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Theriogenology 81 (2014) 138151

Contents lists available at ScienceDirect

Theriogenology
journal homepage: www.theriojournal.com

40th Anniversary Special Issue

Ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear
transfer in cattle, buffalo and horses: from the research laboratory to
clinical practice
Cesare Galli a, b, c, *, Roberto Duchi a, Silvia Colleoni a, Irina Lagutina a, Giovanna Lazzari a, c
a
b
c

Avantea, Laboratory of Reproductive Technologies, 26100 Cremona, Italy


Department of Veterinary Medical Sciences, University of Bologna, Italy
Fondazione Avantea, Cremona, Italy

a r t i c l e i n f o

a b s t r a c t

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

Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick
up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have
been transferred and adapted to buffalo and horses. The successful clinical applications of
these techniques require both the clinical skills specic to each animal species and an
experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be
considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the only possibility for embryo production 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 an
established 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 very
efcient and the only option because conventional IVF does not work. Somatic cell nuclear
transfer is destined to ll a very small niche for generating animals of extremely high
commercial value. The efciency is low, but because normal animals can be generated it is
likely that advancing our knowledge in that eld might improve the technology and
reduce its cost.
2014 Elsevier Inc. All rights reserved.

Keywords:
Assisted reproduction
Embryo technologies
Nuclear transfer
Cattle
Buffalo
Horse

1. Introduction
Breeding techniques have always been at the center of any
livestock enterprise, motivated by curiosity and consolidated
by breeders needs and interests. This has been the case, for
example, when articial insemination was developed as an
hygienic measure to prevent disease transmission or at present in the genomics era, where embryos can be genotyped
and/or propagated by somatic cell nuclear transfer (SCNT)
[1,2] to reproduce the desired genotype from selected parents.
Livestock species have provided for decades the knowledge

* Corresponding author. Tel.: 39 0372 437242; fax 39 0372 436133.


E-mail address: cesaregalli@avantea.it (C. Galli).
0093-691X/$ see front matter 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2013.09.008

for assisted reproduction techniques that have been translated to humans. Because of the easy availability of both
gametes and pre-implantation embryos, the closer similarities with the human counterpart and the different ethical
requirements, scientists working with livestock have
contributed to the advancement and consolidation of the
human eld [3]. In turn, many of the more advanced techniques developed for humans have provided a model for animal scientists after the birth of the rst human being
conceived in vitro [4] and following the subsequent developments, including transvaginal ovum pick up (OPU) [57].
The practical application of assisted reproduction technologies in livestock requires the integration of the laboratory techniques with the clinical management of donors,
recipients and newborn animals, because what matters to

C. Galli et al. / Theriogenology 81 (2014) 138151

the client requesting such services is a live offspring. The


drive behind the developments of advanced assisted
reproduction techniques discussed in this article for cattle
and buffalo is the need to generate large numbers of embryos, preferably of predetermined sex and known genotype, for genetic improvement of the herds. On the contrary,
in horses the main reason for applying these technologies
has been for addressing both female as well as male infertility [8], very much as it is done in human assisted reproduction, including pre-implantation genetic diagnosis.
In this paper, we revisit the major developments that
played a role in turning experimental ndings into specialized clinical practices in the breeding of high genetic merit
livestock. We discuss the impact of the techniques as well as
the bottlenecks and the possible future developments. It is
more of a personal view and experience than a comprehensive review of all the literature.
2. Ovum pick up
2.1. Cattle
The development of OPU in cattle followed the establishment of reproducible techniques for in vitro maturation
[9], fertilization [10,11] and culture of sheep [12] and cattle
embryos [13], bringing to the birth of normal offspring [14],
techniques. All the experimental work done to develop
in vitro embryo production (IVP) procedures was essentially based on oocytes that were recovered from slaughterhouse ovaries in large numbers. However, for practical
application, it was clearly desirable to recover oocytes from
living donors of known genetic value.
According to the International Embryo Transfer Society
statistics (Fig. 1), the number of embryos produced in vitro
and transferred into recipients has increased more than 10
times [15] in the last dozen years and are now approaching
the numbers of embryos produced in vivo by superovulation. This indicates that OPU and IVP is considered a reliable
and cost-effective technique and has acquired a role in
cattle breeding.
The rst attempts to use ultrasound-guided follicular
aspiration for embryo production in vitro were reported by
Pieterse and other authors in the late 1980s [1618] by
using a human endovaginal probe adapted for the use in

Fig. 1. Summary of the International Embryo Transfer Society statistical data


collected by the data retrieval committee. In vitro production of embryos is
steadily growing and the majority of them are now produced by ovum pick
up (OPU).

