The limits to sustained energy intake set physiological upper boundaries that affect many aspects of human and animal performance. The mechanisms underlying these limits, however, remain unclear. We exposed Swiss mice to either supplementary thyroid hormones (THs) or the inhibitor methimazole during lactation at 21 or 32.5°C, and measured food intake, resting metabolic rate (RMR), milk energy output (MEO), serum THs and mammary gland gene expression of females, and litter size and mass of their offspring. Lactating females developed hyperthyroidism following exposure to supplementary THs at 21°C, but they did not significantly change body temperature, asymptotic food intake, RMR or MEO, and litter and mass were unaffected. Hypothyroidism, induced by either methimazole or 32.5°C exposure, significantly decreased asymptotic food intake, RMR and MEO, resulting in significantly decreased litter size and litter mass. Furthermore, gene expression of key genes in the mammary gland was significantly decreased by either methimazole or heat exposure, including gene expression of THs and prolactin receptors, and Stat5a and Stat5b. This suggests that endogenous THs are necessary to maintain sustained energy intake and MEO. Suppression of the thyroid axis seems to be an essential aspect of the mechanism by which mice at 32.5°C reduce their lactation performance to avoid overheating. However, THs do not define the upper limit to sustained energy intake and MEO at peak lactation at 21°C. Another, as yet unknown, factor prevents supplementary thyroxine exerting any stimulatory metabolic impacts on lactating mice at 21°C.

Lactation is one of the most energy demanding periods in the life cycle of small mammals (Loudon and Racey, 1987; Wade and Schneider, 1992; Hammond and Diamond, 1997; Butte and King, 2005; Speakman and Król, 2005a,b). During lactation, females increase their food intake enormously to meet the energy requirements of the offspring, but at peak lactation they reach a limit to either food intake or milk production, or both, i.e. the sustained maximal rate of energy intake (Drent and Daan, 1980; Hammond and Diamond, 1992, 1994, 1997; Peterson et al., 1990; Speakman and Król, 2005a,b; Weiner, 1992; Król and Speakman, 2003a; Laurien-Kehnen and Trillmich, 2003). The limits to sustained energy intake set physiological upper boundaries that affect many aspects of human and animal performance, such as reproductive performance at peak lactation, as well as thermoregulatory capability under extreme cold temperatures, and running performance for predation (Hammond and Diamond, 1997; Speakman and Król, 2005b, 2011).

The nature of these limits, has been a subject of debate for at least 30 years (reviewed in Speakman and Król, 2005b, 2011; Król et al., 2011). Twenty to thirty years ago, the debate focused on whether the limits to sustained energy intake during lactation are imposed ‘centrally’, by the capacity of the gut and other central energy-supplying organs, or ‘peripherally’, by the milk-producing capacity of mammary glands, i.e. the peripheral hypothesis (Koteja, 1996; Hammond and Diamond, 1997; Ohrnberger et al., 2018). In the last 15 years, the heat dissipation limitation (HDL) theory has provided an alternative explanation, i.e. at a given ambient temperature, the capacity to dissipate heat may limit sustained energy intake during lactation (Speakman and Król, 2005a). When exposing female mice to the cold at peak lactation, they ingest more food, synthesise more milk and wean larger pups, as cold exposure removes the constraint on their ability to dissipate heat (Hammond and Kristan, 2000; Johnson and Speakman, 2001). Conversely, exposing lactating females into hot conditions reduces their capacity to dissipate heat, enforces a reduction in food intake and milk synthesis, and leads to stunted offspring (Speakman and Król, 2011; Król and Speakman, 2003a,b; Zhao et al., 2016, 2020). So far, the studies extensively performed in rodents have provided evidence either for the peripheral hypothesis (Hammond and Diamond, 1992, 1994; Zhao et al., 2010; Wen et al., 2017; Huang et al., 2020) or for the HDL theory (Król et al., 2007; Wu et al., 2009; Valencak et al., 2010, 2013; Simons et al., 2011; Yang et al., 2013; Sadowska et al., 2016). An alternative interpretation has been also provided for the data that may be not resolved only by the peripheral hypothesis or the HDL theory, i.e. the two limitations may exist in a female with strain differences in the maximum capacity to synthesise milk or the maximum heat dissipation capacity related to ambient temperature (Speakman and Król, 2011). In addition, the peripheral limitation is proposed to be dominant at room temperature, but heat dissipation is more significant at warm temperatures, by which the level of the heat dissipation limits may be temperature dependent, shifting down with increasing temperature (Wen et al., 2017). This suggests that the factors imposing the limits to sustained maximum energy intake during lactation may be different at different ambient temperatures. However, the basis of these different limits remains uncertain.

High temperatures adversely impact the body by interfering with its ability to dissipate heat and thermoregulate, leading to increased risk of hyperthermia, possibly heat stress, and even heat shock and death (Quiniou and Noblet, 1999). The HDL theory predicts that this problem is worse in mammals during high energy demand periods, such as periods of lactation (Hammond and Diamond, 1997; Król and Speakman, 2003a,b; Speakman and Król, 2005a,b, 2011; Simons et al., 2011; Sadowska et al., 2016). This has been partly confirmed in a diversity of lactating females exposed to warm ambient temperatures, including laboratory mice, hamsters, voles, gerbils and hares (Król and Speakman, 2003a,b; Wu et al., 2009; Valencak et al., 2010, 2013; Yang et al., 2013; Simons et al., 2011; Sadowska et al., 2016; Zhao et al., 2016; Huang et al., 2020). Under conditions of reduced heat dissipation capacity in higher temperatures, it is expected that animals would have to decrease metabolic rate, and thus reduce heat production as a by-product, during which the neuroendocrine hormones related to the control of metabolic rate and heat production may be involved. Thyroid hormones (THs: 3,5,3′,5′-tetraiodothyronine or thyroxine, T4; and 3,5,3′-triiodothyronine, T3) exert many physiological and metabolic effects (Bauer, 1987). Physiologically, they regulate skeletal, cardiovascular and nervous system homeostasis; metabolically, they stimulate cellular metabolism in most tissues through acceleration of protein, carbohydrate and lipid metabolism (Samuels and Tsai, 1973; Romero et al., 2000; Bassett and Williams, 2003; Mittag et al., 2010; Martinez et al., 2017). THs have significant effects on mammary function and lactation, and, acting through their nuclear receptors (TRs) of mammary epithelial cells, regulate STAT5 protein activity and prolactin (PRL) target genes such as α-lactalbumin (Lalba) and β-casein (csn2), etc. (Favre-Young et al., 2000; Campo Verde Arbocco et al., 2016). Hypothyroidism (HypoT) has a deleterious effect on lactation in rats, causing increased pup mortality, reduced growth rate of the litter and diminished milk nutritional quality (Hapon et al., 2003, 2007; Campo Verde Arbocco et al., 2015, 2016).

It's known that THs are physiologically adjusted in relation to environmental factors, in particular ambient temperature (Zhao et al., 2022b). Animals under warm temperature show reduced TH secretion, resulting in decreased metabolic rate and heat production (Yehuda-Shnaidman et al., 2014). Based on the HDL hypothesis, we hypothesised that animals under higher temperatures would have limited heat dissipation capacity; thus, the decreased heat production mediated by TH reduction would decrease the risk of hyperthermia and consequently increase the survival rate in warming climates.

