Materials and methods
ANIMALS
Striped hamsters were obtained from the laboratory-breeding colony, the founders of which were captured in farmland at the center of Hebei province (115°13′E, 38°12′S) on the North China Plain. The climate of this region is arid and characterized by warm, dry summers with a maximum temperature of 42°C, and cold winters in which the temperature can fall below -20°C (Zhao et al. 2010a; Zhao et al. 2010b). The animals were kept at 21±1°C under a 12L:12D (light: dark, lights on at 0800h) photoperiod. Food (standard rodent chow, 17.6 kJ/g; Beijing Keao feed company, Beijing, China) and water were provided ad libitum .
Experiment 1 was designed to examine the effect of wind on the energy intake and reproductive performance of lactating female striped hamsters. Seventy 3 to 3.5-month-old, virgin, female hamsters were housed individually in plastic cages (29 × 18 × 16 cm) provided with sawdust bedding. Each female was paired with a male for two weeks after which the males were removed. Females that subsequently gave birth were allowed to raise their offspring for four days from parturition (day 1 to day 4 of the experiment), during which no measurements were made on either females or pups. On day 5 litter sizes were artificially adjusted so that each female had 8 pups which they were allowed to suckle for 16 days. Females were also randomly assigned to one of four treatment groups:
  1. A 21°C group (21°C, n=9), in which females and pups were kept at an ambient temperature of 21 ± 1°C and not exposed to wind.
  2. A 21°C plus wind group (21°C+W, n=9), in which females and pups were kept at an ambient temperature of 21 ± 1°C and exposed to simulated wind generated by an electric fan.
  3. A 32.5°C group (32.5°C, n=18) in which females and pups were kept at an ambient temperature of 32.5 ± 1°C and not exposed to wind.
  4. A 32.5°C plus wind group (32.5°C+W, n=18), in which females and pups were kept at an ambient temperature of 32.5 ± 1°C and exposed to simulated wind.
All treatments began on day 6 and continued until the end of the experiment on day 16. Wind was simulated using an electric fan (AUX FS1605, AUX Electrical Appliances Co., Ltd. China) to create a wind speed of 2m/s, ranging from 1.6 to 3.3m/s around inside the cages (Anemometer, Testo 405-V1, Testo Instruments International Trading Ltd. Germany).
BODY MASS AND FOOD INTAKE
The body mass and food intake of females were measured daily from day 5 to day 15. Food intake was calculated as the difference between the mass of the food provided and uneaten food on the following day, minus any food residue mixed with bedding material. Litter size and litter mass were also measured daily from day 5 to day 16.
ENERGY INTAKE AND DIGESTIBIITY
Gross energy intake (GEI) and digestibility were measured between days 13 and 14 of the experiment using the food balance method described previously (Grodzinski & Wunder 1975; Wen et al. 2018a; Wen et al. 2018b). In brief, a known quantity of food was provided, and any uneaten food and orts mixed with the bedding material were collected, together with feces, every 24h. Food and feces were separated manually after drying to constant mass at 60°C. The gross energy content of food and feces were then determined using an IKA C2000 oxygen bomb calorimeter (IKA, Germany). GEI, gross energy of feces (GEF), digestive energy intake (DEI) and digestibility were calculated using the following equations:
GEI (kJ/d) = [food provided (g/d) × dry matter content of food (%) – dry spillage of food and uneaten food] × gross energy content of food (kJ/g); GEF (kJ/d) =dry feces mass (g/d) × energy content of feces (kJ /g); DEI (kJ/d) =GEI–GEF; and digestibility (%) = DEI/GEI × 100%
MILK ENRGY OUTPUT
Milk energy output (MEO) during the peak of lactation (days 13-14) was assessed from the energy budget of litters, as described previously (Król & Speakman 2003b). Pups obtain all their energy from their mother’s milk, so total energy was calculated as the sum of the energy allocated for pups’ daily energy expenditure (daily energy expenditure, DEE) and the growth of new tissue (Zhao et al. 2011). DEE was predicted from pup body mass on the basis of the relationship between the resting metabolic rate (RMR) and body mass, under the assumption that DEE = 1.4×RMR to take into account the energetic costs of pups’ activity.
The equation used was (Król & Speakman 2003b):
MEO = [(7.28+0.17×LM) × CF + LMinc × GEpups] ×100/dmilk,
where LM (g) is the litter mass on day 13; CF is the correction factor (CF=1.4, the mean ratio of DEE to RMR) and GEpups (kJ/g wet mass) is the gross energy content of the pups. The mean GEpups values used in this formula were determined using an IKA C2000 oxygen bomb calorimeter. LMinc (g/d) was the increase in litter mass between days 13 and 14, and dmilk was the apparent digestibility of milk (dmilk-96%) (Król & Speakman 2003b).
