Journal of Agricultural Science and Technology A 6 (2016) 301-313
`doi: 10.17265/2161-6256/2016.05.002
`
`D
`Animal Reproduction Technologies—Future
`Perspectives
`
`DAVID PUBLISHING
`
`Jane Morrell and Patrice Humblot
`Department of Clinical Sciences, Swedish University of Agricultural Sciences (SLU), Box 7054, SE-75007, Uppsala, Sweden
`
`Abstract: Reproductive biotechnologies, such as artificial insemination, sperm selection and embryo technologies, offer possibilities
`for animal producers to increase reproductive efficiency. There have been many significant developments in reproductive
`biotechnologies over the last few decades, e.g., in sperm handling and preservation, in vitro embryo production and preservation,
`sexing and cloning. This review discusses some of the key changes that have occurred and explores their potential for increasing the
`reproductive efficiency of domestic animals in the future. As a consequence, they also offer opportunities to facilitate or accelerate
`genetic selection. If properly used, they may contribute to increase the sustainability of animal production. The role of epigenetics in
`influencing phenotype is also considered.
`
`Key words: Animal reproduction technologies, livestock, genetic selection, conservation breeding, sperm, embryo production,
`epigenetics, future perspectives.
`
`1. Introduction
`
`Animal producers are increasingly expected to
`produce “more for less”, i.e., to have healthy animals
`that achieve target production levels faster than
`previously, while consuming fewer resources and
`producing less waste and fewer greenhouse gases.
`Producers must meet these high expectations, despite
`increasingly scarce resources, declining fertility in
`some species, emerging diseases, environmental
`toxicology and climate change. Since all animal
`production starts with reproduction, reproductive
`biotechnologies can bring about improvements in
`reproductive efficiency [1]. They can also facilitate
`the implementation of breeding schemes especially in
`the context of genomic selection [2]. Importantly,
`when coupled to genomic selection, reproductive
`technologies applied to livestock production are key to
`improving
`the
`sustainability of
`farm
`animal
`production [3]. However, the level of development
`achieved suffers from severe limitations in many
`species, leading to bottlenecks, especially for the
`
`Corresponding author: Jane Morrell, professor, research
`field: veterinary reproductive biotechnologies.
`
`
`conservation of endangered species and livestock
`breeds [4]. The purpose of this review is to look at
`current trends in assisted reproduction technologies
`(ART) to determine possible future developments.
`
`2. Animal Reproduction Technologies
`
`include artificial
`The ART considered here
`insemination (AI), in vitro production of embryos
`(IVP), embryo
`transfer
`(ET), embryo
`sexing,
`genotyping and cloning,
`together with other
`approaches, such as epigenetics. The advantages and
`disadvantages of these technologies are presented in
`Table 1.
`
`2.1 AI
`
`Certain pre-requisites are necessary for AI to be
`successful, namely, a supply of semen of good quality,
`reliable methods of oestrus detection in the female or
`efficient protocols for fixed-time AI, and a means of
`depositing the semen into a suitable part of the female
`reproductive tract. Of these factors, only sperm quality
`will be considered in detail here, since there are
`several recent reviews providing an update on oestrus
`
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`Animal Reproduction Technologies—Future Perspectives
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`Sperm sexing
`
`Embryo transfer
`
`Ovum pick-up and
`in vitro production
`of embryos
`Intracytoplasmic
`sperm injection
`
`in
`
`semen
`
`Disadvantages
`Only genetic contribution from sire; pathogens
`can be disseminated in semen
`step
`Colloids expensive; extra
`preparation for insemination doses
`than
`rates
`Expensive;
`lower
`pregnancy
`conventional semen; not feasible for all breeds or
`all males
`Specialist skills and equipment required; welfare
`considerations
`important; specialist
`Welfare considerations
`skills and equipment required; not possible in all
`species, e.g., horse
`Not useful
`in cattle (disruption of oocyte
`cytoplasm, lack of activation)
`Low
`success
`rate;
`considerable welfare
`implications; not permitted in some countries;
`does not favour genetic progress
`
`
`Table 1 Advantages and disadvantages of various ART.
