Histological characterisation of the horn bud region in 58 day old bovine fetuses.

The presence of horns in domestic ruminants, such as cattle, sheep and goats, has financial and welfare implications. The genetic interactions that lead to horn development are not known. Hornless, or polled, cattle occur naturally. The known causative DNA variants (Celtic, Friesian, Mongolian and Guarani) are in intergenic regions on bovine chromosome 1, but their functions are not known. It is thought that horns may be derived from cranial neural crest stem cells and the POLLED variants disrupt the migration or proliferation of these cells. Relaxin family peptide receptor 2 (RXFP2) is more highly expressed in developing horns in cattle compared to nearby skin and has been shown to play a role in horn development in sheep. However, the role of RXFP2 in horn formation is not understood. Histological analyses of cranial tissues from homozygous horned and polled cattle fetuses at day 58 of development was carried out to determine the differences in the structure of the horn bud region. Condensed cells were only observed in the horn bud mesenchyme of horned fetuses and could be the progenitor horn cells. The distribution of neural crest markers (SOX10 and NGFR) and RXFP2 between horned and polled tissues by immunohistochemistry was also analysed. However, SOX10 and NGFR were not detected in the condensed cells, and therefore, these cells are either not derived from the neural crest, or have differentiated and no longer express neural crest markers. SOX10 and NGFR were detected in the peripheral nerves, while RXFP2 was detected in peripheral nerves and in the horn bud epidermis. Previous research has shown that RXFP2 variants are associated with horn phenotypes in cattle an sheep. Therefore, the RXFP2 variants may affect the development of the epidermis or peripheral nerves in the horn bud.

Meta-Analysis on the Prevalence of Failed Transfer of Passive Immunity in Calves from Pasture-Based Dairy Farms in Australasia.

Monitoring and minimizing the prevalence of failed transfer of passive immunity (FTPI) in dairy replacement calves within the first week of life is crucial for calf health and farm profitability. In this study, a systematic literature search and meta-analysis were conducted on papers reporting the prevalence of FTPI in calves from pasture-based dairy farms in Australia and New Zealand. Two search methods, a "traditional method" and a "search engine method", were conducted to identify published studies on FTPI in Australia and New Zealand. Data from a total of 13,430 calves from eight studies in Australasia were included in the analysis for FTPI within 8 days of birth. The meta-analysis revealed that the average prevalence of FTPI was 33% across the two countries, with the lowest FTPI (9%) in Western Australia and the highest FTPI (59%) in New Zealand. Using farm data from three studies, the average prevalence of FTPI at the farm level in Australasia was 38%, with the lowest prevalence found in a farm in South Australia (6%). In conclusion, the meta-analysis confirmed the need for good management of cows and newborn calves after birth in pasture-based systems to reduce FTPI in calves. Collecting newborn calves from pasture at least twice per day after birth and providing colostrum of sufficient quantity and quality as soon as possible were the best practices for preventing FTPI in Australasian dairy systems.

Analysis of immune-related microRNAs in cows and newborn calves.

Bovine colostrum contains a high concentration of immune-related microRNAs (miRNAs) that are packaged in exosomes and are very stable. In this study, 5 immune-related miRNAs (miR-142-5p, miR-150, miR-155, miR-181a, and miR-223) were quantified in dam blood, colostrum, and calf blood using reverse transcription quantitative PCR. Their levels in calf blood after colostrum ingestion were investigated to assess whether miRNAs are transferred from the dam to newborn calves. Three groups of Holstein-Friesian bull calves were bottle-fed 2 L of colostrum or milk from different sources twice per day. The group A calves received colostrum from their own dam and the group B calves were fed foster dam colostrum. Each pair of group A and group B calves were fed identical colostrum from the same milking of the corresponding group A dam for 3 d and then bulk tank milk for 7 d after birth. Group C calves were fed only 2L of "pooled colostrum" from multiple dams d 0 to 4 postpartum, and then fed bulk tank milk thereafter for 7 d after birth. The groups were fed colostrum from different sources and different amounts to assess possible miRNA absorption from the colostrum. All miRNAs were at the highest level in colostrum at d 0 and then decreased rapidly after d 1. The level of miR-150 had the largest decrease from 489 × 106 copies/µL (d 0) to 78 × 106 copies/µL (d 1). MicroRNA-223 and miR-155 were the most abundant in both colostrum and milk. Dam colostrum had significantly higher levels of miR-142-5p, miR-155, and miR-181a than the bulk tank milk. However, only the miR-155 concentration was significantly higher in the dam colostrum than in the pooled colostrum. The concentrations of miRNAs in the colostrum were less than in the cow blood (100- to 1,000-fold less). There was no significant correlation between the level of miRNAs in the dam blood and their colostrum, suggesting that miRNA is synthesized locally by the mammary gland rather than being transferred from the blood. MicroRNA-223 had the highest level in both calf and cow blood compared with the other 4 immune-related miRNAs. Calves were born with high levels of immune-related miRNAs in their blood, and there were no significant differences in miRNA levels between the 3 calf groups at birth or after they were fed different colostrum. This suggests that these miRNAs were not transferred from the colostrum to the newborn calves.

