ilable at ScienceDirect

Theriogenology 81 (2014) 138–151

Contents lists ava

Theriogenology

journal homepage: www.theriojournal .com

40th Anniversary Special Issue

Ovum pick up, intracytoplasmic sperm injection and somatic cell nucleartransfer in cattle, buffalo and horses: from the research laboratory toclinical practice

Cesare Galli a,b,c,*, Roberto Duchi a, Silvia Colleoni a, Irina Lagutina a, Giovanna Lazzari a,c

aAvantea, Laboratory of Reproductive Technologies, 26100 Cremona, ItalybDepartment of Veterinary Medical Sciences, University of Bologna, Italyc Fondazione Avantea, Cremona, Italy

a r t i c l e i n f o

Article history:Received 8 August 2013Received in revised form 6 September 2013Accepted 7 September 2013

Keywords:Assisted reproductionEmbryo technologiesNuclear transferCattleBuffaloHorse

* Corresponding author. Tel.: þ39 0372 437242; fE-mail address: cesaregalli@avantea.it (C. Galli).

0093-691X/$ – see front matter � 2014 Elsevier Inchttp://dx.doi.org/10.1016/j.theriogenology.2013.09.0

a b s t r a c t

Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pickup (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, havebeen transferred and adapted to buffalo and horses. The successful clinical applications ofthese techniques require both the clinical skills specific to each animal species and anexperienced laboratory team to support the in vitro phase of the work. In cattle, OPU can beconsidered a consolidated technology that is rapidly outpacing conventional superovula-tion for embryo transfer. In buffalo, OPU represents the only possibility for embryo pro-duction to advance the implementation of embryo-based biotechnologies in that industry,although it is still mainly in the developmental phase. In the horse, OPU is now anestablished procedure for breeding from infertile and sporting mares throughout the year.It requires ICSI that in the horse, contrary to what happens in cattle and buffalo, is veryefficient and the only option because conventional IVF does not work. Somatic cell nucleartransfer is destined to fill a very small niche for generating animals of extremely highcommercial value. The efficiency is low, but because normal animals can be generated it islikely that advancing our knowledge in that field might improve the technology andreduce its cost.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

Breeding techniques have always been at the center of anylivestock enterprise, motivated by curiosity and consolidatedby breeder’s needs and interests. This has been the case, forexample, when artificial insemination was developed as anhygienic measure to prevent disease transmission or at pre-sent in the genomics era, where embryos can be genotypedand/or propagated by somatic cell nuclear transfer (SCNT)[1,2] to reproduce thedesiredgenotype fromselectedparents.Livestock species have provided for decades the knowledge

ax þ39 0372 436133.

. All rights reserved.08

for assisted reproduction techniques that have been trans-lated to humans. Because of the easy availability of bothgametes and pre-implantation embryos, the closer similar-ities with the human counterpart and the different ethicalrequirements, scientists working with livestock havecontributed to the advancement and consolidation of thehuman field [3]. In turn, many of the more advanced tech-niques developed for humans have provided a model for an-imal scientists after the birth of the first human beingconceived in vitro [4] and following the subsequent deve-lopments, including transvaginal ovum pick up (OPU) [5–7].

The practical application of assisted reproduction tech-nologies in livestock requires the integration of the labo-ratory techniques with the clinical management of donors,recipients and newborn animals, because what matters to

C. Galli et al. / Theriogenology 81 (2014) 138–151 139

the client requesting such services is a live offspring. Thedrive behind the developments of advanced assistedreproduction techniques discussed in this article for cattleand buffalo is the need to generate large numbers of em-bryos, preferably of predetermined sex and known geno-type, for genetic improvement of the herds. On the contrary,in horses the main reason for applying these technologieshas been for addressing both female as well as male infer-tility [8], very much as it is done in human assisted repro-duction, including pre-implantation genetic diagnosis.

In this paper, we revisit the major developments thatplayed a role in turning experimental findings into special-ized clinical practices in the breeding of high genetic meritlivestock.We discuss the impact of the techniques aswell asthe bottlenecks and the possible future developments. It ismore of a personal view and experience than a compre-hensive review of all the literature.

2. Ovum pick up

2.1. Cattle

The development of OPU in cattle followed the estab-lishment of reproducible techniques for in vitromaturation[9], fertilization [10,11] and culture of sheep [12] and cattleembryos [13], bringing to the birth of normal offspring [14],techniques. All the experimental work done to developin vitro embryo production (IVP) procedures was essen-tially based on oocytes that were recovered from slaugh-terhouse ovaries in large numbers. However, for practicalapplication, it was clearly desirable to recover oocytes fromliving donors of known genetic value.

According to the International Embryo Transfer Societystatistics (Fig. 1), the number of embryos produced in vitroand transferred into recipients has increased more than 10times [15] in the last dozen years and are now approachingthe numbers of embryos produced in vivo by superovula-tion. This indicates that OPU and IVP is considered a reliableand cost-effective technique and has acquired a role incattle breeding.

The first attempts to use ultrasound-guided follicularaspiration for embryo production in vitrowere reported byPieterse and other authors in the late 1980s [16–18] byusing a human endovaginal probe adapted for the use in

Fig. 1. Summary of the International Embryo Transfer Society statistical datacollected by the data retrieval committee. In vitro production of embryos issteadily growing and the majority of them are now produced by ovum pickup (OPU).

cattle. They reported a recovery rate of 55%, repeatability ofthe procedure, and absence of side effects on the donorcows. Although the procedures for embryo production inthose days still required major refinements that came later[19], the basics of OPU described by Pieterse et al. are stillthe same used today by many practitioners. Recovery rateshave been improved in the excess of 70% owing to the useof better ultrasound equipment with 6- or 7-MHz convexarray probes that provide a better resolution on smallerfollicles and the use of gonadotrophin priming that in-creases the size of smaller follicles that otherwise wouldnot be picked up by inexperienced operators. Nativeendovaginal probes are available essentially for human useand need to be adapted to hold longer 60 cm long singlelumen needles (Fig. 2). Several manufacturers of ultrasoundequipment have provided custom-made plastic holdersthat can house generic convex array transducers togetherwith the needle guide. A partial simplification of theequipment was described by Bols, et al. [20] that usedsimple hypodermic disposable needles. However, thissimpler setting has the disadvantage of a bigger size that,although it is fine for cows, can be traumatic for very youngheifers. The use of native endovaginal probes allows thecomplete replacement, for each donor animal, not only ofthe probe latex cover, but also of the needle guide togetherwith the needle. This solution uses one sterile long needleand one needle guide for each OPU session/donor, requiringmore equipment and more labor for cleaning, sterilizing,and packaging; as a consequence, it is more expensive.However, from an hygienic standpoint and quality control,it is a system that provides no cross-contamination bet-ween donors and therefore provides the highest biosafetystandards, as it is done in humans. On the contrary, thesetting with a generic probe enclosed in a plastic adaptertogether with the needle guide make it convenient toreplace the disposable and less expensive needle used foraspiration. However, the needle guide is normally notreplaced from donor to donor, being enclosed in the plasticadapter. Therefore, there is always some blood that bycapillarity will infiltrate in the needle guide and in thesurroundings of the probe cover when the vaginal wall ispunctured. The needle in both settings is connectedthrough a tubing and a test tube to a vacuum pump thatusually provides a vacuum set at flow rate between 15 and25mL/min [21] to ensuremaximum recoverywith the leastdamage to the cumulus oocytes complexes. Flow rates aremore indicative than vacuum pressure because the gaugeof the needle and the length of the tubing can make a bigdifference. Simple flushing media for embryo transfer,supplemented with heparin to avoid follicular fluid orblood clotting, are currently used for oocyte recovery.

The OPU technique was initially applied on problemcows that did not respond to superovulation [22,23], butlater on it was applied on a wider scale also on pregnantcows and heifers, including prepubertal heifers [21]. It isdifficult and often not relevant to make comparisons be-tween different dataset because there are somany variablesinvolved, most of which are not even manageable. Beefbreeds perform better than dairy cows, dry cows do betterthan lactating ones, and cows perform better than heifers(Table 1). Climate conditions with high temperature and

Fig. 2. Set up for ovum pick up (OPU) in a residential center. (A) Pregnant cow with the front legs sitting on a step to facilitate the access to the ovaries. (B)Prepubertal, heifer (6 month old). (C) Buffalo cow subjected to OPU with the same equipment. (D) Human 6-MHz endovaginal probe with a home-made adapterto extend its length, needle guide, and needle. Note the small size of the transducer that will fit into very small animals.

C. Galli et al. / Theriogenology 81 (2014) 138–151140

heat stress reduce the number of follicles and the quality ofoocytes. Bos Indicus cows have far greater number of folli-cles compared with Bos Taurus and as a consequence pro-duce proportionally more embryos [24]. In our experience,the donor animal is a major source of variation both fromthe management and genetic point of view. There is nowstrong evidence that nutrition plays an important role onoocyte and embryo quality [25–27], as well as the geneticbackground [28]. This last aspect has become evident withthe studies on the plasma level of anti-Müllerian hormone[29], a parameter that can be used in practice to assess thefollicular population present on the ovary of any givendonor [30] to optimize embryo production. However, thepreselection of donors is not always an option, especiallywhen specific genetic programs require selected individualdonors. For this reasons, much work has been performed toimprove embryo yield. Twice weekly OPU is the mosteffective protocol to maximize oocyte recovery withouthormonal stimulation [21,31,32] without any side effects[33] on donor well-being and avoiding the presence of thedominant follicle that could negatively affect develop-mental competence [34]. To increase embryo productionand reduce the labor required in twice weekly OPU pro-tocols, gonadotropin stimulation has been introduced.However, this technique is not the best solution for problemdonors that usually are subjected to OPU because they havealready failed to produce embryos by superovulation.Therefore, the choice of combining superovulatory treat-ment with OPU should be made depending on the type ofdonor available and the clinical assessment of the previoustreatments. Several investigators have used gonadotropinstimulation in OPU protocols [23,35–37] ranging from a fullsuperovulatory dose to shorter treatments for 2 to 3 days

with one or two injections per day in presence of a pro-gesterone releasing device or a corpus luteum. Sirard, et al.[38] reported that after follicular wave synchronization and3 days of treatment with six injections of a constant dose ofgonadotropin, an interval of 48 hours before OPU wasoptimal for subsequent developmental competence [38].The positive effect of the gonadotropins can be attributed tothe increase in size of small follicles and to the acquisition ofa higher developmental competence of the oocytes as itoccurs with in vivo-matured oocytes [32]. In prepubertalcalves gonadotropin stimulation is required to obtain anacceptable level of developmental competence [21]. How-ever, use of gonadotropin stimulation does not seem to beeffective in Bos Indicus donors [39]; it is not used in large-scale programs [24,40]. The implementation of an OPUprogram requires always the support of a specialized lab-oratory for embryo production. Moreover, because of theeconomic value of the calves born, adequate veterinaryassistance is recommended tominimize losses owing to thepossible incidence of the large offspring syndrome or othercommon perinatal pathologies.

Altogether, the cost of producing an embryo by OPU indairy cows in Europe could be 50% to 100% greater than bymultiple ovulation and embryo transfer. This greater costand the current breeding context in Europe allows for theuse of OPU for a very specialized niche market. Otherconditions in other countries offer different opportunitiesalso dictated by economics: for example, the large use inBrazil is certainly determined by the fact that OPU in gen-eral works better and it is more cost effective than super-ovulation in Bos Indicus beef donors. Table 2 estimatesexpected efficiencies of the various procedures available toproduce embryos in dairy cattle.

Table 1Embryo production by ovum pick up (OPU) from Holstein heifers and cows.

Donor No. ofOPU

No. offollicles

No. ofoocytes

No. ofoocytesper OPU

No. cleaved Cleavage (%) No. of embryos No. ofembryosper OPU

% embryos/oocytes

% embryos/cleaved

Cow 2251 41,983 28,852 12.81 19,711 68.32 5601 2.49 19.41a 28.42aPregnant heifers 97 1103 705 7.26 494 70.07 139 1.43 19.72a 28.14aPubertal heifers 566 9048 5900 10.42 4052 68.68 1054 1.86 17.86b 26.01bPrepubertal heifers 258 4110 2800 10.85 1885 67.32 447 1.73 15.96c 23.71b

Values with different letters within columns differ significantly (chi square test; P < 0.05). Data from Galli et al. (unpublished).

C. Galli et al. / Theriogenology 81 (2014) 138–151 141

2.2. Buffalo

In the buffalo, OPU has great potential because super-ovulation (multiple ovulation and embryo transfer) hasgiven poor results compared with those in cattle [41,42]and it has never made an impact on buffalo breeding pro-grams, both because of the limited number of embryos thatcan be recovered and also because of the low survival aftercryopreservation [43]. Because of these limits, the OPUtechnology has always been of great interest also in buffalobreeding. The first OPUs in buffalo were reported by Boni,et al. [44]. The procedure is performed exactly in the sameway as in cows (Fig. 2C). Oocyte recovery, embryo pro-duction, and offspring obtained have been described inseveral publications [21,45], but only about 10% to 15% ofthe oocytes recovered develop to transferable embryos(Table 3). In general, the ovaries of buffalo cows and heifersare small; in addition, the follicles tend to be fewer and ofsmall diameter (Fig. 3). Therefore, few follicles are availablefor OPU and the follicular population is influenced by theseasonality reported in the buffalo [46]. Gonadotrophinstimulation might be beneficial [47] for increasing thesmall size and paucity of follicles found especially inanestrous donors. Because of the great value of the femaleoffspring in buffalo herds, the combination of OPU withsexed semen [48] and cryopreservation [49] offer the op-portunity to accelerate the genetic gain in the buffaloindustry.

Table 2Embryo production efficiencies of current embryo technologies in dairycattle.

Treatment before oocyte collection Number of embryos

Per session Per wk

Natural cycle, AI and flushing at day 7 0.7 0.23MOET, standard superovulation 6 1DFR-MOET, superovulation preceded by

removal of dominant follicle5.5 1.1

OPU (twice weekly) at 3- and 4-dayintervals

1 2

OPU after FSH prestimulation at 2-wkintervals

3 1.5

(DFR) MOET-OPU, superovulation withcollection before ovulation

5.5 1.1

Modified from Merton et al. 2003 [94].Abbreviations: DFR, dominant follicle removal; MOET, multiple ovulationand embryo transfer; OPU, ovum pick up.

2.3. Horse

The recovery of oocytes from the mare was first appliedto the preovulatory follicle using different procedures,including laparotomy under general anesthesia, colpotomy,and aspiration using a long needle placed through the flankin the paralumbar fossa. However, these approaches wereinvasive and their efficacy was limited. The most practical,less invasive, efficient, and repeatable technique now usedis the ultrasound-guided transvaginal follicular aspirationusing a double-lumen, 12-ga needle [50]. Oocytes can becollected from the pre-ovulatory follicle that has reached atleast 35 mm in diameter, 24 hours after HCG injection withthe donor showing signs of uterine edema. Only mildsuperovulatory treatment has improved oocyte recovery[51]. Ovulation normally takes place 36 to 40 hours afterhCG administration. Therefore, oocytes collected at 24hours or later have resumed meiosis, have an expandedcumulus that facilitate their recovery but require 16 hoursof additional culture before being inseminated. After

recovery from the donor mare and a few hours of culture tocomplete maturation, oocytes can be surgically transferredto inseminated recipients [52] or can be subjected tointracytoplasmic sperm injection (ICSI) and again trans-ferred surgically to the oviduct of a synchronized recipientor cultured in vitro up to the blastocyst stage and finallytransferred nonsurgically. In the clinical context, the suc-cess rate of establishing a pregnancy with surgical oocytetransfer to the oviduct for in vivo fertilization or shortlyafter ICSI, is in the range of 40% [52]. There is a limit to thenumber of in vivo–matured oocytes, although of highquality, that can be recovered from any given donor duringthe year; normally, one preovulatory follicle is present atany cycle and only during the breeding season. For thesereasons, based on previously published studies [53,54] andon the experience accumulated in cattle, in our laboratorywe refined the procedures for OPU of equine immatureoocytes (Fig. 4A, B) as it is done in cattle [8,21], in vitromaturation, ICSI and embryo culture and we demonstratedtheir efficacy and reproducibility. During OPU, we aspirateall follicles that are at least 1 cm, also in the nonbreedingseason, thereby increasing the number of oocytes that canbe recovered from a donor over a given time. Oocyte re-covery rates from immature follicles can be disappointingfor the beginners. Because of the large size of the folliclesand the strong attachment to the follicle wall of thecumulus oocyte complex, it is necessary to use double-lumen needles that allow for repeated flushing, up to 8 to10 times for each follicle. Early reports of recovery ofimmature oocytes [55,56] provided a very limited assess-ment of the developmental potential of such oocytes.

Table 3Embryo production by ovum pick up (OPU) in Mediterranean Buffalo (Galli et al. unpublished).

No. ofOPU

No. offollicles

No. ofoocytes

No.cleaved

Cleavage(%)

No. ofembryos

No. of embryosper OPU

% embryos/oocytes

% embryos/cleaved

123 1392 815 389 47.73 132 1.07 16.20 33.93

Data from Galli et al. (unpublished).

C. Galli et al. / Theriogenology 81 (2014) 138–151142

Subsequently, we performed in our laboratory, over severalyears, a large study to assess the developmental compe-tence of immature oocytes recovered by OPU from bothexperimental and commercial donors within our OPUclinical program [57]. The data reported in Table 4 refer torepeated OPU collections from donors of various breedswithin our current OPU service. To ensure the highest rateof oocyte collection, we monitor the donors by ultrasoundto choose the most suitable time for OPU based on thepresence of a sufficient number of follicles (normally �8–10 follicles >1 cm) and preferably in the absence of adominant follicle. This situation is particularly favorableduring the transition season (no dominant follicle with aprevalence of medium sized antral follicles). For these

Fig. 3. (A) Buffalo ovaries. note the small size and fewer follicles than cows. (B) OoCleaved embryos 48 hours after IVF. (E) Blastocysts on day Dþ6 after fertilization. Noabundance of lipid content. This makes the embryos less tolerant to cryopreservat(Galli et al., 1998).

reasons, the average numbers of follicles aspirated andoocytes collected by our group are higher than those inmost published studies, although the recovery rate hasbeen steadily increasing with experience and practice.Development to blastocyst for this commercial work iscarried out completely in vitro. The outcome is very variableowing to the extreme differences between both female andmale donors and to the different causes of infertility lead-ing to the use of these procedures. A breed effect can also beobserved. Mares can be subjected to repeated collectionswithout any side effect, under detomidine sedation,epidural anesthesia, to avoid the contraction of the rectum,and with catheterization of the bladder [58]. At the endof the procedure, the donor is treated with antibiotics for

cytes before in vitro maturation and (C) at the end of in vitro maturation. (D)te the dark appearance, like in oocytes and early stages embryos owing to theion. (D) First calves born in 1998 from OPU embryos after cryopreservation

Fig. 4. (A) Mare subjected to ovum pick up (OPU). Three operators are required; the third operates the syringe to inject the flushing media to repeatedly flush thefollicles. (B) Oocytes recovered from one OPU session before in vitro maturation. (C) The piezoelectric manipulator used for intracytoplasmic sperm injection(ICSI). (D) Metaphase II oocyte being injected with a sperm. The arrow points to the sperm being delivered to the oocyte. (E) Blastocysts resulting from ICSIaround the time of expansion. Some cells protrude from the hole made by the ICSI pipette. Embryos are frozen or transferred at this stage before they enlarge toomuch. (F) Foal produced by ICSI with the foster mother.

C. Galli et al. / Theriogenology 81 (2014) 138–151 143

3 days as a preventive measure. Other programs have re-ported a fixed, biweekly schedule [59] on experimentalyoung animals; however, for aged or infertile donors, as arethose enrolled in the clinical OPU program, it is difficult toestablish a fixed schedule and we prefer to adapt thecollection to the need of the individual donors.

3. Intracytoplasmic sperm injection

The clinical use of ICSI was developed many years ago asa solution to some sperm-relatedmale infertility in human-assisted reproduction [60]. The technique now works soefficiently that in many clinics it is the preferred way ofin vitro fertilization in humans. There are suggestions that,because sperm selection does not occur in a physiologicway, it may carry abnormalities to the offspring. However,when data analysis is adjusted for age, twin pregnancies,infertility, and other patient-related factors, ICSI offspringdo not present a greater incidence of abnormalities thannormal offspring [61,62].

3.1. Cattle

The use of ICSI in cattle has never attractedmuch interestand has mainly been used for research purposes, essentiallybecause IVF after heparin capacitationworks very efficientlywith the majority of bulls. On the other hand, it would bevery time consuming and expensive to fertilize the numbersof oocytes that are dealt with in a cattle OPU/IVF program tobe viable in a commercial setting. Moreover, the efficiency incattle is not comparable with that in humans [63]. Afterinitial experiments, where blastocyst formation [64–66]rates were generally lower than with IVF, the work wasdirected toward improving activation protocols to increaseembryo development [67]; few publications reported thebirth of offspring [63,68]. It is not clear whether the reduceddevelopment to blastocyst, after ICSI, is owing to inadequateoocyte activation [69] or cytoskeletal damage induced by theinjection procedure [70]. Most of the researchers usepiezoelectric manipulator [71] (Fig. 4C) that greatly facilitatethe penetration of the oolemmawith little pressure. The sizeof bovine spermatozoa require a pipette with a relatively

Table 4Comparison of the ovum pick up (OPU) technique performed on mares of different breed in a clinical context.

Breed No. ofdonors

No.OPU

No. offollicles

No. ofoocytes

Recovery(%)

No. of MIIinjections

MII (%) No. cleaved Cleavage rate No. of embryos %/oocytes

Arabian 56 214 3726 2607 69.97 1526 58.53a 680 44.56a 63 2.42aPer OPU 17.41 12.18 7.13 3.18 0.29

Warmblood 54 110 1683 1170 69.52 813 69.49b 509 62.61b 92 7.86bPer OPU 15.30 10.64 7.39 4.63 0.84

Quarter 22 43 611 404 66.12 265 65.59b 154 58.11b 26 6.44bper OPU 14.21 9.40 6.16 3.58 0.6

Values with different letters within columns differ significantly (chi square test; P < 0.05).Abbreviation: MII, metaphase II.

C. Galli et al. / Theriogenology 81 (2014) 138–151144

large outer diameter of 10 mm that could be responsible forthe damage on the cytoskeleton reducing the developmentalpotential [63]. Bovine ICSI could be valuable as a model forconservation biology of other ruminants by using subopti-mal or dead sperm samples that cannot be used successfullyin IVF [72], or for genetic engineering [73].

3.2. Buffalo

In buffalo, ICSI has only been attempted very recently,demonstrating that, contrary to bovine, without oocyteactivation there is no pronuclear formation [74]. The sameauthors reported, however, that the embryos resultingfrom ICSI and partenogenetic activation were in fact par-thenotes [75] because they did not express genes of thepaternal genome. Different sperm pretreatment, like theuse of dithiothreitol found to be effective on bovine sperm,stimulated the male pronuclear formation [76]. With thepresent results, there is no foreseeable possibility of usingICSI for buffalo embryo production.

3.3. Horse

The use of ICSI in the horse has been fundamental to thedevelopment of assisted reproduction techniques becauseof the difficulties to achieve IVF by simple co-incubation ofthe female and males gametes. In fact, only two foals havebeen reported from IVF of in vivo-matured oocytes [77,78].The first pregnancy, derived from an in vitro-maturedoocyte fertilized by ICSI, was carried successfully to term asreported by Squires, et al. [79]. This success was followedby a period of variable results until the development of ICSIusing the piezo drill (Fig. 4C), which eliminated most of theinconsistency of the technique owing to a heterogeneousand thick zona pellucida difficult to penetrate with con-ventional ICSI pipettes, especially of in vitro matured oo-cytes. Moreover, the additional advantage of ICSI comparedwith IVF is the possibility to widen the choice of the stal-lions to be used, including those with poor sperm motilityand inferior reproductive performance in vivo, which is themain reason why ICSI was developed (Fig. 4D). In an orig-inal study, Lazzari, et al. [80] compared the developmentalpotential of in vitro matured oocytes fertilized by ICSI withfrozen stallion semen of different motility and/or differentfertility in the field. They found no differences in eithercleavage or blastocyst development rates among oocytesinjected with sperm from stallions of good, poor, and nofertility in the field, as long as a motile sperm was selected

for ICSI. In contrast, when at the time of ICSI it was notpossible to select a live sperm before injection, there wasno development to blastocyst. In a different study [81],oocytes injected with nonmotile sperm isolated fromsemen subjected to two freeze–thaw cycles led to blasto-cyst development. In a further study, the same authors [82]have even used lyophilized sperm and activation of theoocyte with sperm extract to generate offspring.

To date, there are a growing number of laboratories thathave consistently and reproducibly reported the birth ofICSI foals both from in vivo- and in vitro-matured oocytes;presently, a few equine practices are starting to use it forclinical treatment of infertility. Some groups have reportedthe occasional birth of foals after ICSI of in vitro-maturedoocytes, after either surgical transfer of early embryos tothe oviduct [83] or in vitro embryo culture to the blastocyststage and transcervical transfer to the uterus [84]. Theprogress of in vitro maturation and ICSI technology hasincreased efforts to design suitable culture systems forearly cleavage stage embryos. Many different culture con-ditions have been reported for pre-implantation develop-ment of ICSI fertilized horse oocytes, including definedmedia such as G1.2 [85], Dulbecco’s Modified Eagle Me-dium/Nutrient Mixture F-12 and CZB [86], and modifiedsynthetic oviduct fluid [87]. Co-culture with somatic cellsin earlier work evaluated the use of Vero cells [88], oviductepithelial cells [89], cumulus cells [84], granulosa cells [90],or culture in conditioned media [91]. In most of thesesystems, however, the blastocyst rates remained low,ranging from 4% to 16%, a rate that is comparable withparthenogenetic activation [92]. A comparison between thepublished reports on in vivo culture of ICSI early cleavagestage embryos in the oviducts of mares [86] or temporaryrecipient sheep [80,93] and in vitro culture in various cul-ture media clearly demonstrated that the culture in vivosupports greater blastocyst development, being approxi-mately 36% of injected oocytes in both the mare oviductand the surrogate sheep oviduct. In another study, anin vitro culture system based on Dulbecco’s Modified EagleMedium/Nutrient Mixture F-12 medium under a mixed gasatmosphere provided blastocyst development similar tothat seen in vivo (27%–38%) [59,94]. However, when day 7blastocyst cell numbers are accounted for [95], in vitro-produced embryos cultured in a modified synthetic oviductfluid had significantly fewer cells, being more similar to aday 5 in vivo embryo. This difference has to be taken intoaccount when in vitro produced embryos are transferred,by using recipients 5 day after ovulation. There is, however,

C. Galli et al. / Theriogenology 81 (2014) 138–151 145

an advantage of having embryos at an earlier develop-mental stage, that is the improvement of their survival atcryopreservation. In fact, it is well-known that in vivo-recovered equine embryos, because of their large size whenthey are normally flushed from the uterus, do not freezesuccessfully. Cryopreservation of in vitro-produced em-bryos at the early blastocyst or blastocyst stage (Fig. 4E)leads to a very high post-thaw survival and pregnancy ratethat exceed 60% [8]. This efficiency with in vitro-producedembryos is not documented in other livestock species.

4. Somatic cell nuclear transfer

4.1. Bovine

Nuclear transfer in general and SCNT in particular areroutine techniques in many laboratories worldwide, bothfor farm animals and laboratory species. Among livestock,cattle have been cloned in the greatest number because ofthe potential commercial interests of the cattle industryand breeders. As a technique, SCNT is demanding. It iscomplicated to master all the procedures involved to levelsof efficiency and reproducibility that are scientifically solid[96,97] and have a practical relevance to the cattle industryproducing live offspring consistently.