`Technique
`Advantages
`Artificial
`Allows maximal use of superior sires; reduces disease
`insemination
`transmission
`Selects high quality spermatozoa; removes poor quality
`Sperm selection
`spermatozoa, debris and pathogens
`Maximal use of superior sires; majority of offspring of
`desired gender
`Genetic contributions from both sire and dam; increases
`genetic progress and availability of commercial products
`Genetic contributions from both sire and dam; increase
`genetic progress and availability of commercial products;
`it can be used to deal with genetic variability in selection
`schemes
`Uses only one spermatozoon; useful for horses (and
`humans)
`Increases commercial availability of animals with high
`genetic merits
`
`Cloning
`
`
`detection and fixed-time AI, such as Ref. [5]. With
`regard to sperm quality, relationships have been
`established between sperm motility, morphology,
`membrane
`integrity,
`chromatin
`integrity,
`mitochondrial membrane potential and the likelihood
`of an inseminated female becoming pregnant and
`carrying that pregnancy to term. In the past, selective
`breeding was used in species, such as cattle and pigs,
`to select males with good sperm quality to be used as
`breeding sires. There was also a passive selection in
`bulls for “freezability”,
`in
`that animals whose
`ejaculates did not freeze well were not chosen as
`breeding sires, regardless of other desirable traits.
`Genomics is now being used extensively to select
`breeding sires, at least in cattle, which in theory will
`lead to a wider genetic base and faster rate of
`improvement. There is interest for genomic selection
`in other species as well, e.g., dairy breeds of sheep and
`goats, where it is considered to be more profitable if
`efficient breeding schemes are already available 2.
`However, with less emphasis on selection for sperm
`quality, there will be greater reliance on selecting
`good quality spermatozoa. In other species, such as
`horses and camels, males are still chosen on the basis
`of their appearance or performance in competition,
`
`
`
`that advanced semen handling
`the result
`with
`techniques are needed in these species to select good
`quality spermatozoa and separate them from the rest
`of the ejaculate.
`2.1.1 Sperm Selection
`Advanced sperm handling and selection techniques
`have been reviewed previously [6-8]. Briefly, they are
`techniques
`that
`separate
`the most
`“robust”
`spermatozoa from the rest of the ejaculate. Robustness
`could include the most motile spermatozoa, or those
`that are morphologically normal, or with intact
`acrosomes, or those that survive cryopreservation.
`Techniques for selection include swim-up, filtration
`and colloid centrifugation. The most promising of
`these selection techniques is undoubtedly colloid
`centrifugation, since it can be used at semen collection
`stations, particularly the variant called single layer
`centrifugation (SLC) which uses only one layer of a
`species-specific colloid instead of several, as in a
`density gradient. With SLC, it is possible to select
`highly motile spermatozoa with normal morphology,
`intact membranes and good chromatin integrity, and
`to separate them from seminal plasma and the rest of
`the ejaculate [6-8]. Scaling-up the original technique
`allows whole ejaculates to be processed, even for
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`303
`
`boars where the ejaculate is voluminous [9]. Fertility
`of the selected sperm samples is improved, compared
`to unselected samples, e.g., in problem stallions [10]
`as well as in “normal” ejaculates [11]. Selected
`spermatozoa also survive cryopreservation better than
`non-selected spermatozoa, at least in horses [12, 13]
`and pigs [14]. However, recent studies with bull
`spermatozoa in a lecithin-containing extender suggest
`that the selected spermatozoa show increased levels of
`tyrosine phosphorylation after colloid centrifugation,
`suggesting that the removal of the seminal plasma
`leads to the initiation of capacitation, which might not
`be desirable
`in
`sperm
`samples
`for artificial
`insemination [15]. However, it is not yet clear whether
`this is a feature of bull spermatozoa in general or
`whether the soy bean lecithin-containing extender
`plays a role in permitting initiation of capacitation.
`The SLC method has also been scaled-down to
`facilitate processing small volumes (microliters) of
`semen, e.g., thawed sperm samples for in vitro
`fertilization (IVF). Thus, thawed red deer semen was
`processed on 1 mL colloid in an Eppendorf tube [16].
