Condor 85:350-354 ‘2‘ The Cooper Ornithological Soc~ely 1983 FIRST OBSERVATIONS OF BROWN FAT IN BIRDS L. W. OLIPHANT ABSTRACT.-Brown fat, the multilocular fat involved in non-shivering ther- mogenesisin many mammals, is generally believed to be absent in birds. Phys- iological data indicate that non-shivering thermogenesisdoes not occur in adult birds. Adipose tissuesfrom a Ruffed Grouse (Bonasa umbellus) and two Black- capped Chickadees (Parus atricapillus) were found to show typical histological and ultrastructural features of mammalian brown fat. The cells of these tissues were polygonal with central nuclei, multiple lipid droplets and numerous well- developed mitochondria. An extensive capillary bed permeated the tissue. Al- though no direct demonstration of thermogenesisby this tissue was attempted, its remarkable similarity to mammalian brown fat suggeststhat non-shivering thermogenesisin birds should be reinvestigated. Typical white fat of birds and mammals is trated by Lucas and Stettenheim (1972:594). white to yellow in color and comprised of large None of these authors suggestedthat these tis- cells containing a single lipid droplet that oc- sueswere brown fat or presented other struc- cupies most of the cell volume. The nucleus is tural features compatible with their being flattened againstthe cell periphery by the lipid. brown fat. It is known that white fat can often Extraction of the lipid during routine histo- take on multilocular appearance. Luckenbill logical processingresults in the typical “signet and Cohen (1966) presentedpositive evidence ring” appearance of these cells. Many mam- that the “mulberry” cells they observed dif- mals, especially those that hibernate, have a fered from mammalian brown fat in lacking second distinct type of adipose tissue that is numerous mitochondria. brown and appearsalmost glandular, the cells During a routine examination of tissuesfrom being smaller and more closely packed than in a Ruffed Grouse (Bonasa umbellus), I noticed white fat (Smith and Horwitz 1969). Cells of tissue adjacent to the adrenal gland that had this “brown fat” contain much more cyto- the histological appearanceof brown fat. Sub- plasm and the fat is distributed as numerous sequently I examined a Black-capped Chick- smaller lipid droplets associated with abun- adee (Parus atricapillus) and found that its fat dant large mitochondria. The nuclei are more was grosslyand histologically similar to mam- or less spherical and are not displaced to the malian brown fat. A secondchickadee was col- cell periphery. Brown fat also possessesa richer lected to confirm the nature of this tissue by vascular supply and innervation than white fat electron microscopy. This paper describesthe (Napolitano 1965). structure of the fat from these three birds. In mammals, brown fat has been implicated as the primary site of non-shivering thermo- METHODS genesisduring neonatal life (Dawkins and Hull The Ruffed Grouse (adult female) was collect- 1965) during cold acclimation in adults(Foster ed in November 1971 near Saskatoon, Sas- and Frydman 1978) and during arousal from katchewan. A variety of organswere removed hibernation (Hayward and Lyman 1967). Adult in the field and placed in standard Bouin’s fix- birds are n& known to exhibit non-shivering ative. These tissueswere processedroutinely thermogenesis(West 1965, Chaffee and Rob- and 7-pm sections were stained with hema- erts 197 1). Freeman (197 1) presentedevidence toxylin and eosin. of non-shivering thermogenesis in newly The first chickadee was obtained in March hatched domestic chickens(Gallus gallus, var. 1980 (Ithaca, New York) and the second in domesticus)but could not find brown fat in March 1982 (Saskatoon). I made a complete these birds. Histological examination of tis- gross dissection of both chickadees and ex- sues from 11 species of adult and neonatal amined their fat tissue under a dissectingmi- birds known to exhibit poor thermoregulation croscope. Small representative pieces of fat and/or torpor also disclosed no brown fat were removed and placed in fixative (either (Johnston 197 1). 5% glutaraldehyde in 0.2 M S-collidine buffer Multilocular or “plurivacuolar” fat cells in or 2% osmium tetroxide in 1.25% sodium bi- birds have been reported (Clara 1923, 1929; carbonate buffer at pH 7.4). The tissueswere Luckenbill and Cohen 1966) and such fat from processed routinely and embedded in epon, the cere of a pigeon was described and illus- except for some larger glutaraldehyde-fixed [3501 BROWN FAT IN BIRDS 351 FIGURE I. Periadrenal fat from a Ruffed Grouse. The FIGURE 3. Fat from the neck of a Black-cappedChick- sphericalnuclei(N) are centrally placedand contain a large adee. The abundant mitochondria (M; in center of figure) densenucleolus. The cytoplasmsurrounding the clear lipid fill the cytoplasm between the more densely stained lipid droplets is filled with eosinophilicgranules. Capillaries (C) droplets. One-micron epon section, light micrograph. surround the tightly packed cells. Paraffin section, light FIGURE 4. Thoracic fat from a chickadee. The exten- micrograph. sive