Developmental synergism of steroidal estrogens in sex determination.

Gonadal sex in the red-eared slider turtle, Trachemys scripta, is determined by incubation temperature during embryonic development. Evidence suggests that temperature determines sex by influencing steroid hormone metabolism and/or sensitivity: steroidogenic enzyme inhibitors or exogenous sex steroid hormones and their man-made analogs override (or enhance) temperature effects on sex determination. Specifically, nonaromatizable androgens and aromatase inhibitors induce testis differentiation at female-producing temperatures, whereas aromatizable androgens and estrogens induce ovary differentiation at male-producing temperatures. Moreover, natural estrogens and temperature synergize to produce more females than would be expected if estrogens and temperature had purely additive effects on sex determination. In this study, we use sex reversal of turtle embryos incubated at a male-producing temperature to examine synergism among steroidal estrogens: estrone, 17ss-estradiol, and estriol. A low dose of 17ss-estradiol (200 ng) showed significant synergism when administered with a single low dose of estriol (10 ng). Likewise, a single low dose of estrone (250 ng) had a synergistic effect when combined with the same low dose of estriol (10 ng). We conclude that the weak natural estrogens estrone and 17ss-estradiol synergize with a low dose of the more potent estriol to reverse gonadal sex during the critical period of sexual differentiation. These results suggest that weak environmental estrogens may also synergize with stronger natural estrogens.

No threshold dose for estradiol-induced sex reversal of turtle embryos: how little is too much?

Risk assessments for nongenotoxic chemicals assume a threshold below which no adverse outcomes are seen. However, when an endogenous chemical, such as 17ss-estradiol (E2), occurs at a concentration sufficient to cause an effect, the threshold is already exceeded. Under these circumstances, exogenous estradiol is not expected to provide a threshold dose. This principle is demonstrated for E2 in the red-eared slider, a turtle with temperature-dependent sex determination. In this species, gonadal sex is determined by egg incubation temperature; female development requires endogenous estrogen produced by elevated temperature. While normal production of females by endogenous estrogens is not an adverse effect, exogenous estrogens can sex reverse presumptive males, which can be an adverse effect. A large dose-response study was conducted using seven doses and a vehicle control (starting n = 300/group); a single E2 dose was applied to the eggshell of recently laid eggs. Animals were sexed after hatching. The incubation temperature chosen, 28.6 degrees C, generates a minority of females. Thus, the criteria for testing the threshold hypothesis were met, i.e., there is evidence that there is endogenous estrogen and that it generates an irreversible response. The lowest E2 dose tested, 400 pg/egg (40 ng/kg), sex reversed 14.4% of the animals, demonstrating very low dose sensitivity. The data were fit with a modified Michaelis-Menten equation, which provided an estimate of 1.7 ng/egg for endogenous estradiol. The median effective dose (ED50) was 5.0 +/- 2.0 ng/egg (95% confidence limits), of which 1.7 ng/egg was endogenous estradiol and 3.3 ng/egg came from the applied estradiol. There was no apparent threshold dose for E2. A smaller replication confirmed these results. These results provide a simple biologically based dose-response model and suggest that chemicals which act mechanistically like E2 may also show no threshold dose. If so, even low environmental concentrations of such chemicals may carry risk for sex reversal.

In vitro microbiological characterization of novel cyclic homopentapeptides, CP-101,680 and CP-163,234, for animal health use.

Two cyclic homopentapeptides, CP-101,680 and CP-163,234 [6a-(3',4'-dichlorophenylamino) analogs of viomycin and capreomycin, respectively], were identified as novel antibacterial agents for the treatment of animal disease, especially for livestock respiratory disease. The in vitro microbiological characterization of both CP-101,680 and CP-163,234 was carried out using their parent compounds, viomycin and capreomycin, as controls. This characterization included antibacterial spectrum, influence of media, inoculum size, pH, EDTA, polymixin B nonapeptide (PMBN), serum, cell-free protein synthesis inhibition, and time-kill kinetics. Our results indicated that the capreomycin analog, CP-163,234, showed slightly improved in vitro potency over the viomycin analog, CP-101,680. Both analogs showed very potent cell-free protein synthesis inhibition activity and were bactericidal against Pasteurella haemolytica, P. multocida and Actinobacillus pleuropneumoniae at the level of 4 times and 8 times MICs. CP-163,234 was bactericidal at the level of 4x and 8x MIC against E. coli, but re-growth was observed after 24 hours incubation at both concentrations of CP-101,680.

Cloning and in situ hybridization analysis of estrogen receptor in the developing gonad of the red-eared slider turtle, a species with temperature-dependent sex determination.

Many reptiles exhibit temperature-dependent sex determination where the incubation temperature of the egg determines the gonadal sex of the individual. If exogenous estrogen is administered during the temperature-sensitive period to embryos incubating at a male-producing temperature, the temperature effects can be overridden and females will be produced. Inhibiting production of endogenous estrogens at female-biased incubation temperatures results in embryos developing as males rather than females. Thus, estrogen-estrogen receptor-dependent mechanisms appear to play a key role in female sex determination. The present study characterized the expression of the estrogen receptor during the critical period of temperature sensitivity in the red-eared slider turtle, Trachemys scripta. Polymerase chain reaction was used to amplify estrogen receptor cDNA. A portion of the estrogen receptor cDNA was used to produce probes for in situ hybridization analyses to localize and quantitate levels of estrogen receptor mRNA at different stages of development in embryos from different incubation temperatures. Estrogen receptor mRNA is expressed in the gonadal tissues of both putative males and putative females even before the gonads begin to resolve as ovaries or testes. There is a greater abundance of estrogen receptor mRNA in putative females at the beginning of the temperature-sensitive period as compared to putative males. In embryos from a female-producing incubation temperature, levels of estrogen receptor mRNA are higher in the beginning of the temperature-sensitive window compared to levels after the ovary is differentiated. These results support the hypothesis that estrogen-estrogen receptor dependent processes are important during sex determination and gonadal differentiation in temperature-dependent sex determination.

In vitro microbiological characterization of novel macrolide CP-163,505 for animal health specific use.

A novel 16-membered-ring macrolide agent (CP-163,505, a reductive amination derivative of repromicin) was identified as an antibacterial against Pasteurella haemolytica, P. multocida and Actinobacillus pleuropneumoniae, important etiological agents of livestock respiratory disease. In vitro MIC50/90 analysis revealed that CP-163,505 was more potent (4x) than tilmicosin against P. multocida, and equivalent to tilmicosin against P. haemolytica and A. pleuropneumoniae. In time kill kinetic studies, CP-163,505 showed bactericidal activity against P. haemolytica, P. multocida and A. pleuropneumoniae and bacteriostatic activity against E. coli at 8 times its MIC. In vitro, CP-163,505 was more potent in alkaline pH (16 approximately 32 x ) and less potent in the presence of excess cations (Mg+2 and Ca+2, 4x). EDTA and PMBN increased CP-163,505 potency against E. coli (4x) but not against the other species. Similar results were obtained with erythromycin A and tilmicosin, which were used as controls. From our data, we hypothesize that Pasteurella and Actinobacillus have an outer membrane significantly different from that of the typical enteric Gram-negative bacterium E. coli.

Synergistic responses of steroidal estrogens in vitro (yeast) and in vivo (turtles).

Many environmental agents exert estrogenic activity. Previous studies from our laboratories demonstrated that certain combinations of environmental estrogens (i) reverse the sex of male turtle embryos in a synergistic manner (Bergeron et al., (1994) Environ. Hlth Perspect. 102, 780-782), and (ii) synergistically transactivate the human estrogen receptor (hER) in yeast and mammalian cells (Arnold et al., (1996) Science 272, 1489-1492). Because our findings with synthetic estrogens suggested that combinations of naturally-occurring steroidal estrogens might also produce synergistic activity of the ER, we used the same model systems to measure the activity of combinations of steroidal estrogens. The activity of combinations of estrone, estradiol-17beta or estradiol-17alpha in yeast strains expressing hER was synergistic at submaximal concentrations of both estrogenic compounds. However, synergy was not observed with mixtures of estrogens when the concentration of one of the estrogens alone was maximally active in yeast. Ligand-binding assays in yeast performed with various radiolabeled estrogens suggested that multiple estrogens may interact with the receptor. The estrogen-dependent process of sex-reversal of turtle embryos incubated at a male-producing temperature was used to determine whether steroidal estrogens also had synergistic activity in vivo. In this instance, a combination of estriol and estradiol-17beta was effective in reversing the gonadal sex of turtle embryos from males to females in a synergistic manner. Our results suggest that the synergy of some combinations of estrogens, synthetic or steroidal, may play a role in the estrogen-dependent process of sexual development in certain species.