139

cattle. They reported a recovery rate of 55%, repeatability of


the procedure, and absence of side effects on the donor
cows. Although the procedures for embryo production in
those days still required major renements that came later
[19], the basics of OPU described by Pieterse et al. are still
the same used today by many practitioners. Recovery rates
have been improved in the excess of 70% owing to the use
of better ultrasound equipment with 6- or 7-MHz convex
array probes that provide a better resolution on smaller
follicles and the use of gonadotrophin priming that increases the size of smaller follicles that otherwise would
not be picked up by inexperienced operators. Native
endovaginal probes are available essentially for human use
and need to be adapted to hold longer 60 cm long single
lumen needles (Fig. 2). Several manufacturers of ultrasound
equipment have provided custom-made plastic holders
that can house generic convex array transducers together
with the needle guide. A partial simplication of the
equipment was described by Bols, et al. [20] that used
simple hypodermic disposable needles. However, this
simpler setting has the disadvantage of a bigger size that,
although it is ne for cows, can be traumatic for very young
heifers. The use of native endovaginal probes allows the
complete replacement, for each donor animal, not only of
the probe latex cover, but also of the needle guide together
with the needle. This solution uses one sterile long needle
and one needle guide for each OPU session/donor, requiring
more 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 between donors and therefore provides the highest biosafety
standards, as it is done in humans. On the contrary, the
setting with a generic probe enclosed in a plastic adapter
together with the needle guide make it convenient to
replace the disposable and less expensive needle used for
aspiration. However, the needle guide is normally not
replaced from donor to donor, being enclosed in the plastic
adapter. Therefore, there is always some blood that by
capillarity will inltrate in the needle guide and in the
surroundings of the probe cover when the vaginal wall is
punctured. The needle in both settings is connected
through a tubing and a test tube to a vacuum pump that
usually provides a vacuum set at ow rate between 15 and
25 mL/min [21] to ensure maximum recovery with the least
damage to the cumulus oocytes complexes. Flow rates are
more indicative than vacuum pressure because the gauge
of the needle and the length of the tubing can make a big
difference. Simple ushing media for embryo transfer,
supplemented with heparin to avoid follicular uid or
blood clotting, are currently used for oocyte recovery.
The OPU technique was initially applied on problem
cows that did not respond to superovulation [22,23], but
later on it was applied on a wider scale also on pregnant
cows and heifers, including prepubertal heifers [21]. It is
difcult and often not relevant to make comparisons between different dataset because there are so many variables
involved, most of which are not even manageable. Beef
breeds perform better than dairy cows, dry cows do better
than lactating ones, and cows perform better than heifers
(Table 1). Climate conditions with high temperature and

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C. Galli et al. / Theriogenology 81 (2014) 138151

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 adapter
to extend its length, needle guide, and needle. Note the small size of the transducer that will t into very small animals.

heat stress reduce the number of follicles and the quality of


oocytes. Bos Indicus cows have far greater number of follicles compared with Bos Taurus and as a consequence produce proportionally more embryos [24]. In our experience,
the donor animal is a major source of variation both from
the management and genetic point of view. There is now
strong evidence that nutrition plays an important role on
oocyte and embryo quality [2527], as well as the genetic
background [28]. This last aspect has become evident with
the studies on the plasma level of anti-Mllerian hormone
[29], a parameter that can be used in practice to assess the
follicular population present on the ovary of any given
donor [30] to optimize embryo production. However, the
preselection of donors is not always an option, especially
when specic genetic programs require selected individual
donors. For this reasons, much work has been performed to
improve embryo yield. Twice weekly OPU is the most
effective protocol to maximize oocyte recovery without
hormonal stimulation [21,31,32] without any side effects
[33] on donor well-being and avoiding the presence of the
dominant follicle that could negatively affect developmental competence [34]. To increase embryo production
and reduce the labor required in twice weekly OPU protocols, gonadotropin stimulation has been introduced.
However, this technique is not the best solution for problem
donors that usually are subjected to OPU because they have
already failed to produce embryos by superovulation.
Therefore, the choice of combining superovulatory treatment with OPU should be made depending on the type of
donor available and the clinical assessment of the previous
treatments. Several investigators have used gonadotropin
stimulation in OPU protocols [23,3537] ranging from a full
superovulatory dose to shorter treatments for 2 to 3 days

with one or two injections per day in presence of a progesterone releasing device or a corpus luteum. Sirard, et al.
[38] reported that after follicular wave synchronization and
3 days of treatment with six injections of a constant dose of
gonadotropin, an interval of 48 hours before OPU was
optimal for subsequent developmental competence [38].
The positive effect of the gonadotropins can be attributed to
the increase in size of small follicles and to the acquisition of
a higher developmental competence of the oocytes as it
occurs with in vivo-matured oocytes [32]. In prepubertal
calves gonadotropin stimulation is required to obtain an
acceptable level of developmental competence [21]. However, use of gonadotropin stimulation does not seem to be
effective in Bos Indicus donors [39]; it is not used in largescale programs [24,40]. The implementation of an OPU
program requires always the support of a specialized laboratory for embryo production. Moreover, because of the
economic value of the calves born, adequate veterinary
assistance is recommended to minimize losses owing to the
possible incidence of the large offspring syndrome or other
common perinatal pathologies.
Altogether, the cost of producing an embryo by OPU in
dairy cows in Europe could be 50% to 100% greater than by
multiple ovulation and embryo transfer. This greater cost
and the current breeding context in Europe allows for the
use of OPU for a very specialized niche market. Other
conditions in other countries offer different opportunities
also dictated by economics: for example, the large use in
Brazil is certainly determined by the fact that OPU in general works better and it is more cost effective than superovulation in Bos Indicus beef donors. Table 2 estimates
expected efciencies of the various procedures available to
produce embryos in dairy cattle.

C. Galli et al. / Theriogenology 81 (2014) 138151

141

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

No. of
OPU

No. of
follicles

No. of
oocytes

No. of
oocytes
per OPU

No. cleaved

Cleavage (%)

No. of embryos

No. of
embryos
per OPU

% embryos/
oocytes

% embryos/
cleaved

Cow
Pregnant heifers
Pubertal heifers
Prepubertal heifers

2251
97
566
258

41,983
1103
9048
4110

28,852
705
5900
2800

12.81
7.26
10.42
10.85

19,711
494
4052
1885

68.32
70.07
68.68
67.32

5601
139
1054
447

2.49
1.43
1.86
1.73

19.41a
19.72a
17.86b
15.96c

28.42a
28.14a
26.01b
23.71b

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

2.2. Buffalo
In the buffalo, OPU has great potential because superovulation (multiple ovulation and embryo transfer) has
given poor results compared with those in cattle [41,42]
and it has never made an impact on buffalo breeding programs, both because of the limited number of embryos that
can be recovered and also because of the low survival after
cryopreservation [43]. Because of these limits, the OPU
technology has always been of great interest also in buffalo
breeding. The rst OPUs in buffalo were reported by Boni,
et al. [44]. The procedure is performed exactly in the same
way as in cows (Fig. 2C). Oocyte recovery, embryo production, and offspring obtained have been described in
several publications [21,45], but only about 10% to 15% of
the oocytes recovered develop to transferable embryos
(Table 3). In general, the ovaries of buffalo cows and heifers
are small; in addition, the follicles tend to be fewer and of
small diameter (Fig. 3). Therefore, few follicles are available
for OPU and the follicular population is inuenced by the
seasonality reported in the buffalo [46]. Gonadotrophin
stimulation might be benecial [47] for increasing the
small size and paucity of follicles found especially in
anestrous donors. Because of the great value of the female
offspring in buffalo herds, the combination of OPU with
sexed semen [48] and cryopreservation [49] offer the opportunity to accelerate the genetic gain in the buffalo
industry.
2.3. Horse
The recovery of oocytes from the mare was rst applied
to the preovulatory follicle using different procedures,
including laparotomy under general anesthesia, colpotomy,
and aspiration using a long needle placed through the ank
in the paralumbar fossa. However, these approaches were
invasive and their efcacy was limited. The most practical,
less invasive, efcient, and repeatable technique now used
is the ultrasound-guided transvaginal follicular aspiration
using a double-lumen, 12-ga needle [50]. Oocytes can be
collected from the pre-ovulatory follicle that has reached at
least 35 mm in diameter, 24 hours after HCG injection with
the donor showing signs of uterine edema. Only mild
superovulatory treatment has improved oocyte recovery
[51]. Ovulation normally takes place 36 to 40 hours after
hCG administration. Therefore, oocytes collected at 24
hours or later have resumed meiosis, have an expanded
cumulus that facilitate their recovery but require 16 hours
of additional culture before being inseminated. After

recovery from the donor mare and a few hours of culture to


complete maturation, oocytes can be surgically transferred
to inseminated recipients [52] or can be subjected to
intracytoplasmic sperm injection (ICSI) and again transferred surgically to the oviduct of a synchronized recipient
or cultured in vitro up to the blastocyst stage and nally
transferred nonsurgically. In the clinical context, the success rate of establishing a pregnancy with surgical oocyte
transfer to the oviduct for in vivo fertilization or shortly
after ICSI, is in the range of 40% [52]. There is a limit to the
number of in vivomatured oocytes, although of high
quality, that can be recovered from any given donor during
the year; normally, one preovulatory follicle is present at
any cycle and only during the breeding season. For these
reasons, based on previously published studies [53,54] and
on the experience accumulated in cattle, in our laboratory
we rened the procedures for OPU of equine immature
oocytes (Fig. 4A, B) as it is done in cattle [8,21], in vitro
maturation, ICSI and embryo culture and we demonstrated
their efcacy and reproducibility. During OPU, we aspirate
all follicles that are at least 1 cm, also in the nonbreeding
season, thereby increasing the number of oocytes that can
be recovered from a donor over a given time. Oocyte recovery rates from immature follicles can be disappointing
for the beginners. Because of the large size of the follicles
and the strong attachment to the follicle wall of the
cumulus oocyte complex, it is necessary to use doublelumen needles that allow for repeated ushing, up to 8 to
10 times for each follicle. Early reports of recovery of
immature oocytes [55,56] provided a very limited assessment of the developmental potential of such oocytes.