capillary beds(C) typical of brown fat are well shown. FIGURE 2. White fat from the ovarian mesentery of a Two-micron methacrylate section, light micrograph. Ruffed Grouse. Compare the large unilocular cells typical of white fat with the multilocular fat in Figure I. Paraffin section, light micrograph. The nuclei of the multilocular cells were large, spherical and centrally located. The pieces that were embedded in glycol methac- chromatin was primarily euchromatic and rylate. One-pm epon sectionswere stainedwith many cells had a single large nucleolus. The 1% toluidine blue 0 and thin sectionsfor elec- cytoplasm contained a variable number of lip- tron microscopy were stained with uranyl ace- id droplets. A few cells contained one or more tate and lead citrate. Glycol methacrylate sec- large droplets, most cells contained many tions were cut at 2 pm and stained with droplets the size of the nucleusor smaller, and hematoxylin and eosin. some cells had only a few small droplets. The cytoplasm between the lipid droplets was filled OBSERVATIONS AND DISCUSSION with small eosinophilic granules 0.5-l pm in Sectionsfrom the adrenal gland (only one col- diameter. These granules were most evident lected) of the Ruffed Grouse included a 3 x 4 in cells containing small amounts of lipid and mm lobular tissuemass adjacent to the adrenal were presumedto be mitochondria. It hasbeen consistingof tightly packed polygonal cells. A my experience that mitochondria are often ad- few small lobules consisted of unilocular fat equately preserved with Bouin’s fixative and cells but most of the lobules contained what are seenas intensely stained eosinophilic gran- appeared to be typical brown fat (Fig. 1). Adi- ules. The cytological appearance of these cells pose tissueassociated with the kidney, spleen, was similar to that of brown fat from thirteen- ovary and caeca of this bird consistedentirely lined ground squirrels(Citellus tridecemlinea- of larger, typical white fat cells (Fig. 2). tus), hamsters, (Mesocricetus auratus), and 352 L. W. OLIPHANT BROWN FAT IN BIRDS 353 cold-acclimated laboratory mice (Mus mus- central nuclei, multilocular lipid, and numer- culus) (Oliphant 1967). ous large mitochondria (Napolitano 1965, Scatteredamong the fat cellswere numerous Smith and Horwitz 1969). smaller, more angular and darkly stained nu- The distribution of this multilocular fat clei belonging primarily to endothelial cells of throughout the body of the chickadees,to the a densecapillary network. Larger blood vessels apparent exclusion of typical white fat, is in and small myelinated nerves were present in distinct contrast to the situation in mammals the loose connective tissue between the lob- where brown fat is restricted to certain local- ules. ized body areas (Smith and Horwitz 1969). The chickadeesboth possessedfat at the base The apparent restriction of brown fat to the of the neck, along the posterior edge of the periadrenal area of the Ruffed Grouse is just thorax, in the intestinal mesentery and base of as unusual. Although many mammals have the heart. The color of this fat varied from brown fat beside the adrenals(Smith and Hor- pinkish to a light brown rather than the more witz 1969; Oliphant, unpubl. observ.) they typical bright yellow of most wild birds. Under generally have brown fat in other body regions the dissectingmicroscope the tissue appeared as well. Since I did not make a grossdissection well vascularized. A small piece of fat that was of the grouse it is possible that other pads of dehydrated and cleared in xylene showed an multilocular fat were missed. extensive capillary network made visible by The apparent absenceof brown fat in other the red blood cells within the vessels. birds examined previously (Johnston 197 1) The fat from all body areasof the chickadees may be due to the types of birds chosen and was histologically similar to the multilocular the conditions under which they were collect- fat of the Ruffed Grouse (Fig. 3). The cells were ed. Johnston’s choice of birds that exhibit poor polygonal and tightly packed into an epithelial- thermoregulatory ability and/or torpor was like mass. Capillaries formed a dense network logical, since hibernating mammals have well surrounding each cell (Figs. 4 and 5). Abun- developed brown fat. His specimenswere col- dant, small densely-stained granules were lected, however, in the southern United States presentin the cytoplasm around the lipid drop- from late March to September, a region and lets. These were more distinct than in the time in which little thermal stressmight occur. Ruffed Grouse tissuedue to better fixation and Johnston pointed out the possible conversion thinner sections. of typical brown fat to a unilocular state which Electron micrographs showed that these cy- would be difficult or impossible to distinguish toplasmic granuleswere indeed mitochondria from white fat with routine histological meth- (Figs. 5 and 6). They were large, spherical to ods. This could account for the apparent lack ovoid, and contained abundant plate-like cris- of brown fat observed in these birds, if indeed tae.