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The expression of three CCAAT/enhancer-binding proteins (C/EBPs) was examined with immunocytochemistry and Western blot analysis during preadipocyte differentiation in porcine stromal vascular (S-V) cell cultures. Regardless of treatment and time in culture, immunoreactivity for all three C/EBP isoforms was restricted to cell nuclei. At day 1, 50 +/- 6% of S-V cells were C/EBPdelta positive, whereas 13 +/- 3 and 11.7 +/- 3% of S-V cells were AD-3 and C/EBPalpha positive, respectively. After 3 days of seeding in fetal bovine serum (FBS) and dexamethasone (DEX), C/EBPdelta; AD-3, and C/EBPalpha-positive cells increased to 67 +/- 5, 42 +/- 4, and 32 +/- 3%, respectively. Double staining clearly showed that most of the C/EBPalpha reactive cells had not accumulated appreciable lipid after 3 days of FBS + DEX. Following 3 days of insulin treatment, the percentage of C/EBPdelta cells was 50 +/- 6, whereas the percentage of AD-3- and C/EBPalpha-positive cells was 41 +/- 4 and 31 +/- 3, respectively. After insulin treatment all fat cells were AD-3, C/EBPalpha, and C/EBPdelta positive. Double staining demonstrated that fat cells were C/EBPdelta reactive throughout the culture period. Western blotting showed changes in C/EBP isoform expression that were consistent with the immunocytochemical results. We conclude that C/EBPalpha is a terminal differentiation marker which is expressed later than AD-3 but further studies are needed to determine the relationship between C/EBPdelta and adipogenesis in porcine S-V cultures.

The discovery of leptin has clearly demonstrated a relationship between body fat and the neuroendocrine axis since leptin influences appetite and the reproductive axis. Since adipose tissue is a prima

Grazing animals cause major alterations to vegetation structure and botanical composition through their selective grazing, trampling and excretal deposition (Hester et al., 2005). Through these effects they modify habitats and thus the populations of invertebrates and other organisms at higher trophic levels. Herbivores are thus key drivers of ecosystem function and nutrient dynamics within grazed plant communities. Changes in grazing intensity and the species mix of grazing livestock can therefore have important implications for resulting biodiversity. Ongoing reform of the European Union Common Agricultural Policy (CAP) will lead to a shift in the way financial support for livestock is distributed and hence to changes in grazing management practices. Farmers will increasingly receive financial support subject to cross-compliance with various environmental conditions and for delivery of specific environmental and social objectives. In some areas, livestock are likely to be increasingly viewed as tools for habitat management rather than solely as producers of food and other commodities. Against this backdrop of policy change the British Society of Animal Science and the British Ecological Society organized a joint symposium at the BSAS Annual Meeting in April 2005 on the links between farm animals and biodiversity. Four papers from this symposium are presented as mini-reviews in the current issue of Animal Science. David Oglethorpe set the policy context in his review of the environmental implications of CAP reform (Oglethorpe, 2005). His paper highlighted the likely changes in the livestock sector that will ensue including a polarization of agriculture into intensive producers versus environmental managers, increasing extensification in the uplands and some substitution of beef with sheep. Jerry Tallowin then presented a review of the impact of grazing management on grassland biodiversity (Tallowin et al., 2005). His paper showed that lenient grazing pressure by cattle in species-rich grassland was sufficient to maintain botanical diversity but did not enhance it over a 5-year period. For species-poor grassland, grazing management could alter sward structure but, in the absence of seed sources, botanical diversity was resistant to change. There is obviously much research still be done in this area to support the development of suitable agri-environment measures under Pillar 2 support mechanisms. David Buckingham went on to consider the extent to which grassland management might influence habitat quality for farmland birds (Buckingham and Peach, 2005). His paper showed that the exacting requirements of declining granivorous birds pose the greatest challenges while the needs of soil invertebrate feeding species are more easily met under agri-environmental schemes. In the final paper by Bruno Martin, the influence of pasture diversity on cheese quality was the theme (Martin et al., 2005). This paper reviewed recent work, primarily from France, which has examined the links between the diet of grazing animals and the sensory characteristics of various Protected Designation of Origin cheeses. The review highlighted the sometimes subtle, but none the less important influence of the grazing environment on food quality. The purpose of the symposium was to draw together animal scientists, conservation biologists, ecologists and socio-economists to consider the changing role of farm livestock within the new ‘decoupled’ economic environment. Judging by the popularity of the symposium and the vibrant nature of the discussion that followed each paper, there are plenty of issues still to consider and the hope is that some of the contacts made at the meeting will yield fruitful collaborations in the future.

