INVITED REVIEW: Evolution of meat animal growth research during the past 50 years: Adipose and muscle stem cells.

If one were to compare today's animal growth research to research from a mere 50 yr ago, one would see programs with few similarities. The evolution of this research from whole-animal through cell-based and finally molecular and genomic studies has been enhanced by the identification, isolation, and in vitro evaluation of adipose- and muscle-derived stem cells. This paper will highlight the struggles and the milestones that make this evolving area of research what it is today. The contribution of adipose and muscle stem cell research to development and growth, tissue regeneration, and final carcass composition are reviewed.

Hydration profile and influence of beverage contents on fluid intake by women during outdoor recreational walking.

This study examined hydration status, sweat losses, and the effects of flavoring and electrolytes on fluid intake for women (n = 27, age = 24 ± 4 years) walking at a self-selected pace for ~1 h on a 1 km outdoor path during summer mornings or evenings. Over five consecutive days, participants consumed ad libitum one non-caloric beverage containing: (1) water (W), (2) acidified water (AW), (3) acidified water with electrolytes (AWE), (4) acidified water with flavor (AWF), and (5) acidified water with flavor and electrolytes (AWFE) in a counter-balanced order during walks and a 1-h recovery period. Walk Wet bulb globe temperature (26.2 ± 1.8 °C) and pace (6.0 ± 0.5 km/h) did not differ among beverages (P > 0.05). Thirty-four percent of pre-walk urine specific gravity samples exceeded 1.020. Flavoring (AWF 700 ± 393 mL; AWFE 719 ± 405 mL) did not result in greater consumption (P > 0.05) over W (560 ± 315 mL), with all three beverages exceeding grand mean sweat losses (528 ± 208 mL). Addition of electrolytes did not influence (P > 0.05) the intake between AW versus AWE or AWF versus AWFE. The results of this study indicate that the majority of women will consume fluids in excess of their sweat losses within 1 h post-walk. Over half of consumption took place during walks, highlighting the importance of fluid availability during exercise. Great among-subjects variability in sweat losses and fluid intake support the need for promoting individualized hydration strategies based on the changes in body mass for athletic populations.

Examination of adipose depot-specific PPAR moieties.

Molecular mechanisms of peroxisome proliferator activated receptors (PPARs) are being defined rapidly, as illustrated by the volume of papers published. Much of the research is directed towards a clinical end-point/application; however, the non-homogeneous nature of adipose depots in laboratory animals is spurring similar research in domestic meat animals (such as beef cattle). Moreover, the size of adipose depots in meat animals remains an attractive feature for using them to obtain cells for PPAR research. Examination of meat-animal depot-specific PPAR moieties may provide novel information about adipocyte regulation that might be extrapolated to all animals.

Board-invited review: the biology and regulation of preadipocytes and adipocytes in meat animals.

The quality and value of the carcass in domestic meat animals are reflected in its protein and fat content. Preadipocytes and adipocytes are important in establishing the overall fatness of a carcass, as well as being the main contributors to the marbling component needed for consumer preference of meat products. Although some fat accumulation is essential, any excess fat that is deposited into adipose depots other than the marbling fraction is energetically unfavorable and reduces efficiency of production. Hence, this review is focused on current knowledge about the biology and regulation of the important cells of adipose tissue: preadipocytes and adipocytes.

The influence of thiazolidinediones on adipogenesis in vitro and in vivo: potential modifiers of intramuscular adipose tissue deposition in meat animals.

Thiazolidinediones (TZD) are insulin sensitizing agents currently used for the treatment of type 2 diabetes and are widely used as adipogenic agents because they are ligands of peroxisome proliferator-activated receptor gamma (PPARgamma), a key adipogenic transcription factor. In vivo and in vitro studies of TZD as potential modifiers of intramuscular or marbling adipogenesis are reviewed. Thiazolidinedione-induced adipogenesis has been reported in numerous cell culture systems, including rodent, human, bovine, and porcine adipose tissue stromal-vascular (S-V) cell cultures. Studies of porcine S-V cell cultures derived from semitendinosus muscle show that TZD can potentially modify intramuscular or marbling adipogenesis. Preadipocyte recruitment was TZD-dependent in muscle S-V cultures but TZD-independent in adipose S-V cultures. There appear to be differences between adipocytes in muscle and subcutaneous adipose tissue, reminiscent of differences observed in adipocytes from different adipose tissue depots. Troglitazone, a TZD, induces marbling adipogenesis without inhibiting myogenesis when cells are grown on laminin precoated culture dishes. Additionally, troglitazone treatment does not increase lipid content in porcine adipose tissue or muscle S-V cell cultures. Thiazolidinedione treatment increases lipid content of muscle in rodents and humans; however, rosiglitazone treatment for 49 d in pigs did not influence muscle lipid content and meat quality, but several significant changes in muscle fatty acid composition were observed. Although timing of treatment with TZD needs to be optimized, evidence suggests these compounds may enhance marbling deposition in swine.

