Conservation Biology and Reproduction in a Time of Developmental Plasticity

Abstract

The objective of this review is to ask whether, and how, principles in conservation biology may need to be revisited in light of new knowledge about the power of epigenetics to alter developmental pathways. Importantly, conservation breeding programs, used widely by zoological parks and aquariums, may appear in some cases to reduce fitness by decreasing animals’ abilities to cope when confronted with the ‘wild side’ of their natural habitats. Would less comfortable captive conditions lead to the selection of individuals that, despite being adapted to life in a captive environment, be better able to thrive if relocated to a more natural environment? While threatened populations may benefit from advanced reproductive technologies, these may actually induce undesirable epigenetic changes. Thus, there may be inherent risks to the health and welfare of offspring (as is suspected in humans). Advanced breeding technologies, especially those that aim to regenerate the rarest species using stem cell reprogramming and artificial gametes, may also lead to unwanted epigenetic modifications. Current knowledge is still incomplete, and therefore ethical decisions about novel breeding methods remain controversial and difficult to resolve.

Full text

Citation: Holt, W.V.; Comizzoli, P.
Conservation Biology and
Reproduction in a Time of
Developmental Plasticity.
Biomolecules 2022,12, 1297. https://
doi.org/10.3390/biom12091297
Academic Editor: Haengseok Song
Received: 12 August 2022
Accepted: 12 September 2022
Published: 14 September 2022
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biomolecules
Review
Conservation Biology and Reproduction in a Time of
Developmental Plasticity
William V. Holt 1,* and Pierre Comizzoli 2
1Department of Oncology & Metabolism, The Medical School Beech Hill Road, Sheffield S10 2RX, UK
2Smithsonian’s National Zoo and Conservation Biology Institute, Washington, DC 20008, USA
*Correspondence: bill2holt@gmail.com
Abstract:
The objective of this review is to ask whether, and how, principles in conservation biology
may need to be revisited in light of new knowledge about the power of epigenetics to alter develop-
mental pathways. Importantly, conservation breeding programmes, used widely by zoological parks
and aquariums, may appear in some cases to reduce fitness by decreasing animals’ abilities to cope
when confronted with the ‘wild side’ of their natural habitats. Would less comfortable captive condi-
tions lead to the selection of individuals that, despite being adapted to life in a captive environment,
be better able to thrive if relocated to a more natural environment? While threatened populations may
benefit from advanced reproductive technologies, these may actually induce undesirable epigenetic
changes. Thus, there may be inherent risks to the health and welfare of offspring (as is suspected
in humans). Advanced breeding technologies, especially those that aim to regenerate the rarest
species using stem cell reprogramming and artificial gametes, may also lead to unwanted epigenetic
modifications. Current knowledge is still incomplete, and therefore ethical decisions about novel
breeding methods remain controversial and difficult to resolve.
Keywords:
animal conservation; reproduction; conservation breeding; epigenetics; developmental
plasticity; assisted reproductive technologies
1. Introduction
The last two decades have seen a revolution in the way that scientists understand
how organismal development takes place, and a realization that the DNA sequence itself
represents only one part of the process. Expressing the genomic DNA sequence must be
controlled by an exquisitely refined and complex series of interactions that are collectively
known as epigenetic mechanisms. These typically involve important enzyme-mediated
chemical modifications, such as methylation and acetylation, not only of the DNA itself
and various proteins with which the DNA interacts, but also of a series of regulatory
and non-coding RNA species [
1
]. These emerging discoveries have revolutionized our
understanding of the various ways in which finely controlled gene expression influences
not only offspring development, but the inheritance of some newly acquired phenotypic
characteristics, much as hypothesized by Lamarck in the 19th century [
2
,
3
]. The fact
that multiple phenotypes may be generated from a single genotype is often known as
‘developmental plasticity’, and typically occurs in response to external environmental cues
experienced during the more plastic early stages of development [4].
Here, we aim to ask whether, and how, some of these novel discoveries might affect
the ways in which some of the principles that underpin conservation biology may need to
be reviewed or refocused in the light of epigenetics.
2. Conservation and Adaptations
The general aim of conservation biology is to inhibit population declines, minimize
inbreeding as far as possible, and maintain good population health. Therefore, attempting
Biomolecules 2022,12, 1297. https://doi.org/10.3390/biom12091297 https://www.mdpi.com/journal/biomolecules