All techniques used in cattle today are based, in princi-ple, on those described byWilladsen [98]. Basically, there isa requirement for large numbers of matured, good qualityoocytes to be enucleated. Then, the donor cell must befused to or injected in the enucleated oocyte. Finally, thereconstructed embryo needs to be activated and developedin vitro. For large animals like cattle, to perform a nonsur-gical uterine transfer the cloned embryos need to becultured to the blastocyst stage and/or cryopreservedbefore embryo transfer into recipients [96]. As the tech-nology of SCNT stands today, a considerable source ofvariation comes from the laboratory experience, the tech-nician performing the work, and the biological materialused. Essentially, all SCNT work is done with metaphase IIoocytes [99,100] that are used as recipient cytoplasts,although one study reported success with cytoplastsderived after enucleation of zygotes [101] that are physio-logically activated by sperm. Quiescent G0 is the preferredcell-cycle stage of the donor somatic cells, but also cyclingcells [102] and blood leukocytes [103] have been used asnuclear donors. Generally, donor cells come from primarycell lines established from a tissue biopsy and cry-opreserved for successive, repeated uses. Many cell typespresent in the body have been used for SCNT with differentdegree of success; however, there has not been a singlesomatic cell type that consistently performs better thanothers [104]. Moreover, the differentiation status of thesomatic cells has no correlationwith the ability to generateoffspring [105]. Technical modifications such as the zona-free manipulation have improved the efficiency of enucle-ation and fusion and have reduced the labor required[106,107] in several species beside cattle [108], and limitedavailable data indicate that development to term is thesame as conventional zona-enclosed methods.

In vitro culture of SCNT embryos is performed underconditions substantially similar to those used for IVF

embryos [108]; however, when the zona-free system isused, it is necessary to prevent contact between individualembryos during culture. This can be achieved using thewell of the well system [109]. Culture conditions can alterthe epigenetic status to the extent that subpopulations ofsomatic cells can be selected with different ability to sus-tain normal development into viable offspring after nucleartransfer. Culture conditions can also affect the karyotype ofthe cells and chromosomal abnormalities can accumulatein aged cells or after a time in culture resulting in abnormalembryos [110]. After nuclear transfer, the nucleus un-dergoes so-called nuclear reprogramming, a series ofevents that, by interacting with the oocyte cytoplasm, in-duces changes to the structure of the chromatin toward apluripotent pattern that is more representative of embry-onic development. Empirical attempts to facilitate thisreprogramming process have been made using chemicalsthat are known to alter the methylation status of thechromatin, such as trichostatin A, azacytidine, or Scriptaid,either before or after nuclear transfer. In the mouse, the useof trichostatin A (an histone deacetylase inhibitor) signifi-cantly increased the success rate of mouse cloning[111,112]. On the other hand, this finding remains contro-versial in other species like cattle [113–116]. The majorlimiting factor to the widespread use of SCNT in cattlefarms is the high rate of pregnancy losses throughoutpregnancy. The most problematic are late abortions (after 5or 6 months of gestation), and greater perinatal mortalitythat create economic losses and welfare issues. A compar-ative embryo transfer study between IVP embryos andcloned embryos derived from embryonic, fetal, and adultcells demonstrated that, although the initial pregnancy rateat 21 days is similar (from 55.6% to 62.7%), significant dif-ferences becomes evident by 70 days (49% vs. 37.3% vs.22.5% vs. 14.3% for IVP embryos and embryonic, fetal, andadult cell clones, respectively) and at calving (49% vs. 34.3%vs. 15% vs. 6.8%) [117]. Several studies have demonstratedthat SCNT embryos present an altered gene expressionprofile [118] and epigenetic status [119] compared with IVPembryos, and that the high rate of pregnancy loss has beenclinically associated with hydrops and cotyledonary hy-perplasia [120,121]. These problems are, however, notobserved in the offspring of clones, which are normal [122].It is possible to detect and interrupt earlier on in gestationpregnancies at risk by measuring the maternal concentra-tion of pregnancy associated glycoproteins and or by ul-trasound monitoring [123,124]. These preventive measureshave prompted the International Embryo Transfer Societyto issue guidelines on the management of cloned preg-nancies (available from: http://www.iets.org/pdf/HASAC-HealthAssessmentCare.pdf) to control possible recipientwelfare problems. From a practical perspective for thecattle breeding industry, SCNT is characterized by a lowefficiency owing to high pregnancy losses and perinatalmortality documented in extensive field studies [124,125].This makes the production of offspring expensive andjustified today only when progeny-tested bulls or cham-pion dams are cloned for reproductive purposes. Manytop-ranking bulls have been cloned by AI companies as aninsurance for the bull or because the original bull stoppedproducing semen because of accident or disease. The same

C. Galli et al. / Theriogenology 81 (2014) 138–151146

rationale is applied in the case of outstanding dams. InEurope, the implementation of cattle cloning is facingstrong opposition to the limit that all cattle cloning workhas been stopped at both scientific and practical levels. Inother parts of the world (e.g., South America or China),there is great interest in SCNT for cattle breeding. The safetyof the cloned animals and their products has beendemonstrated in several studies [126,127] and regulatoryagencies have carried out extensive risk assessment [128],concluding that cloned animals and their product do notpose any health risk to consumers. Still in Europe, opposi-tion to the use of cloned cattle is based on ethical, ideo-logical, and welfare arguments. It will take years to changeattitudes toward new biotechnologies like cloning within afavorable regulatory framework [129]. In Japan, just like inEurope, there is an absolute governmental ban on thecommercial use of any cloning-related products. All thishappened after a Japanese investigation that concludedthat products from cloned cattle are harmless.

4.2. Buffalo

Attempts to clone buffalo by nuclear transfer were firstreported by Kitiyanant, et al. [130]; however, despite theagricultural interest for this species in tropical and sub-tropical regions, only two studies from the same grouphave reported the birth of live offspring [131,132]. Thisdifficulty summarizes the problems encountered in generalwith assisted reproduction in the buffalo and with SCNT inparticular. The technique used in buffalo is the same as incattle and the limits encountered with the buffalo seems tobe similar [133]. Several investigators, to overcome the lowdevelopmental competence of buffalo oocytes, have usedcattle oocytes. Some initial studies reported blastocystdevelopment of these interspecies nuclear transfer em-bryos [130,134], but never presented data about embryotransfer of such embryos. A more recent and detailed studyhas failed to replicate these findings, demonstrating thatbuffalo somatic cell nuclei do not activate the embryonicgenome in cattle oocytes and arrest at the 8- to 16-cellstage [135,136].

4.3. Equine

Equine cloning by SCNT was first described in 2003[137]. In the same year, three mules were produced fromfetal somatic cells and in vivo-matured oocytes recoveredby OPU from superovulated preovulatory follicles [138] andtransferred to the oviducts of a recipient immediately afteractivation. The first horse, a filly, was originated by nucleartransfer of adult somatic cells into in vitro-matured oocytesand culture to the blastocyst stage before nonsurgicaltransfer to recipient mares [137] (Fig. 5B). In that study, thefoal was carried by same mare that was the source of thecells used for cloning, representing an exclusive example ofautologous pregnancy successfully gone to term in equines.Two years later, other cloned foals were born [139,140]from selected champion donors, demonstrating the po-tential use of this technology to rescue gelded genotypeswith high sporting value that are clearly unable to generateany offspring (Fig. 5C). More foals have been produced since

then [141] in the same laboratories and in another labora-tory [142]. There is also SCNT activity carried out in com-mercial laboratories reported in the popular press, butthere is no published scientific information about thenumbers performed and the results. The success of SCNT inthe horse is the result of the optimization of many stepsinvolved in the cloning procedure, the principles being thesame as in other species. The first step is the derivation andcryopreservation of a cell line, usually starting from a skinbiopsy of the donor animal to be cloned. Blastocyst devel-opment of SCNT embryos is influenced by the cell line[140], which is well demonstrated in other species andvaries from 0% to 17%. In a large dataset obtained in ourlaboratory, the overall blastocyst rate was 4.4% (83 blasto-cysts from 1866 cleaved embryos from a total of 2099reconstructed embryos). Oocyte maturation in vitro is theonly sustainable source of oocytes for cloning and theirquality is as critical as it is for ICSI. Limitations include thenumber of ovaries that can be recovered at the slaughter-house, recovery that is possible only in countries that allowthe slaughter of equines, and the small number of folliclesthat are present at any time on the ovaries. In our labora-tory, on average we recover 3 to 5 oocytes per ovary suit-able for in vitromaturation. In our experience, the use of thezona-free method maximizes the limited number of oo-cytes available for any given day by obtaining highenucleation and cell fusion rates of the somatic cells tothe oocyte [108] or by using a piezoelectric device bothfor enucleation and microinjection of the somatic celldirectly into the oocyte [139]. Equine reconstructed em-bryos are difficult to activate. We use a combination of thetwo most common chemicals used in other species: 6-dimethylaminopurine and cycloheximide [92], obtainingan activation rate exceeding 90%. Other workers [139]used a combination of injection of sperm extract and cul-ture in 6-dimethylaminopurine to produce embryosresulting in cloned offspring. Culture requirement forcloned embryos are the same as for ICSI embryos. Using thezona-free method (Fig. 5A), we were able to obtain pre-implantation development in the range of 17% to 25%,comparable with the rates obtained by ICSI of slaughter-house oocytes (Lagutina I, unpublished data). Cloned em-bryos can also be successfully cryopreserved and we haveobtained offspring after embryo transfer (Galli C, unpub-lished data). We found that cloned embryos have a lesserability to establish pregnancies compared with ICSI em-bryos [140]. For this reason, we normally implant twocloned embryos per recipient; occasionally, twin pregnan-cies result that can be reduced to singleton by transrectalmanipulation or transvaginal ultrasound-guided ablation.The development to term of cloned pregnancies is low as inother species; however, most of the pregnancy losses occurearly in gestation (before day 50), thus creating fewerproblems with recipient management. Moreover, the foal-ings are normal and there are no reports of the problemsdescribed in ruminants, such as hydrops, placenta hyper-plasia, and large offspring syndrome. Although perinatalmortality has been reported, most of the foals are normal orrequire minor assistance at birth [143] and develop intoadult fertile animals [1] (Fig. 5D). So far, the number ofanimals generated by SCNT is very small and equine

Fig. 5. (A) Horse cloned embryos produced with the zona-free method. (B) Prometea, the first cloned horse, standing in front of her mother and twin sister. (C)Pieraz, a colt cloned from a gelded endurance champion. (D) Prometea with her foal born after artificial insemination.

C. Galli et al. / Theriogenology 81 (2014) 138–151 147

cloning is likely to remain a very specialized niche marketfor a few outstanding champions [144] that cannot repro-duce otherwise, to be able to generate offspring from them.Equine cloning has not raised the issues about food safetyor animal welfare that have limited cattle cloning; how-ever, some association like the Jockey Club or the AmericanQuarter Horse Association oppose the use of cloning forbreeding purposes. On the contrary last year the FédérationEquestre Internationale announced that it will allow clonedhorses to compete in international events. The FédérationEquestre Internationale position is the first concrete signthat the industry is considering cloning of potential valuefor horse breeding and sporting. This decision will stimu-late the further development of horse cloning and arenewed interest in some branches of the horse industry.

In this article, we have discussed the contribution of OPU,ICSI, and SCNT in practical terms to the breeding of cattle,buffalo, and horses in the last 25 years. We did not addressthe potential use of these technologies for biotechnologyand biomedical purposes. It is remarkable that Theriogenol-ogy has provided the main vehicle for the publication ofresearch and development in these areas, as attested to byour reference list. Ovum Pick Up and subsequent embryoproduction in cattle has reached a mature stage and willgradually overtake in vivo production by superovulation, asshown by the trend of the International Embryo TransferSociety statistics. In buffalo, the translation and imple-mentation of these technologies is relatively recent; how-ever, given the limits of superovulation and embryo flushing,OPUwill be, even at the current efficiency, themain route forgenetic improvement and breeding of elite animals. Thehorse is also benefiting from OPU essentially tailored onindividual clinical needs of mares and stallions. Embryo

culture and ICSI provide practical solutions both for IVF andembryo cryopreservation. This can create new opportunitiesfor horse breeders and open amarket for horse embryos andfor banking the best genetics. In the last 15 years, SCNT hasnot improved much in cattle, buffalo, or horses, and it willremain a very small niche to generate copies of outstandinganimals. Future work to advance the field will concentrateon understanding nuclear reprogramming as well as theidentification of possible markers that will allow selection ofthe few normal embryos for transfer. The evolution andapplication of these new techniques will follow not only thetechnical feasibility and the economics, but also the publicperception, which could be quite different in different partsof the world.

Acknowledgments

The authors acknowledge the technical support over theyears, to generate the data discussed in the paper, ofGabriella Crotti, Paola Turini, Massimo Iazzi, and the manycolleagues, too many to be mentioned, who have contrib-uted ideas, discussions, and information. This manuscriptwas prepared while funded by EU grant FP7-KBBE-2012n�312097 Fecund by grants InnovaB and Superpig fromLombardy Region, by grant Ex Ovo Omnia from Sardiniaand Lombardy Region.

References

[1] Galli C, Lagutina I, Duchi R, Colleoni S, Lazzari G. Somatic cell nu-clear transfer in horses. Reprod Domest Anim 2008;43(Suppl. 2):331–7.

C. Galli et al. / Theriogenology 81 (2014) 138–151148

[2] Humblot P, Le Bourhis D, Fritz S, Colleau JJ, Gonzalez C, GuyaderJoly C, et al. Reproductive technologies and genomic selection incattle. Vet Med Int 2010;2010:192787.

[3] Betteridge K, Rieger D. Embryo transfer and related techniques indomestic animals, and their implications for human medicine.Hum Reprod 1993;8:147–67.

[4] Steptoe PC, Edwards RG. Birth after the reimplantation of a humanembryo. Lancet 1978;2:366.

[5] Dellenbach P, Nisand I, Moreau L, Feger B, Plumere C, Gerlinger P,et al. Transvaginal, sonographically controlled ovarian folliclepuncture for egg retrieval. Lancet 1984;1:1467.

[6] Dellenbach P, Nisand I, Moreau L, Feger B, Plumere C, Gerlinger P,et al. Transvaginal, sonographically controlled ovarian folliclepuncture for egg retrieval. Fertil Steril 1985;44:656–62.

[7] Gleicher N, Friberg J, Fullan N, Giglia RV, Mayden K, Kesky T, et al.EGG retrieval for in vitro fertilisation by sonographicallycontrolled vaginal culdocentesis. Lancet 1983;2:508–9.

[8] Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G. Developmentalcompetence of equine oocytes and embryos obtained by in vitroprocedures ranging from in vitro maturation and ICSI to embryoculture, cryopreservation and somatic cell nuclear transfer. AnimReprod Sci 2007;98:39–55.

[9] Staigmiller R, Moor R. Effect of follicle cells on the maturation anddevelopmental competence of ovine oocytes matured outside thefollicle. Gamete Res 1984;9:221–9.

[10] Brackett BG, Bousquet D, Boice ML, Donawick WJ, Evans JF,Dressel MA. Normal development following in vitro fertilization inthe cow. Biol Reprod 1982;27:147–58.

[11] Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES,Eyestone WH, First NL. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 1986;25:591–600.

[12] Gandolfi F, Moor RM. Stimulation of early embryonic developmentin the sheep by co-culture with oviduct epithelial cells. J ReprodFertil 1987;81:23–8.

[13] Tervit HR, Whittingham DG, Rowson LE. Successful culture in vitroof sheep and cattle ova. J Reprod Fertil 1972;30:493–7.

[14] Lu K, Gordon I, Gallagher M, McGovern H. Pregnancy established incattle by transfer of embryos derived from in vitro fertilisation ofoocytes matured in vitro. Vet Rec 1987;121:259–60.

[15] Stroud B. The year 2011 worldwide statistics of embryo transfer indomestic farm animals. Embryo Transfer Newsletter A Publicationof the International Embryo Transfer Society 2012;30:16–26.

[16] Callesen H, Greve T, Christensen F. Ultrasonically guided aspirationof bovine follicular oocytes. Theriogenology 1987;27:217. abstract.

[17] Pieterse MC, Kappen KA, Kruip TAM, Taverne MAM. Aspiration ofbovine oocytes during transvaginal ultrasound scanning of theovaries. Theriogenology 1988;30:751–62.

[18] Pieterse MC, Vos PLAM, Kruip TAM, Wurth YA, van Beneden TH,Willemse AH, et al. Transvaginal ultrasound guided follicularaspiration of bovine oocytes. Theriogenology 1991;35:857–62.

[19] Galli C, Lazzari G. Practical aspects of IVM/IVF n cattle. AnimReprod Sci 1996;42:371–9.

[20] Bols PE, Vandenheede JM, Van Soom A, de Kruif A. Transvaginalovum pick-up (OPU) in the cow: a new disposable needle guidancesystem. Theriogenology 1995;43:677–87.

[21] Galli C, Crotti G, Notari C, Turini P, Duchi R, Lazzari G. Embryoproduction by ovum pick up from live donors. Theriogenology2001;55:1341–57.

[22] Kruip TAM, Boni R, Wurth YA, Roelofsen MWM, Pieterse MC. Po-tential use of ovum pick-up for embryo production and breedingin cattle. Theriogenology 1994;42:675–84.

[23] Looney CR, Lindsey BR, Gonseth CL, Johnson DL. Commercial as-pects of oocyte retrieval and in vitro fertilization (IVF) for embryoproduction in problem cows. Theriogenology 1994;41:67–72.

[24] Pontes JH, Silva KC, Basso AC, Rigo AG, Ferreira CR, Santos GM,et al. Large-scale in vitro embryo production and pregnancy ratesfrom Bos taurus, Bos indicus, and indicus-taurus dairy cows usingsexed sperm. Theriogenology 2010;74:1349–55.

[25] Leroy J, Van Soom A, Opsomer G, Goovaerts I, Bols P. Reducedfertility in high-yielding dairy cows: are the oocyte and embryo indanger? Part II. Mechanisms linking nutrition and reduced oocyteand embryo quality in high-yielding dairy cows. Reprod DomestAnim 2008;43:623–32.

