Life Cycle Assessments Guide Aquaculture Sustainability

Life Cycle Assessments and Their
Applications to Aquaculture
Production Systems
PATRIK J. G. HENRIKSSON
1,2
, NATHAN L. PELLETIER
3
,
MAX TROELL
4,5
, PETER H. TYEDMERS
3
1
Institute of Environmental Sciences, Leiden
University, Leiden, The Netherlands
2
Department of System Ecology, Stockholm University,
Stockholm, Sweden
3
Dalhousie University, Halifax, Nova Scotia, Canada
4
The Royal Swedish Academy of Sciences, The Beijer
Institute of Ecological Economics, Stockholm, Sweden
5
Stockholm Resilience Centre, Stockholm University,
Stockholm, Sweden
Article Outline
Glossary
Definition of the Subject
Introduction
LCA – The Method and Its Applicability in
Aquaculture
LCA in Food Production
Guiding the Way for More Sustainable Aquaculture
and Alternative Farming Methods
Discussion
Future Directions
Bibliography
Glossary
Aquaculture The farming of aquatic organisms,
including fish, mollusks, crustaceans, and aquatic
plants. Farming implies some form of intervention
in the rearing process to enhance production, such
as regular stocking, feeding, and protection from
predators. Farming also implies individual or cor-
porate ownership of the stock being cultivated.
Co-product allocation Partitioning the input or out-
put flows of a process or a product system between
the product system under study and one or more
other product systems.
Functional unit The quantified function provided by
the product system(s) under study, for use as
a reference basis in an LCA, e.g., 1,000 h of light.
Life cycle assessment (LCA) An ISO-standardized ana-
lytical tool developed to evaluate environmental
performance of products and processes. It constitutes
a compilation and evaluation of the inputs, outputs,
and potential environmental impacts of a product
system throughout its life cycle; the term may refer
to either a procedural method or a specific study.
System boundary Defines the inputs and outputs that
are included in the study. System boundaries
should be set depending on what will be relevant
to the aim of the study.
Definition of the Subject
Aquaculture production has grown three times faster
than the livestock sector since the 1970s, becoming
a major source of edible seafood and other products.
This rapid expansion has, however, had a combination
of positive and negative environmental, social, and
economic effects. A variety of tools are available to
evaluate these impacts in an attempt to identify the
most sustainable practices. One of the more recent
tools that has been applied to the evaluation of aqua-
culture production is Life Cycle Assessment (LCA), an
ISO-standardized biophysical accounting framework
that allows for multi-criteria environmental perfor-
mance assessments. This chapter reviews studies that
have applied LCA to studying the environmental
dimensions of aquaculture production to date. Meth-
odological differences and alternative approaches are
discussed, along with their influence on research
outcomes. There is little homogeneity between the
studies when it comes to the choice of functional
unit, system boundaries, and basis for allocation. How-
ever, several clear trends do emerge that point toward
imperatives for sustainable practices in aquaculture
and considerations for sustainable development
of the industry moving forward. Recommendations
for further methodological development of LCA for
application to seafood sustainability research are
advanced.
Introduction
Society is increasingly aware of both the drivers and
consequences of natural resource depletion and envi-
ronmental degradation. Various analytical frameworks
1050 Life Cycle Assessments and Their Applications to Aquaculture Production Systems
P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,
# Springer Science+Business Media New York 2013
Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology, # 2012, DOI 10.1007/978-1-4419-0851-3

have therefore been developed for the purpose of eval-
uating the environmental performance of products and
processes. Finding a suitable tool for assessing sustain-
ability in the rapidly developing aquaculture sector has
gained increasing profile over the past 2 decades. What
has emerged is the need for a tool that can incorporate
multiple environmental performance criteria in the
evaluation of diverse aquaculture production technol-
ogies. For this reason, there is increasing interest in,
and application of, life cycle assessment (LCA) as
a research framework to better understand environ-
mental performance in this sector. The interest has,
however, not been coming from within the aquaculture
industry itself, but rather outside. LCA is a versatile
methodology that is well suited to address a broad
suite of resource use and emissions-related issues. Over
the last decade, it has become an increasingly common
tool for characterizing an important subset of environ-
mental impacts in aquaculture and elsewhere.
Aquaculture Development
Even though global capture fisheries landings have
declined since the late 1980s, total production of
marine fisheries products has increased 67% between
1970 and 2007 (including brackish water fish). This has
only been possible through a large increase in aquacul-
ture production over the last 4 decades. Aquaculture
currently provides half of all finfish destined for human
consumption. Seafood from all sources accounts for
about 20% of all animal proteins consumed by humans,
and demand continues to grow [1]. The aquaculture
industry is the fastest growing animal products’ sector,
with an average annual growth rate of 6.9%. At present,
it provides almost 8 kg of seafood per capita yearÀ1
globally [1]. In 2006, aquaculture accounted for
more than 70% of global shrimp and prawn produc-
tion, 47% of total food fish production, and 36% of
total fish production. Mariculture of finfish dominates
production in developed countries [2]. By mass, how-
ever, the majority of global production is accounted
for by carp farmed in extensive and semi-intensive
farms in Asia.
Aquaculture comprises an enormous diversity of
farming technologies, culture settings, and species.
From monoculture to polyculture systems operated in
ponds, raceways, land-based tanks, along with cages,
pens, poles, rafts, and longlines in open water settings,
well over 250 species are currently in culture. Produc-
tion technologies may also reflect traditional farming
methodologies or more modern systems [3–5]. In turn,
post-production processing yields a diverse range of
products, including salted, dried, smoked, and various
kinds of preserved fish. About 37% of all fish and
fishery products are traded internationally, with the
major importers being Japan, the USA, and Spain [1].
Aquaculture and the Environment
It has been proposed that aquaculture represents the
most viable option for meeting future demands for fish,
as well as providing economic and nutritional benefits
to millions [1]. The recent rapid expansion of this
sector has, however, been accompanied by a range of
environmental and social concerns, including localized
nutrient enrichment or depletion, chemical pollution,
genetic pollution, introduction of non-indigenous spe-
cies, habitat destruction, greenhouse gas (GHG) emis-
sions, depletion of wild fish stocks, inefficient energy
and biotic resource usage, and spread/amplification of
diseases and parasites [2, 3, 6–9]. Of these, local-scale
interactions have traditionally attracted the most
attention. However, global scale interactions such as
greenhouse gas emissions associated with intensive
production strategies are of increasing interest. What
has become clear is that each production strategy is
characterized by a unique suite of environmental inter-
actions at local, regional, and global scales. Informed
decision making for improved environmental manage-
ment in aquaculture, therefore, requires tools, which
can provide multi-criteria environmental performance
assessments and make clear the environmental
trade-offs associated with specific aquaculture technol-
ogies and products.
Sustainability Tools in Aquaculture
Increasing aquaculture production to meet future
demands is clearly attractive from a policy and devel-
opment perspective. However, a number of critical
questions related to growth in this sector must be
addressed. These questions encompass complex issues
associated with sustainability objectives at local,
regional, and international scales. For example,
a spectrum of negative ecological and social
1051Life Cycle Assessments and Their Applications to Aquaculture Production Systems

externalities associated with aquaculture and other
food production systems bear careful scrutiny and
must be weighed against anticipated benefits [10].
Such comparisons need to extend beyond short-term
gains and localized impacts and incorporate a long-
term social-ecological resilience perspective. This
requires tools to identify the most sustainable aquacul-
ture practices, drawing knowledge from both new and
traditional culture systems [5].
A wide range of tools/frameworks for assessing
various aspects of environmental performance have
been advanced [11], some focusing especially on food
production. These include techniques such as Risk
Assessment, Ecological Footprint, and Energy Analysis.
Frameworks more specific to assessing seafood produc-
tion systems are Fishprint and the Global Aquaculture
Performance Index (GAPI) [10, 12, 13]. Most of these,
however, encompass a limited range of the environ-
mental concerns associated with aquaculture and
some suffer from a lack of methodological standardi-
zation [10]. Moreover, the degree of scientific rigor in
both the methods and their application is also variable.
Data limitations and analytical scope have, therefore,
often led to misrepresentation of the environmental
consequences of specific management decisions.
LCA – The Method and Its Applicability in
Aquaculture
History of LCA
LCA has, since its emergence in the 1970s, evolved from
a tool whose primary application was waste manage-
ment and energy efficiency management to a more
general eco-efficiency measurement framework. It has
close links to energy analysis, but is unique among
biophysical accountancy-type tools in that it has been
internationally standardized (ISO 14040-14044)
[14, 15]. An LCA typically begins at the “cradle” of
a product or service life cycle (i.e., at the point of
primary resource extraction), and extends along the
supply chain to encompass all life cycle stages of inter-
est to a particular analysis. Single or multiple impact
assessment methods may be applied. Estimation of the
cumulative environmental impacts along supply chains
permits attention to spatially and temporally discrete
impacts not typically considered in more traditional
environmental impact analyses. Impact categories
range from highly quantifiable effects, such as green-
house gas emissions or energy use, to (less frequently)
more diverse social consequences, such as human
health effects [16].
The outcome of a LCA is highly influenced by the
ambition, skill, and objectives of the practitioner. Mod-
ern software with built-in inventory databases and
impact assessment methods has simplified the LCA
process, to the extent that an aquaculture system may
be modeled in hours. However, the rigor of such
models is to a large extent dependent on data quality.
While use of generic data available in many public and
commercial life cycle inventory databases may provide
a starting point for scoping analyses, more context-
specific data is required for robust modeling of specific
production systems and technologies. Unfortunately,
the former (simplified analyses) are increasingly com-
mon in the peer-reviewed literature, providing what
may be misleading signals and eroding the credibility
of the research framework, generally.
Software Tools and ISO
The rapid evolution and adoption of LCA have been
accompanied by the creation of a variety of guidelines,
manuals, and dedicated software [17]. The most com-
monly used LCA software platforms are GaBi and
SimaPro, which are commercial products. Others,
such as CMLCA and openLCA, are available free of
charge. There are also several life cycle inventory data-
bases, which have been developed, with the most exten-
sive being the EcoInvent database (www.ecoinvent.ch).
Such databases provide inventory data for materials
and processes common to most product systems – for
example, the production of materials such as concrete,
steel, and plastic; the provision of energy carriers such
as diesel or regionally specific electricity mixes; or
transportation modes like air freight, rail freight, or
private automobile transport. As these “background”
data are often premised on different methods, assump-
tions, and rigor, sourcing data should be done with care
as different databases apply different methodologies.
It is therefore recommended to be consistent when
choosing sources of background systems data and also
to be aware of the methodology used before making
comparisons between studies. ISO compliance does
not require that studies are comparable, only that

they follow the same requirements. Individual studies
also often differ in functional unit (the unit of output
against which impacts are quantified), system bound-
aries, and allocation criteria.
A LCA study is, in accordance to ISO standards,
carried out methodically through four phases (Fig. 1).
As an initial phase, the goal and scope definition will
specify the main characteristics of the study. Goal is
specified as the application, audience, and reason for
the study, while scope outlines the product system to be
studied, functional unit, system boundaries, allocation
procedures, impact categories, data requirements,
assumptions, limitations, data quality, and format of
the report. System boundaries specify the processes
that are to be included in the product system and are
in turn set by the cutoff criteria. This is followed by
a Life Cycle Inventory Analysis (LCI), which involves
the collection and calculation of data as well as alloca-
tion of burdens, in cases where input or output flows
involve more products or processes than the defined
unit. The impacts of these results are then, in the third
phase (Life cycle impact assessment, LCIA), assessed
according to their contribution to the impact catego-
ries defined in the goal and scope phase. Finally, the
three previous phases are interpreted and communi-
cated to the anticipated audience.
Functional Unit
Seafood commodities are farmed for different pur-
poses, which complicates the choice of functional
unit. Most LCA studies to date have used live weight
mass at the farmgate or mass of processed product as
the functional unit. Variable protein contents and edi-
ble portions between aquatic animals therefore may
complicate direct comparisons. For example, the edible
portion of an oyster is only about 18% of its wet weight,
a number that in turn is subject to local variation [18]
while the edible yield from an Atlantic salmon (Salmo
salar) typically exceeds 50%. Local customs may fur-
ther confound the decision as certain parts of the fish
that may be discarded as inedible in one region are
considered good for human consumption in another
region. Two such examples are herring roe and catfish
Goal and scope
definition
- Product development
and improvement
- Strategic planning
- Public policy making
- Marketing
- Other
Life cycle assessment framework
Direct applications:
Inventory
analysis
Interpretation
Impact
assessment
Life Cycle Assessments and Their Applications to Aquaculture Production Systems. Figure 1
The general methodological framework for LCA studies according to ISO 14040 (2006) and its four comprised phases

stomachs; both of which are considered offal in the
western world but are prized in parts of Asia [19, 20].
Ideally, the functional unit should reflect the function
of the product system. For aquaculture products
intended for consumption as food, such functions
might include the provision of caloric energy, protein,
or omega fatty acids, etc.
Setting System Boundaries
System boundaries delineate those processes formally
included in an analysis from those that are excluded.
There are only general requirements on how to set these
boundaries, though they should be decided relative to
the research objectives and include all elements having
a non-trivial influence on research results. Most LCA
studies in aquaculture limit their system boundary
to the farmgate, whereas some include processing,
packaging, marketing, and consumption phases [21–
23]. Thrane [24] evaluated the effects of post-harvest
stages of the life cycle of seafood products and con-
cluded that they have a significant impact on the overall
environmental performance of most seafood products.
For example, inclusion of the processing phase
contributed an additional 10% to life cycle energy
use, while inclusion of the consumption phase resulted
in an average 25% increase in cumulative energy
demand [24].
Allocation
A common issue faced by many LCA practitioners is
how to allocate environmental burdens to products
and materials that are co-produced with other prod-
ucts [25]. Examples in aquaculture include the alloca-
tion of environmental burdens between targeted catch
and bycatch used in fishmeal production, the multiple
products derived from corn and other commodity
crops used in feeds, and alternative uses of fish
by-products or aquaculture wastewater used to fertilize
other crops. The ISO standards for LCA do provide
guidelines for an allocation decision hierarchy, but
leave considerable room for interpretation. According
to the standard, environmental burdens are primarily
to be allocated according to an underlying physical
relationship, if subdivision is unavoidable. The stan-
dard further states that where such relationships can-
not be established, the allocation should reflect other
relationships between the input system and output
system. In reality, the final choice of allocation basis
will likely reflect the goals of the study, as well as
the worldview of the practitioner. Some of the more
common bases for allocation in aquaculture and their
characteristics are summarized in Table 1. Accessibility
of the different allocation factors differs depending
on location and situation, where regional differences
will play a large role in making generalizations. Time
and spatial scales will also have a great influence on
the allocation factors that do not represent physical
relationships, as these will not remain static.
There are several things to keep in mind when
choosing a basis for allocation. The first is that nothing
limits a study to only one allocation factor; each allo-
cation scenario may be treated differently depending
on the circumstances. Results may also be presented
using several of the allocation factors, thus enabling
readers to interpret results according to their own
perspective or worldview. No matter how the alloca-
tion problem is approached, it is very important to be
clear in the supporting text about which methods have
been used. It is, however, important to keep in mind
Life Cycle Assessments and Their Applications to Aquaculture Production Systems. Table 1 The most commonly used
bases for allocation and their characteristics
Allocation factor Accessibility Physical relationship Static Market oriented
Mass Good Yes Yes No
Value Average No No Yes
Nutritional energy content Average Yes Yes No
System expansion Poor Sometimes Sometimes Sometimes

that the basis of allocation can influence the allocated
burden by as an order of magnitude [26].
Impact Categories and Impact Assessment
Any quantifiable performance measure can be included
within the LCA methodology, from emissions to the
well-being of workers, as long as: (a) a causal relationship
between the variable of interest and the provision of the
functional unit can be established and (b) a defensible
impact assessment methodology is available [27]. Awide
variety of impact assessment methods are available for
use in LCA, most of which have been applied to assess-
ments of seafood production systems [27]. These
methods may describe environmental interactions that
have relevance at local (e.g., eutrophication), regional
(e.g., acidification), or global (e.g., greenhouse gas emis-
sions) scales. Generally, impact assessment methods are
based on peer-reviewed, internationally accepted envi-
ronmental accounting protocols. Some of these continue
to evolve, and novel methods emerge in response to
newly identified issues [27, 28].
Labor
Labor is rarely accounted for in LCA due to the difficulty
of establishing defensible system boundaries and quan-
tifying associated environmental impacts. It can also be
debatable if the number of employees should be consid-
ered as a negative or positive input. It is, however,
important to keep labor in mind when comparing tra-
ditional and modern production systems. Labor can be
presented in a number of different ways, either sepa-
rately or incorporated into the individual impact cate-
gories [28, 29]. Several methods have been suggested on
how to quantify the environmental impacts of labor.
Suggested reference units include metabolic energy,
calorific content of food consumed, national fuel
share, or other more complex equations [29].
LCA in Food Production
Working with non-Static Systems
The adaptation of LCA from the characterization of
static industrial systems to food production systems
typified by significant variability has brought with it
new challenges. Annual fluctuations occur both on the
farm, as productivity will depend on water
temperature, extreme weather events, algae blooms,
etc., as well as on indirect variables such as oil prices
and public demand. Fisheries providing fishmeal and
fish oil for use in aquafeeds offer a good example of such
variability, as fuel inputs per tonne of fish landed will
fluctuate with season, stock status, gear type, and skip-
per [30, 31]. The aquaculture sector is particularly
dependant on annual production of the anchoveta
fishery off South America for both of these commod-
ities, which in turn is strongly influenced by El Ninõ-
Southern Oscillation (ENSO) events [32]. Increased
use of compound feeds and higher oil prices have also
boosted prices of both fishmeal and oil over the last
decade [1]. Such fluctuations not only affect economic
allocation but also catch per unit effort. It can therefore
be hard to set average fuel consumption for fishing
fleets, especially since the species and status of the stocks
used for fishmeal production often are unknown [33].
The situation is further complicated in certain parts of
the world where low value fish are used directly as fish
feed [33].
Agricultural crops – the other major source of
aquafeed inputs – may also experience significant
annual fluctuations, with larger variability in devel-
oped countries, for crops such as maize and wheat
[1, 34]. Farmgate prices will further affect the LCA as
many feed formulators and aquaculturists quickly
adjust to price trends in their choice of feed inputs or
cultured species in efforts to maximize profits [20, 35].
One such example is the constant push toward higher
stocking densities of shrimp in SE Asian polyculture
systems, where the large profits that are to be made
often outweigh the risk of white-spot disease [36].
Farming practices, as for feed composition, are also
under constant change, which emphasizes the impor-
tance of considering the time scales used in LCA stud-
ies. Ultimately, a balance must be struck between
feasibility and the goals of the study in pursuing repre-
sentative data and models.
The relevance of such variability will, in part, be
determined by the scope of the analysis and specific
research questions of interest. Variability might be
accommodated when modeling at regional scales by
applying average data over specified spatial and tem-
poral horizons. It is increasingly common to account
for and report such variability in published outcomes.
In other cases, quantifying variability might comprise

research foci – for example, understanding the influ-
ence of variable field-level nitrous oxide emissions on
the overall greenhouse gas intensity of crop production
at local scales.
LCA in Aquaculture
The first LCA studies focusing on aquaculture systems
were conducted in the beginning of the new millen-
nium, with an increasing application of the methodol-
ogy to aquaculture issues toward the end of its first
decade [37–39]. As the number of studies increased, so
too did the seeming detail of analysis and possibly also
the accuracy of the results. Most have focused on pro-
duction systems in developed countries (see Table 2).
The methodological detail used between studies does,
however, vary widely and makes broad comparisons
between studies difficult. This includes differences in
functional unit, system boundaries, data sources and
quality, and choice of allocation criteria.
A common theme that has emerged from LCA
research of intensive, finfish aquaculture production
systems is the importance of feed provision in supply
chain environmental impacts [21, 22, 42–44]. For
example, Pelletier et al. (2009) found that feed provi-
sion accounted for, on average, 92% of quantified
impacts in global salmon farming systems. Of particu-
lar importance are fisheries and livestock products,
which typically have higher impacts per unit mass
relative to crop-derived feed inputs (Fig. 2). Also of
note is that on-site processes have only made
a substantial contribution in highly mechanized sys-
tems, where industrial energy inputs are required to
maintain water quality [3, 20, 21, 40, 44].
Although animal-derived feed ingredients usually
have a higher impact per unit mass compared to crop-
derived inputs [22, 43, 45, 46], their inclusion may
support more rapid growth of the cultured organisms
and, in some cases, result in a higher quality product
[47]. By including a larger portion of agriculturally
sourced materials in feeds, environmental burdens
may be reduced. In this light, it might be anticipated
that rearing herbivorous or omnivorous species is envi-
ronmentally preferable. However, both feed composi-
tion and feed conversion efficiency must be considered
in determining and comparing impacts between cul-
tured organisms [44].
The choice of impact categories in aquaculture
LCAs varies widely (Table 2). These include resource
depletion and emissions-related environmental con-
cerns, as well as toxicological potentials. The only cat-
egories almost consistently applied are global warming
potential, acidification, and eutrophication, while
cumulative energy demand is also very commonly eval-
uated. There is an expected but imperfect correlation of
cumulative energy demand and global warming poten-
tial (Table 3), since much of the feed-related emissions
for agricultural inputs do not arise from fossil fuel
combustion. Rather emissions of nitrous oxide and
methane are typically as or more important. It is only
in systems where ecosystem services have been replaced
to a large extent by anthropogenic processes that on-
farm energy demand has a significant impact on the
total energy consumption.
Guiding the Way for More Sustainable
Aquaculture and Alternative Farming Methods
Feed Production
Improvements of feed conversion ratios (FCRs) for
piscivorous fish in addition to an increased inclusion
of non-fish ingredients has led to great environmental
improvements over the last decades [46]. Additional
improvements are to be made by identifying and sourc-
ing for the least-environmental-cost feed formulations;
especially for sources of fishmeal and oil [22]. In reduc-
tion fisheries, effective management will play a major
role in reducing fuel consumption along with associ-
ated environmental impacts while sustaining output
from the industry [30, 50]. In many fisheries, boats
today have to travel further to find productive fishing
grounds and invest more fishing effort to maintain
catches. This has resulted in a sixfold increase in energy
consumption for some capture fisheries over the last
two decades [51]. A collapse of one of the large reduc-
tion fisheries, of the scale that occurred to the ancho-
veta fishery in the 1970s, would further drive up the
energy intensity of aquafeeds and culture products as
well as have devastating effects on the aquaculture
industry as supply of fishmeal and oil supplies already
are outpaced by demand [1, 52].
Decreasing fishmeal inclusion has, to a large extent,
been driven by increasing public awareness about “fish-
in to fish-out” ratios, as a result of labeling and

LifeCycleAssessmentsandTheirApplicationstoAquacultureProductionSystems.Table2Overviewofspecies,functionalunit,systemboundary,allocation
factor,andimpactcategoriesappliedforaselectionofpublishedLCAstudiesonaquaculturesystems.Onetonatfarmgateisthemostprominentfunctionalunit
whileglobalwarmingpotential,acidification,andeutrophicationaretheonlyimpactcategoriesthatareincludedinallstudies
SpeciesReference
Functional
unit
System
boundary
Allocation
factor
Cumulativeenergydemand
Fossilfueluse
Bioticresourceuse
Abioticdepletionpotential
Waterdependence
Surfaceuse
Globalwarmingpotential
Ozonedepletionpotential
Acidification
Eutrophication
Phtotochemicaloxidant
formation
Freshwateraquatic
ecotoxicity
Marineaquaticecotoxicity
Terrestrialecotoxicity
Humantoxicity
Respiratoryimpactsfrom
inorganics
Carcinogens
Blue
mussels
Iribarren
etal.[23]
1kgofdry
edible
mussel
flesh
Post
consumption
waste
System
expansion
✖✖✖✖✖✖✖✖✖✖
ShrimpsMungkung
[70]
1.8kg
blockof
frozen
shrimp
Post
consumption
waste
Monetary✖✖✖✖✖✖✖
Rainbow
trout,sea-
bass,and
turbot
Aubinetal.
[21]
1tlive
weight
FarmgateMonetary✖✖✖✖✖✖
Salmon,
different
farming
methods
Ayerand
Tyedmers
[40]
1tlive
weight
FarmgateGross
nutritional
energy
content
✖✖✖✖✖✖✖✖
ArcticcharAyerand
Tyedmers
[40]
1tlive
weight
FarmgateGross
nutritional
energy
content
✖✖✖✖✖✖✖✖

LifeCycleAssessmentsandTheirApplicationstoAquacultureProductionSystems.Table2(Continued)
SpeciesReference
Functional
unit
System
boundary
Allocation
factor
Cumulativeenergydemand
Fossilfueluse
Bioticresourceuse
Abioticdepletionpotential
Waterdependence
Surfaceuse
Globalwarmingpotential
Ozonedepletionpotential
Acidification
Eutrophication
Phtotochemicaloxidant
formation
Freshwateraquatic
ecotoxicity
Marineaquaticecotoxicity
Terrestrialecotoxicity
Humantoxicity
Respiratoryimpactsfrom
inorganics
Carcinogens
Atlantic
salmon,
different
feeds
Pelletierand
Tyedmers
2007[43]
1tlive
weight
FarmgateGross
nutritional
energy
content
✖✖✖✖✖✖
Trout,flow
through/
recirculating
system
d’Orbcaster
etal.[41]
1tlive
weight
FarmgateMonetary✖✖✖✖✖✖✖
TroutPapatryphon
etal.[37]
1tonne
liveweight
FarmgateMonetary✖✖✖✖✖
Atlantic
salmon
Ellingsen
and
Aanondsen
[39]
200gfilletProcessed
fillets
Mass/
Monetary
✖✖✖✖✖✖✖✖
Atlantic
salmon
Pelletier
etal.[22]
1tonne
liveweight
FarmgateGross
nutritional
energy
content
✖✖✖✖✖

certification initiatives [9, 46]. It has also been strongly
influenced by rising prices for these commodities, due
to increased competition from the aquaculture sector
[1]. The overall demand for fishmeal and oil in aqua-
culture has, despite this, increased due to a larger share
of farmers using compound feeds and increasing aqua-
culture production over time [46, 53]. At present, in
most aquaculture LCAs, the amount of fishmeal and oil
used is only reported as life cycle inventory data.
Beyond the standard impact categories, methods to
account for the ecological impacts of producing these
products are underdeveloped. To date, only a few
researchers have, e.g., applied a measure of biotic
resource use in life cycle assessment, following the
methods originally advanced by Pauly and Christensen
[54]. This method quantifies the net primary productiv-
ity, as measured in carbon, required to support the
provision of a specified amount of fish-derived material,
taking into account trophic level and species-specific
meal and oil yield rates. Biotic resources used can differ
by as much as an order of magnitude between different
sources of fishmeal and oil [22]. Alternative sources of
proteins that could in part replace fishmeal include
soy meal, wheat gluten, bone, feathers, blood, livestock
co-product meal, and seafood processing materials
[46, 55]. A variety of vegetables oils may also be par-
tially substitutable for fish oils. The environmental
performance of systems using, e.g., agriculture alterna-
tives for fishmeal and oil needs, however, to be carefully
analyzed as such substitution does not guarantee
improved performance. Switching to the culture of
low-trophic species is often described as a solution for
more sustainable aquaculture [6]. While this would
allow for great reductions in fish inclusion rates, the
higher FCRs associated with lower quality feeds may
result in only marginal improvements in GHG and
related life cycle impacts [44]. In contrast, a switch to
more energy and climate-friendly fertilizer production
either through efficiency improvements in existing
fertilizer plants or the use of biological nitrogen fixa-
tion in place of conventional N fertilizers could, how-
ever, offset some of the impacts associated with crop
production [56, 57].
Improvements of feeding practices on farms can
both reduce costs, emissions, and FCR [58]. The
amount of feed added is often calculated according to
feeding charts or as a percentage of the fish biomass.
These generalizations often result in inefficient feed
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Cumulative energy use,
MJ
GHG emissions, kg CO2
eq.
Farm energy
Smolt production
Feed tranport
Feed, milling
Feed, livestock
Feed, fisheries
Feed, crop derived
Life Cycle Assessments and Their Applications to Aquaculture Production Systems. Figure 2
Cumulative Energy Use and Greenhouse gas emissions from salmon production. Feed production is by far the largest
contributor to environmental concerns, constituting 93% of the energy use and 94% of the GHG emissions. The feed
represents an average from farms in Norway, UK, Canada, and Chile with 41.8% of the ingredients derived from crops,
49.4% from fish, and 8.8% from livestock. Data from: Pelletier et al. [22]

utilization as it does not take factors such as species,
genetic stock, feed composition, water temperature, or
growth rate into account [58]. In addition, feeding
efficiency is also influenced by the type of farming
facility as confined bodies of water, such as raceways
or bags, allow for more efficient feeding practices and
effluent management [59].
Replacing Ecosystem Services with Anthropogenic
Processes at Farm Site
Traditional extensive aquaculture systems depend, to
a large extent, on labor and natural energy inputs [3].
Solar energy inputs promote in-situ production utilizable
by some farmed animals while tidal energy or other nat-
ural watercourse flows provide means for water exchange.
Life Cycle Assessments and Their Applications to Aquaculture Production Systems. Table 3 Summary of the different
results for 1 t of seafood product at farmgate
Species Country Source CED (MJ) tÀ1
GWP kg CO2-e tÀ1
Turbot, recirculating France Aubin et al. [21] 290,986 6,017
Sea-bass, cages Greece Aubin et al. [21] 54,656 3,601
Rainbow trout, flow through France Aubin et al. [21] 78,229 2,753
Atlantic salmon, net-pen Canada Ayer and Tyedmers [40] 26,900 2,073
Atlantic salmon, Land base Canada Ayer and Tyedmers [40] 97,900 2,770
Atlantic salmon, Bag Canada Ayer and Tyedmers [40] 32,800 1,900
Atlantic char, land-based recirculating Canada Ayer and Tyedmers [40] 353,000 28,200
Trout, recirculating system Denmark d’Orbcastel et al. [41] 63,202 2,043
Trout, flow through Denmark d’Orbcastel et al. [41] 34,869 2,015
Atlantic salmon Norway Ellingsen and
Aanondsen [39]
65,000a
N.A.
Blue mussels, fresh Spain Iribarren et al. [23] N.A. 472
White-legged shrimps Thailand Zimmo et al. [49] and
Mungkung [70]
45,600 N.A.
Trout, portion sized France Papatryphon et al. [37] 37,842 1,851
Trout, large sized France Papatryphon et al. [37] 62,774 2,499
Atlantic salmon, organic crop/25% soy meal
and 100% canola oil substitute
Canada Pelletier and
Tyedmers [43]
9,860 690
Atlantic salmon, organic crop ingredients/
fisheries by-product meals and oils
Tyedmers [43]
26,900 1,810
Atlantic salmon, organic crop/conventional
animal meals and oils
Tyedmers [43]
17,100 1,250
Atlanti salmon, conventional Canada Pelletier and
Tyedmers [43]
18,100 1,400
Atlantic salmon Worldwide Pelletier et al. [22] 31,100 2,160
Tilapia, Lake Indonesia Pelletier and
Tyedmers [44]
18,200 1,520
Tilapia, Pond Indonesia Pelletier and
Tyedmers [44]
26,500 2,100
Energy use in Norwegian salmon farming reported by Ellingsen and Aanondsen [39] was calculated by assuming that 15% of total
reported energy use was used in the processing and distribution phases

As forenergyconsumed inhighly mechanized production
systems, such as most intensive land-based systems, the
energy for farm-site activities often originates from fuel as
farm-sites typically lack access to alternative sources of
energy. It is therefore recommended to consider the loca-
tion characteristics of the farm-site before implementing
artificial services. Farms situated in areas with high water
turnover, i.e., in streams, tidal zones, or exposed coasts,
may cause little or no impact on the local ecosystem.
Farms situated in areas without such hydrodynamic con-
ditions need to treat their wastewater to avoid negative
impact on the environment or run as closed systems. It is,
however, important to acknowledge that environmental
impacts from farm release need to be analyzed from an
ecosystem perspective, which implies considering addi-
tional pollution sources and more regional scale effects
and thresholds.
LCA has limited capacity to predict the actual con-
sequences of many of the estimated impact potentials
[60]. It may be justifiable to have such an approach for
the impact categories that are operational on a global
scale, such as global warming potential or ozone deple-
tion. For more regional consequences, however, it can
be highly misleading to make comparisons of the
impacts between two localities. To address this in
a more justifiable manner, several country-specific fac-
tors, such as RECIPE, TRACI, EDIP2003, and LUCAS
[61], have been developed and arguments have been
raised for similar factors on regional scales [62].
Transmission of disease and parasites between wild
and farmed stock and introduction of non-indigenous
species are both major concerns associated with aqua-
culture that have yet have not been addressed by LCA
methodology [63, 64]. Nor is the framework necessar-
ily conducive to accommodating such interactions
since it is necessary to be able to link the impact to
the production of a functional unit following a clear
and quantifiable cause-effect pathway. Both of these
types of impacts have attracted much public attention
and have been major incentives for closed farming
facilities. Transmission of sea lice has, apart from hav-
ing potentially detrimental effects on wild fish stocks,
been estimated to account for 6% of the product cost in
salmon farming [64]. Floating cages and net cultures
allow for free movement of pathogens from farmed to
wild stocks [64]. They also run an increased risk of
large escape events of domesticated fish due to their
vulnerability to extreme weather events, marine mam-
mal interactions, failing infrastructure, and manage-
ment errors. Such events can lead to the introduction
of non-indigenous species as well as undermining the
genetic fitness of wild stocks. It has been estimated that
aquaculture is responsible for 16% of all introductions
of non-indigenous species to European coastal waters,
and further introductions are to be expected with
changing climate [63].
The use of chemicals and antibiotics on farm sites is
still only generally covered by existing impact categories.
These practices can result in long-term effects such as
antibiotic-resistant bacteria or other public health risks
[10]. They may also lead to contaminated water sources
and loss of biodiversity [10]. Shrimp farms are impli-
cated most frequently for using large amounts of anti-
biotics to reduce stock losses. On several occasions, this
has resulted in product recalls and import bans by the
EU, Canada, and the USA [10]. The social conse-
quences of this might be direct (e.g., lower water qual-
ity) or indirect, as import bans can seriously affect the
financial viability of many farmers [65]. Even if LCA has
the potential to account for such social and economic
consequences, there is a lack of metrics to describe how
to include socioeconomic indicators [28]. Overcoming
the hurdles associated with the development of such
impact categories would allow for better estimations of
overall sustainability, including environmental, social,
and economic variables [28]. However, it should be
recognized that LCA is not necessarily conducive to
accounting for the full spectrum of sustainability con-
cerns. As such, it should be considered a complement to,
rather than a replacement for, other metrics.
Discussion
As the number of LCA studies describing aquaculture
systems increases, so too does our understanding of
a broader suite of the environmental costs of aquaculture
production. In some cases, it would appear that aquacul-
ture may indeed provide an inexpensive and sustainable
source of food and other products; however, this will
depend on numerous factors including cultured species,
production technology, and socioeconomic characteris-
tics. To date, LCA research has helped to identify those
aspects of the life cycle that contribute disproportionately
to environmental degradation, allowing for the

identification of improvements opportunities. There is,
however, always the danger of oversimplification where
results get misinterpreted as a result of inaccurate data or
where results are not put into their relevant context. LCA
should therefore not be seen as an all-encompassing tool,
but rather as a screening tool, which allows for the map-
ping of good practices. Additional environmental and
socioeconomic analyses can thereafter be applied to
strengthen assessment of sustainability.
As for advancements that can be made within the
sector, the major challenge will be to find good sources
of low impact feed inputs for fed aquaculture systems,
especially for fish oil that currently drives the demand
for wild marine resources [46]. This would, of course,
ideally be combined with further advances in feed
utilization by fish in culture. A shift toward organically
produced crop inputs may also reduce the impacts of
fed aquaculture, while bringing other benefits such as
biodiversity improvements and superior soil quality
[57, 66]. However, the choice of some resource inten-
sive “organic” inputs can negate much of the life cycle
environmental benefits associated with organic crop
production [57]. Another alternative protein source is
offal meal from fish processing. Tilapia, for example,
has an offal yield of about 67% of live weight [59]. This
does, however, increase the risk of disease transmission
and/or recycling of environmental contaminants [53].
Moreover, the environmental costs of producing these
materials from a life cycle perspective may be high [22].
Recently, there has been increased interest in the use
of fish processing co-products for biofuel feedstock
[67]. Conversion of high-quality protein into biofuels
appears rather wasteful when the environmental bur-
dens associated with producing certain high protein
feed inputs are taken into account. It is clearly desirable
to identify and implement optimal uses of high-quality
protein toward the overarching sustainability objective.
This must include, among other things, attention to the
environmental dimensions of alternative protein pro-
duction and use strategies. Since our ability to make
informed decisions will be strongly influenced by the
robustness of our models and the extent to which they
actually reflect the environmental impacts associated
with the products and systems of interest, methodo-
logical decisions such as choice of allocation criterion
should be clearly communicated and defended in the
context of each analysis.
Increasing trade flows of aquatic products from
developing to developed countries highlights the need
for more LCA work beyond production systems in the
developed world. It also indicates that there are sub-
stantial opportunities for expansion of aquaculture in
developed countries. Tyedmers et al. [2] points out that
the USA only accounts for 1% of global aquaculture
production, half of which is made up by channel cat-
fish. Future developments may also to be expected
within mariculture, as much recent effort has focused
on the development of marine fin-fish hatcheries and
offshore cages.
Impacts involved with on-site activities can more
easily be avoided by selecting for better farm locations,
use of renewable energy, improved utilization of
ecosystem services, and farming of more tolerant spe-
cies. One example of such a species is Pangasius catfish,
which has reached high production levels in Vietnam
over the last decade. This fish does not require aeration
as it can utilize aerial respiration when the oxygen level
drops. The farms also often utilize tidal floods for water
exchange [20]. As for tilapia, the tolerance toward
hypoxia and changes in pH is much higher than other
species normally found in western aquaculture. This
would, again, lower the inputs needed for maintaining
water quality. Wastewater quality can also be improved
by the use of settlement ponds and/or plant production
to remove nutrients. Cultivation of plants such as
Azolla spp. and duckweed within ponds can also
enhance carbon fixation and be used as feed inputs
[48, 68]. Duckweed may further reduce GHG emis-
sions as it will shade the pond and thereby limit the
ammonia volatilization rate [49]. Some of these feed
inputs may, however, negatively influence the color and
the taste of the final product, which makes them less
suitable as feed inputs [48].
Converting to energy conserving practices will not
only have environmental benefits, it can also improve
the economic profitability of farms and reduce vulner-
ability to peaks in the price of oil. Lower intensity
systems are also less sensitive to mechanical or infra-
structure failures, such a black outs, which otherwise
can cause mass mortality. This is especially important
in developing countries where there is less access to
spare parts and power failures are recurring events.
Open systems are, on the other hand, more vulnerable
to other events such as algae blooms, pollution, and

extreme weather events, which may cause the loss of an
entire crop. Areas ravaged by extreme storm events,
such as typhoons, may suffer from weeks of poor
water quality as large quantities of sediments may
lead to high turbidity for long periods of time.
More knowledge on the true carbon emissions asso-
ciated with aquaculture is needed as there is increasing
interest in its potential as a climate-friendly source for
food and biofuels [68, 69]. LCA can play an essential part
in the screening for sustainable farming practices and also
provide information for the implementation of carbon
credit schemes. This need is especially critical for develop-
ing countries, as this is where the majority of production
occurs and exports are increasing. This will, however, pose
a challenge as farming methods are highly diverse and data
on farm practices are usually limited.
Also, some important environmental impacts from
aquaculture are at present not quantifiable using the
LCA framework, such as spread of diseases and para-
sites. These impacts have been attracting widespread
public concerns and have influenced development of
farming methods in many countries. The associated
consequences could, for example, be accounted for as
biotic resource use. Concerns associated with defores-
tation to produce agricultural land should also be fur-
ther discussed, as it is only partially covered in current
LCA practice and literature.
Future Directions
LCA provides a robust tool for dealing with an impor-
tant subset of sustainability concerns, many of which
have historically been overlooked in discussions of
environmental management in aquaculture. However,
it must be kept firmly in mind that decisions made
during the analytical process strongly influence
research outcomes.
It is, therefore, important to continue the discus-
sion about such methodological decisions, including
the kind of LCA framework applied (i.e., attributional
versus consequential LCA), systems boundaries, and
choice of allocation methods for LCAs of aquaculture
production. In the least, it would be constructive for all
practitioners to clearly communicate and defend all
methodological choices, as they may ultimately send
different signals to the industry and policy makers
working with sustainability issues.
A more standardized methodology within the sec-
tor would certainly facilitate the advancement of the
field. This could be achieved by better communications
between major practitioners or by the development of
a manual to guide the community toward one common
framework. Even still, there will always be deviations
from common practice as each study serves a unique
purpose, stressing the need for more transparency.
Even though ongoing initiatives for developing the
LCA framework exist, it is important to acknowledge
that the present framework has limited ability to
accommodate the other two pillars of sustainability,
namely, social and economic impacts [28]. Thus, even
if LCA provides a tool with great potential to guide the
aquaculture industry toward more sustainable produc-
tion, its framework needs to be reinforced by other
analytical tools to capture a wider range of sustainabil-
ity concerns. The need to include other tools alongside
LCA has already been recognized and recent projects,
such as the SEAT project (www.seatglobal.eu), include
complementary tools to give a more holistic measure-
ment of sustainability. Limitations aside, however, the
rapid development of this sector, coupled with the
diverse range of possible culture species and technolo-
gies, demands careful attention to environmental
effects at all relevant scales. LCA can and should play
an important role in guiding decisions oriented toward
sustainability in aquaculture.
Bibliography
Primary Literature
1. FAO (2009) State of world fisheries and aquaculture 2008. FAO,
Rome
2. Tyedmers P, Pelletier N, Ayer N (2007) Biophysical sustainabil-
ity and approaches to marine aquaculture development pol-
icy in the United States, A Report to the Marine Aquaculture
Task Force
3. Troell M, Tyedmers P, Kautsky N, Ro¨nnba¨ck P (2004)
Aquaculture and energy use. Encyclopedia Energy 1:97–108
4. Troell M, Joyce A, Chopin T, Neori A, Buschmann A, Fang J-G
(2009) Ecological engineering in aquaculture – potential for
integrated multi-trophic aquaculture (IMTA) in marine off-
shore systems. Aquaculture 297:1–9
5. Troell M (2009b) Integrated marine and brackish water
aquaculture in tropical regions: research, implementation
and prospects. In: Soto D (ed) Integrated mariculture:
a global review. FAO Fisheries and Aquaculture Technical
Paper, 529. FAO, Rome, pp 47–131

6. Naylor R, Goldburg R, Primavera J, Kautsky N, Beveridge M,
Clay J, Folke C, Lubchenco J, Mooney H, Troell M (2000) Effect
of aquaculture on world fish supplies. Nature 405:1017–1024
7. Naylor R, Williams S, Strong D (2001) Aquaculture – a gateway
for exotic species. Science 294:1655–1656
8. Naylor R (2005) Aquaculture and ocean resources: raising
tigers of the seat. Annu Rev Environ Resources 30:185–218
9. Pelletier N, Tyedmers P (2008) Life cycle considerations for
improving sustainability assessments in seafood awareness
campaigns. Environ Manage 42:918–941
10. Bartley D, Bruge´re C, Soto D, Gerber P, Harvey B (2007)
Comparative assessment of the environmental costs of
aquaculture and other food production sectors, Methods for
meaningful comparisons, FAO/WFT Expert Workshop, 24–28
April 2006, Vancouver, Canada. FAO Fisheries Proceedings,
no. 10, Rome
11. Finnveden G, Moberg A˚ (2005) Environmental systems analy-
sis tools – an overview. J Cleaner Prod 13:1165–1173
12. Folke C, Kautsky N, Berg H, Jansson A˚, Troell M (1998) The
ecological footprint concept for sustainable seafood
production: a review. Ecol Soc Am 8(1):S63–S71
13. Volpe JP, Beck M, Ethier V, Gee J, Wilson A (2010) Global
aquaculture performance index. University of Victoria, Victo-
ria, British Columbia, Canada
14. International Organization for Standardization (2006) Environ-
mental management – life cycle assessment – principles and
framework (ISO 14040:2006). International Organization for
Standardization, Geneva, Switzerland
15. International Organization for Standardization (2006) Environ-
mental management – life cycle assessment – requirements
and guidelines (ISO 14044:2006), Geneva, Switzerland
16. Cordella M, Tugnoli A, Spandoni G, Santarelli F, Zangrando T
(2008) LCA of an Italian Beer. Int J LCA 13(2):133–139
17. Baumann H, Tillman A-M (2004) The Hitch Hicker’s Guide to
LCA. Studentlitteratur, Lund
18. Wright DA, Hetzel EW (1985) Use of RNA: DNA ratios as an
indicator of nutritional stress in the American oyster
Crassostrea virginica. Mar Ecol Prog Ser 25:199–206
19. Thrane M, Nielsen E, Christensen P (2009) Cleaner production
in Danish fish processing – experiences status and possible
future strategies. J Cleaner Prod 17:380–390
20. Henriksson P (2009) Energy Intensity in Tropical Aquaculture,
MSc thesis, Stockholm University
21. Aubin J, Papatryphon E, van der Werf H, Chatzifotis S (2009)
Assessment of the environmental impact of carnivorous
finfish production systems using life cycle assessment.
J Cleaner Prod 17(3):354–361
22. Pelletier N, Tyedmers P, Sonesson U, Scholz A, Zeigler F,
Flysjo A, Kruse S, Cancino B, Silverman H (2009) Not all
salmon are create equal: life cycle assessment (LCA) of
global salmon farming systems. Environ Sci Technol
43(23):8730–8736
23. Iribarren D, Moreira M, Gumersindo F (2010) Revisiting the life
cycle assessment of mussels from a sectorial perspective.
J Cleaner Prod 18(2):101–111
24. Thrane M (2004) Environmental impacts from Danish fish
products – hot spots and environmental policies, PhD thesis,
A˚lborg University, A˚lborg, Denmark
25. Reap J, Roman F, Duncan S, Bras B (2008) A survey of
unresolved problems in life cycle assessment. Int J Life Cycle
Assess 13:290–300
26. Thrane M (2006) LCA of Danish fish products. Int J Life Cycle
Assess 11(1):66–74
27. Pelletier N, Ayer N, Tyedmers P, Kruse S, Flysjo A, Robillard G,
Ziegler F, Scholz A, Sonesson U (2007) Impact categories for
life cycle assessment research of seafood production: Review
and prospectus. Int J Life Cycle Assess 12(6):414–421
28. Kruse S, Flygsjo¨ A, Kasperczyk N, Scholz A (2009) Socioeco-
nomic indicators as a complement to life cycle assessment –
an application to salmon production systems. Int J Life Cycle
Assess 14:8–18
29. Zhang T, Dornfeld D (2007) Energy use per worker-hour, eval-
uating the contribution of labor to manufacturing energy use,
part 3:B1, pp 189–193. In: Takata S, Umeda Y (eds) Advances in
life cycle engineering for sustainable manufacturing busi-
nesses, Proceedings of the 14th CIRP conference on life cycle
engineering, Waseda University, Tokyo
30. Tyedmers P, Watson R, Pauly D (2005) Fueling global fishing
fleets. Ambio 34(8):635–638
31. Ruttan L, Tyedmers P (2007) Skippers, spotters and seiners:
analysis of the “skipper effect” in US menhaden (Brevoortia
spp.) purse-seine fisheries. Fish Res 83(1):73–80
32. Sandweiss D, Maasch K, Chai F, Andrus C, Reitz E (2004)
Geoarchaeological evidence for multidecadal natural climatic
variability and ancient Peruvian fisheries. Quatern Res 61:
330–334
33. Tacon A, Metian M (2009) Fishing for aquaculture: non-food
use of small pelagic forage fish – a global perspective. Rev Fish
Sci 17(3):305–317
34. Naylor R, Falcon W, Zavaleta E (1997) Variability and growth in
grain yields, 1950-94: does the record point to greater insta-
bility? Populat Dev Rev 23(1):41–58
35. Tacon A, Silva S (1997) Feed preparation and feed manage-
ment strategies within semi-intensive fish farming systems in
the tropics. Aquaculture 151:379–404
36. Kautsky N, Ro¨nnba¨ck P, Tedengren M, Troell M (2000) Ecosys-
tem perspectives on management of disease in shrimp pond
farming. Aquaculture 191:145–161
37. Papatryphon E, Petit J, Kaushik S, van der Werf H, Kaushik S
(6–8 October 2003) Life cycle assessment of trout farming in
France: a farm level approach. In: Halberg N (ed) Life Cycle
Assessment in the Agri-food sector, Proceedings from the 4th
International Conference. Bygholm, Denmark
38. Aubin J, Papatryphon E, van der Werf H, Petit J, Morvan Y (2006)
Characterisation of the environmental impact of a turbot
(Scophtalmus maximus) re-circulating production systems
using Life Cycle Assessment. Aquaculture 261:1259–1268
39. Ellingsen H, Aanondsen S (2006) Environmental impacts of
wild caught cod and farmed salmon – a comparison with
chicken. Int J Life Cycle Assess 1:60–65

40. Ayer N, Tyedmers P (2009) Assessing alternative aquaculture
technologies: life cycle assessment of salmonid culture sys-
tems in Canada. J Cleaner Prod 17:362–373
41. d’Orbcaster RE, Blancheton J-E, Belaud A (2009) Water quality
and rainbow trout performance in a Danish Model Farm
reciculating system: comparison with a flow through system.
Aquacult Eng 40:135–143
42. d’Orbcaster RE, Blancheton J-E, Aubin J (2009) Towards envi-
ronmentally sustainable aquaculture: comparison between
two trout farming systems using Life Cycle Assessment.
Aquacult Eng 40:113–119
43. Pelletier N, Tyedmers P (2007) Feeding farmed salmon: Is
organic better? Aquaculture 272:399–416
44. Pelletier N, Tyedmers P (2010) A life cycle assessment of frozen
Indonesian tilapia fillets from lake and pond-based production
systems. J Ind Ecol (in press)
45. Alder J, Campbell B, Karpouzi V, Kaschner K, Pauly D (2008)
Forage fish: from ecosystems to markets. Annu Rev Environ
Res 33:7.1–7.14
46. Naylor R, Hardy R, Bureau D, Chiu A, Elliott M, Farrell A,
Forster I, Gatlin D, Goldburg R, Hua K, Nichols P (2009) Feeding
aquaculture in an era of finite resources. Proc Natl Acad Sci
106(36):15103–15110
47. de Francescoa M, Parisia G, Me´daleb F, Lupia P, Kaushikb S,
Poli B (2004) Effect of long-term feeding with a plant protein
mixture based diet on growth and body/fillet quality traits of
large rainbow trout (Oncorhynchus mykiss). Aquaculture
236(1–4):413–429
48. Edwards P, Anh Tuan L, Allan G (2004) A survey of marine trash
fish and fishmeal as aquaculture feed ingredients in Vietnam.
ACIAR Working Paper No. 57
49. Zimmo O, van der Steer N, Gijzen J (2003) Comparison of
ammonia volatilization rates in algae and duckweed-based
waste stabilization ponds treating domestic wastewater.
Water Res 37:4587–4594
50. Driscoll J, Tyedmers P (2010) Fuel use and greenhouse gas
emission implications of fisheries management: the case of
the New England Atlantic herring fishery. Marine Policy
34(3):353–359
51. Tlusty M, Lagueux K (2009) Isolines as a new tool to assess the
energy costs of the production and distribution of multiple
sources of seafood. J Cleaner Prod 17:408–415
52. Pauly D, Christensen V, Gueńette S, Pitcher T, Sumaila U,
Walters C, Watson R, Zeller D (2002) Towards sustainability in
world fisheries. Nature 418:689–695
53. Tacon A, Metian M (2008) Global overview on the use of
fishmeal and fish oil in industrially compounded aquafeeds:
trends and future prospects. Aquaculture 285:146–158
54. Pauly D, Christensen V (1995) Primary production required to
sustain global fisheries. Nature 374:255–257
55. Tacon A, Hasan M, Subasinghe R (2006) Use of fishery
resources as feed inputs for aquaculture development: trends
and policy, implications. FAO Fisheries Circular. No. 1018.
Rome, FAO, 99 p
56. Rafiqul I, Weber C, Lehmann B, Voss A (2005) Energy efficiency
improvements in ammonia production-perspectives and
uncertainties. Energy 30:2487–2504
57. Pelletier N, Arsenault N, Tyedmers P (2008) Scenario-
modeling potential eco-efficiency gains from a transition to
organic agriculture: life cycle perspectives on Canadian
canola, corn, soy and wheat production. Environ Manage
42:989–1001
58. Cho C, Bureau D (2001) A review of diet formulation strategies
and feeding systems to reduce excretory and feed wastes in
aquaculture. Aquac Res 32:349–360
59. Boyd C, Tucker C, McNevin A, Bostick K, Clay J (2007) Indica-
tors of resource use efficiency and environmental perfor-
mance in fish and Crustacean aquaculture. Rev Fish Sci
15:327–360
60. Potting J, Hauschild M (1997) Spatial differentiation in life-
cycle assessment via the site-dependent characterisation of
environmental impact from emissions. Int J Life Cycle Assess
2(4):209–216
61. ILCD handbook (International Reference Life Cycle Data
System) (2010) Analysis of existing environmental
impact assessment methodologies for use in life cycle
assessment, JRC background document, 1st edn, European
Union
62. Gallego A, Rodrı´guez L, Hospido A, Moreira1 M, Feijoo G (2010)
Development of regional characterization factors for aquatic
eutrophication. J Life Cycle Assess 15(1):32–43
63. Occhipinti-Ambrogi A (2007) Global change and marine com-
munities: alien species and climate change. Mar Pollut Bull
55:342–352
64. Costello M (2009) How sea lice from salmon farms may
cause wild salmonid declines in Europe and North America
and be a threat to fishes elsewhere. Proc R Soc 276:
3385–3394
65. Cato J, Lima dos Santos C (1998) European Union 1997
seafood-safety ban: the economic impact on Bangladesh
shrimp processing. Mar Resource Econ 13(3):215–227
66. Reijnders L, Soret S (2003) Quantifications of the environmen-
tal impacts of different dietary protein choices. Am J Clin Nutr
78:664S–668S
67. Wiggers V, Wisniewski A, Madureira L, Chivanga Barros A,
Meier H (2009) Biofuels from waste fish oil pyrolysis: continu-
ous production in a pilot plant. Fuel 88(11):2135–2141
68. Bunting S, Pretty J (2007) Global carbon budgets and aqua-
culture – emissions, sequestration and management options.
Centre for Environment and Society Occasional Paper 2007-1.
University of Essex, Essex
69. Hossain S, Salleh A, Boyce A, Chowdhury P, Naqiuddin
M (2008) Biodiesel fuel production from algae as renewable
energy. Am J Biochem biotechnol 4(3):250–254
70. Mungkung R (2005) Shrimp aquaculture in Thailand: applica-
tion of life cycle assessment to support sustainable develop-
ment, PhD thesis, Center for Environmental Strategy, School of
Engineering, University of Surrey, Surrey, UK

Books and Reviews
Mungkung R, Gheewala SH (2006) Use of life cycle assessment
(LCA) to compare the environmental impacts of aquaculture
and agri-food products. In: Barley DM, Bruge´re C, Soto D,
Gerber P, Harvey B (eds) Comparative assessment of the envi-
ronmental costs of aquaculture and other food production
sectors: methods for meaningful comparisons, FAO/WFT
Expert Workshop, 24–28 April 2006, Vancouver, Canada. FAO
Fisheries Proceedings, no. 10, Rome, FAO, pp 87–96, 207
Ayer N, Tyedmers P, Pelletier N, Sonesson U, Scholz A (2007)
Co-product allocation in life cycle assessments of seafood
production systems: review of problems and strategies. Int
J Life Cycle Assess 12(7):480–487
Baumann H, Tillman A-M (2004) The hitchhiker’s guide to LCA: an
orientation in life cycle assessment methodology and applica-
tion. ISBN 91-44-02364-2
Guineé J (ed), Gorreé M, Heijungs R, Huppes G, Kleijn R,
de Koning A, van Oers L, Wegener Sleeswijk A, Suh S, Udo de
Haes H, de Bruijn J, van Duin R, Huijbregts M (2002) Handbook
on life cycle assessment: operational guide to the ISO
Standards. Series: Eco-efficiency in industry and science.
Springer, Dordrecht

Livestock Somatic Cell Nuclear
Transfer
SERGIO D. GERMAN, KEITH H. S. CAMPBELL
School of Biosciences, University of Nottingham,
Sutton Bonington, Loughborough, Leicestershire, UK
Article Outline
Glossary
Definition of Cloning and SCNT
Introduction: Embryo Development and Highlights
of NT
Nuclear Reprogramming
Factors Affecting Development of Embryos Produced
by SCNT
Improving Development
Summary Points
Future Directions
Bibliography
Glossary
Aneuploid An unbalanced number of chromosomes.
Blastocyst A stage of early development where the
embryo contains a fluid-filled cavity and two cell
populations: an inner cell mass and an outer layer of
trophectoderm cells.
Cell cycle The period between the birth of a cell and its
division. During a single cell cycle, the cell must
duplicate all of its components, including DNA, to
form two equal daughter cells.
Chromatin The combination of DNA and proteins,
mostly histones.
Cytoplast An enucleated cell used as a recipient for
a donor nucleus. Generally in SCNT, the recipient
cytoplast is an enucleated oocyte.
Diploid The cell or nucleus contains two complete
copies of the genome or a complete complement
of chromosomes (2n), in general, a single maternal
and a single paternal.
Donor cell The cell that provides the genetic material
(nucleus) for SCNT. The resulting animal will be a
genomic copy of the animal from which this cell was
collected.
Haploid The cell or nucleus contains only a single
copy of the genome or half of the chromosome
complement (n). Pronuclei are, in general, haploid,
containing either a single maternal genome or
a single paternal genome.
Karyoplast A cell or a membrane-bound portion of a
cell containing the donor nucleus enclosed. In live-
stock SCNT, the karyoplast is generally an intact cell.
Meiosis The process of reduction in the number
of chromosomes that occurs during germ cell for-
mation. Following a round of DNA synthesis,
a single cell undergoes two rounds of division
resulting in four cells, each containing a haploid
genome. During division, independent assortment
of parental chromosomes and homologous recom-
bination generate a unique haploid genotype in
each of the germ cells.
Metaphase II (MII) Stage during the second meiotic
division where the chromosomes are aligned at the
metaphase plate prior to segregation of sister chro-
matids to opposite poles. In most mammalian spe-
cies, mature oocytes arrest at MII and meiosis is
reinitiated and completed upon fertilization.
Parthenote An unfertilized zygote produced by acti-
vation of an oocyte. A parthenote may be haploid
or diploid for maternal DNA, a gynogenote, or
following enucleation and replacement with pater-
nal DNA, an androgenote.
Tetraploid The cell or nucleus contains four haploid
copies of the genome (4n).
Zygote The 1-cell stage of development of a fertilized
embryo. During most of the first cell cycle, the
zygote will contain two pronuclei containing
the maternal and paternal DNA.
Definition of Cloning and SCNT
Cloning is the production of genetically identical indi-
viduals by the process of asexual reproduction. In ani-
mals, the term has been applied to offspring that are
produced by the technique of nuclear transfer (NT).
The process of nuclear transfer involves the production
of an embryo by transferring nuclear genetic material
from a donor cell (karyoplast) into a recipient cell from
which the genetic material has been removed (cytoplast).
Two factors determine the clonality of the resultant
offspring. One is the recipient cell, generally an oocyte
or unfertilized egg obtained from an unrelated animal.
1067Livestock Somatic Cell Nuclear Transfer
P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,
# Springer Science+Business Media New York 2013
Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology, # 2012, DOI 10.1007/978-1-4419-0851-3

The second is the donor cell, which can be obtained from
a variety of sources including embryos, germ line, and
somatic tissues of fetuses and adult organisms. When
cells from somatic tissues are used, the procedure is
termed somatic cell nuclear transfer (SCNT). In most
species, transfer of the donor nucleus is primarily carried
out by cell fusion with fewer reports of direct injection.
In the case of fusion, the complete contents of the donor
cell are introduced into the recipient cell, while in the
case of injection, the donor nucleus might be accompa-
nied with a proportion of the donor cytoplasm.
While SCNT became publicly known with the cre-
ation of Dolly the sheep in 1997, NTwas already in the
realm of science since 1928, with the first successful
SCNT experiments carried out in frogs in the late
1950s. By creating a genetic copy of a known animal,
SCNT opens many opportunities in several fields
including biomedical research and agriculture.
Introduction: Embryo Development and
Highlights of NT
In animals, reproduction occurs primarily by sexual
means, resulting in the creation of a new individual
that contains genetic material (DNA) from two con-
tributing parents of different sexes, each parent con-
tributing half of the genetic material. Conception of
a new individual occurs by the fusion of specialized
cells or gametes, sperm in males and ova in females.
Sperm are haploid (n) and contain a single copy of the
genome, meanwhile ova, in most species, at the time of
fertilization are arrested in metaphase of the second
meiotic division and are diploid (2n). After fusion,
however, the ovum completes meiosis to become
haploid, and the resultant zygote is diploid (2n),
containing a full set of chromosomes derived from
both parents. The ovum is a large cell that contains
sufficient maternally derived proteins and mRNAs to
control early development. Following fertilization, dur-
ing the early stages of development, no net growth
occurs (Fig. 1); transcription from the embryonic
genome is low but subsequently increases rapidly at
a particular stage termed maternal to zygotic transition
(MZT) [1]. The timing of the MZT is species depen-
dent, for example, in mice, it occurs at the 1–2-cell
stage; in pigs and humans at the 4–8-cell stage; in cattle
and sheep at the 8–16-cell stage; while in the amphibian
Xenopus laevis, it occurs approximately at the 4,000-cell
stage [2]. After this transition, the embryonic genome
orchestrates its own development. The embryo con-
tinues to increase in cell number within the confines
of the zona pellucida (ZP), which acts as a protective
shell. By the blastocyst stage, a fluid-filled cavity,
called blastocoel, has formed and two distinct cell
populations have emerged: the inner cell mass
(ICM) and the trophectoderm (TE). As the blastocyst
expands, build-up pressure together with the secretion
of enzymes causes the ZP to break, enabling the blas-
tocyst to hatch. Now the blastocyst is ready to interact
with the endometrium and eventually to implant. By
cell proliferation and differentiation, the ICM will form
the fetus and some extraembryonic tissues while the TE
will give rise to most of the placenta (Fig. 1).
The zygote is a totipotent cell, having the capacity
to produce the fetus and the placenta. In contrast,
sperm and ova are specialized cells. How do they
acquire totipotency? Following fertilization, unknown
factors present in the ovum’s cytoplasm reprogram the
paternal and maternal genomes so that their specialized
gamete functions are erased to give way to an embry-
onic totipotent genome. This capacity to reset
a specialized genome into a less specialized or totipo-
tent genome is termed nuclear reprogramming. It is
this nuclear reprogramming ability of the ovum that is
harnessed in NT technology.
The first rudimentary NT experiments were
performed by Hans Spemann in 1928. He used a
donor nucleus from a 16-cell embryo and a presump-
tive zygotic cytoplast, resulting in a normal salamander
[3]. Interestingly, Spemann had already predicted the
possibility of cloning an adult animal by doing NTwith
an adult donor nucleus in what he termed the “fantas-
tical experiment” [3].
In 1952, Briggs and King used NT to address the
question of nuclear equivalence, that is, whether or not
the different cells of a multicellular organism have the
same genome. They developed the NT procedure by
transplanting nuclei from frog blastula cells into enu-
cleated oocytes [4]. Later on, Briggs and King tested the
nuclear equivalence of more developmentally advanced
frog cells and found that the developmental potential
of reconstructed embryos gradually declined with
increased donor cell differentiation [5]. However,
John Gurdon pushed the field further by generating
1068 Livestock Somatic Cell Nuclear Transfer

cloned adult frogs from differentiated epithelial
somatic cells at the tadpole stage [6, 7], providing
strong evidence for nuclear equivalence. Thus, the
first successful SCNT had been achieved, but attempts
to clone adult frogs failed [8].
Nuclear transfer in mammals lagged behind, with
the first attempt in 1975 using morula-stage blastomere
donors and unfertilized rabbit eggs. Even though the
extent of reprogramming required for a blastomere,
compared with a fully differentiated cell, is quite
reduced, the reconstructed cloned embryos did not
develop beyond the early cleavage stages [9]. In 1983,
McGrath and Solter transplanted zygotic nuclei into
enucleated zygotes resulting in mice developing to
term, showing that the technique of nuclear transfer
in mammals was feasible [10]. However, such a zygotic
nuclei-replacement experiment did not involve any
nuclear reprogramming, and when the same approach
used more developmentally advanced blastomere
nuclei as donors, it failed [11]. This failure in mice
NT was followed by several breakthroughs in which
enucleated oocytes were used as recipients of blasto-
mere nuclei, leading to the production of cloned ani-
mals in sheep [12], cattle [13], and pigs [14]. In
contrast to McGrath and Solter’s failure in cloning
mice, these results suggested that the enucleated oocyte
is a better recipient than the enucleated zygote for
reprogramming the donor blastomere nucleus.
Ten years passed until another breakthrough
advanced the field of mammalian nuclear transfer:
Keith Campbell and colleagues at the Roslin Institute
succeeded in cloning a lamb by NT using donor nuclei
from an established cell line derived from embryos
[15]. In this study, cell cycle coordination between
the donor cell and recipient oocyte was carried out
Livestock Somatic Cell Nuclear Transfer. Figure 1
Flow diagram illustrating mammalian development.
Following fertilization of the MII oocyte by sperm, a zygote
with the two parental pronuclei is formed. By cleavage, the
embryo develops sequentially through the 2-cell, 4-cell,
8-cell, 16-cell, morula, and blastocyst stages. By the
blastocyst stage, the first differentiation event has formed
the inner cell mass (ICM) and the trophectoderm (TE).
These two cell populations will form the foetus and
placenta, respectively
Cumulus-oocyte-complex
Zygote
2-cell embryo
Morula
Blastocoel
Cell segregation
Further
Cleavage
Cleavage
ZP
Fertilization
Sperm cell
Early blastocyst
Expanding blastocyst
Hatched blastocyst
Placenta
TEICM
Foetus
(Continued)

prior to NT in order to improve the development of
reconstructed embryos as previously determined by
this group [16, 17]. One year later, this same group,
led by Ian Wilmut, used a similar approach and shook
the world’s news with the creation of “Dolly” the sheep,
the first mammal to be cloned from an adult somatic
cell [18]. This study showed that an adult somatic cell
retains all the information necessary to generate a new
organism. After the creation of Dolly, much research
has been done in SCNT and several species have been
cloned from adult cells. Despite advances in the field,
the frequency of development of embryos produced by
SCNT remains low, with only 1–10% reaching term,
depending on the species. Moreover, many cloned ani-
mals are born with abnormalities.
Applications of Livestock SCNT
Soon after the arrival of Dolly came the realization that
SCNT had several potential applications. For instance,
derivation of embryonic stem (ES) cells from human
blastocysts was eagerly sought for regenerative medi-
cine research. The rationale was that cells from a
patient could be used for SCNT to derive ES cells,
which in turn could be induced to differentiate into
a specific tissue. Since such a tissue would be genetically
identical to the patient, it could replace a damaged
tissue in the same patient without the risk of immune
rejection. Due to the ethical concerns of destroying
human embryos, SCNT for regenerative medicine has
been fiercely opposed. Fortunately, this problem seems
mostly circumvented with the revolutionary creation of
induced pluripotent stem (iPS) cells [19]. These cells
have similar properties to ES cells and can also be
derived from the somatic cells of a patient without
the use of human oocytes or embryos. Since then,
much of the field of regenerative medicine research
has shifted from SCNT to iPS cell technology. However,
recent studies have found genetic and epigenetic abnor-
malities in iPS cells [20–22], making them potentially
unsafe for medical uses. Whether or not iPS cells are
inferior to ES cells is currently the focus of heated
debate. Since some of the problems found with iPS
cells have not been reported with ES cells derived
from SCNT embryos [23, 24], the process of nuclear
reprogramming occurring in the oocyte appears to be
of better quality than the one taking place during
the derivation of iPS cells. Very little is known about
the mechanism of nuclear reprogramming and the
factors present in the oocyte responsible for triggering
or carrying out such process. If these factors are found,
they could potentially contribute to generate safer iPS
cells for regenerative medicine. Thus, further research
in the mechanism of nuclear reprogramming following
SCNT is needed.
Apart from being a useful tool for basic research
with potential indirect applications in regenerative
medicine, SCNT has wide applications in agriculture.
For instance, an animal of exceptional productivity can
be multiplied by SCNT to increase commercial output
or to reduce environmental impact. However, caution
must be taken with such approach. First, consumption
of products derived from cloned animals is not yet
widely accepted by the public, and in some countries,
there are regulations forbidding their entry into the
human food chain. Second, the SCNT technology is
so inefficient at present that its application for livestock
multiplication might not be economically justified.
Finally, it is important to keep in mind that clones are
not necessarily true copies of their donor because of
epigenetic differences, which result from faulty nuclear
reprogramming following SCNT (discussed later).
Slight epigenetic differences can affect the physiology
of the cloned animal and thus its productivity. Such
consideration should especially warn breeders of race-
winner horses, as a cloned horse is unlikely to be a
winner again.
A more realistic agricultural application of SCNT is
to reproduce animals of high genetic value for breeding
purposes. For instance, a prizewinning bull could be
cloned and subsequently produce thousands of off-
spring by artificial insemination. To a lower extent,
the genetic value of a cow could also be disseminated
by SCNT followed by artificial reproductive technolo-
gies. While there might be risks associated with con-
sumption of products from cloned animals due to
potential epigenetic errors originated during nuclear
reprogramming, their offspring should be safe since
epigenetic and imprinting errors are normally erased
during gametogenesis.
More importantly, SCNT has represented
a great technical advance in the production of trans-
genic animals (this is discussed in detail in the entry
▶ Nuclear Transfer to Produce Transgenic Mammals,

this volume). In turn, transgenic animals have a myriad
of other applications, which are discussed throughout
this volume. Furthermore, transgenic animals produc-
ing pharmaceutical or novel proteins are good candi-
dates for multiplication by SCNTsince the cost of using
this technology would be relatively small compared to
the product harvested from the cloned animals.
SCNT can also be used for cloning individuals from
an endangered species population. Examples of this
application include the cloning of wild cattle (Gaur and
Banteng) and wild sheep (European Mouflon) [25].
These examples resorted to interspecies SCNT in
which a close-related species is used as cytoplasmic
recipients and surrogates for the donor. Therefore,
the resultant offspring will be a hybrid containing
a genomic copy of the endangered donor and most
mitochondrial DNA (mtDNA) from the close-related
recipient. While the physiological consequences for
this are unclear, it certainly raises ethical issues.
Besides, other efforts such as restoration of the
natural habitat and assisted breeding programs are
likely to be more effective at helping an endangered
population.
Taken together, SCNT has some interesting appli-
cations, but the ethical implications surrounding this
technology must be fully considered before rushing
into implementing SCNT in the field. Rather than
an end, SCNT is especially useful when used as a tool
in research, production of transgenic animals, and in
cloning of prizewinning animals for breeding purposes
in agriculture.
NT Techniques
NT is a complex multistep procedure, with several alter-
natives at some steps, which include oocyte maturation,
cumulus cell removal, enucleation, nuclear transfer, acti-
vation, and embryo culture. Culture of the donor cells is
important and usually accompanied with cell-cycle syn-
chronization prior to NT. Enucleation and nuclear
transfer are the most important and difficult steps. To
carry out these steps, most laboratories use microma-
nipulators. A micromanipulator is a relatively complex
instrument composed of a microscope, micromanipu-
lators at both sides to hold the holding and injection
micropipettes, micromanipulator joysticks to precisely
maneuver the micropipettes in three dimensions, and
microinjectors to precisely aspirate and expel fluid and
cellular material from the micropipettes. For mice
cloning, microinjectors are usually equipped with pie-
zoelectric devices to prevent lysis of the fragile mouse
oocyte during enucleation and nuclear transfer. An
alternative to micromanipulators is a technique called
handmade cloning (HMC) and seems equally efficient
as micromanipulator-based NT (for a review of HMC,
see [26]). Following is a brief discussion of the different
steps involved in the entire NT procedure with focus on
livestock species unless stated otherwise.
Oocyte maturation is usually required in livestock
cloning as immature oocytes are commonly obtained
from ovaries collected from slaughterhouses. Alterna-
tively, in vivo–matured oocytes can be obtained by
stimulating superovulation, which is followed by ovi-
duct flushing after slaughtering the animal. In vitro
maturation (IVM) entails mimicking to some extent
the in vivo environmental conditions found in the
ovary at the time of the LH (luteinizing hormone)
surge. Thus, three key hormones, LH, FSH (follicular
stimulating hormone), and estradiol, are added to the
IVM medium to stimulate maturation. Oocytes are
surrounded by cumulus cells, forming a cumulus-
oocyte complex (COC); cumulus cells aid oocyte mat-
uration. COCs are incubated at about 39
C in livestock
species and for 24 h (e.g., cow and sheep). Following
the required maturation period, oocytes are denuded
from the cumulus cells using hyaluronidase to digest
the extracellular matrix that holds cumulus cells
together. Removal of cumulus cells is necessary to
allow visualization of the oocyte’s chromatin during
enucleation and to prevent inserting a cumulus cell
accidentally inside the oocyte during enucleation and
cell/nuclear transfer procedures. Now the denuded
oocytes are ready for enucleation.
A few enucleation methods exist. The most com-
monly employed method uses micromanipulators to
aspirate the chromatin. In this method, the oocyte is
held steady at one side with the holding pipette while the
enucleation pipette is inserted from the opposite side to
remove, depending on the maturation state, the extrud-
ing spindle or first polar body together with the meta-
phase plate (Fig. 2). In order to confirm enucleation, it
is desirable to visualize the chromosomal material. In
mice and human oocytes, which have a clear cyto-
plasm, chromatin can be readily observed under bright

field illumination. However, the oocytes of livestock
species have high lipid contents that hamper chromatin
visualization, and therefore, enucleations must be
made “blind” (Fig. 2a). Generally, to aid enucleation
of livestock oocytes, they are briefly preincubated with
the vital DNA stain Hoechst. The stained karyoplast is
confirmed in the enucleation pipette by UV light fol-
lowing enucleation. Although this procedure does not
expose the oocyte to direct UV irradiation, conflicting
evidence exist whether Hoechst diminishes the devel-
opmental competence of the oocyte.
Alternatively, a blind-enucleation method without
DNA staining uses the first polar body as a point of
reference to subsequently remove the underlying meta-
phase II (MII) plate. Unfortunately, the MII plate can
shift away from the polar body, rendering its removal
inaccurate. However, when blind enucleation is
performed earlier at anaphase I/telophase I (AI/TI)
stages of the cell cycle, removal of the complete spindle
is close to 100% accurate (Fig. 2b) [27]. A newer
microscopy technology based on birefringence, the
PolScope, allows noninvasive visualization of spindles
not only in human oocytes but in other species as well.
Probably due to the high cost of the PolScope, this
enucleation method has not gained much popularity.
Direct enucleation by aspiration can be chemically
assisted with cytoskeleton-relaxing agents, resulting in
a protrusion cone containing the oocyte’s chromosomes.
Drugs used for this purpose include demecolcine and
nocodazole, both interfering with microtubule polymer-
ization. Other enucleation methods that have been
attempted are centrifugation and laser ablation. In
HMC, the zona pellucida is digested, and one third
of the oocyte containing the extruding polar body and
metaphase plate is manually bisected with a microblade
under a stereomicroscope.
Donor cells are usually cultured in vitro and syn-
chronized in a specific phase of the cell cycle prior to
NT in order to ensure a known ploidy content. In
livestock species, donor cells are preferentially used in
G0 and, to a lower extent, in G1. Quiescent G0 cells are
arrested in the G1 phase of cell cycle by either serum
starvation or cell-contact inhibition. The advantage of
these two methods is that they are drug independent,
while methods to arrest cells at G1 usually require the
use of pharmacological agents. Alternatively, actively
dividing presumptive G1 cells can be selected by
PB
Holding
pipette
a
b
AI/TI
MII
UV exposure
UV check
Enucleation
pipette
Diagram illustrating enucleation of livestock oocytes.
(a) When enucleation is carried out at metaphase II (MII),
the polar body (PB) and a portion of cytoplasm is blindly
aspirated to remove the underlying MII plate. Chromatin
cannot be observed under bright field illumination, thus
UV light exposure (dark field) is essential to confirm
enucleation of the pre-stained chromosomes. (b) When
enucleation is carried out earlier at anaphase I/telophase I
(AI/TI), the extruding spindle is aspirated. In contrast to MII
enucleation, the AI/TI spindle can often be observed
directly inside the enucleation pipette. However,
enucleation of pre-stained DNA can be checked with UV
light. In both enucleation methods, the oocyte is removed
from the field of view to avoid damage by direct UV
irradiation when exposing the enucleation pipette

collecting small-sized cells. A more accurate drug-free
method to select for G1 cells from actively dividing
cultures involves using the eukaryotic “baby machine”,
which briefly consists of an effluent that collects newly
divided and floating daughter G1 cells from the culture
medium (this methodology is described in [28]). Such
a methodology would be especially useful when it is
desirable to clone ES cells in G1 (discussed further in
the section Coordination of Donor and Recipient Cell
Cycles). In mice cloning, ES cells are often arrested in
metaphase by nocodazole or colcemid treatments.
A few alternatives exist for inserting the donor
nuclear material into the enucleated oocyte (Fig. 3).
In livestock species, the most common method is to
insert the whole donor cell (cell transfer) into the
perivitelline space to achieve contact between the
membranes of donor and recipient cells. The donor
DNA is then incorporated into the enucleated oocyte
by cell fusion (Fig. 3a) (discussed below). Another
method is to directly insert the nucleus (nuclear micro-
injection) into the enucleated oocyte, a common prac-
tice in mice cloning (Fig. 3b). The advantage of this
method is that it bypasses the need for fusion. Nuclei
are isolated using a micropipette with an inner diame-
ter smaller than the donor cell. Pipetting the cell in and
out breaks the membrane and releases the nucleus.
Interestingly, it is also possible to microinject the
whole cell into the enucleated oocyte cytoplasm, thus
bypassing the need for nuclear isolation or fusion
(Fig. 3c). Although it is unclear how the donor plasma
membrane is degraded, high blastocyst development
has been reported with this technique [29]. Another
alternative method is to directly microinject a broken
cell containing mitotic chromatin and cytoplasm,
known as chromosome transfer (Fig. 3d). In HMC,
phytohemagglutinin is used to glue the donor cell to
the enucleated oocyte, followed by fusion.
When cell transfer is employed, the recipient-donor
couplet must be fused to deliver the donor chromatin
into the recipient oocyte. This is commonly achieved by
electrofusion. In this method, the couplet is placed
between two electrodes and short electric pulses are
applied to fuse the recipient-donor membranes together.
A second method involves the use of inactivated
sendai virus (SV). Donor cells are briefly incubated in
a solution containing SV prior to cell transfer, achieving
oocyte-cell fusion by viral particles. SV-mediated fusion
is less laborious and might be less stressful to the
reconstructed embryo than electrofusion [30].
Once the donor chromatin has been incorpo-
rated into the enucleated oocyte, the reconstructed
embryo is activated to begin development. The
reconstructed embryo is then ready for embryo cul-
ture. Different methods of activation and embryo
culture are discussed later in this entry in relation
to how they affect NT outcomes. When cloned
embryos reach the blastocyst stage in vitro, they
are transferred to surrogate females to carry the
pregnancy.
Hormonal treatments must be administered to the
surrogate so that the “uterine cycle” is synchronized
with the developmental stage of the transferred
embryo. If this is not properly planned, the uterus
will be “out of phase” compared to the blastocyst and
implantation will likely fail. To achieve synchrony, in
farm animals, the surrogate must be induced to enter
estrus on the day of NT. Following transfer to surro-
gates, care of the embryos is shifted to the hands of the
veterinarian. Management of pregnancy includes ultra-
sonography, to check pregnancy status, as well as mea-
suring maternal levels of pregnancy proteins to assess
placental development.
Problems of Cloning
Despite advances in the field, the efficiency of SCNT
technology remains very low, with only 1–10% of
transferred embryos reaching term, depending on the
species. More than 50% of pregnancies are lost in the
first trimester, and in contrast to IVF pregnancies,
losses of clone pregnancies continue throughout gesta-
tion (e.g., [31]). Additionally, developmental abnor-
malities are commonly observed in cloned fetuses and
in their placentas.
During mammalian embryogenesis, the fetus is
derived exclusively from the inner cell mass (ICM)
while extraembryonic tissues, including the placenta,
are mostly derived from the trophectoderm (TE). It is
thought that the extraembryonic membranes are the
most negatively affected by SCNT, including the
placentas of full-term clones. Although cloned fetuses
and offspring can present abnormalities as well as
large offspring syndrome (LOS), these seem to be
secondary to placental dysfunction, which leads

to imbalances in placental ﬂuid and in kidney func-
tion [32]. In sheep, placental abnormalities include
a hypotrophic trophoblastic epithelium, reduced
vascularization, and thickening of the trophoblast
basement membrane [33]. Placentomegaly (enlarged
placenta) is a common occurrence in mice and cattle
cloning. Other placental abnormalities in bovine
cloning include edema and hydroallantois. Develop-
mental failure can account for fetal losses, stillbirth,
and even postnatal mortality of mice, cattle, and
sheep. Postnatal mortality is particularly pronounced
in cloned sheep [34].
A recent study showed that indeed defects in the
trophectoderm lineages rather than in the ICM are
the main cause of low cloning efﬁciencies, at least
in mice [35]. This experiment used chimeric aggre-
gates of diploid and tetraploid embryos (Fig. 4).
Tetraploid embryos are obtained by fusing the
two blastomeres of a two-cell embryo. NT-ICMs were
aggregated with tetraploid fertilization-derived (4nFD)
early-cleavage-stage embryos. In this diploid/tetraploid
“chimera,” the 2n NT-ICM cells can only develop
into the fetus while the 4n cells can only give rise to
the extraembryonic tissues (although minimal cross-
contribution was observed). By this approach, a sixfold
increase in development to term was obtained com-
pared with the nonaggregated NT control. Moreover,
when ICMs from normally fertilized embryos were
Holding
pipette
Injection
pipette
Cell transfer
Cell fusion
Nuclear
microinjection
a
b d
c
Chromosome
transfer
Whole-Cell
microinjection
Diagram illustrating different methods for donor genome transfer into recipient enucleated oocytes. (a) In cell transfer,
an intact donor cell is transferred into the perivitelline space and the genome is then incorporated into the recipient by
cell fusion. (b) In nuclear microinjection, nuclei are previously isolated and a single nucleus is then microinjected into the
recipient. (c) In whole-cell microinjection, the whole cell is injected into the recipient. Eventually, incorporation of the
genome into the recipient occurs by spontaneous degradation of the donor plasma membrane. (d) In chromosome
transfer, a broken mitotic cell is microinjected into the recipient to transfer the mitotic spindle

aggregated with tetraploid NT embryos, the birth rate
was similar to that of the NT control, indicating that
a normal ICM does not rescue the developmental
potential of the reconstructed embryo. However, FD-
ICM/4nFD aggregates showed twice the developmental
rate than NT-ICM/4nFD. Thus, while trophectoderm
defects are mainly responsible for low cloning effi-
ciencies in mice, the ICM has a minor contribution
as well.
It is generally thought that the low cloning efficien-
cies and developmental problems arise from an incom-
plete reprogramming of the donor nucleus.
Nuclear Reprogramming
Successful NTexperiments demonstrated that differen-
tiated cells (with a few exceptions) retain the same
intact genome (i.e., nuclear equivalence) within an
organism. In other words, these experiments supported
that cell differentiation is not achieved through genetic
changes or deletions but rather through epigenetic
changes. Epigenetics regulates cell identity through
chromatin modifications that are heritable through
cell divisions, without altering the DNA sequence.
Thus, chromatin modifications establish an epigenetic
“code” that results in different gene expression pro-
grams between cell types.
Chromatin is formed by DNA associated with
histone and nonhistone proteins. The chromatin
template is epigenetically modified at the DNA
and histone levels. DNA modification is limited to
methylation (or demethylation), while histone
modifications are numerous, including methylation,
acetylation, phosphorylation, ubiquitination, ADP-
ribosylation, biotinylation, and sumoylation. In turn,
such modifications affect gene expression and the
overall chromatin structure, which can be described
as open or compact. During normal development,
global changes in DNA methylation and histone
modifications take place; much effort is being dedi-
cated in understanding how these changes are altered
or preserved in cloned embryos.
For SCNT to be successful, it is essential that the
chromatin of the donor somatic nucleus is remodeled
so that it becomes compatible not only with the toti-
potent gene expression program of development but
SCNT Donor
SCNT Control (1-fold)
Developmental Outcome
of Aggregates (x-fold)
Fertilization-derived
(FD) Donor
2n blastocyst 2n blastocyst
4n embryos
4n embryos
12-fold
~1-fold
6-fold
1 ICM
x2
x2
x2
1 ICM
1
ICM
Experimental evidence shows that the trophectoderm lineage is severely affected in mice cloning. A diploid (2n) ICM
and two tetraploid (4n) 4-cell embryos were aggregated from cloned and fertilized embryos in different combinations.
The ICM forms the embryo while tetraploid blastomeres can only give rise to extraembryonic tissues. Development
to term demonstrated that replacing the trophectoderm lineage in cloned embryos dramatically improves development

Life Cycle Assessments Guide Aquaculture Sustainability

Life Cycle Assessments Guide Aquaculture Sustainability

Empfohlen

Empfohlen

Weitere ähnliche Inhalte

Was ist angesagt?

Was ist angesagt? (20)

Andere mochten auch

Andere mochten auch (16)

Ähnlich wie Life Cycle Assessments Guide Aquaculture Sustainability

Ähnlich wie Life Cycle Assessments Guide Aquaculture Sustainability (20)

Mehr von Springer

Mehr von Springer (20)

Kürzlich hochgeladen

Kürzlich hochgeladen (20)

Life Cycle Assessments Guide Aquaculture Sustainability