Recent Advances
in In Vitro
Fertilization
Avid Science
Recent Advances in In Vitro Fertilization
Abstract
Chapter 1
Recent Developments in In Vitro
Fertilization Technologies in Livestock
Andrew W Taylor-Robinson1* and Van Huong Do1,2
School of Medical & Applied Sciences, Central Queensland University, Australia
2
National Key Laboratory of Animal Cell Technology,
National Institute of Animal Sciences, Vietnam
1
Corresponding Author: Prof. AW Taylor-Robinson,
School of Medical & Applied Sciences, Central Queensland University, Bruce Highway, Rockhampton, QLD
4702, Australia, Tel: +61749232008; E-mail: [email protected]
*
First Published January 27, 2016
Copyright: © 2016 AW Taylor-Robinson & VH Do.
This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted use, distribution, and reproduction
in any medium, provided you give appropriate credit to
the original author(s) and the source.
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Recent Advances in In Vitro Fertilization
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Mammalian oocytes are collected from abattoirderived ovaries or from live donors using ovum pick-up
aspiration. Subsequently, they experience three primary
steps of in vitro embryo production: maturation of oocytes; fertilization of matured oocytes with capacitated
sperm; and in vitro culture (IVC) of zygotes for one week.
The blastocysts formed during this period are transferred
to synchronized recipients or are frozen for future use.
Although in vitro production (IVP) has been achieved
in farm animals, several existing limitations have led to
varying degrees of success among different livestock. In
order to optimize the efficiency of embryo IVP, along with
seeking to improve procedural steps it is a priority to establish the best materials to use. For example, through the
exclusive use of a defined in vitro maturation medium it is
possible to eliminate the variable effects of the unknown
composition of non-defined media supplemented with
extracts of animal origin such as serum or follicular fluids.
Similarly, while still achieving a desirable outcome IVC
requires a specified medium that does not employ the assistance of somatic cells. Further investigation into refining such protocols is needed to establish an effective IVP
system for use with a broad range of domestic animals.
Introduction
The technology of in vitro fertilization (IVF) was first
developed in the 1980s, at which time it was restricted
to research investigations, but it has since become an inwww.avidscience.com
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Recent Advances in In Vitro Fertilization
creasingly important tool employed commercially for assisted production in many farm species [1]. This is due
to its low cost [2-4], combined with the increase that it
offers in transferable embryos and pregnancies per estrous
cycle [5]. In particular, in vitro production (IVP) of cattle
has assumed progressive popularity in tropical countries
like Brazil [6]. Moreover, IVF is considered as a desirable tool to use to gain knowledge of oocyte and embryo
development [7]. Most importantly, in combination with
the utilization of sex-sorted sperm, IVF improves the efficiency of such expensive sperm to produce wanted gender
offspring [8,9]. These striking features arise from escalating improvements to IVF in livestock. Nevertheless, the
many discrepant IVF results attained from laboratories
around the world, as well as between different domesticated animal species, indicate the need to unify IVP protocols into a simplified, inexpensive and highly efficient
method. Over the last decade considerable effort has been
invested to increase IVF outcomes. In this chapter, the
published literature on advancements in IVF in livestock
is reviewed. Particular attention is payed to improvements
to parameters of assisted reproduction in the laboratory,
including oocyte collection, in vitro maturation (IVM)
medium, IVF and in vitro culture (IVC).