Table 2
Embryo production efciencies of current embryo technologies in dairy
cattle.
Treatment before oocyte collection

Number of embryos
Per session

Per wk

Natural cycle, AI and ushing at day 7


MOET, standard superovulation
DFR-MOET, superovulation preceded by
removal of dominant follicle
OPU (twice weekly) at 3- and 4-day
intervals
OPU after FSH prestimulation at 2-wk
intervals
(DFR) MOET-OPU, superovulation with
collection before ovulation

0.7
6
5.5

0.23
1
1.1

1.5

5.5

1.1

Modied from Merton et al. 2003 [94].


Abbreviations: DFR, dominant follicle removal; MOET, multiple ovulation
and embryo transfer; OPU, ovum pick up.

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C. Galli et al. / Theriogenology 81 (2014) 138151

Table 3
Embryo production by ovum pick up (OPU) in Mediterranean Buffalo (Galli et al. unpublished).
No. of
OPU

No. of
follicles

No. of
oocytes

No.
cleaved

Cleavage
(%)

No. of
embryos

No. of embryos
per OPU

% embryos/
oocytes

% embryos/
cleaved

123

1392

815

389

47.73

132

1.07

16.20

33.93

Data from Galli et al. (unpublished).

Subsequently, we performed in our laboratory, over several


years, a large study to assess the developmental competence of immature oocytes recovered by OPU from both
experimental and commercial donors within our OPU
clinical program [57]. The data reported in Table 4 refer to
repeated OPU collections from donors of various breeds
within our current OPU service. To ensure the highest rate
of oocyte collection, we monitor the donors by ultrasound
to choose the most suitable time for OPU based on the
presence of a sufcient number of follicles (normally 8
10 follicles >1 cm) and preferably in the absence of a
dominant follicle. This situation is particularly favorable
during the transition season (no dominant follicle with a
prevalence of medium sized antral follicles). For these

reasons, the average numbers of follicles aspirated and


oocytes collected by our group are higher than those in
most published studies, although the recovery rate has
been steadily increasing with experience and practice.
Development to blastocyst for this commercial work is
carried out completely in vitro. The outcome is very variable
owing to the extreme differences between both female and
male donors and to the different causes of infertility leading to the use of these procedures. A breed effect can also be
observed. Mares can be subjected to repeated collections
without any side effect, under detomidine sedation,
epidural anesthesia, to avoid the contraction of the rectum,
and with catheterization of the bladder [58]. At the end
of the procedure, the donor is treated with antibiotics for

Fig. 3. (A) Buffalo ovaries. note the small size and fewer follicles than cows. (B) Oocytes before in vitro maturation and (C) at the end of in vitro maturation. (D)
Cleaved embryos 48 hours after IVF. (E) Blastocysts on day D6 after fertilization. Note the dark appearance, like in oocytes and early stages embryos owing to the
abundance of lipid content. This makes the embryos less tolerant to cryopreservation. (D) First calves born in 1998 from OPU embryos after cryopreservation
(Galli et al., 1998).

C. Galli et al. / Theriogenology 81 (2014) 138151

143

Fig. 4. (A) Mare subjected to ovum pick up (OPU). Three operators are required; the third operates the syringe to inject the ushing media to repeatedly ush the
follicles. (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 ICSI
around 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 too
much. (F) Foal produced by ICSI with the foster mother.

3 days as a preventive measure. Other programs have reported a xed, biweekly schedule [59] on experimental
young animals; however, for aged or infertile donors, as are
those enrolled in the clinical OPU program, it is difcult to
establish a xed schedule and we prefer to adapt the
collection to the need of the individual donors.

3. Intracytoplasmic sperm injection


The clinical use of ICSI was developed many years ago as
a solution to some sperm-related male infertility in humanassisted reproduction [60]. The technique now works so
efciently that in many clinics it is the preferred way of
in vitro fertilization in humans. There are suggestions that,
because sperm selection does not occur in a physiologic
way, it may carry abnormalities to the offspring. However,
when data analysis is adjusted for age, twin pregnancies,
infertility, and other patient-related factors, ICSI offspring
do not present a greater incidence of abnormalities than
normal offspring [61,62].

