Hematological and serum biochemical profile of farm emus (Dromaius novaehollandiae) at the onset of their breeding season.

Blood profiling is a helpful tool in detecting the health status, metabolic diseases, nutritional deficiencies, and welfare of animals. Body weights, body temperatures, hematological and serum biochemical parameters, enzymes, and electrolytes in both sexes of farm emus at the beginning of their breeding season (November in Canada), were determined. The reference interval for each analyte was also calculated. Emus have lower body temperatures (37.2 ± 0.2) than other poultry species. There was no significant between-sex difference in BW, body temperature, and all the hematological and enzyme parameters measured. However, females had significantly (P < 0.001) higher serum calcium, phosphorus, albumin, total protein, globulin, and triglyceride levels than males, probably in preparation for egg laying. We also examined our findings in light of their sex-role reversal in incubation and brooding. Contrary to other avian species in which only females incubate and brood, there was no sex difference in the hematological and enzyme parameters measured in emus. We found that emus are similar to other ratite species with respect to the changes in protein, globulin, triglyceride, and calcium levels. The findings from our study contribute to the database for reference emu hematological and serum enzyme, metabolite, and electrolyte values.

Ivermectin residues in squab.

No drugs have been approved for the treatment of parasitic nematodes in pigeons, but ivermectin, a broad-spectrum endectocide, has been used extra-label by prescription. Producers currently allow for a 2-wk withdrawal time before marketing squabs. However, because its use is extra-label there is no legal maximum residue limit for ivermectin in squab meat. The purpose of this study was to examine the depletion of ivermectin (passed by the parents to the squabs) from the tissues of squab. Adult pigeons brooding squab were treated with ivermectin in their drinking water (3.3 µg/mL) for 3 d. After dosing the parents, the ivermectin concentration of the breast meat and liver of squabs was found to be greater than the maximum residual limits established for livestock, indicating that ivermectin was transferred from the parents to the squabs. However, ivermectin was not detected in either the breast meat or the livers of squabs 1 wk after dosing. These results indicate that there is a rapid decline in tissue levels of ivermectin in squab.

Maintenance nitrogen requirements of adult female ostriches (Struthio camelus).

Successful ostrich farming requires knowledge of the nutritional needs of the birds. While much information is available on the nutritional value of various feed ingredients fed to ostriches, there is little known about their specific nutrient requirements. In this study, we measured the maintenance nitrogen requirements (MNR) of ostriches by nitrogen balance. We predict, based on the previous analysis of nitrogen requirements of various species of birds, that ostriches would have a MNR of 13.6-19.1 g N/day and a total endogenous nitrogen loss (TENL) of 2.8-5.1 g N/day. Three adult female ostriches were fed five pelleted diets containing 0.6-2.3% N [4-14.6% crude protein (CP)], 17.5 kJ/g gross energy (11.4 kJ/g ME) and 30% neutral detergent fibre. Each dietary trial consisted of a 10-day adaptation period, followed by a 5-day total excreta collection period. Body mass (109 ± 3 kg) and metabolizable energy intake (20.5 ± 0.7 MJ/day) were unaffected by dietary nitrogen levels. After correcting for excreta nitrogen losses during drying, MNR was calculated to be 481 mg N/kg(0.75) /day or 16.2 g N/day (100 g CP/day), and TENL as 310 mg N/kg(0.75) /day or 10.5 g N/day. Failure to correct for the 10.9 ± 4.1% average N losses during drying would underpredict the 'true' MNR by 35% and TENL by 46%. Our estimate for MNR of ostriches predicts a dietary requirement of 6.7% protein. Our estimate of TENL was nearly twice that predicted, possibly reflecting the high fibre content of their diet.

Effect of diatomaceous earth on parasite load, egg production, and egg quality of free-range organic laying hens.