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Recent Advancements in IVF in
Cattle
It is apparent that major IVF technologies play an important role in providing and preserving male and female
genetics [10]. Moreover, the advantages of IVP-embryo
transfer systems outweigh the use of conventional multiple ovulation embryo transfer [11]. The focus of bovine
embryo production has changed from in vivo to in vitro
because of the increase in blastocyst rates and pregnancies that are achieved by using IVF [5]. Although there
is no significant change in bovine blastocyst rate, which
ranges between 30-40%, various other unfavourable factors inherent in IVP-embryos such as the skewed sex ratio
and heavier IVF-derived offspring have been gradually
eliminated. Pontes et al. [5], conducting experiments to
compare embryo transfer in vivo and in vitro in Nelore
cattle reported that the sex ratio of calves derived in vivo
and in vitro is similar; however, using Gyr cattle Camargo et al. [6] claimed that IVP results in a greater proportion of male calves. Yet, with the recent innovation of X
chromosome-sorted semen, it is possible to produce sexdetermined IVF embryos [8,9,12], with a reported prediction accuracy of 94% [13]. Nevertheless, to sex-sort semen
is currently expensive while its application in producing
pre-determined bovine embryos in vitro might be hindered due to low blastocyst rates resulting from the low
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Recent Advances in In Vitro Fertilization
fertility of sperm sorted by flow cytometry [14]. In addition, different bulls vary in fertile competence [13,15].
results, both COCs and DOs benefit from nuclear and cytoplasmic maturation and polyspermic reduction [20].
The factors that determine the success of IVF are derived from the female lineage. There is no doubt that the
source of oocytes affects the developmental competence
of cattle embryos [16-19]. However, recent innovations
in IVM medium may enhance the development of bovine
oocytes. Typically, immature cumulus-oocyte complexes
(COCs) are cultured in tissue culture medium 199 supplemented with fetal calf serum (FCS), follicle-stimulating
hormone, luteinizing hormone and essential amino acids
in 5% CO2 at 38.5oC for 24h. Ideally, an oocyte experiences full nuclear and cytoplasmic maturation. Oliveira e
Silva et al. [20] modified IVM culture conditions so that
bovine oocytes must undergo a two-step process in order
to synchronize nuclear and cytoplasmic maturation. They
proposed that in the first step immature COCs are introduced into defined, serum-free medium for 24h, when
oocyte meiosis arrest is prolonged, whereas in the second
step each oocyte is cultured in non-defined medium for
a further 24h, achieving meiosis reversibility. Of interest,
Dey et al. [21] promoted the effective co-culture system in
which immature COCs and denuded oocytes (DOs) are
incubated together for 24h. Choi et al. [22] reported that
group culture increased blastocyst rates as an improved
micro-environment may be associated with changes in ultrastructure of the zona pellucida. Consistent with these
Co-culture with somatic cells is also recommended for
in vitro culture. Senatore et al. [23] developed a co-culture
IVC system with the support of agarose-embedded helper
embryos derived from abattoir oocytes. This is especially
useful when the number of bovine oocytes collected is restricted. Perhaps surprisingly, they obtained remarkably
high blastocyst rates, from 39.3-49.5%. In contrast, Goovaerts et al. [24] proposed an individual embryo culture
system in which a zygote is cultured singly in combination
with co-cultured cumulus cells at low oxygen tension (5%
O2). They achieved satisfactory results with the blastocyst
rate and cell numbers not significantly different to those
attained for group culture. Although this new system is
costly and labour intensive, it is of particular value when
the number of COCs available in superior cows is limited. For example, for Bos taurus cattle, the average oocyte
numbers per donor retrieved by ovum pick-up (OPU) is
less than eight [5,23].
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While there have been relatively few experimental
investigations aimed at increasing bovine oocyte maturation rates, research to improve post-fertilization culture
conditions is now abundant and the findings proving
controversial. The addition of modified synthetic oviduct
fluids (mSOF) into IVM medium is popular. Besenfelder
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et al. [7] state that the oviduct plays an important role
in providing a microenvironment conducive to various
stages of early embryogenesis. In contrast, enormous IVC
systems have been proposed. Leivas et al. [25] contend
that supplementing media with mSOF, bovine serum albumin (BSA) and FCS raises not only the blastocyst rate
but also achieves an elevated number of high quality grade
1 blastocysts. It is indisputable that adding serum supplements to a non-defined medium leads to desirable blastocyst yields. However, serum culture medium contains
unknown factors [20]; therefore, in order to simplify and
to avoid contamination it is preferable to develop an IVC
system of defined composition, free of blood components
or cell constituents [17,26,27]. Dhali et al. [16] concluded
that addition of growth factors to serum-free culture results in similar blastocyst rates compared to culture medium supplemented with FCS. Similarly, Neira et al. [26]
indicated that the use of recombinant growth factors such
as insulin-like growth factor (IGF)-1, IGF-2, basic fibroblast growth factor and cytokines achieves the same developmental competence of bovine embryos in vitro compared to those incubated in IVC medium with 10% FCS.
