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Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
Sustainable food production
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Sustainable food production

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  • 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. 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. externalities associated with aquaculture and other food production systems bear careful scrutiny and must be weighed against anticipated benefits [10]. Such comparisons need to extend beyond short-term gains and localized impacts and incorporate a long- term social-ecological resilience perspective. This requires tools to identify the most sustainable aquacul- ture practices, drawing knowledge from both new and traditional culture systems [5]. A wide range of tools/frameworks for assessing various aspects of environmental performance have been advanced [11], some focusing especially on food production. These include techniques such as Risk Assessment, Ecological Footprint, and Energy Analysis. Frameworks more specific to assessing seafood produc- tion systems are Fishprint and the Global Aquaculture Performance Index (GAPI) [10, 12, 13]. Most of these, however, encompass a limited range of the environ- mental concerns associated with aquaculture and some suffer from a lack of methodological standardi- zation [10]. Moreover, the degree of scientific rigor in both the methods and their application is also variable. Data limitations and analytical scope have, therefore, often led to misrepresentation of the environmental consequences of specific management decisions. LCA – The Method and Its Applicability in Aquaculture History of LCA LCA has, since its emergence in the 1970s, evolved from a tool whose primary application was waste manage- ment and energy efficiency management to a more general eco-efficiency measurement framework. It has close links to energy analysis, but is unique among biophysical accountancy-type tools in that it has been internationally standardized (ISO 14040-14044) [14, 15]. An LCA typically begins at the “cradle” of a product or service life cycle (i.e., at the point of primary resource extraction), and extends along the supply chain to encompass all life cycle stages of inter- est to a particular analysis. Single or multiple impact assessment methods may be applied. Estimation of the cumulative environmental impacts along supply chains permits attention to spatially and temporally discrete impacts not typically considered in more traditional environmental impact analyses. Impact categories range from highly quantifiable effects, such as green- house gas emissions or energy use, to (less frequently) more diverse social consequences, such as human health effects [16]. The outcome of a LCA is highly influenced by the ambition, skill, and objectives of the practitioner. Mod- ern software with built-in inventory databases and impact assessment methods has simplified the LCA process, to the extent that an aquaculture system may be modeled in hours. However, the rigor of such models is to a large extent dependent on data quality. While use of generic data available in many public and commercial life cycle inventory databases may provide a starting point for scoping analyses, more context- specific data is required for robust modeling of specific production systems and technologies. Unfortunately, the former (simplified analyses) are increasingly com- mon in the peer-reviewed literature, providing what may be misleading signals and eroding the credibility of the research framework, generally. Software Tools and ISO The rapid evolution and adoption of LCA have been accompanied by the creation of a variety of guidelines, manuals, and dedicated software [17]. The most com- monly used LCA software platforms are GaBi and SimaPro, which are commercial products. Others, such as CMLCA and openLCA, are available free of charge. There are also several life cycle inventory data- bases, which have been developed, with the most exten- sive being the EcoInvent database (www.ecoinvent.ch). Such databases provide inventory data for materials and processes common to most product systems – for example, the production of materials such as concrete, steel, and plastic; the provision of energy carriers such as diesel or regionally specific electricity mixes; or transportation modes like air freight, rail freight, or private automobile transport. As these “background” data are often premised on different methods, assump- tions, and rigor, sourcing data should be done with care as different databases apply different methodologies. It is therefore recommended to be consistent when choosing sources of background systems data and also to be aware of the methodology used before making comparisons between studies. ISO compliance does not require that studies are comparable, only that 1052 Life Cycle Assessments and Their Applications to Aquaculture Production Systems
  • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. extreme weather events, which may cause the loss of an entire crop. Areas ravaged by extreme storm events, such as typhoons, may suffer from weeks of poor water quality as large quantities of sediments may lead to high turbidity for long periods of time. More knowledge on the true carbon emissions asso- ciated with aquaculture is needed as there is increasing interest in its potential as a climate-friendly source for food and biofuels [68, 69]. LCA can play an essential part in the screening for sustainable farming practices and also provide information for the implementation of carbon credit schemes. This need is especially critical for develop- ing countries, as this is where the majority of production occurs and exports are increasing. This will, however, pose a challenge as farming methods are highly diverse and data on farm practices are usually limited. Also, some important environmental impacts from aquaculture are at present not quantifiable using the LCA framework, such as spread of diseases and para- sites. These impacts have been attracting widespread public concerns and have influenced development of farming methods in many countries. The associated consequences could, for example, be accounted for as biotic resource use. Concerns associated with defores- tation to produce agricultural land should also be fur- ther discussed, as it is only partially covered in current LCA practice and literature. Future Directions LCA provides a robust tool for dealing with an impor- tant subset of sustainability concerns, many of which have historically been overlooked in discussions of environmental management in aquaculture. However, it must be kept firmly in mind that decisions made during the analytical process strongly influence research outcomes. It is, therefore, important to continue the discus- sion about such methodological decisions, including the kind of LCA framework applied (i.e., attributional versus consequential LCA), systems boundaries, and choice of allocation methods for LCAs of aquaculture production. In the least, it would be constructive for all practitioners to clearly communicate and defend all methodological choices, as they may ultimately send different signals to the industry and policy makers working with sustainability issues. A more standardized methodology within the sec- tor would certainly facilitate the advancement of the field. This could be achieved by better communications between major practitioners or by the development of a manual to guide the community toward one common framework. Even still, there will always be deviations from common practice as each study serves a unique purpose, stressing the need for more transparency. Even though ongoing initiatives for developing the LCA framework exist, it is important to acknowledge that the present framework has limited ability to accommodate the other two pillars of sustainability, namely, social and economic impacts [28]. Thus, even if LCA provides a tool with great potential to guide the aquaculture industry toward more sustainable produc- tion, its framework needs to be reinforced by other analytical tools to capture a wider range of sustainabil- ity concerns. The need to include other tools alongside LCA has already been recognized and recent projects, such as the SEAT project (www.seatglobal.eu), include complementary tools to give a more holistic measure- ment of sustainability. Limitations aside, however, the rapid development of this sector, coupled with the diverse range of possible culture species and technolo- gies, demands careful attention to environmental effects at all relevant scales. LCA can and should play an important role in guiding decisions oriented toward sustainability in aquaculture. Bibliography Primary Literature 1. FAO (2009) State of world fisheries and aquaculture 2008. FAO, Rome 2. Tyedmers P, Pelletier N, Ayer N (2007) Biophysical sustainabil- ity and approaches to marine aquaculture development pol- icy in the United States, A Report to the Marine Aquaculture Task Force 3. Troell M, Tyedmers P, Kautsky N, Ro¨nnba¨ck P (2004) Aquaculture and energy use. Encyclopedia Energy 1:97–108 4. Troell M, Joyce A, Chopin T, Neori A, Buschmann A, Fang J-G (2009) Ecological engineering in aquaculture – potential for integrated multi-trophic aquaculture (IMTA) in marine off- shore systems. Aquaculture 297:1–9 5. Troell M (2009b) Integrated marine and brackish water aquaculture in tropical regions: research, implementation and prospects. In: Soto D (ed) Integrated mariculture: a global review. FAO Fisheries and Aquaculture Technical Paper, 529. FAO, Rome, pp 47–131 1063Life Cycle Assessments and Their Applications to Aquaculture Production Systems
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  • 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. 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. 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. 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. 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. 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. 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. 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. 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. 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
  • 27. also with the rapid cell-cycle dynamics of early embryo cleavage. Nuclear reprogramming, either following fertiliza- tion or SCNT, is the result of active and passive processes. For instance, in most mammals after fertili- zation, the sperm compact chromatin undergoes active remodeling in which protamines are exchanged by ooplasmic histones followed by global loss of DNA methylation. The sperm pronucleus also acquires dis- tinct histone marks such as histone hyperacetylation. However, it is not clear whether such modification is acquired actively through the action of enzymes or it is the result of passive acquisition from the ooplasmic histone pool already rich in acetylation marks [36]. The timing required for completing nuclear reprogramming is unclear. Some researchers have suggested that the donor nucleus should be reprogram- med upon reaching embryonic genome activation. Such a restrictive reprogramming timing would be consistent with the lower cloning efficiencies usually observed in mice compared to bovine, two of the most cloned ani- mals, the latter having more time for reprogramming the differentiated donor nucleus before genome activation. However, it has also been suggested that reprogramming continues up to the blastocyst stage. To better understand the nuclear reprogramming process, the dynamics of the most important epige- netic marks and of gene expression during early embryogenesis in fertilized and cloned embryos are discussed next. Reprogramming of DNA Methylation DNA methylation is a reversible modification of the chromatin template at the DNA level in which a methyl group is predominantly added to cytosine of the dinu- cleotide sequence CpG. DNA methylation is a widely used mechanism of epigenetic regulation commonly associated with stable heritable gene silencing, hetero- chromatin, chromosome stability, genomic imprint- ing, X-chromosome inactivation, and inactivation of retroviral sequences. It has been shown that DNA methylation is essential for embryonic development, especially at postimplantation stages [37]. DNA meth- ylation is carried out by DNA methyltransferases (DNMTs). DNMT1 maintains DNA methylation pat- terns after DNA replication by using the old strand as a template to methylate the new strand. De novo DNA methylation is carried out by DNMT3a and DNMT3b. DNA methylation is dynamically regulated. Removal of methyl groups can be an active process when specific DNA demethylases are involved to remove the methyl groups, or it can be a passive process when failure to copy the methylation marks on the newly synthesized DNA leads to erasure of those marks during cell divisions. In contrast to DNA demethylation of the sperm pronucleus, which is thought to be an active process [38], passive DNA demethylation of the embryonic genome takes place following the zygotic stage. After SCNT, DNA methylation reprogramming is usually inefficient. Mice and bovine cloned embryos have aberrantly hypermethylated DNA [39]. In sheep, SCNT embryos also tend to be significantly hypermethylated compared to fertilized embryos [40]. Such abnormal hypermethylation was observed from the 1-cell to the 8-cell stage and then at the blastocyst stage, in which the trophectoderm was hypermethylated relative to the fertilized control. Interestingly, the rate of demethylation is similar in both SCNT and fertilized embryos, suggesting that the hypermethylated DNA pattern of SCNT embryos is probably inherited from the hypermethylated donor cell [38]. Alternatively, it has been suggested that cloned embryos undergo precocious DNA methylation due to the presence of DNMT1 associated with the donor nucleus, thus preventing passive demethylation [39]. DNA methylation reprogramming appears to be an indicator of developmental potential. In sheep, the proportion of SCNT embryos that were normally methylated at the 2-cell stage coincided with the pro- portion of surviving embryos reaching the 16-cell stage [38]. Similarly in mice, aberrant methylation of early- cleavage cloned embryos was proportionally associated to developmental failure to the blastocyst stage [41]. Reprogramming of Histone Modifications The tails of the histone subunits H3 and H4 are targets for chromatin modifier enzymes, with histone methyl- ation and acetylation modifications the most exten- sively studied. These modifications set epigenetic “marks” associated with either gene silencing or gene activity. 1076 Livestock Somatic Cell Nuclear Transfer
