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Daily milk intake and body water turnover in suckling mink (Mustela vison) kits1
Comparative Biochemistry and Physiology Part A 119 (1998) 931 – 939
Daily milk intake and body water turnover in suckling mink (Mustela 6ison) kits1 Søren Wamberg a,*, Anne-Helene Tauson b b
a Department of Physiology, Institute of Medical Biology, Odense Uni6ersity, Winsløwparken 19, DK-5000 Odense C., Denmark Di6ision of Nutrition and Production, Department of Animal Science and Animal Health, The Royal Veterinary and Agricultural Uni6ersity, DK-1870 Frederiksberg C., Denmark
Received 19 August 1997; accepted 3 December 1997
Abstract Daily (24 h) milk intake and body water turnover were measured in eight litters of suckling mink (Mustela 6ison) kits (6–9 kits litter − 1) during weeks 1–4 post partum using the tritiated water (3HHO) dilution technique. The biological half-life of body water turnover in the mink kits increased linearly from 0.9 days in week 1 (3 – 5 days post partum) to 1.9 days in week 4 (22 –24 days post partum). The daily milk intake varied markedly among the mink kits within a litter and increased significantly with increasing body mass from (mean9 SEM) 10.990.4 g per kit during week 1 to 27.79 1.0 g per kit during week 4. Throughout the study, male kits were 10% heavier and had a significantly higher milk intake than female kits. The results were corrected for water recycling between the dam and her kits, ranging from 4 to 15% of the daily milk water intake, and the calculated daily milk yield of the 2 year old lactating mink dams increased from 87 9 7 g day − 1 in week 1 to 190 915 g day − 1 in week 4 post partum. The average body growth rate of the mink kits ranged from 2.9 g kit − 1 per day in week 1 to 5.4 g kit − 1 per day in week 4, and the calculated mean intake of mink milk per unit of body weight gain was remarkably stable at 4.0 (g g − 1) during weeks 1–3 post partum, but increased to 5.6 (g g − 1) in week 4 post partum. The amount of metabolizable energy supplied to the kits by the daily milk yield of the dam increased from :450 to :990 kJ day − 1, which corresponded well with the calculated daily energy requirements of the kits. The tritiated water dilution technique was found feasible and reliable for repeated measurements of milk intake in suckling mink kits up to 4 weeks of age. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Body composition; Carnivores; Milk intake; Milk yield; Mink kits; Mustela 6ison; Tritiated water; Water turnover
1. Introduction The mink is a strict carnivore and a seasonal breeder with one annual breeding season. In the Northern hemisphere, the kits are born over a short period of time from late April until mid May. Mink kits are born blind, almost hairless and devoid of thermoregulatory capacity and, since they virtually * Corresponding author. Fax: + 45 66 133479; e-mail: [email protected] 1 Part of this study was presented at the Waltham International Symposium on Pet Nutrition and Health in the 21st Century, Orlanda, Florida, May 26 –29, 1997. 1095-6433/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(98)00007-5
lack mobilisable energy reserves, it is of the utmost importance for their survival that lactation is established immediately after birth [35]. Mink kits are capable of very rapid growth with a maximum relative growth rate of 23% per 24 h, recorded from the first to the second day of life, and an average relative growth rate of 12% during the first 3 weeks of life [35]. During this period, their body weight increases from 8–10 g to 120–150 g and normally, the body weight of the male kits exceeds that of the females by up to 10% [14]. The kits are completely dependent on mother’s milk for nourishment during the first 24–25 days of life; from then until weaning at : 6 weeks, they consume solid feed in addition to milk.
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The lactation period places very high energetic demands on the dams, and usually they are unable to cover their total energy expenditure by feed consumption, resulting in a situation of negative energy balance [36] and weight loss [14,34], due to mobilisation of body fat [36]. In some cases, this leads to nursing sickness, a condition characterised by heavy weight loss, emaciation, severe metabolic disorders and a high mortality rate [7,42]. Despite the vital importance of a high milk yield for kit survival and growth performance, and the challenge of food energy requirements for milk production and sustenance of female health [7,42], direct measurements of milk production in mink have, to our knowledge, been reported in only a single study with few animals at peak lactation [25]. The main objective of the present study was to use the isotopic water dilution technique as described by Coward et al. [9] for measurements of milk consumption by suckling mink kits during the first 4 weeks of life, during which mother’s milk is thought to be the only source of water intake, and to evaluate the applicability of the method to studies in mink. Secondly, data on body growth, and the size and turnover rate of the total body water pool of each individual mink kit, were recorded during the first 4 weeks of life and used to calculate the energy requirement of the kits for comparison with the amount of metabolizable energy (ME) supplied by their daily milk intake (MI).
