Life Cycle Assessments and Their
Applications to Aquaculture
Production Systems
have therefore been developed for the purpose of eval-
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processes. Fin...
externalities associated with aquaculture and other
food production systems bear careful scrutiny and
must be weighed agai...
they follow the same requirements. Individual studies
also often differ in functional unit (the unit of output
against whi...
stomachs; both of which are considered offal in the
western world but are prized in parts of Asia [19, 20].
Ideally, the f...
that the basis of allocation can influence the allocated
burden by as an order of magnitude [26].
Impact Categories and Imp...
research foci – for example, understanding the influ-
ence of variable field-level nitrous oxide emissions on
the overall gr...
certification initiatives [9, 46]. It has also been strongly
influenced by rising prices for these commodities, due
to incre...
utilization as it does not take factors such as species,
genetic stock, feed composition, water temperature, or
growth rat...
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systems, such as most intensive land-based systems, the
energy for far...
identification of improvements opportunities. There is,
however, always the danger of oversimplification where
results get m...
extreme weather events, which may cause the loss of an
entire crop. Areas ravaged by extreme storm events,
such as typhoon...
6. Naylor R, Goldburg R, Primavera J, Kautsky N, Beveridge M,
Clay J, Folke C, Lubchenco J, Mooney H, Troell M (2000) Effe...
40. Ayer N, Tyedmers P (2009) Assessing alternative aquaculture
technologies: life cycle assessment of salmonid culture sy...
Books and Reviews
Mungkung R, Gheewala SH (2006) Use of life cycle assessment
(LCA) to compare the environmental impacts o...
Livestock Somatic Cell Nuclear
School of Biosciences, University of Nottin...
The second is the donor cell, which can be obtained from
a variety of sources including embryos, germ line, and
somatic ti...
cloned adult frogs from differentiated epithelial
somatic cells at the tadpole stage [6, 7], providing
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prior to NT in order to improve the development of
reconstructed embryos as previously determined by
this group [16, 17]. ...
this volume). In turn, transgenic animals have a myriad
of other applications, which are discussed throughout
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field illumination. However, the oocytes of livestock
species have high lipid contents that hamper chromatin
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to imbalances in placental fluid and in kidney func-
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Sustainable food production
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Sustainable food production

  1. 1. 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
  2. 2. 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
  3. 3. 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 ( 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 1052 Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  4. 4. 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 1053Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  5. 5. 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 1054 Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  6. 6. 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 Nin˜o- 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 1055Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  7. 7. 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 1056 Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  8. 8. 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 ✖✖✖✖✖✖✖✖ 1057Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  9. 9. 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 ✖✖✖✖✖ 1058 Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  10. 10. 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] 1059Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  11. 11. 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 Canada Pelletier and Tyedmers [43] 26,900 1,810 Atlantic salmon, organic crop/conventional animal meals and oils Canada Pelletier and 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 1060 Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  12. 12. 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 1061Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  13. 13. 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 1062 Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  14. 14. 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 (, 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 1063Life Cycle Assessments and Their Applications to Aquaculture Production Systems
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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 1065Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  17. 17. 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 Guine´e J (ed), Gorre´e 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 1066 Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  18. 18. 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
  19. 19. 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
  20. 20. 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 Livestock Somatic Cell Nuclear Transfer. Figure 1 (Continued) 1069Livestock Somatic Cell Nuclear Transfer
  21. 21. 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, 1070 Livestock Somatic Cell Nuclear Transfer
  22. 22. 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 1071Livestock Somatic Cell Nuclear Transfer
  23. 23. 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 Livestock Somatic Cell Nuclear Transfer. Figure 2 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 1072 Livestock Somatic Cell Nuclear Transfer
  24. 24. 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 1073Livestock Somatic Cell Nuclear Transfer
  25. 25. 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 Livestock Somatic Cell Nuclear Transfer. Figure 3 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 1074 Livestock Somatic Cell Nuclear Transfer
  26. 26. 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 Livestock Somatic Cell Nuclear Transfer. Figure 4 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 1075Livestock Somatic Cell Nuclear Transfer