  • 28. Histone acetylation is associated with open chro- matin and gene activity. This modification neutralizes positive charges on histone tails and thus increases the overall repulsive negative charge of DNA on the chro- matin structure. In turn, an open chromatin favors the binding of transcription factors to DNA. In contrast, histone methylation can be either repressive or activating, depending in the lysine residue modified. These modifications act as signals that are “read” by proteins, which in turn can trigger down- stream chromatin-modulating events. Together with methylated DNA, methylation of lysine 9 of histone 3 (H3K9), H3K27, and H4K20 has been implicated with heterochromatin and gene repression, while methylation of H3K4, H3K36, and H3K79 is associated with active gene expression. In addition to abnormal DNA methylation, embryos produced by SCNT show aberrant reprogramming of histone modifications. Asymmetry of H3K9me3 fails to be established between ICM and TE in bovine cloned blastocysts compared with fertilized controls [42]. When histone acetylation marks are compared, however, cloned and normal blastocysts look similar [43, 44], suggesting that these marks are well reprogrammed. Yet, at earlier embryonic stages, the same histone acetylation marks show significant differences, with cloned embryos being hyperacetylated [42, 43]. Deregulation of DNA methylation and H3K9 methylation might be major problems during nuclear reprogramming [38, 42]. Their aberrant repro- gramming leads to hypermethylated trophectoderm and extraembryonic tissues. As these epigenetic marks promote inactive chromatin, they are likely to cause downregulation of a substantial number of placental genes, explaining the placental abnormalities fre- quently reported in cloned embryos. Reprogramming of Gene Expression After fertilization or SCNT, the ooplasm induces global remodeling of chromatin, genome silencing, erasure of permissive epigenetic marks of differentia- tion-associated genes, and resetting the genome to an embryonic state [45–47]. Four classes of genes play an essential role to ensure normal embryogenesis following NT: (1) pluripotency-associated genes, (2) trophectoderm genes, (3) imprinted genes, and (4) differentiation-associated genes. Re- activation of pluripotent and trophectoderm genes must take place, whereas differentiation-associated genes must be silenced. Imprinted genes should be reactivated without altering the normal imprinting of somatic cells. Pluripotent Genes Pluripotency is the capacity to differentiate into all embryonic cells. The cells of the ICM are pluripotent because they form the embryo. To recognize the state of pluripotency, landmarks of the ICM have been sought, such as stage-specific gene expression or antigens. These landmarks should be associated only with those stages and tissues known to have pluripotent potential such as the cleavage- stage embryo and ICM of the blastocyst. They should not be expressed in differentiated tissues of later development. Key genes associated with the ICM vary with species. In mice, Oct4 is highly expressed and restricted to the ICM. It plays an essential role in early development as Oct4-depleted mouse embryos fail to form the ICM and are developmentally incompetent [48, 49]. Inducing overexpression of Oct4 is key to revert the gene expres- sion profile of an adult differentiated somatic cell to one of embryonic and pluripotent characteristics [19]. Thus, in the mouse, Oct4 appears to be an ICM-specific marker that can be used to assess pluripotency. Also in mice, other important pluripotent factors restricted to the ICM are Nanog and Fgf4 [50, 51]. Nanog is involved in maintenance of the pluripotent state of ES cells, for instance, by repressing differentia- tion into primitive endoderm [50]. Fgf4, a target gene of Oct4 [52], is necessary for proliferation and differ- entiation of both the ICM into embryonic tissues [53] and the TE into placental tissues [54]. In mouse SCNT blastocysts, Oct4 and Oct4-related genes often fail to be reactivated [55, 56]. Frequently, when Oct4 is expressed, aberrant spatial expression in the trophectoderm indicates faulty reprogramming [55]. Interestingly, when ES cells are used as donors, instead of adult somatic cells, Oct4 and related genes were always expressed in cloned blastocysts [56]. Since pluripotent genes are already active in ES cells, these genes do not need to be reactivated in ES-cloned embryos. 1077Livestock Somatic Cell Nuclear Transfer
  • 29. In human and large ungulate mammals, however, it is less clear what master regulatory factors are involved in the segregation of the ICM. For instance, Oct4 is expressed in both ICM and TE of bovine, goat, and human blastocysts [57–60]. Thus, for nonmurine spe- cies, we are left with less information to assess the extent of ICM-lineage reprogramming. Yet, Nanog does seem to be restricted to the ICM in bovine [61] and goats [59]. Similarly, Fgf4 is restricted to the ICM of bovine blastocysts [57]. Therefore, Nanog and Fgf4 are preferred ICM markers to Oct4 depending on the species. To assess reprogramming of ICM, expression of Nanog and Fgf4 has been measured in bovine cloned blastocysts. Differences between studies were found, with similar [62, 63] and lower levels [57, 64] com- pared with fertilized controls. The disparity of results may reflect differences in nuclear transfer procedures leading to variable success in reprogramming. Trophectoderm Genes In mice, the transition from morula to blastocyst that results in the seg- regation of ICM and TE is regulated by the antag- onistic activity of Oct4 and Cdx2, respectively [65]. Together with Cdx2, Taed4, Eomes, and Gata3 are required for specification and development of the TE lineage [66, 67]. Tead4 appears to be a master factor in TE-lineage induction since it activates Cdx2 and Gata3 [67, 68]. Less is known about the mechanism of segregation of the trophectoderm lineage in other mammals. Although CDX2 protein seems to be a conserved TE- restricted marker, as observed in bovine and porcine blastocysts [61], transcripts levels of Cdx2 were also found in the ICM [57]. It is possible that a posttranscriptional regulation mechanism accounts for the difference between gene expression and gene product levels. Thus, to assess reprogramming of Cdx2, it might be more appropriate to look at the protein level. In contrast to mice, Cdx2 does not repress Oct4 expression in bovine [69], explaining why OCT4 is detected in the bovine TE [58]. Another difference between these two species is the expression of Tead4 and Gata3 transcripts, which are found in both ICM and TE of bovine blastocysts, and thus, their role in TE lineage segregation is not clear [57]. Further evidence that bovine TE formation is quite different to mice is the observation that Eomes is not detected in tropho- blast tissue [70]. Surprisingly, although bovine TE cells will only contribute to the trophectoderm lineage, they are not committed to it before pregastrulation stages since they were observed to contribute to ICM in aggregation assays [69]. Taken together, these observa- tions suggest that differences in expression of lineage markers between species might be the result of differ- ent implantation requirements. For instance, while mouse blastocysts implant readily at this stage, domes- tic blastocysts undergo elongation and have delayed implantations. Indeed, while OCT4 is expressed in both the ICM and TE of the bovine blastocyst, it is restricted to the ICM lineage at a later elongated stage [71]. Few studies have assessed reprogramming of the TE lineage in cloned embryos. In mice, CDX2 protein was frequently expressed normally at the blastocyst stage [72]. A similar result was found in the bovine [57]. Although reprogramming of Taed4 and Gata3 was faulty in bovine cloned blastocysts [57], these are not specific markers of the TE lineage in the bovine. There- fore, in nonmurine species, assessing reprogramming outcome of the TE lineage is currently limited to measuring CDX2 protein. Imprinted Genes Imprinted genes are genes expressed either from the paternal or maternal chro- mosomes, but not both. Gene imprinting plays an essential role in development. When androgenetic (two paternal pronuclei) or gynogenetic (two maternal pronuclei) embryos are produced, embryogenesis is arrested shortly after implantation [73]. Thus, the paternal and maternal genomes have different contri- butions and must complement each other at fertiliza- tion for normal development to occur. Imprinting marks originate during gametogenesis and are gener- ally protected from genome-wide reprogramming, such as DNA demethylation/methylation, during embryonic development [36]. Many imprinted genes regulate development, growth, and function of embryonic and extraembry- onic tissues. Following SCNT, many imprinting errors can occur during reprogramming of the somatic chro- matin. For an imprinted gene, simultaneous expression from the paternal and maternal alleles or lack of expres- sion is the result of aberrant reprogramming. Because 1078 Livestock Somatic Cell Nuclear Transfer
  • 30. of their regulatory growth function, deregulation of imprinted genes might explain several placental abnor- malities and “large offspring syndrome” often observed in cloned animals. Some researchers have proposed that long-term culture of donor cells, the usual scenario for embryonic stem (ES) cells, is an additional source for imprinting errors, and thus, it has been suggested that ES cells are a poor choice for NT [74]. However, using microarray technologies, Humpherys et al. [75] found no signifi- cant difference between ES-NT and noncultured cumulus cell-NT clones in terms of gene expression abnormalities including imprinted genes. Therefore, it is not clear whether extensive in vitro culture is a major problem for NT in terms of imprinting deregulation. Moreover, mammals seem rather tolerant to imprint- ing errors [76]. Differentiation-Associated Genes Following SCNT, silencing of differentiation-associated genes is often incomplete. For instance, cloned embryos produced with myoblast nuclei still expressed the myoblast marker GLUT4 [77]. Similar somatic epigenetic mem- ory was also observed in Xenopus cloned embryos, which overexpressed endoderm or ectoderm markers according to the origin of the donor cells [78]. While insufficient silencing of differentiation-associated genes may alter metabolic demands, the functional consequences of retaining expression of somatic genes on the developmental potential of cloned embryos are unclear [46]. Several studies have compared global gene expres- sion profiles between cloned and fertilized embryos. At the 1-cell stage of murine embryos, most transcripts (98%) were similarly abundant between clones and IVF groups [45], although such similarity might be the result of maternal transcripts abundantly present in the oocyte. Nonetheless, similar expression profiles were obtained between cloned and fertilized blastocysts in bovine (e.g., [63, 79]). Thus, overall gene expression of cloned embryos appears normal. However, it is possible that small alterations in gene expression are amplified during postimplantation stages, which would contrib- ute to the low developmental potential of cloned embryos [79]. In summary, perfect gene expression reprogramming may be unlikely following SCNT. Cell-type-specific genes may be incompletely silenced while imprinted genes are subject to deregulation. Reactivation of ICM- or TE-lineage genes might be problematic depending on the species. The mechanism of nuclear reprogramming is still a black box, and much further research is needed to understand where the bottleneck of NT is. Factors Affecting Development of Embryos Produced by SCNT Frequently, reports have shown that there is ample room for optimization in NT technology. Major inves- tigated factors affecting NT success include quality of recipient oocyte, time of enucleation, nuclear donor source, cell-cycle coordination of donor and recipient cells, alterations in the timing and inducer of oocyte activation, and culture of reconstructed embryos. Sources of Recipient Oocytes Mammal females produce mature oocytes (oogenesis) during the ovarian cycle. The earliest stage of oogenesis is the primary oocyte within the primordial follicle. Pri- mary oocytes are arrested at the first meiotic prophase in a quiescent state. Some of these are regularly recruited for further growth since puberty and thereafter. As primary oocytes resume growth, they increase in size and undergo cytoplasmic maturation. An oocyte must undergo cyto- plasmic and nuclear maturation to be developmentally competent. Cytoplasmic maturation involves mRNA synthesis, translation into protein, and posttranslational modifications. Many mRNA and protein molecules are stored in the cytoplasm to function later during early embryo cleavage, before embryonic-genome activation. Some of the proteins produced, such as maturation pro- moting factor (MPF), are necessary for meiotic progres- sion during nuclear maturation. Within the follicle, oocyte growth is accompanied with follicular growth. Indeed, oocytes obtained from medium- and large- sized follicles are developmentally better than those obtained from small-sized follicles. It seems that oocytes from the latter group have not completed cyto- plasmic maturation [80]. Oocytes are surrounded by cumulus cells, and mutual communication is required for complete oocyte maturation. Oocytes can be matured in vivo or in vitro. In vivo maturation involves collecting oocytes that have 1079Livestock Somatic Cell Nuclear Transfer
  • 31. naturally matured within the ovary of a live animal. In contrast, in vitro maturation involves aspirating imma- ture oocytes from the ovaries of slaughtered animals and placing them on favorable culture conditions to complete the maturation process in the incubator. Although much understanding has been gained in oocyte maturation, in vitro maturation is still suboptimal at best. In vitro–matured oocytes have altered energy metabolism [80], higher chromosomal abnormalities [81], and lower developmental compe- tence than in vivo–matured oocytes [82, 83]. For convenience, in livestock cloning, in vitro mat- uration is widely used as very large numbers of imma- ture oocytes can be obtained from slaughterhouses at low cost. However, the poorer quality of these oocytes is likely to contribute to the low frequency of develop- ment usually observed in cloned embryos. Enucleation of Recipient Oocytes Nuclear maturation is resumed at the time of the LH surge in vivo or during in vitro conditions. The oocyte continues meiosis to progress from the arrested prophase I to metaphase I, anaphase I, telophase I, and cytokinesis with unequal cytoplasmic distribution, when half of the chromosomes are discarded in the first polar body. The oocyte progresses to meiosis II and is arrested at metaphase II. Following fertilization or arti- ficial activation, the metaphase II–arrested oocyte resumes meiotic progression once more to complete metaphase II, anaphase II, and telophase II, discarding again half the number of chromosomes in a second polar body, achieving a haploid number of chromo- somes. Enucleation can be carried out at any of these meiotic stages, although with possible different conse- quences (discussed later). Table 1 provides some Livestock Somatic Cell Nuclear Transfer. Table 1 Examples of cloning experiments using recipients at different meiotic and mitotic stages of the cell cycle Recipient cytoplast Donor cell type Species Offspring References Enucleated GV oocytes (subsequently matured) ES cells Mouse No [84] Enucleated Pro-MI oocyte (subsequently matured) ES cells Mouse Nd [84] Enucleated AI/TI oocyte (subsequently matured) Fetal Sheep Yes [85] Adult Sheep Yes Enucleated MII oocyte Embryonic, fetal, adult Sheep Yes [18] Unenucleated MII oocyte (subsequent enucleation) Cumulus cells Mouse Yes [86] Enucleated activated MII oocyte Blastomeres Sheep Yes [17] Enucleated PN Zygote PN karyoplast from zygote Mouse Yes [10] PN Karyoplast from first NT embryo Pig Yes [87] Blastomeres Mouse No [11] Cumulus cells Mouse No [88] TII enucleated oocyte Blastomeres Goat Yes [89] M-phase arrested zygote ES cells Mouse Yes [90] Fibroblast Mouse ND 2-cell embryo Lymphocyte Mouse Yes [91] ND not determined 1080 Livestock Somatic Cell Nuclear Transfer
  • 32. examples of cloning experiments carried out using recipients enucleated at different stages of meiosis or mitosis. For NT, it is most important that the oocyte undergoes cytoplasmic maturation to construct a developmentally competent embryo, while nuclear maturation is less important because the oocyte DNA is eliminated at enucleation and does not form part of the embryo. Enucleation of prophase I oocytes, also known as germinal vesicle (GV) stage, has been attempted in the mouse, and the resultant recipients were capable of reprogramming donor nuclei [84, 92]. However, the reconstructed embryos are developmen- tally incompetent even when recipients are further matured in vitro prior to NT [84]. It is possible that GV oocytes are poor recipients because removal of their transcriptionally active chromatin [93] prevents synthesis of new mRNA necessary for completing cytoplasmic maturation. Similarly, enucleation of pro-metaphase I (pro-MI) oocytes also results in poor recipients [84]. Beyond interfering with mRNA synthe- sis, it is also likely that enucleated GV and pro-MI oocytes are poor recipients because enucleation disag- gregates the cumulus-oocyte complex, thus depriving the oocyte of the beneficial communication with cumulus cells for completing cytoplasmic maturation during in vitro maturation. Enucleation of metaphase II–arrested oocytes is the most common practice in cloning experiments. The rationale of using such recipients is evident as the COC is left undisturbed during the entire in vitro maturation period, thus maximizing the probability of cytoplasmic maturation. However, a practical alter- native is to enucleate at anaphase I/telophase I (AI/TI). By doing so, the spindle is more efficiently removed along with less cytoplasm compared to MII enucleation [27]. AI/TI cytoplasts are further cultured to complete the maturation period and have produced lambs after SCNT [85]. Other recipients have been used for NT. Activation of MII oocytes with subsequent enucleation at ana- phase II/telophase II has been done for practical reasons similar to those of AI/TI enucleation [89, 94]. Zygotes and two-cell embryos have also been enucle- ated and used as recipients for NT. These will be better discussed below in relation to the “potential loss of reprogramming factors” during enucleation and later in relation to the “coordination of donor and recipient cell cycles.” Potential Loss of Ooplasmic Reprogramming Factors Enucleation involves removing the DNA material from the unfertilized or fertilized oocyte plus an unavoidable volume of ooplasm. It has been speculated that loss of ooplasmic volume is accompanied with a reduction of developmental potential, possibly due to the removal of cytoplasmic reprogramming factors. However, fusion of two or more cytoplasts prior to NT did not improve the frequency of development in mice [95]. Apparently, the amount of cytoplasmic factors in the oocyte is not critical for cloning success. However, the above discussion does not take into account that the content of reprogramming factors in the cytoplast is dependent on the cell-cycle phase. The genome can be removed as interphase pronuclei in zygotes or with the spindle if recipients are mitotic zygotes or meiotic oocytes. In the latter case, the com- ponents of the nucleoplasm stay in the recipient as they are diluted in the cytoplasm following nuclear envelope breakdown. Recipient zygotes enucleated at interphase can only support development of nuclei obtained from 1-cell or 2-cell embryos [11] but fail consistently when more differentiated donor nuclei are transferred [11, 96, 97]. Kevin Eggan and colleagues have shown that zygotes and 2-cell embryos at the mitotic phase regain the reprogramming ability [90, 91]. These authors have proposed that reprogramming factors are sequestered in the nucleus during interphase but then redistributed throughout the cytoplasm during M phase [98]. Therefore, reprogramming factors would be lost when enucleation is carried out during interphase [99]. Indeed, Brg1, a component of the Swi/SNF chromatin remodeling complex necessary for embryonic genome activation, was absent in 2-cell cloned embryos when using enucleated interphase zygotes as recipients [99]. Further proof of this idea is supported by the observation that oocyte recipients at the germinal vesicle (GV) stage fail to develop after NT [84]. A possible contributing factor for the low devel- opmental potential of enucleated GV oocytes and interphase zygotes is the low levels of maturation pro- moting factor (MPF) in these recipients (discussed below). Taken together, while the mechanism of 1081Livestock Somatic Cell Nuclear Transfer
  • 33. reprogramming remains largely unknown, essential reprogramming factors are present in the recipient’s cytoplasm during M phase, but absent during inter- phase, at least in the mouse model [99]. It is still possible that some unknown factors bene- ficial for reprogramming might be stably associated with chromatin and lost during enucleation. It is well known that spindle factors are associated with the meiotic chromosomes, and therefore, enucleation could impair spindle function. Indeed, in human SCNT, it is apparently necessary to leave the recipient genome to achieve development to the blastocyst stage, at the cost of producing a triploid embryo [100]. How spindle function is affected by SCNT in livestock spe- cies will be discussed later in this entry. Coordination of Donor and Recipient Cell Cycles During a single cell cycle, a cell must double all of its components, segregate its genetic material equally to the two daughter cells, and undergo cell division. One exception occurs during the first few cell cycles of early embryo development where no net growth occurs. However, cell-cycle events associated with duplication and segregation of the nuclear DNA still occur. Another exception to the mitotic cell cycle is meiosis, which results in halving the chromosome number. Only germ cells undergo meiosis, while somatic cells only mitosis. The mitotic cell cycle has four sequential phases: G1, S, G2, and M. During S phase (S for DNA synthesis), replication of DNA takes place. During M phase (M for mitosis), the cell divides all its components equally into two daughter cells. Cell growth occurs only during interphase, which constitutes G1, S, and G2. In a typical in vitro–cultured mammalian cell, interphase lasts about 23 hours (of a 24-hour cell cycle) while M phase is very short, lasting about one hour. S phase takes about half of the cell-cycle time (reviewed in [101]). The gap phases G1 and G2 are designed to provide extra time for the cell to grow and checkpoints for favorable growth conditions as well as for DNA damage. At fertilization, the oocyte is arrested at MII, in a diploid state, while the sperm cell has completed mei- osis and is haploid. Following oocyte activation by the sperm cell, the oocyte resumes meiosis to become hap- loid, thus matching the chromosome content and forming a viable diploid embryo. In NT, of course, it is not important that the donor chromosome content matches the recipient’s since the oocyte chromatin is removed. Rather, the donor’s cell-cycle phase should be compatible with the recipient cytoplasmic reprogramming content, which in turn depends on the recipient’s cell-cycle phase. An important cytoplasmic reprogramming factor that has been extensively stud- ied and has a profound effect on the donor nucleus is maturation promoting factor (MPF). Campbell and colleagues, at the Roslin Institute (Scotland), carried out pioneer work in cell-cycle coor- dination between the donor cell (karyoplast) and recip- ient cytoplast prior to NT [16, 17, 102]. Their work suggests that cell-cycle coordination is a very important factor that should be taken into account prior to NT experiments to avoid damage of the donor chromatin. For NT, mature MII cytoplasts are the recipients most frequently used. These contain high levels of MPF (also known as M-Cdk). MPF is a complex of two factors, protein kinase p34cdc2 and cyclin B. MPF activ- ity is regulated through synthesis and degradation of cyclin B and through phosphorylation of p34cdc2 . MPF activity is responsible for nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) of the donor nucleus following activation of reconstructed embryos. MPF declines progressively after activation or fertilization and remains low during interphase. Declining MPF levels are followed by nuclear membrane reformation and DNA synthesis. NEBD and PCC occur independent of the cell-cycle stage of the donor nucleus in MII-arrested oocytes. In contrast, when activation precedes NT for a few hours, MPF activity is low and nuclear envelope integrity is maintained because NEBD and PCC do not occur. An intact nuclear membrane prevents replication of previously replicated DNA [16, 103]. Transferring donor nuclei in S or G2 phases into MII cytoplasts followed by activation leads to NEBD and eventually to DNA rereplication, likely resulting in ploidy abnor- malities. In addition, PCC could also cause chromo- somal damage of S-phase nuclear donors. These early observations suggested that only G1 embryonic donor nuclei should be transferred into unactivated MII cytoplasts. In contrast to unactivated MII oocytes, acti- vated oocytes serve as “universal recipients” since MPF activity is low and NEBD/PCC do not occur, allowing 1082 Livestock Somatic Cell Nuclear Transfer
  • 34. the transfer of any nuclear donor without the risk of DNA rereplication. In these reconstructed embryos, coordinated replication of nuclear DNA should occur. Indeed, unsynchronized sheep blastomere donors developed to the blastocyst stage more fre- quently when using activated cytoplasts (low MPF) compared with cytoplasts activated following NT (high MPF) [17]. For differentiated cells, however, activated oocytes might be poor recipients because NEBD and PCC are thought to be beneficial for reprogramming the donor nucleus. In bovine, for instance, when activated enu- cleated MII oocytes were used as recipients for somatic donors at different stages of the cell cycle (except S phase), development did not proceed to the 8-cell stage. However, unactivated MII oocyte recipients resulted in successful in vitro development to blastocyst [104]. Similarly, activated oocytes were shown in mice to be ineffective for development to term when using cumulus cells as donors [105]. These results suggest that activated oocytes, with low levels of MPF, fail to repro- gram somatic donor nuclei. Blastomeres, on the other hand, probably require little or no reprogramming and thus can be cloned successfully with activated recipi- ents as in Campbell et al. [17]. Not surprisingly, for somatic nuclei donors, the most common recipient is the unactivated enucleated MII oocyte. Later research showed that donor genomes at G0, G1, and M phases of the cell cycle can be transferred into unactivated oocytes and produce offspring. For instance, embryonic stem (ES) cells arrested at meta- phase produced live mice [106]. Metaphase-arrested chromatin donors are ready to segregate and form a pseudo-second polar body, thus eliminating the extra set of chromatin. However, G2 donors were observed to fail at extruding the pseudo-second polar body resulting in tetraploidy [104], probably because they are “out of phase” with the meiotic cytoplast. This, combined with the rereplication event, could explain the consistent failure to produce viable off- spring with G2 donors (reviewed in [107]). For G0 and G1 donors, spindle function inhibitors are required to prevent pseudo-polar body formation and loss of chromosomes. There is no apparent advantage between nuclei at G0, G1, or M phases for NT. Somatic cell donors are routinely synchronized at the G0 phase of the cell cycle prior to NT. However, synchronized G0 or G1 fetal fibroblasts were compared and no clear superiority for either donor was found. Their relative cloning effi- ciencies were cell-line dependent in cattle [107]. Syn- chronization of ES cells at G0 or G1 might be problematic since they have a very short G1 phase, and if induced to become quiescent (G0), they likely differentiate. The most successful studies in cloning mice with ES cells used unsynchronized cell populations [108, 109], but others were less successful [110, 111]. ES cells at presumptive G1 (by selecting small cells) were used with cloning success [112] and without success [113]. ES cells were also synchronized at M phase with moderate success in several occasions [106, 114, 115]. Taken together, enucleated MII oocytes seem to be the best recipients. However, the cell cycle of the donor nuclei might be less important since cloning success is achieved with G0, G1, and M donors, adapting the NT protocol accordingly. Sources of Nuclear Donor Cells Donor cells of different ages including embryonic, ES, fetal, and adult cells have been used for NT. It has been suggested that the age of an adult somatic donor cell could be transmitted to the clone. Such belief origi- nated from a study showing that Dolly and other cloned sheep had shorter telomeres compared to age- matched controls [116]. The belief was later empha- sized by the premature death of Dolly. However, several studies in cattle and mice showed that telomere length was normal in clones; telomerase activity was found to be reactivated in cloned bovine preimplantation embryos at similar levels to fertilized controls. Yet, further studies in sheep showed that indeed clones have shorter telomeres [117], suggesting that the mech- anism of telomerase reactivation differs in this species. Nonetheless, studies have not observed premature aging in clones produced by SCNT. Beyond age, a more interesting discussion relies on the epigenetic differences between donor cells. Differ- entiated somatic cell types diverge in their gene expres- sion programs but have similar global chromatin configurations and modifications, or epigenomes. However, the epigenome of differentiated cells con- trasts with that of undifferentiated pluripotent cells. 1083Livestock Somatic Cell Nuclear Transfer
  • 35. It is probably the epigenome features that render one cell more amenable to reprogramming following NT rather than the cell’s specific gene expression program. Epigenomes Differentiated cells are different to plu- ripotent cells in several epigenetic features. Compared to pluripotent cells, differentiated cells show expansion of repressive domains marked by H3K9me3 and H3K27me3 [118]. Similarly, differentiated cells show hypermethylated DNA compared to ES cells [119] and have lower levels of histone acetylation, consistent with heterochromatin configurations [120]. In agreement, electron microscopy showed that ES cells have dis- persed global chromatin architecture, while the chro- matin of the differentiated cells is more compact [121]. The epigenome of a differentiated cell can be reverted to that of an ES cell by overexpression of master-regulatory genes of pluripotency, thus forming iPS cells [19]. It has been shown that iPS and ES cells have similar global DNA methylation [20, 119] and similar global levels of the histone mod- ifications H3K4me3, H3K9me3, and H3K27me3 [118, 119, 122]. Differentiated Versus Undifferentiated Donors It is generally postulated that an inverse relationship exists between the differentiated state of a donor cell and its “reprogrammability.” This hypothesis is based on NT efficiencies obtained with different types of donor nuclei ranging in their differentiation status from the totipotent zygote to the terminally differentiated cell. It has been suggested that a small proportion of somatic stem cells are present in a tissue sample, and these cells, rather than differentiated cells, account for the successful cases of SCNT. Studies were performed to test whether terminally differentiated cells are “clonable.” First attempts to clone mice with donor lymphocyte cells failed [123]. Thus, a two-step NT procedure, in which ES cells are derived from cloned embryos and used for embryo tetraploid complemen- tation, was later adopted for lymphocyte and olfactory neurons, resulting in viable mice offspring [124, 125]. Later on, cloned mice were produced by direct NT of T lymphocyte cells, albeit with a very low efficiency [126]. These results show that terminally differentiated cells can be cloned, but does not reject the possibility that less differentiated cells might be better donors. In contrast to terminally differentiated cells, blasto- meres give high frequencies of development after NT. Yet, the developmental potential of donor blastomeres decreases as the cleavage stage of the embryo increases. In mice, a gradual decline is observed from the 1- to the 4-cell-stage embryo followed by a steep decline from the 4- to the 8-cell stage, remaining low thereafter (reviewed in [127]). In cattle, such a significant decline in developmental potential is observed after the 8-cell stage (e.g., [13]). Apparently, the greatest decline in developmental potential after blastomere NT occurs following embryonic genome activation. Literature reviews on NT generally support that ES cells are better than somatic cells in terms of the devel- opmental potential of the reconstructed embryos. This view is based on experimental evidence gathered from a few early studies in mice, where good outcomes were achieved using ES cells as donors [108, 109, 112]. In these studies, the cloning efficiency (here defined as development to term after blastocyst transfer to surro- gate females) ranges from 8% to 21%. These values are much higher than those obtained previously with SCNT by Wakayama and colleagues, where cloning efficiencies ranged from 0.4% to 1.6% using cumulus cells [128] and fibroblasts [129], respectively. Other studies also found similarly low mice cloning efficien- cies (0–2.5%) with fibroblast cells (e.g., [130]). However, lower efficiencies (0–5.4%) were also reported with ES cell donors (e.g., [111, 113–115]). These studies question whether ES cells have indeed better developmental potential than somatic cells fol- lowing NT and suggest that major reviews are relying too heavily on positive results while overlooking the negative ones. In a critical review, Oback and Wells [127] argue that postimplantation sample sizes are generally small in animal cloning and thus insufficient to draw robust conclusions. Furthermore, they claim that many confounding factors exist in nuclear transfer experiments, rendering comparisons difficult. These factors include cell line and sex, confluency of donor cell culture, cell-cycle, passage number, and nuclear transfer procedure. Oback and Wells conclude that to determine whether ES cells have a better developmental potential than differentiated cells, it is necessary to carry out large NT experiments in which ES cells and the same cells induced to differentiate prior to NT are directly compared while keeping other parameters 1084 Livestock Somatic Cell Nuclear Transfer
  • 36. constant. Taken together, the published data are incon- clusive but suggest that ES cells give better cloning efficiencies than somatic cells. Indeed, only one study directly compared the developmental potential of ES cells to somatic cells, resulting in a cloning efficiency of about four times greater with ES cells [112]. According to Jaenisch’s group, the cloning efficien- cies with ES cells are 10- to 20-fold higher than with somatic cells after embryo transfer [76]. These authors have proposed that ES cells have better developmental potential following NT because they are undiffer- entiated and thus require less reprogramming than differentiated cells [76]. For instance, ES cells already express pluripotency-associated genes such as Oct4, Nanog, and Sox2, needed for early embryogenesis. In contrast, these genes need to be reactivated in differen- tiated cells. A second hypothesis could be that ES cells can be more easily reprogrammed as they have overall open chromatin configurations compared to differen- tiated cells, which typically have compact heterochro- matin. The open and accessible chromatin of ES cells might facilitate the binding of nuclear remodeling fac- tors found in the oocyte’s cytoplasm. Unfortunately, ES cells cannot be stably derived in livestock species yet. However, it is probably a matter of time until the mechanism and factors that govern pluripotency are discovered in these species. When derivation of ES cells in farm animals becomes possible, it would be of interest to further test their developmen- tal potential compared to that of somatic cells follow- ing NT. Differential Organelle Contribution During fertilization, the oocyte and sperm each con- tribute a haploid set of chromosomes to form a diploid zygote. However, most of the cytoplasm and organelles are inherited from the oocyte. Among these are mitochondria, with their own genetic material. An exception to this is the centrosome, which is partly contributed by sperm. The differential contribution of the centrosome and mitochondria can affect NT outcomes. These contributions can be altered by the method used to transfer the donor chromatin. Centrosome Inheritance The centrosome is the main microtubule-organizing center (MTOC) of animal cells involved, among other functions, in assem- bly of the mitotic spindle for cell division. This organ- elle is composed of two centrioles surrounded by abundant centrosomal proteins, which are often referred to as pericentriolar material (PCM). The cen- trosome is replicated during the S phase of the cell cycle from material stored in the cytoplasm. Mammalian oocytes have no centrioles but contain PCM that is recruited by the sperm centriole after fertilization, restoring a functional centrosome. Mice, however, are acentriolar until late preimplantation stages, achieving embryo cleavage by using maternal MTOCs. In SCNT, the MTOCs associated with the oocyte meiotic spindle are removed while the centrosome from the donor cell is introduced [131]. It is not well understood the composition of the donor centrosome once within the recipient. Also, it is unclear whether the donor centrosome recruits leftover PCM from the recipient oocyte [131]. Nevertheless, it is thought that the donor centrosome needs to be remodeled into a zygotic centrosome for normal embryonic cleavage [131]. Centrosomes from donor cells at S phase might be reduplicated following oocyte activation in the subse- quent S phase [132]. This would lead to the formation of extra spindles followed by aberrant segregation of chromosomes, a phenomenon frequently observed with S-phase donors. Even when using G0 donors, 35% of bovine reconstructed embryos had abnormal cleavage correlated with abnormal centrosome number and distribution [133]. This suggests that providing only one centrosome does not guarantee normal cen- trosome function following NT. An early NT study in monkeys suggested that removing the MII meiotic spindle depletes the oocyte of spindle proteins NuMA and HSET [134]. These proteins closely associate with maternal chromosomes and are required for mitotic spindle function. This study showed disorganized spindles with misaligned chromosomes in all reconstructed embryos, which were unable to produce pregnancies. However, in a later study using a different nuclear transfer proce- dure, NuMA location and spindle formation were nor- mal in most reconstructed monkey embryos [135]. Taken together, these results suggest that different parameters of the NT procedure can affect centrosome function. 1085Livestock Somatic Cell Nuclear Transfer
  • 37. Mitochondrial Inheritance The mitochondrion is a special organelle, having a double membrane and containing its own genome (mtDNA). The main func- tion of mitochondria is to produce energy in the form of ATP. Other functions include production of reactive oxygen species (ROS), calcium signaling, and apopto- sis. mtDNA encodes for 13 mitochondrial proteins involved in ATP synthesis, for 22 tRNAs, and for 2 rRNA molecules involved in translating mitochon- drial proteins [136]. Replication and expression of mtDNA are regulated by the nuclear genome through the expression of transcription and replication factors that translocate into mitochondria. Mitochondria are maternally inherited because sperm mitochondria are targeted for destruction upon fertilization [137]. In SCNT, the donor cell mitochondria can persist and contribute to the reconstructed embryo, thus resulting in heteroplasmy (two different populations of mtDNA). Donor mito- chondrial contribution is quite variable, ranging from 0% to 59% (reviewed in [138]). A bovine oocyte contains about 250,000 mitochondria, a 100-fold compared to a somatic cell [139]. Therefore, high heteroplasmy probably results from preferential amplifi- cation of donor mitochondria. Compatibility between nuclear and the recipient’s mtDNA seems to play an important role in SCNT. It has been shown that autologous SCNT (a female donor cell is transferred to its own enucleated oocyte) improves the frequency of pre- and postimplantation development when compared with heterologous SCNT [140]. Likewise, bovine reconstructed embryos devel- oped more frequently as the similarity between the mtDNA haplotype of the donor and of the recipient cell increased [141]. Moreover, it is thought that the developmental block commonly observed in interspe- cies-SCNT embryos is due to genomic-mitochondrial incompatibilities [142]. Transfer of Donor Nuclei From the above discus- sion, it is apparent that the method chosen to deliver the donor chromatin into the cytoplast can have impli- cations in terms of organelle inheritance of the reconstructed embryo. By whole-cell fusion, the donor cell contributes the nuclear and cytoplasmic material including mitochondria and centrosome, while nuclear microinjection does not. This would not seem to be problematic in mice since maternal MTOCs suffice to form the spindle-chromosome com- plex during cell division. In livestock species, however, centrioles function in spindle formation during early embryo cleavage. Nonetheless, pigs have been cloned by nuclear microinjection (e.g., [83]), suggesting that the presence of a centriole is not necessary for embryo cleavage in pigs. While cell fusion contributes mitochon- dria and cytoplasm, there is no evidence that either heteroplasmy or the amount of somatic cytoplasm con- tributed by a single donor cell is enough to reduce the developmental potential of the reconstructed embryo. Cell fusion has been directly compared to nuclear microinjection in cloning experiments. In pigs and cattle, greater preimplantation development with the cell fusion method has been reported [143, 144]. Unless centrioles are often transferred along with residual cytoplasm during nuclear microinjection, these results suggest that centrioles are not strictly necessary for embryo cleavage, although their presence enhance pre- implantation development. In mice, conflicting data have been reported when comparing the two methods. These mixed results are consistent with the exception that centrioles are dispensable in mice during preim- plantation stages. Interestingly, it has been reported that the quality of blastocyst produced by cell fusion was better; analysis showed that piezo-assisted micro- injection can cause DNA damage by shear forces [145]. Activation of Reconstructed Embryos In mammals, egg activation is achieved when summa- tion of intracellular Ca2+ oscillations reach a minimum threshold. Egg activation leading to Ca2+ oscillations is thought to be triggered by a sperm-specific isoform of PLC, known as PLC zeta (PLCz), introduced into the oocyte following fertilization [146]. It has been suggested that summation of these Ca2+ oscillations encodes information for directing later development. For instance, few Ca2+ oscillations lead to decreased implantation, while excess Ca2+ oscillations, to increased postimplantation failure in mice [147]. Maturation promoting factor (MPF) also plays an important role in MII arrest and in regulating Ca2+ oscillations (reviewed in [148]). Inhibiting MPF activ- ity enhances oocyte activation. Inhibition of protein synthesis (by cyclohexamide) or inhibition of 1086 Livestock Somatic Cell Nuclear Transfer
  • 38. phosphatases (by 6-dimethylaminopurine [6-DMAP]) can decrease the activity of MPF and induce meiotic progression. These inhibitors are usually combined with stimulation of Ca2+ oscillations to efficiently activate reconstructed embryos. However, it is important to bear in mind that these chemicals have broad spectrum actions and can interfere with other cellular processes [148]. In the absence of the natural inducer of egg activa- tion, several artificial methods exist to trigger activation of reconstructed SCNT embryos, including electrical pulses, ethanol, calcium ionophore A23187, ionomycin, strontium, and thimerosal (Thi)⁄dithiothreitol (DTT). With the exceptions of strontium and Thi/DTT, the other treatments induce a single Ca2+ oscillation [149]. Comparisons of several activation methods in mice cloning could not found a preferred method [95]. To mimic more physiological activation events, sperm-mediated activation methods have been used. Fertilized oocytes were used as recipients, but such approach has not produced any significant improve- ment in developmental potential to term in mice or bovine cloning [150, 151]. To avoid the added complication of removing the sperm chromatin, an alternative activation protocol involved directly injecting the sperm activating factor PLCz, in the form of mRNA, resulting in long-term Ca2+ oscilla- tions [152]. This approach improved gene expression patterns of several genes and reprogramming of the repressive histone mark H3K27me3 [153]. Therefore, mimmicking sperm-activation events might improve reprogramming of the donor nucleus. Another factor that can be controlled in activation protocols is the timing of activation following embryo reconstruction. It is common practice to delay activa- tion for 1–3 h to extend the time for nuclear remodeling after nuclear envelope breakdown. This “nuclear exposure” has been shown to be beneficial in bovine [104]. However, the effectiveness of this approach likely varies between species since in mon- keys, it was shown that development improves by immediate activation [154]. Culture of Reconstructed Embryos For animal cloning, the reconstructed embryos can be directly transferred to a surrogate female or cultured in vitro to the blastocyst stage followed by transfer. Since in vitro culture functions as a first filter to select grow- ing embryos with exclusion of the developmentally arrested ones, it allows transferring fewer embryos to surrogates and is, therefore, widely used. Many factors can be manipulated in an in vitro culture system to affect the developmental outcome of cloned embryos. These factors include incubation temperature, media composition and osmolarity, oxygen tension, culture substrate, communal or individual culture, embryo concentration, cocultures, medium renewal, and embryo stress. Analyzing each of these factors is outside the scope of this entry, but the reader is encouraged to read Vajta et al. [155] for a comprehensive review on embryo culture. Instead, an eccentric culture prefer- ence of cloned embryos will be highlighted. Usually, NTexperiments culture cloned embryos in conditions designed for normal embryos. However, as discussed during nuclear reprogramming, a cloned embryo is usually not a normal one. Several reports support that reconstructed embryos have altered metabolism and culture requirements compared to normal embryos (e.g., [77, 156]). For instance, due to incomplete reprogramming of the donor chromatin, mice cloned embryos produced with muscle nuclei overexpress the glucose transporter GLUT4 and thus exhibit enhanced rates of glucose uptake and benefit from somatic cell culture media instead of standard embryo culture media [77]. The benefits of using somatic cell culture media included improved blastocyst formation rate and increased total cell numbers in the resultant blastocysts. These results suggest that normal embryo culture conditions might subject cloned embryos to a harsh selection process, while somatic- like culture media seem to maintain the viability of reconstructed embryos, allowing them more time to complete nuclear reprogramming. Nonetheless, sequen- tial use of different embryo culture media has been shown to dramatically improve blastocyst development of mice reconstructed embryos [156]. Taken together, culture media for reconstructed embryos should be optimized to match the donor cell preferences. Perinatal/Neonatal Care Once cloned blastocysts have been transferred to surrogate animals, work is usually limited to monitoring 1087Livestock Somatic Cell Nuclear Transfer
  • 39. the progress of pregnant surrogates of livestock species. However, it is important to bear in mind that cloned animals are prone to suffer health problems arising from epigenetic errors caused by incomplete nuclear reprogramming of the donor chromatin. Respiratory difficulties seem to be the main problem in cloned neo- nates. Other health problems include myoarthroskeletal malformations and metabolic abnormalities. It is advis- able that cloned neonates are regarded as being at high risk [157], and thus, intensive care should be provided to increase survival. Some studies suggest that veteri- nary intervention during the perinatal and neonatal periods can improve the survival rates of cloned live- stock animals (for a review, see [158]). Perinatal care involves monitoring readiness for birth, induced parturition, and induction of final pul- monary maturation. For yet unclear reasons, pregnan- cies of cloned fetuses often extend beyond the normal gestation period [157]. While this might indicate that cloned fetuses require more time in utero to complete maturation, prolonged gestations are associated with increased birth weight, dystocia, and increased mor- bidity and mortality [157]. To prevent these problems, induction of parturition is commonly carried out. In parallel, to aid pulmonary maturation, pharmaceutical drugs are often administered to promote production of lung surfactant necessary for alveoli inflation. Due to respiratory deficiencies, cloned neonates can quickly become hypoxic and acidotic. Lack of vigor and weak suckling reflexes are other common symptoms of cloned neonates. Thus, intensive care of the cloned neonate is crucial, even for preventative measures. Good care practice includes providing oxygen for at least one hour, heat, mechanical ventila- tion for more severe cases, and monitoring blood parameters [158]. Improving Development From the above discussion, it is clear that veterinary science can play an important role in the management of pregnancy and neonatal care to improve the survival rate of cloned animals. In the laboratory, researchers have attempted many things to improve cloning effi- ciencies. The two most promising areas that have yielded the best results are chromatin remodeling treat- ments and embryo aggregation. Chromatin Remodeling Treatments Relaxation of the donor chromatin could enhance the reprogramming of the donor nucleus. To this end, a few different approaches have been implemented to treat donor nuclei or SCNT embryos. Well-defined chemical treatments include trichostatin A (TSA) and 5-azacytidine (5-Aza). Early in the mouse zygote, both parental genomes are rich in histone acetylations, suggesting that these epige- netic marks are important for reprogramming. Treatment of donor cells with TSA, a histone deacetylase inhibitor, has been used to increase histone acetylation and promote opening of the chromatin. Such treatment led to increased development to blastocyst stage in bovine [159]. Later on, optimized TSA treatments resulted in significant increases (up to tenfold) in development to term com- pared to untreated groups in mice cloning (e.g., [160]). Remodeling of the donor chromatin with TSA has been the single most important innovation for improving consistently the efficiency of SCNT [95, 161]. DNA methylation patterns have been observed to be abnormally high in cloned embryos [36], and there- fore, some researchers have attempted to correct this epigenetic abnormality by treating the donor cells with 5-Aza, a DNA demethylating drug. Such treatment, however, has led to poor blastocyst development [159]. A similar decrease in developmental potential was also observed when using 5-Aza in cloned embryos at the 2-cell stage [162]. It is thought that such failure is due to the effects of 5-Aza on massive DNA demethyl- ation and subsequent DNA rearrangements and forma- tion of pronuclei [163]. Chromosomal abnormalities resulting from 5-Aza treatment would be consistent with the regulatory function of DNA methylation on chromosome stability. These unsuccessful results also suggest that following fertilization, active global demethylation of the sperm chromatin must be well regulated to prevent chromosomal damage. Indeed, the sperm genome retains some methylated regions includ- ing centromeres, which contribute to chromosomal stability. When 5-Aza and TSA are used together, how- ever, a synergistic effect has been observed in cloned bovine preimplantation development compared to TSA treatment alone [164]. Another approach to induce chromatin relaxation involves using cell extracts from Xenopus eggs to treat 1088 Livestock Somatic Cell Nuclear Transfer
  • 40. differentiated donor cells prior to NT. The rationale is that many of the reprogramming factors present in the mam- malian oocyte might also be present in the frog egg. This approach showed a significant increase in development to term in sheep [165]. Treated cells had lower global levels of the heterochromatic epigenetic mark H3K9me3, thus probably contributing to more relaxed chromatin configurations and to improved cloning efficiencies. Notoriously, somatic cells have even been preheated at nonphysiological temperatures prior to NT in order to relax higher order chromatin; however, development to term was not significantly higher than nontreated control [166]. Overall, when the “right” treatment is used (such as TSA), remodeling of donor chromatin appears to improve development of cloned embryos. A better understanding of chromatin remodeling following fer- tilization or NTmight help design a “cocktail” of drugs to efficiently remodel the differentiated chromatin of somatic donor cells. Embryo Aggregation Two hypotheses exist supporting the rationale of embryo aggregation. One involves the community effect in which the ability of a cell to take a specific differen- tiation pathway is enhanced when more neighboring cells are differentiating in the same way [167]. Since cloned embryos tend to have lower cell numbers than fertilized controls, at least in mice [168], the community effect obtained by aggregation might enhance the formation of the ICM and/or TE lineage [169]. The second hypothesis involves epigenetic embryo comple- mentation [168, 170]. While the aggregated embryos are genetically identical, reprogramming defects of one embryo can be compensated by another embryo, and vice versa. Although embryo complementation is largely unknown, it is thought that cell-cell communi- cation between blastomeres, by permeable gap junctions or by autocrine and paracrine factors, com- pensates for deficiencies between blastomeres [170]. It is possible that both hypotheses work together since greater cell numbers will increase the opportunities for epigenetic embryo complementation. Aggregation of four-cell cloned embryos improved developmental potential and gene expression. In mice, expression of Oct4 increased to normal levels, the number of cells was higher at the blastocyst stage, and development to term was increased eightfold compared to single-clone embryos [168]. In bovine, embryo aggregation resulted in blastocysts with double the number of cells and in upregulation of a subset of differentially expressed genes involved in transcription, biosynthesis, and signaling compared with single-clone embryos [170]. Overall, embryo aggregation is an interesting approach to improve the quality of a cloned blastocyst. RNA interference While this entry was in production, an article was published in which mice cloning efficiencies were increased tenfold and the gene expression of the resul- tant offspring was similar to IVF controls [171]. Such impressive outcome was simply achieved by knock- down of a single gene, Xist, by RNA interference. This study showed that cloned mouse embryos usually undergo permature overexpression of Xist as well as aberrant X chromosome inactivation. Although regu- lation of X chromosome inactivation differs between mammals [172], cloned bovine embryos were also observed to overexpress Xist [173]. Therefore, the next logical step would be to try the same Xist knock- down approach in livestock species. Summary Points ● SCNT involves using a somatic donor nucleus and an enucleated oocyte to produce an animal geneti- cally identical to the donor. ● The first NT experiments were carried out in 1928, the first successful SCNTs in the late 1950s, and the first cloned adult mammal (Dolly) in 1996. ● Basic research in the nuclear reprogramming mech- anism following SCNT could yield transferable knowledge to produce iPS cells safer for regenera- tive medicine. Realistic pharmaceutical/agricultural applications of SCNT include production of trans- genic animals and cloning prizewinning animals for breeding purposes. ● NT is commonly carried out with micromanipula- tors, although micromanipulator-free NT is possible and effective. NT steps include oocyte maturation, enucleation, nuclear transfer, fusion, activation, embryo culture, and embryo transfer. 1089Livestock Somatic Cell Nuclear Transfer
  • 41. ● Common problems in SCNT include low cloning efficiencies and developmental abnormalities. The placenta is mostly affected by the technique. ● Problems in SCNT probably originate from incom- plete reprogramming of the donor chromatin. Faulty chromatin reprogramming in cloned embryos includes hypermethylated DNA and increased H3K9me3, especially in the TE. ● Gene expression reprogramming is usually aberrant in cloned embryos, with differences between spe- cies. Pluripotent genes often fail to be reactivated in mice, although reactivation is normal when ES cells are used as donors. Fewer markers exist to assess reprogramming of pluripotency and TE lineage in livestock species, and the existing data are rather conflicting. Imprinted genes are often deregulated and differentiation-associated genes incompletely silenced. ● MII oocytes are the best recipients as they have high MPF levels. Reprogramming factors are lost when enucleating interphase recipients, which also have low MPF levels. ● Donor nuclei can be in G0, G1, or M phase of the cell cycle. No specific cell type has been found to be advantageous for NT; however, inconclusive data suggest that ES cells are superior donors than somatic cells. ● Centrosomal structures are recovered by introduc- tion of centrioles along with donor cell in livestock species, although normal spindle function is not always observed in reconstructed embryos. ● Increased compatibility between genomic DNA and mtDNA is beneficial for SCNT. ● In livestock species, cell fusion appears to result in better development than nuclear microinjection, consistent with centrosomal function recovery. ● There is no preferred method for oocyte activation. ● Normal embryo culture media are not optimal for reconstructed embryos. ● Cloned neonates are at high risk, and thus, intensive care should be provided to improve survival. ● Remodeling of the donor chromatin with TSA has been the single most successful innovation for improving consistently the efficiency of SCNT. ● Aggregation of cloned embryos seems to improve embryo quality by two working models: community effect and epigenetic embryo complementation. ● X chromosome inactivation appears to be deregulated in cloned embryos and correcting this by Xist knockdown can dramatically improve development. Future Directions Although efforts are being made toward dissecting the mechanism and the factors that bring about nuclear reprogramming of the donor chromatin, scientists are still quite far away from gaining a clear understand- ing. Even if there is general consensus that the low efficiency of SCNT originates from faulty nuclear reprogramming, there is no common agreement in where the bottleneck is. Does it lie in the reprogramming of the trophectodermal lineage or is the pluripotent lineage to blame? Is deregulation of imprinted genes the culprit of low cloning efficiencies or is it the incomplete silencing of differentiation- associated genes? Is incomplete remodeling of the overall chromatin structure preventing the SCNT technology from thriving? Does the bottleneck lie in the misregulation of X chromosome inactivation? Perhaps all these are contributing factors responsible for the low SCNToutcomes. Unless much further efforts are dedicated toward understanding the mechanism of nuclear reprogramming inside the oocyte, scientists will not have clear answers to such questions. The great improvement in cloning efficiencies observed with TSA treatment and Xist knockdown gives much hope and emphasizes that innovations can radically improve the technology. In basic science, SCNT is not limited to nuclear reprogramming. For instance, SCNT interferes with centrosomal function in livestock species, and more research is needed to understand donor centro- some behavior, spindle formation, and embryo cleav- age following NT in these species. SCNT can also provide insight into genomic-mitochondrial interac- tions, the effects of heteroplasmy, and preferential mitochondrial amplification. Such studies might war- rant a change in technique such as the method by which the donor chromatin is delivered into the cytoplast. As the science behind SCNT will be better under- stood through years of research, it is likely that the outcomes of this technology will closely match those 1090 Livestock Somatic Cell Nuclear Transfer
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  • 47. Lodging Resistance in Cereals PETE M. BERRY Sustainable Crop Management, ADAS UK Ltd, Malton, North Yorkshire, UK Article Outline Glossary Definition of the Subject Introduction Impact of Lodging on Grain Yield and Quality Mechanisms of Lodging Methods for Controlling Lodging Risk Future Directions Bibliography Glossary Anchorage failure moment Anchorage failure at the point of failure. Also described as anchorage strength. Base bending moment Wind-induced force acting on the base of the shoot or the anchorage system. Also described as leverage force. Brackling Lodging resulting from buckling of the upper half of the stems. Crop management Agronomic methods of growing crops. Failure wind speed Wind speed at which a plant will lodge. Hagberg falling number (HFN) Measure of bread making quality. Lodging Permanent displacement of cereal stems from their vertical position. Lodging-proof ideotype Plant dimensions required to achieve a lodging-return period of 25 years. Necking Lodging resulting from buckling of the stem just below the ear. Plant growth regulators – (PGRs) Chemical growth regulators that reduce the rate of stem extensions. Root lodging Lodging resulting from failure of the anchorage system. Stem failure moment Stem strength at the point of failure. Also described as stem strength. Stem lodging Lodging resulting from buckling of the lower stems. Definition of the Subject Lodging is the process by which the shoots of small grained cereals are permanently displaced from their vertical stance. Lodging limits yield potential and reduces grower profits, but it is difficult to control because it is a complex process that is influenced by many factors including wind, rain, topography, soil type, previous crop, crop management, and disease. Significant progress was made during the 1950s, 1960s, and 1970s to reduce lodging risk by the intro- duction of semi-dwarf varieties. The reduced lodging risk of these shorter varieties enabled them to respond to greater amounts of fertilizers and this was a signifi- cant reason for the steady improvement in global cereal grain yields starting in the late 1960s. However lodging is still a major problem in many countries and there is an urgent need to improve lodging resistance to further increase the yield of cereal species. Introduction Lodging is the permanent displacement of cereal stems from their vertical position (Fig. 1) and usually only occurs after the ear or panicle has emerged. This can reduce yield by up to 80% and causes several knock-on effects including reduced grain quality, greater drying costs, and slower harvest. It is a problem that limits cereal productivity in both developed and developing countries. Lodging is a complicated phenomenon that is influenced by many factors including wind, rain, topography, soil type, previous crop, husbandry, and disease. It is frequently associated with conditions that promote plant growth such as an abundant supply of nutrients. Significant progress was made during the 1950s, 1960s, and 1970s to reduce lodging risk by the introduction of semi-dwarf varieties. These shorter varieties had a greater lodging resistance and could respond to greater amounts of fertilizers. For these reasons the introduction of semi-dwarf varieties was one of the most significant reasons for the steady improvement in grain yields starting in the late 1960s, which has resulted in cereal yields increasing by as much as 1 t haÀ1 per decade in western Europe and 0.5 t haÀ1 in many American and Asian countries [1]. The continued improvement in yields in some 1096 Lodging Resistance in Cereals 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
  • 48. countries has been significantly aided by the use of plant growth regulators (PGRs) that further reduce crop height making cereals even more resistant to lodg- ing. Four major types of PGRs have been introduced including chlormequat chloride during the mid-1960s, ethephon during the late 1980s, trinexapac-ethyl during the mid-1990s, and prohexadione-calcium in the 2000s. In France, Germany, and the UK, which have among the highest cereal yields in the world, PGRs are now applied to more than 70% of wheat area (W. Rademacher 2004, personal communication). Dwarfing genes and PGRs have been very effective tools for reducing lodging risk and maintaining steady improvements in yield. However, they have not eradi- cated lodging and there is evidence that farmers will not be able to rely on these tools for further reductions in lodging risk in order to counter the escalating lodg- ing risk resulting from continued yield increases. Sev- eral studies have shown that yield is reduced when crops are shortened too much [2–7]. The reduction in yield appears to be exacerbated by high temperatures or drought stress. Several of these studies indicate that the minimum crop height for optimum yield lies between 0.7 m and 1.0 m. Many modern varieties are already within this height range. While there is scope for fur- ther shortening with PGRs through sequential applica- tions, pressure may be brought to bear to reduce their use because some PGRs leave residues in the grain [8]. It is therefore clear that new methods of improving lodging resistance in cereals must be developed. During the 1990s and 2000s collaborative studies by biologists and engineers have elucidated the mecha- nisms of lodging in cereals [9]. Crucially it has been demonstrated that lodging may occur by two mecha- nisms: stem lodging and root lodging. These studies have culminated in models of the lodging process which help to understand how the plant interacts with its environment during the lodging process and identifies the most important plant traits that must be targeted to improve lodging resistance. Several studies have also explained how variety, sowing date, seed rate, nitrogen fertilizer, and PGRs affect lodging. This improved understanding offers the prospect of design- ing a lodging proof ideotype for cereals which may be achieved through a combination of crop management and plant breeding. This entry on lodging resistance in cereals describes (1) the impact that lodging in different cereal species has on crop yield and grain quality, (2) the mechanisms of the lodging process, (3) the effect of cultivar choice and crop management on lodg- ing resistance, and (4) prospects for improving lodging resistance. This entry is intended to be a concise sum- mary of the most important aspects of lodging resis- tance in cereals, and for a more comprehensive review readers are directed to Berry et al. [10]. Impact of Lodging on Grain Yield and Quality Grain Yield Lodging can reduce cereal yield by reducing the grain size and number or through reducing the amount of crop that can be recovered at harvest. This section deals only with physiological reductions in yield associated with lodging. The greatest lodging-induced reductions in grain yield occur when crops are lodged flat at anthesis or early on in grain filling. Yield reductions from this type of lodging have been reported to reduce yields of wheat by [11] 31–80% [12], barley by 28–65% [13–15], oats by 37% [16], and rice by 38% [17]. All of the above studies, apart from Easson et al. [12], artifi- cially lodged the plants. This was achieved by growing the plants through wire netting and then moving the wires to effect lodging. This method has the advantage of lodging the crops at specific dates and at different angles, but may induce damage not normally incurred with natural lodging. Easson et al. [12] compared the yields of crops grown at high seed rate, which lodged Lodging Resistance in Cereals. Figure 1 Lodging in wheat 1097Lodging Resistance in Cereals
  • 49. naturally, with those at low seed rate, which experi- enced negligible lodging. Smaller yield losses have been observed when the angle of lodging is less than 90 from the vertical. Lodging at 45 causes between one quarter and one half of the yield losses incurred from 80 lodging in wheat [18], barley [14], and oats [16]. Smaller yield losses also occur when lodging occurs at a later stage of development. Artificial lodging at the ear emergence, milk, soft dough, and hard dough stages reduced yield by 31%, 25%, 20%, and 12%, respectively [11]. Stapper and Fischer [19] supported these observations by showing that about 0.5% of the potential yield was lost for each day of the grain filling period that a crop was lodged flat. Crops that lodge before anthesis often have smaller yield losses than crops that lodge soon after anthesis [18]. This appears to be associated with the upper one of two internodes bending upward to partially re-erect the crop. Crops that lodge after anthe- sis have completed stem extension and are unable to re- erect themselves. In natural situations, the re-erected crops are very unstable and are usually re-lodged by unexceptional weather conditions [12]. Grain Quality Artificial lodging has been observed to cause significant reductions in grain quality in terms of the bread mak- ing quality (measured as the Hagberg falling number [HFN]), individual grain weight, and the specific weight of the grain [10]. Lodging increases the likeli- hood of grain sprouting in the ear due to the more moist environment (Fig. 2) and this reduces HFN. Lodging induced during early grain filling reduced grain quality by reducing the HFN from 289 s to 114 s, reducing individual grain weight from 42.2 mg to 37.2 mg, and reducing specific weight from 70.3 kg/hl to 65.8 kg/hl. Lodging after early grain filling caused smaller effects on quality. A HFN of at least 250 s is required to produce good quality bread. Similar effects on grain weight and specific weight have also been observed in wheat [11, 20], barley [13, 21], and oats [22]. In the UK, the harvest year of 1992 was a severe lodging year with 16% of the wheat area lodged [23]. In this year the national average HFN fell from a 5-year average of 287 s to 254 s, thus significantly reducing the amount of bread making grain produced in this year. Also in this year, the specific weight of wheat grains fell from 77 kg hlÀ1 to 73 kg hlÀ1 and the number of small grains (2.0 mm) increased from 1.9% to 2.6% [24]. It is likely that at least a proportion of these effects were caused by the greater than usual lodging experienced in this country during this year. Mechanisms of Lodging Lodging can either occur through stem buckling (stem lodging) or displacement of the roots within the soil (root lodging) (Fig. 3). During stem lodging the roots are held firmly in a strong soil and the wind force buckles the stem. Stem lodging can occur due to buck- ling of the lower internodes. Buckling of the middle internodes is commonly known as “brackling” and is often observed in barley (Fig. 4) [25] and oats. Buck- ling of the peduncle just below the ear is known as “necking” and occurs most frequently in barley [26]. Root lodging becomes more likely when the anchorage strength is reduced by weak soil or poorly developed anchorage roots. Rainfall can reduce soil strength by several fold and has an important influence on the anchorage strength of cereals. Very few observations have been reported of the lodging process as it occurs and conjecture exists as to whether stem lodging or root lodging predomi- nates in cereals. Wheat and barley have been observed to root lodge [27, 28] and to stem lodge [25]. However, recently a quantitative understanding of root and shoot Lodging Resistance in Cereals. Figure 2 Sprouting in a lodged wheat crop 1098 Lodging Resistance in Cereals
  • 50. lodging has been developed for wheat and barley show- ing that both types of lodging are possible depending on the circumstances of a particular crop [29, 30]. This has been confirmed in wheat by direct observations of both mechanisms occurring during wind-tunnel experiments on field-grown winter wheat [31]. The lodging models described by Baker et al. [29] and Berry et al. [30] calculate the wind-induced base bending moment of a shoot from plant characteristics and meteorological data. The base bending moment is then compared to the failure moments (strength at the point of failure) of the stem base and of the anchorage system. Stem lodging is assumed to occur when the base bending moment of a single shoot exceeds the failure moment of the stem base. Root lodging is assumed when the base bending moment of all the shoots belonging to a single plant exceeds the failure moment of the anchorage system. The following sections describe current understanding about how the components of lodging (base bending moment, stem failure moment, and anchorage failure moment) may be calculated and what factors influence them. Base Bending Moment The wind-induced force acting on the upper sections of a shoot or plant results in a bending moment at the plant’s base. This can be described as the shoot leverage. The coherent waving of cereal shoots, apparent even in light winds, provides evidence that cereal shoots are subjected to varying forces and illustrates the impor- tance of including the shoot’s motion in any calculation of the applied base bending moment. Baker [32] attempted to account for the dynamic nature of shoot movement by considering the forces that act on an idealized shoot and assuming that the shoot’s move- ment could be modeled as damped harmonic oscillator. The theoretical modeling work described in [32] and [29], which was later validated in wind-tunnel experiments using field crops [31], showed that the wind-induced base bending moment of wheat could be calculated from a range of environmental and plant inputs. These include: the wind speed at crop height, the natural frequency of the shoot (rate at which it oscillates), the damping ratio of the shoot (which describes the rate at which oscillations die out), the height at center of gravity of the shoot, and the projected area and drag coefficient of the ear. These parameters can be used to estimate the bending moment at the base of the shoot for a shoot with a stiff stem such as wheat. Additional parameters are required to estimate the bending moment of more flexible stems such as for barley and include the flex- ural rigidity of the stem and its fresh weight. A method for calculating the bending moment of flexible stems is described in [30]. Lodging Resistance in Cereals. Figure 3 Wheat plants leaning as a result of root lodging Lodging Resistance in Cereals. Figure 4 Brackling in barley 1099Lodging Resistance in Cereals
  • 51. Anchorage Failure Moment There is uncertainty about the exact mechanism of anchorage failure in cereals due to the obvious diffi- culty associated with observing the process in field conditions. Ennos [33] showed that anchorage failure of spring wheat involved bending of the crown roots and resistance to axial movement through the soil. Crook and Ennos [27] showed that the upper portions of the crown root system of winter wheat form a cone (Fig. 5) and anchorage failure occurred when the root- soil cone rotated at its windward edge, the soil inside the cone moved as a block and compressed the soil beneath. This idea supported earlier observations by Pinthus [34], who showed that a wider angle of root spread was related to greater resistance to root lodging. Easson et al. [35] suggested that winter wheat roots acted like ropes to withstand root lodging and that anchorage strength would therefore be a function of the tensile strength of the roots on the windward side of the plant. The model developed by Crook and Ennos [27] has been tested and calibrated with field experiments on wheat [29] and on barley [30] (Fig. 6). These experi- ments showed that the anchorage strength was linearly related to the product of the diameter of the root cone cubed, the shear strength of the surrounding soil, and a constant specific to wheat or barley. The size of the root plate is identified by the parts of the crown roots that are surrounded by a rhizosheath. The rhizosheath is a dense mat of hairs that cover the upper sections of crown roots. These sections of roots have been shown to have an outer ring of lignified tissue in addition to the lignified central stele [27], which is why the rhizosheath can be used to estimate the length of root that provides anchorage. The observation that anchorage strength was line- arly related to the spread of the diameter of the root cone for both wheat and barley strongly suggests that both species have the same mechanism of anchorage failure first described by Crook and Ennos [27]. However, it was apparent that the constant factor was different between species with a value of 0.39–0.43 for wheat [29, 30] compared with 0.58 for barley demon- strating a greater anchorage strength for a given root plate spread for barley. This may have been caused by the greater number of stems per mm of root plate for barley compared with wheat. Up to 20 mm of the stem base is below ground, so it seems likely that a greater number of stems will increase the rotational resistance of the anchorage system. A model of soil strength developed by Baker et al. [29] showed that variation in clay content, moisture content, and compaction that is normally found within farmer’s fields could each be expected to alter the soil Lodging Resistance in Cereals. Figure 5 Upper portions of the root system of winter wheat 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 sd3 (Nm) BR(Nm) Lodging Resistance in Cereals. Figure 6 The product of soil shear strength (s) and root plate spread cubed (d3 ) plotted against failure moment (BR) for winter barley (ο; y = 0.58x, R2 = 0.95) and for winter wheat (5; y = 0.39x, R2 = 0.69) (Adapted from [30]) 1100 Lodging Resistance in Cereals
  • 52. shear strength by several fold. This indicates that the state of the soil is likely to be of paramount importance for determining lodging given that it has been predicted in the anchorage model described above to be directly proportional to anchorage strength. Stem Failure Moment Assuming that a typical stem can be considered to be analogous to a cylinder, Baker [32] showed that the stem failure moment (BS) can be expressed as: BS ¼ spa3 4 1 À a À t a 4 where s is the stem failure yield stress (material strength), a is the external radius of the stem, and t is the wall thickness. This formula assumes that the pith in the center of the stem does not contribute to the structural properties of the stem. Experiments by Neenan and Spencer-Smith [25] have shown that the stems of wheat and barley buckle at a certain critical ratio of radius of curvature to the outside diameter of the stem. Buckling was shown to occur suddenly with negligible amounts of plastic deformation. The Young’s modulus of wheat and barley was shown to remain reasonably constant for a range of stem curvatures which indicates that negligible plastic deformation occurs and that the limit of proportionality between the applied stress and corresponding strain is seldom exceeded. It may therefore be concluded that stem lodging occurs abruptly and will result in complete structural failure of the stem. Influence of Crop and Environmental Characteristics on Lodging Risk Lodging is a complex process that involves several environmental and plant characteristics. Any assess- ment of the effect of individual characteristics on lodg- ing risk must account for interrelationships between characteristics. To date the only study to have achieved this involves a sensitivity analysis of the lodging model of Baker et al. [29] and this is described in [9]. This study further developed the model of Baker et al. [29] to account for spatial non-uniformity between plants and temporal changes in plant structure during the growing season. A sensitivity analysis using this model showed that the risk of stem lodging is influenced most by changes in stem diameter and the risk of root lodging is affected most by changes to the diameter of the root cone (also referred to as the spread of the root plate) (Fig. 7). In these analyses, the risk of lodging is measured in terms of the wind speed required to cause lodging, which is termed the “stem” or “root” “failure wind speed.” The large effect on the chance of lodging caused by small changes in the failure wind speed is illustrated by the probabilities of experiencing different wind speeds above a UK wheat crop (Fig. 8). Methods for Controlling Lodging Risk Crop Management Cultivations The use of minimal cultivations or direct drilling to prepare seed beds have been shown to reduce lodging compared with more traditional methods which usually involve plowing to about 20 cm depth [36]. It seems likely that observations for direct drilling or minimal cultivations to reduce lodging are mainly caused directly by increased soil strength resulting from greater bulk density [37, 38]. The common observations for high bulk density to impede root extension and increase root thickness [39, 40] appear to be restricted to sections of the cereal root system that play little part in anchorage, namely, the seminal roots or the distal sections of the crown roots. Rolling to consolidate the soil is another manage- ment practice that has been shown to reduce lodging [34, 41–43]. This can be done immediately after the primary cultivations or can be done in spring to re- consolidate the top-soil after it has been loosened by cycles of freezing and thawing. Berry et al. [43] showed that rolling a sandy loam in the spring increased shear strength in the top 5 cm by 25% and this effect persisted until harvest. No effects were observed on the bio- mechanical properties of the wheat roots. This study also showed that rolling before growth stage (GS) 30 [44] reduced lodging, but rolling after GS31 had no effect on lodging. It was hypothesized that this treat- ment damaged the extending stems, which encouraged extra tillering, and these extra shoots countered the effects of the stronger soil. This theory was supported by rolling experiments to break cereal stems by Peltonen and Peltonen-Sainio [45]. 1101Lodging Resistance in Cereals
  • 53. Sowing Date and Seed Rate The lodging risk of wheat is almost always reduced by delaying sowing [19, 46–49]. Pinthus [50] cites two studies that show reduced lodging in barley when it is sown later, but [47] observed that early sowings could reduce or increase lodging in barley. Reducing the number of plants established also causes a large reduction in the lodging risk of wheat [12, 19, 48, 51–53], and of barley [54]. 0 10 20 30 10.50 Range Stemfailurewindspeed(ms-1) Stem radius (1–3mm) Stem wall width (0.3–1.6mm) Stem failure yield stress (15–80MPa) Centre of gravity (0.3–0.8m) Natural frequency (0.4–1.8 Hz) Ear area (5–20 cm2 ) Drag coefficient (0.5–1.5) Damping ratio (0.03–0.13) 0 5 10 15 20 10.50 Rangeb a Rootfailurewindspeed(ms-1) Shoot number (1–9) Root spread (15–80mm) Root depth (15–70mm) Centre of gravity (0.3–0.8m) Natural frequency (0.4–1.8 Hz) Ear area (5–20 cm2 ) Drag coefficient (0.5–1.5) Damping ratio (0.03–0.13) Lodging Resistance in Cereals. Figure 7 Failure wind speeds after 7 mm rain for (a) internode 1 and (b) anchorage. The ranges (0–1) are judged to represent the combined genetic and environmental range of each parameter within a high yielding wheat crop (Adapted from [9]). Stem radius – radius at the mid-point of the lowest internode; stem wall width – wall width at the mid-point of the lowest internode; stem failure yield stress – material strength of the stem wall of the bottom internode; center of gravity – height at center of gravity of the main shoot; natural frequency – rate at which the shoot oscillates; ear area – projected area of the ear; drag coefficient – resistance offered by the ear to wind; damping ratio – rate at which the shoot’s oscillations stop; shoot number – number of fertile shoots per plant; root spread – diameter of the root plate defined by the thickened regions of the crown roots; root depth – depth of the root plate defined by the thickened regions of the crown roots 1102 Lodging Resistance in Cereals
  • 54. The sowing date and seed rate effects described above are caused by changes to the structure of the crop. Berry et al. [48] showed that sowing winter wheat 6 weeks earlier increased both root and stem lodging risk by increasing the base bending moment of the shoot by about 30% and by reducing the strength of the stem base and the strength of the anchorage system by about 50%. Stapper and Fischer [19] have shown that early sowing results in a greater number of extended internodes, and this probably caused the lon- ger stems which gave rise to the greater base bending moment. Establishing 200 plants mÀ2 compared with 400 plants mÀ2 reduced lodging risk by increasing the strength of the anchorage system by more than 50% and the strength of the stem base by 15% [48]. The increase in anchorage strength more than compensated for the increase in shoot number on these plants. The greater anchorage strength has been attributed to sev- eral morphological changes including more roots per plant [12], stronger and thicker roots [55], and a wider and deeper root cone [48]. Sowing earlier or establishing more plants resulted in weaker stems because the stems were narrower and had thinner walls [48]. The mechanism by which weak stems develop is thought to be due to a greater number of shoots competing for limited photo-assimilate dur- ing early stem extension, which reduces the dry matter per unit length of the lower internodes [56]. Sparsely populated plants have many tillers [57] each of which develops up to four crown roots from each of their subterranean nodes. Therefore, it should be of no sur- prise that establishing fewer plants results in plants with more crown roots. Thicker and stronger roots may be caused by the absence of a strong shade avoid- ance response by the plant, which stimulates a greater proportion of assimilate to be partitioned to the roots [58]. Similar effects on anchorage strength were observed after later sowing as a result of fewer plants established. Drilling Depth and Seed Treatment Deeper sowing has also been found to reduce lodging in barley [50], but in general published evidence for sowing depth effects is scarce. This is probably caused by the plants ability to adjust its crown depth to about 40 mm for sowing depths of between 40 and 70 mm [59]. This means that sowing depths over this range are unlikely to affect the depth of the structural roots. However, drilling more shallowly than 40 mm may be expected to raise the crown and its structural roots, thus weakening anchorage. Evidence that altering crown depth can affect lodg- ing can be found from the effect of seed treatments: fluquinconazole [60] and triadimenol [61]. Studies on fluquinconazole showed that it shortened the sub- crown internode linking the seed to the crown (the part of the plant where the crown roots and tillers emerge). This deepened the crown and the depth of the root plate, which in turn increased anchorage strength and the resistance to root lodging. Observed natural root lodging also showed that the plots treated with the triazole seed treatment were less susceptible to root lodging [60]. Disease Scott and Hollins [62] showed that wheat crops with a greater incidence of sharp eyespot (Rhizoctonia), brought about through inoculation, had more lodging. It has been shown that severe levels of either disease can reduce the failure moment of the lower internodes by between 30% and 40%, thus increasing the likelihood of stem lodging [10]. Inter- estingly, slight or medium levels of disease did not appear to weaken the stems. There is no evidence that take-all root disease increases the risk of lodging. 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 15 20 25 Maximum summer wind speed (ms-1 ) Probability Lodging Resistance in Cereals. Figure 8 Probabilities of experiencing wind gusts independent of rainfall (—) and wind gusts with ! 7 mm daily rain (‐‐‐) between mid-June and mid-August within the main wheat-growing regions of the UK (Adapted from [9]) 1103Lodging Resistance in Cereals
  • 55. Nutrition An increased supply of available nitrogen from either mineralization of organic matter or from inorganic fertilizer has frequently been shown to increase lodging in wheat [48, 63, 64], barley [26, 65], and oats [66]. In wheat, the greatest increase in lodging is usually observed in response to early applications of nitrogen fertilizer before the onset of stem elongation [22, 23, 67], with applications after anthesis having no effect [52]. Contrary to this, Chalmers et al. [66] found that lodging in winter oats was reduced by applications of nitrogen before the onset of stem extension com- pared with later applications at GS30/31. Both Crook and Ennos [68] and Berry et al. [48] showed that increasing the nitrogen supply to winter wheat, through either greater amounts of soil residual nitrogen at sowing or through larger applications of fertilizer in the spring, reduced the strength of the stem base and, to a lesser extent, reduced the strength of the anchorage system. Increases in crop height were gener- ally small. Reductions in stem strength could be as much as 50% when high levels of residual nitrogen were combined with applications of fertilizer early in the spring [48]. Greater nitrogen supply almost always decreases the dry weight per unit length of the basal internodes of wheat [27, 48], barley [26], oats, and rye [22]. In relation to this, stem diameter and stem wall width are also frequently reduced. Berry et al. [48] showed that high levels of residual nitrogen reduced the strength of the stem wall material. These findings were supported by Crook and Ennos [27], who showed that a component of material strength, Young’s modu- lus (which approximates to the stiffness of the stem), was also reduced by more fertilizer in spring. The cause of these effects may have resulted from a reduced amount of lignified tissue within the sclerenchyma zone and the thickness of the sclerenchyma cell walls [22]. Reductions in anchorage strength in response to more nitrogen can be linked with fewer roots, which are thinner with smaller bending and tensile strengths [55, 68]. Mulder [22] showed that the crown roots of oat plants supplied with large amounts of nitrogen were practically free from lignified cells beneath the epidermis, in contrast with plants supplied with mod- erate amounts of nitrogen. In the consideration of nutrition, there should be a differentiation between the effects that result from repairing a deficiency and effects resulting from super-optimal supply. For example, it appears that an increase in nitrogen will increase lodging risk, but the mechanism by which this occurs will depend on the level of nitrogen supply. If a nitrogen deficiency is being repaired then lodging risk increases because the lever- age of the shoot and ear increases. It seems likely that the stem strength, and possibly the anchorage strength, will be increased by correcting the deficiency but these effects are outweighed by the greater leverage. Addi- tional nitrogen increases the shoot leverage by progres- sively smaller amounts, but lodging risk continues to rise because the strength of the stem base and root system begins to decrease as a result of the indirect effects of shading. It is possible that phosphorus behaves in a similar way to nitrogen [67]. However, potassium might be different as the evidence [22, 50, 69] indicates that it can reduce lodging when repairing a deficiency and additional amounts have no effect. This may be due to the important role that this element plays in regulating the turgor of plant tissues. Plant Growth Regulators Plant growth regulators (PGRs) are synthetic compounds that can be used to reduce lodging in cereal species. They are most com- monly used for this purpose in north and western European countries and in North America. In the UK, 89% of the winter wheat is treated with PGRs [70]. PGRs can be classified into two main groups: inhibitors of gibberellic acid biosynthesis and ethylene-releasing compounds. The most commonly used inhibitors of gibberellic acid biosynthesis in cereal crops are chlormequat chloride, mepiquat chloride, trinexapac- ethyl and prohexadione-calcium [71]. Ethephon is the most commonly used ethylene-releasing compound used on cereals [72]. Plant growth regulators have been shown to be a cost-effective method of reducing the incidence of lodging. PGRs applied before the emergence of the ear reduced lodging in almost all of the vast number of published experiments that have studied their effect and in which lodging occurred, with reductions in the percentage area lodged of anything up to 70% [10]. The primary mechanism by which PGRs have been shown to reduce lodging risk is by reducing crop height, with height reductions of up to 40% [10]. The variation on plant height reduction is probably caused by interac- tions between the type of active ingredient, the cereal 1104 Lodging Resistance in Cereals
  • 56. species together with the stage of plant development, and the environmental conditions when the chemical is applied. No evidence has been found for PGRs to increase the strength of the stem or of the anchorage system [48, 68], although it must be recognized that only two studies have investigated the effects of PGRs by directly measuring stem and anchorage strength. Chlormequat has been shown to be effective at reduc- ing lodging in winter and spring wheat, oats, and rye, but less effective on barley [73, 74]. Barley undergoes large height reductions in response to a mixture of ethephon and mepiquat chloride [13, 75]. Summary of Crop Management Effects Many crop management practices result in large changes in lodg- ing risk by either affecting the wind-induced leverage of the shoot, the strength of the stem base, the strength of the anchorage system, or a combination of all three mechanisms. Furthermore, the strengths of the stem base and anchorage system are often changed by differ- ent amounts for any change in crop management. This means that certain types of crop management would be expected to reduce one type of lodging (stem or root) more than the other. The effects of several crop manage- ment practices on the risk of stem and root lodging have been summarized by Berry et al. [76] in terms of changes to the failure wind speed (Table 1). This shows that stem lodging is best reduced by sowing on soils with less residual nitrogen and by reducing and delaying the amount of fertilizer applied in the spring. Root lodging is best reduced by establishing fewer plants, using a seed treatment with growth regu- latory properties and by rolling in the spring to con- solidate the soil. Delayed sowing and growth regulators were estimated to reduce stem and root lodging by equal amounts. Plant Breeding The Rht (Reduced height) alleles began to be intro- duced into wheat varieties during the 1960s and 1970s and are now part of the germplasm of most high yielding semi-dwarf varieties. In UK and German wheats, Rht1 and Rht2 alleles can reduce height by 14–17% independently of each other and by 42% when in combination [2]. Rht3 can reduce height by 59%, but has not yet been used in commercial varieties. In the UK, the Rht1 and Rht2 alleles have helped to reduce the height of wheat cultivars from over 1 m to about 0.7–0.9 m between the early 1970s and the mid- 1990s. This reduction in height and consequent reduc- tion in leverage has enabled the amount of nitrogen fertilizer applied to wheat to be increased from less than 100 kg haÀ1 in the early 1970s to nearly 200 kg haÀ1 in the 1990s [77] without a dramatic increase in the incidence of lodging. Pleiotropic effects have further added to the yield improvements associated with these Rht genes in wheat. Reduced stem growth rates allow Lodging Resistance in Cereals. Table 1 Effect of crop management on the wind speed required to cause stem or root lodging (Adapted from [60, 76] Factor Increase in stem failure wind speed msÀ1 Increase in root failure wind speed msÀ1 Less soil residual N (116–71 kg N haÀ1 ) 2.3 (3.9 a ) 1.3 Seed treatment with PGR activity (e.g., Fluquinconazole [60]) 0 0.7 Delayed sowing (per week, between 20 September and 1 November) 0.5 0.5 Less plants established (per 100 plants mÀ2 , between 400 and 200 plants mÀ2 ) 0.8 1.8 PGRs (split chlormequat @ GS30/31) 1.4 1.4 Delayed and less fertilizer N (Target GAI of 5) 1.4 0.8 Spring rolling (pre-GS30) 0 0.8 a At 400 plants mÀ2 1105Lodging Resistance in Cereals
  • 57. more resources to be allocated to the developing ear which results in a greater number of fertile florets and grains per year. In oats, the variety S172, released in 1939, has been reported to be Europe’s first dwarf cereal variety [78]. However, dwarfness in this and several derived varieties was associated with small grains and a yield penalty. Recently varieties have been released that contain the DW-6 dwarfing gene which was discovered in a mutation program in Canada [79] and has been shown to shorten the peduncle [80]. This gene has been shown to reduce height by 20–75 cm, have thicker stems, and to reduce lodging by large amounts [81]. Major dwarfing genes are common in spring barley. The ari-eGP dwarfing gene was found in cvs Golden Promise and Midas, which comprised over 70% of the Scottish barley crop from the mid-1970s to early 1980s. The ari-eGP gene was then superseded by the sdw1 dwarfing gene, such that by 1989 the percentages of certified seed carrying the sdw1 and ari-eGP genes were 74 and 8, respectively [82]. There is great potential to continue increasing lodg- ing resistance through further height reductions via the introduction of more extreme dwarfing genes such as Rht3 in wheat. However, several studies have shown that yield is reduced when crops are shortened too much [2–7]. The reduction in yield appears to be exacerbated by high temperatures or drought stress. Several of these studies indicate that the minimum crop height for optimum yield lies between 0.7 m and 1.0 m, a height which many modern varieties have already achieved. There is evidence that the same prob- lem could occur in oats because the DW-6 dwarfing gene has been associated with small grains, low kernel content and poor extrusion of the panicles from the flag leaves in some genetic backgrounds [81]. It there- fore seems unlikely that much further improvement in lodging resistance can be made by continuing to shorten wheat crops and there may be only limited further shortening possible in other cereal species. As a result breeders must target other plant traits, namely, stem strength and anchorage strength, to improve lodging resistance and counter greater yields. The lodging model of Baker et al. [29] has been used with preliminary datasets describing the dry matter costs of improving traits associated with stem strength and anchorage strength to estimate the dimensions of a wheat plant to make it lodging-proof for the least investment of biomass in the supporting stem and root system [83]. The characteristics required to give a crop yielding 8 t haÀ1 with 500 shoots mÀ2 and 200 plants mÀ2 a lodging return period of 25 years in a UK environment include a height of 0.7 m, a root plate spread of 57 mm, and for the bottom internode a wall width of 0.65 mm, a stem diameter of 4.94 mm, and a material strength of 30 Mpa (Fig. 9). Observa- tions of a range of varieties grown in the UK showed that the root plate of the best variety was 7 mm less than the ideotype target, the widest stem was 0.5 mm below the ideotype target, other stem character targets were achieved but not all in one variety, and the height target was achievable with the use of plant growth regulators. It is possible that the lodging-proof ideotype traits could be achieved because large differences among wheat varieties have been observed for the traits that determine stem strength and anchorage strength [42, 76, 84]. The latter study showed that anchorage strength could vary from 206 Nmm to 587 Nmm and stem strength could vary from 122 Nmm to 175 Nmm 50 mm 700 mm 9.4 cm2 0.95 Hz 57 mm BarleyWheat 0.65 mm 4.94 mm 4.77 mm Material strength 30 MPa Lodging Resistance in Cereals. Figure 9 Description of a lodging-proof ideotype for wheat grown in a UK environment as defined in [83] 1106 Lodging Resistance in Cereals
  • 58. between varieties. These differences were caused by a combination of wider, deeper root plates and stiffer roots for anchorage strength, and wider, thicker walled stems with a greater material strength for overall stem strength. Subsequent studies using more varieties and breeding lines showed even greater genetic variation in the traits which determine stem strength and anchor- age strength [83, 85]. In barley, differences in culm wall thickness have frequently been positively correlated with varietal differences in lodging resistance [86–88]. If oats and barley have a similar level of genetic varia- tion in stem and anchorage strength as has been observed in wheat then there appears to be significant scope for breeders to improve the strength of the stems and anchorage systems of cereals. However, these traits are time-consuming to measure; therefore, new tech- nologies will be required to help plant breeders to rapidly select them. Future Directions The majority of lodging research has concentrated on wheat. This has enabled models of the lodging mecha- nism to be developed, which have been used to identify the critical plant characters, to quantify the effects of factors on lodging and to elucidate the mechanisms by which these effects are caused. A rudimentary model of lodging has been developed for barley, but further work is required to fully validate this model, particularly the way in which flexural rigidity of the stem is considered. Understanding about lodging in other cereals, such as rice, maize, and oats, lags far behind wheat. In order to replicate the advances made in wheat the next step must be to model the lodging mechanisms in these cereals. This may require fundamentally different types of lodging model due to the different plant struc- tures of these species. Historically reducing crop height has been the main avenue by which the lodging risk of wheat has been reduced. However, several studies on wheat indicate that the minimum height compatible with high yields is around 0.7 m. This height has been achieved by many wheat varieties. Crop height also has a major impact on the structural dry matter requirements for lodging resistance with each additional centimeter in height increasing the stem dry matter required by 0.23 t/ha [83]. Further work must investigate why there appears to be a minimum height for high yield, whether this barrier can be overcome and whether the minimum height for high yield varies between environments. Preliminary studies with wheat have indicated that increasing stem strength has a significant dry matter cost which could compete with the formation of grain yield [83]. Further work must quantify the dry matter cost of increasing stem strength, understand the opti- mum combination of stem diameter, wall width, and material that is required to minimize the dry matter cost of increasing stem strength, and quantify the extent to which this competes with yield formation. It has been predicted that breeders must increase the stem strength and anchorage strength of wheat in order to achieve a lodging-proof ideotype for wheat. These traits are time-consuming to measure; therefore methods must be developed for rapidly assessing these traits. Berry et al. [89] and Keller et al. [90] have identified more than one quantitative trait loci associ- ated with these traits and indicated that they are con- trolled by several genes. Further work will be required to better understand the genetic control of the traits associated with lodging and to investigate whether reli- able genetic markers can be identified which work across a range of genotypes and environments. Pheno- typic screens must also be investigated to assess whether they can offer an alternative method to genetic markers for selecting germplasm. Recent work has shown that although a wide genetic variation for the key lodging traits is present within UK wheat breeding material [85], few of the target traits required for complete lodging proof have been identified. This indicates that a wider range of germplasm must be assessed to find the target traits. Currently there is not a reliable method predicting the likelihood of lodging from earlier stages of crop development, when growers can alter lodging risk through their crop management (PGRs, fertilizer, rolling). Therefore, further research must study the development of the lodging-associated plant characters with the objective of predicting lodging from early assessments of the crop. Any prediction scheme must predict lodging risk before, or soon after, the onset of stem elongation to enable growers to alter their crop management accordingly. PGRs reduce lodging risk by shortening crops, but there is little published evidence that they can 1107Lodging Resistance in Cereals
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