Temperature and non-aromatizable androgens: a common pathway in male sex determination in a turtle with temperature-dependent sex determination?

This study addressed the hypothesis that, in the red-eared slider turtle, Trachemys scripta, non-aromatizable androgens are the physiological equivalent of temperature in determining male development. In the first experiment, eggs were treated in the middle of the temperature-sensitive period with 1.0 or 10.0 micrograms androsterone, 5 alpha-dihydrotestosterone, 3 alpha-androstanediol, or 3 beta-androstanediol, while at an all-male, male-biased, or one of two female-biased incubation temperatures. In the second experiment, eggs were treated with the same dosages of dihydrotestosterone at different stages of embryonic development while at a male-biased, threshold, or a female-biased incubation temperature. Results of experiment one indicated that hormone-induced masculinization is specific to non-aromatizable androgens. Results of experiment two indicated that the sensitivity to dihydrotestosterone corresponds to the temperature-sensitive window during development. Further, there is a dose-response relationship but no apparent synergism between exogenous dihydrotestosterone and incubation temperature. When considered with other research, it is suggested that non-aromatizable androgens and their products are involved in the initiation of male sex determination whereas oestrogens and their aromatizable androgen precursors are involved in the initiation of female sex determination.

The relative effectiveness of androstenedione, testosterone, and estrone, precursors to estradiol, in sex reversal in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination.

In many turtles the temperature during the middle of incubation determines the gonadal sex of the hatchling. Sex steroid hormones have been implicated in temperature-dependent sex determination in the red-eared slider turtle, Trachemys scripta; androgen is involved in male sex determination and estradiol in female sex determination. Administration of exogenous estradiol and its agonists to eggs incubating at a male-producing temperature can overcome the effect of temperature and result in all-female offspring. Exogenous testosterone will also result in some female hatchlings if administered to eggs incubated at a male-producing temperature, an effect due to the aromatization of testosterone to estradiol. This study demonstrates that in the red-eared slider, androstenedione, the precursor to both testosterone and estradiol, has a similar effect. In addition, both testosterone and androstenedione synergize with incubation temperature to exert a greater effect at intermediate incubation temperatures that normally produce mixed sex ratios, indicating that as with estradiol, androstenedione and testosterone are involved in the final common pathway of sex determination in this species. At the single dosage administered, estrone and estradiol produced all females at a male-producing incubation temperature.

The role of estrogen in turtle sex determination and the effect of PCBs.

In the current model of vertebrate sex determination and sexual differentiation, gonadal sex is fixed at fertilization by specific chromosomes, a process known as genotypic sex determination (GSD). Only after the gonad is formed do hormones begin to exert an influence that modifies specific structures that eventually will differ the sexes. Many egg-laying reptiles do not exhibit GSD but rather depend on the temperature of the incubating egg to determine the gonadal sex of the offspring, a process termed temperature-dependent sex determination (TSD). Research on TSD indicates that sex determination in these species is fundamentally different in at least one way. Gonadal sex is not irrevocably set by the genetic composition inherited at fertilization but depends ultimately on which genes encoding for steroidogenic enzymes and hormone receptors are activated during the midtrimester of embryonic development by temperature. Incubation temperature modifies the activity as well as the temporal and spatial sequence of enzymes and hormone receptors such that sex-specific hormone milieus, created in the urogenital system of the developing embryo, determine gonad type. Estrogen is the physiologic equivalent of incubation temperature and the proximate cue that initiates female sex determination. There is increasing evidence that some polychlorinated biphenyl (PCB) compounds are capable of disrupting reproductive and endocrine function in fish, birds, and mammals, including humans. Reproductive disorders resulting from exposure to these xenobiotic compounds may include reductions in fertility, hatch rate in fish and birds, and viability of offspring, as well as alterations in hormone levels or adult sexual behaviors, all of which have further implications, particularly in wildlife population dynamics. Research on the mechanism through which these compounds may be acting to alter reproductive function indicates estrogenic activity, by which the compounds may be altering sexual differentiation. In TSD turtles, the estrogenic effect of some PCBs reverses gonadal sex in individuals incubating at an otherwise male-producing temperature. Furthermore, certain PCBs are synergistic in their effect at very low concentrations.

Role of reductase and aromatase in sex determination in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination.