2. Materials and methods
respectively, but in two cases (dams no. 418 and 250) one kit was lost in the interval between the first study period and the re-study. The kits were not replaced, resulting in average litter sizes of 7.5 and 6.8 kits in the third and fourth experimental periods, respectively (Table 1). All experimental procedures followed the guidelines approved by the member States of the Council of Europe for the use of live animals for scientific research [1]. Energy metabolism parameters and water turnover in the lactating dams and their progeny were studied in consecutive 1 week balance experiments, each including a 22 h respiration experiment by means of indirect calorimetry in an open-air circulation system. The routines applied in the balance experiments have been described in detail by Wamberg et al. [43] and Tauson et al. [38]. The methods used in the respiration experiments have been given in detail by Tauson et al. [37]. Results from this part of the study will be reported elsewhere.
3. Experimental techniques The isotopic water dilution technique was used to calculate the daily milk intake of the mink kits. Following an intraperitoneal injection of tritiated water (3HHO), the decline in specific activity of plasma water
Table 1 Mink litter dataa Lactation week
2.1. Animals Eight lactating mink dams of the wild colour type, each 2 years old and raising litters of 6 – 9 kits (Fig. 2) were used in this study. The animals were apparently healthy when transferred to the laboratory : 1 week prior to delivery. They were confined to individual metabolism cages equipped with plywood nest-boxes (21 cm long, 19 cm wide and 24 cm high) in a controlled environment (room temperature: 18 – 20°C; relative humidity: 40–60% and a natural spring daylight cycle; 55°N, 12°E). Each morning the females were given a weighed portion of a conventional wet mink diet (DM: 303 g kg − 1; crude protein (N×6.25): 167 g kg − 1; with a calculated metabolizable energy content of 5.60 MJ kg − 1). They had free access to tap water from a closed water bottle system throughout the study. All the kits were born between 5 – 8 May 1996. The day after delivery was designated day 1 of lactation. The kits were reared by the dam throughout the 6 week nursing period. Litter size averaged 7.8 and 7.0 kits for the females studied in week 1 and week 2 post partum,
Week 1
Week 2
No. of littersb 4 4 No. of kits per 7.8 90.53d 7.0 90.4 litterc Dam’s feed in95 9 6 169 914 take, (g d−1) Dam weight 1081 9 7 1133 9 26 (g) Litter weight 141 911 250 934 (g) Litter weight 21.7 9 2.1 31.4 9 4.1 gain (g d−1) Kit weight gain 2.8 90.3 4.4 9 0.3 (g d−1)
Week 3
Week 4
4 7.5 9 0.6
4 6.89 0.5
219 9 11
270 9 13
1064 9 11
1093 9 32
584 926
762 9 89
45.1 92.1
36.0 9 3.4
6.1 9 0.4
5.4 9 0.6
Number of mink (Mustela 6ison) litters studied, average number of kits per litter, dam’s feed intake and body weight, litter weight, and weight gain of litters and individual mink kits during lactation weeks 1 – 4. b The four litters studied in week 1 and week 2 were re-studied in weeks 3 and 4, respectively. For details, see Section 2, P: 000. c For details on number of kits per litter, see Fig. 2(upper panel) and Section 2, P: 000 – 000. d Values are mean 9SEM. a
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was multiplied by the total body water pool, as described by Coward et al. [9]. In order to minimize the influence of experimental procedures on mother – young interaction [15], the study was conducted as follows: Four dams (dams no. 239, 250, 449 and 453) were studied with their litters for a 2 day period in week 1 (days 3–5 post partum) and re-studied 2 weeks later (days 16–18 post partum) and the remaining four dams (dams no. 178, 418, 435 and 486) were studied in week 2 (days 8–10 post partum) and re-studied in week 4 (days 22–24 post partum) as shown in Fig. 2 (upper panel). For each experimental period, the changes in body weight of all dams and their kits were recorded (Table 1). In the morning of the first day of each experimental period, all kits in the litter were weighed, ranked by weight, and marked with blue ink for identification. The kits were then given an intraperitoneal injection of an accurately weighed amount (0.2 – 0.5 ml) of sterile isotonic saline (0.154 M), containing 370 kBq of 3HHO per ml, except for two kits per litter who served as controls for the calculation of water recirculating between the dam and her kits due to the uptake of urinary and faecal water by the dam [4].