They further implied that using a defined culture system
is necessary in order to assure the bio-security of embryo
transfer. It is well established that in vitro embryos are
more susceptible to harmful chilling during cryopreservation than in vivo counterparts. Recently, Stewart et al.
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[12] have developed an IVC medium designed to increase
the yield of bovine embryos in vitro as well as to improve
their cryo-tolerance. This defined, serum-free medium is
called BBH7.
The discussion above indicates that the in vitro culture
system which is currently employed for both bovine oocytes and embryos remains sub-optimal [12]. This may be
due to a still in exact understanding of the culture requirements of embryos in vitro [17].
Recent Advancements in IVF in
Buffalo
The recent developments in IVF in buffalo are quite
limited. There is some interest in how to increase the
number of oocytes available for OPU. Neglia et al. [28],
performing OPU twice-weekly for 9 months continuously, showed that in buffalo, it is possible to collect immature
oocytes by OPU within 6 successive months without suffering a reduction in oocyte yield. They also inferred that
loss of buffalo developmental competence happens when
daily light hours decrease. On the other hand, Francesco et
al. [29], evaluating the effects of different times of year on
follicular populations in buffalo, reported that seasons do
not affect follicular yields. Interestingly, they further contended that although morphologically abnormal oocytes
may exist, in autumn the capacity of oocytes to develop to
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blastocysts is better than during other seasons. Moreover,
in order to increase the availability of buffalo follicles subjected to OPU performance, Sa Filho et al. [30] conducted
a trial using bovine somatotropin (bST) treatments to live
donors. Their findings showed that administering bST is
beneficial in increasing oocyte recovery rates (an average
of 5.2 oocytes per session) as well as in providing good oocyt equality, but there was no difference in blastocyst rates.
Furthermore, it is worth noting that while Mehmood et
al. [31] revealed that in regard to sperm activation for
use with IVF the swimming-up procedure is preferable
to the Percoll gradient method, Vedantam et al. [32] proposed the addition of 100 nM angiotensin-II is optimal for
sperm capacitation.
In addition, the development of IVM for buffalo
oocytes and embryos is slow because these media have
been produced primarily for IVF in cattle. Chandra et al.
[33] noted that supplementing 100 ng/mL IGF-1 to medium containing mSOF enhanced buffalo blastocyst rates
(29.7%) and cell proliferation. In agreement with this
finding, Sharma et al. [34] concluded that IVM medium
plus IGF-1 has a great influence on both the survival and
growth rates of buffalo oocytes. Furthermore, in an effort
to improve the efficacy of co-culture in buffalo, Attanasio et al. [35] demonstrated that denuded vitrified oocytes
cultured with compact COCs during IVF are able to restore their fertility but that the co-cultured cells do not
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improve blastocyst development.
Recent Developments in IVF in Small
Ruminants
In sheep and goats, IVF affords valuable yet inexpensive embryo resources for basic research as well as for
commercial embryo transfer. However, there are a number of innovations in these species aimed at enhancing the
efficiency of ovine and caprine IVF. Paramio [36] states
that although recent progress in IVP in sheep and goats
has been achieved, there is still variability in results between laboratories due to insufficient understanding of
how to achieve oocytes of high quality. Thus, the importance should be stressed of choosing oocytes with potential for developmental competence [37]. Catala et al. [38]
highlights a useful, non-invasive technique to determine
the quality of ovine oocytes for IVM by using the brilliant
cresyl blue (BCB) test. Romaguera et al. [39], however, indicated that follicle diameter is a crucial criterion in oocyte quality evaluation. In addition, although OPU is the
preferred method to retrieve COCs from live donors, in
the goat the use of laparoscopy to collect oocytes is minimally invasive [40], and does not cause gross lesions such
as adherences and fibroses in ovaries and other organs
[41].