Mature adipocytes may not be a terminally differentiated cell form, as previously thought by about everyone in science. Instead, these adipocytes may be capable of re-entering the cell cycle and forming proliferative-competent precursor cells like preadipocytes, adipofibroblasts, or even other forms of cells. Why is this possibility important to animal science? The main reason is in directing the cellularity of adipocyte depots in meat animals. For example, we know that the fat characteristics of beef animals are a direct function of the ‘cellularity’ of cells committed to form adipocytes. Beef steers given a high-concentrate diet commonly possess fewer adipocytes in any given fat depot, but most of the cells are completely filled with lipid to capacity. Alternatively, steers on a high-roughage diet seem to possess more fat cells, but fewer of them are filled to capacity with storage lipid. A variety of systemic and local regulators have been implicated in altering the cellularity of any given fat depot. Major regulators appear to be growth hormone, any number of repartitioning agents, as well as adipocytokines produced by the cells within the fat depot themselves. The specific mechanisms involved in directing cellularity of any specific fat depot, however, are poorly understood.

The development of adipose tissue lipoprotein lipase (LPL) activity was determined cytochemically in sections from normal and decapitated pig fetuses. Cellular LPL activity was first evident in 60-day-old fetuses. Capillaries were stained for LPL activity in 85-day-old and older fetuses and in young pigs. Cellular synthesis of LPL and capillary staining were not concurrent events. LPL activity developed after adipocyte differentiation and after structural development of capillaries. The cellular and capillary staining of fat cell clusters throughout development and in decapitated fetuses indicated a possible inverse relationship between cellular synthesis of LPL and capability of capillaries to transport LPL to luminal sides. These observations indicate that LPL activity develops in a pattern quite distinct from that observed in vitro. This discrepancy may be due to the involvement of blood vessels in the in vivo developmental pattern.

Perirenal adipose tissue samples were obtained from fetuses removed from pregnant (crossbred) sows at 3 stages of gestation (70, 90 and 110 days). Phosphatase histochemistry, succinate dehydrogenase (SDH) histochemistry and factor VIII antigen immunocytochemistry were conducted on fresh-frozen cryostat sections. Age-associated changes in nucleosidediphosphatase (NDPase) reactions in the arteriolar system were correlated with the morphological development of the medial layer of arterioles and arteries. For instance, a strong NDPase reaction in small arterioles was associated temporally with the assumption of a normal smooth-muscle cell morphology and arrangement in the medial layer. Age-associated changes in blood vessel reactions for factor VIII antigen and alkaline phosphatase activity were not correlated with morphological development. In the youngest fetuses, alkaline phosphatase activity was evident in large and small arterioles, but in the oldest fetuses, alkaline phosphatase activity was restricted to the smallest arterioles and vessels associated with them. Arteriolar differentiation was demonstrable with either adenosine triphosphatase (ATPase) or inosine diphosphatase (IDPase) reactions. Primordial stromal cells around differentiated arterioles were reactive for ATPase but not for IDPase activities. In older fetuses, there were large areas that contained ATPase-reactive stromal cells, no adipocytes, differentiated (ATPase and IDPase) arterioles and few capillaries. Positive reactions for SDH were evident in the ATPase-reactive stromal areas that contained no adipocytes. Differentiated adipocytes were SDH- and ATPase-reactive. These data illustrate the utility of differential phosphatase histochemistry to identify adipose tissue primordia.

Subcutaneous adipose tissue was obtained from fetuses removed from pregnant obese (Ossabaw) and lean (crossbred) sows at three stages of gestation (70, 90, and 110 days). Histochemical analysis for nucleoside phosphatase (NPase), alkaline phosphatase (APase), and NADH tetrazoleum reductase (NADH-TR) was conducted on fresh-frozen cryostat sections. Age- associated changes in NPase and NADH-TR reactions in the arteriolar system were correlated with the morphological development of the medial layer of arterioles and arteries. For instance, a strong NPase reaction in small arterioles was associated temporally with the assumption of a normal smooth muscle cell morphology and arrangement in the medial layer. In the youngest fetuses, strong NADH-TR reactions were only evident in small and presumptive arterioles and venules (associated with fat cells). Little NADH-TR reactivity was evident in larger arterioles and venules in 70-day tissue. Arteries and large arterioles were distinguished from veins and venules (strong reactions vs. weak reactions) with NADH-TR and NPase reactions in the oldest fetuses. In the younger fetuses, the NPase distinction (arterioles vs. veinules) was obvious before NADH-TR distinction. Small adipocyte-associated vessels were APase positive in the youngest fetuses, but APase reactivity was limited to short segments of vessel between arterioles and capillaries in the oldest fetuses. With the following exceptions, all the above observations were independent of fetal strain. In obese fetuses (110 day) small venules and small arterioles were equally reactive for NPase activity. Capillaries in obese fetuses (110 day) were NADH-TR reactive, whereas no activity was evident in capillaries from lean fetuses (110 day). The histology of intimal and medial vessel walls was independent of fetal strain. However, at every fetal age the adventitial layer of large arterioles and the collagenous stroma (around fat cell clusters) was thicker and better organized in obese fetuses. In the present study, differential enzyme histochemistry was utilized to distinguish the differentiation of small adipocyte-associated arterioles from larger arterioles. Furthermore, structural evidence of the arteriolar system clearly precedes metabolic and histological differentiation of tunica media cells. Finally, metabolic and structural traits of the stromal vascular cells of adipose tissue were sensitive to a fetal obese pheno-type.