3.1. Blood sampling At the end of the 2 h equilibration and the 48 h experimental period, respectively, the mink kits were placed on a thermostat-controlled heating pad for 10 min before blood sampling by the following procedure: The unanaesthetized animal was fixed with one hand and the tail tip was cleaned with antiseptic tissue. The skin of the outer tail tip was cut off using a pair of sharp surgical scissors and : 60 ml of venous blood was carefully collected in a heparinized micro-haematocrit capillary tube by repeated gently sweepings of the tail. Haemostasis was ensured by compressing the tail tip with gauze containing chloramine dusting powder (20 mg g − 1) before the kit was returned to the nest box. After centrifugation (12000 RPM for 15 min) the haematocrit value was recorded and the capillary tube cut to separate the plasma, which was transferred directly to a calibrated 25 ml micropipette (Vitrex®, Hounisen, Risskov, Denmark) and prepared for radioactivity counting as described below.
3.2. Body composition At the end of the study (24 days post partum) two kits from each litter were randomly selected for blood sampling by heart puncture using a 5 ml heparinized syringe under light ketamine (Ketaminol® Vet, 50 mg ml − 1; 40 mg kg − 1 Lwt.) and xylazine (Narcoxyl® Vet, 20 mg ml − 1; 2 mg kg − 1 Lwt.) anaesthesia. The kits were then killed by exsanguination, the gastrointestinal
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tract excised and emptied, and the carcass and organs weighed, cut into small pieces (B 0.5×0.5× 0.5 cm) using a poultry shears, and homogenised in a Waring® household blender (Waring, Winsted, CT).
3.3. Analytical procedures The total body water of the mink kits was determined by dessication to constant weight at 100°C, whereas plasma water was measured by dessication at 80°C of 1000 ml samples of mink plasma, obtained from eight adult female mink and 16 kits at the end of the study. Carcass fat and protein were determined by petroleum ether (b.p. 40–60°) extraction using a Soxhlet apparatus [41] and the micro-Kjeldahl technique [38], respectively. All samples were assayed in duplicate and the chemicals used were analytical grade, purchased from E. Merck, Darmstadt, FRG. Tritiated water (185 MBq ml − 1; code TRS3) was obtained from Amersham International, Buckinghamshire, UK, and the pharmaceuticals, Ketaminol® Vet. and Narcoxyl® Vet., were obtained from Veterinaria AG, Zu¨rich, Switzerland.
3.4. Measurements of tritiated water The plasma samples were stored for 24 h at 4°C before measuring the radioactivity by liquid scintillation counting, using disposable minivials containing 3 ml of scintillation fluid (Ecoscint-A, National Diagnostics, Manville, NJ) and the Packard Liquid Scintillation Analyzer, model Tri-Carb 2100TR (Packard Instrument, Meriden, CT). Standards were prepared by dilution (1:100) with distilled water of the 3HHO-solution injected and corrections were made for the DM content of plasma, 72.09 1.0 g l − 1 in adult mink and 59.592.1 g l − 1 in mink kits. Corrections for chemical quenching were made by the external standard channels ratio method [19]. The efficiency for tritium counting was in the range of 51–57%.
4. Calculations
4.1. Total body water For each experimental period, it was assumed that the body water pool of the injected kits remained a constant fraction of their body mass during the 48 h observation period. Hence, the change in total body water was calculated from the change in body weight multiplied by a factor relevant to the age of the kits, which was taken from a linear relation between 0.82 (g water g − 1 body weight), the value found in 3 –5 day old mink kits [35], and 0.74 (g g − 1), the value obtained
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in 4 week old mink kits at the end of this study. The biological half-life (T1/2) of total body water turnover in the mink kits was calculated from a semi-logarithmic plot of the corrected plasma activities against time for each experimental 2 day period using the formula, T1/2 =ln 2/k, where k (day − 1) is the rate constant for disappearance of tritium from the body water pool.
4.2. Milk intake For each kit, the daily (24 h) milk water intake (MWI) was calculated from the rate of disappearance of tritiated water injected in the kit by the equation given by Coward et al. [9]: MWI =(Q1 − Q2)× log (C1/C2)/log (Q1/Q2) (ml day − 1)
(1)
where Q1 and Q2 are the volumes of total body water and C1 and C2 are the specific activities of tritium in the body water pool at two different points in time (t1 and t2) after injection of 3HHO into the young. In small mammals, hydrogen isotopes equilibrate in B2 h [8,29]. Thus, for each litter in each experimental period, the tritium-radioactivities of samples of plasma water of the injected kits, obtained at 2 and 48 h after equilibration, were used to calculate the individual rates of body water turnover. In each case, the fraction of isotopic water being recycled between the dam and the kits [4] was accounted for by subtraction of the mean plasma water radioactivity of the two non-injected kits from the corresponding (individual) values of the 3 HHO-injected kits. In a previous study [28], the chemical composition of mink milk was found to vary little during the first 3–4 weeks of lactation, and based on these data, the average chemical composition of mink milk during postnatal weeks 1–4 of the present study was assumed to be: water 78%, protein 7.5%, fat 8.5% and carbohydrate 5%. Furthermore, taking the amount of water produced by complete oxidation of 1 MJ of protein, fat and carbohydrate to be 23.9, 28.7 and 33.4 ml, respectively [17] and assuming a DM metabolisability of 85%, the intake of 100 g of mink milk would provide a total amount of 91 (=78 +13) ml of water. Therefore, for each individual mink kit, the total (preformed+ metabolic) water intake, measured over the 2 day interval, was multiplied by 1/0.91 and by 24/46 in order to calculate the actual MI in g day − 1.
