ABSTRACT The objective of the present study was to develop an immunohistochemical technique to locate preadipocytes in bovine muscle tissue. Preadipocyte factor‐1 (pref‐1) is a transmembrane protein that is part of the family of epidermal growth factor‐like repeat‐containing proteins that are involved in cell fate determination. Pref‐1 is highly expressed in preadipocytes, but expression is completely abolished during differentiation into an adipocyte. In the present study, reverse transcription polymerase chain reaction demonstrated that bovine intramuscular adipose tissue contains the three splice forms of pref‐1 (A, C2 and E). Western blots were used to confirm that the protein for pref‐1 was expressed in intramuscular adipose tissue. Polyclonal antibodies against pref‐1 were tested against a cell culture of bovine preadipocytes from an embryo source to confirm that the antibody would immunolocate bovine preadipocytes. The antibody was applied to sections of longissimus dorsi muscle from Charolais and Holstein cattle. Immunohistochemical results showed that pref‐1 is expressed in the perimycium near mature adipocytes and blood vessels. The pool size of preadipocytes appeared to be low. Previous reports, however, have demonstrated that preadipocytes are known to divide and/or migrate, providing a potentially endless source of adipogenic precursor cells.

Peroxisome proliferator-activated receptor γ(PPARγ) regulates adipogenesis and lipid metabolism-related gene transcripts. The role, however, of PPARγ in different adipose depots and muscle in Holstein and Charolais cattle is still unclear. We used 20 animals (10 from each breed) for semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) to measure PPARγ mRNA levels in subcutaneous (SC), perirenal (PR), omental (OM), and intramuscular (IM) adipose depots as well as longissimus muscle (MU). IM fat was dissected from muscle tissue in MU. Holstein were characterized by their higher OM (P < 0.01) and PR (P < 0.05) fat weights while the Charolais had a higher body weight (P < 0.001) and a larger longissimus muscle area (P < 0.001). The IM fat content and marbling scores tended to be higher in Holstein. No significant differences in PPARγ mRNA expression were observed between these two breeds for any tissue. In both breeds, MU PPARγ had the lowest expressed mRNA level (P < 0.05). In the IM fat depot, expression was higher (P < 0.05) than MU, but lower than the SC, PR, and OM fat depot PPARγ mRNA levels. Only OM PPARγ mRNA levels were higher (P < 0.05) than SC and PR in Charolais. To characterise the role of PPARγ mRNA in bovine adipogenesis, correlations were performed among PPARγ mRNA, carcass characteristics, and adipogenesis-related genes. Key words: Adipose tissue, muscle tissue, PPARγ, cattle

Meaningful estimates of diacylglycerol acyltransferase (EC 2.3.1.20) activity in different tissue samples require effective, unbiased methods of sample storage. Samples of the pars costalis diaphragmatis muscle (skirt muscle of the diaphragm) were obtained from 18- to 20-month-old cattle and assayed for microsomal protein content and diacylglycerol acyltransferase activity after having been stored under various conditions as dissected tissue or microsomes prepared from dissected tissue. There was relative enrichment of diacylglycerol acyltransferase specific activity (p<0.05) when samples prepared from the pars costalis diaphragmatis muscle were dehydrated and stored for 2 weeks, as compared to the control condition (in which the microsome fraction was prepared from fresh pars costalis diaphragmatis muscle and assayed immediately). The results suggested that dehydration was an effective method of storage for bovine muscle samples destined for estimation of the microsomal diacylglycerol acyltransferase activity. The dehydration approach for preparing samples for analysis of diacylglycerol acyltransferase activity might also prove useful to investigators who are interested in obtaining reliable estimates of the activity of other enzymes in tissue samples.