Secreted proteins and genes in fetal and neonatal pig adipose tissue and stromal-vascular cells.

Although microarray and proteomic studies have indicated the expression of unique and unexpected genes and their products in human and rodent adipose tissue, similar studies of meat animal adipose tissue have not been reported. Thus, total RNA was isolated from stromal-vascular (S-V) cell cultures (n = 4; 2 arrays; 2 cultures/array) from 90-d (79% of gestation) fetuses and adipose tissue from 105-d (92% of gestation) fetuses (n = 2) and neonatal (5-d-old) pigs (n = 2). Duplicate adipose tissue microarrays (n = 4) represented RNA samples from a pig and a fetus. Dye-labeled cDNA probes were hybridized to custom microarrays (70-mer oligonucleotides) representing more than 600 pig genes involved in growth and reproduction. Microarray studies showed significant expression of 40 genes encoding for known adipose tissue secreted proteins in fetal S-V cell cultures and adipose tissue. Expression of 10 genes encoding secreted proteins not known to be expressed by adipose tissue was also observed in neonatal adipose tissue and fetal S-V cell cultures. Additionally, the agouti gene was detected by reverse transcription-PCR in pig S-V cultures and adipose tissue. Proteomic analysis of adipose tissue and fetal and young pig S-V cell culture-conditioned media identified multiple secreted proteins including heparin-like epidermal growth factor-like growth factor and several apolipoproteins. Another adipose tissue secreted protein, plasminogen activator inhibitor-1, was identified by ELISA in S-V cell culture media. A group of 20 adipose tissue secreted proteins were detected or identified using the gene microarray and the proteomic and protein assay approaches including apolipoprotein-A1, apolipoprotein-E, relaxin, brain-derived neurotrophic factor, and IGF binding protein-5. These studies demonstrate, for the first time, the expression of several major secreted proteins in pig adipose tissue that may influence local and central metabolism and growth.

A comparison of thiazolidinedione-induced adipogenesis and myogenesis in stromal-vascular cells from subcutaneous adipose tissue or semitendinosus muscle of postnatal pigs.

This study compared the adipogenic potential of porcine stromal-vascular (S-V) cells from semitendinosus muscles and s.c. adipose tissue using thiazolidinediones. Stromal-vascular cells were obtained from s.c. adipose tissue and both semitendinosus muscles from 5- to 7-d-old pigs after collagenase digestion. Preadipocyte recruitment was measured using immunohistological evaluation for AD-3, a preadipocyte antibody. Ciglitazone increased the number of preadipocytes in adipose tissue but not semitendinosus muscle S-V cell cultures, whereas 10 microM troglitazone increased preadipocyte abundance in both adipose and muscle S-V cultures by approximately 3-fold (P < 0.05). Increasing troglitazone doses did not further increase preadipocyte number. Increases in preadipocytes were paralleled by increases in CCAAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) positive cells in adipose tissue S-V cultures, whereas PPARgamma-reactive but not C/EBPalpha-reactive cells were increased in muscle S-V cultures treated with 10 microM troglitazone. Additionally, troglitazone treatment did not increase lipid content in s.c. adipose tissue or muscle S-V cell cultures. Cells plated on laminin-precoated culture dishes were used to determine whether troglitazone influenced adipogenesis or myogenesis in cocultures from muscle S-V cells. There was no effect on the number of myotubes or the average number of nuclei per myotube, suggesting myogenesis was not impaired by troglitazone treatment. These results suggest that regulation of intramuscular adipogenesis differs from that of subcutaneous adipogenesis.

A method to establish co-cultures of myotubes and preadipocytes from collagenase digested neonatal pig semitendinosus muscles.