Histochemical and ultrastructural aspects of adipocyte differentiation in subcutaneous tissue of fetal pigs were analyzed in a longitudinal study. A matrix of collagen fibers surrounding adipocytes developed after the establishment of a distinct and continuous PAS-positive basement membrane. The degree of plasma membrane invagination and specialization was positively correlated with the extent of basement membrane and collagen matrix formation. Close spatial relationships between narrow, smooth endoplasmic reticulum, plasma membrane invaginations, the surface of lipid droplets and mitochondria were observed in differentiating adipocytes. Histochemical and ultrastructural criteria for the identification of preadipocytes are: (1) perivascular location; (2) mitochondria localized in the Golgi zone; (3) cytosolic glycogen; (4) rough endoplasmic reticulum with cisternae uniformly and approximately 600 Å wide; (5) free ribosomes and few polysomes, and (6) lipid droplets encased by microfilaments. These criteria permitted clear distinction from obvious fibroblasts and macrophages. Other stromal cells were morphologically abnormal. Occasionally, adipocytes and perivascular cells exhibited close intercellular contacts that were morphologically distinct from intercellular contacts between contiguous endothelial cells.

The relationship between mammary gland development and adipocyte formation in rat subcutaneous tissue was studied. Tissue samples from newborn rats and young rats were prepared for histological and histochemical analysis. The development of subcutaneous adipocytes in the young rat is associated temporally and spatially with mammary gland development. The growing mammary gland ducts and the blood vessels and nerves that serve the ducts may physically interact with connective tissue cells, resulting in a population of undifferentiated cells. Adipocytes and a wide variety of cell types may be derived from this pool of undifferentiated cells. Histochemical characteristics of cells of immature ends of mammary gland ducts indicated hypoxic conditions. These conditions may have directed a vascularization process that resulted in a network of adipocytes and capillaries around mammary gland ducts. Thus, under normal conditions, adipocyte formation is an integral phase of mammary gland development in the fetal and yount rat.

Connective tissue cells that are histochemically and morphologically distinct from ‘fibroblasts’ are localized around developing hair follicles in the pig and rat. Immature adipose tissue is limited to small areas immediately around fully descended hair follicles in the rat hypodermis. In the present study, connective tissue around large nerves and blood vessels in fetal pig subcutaneous tissue was examined for the presence of enzymes typical of adipocytes. Samples from decapitated pig fetuses were studied so that the effects of an altered hormonal profile could be examined. Samples of dorsal subcutaneous adipose tissue were obtained from fetuses at 65, 70, 85, 90, 110, and 112 days of gestation. Fetuses were decapitated in utero at 45 days of gestation, and adipose tissue samples were obtained from these fetuses at 110 days of gestation. A close spatial relationship was observed between the growth of large blood vessels and nerves and fat cell cluster development in the older ( > 70 days) fetuses. Connective tissue cells that were contiguous with fat cell clusters were histochemically identical to adipocytes. The lipid histochemistry of the reactive connective tissue cells (histochemically identical to adipocyte) was variable in young fetuses. In all 110- and 112-day-old fetuses, the reactive cells contained lipid, whereas the reactive cells in decapitated fetuses were devoid of lipid. The reactive connective tissue cells were not associated with capillaries and did not contain basement membranes. The histochemistry of these cells suggests that they respond to a particular hormonal or metabolic profile as do adipocytes. Connective tissue cells contiguous with fat cell clusters may represent a distinct stage of adipocyte differentiation between the undifferentiated state and the mature adipocyte.