The present study was aimed at elucidating the roles of THs in sustained maximum energy intake during lactation, as well as the lactating performance in Swiss mice that were exposed to warm temperature. We measured energy budgets of females, and litter size and mass, throughout lactation in Swiss mice subjected to supplementary thyroxine or the thyroid-peroxidase inhibitor methimazole at either 21 or 32.5°C. Milk energy output (MEO) was examined at peak lactation. The gene expression in mammary glands related to milk synthesis was also measured. We hypothesised that levels of circulating THs play a role in setting the maximum body temperature (Tb); when the maximum Tb is reached, other energy-producing processes are expected to drop, preventing hyperthermia. We predicted that the asymptotic food intake and MEO at peak lactation, as well as the gene expression of the mammary gland would be increased in the females that were treated with supplementary thyroxine, whereas decreased in the females exposed to methimazole.

Animals

Female Swiss mice, 9–11 weeks of age, were obtained from the breeding colony maintained in the animal house of Wenzhou University. Animals were housed individually in plastic cages (29 cm×18 cm×16 cm) with sawdust bedding and were initially kept at a constant temperature of 21±1°C under a 12 h:12 h light:dark photoperiod (lights on at 08:00 h). Food (standard low-fat diet with 11.8% fat: Beijing KeAo Feed Co., Beijing, China) and water were provided ad libitum. All experimental procedures complied with the Wenzhou University Animal Care and Use Committee's (WU-ACUC) guidelines and were approved by the WU-ACUC.

One hundred and fifteen female mice were paired with males for 11 days, after which the males were removed; 109 females subsequently became pregnant and gave birth. Pups were transferred between females on the day of parturition so that each female ultimately had 12 pups to raise. The litter size was adjusted randomly to correct for any effect of pup sex on growth/body mass. The day on which litter size was equalised for all females became day 0 of the experiment. Females and their pups were kept at 21°C between day 0 and 5. On day 6, females were randomly assigned to one of six treatment groups: a 21°C group (21°C, n=18), a 21°C plus supplementary thyroxine group (21°C+T4, n=15), a 21°C plus methimazole group (21°C+MMI, n=15), a 32.5°C group (32.5°C, n=18), a 32.5°C plus supplementary thyroxine (32.5°C+T4, n=20) group and a 32.5°C plus methimazole group (32.5°C+MMI, n=23). Lactating females in the 21°C, 21°C+T4 and 21°C+MMI groups continued to be kept at 21°C, whereas those in the 32.5°C, 32.5°C+T4 and 32.5°C+MMI groups were transferred to a room where ambient temperature was controlled at 32.5±1°C. Levothyroxine sodium (750 µg kg−1 diet; Merck KGaA, Darmstadt, Germany) was added to the food of 21°C+T4 and 32.5°C+T4 groups for 11 days (from day 6 to 16), which leads to a final dose of approximately 15 µg day−1 animal−1; and 0.1% (w/v) methimazole (Merck KGaA, Darmstadt, Germany) was added to the drinking water of 21°C+MMI and 32.5°C+MMI groups from day 6 to 16. There were no obvious changes in maternal care before and after the treatments based on casual observations but we did not collect systematic data on this. Pups were weaned on day 16 of lactation, and litter size and litter mass were measured daily throughout lactation (from day 1 to 16). Two females in the 32.5°C+T4 group unexpectedly died on day 9 of lactation, and the females in the 32.5°C+T4 were euthanized on day 10. Therefore, the treatment on the females in this group was terminated on day 10. While no additional data were collected for the 32.5°C+T4 mice, data collection continued for the other 32.5°C groups.

Tb of females

The core Tb of lactating females was estimated as the intraperitoneal Tb, which was measured daily from day 1 until day 16 of lactation. In detail, an encapsulated thermo-sensitive passive transponder (diameter 2 mm and length 14 mm, Destron Fearing, South St Paul, MN, USA) was implanted intraperitoneally on day 1 in the females under general anaesthesia [induction 3–5%, maintenance 1–2% isoflurane (RWD Life Science Co., Ltd, Shenzhen, China)] using an R530 Portable Small Animal Anaesthesia Machine (RWD Life Science Co., Ltd). The Tb was recorded with a Pocket Reader by approaching the cage, which did not touch the females or affect their, or their pups', behaviour. The average Tb during peak lactation was calculated by the mean Tb between days 10 and 14 of lactation. A factory-calibrated handheld thermal imaging camera (FLIR C2, FLIR Systems, Inc.; 320×240 pixels) was used to perform infrared thermography on the females at peak lactation (day 13). We recognise that the general anaesthesia and chip implantation on lactation day 1 might have been a stressor for the dams.

Food intake and body mass of females

Food intake and body mass of females were measured daily over the lactation period (from day 2 to 16 of lactation). Food intake was calculated as the difference between the mass of the food provided and that of the uneaten food on the following day, minus any food residue mixed with bedding material. Asymptotic food intake during the peak of lactation was calculated from the food intake from day 10 until day 14.

Gross energy intake and digestibility

Gross energy intake and digestibility were measured between days 13 and 14 of the experiment using the food balance method described previously (Grodzinski and Wunder, 1975; Liao et al., 2023). In brief, a known quantity of food was provided on day 13, and 24 h later any uneaten food and orts mixed with the bedding material were collected, together with faeces. Food orts and faeces were dried to a constant mass at 60°C for 2 weeks, and then were separated manually. The gross energy content of dry food and faeces was determined using an IKA C2000 oxygen bomb calorimeter (IKA Werke GmbH & Co. KG, Staufen, Germany). As described previously (Grodzinski and Wunder, 1975; Zhao et al., 2020; Liao et al., 2023), gross energy intake, gross energy of faeces, digestive energy intake and digestibility were calculated using the following equations:
formula
(1)
where GEI is gross energy intake (kJ day−1), FI is dry food intake (dry food mass−dry orts mass; g day−1) and GEfood is the gross energy content of the food (kJ g−1 dry mass);
formula
(2)
where GEF is gross energy of faeces (kJ day−1), Mfaeces is dry faeces mass (g day−1) and Efaeces­ is the energy content of faeces (kJ g−1);
formula
(3)
where DEI is digestive energy intake (kJ day−1);
formula
(4)
where digestibility is a percentage.

Resting metabolic rate

The resting metabolic rate (RMR) of females was measured on day 16 of lactation, using an open-flow respirometry system (PhenoMaster/LabMaster, TSE systems, Berlin, Germany). In brief, air was pumped at a rate of 1000 ml min−1 through a cylindrical sealed Perspex chamber (diameter 8 cm, length 35 cm), where the temperature was controlled at 30±0.5°C (the thermal neutral zone of the mouse) using an incubator. Gases leaving the chamber were directed through the oxygen analyser at a flow rate of 380 ml min−1 (TSE systems). RMR was measured for 2.5 h in females, during which pups were left alone in their home cages, but after which females were returned to their litters. Oxygen consumption rate was recorded every 10 s, and RMR was calculated from the consecutive minimum rate of oxygen consumption over 10 min. RMR was expressed as ml O2 h−1 after correcting to standard temperature and air pressure (STP) conditions (Zhao et al., 2022a).

