Integrating Multiple Biomarkers of Fish Health: A Case Study of Fish Health in Ports.

Biomarkers of fish health are recognised as valuable biomonitoring tools that inform on the impact of pollution on biota. The integration of a suite of biomarkers in a statistical analysis that better illustrates the effects of exposure to xenobiotics on living organisms is most informative; however, most published ecotoxicological studies base the interpretation of results on individual biomarkers rather than on the information they carry as a set. To compare the interpretation of results from individual biomarkers with an interpretation based on multivariate analysis, a case study was selected where fish health was examined in two species of fish captured in two ports located in Western Australia. The suite of variables selected included chemical analysis of white muscle, body condition index, liver somatic index (LSI), hepatic ethoxyresorufin-O-deethylase activity, serum sorbitol dehydrogenase activity, biliary polycyclic aromatic hydrocarbon metabolites, oxidative DNA damage as measured by serum 8-oxo-dG, and stress protein HSP70 measured on gill tissue. Statistical analysis of individual biomarkers suggested little consistent evidence of the effects of contaminants on fish health. However, when biomarkers were integrated as a set by principal component analysis, there was evidence that the health status of fish in Fremantle port was compromised mainly due to increased LSI and greater oxidative DNA damage in fish captured within the port area relative to fish captured at a remote site. The conclusions achieved using the integrated set of biomarkers show the importance of viewing biomarkers of fish health as a set of variables rather than as isolated biomarkers of fish health.

Management practices associated with conception rate and service rate of lactating Holstein cows in large, commercial dairy herds.

Data from lactating Holstein cows in herds that participate in a commercial progeny testing program were analyzed to explain management factors associated with herd-average conception and service rates on large commercial dairies. On-farm herd management software was used as the source of data related to production, reproduction, culling, and milk quality for 108 herds. Also, a survey regarding management, facilities, nutrition, and labor was completed on 86 farms. A total of 41 explanatory variables related to management factors and conditions that could affect conception and service rate were considered in this study. Models explaining conception and service rates were developed using a machine learning algorithm for constructing model trees. The most important explanatory variables associated with conception rate were the percentage of repeated inseminations between 4 and 17 d post-artificial insemination, stocking density in the breeding pen, length of the voluntary waiting period, days at pregnancy examination, and somatic cell score. The most important explanatory variables associated with service rate were the number of lactating cows per breeding technician, use of a resynchronization program, utilization of soakers in the holding area during the summer, and bunk space per cow in the breeding pen. The aforementioned models explained 35% and 40% of the observed variation in conception rate and service rate, respectively, and underline the association of herd-level management factors not strictly related to reproduction with herd reproductive performance.

Survey of management practices on reproductive performance of dairy cattle on large US commercial farms.

A survey regarding general management, sire selection, reproductive management, inseminator training and technique, heat abatement, body condition scoring, facility design and grouping, nutrition, employee training and management, and animal health and bio-security was carried out from March to September of 2004 in 153 herds in the Alta Genetics (Watertown, WI) Advantage Progeny Testing Program. A total of 103 herds (67.3%) completed the survey. Herd size was 613 +/- 46 cows, with herds located in Wisconsin (26), California (12), New York (11), Minnesota (10), Michigan (7), Washington (6), Pennsylvania (6), Iowa (5), Idaho (5), Texas (4), Ohio (4), and other states (7). These farms sold 34.5 +/- 0.3 kg of milk/d per cow, with an annual culling rate of 34 +/- 1% and a calving interval of 13.8 +/- 0.1 mo. Cows were observed for estrus 2.8 +/- 0.3 times/d, for a duration of 27 +/- 4 min, but 78% of the respondents admitted that detection of estrus was not the employee's sole responsibility at that time. Managers tried to achieve pregnancy until 8.8 +/- 0.9 failed inseminations, 300 +/- 26 d postpartum, or milk yield <17.7 +/- 0.5 kg/d. Nonpregnant cows were culled at 326 +/- 36 d postpartum or milk yield <16.4 +/- 0.3 kg/ d. Mean durations of the voluntary waiting period were 52 +/- 1.3 and 53 +/- 1.4 d for primiparous and multiparous cows, respectively. Hormonal synchronization or timed artificial insemination programs were used in 87% of the herds, with 86% synchronizing first services, 77% resynchronizing repeat services, and 59% treating cystic, anestrous, or anovular cows. Finding good employees was identified as the greatest labor challenge, followed by training and supervising employees. Mastitis and hairy heel warts were noted as the greatest animal health concerns, followed by lameness, abortions, and death losses, whereas the greatest reproductive challenges were artificial insemination service rate, conception rate, twinning, and retained placenta or metritis. Results of this study can provide a useful benchmark or reference with regard to commonly used management practices on large commercial US dairy farms at the present time.