Similar to IVF-related goals in other species, enhancements to IVM and IVC media are required. An efficient
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standard IVC medium for IVP is considered a priority.
In sheep, culturing COCs in IVM medium alone results
in low maturation rates [42]; thus, group co-culture is a
key factor in IVM improvement and the subsequent increase in efficacy of IVP. Kyasari et al. [42] showed that
the maturation rate may increase when ovine COCs are
cultured with somatic cell of cumulus origin (sCC). In addition, Shi et al. [43] indicated that ovine oocytes could be
cultured in IVM medium supplemented with monolayers
of another kind of somatic cell, ovarian cortex, which influence the meiotic resumption and progression of sheep
oocytes during IVM.
Furthermore, Sofi et al. [44] noted that the addition
of cysteamine and epidermal growth factor improves the
maturation rate of ovine oocytes. In contrast to this defined medium, Wan et al. [45] showed that Charles Rosenkrans (CR1) medium plus BSA and FCS is able to support
in vitro development of ovine IVF embryos, but that IVC
medium with SOF supplemented by amino acids and BSA
is more appropriate.
Meanwhile, in the goat, research into IVM and IVC
emphasizes the value of media supplementation rather than the synergic effects of somatic cell co-culture.
Pradeep et al. [46] revealed that for IVM use of oviductin,
an oviductal-specific glycoprotein, improves cleavage rates
and IVF blastocyst yields. This is because for goat embryos oviductin plays a role in protecting the integrity of the
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zona pellucida from oviductal proteases, with consequent
inhibition of polyspermy. Also, De et al. [4] indicated that
adding 100-200µM cysteamine to IVM medium enhances
cleavage rates as well as blastocyst formation. They contended that cysteamine reduces levels of reactive oxygen
species by raising the concentration of glutathione.
Recent Developments in IVF in
Horses
To date, there are relatively few developments in IVF
of horse oocytes, due to comparatively low success rates
of equine IVF [47-49]. Unlike those from cattle and pigs,
abattoir-derived ovaries of horses are in short supply [50].
Moreover, although the OPU technique is applied, recovery rates of immature equine oocytes remain poor [50,51].
In the horse the anatomical proximity of oocytes to the follicle wall reduces the efficiency of oocyte collection in vivo
and in vitro [48]. Blanco et al. [50] promoted the use of
equine pituitary extract combined with human chorionic
gonadotropin (hCG) to increase the availability of oocytes
for OPU/IVP. On the contrary, Jacobson et al. [51] emphasized the importance of OPU without hormone treatments
in horses by determining the appropriate OPU frequency.
Their findings showed that it is efficient to aspirate immature oocytes every two weeks without monitoring ovarian
follicular growth. For evaluation of oocyte developmental
competence for IVM, Mohammadi-Sangcheshmeh et al.
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[49] proposed oocyte assessments based on detection of
glucose-6-phosphate dehydrogenase using the BCB test
which is considered as a non-invasive oocyte evaluation
procedure.
It appears, however, that standard IVF may not work
well in horses [11,52-54]. Fortunately, the intracytoplasmic sperm injection (ICSI) technique is well suited to
equine embryos [11,55]. Recently, Sessions-Bresnahan
et al. [56] achieved an impressive rate (85%) of highly
cleaved equine embryos by ICSI of sperm pre-treated with
dilauroyl phosphatidyl choline.
Recent Innovations in IVF in Pigs
In pigs, there is a high incidence of polyspermic penetrations and, thus far, disappointing results have been
obtained for IVF [57-59]. Despite this, there have been
several developments in IVP systems. Blastocyst rates
remain at 10-25%. The origin of oocytes is crucial to the
developmental competence of porcine IVF embryos, implying an increasing need to identify accurately oocytes of
good quality.