The effectiveness of diatomaceous earth (DE) as a treatment against parasites and to increase feed efficiency and egg production of organically raised free-range layer hens was evaluated in 2 breeds of commercial egg layers [Bovan Brown (BB) and Lowmann Brown (LB)] that differ in their resistance to internal parasitic infections. Half the hens of each breed were fed diets supplemented with DE (2%). Their internal parasite loads were assessed by biweekly fecal egg counts (FEC) and by postmortem examination of the gastrointestinal tract. Supplementing DE in diets of LB hens, the more parasite-resistant breed, did not significantly affect their FEC and adult parasite load. However, BB hens treated with dietary DE had significantly lower Capillaria FEC, slightly lower Eimeria FEC, fewer birds infected with Heterakis, and significantly lower Heterakis worm burden than control BB hens. Both BB and LB hens fed the diet containing DE were significantly heavier, laid more eggs, and consumed more feed than hens fed the control diet, but feed efficiency did not differ between the 2 dietary treatments. Additionally, BB hens consuming the DE diet laid larger eggs containing more albumen and yolk than hens consuming the control diet. In a subsequent experiment, the effectiveness of DE to treat a Northern fowl mite (Ornithonyssus sylviarum) infestation was tested. Relative to controls, both breeds of hens that were dusted with DE had reduced number of mites. The results of this study indicate the DE has the potential to be an effective treatment to help control parasites and improve production of organically raised, free-range layer hens.

Selenium enrichment of table eggs.

Selenium is an essential trace element with a recommended dietary allowance for human adults of 55 µg/d. However, there is evidence that greater dietary intakes may have possible health benefits, including a reduction in the risk of cancer. Several studies have shown the feasibility of enriching eggs using organic Se and that Se-enriched eggs are an effective way to supplement human diets. However, few studies have examined the response of egg Se concentration to high (>1 µg/g) dietary organic Se intake by the laying hens. The objective of the current study is to examine the effect of higher dietary organic Se levels on production, egg mass, and egg Se levels. These were assessed by feeding 3 breeds of laying hens (Barred Plymouth Rock, Lohmann Brown, Lohmann White) a basal diet containing 0.3 µg of Se/g of diet as Na2SeO3. Into this diet, Se yeast (SelenoSource AF 600), an organic source of Se, was added at 1.0, 2.4, or 5.1 µg of Se/g of diet for 4 wk. Feed consumption, egg production, and egg mass were not affected by the dietary Se concentration in all 3 breeds. Within the range of Se levels employed in the laying hens' diet, egg Se content increased linearly as dietary levels of Se increased. The results of this study indicate that feeding up to 5.1 µg/g of Se will not affect egg production and the welfare of the laying hen and is a practical way of producing Se-enriched eggs for the consumers.

Effects of subzero saline chilling on broiler chilling efficiency, meat quality, and microbial safety.

The poultry industry has attempted to improve carcass chilling efficiency, meat quality, and product safety. The purpose of this research was to investigate the effects of subzero saline chilling on carcass chilling, breast fillet tenderness, and microbial safety. After evisceration, broiler carcasses were chilled using ice slurry control (0% NaCl/0.5°C) or subzero saline solutions (3% NaCl/-1.8°C and 4% NaCl/-2.41°C). Broiler carcasses in the subzero saline solutions were chilled efficiently and reduced the chilling time by 11% in 3% NaCl/-1.8°C and 37% in 4% NaCl/-2.41°C over the ice slurry chilling. The breast fillets of broiler carcasses in 4% NaCl/-2.41°C were significantly tenderized than those in water control (P < 0.05), with an intermediate value observed in 3% NaCl/-1.8°C. Before chilling, broiler carcasses possessed mesophilic aerobic bacteria, Escherichia coli, and total coliforms for 3.81, 0.78, and 1.86 log cfu/g, respectively, which were significantly reduced after chilling in 3% NaCl/-1.8°C or 4% NaCl/-2.41°C solution over the water control (P < 0.05), except the mesophilic aerobic bacteria. Based on these results, chilling of boiler carcass in 4% NaCl/-1.8°C solution appears to improve carcass chilling efficiency, meat tenderness, and bacterial reduction for E. coli and total coliforms.

Improvement of chilling efficiency and meat tenderness of broiler carcasses using subzero saline solutions.