3.1. Cattle
The use of ICSI in cattle has never attracted much interest
and has mainly been used for research purposes, essentially
because IVF after heparin capacitation works very efciently
with the majority of bulls. On the other hand, it would be
very time consuming and expensive to fertilize the numbers
of oocytes that are dealt with in a cattle OPU/IVF program to
be viable in a commercial setting. Moreover, the efciency in
cattle is not comparable with that in humans [63]. After
initial experiments, where blastocyst formation [6466]
rates were generally lower than with IVF, the work was
directed toward improving activation protocols to increase
embryo development [67]; few publications reported the
birth of offspring [63,68]. It is not clear whether the reduced
development to blastocyst, after ICSI, is owing to inadequate
oocyte activation [69] or cytoskeletal damage induced by the
injection procedure [70]. Most of the researchers use
piezoelectric manipulator [71] (Fig. 4C) that greatly facilitate
the penetration of the oolemma with little pressure. The size
of bovine spermatozoa require a pipette with a relatively

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C. Galli et al. / Theriogenology 81 (2014) 138151

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

No. of
donors

No.
OPU

No. of
follicles

No. of
oocytes

Recovery
(%)

No. of MII
injections

MII (%)

No. cleaved

Cleavage rate

No. of embryos

%/oocytes

Arabian
Per OPU
Warmblood
Per OPU
Quarter
per OPU

56

214

63
0.29
92
0.84
26
0.6

2.42a

43

680
3.18
509
4.63
154
3.58

44.56a

22

1526
7.13
813
7.39
265
6.16

58.53a

110

2607
12.18
1170
10.64
404
9.40

69.97

54

3726
17.41
1683
15.30
611
14.21

69.52
66.12

69.49b
65.59b

62.61b
58.11b

7.86b
6.44b

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

large outer diameter of 10 mm that could be responsible for


the damage on the cytoskeleton reducing the developmental
potential [63]. Bovine ICSI could be valuable as a model for
conservation biology of other ruminants by using suboptimal or dead sperm samples that cannot be used successfully
in 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 oocyte
activation there is no pronuclear formation [74]. The same
authors reported, however, that the embryos resulting
from ICSI and partenogenetic activation were in fact parthenotes [75] because they did not express genes of the
paternal genome. Different sperm pretreatment, like the
use of dithiothreitol found to be effective on bovine sperm,
stimulated the male pronuclear formation [76]. With the
present results, there is no foreseeable possibility of using
ICSI for buffalo embryo production.
3.3. Horse
The use of ICSI in the horse has been fundamental to the
development of assisted reproduction techniques because
of the difculties to achieve IVF by simple co-incubation of
the female and males gametes. In fact, only two foals have
been reported from IVF of in vivo-matured oocytes [77,78].
The rst pregnancy, derived from an in vitro-matured
oocyte fertilized by ICSI, was carried successfully to term as
reported by Squires, et al. [79]. This success was followed
by a period of variable results until the development of ICSI
using the piezo drill (Fig. 4C), which eliminated most of the
inconsistency of the technique owing to a heterogeneous
and thick zona pellucida difcult to penetrate with conventional ICSI pipettes, especially of in vitro matured oocytes. Moreover, the additional advantage of ICSI compared
with IVF is the possibility to widen the choice of the stallions to be used, including those with poor sperm motility
and inferior reproductive performance in vivo, which is the
main reason why ICSI was developed (Fig. 4D). In an original study, Lazzari, et al. [80] compared the developmental
potential of in vitro matured oocytes fertilized by ICSI with
frozen stallion semen of different motility and/or different
fertility in the eld. They found no differences in either
cleavage or blastocyst development rates among oocytes
injected with sperm from stallions of good, poor, and no
fertility in the eld, as long as a motile sperm was selected

for ICSI. In contrast, when at the time of ICSI it was not


possible to select a live sperm before injection, there was
no development to blastocyst. In a different study [81],
oocytes injected with nonmotile sperm isolated from
semen subjected to two freezethaw cycles led to blastocyst development. In a further study, the same authors [82]
have even used lyophilized sperm and activation of the
oocyte with sperm extract to generate offspring.
To date, there are a growing number of laboratories that
have consistently and reproducibly reported the birth of
ICSI foals both from in vivo- and in vitro-matured oocytes;
presently, a few equine practices are starting to use it for
clinical treatment of infertility. Some groups have reported
the occasional birth of foals after ICSI of in vitro-matured
oocytes, after either surgical transfer of early embryos to
the oviduct [83] or in vitro embryo culture to the blastocyst
stage and transcervical transfer to the uterus [84]. The
progress of in vitro maturation and ICSI technology has
increased efforts to design suitable culture systems for
early cleavage stage embryos. Many different culture conditions have been reported for pre-implantation development of ICSI fertilized horse oocytes, including dened
media such as G1.2 [85], Dulbeccos Modied Eagle Medium/Nutrient Mixture F-12 and CZB [86], and modied
synthetic oviduct uid [87]. Co-culture with somatic cells
in earlier work evaluated the use of Vero cells [88], oviduct
epithelial cells [89], cumulus cells [84], granulosa cells [90],
or culture in conditioned media [91]. In most of these
systems, however, the blastocyst rates remained low,
ranging from 4% to 16%, a rate that is comparable with
parthenogenetic activation [92]. A comparison between the
published reports on in vivo culture of ICSI early cleavage
stage embryos in the oviducts of mares [86] or temporary
recipient sheep [80,93] and in vitro culture in various culture media clearly demonstrated that the culture in vivo
supports greater blastocyst development, being approximately 36% of injected oocytes in both the mare oviduct
and the surrogate sheep oviduct. In another study, an
in vitro culture system based on Dulbeccos Modied Eagle
Medium/Nutrient Mixture F-12 medium under a mixed gas
atmosphere provided blastocyst development similar to
that seen in vivo (27%38%) [59,94]. However, when day 7
blastocyst cell numbers are accounted for [95], in vitroproduced embryos cultured in a modied synthetic oviduct
uid had signicantly fewer cells, being more similar to a
day 5 in vivo embryo. This difference has to be taken into
account when in vitro produced embryos are transferred,
by using recipients 5 day after ovulation. There is, however,