Incidence of Inadequate Transfer of Passive Immunity in Dairy Heifer Calves in South Australia.

The objective of this observational study was to estimate the incidence of inadequate transfer of passive immunity (ITPI) on five pasture-based dairy farms in South Australia. Heifer calf uptake of colostrum was evaluated within the first 1−7 days of age (n = 2638) using a digital refractometer to estimate each calf's serum total protein concentration, as an indicator of colostrum uptake. Results of <51 g/L indicated inadequate transfer of passive immunity (ITPI). The data showed that the incidence of ITPI on the farms was 6.5%, 31.3%, 48.8%, 49.7% and 52.4%. The incidence of ITPI was calculated in relation to the age of the calf at testing and the breed of calf, and no significant differences were found. A significant difference was found in the incidence of ITPI when comparing the calf's first feed after separation from the dam (colostrum versus a colostrum-transition milk mixture). The farm with the lowest incidence of ITPI collected calves twice a day, measured colostrum quality on farm with a Brix refractometer and ensured that each calf received an appropriate amount of high-quality colostrum soon after collection. Further studies are required to establish the risk factors of ITPI in South Australian dairy heifers.

Canine mast cell tumours part I: Clinical and survival outcomes.

BACKGROUND: Dogs have a species-specific susceptibility for developing mast cell tumours (MCTs). Mutations in the KIT proto-oncogene (KIT) are known to contribute to the neoplastic biology of mast cells. In dogs, the most common KIT mutation is an internal tandem duplication (ITD) in exon 11 which has been considered a useful prognostic supplement to traditional histopathological tumour grading. OBJECTIVE: The aim of this retrospective study was to explore the importance of KIT exon 11 ITD mutation status and known clinical and pathological indices in predicting prognosis in a cohort of Australian dogs diagnosed with MCT. METHODS: Clinical parameters, survival data, and KIT mutation status were collected and assessed for 220 dogs with cutaneous or subcutaneous MCT (n = 189 and n = 31, respectively). RESULTS: In at least one of the multivariable models, tumour grade (cutaneous Kiupel low or high grade) or tumour subcutaneous location, multiple concurrent MCTs, metastasis at the time of surgery, and senior age were statistically significant in predicting the outcome (MCT-related death and/or second MCT diagnosis) at 6- or 12-month post-tumour excision. KIT exon 11 ITD mutation status was not a significant predictor in any of the final multivariable models and was strongly correlated with high histological grade (p < 0.001). CONCLUSION: In this sample of dogs, tumour histological grading remained the single most powerful prognostic indicator for MCT outcome. However, concurrent evaluation of multiple prognostically significant parameters provides information of potential value to inform therapeutic management for each patient.

Predicting colostrum and calf blood components based on refractometry.

Provision of good quality colostrum is essential for the passive immunity and nutrition of newborn calves. In order to better predict the quality of colostrum and the transfer of passive immunity, the relationships between colostrum components and between calf serum components were examined in this study. Samples of bulk tank milk, colostrum pooled from several cows 0-4 d postpartum, and colostrum collected from individual cows twice daily for 3 d post-partum were compared. With the exception of fat percentage, there were strong correlations between the levels of the components in the pooled colostrum and in the individual cow colostrum collected 0-1 d postpartum. The correlations between total solids as measured by Brix refractometry and total protein, immunoglobulin G (IgG), lactose % and protein % in colostrum within 1 d postpartum and pooled colostrum were 0.92, 0.90, -0.88 and 0.98, respectively. These high correlations enabled these colostrum components to be accurately predicted from Brix % and therefore, the volume of colostrum required to feed neonate calves can be optimised based on Brix refractometry to avoid failure of passive immunity transfer. To assess whether the components obtained from colostrum were correlated in calf blood, newborn calves were separated from their dams before suckling and blood sampled before feeding (day 0), and on days 1 and 7, after receiving colostrum or milk twice a day. The correlations between glucose, total protein, IgG, and gamma-glutamyl transferase (GGT) levels in the calf blood were lower than the correlations observed between the colostrum components. The highest correlation was between serum protein measured by refractometer and serum IgG within one week postpartum. GGT activity was not a good indicator of serum IgG levels. However, serum protein refractometer measurements predicted serum IgG level with high accuracy, providing an on-farm test to determine that calves have received sufficient passive immunity and colostrum components.