MEO of lactating females

As described previously (Król and Speakman, 2003b), MEO of females at peak lactation (days 13–14) was estimated from the energy budget of litters. The pups obtain all their energy from their mother's milk before they get access to the diet at weaning. Thus, total energy of the pups was quantified as the sum of the energy allocated for the pups' growth of new tissue and their daily energy expenditure (DEE). As RMR of the pup was correlated with DEE on the basis of its body mass, DEE was estimated under the assumption that DEE=1.4×RMR to take into account the energetic costs of the pup's activity (Król and Speakman, 2003b; Deng et al., 2020). The equation used was (Król and Speakman, 2003b):
formula
(5)
where LM (g) is the litter mass on day 13, CFact is the correction factor (CFact=1.4, the mean ratio of DEE to RMR) and GEpups (kJ g−1 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. LMincrease (g d−1) is the increase in litter mass between days 13 and 14, and dmilk is the apparent digestibility of milk (dmilk ∼96%) (Król and Speakman, 2003b; Zhao et al., 2010; Wen et al., 2017).

Serum levels of T3 and T4

At the end of day 16 of lactation, females were killed by decapitation. Trunk blood was collected in each female at this point, centrifuged and the serum stored at −20°C for subsequent radioimmunoassay. Serum T3 and T4 concentrations were determined using I125 RIA kits according to the manufacturer's instructions (Beijing North Institute of Biological Technology, Beijing, China) (Zhao et al., 2022b).

Real-time reverse transcription PCR

After blood was collected, the pituitary, liver and mammary gland were immediately removed and stored at −80°C for subsequent RNA preparation. The expression of genes related to lactoprotein, glucose, lactose and the regulation of milk secretion was measured using real-time (quantitative) reverse transcription-PCR (RT-qPCR). As described previously (Zhao et al., 2014), extraction of total RNA from the mammary gland was performed using TRIzol Reagent (TAKARA, Dalian, China), and then RT-qPCR analysis was carried out. In detail, we used 2 μl of the cDNA samples as a template for the subsequent qPCR reaction (the gene-specific primers are presented in Table S1). β-Actin expression was used as an internal standard to quantify the relative gene expression (Zhao et al., 2014). The comparative cycle threshold method was used to determine the amount of target, normalised to an endogenous reference (β-actin), and relative to a calibrator (2−ΔΔCT) (Yu et al., 2020; Livak and Schmittgen, 2001; Zhao et al., 2014).

Statistics

Data were analysed using SPSS statistical software (v.20.0). All variables were tested for normality with the Kolmogorov–Smirnov test, which confirmed that all data, excluding litter size, were normally distributed. The original plan was to do two-way ANOVA with the main effects of temperature and drug. However, with the unfortune loss of the 32.5°C+T4 group at day 10, the design was no longer fully crossed, and this approach was no longer possible. The one-way ANOVA across groups, followed by a Student–Neuman–Kuells (SNK) post hoc test, was used to examine the group differences in asymptotic food intake, average Tb, GEI, digestibility, RMR and MEO. The group differences in serum T3 and T4, and gene expression in the pituitary, liver and mammary gland, were also examined using one-way ANOVA across groups, followed by a SNK post hoc test where required. The repeated measures in body mass, Tb, food intake, litter size and litter mass were analysed using repeated measures ANOVA (RM-ANOVA). All data are presented as means±s.e.m. or means±s.d. All tests were two-tailed and P<0.05 was considered statistically significant.

Tb

There were considerable statistical differences in Tb between the normothermic and high-temperature treatment groups without any TH manipulation, with Tb being significantly higher in the 32.5°C group on day 6–16 than in the 21°C group (Table S2). Tb of the 21°C+MMI group was significantly lower than that of the 21°C or 21°C+T4 groups on days 8–16 (Fig. 1A; Table S2). Tb of the 32.5°C group was considerably increased following high-temperature exposure on day 6 onwards, whereas Tb in the 32.5°C+MMI group was not increased, being lower than that in the 32.5°C group. Consistently, the average Tb at peak lactation was not significantly different between the 21°C+T4 group and 21°C group, but it was significantly lower in the 21°C+MMI group than in the 21°C and 21°C+T4 groups (Fig. 1B; Table S2). The average Tb at peak lactation in the 32.5°C+MMI group was lower, on average, by 1.7°C compared with that in the 32.5°C group. The effect of TH treatment on Tb was also shown in the infrared thermography estimates of surface temperature of the females at peak lactation (Fig. S1). The dorsal and cephalic Tb was significantly affected by MMI treatment and exposure to warm temperature, being higher at 32.5°C than at 21°C. MMI treatment resulted in a considerable reduction in dorsal temperature at both 21 and 32.5°C (Fig. S1). Over the period of lactation (days 1−16), Tb of 21°C group was not significantly changed, whereas Tb of the 21°C+T4 group was significantly increased, and that of the 21°C+MMI group was significantly decreased. Interestingly, Tb of the 32.5°C group was considerably increased, while Tb of the 32.5°C+MMI group was significantly decreased over the lactation period (Table S3).

Fig. 1.

Body temperature and food intake of Swiss mice during lactation. (A) Body temperature (Tb) over the period of lactation, (B) average Tb at peak lactation and (C) asymptotic food intake (aFI). Solid arrows indicate that females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation [21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+T4, n=20; 32.5°C+MMI, n=15; one-way ANOVA followed by Student–Newman–Kuells (SNK) post hoc tests, two-tailed]. Data are means±s.e.m. Asterisks indicate a significant difference across the groups (***P<0.001). Different letters indicate significant differences between the groups (P<0.05).

Fig. 1.

Body temperature and food intake of Swiss mice during lactation. (A) Body temperature (Tb) over the period of lactation, (B) average Tb at peak lactation and (C) asymptotic food intake (aFI). Solid arrows indicate that females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation [21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+T4, n=20; 32.5°C+MMI, n=15; one-way ANOVA followed by Student–Newman–Kuells (SNK) post hoc tests, two-tailed]. Data are means±s.e.m. Asterisks indicate a significant difference across the groups (***P<0.001). Different letters indicate significant differences between the groups (P<0.05).

Food intake

Food intake was markedly different between the normothermic and high-temperature treatment groups without any TH manipulation, with females in the 32.5°C group consuming significantly less food on days 6–16 compared with those in the 21°C group (Table S2). Supplementary T4 had no significant effect on food intake for the females lactating at 21°C, whereas supplementary MMI resulted in a significantly lower food intake on days 6–16 of lactation compared with that in the 21°C and 21°C+T4 groups (Fig. 2A; Table S2). MMI treatment also resulted in a significantly lower food intake at 32.5°C, with the females in the 32.5°C+MMI group consuming significantly less food on days 6–16 than those in the 32.5°C group. Asymptotic food intake was not different between the 21°C and 21°C+T4 groups, whereas it was lower by 33.2% and 33.3% in the 21°C+MMI group than in the 21°C and 21°C+T4 groups, respectively (Fig. 1C; Table S2). Asymptotic food intake was also significantly affected by MMI treatment, with asymptotic food intake in the 32.5°C+MMI group being lower by 23.0% compared with that in the 32.5°C group. Over the period of lactation (days 2–16), the females in the 21°C and 21°C+T4 groups significantly increased food intake, whereas the females in the 21°C+MMI, 32.5°C and 32.5°C+MMI groups significantly decreased food intake following exposure to warm or MMI treatment (Table S3).

