Effect of different ambient temperatures on reproductive outcome and wellbeing of lactating females in two mouse strains

Ambient temperature is an important non-biotic environmental factor influencing immunological and oncological parameters in laboratory mice. It is under discussion which temperature is more appropriate and whether the commonly used room temperature in rodent facilities of about 21°C represents a chronic cold stress or the 30°C of the thermoneutral zone constitutes heat stress for the animals. In this study we selected the physiological challenging period of lactation to investigate the influence of a cage temperature of 20°C, 25°C, and 30°C, respectively, on reproductive performance and stress hormone levels in two frequently used mouse strains. We found that more pups were weaned from B6D2F1 hybrids compared to C57BL/6N mothers and that the number of weaned pups was strongly reduced if mothers of both strains were kept at 30°C. Furthermore, at 30°C mothers and pups showed reduced body weight at weaning and offspring had longer tails. Despite pronounced temperature effects on reproductive parameters, we did not find any impact on adrenocortical activity in breeding and control mice. Independent of the ambient temperature however, we found that females raising pups showed elevated levels of fecal corticosterone metabolites (FCMs) compared to controls. Increased levels of stress hormone metabolites were measured specially around birth and during the third week of lactation. Our results provide no evidence for reduced or improved wellbeing of lactating mice at different ambient temperatures, but we found that a 30°C cage temperature impairs reproductive performance.

Aiming to study thermoregulatory behavior in mice Gordon and Coworkers [1] started 51 a discussion about the optimal ambient temperature, which culminated in a widely 52 noticed publication of Hylander and Repasky [2]. The authors emphasized in their 53 paper the different results of immunological and oncological studies when conducted 54 at 20°C or at 30°C. Consequently, the results of studies on mouse models for human 55 diseases, performed at 20-26°C standard ambient temperature were questioned and 56 considered to be temperature biased, because of low reproducibility if performed under 57 higher ambient temperatures [3][4][5][6]. It is generally accepted that room temperature can 58 influence experimental results, like many other biotic and non-biotic environmental 59 factors [7]. However, some of the reported effects related to ambient temperature 60 merge only when mice were heated up to a body temperature of 39-40°C for 6 h [8-61 12] or to 42°C for 40 min [13]. 62 Although a comprehensive analysis about the appropriate ambient temperature for 63 laboratory mice in experiments is still missing, the call for housing laboratory mice in 64 their thermoneutral zone as standard ambient temperature arised. The thermoneutral 65 zone is defined as a temperature range in which the general metabolism of the 66 organism, in the absence of any physical activity, generates sufficient heat as a 67 byproduct of the continually ongoing metabolism to maintain the predetermined body 68 temperature [14]. Thermal physiology of nocturnal mice seems to be different between 69 dark and light periods. Influenced by the circadian rhythm two diurnal changing discrete 70 ambient temperatures are proposed as thermoneutral points (TNP): ~29°C in light 71 phase and ~33°C in dark phase [15]. In initial tests mice preferred to stay in warmer 72 areas of experimental settings even if nesting material was provided. These 73 thermoregulatory experiments were conducted using a copper pipe with a wire mesh 4 74 inside [1] or an aluminium channel [16], heated at one side, cooled at the opposite 75 side. This setup led to the assumption that mice prefer an ambient temperature near 76 their homeothermic temperature of 30°C. In later studies, a more common laboratory 77 mouse environment was used [17,18]. By offering bedding and nesting material it 78 became obvious that the preferred ambient temperature depends on the activity of the 79 mice and the amount and quality of nesting material [19][20][21][22][23]. With enough and useful 80 nesting material mice can prevent their body from cooling down during resting periods 81 [24]. Depending on activity, the body core temperature can change between 36°C and 82 37°C [25]. Also, the homeothermic zone seems to be more a temperature point than a 83 zone and varies about 4°C across the day. Temperatures below this homeothermic 84 point lead to increased energy expenditures, whereas temperatures above lead to a 85 rise in body temperature [15].

87
For a naked human being the thermoneutral zone is similar to that of mice and ranges 88 between 28°C and 29°C [26]. But as soon as the human body is covered with light 89 clothing (e.g. long sleeved shirt or blouse and light trousers) this range drops down to 90 23°C -25°C [27]   Unfortunately, there are no studies to our knowledge, regarding the optimal ambient 131 temperature for the wellbeing of lactating mice. Lactation is a highly demanding 132 metabolic process [45,46] accompanied by considerable metabolic heat production as 133 a by-product. Knowing the optimal ambient temperature of lactating mice would be 134 highly valuable to optimize animal keeping and conditions in breeding colonies.

135
In this study we therefore investigated the impact of different ambient temperatures    These females were randomly assigned (12/12/13) to one of the temperature groups 171 (30°/25°/20°C). In addition, 8 B6 and 8 F1 plug negative or non-mated females, and 8 8 172 B6 and 8 F1 males of the same age were used as controls for each temperature group.

173
Seven days after the detection of a vaginal plug the group that was assigned to a 30°C 174 cage temperature was transferred into an identical room next door with 25°C cage 175 temperature for stepwise adaptation. After seven days, this group was finally re-located 176 to an identical room next door with 30°C for the last week of pregnancy, birth and 177 lactation. The second group was transferred to 25°C room 14 days after plug detection.

178
The third group stayed in the room with 20°C cage temperature from the beginning 179 and remained there until the end of the experiment (Fig 1). We expected pup births 180 about 20 days after plug detection. Consequently, one week before the expected birth 181 date all experimental and control animals were in rooms with their assigned cage 182 temperature. Because birth took place between 18 to 21 days after plug detection the 183 exact number of days under increased ambient temperatures before parturition differed 184 slightly between animals of the respective temperature groups.   Additionally, once a week a 24 h sample collection was performed. Therefore, animals 240 were transferred to a fresh cage and after 24 h bedding and feces were collected and 241 frozen. As voided feces were mixed with the fresh bedding we sorted the fecal pellets 242 later by hand before weighing. The total amounts of excreted feces within 24 h was 243 recorded in mice between temperature groups to be able to account for differences in    Mean pup body mass also differed significantly between cage temperatures (F=13.39, 338 p<0.001; Fig 5) and was highest in the 25°C group, followed by the 20°C group and 339 was lowest in the 30°C group (all post-hoc tests p≤0.025). We did not detect any strain 340 specific differences in mean pup body mass (F=3.34, p=0.075; Fig 5), and we did not  Fig 7C). Experimental (breeding) females 367 consumed significantly more food compared to mice from the control groups (p<0.001).

368
No difference was found between male and female control mice (p=0.535). In line with 369 the higher food consumption, F1 hybrids produced significantly more feces per day 370 than B6 mice (F=19.48, p<0.001; Fig 8B). Moreover, feces production significantly