[26] Leroy JL, Opsomer G, Van Soom A, Goovaerts IG, Bols PE. Reducedfertility in high-yielding dairy cows: are the oocyte and embryo indanger? Part I. The importance of negative energy balance andaltered corpus luteum function to the reduction of oocyte andembryo quality in high-yielding dairy cows. Reprod Domest Anim2008;43:612–22.

[27] Leroy JL, Rizos D, Sturmey R, Bossaert P, Gutierrez-Adan A, VanHoeck V, et al. Intrafollicular conditions as a major link betweenmaternal metabolism and oocyte quality: a focus on dairy cowfertility. Reprod Fertil Dev 2011;24:1–12.

[28] Merton JS, Ask B, Onkundi DC, Mullaart E, Colenbrander B,Nielen M. Genetic parameters for oocyte number and embryoproduction within a bovine ovum pick-up-in vitro productionembryo-production program. Theriogenology 2009;72:885–93.

[29] Monniaux D, Barbey S, Rico C, Fabre S, Gallard Y, Larroque H. Anti-Mullerian hormone: a predictive marker of embryo production incattle? Reprod Fertil Dev 2010;22:1083–91.

[30] Rico C, Drouilhet L, Salvetti P, Dalbies-Tran R, Jarrier P, Touze JL,et al. Determination of anti-Mullerian hormone concentrations inblood as a tool to select Holstein donor cows for embryo pro-duction: from the laboratory to the farm. Reprod Fertil Dev 2012;24:932–44.

[31] Hasler JF, Henderson WB, Hurtgen PJ, Jin ZQ, McCauley AD,Mower SA, et al. Production, freezing and transfer of bovine IVFembryos and subsequent calving results. Theriogenology 1995;43:141–52.

[32] Merton JS, de Roos AP, Mullaart E, de Ruigh L, Kaal L, Vos PL, et al.Factors affecting oocyte quality and quantity in commercialapplication of embryo technologies in the cattle breeding industry.Theriogenology 2003;59:651–74.

[33] Chastant-Maillard S, Quinton H, Lauffenburger J, Cordonnier-Lefort N, Richard C, Marchal J, et al. Consequences of transvaginalfollicular puncture on well-being in cows. Reproduction 2003;125:555–63.

[34] Machatkova M, Krausova K, Jokesova E, Tomanek M. Develop-mental competence of bovine oocytes: effects of follicle size andthe phase of follicular wave on in vitro embryo production.Theriogenology 2004;61:329–35.

[35] Bousquet D, Twagiramungu H, Morin N, Brisson C, Carboneau G,Durocher J. In vitro embryo production in the cow: an effectivealternative to the conventional embryo production approach.Theriogenology 1999;51:59–70.

[36] Chaubal SA, Ferre LB, Molina JA, Faber DC, Bols PE, Rezamand P,et al. Hormonal treatments for increasing the oocyte and embryoproduction in an OPU-IVP system. Theriogenology 2007;67:719–28.

[37] De Roover R, Feugang JM, Bols PE, Genicot G, Hanzen C. Effects ofovum pick-up frequency and FSH stimulation: a retrospectivestudy on seven years of beef cattle in vitro embryo production.Reprod Domest Anim 2008;43:239–45.

[38] Sirard MA, Picard L, Dery M, Coenen K, Blondin P. The time in-terval between FSH administration and ovarian aspiration in-fluences the development of cattle oocytes. Theriogenology 1999;51:699–708.

[39] Monteiro FM, Ferreira MMG, Potiens JR, Eberhardt BG, Trinca LA,Barros CM. Influence of superovulatory protocols on in vitro pro-duction of nellore (Bos indicus) embryos. Reprod Domest Anim2010;45:860–4.

[40] Pontes JHF, Melo Sterza FA, Basso AC, Ferreira CR, Sanches BV,Rubin KCP, et al. Ovum pick up, in vitro embryo production,and pregnancy rates from a large-scale commercial programusing Nelore cattle (Bos indicus) donors. Theriogenology 2011;75:1640–6.

[41] Carvalho NA, Baruselli PS, Zicarelli L, Madureira EH, Visintin JA,D’Occhio MJ. Control of ovulation with a GnRH agonist aftersuperstimulation of follicular growth in buffalo: fertilization andembryo recovery. Theriogenology 2002;58:1641–50.

[42] Misra AK, Joshi BV, Agrawala PL, Kasiraj R, Sivaian S,Rangareddi NS, et al. Multiple ovulation and embryo transfer inIndian buffalo (Bubalus buoalis). Theriogenology 1990;33:1131–41.

[43] Techakumph M, Sukavong Y, Yienvisavakul V, Buntaracha B,Pharee S, Intaramongkol S, et al. The transfer of fresh and frozenembryos in an elite swamp buffalo herd. J Vet Med Sci 2001;63:849–52.

[44] Boni R, Roviello S, Zicarelli L. Repeated ovum pick-up in ItalianMediterranean buffalo cows. Theriogenology 1996;46:899–909.

[45] Galli C, Duchi R, Crotti G, Lazzari G. Embryo production by ovumpick up in water buffalo. Theriogenology 1998;49:400. abstract.

[46] Di Francesco S, Novoa MV, Vecchio D, Neglia G, Boccia L,Campanile G, et al. Ovum pick-up and in vitro embryo production(OPU-IVEP) in Mediterranean Italian buffalo performed in differentseasons. Theriogenology 2012;77:148–54.

[47] Techakumphu M, Promdireg A, Na-Chiengmai A, Phutikanit N.Repeated oocyte pick up in prepubertal swamp buffalo (Bubalus

C. Galli et al. / Theriogenology 81 (2014) 138–151 149

bubalis) calves after FSH superstimulation. Theriogenology 2004;61:1705–11.

[48] Liang XW, Lu YQ, Chen MT, Zhang XF, Lu SS, Zhang M, et al. In vitroembryo production in buffalo (Bubalus bubalis) using sexed spermand oocytes from ovum pick up. Theriogenology 2008;69:822–6.

[49] Galli C, Duchi R, Lazzari G, Lagutina I, Colleoni S, Turini P, et al.Pregnancies and calves after transfer of in vitro-produced riverbuffalo embryos. Reprod Fertil Dev 2012;24:211. abstract.

[50] Carnevale EM, Coutinho da Silva MA, Panzani D, Stokes JE,Squires EL. Factors affecting the success of oocyte transfer in aclinical program for subfertile mares. Theriogenology 2005;64:519–27.

[51] Altermatt JL, Suh TK, Stokes JE, Carnevale EM. Effects of age andequine follicle-stimulating hormone (eFSH) on collection andviability of equine oocytes assessed by morphology and develop-mental competency after intracytoplasmic sperm injection (ICSI).Reprod Fertil Dev 2009;21:615–23.

[52] Carnevale EM. Oocyte transfer and gamete intrafallopian transferin the mare. Anim Reprod Sci 2004;82–83:617–24.

[53] Cochran R, Meintjes M, Reggio B, Hylan D, Carter J, Pinto C, et al.Production of live foals from sperm-injected oocytes harvestedfrom pregnant mares. J Reprod Fertil Suppl 2000;56:503–12.

[54] Mckinnon A, Lacham-Kaplan O, Trounson A. Pregnancies producedfrom fertile and infertile stallions by intracytoplamic sperm in-jection (ICSI) of single frozen/thawed spermatozoa into in vitromatured mare oocytes. Paper presented at: 7th InternationalSymposium on Equine Reproduction 137 (abstr.). University ofPretoria. July 12–17, 1998; Pretoria, South Africa.

[55] Bruck I, Raun K, Synnestvedt B, Greve T. Follicle aspiration in themare using a transvaginal ultrasound-guided technique. EquineVet J 1992;24:58–9.

[56] Vanderwall DK, Hyde KJ, Woods GL. Effect of repeated transvaginalultrasound-guided follicle aspiration on fertility in mares. J Am VetMed Assoc 2006;228:248–50.

[57] Colleoni S, Barbacini S, Necchi D, Duchi R, Lazzari G, Galli C.Application of ovum pick-up, intracytoplasmic sperm injectionand embryo culture in equine practice. Proceedings of theAmerican Association for Equine Practitioners (AAEP) 2007;53:554–559.

[58] Mari G, Barbara M, Eleonora I, Stefano B. Fertility in the mare afterrepeated transvaginal ultrasound-guided aspirations. Anim ReprodSci 2005;88:299–308.

[59] Jacobson CC, Choi YH, Hayden SS, Hinrichs K. Recovery of mareoocytes on a fixed biweekly schedule, and resulting blastocystformation after intracytoplasmic sperm injection. Theriogenology2010;73:1116–26.

[60] Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnanciesafter intracytoplasmic injection of single spermatozoon into anoocyte. Lancet 1992;340:17–8.

[61] Palermo GD, Neri QV, Monahan D, Kocent J, Rosenwaks Z. Devel-opment and current applications of assisted fertilization. FertilSteril 2012;97:248–59.

[62] Palermo GD, Neri QV, Takeuchi T, Squires J, Moy F, Rosenwaks Z.Genetic and epigenetic characteristics of ICSI children. ReprodBiomed Online 2008;17:820–33.

[63] Galli C, Vassiliev I, Lagutina I, Galli A, Lazzari G. Bovine embryodevelopment following ICSI: effect of activation, sperm capacita-tion and pre-treatment with dithiothreitol. Theriogenology 2003;60:1467–80.

[64] Catt JW, Rhodes SL. Comparative intracytoplasmic sperm injection(ICSI) in human and domestic species. Reprod Fertil Dev 1995;7:161–6.

[65] Goto K, Kinoshita A, Nakanishi Y, Ogawa K. Blastocyst formationfollowing intracytoplasmic injection of in-vitro derived spermatidsinto bovine oocytes. Hum Reprod 1996;11:824–9.

[66] Goto K, Yanagita K. Normality of calves obtained by intra-cytoplasmic sperm injection. Hum Reprod 1995;10:1554.

[67] Suttner R, Zakhartchenko V, Stojkovic P, Muller S, Alberio R,Medjugorac I, et al. Intracytoplasmic sperm injection in bovine:effects of oocyte activation, sperm pretreatment and injectiontechnique. Theriogenology 2000;54:935–48.

[68] Oikawa T, Takada N, Kikuchi T, Numabe T, Takenaka M, Horiuchi T.Evaluation of activation treatments for blastocyst production andbirth of viable calves following bovine intracytoplasmic sperminjection. Anim Reprod Sci 2005;86:187–94.

[69] Malcuit C, Maserati M, Takahashi Y, Page R, Fissore RA. Intra-cytoplasmic sperm injection in the bovine induces abnormal[Ca2þ]i responses and oocyte activation. Reprod Fertil Dev 2006;18:39–51.

[70] Hara H, Abdalla H, Morita H, Kuwayama M, Hirabayashi M,Hochi S. Procedure for Bovine ICSI, not Sperm Freeze-drying, Im-pairs the Function of the Microtubule-organizing Center. J ReprodDev 2011;57:428–32.

[71] Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in themouse. Biol Reprod 1995;52:709–20.

[72] Garcia-Rosello E, Garcia-Mengual E, Coy P, Alfonso J, Silvestre MA.Intracytoplasmic sperm injection in livestock species: an update.Reprod Domest Anim 2009;44:143–51.

[73] Eghbalsaied S, Ghaedi K, Laible G, Hosseini SM, Forouzanfar M,Hajian M, et al. Exposure to DNA is insufficient for in vitro trans-genesis of live bovine sperm and embryos. Reproduction 2013;145:97–108.

[74] Liang YY, Ye DN, Laowtammathron C, Phermthai T, Nagai T,Somfai T, et al. Effects of chemical activation treatment on devel-opment of swamp buffalo (Bubalus bubalis) oocytes maturedin vitro and fertilized by intracytoplasmic sperm injection. ReprodDomest Anim 2011;46:e67–73.

[75] Chankitisakul V, Tharasanit T, Phutikanit N, Tasripoo K, Nagai T,Techakumphu M. Lacking expression of paternally-expressed geneconfirms the failure of syngamy after intracytoplasmic sperm in-jection in swamp buffalo (Bubalus bubalis). Theriogenology 2012;77:1415–24.

[76] Chankitisakul V, Am-In N, Tharasanit T, Somfai T, Nagai T,Techakumphu M. Sperm pretreatment with dithiothreitol in-creases male pronucleus formation rates after intracytoplasmicsperm injection (ICSI) in swamp buffalo oocytes. J Reprod Dev2013;59:66–71.

[77] Bezard J. In vitro fertilization in the mare. Proceedings of the In-ternational Scientific Conference on Biotechnics in Horse Repro-duction. Cracow, Poland: Agricultural University of Cracow; 1992.p. 12 (abstract).

[78] Palmer E, Bezard J, Magistrini M, Duchamp G. In vitro fertilizationin the horse. A retrospective study. J Reprod Fertil Suppl 1991;44:375–84.

[79] Squires E, Wilson J, Kato H, Blaszczyk A. A pregnancy after intra-cytoplasmic sperm injection into equine oocytes matured in vitro.Theriogenology 1996;306.

[80] Lazzari G, Crotti G, Turini P, Duchi R, Mari G, Zavaglia G, et al.Equine embryos at the compacted morula and blastocyst stage canbe obtained by intracytoplasmic sperm injection (ICSI) of in vitromatured oocytes with frozen-thawed spermatozoa from semen ofdifferent fertilities. Theriogenology 2002;58:709–12.

[81] Choi YH, Love CC, Varner DD, Hinrichs K. Equine blastocystdevelopment after intracytoplasmic injection of sperm subjectedto two freeze-thaw cycles. Theriogenology 2006;65:808–19.

[82] Choi YH, Varner DD, Love CC, Hartman DL, Hinrichs K. Productionof live foals via intracytoplasmic injection of lyophilized sperm andsperm extract in the horse. Reproduction 2011;142:529–38.

[83] Cochran R, Meintjes M, Reggio B, Hylan D, Carter J, Pinto C, et al.Live foals produced from sperm-injected oocytes derived frompregnant mares. J Equine Vet Sci 1998;18:736–40.

[84] Li X, Morris LH, Allen WR. Influence of co-culture during matura-tion on the developmental potential of equine oocytes fertilized byintracytoplasmic sperm injection (ICSI). Reproduction 2001;121:925–32.

[85] Choi YH, Love CC, Love LB, Varner DD, Brinsko S, Hinrichs K.Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm in-jection with fresh or frozen-thawed spermatozoa. Reproduction2002;123:455–65.

[86] Choi YH, Roasa LM, Love CC, Varner DD, Brinsko SP, Hinrichs K.Blastocyst formation rates in vivo and in vitro of in vitro-maturedequine oocytes fertilized by intracytoplasmic sperm injection. BiolReprod 2004;70:1231–8.

[87] Ritchie WA. Nuclear transfer in sheep. Methods Mol Biol 2006;325:11–23.

[88] Dell’Aquila ME, Cho YS, Minoia P, Traina V, Lacalandra GM,Maritato F. Effects of follicular fluid supplementation of in-vitromaturation medium on the fertilization and development ofequine oocytes after in-vitro fertilization or intracytoplasmicsperm injection. Hum Reprod 1997;12:2766–72.

[89] Battut I, Bezard J, Palmer E. Establishment of equine oviduct cellmonolayers for co-culture with early equine embryos. J ReprodFertil Suppl 1991;44:393–403.

[90] Rosati I, Berlinguer F, Bogliolo L, Leoni G, Ledda S, Naitana S. Theeffect of co-culture on the development of in vitro matured equineoocytes after intracytoplastic sperm injection. Equine Vet J 2002;34:673–8.

C. Galli et al. / Theriogenology 81 (2014) 138–151150

[91] Choi YH, Chung YG, Seidel Jr GE, Squires EL. Developmental ca-pacity of equine oocytes matured and cultured in equinetrophoblast-conditioned media. Theriogenology 2001;56:329–39.

[92] Lazzari G, Mari G, Galli C. Synergistic effect of cicloheximide and 6-DMAP on activation of equine and bovine oocytes. J Reprod FertAbstract Series 2002;28:73.

[93] Galli C, Crotti G, Turini P, Duchi R, Mari G, Zavaglia G, et al. Frozen-thawed embryos produced by Ovum Pick Up of immature oocytesand ICSI are capable to establish pregnancies in the horse. Ther-iogenology 2002;58:705–8.

[94] Alm H, Choi YH, Love L, Heleil B, Torner H, Hinrichs K. Holdingbovine oocytes in the absence of maturation inhibitors: kinetics ofin vitro maturation and effect on blastocyst development afterin vitro fertilization. Theriogenology 2008;70:1024–9.

[95] Tremoleda JL, Stout TA, Lagutina I, Lazzari G, Bevers MM,Colenbrander B, et al. Effects of in vitro production on horse em-bryo morphology, cytoskeletal characteristics, and blastocystcapsule formation. Biol Reprod 2003;69:1895–906.

[96] Galli C, Lagutina I, Lazzari G. Introduction to cloning by nucleartransplantation. Cloning Stem Cells 2003;5:223–32.

[97] Galli C, Maclellan LJ, Crotti G, Turini P, Ponderato N, Duchi R, et al.Development of equine oocytes matured in vitro in differentmedia and fertilised by ICSI. Theriogenology 2002;57:718.

[98] Willadsen SM. Nuclear transplantation in sheep embryos. Nature1986;320:63–5.

[99] Campbell KH, Loi P, Otaegui PJ, Wilmut I. Cell cycle co-ordinationin embryo cloning by nuclear transfer. Rev Reprod 1996;1:40–6.

[100] Oback B, Wells DN. Cloning cattle. Cloning Stem Cells 2003;5:243–56.

[101] Schurmann A, Wells DN, Oback B. Early zygotes are suitable re-cipients for bovine somatic nuclear transfer and result in clonedoffspring. Reproduction 2006;132:839–48.

[102] Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, et al.Cloned transgenic calves produced from nonquiescent fetal fibro-blasts. Science 1998;280:1256–8.

[103] Galli C, Duchi R, Moor RM, Lazzari G. Mammalian leukocytescontain all the genetic information necessary for the developmentof a new individual. Cloning 1999;1:161–70.

[104] Colleoni S, Donofrio G, Lagutina I, Duchi R, Galli C, Lazzari G.Establishment, differentiation, electroporation, viral transduction,and nuclear transfer of bovine and porcine mesenchymal stemcells. Cloning Stem Cells 2005;7:154–66.

[105] Oback B, Wells DN. Donor cell differentiation, reprogramming, andcloning efficiency: elusive or illusive correlation? Mol Reprod Dev2007;74:646–54.