Biomolecules 2022,12, 1297 2 of 18
to support reproductive success and future population fitness is an important component
of this aim. However, given that the threatened species live within diverse habitats, it
follows that they will experience, and respond to, the demands of the habitats in question
(Figure 1).
Biomolecules 2022, 12, x FOR PEER REVIEW 5 of 5
2. Conservation and Adaptations
The general aim of conservation biology is to inhibit population declines, minimize
inbreeding as far as possible, and maintain good population health. Therefore, attempting
to support reproductive success and future population fitness is an important component
of this aim. However, given that the threatened species live within diverse habitats, it fol-
lows that they will experience, and respond to, the demands of the habitats in question
(Figure 1).
Figure 1. Factors that induce developmental plasticity.
Habitats vary enormously, and highly localized forms of conservation practice have
developed. These include large-scale projects such as the establishment of marine conser-
vation areas or large national parks, as well as some very small and local projects focused
on a particular species. Captive breeding is also a well-established and widely used con-
servation method, being mainly employed by zoos, aquariums, and some wildlife re-
serves. An estimate published in 2011 suggested that one-seventh of all threatened species
are held, and bred, in zoos and aquariums [5]. In fact, some small populations owe their
continued existence exclusively to captive breeding, and a relatively small number have
even been successfully reintroduced to their original habitats. A classic example of a suc-
cessfully reintroduced captive-bred species is the Père David’s deer (Elaphurus davidianus)
[6], which were returned to China in the 1980s, having been bred in western zoos for
nearly a century. Given the situation and the diversity of species in question, the concept
that species can adapt and respond successfully to all manner of environmental changes
is almost incredible. It is certainly not our intention to suggest that captive-bred mammals
are necessarily unsuitable for reintroduction.
Whether the long-term survival of zoo-bred and reintroduced species is advantaged
or disadvantaged by developmental plasticity is, at present, an unanswerable question.
While reintroduction programs for captive-bred mammals are always managed with
great care, allowing animals to become acclimated to their new environments, their long-
term success is likely not only to be species-dependent, but also only recognizable several
generations later. At present there are few, if any, long-term studies that can shed light on
this question, but the well-established black-footed ferret (Mustela nigripes) captive breed-
ing and assisted reproduction program [7,8] may, in the future, provide some answers.
Genetic studies have shown that artificial insemination with cryopreserved black-footed
Figure 1. Factors that induce developmental plasticity.
Habitats vary enormously, and highly localized forms of conservation practice have
developed. These include large-scale projects such as the establishment of marine conserva-
tion areas or large national parks, as well as some very small and local projects focused on
a particular species. Captive breeding is also a well-established and widely used conserva-
tion method, being mainly employed by zoos, aquariums, and some wildlife reserves. An
estimate published in 2011 suggested that one-seventh of all threatened species are held,
and bred, in zoos and aquariums [
5
]. In fact, some small populations owe their continued
existence exclusively to captive breeding, and a relatively small number have even been
successfully reintroduced to their original habitats. A classic example of a successfully
reintroduced captive-bred species is the Père David’s deer (Elaphurus davidianus) [
6
], which
were returned to China in the 1980s, having been bred in western zoos for nearly a century.
Given the situation and the diversity of species in question, the concept that species can
adapt and respond successfully to all manner of environmental changes is almost incredi-
ble. It is certainly not our intention to suggest that captive-bred mammals are necessarily
unsuitable for reintroduction.
Whether the long-term survival of zoo-bred and reintroduced species is advantaged or
disadvantaged by developmental plasticity is, at present, an unanswerable question. While
reintroduction programs for captive-bred mammals are always managed with great care,
allowing animals to become acclimated to their new environments, their long-term success
is likely not only to be species-dependent, but also only recognizable several generations
later. At present there are few, if any, long-term studies that can shed light on this question,
but the well-established black-footed ferret (Mustela nigripes) captive breeding and assisted
reproduction program [
7
,
8
] may, in the future, provide some answers. Genetic studies have
shown that artificial insemination with cryopreserved black-footed ferret semen, collected
and stored several years prior to use, successfully reduced the level of inbreeding in the
captive population. However, it is not yet known whether and how the levels of inbreeding
may influence the eventual breeding success and survival of the wild populations.

Biomolecules 2022,12, 1297 3 of 18
Some caution is also relevant in this context. A recent meta-analysis of birth-origin
effects on the outcomes of captive breeding across a wide range of species, including
invertebrates, fish, birds, and mammals [
9
], showed that wild-born animals in captivity
have a massive advantage (about 74%) in terms of reproductive success compared to their
captive-born counterparts. These results were based on several reproductive traits, includ-
ing reproductive yield (e.g., litter size), birth weight, offspring survival, and reproductive
phenology (e.g., interbirth interval). Interestingly, when data were examined in relation to
the captive environments, only aquaculture showed a large statistically significant mean
advantage for wild-born over captive-born animals. As aquaculture tends to be focused on
improving reproductive outputs, this effect was regarded as unexpected, and the authors
suggested that supplementation with wild stocks would be required in order to sustain
population fitness and welfare. However, other evidence obtained from aquaculture sug-
gested that the plasticity effects involved in captive breeding could be induced so rapidly
that they might actually cause maladaptation [10].
3. Contemporary Evolution and Developmental Plasticity
The evolutionary literature is replete with examples of species where both males and
females have evolved and adapted their physiological, behavioural, and morphological
characteristics, optimizing their survival, reproductive success and fitness over centuries
and millennia. However, while many of these long-term adaptations have been effected
via gene mutation and various kinds of evolutionary selection, many such adaptations
are induced by changes in the diverse ways in which gene expression is controlled. Such
changes, caused via developmental plasticity, need not be solely determined by the precise
nature of the DNA sequences involved. In fact, by changing the way that the DNA
sequences are controlled, species are able to respond quickly to environmental changes.
For example, the complex social organization of eusocial insect societies (notably, bees,
wasps and ants) is dependent on developmental plasticity, whereby genetically identical
individuals develop differentially into functionally different castes [
11
]. This level of
sophistication involves the control of gene expression during development, via DNA
methylation, histone modifications and other epigenetic mechanisms [
12
,
13
] and is a
beautiful example of developmental plasticity. “Polyphenism” is similar in principle, but
the term is used to describe the remarkable ability of many insects and some fishes, such
as the blue headed wrasse (Thalassoma bifasciatum) [
14
,
15
], to alter their own phenotype in
response to changing environmental conditions (for review, see [
16
]). Locusts are notable
in this context, as they can change their own behaviour and morphology, and those of
their offspring, in response to cues such as crowding, which involves visual, olfactory and
tactile stimuli.
Plasticity under Extreme Conditions
Developmental plasticity allows species to respond in the face of challenging condi-
tions, and some have exploited that facility in ways that allow them to live successfully
under the most extreme environments. Some of the developmental strategies are truly
remarkable and possibly unexpected. For example, some fish species, especially the African
and South American killifishes, which live in so-called “ephemeral” bodies of water that
are prone to both flooding and droughts, survive successfully by enabling their embryos to
undergo a series of physiological mechanisms minimizing water loss and entering a period
of dormancy or diapause [
17
]. This strategy is so extreme that in some cases none of the
adult fish remain alive during the drought, and the entire population is represented by
dormant embryos. Embryos are frequently buried in mud to avoid predation, and when
the drought finishes, the embryos hatch, grow rapidly and reproduce prior to the next dry
period [18–21].
Some species that can survive in extreme habitats, such as hyper-arid deserts and
rocks, oceanic deeps, salt lakes, the north and south poles, volcanoes, high mountains, and
upper atmosphere, provide interesting examples of the resilience that can be attained via