Death of serum-free mouse embryo cells caused by epidermal growth factor deprivation.

Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, are proastroblasts that are acutely dependent on epidermal growth factor (EGF) for survival. Ultrastructurally, an early change found in SFME cells deprived of EGF was a loss of polysomes which sedimentation analysis confirmed to be a shift from polysomes to monosomes. The ribosomal shift was not accompanied by decreased steady-state level of cytoplasmic actin mRNA examined as an indicator of cellular mRNA level. With time the cells became small and severely degenerate and exhibited nuclear morphology characteristic of apoptosis. Genomic DNA isolated from cultures undergoing EGF deprivation-dependent cell death exhibited a pattern of fragmentation resulting from endonuclease activation characteristic of cells undergoing apoptosis or programmed cell death. Flow cytometric analysis indicated that cultures in the absence of EGF contained almost exclusively G1-phase cells. Some of the phenomena associated with EGF deprivation of SFME cells are similar to those observed upon NGF deprivation of nerve cells in culture, suggesting that these neuroectodermal-derived cell types share common mechanisms of proliferative control involving peptide growth factor-dependent survival.

Oncogene transformation frequency of nonsenescent SFME cells is increased by c-myc.

Immortalized, postcrisis mouse embryo cell cultures derived in serum-containing medium display genomic abnormalities and an altered, preneoplastic phenotype. These lines can be transformed with single oncogenes, such as Ha-ras, while efficient transformation of precrisis, genomically unaltered rodent embryo cultures require cooperating oncogenes, such as Ha-ras and the mouse c-myc gene constitutively expressed. Serum-free mouse embryo (SFME) cells, cultured under conditions in which serum is replaced by growth factors and other supplements, are 'immortalized' in the genomically unaltered state. SFME cells do not exhibit growth crisis or gross chromosomal aberration, and are dependent on epidermal growth factor for survival, growth inhibited by serum, and are nontumorigenic. Transformation of SFME cells can be achieved with ras alone, but the introduction of c-myc increased the transfection frequency upon subsequent transfection with ras by as much as twenty fold. Similar results were obtained with mutationally activated neu oncogene and with genomic human tumor DNA. Constitutive expression of c-myc alone did not alter the properties of the SFME cells. These results demonstrate that c-myc alters cellular responses to oncogenes in a culture system in which oncogene-induced immortalization is not a factor, indicating that the effects of myc may extend beyond an 'immortalization' function in these cells.

Serial passage of embryonic human astrocytes in serum-free, hormone-supplemented medium.

We applied serum-free cell culture methods that allow extended proliferation of mouse astrocyte precursor cells to the multipassage culture of embryonic human brain cells. Cells were cultured in nutrient medium supplemented with insulin, transferrin, epidermal growth factor, fibroblast growth factor, heparin, high-density lipoprotein, and fibronectin. Cultures were maintained for a maximum of 70 population doublings before proliferation ceased. The cells synthesized glial fibrillary acidic protein, an astrocyte marker, and expression of this protein was increased by incubation of the cells with transforming growth factor beta or serum. These results identify extracellular factors important for proliferation and differentiation of embryonic human astrocytes and provide a controlled system for multipassage culture.

Serum inhibition of proliferation of serum-free mouse embryo cells.