Fig. 2.

Food intake and body mass of Swiss mice during lactation. (A) Food intake and (B) body mass. Solid arrows indicate that females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+T4, n=20; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Data are means±s.e.m. Asterisks indicate a significant difference across the groups (***P<0.001).

Fig. 2.

Food intake and body mass of Swiss mice during lactation. (A) Food intake and (B) body mass. Solid arrows indicate that females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+T4, n=20; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Data are means±s.e.m. Asterisks indicate a significant difference across the groups (***P<0.001).

Body mass

Body mass was significantly different between the normothermic and high-temperature treatment groups without any TH manipulation, being lower in the 32.5°C group on days 6–16 compared with that in the 21°C group (Table S2). Supplementary T4 had no significant effect on body mass of the females lactating either at 21 or 32.5°C (Fig. 2B; Table S2). Body mass of the females in the 21°C+MMI group was significantly lower on days 6–16 than that of the females in the 21°C and 21°C+T4 groups. Body mass in the 21°C and 21°C+T4 groups showed significant fluctuations over lactation, increasing during early and peak lactation, but decreasing on late lactation days. Body mass in the 21°C+MMI, 32.5°C and 32.5°C+MMI groups significantly decreased following the exposure to warm or MMI treatment (Table S3).

GEI and digestibility

The females in the 32.5°C group showed significantly lower GEI, DEI and GEF than those in the 21°C group, whereas there was no significant difference in digestibility between the normothermic and high-temperature treatment groups without any TH manipulation (Table S4). Supplementary T4 had no significant effect on either GEI or DEI at 21°C (Fig. 3A,B; Table S4). MMI treatment significantly decreased GEI and DEI at 21°C, such that the females in the 21°C+MMI group had 37.7% and 38.9% lower GEI, and 35.0% and 36.2% lower DEI compared with the females in the 21°C and 21°C+T4 groups, respectively. MMI also significantly decreased GEI and DEI at 32.5°C, being lower by 30.1% in GEI and 29.5% in DEI, respectively, in the 32.5°C+MMI group compared with that in the 32.5°C group. There was no significant difference in GEF between the 21°C and 21°C+T4 groups, whereas the females in the 21°C+MMI group produced 47.2% and 48.3% less faeces compared with those in the 21°C and 21°C+T4 groups (Fig. 3C; Table S4). Consistently, the females in the 32.5°C+MMI group produced 27.3% less faeces compared with those in the 32.5°C group. Supplementary T4 and MMI had no significant effect on digestibility at either 21 or 32.5°C (Fig. 3D; Table S4).

Fig. 3.

Energy intake and digestibility of Swiss mice during lactation. (A) Gross energy intake (GEI), (B) digestive energy intake (DEI), (C) gross energy of faeces (GEF) and (D) digestibility. Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Box plots show means±s.e.m. with minimal and maximal values. Different letters indicate significant differences between the groups (P<0.05).

Fig. 3.

Energy intake and digestibility of Swiss mice during lactation. (A) Gross energy intake (GEI), (B) digestive energy intake (DEI), (C) gross energy of faeces (GEF) and (D) digestibility. Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Box plots show means±s.e.m. with minimal and maximal values. Different letters indicate significant differences between the groups (P<0.05).

Litter size and litter mass

The offspring were significantly different in size and mass between the normothermic and high-temperature treatment groups without any TH manipulation, with females in the 32.5°C group raising smaller litters on days 15–16, and lower litter mass and pup mass on days 7–16, compared with that in the 21°C group (Table S2). Litter size was similar between the 21 and 21°C+T4 groups, whereas it was significantly lower in the 21°C+MMI group on days 10–16 compared with that in the 21°C and 21°C+T4 groups (Fig. 4A; Table S2). Litter size in the 32.5°C+MMI group was significantly lower on days 10–16 compared with that in the 32.5°C group. Litter size was not significantly changed over lactation in the 21°C and 21°C+T4 groups, whereas it was significantly decreased in the 21°C+MMI, 32.5°C and 32.5°C+MMI groups following warm exposure or MMI treatment (Table S3).

Fig. 4.

Litter size and mass of Swiss mice during lactation. (A) Litter size, (B) litter mass and (C) mean pup mass. Solid arrows indicate that females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+T4, n=20; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Data are means±s.e.m. Asterisks indicate a significant difference across the groups (***P<0.001).

Fig. 4.

Litter size and mass of Swiss mice during lactation. (A) Litter size, (B) litter mass and (C) mean pup mass. Solid arrows indicate that females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+T4, n=20; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Data are means±s.e.m. Asterisks indicate a significant difference across the groups (***P<0.001).

Consistently, females in the 21°C and 21°C+T4 groups raised litters of similar mass, whereas females in the 21°C+MMI group raised litters of significantly lower mass on days 7–16 than the other two groups, with litter mass in the 21°C+MMI group being lower by 54.7% and 53.9% at weaning, respectively, than that in the 21°C and 21°C+T4 groups (Fig. 4B; Table S2). Litter mass in the 32.5°C+MMI group was significantly lower on days 9–16 than that in the 32.5°C group, and was lower by 54.9% at weaning in the 32.5°C+MMI group than in the 32.5°C group. Litter mass over lactation was considerably increased in the 21°C and 21°C+T4 groups, but it was significantly decreased in the 21°C+MMI, 32.5°C and 32.5°C+MMI groups following warm exposure or MMI treatment (Table S3).

Mean pup mass was significantly affected by MMI treatment on days 6–16, and the pups weaned in the 21°C+MMI group were lighter by 25.5% and 23.4% than the pups in the 21°C and 21°C+T4 groups, respectively (Fig. 4C; Table S2). Mean pup mass, however, was not significantly different between the 32.5°C and 32.5°C+MMI groups. Mean pup mass increased by 239.9% and 227.7% in the 21°C and 21°C+T4 groups over the period of lactation (day 16 versus day 1), while it increased only by 153.4%, 127.9% and 76.1% in the 21°C+MMI, 32.5°C and 32.5°C+MMI groups (Table S3).

RMR

The RMR of females was significant different between the normothermic and high-temperature treatment groups without any TH manipulation, being lower by 16.1% in the 32.5°C group compared with that in the 21°C group (Table S4). RMR of females was significantly affected by supplementary T4 at 21°C, being significantly higher in the 21°C+T4 group compared with that in the 21°C group (Fig. 5A; Table S4). In contrast, MMI treatment resulted in a significant decrease in RMR, with the RMR in the 21°C+MMI group being lower by 41.9% and 51.4% than that in the 21°C and 21°C+T4 groups, respectively. MMI treatment also significantly decreased RMR at 32.5°C, with the RMR in the 32.5°C+MMI group being lower by 23.1% than that in the 32.5°C group.

Fig. 5.

Resting metabolic rate and milk energy output of Swiss mice during lactation. (A) Resting metabolic rate (RMR) and (B) milk energy output (MEO). Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Data are means±s.e.m. Different letters indicate significant differences between the groups (P<0.05).

Fig. 5.

Resting metabolic rate and milk energy output of Swiss mice during lactation. (A) Resting metabolic rate (RMR) and (B) milk energy output (MEO). Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Data are means±s.e.m. Different letters indicate significant differences between the groups (P<0.05).

MEO

MEO was significantly different between the normothermic and high-temperature treatment groups without any TH manipulation, with the females in the 32.5°C group producing 50.3% less milk compared with those in the 21°C group (Table S4). MEO of females in the 21°C+T4 group was not significantly different from that of females in the 21°C group (Fig. 5B; Table S4). MEO was significantly negatively affected by MMI treatment at 21°C, with the females in the 21°C+MMI group producing 46.6% and 44.7% less milk than those in the 21°C and 21°C+T4 groups, respectively. MEO was also significantly affected by MMI treatment at 32.5°C, with the females in the 32.5°C+MMI group producing 54.6% less milk compared with those in the 32.5°C group (Fig. 5B; Table S4).

Serum T3 and T4 levels

The TH levels were significantly different between the normothermic and high-temperature treatment groups without any TH manipulation, with T3, T4 and T3/T4 ratio values in the 32.5°C group being 62.9%, 38.8% and 41.9% those in the 21°C group (Table S4). Serum T3 levels were significantly different between the 21°C, 21°C+T4 and 21°C+MMI groups, being higher by 41.7% in the 21°C+T4 group, and lower by 39.6% in the 21°C+MMI group relative to that in the 21°C group (Fig. 6A), whereas it was not significantly different between 32.5°C and 32.5°C+MMI groups (Table S4). Serum T4 was significantly higher in the 21°C+T4 group, and lower in 21°C+MMI group, compared with the 21°C group (Fig. 6B), whereas it was not significantly different between the 32.5°C+MMI group and 32.5°C group (Table S4). The T3/ T4 ratio was significantly lower in the 21°C+T4 group than in the 21°C group (Fig. 6C), whereas it was not significantly different between the 21°C+MMI and 21°C group (Table S4). The T3/T4 ratio was not significantly different between the 32.5°C and 32.5°C+MMI groups (Table S4).

Fig. 6.

Serum thyroid hormone levels of Swiss mice during lactation. (A) T3, (B) T4 and (C) the ratio of T3 to T4. Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Data are means±s.e.m. Different letters indicate significant difference between the groups (P<0.05).

Fig. 6.

Serum thyroid hormone levels of Swiss mice during lactation. (A) T3, (B) T4 and (C) the ratio of T3 to T4. Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Data are means±s.e.m. Different letters indicate significant difference between the groups (P<0.05).

Gene expression in the pituitary and liver

Gene expression of most of the genes we measured in the pituitary and liver was not significantly different between the normothermic and high-temperature treatment groups without any TH manipulation (Table S5). TSHβ expression in the pituitary was not significantly affected by supplementary T4 or MMI treatment at 21°C, and it was also not significantly different between the 32.5°C and 32.5°C+MMI groups (Fig. 7A; Table S5). We examined the expression of T3 target genes in the liver, which are involved in glycometabolism, lipogenesis and fatty acid β-oxidation. G6pase expression was not significantly affected by supplementary T4 or MMI treatment at 21°C, whereas it was significantly decreased in the 32.5°C+MMI group compared with the 32.5°C group (Fig. 7B; Table S5). Glut2 expression was not significantly changed in the 21°C+T4 and 21°C+MMI groups compared with the 21°C group, and it was significantly increased in the 21°C+MMI group compared with the 21°C+T4 group (Fig. 7C; Table S5). Neither supplementary T4 nor MMI treatment had a significant effect on the expression of PEPCK, Cpt1α or Me at 21°C or 32.5°C (Fig. 7D–F; Table S5). Fasn expression was not significantly affected by supplementary T4, whereas it was significantly decreased by MMI treatment at both temperatures (Fig. 7G; Table S5). Acc expression was also significantly decreased in the 21°C+MMI group compared with the 21°C+T4 group (Fig. 7H; Table S5). PPARγ expression was not significantly different across the groups (Fig. 7I; Table S5). Dio expression was 4.2-fold up-regulated in the 21°C+T4 group, and down-regulated by 84.9% in the 21°C+MMI group, compared with that in the 21°C group (Fig. 7J; Table S5). TRβ1 expression was decreased by 52.6% in the 21°C+MMI compared with the 21°C+T4 group, whereas the difference between the 21°C group and the other four groups was not statistically significant (Fig. 7K; Table S5).

Fig. 7.

Expression of T3 target genes in the pituitary and liver of Swiss mice during lactation. (A) TSHβ (pituitary) and (B–K) G6pase, Glut2, PEPCK, Cpt1α, Me, Fasn, Acc, PPARγ, Dio and TRβ1 (liver) expression (RU, relative units). Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=10; 21°C+T4, n=6; 21°C+MMI, n=7; 32.5°C, n=8; 32.5°C+MMI, n=8; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Data are means±s.e.m. Different letters indicate significant differences between the groups (P<0.05).

Fig. 7.

Expression of T3 target genes in the pituitary and liver of Swiss mice during lactation. (A) TSHβ (pituitary) and (B–K) G6pase, Glut2, PEPCK, Cpt1α, Me, Fasn, Acc, PPARγ, Dio and TRβ1 (liver) expression (RU, relative units). Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=10; 21°C+T4, n=6; 21°C+MMI, n=7; 32.5°C, n=8; 32.5°C+MMI, n=8; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Data are means±s.e.m. Different letters indicate significant differences between the groups (P<0.05).

Gene expression in the mammary gland

Gene expression in the mammary gland of the genes we measured was not significantly different between the normothermic and high-temperature treatment groups without any TH manipulation (Table S5). Csn expression was not significantly different between the 21°C and 21°C+T4, or 21°C+T4 and 21°C+MMI groups, whereas it was down-regulated by 47.6% in the 21°C+MMI group compared with the 21°C group (Fig. 8A; Table S5). There was a significant decrease in Lalba expression in the 21°C+MMI group compared with the 21°C+T4 group (by 53.8%), but it did not significantly differ between the 21°C and 21°C+T4, or between the 21°C and 21°C+MMI groups (Fig. 8B; Table S5). Wap expression was significantly increased in the 21°C+T4 group (by 36.6%), but it was significantly decreased in the 21°C+MMI group (by 61.4%), compared with the 21°C group (Fig. 8C; Table S5).

Fig. 8.

Gene expression in the mammary gland of Swiss mice during lactation. (A–L) Csn, Lalba, Wap, Acc, Acly, Fasn, Slc, Spot, Fads, Aldoc, G6pd2 and Glut-1 expression. Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Data are means±s.e.m. Different letters indicate significant differences between the groups (P<0.05).

Fig. 8.

Gene expression in the mammary gland of Swiss mice during lactation. (A–L) Csn, Lalba, Wap, Acc, Acly, Fasn, Slc, Spot, Fads, Aldoc, G6pd2 and Glut-1 expression. Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Data are means±s.e.m. Different letters indicate significant differences between the groups (P<0.05).

