40 years of bovine IVF in the new genomic selection context Marc-André Sirard Centre de Recherche en Reproduction, Développement et Santé… [609528]

REPRODUCTIONREVIEW
40 years of bovine IVF in the new genomic selection context
Marc-André Sirard
Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI), Département des
Sciences Animales, Faculté des Sciences de l’Agriculture et de l’Alimentation, Université Laval, Québec, Canada
Correspondence should be addressed to M-A Sirard; Email: [anonimizat]
Abstract
The development of a complex technology such as in vitro fertilization (IVF) requires years of experimentation, sometimes comparing
several species to learn how to create the right in vitro environment for oocytes, spermatozoa and early embryos. At the same time,
individual species characteristics such as gamete physiology and gamete interaction are recently evolved traits and must be analysed
within the context of each species. In the last 40 years since the birth of Louise Brown, IVF techniques progressed and are now used
in multiple domestic and non-domestic animal species around the world. This does not mean that the technology is completely
matured or satisfactory; a number of problems remain to be solved and several procedures still need to be optimized. The
development of IVF in cattle is particularly interesting since agriculture practices permitted the commercial development of the
procedure and it is now used at a scale comparable to human IVF (millions of newborns). The genomic selection of young animals or
even embryos combined with sexing and freezing technologies is driving a new era of IVF in the dairy sector. The time has come for a
retrospective analysis of the success and pitfalls of the last 40 years of bovine IVF and for the description of the challenges to
overcome in the years to come.
Reproduction (2018) 156 R1–R7
Introduction
The first human IVF baby was born 40 years ago, and
the technology has since been applied to several other
species including cattle. Initially, the objective of IVF
in cows was similar as in humans: the treatment of
infertility. Some success was achieved with animals
showing pathological infertility, but most of the
development and applications of the methodology
were oriented towards improving genetic selection and
decreasing generation interval ( Bousquet et  al. 1999 ).
The commercial application of these methods has been
fuelled by 40 years of research performed mainly with
oocytes obtained at slaughterhouses and frozen semen
(sperm), resulting in a very powerful model for clinical
application for cows and also for humans. Several
excellent reviews have been already published about
IVF in different species, focussing on the early days of the
technologies ( Bavister 2002 ) or applied industrial aspect
in cattle ( Hasler 2014 ) as well as the problems related
to the use of IVF in cattle ( Hansen 2006 , Lonergan &
Fair 2008 ). The following review will recapitulate the
important steps, pitfalls and progresses made during
these four decades focussing on cattle IVF ( Fig. 1 ).
The initial challenges (1980–1990)
Although reproductive biologists were experimenting
with IVF in rodents since the fifties ( Austin 1951 , Chang 1959 ), the birth of Louise Brown in 1978 created
tremendous interest and an intensification of efforts to
make it work in other large mammals including the cow.
The exceptional work that occurred in the last century
that led to the development of IVF is well reviewed in a
paper by Sir Robert Edwards ( Edwards 1996 ), who was
a pioneer in the observation of immature oocytes from
bovine and other mammals in the sixties ( Edwards 1965 )
and received the Nobel Prize in Physiology or Medicine
in 2010 for the development of IVF.
Since the procedure leading to the birth of the first IVF
baby in humans involved the recovery of mature eggs,
initial efforts focused on also obtaining mature eggs from
cows using laparotomy and eventually laparoscopy.
The world’s first IVF calf was produced by the group of
Benjamin Brackett using mid-ventral surgery for oocyte
recovery and for transfer of embryos to the oviducts
(Brackett et al. 1982 ). It took four more years to develop
laparoscopic recovery of oocytes and surgical transfer of
blastocysts to the uterus after a seven-day incubation in
a rabbit oviduct ( Sirard & Lambert 1986 ). Laparoscopic
recovery of oocytes permitted the use of IVF for the
treatment of infertility and the possibility of transfer into
the uterus, and opened the use of the technology in a
clinical context.
Laparoscopy remained the preferred technique for
the clinical application of IVF to infertile cows during
the first 10 years but was later replaced by ultrasound.