Carcass chilling in subzero saline solutions has the potential to improve chilling efficiency and meat quality. The purpose of this research was to investigate the effects of subzero saline chilling on broiler carcass for chilling efficiency and breast tenderness. Broiler carcasses were chilled using 2 high saline solutions (4% NaCl/-2.41°C and 8% NaCl/-5.08°C) without (Experiment I) and with (Experiment II) a pre-chilling step, and 3 low saline solutions (1% NaCl/-0.6°C, 2% NaCl/-1.2°C, and 3% NaCl/-1.8°C) in Experiment III. Ice water slurry (0% NaCl/0.5°C) was used as a control. In Experiments I and II, the breast fillets of broilers in subzero saline solutions showed significantly lower shear forces than those in water control, regardless of salt content and temperature level (P < 0.05). In Experiment III, 3% NaCl/-1.8°C solution reduced the broiler chilling time by 22% over the water control, with an intermediated reduction (13 to 17%) observed for 1% NaCl/-0.6°C and 2% NaCl/-1.2°C solutions. Shear force was stepwise reduced as the salt concentration increased from 0 to 3% and the solution temperature decreased from 0.5 to -1.8°C. No significant difference was observed for carcass chilling yield, breast pH/R-value, and breast cooking yield/salt content, regardless of chilling method. Based on these results, chilling of boiler carcass in 3% NaCl/-1.8°C or higher NaCl and lower temperature solutions appears to improve carcass chilling efficiency and meat tenderness over the traditional water immersion chilling.

Increased experimental metastatic capacity of a murine melanoma following induction of differentiation.

Because of the interest in possible links between defective differentiation and cellular malignancy, the effects were examined of induced cell differentiation upon the experimental metastatic potential of the sublines F1 and F10 of the B16 mouse melanoma. These cell lines normally have low and high rates, respectively, of colonization of the lungs of mice after i.v. injection. Cellular differentiation was assessed by pigmentation and tyrosinase activity. In both cell lines, low and high levels of differentiation could reproducibly be generated by culture, respectively, at a low extracellular pH and at a higher pH in the presence of a melanocyte-stimulating hormone. Surprisingly, in both lines the cells grown under conditions promoting differentiation showed a markedly higher rate of experimental metastasis, despite their slower proliferation in culture and in subcutaneous tumor implants, than the poorly differentiated cells. Radiolabeled well- and poorly pigmented cells were not initially deposited at significantly different rates in the lungs of mice after i.v. injection. However, subsequent retention in the lungs fell more quickly for the poorly differentiated cells. As indicated by tests in vitro, this difference appears not to be due to differential cytotoxicity by either host macrophages or natural killer cells, and it is under further study.

Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.

Epithelial cell kinase (ECK) is a receptor protein tyrosine kinase, the role of which in melanoma biology is unclear. Here we studied the role of ECK during melanoma progression. ECK mRNA was overexpressed in virtually all melanoma lines tested, and levels were significantly higher in cell lines from distant metastases than primary melanomas; melanocytes were negative. Gene amplification was not detected in melanomas. Levels of ECK protein corresponded well with mRNA levels. B61 or LERK-1, recently identified as an ECK ligand, stimulated the growth of ECK-expressing melanoma cell lines, its first identified biological activity. Melanoma chemotaxis and chemoinvasion were not affected by B61. Growth of normal melanocytes was not affected. mRNA for B61 was detected in both melanoma cell lines and normal melanocytes. B61 was also identified by Western blotting and ECK binding activity with the use of a BIAcore binding assay in melanoma cell-conditioned media. These results suggest that B61 is an autocrine growth factor for melanomas but not normal melanocytes.

The POU domain transcription factor Brn-2: elevated expression in malignant melanoma and regulation of melanocyte-specific gene expression.

Previous work has shown that melanoma cell lines express a distinct octamer binding protein. Given the role of octamer-binding proteins in cell differentiation and development, the role this factor is a key issue in understanding melanocyte differentiation and transformation. Using a proteolytic clipping assay, we show that the melanoma-specific octamer factor is Brn-2/N-Oct3, a POU domain protein previously known to be expressed in adult brain and in the developing nervous system. N-Oct3 mRNA was detected in a range of human melanoma cell lines and was around 10-fold elevated compared to normal human melanocytes while mRNA for Brn-2 was also detected in a mouse melanoblast cell line. Expression of Brn-2/N-Oct3, in melanoma cells in cotransfection assays activated the expression of the MHC class II DR alpha promoter but repressed the activity of the melanocyte-specific tyrosinase promoter. Repression correlated with Brn-2/N-Oct3 binding in a mutually exclusive fashion with basic-helix-loop-helix-leucine-zipper (bHLH-LZ) transcription factor USF in vitro and with Brn-2 expression preventing activation of the tyrosinase promoter by the bHLH-LZ factor Microphthalmia in vivo. The potential role of Brn-2/N-Oct3 in melanocyte differentiation and gene expression is discussed.