Acc expression was not significantly different between the 21°C and 21°C+T4 groups, but it was down-regulated by 53.0% and 57.7%, respectively, in the 21°C+MMI group compared with that of the 21°C and 21°C+T4 groups (Fig. 8D; Table S5). There were no significant differences in Acly expression between the 21°C and 21°C+T4, or between the 21°C+T4 and 21°C+MMI groups, whereas it was significantly down-regulated in the 21°C+MMI group compared with the 21°C group (Fig. 8E; Table S5). The 32.5°C and 32.5°C+MMI groups did not differ in Acly gene expression. Fasn expression at 21°C was not significantly affected by the supplementary T4, but it was significantly affected by MMI treatment, being lower by 70.0% and 76.7% in the 21°C+MMI group than in the 21°C and 21°C+T4 groups (Fig. 8F; Table S5). Fasn expression in the 32.5°C+MMI group was lower by 73.5% compared with that in the 32.5°C group. Slc expression did not differ significantly across the groups (Fig. 8G; Table S5). Spot expression at 21°C was also not significantly affected by the supplementary T4, but it was significantly decreased by MMI treatment (Fig. 8H; Table S5). Fads expression was significantly decreased in the 21°C+T4 and 21°C+MMI groups compared with that in the 21°C group (Fig. 8I; Table S5). MMI treatment significantly decreased Fads gene expression at 32.5°C.

Aldoc expression was not significantly different between the 21°C, 21°C+T4 and 21°C+MMI groups, and it was significantly increased in the 32.5°C+MMI group compared with the 32.5°C group (Fig. 8J; Table S5). Consistently, G6pd2 expression in the 21°C+MMI group was decreased, by 65.5% and 66.4%, respectively, compared with that in the 21°C and 21°C+T4 groups (Fig. 8K; Table S5). G6pd2 expression was not significantly affected by MMI treatment at 32.5°C. Glut-1 expression was not significantly different across the groups (Fig. 8L; Table S5).

Akt gene expression was not significantly affected by either supplementary T4 or MMI treatment at 21°C or 32.5°C (Fig. 9A; Table S5). PrlR expression was not significantly different between the 21°C and 21°C+T4 groups, but it was decreased by 65.2% and 64.9% in the 21°C+MMI group compared with that in the 21°C and 21°C+T4 groups (Fig. 9B; Table S5). MMI treatment at 32.5°C also significantly decreased PrlR expression, being lower by 45.9% in the 32.5°C+MMI group than in the 32.5°C group. Consistently, TRβ1 expression in the 21°C+MMI group was decreased, by 69.9% and 67.8%, respectively, compared with that in the 21°C and 21°C+T4 groups (Fig. 9C; Table S5). TRβ1 expression was also significantly decreased in the 32.5°C+MMI group compared with that in the 32.5°C group (by 59.6%).

Fig. 9.

Gene expression in the mammary gland of Swiss mice during lactation. (A–G) Akt, PrlR, TRβ1, Srebp, Stat5a, Stat5b and Dio expression. Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Data are means±s.e.m. Different letters indicate significant differences between the groups (P<0.05).

Fig. 9.

Gene expression in the mammary gland of Swiss mice during lactation. (A–G) Akt, PrlR, TRβ1, Srebp, Stat5a, Stat5b and Dio expression. Females were subjected to supplementary thyroxine (+T4) or methimazole (+MMI) at either 21 or 32.5°C on days 6–16 of lactation (21°C, n=8; 21°C+T4, n=9; 21°C+MMI, n=8; 32.5°C, n=10; 32.5°C+MMI, n=15; one-way ANOVA followed by SNK post hoc tests, two-tailed). Note, there was no T4 treatment at 32.5°C. Data are means±s.e.m. Different letters indicate significant differences between the groups (P<0.05).

Srebp expression was also significantly decreased by MMI treatment at 21°C, being lower by 76.0% and 65.7% in the 21°C+MMI group compared with that in the 21°C and 21°C+T4 groups (Fig. 9D; Table S5). Stat5a expression was not significantly different across the groups (Fig. 9E; Table S5). Stat5b expression was not significantly affected by supplementary T4 at 21°C, but it was significantly affected by MMI treatment, being lower by 59.0% and 68.5%, respectively, in the 21°C+MMI group than that in the 21°C and 21°C+T4 groups (Fig. 9F; Table S5). MMI treatment at 32.5°C did not affect the expression of Srebp, Stat5a or Stat5b. Dio1 expression was not significantly affected by supplementary T4 or MMI treatment at either 21 or 32.5°C (Fig. 9G; Table S5).

It is well known that THs are major hormonal regulators of metabolic rate and heat production in almost all tissues (Hollenberg, 2008; Polat et al., 2014; Brinkmann et al., 2016). In mammals and birds, hyperthyroidism is associated with increased metabolism and Tb, and vice versa (Silva, 2003, 2006; Mullur et al., 2014; Dittner et al., 2019). In this study, supplementary T4 resulted in a 41.9% increase of T3 and a 443.4% increase of T4 in lactating females at 21°C, and MMI treatment decreased T3 and T4 by 38.5% and 45.6%, respectively. These data show that the experimental manipulations successfully induced hyperthyroidism and hypothyroidism. Unexpectedly, we found that supplementary T4 did not increase Tb in lactating Swiss mice at 21°C compared with the control females, but as expected MMI decreased Tb. A possible explanation is that the females may have been maximised in their metabolic rate and heat production during lactation as the sustained maximum energy intake and milk output have reached a ceiling because of the heat dissipation limitation. Based on the HDL theory, the heat dissipation capacity is maximised at peak lactation (Król and Speakman, 2003a,b), and any increases in metabolic rate and heat production during this period would result in increased risk of further hyperthermia of females and death. This was confirmed in females lactating at 32.5°C in this study, which had on average 1.5°C higher Tb than the females lactating at 21°C. Unexpectedly, at 32.5°C, the females with supplementary T4 showed hyperthermia when they reached peak lactation (day 9 of lactation), and the experiments with this group were terminated. This finding is consistent with that predicted by the HDL theory, i.e. the heat dissipation limits to sustained energy intake would be more considerable in the females lactating at higher temperature than in those females lactating at lower temperature (Wen et al., 2017; Huang et al., 2020). This suggests that supplementary T4 fails to increase Tb in lactating females, which was inconsistent with observations in non-breeding individuals (Renaudeau et al., 2003; Weitzel et al., 2017), indicating that the roles of THs in mediating metabolic thermogenesis and thermal regulation may be associated with reproduction stage. More importantly, for the females lactating at high temperature, supplementary T4 further increased the risk of hyperthermia.

