Temporal dynamics of milk composition of the precocial caviomorph Octodon degus ( Rodentia : Octodontidae )

During lactation, both the nutritional and energetic requirements of suckling change gradually. These changes normally are accompanied by modifications in chemical composition of the milk. We investigated the temporal course of milk composition during lactation in a precocial caviomorph rodent, the “degu” (Octodon degus) under laboratory condition. Female degus were kept in laboratory during gestation and lactation and fed with commercial food pellets. Milk was collected at three stages of lactation: early (days 5-8, n = 12), middle (days 15-21, n = 7) and late (days 26-40, n = 6), and analyzed for protein, carbohydrates, lipids, ash, total solids and energy. On average, carbohydrates decreased from 3.1 ± 0.3 % (early) to 1.1 ± 0.3 % (late) during lactation; lipids, protein, ash, total solids and energy remained about the same. Lipids, the main component of the milk, were 17.3 % and protein remained near 4.4 %. Over lactation, total energy concentration of milk remained near 4.0 kJ mL-1. The maintenance of milk composition during lactation may be related to the initially high energetic and nutritional requirements associated with a precocial reproductive mode.


INTRODUCTION
Mammals are distinguished by two basic developmental strategies: altricial or precocial.Altricial mammals are characterized by short gestation and little somatic development of the newborn.On the contrary, precocial species experience a relatively long pregnancy, and somatic development of their newborn is more advanced (Derrickson 1992).Martin (1984) postulated that precocial animals should have more diluted milk than altricial, along with a different pattern of change in milk composition and production during lactation.
In mammals, milk is the only initial avenue for intake of matter and energy in the young.The increased energy expenditure associated with milk production in small mammals imposes maximal demands of nutrients and energy by mothers (Kenagy 1987, König et al. 1988).There is therefore, a strong selection pressure on physiological performance during lactation, as a result of the high energy turnover (Sadleir 1984).The energy intake and expenditure during lactation have been evaluated in a variety of rodent species (Millar 1979, Glazier 1985, König et al. 1988, Kenagy et al. 1990, Sikes 1995), but little is known about milk composition, its production and allocation to growth of pups in relation to the reproductive mode.
During lactation, both nutritional and energetic requirements of suckling young change gradually (Oftedal 1984), evidenced by changes in the proportion of lipids, proteins, and carbohydrates found in milk (Arman et al. 1974, Oftedal et al. 1983, Cork & Dove 1989, Korine & Arad 1999, Veloso et al. 2003).While lactating, rodents convert food eaten almost immediately into milk (Sadleir 1984).Interspecific variation in milk composition may reflect adaptation to differences in neonatal growth rates (Oftedal 1986).
Octodon degus is the most common Chilean diurnal rodent, of approximately 200 g, and inhabits shrubland areas of arid central Chile.This species has a relatively long gestation period (three months), pups are precocial, and have a relatively short lactation period (30 days) (Rojas et al. 1977, Mann 1978, Morales 1982).Here we investigated the temporal pattern of milk composition during lactation in the precocial caviomorph rodent Octodon degus in laboratory conditions.

MATERIAL AND METHODS
Forty-four animals (38 female and 6 male) were captured between March and May near Lampa, central Chile (33°17'S, 70°53'W, elevation 490 m).All females were allowed to mate and give birth in captivity.
Females were maintained in large cages (1.6 x 0.8 x 1.0 m) in groups of 8-10 individuals with natural outdoor temperature and photoperiod; food and water were provided ad libitum.Between May and August females were inspected to determine vulvar opening, at which time they were placed singly in a breeding cage (1.6 x 1.6 x 2.0 m) with two or three males.After five days each female was separated in to an individual cage (25 x 30 x 15 cm) and maintained during three months of pregnancy and one month of lactation under a 12L:12D photoperiod at 22 ± 3 °C.Only thirteen females (34.2 %) were pregnant.
Pregnant females received water and food ad libitum.Food was commercial rabbit pellet, and chemical composition is shown in Table 1, as analyzed by the Laboratory of Analytical Services at the Faculty of Agronomy, Pontificia Universidad Católica de Chile.Energy content (kJ g -1 )18.4 Milk was collected from females, all of known parturition days, over the course of lactation, ranging from day five to day 40.A total of 25 useful samples was obtained from 13 individuals, from ten of which repeated samples were obtained at different stages of lactation on two to three occasions.To facilitate milk release and collection, females were injected intramuscularly with 2 I.U kg -1 oxytocin, diluted from a stock of 5 I.U.mL -1 (Sanderson S.A. Laboratories).Milk was collected by capillarity, using a Pasteur pipette from each of the eight teats, with gentle manual expression, until no further milk was apparent.All samples were obtained between 11:00 and 13:00 h, after having separated the mother from the young for three hours.The sample volumes ranged from 50 to 200 µL and were stored in 0.6 mL plastic tubes at -20 °C until analysis.
Each sample was analyzed for: energy, total solid, protein, carbohydrate, lipid and ash.Total solid and ash were determined gravimetrically (Sartorius Supermicro S3D, to the nearest 0.5 µg) by weighing a sample of 10 µL and drying it at 100 °C for 24 h in a small aluminum container.Ash content (inorganic matter) was determined using the same sample by burning it twice in a small aluminum container in a muffle furnace (Nabertherm L3/P) at 560 °C.Protein content was determined by analyzing 2 µL of milk by the Coomassie Blue method (Bradford 1976) using bovine serum albumin as a standard.Carbohydrate content was determined on a 2 µL sample by the anthrona method, using lactose as a standard (Yemm & Willis 1954).All microvolumetric measurements were made with quantitative grade micropipettes.Lipid content was calculated for each sample by taking the difference between total solids and the sum of carbohydrates, proteins and ash.Finally, energy content of the milk was estimated from the measured milk composition using energetic equivalents of 24.6 kJ g -1 for proteins,16.5kJ g -1 for carbohydrates, and 38.1 kJ g -1 for lipids (Perrin 1958).Values of percentage concentration are presented on a wet-mass basis, and total energy content is expressed with respect to volume of whole milk.
Changes in milk composition over the course of lactation were analyzed by applying linear regression to the arcsine of square root transformed percentage data (Zar 1996).All values are mean ± 1 SD.

RESULTS
Parturition occurred between 12 September and 11 November.Mean pregnancy time was 87.9 ± 2.6 days with a range of 86 to 93 days.
We obtained a total of 25 milk samples ranging from day 5 to day 40 of lactation between 16 September and 17 November.The temporal distribution of data falls into categories of early (5-8 days, n = 12), middle (15-21 days, n = 7) and late (26-40 days, n = 6) lactation.
Total solids, ashes, lipid and protein in degu milk did not vary significantly during lactation (Fig. 1).Milk was relatively diluted with an average total solid concentration of 26.9 ± 5.8 % through all the lactation period.Lipids were the major constituent of degu milk, averaging 17.3 ± 5.5 % during lactation.Protein average concentration was 4.4 ± 0.4 % during lactation, while ash averaged 2.7 ± 0.8 %.In contrast with other milk components carbohydrate concentration decreased significantly from 3.1 ± 0.3 % to 1.0 ± 0.3 % from early to late lactation.
Total energy content did not change over time (Fig. 2), with an average value of 4.0 ± 0.8 kJ ml -1 .Throughout lactation lipids were the major source of milk energy, supplying 70.0 % of total milk energy content, with protein providing 20.0 %, while carbohydrates ranged between 13.2 % (early lactation) and 5.1 % (late lactation) of total energy.

DISCUSSION
In Octodon degus both energy concentration and nutrient composition of milk were practically constant during lactation.This pattern differs from the normal variation in milk concentration found in altricial rodents and mammals in general, where total solids increase with time principally due to increase of lipids (Oftedal 1984, Veloso et al. 2003).Changes in milk concentration, and particularly in lipid concentration, have been associated with increasing energy and nutrient demands of pups during development (Baverstock et al. 1976).The relatively high initial lipid concentration of degu milk in early lactation may be related with its precocial condition and the high energy demand during the first days of life, when pups consume only milk.
Precocial species produce diluted milk, because their pups begin to eat solid food early in ontogeny, and may thus supplement their energy requirements (Martin 1984).Nevertheless Derrickson et al. (1996) found different milk concentration in two precocial rodents: diluted in the caviomorph Kerodon rupestris and concentrated in the murid Acomys cahirinus.Octodon degus milk concentration ranged between both species.Among caviomorphs, Oftedal (1984) show in two Fig. 2: Total milk energy concentration during lactation of Octodon degus.The line is described by the regression: y = 3.845 + 0.009x (r 2 = -0.03,P = 0.5), where y is energy concentration of whole milk in kJ mL -1 .
Milk energy concentration (kJ mL species (Cavia porcellus and Chinchilla lanigera) similar concentrations to those found in K. rupestris.However, O. degus milk was more concentrated than in the other three caviomorph species.An important point is that C. porcellus (Künkelle & Trillmich 1996), K. rupestris and C. lanigera pups can eat and digest solid food during early lacation (at 3 days of life), however O. degus pups start to digest solid food not until the 15 th day of lactation.In this context, the highest concentration observed in O. degus milk should be associated with a major dependence of the maternal nutrition in relation with the other three caviomorph species shown in Table 2. Within the lineage of caviomorph rodents, the milk of O. degus has the highest lipid and total solid concentration of four species shown in Table 2, and the lowest proportion of protein and carbohydrates.Octodon degus is a precocial species, but pups cannot control body temperature before twenty days and do not digest solid food until the fifteen day of lactation (Veloso 1997).Apparently degus require a high energy and nutrient supply from milk during early lactation, increasing gradually the importance of solid food until late lactation.This conclusion is enforced by changes in disaccharidase activity (decrease of lactose and increase of sucrase activity) during lactation time (Sabat & Veloso 2003), and with the significant reduction in milk carbohydrate concentration observed during lactation, both starting at day 15 approximately.
In short, the pattern of milk concentration observed in O. degus is associated with the precocial life of this rodent.