U.S. Department of Agriculture, Beltsville, Maryland
I. INTRODUCTION
II. GENETIC SELECTION AND MANAGEMENT STRATEGIES
III. GROWTH-PROMOTING AGENTSA. Appetite StimulantsB. Antibacterial Growth PromotersC. Rumen ModifiersD. Anabolic Steroids and Related SubstancesE. Endogenous SomatotropinF. Exogenous SomatotropinG. Growth Hormone Releasing FactorH. SomatostatinI. Beta-Adrenergic Agonists
IV. TRANSGENIC ANIMALSA. Swine with Growth-Related Transgenes
V. CONCLUSIONS
REFERENCES
I. INTRODUCTION
Biotechnology is the implementation of biological techniques to produce or modify prod-ucts and to manipulate cell genome and function. Use of science for the improvement ofmuscle foods has involved natural selection of dominant traits, selection of preferred traitsby cross-breeding, the use of endogenous and exogenous growth factors, and ultimatelygene manipulation and cloning to produce desirable changes in meat/carcass quality andyield.
Until recently, improvements in the quality of meat products that reached the mar-ketplace were largely the result of postharvest technologies. Extensive postharvest effortshave been implemented to improve or to control tenderness, flavor, and juiciness. Tender-
ness, flavor, and juiciness are the sensory attributes that make meat products palatable andare often the attributes that consumers consider when making their selection to purchasemeat products.
Consumers not only have been interested in the quality (palatability) but also havebeen concerned with the nutritional value, safety, and wholesomeness of the meat they con-sume. The public has been inundated with warnings about the health risks of consumingcertain types or classes of foods (in particular, the fat profile). Consumers became morehealth and weight conscious in the 1980s, desiring fewer calories in their diet. In fact, thisdecade was considered the “decade of nutrition.” However, present trends suggest thatthere is less concern among consumers about many of the substances previously viewed asharmful. There is also less concern about calories. Consumers appear to prefer traditionaland familiar food—foods they have always eaten. New technologies (e.g., biotechnology)and alternative production methods appear to hold great promise for improving the qualityand yield attributes of animal products.
A wide range of biotechnology strategies for altering the balance between lean andadipose tissue growth and deposition in meat-producing animals are available. These in-clude genetic selection and management (production) strategies. More recently, the confir-mation of the growth-promoting and nutrient repartitioning effects of somatotropin, so-matomedin, -adrenergic agonists, immunization of animals against target circulatinghormones or releasing factors, myostatin mutations, polar overdominance (callipyge muta-tion), and gene manipulation techniques have given rise to a technological revolution foraltering growth and development in meat producing animals.
II. GENETIC SELECTION AND MANAGEMENT STRATEGIES
The main genetic alteration during the past 40 years has been to decrease carcass fatnessand increase lean tissue deposition. These alterations have been via growth rate and maturesize of meat animals, particularly through genotypic and sex manipulation. There havebeen numerous papers on the effects of breed and sex condition on carcass composition andmeat quality, and therefore this will not reviewed in this chapter.
III. GROWTH-PROMOTING AGENTS
Growth-promoting agents are substances that enhance growth rate of animals without be-ing used to provide nutrients for growth such as nutrient partitioning agents. Growth-pro-moting agents are anabolic; that is, they produce more body tissues and thereby result inmore rapid animal growth. Growth-promoting agents cause changes in carcass composi-tion, mature weight, and efficiency of growth. Many substances qualify as “growth-pro-moting agents” despite their varied origin and chemical nature. Examples of growth pro-moting agents are the following: antibacterial agents (e.g., antibiotics), rumen modifiers(e.g., monensin, lasalocid), steroids (e.g., testosterone, estrogen), -adrenergic agonists(e.g., clenbuterol) and somatotropin (growth hormone). Growth-promoting agents influ-ence growth in three ways: (a) by stimulating feed intake (appetite stimulants) and therebyincreasing the supply of nutrients available for growth, (b) by altering the efficiency of thedigestive process, resulting in improved supply and/or balance of nutrients derived per unitof feed consumed (antibacterial agents, rumen additives, i.e., ionophores), and (c) by alter-ing the manner in which the animals utilize or partition absorbed nutrients for specificgrowth processes.
The effects of various growth-promoting agents on improving efficiency of growth,changing nutrient requirements (thereby increasing lean meat yield), and concomitantly de-creasing fat have been thoroughly reviewed (1–9). It is not the intent of this chapter to re-view all the plethora of literature on growth and development but instead to discuss the im-plications these biotechnological interventions have on muscle and meat quality.
A. Appetite Stimulants
Many growth-promoting agents influence voluntary feed intake; however, responses involuntary intake are generally small and inconsistent. Stimulating appetite (intake) couldresult in improved growth rate and the production of edible protein (lean tissue). Scientistsare investigating the use of drugs to control appetite. If successful substances are devel-oped, it may be possible to enhance meat production in the future by increasing or de-creasing an animal’s appetite.
B. Antibacterial Growth Promoters
A wide variety of antibiotics (antibacterial agents) enhance growth in primarily nonrumi-nant animals. Some common antibiotics that are used to remedy clinical infections are suc-cessful at promoting growth. Included in these common antibiotics are penicillins, tetracy-clines, bacitracin, avoparcin, and virginiamycin. These antibiotics are active againstgram-positive bacteria and, in most instances, are not retained in the tissues of the animal.As much as a 20% improvement in growth rate and feed efficiency have been observedwith the administration of antibacterial agents to nonruminants.
C. Rumen Modifiers
A special class of antibiotics whose principal site of action is the reticulorumen of rumi-nants has been investigated. These include monensin (Rumensin, Romensin) and lasalocid(Bovatec), which are classified as ionophores. Rumen additives are generally administeredto growing ruminant animals. The type of diet fed to the animal receiving rumen modifiershas an effect on the outcome. Voluntary intake is depressed when rumen modifiers are in-cluded in concentrate diets, whereas little depression in intake is observed when includedin forage-based diets. Improvements in growth rate and productivity are generally less than10%. The mode of action of rumen modifiers is poorly understood but is thought to be a re-sult of altering the metabolism (digestive process) of rumen microflora. Rumen modifiersact against gram-positive bacteria in the rumen, causing a shift in patterns of volatile fattyacid production, improved digestive efficiency, reduced bloat and reduced production ofmethane and hydrogen. In a study using lambs reported by Solomon et al. (10) and Fluhartyet al. (11) on the effects of energy source and ionophore (lasalocid) supplementation on car-cass characteristics, lipid composition and meat sensory properties suggested thationophore supplementation had no significant effect on either carcass/meat quality or yieldcharacteristics (10, 11). Diet (forage vs concentrate) offered more opportunities for manip-ulating carcass composition without jeopardizing meat quality than the use of ionophoresupplementation.
D. Anabolic Steroids and Related Substances
The importance of steroids such as androgens and estrogens in regulating growth is evidentfrom distinct differences between male and females in growth and composition. Castration
of males, which removes their primary source of androgens (testosterone), is the oldestmethod of manipulating growth by non-nutritional mechanisms. A number of steroid-re-lated growth-promoting agents that are either available for commercial use or have been ex-tensively researched exist. These are listed in Table 1. These agents are most effective incastrated males or females (especially in ruminants). Naturally occurring and synthetic es-trogens and androgens have been used to improve efficiency of growth and carcass com-position of meat animal for more than 50 years. Steroid-related growth-promoting agentsgenerally increase live weight gain and improve carcass lean-to-fat ratios in ruminants butare not as effective in swine. In fact, many of the growth-promoting agents are not approvedfor use in growing swine in the United States. Their mode of action is unclear but manyhave a direct effect on muscle cells. A review of the biosynthesis and metabolism of thenaturally occurring estrogens and androgens has been published (12). The literature ongrowth-performance responses to anabolic steroids indicates great variability, ranging fromno response in feedlot bulls (13) to a 70% increase in average daily gain in heifers (14). Theefficacy of anabolic steroid implants is summarized in several reviews (12, 15–18).
In a recent study (19) looking at the effects of different growth-promoting implantson muscle morphology in finishing steers, these researchers found Revalor implants to bemost effective, followed by Ralgro implants, in increasing lean mass through muscle fiberhypertrophy. Synovex implant had the least pronounced capacity for muscle growth en-hancement. Other studies (see exogenous somatotropin section) have compared combininganabolic steroids with somatotropin administration in cattle.
E. Endogenous Somatotropin
A group of peptide hormones, i.e., somatotropin (ST), growth hormone-releasing factor(GRF), somatostatin, insulin-like growth factor–I (IGF-I), insulin, and thyrotropic hor-mone, work in harmony to regulate and coordinate the metabolic pathways responsible fortissue formation and development. Even though relationships between growth and circu-lating levels of some of these peptide hormones have often produced conflicting results, themajority of data indicates that the genetic capacity for growth is related to increased circu-lating levels of somatotropin and IGF-I in livestock.
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Table 1 Steroid-Related Growth-Promoting Agents
Class Active agent(s) Trade name
Natural estrogens Estradiol-17B Compudose®Estrogen analogues Diethylstilbesterol DES
The anterior pituitary secretes three hormones (ST, prolactin and thyroid stimulatinghormone) that influence growth and carcass composition. Somatotropin, often calledgrowth hormone (GH), is the most notable, with commercial growth-promoting potential.Somatotropin is a small, single-chain polypeptide, made up of 191 amino acids, secretedby the pars distalis of the pituitary’s adenohypophysis. The structure of somatotropin variesamong species. Release of ST is stimulated by growth hormone releasing factor (GRF orGHRH) produced in the hypothalamus (20). Understanding how to control the productionof these hormones within the meat animal is a long-term goal of scientists.
F. Exogenous Somatotropin
1. Porcine Somatotropin
There is a growing database supporting the use of pituitary-derived porcine somatotropin(pST) as an agent to improve efficiency of growth and carcass composition in swine. Tur-man and Andrews (21) and Machlin (22) were the first to demonstrate that daily exogenousadministration (injection) of highly purified pST dramatically altered nutrient use, result-ing in improved growth rate and feed conversion of growing-finishing pigs. Pigs injectedwith pST had less (35%) fat and more (8%) protein.
However, their original observations were of little practical significance because pu-rification of porcine ST from pituitary glands was not economical. A single dose required25–100 pituitary glands. More recently, the development of recombinant deoxyribonucleicacid (DNA) technology has provided a mechanism for large-scale production of soma-totropin. The gene for ST protein is inserted into a laboratory strain of Escherichia coli,which can be grown on a large scale and from which ST can be purified and concentratedfor use (23). There is also a growing database supporting the use of recombinantly derivedpST (rpST). No significant differences between the effectiveness of pST and rpST havebeen observed.
With greater emphasis on lean tissue deposition and less lipid, the optimal genetic po-tential for protein deposition of an animal is a very important concept in that this potential,or ceiling, defines the protein requirement of the animal. In defining the optimal/genetic po-tential for protein deposition, ST is used as a tool to maximize genetic potential for proteinaccretion. Administration of pST to growing pigs elicits a pleiotropic response that resultsin altered nutrient partitioning. In studies with growing pigs, significant improvements of40% in average daily gain and 30% in feed conversions can be achieved by administrationof pST. Research has also shown that the effect of pST is enhanced by good managementand nutritional practices (24–26). Furthermore, a 60% reduction in carcass fat and a 70%increase in carcass protein content can be attained. The magnitude of response has variedin the various studies performed since the initial classical studies (21,22). Different inter-pretations in response have been attributed to differences in experimental designs. Theseinclude initial and final weight of pigs, length of study, genotype, sex, dose of ST, nutri-tional conditions, and time of injection. However, despite these differences in design, it isquite apparent that pST or rpST increases average daily gain by as much as 40%, decreasescarcass fat deposition by as much as 60%, and concomitantly increases carcass protein(lean) accretion by 70%.
Daily injections of pST has been the method of choice for pST administration.Porcine ST must be administered by injection because it is a protein and would be inacti-vated by digestive enzymes if given orally. The response from pST administration is not re-lated to the site of injection nor to the depth of injection. Recently, researchers (27, 28) in-
vestigated the effects of daily pST administration compared with injecting larger doses ofpST over an extended period of time. Frequency of administration influenced the magni-tude of responsiveness to pST treatment. Results indicated that optimal benefit would berealized by a delivery system that mimicked a daily surge of pST.
Mechanisms of pST action have been reviewed and discussed in numerous reviews(several listed above). Clearly, pST affects many metabolic pathways that influence theflow of nutrients among various tissues of the body. Many have concluded that the mech-anism by which pST decreases fat content in pigs is via the inhibition of lipogenesis (Table2). These changes in metabolism and cell proliferation lead to the alteration of carcass com-position via the reduction of nutrients normally destined to be deposited as lipid to othertissues. Studies have demonstrated that regardless of the stage of growth, pigs respond toexogenous pST at all ages. However, rapidly growing animals do not seem to benefit fromexogenous pST as much as do finishing animals with respect to fat alterations. This is notsurprising, because pigs that are growing rapidly and are more efficient are not producingmuch fat.
a. Meat Tenderness. Administration of pST represents a technology with apromise not only for improving production efficiency but also as a means for packers andretailers to offer leaner pork products. A multitude of pST studies have been performed inevaluating the flexibility of pST technology in conjunction with other variables (e.g., man-agement and environmental conditions) to alter carcass and muscle characteristics of mor-phology as well as meat palatability. These studies typically found that administration ofpST to barrows reduced tenderness (increased shear force) by as much as 39% in the longis-simus (LM) muscle and 15% in the semimembranosus (SM) when compared with controls.Although the reason for the increase in shear values representing reduced tenderness re-mains unclear, Solomon et al. (29) proposed that it may involve an alteration of the musclecomposition and/or the cold shortening phenomena of muscle. Solomon et al. (30) foundthat pST administration to boars (Table 3) and gilts lowered shear force of the LM (13.9%and 17.1%, respectively), thus improving muscle tenderness. Klindt et al. (31) reported thatextended administration of pST for 18 weeks to barrows resulted in reduced tenderness and
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Table 2 Biological Effects of Somatotropin on Adipose and Skeletal Muscle Tissue
Tissue Effect Physiological process affected
Adipose tissue ↓a Glucose uptake↓ Glucose oxidation↓ Lipid synthesis↓ Lipogenic enzyme activity↓ Insulin stimulation of glucose metabolism↑b Basal lipolysis↑ Catecholamine-stimulated lipolysis↑ Ability of insulin to inhibit lipolysis
Table 3 Comparisona of Total Carcass and Lean Tissue Lipid and Cholesterol Content, Longissimus Muscle Area, Shear Force andFiber Characteristics for Transgenic, pST-treated Fed Pigs
Significance
Item T-Control T-bST T-oST pST pST-Control SEM T pST
juiciness, but this was not observed with a 6-week pST administration period. It is difficultto determine whether these tenderness differences found in barrows will be perceived bythe consumer; however, it should be noted that most of the shear force values reported werewithin the shear values associated with normal pork products. Research is needed to deter-mine whether consumers and/or trained sensory panels can detect tenderness differences orother possible problems with pST-treated pork.
In a study (32), time post mortem of sampling muscle from pST-treated barrows forsubsequent shear force analysis had a significant effect on tenderness. Differences in shearforce tenderness between pST and control pigs were virtually eliminated when loin chopswere removed from the carcass and frozen within 1.5 hours post mortem compared withcontrols (frozen 5 days post mortem). Some of the inconsistencies reported in the literaturefor shear force and tenderness as a result of pST administration may be a result of incon-sistencies in the time that the meat sample is removed and subsequently frozen.
Minimal observable differences in processing yields, color retention, or compositionof products from control and pST-treated pigs have been observed. From the wealth of lit-erature, it appears that pST affects carcass composition and not quality (other than possi-bly tenderness). However, pale, soft, exudative (PSE) muscle has been observed in a cou-ple of pST studies (discussed in pST Muscle Morphology section).
b. Muscle Morphology. The consensus is that pST exerts a hypertrophic responseon carcass muscles, which can be seen at both the cellular level (fiber types) and with thenaked eye (carcass conformation and loin-eye area). Most of the research demonstrates thatmuscle fiber area increased in size with the use of pST. However, a rate-limiting factor inthe hypertrophic response of muscles to pST administration was the level of dietary proteinused (32) in combination with pST administration. This confirms that the beneficial effectsof pST is dependent on good management and nutritional practices. Porcine ST adminis-tration has little effect on the (percentage) distribution of muscle fiber types (Table 3).Solomon et al. (33, 34) reported that alterations in muscle fiber type populations are oftenassociated with differences in physiological maturation rates of the animals studied.
Sorensen et al. (35) observed that genotypes with relatively large muscle fibers areless responsive to pST treatment than genotypes with relatively small muscle fibers; otherinvestigators (29, 30, 32, 36, 37) observed hypertrophied (giant) fibers in muscles frompST-treated pigs (Table 3). Whether the giant fiber anomalies occurred through increasedactivity associated with compensatory (flux) adaptations or from fibers undergoing degen-erative changes has yet to be determined. The occurrence of giant muscle fibers has beenassociated with stress-susceptible pigs, which exhibit pale, soft, exudative (PSE) muscle.In two pST studies (30, 37), pST-treated pigs exhibited PSE muscle (30% and 62% inci-dence, respectively), which is much higher than the 12% that is reported as normal occur-rence by packers. One explanation offered for the increased incidence of PSE in these stud-ies was a seasonal effect. The experiments were conducted during the summer with averagetemperatures ranging above 35°C for the duration of the experiments. Perhaps pST admin-istration in conjunction with elevated environmental temperatures may induce the PSE syn-drome. However, one cannot discount the possibility that the occurrence of PSE in thesetwo studies and the absence of PSE in other studies could be due to genetic differences be-tween pigs used in the different studies. Aalhus et al. (38) did not observe an increase in theproportion of giant fibers or an interactive effect with the halothane genotype. They indi-cated that the effects of pST appear to be muscle and gender specific, which may be the re-sult of differences in maturity and rates of growth at the time of pST administration. Al-though they observed minor reductions in the muscle color (paler) and increased drip as
well as higher shear values, these quality differences were not always significant nor didthey result in PSE meat.
Ono et al. (39) reported on the effects of pST administration to barrows on muscleslocated within different regions of the body (i.e., locomotive vs postural muscles). Basedon the analysis of changes of the percentage of muscle fiber types from 20 to 90 kg bodyweight, they suggested that the fiber type transformation from small slow-twitch oxida-tive (SO) to large fast-twitch glycolytic (FOG) fibers plays an important role in musclesize enlargement as animals grow. Treatment with pST showed different effects on mus-cle fiber growth among the different fiber types and different locations of muscles in thebody, suggesting some relationship between pST and muscle function and metabolismand/or muscle maturation. Ji et al. (40) found that the enhanced muscle growth achievedby pST was not associated with altered expression of the p94 or �-actin gene (key genesrelative to muscle growth), or with an increase in the abundance of any calpastatin tran-scription product.
c. Carcass Composition. Lipid composition studies have demonstrated that thelipid content from pST-treated pigs was as much as 27% less in the lean tissue and 23% lessin the subcutaneous fat compared to controls. Carcasses (Fig. 1) from pST-treated pigs con-tained 22% less saturated fatty acids (SFA), 26% less monounsaturated fatty acids(MUFA), and no difference in polyunsaturated fatty acids (PUFA) compared with controls(41). The administration of pST resulted in lean tissue (Fig. 2) containing as much as 40%less SFA, 37% less MUFA and no difference in PUFA compared to controls. The subcuta-neous fat from pST-treated pigs contained 33% less SFA, 24% less MUFA, and 9% lessPUFA than controls. Cholesterol content in the subcutaneous fat from pST pigs was 12%higher than from control pigs. Cholesterol content of intramuscular fat was similar. Theseresults indicate that significant reductions in total lipid and all three classes of fatty acidscan be achieved using pST. This represents a favorable change in regard to human dietaryguidelines. Few differences in fatty acid profiles of the intramuscular fat extracted fromcooked pork rib chops as a result of pST administration have been observed (42). Choles-
Meat Biotechnology 135
Figure 1 Relative fatty acid composition for carcasses from transgenic and pST administered pigs.
terol content of cooked chops from pigs receiving pST in their study were greater than con-trols. Differences in cholesterol values may have resulted from less concentration of thecholesterol during cooking (heating).
Lonergan et al. (43) reported that pST treatment did not alter the overall fatty acidsaturation in subcutaneous or perirenal fat depots, but resulted in a greater reduction of themore saturated middle and inner layers of subcutaneous fat at the 10th rib location. As a re-sult, the more unsaturated outer layer of subcutaneous fat was present in a greater propor-tion in pST-treated pigs. Oksbjerg et al. (44) found a change to more polyunsaturated fattyacids and less saturated fatty acids in the backfat of pST treated gilts.
The decrease in total SFA and MUFA, and virtual no change in PUFA, support theconclusion that the mechanism by which pST decreases carcass fat content in pigs is by theinhibition of lipogenesis. A decrease in fat synthesis would lead to a decrease in the pro-duction of SFA and MUFA with little effect (change) in the amount of PUFA. The major-ity of PUFA in pig tissues are the result of dietary fatty acids linoleic and linolenic, and arenot synthesized. However, the possibility of an increased turnover of storage lipids, at thelevel of triacylglycerol synthesis or hydrolysis, exists (45).
Use of pST is similar to cattle implants and anabolic agents used to enhance growthin that all cause increases in growth and more efficient utilization of feed. However, theyare different in chemical structure. Porcine ST is a protein that is not active orally and isreadily digested like any protein. Since pST is a protein and is broken down in the gas-trointestinal tract, human ingestion of pST would present no dangers because digestiveprocesses would inactivate the protein and provide no residues. Digestion would breakthe protein down into its component amino acids, making it available for normalmetabolic processes. This has been one of the concerns for the acceptance of the use ofpST in pigs.
Caperna et al. (46) reported that daily pST treatment to growing barrows enhancedcollagen deposition in the skin, head, and viscera, whereas non-collagen protein depositionand collagen maturation were enhanced in the carcass tissues.
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Figure 2 Relative fatty acid composition for lean from transgenic and pST administered pigs.
There is evidence that exogenous bovine and ovine ST improves efficiency of growth andlean-to-fat ratio in ruminant animals. The database is much less extensive than it is forswine. Bovine somatotropin has been found to increase gain and protein deposition in feed-lot steers, but reports in the literature suggest the greatest effect on steers is in the noncar-cass fraction (47,48). An additive effect of estrogen and somatotropin treatment on weightgain and protein deposition of steers has been suggested (49,50). Rumsey et al. (51) foundthat rbST (Somavubove) and the estrogenic growth promoter Synovex-S independently in-creased growth and protein deposition in young beef steers. The combined treatments con-sistently demonstrated an additive effect on growth rate, carcass growth, protein deposi-tion, and the energy efficiency of protein deposition. The combination of these twogrowth-promoting agents was also effective in some muscles beyond the response obtainedwith either treatment alone (52,53). The overall muscle growth effects were greater whenthe two growth-promoting agents were combined than when they were administered sin-gularly. Similar results were observed (54) using rbST (Posilac) and Revalor-S (a tren-bolone acetate and estrogen implant).
Vann et al. (55) observed that rbST alone administered to creep-fed beef calves in-creased muscle mass but did not affect satellite cell number or concentration of myosinlight chain-1f mRNA. The increased muscling appeared to be the result of a greater distri-bution of FG fibers, which possess larger cross-sectional areas than the other fibers (56).
G. Growth Hormone Releasing Factor
Somatocrinin, often called growth hormone releasing factor (GRF) or growth hormone re-leasing hormone (GHRH), is a peptide hormone belonging to the glucagon family of thegut. Effects on growth of the administration or manipulation of GHRH are likely to be dueto direct effects on the secretion of ST. Exogenous GRF administration increases concen-trations of ST in serum of meat animals (57). Long-term administration of GRF has beenshown to stimulate growth in rats and in humans by increasing both the secretion and syn-thesis of pituitary ST (58).
H. Somatostatin
Somatostatin, another hormone produced by the hypothalamus, acts directly on the adeno-hypophysis of the pituitary gland to inhibit ST release (20). Endogenous ST secretion couldbe enhanced by GRF agonists or somatostatin antagonists. Enkephalins, also hypothalamicpeptides, stimulate ST release, and it is likely that enkephalin agonists could also be usedto enhance endogenous secretion (59).
Although somatostatin was originally purified from the hypothalamus, it is now rec-ognized that many cells and tissues throughout the body secrete this peptide. One of thesenetworks releases somatostatin to affect ST release (60). Several studies [reviewed (61)]used somatostatin antibodies to attempt to neutralize circulating levels of somatostatin inblood and promote growth by increasing blood ST concentrations. Although this techniqueappeared to be an attractive possibility for manipulating growth, immunization against so-matostatin did not consistently stimulate whole body growth. Lack of a response may re-sult from the multitude of sources (cells and tissues) that secrete somatostatin. Immuniza-tion against somatostatin has led to other strategies based on immunization techniques thatmay neutralize or amplify hormonal signals via receptors for hormones.
Passive (62) and active (63) immunization against somatostatin markedly increasedserum concentrations of ST. As a result of the increased concentration of ST, researchersset out to determine if immunization against somatostatin would improve growth in carcasslean:fat ratio. As much as a 22% improvement in ratio of gain and 13% improvement in ef-ficiency of gain in lambs was reported (64). However, no significant effect on carcass com-position was realized. This would be expected if the effect were mediated via ST or ST re-ceptors (65).
I. Beta-Adrenergic Agonists
The discovery of -adrenergic agonists, which are chemical analogues of epinephrine, nor-epinephrine, and catecholamines, is a promising development in growth-promotion appli-cations in animals. Included in this class of compounds are the following: clenbuterol,cimeterol, ractopamine, L-644,969 and L-640,033, and isoproterenol. -adrenergic ago-nists are orally active and thus may be administered in the feed. Many are chemically sta-ble and extremely potent, making successful development of implants for cattle, sheep, andswine likely. -agonists improved live weight gain (15%) and feed conversion efficiency(15%) in meat-producing animals in research trials. Carcass protein (muscle) content issubstantially increased (25%) while carcass fat is decreased (30%). These changes were ob-served in intact and castrated males and females. Unlike the effects of anabolic steroids, theeffects of -agonists were not dependent on sex of the animal. They represent an alteredpattern of metabolism such that nutrients are directed or partitioned away from adipose (fat)tissue and directed toward lean (muscle) tissue. For this reason, the term “nutrient reparti-tioning agents” is commonly applied to adrenergic agonists. Mechanisms (Table 4) bywhich -agonists influence growth have been reviewed (66–68).
Changes in carcass lean content is primarily the result of hypertrophy (increased cellsize), rather than hyperplasia (increased cell number). -agonists appear to exert their ef-fects on skeletal muscle by reducing degradation rate without altering the rate of protein
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Table 4 Biological Effects of -Adrenergic Agonists on Adipose and Skeletal MuscleTissue
synthesis. Muscle tissue is continually being degraded and resynthesized in animals. Ni-trogen retention in skeletal muscle is increased by -agonists yet decreased in skin/hide andvisceral organs. This decrease in nitrogen retention of non-carcass components, togetherwith reduced fat deposition, accounts for the increased carcass yield in animals treated with-agonists. Hypertrophy of cross-sectional area of skeletal muscle fiber types is typical ofall red-meat-producing animals treated with -agonists. Stimulating increased rates oflipolysis (fat breakdown) and decreased rates of lipogenesis (fat synthesis) reflect de-creased carcass fat deposition (69). Additionally, the supply of energy available for fat syn-thesis may be reduced in treated animals, both because an increased proportion of dietaryenergy is used for protein synthesis and because the -agonists elicit a general increase inmetabolic rate.
Growth-promoting -agonists were found to reduce plasma levels of insulin and so-matomedin-C, but did not elevate plasma ST levels. This suggests the concept that growth-promoting -agonists work directly through skeletal muscle cell receptors and not indi-rectly through the elevation of plasma ST or insulin concentrations (69). A critical factor inthe usefulness of -agonists is likely to be the degree to which their effects are retained af-ter withdrawal, because a recommended withdrawal period is likely to be required. Growthrates decline following withdrawal. Effects on the carcass are not as rapid but the advan-tages are not sustained after prolonged periods of withdrawal.
1. Porcine
In the majority of studies, daily gain was not significantly increased by feeding of -ago-nists. In fact, some studies showed a depression in growth rate. The repartitioning effectsof -agonists appear to increase with dose rate in pigs and result in a decrease in carcassfat. Minimal differences according to sex (gilt vs. barrow vs. boar) have been observed. Mi-nor increases in meat toughness have been reported for pigs treated with -agonists. In arecent review, Warriss (70) concluded that -agonists fed to pigs do not promote pale, soft,exudative (PSE) meat but might lead to a greater propensity for dark, firm, dry meat. -ag-onists appear to have no effect on water-holding capacity and ultimate muscle pH. Rumi-nants appear to be more responsive than swine to -agonists.
2. Bovine and Ovine
Response to treatment with -agonists has been studied in bulls, steers, and heifers as wellas rams, wethers, and ewes in different trials. Responses have ranged from no response toan improvement of 48% in growth rate and feed conversion. Beef carcasses treated with -agonists during the finishing phase contain less fat and more lean than controls. -agonistsare very effective in repartitioning utilizable energy away from fat deposition and towardprotein accretion in rams, wethers and ewes. Ruminants seem to be quite susceptible tomeat toughening when treated with -agonists.
Hamby et al. (71) were among the first to report that toughening of meat (lamb) oc-curs in -agonist–treated animals. Shear force in the longissimus muscle increased 114%in treated lambs. Although treated lambs had less carcass fat, Hamby et al. (71) concludedthat some factor other than cold-shortening was involved in the 114% increase in toughnessas a result of a low r2 value for linear regression of shear force on neutral lipid content. Thehypothesis that reduced tenderness is due to reduced protein degradation has been consid-ered (68). Collagen and its respective cross-linking is a major determinant in the texture(tenderness) of cooked meat (72). If -agonists cause an increase in growth by reducingprotein degradation, this would allow the collagen molecules more time to cross-link, (72).
The increase in cross-linking would enhance the toughness independently of postmortemproteolytic degradation. Beermann et al. (73) demonstrated a significant (as much as 70%)reduction of �M calcium dependent proteinase (CDP) activity in skeletal muscle of lambsfed a -agonist for 3 or 6 weeks. This reduction was synonymous with a dose-dependentincrease in shear-force values of treated lambs.
IV. TRANSGENIC ANIMALS
Development of recombinant DNA technology has enabled scientists to isolate singlegenes, analyze and modify their nucleotide structures, make copies of these isolated genes,and transfer copies into the genomes of livestock species. Such direct manipulation of ge-netic composition is referred to as genetic engineering, and the term transgenic animal de-notes an animal whose genome contains recombinant DNA.
The dramatic achievements in molecular biology during the past decade and the de-velopment of micromanipulation for early-stage embryos provided the combined capabili-ties for introducing cloned genes into the mouse genome in 1980 (74,75). The transfer ofgenes was immediately recognized as an important scientific achievement. However, thesubsequent creation of a “super” mouse by the transfer of a rat somatotropin gene providedthe convincing evidence that demonstrated the potential offered by gene transfer (76). Atthe Ohio University (Athens), transgenic mice carrying a modified version of the bovinesomatotropin gene that originally created the “super” mouse were found to produce “mini”mice (approximately half the size as the controls). Modifying a somatotropin gene and in-corporating it into mouse DNA that in turn prevents stimulating growth lends itself as apowerful tool for probing the hormone’s function. This suggests that somatotropin doesmore than promote growth. The first U.S. patent for a transgenic animal, a mouse express-ing a foreign oncogene created by Harvard University researchers, was issued in 1988. Theissue of that patent triggered intense criticism from animal rights activists, ethicists, and en-vironmentalists. As a result, the government did not issue any further patents. Environ-mentalists and others argue that there are dangers in releasing transgenic animals into theenvironment, as well as ethical issues that have not been fully explored.
Scientists had been struggling with practical problems involved in transferring genesfrom one animal to another. Scientists inserting genes from a variety of different species,for example the pig, have encountered significant problems. One unresolved major diffi-culty is the inability to insert genes precisely into animals’ DNA, the building block of ge-netics. Instead, the gene is inserted into the nucleus of a cell with the hope that the genelands in an appropriate location. In addition, many pigs develop severe health problems,e.g., ulcers, pneumonia, arthritis, cardiomegaly, dermatitis, and renal disease. The success-ful use of genetic engineering to enhance carcass composition and the efficiency of meatproduction in livestock depends on many factors. These include identification, isolation,and modification of useful genes or groups of genes that influence meat quality and quan-tity. Control of the time and level of expression of the inserted genes in transgenic animalsso that their health status is either improved or not diminished affects the successful inser-tion of these genes into the genome. The progress and methods of transferring genes in farmanimals was published in the 1995 ACS Symposium Series—Genetically Modified Foods:Safety Issues (77). Recently, Pursel et al. (78) reported on the successful expression of in-sulin-like growth factor–I (IGF-I) in skeletal muscle of transgenic swine. The generalhealth of the IGF-I transgenic and control pigs did not differ in physical appearance, be-
havior, ability to tolerate summertime heat stress, or reproductive capacity. These types ofproblems were observed for the transgenic pigs expressing bovine growth hormone (79).
In April 1997, a Scottish scientist, Ian Wilmut, successfully cloned a fully grownsheep. Named Dolly, the sheep represented the first time DNA from a mammal had beenused to produce an exact genetic replica. The Roslin Institute research team in Edinburghtook DNA from single mammary cells from a 6-year-old ewe, inserted it into fertilizedeggs, then implanted the eggs in 13 ewes, one of which became pregnant and gave birth tothe cloned lamb, Dolly. This method proved that mature cells other than reproductive cellscontain DNA capable of programming regeneration of an entire animal. In August of 1997,a Wisconsin-based company, Deforest, successfully cloned a Holstein bull calf. In June of1999, researchers at the University of Connecticut announced the birth of a cloned calffrom an adult farm animal using cells from the ear of the adult, not the reproductive organs.Similarly, scientists in Hawaii announced the successful cloning of mice using tail cellsfrom adult males, again the successful exhibition of cloning without using reproductive or-gans/cells. With regards to the cloning of farm animals, most of the research focuses onstate-of-the-art science and cutting-edge methodologies, technical improvements, and cur-rent progress toward producing transgenic animals for medical and agricultural applica-tions. To date, the effects of cloning farm animals on carcass and meat composition andquality have not been investigated.
A tremendous amount of variation in carcass components, such as muscle develop-ment, fat content distribution, tenderness, and flavor, exists among and within breeds ofeach species. Animal breeders have successfully utilized selection from this genetic varia-tion to improve farm animals for many years. Unfortunately, the quantitative genetic ap-proach has yielded few clues regarding the fundamental genetic changes that accompaniedthe selection of animals for superior carcass attributes. Few single genes have been identi-fied that have major effects on carcass composition. A national effort to map the genes ofmeat animals is under way. In cattle, the double-muscle gene is responsible for muscle hy-perplasia and hypertrophy and for enhanced lean tissue deposition. In sheep, the callipygemutated gene is responsible for muscle hypertrophy and enhanced lean tissue accretion. Inpigs, the halothane sensitivity gene (Hal) is associated with increased yield of lean meat andporcine stress syndrome. Pigs homozygous for “Hal” are susceptible to stress and have ahigh incidence of pale, soft, exudative (PSE) meat. These genes offer considerable poten-tial for investigation of carcass composition in meat-producing animals. However, exceptfor the Hal gene, which has been identified as a single mutation in the ryanidine receptorgene, the specific product of each gene remains to be identified.
A. Swine with Growth-Related Transgenes
A number of transgenic pigs containing various ST transgenes have been raised (80). Pro-duction of excess ST in transgenic animals caused multiple physiological affects but did notresult in “giantism” as was expected based on the earlier production of “super” mice as de-scribed (76). However, transgenic pigs that have excess ST levels exhibited numerousunique carcass traits. Reduced carcass fat, alteration of muscle fiber profiles, thickening ofthe skin, enlargement of bones, and redistribution of major carcass components occurred intransgenic pigs. Some of these effects are similar to those observed after daily injections ofpST; others are considerably different (Table 4). Possibly, these differences are the conse-quence of continual presence of excess ST in the transgenics whereas injections of pST pro-vide a daily pulse of excess ST.
The first transgenic animals for which carcass and meat quality was evaluated were the T-bST pigs at the USDA-ARS-Beltsville, MD, research facility. Carcass fat was dramaticallyreduced (Fig. 3) in transgenic pigs that expressed a bST transgene at five different liveweights (81). This difference in fat became greater among transgenic and non-transgeniclittermates as the pigs approached market weight. Total cholesterol content of ground car-cass tissue of bST transgenic pigs was not different from sibling control pigs at any of thedesignated weights. However, as body weight increased from 14 to 92 kg, the cholesterolcontent decreased for both groups of pigs. Analysis of fatty acids showed that carcasses ofbST transgenic pigs consistently contained less total (expressed as g/100 g tissue) SFA thansibling control pigs at each body weight. These differences in SFA were primarily a resultof reductions in palmitic, stearic and myristic acid. Both myristic and palmitic acids havebeen reported to be hyperlipidemic and hypercholesterolemic in humans. Consumption ofhypercholesteremic fatty acids by humans has come under attack by health professionals inthe United States. Carcasses from bST transgenic pigs contained less total MUFA andPUFA fatty acids than sibling control pigs. Similar observations (Fig. 2) were found for T-oST (transgenic pigs with an ovine somatotropin gene). Carcasses and lean tissue fromtransgenic pigs (Figs. 1 and 2) had near the optimum ratio of 1:1:1 for SFA:MUFA:PUFA,as recommended (82).
When carcasses were separated into the four primal (pork) cuts, the hams of the bSTtransgenic pigs were significantly larger and the loins were significantly smaller than thoseof the sibling control pigs (83). The intramuscular fat for each primal cut (lean portion only)showed large differences between bST transgenic pigs and the controls. In spite of thesedramatic reductions of fat throughout all primal cuts, evaluation of tenderness by shear-force determination indicated there were no significant differences between the two groupsof pigs for the longissimus (loin) muscle (Table 3).
Although somatotropin is considered the primary growth-promoting hormone inmammals, many of its effects are thought to be mediated by insulin-like growth factor-I(IGF-I). Transgenic pigs have been produced using a skeletal �-actin regulatory sequenceto direct expression of an IGF-I gene specifically in skeletal muscle (78). The underlying
142 Solomon
Figure 3 Carcass lipid accretion in control and transgenic pigs from 14 to 92 kg.
rationale was to initiate a paracrine response with IGF-I to enhance muscle developmentwithout altering the general physiology that might occur from an endocrine response.Founder T pigs were mated to non-T pigs to produce G1 transgenic and sibling controlprogeny. In comparison to sibling controls, T females and intact males had less carcass fat(9.9% and 8.1%, respectively), and more muscle (8.6% and 3.6%, respectively) (78). In afollow-up study, using barrows and gilts (84), IGF-I transgene pigs had larger (34%) loineye areas and heavier (range 9–24%) muscle weights of five major muscles of the carcass(84). Neither average daily gain nor feed efficiency differed for T and control pigs. T andcontrol pigs did not differ in general appearance, and no gross abnormalities, pathologies,or health-related problems were encountered as was observed for transgenic pigs with so-matotropin genes. Thus, enhancing IGF-I specifically in skeletal muscle had a positive ef-fect on carcass composition of swine. Shear force values for control pigs was 6.52 kg and6.42 kg for IGF-I T pigs (84).
2. Muscle Morphology
Morphological evaluation (Table 3) of bST transgenic-pig skeletal muscles revealed bST transgenic pigs had fewer SO fibers and more FOG fibers than control pigs. The population of FG was similar for the transgenic and control pigs; however, the classicalporcine fiber arrangement with SO fibers grouped in clusters surrounded by FOG and FG fibers was less evident in the transgenic muscle (85). Morphological fiber profiles for T-bST pigs resembled that of bovine muscle rather than porcine muscle. Hypertrophied(giant) fibers, which were identified in pST-injected pigs, were not observed in bST transgenic pigs (Table 3). The shift in the percentage of SO fibers to FOG fibers in the bST transgenic pigs has not been identified in pigs that have received daily injections of pST.
Muscle fiber growth patterns in bST and oST transgenic pigs differ markedly fromthat seen in muscle of pigs injected daily with pST. All three fiber types are enlarged inpST-treated pigs, whereas in bST transgenic pigs, only SO fibers appear to hypertrophyduring growth compared to controls. In the T-oST pigs, both the SO and FG fibers hyper-trophy similar to controls during growth, whereas the FOG fibers remain much smaller thancontrols (Table 3). No giant fibers were observed in muscle tissue from bST transgenicpigs. Even though bST transgenic pigs were highly stress sensitive, there were no signs ofpale, soft, exudative meat.
The IGF-I transgene pigs from the Pursel et al. (78) study as reported in Bee et al.(86) exhibited an increase in FG fibers and a decrease in FOG fibers. All fibers increasedin size, with the hypertrophic response being greatest for the SO fibers, followed by theFOG and FG fibers. The IGF-I transgene pigs from the Eastridge et al. (84) study showedthat there was no difference in fiber type percentages between the T pigs and controls, butthat the increase in muscle mass was due to an increase in muscle fiber area (hypertrophy)for all three fiber types. Bee et al. (86) also reported that IGF-I transgene expression alteredthe distribution of slow and fast isomyosin forms.
3. Bovine and Ovine with Growth-Related Transgenes
To date, producing transgenic cattle by microinjection of DNA into pronuclei is inefficientand extremely costly, in large part due to the cost of maintaining numerous pregnancies toterm. Many pregnancies result in non-transgenic progeny. The success rate in both bovineand ovine is significantly less than that for swine. No carcass data are available for trans-genic bovine or ovine. The cloning technology described by Wilmut in 1997 has introduced
the successful cloning of cattle and sheep, however, these research programs have notlooked at carcass data as well.
V. CONCLUSIONS
The potential for manipulation of growth and composition of farm animals has never beengreater than at present because of the wide array of strategies for altering the balance be-tween lean and fat. Recent discoveries of repartitioning effects of somatotropin, select -adrenergic agonists, as well as the variety of growth-promoting agents, and gene manipu-lation techniques offer a wide range of strategies. Although progress is being made, muchmore needs to be accomplished. Eating quality and safety must not be sacrificed as leaneranimals are developed. We are still a long way from fully understanding the integratedmechanisms resulting from manipulation of growth and carcass composition and possibleeffects on meat quality (either positive or negative) as a result of the techniques describedin this chapter.
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