Biomolecules 2022,12, 1297 4 of 18
evolutionary plasticity. It is not surprising that some researchers are currently exploiting
biological plasticity for the development of novel technologies and applications [
22
]. One
well-known and notable example is the widely used enzyme for DNA amplification,
Taq DNA polymerase. This enzyme, which is derived from the thermophilic eubacterial
microorganism Thermus aquaticus, is unusual because of its extreme heat resistance. With
a half-life of 40 min at 95
◦
C, it can be used in the polymerase chain reaction technique,
where other less stable enzymes would be rapidly denatured.
4. Developmental Plasticity Is a Continuing Process
A contemporary example of developmental plasticity involves the food fish steelhead
trout (Oncorhynchus mykiss) [
23
,
24
]. Comparison of wild and hatchery-raised trout found
evidence that adaptation to a life in freshwater, rather than the ocean, could occur rather
quickly. The authors identified three chromosomal regions that were associated with rapid
genetic and functional adaptation to environmental change. The environmental change
in question relates to historical relocation of these particular fish from native rivers in
California, where a period of developmental time is normally spent in the ocean, but where,
following geographical translocation to a freshwater habitat in Lake Michigan, the fishes
treat the freshwater lake as a surrogate ocean. One chromosomal region was found to
contain functional changes to ceramide kinase, which may have altered metabolic and
wound-healing rates in the Lake Michigan steelhead. The second and third chromosomal
regions encoded carbonic anhydrases and a solute carrier protein, both of which are critical
for osmoregulation. Importantly, these authors found that despite their high reproductive
success in captivity, the hatchery-bred fishes exhibited low reproductive success and high
mortality in the wild. The authors suggested several potential reasons for this difference,
including changes in egg size, growth rate, and ability to avoid predators, all of which
represent selection pressures that govern the ability of populations to withstand environ-
mental stresses and thrive into the future. A separate study [
25
] of the role of phenotypic
plasticity in the adaptation of wild Trinidadian guppies (Poecilia reticulata) to a completely
new environment, found that dramatically improved reproductive rates could be detected
within 20–30 generations and supported a previous suggestion that phenotypic plasticity
could be regarded as “contemporary evolution” [26].
5. Reproductive Forecasting and Development
Reproductive and developmental success both involve some degree of forecasting
in the face of changing future environments. This helps the organism to match the path
of embryonic development against expectations [
27
], and thus optimize the survival of
subsequent generations. In fact, this relationship is at the heart of reproductive seasonality,
where species have developed physiological strategies that typically synchronize breeding
and parturition with climatic conditions for the optimal survival of their offspring [
28
].
If the forecast turns out to be inaccurate because the environment changes and defies
expectations, the mismatch can have unfortunate and lifelong health consequences.
Several amphibian species that are vulnerable to drought exploit forecasting and
developmental plasticity to protect themselves in case their aquatic habitats are likely
to dry out. Some may be able to speed up the rate of larval development if they detect
increased salinity [
29
–
32
], while others pay attention to their choice of nest site [
33
]. The
exact responses appear to be species specific, and not always successful if features of their
environment, such as rainfall, are unpredictable [34].
Organisms are now known to use such developmental forecasting to predict how to
prepare for possible and stressful changes in nutrition, temperature, salinity, and even
the presence of environmental toxicants. Such relationships are now well recognized in
humans and other mammals [
35
], owing mainly to a series of studies linking early life
nutrition to the onset of diseases in adulthood (see, for example, [
36
,
37
]). The realization
that there are systematic relationships between pre- and/or peri-conception nutritional
conditions and chronic disease conditions in adults has stimulated a great deal of research

Biomolecules 2022,12, 1297 5 of 18
in humans and other species. It has even been shown, in humans, that transgenerational
effects on body condition in males are related to the father’s and grandfather’s smoking
habits [
38
]. Similarly, experimental studies in rats have demonstrated the occurrence of
transgenerational impacts of experimentally administered agricultural chemicals [
39
,
40
].
Transgenerational reproductive forecasting, sometimes known as “anticipatory transgen-
erational phenotypic plasticity” was also observed in relation to rates of flea infestation
in a desert rodent (Meriones crassus) [
41
]. Maternal reproductive success was best when
mothers and grandmothers experienced similar risks of parasitism. When both were either
infested or non-parasitized, the pups would reach sexual maturity more quickly than those
pups whose mothers’ infestation status did not match that of their grandmothers.
6. Wildlife Conservation, Captive Breeding, and Mammalian Sex Ratios
Vertebrate populations have evolved variously sophisticated methods of adjusting
the sex ratios of their offspring in response to current environmental conditions, or in
anticipation of future conditions. Achieving these outcomes at the population level can
only occur if individuals are able detect and respond to environmental cues experienced
very early in the reproductive process, even before conception in the case of species with
internal fertilization. Although many mammalian species possess the ability to skew
offspring sex ratios [
42
], the biological mechanisms seem, at first sight, to defy statistical
logic. Spermatogenesis in mammals results in two distinct, and equally sized, populations
of spermatozoa, with each cell either carrying an X or a Y-chromosome. Once fertilization
takes place and embryogenesis begins, the presence of the Y-chromosome within the
zygote activates the molecular processes involved in sex determination, and a male embryo
develops. Statistically, a 50:50 ratio of male to female embryos would normally be expected,
unless the females’ physiology can be influenced to interfere with sperm transport, impose
sperm selection prior to fertilization, or even selectively prevent embryonic development.
Captive-bred pigmy hippopotamus (Choeropsis liberiensis) present an extreme example
of sex ratio skewing, with highly female-biased sex ratios at birth described in two different
studies (41% and 43% males) [
43
,
44
]. This outcome appears to support the hypothesis
proposed by Trivers and Willard in the 1970s [
45
], that as maternal condition declines,
adult females tend to produce more female than male offspring. Conversely, a significantly
male-biased sex ratio of Asian elephants born in European zoos between 1962 and 2006
(ratio: 0.61, p= 0.044) was reported in 2009 [
46
], as well as an even more striking male bias
in elephant births following artificial insemination (0.83, p= 0.003). It has clearly been very
difficult for researchers to find a unifying theory that explains the differing reports of biased
mammalian sex ratios at birth in diverse wild and captive species (see, for example [
47
–
53
]),
in relation to environmental conditions. However, that such mechanisms exist and may
sometimes, but not necessarily always, be adaptive, is now beyond dispute.
The mechanisms involved in sex ratio biasing are likely to involve sperm selection
within the female reproductive tract, thus biasing the genetic properties of the sperma-
tozoa that eventually fertilize the oocyte(s). Cameron et al. [
54
] and others [
55
–
57
] have
critically analysed possible stages in the mammalian reproductive process that could lead
to skewed sex ratios, with a focus on mechanisms that might affect sperm transport and
fertilization. They concluded that the female reproductive tract employs many effective
sperm-selection mechanisms that are capable of preventing, or enabling, the passage of
specific sperm subpopulations. Cervical and oviductal mucus can act as physical barriers
to sperm progression, and some anti-microbial defensins appear to be influential in both
inhibiting or assisting sperm transport [
58
]. Recent studies of mouse sperm progression
in vivo
by direct examination of whole reproductive tracts [
59
] have demonstrated clearly
that the spermatozoa travel as clusters, rather than as individuals, a cooperative behaviour
that is also observed in other mammalian species [
60
]. Spermatozoa are highly responsive
to a range of molecules, some as simple as the bicarbonate ion, which activates cyclic
adenosine monophosphate (cAMP), induces protein phosphorylation and stimulates both
capacitation and rapid flagellar movement [
61
,
62
]. Sperm entry into the oviductal isth-

Biomolecules 2022,12, 1297 6 of 18
mus from the utero-tubal junction is not only dependent on progressive motility, but is
critically dependent on the exact molecular nature of the proteins expressed at the sperm
surface, especially the A-disintegrin and metalloprotease group, widely known as ADAM
proteins [
63
–
65
]. Moreover, the epithelial cell products typically prevent sperm motility
and capacitation until they register that ovulation is under way [
66
], whereupon motility
and directionality are influenced by chemotaxis [
67
,
68
] and thermotaxis [
69
]. Furthermore,
components of oviductal fluid cause significant hardening of the zona pellucida [
70
–
72
], the
proteinaceous coat the surrounds the oocyte, and reduce polyspermic fertilization. Seminal
plasma is also known for its ability to influence the responses that the female reproductive
tract to the arrival of spermatozoa [
73
], including the induction of de novo gene expression.
Experimental ablation of the seminal vesicles in mice resulted not only in the impairment of
pregnancy, but also abnormal placental function in late pregnancy, and deleterious impacts
on the health of male offspring after birth [74].
Oviductal Cells Can Discriminate between X- and Y-Chromosome-Bearing Spermatozoa
Those spermatozoa that successfully enter the oviductal environment are known to
bind and interact with epithelial cells, which respond by stimulating de novo gene transcrip-
tion [
75
–
77
]. Some of the newly synthesized proteins, such as heat shock proteins, support
prolonged sperm viability [
78
], while others are more directly involved in fertilization itself.
Although these effects may not necessarily result in biased sex ratios, recent evidence [
79
]
has shown that the oviductal cells themselves are capable of differentially responding to
the presence of X- and Y-chromosome-bearing spermatozoa. The authors used laparoscopic
surgery to isolate the oviducts of female pigs that were in heat. Sex-sorted [
80
] X- and
Y-bearing spermatozoa (3
×
10
5
sperm in 100
µ
L volumes) were separately inseminated
into the left and right oviducts, and the oviductal tissues were subsequently recovered after
24 h for genomic analysis (Figure 2).
Biomolecules 2022, 12, x FOR PEER REVIEW 5 of 5
Figure 2. Schematic diagram showing the method used to establish that the oviducts can distinguish
between X- and Y-chromosome-bearing spermatozoa.
7. Impacts of Gamete Cryopreservation on Offspring Development and Survival
At present, it is probably too soon to evaluate the conservation relevance of epige-
netics, and especially the epigenetically mediated inheritance of phenotypic characteris-
tics, on the numerous threatened species receiving some kind of protection. However,
given the current and increasing interest in the potential harnessing of various biotech-
nologies in support of breeding programmes, it is worth exploring what lessons have been
learned from decades of research in both human clinical medicine and agriculture.
The cryopreservation of spermatozoa has been used successfully in agriculture and
human medicine for more than five decades, but applications in conservation biology
have remained somewhat unpredictable. Semen cryopreservation and successful animal
breeding have been undertaken in a range of fishes, mammals, birds, reptiles and amphib-
ians, and this experience has led to the currently increasing interest in semen cryopreser-
vation and the establishment of genetic resource banks (GRBs), often known as biobanks
[82–84]. The breeding of the Giant panda (Ailuropoda melanoleuca) and Black-footed ferret
(Mustela nigripes) populations provides two, and possibly the only two, examples in which
endangered species populations have been successfully boosted through the use of cryo-
preserved semen, biobanking and artificial insemination [85,86]. Within the GRB context,
one important question yet to be resolved, and for which the answers are probably species
Figure 2.
Schematic diagram showing the method used to establish that the oviducts can distinguish
between X- and Y-chromosome-bearing spermatozoa.

Biomolecules 2022,12, 1297 7 of 18
The outcome indicated that the oviducts possess a recognition system that distin-
guishes the chromosomal nature of spermatozoa (out of 501 transcripts, 54.1% were down-
regulated by the presence of Y-bearing spermatozoa, and 45.9% were upregulated); these
data indicate that the oviducts could therefore affect gender selection prior to fertilization.
The biological systems most affected by these changes were (in descending order) signal
transduction, the immune system, the digestive system, and the endocrine system.
A recent review [
81
] presented a summary table derived from six independent studies,
showing proteins that have been reported as differentially expressed in X- and Y-bearing
spermatozoa (31 proteins were upregulated in X-spermatozoa, while only 10 were upreg-
ulated in Y-spermatozoa). The authors were somewhat reticent in their interpretation of
these outcomes, and concluded that further research would be required before they could
be entirely convinced of the veracity of the data.
7. Impacts of Gamete Cryopreservation on Offspring Development and Survival
At present, it is probably too soon to evaluate the conservation relevance of epigenetics,
and especially the epigenetically mediated inheritance of phenotypic characteristics, on
the numerous threatened species receiving some kind of protection. However, given the
current and increasing interest in the potential harnessing of various biotechnologies in
support of breeding programmes, it is worth exploring what lessons have been learned
from decades of research in both human clinical medicine and agriculture.
The cryopreservation of spermatozoa has been used successfully in agriculture and
human medicine for more than five decades, but applications in conservation biology
have remained somewhat unpredictable. Semen cryopreservation and successful ani-
mal breeding have been undertaken in a range of fishes, mammals, birds, reptiles and
amphibians, and this experience has led to the currently increasing interest in semen cry-
opreservation and the establishment of genetic resource banks (GRBs), often known as
biobanks [
82
–
84
]. The breeding of the Giant panda (Ailuropoda melanoleuca) and Black-
footed ferret (Mustela nigripes) populations provides two, and possibly the only two, ex-
amples in which endangered species populations have been successfully boosted through
the use of cryopreserved semen, biobanking and artificial insemination [
85
,
86
]. Within the
GRB context, one important question yet to be resolved, and for which the answers are
probably species dependent, concerns the future health and fitness of offspring derived
from cryopreserved semen. However, because relatively small numbers of offspring are
produced following artificial insemination in threatened birds and mammals, there has
been little opportunity to find out whether sperm cryopreservation produces any unwanted
effects on offspring.
Recent advances in functional genomics now offer interesting insights into the ways in
which cryopreserved and stored spermatozoa need to be used with great care. Just as some
wild mammal populations (for example, gazelles, manatees, and marsupial gliders) are
capable of anthropogenically influenced interspecific hybridization, often leading to low-
ered fertility or even sterility [
87
–
90
], there is an ongoing risk of creating hybrids through
the use of poorly organized and inadequately characterized collections of stored gametes.
Although it is not yet technically feasible to cryopreserve, recover and use marsupial sper-
matozoa for artificial insemination, some scientists advocate keeping such frozen collections
just in case it ever becomes feasible to undertake techniques such as intracytoplasmic sperm
injection (ICSI) [
91
]. A relevant, and outstanding, example of the care that would be needed
involves attempts to conserve the Tasmanian devil (Sarcophilus harrisii). This Australian
carnivorous marsupial is under considerable threat from inbreeding depression [
92
], as
well as a contagious and fatal form of facial tumour. In 2012, an insurance subpopulation
of uninfected animals, derived from two separate founder populations, was established
on Maria Island, Tasmania, as one of the conservation measures. Using a combination of
microsatellite and single nucleotide polymorphism (SNP) analysis, the researchers showed
not only the existence of genetic differences between the two geographically separated
founder populations, but an increased level of genetic diversity in the resultant translo-

Biomolecules 2022,12, 1297 8 of 18
cated population [
93
]. Some aspects of the genomic study also pointed to correlations
between genes, their respective SNPs, and their functional roles. These included genes
involved in embryogenesis, fertilization and the hormonal regulation of reproduction [
94
]
and confirmed earlier findings that inbred Tasmanian devils produce lower litter sizes than
expected [
92
,
95
]. This conveys a clear and general message about the need for long-term
somatic cell, embryo, tissue and gamete repositories to accompany their stored samples
with detailed genetic and geographic metadata.
Data from studies with fishes and amphibians have provided useful and valid in-
formation about the survival and fitness of offspring derived from cryopreserved sper-
matozoa. Recent experimental conservation breeding studies with amphibians (Fowler
toads; Anaxyrus fowleri) indicated that cryo-derived tadpoles were unusually small and
showed poorer post-release survival than those produced without the use of cryopreserved
spermatozoa [
96
,
97
]. When these authors modelled the likely long-term population impacts
of the cryo-derived offspring, they predicted that naturally bred populations would remain
stable over a 30-year period, and that populations derived via sperm cryopreservation
would also remain stable, but with lower population numbers. A critically important
but currently unknown aspect of this work concerns the possibility that the effects of
cryopreservation might be heritable across several generations. The authors modelled this
possibility as part of their study and found that the population would probably become
extinct in approximately 17 years.
It is not unlikely that the effects of amphibian sperm cryopreservation are herita-
ble, but at present, this is an open question that demands further investigation. This is
because the successful use of cryopreserved spermatozoa in breeding programmes for
threatened amphibian species, and for establishing biobanks and insurance populations
as a hedge against extinction, is widely regarded as an important aspect of amphibian
conservation [
98
,
99
]. Although there are now several other studies in which pheno-
typically normal embryos were produced by artificial fertilization with cryopreserved
frog and toad spermatozoa, and in which metamorphosis resulted in normal adults
(i.e., (Xenopus laevis [
100
] and Golden Bell frog (Litoria aurea) [
101
]), it seems that more
research into the heritability question is needed.
To some extent, these results with amphibian spermatozoa mirror those found by
Nusbaumer and colleagues [
102
] in an experiment in which semen from wild-caught
brown trout (Salmo trutta) was frozen using a method [
103
] that produced fertilization
rates comparable to those of non-cryopreserved spermatozoa. Cryopreservation did not
reduce fertilization rates or affect embryo viability, but did reduce larval growth by about
4%. One possible explanation for the suboptimal success of cryopreserved brown trout
spermatozoa is the inhibition of DNA repair within the zygotes [
104
]. Inhibition of DNA
repair mechanisms within oocytes suggested that oocytes defective in some transcripts
or proteins involved in repair could block development after fertilization by damaged
spermatozoa. As cryopreservation is usually accompanied by the production of reactive
oxygen species (ROS) that generate single and double DNA strand breaks [
105
], efficient
cytoplasmic repair mechanisms within the oocyte cytoplasm are necessary to overcome
the problem.
These results confirmed the outcome of a previous experiment undertaken by Kopeika
et al. [
106
]. Embryos were produced from spermatozoa and oocytes collected from a fresh-
water fish species, the weather loach (Misgurnus fossilis). The embryos derived from the
cryopreserved spermatozoa were exposed to a DNA-repair inhibitor (3-aminobenzamide—
3-AB), and their developmental progress was monitored. Parthenogenically developing
embryos were excluded from the analysis, as they do not survive beyond 20 h after fer-
tilization. Sperm cryopreservation significantly reduced embryonic survival during the
post-fertilization period. However, to assess the significance of DNA-repair inhibition, the
developmental ability of embryos treated with and without, 3-AB was compared after the
20 h post-fertilization stage. The results showed that DNA-repair inhibition significantly
reduced the ability of embryos to continue their development (the outcome with 3-AB

Biomolecules 2022,12, 1297 9 of 18
treated embryos was 84% survival, compared to 91% survival without 3-AB treatment).
These results supported the hypothesis that sperm cryopreservation induces DNA damage
and interferes with subsequent embryonic development, but also showed that oocytes
possess the ability to repair the damage. Significantly, differences in the DNA-repair ability
of embryos derived from different females were also observed in this study.
In their 2014 study, Nusbaumer et al. [
102
] included an interesting extra treatment in
their experiment to evaluate the morphology of juvenile brown trout obtained using cryop-
reserved spermatozoa. Individual embryos derived from the cryopreserved spermatozoa
were either exposed to a pathogenic bacterial suspension (Aeromonas salmonicida), or only
sham exposed as a control treatment. Significantly, exposure to the pathogen increased the
incidence of embryo mortality by about fourfold, and those that survived were smaller
when they hatched.
Delayed and abnormal embryonic development was also reported when spermatozoa
from the south American freshwater fish species, Colossoma macropomum, were cryopre-
served using DMSO, dimethylformamide, methanol, ethyl glycol and glycerol as cryopro-
tectants [
107
]. All of the cryopreserved spermatozoa had lower methylation levels and
exhibited more delays and abnormalities during embryonic development than the control
embryos derived with non-frozen spermatozoa. In this experiment the developmental
delay started 4 h after fertilization, and glycerol showed the highest incidence of embryonic
abnormality with >90% embryonic mortality 11.5 h after fertilization.
The possibility that fish sperm cryopreservation might lead to inappropriate methyla-
tion owing to the use of cryoprotectants was recently discussed in some detail [
108
], where
it was pointed out that the issue is not straightforward. Goldfish sperm DNA methylation
was not affected when cryopreserved using methanol, in contrast to the effects on zebrafish
spermatozoa, which experienced increased DNA methylation; conversely, DMSO and
1.2 propanediol caused a decrease in DNA methylation. Moreover, these and other authors
(e.g., [
109
]) reminded readers that sperm suspensions from fishes, and indeed from many
other species, are not homogeneous, and that sperm subpopulations are not only detectable
by a variety of methods, but are likely to be differentially affected by cryopreservation.
8. ART and Human Infertility Treatment
The application of ARTs in various human infertility treatments is estimated to have
resulted in the birth of over 8 million children worldwide [
110
,
111
]. Evidence suggests
that a small proportion of the children born following
in vitro
fertilization (IVF) and em-
bryo transfer suffer from genomic imprinting diseases (Beckwith–Wiedemann, Angelman,
Prader–Willi and Silver–Russell syndromes), slightly elevated risk of infant mortality in the
first year of life, exhibit signs of large size for gestational age, high birthweight and other
problems (for reviews, see [
110
,
112
,
113
]). While some of these problems may be related
to the original causes of the infertility, the various technologies used in clinical ART have
also been implicated. These include sperm and embryo cryopreservation, embryo culture,
and the nature of the culture media used. As many of these technologies are essentially the
same as those used in animal studies, it is likely that problems with conservation-relevant
animal species would be affected in the same ways but are unlikely to be detectable in the
near future as comparatively few such procedures are performed.
Nevertheless, although it is possible that the sperm cryopreservation process might
skew the success of assisted reproduction methods in general, it is encouraging that the
extensive biomedical and research use of cryopreserved, and banked, mouse spermatozoa
(where ICSI is widely preferred as the fertilization method) do not appear to have produced
the range of problems seen with fishes and amphibians [114,115].
9. Stem Cells, Conservation and Ethics
The cryopreservation of fish and amphibian oocytes presents a difficult problem,
because these are large cells filled with aqueous cytoplasm, typically enclosed within imper-
meable and tough outer coatings that inhibit, or prevent, the penetration of cryoprotectants.

Biomolecules 2022,12, 1297 10 of 18
An alternative approach that works for fishes involves the cryopreservation and transplan-
tation of primordial germ cells (PGCs) into sterilized surrogate recipient individuals at
different life stages [
116
–
119
]. PGCs are the precursors of gonocytes, the stem cells that
migrate to, and colonize, the genital ridges of early embryos, where they populate the testes
and ovaries, and are eventually responsible for producing spermatozoa and oocytes. When
the PGCs have originally been obtained from a donor species that differs from the surrogate
recipient, the spermatozoa and oocytes produced thereafter are derived from the PGC
donor. Thus, PGCs are widely considered to represent a practical solution for the cryobank-
ing of fish species, as they conserve both the paternal and maternal genomes. Fewer authors
have reported undertaking successful PGC transfers in amphibians. Rana pipiens PGCs,
dissected from tadpole genital ridges [
120
], were successfully transferred, into enucleated
eggs, obtaining normal development in about 40% of the transfers. and demonstrating the
totipotency of the germ cells. A later study [
121
] also demonstrated the principle of am-
phibian germ cells totipotency using salamanders. These authors transferred diploid germ
cell nuclei into irradiated eggs, and obtained not only an adult male and female, but also
some normal offspring. Despite the current catastrophic extinction rates being experienced
globally by amphibian populations, there has been very little interest in developing PGC
transplantation methods in aid of amphibian conservation. This may simply have been a
matter of scientific priorities, with aquaculture research providing the stronger motivation.
However, a recent review [
122
] nevertheless expressed the view that these approaches
might still be worthwhile exploring.
In practice, there are several variations of the techniques involved in gonocyte trans-
plantation, some involving transfer of PGCs into recipient blastulae, while others involve
transplantation of PGCs, spermatogonia or oogonia into hatchlings, or spermatogonial
transfer into adults [
123
]. Once the cell suspensions are microinjected into the intraperi-
toneal cavity, the PGCs migrate to the genital ridges where they develop and produce either
oocyte or spermatozoa. Rivers et al. [
117
] pointed out that the PGC transplantation method
appears to be more suitable for conservation biobanking than other conventional methods
of gamete storage.
Spermatogonia [
124
], as well as PGCs, can be cryopreserved easily and, when trans-
planted, they have the potential to produce millions of gametes and offspring throughout
the life of the recipient. Spermatogonial transplantation is widely seen as a potentially
successful method for treating testicular cancer in prepubertal and adolescent humans,
who have to undergo chemotherapy or radiotherapy. Some approaches require that the
patient’s spermatogonia are recovered before treatment and subsequently used to recolo-
nize the testes. Other approaches require the spermatogonial recovery to include various
intermediate steps, including the temporary culture of cells within the bodies of immunod-
eficient animals such as nude mice. All of these options have been summarized in a recent
review [
125
]. With a view to conservation, some of the techniques have been applied to
non-human mammalian [126–130] and avian species [131,132], as well as fishes.
Subjecting one group of (non-threatened) animals (typically immunodeficient and
sterilized mice or their equivalent) to highly invasive surgery, such as inserting testicular
cell aggregates or cell cultures beneath the kidney capsule, is ethically dubious if the
objective is to protect a different group of animals, even though they may be considered
to be at heightened risk of extinction (see [
129
,
133
–
135
] for examples of these techniques).
Careful ethical justification is needed when proposing such steps for species conservation,
including the valid statistically based reassurance that the offspring are not adversely
affected because of the varied technologies required.
The explosion of interest in the exploitation of various stem cell technologies for the
production of spermatozoa and oocytes
in vivo
, in order ultimately to produce embryos, has
not only raised new technical possibilities for species conservation, but has also generated
considerable ethical debate. The discovery that pluripotent stem cells (iPSCs) can be
generated from mouse embryonic cells and adult fibroblasts [
136
] has been followed by the
generation of iPSCs in multiple species [
137
,
138
]. This has led many biotechnologists to

Biomolecules 2022,12, 1297 11 of 18
hypothesize that it will eventually be possible to produce functional gametes, and therefore
also embryos, from rare and endangered species without the need to involve other animals
such as the immunocompromised and ovariectomized mouse [
134
,
137
,
139
]. At present, the
success rate would likely be rather low, and given the number of technical steps needed,
the health and survival of any offspring might also be low, especially as the detailed
reproductive biology of most threatened species is poorly documented.
Although accessing suitable tissue and cell samples from rare and endangered species
for the purpose of creating stem cells is not a problem with captive and domestic animals, it
would undoubtedly present problems with free-roaming wild species. Deriving stem cells
from skin samples might provide a more practical solution to this problem [
140
], as wild
species, including marine mammals [
141
], are often biopsied remotely for genetic studies,
using puncture darts and without the use of anaesthetics. Although this approach still
requires further development, stem cells successfully derived from porcine and murine skin
have been shown to possess the capacity to form oocyte-like and PGC-like cells [
142
,
143
],
as well as other non-germline cell types.
10. Conclusions
To survive on earth, all organisms have developed mechanisms that permit them to
cope with the environment in which they find themselves. Newborns cannot usually choose
the locations in which they would prefer to live, but some species have evolved mechanisms
that allow for some degree of parental choice in this matter. For example, many reptiles
have evolved temperature-dependent sex determination, whereby the females are able to
predict the expected temperature that eggs will experience in the nest, thus skewing the
offspring sex ratio [
144
]. In some reptiles, this mechanism operates alongside a genetically
based sex determination system, and recent evidence has shown that the two systems can
even be operated alternately and independently [
145
]. This is an astonishing example of
adaptive developmental plasticity but, as described above, it is only one among many
different solutions to the problem of forecasting the immediate environmental future and
responding appropriately.
In this sense, environmental change includes not only the warming climate and its
consequences, but even some of the well-meant mitigation measures that are under human
control. As discussed above, the captive breeding programmes used widely by zoos,
wildlife parks and aquariums as conservation measures, or even simply providing food
to wild animals such as bottle nose dolphins [
146
], appear in some cases to reduce the
fitness of the animals that are being protected. Should zoos and nature reserves respond to
such findings by reducing welfare and introducing competitors, predators and parasites,
thus creating more adverse conditions? This would undoubtedly be a counter-intuitive,
unpopular, unethical, and even illegal measure, but it would presumably lead to the
selection of individuals that are (a) adapted to life in a captive environment, but (b) also
able to survive if relocated to a more natural but possibly less comfortable environment.
On a related topic, it is evident from the preceding discussion that while the threat-
ened populations may benefit from technologies such as genetic supplementation with
cryopreserved and stored spermatozoa, there may be an inherent risk to the health and
welfare of offspring. Whether the introduction of more advanced breeding technologies,
especially those that will aim to regenerate the rarest species using stem cell reprogram-
ming and artificial gametes, would exacerbate the risk is largely unknown at present given
that numerically large datasets are needed before minor effects can be detected. Detailed
analyses of developmental problems in laboratory and agricultural mammals derived from
somatic cell nuclear transfer, and more recently the use of iPSCs, have revealed multiple
problems (summarized in [
147
]) that frequently lead to an abnormally high incidence of
perinatal mortality, inadequate placental function and abnormal offspring [148].
Whether and when to use such advanced technologies in conservation, where the
objective is to produce viable and healthy offspring with the capacity to survive and live a
healthy life, raises many difficult and controversial decisions. For example, is it acceptable

Biomolecules 2022,12, 1297 12 of 18
to deliberately produce animals that are highly likely to be unhealthy and abnormal? As
some of the species of interest require several years to reach breeding age, where might
such unhealthy, and possibly disabled, animals be kept? In the exceptional circumstance
that one or two members of an extinct species, such as the woolly mammoth, is successfully
regenerated using advanced technologies [
149
], would the absence of appropriate maternal
care and social interaction be a problem? Detailed ethical discussions around this topic
are outside the scope of this article, and the reader is referred to other articles for further
information [150–153].
Author Contributions:
Conceptualization, W.V.H. and P.C.; writing—original draft preparation,
W.V.H. and P.C.; writing—review and editing, W.V.H. and P.C.; visualization, W.V.H. and P.C.;
supervision, W.V.H. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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