C. Galli et al. / Theriogenology 81 (2014) 138151

an advantage of having embryos at an earlier developmental stage, that is the improvement of their survival at
cryopreservation. In fact, it is well-known that in vivorecovered equine embryos, because of their large size when
they are normally ushed from the uterus, do not freeze
successfully. Cryopreservation of in vitro-produced embryos at the early blastocyst or blastocyst stage (Fig. 4E)
leads to a very high post-thaw survival and pregnancy rate
that exceed 60% [8]. This efciency with in vitro-produced
embryos is not documented in other livestock species.
4. Somatic cell nuclear transfer
4.1. Bovine
Nuclear transfer in general and SCNT in particular are
routine techniques in many laboratories worldwide, both
for farm animals and laboratory species. Among livestock,
cattle have been cloned in the greatest number because of
the potential commercial interests of the cattle industry
and breeders. As a technique, SCNT is demanding. It is
complicated to master all the procedures involved to levels
of efciency and reproducibility that are scientically solid
[96,97] and have a practical relevance to the cattle industry
producing live offspring consistently.
All techniques used in cattle today are based, in principle, on those described by Willadsen [98]. Basically, there is
a requirement for large numbers of matured, good quality
oocytes to be enucleated. Then, the donor cell must be
fused to or injected in the enucleated oocyte. Finally, the
reconstructed embryo needs to be activated and developed
in vitro. For large animals like cattle, to perform a nonsurgical uterine transfer the cloned embryos need to be
cultured to the blastocyst stage and/or cryopreserved
before embryo transfer into recipients [96]. As the technology of SCNT stands today, a considerable source of
variation comes from the laboratory experience, the technician performing the work, and the biological material
used. Essentially, all SCNT work is done with metaphase II
oocytes [99,100] that are used as recipient cytoplasts,
although one study reported success with cytoplasts
derived after enucleation of zygotes [101] that are physiologically activated by sperm. Quiescent G0 is the preferred
cell-cycle stage of the donor somatic cells, but also cycling
cells [102] and blood leukocytes [103] have been used as
nuclear donors. Generally, donor cells come from primary
cell lines established from a tissue biopsy and cryopreserved for successive, repeated uses. Many cell types
present in the body have been used for SCNT with different
degree of success; however, there has not been a single
somatic cell type that consistently performs better than
others [104]. Moreover, the differentiation status of the
somatic cells has no correlation with the ability to generate
offspring [105]. Technical modications such as the zonafree manipulation have improved the efciency of enucleation and fusion and have reduced the labor required
[106,107] in several species beside cattle [108], and limited
available data indicate that development to term is the
same as conventional zona-enclosed methods.
In vitro culture of SCNT embryos is performed under
conditions substantially similar to those used for IVF