Changes in body mass reflect the balance between energy intake and expenditure. In this study, the females at 21°C increased food intake considerably to meet the energy requirements of their offspring, resulting in energy balance and constant body mass across the period of lactation. However, for the females at 32.5°C this was not the case, suggesting they may be not capable of consuming sufficient food to meet the requirements of their offspring, consequently mobilising their fat and protein storage, resulting in a considerable decrease in body mass. The key question is why are they not capable of increasing food intake at 32.5°C, and what are the factors limiting that food intake? As the heat dissipation of the females is largely decreased at 32.5°C compared with that at 21°C, the capacity for heat dissipation probably constrains their food intake, and consequently energy intake does not meet the energy expenditure for MEO, resulting in a lower body mass. This suggests that heat dissipation limitation is possibly the factor limiting sustained energy intake (consistent with several previous studies: Król and Speakman, 2003a,b; Król et al., 2007, 2011; Wu et al., 2009; Yang et al., 2013; Al Jothery et al., 2014; Wen et al., 2017), but this does not exclude other potential factors that are involved in the limitation to energy intake for the females lactating at 32.5°C.

In this study, supplementary T4 also failed to increase food intake at peak lactation, whereas asymptotic food intake of females exposed to MMI was lower by 33.2% at 21°C, and 23.0% at 32.5°C. It is known that THs have roles in stimulating metabolism in most tissues (Samuels and Tsai, 1973; Romero et al., 2000; Mittag et al., 2010; Martinez et al., 2017). It has been observed that in the same strain of Swiss mice, serum T3 is considerably increased following a gradient decrease in ambient temperature, with food intake being significantly increased (Zhao et al., 2022b). However, in the present study, supplementary T4 had no effect on GEI at peak lactation, and GEI was significantly reduced following MMI treatment. In addition, the digestive efficiency was not significantly affected by either supplementary T4 or MMI. These findings suggest that endogenous THs may be necessary for the lactating females to maintain sustained energy intake. But critically, THs do not appear to define the upper limits to the sustained maximum energy intake during lactation at 21°C.

Although it has been widely reported that THs have important roles in stimulating metabolic rate (Silva, 2003, 2006; Hollenberg, 2008; Mullur et al., 2014; Polat et al., 2014; Brinkmann et al., 2016; Dittner et al., 2019), we found that supplementary T4 did not significantly change RMR of the females at peak lactation at 21°C. The reason why the supplementary T4 failed to increase metabolic rate during lactation is presently unknown but it suggests that the thyroid axis is already maximally stimulated under these conditions and hence adding more had no appreciable impact. This maximal stimulation may be because females during lactation are limited by their heat dissipation capacity (Król and Speakman, 2003a,b), meaning that the specific metabolic rate of active organs is unable to increase further after exposure to supplementary T4. RMR is an obligatory thermogenesis of mammals, with higher basal metabolic rate (BMR; approximately equal to RMR in the thermoneutral zone) being related to higher Tb. It has been observed that BMR and Tb are positively correlated across 267 small mammals (Lovegrove, 2003). The females during lactation had a considerable increases in RMR, contributing to a significantly increased Tb, compared with non-breeding controls (Speakman and McQueenie, 1996; Johnson et al., 2001a,b; Al Jothery et al., 2014; Sadowska et al., 2019; Zhao et al., 2020). If supplementary T4 increased RMR further without also changing the heat dissipation limit, the increased obligatory thermogenesis would considerably increase the risk of hyperthermia at peak lactation. Therefore, these results provide strong support for females at peak lactation having a fixed maximum heat dissipation capacity that is not affected by TH levels.

The mechanism explaining why supplementary T4 did not stimulate RMR is presently unknown. The females exposed to supplementary T4 had 4.4-times higher serum T4 (P<0.001), and 41.9% higher serum T3 levels (P=0.149), compared with control females. One possible explanation is that TH receptor gene expression may be not upregulated in the lactating female in response to supplementary T4. For example, in this study we observed that TH receptor gene expression in mammary glands was not significantly changed following exposure to supplementary T4. However, in this study, TH receptor gene expression was determined in the mammary glands only. Interestingly, we found that RMR of the lactating females was decreased by 43.4% following MMI treatment at 21°C. The decreased RMR was also observed in the females lactating at 32.5°C after exposure to MMI. This suggests that endogenous THs are involved in the high RMR in lactating females relative to non-breeding females, and that this level can be reduced but not further increased.

In lactating animals, THs are reported to be galactopoietic and help to establish the mammary glands' metabolic priority during lactation (Capuco et al., 2008). However, in this study, the females with supplementary T4 did not elevate MEO and, in consequence, these supplemented females also did not raise heavier litters or larger litter sizes, compared with un-supplemented control females. Based on the peripheral limitation hypothesis, the mammary glands may have been working maximumly at peak lactation (Hammond and Diamond, 1992, 1997; Speakman and Król, 2005a, 2011). This may be a possible explanation for the absence of an effect of supplementary T4 on the performance of the mammary glands. Alternatively, the females were probably limited by their capacity to dissipate heat, and they would increase the risk of hyperthermia if the supplementary T4 stimulated the metabolic rate of the mammary glands and other tissues. Importantly, we observed that maternal MEO and litter growth were significantly decreased in females following MMI treatment or in females lactating at high temperature.

Circulating levels of THs are controlled by a negative feedback system involving the hypothalamic-pituitary–thyroid axis (Costa-e-Sousa and Hollenberg, 2012). Thyroid stimulating hormone (TSH) produced by pituitary thyrotrophs stimulates the thyroid gland to synthesise and secrete THs. Importantly, THs negatively regulate their own production by inhibiting TSH synthesis and secretion (Yen, 2001; Aninye et al., 2014; Brûlé et al., 2022). However, in this study, expression of TSHβ was not significantly changed following exposure to supplementary T4 or MMI treatment in lactating females at either 21°C or 32.5°C, which is inconsistent with findings in non-reproductive animals (Aninye et al., 2014; Zhang et al., 2016; Brûlé et al., 2022). In addition, in this study, we examined mRNA expression levels of eight different T3 target genes, G6pase, Glut2, PEPCK, Cpt1α, Me, Fasn, Acc, PPARγ in liver. Neither supplementary T4 nor MMI treatment had a consistent effect on the expression of G6pase, Glut2 or PEPCK genes, which are known to be involved in hepatic glucose production and transport, and gluconeogenesis, and are up-regulated in non-reproductive animals following TH treatment (Khan et al., 1987; Weinstein et al., 1994; Thakran et al., 2013). The expression of Cpt1α, Me, Fasn or Acc was also not significantly changed in lactating females following supplementary T4, whereas expression of Fasn and Acc was significantly decreased by MMI treatment or exposure to warm temperature. These genes, associated with fatty acid synthase or β-oxidation in hepatocytes, have previously been reported to be increased in animals with hyperthyroid states compared with hypothyroid animals (Mynatt et al., 1994; Zabrocka et al., 2006; Bates et al., 2020). PPARγ is a master regulator of adipogenesis rather than fatty acid oxidation (Lowell, 1999), and its expression has been reported to be significantly down-regulated in rats after the administration of T3 (Weitzel et al., 2003; Weitzel and Iwen, 2011). However, in the present study, we did not observe a negative effect of supplementary T4 on PPARγ. These findings suggest that the role of THs in regulating glucose production, transport, fatty acid synthase or β-oxidation in liver during lactation may be somewhat inconsistent with that in non-lactating animals. The mechanisms underpinning the inconsistency will be important avenues for further study.

It has previously been reported that THs levels in the mammary gland are associated with the rate of T4 to T3 conversion, which is catalysed by type 1 deiodinase (Dio1) (Anguiano et al., 2004). In rats, expression of mammary Dio1 (MDio1) in mid-lactation is at its peak and may locally provide significant amounts of T3 and iodine (Aceves et al., 1995). In the present study, expression of mammary Dio1 at 21°C was not significantly affected by supplementary T4 or MMI treatment. However, the expression of liver Dio1 at 21°C was increased by 4.2-fold by supplementary T4, but it was decreased by 84.9% by MMI treatment. In addition, it has been reported that the expression of mammary TRβ1 is significantly increased in lactating cows compared with that in prepartum individuals, which provides a mechanism to increase TH activity within the mammary gland during lactation (Capuco et al., 2008). In the present study, TRβ1 expression was significantly decreased in the mammary glands in females following MMI treatment or in females lactating at high temperature. Here, Csn2, Lalba and Wap gene expression of the mammary glands was significantly reduced following MMI treatment or exposure to high temperature, compared with that in the 21°C group, indicating that the synthesis of β-casein, α-lactalbumin and whey acidic protein might be attenuated. In addition, it has previously been found that lactating HypoT rats have premature mammary involution, in parallel with decreased β-casein and α-lactalbumin gene expression (Campo Verde Arboccó et al., 2016). This suggests that there may be a link between lower T3 levels and impaired milk output in lactating females which have a fixed heat dissipation limitation.

The signal transducer and activator of transcription (STAT) family of transcription factors have a spectrum of functions in mammary gland development and milk synthesis (Hughes and Watson, 2012). In this study, we observed that the lactating mice showed significant down-regulation of Stat5a and Stat5b gene expression following exposure to MMI or high temperature. STAT5 is essential for the regulation of mammary epithelial cells and plays an essential role in regulating and ensuring female fertility, and is functionally important for the mammary gland (Akira, 1999). STAT5 is activated by a wide spectrum of external stimuli including prolactin, and is probably involved in controlling milk synthesis (Darnell et al., 1994; Darnell, 1996; Campo Verde Arboccó et al., 2017). In this study, prolactin receptor gene expression was significantly reduced in lactating mice exposed to MMI or high temperature. Consistently, lactating HypoT rats showed decreased mRNA levels of prolactin receptor and Stat5a/b (Campo Verde Arbocco et al., 2017). The mammary involution in lactating rats due to a lack of suckling at weaning is characterised by a loss of STAT5a/b activity, decreased mRNA levels of prolactin target genes such as α-lactalbumin (Lalba) and β-casein (Li et al., 1997). Stat5a/b has functional TH response elements in the regulatory regions that bind TRβ differentially and in a TH-dependent manner. The overall decrease in the prolactin signalling pathway and consequently in target gene (Lalba) mRNA transcription explains the profound negative impact of HypoT on mammary function through lactation (Campo Verde Arbocco et al., 2017). These findings suggest that the lower THs in the lactating females following MMI treatment and heat exposure might down-regulate the STAT5 signalling pathway related to prolactin and its receptor, resulting in the decline in expression of target genes associated with milk protein and lipid synthesis in the mammary glands. Furthermore, this indicates that female mammals may have to impair their maximum capacity of thermogenesis and in particular lactation performance in response to decreased heat dissipation capacity at high temperatures. This would decrease the risk of hyperthermia, and consequently increase the survival rate and fitness at high temperatures, with which the down-regulated STAT5 signalling pathway as well as the prolactin and target genes mediated by TH reduction might be involved.

One possible explanation is that THs in physiological concentrations are essential for mammary gland function (Campo Verde Arbocco et al., 2017). Exposure to insufficient quantities of THs during lactation diminishes milk production and quality and advances mammary involution (Varas et al., 2001, 2002; Campo Verde Arbocco et al., 2015, 2016, 2017). Another explanation is that the excessive THs may result in additional heat production and thus increase the risk of hyperthermia, provided the heat dissipation capacity is fixed, which in turn leads to a limitation to reproductive performance at peak lactation. This suggests that the impaired reproductive performance resulting from the limited heat dissipation capacity at high temperatures may also be related to hypothyroidism.

We compared treatment groups across related variables, and found that the supplementary T4 treatment of lactating females at 21°C had no significant effects on food intake, RMR, body mass or lactation performance. Further, T4 treatment did not increase Tb of the lactating mice. Although we provide above several potential explanations for the absence of a Tb response, the exact reasons are currently unknown. The MMI treatment of lactating females at room and high temperature resulted in a reduction of serum THs, which was paralleled by significant decreases in Tb, food intake, RMR and MEO of females and litter mass in the treatment groups compared with that in the control group at 21°C. These findings suggest that Tb, RMR, food intake and lactation performance may be closely tied to one another. So, if Tb does not respond to a TH treatment, it is unlikely that RMR, food intake and lactation performance will respond. Likewise, if Tb responds, it is likely the other variables will follow.

Hyperthyroidism induced by supplementary T4 did not show altered Tb, asymptotic food intake or GEI of Swiss mice at peak lactation at 21°C. These females also did not significantly change RMR, MEO or litter size and litter mass during the entire lactation period. In contrast, hypothyroidism induced by either MMI treatment or heat exposure significantly decreased asymptotic food intake, RMR and MEO in the females at peak lactation, resulting in a significantly decreased litter size and litter mass. The thyroid axis seems therefore to be an important mediator of the reduced lactation performance at 32.5°C compared with that at 21°C. These decreases were even more considerable in the females exposed to MMI at high temperature. Furthermore, the synthesis of β-casein, α-lactalbumin, whey acidic protein and milk fat acid production was adversely affected by hypothyroidism induced by either MMI or heat exposure, which was associated with decreased gene expression of the prolactin receptor, Stat5a and Stat5b. These findings suggest that endogenous THs may be necessary for the lactating females to maintain sustained energy intake and MEO. Reduced THs and STAT5 signalling pathway would impair reproductive performance. THs may not define the upper limit to sustained energy intake and MEO at peak lactation at 21°C, but inhibiting the thyroid axis may be an important mechanism by which mice in hotter (32.5°C) conditions keep their MEO and heat production below the lowered heat dissipation limit.

We thank Shasha Liao and Wei Liu for their support and assistance with this project. We also would like to thank the anonymous reviewers for useful comments and criticisms on an earlier version of the publication.

Author contributions

Conceptualization: Z.Z.; Methodology: R.Y., Z.Z.; Investigation: R.Y., J.C.; Data curation: R.Y., J.C., Z.Z.; Writing - original draft: R.Y., J.R.S., Z.Z.; Writing - review & editing: J.R.S., Z.Z.; Supervision: J.R.S., Z.Z.; Funding acquisition: Z.Z.

Funding

This work was partly supported by grants from the National Natural Science Foundation of China (31670417 and 31870388 to Z.Z., and 92057206 to J.R.S.) and the National Key R&D Program of China (2019YFA0801900 to J.R.S.), and the Master's Innovation Foundation of Wenzhou University (3162023003047).

Data availability

All relevant data can be found within the article and its supplementary information.

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Competing interests

The authors declare no competing or financial interests.

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