[106] Oback B, Wiersema AT, Gaynor P, Laible G, Tucker FC, Oliver JE,et al. Cloned cattle derived from a novel zona-free embryoreconstruction system. Cloning Stem Cells 2003;5:3–12.

[107] Vajta G, Lewis IM, Trounson AO, Purup S, Maddox-Hyttel P,Schmidt M, et al. Handmade somatic cell cloning in cattle: analysisof factors contributing to high efficiency in vitro. Biol Reprod 2003;68:571–8.

[108] Lagutina I, Lazzari G, Duchi R, Turini P, Tessaro I, Brunetti D, et al.Comparative aspects of somatic cell nuclear transfer with con-ventional and zona-free method in cattle, horse, pig and sheep.Theriogenology 2007;67:90–8.

[109] Vajta G, Peura TT, Holm P, Paldi A, Greve T, Trounson AO, et al. Newmethod for culture of zona-included or zona-free embryos: theWell of the Well (WOW) system. Mol Reprod Dev 2000;55:256–64.

[110] Balbach ST, Jauch A, Bohm-Steuer B, Cavaleri FM, Han YM,Boiani M. Chromosome stability differs in cloned mouse embryosand derivative ES cells. Dev Biol 2007;308:309–21.

[111] Kishigami S, Mizutani E, Ohta H, Hikichi T, Thuan NV, Wakayama S,et al. Significant improvement of mouse cloning technique bytreatment with trichostatin A after somatic nuclear transfer. Bio-chem Biophys Res Commun 2006;340:183–9.

[112] Rybouchkin A, Kato Y, Tsunoda Y. Role of histone acetylation inreprogramming of somatic nuclei following nuclear transfer. BiolReprod 2006;74:1083–9.

[113] Iager AE, Ragina NP, Ross PJ, Beyhan Z, Cunniff K, Rodriguez RM,et al. Trichostatin A improves histone acetylation in bovine so-matic cell nuclear transfer early embryos. Cloning Stem Cells 2008;10:371–9.

[114] Sangalli JR, De Bem TH, Perecin F, Chiaratti MR, Oliveira Lde J, deAraujo RR, et al. Treatment of nuclear-donor cells or cloned zy-gotes with chromatin-modifying agents increases histone acety-lation but does not improve full-term development of clonedcattle. Cell Reprogram 2012;14:235–47.

[115] Wang YS, Xiong XR, An ZX, Wang LJ, Liu J, Quan FS, et al. Pro-duction of cloned calves by combination treatment of both donorcells and early cloned embryos with 5-aza-2/-deoxycytidine andtrichostatin A. Theriogenology 2011;75:819–25.

[116] Wu X, Li Y, Li GP, Yang D, Yue Y, Wang L, et al. Trichostatin Aimproved epigenetic modifications of transfected cells but did notimprove subsequent cloned embryo development. Anim Bio-technol 2008;19:211–24.

[117] Heyman Y, Chavatte-Palmer P, LeBourhis D, Camous S, Vignon X,Renard JP. Frequency and occurrence of late-gestation losses fromcattle cloned embryos. Biol Reprod 2002;66:6–13.

[118] Smith SL, Everts RE, Tian XC, Du F, Sung LY, Rodriguez-Zas SL, et al.Global gene expression profiles reveal significant nuclear reprog-ramming by the blastocyst stage after cloning. Proc Natl Acad SciUSA 2005;102:17582–7.

[119] Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E,et al. Conservation of methylation reprogramming in mammaliandevelopment: aberrant reprogramming in cloned embryos. ProcNatl Acad Sci USA 2001;98:13734–8.

[120] Everts RE, Chavatte-Palmer P, Razzak A, Hue I, Green CA, Oliveira R,et al. Aberrant gene expression patterns in placentomes areassociated with phenotypically normal and abnormal cattle clonedby somatic cell nuclear transfer. Physiol Genomics 2008;33:65–77.

[121] Heyman Y, Zhou Q, Lebourhis D, Chavatte-Palmer P, Renard JP,Vignon X. Novel approaches and hurdles to somatic cloning incattle. Cloning Stem Cells 2002;4:47–55.

[122] Heyman Y, Richard C, Rodriguez-Martinez H, Lazzari G, Chavatte-Palmer P, Vignon X, et al. Zootechnical performance of clonedcattle and offspring: preliminary results. Cloning Stem Cells 2004;6:111–20.

[123] Chavatte-Palmer P, de Sousa N, Laigre P, Camous S, Ponter AA,Beckers JF, et al. Ultrasound fetal measurements and pregnancyassociated glycoprotein secretion in early pregnancy in cattle re-cipients carrying somatic clones. Theriogenology 2006;66:829–40.

[124] Panarace M, Aguero JI, Garrote M, Jauregui G, Segovia A, Cane L,et al. How healthy are clones and their progeny: 5 years of fieldexperience. Theriogenology 2007;67:142–51.

[125] Watanabe S, Nagai T. Survival of embryos and calves derived fromsomatic cell nuclear transfer in cattle: a nationwide survey inJapan. Anim Sci J 2011;82:360–5.

[126] Heyman Y, Chavatte-Palmer P, Fromentin G, Berthelot V, Jurie C,Bas P, et al. Quality and safety of bovine clones and their products.Animal 2007;1:963–72.

[127] Yamaguchi M, Ito Y, Takahashi S. Fourteen-week feeding test ofmeat and milk derived from cloned cattle in the rat. Ther-iogenology 2007;67:152–65.

[128] Rudenko L, Matheson JC. The US FDA and animal cloning: risk andregulatory approach. Theriogenology 2007;67:198–206.

[129] Niemann H, Lucas-Hahn A. Somatic cell nuclear transfer cloning:practical applications and current legislation. Reprod Domest Anim2012;47(Suppl. 5):2–10.

[130] Kitiyanant Y, Saikhun J, Chaisalee B, White KL, Pavasuthipaisit K.Somatic cell cloning in Buffalo (Bubalus bubalis): effects of inter-species cytoplasmic recipients and activation procedures. CloningStem Cells 2001;3:97–104.

[131] Shi D, Lu F, Wei Y, Cui K, Yang S, Wei J, et al. Buffalos (Bubalusbubalis) cloned by nuclear transfer of somatic cells. Biol Reprod2007;77:285–91.

[132] Yang BZ, Yang CY, Li RC, Qin GS, Zhang XF, Pang CY, et al. An inter-subspecies cloned buffalo (Bubalus bubalis) obtained by trans-ferring of cryopreserved embryos via somatic cell nuclear transfer.Reprod Domest Anim 2010;45:e21–5.

[133] Suteevun T, Smith SL, Muenthaisong S, Yang X, Parnpai R, Tian XC.Anomalous mRNA levels of chromatin remodeling genes in swampbuffalo (Bubalus bubalis) cloned embryos. Theriogenology 2006;65:1704–15.

[134] Saikhun J, Pavasuthipaisit K, Jaruansuwan M, Kitiyanant Y. Xen-onuclear transplantation of buffalo (Bubalus bubalis) fetal andadult somatic cell nuclei into bovine (Bos indicus) oocyte cyto-plasm and their subsequent development. Theriogenology 2002;57:1829–37.

[135] Lagutina I, Zakhartchenko V, Fulka H, Colleoni S, Wolf E, Fulka Jr J,et al. Formation of nucleoli in interspecies nuclear transfer em-bryos derived from bovine, porcine, and rabbit oocytes and nucleardonor cells of various species. Reproduction 2011;141:453–65.

[136] Srirattana K, Matsukawa K, Akagi S, Tasai M, Tagami T, Nirasawa K,et al. Constant transmission of mitochondrial DNA in intergenericcloned embryos reconstructed from swamp buffalo fibroblasts andbovine ooplasm. Anim Sci J 2011;82:236–43.

C. Galli et al. / Theriogenology 81 (2014) 138–151 151

[137] Galli C, Lagutina I, Crotti G, Colleoni S, Turini P, Ponderato N, et al.Pregnancy: a cloned horse born to its dam twin. Nature 2003;424:635.

[138] Woods GL, White KL, Vanderwall DK, Li GP, Aston KI, Bunch TD,et al. A mule cloned from fetal cells by nuclear transfer. Science2003;301:1063.

[139] Hinrichs K, Choi YH, Love CC, Chung YG, Varner DD. Production ofhorse foals via direct injection of roscovitine-treated donor cellsand activation by injection of sperm extract. Reproduction 2006;131:1063–72.

[140] Lagutina I, Lazzari G, Duchi R, Colleoni S, Ponderato N, Turini P,et al. Somatic cell nuclear transfer in horses: effect of oocytemorphology, embryo reconstruction method and donor cell type.Reproduction 2005;130:559–67.

[141] Hinrichs K, Choi YH, Varner DD, Hartman DL. Production of clonedhorse foals using roscovitine-treated donor cells and activationwith sperm extract and/or ionomycin. Reproduction 2007;134:319–25.

[142] Gambini A, Jarazo J, Olivera R, Salamone DF. Equine cloning:in vitro and in vivo development of aggregated embryos. BiolReprod 2012;87:15.

[143] Johnson AK, Clark-Price SC, Choi YH, Hartman DL, Hinrichs K.Physical and clinicopathologic findings in foals derived by use ofsomatic cell nuclear transfer: 14 cases (2004-2008). J Am Vet MedAssoc 2010;236:983–90.

[144] Church SL. Nuclear transfer saddles up. Nat Biotechnol 2006;24:605–7.