`However, a comparison of 1 mL of colloid in a 15 mL
`centrifuge tube and 1 mL of colloid in an Eppendorf
`tube for thawed bull semen indicated that a higher
`sperm yield was obtained when the 15 mL centrifuge
`tube was used [17]. Since the area of the interface
`between the colloid and semen is greater in the 15 mL
`tube, presumably there is less competition for the
`spermatozoa to pass into the colloid than in the
`Eppendorf tube. This possibility of using less colloid
`for sperm preparation makes the use of colloids more
`attractive when preparing sperm samples for IVF.
`Apart from sperm quality, improvements in sperm
`survival during storage are to be expected in the future.
`There has been interest recently in the influence of
`season on seminal plasma with its attendant effects on
`sperm quality. These seasonal changes were observed
`at least 20 years ago [18], but have received renewed
`interest recently. It may well be that changes in diet,
`
`
`
`particularly lipid content, affect the composition of
`sperm membranes, rendering them more susceptible to
`damage during freezing. Studies of these effects may
`lead to identification of additives for diets or for
`semen extenders to overcome seasonally-induced
`dietary deficiencies in the composition of sperm
`membranes, thus improving cryosurvival.
`Another suggestion to improve sperm quality is that
`adding oviductal components, such as heat shock
`proteins (HSP), may have a beneficial effect on sperm
`membranes during storage [19]. However, since sperm
`capacitate under physiological conditions
`in
`the
`oviducts, it is not clear whether adding oviductal
`components to sperm doses for insemination would
`necessarily be beneficial, although potential benefits
`do exist for IVF.
`Cryopreservation is the most effective method of
`extending
`sperm
`life. Recent
`advances
`in
`cryopreservation
`techniques
`suggest
`that
`even
`spermatozoa from species
`that were previously
`considered difficult
`to
`freeze can be
`frozen
`successfully in the research laboratory. In the near
`future, it may be possible to develop practical methods
`for use in the field. New forms of sperm packaging
`may help
`to extend sperm
`life. Recently,
`the
`possibility of encapsulating spermatozoa was suggested
`as a means of providing slow release of spermatozoa
`into
`the
`female
`reproductive
`tract. Although
`preliminary fertility data in cattle and pigs look
`promising [20], it remains to be seen whether this
`form of packaging will be useful for the AI industry.
`2.1.2 Sperm Sexing
`Pre-conception gender selection has been a goal for
`many years. Many animal production systems make
`use of only one gender, e.g., dairy cows for milk
`production and hens for egg-laying, but even in pig
`production units, the ability to select for female
`offspring would obviate
`the need for surgical
`castration of male piglets. Some species of fish stop
`growing on attaining sexual maturity, which means
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`Animal Reproduction Technologies—Future Perspectives
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`that females (which mature later than males) tend to
`achieve larger body weights and therefore have a
`higher carcass value than males. In many instances,
`the birth of offspring that are not of the desired gender
`leads to production inefficiencies and wastage, and
`hence pre-conception gender selection is likely to be
`one of the important areas of focus in the coming
`decades. Although sperm sexing would not allow
`gender selection of poultry (where the female is the
`heterogametic sex), it would be effective for mammals
`and has therefore received a lot of attention in the last
`few decades. The only method of sexing spermatozoa,
`which has been shown to work reliably so far, is the
`flow sorting of spermatozoa [21, 22] stained with the
`vital dye, H33342 [23]. However, the process of
`sorting sufficient sperm numbers for an insemination
`dose takes a long time, since the stained spermatozoa
`must pass individually through a laser beam for
`detection of their DNA content. Although flow
`cytometric sexing of spermatozoa is 70%-90% reliable,
`the method is slow and expensive, and sperm fertility
`may be reduced. Preliminary data on sexing of boar
`spermatozoa using antibodies
`to
`the
`recently
`discovered sperm-surface proteins [24] suggest that an
`alternative route to achieve sperm sexing may be
`available in the future.