Colostrum source and passive immunity transfer in dairy bull calves.

Colostrum is essential for good neonate health; however, it is not known whether different calves absorb the nutrients from colostrum equally well. In this study, the absorption of protein, IgG, and γ-glutamyl transferase was compared in newborn dairy bull calves for 1 wk after feeding colostrum from different sources. Thirty-five Holstein-Friesian bull calves were randomly allocated into 3 groups and fed colostrum within 4 h after birth. Group A calves (n = 12) were bottle fed colostrum from their own dam for 3 d. Colostrum from these group A cows was also used as foster cow colostrum for the group B calves (n = 12), such that each group A and B calf pair received identical colostrum from each milking of the respective group A dam (10% of birth weight per day). The group C calves (n = 11) were fed 1 bottle (2 L) of pooled colostrum and transition milk (referred to as pooled colostrum), as was the standard practice on the dairy farm. The pooled colostrum was collected from the other dairy cows on the farm 0 to 4 d postpartum and stored at 4°C for less than 12 h. Blood was sampled from calves before the first feeding and at 1, 2, 3, and 7 d after birth. Levels of total solids, total protein, and IgG were higher in the dam colostrum than in the pooled colostrum. At birth, there were no differences between the calf groups for any measurements, and all calves had very low IgG levels. After receiving colostrum, the glucose, plasma γ-glutamyl transferase, serum total protein, and IgG concentrations increased significantly in all calves. There were no differences in any blood measurements at any time point between the pairs of group A and group B calves that received colostrum from the same cow except for the IgG concentration 2 d after birth. However, the group A calves had a higher total serum protein level and IgG concentration than the group C calves for all the time points after the first feeding. The group B calves had a higher IgG concentration than the group C calves on d 1, 2, and 7 after birth. Compared with groups A and B, there was no difference in the proportion of calves in group C that failed to have passive immunity transferred adequately based on the IgG threshold (<10 g/L). Thus, the calves receiving identical colostrum from the same cow had the same levels of IgG, and even the pooled colostrum provided sufficient transfer of IgG as the calves were fed within 4 h after birth.

KIT mutations in mast cell tumours from cheetahs (Acinonyx jubatus) and domestic cats (Felis catus).

Mast cell tumours (MCT) have been documented in numerous species and mutations within the KIT proto-oncogene are implicated in the neoplastic biology of mast cells in humans, dogs and cats. This study determined high KIT gene nucleotide and Kit amino acid sequence homology between several species known to suffer mast cell neoplasia and especially high sequence conservation between the cheetah (Acinonyx jubatus) and domestic cat (Felis catus) KIT sequences. As a result, we hypothesised that KIT mutations would exist in the neoplastic DNA of four cheetahs diagnosed with MCT from a recent case series. PCR and Sanger sequencing identified conservative exon 6 KIT mutations in two of the four cheetahs. The mutations were different between the two cheetahs. Only wild-type DNA in exons 6, 8, 9 and 11 of KIT was observed in the MCTs of the remaining two cheetahs. Twenty cutaneous MCTs from domestic cats were collected for KIT mutation comparison. Twelve tumours possessed a mutation within KIT exons 6, 8 or 9 (60%, 95% CI 38.5%-81.5%). No mutations were detected in exon 11. There was no significant association between domestic feline MCT KIT mutation status and tumour histological grade (traditional schematic, P = .934; Sabattini 2-tier schematic, P = .762) or mitotic index (P = .750). KIT mRNA and Kit protein sequences are conserved across species but the role of KIT in feline MCT pathogenesis is not completely understood.

Comparative aspects of mast cell neoplasia in animals and the role of KIT in prognosis and treatment.