The relationships between adipocyte and muscle cell development within muscle are important in the study of factors or agents that may improve meat quality. Neonatal porcine muscle has the potential to yield both cell types for cell culture because it contains developing adipocytes and a high number of muscle satellite cells. Therefore, we modified a conventional collagenase-based procedure to digest neonatal porcine muscle and subsequently cultured the resultant muscle stromal-vascular (SV) cells on several substrata in basal and dexamethasone (DEX)-containing media. Developing myotubes and preadipocytes were present in muscle SV cell cultures on laminin substrata following seeding and plating with fetal bovine serum (FBS) with or without DEX. Myotube number was much higher (P < 0.05) on laminin substrata compared with all other substrata, whereas preadipocyte number in muscle SV cell cultures was independent of substrata, as we have shown previously. This approach can be used to establish co-cultures of differentiating adipocytes and myotubes from collagenase-digested neonatal pig muscle. Because the comparison is within the same culture dish, this method allows for a direct comparison of the responses of adipogenic and myogenic cells to growth and differentiation factors. For example, DEX did not alter myogenesis (i.e., 11 +/- 3 vs. 11 +/- 4 myotubes per unit area for control and DEX-treated cultures, respectively), but it has been shown to markedly increase preadipocyte number in muscle SV cell cultures.

Recruitment and differentiation of intramuscular preadipocytes in stromal-vascular cell cultures derived from neonatal pig semitendinosus muscles.

The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte recruitment and expression of CCAAT/enhancing binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) proteins in stromal-vascular (SV) cell cultures derived from neonatal subcutaneous adipose tissue and semitendinosus muscles. One adipose tissue SV cell culture and one semitendinosus muscle SV cell culture were established from each of six young pigs (5 to 7 d of age). Conventional SV cell-culture procedures were used to digest adipose and muscle tissue and to harvest and culture adipose and muscle SV cells. Muscles were digested after the removal of all visible connective tissue from the excised muscle. One hour after seeding, muscle SV cell cultures were rinsed and refed new media to remove debris and insoluble muscle protein. The SV cell cultures were double-stained for lipid and the AD-3 antibody, a preadipocyte marker, at 1, 3, and 6 d and were double-stained for lipid and C/EBPalpha or PPARgamma at d 6. Preadipocytes were randomly distributed and not clustered after 1 d in muscle and adipose SV cultures. Regardless of treatment, relative and absolute fat cell numbers were lower (P < 0.05) in muscle than in adipose-SV cell cultures. The DEX treatments produced similar magnitudes of increase in relative and absolute preadipocytes and adipocytes in muscle- and adipose-SV cultures. Several extracellular matrix substrata had no influence on adipogenesis in muscle-SV cell cultures. These studies indicate that muscle-SV cultures are characterized by a low number of adipocytes under basal conditions and a low number of glucocorticoid-responsive preadipocytes.

Intravenous versus intrastriatal cord blood administration in a rodent model of stroke.

Human umbilical cord blood (hUCB) is a rich source of hematopoietic stem cells that have been used to reconstitute immune cells and blood lineages. Cells from another hematopoietic source, bone marrow, have been found to differentiate into neural cells and are effective in the treatment of stroke. In this study, we administered hUCB cells intravenously into the femoral vein or directly into the striatum and assessed which route of cell administration produced the greatest behavioral recovery in rats with permanent middle cerebral artery occlusion (MCAO). All animals were immunosuppressed with cyclosporine (CSA). When spontaneous activity was measured using the Digiscan automated system, it was found to be significantly less when hUCB was transplanted 24 hr after stroke compared with nontransplanted, stroked animals (P < 0.01). Furthermore, behavioral recovery was similar with both striatal and femoral hUCB delivery. This is in contrast to the step test, in which significant improvements were found only after femoral delivery of the hUCB cells. In the passive avoidance test, transplanted animals learned the task faster than nontransplanted animals (P < 0.05). Together, these results suggest that hUCB transplantation may be an effective treatment for brain injuries, such as stroke, or neurodegenerative disorders. In addition, intravenous delivery may be more effective than striatal delivery in producing long-term functional benefits to the stroked animal.

Pre- and postnatal dietary conjugated linoleic acid alters adipose development, body weight gain and body composition in Sprague-Dawley rats.