`
`2.2 Embryo Technologies
`
`Embryos can be obtained from IVP, IVF or
`intracytoplasmic sperm injection (ICSI). IVP typically
`involves the in vitro maturation of immature oocytes
`obtained from slaughterhouse material, followed by
`IVF and culture of the resulting zygotes before
`transferring to recipients or freezing for later transfer.
`Although these techniques have been available for
`several decades, their use in animal production
`systems is still limited by the cost of techniques, and
`is almost exclusive in cattle. In Europe (France,
`Germany, the Netherlands), they are now extensively
`used in dairy cow selection schemes. In South
`
`
`
`America, mainly Brazil, there has been a huge
`commercial development for beef production over the
`last decade, although there has recently been a shift in
`predominance towards dairy breeds [5]. Although the
`production of large offspring and associated syndrome
`of the neonate is now avoided by use of media without
`fetal calf serum (FCS), there are still some problems
`to be resolved and the efficiency of the system
`requires optimization to reduce the cost. Oocyte
`quality varies considerably depending on a huge
`number of factors, which influences the subsequent
`outcome of ET. Many epigenetic factors known to
`affect oocyte
`function
`and
`early
`embryonic
`development (see section on epigenetics) are also
`thought to affect the health of the resulting offspring.
`It is possible that IVP embryos are more at risk of
`adverse effects because of lack of control mechanisms
`that would be present in vivo.
`Despite the considerable interest in using IVP in the
`production of transgenic pigs during the 90s, the
`technique of IVP for pig embryos is currently not as
`well developed as for cattle embryos. The problem of
`polyspermy is still a limiting factor in this species and
`in vitro culture is not considered to be optimal yet [25].
`On-going research may overcome the polyspermy
`problem in the future, thus enabling more use to be
`made of IVP in pigs.
`Ovum pick-up (OPU) for subsequent IVF and ET is
`increasing in Europe and North America, and has the
`potential for obtaining many more offspring from
`cows of known genetic value than they could produce
`physiologically. It is rapidly outpacing conventional
`superovulation for ET, having increased more than
`ten-fold since the turn of century [26]. The technique
`uses ultrasound-guided follicular aspiration to recover
`oocytes from pre-ovulatory follicles in situ, and can
`even be used on pregnant cows. Twice weekly OPU
`can be practiced without any side effects that could
`compromise subsequent embryo development. In
`some circumstances, OPU can be combined with
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`
`superovulation to increase the number of oocytes
`recovered. The remarkable increase in its use in Brazil
`is attributed to it being more effective and cost
`efficient than superovulation in Bos indicus cattle.
`In Europe, some companies work mainly with in vivo
`produced embryos, whereas others have integrated IVP
`in their systems. However, there is a strong need for
`companies to develop and optimize the efficiency of
`embryo technologies in order to be competitive. Most
`particularly, the variation in pregnancy rates obtained
`after the transfer of IVP, biopsied and cryopreserved
`embryos (the most interesting strategy from a genetic
`point of view) still represents a major bottleneck in the
`use of these technologies.
`OPU has been used in other ruminants, such as
`buffalo, although the ovaries are small compared to
`cows and contain fewer follicles. The follicular
`population may be influenced by season [27]. In
`contrast, superovulation with subsequent ET has no
`great impact in this species, because of the limited
`number of embryos recovered and their low survival
`after cryopreservation [28]. However, OPU is still the
`only reliable method for obtaining oocytes for equine
`IVF, since IVP of horse oocytes tends to result in
`hardening of
`the zona,
`thus preventing sperm
`penetration. Normally only one pre-ovulatory follicle
`is present in each cycle during the breeding season,
`thus making the yield from this technique less
`effective than in cattle. However, the yield of useable
`oocytes can be increased by aspiration from all
`follicles greater than 1 mm in diameter [26, 29].
`Researchers are still continuing their attempts to
`develop equine IVF, although, to date, only a few
`foals have been born using the technique, despite
`many attempts. Moreover, IVP allows the production
`of some hybrids, e.g., camas (camel/llama hybrids),
`although such applications are research-based rather
`than being of practical use for animal production at
`present. Similarly, the techniques may be used for
`species conservation. In contrast to equine ICSI,
`
`
`
`bovine ICSI has not progressed into clinical practice.