Experiment 2 was designed to examine the effect of wind on the thermal conductance of fur and the rate of water evaporation at both a moderate (21°C) and high (32.5°C) temperature. Twelve female hamsters maintained individually at 21 ± 1°C were killed by CO2 overdose as described previously (Zhao et al. 2013), and the entire pelage, except for the head, limbs and tail, immediately removed. Pelage from each hamster was then stitched around a 10mL (2cm diameter x 5.5cm long) glass vessel containing water. These pelage were randomly assigned to either a 21°C (21°C, n =6) or a 32.5°C (32.5°C, n=6) treatment groups.
THE EFFECT OF WIND ON WATER EVAPORATION
Vessels were warmed to 60°C and transferred to one of two temperature controlled rooms kept at either 21°C or 32.5°C, respectively. Water temperature was monitored using an encapsulated thermo-sensitive passive transponder (diameter 2 mm and length 14 mm; Destron Fearing, South St Paul, USA) in each vessel, and recorded on a Pocket Reader (Destron Fearing, South St Paul, USA) at one-minute intervals. Wind (2m/s) was simulated as in experiment 1. The temperature of glass vessels without pelage was also monitored under both temperatures to serve as a control.
THE EFFECT OF WIND ON WATER THERMOREGULATION
The rate of water evaporation was monitored using a glass plate (diameter, 10cm) filled with 40 g tap water at 37°C. This was placed on a balance in temperature controlled rooms at 21°C and 32.5°C. The simulated wind treatment at both temperatures was carried out as described above. The change in water weight was measured over 30 min at one-minute intervals.
Experiment 3 was designed to examine the effect of temperature on the preference of females for cages that were exposed to simulated wind or sheltered cages during the peak of lactation. Thirty lactating hamsters were organized as described in experiment 1. Females and their pups were housed in two plastic cages (29 × 18 × 16 cm) connected with a 15cm-long plastic tube of 5cm diameter that allowed females to move freely from one cage to the other. Females had free access to food and water in both cages. All females were kept at 21°C from days 0 to 5 until day 6 when they were randomly assigned to either a 21°C group (n =15) or a 32.5°C group (n =15). From day 10 till day 14, an electric fan was used to expose one of each pair of cages to an air speed of 1.6 – 3.3m/s. The preference of each female for the cage exposed to simulated wind or the sheltered cage was recorded by observing each female in succession for 40 s over a period of 10 min. The number of pups that stayed in the cage exposed to simulated wind or the sheltered cage was also recorded. Observations were carried out over 8 hours both day (8:00-10:00 and 18:00-20:00) and night (6:00-8:00 and 20:00-22:00). Observations during the night were made using a 30 W red light. The preference of females for the cages exposed to simulated wind or sheltered cages was assessed from the amount of time they spent each cage type and expressed as min/h.
Experiment 4 was designed to examine the preferences of wild female hamsters for windy vs calm days during spring and summer. This experiment was performed in late April and mid-July in a dry river bed in Shenze County, Hebei province (115°13′E, 38°12′S) on the North China Plain. A sparse growth of weeds covered the dry, sandy, river bed. The average daily maximum and minimum temperatures in late April were 27.2°C and 15.1°C, respectively, and 36.6°C and 27.1°C in mid-July. Windy days were defined as those on which the wind speed 20cm above the ground was more than 1.6 m/s during the night, and calm days were defined as those on which the wind speed was less than 1.6 m/s during the night. Wind speed was measured with an anemometer (Testo 405-V1, Testo Instruments International Trading Ltd. Germany). Hamsters were captured in 85 live-capture traps placed at 20 m intervals. The total number of adult females and the number of lactating females captured was recorded daily. Lactating females can be easily distinguished by their relatively large nipples. The percentage of lactating females was calculated as the number of lactating females divided by total number of adult females captured. All hamsters were released after their sex and lactation status had been recorded.
STATISTICS
Data were analyzed using SPSS statistical software (V 20.0). In Experiment 1, the effects of temperature and wind on body mass, food intake and the energy parameters of females, as well as on litter size and liter mass, were examined using a two-way ANOVA (temperature x wind), followed by Tukey’s post hoc tests where required. Correlation coefficients between different variables were estimated using Pearson’s correlation coefficient. In Experiment 2, the statistical significance of differences in water temperature and weight were analyzed using a two-way ANOVA (temperature x wind). In Experiment 3, the preference of females for cages exposed to simulated wind vs sheltered cages was assessed using a two-way ANOVA (temperature x wind). In Experiment 4, the statistical significance of differences in the total number of females and proportion of lactating females captured on calm and windy days in spring and summer was also assessed using a two-way ANOVA (season x wind). All data are presented as means ± SEM; P -values <0.05 were considered statistically significant.