Sprague-Dawley rats were fed either a control diet (7 g/100 g soybean oil) or a conjugated linoleic acid (CLA) diet (6.5 g/100 g soybean oil and 0.5 g/100 g CLA) beginning on d 7 of gestation to determine whether pre- and postnatal CLA affects short- and long-term growth and adiposity. At weaning (d 21), progeny were assigned control or CLA diet and fed until 11 wk of age. At birth, litter size and weight were not different between treatments. There were age- and sex-dependent changes in inguinal adipose fatty acid composition at birth and weaning, whereas there were no differences in lipid accretion or adipocyte proliferation. At weaning, CLA did not alter inguinal adipocyte proliferation but increased (P < 0.01) CCAAT/enhancer binding protein alpha expression in inguinal adipose tissue from females, whereas there was no difference in expression in males. Significant differences in size distribution of inguinal adipocytes at weaning and retroperitoneal adipocytes at 11 wk of age were observed. In general, CLA increased the proportion of smaller cells and decreased the proportion of larger cells. The main long-term effect of the dams' diet was the significantly heavier gastrocnemius and soleus muscles, and significantly longer tail lengths, an indication of skeletal growth, of male pups whose dams were fed CLA. Postweaning diet reduced fat pad weights in female but not male pups fed CLA. This response was due to differences in cell size rather than number. Response to CLA treatment may depend on the sex and age of the animal as well as duration of feeding.

Artificial lighting conditions and melatonin alter motor performance in adult rats.

Entrained circadian rhythms may modulate many behavioral activities of animals and humans. In the present study, we examined whether lighting conditions and melatonin treatment participate behaviorally in the entrainment of circadian rhythms in the rodent. In experiment one, Sprague-Dawley rats were introduced to the Rotorod test apparatus at nighttime or daytime and either with the lights on (4 lux) or in the dark. During nighttime tests, the exposure of rats to dark or light condition did not alter mean rev./min or length of times spent on the Rotorod. Interestingly, during daytime tests, animals exposed to light condition displayed significantly reduced mean rev./min (7.95 +/- 1.68), as well as length of time on the Rotorod (41.07 +/- 3.45 s) compared with their performance in the dark condition (mean rev./min, 11.16 +/- 1.52; length of time spent on the Rotorod, 66.94 +/- 6.15 s). In experiment two, treatment with melatonin (1.5 mg/kg, orally administered at 1 h prior to testing) in animals introduced to the daytime test with exposure to light condition, restored the rev./min (12.90 +/- 1.26) and the time spent on the Rotorod (63.21 +/- 2.73 s) to near normal levels. Thus, we demonstrated here that exposure of nocturnal animals to their preferred dark condition and treatment with melatonin could enhance motor coordination.

Sertoli cells decrease microglial response and increase engraftment of human hNT neurons in the hemiparkinsonian rat striatum.

Sertoli cells (SCs) provide immune protection and nutritive support to the developing germ cells in the testis. Sertoli cells have also been shown to provide immune protection to islets transplanted outside the testes. In this study, the ability of these cells to diminish the infiltration/activation of microglia into a neural graft implanted in the lesioned striatum of a hemiparkinsonian rat was investigated. Human neuron-like cells (hNT neurons) were implanted either alone or in combination with rat SCs. Three months later, the animals were sacrificed and immunohistochemistry was performed to determine the survival of the xenografted neurons as well as microglial infiltration/activation. Cotransplantation of the SCs with the hNT neurons increased graft survival and was associated with an increase in graft size. Furthermore, there were fewer microglia present in the grafted tissue of the cotransplantation groups. These results show that SCs retain their immunosuppressive ability even within the brain. As immune responses to grafted neural tissue within the central nervous system become better understood, this ability of the SCs to provide localized immunosuppression to the transplanted tissue may become more important. This is particularly true as the search for alternative sources of neural tissue to treat neurodegenerative diseases expands to encompass other species.

Sertoli cells enhance the survival of co-transplanted dopamine neurons.

One of the major issues in neural transplantation is the low survival rate (<5%) of transplanted dopamine (DA) neurons [3]. Recently it has been shown that it is possible to enhance the survival of these neurons, which in turn may decrease the amount of tissue that is required for each transplantation patient. The present paper demonstrates a novel approach for enhancing neuronal survival by co-transplantation of neuronal tissue with Testis-derived Sertoli cells (SC). This strategy could improve neuronal survival through the provision of trophic support.