`The lack of success in producing offspring is generally
`attributed to inadequate oocyte activation [30] or to
`cytoskeletal damage, e.g., from the large diameter
`pipette needed to accommodate the bull spermatozoon
`[31]. Although advances in oocyte activation may
`allow more embryos to develop after ICSI, the ease
`and availability of bovine IVF provides little incentive
`to develop the more laborious technique of ICSI in
`this species.
`Use of sperm sexing in association with IVF-IVP
`may also avoid some of the present limitations in
`selection schemes, due to the high number of
`spermatozoa that must be discarded and the large
`individual variation associated with the sexing process
`by flow cytometry [32]. Sexing of embryos enables
`only those of the desired gender to be transferred.
`However,
`the procedure
`is
`time-consuming and
`requires considerable skill to avoid harming the
`embryo. The procedure involves removing some cells
`from the embryo; the DNA from the biopsy is then
`processed by polymerase chain reaction (PCR) for
`identification of X- or Y-chromosome specific
`sequences, whilst the embryo is frozen or vitrified.
`Once the gender of the embryo has been determined,
`the embryo can be transferred to a recipient or
`destroyed. New techniques have been used with horse
`embryos that permit sexing to be done with a
`reasonable degree of accuracy in 6-10 h, whilst the
`embryo is maintained in cultured. Thus, the embryo
`can be transferred on the same day as the biopsy [33],
`which is a significant development in this species.
`Presumably the technique could also be modified for
`use in other domestic species.
`
`2.3 Cloning
`
`Somatic cell nuclear transfer (SCNT) has been
`carried out in cattle and other livestock, but the
`procedure
`remains
`technically demanding and
`expensive. A donor cell is injected into or fused with a
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`Animal Reproduction Technologies—Future Perspectives
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`mature, good quality, enucleated oocyte, followed by
`activation and culture of the product. However, there
`are high rates of pregnancy loss and also abnormal
`placental development and pathologies during the
`neonatal period, which is a major limiting factor in the
`adoption of this technique.
`This technique may allow a wider diffusion of
`animals of interest, and some results have been
`obtained in the preservation of endangered species.
`However, the efficiency of the process is not high
`enough
`to
`reconstitute viable populations. The
`technique also has considerable limitations with
`regard to genetic progress, since genetic variability is
`an important component even in the context of
`genomic selection [34]. It is possible that future
`developments will enable the technique to achieve the
`levels of efficiency and reproducibility necessary to
`produce live offspring consistently. Considering the
`need to maximize genetic variability, cloning is
`unlikely, at least at present, to represent a useful tool
`in
`selection
`schemes organized by breeding
`associations and companies due to limitations in
`reproductive efficiency. However, individual farmers
`with access to genomic selection may be interested in
`the duplication of their best cows through cloning for
`commercial purposes in countries allowing the use of
`this process.
`Much attention has been focused on cloning in pigs
`but with relatively little success in terms of live piglets.
`Currently, only 1%-5% of SCNT embryos develop to
`become piglets [25]. However, increased success has
`been reported using zona-free reconstructed embryos,
`which may eventually lead to improvements and
`eventual optimization of the process.
`
`3. Use of Embryo-Based Biotechnologies in
`Genomic Selection
`
`In an attempt to improve numerous traits by
`genomic selection, knowledge of the relationships
`between genome information and phenotypic criteria
`
`
`
`is of crucial importance. Initially, microarrays were
`used
`to characterize
`the
`relationships between
`genotype and phenotype [35, 36]. More recently,
`high-throughput technologies for DNA and RNA
`sequencing analysis have been used
`to study
`relationships between genotype, phenotype and gene
`expression. With
`these objectives, phenotyping
`(animal models, precise criteria and methods)
`becomes the main bottleneck to achieve this goal. As a
`consequence, research is needed to phenotype new
`critical traits and to improve the precision of the
`phenotypes for existing traits, such as fertility and
`reproductive
`traits [37, 38]. For
`this purpose,
`proteomics, lipidomics and metabolomics may be
`particularly appropriate to find new markers for
`fertility [38] or for predicting embryo survival
`potential and the ability of recipient to sustain
`pregnancy to term [39, 40].
