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Changes to food intake and nutrition of female red-tailed phascogales (Phascogale calura) during late lactation
Physiology & Behavior 151 (2015) 398–403
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Changes to food intake and nutrition of female red-tailed phascogales (Phascogale calura) during late lactation Hayley J. Stannard ⁎, Julie M. Old Water and Wildlife Ecology, School of Science and Health, University of Western Sydney, NSW 2751, Australia
H I G H L I G H T S • • • •
Nutrient requirements were 2–2.5× higher for lactating red-tailed phascogales. Young in smaller litters grew faster than those in larger litters. Nutrient absorption did not differ between lactating and non-lactating animals. Maternal age did not influence nutrient requirements.
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Article history: Received 9 May 2015 Received in revised form 4 August 2015 Accepted 5 August 2015 Available online 7 August 2015 Keywords: Marsupial Dasyurid Litter size Pouch young Energy requirements Litter growth
a b s t r a c t Reproduction and especially lactation are nutritionally costly for mammals. Maternal access to adequate and optimal nutrients is essential for fecundity, survival of offspring, and offspring growth rates. In eutherian species energy requirements during lactation can be heavily dependent on litter size and the body mass of the female. In marsupials litter size does not appear to affect nutritional requirements during lactation; however, studies of marsupial nutritional requirements during lactation are rare. Marsupials are distinct from eutherians as they give birth to young at a much more underdeveloped state and the majority of their investment into the growth of their offspring occurs postnatally. Nutritional requirements of adult female red-tailed phascogales (Phascogale calura) were measured to determine the differences between those lactating and not lactating. On average females that were lactating had maintenance energy requirements of 1728 ± 195 kJ kg−0.75 d−1, double that of non-lactating animals. There was no significant correlation between energy requirements and litter size among lactating female phascogales. Apparent absorption of macronutrients did not differ between lactating and non-lactating individuals. The study has shown that food needs to be increased by at least double during late lactation. Litter size appears to have no influence on maternal nutrient requirements when food is available ad libitum and offspring in smaller litters grow faster than those in larger litters. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Lactation is one of the most energetically expensive physiological processes experienced by female mammals, especially for those rearing multiple offspring. If a female is unable to meet her nutritional requirements while lactating, she may lose offspring, resulting in a substantial loss of time and energy (investment) [1]. Energy requirements during lactation can increase by 100–480% above non-lactating values in small mammal species [2,3,4,5,6,7,8,9,10]. These increased energy requirements are dependent on maternal size, litter size and diet. Females maintained on protein restricted diets have poor reproductive output, smaller offspring and reduced litter sizes [11,12]. It has been suggested ⁎ Corresponding author at: Water and Wildlife Ecology, School of Science and Health, University of Western Sydney, Hawkesbury Campus Bldg. M15, Locked Bag 1797, Penrith, NSW 2751, Australia. E-mail address: [email protected] (H.J. Stannard).
http://dx.doi.org/10.1016/j.physbeh.2015.08.012 0031-9384/© 2015 Elsevier Inc. All rights reserved.
that during lactation 6% of total calories should come from protein for human and non-human primates; however, energy intake has a significant influence over milk production [13]. For marsupials the majority of maternal investment occurs during lactation due to the majority of neonate growth occurring post-partum [14]. Generally marsupials have a 30% lower metabolic rate than eutherians and this may indicate that there is a similar difference in nutritional requirements during lactation [15,16]. In the case of one herbivorous marsupial, the tammar wallaby (Macropus eugenii), energy requirements during reproduction appear to be lower than comparatively sized eutherians [17]. The grey short-tailed opossum (Monodelphis domestica) is more energy efficient (per young per day) than a similar sized eutherian; however opossums invest a longer period of time raising their young [18]. Previous studies addressing changes to nutritional requirements of marsupials during pregnancy and lactation have established an increase in resting metabolic rate, total daily energy expenditure, food intake and
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also adjustments to behaviour and digestive strategies [19,20,21,22,23, 24]. However, detailed measurements of dietary requirements under controlled laboratory conditions are limited to only three species [25, 26,27] and additional information specifically on smaller species and the influence of litter size on energy requirements are sorely needed. Nevertheless the available data suggest that during pregnancy and early lactation there appears to be only a small change in nutritional requirements for marsupials. During late lactation however, nutrient requirements increase significantly [25,26]. For example, in the tammar wallaby early lactation energy requirements are similar to nonlactating requirements, and requirements increase after 105 days of lactation (approximately one third of the way through lactation) and become significantly greater after 205 days [25]. For the bare-tailed woolly opossum (Caluromys philander), females with only one offspring (normally raise eight offspring) energy requirements are not significantly different to non-lactating females throughout the entire lactation period [26]. However, litters of two or more are energetically demanding, and there is no significant difference between energy requirements of varying litter sizes of two or more young [26]. It appears that the additional nutrients available to smaller litters are used by the young for faster growth. The red-tailed phascogale (Phascogale calura) is a small dasyurid that feeds primarily on insects [28,29]. It inhabits a small area of south-west Western Australia and is listed as endangered [30]. The red-tailed phascogale is a monoestrus breeder with a very short (3 week) synchronised breeding season [31,32]. Females give birth to supernumerary young after a 28 day gestation, but can only accommodate eight young in the pouch [32]. Young are fixed to the nipple for 44 days and are weaned at 90–110 days [33]. Males exhibit semelparity in the wild and can live longer in captivity but are infertile after 11.5 months [32]. The red-tailed phascogale was used as a model in this study to: 1) determine the difference in macronutrient requirements between lactating and non-lactating female dasyurids in captivity; 2) quantify the increased energy requirements of lactating female red-tailed phascogales; 3) determine the optimal litter size and whether more energy is needed to raise a larger litter; and 4) determine the influence of maternal age on nutrition requirements. 2. Methods 2.1. Ethical approval
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weight) and stored at −20 °C for later analysis. Animals were weighed at the start, at the five day mark, and at the end of each trial, with the lactating females weighed with and without their pouch young (where possible). Pouch young were weighed together and means determined based on the number of young within the litter (if they were unattached from the nipple). There was a rest period in-between (four days) the trials to allow animals to return to their normal body mass, whereby they were fed their normal daily scheduled diet (either adult mouse, crickets, mealworms, cockroaches, or neonate mice). In the first trial the animals were fed neonate mice (Mus domesticus) and in the second trial they were fed adult crickets (Acheta domesticus). These diet items form part of their normal diet in captivity. The neonate mice were purchased from DoLittle Farm (Molong, NSW) and frozen at −20 °C for a minimum of 4 weeks to prevent toxoplasmosis transmission. Crickets were purchased live from Pisces Enterprises (Brookfield, QLD) and fed on carrots.
2.4. Nutritional analysis Daily food samples, faecal material and leftover food samples for individuals were pooled for the week. These samples were then oven dried at 65 °C to a constant mass and weighed to determine the dry matter. Samples were then ground and blended into a homogenous mixture for further analysis. Samples were placed in a muffle furnace for 24 h at 500 °C to determine ash and organic matter composition. Energy was determined using an oxygen bomb calorimeter (Parr 6200; Parr Instrument Company, Moline, Illinois). Crude protein was determined using the Kjeldahl method, calculated as nitrogen × 6.25 [34, 35]. Lipids were extracted using a 1:1 volume of chloroform and methanol extraction technique [36]. Total carbohydrates were digested in 2.5 M HCl at 65 °C for 1 h then analysed using an anthrone colour reaction as per AOAC method 931.02 [37].
2.5. Statistical analysis Repeated measures ANOVAs were used to determine significant changes in the body mass of females and pouch young over the course of the trials. Single factor ANOVAs were used to compare diet composition and diet digestibility. Analysis of Covariance (ANCOVA) analyses were conducted on intake and output values for macronutrients, using SPSS [38].
The research was approved by the University of Western Sydney Animal Care and Ethics Committee, A10780 and animals were held under a NSW NPWS licence, SL100760.
3. Results
2.2. Animals
3.1. Dietary analysis
Sixteen adult female red-tailed phascogales (8 lactating and 8 nonlactating) from a captive colony held at the University of Western Sydney, Richmond, New South Wales, Australia, were used in this study. Of the lactating females four had four pouch young and four had eight pouch young. The pouch young were between 51 and 63 days old at the beginning of the trials. Each animal was housed individually in a wooden enclosure (1 m × 1 m × 0.58 m) with a paper substrate and wooden nest box, under natural spring photocycles. Room temperature was maintained at 22 ± 4 °C.
Compared with the neonate mice, the cricket diet was higher in dry matter, crude protein and organic matter composition (Table 1).
2.3. Nutrition trials During the nutrition trials, the animals were fed (ad libitum) one food type during the day (before the animals became active) for a total of 12 days. Water was available ad libitum. The first five days of the trial were an adjustment period; during the last seven days all faecal matter and leftover food were collected daily, weighed (± 0.1 g, wet
Table 1 Nutritional composition (mean ± SD) of the diets fed to the phascogales during the nutrition trials (n = 3, values are on a dry matter basis).
Dry matter (% of wet weight) Ash, % Organic matter, % Gross energy, kJ g−1 Nitrogen, % Crude protein, % Lipids, % Carbohydrates, % a n = 6. ⁎ Significantly different, P b 0.01.
Neonatal mice
Crickets
23.2 ± 0 7.5 ± 0.5 92.5 ± 0.5 23.9 ± 0.2 9.2 ± 0.1 57.4 ± 0.5 29.2 ± 4.2a 0.8 ± 0.1
29.6 ± 0⁎ 4.1 ± 0.1⁎ 95.9 ± 0.1⁎ 21.9 ± 0.3⁎ 10.4 ± 0.1⁎ 64.9 ± 0.6⁎ 25.5 ± 1.8⁎ 2.8 ± 0.1⁎
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3.2. Female body mass There was no significant change in the body mass of the adult females over the duration of the trials (lactating females: F3, 24 = 3.0, P = 0.05; non-lactating females: F2,15 = 2.3, P = 0.13). The body mass of lactating and non-lactating females was similar and showed no significant difference (F11,95 = 1.4, P = 0.18) (Table 2). 3.3. Pouch young growth A repeated measures analysis of the mean pouch young weights over the course of the trials showed that they increased significantly (F3,21 = 10.7, P b 0.05) (Table 2). Young in litters of four were significantly heavier than those in litters of eight on day 12 — neonate mice (F1,7 = 56.0, P b 0.01), day 1 — crickets (F1,7 = 36.1, P b 0.01) and day 5 — crickets (F1,7 = 51.6, P b 0.01). There was no significant difference between litter size and mean body mass on the final experiment day, day 12 — cricket diet (F1,7 = 1.1, P = 0.36). The difference in the mean body mass of young between the two differing litter sizes on this day was less than on previous days. It appears that the young in larger litters increased their body mass substantially in a week and had similar masses to those in the smaller litters on the last day of the trial. It is possible that towards the end of the cricket trial young were eating solid food; however no observations of their feeding behaviours were made and all pouch young included in the trials were less than 90 days old, which is the onset of weaning for phascogales [33]. Due to difficulties separating pouch young and mothers we could not get data for all litters for days 1 and 5 of the neonate mouse trial and thus data is not included in Table 2 for those days. 3.4. Dietary trials Females with pouch young were consuming 234–268% more than their non-lactating counterparts, on a dry matter basis. Lactating females consumed 14.0 ± 1.2% of their body mass per day and nonlactating females 6.0 ± 0.4% on the neonatal mouse diet. Lactating females consumed 15.5 ± 2.6% of their body mass per day and nonlactating females 5.9 ± 0.6% on the cricket diet. Individual phascogales showed preferences for certain body parts of the neonatal mice. Some animals, as well as consuming whole neonate mice, would decapitate and consume the heads, some would leave the heads and consume the bodies, and many would consume the whole neonate mouse with the exception of the stomach. The stomach was easily identified as it was full of milk. Selection and avoidance to certain body parts did not appear to be related to overall daily food intake. Mean daily energy intake for lactating females was double that of non-lactating females, 1728 ± 195 kJ kg−0.75 d−1, and 812 ± 109 kJ kg−0.75 d−1, respectively (Table 3). The difference between protein intake and lipid intake between non-lactating and lactating animals was around double (a 2× to 2.2× increase). Energy intake did not significantly differ between one and two year old animals for both diets: neonate mice (1 vs 2 year old lactating: F1,7 = 4.5, P = 0.08; non-lactating animals: F1,7 = 1.0, P = 0.36) and crickets (1 vs 2 year old lactating: F1,7 = 1.2, P = 0.31; non-lactating animals: F1,7 = 0.3, P = 0.59).
Table 3 Digestible energy intake values for lactating and non-lactating female red-tailed phascogales on a neonate mouse and cricket diet (mean ± SD). Neonatal mice
−1
−1
kJ g d kJ kg−0.75 d−1
Crickets
Lactating
Non-lactating
Lactating
Non-lactating
4.4 ± 0.5 1986.9 ± 172.3
2.0 ± 0.2 906.7 ± 64.2
3.3 ± 0.5 1469.6 ± 217.0
1.6 ± 0.4 717.2 ± 151.9
ANCOVA analysis shows that there was a significant linear relationship between intake and output on the neonate mouse diet for dry matter (F1,15 = 79.7, P b 0.01), ash (F1,15 = 70.3, P b 0.01), protein (F1,15 = 39.5, P b 0.01), carbohydrates (F1,15 = 53.7, P b 0.01) and energy (F1,15 = 78.1, P b 0.01) (Fig. 1). There was no relationship for lipid intake and output for lactating (F1,7 = 2.7, P = 0.16) and non-lactating individuals (F1,7 = 0.99, P = 0.36) on the neonatal mouse diet. However, lipid values were clustered at the lower end of the scale for non-lactating animals and at the higher end for lactating animals (Fig. 1E). ANCOVA analysis of the cricket diet shows that there was a significant linear relationship between intake and output for dry matter (F1,15 = 639.8, P b 0.01), ash (F1,15 = 90.7, P b 0.01), protein (F1,15 = 158.2, P b 0.01), energy (F1,15 = 771.0, P b 0.01), carbohydrates (F1,15 = 44.7, P b 0.01) and lipids (F1,15 = 43.3, P b 0.01) (Fig. 1). There was no significant difference between the apparent digestibility values exhibited by the lactating and non-lactating animals for any nutrients on either diet, and hence the values were pooled in Table 4. Apparent digestibility showed significant variation between the two diets for dry matter (F1,31 = 848.0, P b 0.01), lipids (F1,31 = 27.4, P b 0.01), energy (F1,31 = 1399.4, P b 0.01), protein (F1,31 = 412.2, P b 0.01) and carbohydrates (F1,31 = 19.7, P b 0.01). Ash digestibility showed no significant difference (F1,31 = 1.4, P = 0.24) between diets. The neonate mouse diet was more digestible than the cricket diet, as shown by the higher apparent digestibility values (Table 4).
4. Discussion Lactating adult female red-tailed phascogales require around a 250% increase in food on a dry matter basis. Despite using animals with four and eight pouch young there was no significant difference in the requirements of the females with differing pouch young numbers. Pouch young in smaller litters were growing quicker than those in larger litters. The body mass of lactating versus non-lactating animals was not significantly different during the trials. As the lactating females were consuming more than double (in mass and energy) than non-lactating females, it can be assumed that they were using the extra nutrients and energy for lactation, and hence maintained a similar body mass. All animals appeared to show some aversion to the stomachs of neonate mice, presumably because they disliked the texture or smell. On the second collection day of the neonate mouse trial there was a high level of food refusal (60–100% of food fed) by non-lactating females. It is possible that these females used torpor to compensate for the low energy consumption on that day. Non-lactating females were found to consume a similar percentage of body mass in food per day (31–34% on a wet matter basis) to those
Table 2 Changes in the body mass (g) of adult female red-tailed phascogales and pouch young, when unattached to the mother's nipples, during the nutrition trials. All data are mean ± 1 SD.
Non-lactating (n = 8) Lactating (n = 8) Pouch young (litter of 4) (n = 16) Pouch young (litter of 8) (n = 32)
Neonate mouse trial day 1
Neonate mouse trial day 5
Neonate mouse trial day 12
Cricket trial day 1
Cricket trial day 5
Cricket trial day 12
43.3 ± 3.4 40.1 ± 1.7 – –
41.5 ± 5.4 41.1 ± 3.2 – –
42.5 ± 3.9 43 ± 4.5 13.8 ± 0.9 7.8 ± 1.3
41.1 ± 3.4 39 ± 4.1 14.4 ± 1.2 8.9 ± 1.4
43.3 ± 3.8 38.9 ± 4.1 14.8 ± 0.2 8.7 ± 1.7
39.6 ± 5.6 39.1 ± 3.5 16.1 ± 1.0 14.5 ± 3.0
Due to difficulties separating pouch young and mothers (they are permanently attached to the nipple until 44 days) we could not get data for all litters for days 1 and 5 of the neonatal mouse trial.
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Fig. 1. Intake versus output data of lactating and non-lactating red-tailed phascogales during the cricket and neonate mouse trials. The linear relationship between intake and excretion is shown by the linear line of best fit. Slopes: Cricket diet — lactating: A: 0.3, B: 0.5, C: 0.3, D: 0.5, E: 0.5, and F: 1.2; non-lactating: A: 0.4, B: 0.5, C: 0.3, D: 0.5, E: 0.4, and F: 1.8. Neonate mouse diet — lactating: A: 0.2, B: 0.5, C: 0.1, D: 0.1, and F: 0.9; non-lactating: A: 0.2, B: 0.9, C: 0.1, D: 0.1, and F: 1.
values previously reported for small dasyurids, between 17 and 53%, on a wet matter basis [39,40,41,42]. The previously reported values are for insect, whole vertebrate and manufactured diets, the latter two being generally consumed in higher quantities per unit of body mass. Values for lactating females however, exceeded the values previously reported Table 4 Apparent digestibility (%) values for female red-tailed phascogales on a neonate mouse and cricket diet (mean ± SD).
Dry matter Ash Crude protein Gross energy Lipids Carbohydrates ⁎ Significantly different, P b 0.01.
Neonatal mice
Crickets
87.8 ± 2.0 52.0 ± 8.4 89.0 ± 2.2 93.0 ± 1.1 89.2 ± 4.9 49.0 ± 8.4
67.9 ± 1.9⁎ 48.5 ± 7.7 63.1 ± 4.6⁎ 72.4 ± 1.9⁎ 79.7 ± 5.4⁎
30.8 ± 14.1⁎
for dasyurids mentioned above [39,40,41,42], with lactating females consuming 70–87% of their body mass in food per day, on a wet matter basis. The daily digestible energy intake (DEI) for non-lactating redtailed phascogales is lower than that reported previously for mostly senescent male animals (1033 ± 192 kJ kg−0.75 d− 1) maintained on a cricket diet, when scaled for body mass [41]. Lactating females (body mass b 48 g) have a higher DEI than previously reported values for males (body mass b 68 g) maintained on a cricket diet, when scaled for body mass [41]. As expected the DEI of lactating phascogales was higher thanthelargereasternquoll(740±117kJkg−1 d−1 (~250kJkg−0.75 d−1)) 1)) during late lactation [27], as generally mass specific energy requirements increase with decreased body mass [16]. All females maintained a consistent body mass and showed no significant changes throughout the trials. Thus lactating animals maintained a positive energy balance during late lactation and were converting excess energy and nutrients provided (above that of nonlactating requirements) into milk for their young. Phascogales appear
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not to gain or mobilise excess fat stores to aid late lactation. They have been shown to gain 6–36% of their body mass in the weeks prior to mating but return to a base body mass two weeks prior to pregnancy [43]. Similarly some small eutherian mammals rely on food intake to supply energy and nutrients for lactation [7,44]. There are however, other eutherian species that rely on mobilising fat stores to support lactation [6,9,45,46]. Lactating female red-tailed phascogales required a 205–219% increase of energy intake above that of non-lactating animals. This increased energy requirement during lactation is similar to published values for a carnivorous marsupial, the eastern quoll (222%) [27]. Phascogale values are slightly higher than an omnivorous marsupial, the bare-tailed woolly opossum (112–199%) and the herbivorous tammar wallaby (174%) [25,26]. The increased energy need value falls within the range of some small insectivorous eutherian species, such as shrews (Crocidura spp.), between 100 and 200% [4]. However, values for species such as white-toothed shrews (Crocidura russula monacha) and Sorex spp. exceed the values determined for phascogales, 230– 480% [3] and 300% [4], respectively. There was no significant difference in nutritional requirements of lactating female phascogales with four or eight pouch young. Phascogales with smaller litters (four pouch young) were transferring the same quantity of macronutrients to the young as those with eight young, leading to an increased growth rate for those in smaller litters. Similarly, Atramentowicz [26] found that young bare-tailed woolly opossums in larger litters had a smaller body mass. However in eutherian mammals energy requirements during lactation can be heavily dependent on litter size [4,10,44]. For example, in grasshopper mice (Onychomys leucogaster) with one offspring, energy requirements above that of non-lactating females were 155% and 214% for those with three offspring and 293% for those with six offspring [44]. By day 16 mice in smaller litters had a higher body mass than those in larger litters. Grasshopper mice show a much faster juvenile growth rate than dasyurids and hence the potential maximum growth rate can become a limiting factor. The most beneficial litter size, in terms of energy turnover and reproductive success for grasshopper mice is four offspring [44]. It appears that lactating female phascogales are operating at a maximum digestive capacity when provided with food ad libitum. Energy transfer to the litter is at a maximum, and thus young in smaller litters are able to grow quicker. Hence, when food availability and quality are not an issue transfer of nutrients to the litter is limited by the digestive capacity of the female rather than the capacity for growth in juveniles. In the bare-tailed woolly opossum, it has been shown that food intake during late lactation correlates to the total mass of the litter at weaning [26]. Likewise for phascogales, food intake was positively correlated to the total mass of the litter (at age 76–88 at the end of the trial)
during the cricket trial; however food intake during the neonate mouse trial was not correlated to the final total litter mass (at the end of the trials) (Fig. 2). At the end of the trial, the young were weighed, rather than at the time of weaning, even though the young were close to weaning age. Food intake had a linear relationship with litter mass. Food intake was higher for those females with a heavier litter mass. ANCOVA analysis showed a linear relationship between intake and output for both diets for the macronutrients studied, with the exception of lipids on the neonate mouse diet. During the cricket trial the linear relationships (gradient of the slopes) were consistent across both nonlactating and lactating females for the macronutrients studied, with the exception of carbohydrates. It shows that the lactating females consumed more and excreted more than non-lactating animals but were utilising the macronutrients in the same manner just at a higher quantity to meet the needs of lactation. For the cricket diet the carbohydrate slopes were 1.2 for lactating females and 1.8 for non-lactating females. During the neonatal mouse trial there was a linear relationship between intake and output; however the slope/gradient differed between nonlactating and lactating animals, particularly for ash and carbohydrates. There was no relationship between intake and output for lipids. The variation of intake on the neonate mouse diet is likely due to females being able to select which body parts of mice they want to ingest. Some animals showed a preference for certain body parts, and most animals showed an aversion to stomachs, which would have impacted the macronutrient intake of individual females. Consistent utilisation of macronutrients is also reiterated by the apparent digestibility values with no significant difference between data for lactating and non-lactating animals. Apparent digestibility of macronutrients on the cricket diet was lower than those previously reported for red-tailed phascogales and other small dasyurid species (dry matter: 79–90%, protein: 80–91%, energy: 73–84% and lipids: 83–92%) [41,42]. It is possible that animals reached their upper limit of intake for protein in this diet thus explaining the lower digestibility values observed. On an energy basis the protein in the cricket diet provided 53% (protein 17 kJ g−1) of the energy and although this is the protein target of a strict carnivore, the cat (Felis catus) [47], more data is needed to explore these limits in insectivores. Mink (Mustela vison) target for a lower protein level of 35% of metabolisable energy [48], which is lower than the neonate mouse diet fed to the phascogales (protein: 47% of energy) and it may be that phascogales target for a lower level; however more research is required. The data in this study shows that during late lactation, phascogales on average require a two fold increase in energy, protein and lipids. Litter size does not strongly influence nutritional needs (when food is available ad lib) and thus it is advantageous for phascogales to produce a full litter (eight pouch young), which aligns with their life history strategy to produce supernumerary young at birth [32]. Age (one and two year old females) does not influence nutritional requirements in either lactating or non-lactating phascogales. This study provides data on the increased needs of an endangered dasyurid and it is likely that these increased requirements are similar in other small dasyurid species, and thus can be used for management of other captive dasyurid species. Further research encompassing the full reproductive cycle (gestation, lactation and post-weaning) would quantify the changes across the whole female reproductive period. As current nutritional data is only available for senescent males [41], further investigation into the nutrition of reproductively active males is required.
Acknowledgements
Fig. 2. Relationship between litter mass at age 76–88 days and daily food intake by lactating females; line of best fit shows linear relationship between food intake and litter mass for the cricket diet.
Thank you to Ben Stepkovitch for his assistance with the sample collection and laboratory analyses and to Mark Emanuel for assistance with laboratory analyses. The School of Science and Health, University of Western Sydney financially support the Small Native Mammal Teaching and Research Facility.
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Changes to food intake and nutrition of female red-tailed phascogales (Phascogale calura) during late lactation Hayley J. Stannard ⁎, Julie M. Old Water and Wildlife Ecology, School of Science and Health, University of Western Sydney, NSW 2751, Australia
H I G H L I G H T S • • • •
Nutrient requirements were 2–2.5× higher for lactating red-tailed phascogales. Young in smaller litters grew faster than those in larger litters. Nutrient absorption did not differ between lactating and non-lactating animals. Maternal age did not influence nutrient requirements.
a r t i c l e
i n f o
Article history: Received 9 May 2015 Received in revised form 4 August 2015 Accepted 5 August 2015 Available online 7 August 2015 Keywords: Marsupial Dasyurid Litter size Pouch young Energy requirements Litter growth
a b s t r a c t Reproduction and especially lactation are nutritionally costly for mammals. Maternal access to adequate and optimal nutrients is essential for fecundity, survival of offspring, and offspring growth rates. In eutherian species energy requirements during lactation can be heavily dependent on litter size and the body mass of the female. In marsupials litter size does not appear to affect nutritional requirements during lactation; however, studies of marsupial nutritional requirements during lactation are rare. Marsupials are distinct from eutherians as they give birth to young at a much more underdeveloped state and the majority of their investment into the growth of their offspring occurs postnatally. Nutritional requirements of adult female red-tailed phascogales (Phascogale calura) were measured to determine the differences between those lactating and not lactating. On average females that were lactating had maintenance energy requirements of 1728 ± 195 kJ kg−0.75 d−1, double that of non-lactating animals. There was no significant correlation between energy requirements and litter size among lactating female phascogales. Apparent absorption of macronutrients did not differ between lactating and non-lactating individuals. The study has shown that food needs to be increased by at least double during late lactation. Litter size appears to have no influence on maternal nutrient requirements when food is available ad libitum and offspring in smaller litters grow faster than those in larger litters. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Lactation is one of the most energetically expensive physiological processes experienced by female mammals, especially for those rearing multiple offspring. If a female is unable to meet her nutritional requirements while lactating, she may lose offspring, resulting in a substantial loss of time and energy (investment) [1]. Energy requirements during lactation can increase by 100–480% above non-lactating values in small mammal species [2,3,4,5,6,7,8,9,10]. These increased energy requirements are dependent on maternal size, litter size and diet. Females maintained on protein restricted diets have poor reproductive output, smaller offspring and reduced litter sizes [11,12]. It has been suggested ⁎ Corresponding author at: Water and Wildlife Ecology, School of Science and Health, University of Western Sydney, Hawkesbury Campus Bldg. M15, Locked Bag 1797, Penrith, NSW 2751, Australia. E-mail address: [email protected] (H.J. Stannard).
http://dx.doi.org/10.1016/j.physbeh.2015.08.012 0031-9384/© 2015 Elsevier Inc. All rights reserved.
that during lactation 6% of total calories should come from protein for human and non-human primates; however, energy intake has a significant influence over milk production [13]. For marsupials the majority of maternal investment occurs during lactation due to the majority of neonate growth occurring post-partum [14]. Generally marsupials have a 30% lower metabolic rate than eutherians and this may indicate that there is a similar difference in nutritional requirements during lactation [15,16]. In the case of one herbivorous marsupial, the tammar wallaby (Macropus eugenii), energy requirements during reproduction appear to be lower than comparatively sized eutherians [17]. The grey short-tailed opossum (Monodelphis domestica) is more energy efficient (per young per day) than a similar sized eutherian; however opossums invest a longer period of time raising their young [18]. Previous studies addressing changes to nutritional requirements of marsupials during pregnancy and lactation have established an increase in resting metabolic rate, total daily energy expenditure, food intake and
H.J. Stannard, J.M. Old / Physiology & Behavior 151 (2015) 398–403
also adjustments to behaviour and digestive strategies [19,20,21,22,23, 24]. However, detailed measurements of dietary requirements under controlled laboratory conditions are limited to only three species [25, 26,27] and additional information specifically on smaller species and the influence of litter size on energy requirements are sorely needed. Nevertheless the available data suggest that during pregnancy and early lactation there appears to be only a small change in nutritional requirements for marsupials. During late lactation however, nutrient requirements increase significantly [25,26]. For example, in the tammar wallaby early lactation energy requirements are similar to nonlactating requirements, and requirements increase after 105 days of lactation (approximately one third of the way through lactation) and become significantly greater after 205 days [25]. For the bare-tailed woolly opossum (Caluromys philander), females with only one offspring (normally raise eight offspring) energy requirements are not significantly different to non-lactating females throughout the entire lactation period [26]. However, litters of two or more are energetically demanding, and there is no significant difference between energy requirements of varying litter sizes of two or more young [26]. It appears that the additional nutrients available to smaller litters are used by the young for faster growth. The red-tailed phascogale (Phascogale calura) is a small dasyurid that feeds primarily on insects [28,29]. It inhabits a small area of south-west Western Australia and is listed as endangered [30]. The red-tailed phascogale is a monoestrus breeder with a very short (3 week) synchronised breeding season [31,32]. Females give birth to supernumerary young after a 28 day gestation, but can only accommodate eight young in the pouch [32]. Young are fixed to the nipple for 44 days and are weaned at 90–110 days [33]. Males exhibit semelparity in the wild and can live longer in captivity but are infertile after 11.5 months [32]. The red-tailed phascogale was used as a model in this study to: 1) determine the difference in macronutrient requirements between lactating and non-lactating female dasyurids in captivity; 2) quantify the increased energy requirements of lactating female red-tailed phascogales; 3) determine the optimal litter size and whether more energy is needed to raise a larger litter; and 4) determine the influence of maternal age on nutrition requirements. 2. Methods 2.1. Ethical approval
399
weight) and stored at −20 °C for later analysis. Animals were weighed at the start, at the five day mark, and at the end of each trial, with the lactating females weighed with and without their pouch young (where possible). Pouch young were weighed together and means determined based on the number of young within the litter (if they were unattached from the nipple). There was a rest period in-between (four days) the trials to allow animals to return to their normal body mass, whereby they were fed their normal daily scheduled diet (either adult mouse, crickets, mealworms, cockroaches, or neonate mice). In the first trial the animals were fed neonate mice (Mus domesticus) and in the second trial they were fed adult crickets (Acheta domesticus). These diet items form part of their normal diet in captivity. The neonate mice were purchased from DoLittle Farm (Molong, NSW) and frozen at −20 °C for a minimum of 4 weeks to prevent toxoplasmosis transmission. Crickets were purchased live from Pisces Enterprises (Brookfield, QLD) and fed on carrots.
2.4. Nutritional analysis Daily food samples, faecal material and leftover food samples for individuals were pooled for the week. These samples were then oven dried at 65 °C to a constant mass and weighed to determine the dry matter. Samples were then ground and blended into a homogenous mixture for further analysis. Samples were placed in a muffle furnace for 24 h at 500 °C to determine ash and organic matter composition. Energy was determined using an oxygen bomb calorimeter (Parr 6200; Parr Instrument Company, Moline, Illinois). Crude protein was determined using the Kjeldahl method, calculated as nitrogen × 6.25 [34, 35]. Lipids were extracted using a 1:1 volume of chloroform and methanol extraction technique [36]. Total carbohydrates were digested in 2.5 M HCl at 65 °C for 1 h then analysed using an anthrone colour reaction as per AOAC method 931.02 [37].
2.5. Statistical analysis Repeated measures ANOVAs were used to determine significant changes in the body mass of females and pouch young over the course of the trials. Single factor ANOVAs were used to compare diet composition and diet digestibility. Analysis of Covariance (ANCOVA) analyses were conducted on intake and output values for macronutrients, using SPSS [38].
The research was approved by the University of Western Sydney Animal Care and Ethics Committee, A10780 and animals were held under a NSW NPWS licence, SL100760.
3. Results
2.2. Animals
3.1. Dietary analysis
Sixteen adult female red-tailed phascogales (8 lactating and 8 nonlactating) from a captive colony held at the University of Western Sydney, Richmond, New South Wales, Australia, were used in this study. Of the lactating females four had four pouch young and four had eight pouch young. The pouch young were between 51 and 63 days old at the beginning of the trials. Each animal was housed individually in a wooden enclosure (1 m × 1 m × 0.58 m) with a paper substrate and wooden nest box, under natural spring photocycles. Room temperature was maintained at 22 ± 4 °C.
Compared with the neonate mice, the cricket diet was higher in dry matter, crude protein and organic matter composition (Table 1).
2.3. Nutrition trials During the nutrition trials, the animals were fed (ad libitum) one food type during the day (before the animals became active) for a total of 12 days. Water was available ad libitum. The first five days of the trial were an adjustment period; during the last seven days all faecal matter and leftover food were collected daily, weighed (± 0.1 g, wet
Table 1 Nutritional composition (mean ± SD) of the diets fed to the phascogales during the nutrition trials (n = 3, values are on a dry matter basis).
Dry matter (% of wet weight) Ash, % Organic matter, % Gross energy, kJ g−1 Nitrogen, % Crude protein, % Lipids, % Carbohydrates, % a n = 6. ⁎ Significantly different, P b 0.01.
Neonatal mice
Crickets
23.2 ± 0 7.5 ± 0.5 92.5 ± 0.5 23.9 ± 0.2 9.2 ± 0.1 57.4 ± 0.5 29.2 ± 4.2a 0.8 ± 0.1
29.6 ± 0⁎ 4.1 ± 0.1⁎ 95.9 ± 0.1⁎ 21.9 ± 0.3⁎ 10.4 ± 0.1⁎ 64.9 ± 0.6⁎ 25.5 ± 1.8⁎ 2.8 ± 0.1⁎
400
H.J. Stannard, J.M. Old / Physiology & Behavior 151 (2015) 398–403
3.2. Female body mass There was no significant change in the body mass of the adult females over the duration of the trials (lactating females: F3, 24 = 3.0, P = 0.05; non-lactating females: F2,15 = 2.3, P = 0.13). The body mass of lactating and non-lactating females was similar and showed no significant difference (F11,95 = 1.4, P = 0.18) (Table 2). 3.3. Pouch young growth A repeated measures analysis of the mean pouch young weights over the course of the trials showed that they increased significantly (F3,21 = 10.7, P b 0.05) (Table 2). Young in litters of four were significantly heavier than those in litters of eight on day 12 — neonate mice (F1,7 = 56.0, P b 0.01), day 1 — crickets (F1,7 = 36.1, P b 0.01) and day 5 — crickets (F1,7 = 51.6, P b 0.01). There was no significant difference between litter size and mean body mass on the final experiment day, day 12 — cricket diet (F1,7 = 1.1, P = 0.36). The difference in the mean body mass of young between the two differing litter sizes on this day was less than on previous days. It appears that the young in larger litters increased their body mass substantially in a week and had similar masses to those in the smaller litters on the last day of the trial. It is possible that towards the end of the cricket trial young were eating solid food; however no observations of their feeding behaviours were made and all pouch young included in the trials were less than 90 days old, which is the onset of weaning for phascogales [33]. Due to difficulties separating pouch young and mothers we could not get data for all litters for days 1 and 5 of the neonate mouse trial and thus data is not included in Table 2 for those days. 3.4. Dietary trials Females with pouch young were consuming 234–268% more than their non-lactating counterparts, on a dry matter basis. Lactating females consumed 14.0 ± 1.2% of their body mass per day and nonlactating females 6.0 ± 0.4% on the neonatal mouse diet. Lactating females consumed 15.5 ± 2.6% of their body mass per day and nonlactating females 5.9 ± 0.6% on the cricket diet. Individual phascogales showed preferences for certain body parts of the neonatal mice. Some animals, as well as consuming whole neonate mice, would decapitate and consume the heads, some would leave the heads and consume the bodies, and many would consume the whole neonate mouse with the exception of the stomach. The stomach was easily identified as it was full of milk. Selection and avoidance to certain body parts did not appear to be related to overall daily food intake. Mean daily energy intake for lactating females was double that of non-lactating females, 1728 ± 195 kJ kg−0.75 d−1, and 812 ± 109 kJ kg−0.75 d−1, respectively (Table 3). The difference between protein intake and lipid intake between non-lactating and lactating animals was around double (a 2× to 2.2× increase). Energy intake did not significantly differ between one and two year old animals for both diets: neonate mice (1 vs 2 year old lactating: F1,7 = 4.5, P = 0.08; non-lactating animals: F1,7 = 1.0, P = 0.36) and crickets (1 vs 2 year old lactating: F1,7 = 1.2, P = 0.31; non-lactating animals: F1,7 = 0.3, P = 0.59).
Table 3 Digestible energy intake values for lactating and non-lactating female red-tailed phascogales on a neonate mouse and cricket diet (mean ± SD). Neonatal mice
−1
−1
kJ g d kJ kg−0.75 d−1
Crickets
Lactating
Non-lactating
Lactating
Non-lactating
4.4 ± 0.5 1986.9 ± 172.3
2.0 ± 0.2 906.7 ± 64.2
3.3 ± 0.5 1469.6 ± 217.0
1.6 ± 0.4 717.2 ± 151.9
ANCOVA analysis shows that there was a significant linear relationship between intake and output on the neonate mouse diet for dry matter (F1,15 = 79.7, P b 0.01), ash (F1,15 = 70.3, P b 0.01), protein (F1,15 = 39.5, P b 0.01), carbohydrates (F1,15 = 53.7, P b 0.01) and energy (F1,15 = 78.1, P b 0.01) (Fig. 1). There was no relationship for lipid intake and output for lactating (F1,7 = 2.7, P = 0.16) and non-lactating individuals (F1,7 = 0.99, P = 0.36) on the neonatal mouse diet. However, lipid values were clustered at the lower end of the scale for non-lactating animals and at the higher end for lactating animals (Fig. 1E). ANCOVA analysis of the cricket diet shows that there was a significant linear relationship between intake and output for dry matter (F1,15 = 639.8, P b 0.01), ash (F1,15 = 90.7, P b 0.01), protein (F1,15 = 158.2, P b 0.01), energy (F1,15 = 771.0, P b 0.01), carbohydrates (F1,15 = 44.7, P b 0.01) and lipids (F1,15 = 43.3, P b 0.01) (Fig. 1). There was no significant difference between the apparent digestibility values exhibited by the lactating and non-lactating animals for any nutrients on either diet, and hence the values were pooled in Table 4. Apparent digestibility showed significant variation between the two diets for dry matter (F1,31 = 848.0, P b 0.01), lipids (F1,31 = 27.4, P b 0.01), energy (F1,31 = 1399.4, P b 0.01), protein (F1,31 = 412.2, P b 0.01) and carbohydrates (F1,31 = 19.7, P b 0.01). Ash digestibility showed no significant difference (F1,31 = 1.4, P = 0.24) between diets. The neonate mouse diet was more digestible than the cricket diet, as shown by the higher apparent digestibility values (Table 4).
4. Discussion Lactating adult female red-tailed phascogales require around a 250% increase in food on a dry matter basis. Despite using animals with four and eight pouch young there was no significant difference in the requirements of the females with differing pouch young numbers. Pouch young in smaller litters were growing quicker than those in larger litters. The body mass of lactating versus non-lactating animals was not significantly different during the trials. As the lactating females were consuming more than double (in mass and energy) than non-lactating females, it can be assumed that they were using the extra nutrients and energy for lactation, and hence maintained a similar body mass. All animals appeared to show some aversion to the stomachs of neonate mice, presumably because they disliked the texture or smell. On the second collection day of the neonate mouse trial there was a high level of food refusal (60–100% of food fed) by non-lactating females. It is possible that these females used torpor to compensate for the low energy consumption on that day. Non-lactating females were found to consume a similar percentage of body mass in food per day (31–34% on a wet matter basis) to those
Table 2 Changes in the body mass (g) of adult female red-tailed phascogales and pouch young, when unattached to the mother's nipples, during the nutrition trials. All data are mean ± 1 SD.
Non-lactating (n = 8) Lactating (n = 8) Pouch young (litter of 4) (n = 16) Pouch young (litter of 8) (n = 32)
Neonate mouse trial day 1
Neonate mouse trial day 5
Neonate mouse trial day 12
Cricket trial day 1
Cricket trial day 5
Cricket trial day 12
43.3 ± 3.4 40.1 ± 1.7 – –
41.5 ± 5.4 41.1 ± 3.2 – –
42.5 ± 3.9 43 ± 4.5 13.8 ± 0.9 7.8 ± 1.3
41.1 ± 3.4 39 ± 4.1 14.4 ± 1.2 8.9 ± 1.4
43.3 ± 3.8 38.9 ± 4.1 14.8 ± 0.2 8.7 ± 1.7
39.6 ± 5.6 39.1 ± 3.5 16.1 ± 1.0 14.5 ± 3.0
Due to difficulties separating pouch young and mothers (they are permanently attached to the nipple until 44 days) we could not get data for all litters for days 1 and 5 of the neonatal mouse trial.
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Fig. 1. Intake versus output data of lactating and non-lactating red-tailed phascogales during the cricket and neonate mouse trials. The linear relationship between intake and excretion is shown by the linear line of best fit. Slopes: Cricket diet — lactating: A: 0.3, B: 0.5, C: 0.3, D: 0.5, E: 0.5, and F: 1.2; non-lactating: A: 0.4, B: 0.5, C: 0.3, D: 0.5, E: 0.4, and F: 1.8. Neonate mouse diet — lactating: A: 0.2, B: 0.5, C: 0.1, D: 0.1, and F: 0.9; non-lactating: A: 0.2, B: 0.9, C: 0.1, D: 0.1, and F: 1.
values previously reported for small dasyurids, between 17 and 53%, on a wet matter basis [39,40,41,42]. The previously reported values are for insect, whole vertebrate and manufactured diets, the latter two being generally consumed in higher quantities per unit of body mass. Values for lactating females however, exceeded the values previously reported Table 4 Apparent digestibility (%) values for female red-tailed phascogales on a neonate mouse and cricket diet (mean ± SD).
Dry matter Ash Crude protein Gross energy Lipids Carbohydrates ⁎ Significantly different, P b 0.01.
Neonatal mice
Crickets
87.8 ± 2.0 52.0 ± 8.4 89.0 ± 2.2 93.0 ± 1.1 89.2 ± 4.9 49.0 ± 8.4
67.9 ± 1.9⁎ 48.5 ± 7.7 63.1 ± 4.6⁎ 72.4 ± 1.9⁎ 79.7 ± 5.4⁎
30.8 ± 14.1⁎
for dasyurids mentioned above [39,40,41,42], with lactating females consuming 70–87% of their body mass in food per day, on a wet matter basis. The daily digestible energy intake (DEI) for non-lactating redtailed phascogales is lower than that reported previously for mostly senescent male animals (1033 ± 192 kJ kg−0.75 d− 1) maintained on a cricket diet, when scaled for body mass [41]. Lactating females (body mass b 48 g) have a higher DEI than previously reported values for males (body mass b 68 g) maintained on a cricket diet, when scaled for body mass [41]. As expected the DEI of lactating phascogales was higher thanthelargereasternquoll(740±117kJkg−1 d−1 (~250kJkg−0.75 d−1)) 1)) during late lactation [27], as generally mass specific energy requirements increase with decreased body mass [16]. All females maintained a consistent body mass and showed no significant changes throughout the trials. Thus lactating animals maintained a positive energy balance during late lactation and were converting excess energy and nutrients provided (above that of nonlactating requirements) into milk for their young. Phascogales appear
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not to gain or mobilise excess fat stores to aid late lactation. They have been shown to gain 6–36% of their body mass in the weeks prior to mating but return to a base body mass two weeks prior to pregnancy [43]. Similarly some small eutherian mammals rely on food intake to supply energy and nutrients for lactation [7,44]. There are however, other eutherian species that rely on mobilising fat stores to support lactation [6,9,45,46]. Lactating female red-tailed phascogales required a 205–219% increase of energy intake above that of non-lactating animals. This increased energy requirement during lactation is similar to published values for a carnivorous marsupial, the eastern quoll (222%) [27]. Phascogale values are slightly higher than an omnivorous marsupial, the bare-tailed woolly opossum (112–199%) and the herbivorous tammar wallaby (174%) [25,26]. The increased energy need value falls within the range of some small insectivorous eutherian species, such as shrews (Crocidura spp.), between 100 and 200% [4]. However, values for species such as white-toothed shrews (Crocidura russula monacha) and Sorex spp. exceed the values determined for phascogales, 230– 480% [3] and 300% [4], respectively. There was no significant difference in nutritional requirements of lactating female phascogales with four or eight pouch young. Phascogales with smaller litters (four pouch young) were transferring the same quantity of macronutrients to the young as those with eight young, leading to an increased growth rate for those in smaller litters. Similarly, Atramentowicz [26] found that young bare-tailed woolly opossums in larger litters had a smaller body mass. However in eutherian mammals energy requirements during lactation can be heavily dependent on litter size [4,10,44]. For example, in grasshopper mice (Onychomys leucogaster) with one offspring, energy requirements above that of non-lactating females were 155% and 214% for those with three offspring and 293% for those with six offspring [44]. By day 16 mice in smaller litters had a higher body mass than those in larger litters. Grasshopper mice show a much faster juvenile growth rate than dasyurids and hence the potential maximum growth rate can become a limiting factor. The most beneficial litter size, in terms of energy turnover and reproductive success for grasshopper mice is four offspring [44]. It appears that lactating female phascogales are operating at a maximum digestive capacity when provided with food ad libitum. Energy transfer to the litter is at a maximum, and thus young in smaller litters are able to grow quicker. Hence, when food availability and quality are not an issue transfer of nutrients to the litter is limited by the digestive capacity of the female rather than the capacity for growth in juveniles. In the bare-tailed woolly opossum, it has been shown that food intake during late lactation correlates to the total mass of the litter at weaning [26]. Likewise for phascogales, food intake was positively correlated to the total mass of the litter (at age 76–88 at the end of the trial)
during the cricket trial; however food intake during the neonate mouse trial was not correlated to the final total litter mass (at the end of the trials) (Fig. 2). At the end of the trial, the young were weighed, rather than at the time of weaning, even though the young were close to weaning age. Food intake had a linear relationship with litter mass. Food intake was higher for those females with a heavier litter mass. ANCOVA analysis showed a linear relationship between intake and output for both diets for the macronutrients studied, with the exception of lipids on the neonate mouse diet. During the cricket trial the linear relationships (gradient of the slopes) were consistent across both nonlactating and lactating females for the macronutrients studied, with the exception of carbohydrates. It shows that the lactating females consumed more and excreted more than non-lactating animals but were utilising the macronutrients in the same manner just at a higher quantity to meet the needs of lactation. For the cricket diet the carbohydrate slopes were 1.2 for lactating females and 1.8 for non-lactating females. During the neonatal mouse trial there was a linear relationship between intake and output; however the slope/gradient differed between nonlactating and lactating animals, particularly for ash and carbohydrates. There was no relationship between intake and output for lipids. The variation of intake on the neonate mouse diet is likely due to females being able to select which body parts of mice they want to ingest. Some animals showed a preference for certain body parts, and most animals showed an aversion to stomachs, which would have impacted the macronutrient intake of individual females. Consistent utilisation of macronutrients is also reiterated by the apparent digestibility values with no significant difference between data for lactating and non-lactating animals. Apparent digestibility of macronutrients on the cricket diet was lower than those previously reported for red-tailed phascogales and other small dasyurid species (dry matter: 79–90%, protein: 80–91%, energy: 73–84% and lipids: 83–92%) [41,42]. It is possible that animals reached their upper limit of intake for protein in this diet thus explaining the lower digestibility values observed. On an energy basis the protein in the cricket diet provided 53% (protein 17 kJ g−1) of the energy and although this is the protein target of a strict carnivore, the cat (Felis catus) [47], more data is needed to explore these limits in insectivores. Mink (Mustela vison) target for a lower protein level of 35% of metabolisable energy [48], which is lower than the neonate mouse diet fed to the phascogales (protein: 47% of energy) and it may be that phascogales target for a lower level; however more research is required. The data in this study shows that during late lactation, phascogales on average require a two fold increase in energy, protein and lipids. Litter size does not strongly influence nutritional needs (when food is available ad lib) and thus it is advantageous for phascogales to produce a full litter (eight pouch young), which aligns with their life history strategy to produce supernumerary young at birth [32]. Age (one and two year old females) does not influence nutritional requirements in either lactating or non-lactating phascogales. This study provides data on the increased needs of an endangered dasyurid and it is likely that these increased requirements are similar in other small dasyurid species, and thus can be used for management of other captive dasyurid species. Further research encompassing the full reproductive cycle (gestation, lactation and post-weaning) would quantify the changes across the whole female reproductive period. As current nutritional data is only available for senescent males [41], further investigation into the nutrition of reproductively active males is required.
Acknowledgements
Fig. 2. Relationship between litter mass at age 76–88 days and daily food intake by lactating females; line of best fit shows linear relationship between food intake and litter mass for the cricket diet.
Thank you to Ben Stepkovitch for his assistance with the sample collection and laboratory analyses and to Mark Emanuel for assistance with laboratory analyses. The School of Science and Health, University of Western Sydney financially support the Small Native Mammal Teaching and Research Facility.
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