Mast cell neoplasia clinical presentation and biological behaviour vary considerably across mammalian species, ranging from a solitary benign mass to an aggressive systemic malignancy. Mutations in the KIT Proto-Oncogene Receptor Tyrosine Kinase (KIT) gene are common molecular abnormalities involved in mast cell tumorigenesis. KIT mutations often occur in dog, cat and human neoplastic mast cells and result in altered Kit protein structure and function. In dogs, certain KIT mutations are associated with more malignant and lethal disease. In contrast, KIT mutations in feline and human mast cell neoplasms are not correlated with prognosis, but are of value in diagnosis and treatment planning in humans. KIT genetic abnormalities have not been well investigated in other species, although aberrant cytoplasmic Kit protein staining detected in neoplasms of the ferret, horse and cow resembles aberrant Kit staining patterns detected in neoplastic mast cells of dogs, cats and humans. Mutations within KIT are classified as either regulatory-type or enzymatic pocket-type mutations according to their location within the KIT Proto-Oncogene. Mutations within the enzymatic pocket domain confer tumour resistance to tyrosine kinase inhibitors (TKIs). Hence, knowledge of tumour KIT mutation status adds valuable information for optimizing patient treatment strategies. The use of TKIs in combination with conventional chemotherapeutics has opened a new treatment avenue for patients unresponsive to existing drugs. This review highlights the similarities and differences of mast cell neoplasia in mammals with a special focus on the involvement of KIT in the canine and feline forms in comparison to human mast cell neoplasia.

Evidence of marbling as a single connected entity in beef striploins.

Serial sections of Angus striploins that varied in marbling were analysed in three dimensions to assess potential differences in intramuscular fat structure. The majority of the intramuscular fat appeared to be connected along the 100 mm of muscle in both the highly marbled and less marbled striploins. Thus, rather than having dispersed individual flecks of marbling, the intramuscular fat was a single entity within the striploin. The local shape patterns of this entity varied with marbling level in that the structure had an increased diameter in the highly marbled striploins. However, the amount of branching in the intramuscular fat did not vary with the level of marbling. The results suggest that marbling may occur along an internal structure, such as the vascular system or interstitium, in the longissimus muscle. It is postulated that when beef marbling increases, additional intramuscular fat is not deposited in isolated sites but along this internal structure, widening the existing entity rather than changing the shape.

Precision of spinal radiographs as a screening test for intervertebral disc calcification in Dachshunds.

Among dog breeds, the Dachshund has the highest lifetime incidence of intervertebral disc disease (IVDD). Intervertebral disc (IVD) calcification is an indicator of severe degeneration that predisposes to disc herniation. IVDD is heritable in Dachshunds, and in some countries, breeding candidates are screened to reduce IVDD occurrence by selecting dogs according to their score of radiographically detectable intervertebral disc calcification (RDIDC) and excluding dogs with ≥5 RDIDCs from breeding. This study evaluated the precision of scoring spinal radiographs for IVD calcification and subsequent classification of Dachshund dogs for breeding based on their RDIDC score. Digital radiographs of the spine were obtained in 19 clinically healthy, young adult Dachshunds, and scored for RDIDC independently by five scorers with varying levels of experience, three times each. Within scorer (repeatability) and between scorer (reproducibility) variability was estimated both at the individual IVD level and at the whole dog level for breeding classification purposes. At the IVD level, some degree of scorer effect was supported by the pairwise repeatability (92.3%; 95% CI: 88.8-94.7%) being marginally higher than the reproducibility (89.2%; 95% CI: 85.7-91.8%). Scorer-specific patterns confirmed the presence of scorer subjectivity. Repeatability significantly increased with scorer experience but the reproducibility did not. RDIDC scoring repeatability and reproducibility substantially decreased at the cervicothoracic spine region, likely due to anatomical superimpositions. At the dog level, a breeding classification could be repeated by the same scorer for 83.6% (95% CI: 73.8-90.2%) of the dogs, and was reproduced between two scorers for 80.2% (95% CI: 66.6-89.1%) of the dogs. The repeatability of breeding classification also seemed to increase with scorer experience but not the reproducibility. Overall, RDIDC scoring revealed some degree of inconsistency explained by scorer subjectivity and inexperience, and anatomical superimpositions. Scorer training and experience is strongly recommended to improve test precision and ensure appropriate classification of Dachshunds for breeding.

Radiographic scoring for intervertebral disc calcification in the Dachshund.