`One of the most important features of the new
`selection procedures will be to increase the number of
`candidates submitted
`for genomic selection,
`to
`maximize
`the chances of producing
`interesting
`individuals that will be evaluated positively for a large
`number of traits. As mentioned before, this will allow
`an increase in the selection pressure for those traits. In
`addition, it will be possible to use bulls for AI at a
`younger age, thereby lowering the generation interval.
`Finally, the use of groups of bulls with a favourable
`genomic index will improve the precision of indices,
`when compared to the use of a very limited number of
`older sires, as was the case in the past. This may be
`favourable to genetic variability if adequate and wise
`breeding
`schemes are
`implemented; otherwise
`shortening the generation interval may also lead to an
`increased inbreeding rate.
`The method of producing the large numbers of
`animals to be genotyped may become a rate-limiting
`factor. Thus AI alone may be inadequate to generate
`sufficient animals in a given period of time, and the
`efficiency of multiple ovulation and embryo transfer
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`
`(MOET) and OPU-IVP will become more important.
`With these “intensive” embryo-based reproductive
`techniques, it is relatively easy to increase the number
`of candidates by increasing the number of flushes in
`MOET schemes. However, compared to MOET, two
`to three times the number of embryos can be produced
`in a given period of time by use of repeated OPU-IVF
`sessions
`[41, 42]. This method also presents
`advantages in preserving genetic variability. Much
`research has been done to improve in vitro culture
`systems. This allows most teams working with IVF to
`obtain overall development rates up to the blastocyst
`stage of between 30% and 40%.
`The effect of a previous superovulation on
`fertilization and subsequent embryonic development is
`still controversial [43]. It has been shown very clearly
`from most studies that there is a significant decrease
`in embryo production when oocytes are matured in
`vitro in standard medium compared to in vivo
`conditions [43, 44]. This emphasizes the roles of the
`final steps of oocyte growth and maturation on
`subsequent embryo development, which have also
`been illustrated by epidemiological studies showing
`relationships between some factors influencing these
`steps and embryonic mortality [45]. It is likely that
`much progress can be achieved in embryo production
`by optimizing the conditions under which the oocytes
`are growing within follicles
`in donor females.
`Handling at the time of collection, and thereafter, as
`in vitro maturation,
`well as during
`requires
`optimization since dramatic metabolic changes occur
`very quickly after oocyte recovery [45].
`Despite these limitations, the work that has been
`done in the past 15 years to improve oocyte collection
`and in vitro embryo production systems has enabled
`the most advanced breeding companies to produce
`more embryos in their genetic schemes [41, 46].
`However, there are also disadvantages, such as
`miss-management of the use of these techniques, that
`may lead to a significant increase in inbreeding,
`
`
`
`especially if bull dams are over-exploited. Moreover,
`due
`to new
`requirements
`in
`relation
`to
`the
`implementation of genomic selection
`(increased
`number of candidates), additional limitations exist for
`producing the very large number of calves that would
`be genotyped after birth. Effectively, one of the main
`bottlenecks experienced by breeding organizations
`working with dairy cattle in Europe is the limited
`availability of recipients. This is reinforced by the fact
`that not only are lower pregnancy rates achieved when
`using cows as recipients instead of heifers, but also
`the efficiency of ET is much lower if heifers are used
`as donors 42. In addition, high costs will be induced
`by the transfer of a very large number of embryos into
`recipients that must be maintained until birth of the
`progeny, and
`the economic potential of
`the
`non-selected calves will be low. When producing
`these candidate animals on farms, the amount of field
`work in relation to ET and in vitro production will be
`even greater than at present and will generate high
`logistical costs.
`Limitations
`in embryo production are also
`encountered due to use of young animals to reduce the
`generation
`interval. Genomics
`allow
`early
`identification of candidates for the selection scheme,
`but the challenge is now to produce enough good
`quality gametes and embryos from young animals.