In many turtles the temperature during the middle of incubation determines the gonadal sex of the hatchling. In the red-eared slider turtle (Trachemys scripta), an incubation temperature of 26 degrees C results in all male offspring, whereas an incubation temperature of 31 degrees C results in all female offspring; at temperatures intermediate to these (e.g. 29, 29.2, 29.4 degrees C) a mixed sex ratio is obtained. Administration of exogenous oestrogens will overcome the effects of an all-male producing incubation temperature to cause female sex determination, whereas administration of exogenous dihydrotestosterone (DHT) or testosterone to eggs incubating at an all-female temperature will have no discernible effect. Administration of DHT will cause male sex determination only if administered at intermediate incubation temperatures whereas administration of testosterone to eggs incubating at all male-producing and male-biased intermediate temperatures results in a significant number of female offspring, an effect presumably due to aromatization of testosterone to oestradiol (OE2). Since testosterone serves as the precursor to both DHT and OE2, being metabolized by reductase and aromatase respectively, three experiments were conducted to determine whether various putative reductase and aromatase inhibitors would overcome the effect of incubation temperature. First, while administration of testosterone to eggs incubating at all male-producing and male-biased intermediate temperatures produced females in a dose- and temperature-dependent manner, significant numbers of intersex individuals resulted from high dosage testosterone treatment to eggs incubating at a female-biased intermediate temperature. The reductase inhibitors 4MA and MK906 were capable of producing female offspring if administered at intermediate temperatures, but not in a dose-dependent fashion. Administration of the aromatase inhibitors CGS16949A and CGS20267 resulted in male offspring at both female-biased intermediate and at all female-producing temperatures in a dose-dependent fashion. Second, similar findings were obtained with combined doses of testosterone and reductase or aromatase inhibitors. Combined treatment of eggs at male-biased intermediate incubation temperatures with testosterone and reductase inhibitor resulted in female hatchlings, whereas combined treatment of testosterone and aromatase inhibitor at both female-biased intermediate and at all female-producing temperatures resulted in male hatchlings.(ABSTRACT TRUNCATED AT 400 WORDS)

PCBs as environmental estrogens: turtle sex determination as a biomarker of environmental contamination.

Polychlorinated biphenyls (PCBs) are widespread, low-level environmental pollutants associated with adverse health effects such as immune suppression and teratogenicity. There is increasing evidence that some PCB compounds are capable of disrupting reproductive and endocrine function in fish, birds, and mammals, including humans, particularly during development. Research on the mechanism through which these compounds act to alter reproductive function indicates estrogenic activity, whereby the compounds may be altering sexual differentiation. Here we demonstrate the estrogenic effect of some PCBs by reversing gonadal sex in a reptile species that exhibits temperature-dependent sex determination.

Temperature-dependent sex determination in reptiles: proximate mechanisms, ultimate outcomes, and practical applications.

In many egg-laying reptiles, the incubation temperature of the egg determines the sex of the offspring, a process known as temperature-dependent sex determination (TSD). In TSD sex determination is an "all or none" process and intersexes are rarely formed. How is the external signal of temperature transduced into a genetic signal that determines gonadal sex and channels sexual development? Studies with the red-eared slider turtle have focused on the physiological, biochemical, and molecular cascades initiated by the temperature signal. Both male and female development are active processes--rather than the organized/default system characteristic of vertebrates with genotypic sex determination--that require simultaneous activation and suppression of testis- and ovary-determining cascades for normal sex determination. It appears that temperature accomplishes this end by acting on genes encoding for steroidogenic enzymes and steroid hormone receptors and modifying the endocrine microenvironment in the embryo. The temperature experienced in development also has long-term functional outcomes in addition to sex determination. Research with the leopard gecko indicates that incubation temperature as well as steroid hormones serve as organizers in shaping the adult phenotype, with temperature modulating sex hormone action in sexual differentiation. Finally, practical applications of this research have emerged for the conservation and restoration of endangered egg-laying reptiles as well as the embryonic development of reptiles as biomarkers to monitor the estrogenic effects of common environmental contaminants.

Identification of an isoform of the estrogen receptor messenger RNA lacking exon four and present in the brain.

An isoform of the estrogen receptor messenger RNA (ER-mRNA) was identified in RNA from the brain of lizards and rats. Poly(A)+ RNA from brain and uteri was reverse transcribed using gene-specific primer for the ER. The resulting complementary DNA was amplified in a polymerase chain reaction followed by cloning and sequencing of the amplified products. This isoform lacks exon four and is designated delta 4 ER-mRNA. Although several isoforms of the ER have been described from cancerous cells, to our knowledge, none has been identified previously in the brain. Furthermore, the delta 4 isoform is the only isoform detected in normal tissue. The delta 4 isoform appeared most abundant in RNA from brain tissue, whereas uterine RNA contained only trace amounts of the isoform. Apparently, tissue-specific alternative splicing accounts for these differences in abundance. Because exon four encodes a part of the steroid-binding domain, we predict that the corresponding protein encoded by the isoform will not bind estradiol and may therefore belong to a growing subclass of the steroid/thyroid/vitamin superfamily known as orphan receptors. We predict that the putative delta 4 protein may function as a ligand-independent transcription factor that acts on the same DNA response elements as the conventional ER. The abundance of this isoform in the brain relative to the uterus raises fundamental questions regarding the regulation of estrogen-responsive genes in different tissues.

Comparison of azithromycin, roxithromycin, and cephalexin penetration kinetics in early and mature abscesses.

During the process of abscess formation, a myriad of changes are observed histologically that impede the penetration of antimicrobial agents into infection loci. A Staphylococcus aureus foreign body abscess, developed in rats, was employed to evaluate the penetration kinetics of azithromycin, roxithromycin and cephalexin at various stages of abscess development; the progressive patho-histological changes of abscess formation were also characterized in this model. In an early abscess (18 h post-challenge), azithromycin penetration into inflammatory fluid was enhanced (AUC of 351 vs 130 mg.h/kg) and residence prolonged relative to an inflammation control (half-life of 88 vs 27 h). In contrast, roxithromycin and cephalexin penetration into, and residence in, inflammatory fluid were unaltered in the early abscess. However, penetration into, and egress from, a mature abscess (ten days post-challenge) were impeded for all three antimicrobials (P < or = 0.03). The penetration kinetics of azithromycin into inflammatory fluid in an early abscess were independent of the dose regimen, but dependent on the total dose. The persistently high concentrations of azithromycin in inflammatory fluid within abscess were associated with the infiltration of phagocytic cells and encapsulation by fibrous tissue. These data are consistent with a phagocytic delivery mechanism for azithromycin, whereby the presence of high concentrations of azithromycin in inflammatory fluid are a consequence of augmented drug distribution via the release of accumulated intracellular drug from the infiltrating phagocytic cells and fibroblasts associated with abscess formation.

Preferential concentration of azithromycin in an infected mouse thigh model.

The possibility of augmentation of azithromycin delivery to infection loci was evaluated by the use of Staphylococcus aureus thigh infection models with CD-1 mice. The intramuscular infections that developed were characterized by rapid growth of bacteria and induction of a localized oedema that was assessed gravimetrically. Microscopic examination of infected thighs showed massive infiltration of polymorphonuclear leucocytes (viable and degranulated), when compared to saline-injected thighs, from 24 to > or = 72 h after infection. Azithromycin concentrations were enhanced significantly (P < or = 0.02) in infected thigh tissues compared with contralateral non-infected tissues, and correlated with oedema from 24-72 h after challenge and dosing. The azithromycin levels in infected tissue after a 5 mg/kg dose were sufficient to cause a significant reduction in the number of cfu. If azithromycin administration was delayed until inflammation was more severe, the result was an even greater preferential concentration of azithromycin into the infected thigh. Preferential concentration of azithromycin was not observed when extensive oedema was produced by injection of histamine. However, this oedema was not associated with a significant influx of polymorphonuclear leucocytes. In comparative studies, macrolide antibiotics known to be concentrated in phagocytes, such as erythromycin, roxithromycin, and clarithromycin, were not concentrated preferentially in infected tissues under the experimental conditions used; tissue levels were above or at the in-vitro MIC level for < or = 24 h. The data indicate that delivery of biologically available azithromycin to infected tissues is enhanced by cellular inflammatory processes.

Human alpha 2-HS-glycoprotein/bovine fetuin homologue in mice: identification and developmental regulation of the gene.

Human alpha 2-HS-glycoprotein (AHSG) is a plasma protein synthesized in liver and selectively concentrated in bone matrix. It has been reported to be involved in bone formation and resorption as well as immune responses. Recently, AHSG was found to be the species equivalent protein of fetuin, the major fetal serum protein in cattle and sheep. The function and regulation of AHSG/fetuin in different species are not understood. We have isolated a liver cDNA clone that encodes the human AHSG/bovine fetuin homologue in the mouse. The AHSG/fetuin gene may have a role in differentiation since it is expressed in mouse limb buds and brain only at certain stages during development. Mouse liver AHSG/fetuin mRNA was present at low level at 12 days gestation but its level increased during the late part of gestation and peaked between 1 to 3 months after birth. The regulation of mouse AHSG/fetuin synthesis during development was found to be significantly different from that of sheep and bovine fetuin. Compared to fetuin, which is reduced in adult to 1 to 2% of the fetal level, mouse AHSG synthesis subsides only 50% 4 months after birth.

Molecular properties of monoamine oxidases A and B.

Monoamine oxidases A and B (MAO-A and B) catalyze the oxidative catabolism of biogenic amines and xenobiotics. Investigation of these mitochondrial membrane proteins shows that they differ in substrate preference, inhibitor specificity, tissue and neuronal cell distribution, immunological properties, and nucleotide and deduced amino acid sequences. Comparisons of MAO-A and B from the human, bovine, and rat species show strikingly high similarity (85-88%) in the amino acid sequences of each enzyme. Furthermore, three regions in MAO-A and B have sequence identities across species of 78, 88, and 86%. These regions correspond to a nucleotide-binding site near the N-terminal end that is found in the vast majority of enzymes that require flavin adenine dinucleotide (FAD), a region of unknown function, and the FAD-binding site toward the C-terminal end. Genomic clones of MAO-B which span almost the entire gene (greater than 40 kb) have been isolated, restriction mapped, and partially sequenced. Likewise, genomic clones of MAO-A that correspond to the 3'-flanking region have also been investigated. Current studies which focus on identification of the promoter and regulatory sequences should help to establish why MAO-A and B are localized in different subsets of neurons in brain.

Mapping and conservation of the group-specific component gene in mouse.

The group-specific component (GC), also known as the vitamin D-binding protein, transports vitamin D and its metabolites in plasma to target tissues throughout the body. The GC gene shares an evolutionary origin with genes encoding albumin (ALB) and alpha-fetoprotein (AFP). All three genes are descendants of an evolutionary ancestor that arose from an intragenic triplication. As a result, each gene is composed of three homologous domains. The study described here characterizes and compares mouse GC to the corresponding nucleotide and amino acid sequences of GC from human and rat. The deduced amino acid sequence of mouse GC was 78% identical to human and 91% identical to rat GC. The results suggest that, unlike the corresponding sequences in the ALB and AFP genes, chromosomal sequences encoding the first domain and the leader sequence of the GC gene have specifically been conserved throughout vertebrate evolution. Protection of domain I during evolution may correlate with an important functional aspect of its sequence. The mouse GC gene was mapped to chromosome 5, where the ALB and AFP genes are also located, demonstrating conservation of the three genes in vertebrate species.

Branch-cutting behavior by the vole (Microtus pennsylvanicus) : A mechanism to decrease toxicity of secondary metabolites in conifers.

While investigating the preferences of meadow voles (Microtus pennsylvanicus) toward various coniferous species, we noted they often cut the branches of young trees, and then left them lying on the snow for two days or more before eating them completely. We tested whether this "behavioral manipulation" of a potential food resource resulted in a decrease in the level of secondary metabolites in branches. We found that after being cut for more than three days, the branches of young conifers had significantly less phenolics and condensed tannins. These postmanipulation levels of secondary metabolites were similar to those found in preferred summer food resources. Voles were thus capable of transforming a low-quality resource into one that was more compatible with their feeding patterns and detoxification mechanisms.

Role of phenolics of coniferous trees as deterrents against debarking behavior of meadow voles (Microtus pennsylvanicus).

Preferences of meadow voles (Microtus pennsylvanicus) toward various coniferous species were tested under winter conditions. Cafeteria tests showed preferences for Norway spruce (Picea abies) and Norway pine (Pinus resinosa) and rejection of white pine (Pinus strobus) and white spruce (Picea glauca). When phenolic extracts of these species were prepared and added to a low 8% protein diet, food intake of voles and preferences by rank followed the same patterns. Determination of the concentration of phenolics in branch samples using the Singleton and Rossi procedure showed that Norway pines contained the smallest amount (2.15%/g dry matter) while spruces yielded the most (2.76%/g dry matter). These experiments lead us to expect high losses of Norway pine and Norway spruce in habitats harboring high vole densities.