is statistically significantand in agreement with other studies (Hinrichs andWilliams 1997; Hinrichs and Schmidt 2000), in which,oocytes with expanded cumulus were found to be morecapable to complete maturation than were oocytes witha compact cumulus. Yet, the ability of oocytes withexpanded cumuli to develop to the blastocyst stage wasnot statistically different from oocytes with a compactcumulus (see Table 2). In calculating the maturationrate, the authors included the large number of degeneratingoocytes that are found after IVM. In fact, at thetime of collection, the oocytes that will degenerateduring maturation cannot be identified; this is anotherpeculiarity of the equine species when oocytes arecollected from abattoir ovaries. At the end of maturation,after the removal of the surrounding somatic cells,degenerated oocytes are easily detected. If these oocytesthat degenerate in culture are not included in thecalculation, the percentage of mature oocytes increasesconsiderably into the range of 70–85% and is comparableto that obtained from OPU oocytes from livemares. As no lysis is observed with OPU oocytes, thisfinding implies that the postmortem modificationsoccurring in the large equine ovaries are responsiblefor the degeneration of abattoir oocytes. Indeed, whenequine oocytes were placed into maturation immediatelyafter slaughter, the rate of maturation was higher andthe rate of degeneration lower than those for oocytesrecovered after transport of ovaries to the laboratoryTable 2. Effect of cumulus morphology on the developmental competenceof equine oocytes matured in vitro, fertilized by ICSI andcultured in vivo in the sheep oviduct (Galli et al. 2007)CumulusmorphologyNo. injected(MII)No.cleavedCleavagerate (%)No. ofblastocysts% Blastocyst ⁄MIIinjectedCompact 150 110 73.3 a 43 28.6 aExpanded 73 43 58.9 b 13 17.8 aChi square test: values within columns with different letters differ (p < 0.05).Table 1. Maturation competence of horse oocytes derived from expanded or compact COCs (Galli et al. 2007)No. ofovariesNo. of follicles(per ovary)No. of COCsexpanded(per ovary)No. of COCscompact(per ovary)No. of COCs matured after IVMNo. of COCs degenerated after IVMExpanded (%) Compact (%) Expanded (%) Compact (%)603 3204 (5.3) 590 (1) 1672 (2.8) 354 (60.0) a 855 (51.1) b 177 (30.0) 558 (33.4)Chi square test: values with different letters differ (p < 0.05).Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag