Sampling Adipose and Muscle Tissue following Post-Harvest Scalding Does Not Affect RNA Integrity or Real-Time PCR Results in Market Weight Yorkshire Pigs.

Improving production efficiency while enhancing pork quality is pivotal for strengthening sustainable pork production. Being able to study both gene expression and indices of pork quality from the same anatomical location of an individual animal would better enable research conducted to study relationships between animal growth and carcass merit. To facilitate gene expression studies, adipose and muscle tissue samples are often collected immediately following exsanguination to maximize RNA integrity, which is a primary determinant of the sensitivity of RNA-based assays, such as real-time PCR. However, collecting soft tissue samples requires cutting through the hide or skin. This leaves the underlying tissue exposed during scalding, poses possible food safety issues, and potentially confounds pork quality measures. To overcome these limitations, the effect of tissue sample timing post-harvest on RNA integrity, real-time PCR results, and pork quality measurements was investigated by sampling subcutaneous adipose tissue and longissimus thoracis et lumborum muscle immediately following either exsanguination, scalding, or chilling. Sampling time did not affect RNA quality, as determined by the RNA integrity number of RNA samples purified from either adipose (RIN; p > 0.54) or muscle tissue (p > 0.43). Likewise, the sampling time did not influence the results of real-time PCR analysis of gene expression when comparing RNA samples prepared from adipose or muscle tissue immediately following either exsanguination or scalding (p > 0.92). However, sampling tissue prior to scalding resulted in a greater visual color score (p < 0.001) and lesser L* (p < 0.001) and b* (p < 0.001) values without impacting the 24 h pH (p < 0.41). These results suggested that if both RNA-based assays and meat quality endpoints are to be performed at the same anatomical location on an animal, tissue sampling to facilitate RNA-based assays should occur at a time point immediately following scalding. These findings demonstrated that sampling of adipose and muscle tissue can be delayed until after scalding/dehairing without decreasing the RNA integrity or altering the results of real-time PCR assays, while doing so was associated with little impact on measures of pork quality.

Pigs (Sus Scrofa) in Biomedical Research.

Much of biomedical oriented research is conducted with animal models. Over the years, rodents (primarily rats and mice) have emerged as the preferred species for basic biochemistry, cell biology, physiology and nutrition studies. In the past, dogs have been used for the evaluation of dietary protein quality and other aspects of animal nitrogen metabolism and physiology, cardiovascular and endocrine research. At an increasing rate, pigs have also been used as a model species in biomedical research. Pigs are readily available in various mature sizes and genotypic/phenotypic traits, and there are many anatomic, nutritional and physiologic similarities between human beings and pigs. Many notable reviews summarizing the role of pigs in biomedical studies have already been published and these are cited below. The present review focuses on characteristics that make pigs an excellent biomedical animal model in particular in obesity, diabetes and cardiovascular research. To procure an animal model for obesity, irrespective of species used, these animals must be fed a dense caloric diet (high fat) to achieve an experimental working model within a reasonable period. This review also focuses on a putative role of gastrointestinal microbiota in obesity as obese animals exhibit a shift in the distribution of gastrointestinal microbial phyla from lean animals. But to date such results have not pinpointed a treatable cause for obesity. Sometimes, the choice of sampling sites for microbial assessment in many reports can be questioned as the microbial content and phyla distribution in easily collected fecal samples may differ from those obtained directly from the small intestine and upper colon. While pigs are still utilized in many countries for medical surgery practice, this has been discontinued in US medical schools.

Amino Acids in Beef Cattle Nutrition and Production.

Proteins have been recognized for a long time as an important dietary nutritional component for all animals. Most amino acids were isolated and characterized in the late nineteenth and early twentieth century. Initially dietary proteins were ranked high to low quality by growth and N balance studies. By the 1950s interest had shifted to studying the roles of individual amino acids in amino acid requirements by feeding studies with non-ruminants as rodents, poultry and pigs. The direct protein feeding approaches followed by measurements of nutritional outcomes were not possible however in ruminants (cattle and sheep). The development of measuring free amino acids by ion exchange chromatography enabled plasma amino acid analysis. It was thought that plasma amino acid profiles were useful in nutritional studies on proteins and amino acids. With non-ruminants, nutritional interpretations of plasma amino acid studies were possible. Unfortunately with beef cattle, protein/amino acid nutritional adequacy or requirements could not be routinely determined with plasma amino acid studies. In dairy cows, however, much valuable understanding was gained from amino acid studies. Concurrently, others studied amino acid transport in ruminant small intestines, the role of peptides in ruminant N metabolism, amino acid catabolism (in the animal) with emphasis on branched-chain amino acid catabolism. In addition, workable methodologies for studying protein turnover in ruminants were developed. By the 1990s, nutritionists could still not determine amino acid requirements with empirical experimental studies in beef cattle. Instead, computer software (expert systems) based on the accumulated knowledge in animal and ruminal amino acids, energy metabolism and protein production were realized and revised frequently. With these tools, the amino acid requirements, daily energy needs, ruminal and total gastrointestinal tract digestion and performance of growing beef cattle could be predicted.

The history of adipocyte and adipose tissue research in meat animals.

Research in growth and development, accumulation of lean, and fat metabolism in farm animals was gaining attention principally from a carcass perspective by meat scientists and animal nutritionists about a century ago. Under the auspices of the USDA Cooperative State Research Service, State Agricultural Experiment Stations, and the Land Grant University system, researchers from various universities embarked on forming combined regional research projects (across states) with unifying specific aims. In the North Central region, this included states in the upper and lower Mid-West region. For those interested in improving production and eating quality of meats, initially a single multistate committee was formed in the North Central region which was active for many years. However, these efforts were later split into two committees with one addressing lipids and the other muscle biology. Herein we reviewed research of workers in the North Central region in the 1940s and 1950s and to a limited extent in the 2000s on meat animal's lipid metabolism. We further reviewed the history of meat animal carcass composition research and the influence of the Word War II (WWII) period on porcine carcass composition. The development and utilization of adipocyte cellularity research methodology in meat animals was demonstrated. The history of the progression of adipose tissue metabolism research in meat animals was also reviewed. Finally, the history of research on lipid deposition in muscle that ultimately precipitated the expanded marbling and the intramuscular research was delineated. By the 1970s, great interest had emerged on how to curtail excessive fat deposition in meat-producing animals. Thus, for some segments of the animal lipid metabolism community, the focus then shifted to exploring the processes of lipogenesis and lipolysis in farm animals. These efforts morphed into research efforts in fat cell biology and cellularity. Today adipocyte biology is studied by many in the biomedical and agricultural-life sciences communities. In this article, we present a history of this research and notable achievements up to the 1980s. Herein we revisit these research efforts and results that have become an important knowledge base for growth and development, nutrition, and meat science research.

Conserved forage-based systems for backgrounding weaned beef calves.

A 45-d backgrounding study was conducted to compare animal performance, forage nutritive value, digestion dynamics, and diet costs of conserved forage systems for weaned beef calves. One hundred and eight weaned Angus × Simmental beef calves (initial BW 279 ± 34 kg) were randomly assigned to one of three diets (n = 3 pens/treatment): 1) free-choice annual ryegrass (RB; cv. 'Marshall') baleage and 4 kg of a 50/50 mixture of pelleted soybean hulls and corn gluten feed, 2) free-choice Tifton 85 bermudagrass (BH) and 3 kg of a 50/50 mixture of pelleted soybean hulls and corn gluten feed, or 3) free-choice corn silage (CS; cv. Pioneer P1662YHR) and 2 kg of a 85% cracked corn and 15% cottonseed meal mixture. Diets were formulated to achieve a target gain of 0.9 kg/d based on the NRC (2000) requirement for a 270 kg growing calf. Animal performance (initial BW, final BW, and ADG) was measured on days 0 and 45 of the study. Forage nutritive value and an in vitro digestion trial were conducted to evaluate supplementation effects on forage diet digestion dynamics. Data were analyzed using PROC Mixed in SAS 9.4 as a completely randomized design. Pen was the experimental unit. Mean initial and final BW of the animals did not differ (P = 0.50 and P = 0.99, respectively) across treatments. Calf ADG for RB, BH, and CS diets were 0.61, 0.72, and 0.72 kg/d, respectively, and did not differ across treatments (P = 0.57). Based on these results, these forage options supported a similar level of gain when used for backgrounding beef calves. Forage in vitro DM digestibility differed 48 h after digestion, and BH + 50:50 had greater 48-h digestibility than when unsupplemented, which may be related to complementary forage-supplement interactions. In diets containing RB and CS, digestibility was greater with no supplementation at the 48-h time point. These data support the observation that supplementation type and level influence conserved forage diet digestibility compared with forage alone. The cost of feeding a baleage-based diet in this system was higher ($1.37/d) than CS or BH diets ($1.02 and $0.95/d, respectively). Results suggest that RB baleage-based diets may support a similar level of gain to BH or CS diets in growing beef calves, but supplement type, level, and ration costs should be evaluated when determining cost-effective backgrounding options in the Southeastern United States.

Peptide-Based Regulation of Metabolism as Related to Obesity.

Currently there are few, if any, approved and effective medicines for the attenuation of obesity, diabetes, and insulin resistance. This commentary addresses a communication describing the effect on glucose dynamics and obesity of a peptide monoclonal antibody for fibroblast growth factor (FGF) receptor 1c isoform (FGFR1c). The general lack of suitable, effective drugs is discussed, as is the treatment potential of FGF family receptors. The FGFR1c monoclonal antibody developed by the authors lowers body weight gain, blood glucose, and adipose tissue weight. It also enhances glucose uptake by fat cells [white adipose tissue (WAT) and 3T3-L1]. The robust weight loss, fat loss, and lower blood glucose were attributed to an observed potential futile cycle of continuous lipogenesis and lipolysis and adenosine triphosphate use in WAT.

Regulation of lipid deposition in farm animals: Parallels between agriculture and human physiology.

For many years, clinically oriented scientists and animal scientists have focused on lipid metabolism and fat deposition in various fat depots. While dealing with a common biology across species, the goals of biomedical and food animals lipid metabolism research differ in emphasis. In humans, mechanisms and regulation of fat synthesis, accumulation of fat in regional fat depots, lipid metabolism and dysmetabolism in adipose, liver and cardiac tissues have been investigated. Further, energy balance and weight control have also been extensively explored in humans. Finally, obesity and associated maladies including high cholesterol and atherosclerosis, cardiovascular disease, insulin resistance, hypertension, metabolic syndrome and health outcomes have been widely studied. In food animals, the emphasis has been on regulation of fatty acid synthesis and lipid deposition in fat depots and deposition of intramuscular fat. For humans, understanding the regulation of energy balance and body weight and of prevention or treatment of obesity and associated maladies have been important clinical outcomes. In production of food animals lowering fat content in muscle foods while enhancing intramuscular fat (marbling) have been major targets. In this review, we summarize how our laboratories have addressed the goal of providing lean but yet tasty and juicy muscle food products to consumers. In addition, we here describe efforts in the development of a new porcine model to study regulation of fat metabolism and obesity. Commonalities and differences in regulation of lipid metabolism between humans, rodents and food animals are emphasized throughout this review.

Small-intestinal or colonic microbiota as a potential amino acid source in animals.

Factors affecting physiological impacts of the microbiome on protein nutrition are discussed for hind-gut fermenters (humans, pigs, rodents). The microbiome flourishes in all gastrointestinal organs, and is a major source of amino acids to fore-gut fermenting animals. In humans, rats and pigs the net effect of microbiome biomass synthesis on amino acid requirements is much less certain. Dietary proteins, amino acids, peptides, endogenous-secreted protein and recycled urea may all be utilized as nitrogen source by growing bacteria in the small intestine and colon. The inclusions of radiolabelled amino acid precursors will result in labeled bacteria which can be digested and absorbed in the ileum and to some degree in the colon. This does not necessarily indicate a significant nutritional role of the microbiome in humans, pigs and rodents. The physiological attributes required for small-intestinal and colon microbiome utilization are a vigorous proteolytic digestion with pancreatic or intestinal enzymes and the presence of amino acid transporters. Findings to date seem to suggest that these two physiological attributes for effective bacterial protein utilization are present in the small intestine; however, these attributes have a much lower capacity/impact in the colon. The gastrointestinal microbiome is likely a protein source of medium to high nutritional quality, but overall the microbiome is not an important amino acid source in humans and animals fed amino acids at requirement levels.

Adipose depots differ in cellularity, adipokines produced, gene expression, and cell systems.

The race to manage the health concerns related to excess fat deposition has spawned a proliferation of clinical and basic research efforts to understand variables including dietary uptake, metabolism, and lipid deposition by adipocytes. A full appreciation of these variables must also include a depot-specific understanding of content and location in order to elucidate mechanisms governing cellular development and regulation of fat deposition. Because adipose tissue depots contain various cell types, differences in the cellularity among and within adipose depots are presently being documented to ascertain functional differences. This has led to the possibility of there being, within any one adipose depot, cellular distinctions that essentially result in adipose depots within depots. The papers comprising this issue will underscore numerous differences in cellularity (development, histogenesis, growth, metabolic function, regulation) of different adipose depots. Such information is useful in deciphering adipose depot involvement both in normal physiology and in pathology. Obesity, diabetes, metabolic syndrome, carcass composition of meat animals, performance of elite athletes, physiology/pathophysiology of aging, and numerous other diseases might be altered with a greater understanding of adipose depots and the cells that comprise them-including stem cells-during initial development and subsequent periods of normal/abnormal growth into senescence. Once thought to be dormant and innocuous, the adipocyte is emerging as a dynamic and influential cell and research will continue to identify complex physiologic regulation of processes involved in adipose depot physiology.

Dedifferentiated adipocyte-derived progeny cells (DFAT cells): Potential stem cells of adipose tissue.

Analyses of mature adipocytes have shown that they possess a reprogramming ability in vitro, which is associated with dedifferentiation. The subsequent dedifferentiated fat cells (DFAT cells) are multipotent and can differentiate into adipocytes and other cell types as well. Mature adipocytes can be easily obtained by biopsy, and the cloned progeny cells are homogeneous in vitro. Therefore, DFAT cells (a new type of stem cell) may provide an excellent source of cells for tissue regeneration, engineering and disease treatment. The dedifferentiation of mature adipocytes, the multipotent capacity of DFAT cells and comparisons and contrasts with mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPS) are discussed in this review.

Bovine dedifferentiated adipose tissue (DFAT) cells: DFAT cell isolation.

Dedifferentiated fat cells (DFAT cells) are derived from lipid-containing (mature) adipocytes, which possess the ability to symmetrically or asymmetrically proliferate, replicate, and redifferentiate/transdifferentiate. Robust cell isolation and downstream culture methods are needed to isolate large numbers of DFAT cells from any (one) adipose depot in order to establish population dynamics and regulation of the cells within and across laboratories. In order to establish more consistent/repeatable methodology here we report on two different methods to establish viable DFAT cell cultures: both traditional cell culture flasks and non-traditional (flat) cell culture plates were used for ceiling culture establishment. Adipocytes (maternal cells of the DFAT cells) were easier to remove from flat culture plates than flasks and the flat plates also allowed cloning rings to be utilized for cell/cell population isolation. While additional aspects of usage of flat-bottomed cell culture plates may yet need to be optimized by definition of optimum bio-coating to enhance cell attachment, utilization of flat plate approaches will allow more efficient study of the dedifferentiation process or the DFAT progeny cells. To extend our preliminary observations, dedifferentiation of Wagyu intramuscular fat (IMF)-derived mature adipocytes and redifferentiation ability of DFAT cells utilizing the aforementioned isolation protocols were examined in traditional basal media/differentiation induction media (DMI) containing adipogenic inducement reagents. In the absence of treatment approximately 10% isolated Wagyu IMF-mature adipocytes dedifferentiated spontaneously and 70% DFAT cells displayed protracted adipogenesis 12 d after confluence in vitro. Lipid-free intracellular vesicles in the cytoplasm (vesicles possessing an intact membrane but with no any observable or stainable lipid inside) were observed during redifferentiation. One to 30% DFAT cells redifferentiated into lipid-assimilating adipocytes in the DMI media, with distinct lipid-droplets in the cytoplasm and with no observable lipid-free vesicles inside. Moreover, a high confluence level promoted the redifferentiation efficiency of DFAT cells. Wagyu IMF dedifferentiated DFAT cells exhibited unique adipogenesis modes in vitro, revealing a useful cell model for studying adipogenesis and lipid metabolism.

A long journey to effective obesity treatments: is there light at the end of the tunnel?

As the obesity epidemic continues, more Americans are getting fatter, having more weight-related problems such as cardiovascular disease, and are experiencing new metabolic dysfunctions. For over 50 years, the adipose tissue (AT), commonly referred to as fat, has been of interest to academic and clinical scientists, public health officials and individuals interested in body composition and image including much of the average public, athletes, parents, etc. On one hand, efforts to alter body shape, weight and body fat percentage still include bizarre and scientifically unfounded methods. On the other hand, significant new scientific strides have been made in understanding the growth, function and regulation of anatomical and systemic AT. Markers of transition/conversion of precursor cells that mature to form lipid assimilating adipocytes have been identified. Molecular 'master' regulators such as peroxisome proliferator-activated receptor gamma and CCAAT-enhancer-binding proteins were uncovered and regulatory mechanisms behind variables of adiposity defined and refined. Interventions including pharmaceutical compounds, surgical, psychosocial interventions have also been tested. Has all of the preceding research helped alleviate the adverse physiologies of overweight and/or obese people? Does research to date point to new modalities that should be the focus of efforts to rid the world of obesity-related problems in the 21st century? This review provides a general overview of scientific efforts to date and a provocative view of the future for adiposity.

Emerging roles of zinc finger proteins in regulating adipogenesis.

Proteins containing the zinc finger domain(s) are named zinc finger proteins (ZFPs), one of the largest classes of transcription factors in eukaryotic genomes. A large number of ZFPs have been studied and many of them were found to be involved in regulating normal growth and development of cells and tissues through diverse signal transduction pathways. Recent studies revealed that a small but increasing number of ZFPs could function as key transcriptional regulators involved in adipogenesis. Due to the prevalence of obesity and metabolic disorders, the investigation of molecular regulatory mechanisms of adipocyte development must be more completely understood in order to develop novel and long-term impact strategies for ameliorating obesity. In this review, we discuss recent work that has documented that ZFPs are important functional contributors to the regulation of adipogenesis. Taken together, these data lead to the conclusion that ZFPs may become promising targets to combat human obesity.

Cell culture purity issues and DFAT cells.

Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

Topics in transcriptional control of lipid metabolism: from transcription factors to gene-promoter polymorphisms.

The central dogma of biology (DNA>>RNA>>Protein) has remained as an extremely useful scaffold to guide the study of molecular regulation of cellular metabolism. Molecular regulation of cellular metabolism has been pursued from an individual enzyme to a global assessment of protein function at the genomic (DNA), transcriptomic (RNA) and translation (Protein) levels. Details of a key role by inhibitory small RNAs and post-translational processing of cellular proteins on a whole cell/global basis are now just emerging. Below we emphasize the role of transcription factors (TF) in regulation of adipogenesis and lipogenesis. Additionally we have also focused on emerging additional TF that may also have hitherto unrecognized roles in adipogenesis and lipogenesis as compared to our present understanding. It is generally recognized that SNPs in structural genes can affect the final structure/function of a given protein. The implications of SNPs located in the non-transcribed promoter region on transcription have not been examined as extensively at this time. Here we have also summarized some emerging results on promoter SNPs for lipid metabolism and related cellular processes.

Lipid metabolism, adipocyte depot physiology and utilization of meat animals as experimental models for metabolic research.

Meat animals are unique as experimental models for both lipid metabolism and adipocyte studies because of their direct economic value for animal production. This paper discusses the principles that regulate adipogenesis in major meat animals (beef cattle, dairy cattle, and pigs), the definition of adipose depot-specific regulation of lipid metabolism or adipogenesis, and introduces the potential value of these animals as models for metabolic research including mammary biology and the ontogeny of fatty livers.

Skeletal muscle stem cells from animals I. Basic cell biology.

Skeletal muscle stem cells from food-producing animals are of interest to agricultural life scientists seeking to develop a better understanding of the molecular regulation of lean tissue (skeletal muscle protein hypertrophy) and intramuscular fat (marbling) development. Enhanced understanding of muscle stem cell biology and function is essential for developing technologies and strategies to augment the metabolic efficiency and muscle hypertrophy of growing animals potentially leading to greater efficiency and reduced environmental impacts of animal production, while concomitantly improving product uniformity and consumer acceptance and enjoyment of muscle foods.

Intestinal nitrogen recycling and utilization in health and disease.

The role of intestinal microflora in digestive and metabolic processes has received increasing attention from researchers and clinicians. Both enterocytes and small intestine luminal microorganisms can degrade peptides and amino acids (AA). Further, enterocytes can utilize ammonia via glutamate, glutamine, citrulline, and urea synthesis, whereas luminal microbes will deaminate AA, hydrolyze luminal urea, and recycle this ammonia by synthesis of new microbial cells. Although, undoubtedly, some indispensable AA may arise from N cycling and microbial synthesis in the intestinal lumen, the actual net impact on protein nutrition status appears to be limited in humans and animals. Moreover, potential contributions of the recycled N as colonic luminal microbial proteins to AA in blood depend on colonic protein digestion and AA absorption. Finally, new evidence indicates that gut microbial metabolism may be enhanced by prebiotics and probiotics, with the prospects of new treatment paradigms for eliminating undesirable secondary N metabolites and ameliorating complications in whole-body N metabolism under the conditions of intestinal stress, liver disease, and kidney failure.

Contribution of research with farm animals to protein metabolism concepts: a historical perspective.

The roles of proteins, carbohydrates, fats, and micronutrients in animal and human nutrition were broadly described during the late 18th and 19th centuries, and knowledge in protein nutrition evolved from work with all species. Although much of the fundamental and theoretical research in protein metabolism during the 20th century was conducted with laboratory animals, basic protein nutrition research in farm animals complemented those efforts and led to the development and use of new investigative methods (particularly in amino acid nutrition) as well as use of animal models in furthering the understanding of human protein metabolism. All these efforts have led to a contemporary hybrid model of protein nutrition and metabolism applicable to both humans and animal species. Now in the 21st century, farm animals are used in fetal and pediatric nutrition research, and data accruing for excess amino acid feeding in research with farm animals provide direction for assessment of pharmacological effects of amino acids when consumed in excessive quantities. Thus, as nutritional science is moving forward into nutrigenomics, nutriproteomics, and metabolomics, farm animal and human nutrition research interactions will likely continue with genetically modified farm animals produced for agricultural reasons (improved function and product quality) or those produced with human genes introduced to generate even better models of human protein metabolism.