145

embryos [108]; however, when the zona-free system is


used, it is necessary to prevent contact between individual
embryos during culture. This can be achieved using the
well of the well system [109]. Culture conditions can alter
the epigenetic status to the extent that subpopulations of
somatic cells can be selected with different ability to sustain normal development into viable offspring after nuclear
transfer. Culture conditions can also affect the karyotype of
the cells and chromosomal abnormalities can accumulate
in aged cells or after a time in culture resulting in abnormal
embryos [110]. After nuclear transfer, the nucleus undergoes so-called nuclear reprogramming, a series of
events that, by interacting with the oocyte cytoplasm, induces changes to the structure of the chromatin toward a
pluripotent pattern that is more representative of embryonic development. Empirical attempts to facilitate this
reprogramming process have been made using chemicals
that are known to alter the methylation status of the
chromatin, such as trichostatin A, azacytidine, or Scriptaid,
either before or after nuclear transfer. In the mouse, the use
of trichostatin A (an histone deacetylase inhibitor) signicantly increased the success rate of mouse cloning
[111,112]. On the other hand, this nding remains controversial in other species like cattle [113116]. The major
limiting factor to the widespread use of SCNT in cattle
farms is the high rate of pregnancy losses throughout
pregnancy. The most problematic are late abortions (after 5
or 6 months of gestation), and greater perinatal mortality
that create economic losses and welfare issues. A comparative embryo transfer study between IVP embryos and
cloned embryos derived from embryonic, fetal, and adult
cells demonstrated that, although the initial pregnancy rate
at 21 days is similar (from 55.6% to 62.7%), signicant differences becomes evident by 70 days (49% vs. 37.3% vs.
22.5% vs. 14.3% for IVP embryos and embryonic, fetal, and
adult cell clones, respectively) and at calving (49% vs. 34.3%
vs. 15% vs. 6.8%) [117]. Several studies have demonstrated
that SCNT embryos present an altered gene expression
prole [118] and epigenetic status [119] compared with IVP
embryos, and that the high rate of pregnancy loss has been
clinically associated with hydrops and cotyledonary hyperplasia [120,121]. These problems are, however, not
observed in the offspring of clones, which are normal [122].
It is possible to detect and interrupt earlier on in gestation
pregnancies at risk by measuring the maternal concentration of pregnancy associated glycoproteins and or by ultrasound monitoring [123,124]. These preventive measures
have prompted the International Embryo Transfer Society
to issue guidelines on the management of cloned pregnancies (available from: http://www.iets.org/pdf/HASACHealthAssessmentCare.pdf) to control possible recipient
welfare problems. From a practical perspective for the
cattle breeding industry, SCNT is characterized by a low
efciency owing to high pregnancy losses and perinatal
mortality documented in extensive eld studies [124,125].
This makes the production of offspring expensive and
justied today only when progeny-tested bulls or champion dams are cloned for reproductive purposes. Many
top-ranking bulls have been cloned by AI companies as an
insurance for the bull or because the original bull stopped
producing semen because of accident or disease. The same

146

C. Galli et al. / Theriogenology 81 (2014) 138151

rationale is applied in the case of outstanding dams. In


Europe, the implementation of cattle cloning is facing
strong opposition to the limit that all cattle cloning work
has been stopped at both scientic and practical levels. In
other parts of the world (e.g., South America or China),
there is great interest in SCNT for cattle breeding. The safety
of the cloned animals and their products has been
demonstrated in several studies [126,127] and regulatory
agencies have carried out extensive risk assessment [128],
concluding that cloned animals and their product do not
pose any health risk to consumers. Still in Europe, opposition to the use of cloned cattle is based on ethical, ideological, and welfare arguments. It will take years to change
attitudes toward new biotechnologies like cloning within a
favorable regulatory framework [129]. In Japan, just like in
Europe, there is an absolute governmental ban on the
commercial use of any cloning-related products. All this
happened after a Japanese investigation that concluded
that products from cloned cattle are harmless.
4.2. Buffalo
Attempts to clone buffalo by nuclear transfer were rst
reported by Kitiyanant, et al. [130]; however, despite the
agricultural interest for this species in tropical and subtropical regions, only two studies from the same group
have reported the birth of live offspring [131,132]. This
difculty summarizes the problems encountered in general
with assisted reproduction in the buffalo and with SCNT in
particular. The technique used in buffalo is the same as in
cattle and the limits encountered with the buffalo seems to
be similar [133]. Several investigators, to overcome the low
developmental competence of buffalo oocytes, have used
cattle oocytes. Some initial studies reported blastocyst
development of these interspecies nuclear transfer embryos [130,134], but never presented data about embryo
transfer of such embryos. A more recent and detailed study
has failed to replicate these ndings, demonstrating that
buffalo somatic cell nuclei do not activate the embryonic
genome in cattle oocytes and arrest at the 8- to 16-cell
stage [135,136].
4.3. Equine
Equine cloning by SCNT was rst described in 2003
[137]. In the same year, three mules were produced from
fetal somatic cells and in vivo-matured oocytes recovered
by OPU from superovulated preovulatory follicles [138] and
transferred to the oviducts of a recipient immediately after
activation. The rst horse, a lly, was originated by nuclear
transfer of adult somatic cells into in vitro-matured oocytes
and culture to the blastocyst stage before nonsurgical
transfer to recipient mares [137] (Fig. 5B). In that study, the
foal was carried by same mare that was the source of the
cells used for cloning, representing an exclusive example of
autologous pregnancy successfully gone to term in equines.
Two years later, other cloned foals were born [139,140]
from selected champion donors, demonstrating the potential use of this technology to rescue gelded genotypes
with high sporting value that are clearly unable to generate
any offspring (Fig. 5C). More foals have been produced since