`Thus, a very important research area is how to obtain
`gametes from pre-pubertal animals and/or to hasten
`puberty. However, it is also important to balance the
`desire for early puberty against the long term health
`and productivity of the animal [47. Thus females
`should not be bred at their first ovulation, but should
`be given time to enable regular ovarian cycles to
`become established and for the uterus to become
`capable of supporting a pregnancy. In parallel, it is
`critical to develop tools to predict the donor’s
`superovulation responses to avoid the inefficient
`treatment of poor responders, and thus to decrease the
`costs of selection schemes.
`
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`Location
`
`
`
`Table 2 Pregnancy rates (day 90 post transfer) following transfer of fresh or frozen biopsied embryos.
`Pregnancy rates
`Transfer of frozen biopsied embryos
`Transfer of fresh biopsied embryos
`61/116 (52.3%)
`109/171 (63.7%)
`Farm
`46/83 (55.4%)
`
`Station 1
`28/54 (52.0%)
`
`Station 2
`135/253 (53.0%)
`109/171 (63.7%)
`Total
`Results from Farm and Station 1 were obtained after embryo sexing. Results from Station 2 were obtained after biopsy and typing for
`45 microsatellites (detection rate 98%), modified from Ref. [30]. Farm refers to field conditions, whereas Station 1 and 2 refer to
`research stations where the conditions may be different to those found in the field.
`
`
`Finally, this process may increase the contractual
`cost for individual farmers especially due to the
`potential existence of very interesting candidates
`identified by genomics. For these reasons, genotyping
`the embryos and selecting them before transfer appear
`to be an attractive scenario to maximize the chances of
`finding interesting individuals for multiple traits,
`while transferring a “reasonable” number of embryos.
`
`4. Embryo Typing
`
`typing for breeding
`interest of embryo
`The
`companies was discussed long before the emergence of
`the new
`techniques for genomic selection
`that
`currently includes thousands of markers [41]. Typing
`and selection early in life was expected to be a
`solution to shorten the generation interval, to limit the
`costs of producing high numbers of calves and
`progeny testing to achieve multi-character selection.
`Today,
`the potential advantages of combining
`intensive embryo production and genotyping are even
`higher. Results reported initially in the literature for
`ruminants were based on typing for a limited number
`of markers [48]. Field studies with in vivo produced,
`biopsied embryos (either fresh or frozen) have shown
`that pregnancy rates following transfer on-farm were
`compatible with field use [34] (Table 2).
`Initially, typing was envisaged from a large
`number of cells obtained from reconstituted embryos
`following cloning of blastomeres. Since
`then,
`preliminary studies from limited numbers of biopsies
`and typing have shown that the use of pre-amplified
`
`
`DNA is possible [34] and also compatible with
`typing from high density marker chips. Thus, it may
`be useful
`to perform economic and genetic
`simulations to evaluate the costs and advantages for
`the genetic schemes of such procedures based on
`embryo typing.
`
`5. Epigenetics: Perspectives for Its Use
`
`Epigenetic changes are stable alterations of
`DNA-associated molecules
`that do not
`involve
`changes of the actual genes. The expression of genes
`can be altered due
`to modifications of active
`regulatory sequences inducing alterations of
`the
`cellular phenotype, which can
`in
`turn
`lead
`to
`modifications of animal phenotype. Epigenetic
`alterations involve changes in DNA methylation
`(including global hypomethylation and more locus
`specific hypermethylation) and methylation or
`acetylation of histones [49, 50]. They can occur in all
`cells in the body, but if they occur in oocytes or
`spermatozoa, they may be passed on to the next
`generation. The epigenome is especially vulnerable
`during embryogenesis, fetal and neonatal life and at
`puberty
`[51, 52]. Effectively,
`the epigenome
`undergoes extensive
`reprogramming when
`the
`gametes fuse at fertilization (during the final stages of
`meiosis and around fertilization due to chromosomal
`decondensation and intense remodelling) and during
`the preimplantation period. These epigenetic changes
`are necessary for normal embryonic development and
`survival [53]. The modification of epigenetic marks
`
`Exhibit 1038
`Select Sires, et al. v. ABS Global
`
`

`

`Animal Reproduction Technologies—Future Perspectives
`
`309
`
`that occurs during preimplantation development are
`