Reproduction in Domestic Animals

Embryo Biotechnologies in Farm Animals 3intracytoplasmic sperm injection (ICSI) in humans(Palermo et al. 1992). The use of intracytoplasmicsperm injection (ICSI) has been very efficient as a wayto circumvent failure of IVF (Galli et al. 2007). In otherfarm animals like cattle (Goto and Yanagita 1995; Galliet al. 2003c), sheep (Catt et al. 1996) and pigs (Kolbeand Holtz 2000), the efficiency of ICSI for embryoproduction remains lower than for IVF and thereforethe technique is not used routinely. In pigs, IVF ischaracterized by a high incidence of polyspermy thatcompromises embryonic development. This problem hasbeen overcome in part by improving the quality ofin vitro matured oocytes rather than by changing IVFconditions.In recent years, separation of X and Y-bearing spermby flow cytometry is finding wide application in reproductivetechnologies. When combined with IVF or ICSI,a synergy can occur to increase the number of embryosof the desired sex combination (Seidel 2003; Cran 2007)produced by assisted reproductive techniques moreeffectively than older technologies based on sexing ofcells collected by embryo biopsy.Manipulaton of Embryo DevelopmentThe goal of increasing efficiency of in vitro embryoproduction (IVP), especially in cattle, has been thedriving force for much of the applied research in embryobiology and culture; scores of papers have beenpublished on methods for improving the yield of IVPembryos in cattle. Yet, it became soon obvious thatmerely counting the number of blastocysts was not anaccurate measure of the quality of the overall procedureand of the viability of the embryos (Gandolfi and Moor1987). For many years, these constraints on the cultureof viable cattle embryos in vitro were overcome by usingin vivo culture in the oviduct of surrogate sheep (Galliand Lazzari 1996). In particular, a major concern forembryos produced by IVP was the so-called largeoffspring syndrome (LOS), first in sheep and then incattle (Young et al. 1998). The use of serum supplementationand coculture were recognized as the primarycause of LOS. Later, an extensive field study (vanWagtendonk-de Leeuw et al. 2000) demonstrated thatthe incidence of LOS was greatly reduced using a culturemedium based on Synthetic oviduct Fluid (Tervit et al.1972) and the industry took up this method in mostinstances. These observations were accompanied by alarge set of studies looking at the effects of in vitroculture on molecular and cellular markers of embryodevelopment, metabolism, gene expression and viability.Together with others, we investigated the cause of LOSby comparing different in vitro culture systems within vivo culture of bovine embryos in the sheep oviduct.We set up an experiment comparing in vivo culture in thesheep oviduct with in vitro culture in the presence ofserum or high levels of bovine serum albumin (BSA)(Lazzari et al. 2002). Results indicated that embryosgrown in serum but also embryos grown in high levels ofBSA have alterations in the gene expression levels ofseveral developmentally important transcripts. In addition,we demonstrated that these embryos have morecells at the blastocyst stage on day 7 than IVM–IVFembryos developed in the sheep oviduct. Followingtransfer of day 7 blastocysts to recipients and recoveryafter 5 days, we showed that in vitro cultured embryosare larger on day 12 than those derived from sheepoviduct cultured up to day 7. Moreover, when the day12 embryos derived from in vitro culture, werere-transferred to new recipients, they gave rise tooffspring with an average birth weight higher than thecorresponding offspring derived from embryos culturedin the sheep oviduct. These findings clearly indicatedthat in vitro culture can alter development at very earlystages and that the LOS is correlated to abnormallyadvanced embryonic growth and gene expressionpatterns at very early stages.Besides LOS, another important issue that has limitedthe use of in vitro produced embryos is their inconsistentsurvival to freezing and thawing. This limitation wasanother reason why we used for many years the sheepoviduct culture system because it allowed production ofIVM–IVF embryos that were undistinguishable fromin vivo produced embryos in terms of viability andfreezability (Galli and Lazzari 1996; Lonergan and Fair2008). At present, recent improvements in mediaformulation (Gardner et al. 1994) have removed almostentirely the barrier of embryo freezability; pregnancyrate following transfer of frozen-thawed in vitro producedcattle embryos is commercially viable. Importantvariations still exist across laboratories but those thathave really mastered the technology are now wellestablishedcommercial producers of IVP embryos.In vitro produced embryos account for approximatelyone-third of the cattle embryos produced worldwide(http://www.iets.org).A slightly different story has unfolded in the horse.For many years, equine-assisted reproduction laggedbehind other species. This was due in part to the lack ofinterest of the horse industry in developing this technologybut also to a sort of reluctance to really translatequestions that had been asked and answered before inother species. Our work on equine reproduction datesback to the Cambridge period, in the late 1980s, whenwe obtained equine embryos from in vitro maturedoocytes transferred to the oviduct of inseminated mares(Zhang et al. 1989). The most recent work is summarizedin a paper (Galli et al. 2007) where all the relevantsteps have been described including oocyte maturation,fertilization by ICSI, embryo culture and freezing, nonsurgicaltransfer, and somatic cell nuclear transfer.Prometea, the first cloned horse, was derived totallyfrom in vitro procedures that had been optimized step bystep with dedication and perseverance, taking advantageand translating the experience derived from the establishedbovine technology. Today, viable and freezableequine embryos can be produced efficiently by in vitrotechnologies (Colleoni et al. 2007). This success story isexpected to finally open up the horse industry to thesame opportunities that the cattle industry has exploitedfor several years.Nuclear Transfer and CloningThe birth of Dolly the sheep through cloning by nucleartransfer (Wilmut et al. 1997) is an astonishing accom-Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag