2889 INTRODUCTION In mammals, lactation is the most energetically demanding period of a female’s life and is characterized by a dramatic increase in the energy and nutrient requirement of the organism for milk production (Gutgesell et al., 2009). It is well known that several metabolic adaptations occur in the different tissues of a lactating animal (Williamson, 1980; Smith and Grove, 2002; Gutgesell et al., 2009). For example, rates of fatty acid oxidation and ketogenesis, which predominantly occur in the liver, are reduced during lactation and this reduction helps to spare fatty acids for milk production in the mammary gland (Whitelaw and Williamson, 1977). Reproduction also requires particularly high levels of metabolism. In lactating laboratory mice, for example, an elevated food intake as well as a metabolic rate increase of 400% have been observed when compared with non-reproductive mice (Hammond and Diamond, 1992; Hammond, 1997; Cretegny and Genoud, 2006). Although food consumption can increase dramatically during lactation, a point is reached where this intake is maximized and females appear unable, or unwilling, to increase their energy intake any further (Speakman, 2008). Several theories have been put forward to explain limits on the maximum rate at which animals can ingest and expend energy (Hammond and Diamond, 1992; Hammond et al., 1994; Speakman and Król, 2005; Speakman and Król, 2010; Speakman and Król, 2011). The original ‘central limitation’ hypothesis suggests a limitation in the capacity of the alimentary tract to assimilate energy (including the liver), although this has been refuted by several studies (Król and Speakman, 2003a; Król and Speakman, 2003b; Król et al., 2003; Wu et al., 2009). The ‘peripheral limitation’ hypothesis states that the sustained energy intake is set by the energy-consuming organs such as the mammary glands during lactation (Hammond et al., 1994; Speakman and Król, 2005; Zhao and Cao, 2009; Speakman and Król, 2011; Zhao, 2011). Alternatively, the ‘heat dissipation limit’ theory postulates that the sustained energy intake is driven by the capacity of animals to dissipate heat (Król and Speakman, 2003a; Król and Speakman, 2003b; Król et al., 2003; Speakman and Król, 2011). Both hypotheses are likely to be important in all animals, but to different extents (Speakman and Król, 2011; Zhao, 2011), and suggest that energy intake and expenditure are central to the ability of animals to reproduce. Additionally, the ‘saturated neural control’ hypothesis suggests that there may be limits in the ability of peripheral signals such as leptin to stimulate factors in the brain that regulate the intake of food, and contribute to energy intake during lactation (Woodside et al., 2000; Mercer and Speakman, 2001; Denis et al., 2003; Speakman and Król, 2011). The abilities of animals to increase metabolic rates and food intake have therefore been fairly well investigated. However, the cellular changes that occur during periods of high energy demand such as lactation have received less attention. Mitochondria are fundamental for energy production and their ability to generate ATP in relation to tissue or cell demands could potentially dictate reproductive investment. Cellular adaptation to SUMMARY Reproduction imposes significant costs and is characterized by an increased energy demand. As a consequence, individuals adjust their cellular structure and function in response to this physiological constraint. Because mitochondria are central to energy production, changes in their functional properties are likely to occur during reproduction. Such changes could cause adjustments in reactive oxygen species (ROS) production and consequently in oxidative stress levels. In this study, we investigated several mechanisms involved in energy production, including mitochondrial respiration at different steps of the electron transport system (ETS) and related the results to citrate synthase activity in the liver of non-reproductive and reproductive (two and eight pups) female house mice at peak lactation. Whereas we did not find differences between females having different litter sizes, liver mitochondria of reproductive females showed lower ETS activity and an increase in mitochondrial density when compared with the non-reproductive females. Although it is possible that these changes were due to combined processes involved in reproduction and not to the relative investment in lactation, we propose that the mitochondrial adjustment in liver might help to spare substrates and therefore energy for milk production in the mammary gland. Moreover, our results suggest that these changes lead to an increase in ROS production that subsequently upregulates antioxidant defence activity and decreases oxidative stress. Key words: reproduction, metabolism, mitochondrial respiration, liver, citrate synthase. Received 4 November 2012; Accepted 14 February 2013 The Journal of Experimental Biology 216, 2889-2895 © 2013. Published by The Company of Biologists Ltd doi:10.1242/jeb.082685 RESEARCH ARTICLE Physiological adaptations to reproduction. II. Mitochondrial adjustments in livers of lactating mice Nicolas Pichaud 1,2, *, Michael Garratt 1 , J. William O. Ballard 2 and Robert C. Brooks 1 1 Evolution and Ecology Research Centre and School of Biological, Earth and Environmental Sciences, The University of New South Wales, Sydney, New South Wales 2052, Australia and 2 School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales 2052, Australia *Author for correspondence (pichaud.nicolas@wanadoo.fr) THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY