Experimental Section / Mini-Review
`
` Gerontology 2017;63:417–425
` DOI: 10.1159/000452444
`
` Received: May 31, 2016
` Accepted: October 12, 2016
` Published online: November 8, 2016
`
` Aging of Cloned Animals: A Mini-Review
`
` Jörg Patrick Burgstaller Gottfried Brem
`
` Institute of Biotechnology in Animal Production, Department of Agrobiotechnology, IFA Tulln, Tulln , and Institute
`of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna , Austria
`
`
`
` Keywords
` Aging · Somatic cell nuclear transfer · Cloned animals ·
`Telomere length
`
`dence of cloned animals reaching high age is available. We
`therefore encourage reports on the aging of cloned animals
`to make further analysis on the performance of SCNT pos-
`sible.
` © 2016 S. Karger AG, Basel
`
` Abstract
` The number of species for which somatic cell nuclear trans-
`fer (SCNT) protocols are established is still increasing. Due to
`the high number of cloned farm, companion, and sport ani-
`mals, the topic of animal cloning never ceases to be of public
`interest. Numerous studies cover the health status of SCNT-
`derived animals, but very few cover the effects of SCNT on
`aging. However, only cloned animals that reach the full ex-
`tent of the species-specific lifespan, doing so with only the
`normal age-related afflictions and diseases, would prove
`that SCNT can produce completely healthy offspring. Here,
`we review the available literature and own data to answer
`the question whether the aging process of cloned animals is
`qualitatively different from normal animals. We focus on 4
`main factors that were proposed to influence aging in these
`animals: epigenetic (dys)regulation, accumulation of dam-
`aged macromolecules, shortened telomeres, and (nuclear
`donor-derived) age-related DNA damage. We find that at
`least some cloned animals can reach the species-specific
`maximum age with a performance that matches that of nor-
`mal animals. However, for most species, only anecdotal evi-
`
` Introduction
`
` It is a basic, yet still quite mysterious fact that at fertil-
`ization the aging clock in metazoans is “reset to zero.”
`While every individual “ages” over time, and consequent-
`ly dies at some point, the cells in the germline seem com-
`pletely resistant to age-related changes – otherwise a spe-
`cies would age as quickly as the individual itself [1] . While
`individual germ cells do age along with its organism, var-
`ious control and selection mechanisms assure that the
`next generation starts relatively “unchanged” and healthy
` [2, 3] . It is, for example, now known that both nuclear and
`mitochondrial genomes are likely to acquire a small num-
`ber of mutations between parents and offspring [4] . We
`regard this minimal change that occurs during natural
`reproduction, within the physiological reproductive life-
`span of the parents, as the ideal ‘reset to zero’ of the aging
`clock, against which the aging of cloned animals has to be
`compared.
`
` © 2016 S. Karger AG, Basel
`
`
`E-Mail karger@karger.com
` www.karger.com/ger
`
` Jörg Patrick Burgstaller
` Institute of Biotechnology in Animal Production, Department of Agrobiotechnology
`IFA Tulln, Konrad Lorenzstrasse 20
` AT–3430 Tulln (Austria)
` E-Mail joerg.burgstaller   @   vetmeduni.ac.at
`
`Exhibit 1036
`Select Sires, et al. v. ABS Global
`
`

`

` In somatic cell nuclear transfer (SCNT), the nucleus of
`an adult cell is transferred to an enucleated oocyte, and is
`thought to not only regain pluripotency, but is also “reju-
`venated” by factors in the ooplasm. Starting with works
`based on frogs [5] , SCNT fully took off with the birth of
`Dolly the sheep [6] . Since then, SCNT has been applied
`successfully in numerous species (mouse, cattle, goat, pig,
`mouflon, domestic cat, rabbit, horse, mule, rat, African
`wildcat, dog, ferret, wolf, red deer, buffalo, camel, and
`coyote) (see online suppl. Table S1; for all online suppl.
`material, see www.karger.com/doi/10.1159/000452444
`for details). Efficiency of SCNT is still rather low, with
`success rates of 0.3–1.7% per reconstructed oocyte and
`3.4–13% per transferred SCNT embryo (as reviewed in
` [7] for farm animals). There are relatively high losses of
`individuals derived from SCNT during their perinatal
`and early postnatal development, but they are thought to
`be indistinguishable from controls once they reach high-
`er age. In fact, they are reported to have comparable per-
`formance on traits like beef and milk production [8] .
`While there are clearly factors that limit the efficiency of
`cloning, at least some nuclei seem to be completely repro-
`grammed and rejuvenated to result in a completely “nor-
`mal” adult individual. However, is it possible with a nu-
`cleus derived from a somatic cell, to completely start at
`time point zero, like gametes after a conventional fertil-
`ization?
`
` Factors That Influence Aging of SCNT-Derived
`Animals
`
` The effects of aging are quite complex, and cellular
`biomarkers of aging remain somewhat elusive [1] . Nev-
`ertheless, 4 main factors were proposed to underlie (pos-
`sible) changes in the aging characteristics in cloned ani-
`mals. They are (a) epigenetic (dys)regulation, (b) accu-
`mulation of damaged macromolecules, (c) shortened
`telomeres, and (d) age-related DNA damage.
`
` Epigenetic Reprogramming
` Epigenetic regulation is a process that tells genetically
`identical cells which role to adopt. Epigenetic regulation
`happens on multiple levels. “Cis-epigenetics” refers to di-
`rect methylation and demethylation of DNA bases, as
`well as to chromatin modifications. “Trans-epigenetics”
`covers proteins and RNAs. Both cis-and trans-epigenetics
`lead to the expression and repression of genes. In SCNT,
`the “cis-acting” nucleus of the differentiated donor cell
`gets exposed to the “trans-acting” factors of the recipient.
`
`In consequence, a sufficient number of genes to allow
`SCNT of the donor cells are reprogrammed to resemble
`that of the recipient cell. However, other genes remain in
`the expression pattern of the donor cell. This incomplete
`“reprogramming” is thought to be responsible for most
`of the SCNT-associated problems like fetal and placental
`anomalies (reviewed, e.g., in [9, 10] ). Our understanding
`of these reprogramming effects greatly increased with the
`introduction of induced pluripotent stem cells (iPSCs)
` [11] . Terminally differentiated adult cells, initially from
`mouse and human and more recently from other species,
`can be converted to pluripotent stem cells by the intro-
`duction of a small number of transcription factors such
`as Oct4, Sox2, and Klf4 (reviewed in [12] ). Like in SCNT,
`the reprogramming efficiency is still very low. Important-
`ly, it has recently been shown that this low efficiency
`is not based on few ‘elite’ cells in a tissue that respond
`to reprogramming. Virtually every cell can be repro-
`grammed, but stochastic effects mean that only a small
`number of the cells finish the process [12] . It has been
`speculated that SCNT protocols lead to a selection pro-
`cess, as only a rather small portion of cells can support the
`development of a healthy animal [13] . Therefore, further
`optimization of SCNT and iPSC protocols will very likely
`be able to increase the efficiency of both approaches. In
`fact, the application of a histone deacetylase inhibitor,
`trichostatin A, improved the success rate of mouse clon-
`ing up to 5-fold. Moreover, with this protocol, the mice
`could be serially re-cloned for 25 generations, a feat that
`had not been previously achieved [13] (see below). It will
`be very interesting to see whether this success can be re-
`peated in other, larger (domestic) species.
` What are the consequences of incomplete reprogram-
`ming on aging? The relatively high losses of cloned ani-
`mals during their perinatal and early postnatal develop-
`ment are thought to be mainly caused by failed epigenetic
`reprogramming [9] . Later in life, a certain dilution effect is
`expected to act upon epigenetic markings: if the enzymes
`that modify DNA and histones fail to reinforce the modi-
`fications during replications and cell division, both cis-
`and trans-epigenetics of the cells can be altered [1] . This
`might lead to a completely normal phenotype later in life.
`Moreover, successful clones are presumably derived from
`nuclei that, by chance, fulfilled all or most of the steps that
`lead to sufficient/complete reprogramming [12] .
` It is of course difficult to distinguish the overlapping
`areas of disease and aging. It is noteworthy, however, that
`perfectly reprogrammed cloned animals seem to be pos-
`sible by optimizing the cloning protocols. Diseases of
`cloned animals are beyond the scope of this review (for a
`
`418
`
` Gerontology 2017;63:417–425
`DOI: 10.1159/000452444
`
` Burgstaller/Brem
`
`
`
`Exhibit 1036
`Select Sires, et al. v. ABS Global
`
`

`

` Table 1. Telomere length of cloned animals (see also Table S2 for details)
`
`Species
`
`Cattle
`
`Pig
`
`Sheep
`
`Goat
`
`Mouse
`
`Wolf
`
`Dog
`
`Relative telomere
`length compared to
`control animals
`
`Studies,
`n
`
`Cloned animals,
`n
`
`Cloned animals
`with normal
`telomeres, %
`
`Normal/longer
`Shorter
`Normal/longer
`Shorter
`Normal/longer
`Shorter
`Normal/longer
`Shorter
`Normal/longer
`Shorter
`Normal/longer
`Shorter
`Normal/longer
`Shorter
`
`5
`3
`3
`2
`3
`3
`2
`3
`2
`No
`No
`2
`2
`No
`
`42
`23
`32
`14
`6
`10
`8
`12
`535
`No
`No
`5
`2
`No
`
`64.6
`
`69.6
`
`37.5
`
`36.4
`
`100.0
`
`0.0
`
`100.0
`
`Reference
`
`[27 – 31]
`[8, 30, 32]
`[26, 33, 34]
`[26, 35]
`[22, 36, 37]
`[22, 36, 37]
`[38, 39]
`[38 – 40]
`[13, 41]
`No
`No
`[42, 43]
`[44]
`No
`
`detailed review on this topic see, e.g., [10] ); therefore, we
`focus on differences between cloned and control animals
`towards the end of the natural lifespan of the respective
`species (see below).
`
` Accumulation of Damaged Macromolecules
` Another cellular mediator of aging is accumulation of
`damaged macromolecules, including proteins and lipids,
`and highly stable aggregates of those molecules [14] . Like
`epigenetic regulation, damaged macromolecules are the-
`oretically reversible by dilution, i.e. by cell division and
`new synthesis of macromolecules. So, this mechanism
`has very likely limited influence on the rejuvenation effect
`on SCNT; at least at a higher age damaged macromole-
`cules derived from the nuclear donor cell should be di-
`luted sufficiently to not cause any harm. If cloned animals
`produced a higher amount of damaged macromolecules
`and/or were less adept to handle them during their aging
`process, this mechanism could nevertheless play an indi-
`rect role in the aging process. Aggregation-associated de-
`generative disorders are well studied in humans (and an-
`imal models) as they cause severe, age-related degenera-
`tive diseases [15] . It has yet to be determined whether
`similar mechanisms could influence the efficiency and
`long-term outcome of SCNT.
`
` Shortened Telomeres
` Mammalian telomeres are repeated guanine-rich se-
`quences that cap the end of chromosomes to preserve ge-
`nome stability [16, 17] . The telomere length is different
`
`among individuals and species [18] . At each cell division,
`the telomere length is shortened in normal cells, leading
`to irreversible growth arrest (varying in a tissue-specific
`way) when reaching a certain threshold [18] . In active
`germ cells and during early embryogenesis, the enzyme
`telomerase is active, guaranteeing restoration of telomere
`length for the next generation. Consequently, it could be
`expected that when using an aged somatic cell with short-
`ened telomeres for cloning, the offspring might start with
`a diminished replicating capability of its cells and conse-
`quently age, or at least reach senescence, faster. More-
`over, such offspring might also suffer from telomere dys-
`function-induced diseases such as cancer or dyskeratosis
`congenita [19] . While such diseases seem not to be abun-
`dant at least earlier in life, for which time-sufficient data
`are available [20] , premature aging was a serious concern
`from the beginning. In fact, the telomeres of the first adult
`clone [21] Dolly were 20% shorter when compared with
`age-matched controls [22] , and she died (of a viral illness,
`but also suffering from arthritis that was speculated to be
`SCNT-derived [23] ) at the age of 6 years, while the life
`expectancy of her breed would have been 12 years [1] .
`However, further work on several species showed that at
`least in some clones telomere length was normal or even
`elongated when compared to age-matched controls. It
`was found that telomere length can be restored in the em-
`bryo during SCNT [13, 24, 25] . It is still unclear why this
`does not happen in all cases. We have summarized the
`results of various studies with regard to telomere length
`in Table 1 (see also online suppl. Table S2 for further de-
`
` Aging of Cloned Animals
`
` Gerontology 2017;63:417–425
`DOI: 10.1159/000452444
`
`419
`
`Exhibit 1036
`Select Sires, et al. v. ABS Global
`
`

`

`tails). In one-third to half of the studies (and cloned ani-
`mals), the telomere length is reduced, either compared to
`the cell line used for SCNT or (more significant) to age-
`matched control animals. In cattle, several studies report
`normal or even elongated telomere length, while others
`find shortened telomeres. Also in pig, several studies re-
`port normal telomere length, while other studies find
`shortened telomeres. Curiously, in sheep almost all re-
`ported cloned animals show shorter telomeres than con-
`trol animals (in absolute numbers, if not significantly. See
`online suppl. Table S2). In goats, 3 reports show both nor-
`mal and shortened telomeres. Telomeres of 2 cloned dogs
`were normal, while in 5 cloned wolves, telomere length
`was reduced. In mouse (a species with notably long telo-
`meres) telomere length was found normal, even after re-
`cloning of 25 generations (as described below).
` Elongation of telomeres during SCNT takes place be-
`tween the morula and blastocyst stage [24, 25] . However,
`this clearly does not work perfectly and might be a critical
`issue for the long-term outcome of SCNT. The reasons
`for these ambiguous results are currently unclear. Possi-
`ble factors are species differences, donor cell origin and
`of course the NT protocol itself. Incidentally, the degree
`of telomere lengthening was found to be associated with
`nuclear reprogramming [16] . Currently, the application
`of trichostatin A, as already mentioned, seems not only to
`improve the success rate of cloning but also to favourably
`influence the telomere length [26] .
`
` Genomic Changes in Nuclear and Mitochondrial DNA
` The primordial germ cells in the embryo are separated
`from the somatic cells at a very early stage. Moreover, at
`least in the ovary there seems to be a selection mechanism
`that ensures the “fitness” of a developing oocyte, resulting
`in the degradation of numerous follicles [2] . Also for mi-
`tochondrial DNA (mtDNA), a purifying selection exists
`at the oocyte level (and possibly during gestation) [3] .
`Therefore, DNA in the germ line is preserved at a very
`high level, and harmful mutations are likely to get sorted
`out ensuring the genetic fitness of the offspring. In con-
`trast, somatic cells accumulate a high number of muta-
`tions both in nuclear and mtDNA. While in postmitotic
`tissues these mutations get fixed, in dividing tissues cells
`that are dysfunctional are thought to be replaced by oth-
`ers that have no or less detrimental mutations, counter-
`acting loss of tissue function [3] . But how high is the dan-
`ger of selecting a somatic cell with detrimental muta-
`tions?
` The mutation rate of mtDNA is believed to be at least
`100-fold higher than that of the nuclear genome [3] . In a
`
`mouse model, somatic mutations of mtDNA were shown
`to potentially aggravate aging [45] . Also iPSCs of adult
`individuals were recently found to harbour age-related
`mutations [46] . While these mutations are potentially
`harmful to the somatic cell, in SCNT the mtDNA of the
`somatic cell is largely replaced or outnumbered by the
`vast majority of mtDNAs derived from the donor oocyte
` [47, 48] . However, the mtDNA of the recipient oocyte is
`foreign to the donor cell nucleus. This could theoretically
`lead to nucleo-mitochondrial incompatibility, i.e. errors
`in the normally fine-tuned orchestration of gene expres-
`sion and replication between the nuclear and mitochon-
`drial genome that guaranties optimal energy supply. The
`extent of this incompatibility and its physiological influ-
`ence is currently debated [49] . Even if present in small
`amounts, the mtDNA of the nuclear donor cell could the-
`oretically have a “replicative advantage,” thereby domi-
`nating the cells after some time [50] . First evidence of
`such a mechanism was found in cloned sheep [47] . Also
`in mice ( [50] and references therein) and cattle [51] that
`harbour 2 types of mtDNA, biased segregation towards 1
`of the 2 mtDNA types in the cell (heteroplasmy) was ob-
`served. However, no extensive analysis of this phenome-
`non has been conducted to date on cloned animals.