then [141] in the same laboratories and in another laboratory [142]. There is also SCNT activity carried out in commercial laboratories reported in the popular press, but
there is no published scientic information about the
numbers performed and the results. The success of SCNT in
the horse is the result of the optimization of many steps
involved in the cloning procedure, the principles being the
same as in other species. The rst step is the derivation and
cryopreservation of a cell line, usually starting from a skin
biopsy of the donor animal to be cloned. Blastocyst development of SCNT embryos is inuenced by the cell line
[140], which is well demonstrated in other species and
varies from 0% to 17%. In a large dataset obtained in our
laboratory, the overall blastocyst rate was 4.4% (83 blastocysts from 1866 cleaved embryos from a total of 2099
reconstructed embryos). Oocyte maturation in vitro is the
only sustainable source of oocytes for cloning and their
quality is as critical as it is for ICSI. Limitations include the
number of ovaries that can be recovered at the slaughterhouse, recovery that is possible only in countries that allow
the slaughter of equines, and the small number of follicles
that are present at any time on the ovaries. In our laboratory, on average we recover 3 to 5 oocytes per ovary suitable for in vitro maturation. In our experience, the use of the
zona-free method maximizes the limited number of oocytes available for any given day by obtaining high
enucleation and cell fusion rates of the somatic cells to
the oocyte [108] or by using a piezoelectric device both
for enucleation and microinjection of the somatic cell
directly into the oocyte [139]. Equine reconstructed embryos are difcult to activate. We use a combination of the
two most common chemicals used in other species: 6dimethylaminopurine and cycloheximide [92], obtaining
an activation rate exceeding 90%. Other workers [139]
used a combination of injection of sperm extract and culture in 6-dimethylaminopurine to produce embryos
resulting in cloned offspring. Culture requirement for
cloned embryos are the same as for ICSI embryos. Using the
zona-free method (Fig. 5A), we were able to obtain preimplantation development in the range of 17% to 25%,
comparable with the rates obtained by ICSI of slaughterhouse oocytes (Lagutina I, unpublished data). Cloned embryos can also be successfully cryopreserved and we have
obtained offspring after embryo transfer (Galli C, unpublished data). We found that cloned embryos have a lesser
ability to establish pregnancies compared with ICSI embryos [140]. For this reason, we normally implant two
cloned embryos per recipient; occasionally, twin pregnancies result that can be reduced to singleton by transrectal
manipulation or transvaginal ultrasound-guided ablation.
The development to term of cloned pregnancies is low as in
other species; however, most of the pregnancy losses occur
early in gestation (before day 50), thus creating fewer
problems with recipient management. Moreover, the foalings are normal and there are no reports of the problems
described in ruminants, such as hydrops, placenta hyperplasia, and large offspring syndrome. Although perinatal
mortality has been reported, most of the foals are normal or
require minor assistance at birth [143] and develop into
adult fertile animals [1] (Fig. 5D). So far, the number of
animals generated by SCNT is very small and equine

C. Galli et al. / Theriogenology 81 (2014) 138151

147

Fig. 5. (A) Horse cloned embryos produced with the zona-free method. (B) Prometea, the rst 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 articial insemination.

cloning is likely to remain a very specialized niche market


for a few outstanding champions [144] that cannot reproduce otherwise, to be able to generate offspring from them.
Equine cloning has not raised the issues about food safety
or animal welfare that have limited cattle cloning; however, some association like the Jockey Club or the American
Quarter Horse Association oppose the use of cloning for
breeding purposes. On the contrary last year the Fdration
Equestre Internationale announced that it will allow cloned
horses to compete in international events. The Fdration
Equestre Internationale position is the rst concrete sign
that the industry is considering cloning of potential value
for horse breeding and sporting. This decision will stimulate the further development of horse cloning and a
renewed 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 address
the potential use of these technologies for biotechnology
and biomedical purposes. It is remarkable that Theriogenology has provided the main vehicle for the publication of
research and development in these areas, as attested to by
our reference list. Ovum Pick Up and subsequent embryo
production in cattle has reached a mature stage and will
gradually overtake in vivo production by superovulation, as
shown by the trend of the International Embryo Transfer
Society statistics. In buffalo, the translation and implementation of these technologies is relatively recent; however, given the limits of superovulation and embryo ushing,
OPU will be, even at the current efciency, the main route for
genetic improvement and breeding of elite animals. The
horse is also beneting from OPU essentially tailored on
individual clinical needs of mares and stallions. Embryo

culture and ICSI provide practical solutions both for IVF and
embryo cryopreservation. This can create new opportunities
for horse breeders and open a market for horse embryos and
for banking the best genetics. In the last 15 years, SCNT has
not improved much in cattle, buffalo, or horses, and it will
remain a very small niche to generate copies of outstanding
animals. Future work to advance the eld will concentrate
on understanding nuclear reprogramming as well as the
identication of possible markers that will allow selection of
the few normal embryos for transfer. The evolution and
application of these new techniques will follow not only the
technical feasibility and the economics, but also the public
perception, which could be quite different in different parts
of the world.

Acknowledgments
The authors acknowledge the technical support over the
years, to generate the data discussed in the paper, of
Gabriella Crotti, Paola Turini, Massimo Iazzi, and the many
colleagues, too many to be mentioned, who have contributed ideas, discussions, and information. This manuscript
was prepared while funded by EU grant FP7-KBBE-2012
n 312097 Fecund by grants InnovaB and Superpig from
Lombardy Region, by grant Ex Ovo Omnia from Sardinia
and Lombardy Region.

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