The ‘explosion’ in the use of IVF for research purposes
-18-00081561
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M-A SirardR2
Reproduction (2018) 156 R1–R7 www.reproduction-online.orgresulted from the possibility of using oocytes obtained
from slaughterhouses. This was developed in several
locations in Europe and the United States at about
the same time with quite similar protocols (reviewed
by Greve & Madison 1991 ). The discovery of the
stimulatory effect of heparin on the capacitation of
bovine spermatozoa in vitro was also instrumental in the
rapid increase in fertilization and success rates ( Parrish
et  al. 1985 ) review in 2014 ( Parrish 2014 ). The effect
of heparin also partially explained the great success
of laparoscopic oocyte recovery since this procedure
requires the use of an anticoagulant in the needle
rinsing medium to prevent coagulation in the 60-cm
laparoscope used on cows. The discovery of the heparin
effect was accidental and was understood only two years
later when John Parrish published his work ( Parrish et al.
1986 ). The heparin although not truly present in the
oviduct could mimic some follicular fluid constituents
like proteoglycans ( Parrish et al. 1985 ). The initial work
came for the study of follicular fluid components by
the group of Roy Ax ( Handrow et  al. 1982 ). On the
oocyte side, the challenge was to find optimal culture
conditions as Robert Edwards had already shown the
capacity of oocytes to resume meiosis spontaneously
in vitro . Surprisingly, the first media to be largely used
have remained the same for over 30  years. Indeed,
most laboratories still use TCM-199 supplemented with
serum, FSH and oestradiol for IVM as developed in Neal
First’s laboratory ( Ball et al. 1984 , Sirard et al. 1988 ), as
well as Tyrode-Lactate medium for sperm washing and
sperm/egg co-incubation. The situation is quite different
for the embryo culture medium which was modified
several times in the first ten years (for review see: Greve &
Madison 1991 ) and is still changing today. The challenge
was and remains the dynamic condition during the first
few days of embryonic development in the oviduct and
the adaptation of the embryo metabolism to the pre-
and post-genome activation transition. In fact, the initial
successes of bovine IVF were obtained with a transient incubation of embryos in the oviducts of rabbits ( Sirard
& Lambert 1986 ) or sheep ( Lu et al. 1987 ). Incubation
into oviducts was eventually replaced with co-culture
with oviductal cells which was initially required to pass
the 8-cell block in bovine embryos ( Gandolfi & Moor
1987 , Eyestone & First 1989 ). Therefore, a sequential
approach as in humans, where the medium composition
is modified at the time of embryonic genome activation,
has been privileged with two or three medium changes
in the period of development from one-cell embryos to
blastocyst ( Felmer et al. 2011 ). The progressive definition
of the optimal components of the ideal medium began
with the analysis of the oviductal environment and the
preparation of a synthetic oviductal fluid (SOF) ( Tervit
et  al. 1972 ) which was rapidly adopted ( Bondioli &
Wright 1980 ) and initially supplemented with serum or
BSA ( Thompson et  al. 1989 ), but eventually enriched
with specific amino acids ( Keskintepe et  al. 1995 ) to
allow the development of one-cell embryos to blastocysts
in defined or semi-defined (BSA) conditions. What has
remained puzzling and to some extent unique to bovine
is the requirement for group culture ( O’Doherty et al.
1997 ) although some success has been obtained with
single culture in presence of serum ( Hagemann et  al.
1998 ). Although some mechanical conditions like micro
fluidics ( Krisher & Wheeler 2010 ) or very small culture
drops or using the micro well approach ( Rienzi et  al.
2011 ) have been used to decrease the dispersion of
autocrine factors, such factor has not been identified yet.