Serum-free mouse embryo (SFME) cells, derived in medium supplemented with insulin, transferrin, high density lipoprotein, epidermal growth factor, and fibronectin, do not undergo crisis, maintain a predominantly diploid karyotype with no detectable chromosomal abnormalities for well over 100 population doublings in vitro, and are growth inhibited by concentrations of serum that are growth-stimulatory for most cell lines in culture. Serum inhibition of SFME cell proliferation was reversible and was not prevented by addition of the supplements of the serum-free medium, even when added repeatedly during the culture period. The serum effect on SFME cell proliferation could be detected after incubation in serum-containing medium for as little as 8 h. SFME cells in serum-containing medium were arrested in the G1 phase of the cell cycle with a greatly reduced rate of incorporation of precursors into DNA and thymidine kinase activity, while a reduction in rate of incorporation of amino acids into protein was not observed. SFME cultures maintained for extended periods in serum-containing medium underwent a crisis-like period followed by the appearance of variant cells capable of growing in serum-supplemented medium. These cells exhibited abnormal karyotype and were resistant to several inhibitors of proliferation active on the parent SFME cell type.

Serum and transforming growth factor beta regulate glial fibrillary acidic protein in serum-free-derived mouse embryo cells.

Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, display distinctive properties: (i) SFME cells do not lose proliferative potential or show gross chromosomal aberration upon extended culture, (ii) these cells depend on epidermal growth factor for survival; and (iii) SFME cell proliferation is reversibly inhibited by serum. Treatment of SFME cells with serum or transforming growth factor beta led to the appearance of glial fibrillary acidic protein, a specific marker for astrocytes. The appearance of glial fibrillary acidic protein in cultures was reversed upon removal of transforming growth factor beta or serum. Cells with properties similar to SFME cells were also isolated from adult mouse brain. These results suggest a role for transforming growth factor beta in astrocyte differentiation in developing organisms and in response to injury and identify the cell type that has the unusual properties of SFME cells.

Transforming growth factor beta regulates cystatin C in serum-free mouse embryo (SFME) cells.

Differential screening of a cDNA library derived from mRNA of TGF beta-treated serum-free mouse embryo (astrocyte precursor) cells isolated a strongly TGF beta-regulated mRNA that codes for cystatin C, a cysteine protease inhibitor. Increase in cystatin C mRNA level was observed within four hours after treatment with picomolar concentrations of TGF beta. The increase was reversible upon removal of TGF beta and was not prevented by cycloheximide. These results suggest that cystatin C expression may represent a developmentally regulated differentiated function of astrocytes, and also suggest that cystatin C expression may be involved in the response of brain cells to platelet release of TGF beta after trauma or injury.

ras and neu oncogenes reverse serum inhibition and epidermal growth factor dependence of serum-free mouse embryo cells.

Serum-free mouse embryo cells, cultured in basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor, fibronectin, and high-density lipoprotein, do not exhibit growth crisis, lack detectable chromosomal aberrations, are nontumorigenic in vivo, are dependent on epidermal growth factor for survival, and are growth inhibited by serum or platelet-free plasma. These cells after transfection with the human Ha-ras or rat neu oncogenes no longer required epidermal growth factor for survival, were tumorigenic in vivo, and also proliferated in serum-containing medium. Autocrine activity capable of replacing epidermal growth factor was detected in conditioned medium from ras-transformed cultures, but little such activity was detected in medium from neu-transformed cultures. In addition, the capability of ras or neu-transformed cells to grow in serum-containing medium could not be mimicked in untransformed cells by the addition of growth factors or conditioned medium from transformed cells. These results suggest that the known structural similarity of the neu gene product to the EGF receptor is also reflected in a functional similarity by which the mutationally activated neu protein can replace the ligand-activated EGF receptor. These results also suggest that the ability of ras- and neu-transformed cells to escape the effect of the inhibitory serum activity is a nonautocrine property distinct from the acquisition of EGF autonomy.

Glucocorticoid and thyroid hormones inhibit proliferation of serum-free mouse embryo (SFME) cells.

Mouse embryo cells derived in a serum-free medium formulation (SFME cells) do not exhibit growth crisis or chromosomal abnormalities and are nontumorigenic in vivo; these cells are also reversibly growth inhibited by serum or platelet-free plasma (Loo et al.; Science, 236:200-202, 1987). A portion of the inhibitory activity of serum could be extracted by charcoal, a procedure that removes steroid and thyroid hormones. Both L-3,5,3'-triiodothyronine (T3) and hydrocortisone inhibited growth of SFME cells in a reversible manner. The inhibitory activity of serum also was partially removed by treatment with anion exchange resin in a procedure designed to deplete serum of thyroid hormone. However, the effect of serum on untransformed SFME cells could not be prevented by addition of the antiglucocorticoid RU38486, and ras-transformed clones of SFME cells, which are capable of growing in serum-containing medium, retained inhibitory responses to glucocorticoid and, with some clonal variability, to T3. These results suggest that glucocorticoid or thyroid hormones may contribute to the inhibitory activity of serum on SFME cells, but additional factors are also involved.

Death of serum-free mouse embryo cells caused by epidermal growth factor deprivation is prevented by cycloheximide, 12-O-tetradecanoylphorbol-13-acetate, or vanadate.

Serum-free mouse embryo cells cultured in medium supplemented with insulin, transferrin, high-density lipoprotein, and fibronectin are dependent on epidermal growth factor for survival. Cycloheximide or actinomycin D prevented cell death caused by growth factor deprivation, suggesting that cell death required the synthesis of RNA and protein, a phenomenon similar to that reported for neuronal cell death in the absence of nerve growth factor. Orthovanadate, an inhibitor of phosphotyrosine phosphatases, and 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, also prevented serum-free mouse embryo cell death in the absence of epidermal growth factor.

Cardiovascular performance in anesthetized rats pretreated with 1,3-bis(2-chloroethyl)-1-Nitrosourea (BCNU).

Cardiovascular function was studied in anesthetized rats at weekly intervals following their pretreatment with single i.p. doses of BCNU (20 mg/kg). Over the first two weeks post-treatment the predominant effects were on total peripheral resistance. Femoral artery diastolic and mean pressures increased with a corresponding decrease in pulse pressure. Thereafter, apparent cardiac effects were also manifest by decreases in systolic and mean pressures, and possibly heartrate. Diastolic and pulse pressures also declined in this second phase. The water content of caudal artery segments from these rats decreased from 82.3 +/- 0.6% in controls to 78.4 +/- 0.6% (P less than 0.05) at 4 weeks post treatment. The total collagen content of these segments had increased 35% over controls as early as 2 weeks after BCNU while total protein content remained unchanged. Whole body responsiveness to the pressor effects of bolus injections of norepinephrine was significantly diminished only at the 4-week sampling time. The cardiovascular toxicity of BCNU may be a complex of direct effects of the drug on vascular endothelia and secondary effects associated with the cholestasis which develops over a similar time course.

Serum-free mouse embryo cells: growth responses in vitro.

We have derived serum-free mouse embryo (SFME) cultures in a basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and fibronectin. These cells are nontumorigenic, lack gross chromosomal aberrations, and exhibit several other unique properties, including dependence on EGF for survival and growth inhibition by serum. We have examined the concentration dependence of the growth stimulatory effects of protein supplements used in the SFME medium formulation and surveyed other supplements that might act as alternative or complementary additions to the culture medium. Insulin could be replaced by insulin-like growth factor I and EGF could be replaced by transforming growth factor alpha in the same concentration range. Transferrin could be replaced by higher concentrations of lactoferrin. Deterioration of cultures in the absence of EGF began within 8 hours of the removal of the growth factor, and could be prevented by the addition of fibroblast growth factor/heparin-binding growth factor. Attachment proteins other than fibronectin were effective on SFME cells, but limited success was obtained when substituting other lipid preparations for HDL. These data introduce a precise system for exploring the unusual characteristics of SFME cells and contribute additional information that may be useful in the extension of these approaches to other cell types and species.

Extended culture of mouse embryo cells without senescence: inhibition by serum.

Mouse embryo cells cultured in vitro in serum-supplemented media undergo growth crisis, resulting in the loss of genomically normal cells prior to the appearance of established, aneuploid cell lines. Mouse embryo cells established and maintained for multiple passages in the absence of serum did not exhibit growth crisis or gross chromosomal aberration. Cells cultured under these conditions were dependent on epidermal growth factor for survival. Proliferation was reversibly inhibited by serum or platelet-free plasma, suggesting that mouse embryo cultures maintained by conventional procedures are under the influence of inhibitory factors.