| Predation is one of the strongest selective pressures. Mustelid odors have been shown to suppress breeding in voles from cyclic populations. The mechanism behind the suppression is unknown. Predation risk has been shown to alter animal behavior in several ways based on genotypic responses and/or phenotypic flexibility of individual's feeding behavior or habitat use. In most studies, predation risk reduced the general activity and feeding of prey individual. Breeding activities, such as mate-searching, copulation and egg-laying and parental activity, can also increase an individual's susceptibility to predation. Despite numerous studies on other taxonomic groups and behavioral traits, little is known about the effects of predation on mating behavior of higher verebrates, especially mammals. One more possible mechanism for breeding suppression is a physiological one based on hormonal changes caused by predation risk. However, relatively few studies on physiological and psychological stress have worked with predators.Measuring steroid hormone metabolites in fecal samples has become a widely appreciated technique, because it has been proved to be a powerful noninvasive tool that provides important information about an animal's endocrine status (adrenocortical activity and reproductive status). Measurement of these hormones in fecal extracts by high-performance liquid chromatography (HPLC) was validated using chemical derivatization, mass spectrometry.The objectives of the present study were (1) to develop a method for assessment of fecal glucocorticoids (specifically cortisol and corticosterone) as ameasure of stress in rodent, specifically striped field mice (Apodemus agrarius), and (2) to discuss the mechanism for breeding suppression based on hormonal changes caused by predation risk, and (3)to investigate the possible change in the mating behavior between experimental and control groups and to examine whether paired mice show intersexual differences in their response to predation risk. The main results of this are as follow.1. A simple and sensitive method for the determination of steroid compounds extracted from mice feces using 1, 2-benzo-3, 4-dihydrocarbazole-9-ethyl-carbonylhydazine (BCEC) as pre-column derivatization reagent by high-performance liquid chromatography in combination with a gradient elution with fluorescence detection and mass spectrometric identification has been developed. Studies on derivatization conditions indicate that steroid compounds react with BCEC at 65 °C within a two-hour period in the presence of trichloroacetic acid (TCA) catalyst with acetonitrile as reaction co-solvent to give the corresponding sensitive fluorescence derivatives. The fluorescence excitation and emission wavelengths are set at A. ex 333 and ^ em390nm, respectively. The identification of steroid derivatives is performed under APCI source in positive-ion mode. Correlation coefficients for steroid derivatives are >0.9999, and detection limits (at signal-to-noise ratio of 3:1) are 47.3—71.2 finol. The established method is sensitive and reproducible for the determination of steroid compounds from real samples with satisfactory results.2. The weights of both females and males differed significantly between during the days of stress treatment, but females were more frangibility. Males lost weight under predator odor significantly on dayl4 (P=0.016) and day21 (P=0.031) contrast to baseline, while female mice body mass began to decrease significantly on day7, lasted to day21 (day7, P=0.003;day 14, P=0.006;day21, P=0.01).3. Our data suggest that there is difference in mechanism between male and female mice to adjust endocrine responses to predator odor. A one-way ANOVA revealed that long-term predator odor (21 day) treatment did not altercortisol[F(4,35)=1.35,P=0.27] and testosterone[F(4,35)= 0.424,P=0.79] levels in male striped field mice, but corticosterone level on day21 was higher than that of baselines [F (l,39)=6.923;P=0.013]. Cortisol levels in female mice were significantly higher that those of baselines [F (1, 39) =5.84, P=0.021];LSD post hoc tests indicated that average cortisol level in individuals on baselines was significantly lower than that of dayl4 (P=0.037), and individuals on day21 (P=0.032). Long-term stress treatment did not alter corticosterone levels in female mice [F (4, 35) =2.39, P=0.069], but LSD post hoc tests indicated that average corticosterone level in individuals on day7 was significantly higher than that of baselines (P=0.004). One-way ANOVA analyses revealed that long-term predator odor treatment restrained female mice from secreting progesterone [F (4, 35) =2.87, P=0.037], LSD post hoc tests indicated that average progesterone level in individuals on day21 was significantly lower than that of baselines (P=0.002).4. Our results suggest that under predator odor treatment female behavior have most changed. This difference in the reaction between the sexes is in accordance with predictions for a situation where reproductive costs are divided unequally between the two sexes. Predation risk simulated by odor decreased the investigating (Z=2.38, P=0.015), self-grooming behavior (Z=2.42, P=0.015) and mating behavior (Z=2.83, P=0.004) of the experimental male mice compared to control ones, and the accumulated time of investigating (Z=2.38, P=0.017), self-grooming behavior (Z=2.08, P=0.037) and mating behavior (Z=3.36, P=0.0007) were significant decreased. While the general activity, chasing, aggressive behavior and the accumulated time were not observed significant differences. Under predator odor treatment, the self-grooming, investigating, aggressive, solciation walk and lordosis behavior frequencies and accumulated times were significantly decreased.
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