Intervertebral disc disease is a common, painful and debilitating neurological condition of dogs, causing substantial morbidity and mortality. The Dachshund is particularly susceptible to this disorder. The goal of this article is not to duplicate previously published reviews on canine intervertebral disc degeneration and degenerative diseases. Rather, the aims are threefold: (1) to reflect on selected clinical and pathophysiological aspects of intervertebral disc degeneration and disc disease that are pertinent to the Dachshund breed; (2) to review a radiographic spinal scoring scheme developed to reduce the prevalence of intervertebral disc disease in Dachshunds; and (3) to suggest further areas of research to improve upon the currently established scoring scheme in an attempt to address this breed's greatest health problem.

Major effect of retinal short-chain dehydrogenase reductase (RDHE2) on bovine fat colour.

Beef with yellow fat is considered undesirable by consumers in most European and Asian markets. ß-Carotene is the major carotenoid deposited in the adipose tissue and milk fat of cattle (Bos taurus), which can result in the yellowness. The effects of retinal short-chain dehydrogenase reductase (RDHE2) and ß, ß-carotene 9',10-dioxygenase (BCO2) were considered jointly as major candidate genes for causing the yellow fat colour, based on their genomic locations in the fat colour quantitative trait loci (QTL) and their roles in the metabolism of ß-carotene. In a secondary pathway, BCO2 cleaves ß-carotene into retinoic acid, the most potent form of vitamin A. RDHE2 converts trans-retinol to trans-retinal, a less active form of vitamin A. We evaluated the effects of two amino acid variants of the RDHE2 gene (V6A and V33A) along with a mutation in the BCO2 gene that results in a stop codon (W80X) in seven cattle populations. The RDHE2 V6A genotype affected several fat colour traits but the size of the effect varied in the populations studied. The genotype effect of the RDHE2 V33A variant was observed only in New Zealand samples of unknown breed. In general, the individual effects of RDHE2 V6A and V33A SNPs genotypes were greater in the random New Zealand samples than in samples from pedigreed Jersey-Limousin backcross progeny, accounting for 8-17 % of the variance in one population. Epistasis between the BCO2 W80X and RDHE2 variants was observed, and in some populations this explained more of the variation than the effects of the individual RDHE2 variants.

Mouse model of skeletal muscle adiposity: a glycerol treatment approach.

Fat cell accumulation in skeletal muscle is a major characteristic of various disorders, such as obesity, sarcopenia and dystrophies. Moreover, these fat cells could be involved in muscle homeostasis regulation as previously described for adipocytes in bone marrow. Despite recent advances on the topic, no clearly characterized mouse model is currently available to study fat accumulation within skeletal muscle. Here, we report a detailed characterization of a mouse model of skeletal muscle fat cell accumulation after degeneration induced by intra-muscular injection of glycerol. Information is provided on the kinetics of degeneration/fat deposition, including the quantity of fat deposited based on various parameters such as glycerol concentration, age, sex and strain of mice. Finally, these fat cells are characterized as true white adipocytes morphologically and molecularly. Our study shows that the mouse adipocyte accumulation within skeletal muscle after glycerol degeneration is a reproducible, transposable and easy model to use. This mouse model should allow a more comprehensive understanding of the impact of adipocyte accumulation in skeletal muscle pathophysiology.

Fatty acid synthase effects on bovine adipose fat and milk fat.

A quantitative trait locus (QTL) was identified by linkage analysis on bovine Chromosome 19 that affects the fatty acid, myristic acid (C14:0), in subcutaneous adipose tissue of pasture-fed beef cattle (99% level: experiment-wise significance). The QTL was also shown to have significant effects on ten fatty acids in the milk fat of pasture-fed dairy cattle. A positional candidate gene for this QTL was identified as fatty acid synthase (FASN), which is a multifunctional enzyme with a central role in the metabolism of lipids. Five single nucleotide polymorphisms (SNPs) were identified in the bovine FASN gene, and animals were genotyped for FASN SNPs in three different cattle resource populations. Linkage and association mapping results using these SNPs were consistent with FASN being the gene underlying the QTL. SNP substitution effects for C14:0 percentage were found to have an effect in the opposite direction in adipose fat to that in milk fat. It is concluded that SNPs in the bovine FASN gene are associated with variation in the fatty acid composition of adipose fat and milk fat.

A gene coexpression network for bovine skeletal muscle inferred from microarray data.

We present the application of large-scale multivariate mixed-model equations to the joint analysis of nine gene expression experiments in beef cattle muscle and fat tissues with a total of 147 hybridizations, and we explore 47 experimental conditions or treatments. Using a correlation-based method, we constructed a gene network for 822 genes. Modules of muscle structural proteins and enzymes, extracellular matrix, fat metabolism, and protein synthesis were clearly evident. Detailed analysis of the network identified groupings of proteins on the basis of physical association. For example, expression of three components of the z-disk, MYOZ1, TCAP, and PDLIM3, was significantly correlated. In contrast, expression of these z-disk proteins was not highly correlated with the expression of a cluster of thick (myosins) and thin (actin and tropomyosins) filament proteins or of titin, the third major filament system. However, expression of titin was itself not significantly correlated with the cluster of thick and thin filament proteins and enzymes. Correlation in expression of many fast-twitch muscle structural proteins and enzymes was observed, but slow-twitch-specific proteins were not correlated with the fast-twitch proteins or with each other. In addition, a number of significant associations between genes and transcription factors were also identified. Our results not only recapitulate the known biology of muscle but have also started to reveal some of the underlying associations between and within the structural components of skeletal muscle.

Alpha-tocopherol modulates the low density lipoprotein receptor of human HepG2 cells.

The aim of this study was to determine the effects of vitamin E (alpha-tocopherol) on the low density lipoprotein (LDL) receptor, a cell surface protein which plays an important role in controlling blood cholesterol. Human HepG2 hepatoma cells were incubated for 24 hours with increasing amounts of alpha, delta, or gamma-tocopherol. The LDL receptor binding activity, protein and mRNA, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA, cell cholesterol and cell lathosterol were measured. The effect of alpha-tocopherol was biphasic. Up to a concentration of 50 microM, alpha-tocopherol progressively increased LDL receptor binding activity, protein and mRNA to maximum levels 2, 4 and 6-fold higher than control, respectively. The HMG-CoA reductase mRNA and the cell lathosterol concentration, indices of cholesterol synthesis, were also increased by 40% over control by treatment with 50 microM alpha-tocopherol. The cell cholesterol concentration was decreased by 20% compared to control at 50 microM alpha-tocopherol. However, at alpha-tocopherol concentrations higher than 50 microM, the LDL receptor binding activity, protein and mRNA, the HMG-CoA reductase mRNA and the cell lathosterol and cholesterol concentrations all returned to control levels. The biphasic effect on the LDL receptor was specific for alpha-tocopherol in that delta and gamma-tocopherol suppressed LDL receptor binding activity, protein and mRNA at all concentrations tested despite the cells incorporating similar amounts of the three homologues. In conclusion, alpha-tocopherol, exhibits a specific, concentration-dependent and biphasic "up then down" effect on the LDL receptor of HepG2 cells which appears to be at the level of gene transcription. Cholesterol synthesis appears to be similarly affected and the cell cholesterol concentration may mediate these effects.

Polyunsaturated fatty acids downregulate the low density lipoprotein receptor of human HepG2 cells.

The aim of the study was to investigate the effect of different fatty acids on the low density lipoprotein (LDL) receptor of cultured human liver HepG2 cells. Previous studies investigating the effect of fatty acids on LDL expression have reported conflicting findings and are limited to measurements of LDL receptor binding activity. Therefore, this study is unique in that the relative effects of different fatty acids on the LDL receptor were investigated at three different stages of expression: 1) functional cellular LDL binding activity, 2) amount of LDL receptor protein and 3) LDL receptor mRNA level. The HepG2 cells were incubated for 24 hr with either 100 &mgr;M palmitic, oleic, linoleic or eicosapentaenoic acid (EPA). The measurement of LDL receptor binding activity was with colloidal gold-LDL conjugates, cellular LDL receptor protein was by western blotting and LDL receptor mRNA by Southern blotting of reverse-transcribed, polymerase chain reaction-amplified cDNA. The LDL receptor binding activity, protein and mRNA levels decreased as the degree of unsaturation of the fatty acids increased (palmitic acid greater-than-or-equal oleic acid > linoleic acid > EPA) and the inverse relationship held whether or not cholesterol was included in the culture media. The relative differences were very similar for the three stages of expression indicating that modulation of the LDL receptor by the fatty acids occurred at the level of gene transcription. The increased susceptibility to oxidation of the polyunsaturated fatty acids was unlikely to be a factor in the effect because EPA and linoleic acid (250 &mgr;M) still downregulated the LDL receptor in the presence of the antioxidant vitamin E (50 &mgr;M). In conclusion, the polyunsaturates, linoleic acid and EPA, effectively downregulated the LDL receptor of HepG2 cells compared to palmitic acid. The effects of these fatty acids were observed at the level of LDL receptor binding activity, protein and mRNA, strongly suggesting that the fatty acid effects were at the level of gene transcription.