In general, swine COCs are selected for IVM based on
the morphology of cumulus layers, observation of which
is subjective and inexact. Ishizaki et al. [60] proposed a
non-invasive method to determine porcine oocyte quality for maturation by using the BCB test. Although this
technique does not improve the developmental capacity
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directly, it provides useful information on the developmental competence of a single ovum, enabling appropriate selection of oocytes for IVM. Hulinska et al. [58] also
reported that in pigs high polyspermy is associated with
oocytes aspirated from smaller ovarian follicles.
Similar to improvements in bovine IVP, porcine IVF
research has paid attention to preparation of an efficient
culture medium (either IVM or IVC). Wang et al. [61]
noted that NCSU-23 medium meets metabolism and nutrient requirements for pig oocytes cultured in vitro. This
chemically defined solution is used routinely as an IVM
medium, theoretically providing a standard in regard to
inhibition of contamination but with batch variations
noted [59,60]. NCSU-23 has been promoted as a defined
medium with the addition of porcine granulocyte-macrophage colony stimulating factor, a supplement which
not only increases the blastocyst rate but also improves
porcine IVF embryo quality [61]. Consistent with this
finding, Biswas and Hyun [62] showed that supplementation of vascular endothelial growth factor (VEGF) to
IVM medium not only raised the maturation rate, quality
and quantity of blastocysts but increased significantly the
proportion of monospermic zygotes. It may be that VEGF
causes an elevation of intracellular glutathione, the presence of which is regarded as an indicator of cytoplasmic
maturity of oocytes.
Meanwhile, Wu et al. [63] contended that supplementing either IVM or IVC media with L-carnitine acwww.avidscience.com
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celerates nuclear maturation of swine oocytes, preventing
oxidative damage and embryo apoptosis. Cell apoptosis,
or programmed cell death, is a natural process during
embryo development to remove deviant cells, but a high
occurrence of apoptosis is related to abnormal embryo
development in vitro [45]. Moreover, Mito et al. [57] developed a defined medium for IVM using transforming
growth factor (TGF)-α, thereby eliminating the effects of
unknown and uncontrolled factors as well as excluding
the possible introduction of pathogens from animal-derived materials. The results suggested that supplementation of TGF-α to defined IVM medium not only increases
the blastocyst rate of porcine oocytes (28.1%), but that following embryo transfer they have the potential to develop
to full term. Adding TGF-α had a more potent effect than
IGF in increasing both nuclear and cytoplasmic maturation of pig oocytes in the absence of hCG [57]. On the
other hand, Agung et al. [64] argued that a non-defined
culture medium with porcine follicular fluids, containing
a combination of growth factors, hormones and amino acids, is superior to defined medium. Consistent with this
assertion, porcine zygote medium with added 10% FCS
is reported to be better than other in vitro culture media
[59,65].
Co-culture with somatic cells supports porcine oocytes in vitro [64]. However, Ishizaki et al. [60] indicated
that the maturity of oocytes is not improved by modifying
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IVM culture systems. They recommended an individual
IVM culture with an optimal droplet size of 5µL medium.
In addition, this single culture enables the developmental
capacity of each oocyte to be determined.
In summary, while there are some advances in the use
of IVF in pigs, it remains sub-optimal [61]. Further investigation is required to establish a standard medium for the
in vitro culture of porcine oocytes and embryos.
Conclusion
For species of farm animals, recent progress in embryo production in vitro has facilitated an increase in both
efficacy and application. Although this assisted reproduction now enables embryo maturation in cattle and pigs as
routine, there is still scope for improvement to blastocyst
rates and embryo quality. By comparison, research into,
and commercial use of, IVF in other livestock species is
far less advanced. Therefore, it is recommended that future research should focus on identifying optimal, standard IVP systems in buffalo, horses, sheep and goats, as well
as potentially in new and as yet untested species. Prospective breakthroughs in this technology will promote a wider and more regular application of IVF in domesticated
animals.
Acknowledgements
Financial support for our work is provided by Central Queensland University and Australian Reproductive
Technologies.
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Gyr (Bos indicus) cattle embryos. Anim Reprod
Sci. 2010; 120: 10-15.
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