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Mar 31

Complex effects of testosterone level on ectoparasite load in a ground squirrel: an experimental test for the … – Parasites & Vectors

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Study area

We carried out our field work in the grassland located within the Experiment Demonstration Base, Grassland Research Institute, Chinese Academy of Agricultural Sciences (40 36 N, 111 45 E). This area has a continental temperate monsoon climate, with an average annual rainfall of ca. 400mm and an annual mean temperature of ca. 6.9C. The dominant plant species are Leymus chinensis, Stipa capillata, Cleistogenes squarrosa and Medicago sativa. Based on our own trapping record, S. dauricus has been the dominant rodent species here in recent years. Other rodents, such as striped hamster (Cricetulus barabensis) and Mongolian gerbil (Meriones Unguiculatus), were also recorded but relatively low in abundance. According to our observation, steppe polecats (Mustela eversmanii), red foxes (Vulpes vulpes) and domestic dogs (Canis lupus familiaris) are the major predators feeding on S. dauricus [32]. Cattle grazing is common here in spring and summer, resulting in an average grass height of ca. 20cm and an average vegetation cover of 45%.

In mid-July, 2023, we conducted the first round of live-trapping in two 1-ha sites located in the study area. In this season, most S. dauricus were reproductively inactive. The two sites were comparable in terms of vegetation, topography and rodent density. To ensure independence in sampling among the sites, there was a distance of 400m between the nearest sites. We placed 100 Sherman live traps (arranged in a 1010 grid, with 10-m intervals between neighboring traps) baited with fresh peanuts in each site. Since S. dauricus were diurnal, the traps were set open between 07:00 and 19:00 (Beijing time). This round of live-trapping lasted for four consecutive days. We checked all the traps every 2h and rebaited the traps if needed. All the S. dauricus captured were immediately put in separate cotton bags and taken back to our laboratory.

A total of 59 S. dauricus (27 males and 32 females) were captured during the first round of live-trapping. We anesthetized each individual by a multi-channel anesthesia machine designed for small animals (R550IE, RWD Life Science Co., Ltd., Shenzhen, China) with isoflurane. To collect the ectoparasites, the body surface of each S. dauricus was carefully scanned using a fine-toothed comb and a tweezer. We also checked the inner side of each cotton bag used to contain the ground squirrels. All the ectoparasites collected from a S. dauricus were immediately placed in ethanol (95%) contained within a separate 5-ml centrifuge tube. All the S. dauricus were weighted to the nearest 0.1g using an electronic balance, toe-clipped for individual identification, and then maintained in separate plastic boxes for 72h with access to ad libitum food (peanuts, alfalfa leaves, and commercial pellets) and water. No S. dauricus showed any abnormal behavior or healthy problem during this period.

We used all the 52 adult individuals (defined as those heavier than 100g, 23 males and 29 females) for our formal experiment. Each ground squirrel was randomly assigned to one of two groups: control group (without testosterone injection, 11 males and 14 females) and treatment group (with testosterone injection, 12 males and 15 females). At 15:0016:00 in the next day after capture, we collected a fresh fecal sample (typically 0.20.3g) from each experimental animal. All the fecal samples were placed in separate 5-ml centrifuge tubes and then immediately stored frozen at 80C. About 72h after capture, each individual was injected intramuscularly with either a dose of tea oil (control group) or a dose of testosteroneoil mixture (10mg of testosterone undecanoate per ml of tea oil). A total of 1h after injection, we released all the individuals at the places where they were captured.

A total of 10days later, we conducted the second round (five consecutive days) of live trapping to recapture the experimental animals. The procedures of live-trapping, anesthesia, ectoparasite collection, fecal sample collection, and animal maintenance were similar to the first round. A total of 28 S. dauricus were recaptured (six males and seven females from the control group, and five males and ten females from the treatment group). An experienced taxonomist (Jian-Jun Wang) later identified all the ectoparasites based on dichotomous keys. The whole experimental procedure adhered to the guidelines approved by the American Society of Mammalogists [34] and the Regulations of the Animal Welfare Committee of Beijing Veterinarians of the Agriculture Ministry of China (Beijing, China).

We typically followed the protocol used by Li etal. [35] to extract testosterone from the fecal samples, with some modifications. A total of 56 fecal samples were used for hormone analyses (i.e., samples collected from the 28 individuals with recaptures, two samples per individual). Since we used wet feces, variations in water content among samples must be accounted for. Therefore, we simultaneously weighed two fecal subsamples (each ca. 0.1g in weight, hereafter subsamples A and B) from each fecal sample. Subsample B was used for measuring water content and was weighed before and after 24-h drying in a drying oven. The water content value was then used to translate the wet sample weight of the relevant subsample A into dry weight.

Subsamples A were used for hormone extraction and placed in separate 10-ml centrifuge tubes. For each tube, we added 4ml of methanol and 1ml of distilled water and then vortexed it for 30min. We then added 2.5ml of petroleum ether to each tube to remove lipid from it. After 10min of vortex, each tube was centrifuged at 1500r/min for 15min. A total of 2ml of liquid was drawn from the methanol layer within each tube and then placed into a 5-ml cryopreservation tube. The methanol was dried off under forced air and the remain was used for hormone assay.

We performed testosterone assays with a commercially available enzyme immunoassay kit (Rat Testosterone Elisa Kit, produced by FanYin Biotechnology Co., Ltd., Shanghai, China). This kit has a sensitivity of 1.0 nanomol/l, and<1% cross-reactivity to other steroids (including progestins, corticoids and estrogens). The testosterone levels were reported as nanogram of fecal testosterone per gram of dry feces.

We performed all the statistical work in R platform 4.2.2 [36]. We first adopted a paired-sample t-test to test whether our experimental treatment affected the fecal testosterone level of S. dauricus. We built a negative-binomial generalized linear mixed-effect model (GLMM) on tick load recorded on the recaptured individuals (hereafter TickLoadafter) using the R package lme4 [37] and lmerTest [38]. The fixed terms included treatment (control or treatment group), sex (male or female), body weight (averaged value of the two measurements), tick load recorded in the first round of ectoparasite check (i.e., tick load before the testosterone manipulation, hereafter TickLoadbefore), flea load in the second round of ectoparasite check (hereafter FleaLoadafter), and an interactive term between treatment and sex. Site ID was used as a random term. Similar models were also built for FleaLoadafter, with fixed factors including treatment, sex, body weight, flea load recorded in the first round of ectoparasite check (hereafter FleaLoadbefore), TickLoadafter, and an interaction between treatment and sex. Variance inflation factors (VIFs) were calculated using the R package car [39] to assess multicollinearity. As the VIFs were all smaller than ten (Table1), we retained all the factors in the models. Model selection was performed based on Alkaike Information Criterion corrected for small sample size (AICc) [40] using the R package MuMIn [41]. Since the performance did not differ significantly between top candidate models (i.e., delta AICc smaller than 2), we used conditional model averaging to get an averaged model based on the full set of candidate models [40]. As we detected a significant interactive effect between treatment and sex on TickLoadafter, we also built two GLMMs on TickLoadafter for male and female squirrels separately (Table2). For these two models, the fixed terms included treatment, body weight, TickLoadbefore, and FleaLoadafter.

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