The achievement of IVM (1990–2000)
Following the very few publications on IVF using in-vivo –
matured oocytes, the potential of using oocytes obtained
from slaughterhouses became a very active research
field. It was known that the oocyte maturation process
was different from the follicular maturation process and
that the latter could be reproduced in vitro much more
easily than the former ( Thibault 1977 ). The groups of
Figure 1 The chronology of important steps in bovine IVF, with very early images in-vitro -matured oocyte (1) or of embryos from in-vivo matured
oocytes (2–3–4) and bovine embryos incubated in a rabbit oviduct for several days (4). All included means that it is now possible to add all
these techniques and obtain acceptable success.
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Bovine IVF after 40 yearsR3
www.reproduction-online.org Reproduction (2018) 156 R1–R7I. Gordon in Ireland ( Lu et al. 1987 ) and Neal First in
the United States ( Ball et  al. 1984 , Leibfried-Rutledge
et  al. 1985 , 1987 , Sirard et  al. 1988 ) were amongst
the most active at testing culture conditions for these
oocytes. It was rapidly observed that nuclear maturation
was quite automatic in most culture conditions and
within 24 h, the rate of metaphase II oocytes was over
85% ( Sirard et al. 1989 ). Therefore, as early as the late
eighties, obtaining hundreds of embryos per experiment
using slaughterhouse material was a true possibility.
This almost unlimited source of oocytes in cattle and
pigs was instrumental to the development of better
techniques and culture conditions compared to other
species where progress has been slower like horses
and IVF is still limited by the sperm failure to fertilize
in vitro requiring injection of spermatozoa into the
oocyte ( Leemans et al. 2016 ) or pets where seasons and
physiological peculiarities alter the success of IVF ( Van
Soom et al. 2014 ).
With use of slaughterhouse oocytes to generate
embryos, it was noted that different culture media
could affect the phenotype of offspring. The large
offspring syndrome was initially observed in sheep,
then replicated in cows and was associated with the
presence of serum in culture media post fertilization.
We now know that this phenomenon is epigenetic
in nature and is exacerbated when embryos are
generated by cloning, either with blastomeres fused to
enucleated oocytes ( Willadsen 1989 ) or when fused to
somatic nuclei ( Farin et al. 2004 ). The incidence of the
phenotype is difficult to estimate today as it can be mild
with a few more days in gestation length, or it can be
extreme including stillbirth with abnormal calf weight
and placental anomalies ( Siqueira et  al. 2017 ). In a
large Brazilian operation where oocytes are obtained
by ovarian pick up using ultrasound-guided aspiration
without hormonal stimulation of the donors, a recent
survey based on 15,000 births (including control AI)
indicated an average increase in birth weight of 1 kg for
IVF calves (Pietro Baruselli, unpublished observation).
For many years, doubt remained that defined media,
although allowing the culture up to the blastocyst
stage, were the major cause of limited developmental
competence of oocytes. Indeed, from 1990 to 2000, the
average success rate plateaued at 30–40% of oocytes
developing to the blastocyst stage despite the addition
of hundreds of different products, cytokines, growth
factors, anti-oxidants, in addition to the introduction of
new types of incubators, and reduced oxygen tension.
Very few of these conditions were ever tested with in
vivo transfers ( Hansen 2006 , Lonergan et  al. 2006 ).
Then, attention turned towards the source of oocytes
as a potential explanation for the limited success rates
of IVF.
The morphology of oocyte–cumulus complexes
obtained when all follicles are aspirated form ovaries at
slaughter varies greatly. In cows, follicles are growing in waves principally under the influence of FSH. The
oestrous cycle of 21  days includes two, or sometime
three, waves of growth, each leading to the selection of
one dominant follicle over 9 mm ( Ginther et al. 1997 ).
When FSH starts to rise, the most advanced follicle is
likely the one that will become dominant, while the
smaller ones will grow for a few days under FSH support
and then will start an atresia process as the FSH level
drops ( Ginther et  al. 1989 ). This cohort phenomenon
occurs from puberty to death and even during pregnancy.
Therefore, if ovaries are collected at random at slaughter,
most follicles will be between 3 mm and 9 mm and they
contain the population of oocytes available for IVM-
IVF. The effect of size becomes more important starting
at 8 mm ( Hendriksen et al. 2000 ); but even if oocytes
come from larger follicles, success is not guaranteed.
Because the atresia process is progressive and takes
time, approximately one-third of the antral follicles in
random ovaries will be in the FSH-dependant growing
phase, one-third will be in the plateau phase when FSH
decreases, and one-third will be more advanced in the
atresia process ( Douville & Sirard 2014 ). This situation
is reflected in the morphology of the cumulus–oocyte
complexes recovered from these follicles and is partially
associated with outcome ( Blondin & Sirard 1995 ). A very
compact and bright cumulus indicates that the follicle of
origin was in the growth phase and the oocyte is not
fully competent. Early expansion of the cumulus can be
associated with the plateau phase and the outcome is
better but still far from 100%. Finally, oocytes with a
partially denuded or atretic-like cumulus have a lower
competence compared to the plateau group ( Sirard
2011 ). Oocytes from pre-ovulatory follicles sometimes
demonstrate higher developmental potential ( Dieleman
et al. 2002 ), but because dominant follicles are not all
ovulated, the majority disappear in the atresia process,
and the same phenomenon of variable quality is observed
(Sirard 2011 ). These observations were instrumental in
the development of pre-ovum pick up animal treatments
that began in the nineties.
The improvement of animal
pre-treatments (2000–2010)
When ovarian stimulation was used prior to oocyte
recovery but no hCG given in order to allow in vitro
maturation of these oocytes, the success rate varied
enormously independent of the culture conditions
(Hendriksen et al. 2000 , Dieleman et al. 2002 ). Indeed,
the aspiration of oocytes during the growing phase of
the follicles under FSH stimulation was associated with
much reduced developmental competence ( Blondin
et al. 1996 ), while the arrest of FSH for 2–3 days prior
to aspiration created a window of LH-supported growth
(for follicles over 9 mm), enabling them to generate
oocytes of very high developmental potential ( Blondin
et  al. 1997 a). This later experiment demonstrated the
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M-A SirardR4
Reproduction (2018) 156 R1–R7 www.reproduction-online.orgexcellent performance of the culture medium since with
the right oocyte population, blastocyst rates over 80%
could be reached ( Blondin et al. 2002 ). Surprisingly, it
was also noted during these experiments that the post-
mortem incubation of ovaries at body temperature
(during transport and holding) resulted in a gain of
developmental competence compared to oocytes
aspirated immediately after slaughter ( Blondin et  al.
1997 b). This phenomenon was later associated with
the induction of post-LH conditions, and more recently
with the transfer of RNA molecules from the cumulus to
the oocyte ( Macaulay et al. 2016 ). These results led to
the development of a new protocol to treat cows before
oocyte aspiration. The concept is to create a wave of
dominant follicles using FSH stimulation (ideally after
removing any follicles over 5 mm to synchronize the
population to be stimulated) and continue stimulation
until most of the follicles have reached the stage when
LH receptors appear (8 mm in bovine ( Ginther et  al.
1997 )), and then mimicking the rest of the natural cycle
when follicles continue to grow and differentiate without
FSH but with basal LH. At that time, the follicular
growth pattern changes from a high mitotic period when
the total granulosa and theca, cell number increases
to a period when the volume is expanded mainly by
fluid accumulation with a lower mitotic index ( Girard
et al. 2015 ). This post-FSH period is associated with an
increase in oocyte quality as measured by the blastocyst
rates observed after IVF ( Blondin 2002 ). However,
following these observations, it remained unclear if
competence would be maintained throughout ovulation
or, if ovulation was prevented, if oocyte quality would
drop as the follicle entered atresia as demonstrated by
Dias et al. (2013) .
To further improve our understanding of the effects
of FSH withdrawal on oocyte quality, a stimulation
protocol was built to assess the effects of coasting time
on blastocyst rates. A specific design was created in
which six animals received the same stimulation regimen
followed by four different coasting times (within each
animal), followed by oocyte aspiration, IVF and culture.
The results confirmed that a longer period of coasting
was better than a shorter one (44 and 68 h resulted in
more embryos than 20 h of coasting) and, although not
significant, the longer period of coasting (92 h) resulted
in fewer embryos compared to 44 and 68 h (Nivet et al.
2012 ). In these experiments, some animals generated
groups of oocytes producing 100% blastocysts,
demonstrating unequivocally that culture conditions
are not detrimental when oocyte quality is excellent.
Moreover, tissue samples were obtained from the three
compartments (oocyte–cumulus–granulosa) and were
instrumental in the resolution of the physiological
processes occurring during this final transformation
(Khan et al. 2016 ).
In a paper addressing the limitations of IVF Patrick
Lonergan stated that ‘there is considerable evidence of a significant influence of follicular origin on oocyte
developmental potential and it appears that once the
oocyte is removed from the follicle its developmental
capacity is capped’ ( Lonergan & Fair 2008 ). I totally
agree with the latter statement and I believe that there is
still much improvement possible in the pre-conditioning
follicles prior to oocyte aspiration.
The genomic effect (2010–2020)
In this last section, two aspects of the genomic revolution
will be discussed: first, the genomic analysis of the
follicles, oocytes and the embryos generated from IVF
using transcriptomic and epigenetic tools and second,
the impact of genomic selection on the use of IVF
in cattle.
From the early days of IVF, it was obvious that in-vitro-
matured oocytes and in-vitro -produced embryos looked
and behaved differently. For example, several studies
show that in-vitro -produced embryos are less competent
to implant and more sensitive to cryopreservation
compared to in-vivo -produced embryos ( Pryor et  al.
2011 ). With the development of genomic tools, the
capacity to analyse embryos from the one-cell to
blastocyst stage provided a new angle to compare in vivo
and in vitro aspects. Several studies have used micro-
array or RNAseq technologies on embryos cultured in
multitudes of different conditions to compare to in vivo
control embryos. Most of the studies mentioned in the
following and the conditions used are now summarized
at a web site from the Canadian EmbryoGENE
Network (http://emb-bioinfo.fsaa.ulaval.ca/IMAGE/).
The work of Tesfaye in Bonn ( Gad et al. 2012 ) generated
the most comprehensive data set by analysing each
developmental steps individually (IVM, fertilization,
early cleavage, MET (maternal to embryo transition)
morula and blastocysts)) by comparing gene expression
in embryos alternatively cultured in vitro and in vitro
The results indicated that embryos are particularly
sensitive to in vitro conditions at the 4-cell and morula
stages ( Gad et al. 2012 ). The same group then analysed
the DNA methylation of blastocysts obtained from
different combinations of in vitro and in vivo periods to
demonstrate a progressive increase in DMR (differently
methylated regions) with time spent in vitro (Salilew-
Wondim et  al. 2015 ). The culture conditions also
impacted the transcriptome, and the presence of specific
amounts of sugars, lipids or reactive oxygen species
resulted in changes in gene expression (see review from
Cagnone and Sirard (2016) ) and in DNA methylation
(Tremblay et  al. 2017 ). These tools are obviously very
sensitive, but the differences observed were partially
expected as the in vitro environment will never be
completely similar to in vivo . The changes observed are
part of the adaptation to this different environment and
the better embryos produced in vitro will always have
differences from the in-vivo -produced ones.
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Bovine IVF after 40 yearsR5
www.reproduction-online.org Reproduction (2018) 156 R1–R7Not surprisingly, the pregnancy rates are slightly
affected by IVF as there are more embryonic and foetal
losses with embryos from IVF than artificial insemination
or embryo transfer from in vivo origin ( Taverne et  al.
2002 ), and more recently some post-natal phenotypes
are described but more with sexed sperm and recipient
than IVF per se (Siqueira et al. 2017 ).
In all species studied, offspring obtained by IVF are
somewhat different from naturally conceived ones. A
recent special issue of the Journal of Developmental
Origins of Health and Disease covered the effects of
artificial reproductive technologies in many species
including the cattle ( Sirard 2017 ). As for the serum effect
noted in the nineties, the epigenetic effect of culture is
worrisome even if millions of healthy calves and human
babies have been born so far. Clearly, more research is
needed to better measure, follow-up and understand the
potential consequences for health in later life.
Genomic selection
The selection of the most valuable individuals using
the genomic tools (haplotypes maps) was introduced
in early 2010 and allows rapid modification of specific
breeds. Although the objectives and decisions using
genomic selection remain similar to the phenotype
analysis, the speed of progress is enhanced by the
ability to obtain information at the blastocyst stage.
The precise identification of the characteristics desired
in animals that make up the next generation created
a rapid rise in demand for new artificial reproduction
techniques to rapidly select and multiply the animals
possessing the desired traits. Initially, proven bulls
(5  years and more) and proven cows (3  years and
more) were selected for IVF; but now, with genomic
analysis performed soon after birth, the genetic value
of the bull is known immediately and as soon as semen
can be produced, the bull can be selected as a sire for
IVF, often using sexing technologies to improve the
efficiency of producing females of the right genomic/
genetic background. The same is true on the female
side, with the difference that oocytes can now be
collected before puberty with relatively high success
(Landry et  al. 2016 ). This ‘genomic bubble’ is now
driving the development of several IVF laboratories in
North America and elsewhere in the world. The number
of IVF-produced embryos was on a constant rise in the
last 10  years and following the millions of embryos
transferred in the beef industry in South America, we
now see a rapid increase in dairy embryos generated by
IVF. An additional incentive to use IVF is the possibility
to use sexed semen in vitro , which makes some bulls
more affordable by using much less semen per batch
of embryos ( Blondin et al. 2009 ). The new possibility
of cryopreserving IVF embryos also opens the door to
perform embryo biopsies for genomic analysis and the
selection of only the best combinations for transfer. Suddenly, all of the work invested in the optimization
of the IVF process culminates in a very efficient
procedure to improve genomic merit more rapidly. The
cumulative steps of IVM, IVF with sexed sperm, culture
in defined media, biopsies and cryopreservation must
all work almost perfectly to make an efficient pipeline.
Since many of these steps are technically complex
and sensitive to operators or environment, it remains a
challenge to make it commercially viable.
The future of IVF
Certainly, IVF is not something that will disappear
shortly from the animal selection toolbox especially
with the list of advantages listed earlier such as using
sexed semen and genomic selection at the blastocyst
stage. New approaches, often developed in human,
such as morphokinetics ( Kaser & Racowsky 2014 )
and aneuploidy screening using genomic ( Treff &
Zimmerman 2017 ) or less invasive techniques as free
DNA ( Liu et al. 2017 ), may become affordable in cattle
and improve pregnancy rates in the field.
A few years ago, IVF was instrumental to develop
embryonic ( Westhusin et  al. 1991 ) and then somatic
nuclear transfer procedures, mainly for the purpose of
genetic selection and modification ( Tan et  al. 2016 ).
Today, we can foresee the same use of IVF for CRISPR-
Cas-9 related technologies that will allow precise
genome editing or even epigenome editing. Indeed, the
capacity to access the embryo at the pronuclear stage is
important to minimize mosaicism and improve success
rates of genome editing ( Yum et al. 2018 ).
Conclusions
In the last 40  years, bovine IVF has evolved from an
experimental procedure to treat infertile animals towards
a genomic accelerator for many breeds. The progress
in culture, cryopreservation, oocyte preparation and
laboratory environment made possible the production
of millions valuable animals. Some questions related
to the phenotypes of some offspring still require more
research to better understand the impact of the seven
days spent in vitro , but the technology has matured to a
similar scale as we see in human IVF and will probably
be around for several more decades.
Declaration of interest
I declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of this review.
Funding
This research did not receive any specific grant from any funding
agency in the public, commercial or not-for-profit sector.
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M-A SirardR6
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Received 5 January 2018
First decision 15 February 2018
Revised manuscript received 3 April 2018
Accepted 10 April 2018
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