cDNAs encoding acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), designated BnDGAT1 and BnDGAT2, were obtained from a microspore-derived cell suspension culture of oilseed rape (Brassica napus L. cv Jet Neuf). BnDGAT2 shares a very high level of identity with BnDGAT1, but is a smaller protein lacking the relatively hydrophilic N-terminal segment found in BnDGAT1. Both transcripts were produced in the cell suspension cultures and the cDNAs were functionally expressed in transformed yeast (Pichia pastoris) cells. Sucrose-mediated changes in triacylglycerol (TAG) metabolism and expression of BnDGAT1 were examined in the cell suspension cultures following transfer of cells from media containing 6% (w/v) sucrose to media containing 14% sucrose. TAG content and DGAT activity of the cells increased transiently within the first 12 h after transfer (HAT). The rapid decline in TAG content observed at 12 HAT was inversely related to an increase in TAG lipase (EC 3.1.1.3) activity. The transient increases in TAG content and DGAT activity correlated with the elevated amounts of BnDGAT1 polypeptide. Transcript levels were also induced, but levels of mRNA encoding BnDGAT1 were not tightly correlated with DGAT activity and amount of polypeptide suggesting some control of expression at the post-transcriptional level. In general, the rapid changes in TAG content were closely associated with the changes in the activity of TAG-metabolizing enzymes and expression of BnDGAT1.

Plasma leptin concentrations and beef cattle carcass characteristics in eight Continental Crossbred steers [0% Wagyu Cattle (WC)] were compared to crossbred cattle with 50 and 75% WC (eight steers each) genetic makeup to determine if a relationship exists between plasma leptin concentrations and intramuscular fat content (marbling) in beef cattle. Plasma leptin concentrations were measured at two stages of cattle growth, 16 and 4 wk prior to slaughter (W P S). Beef cattle characteristics including marbling score, ribeye area, i.m. total lipid content, and backfat depth were determined, and correlation coefficients obtained between these traits and leptin concentration at both sampling dates. Plasma leptin concentrations increased relative to the lipid content in the 24 steers based on the significant positive correlation observed between plasma leptin and total lipids (% wet weight) from both pars costalis diaphragmatis (p.c.d.)(16 WPS: r = 0.69, P = 0.0004; 4 WPS: r = 0.35, P = 0.104) and longissimus (16 WPS: r = 0.59, P = 0.002; 4 WPS: r = 0.51, P = 0.011) muscles. A trend was observed, however, at 4 WPS when the groups of varying Wagyu genetics were compared. Plasma leptin was positively correlated with muscle lipid content for the 0% Wagyu cattle (longissimus: r = 0.62, P = 0.103; p.c.d.:r = 0.40, P = 0.410)but there was almost no correlation in these parameters for the 50% WC (longissimus: r = 0.11, P = 0.797; p.c.d.: r = 0.005, P = 0.990). Plasma leptin concentration was negatively correlated with lipid content in the 75% WC (longissimus: r = –0.60, P = 0.120; p.c.d.: r = –0.65, P = 0.164). The results suggest that increasing Wagyu genetics negates any relationship between leptin concentrations and i.m. fat content in cattle. Key words: Wagyu crossbred cattle, meat quality, intramuscular fat, marbling, leptin

The effect of sucrose concentration on triacylglycerol biosynthesis and associated gene expression was examined in a microspore-derived cell suspension culture of oilseed rape (Brassica napus L. cv. Jet Neuf). The triacylglycerol content of the cells increased about 5-fold on a fresh weight basis when the sucrose concentration in the growth medium was raised from 2% to 22% (w/v). The specific activity of microsomal diacylglycerol acyltransferase (EC 2.3.1.20) and its activity per unit fresh weight increased about 2.5-fold and 6-fold, respectively, when sucrose concentration was increased from 2% to 14%. mRNA encoding the major oleosin also appeared to increase in abundance over the 2–14% sucrose concentration range when RNA fractions were analysed by the reverse transcription-polymerase chain reaction. The sucrose-mediated increases in diacylglycerol acyltransferase activity and oleosin mRNA indicated that the cell suspension could be a useful research tool for the identification of cDNAs encoding triacylglycerol biosynthetic enzymes and associated proteins.