Viability and survival of hNT neurons determine degree of functional recovery in grafted ischemic rats.

We recently reported behavioral improvements following intrastriatal transplantation of cryopreserved cultured human neuroteratocarcinoma-derived cells (hNT neurons) in rats with cerebral ischemia induced by occlusion of the middle cerebral artery. In the present study, the viability and survival of hNT neurons were evaluated immediately prior to the transplantation surgery and at 3 months post-transplantation in ischemic rats. Cryopreserved hNT neurons were routinely thawed, and trypan blue exclusion viability counts revealed 52-95% viable hNT neurons before transplantation. Monthly behavioral tests, starting at 1 month and extending to 3 months post-transplantation, revealed that ischemic animals that were intrastriatally transplanted with hNT neurons (approximately 40000) and treated with an immunosuppressive drug displayed normalization of asymmetrical motor behavior compared with ischemic animals that received medium alone. Within-subject comparisons of cell viability and subsequent behavioral changes revealed that a high cell viability just prior to transplantation surgery correlated highly with a robust and sustained functional improvement in the transplant recipient. Furthermore, histological analysis of grafted brains revealed a positive correlation between number of surviving hNT neurons and degree of functional recovery. In concert with similar reports on fetal tissue transplantation, we conclude that high cell viability is an important criterion for successful transplantation of cryopreserved neurons derived from cell lines to enhance graft-induced functional effects.

Bilateral fetal striatal grafts in the 3-nitropropionic acid-induced hypoactive model of Huntington's disease.

We investigated the 3-nitropropionic acid (3-NP)-induced hypoactive model of Huntington's disease (HD) to demonstrate whether fetal tissue transplantation can ameliorate behavioral deficits associated with a more advanced stage of HD. Twelve-week-old Sprague-Dawley rats were introduced to the 3-NP dosing regimen (10 mg/kg, i.p., once every 4 days for 28 consecutive days), and were then tested for general spontaneous locomotor activity in the Digiscan locomotor apparatus. All rats displayed significant hypoactivity compared to their pre-3-NP injection locomotor activity. Randomly selected rats then received bilateral intrastriatal solid grafts of fetal striatal (lateral ganglionic eminence, LGE) tissues from embryonic day 14 rat fetuses. Approximately 1/3 of each LGE in hibernation medium was infused into each lesioned host striatum. In control rats, medium alone was infused intrastriatally. A 3-mo posttransplant maturation period was allowed prior to locomotor activity testing. Animals receiving fetal LGE grafts exhibited a significant increase in locomotor activity compared to their post-3-NP injection activity or to the controls' posttransplant activity. Surviving striatal grafts were noted in functionally recovered animals. This observation supports the use of fetal striatal transplants to correct the akinetic stage of HD. To the best of our knowledge, this is the first study that has investigated the effects of fetal striatal transplantation in a hypoactive model of HD.

Neural transplantation as an experimental treatment modality for cerebral ischemia.

Cerebrovascular disease exemplifies the poor regenerative capacity of the CNS. While there are methods to prevent cerebral infarction, there is no effective therapy available to ameliorate the anatomical, neurochemical and behavioral deficits which follow cerebral ischemia. Focal and transient occlusion of the middle cerebral artery (MCA) in rodents has been reported to result in neuropathology similar to that seen in clinical cerebral ischemia. Using specific techniques, this MCA occlusion can result in a well-localized infarct of the striatum. This review article will provide data accumulated from animal studies using the MCA occlusion technique in rodents to examine whether neural transplantation can ameliorate behavioral and morphological deficits associated with cerebral infarction. Recent advances in neural transplantation as a treatment modality for neurodegenerative disorders such as Parkinson's disease, have revealed that fetal tissue transplantation may produce neurobehavioral recovery. Accordingly, fetal tissue transplantation may provide a potential therapy for cerebral infarction. Preliminary findings in rodents subjected to unilateral MCA occlusion, and subsequently transplanted with fetal striatal tissue into the infarcted striatum have produced encouraging results. Transplanted fetal tissue, assessed immunohistochemically, has been demonstrated to survive and integrate with the host tissue, and, more importantly, ameliorate the ischemia-related behavioral deficits, at least in the short term. Although, this review will focus primarily on cerebral ischemia, characterized by a localized CNS lesion within the striatum, it is envisioned that this baseline data may be extrapolated and applied to cerebral infarction in other brain areas.