` Nuclear DNA, especially that of dividing cells, is also
`very likely to accumulate mutations over time [52] in a
`tissue- and species-specific way [18] . These mutations
`can definitely not be reversed by SCNT, and very likely
`represent the only irreversible differences between an
`aged and a juvenile (donor) cell [1] . For example, a cell
`line with aberrant genetic material led to accelerated ag-
`ing in 3 cloned pigs [35] . On the other hand, whole-ge-
`nome comparison of a cloned dog and its respective nu-
`clear donor showed less de novo differences than between
`2 human monozygotic twins [4] , showing that in SCNT-
`derived animals the DNA can be conserved to a very high
`level.
`
` Can Cloned Animals Reach a Life Expectancy Similar
`to That of Control Animals?
`
` The ultimate outcome of aging is death, and therefore
`life expectancy is perhaps the most easily measurable pa-
`rameter of aging (the question of aging can of course not
`be reduced to life expectancy alone). The time since sev-
`eral species were first cloned outdates, or is at least close
`to, the life expectancy of the respective species by now:
`goat, cattle, dog, sheep, mouse, cat, and pig. Therefore, we
`should be able to finally answer the question of whether
`
`420
`
` Gerontology 2017;63:417–425
`DOI: 10.1159/000452444
`
` Burgstaller/Brem
`
`
`
`Exhibit 1036
`Select Sires, et al. v. ABS Global
`
`

`

` Table 2. Reported maximum lifespans of cloned animals
`
`Species
`
`Breed
`
`Typical life
`expectancy of
`species/breed, years
`
`Reported maximum
`lifespan of cloned animals,
`years
`
`Reference
`
`Goat
`
`Cattle
`
`Dog
`Sheep
`Mouse
`
`Cat
`Pig
`
`Dairy goats
`
`Jersey
`Simmental Fleckvieh
`
`Afghan hound
`Finn Dorset
`C57/BL6, DBA/2,
`129/Sv
`
`Large, white,
`Göttingen, Yucatan
`
`15
`
`15
`
`10 – 12
`<10
`2 – 3
`
`15
`15 – 17
`
`>15
`
`11.8 oldest dairy SCNT cow, 2011
`14.4 “Lara 8” (euthanized due to
`project end)
`>10
`9
`3
`
`10 (in 2011)
`6
`
`[Gavin, pers.
`commun.; 54]
`[55]
`[Brem, unpubl.]
`
`[44]
`[53]
`[13]
`
`[56]
`[57]
`
` We report here the typical life expectancy as reported (often compared to control animals) in the respective
`reference; or in [58] (cattle, cat) and [59] (pig). For maximum lifespans, see [18] and references therein.
`
`at least some cloned animals can reach a life expectancy
`similar to that of the control animals.
` Table 2 shows the reported (maximum) lifespans of
`cloned animals compared to the respective typical life-
`span of the species. In several species, cloned animals
`reach indeed the expected lifespan. Cloned dogs seem to
`reach a high age. Snappy, an Afghan hound and the first
`cloned dog, was 10 in 2015; and cloned female dogs of
`the same breed were 9. Also 3 cloned dairy goats lived to
`a normal age of 15 years, and Yang Yang, China’s first
`cloned goat turned 15 in 2015. Also for cloned mice, sev-
`eral studies report a normal lifespan, most outstanding
`in serial cloning (see below). While Dolly, the first cloned
`sheep, only reached 6 years, very recently, important
`further work on the aging of cloned sheep was published
`by the lab of the late Keith Campbell. Thirteen aged (7–9
`years old) cloned sheep, with 4 of them derived from the
`cell line that gave rise to Dolly, were analyzed. Detailed
`measurements of blood pressure and metabolism, as
`well as musculoskeletal tests showed no significant dif-
`ferences from age matched controls. Notably, these
`cloned sheep are already close to their typical natural
`lifespan (<10 years) [53] . The oldest reported dairy cattle
`reached 11.8 years in 2011. A cloned Simmental Fleck-
`vieh cow reached healthy 14.4 years and was euthanized
`only due the project end [Brem, unpubl. data]. Copycat,
`the first cloned cat turned 10 in 2011, which is at least
`respectable for a cat, if still several years from the maxi-
`mum lifespan. Pigs were first cloned in 2000, but the
`
`highest age reported to the best of our knowledge was 6
`years.
` While the question which age cloned animals can
`reach is asked very often, it is surprising that actual data
`in the scientific literature are scarce, even about the “cel-
`ebrated” first cloned animals of several species. There-
`fore, we had to resort to own data, personal communica-
`tion and even newsletters to finalize Table 2 .
` Nevertheless, including the very recent report about
`the aging of cloned sheep [53] , it is now possible to say
`that at least for those species where the question of lon-
`gevity of cloned animals was addressed (mouse, goat,
`sheep), a normal lifespan is possible. Also cats and dogs
`seem to reach a high age, as well as cloned cattle that reach
`a respectable age at least for dairy cows. For the other
`cloned species, the time is either too short to reach maxi-
`mum lifespan, or we were unable to find reliable data. It
`would be interesting to find out what proportion of
`cloned animals indeed reaches old age, but with the cur-
`rent amount data it is impossible to do so.
`
` Serial Cloning: Summing up the Years?
`
` One of the biggest concerns regarding aging of cloned
`animals is the age of the nuclear donor cell. It was argued
`that if this cell is old, and consequently has shortened
`telomeres, the clone would already start at the age of the
`donor cell. In further consequence, serial cloning, i.e. us-
`
` Aging of Cloned Animals
`
` Gerontology 2017;63:417–425
`DOI: 10.1159/000452444
`
`421
`
`Exhibit 1036
`Select Sires, et al. v. ABS Global
`
`

`

`ing donor cells from already cloned animals for cloning
`should fail once the “summed-up” age of the donor cells
`exceed the maximum lifespan of the species. As shown
`above, the telomere length turned out to be at least partly
`restored during SCNT. Nevertheless, serial cloning of a
`17-year-old bull failed after only 2 generations [28] . Suc-
`cessively cloned mice could be obtained for 5 generations
`in 2 lines, and then both lines stopped [41] . Also pig [34,
`60] , cat [61] , and cattle [28] were serially cloned only up
`to 3 times. Interestingly, after the introduction of tricho-
`statin A mice that were serially cloned from cumulus
`cells, always at the age of 3 months, reached over 25 gen-
`erations. Moreover, the cloned mice showed normal life
`expectancies of up to 3 years [13] . The “nuclear age” of 25
`generations of donor nuclei (taken at 3 months of age),
`sum up to over 6 years – twice the maximum lifespan of
`a mouse. This impressively shows that, at least for this
`species and this cell line, no cumulative effect, be it telo-
`mere length, DNA damage or reprogramming defects ap-
`pear.
`
` Health Status of Cloned Animals during Senescence
`
` Currently, there are cloned animals of only 4 species
`that reached their life expectancy (sheep, goat, dog,
`mouse), with 3 more (cattle, cat, pig) that are close to it.
`The few dogs and cats seem to have reached a high age
`without major difficulties. Dolly the sheep died famously
`at only 6 years of an infection, but other cloned sheep
`seem to have reached a high age without any difficulties
`(mild osteoarthritis being the only significant clinical
`finding) [53] . Also, 3 cloned goats for which data are
`available reached high ages with only clinical findings
`that do not suggest nuclear transfer (or in that case also
`transgenic) aetiology: dermatitis, non-specific musculo-
`skeletal lameness, pneumonia, hydrometra, and dystocia.
`Cloned mice showed the same agility level and memory
`skills while reaching same lifespans as control mice, dif-
`fering only in increased body weight [62] .
` Most reports are available for cattle. This is unsurpris-
`ing as several thousand cattle (no exact number is avail-
`able) have already been cloned [63] . However, cattle are
`rarely kept for longer than their productive lifespan for
`economic reasons, which differs significantly from their
`natural lifespan of up to 20 years.
` Konishi et al. [55] analyzed 4 groups of SCNT cows
`derived from both dairy (Holstein and Jersey) and beef
`(Japanese Black) cattle and compared them with 47 con-
`trol cows (Holstein). Interestingly, the performance of
`
`the cloned cows was better than that of the control, with
`considerably better survival time: of 18 SCNT animals 6
`were still alive at the end of the study, with an average
`age at death of 6 years. In contrast, all 47 Holstein cows
`were dead with an average age of 4.3–4.5 years (includ-
`ing animals that were culled due to reproductive disor-
`der or low ability). Death reasons of SCNT cattle includ-
`ed: accidents (and associated infections), mismanage-
`ment, acute mastitis and hypocalcaemia. All 5 Japanese
`Black-derived clones were alive at the end of the study
`aged around 9 years. This highlights either the advan-
`tage of beef cattle over dairy cattle due to much lighter
`burden of lactations, or might show a difference in the
`cell lines.
` Watanabe and Nagai reviewed (of papers in Japa-
`nese) the health status and productive performance of
`SCNT-cloned cattle [64] . They could not find any sig-
`nificant statistical evidence in haematology, pathology,
`growth performance, reproductive performance, and
`meat production and milk production performance.
`Also Chavatte-Palmer et al. [65] could not find major
`differences in the health status of cloned cattle older
`than 6 months. However, both studies lack data of older
`animals.
` Our own data of 33 SCNT-cloned dairy cattle [66–68]
`show a maximum age of 14.4 years, with an average life-
`span of 7.5 years. The cattle lines were discontinued in
`2014 due to the end of the project. In accordance with the
`other studies, death reasons included accidents, mastitis,
`lameness, pneumonia and also diarrhoea. It is not clear
`whether any of these outcomes are related to SCNT, but
`they are qualitatively not different from conventional
`kept cattle [Brem, unpubl. data].
` This mostly anecdotal evidence shows that the aging
`of cloned animals seems to be qualitatively very similar or
`even the same as that of normal animals. Once the cloned
`animal has reached adulthood, most problems of the
`rather unspecific condition “reprogramming failure of
`the donor nucleus” seem to be overcome. Unfortunately,
`there are by far too little data available to measure possi-
`ble, or even probable quantitative differences. There are
`of course also reports that show opposite outcomes, i.e.
`faster aging: Senescence was accelerated in 3 cloned pigs
`of a genetically abnormal fetal fibroblast cell line [35] . Ge-
`netic dysregulation also led to phenotypical abnormities
`in G0 and G1 generations of serial cloned pigs, but gen-
`erations G2 and G3 were back to normal again [60] . An-
`other report features a cloned Korean native goat that
`showed accelerated growth and development in addition
`to telomere shortening [40] .
`
`422
`
` Gerontology 2017;63:417–425
`DOI: 10.1159/000452444
`
` Burgstaller/Brem
`
`
`
`Exhibit 1036
`Select Sires, et al. v. ABS Global
`
`

`

` Outlook
`
` Since the birth of Dolly, cloning procedures have made
`immense progress, and the number of species for which
`SCNT protocols are established is still increasing. Due to
`the possibilities of companion and sport animal cloning,
`and also due to the increasing number of cloned farm
`animals, the topic of SCNT never ceases to be of public
`interest.
` The success rate of the cloning protocols and the health
`status and survival rate of born SCNT-derived animals
`are the focus of numerous research papers and reviews.
`Once cloned animals reach their reproductive age, they
`seem to have crossed the threshold for a normal lifespan.
`The question whether their aging process is qualitatively
`different from normal animals is often raised, but surpris-
`ingly few studies try to answer it. Early losses and obvious
`abnormalities are easily attributed to more- or less-de-
`fined epigenetic reprogramming errors. Differences later
`in life can be expected to be more subtle, as unsuccessful
`outcomes are already eliminated. Unfortunately, research
`on aged cloned animals seems almost non-existent de-
`spite the public interest in various “safety” questions of
`SCNT. This might partly be explained by the fact that
`SCNT is still a very recent technique when compared to
`the life expectancy of most cloned species. Moreover,
`cloned farm animals are unlikely to be kept longer than
`their productive phase. Cloned sport and companion an-
`imals are mainly being kept in private care, and thus are
`less accessible for scientific studies. Based on the litera-
`ture available so far, and also in our experience, the aging
`of cloned animals seems to proceed very similar to con-
`
`trol animals. However, a thorough clinical study with a
`sufficient number of cloned animals, together with con-
`trol animals over their entire lifespan is clearly needed for
`every species. Very recently, the first detailed clinical
`study on the aging of cloned animals, fittingly about
`cloned sheep and including clones of Dolly, was pub-
`lished [53] . Only more studies such as this will finally an-
`swer the question how SCNT-derived animals age quali-
`tatively, and above all quantitatively (i.e., what percentage
`of the animals ages normally). With the exception of
`mice, this will clearly be a very long and costly undertak-
`ing, especially when maintaining large domestic species
`long-term, well past their prime production age. There-
`fore, it is likely that we still have to rely on mostly anec-
`dotal evidence in the foreseeable future. Short scientific
`“updates” on the fate of reported cloned animals would
`therefore be very desirable. Currently, it is simpler to find
`the age of famous cloned animals as “birthday reports” in
`the public press. We therefore encourage reports on the
`aging of cloned animals to make further analysis on the
`long-term performance of SCNT-produced animals pos-
`sible.
`
` Acknowledgement
`
` We thank W. Gavin (GTC Biotherapeutics, Framingham) for
`data on the life expectancy and health status of cloned goats. We
`thank E. Wolf (Gene Center, LMU Munich), F. Gandolfi (Depart-
`ment of Animal Science, University of Milan) and C. Galli (De-
`partment of Veterinary Medical Sciences, University of Bologna)
`for information on SCNT. We thank Iain Johnston (School of Bio-
`sciences, University of Birmingham) for critical reading of the
`manuscript.
`
`
` References
`
` 1 Rando TA, Chang HY: Aging, rejuvenation,
`and epigenetic reprogramming: resetting the
`aging clock. Cell 2012; 148: 46–57.
` 2 Tilly JL: Commuting the death sentence: how
`oocytes strive to survive. Nat Rev Mol Cell
`Biol 2001; 2: 838–848.
` 3 Wallace DC, Chalkia D: Mitochondrial DNA
`genetics and the heteroplasmy conundrum in
`evolution and disease. Cold Spring Harb Per-
`spect Biol 2013; 5:a021220.
` 4 Kim H-M, Cho YS, Kim H, Jho S, Son B, Choi
`JY, Kim S, Lee BC, Bhak J, Jang G: Whole ge-
`nome comparison of donor and cloned dogs.
`Sci Rep 2013; 3: 2998.
` 5 Gurdon JB: Adult frogs derived from the nu-
`clei of single somatic cells. Dev Biol 1962; 4:
` 256–273.
`
` 6 Wilmut I, Schnieke AE, McWhir J, Kind AJ,
`Campbell KH: Viable offspring derived from
`fetal and adult mammalian cells. Nature 1997;
` 385: 810–813.
` 7 Keefer CL: Artificial cloning of domestic ani-
`mals. Proc Natl Acad Sci USA 2015; 112: 8874–
`8878.
` 8 Konishi K, Yonai M, Kaneyama K, Ito S, Mat-
`suda H, Yoshioka H, Nagai T, Imai K: Rela-
`tionships of survival time, productivity and
`cause of death with telomere lengths of cows
`produced by somatic cell nuclear transfer. J
`Reprod Dev 2011; 57: 572–578.
` 9 Long CR, Westhusin ME, Golding MC: Re-
`shaping the transcriptional frontier: Epi-
`genetics and somatic cell nuclear transfer.
`Mol Reprod