Requirement for focal adhesion kinase in tumor cell adhesion.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase and a major phosphotyrosine-containing protein. FAK is found in cell-matrix attachment sites (focal adhesions), and is activated on integrin-ligand binding and by other signaling pathways. Several roles have been proposed for FAK; here we report a novel function. We observed abundant FAK protein in all human melanoma cell lines tested except COLO839, a line that grows predominantly in suspension and was derived from peripheral blood. Five adherent lines, isolated from solid metastases in the same patient as COLO839, did express FAK. We derived four adherent sublines from COLO839. These did express FAK, even when plated on bacteriological plastic, to which they did not adhere. Thus, substrate attachment was not required for FAK expression. Three of the adherent sublines were then grown in the presence of antisense oligonucleotides to the initial FAK coding sequence. All showed substantially reduced FAK expression and, interestingly, the cells largely detached from the substrate while continuing to grow. Similar results were obtained with an independent melanoma line, DX3. Thus, FAK expression appears to be required by melanoma cells for substrate adhesion.

Characterization of TRP-1 mRNA levels in dominant and recessive mutations at the mouse brown (b) locus.

The mouse brown locus encodes a putative membrane-bound metalloenzyme, tyrosinase-related protein-1 (TRP-1). We have examined the effect on mRNA expression of the locus of a number of mutant alleles. The common null mutant allele, brown, produces wild-type levels of TRP-1 mRNA, which is nonfunctional. Another recessive allele, cordovan-Harwell, has an intermediate, dark-brown phenotype and produces only very low levels of presumably normal TRP-1 mRNA. Two dominant alleles appear to act by killing the melanocyte in which they are expressed. One of them, Light, has normal size and amounts of TRP-1 mRNA. The other, White-based brown, produces no detectable TRP-1 mRNA. It has a gross DNA rearrangement at the 5' end, and we speculate that this results in activation of transcription of sequences not usually seen in melanocytes, and that this is toxic to the cell. The relationship between phenotype and molecular structure at the locus is discussed, and we draw some general principles applicable to other developmental genes.

The murine misty mutation: phenotypic effects on melanocytes, platelets and brown fat.

Although the recessive murine mutation misty (m) is well known, its phenotype has never been reported beyond brief descriptions of a dilution of coat color and white spotting of the belly and extremities, suggesting a developmental mutation. A report in abstract has also suggested effects on white fat and body weight. Here, we report effects of the homozygous misty mutation on an unusual combination of three cell types: melanocytes, platelets, and brown fat. Brown fat appeared to be completely absent from all expected locations in neonatal m/m mice. A prolonged bleeding time was observed; platelet count and platelet serotonin and ATP levels were normal, but the level of ADP in m/m platelets was low. Primary cultures and immortal lines of melanocytes from m/m mice showed several abnormalities. There was a marked deficiency in net proliferation, suggesting that the color dilution and spotting in vivo may result from reduced numbers of melanocytes and their precursors. m/m melanocytes were also hyperdendritic in morphology, overproduced melanin, and had deficient responses to the cAMP agonists cholera toxin and melanocyte-stimulating hormone, which normally promote melanin production. The misty gene product may be involved in adenine nucleotide metabolism or signaling.

Complementation of hypopigmentation in p-mutant (pink-eyed dilution) mouse melanocytes by normal human P cDNA, and defective complementation by OCA2 mutant sequences.

Mutations in the P gene of humans and the homologous p-locus of mice, respectively, result in the homologous disorders oculocutaneous albinism type 2 (OCA2) and pink-eyed dilution. Although clearly required for melanin biosynthesis, the specific function of the P gene product, a melanosomal transmembrane protein expressed in melanocytes of the skin, hair, and eyes, is not yet known. Here we describe lines of immortal melanocytes and melanoblasts from mice of the null genotype p(cp)/p(25H). These p-null melanocytes were severely hypopigmented, although they and the melanoblasts expressed mRNAs for a number of melanosomal proteins. Proliferation of the p-null melanoblasts was normal. Both diploid and immortal p-null melanocytes grew more slowly than wild-type melanocytes, however, and were unusually susceptible to the antibiotic G418; these abnormalities were corrected by culture in high concentrations of L-tyrosine. Transfection of the p-null melanocytes with full-length normal human P cDNA resulted in complementation of deficient melanin biosynthesis and hypopigmentation. In contrast, transfection with mutant human P cDNAs containing amino acid substitutions (A481T, V443I) found in patients with OCA2 resulted in minimal or partial correction, consistent with the corresponding pigmentation phenotypes in patients with these mutations. These results demonstrate the utility of this model system for distinguishing true OCA2 mutations from nonpathologic polymorphisms and for quantitating the effect of these mutations on P function.

Novel and known protein tyrosine kinases and their abnormal expression in human melanoma.

We have used the polymerase chain reaction and Northern blotting to identify protein tyrosine kinases that may play an important role in the process of melanoma initiation and progression. Degenerate primers from the conserved catalytic domain of tyrosine kinase genes were used to amplify and clone partial cDNA sequences from a human melanoma cell line (DX3-LT5.1) and normal human melanocytes. When the melanoma reaction products were sequenced, 13 distinct clones were found, of which one is novel to date and has provisionally been named MEK (for melanocytic kinase). Of the remaining 12 known kinases, only two, ERB-B2 and IGF1-R, have previously been reported in pigment cells. Reaction products from melanocytes included only eight of these 13 sequences. To test for quantitative differences in tyrosine kinase expression between normal and malignant cells, a panel of eight melanoma lines and normal melanocytes was analyzed by Northern blotting. Two tyrosine kinases (JTK-14/TIE and TYRO-9) were detected in some melanomas but were not found in normal melanocytes, whereas others, including MEK, appeared to be overexpressed in some malignant lines. A minority of kinases showed either no change or a reduction in the level of mRNA. Expression of tyrosine kinases varied independently, and individual lines contained various combinations of these enzymes. Our findings are consistent with an increased overall expression of these putative growth factor receptors during melanoma development.

Experimental metastasis and differentiation of murine melanoma cells: actions and interactions of factors affecting different intracellular signalling pathways.

Various factors that modulate the differentiation of malignant cells are known to affect their experimental metastatic potential (EMP), or lung colonization after intravenous injection into syngeneic animals. However, some results and conclusions on the relation between cell differentiation and metastasis have appeared to conflict. We have reanalysed this by measurement of EMP of B16 melanoma sublines after culture with agents or conditions that acted on differentiation through various intracellular pathways. All tested agents did affect the EMP. EMP was usually positively correlated with differentiation under diverse conditions, but exceptions showed that there is no direct causal connection. Nor could all findings be explained in terms of cell proliferation or expression of major histocompatibility antigens. Some data helped to explain disparities between previous reports. Specific novel findings included the following. The stimulation of EMP by melanocyte-stimulating hormone (MSH) as well as all other tested effects of MSH were prevented by extended exposure to 12-O-tetradecanoyl phorbol acetate (TPA), suggesting a requirement for protein kinase C activity as well as G-protein coupling in MSH action. Cells grown with cholera toxin were always more differentiated than untreated cells, but the EMP could be either markedly increased or markedly decreased by cholera toxin under different conditions. The basic culture medium apparently determined this striking reversal. The EMP was also significantly affected by the extracellular pH.

Mechanisms of differentiation in melanoma cells and melanocytes.

Literature is reviewed on the mechanisms of differentiation in mammalian melanoma cells and normal melanocytes. Pigment cells are particularly useful for studies requiring the observation of differentiation in living cells, for example, studies of commitment. Topics discussed include melanin synthesis and other markers of pigment cell differentiation; stochastic models of differentiation and commitment; the lability of early stages of differentiation; extracellular factors affecting pigment cell differentiation, with implications for intracellular controls; the role of proliferation and the cell cycle in differentiation, and the relative roles of changes in transcription, translation, and posttranslational processes.

Protein tyrosine kinases in malignant melanoma.

Protein tyrosyl phosphorylation is an essential component in intracellular signalling, with diverse and crucial functions including mediation of cell proliferation, survival, death, differentiation, migration and attachment. It is regulated by the balance between the activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases. A number of PTKs are encoded by proto-oncogenes or viral oncogenes, and are thus strongly implicated in cancer. While a role for PTKs in human melanoma is less firmly established, human melanomas or melanoma cells have been reported to contain more tyrosine phosphate than normal melanocytes, and some receptor PTKs (EPH-A2/ ECK and EPH-B3) are overexpressed in over 90% of melanoma cell lines. Other specific PTKs are also frequently overexpressed, including KDR and fibroblast growth factor receptor-4 (FGF-R4), while, interestingly, yet others, such as KIT and FES, are consistently downregulated in melanoma cell lines. All of these differentially expressed PTKs are candidates for gene products important in melanoma development. In addition, PTKs expressed in significant amounts in both benign and malignant melanocytes, such as insulin-like growth factor-1 receptor (IGF1-R), FGF-R1, HER2/NEU and FAK, are likely to play a role in melanoma genesis and progression.

Detection of a human DNA sequence correlated with melanocyte-like differentiation and tumour suppression after transfection into murine melanoma cells.

Since there is much indirect evidence for dominant suppressor genes for melanoma, we sought to isolate such a gene. Metastatic B16-F10 murine melanoma cells were lipofected with a normal human genomic library in a cosmid vector that also confers resistance to the drug G418. A few of the G418-resistant colonies acquired combinations of properties resembling those of normal melanocytes, including differentiated features (pigmentation, dendricity), slower growth, flat shape, monolayered colony form, stimulation of proliferation by a phorbol ester, and anchorage dependence. Four out of eight also showed reduced tumorigenicity in mice. Southern blotting indicated the presence of numerous cosmids in the melanocyte-like transfectants. DNA from one such line was used for secondary transfection. One secondary G418-resistant line showed pronounced melanocytic properties and marked tumour suppression in syngeneic and nude mice. A human repetitive sequence detected in this line was used in the polymerase chain reaction (PCR) to isolate intervening unique DNA sequences. One unique human sequence was attenuated in all tumors arising from both primary and secondary transfectants, suggesting close linkage with the sequence responsible for tumour suppression.

Morphological and pathological effects of cadmium ingestion on Pekin ducks exposed to saline.

This study examined the effects of simultaneous exposure to saline and cadmium (Cd) on organ mass and histology of a bird with salt glands, the Pekin duck, Anas platyrhynchos. Three mixed-sex groups, each containing 6 birds, ate duck pellets containing 0, 50, or 300 microg Cd/g, respectively, for 4 1/2 mo and drank 300 mM NaCl. Only females on the high-Cd diet lost body mass. Ingestion of Cd reduced heart mass in females. There was increased mass of Harderian and salt glands in both sexes. Mass of kidneys and liver increased only in males, and the gut mass (also length) increased more in males. Cadmium ingestion also induced (1) inflammation of renal interstitium and degenerative tubular changes, (2) marked degenerative changes in testes, (3) increased heart water content, (4) decreased cytoplasmic volume of liver cells, (5) reduced proportion of basophilic granular cells in chromaffin tissue of the adrenal glands, and (6) in the ileum, increased heterophilia in the lamina propria and, only in females, the apoptosis to mitosis ratio in crypt cells of the epithelium. The ducks' outward appearance gave no indication that ingesting large amounts of cadmium for 4 1/2 mo produced deleterious effects, but the physiological consequences were profound. Both sexes had greatly reduced gonadal mass and the males produced no sperm. The higher dietary level greatly hypertrophied the liver, kidneys, and gut only in males. The cadmium-induced changes in organs, particularly in the gonads, kidneys, and adrenal glands, should greatly impair the health and reproductive capacity of these ducks.