4.3. Total milk and milk energy yields For each period, the daily milk yield produced by each dam was calculated as the sum of the daily milk intake of all tritium-loaded kits in the litter plus the milk intake of the non-injected control kits, calculated
as the average daily milk intake (g kg − 1 body wt.) of the injected kits multiplied by the mean body weight of the non-injected kits during the 2 day observation period (Fig. 2). Using the average data for mink milk composition [28] given above to calculate the gross energy (GE) content in milk, and assuming 85% metabolisability of GE, the amount of metabolizable energy supplied by the intake of 1 g of mink milk would be : 5.2 kJ g − 1. Based on this value, the total daily milk energy output was calculated for each dam and compared to the estimated value for total daily energy requirements of the corresponding litter of suckling kits. It was assumed that the DM content of the body followed the linear relationship mentioned above (Section 4.1), and that all DM retention occurred as protein and fat. Thus, using a value of 53 kJ for each g of protein or fat deposited [30], the energy deposition associated with body growth in the kits corresponded to a calculated efficiency of utilization of ME for deposition of protein (kp) of 0.45 and for fat deposition (kf) of 0.75, efficiencies which are in good agreement with experimental findings in other monogastric species [5]. Estimates of the ME requirement for maintenance (MEm) of newborn mink kits are lacking and therefore, the value of 525 kJ/kg0.75, which is in close agreement with findings in adult male [6] and female [40] mink in the thermoneutral zone, was tentatively taken to represent the MEm requirement of the mink kits during the 4 week experimental period (Fig. 4).
4.4. Statistics All results and literature data are given as means with their standard errors, unless otherwise stated. The equation of the linear regression line (Fig. 1) was calculated by the least squares method [2]. Data on body weight, daily weight gain, milk intake and milk intake per g daily weight gain were evaluated with two way variance analysis according to the GLM procedure in SAS [32]. The fixed effects of kit age (week 1, 2, 3 or 4) and sex (male or female) and the interaction between week and sex were tested for the dependent variables.
5. Results
5.1. Animal performance and kit weight gain The rates of feed intake by the dams and the body weights of the nursing dams and their litters (Table 1) demonstrated that the animals were apparently normal and thrived well throughout the 4 week experimental period. Kit losses were low since only two out of a total of 60 kits were lost during the experiment.
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Fig. 1. Half-life (days) of tritiated water in suckling mink kits measured over a 2 day period plotted as a function of body mass (g). The following equation for the linear regression line was calculated by the least squares method: y= 0.0131× +0.739 (r 2 = 0.828; df = 79). For details, see Sections 2 and 4 P: 000–000.
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sumption. Furthermore, the mean daily MI increased remarkably from 10.9 9 0.4 g per kit per day in week 1 (3–5 days post partum) to 27.79 1.0 g per kit per day in week 4 (22–24 days post partum). MI reflected differences in body weight between sexes by being significantly greater in male than in female kits (PB 0.001). In Fig. 2 (panel A), the individual data for MI were summed for all kits to give the total daily milk yield for each dam. Hence, the calculated milk yield of the lactating dams increased from 879 7 g day − 1 in week 1 to 1909 15 g day − 1 in week 4, indicating a total milk production of 4200 g by an 1100 g mink dam during the first 4 weeks of lactation. In all litters studied, water recirculation accounted for, on average, 9.0% (range: 4.3–14.9%) of the daily MWI by the kits, although the data from individual
The body mass of the lactating dams decreased from a starting level of 1081 and 1133 g (periods 1 and 2, respectively) to 1064 and 1093 g (periods 3 and 4), but this change did not reach statistical significance. Kit body weights increased from an average of 18.49 0.6 g in the first experimental period to 1159 2.8 g in the last period, when the kits were 22 – 24 days old. Throughout the experiment, sexual dimorphism was considerable, male kits being the heavier (P B0.001). The average rate of weight gain of the kits was 2.8 g day − 1 in week 1 and reached 6.1 and 5.4 g day − 1 in weeks 3 and 4, respectively (Table 1), but the rate of weight gain was not different between sexes (P = 0.33).
5.2. Kit body water turno6er The mean biological half-life of total body water turnover in the mink kits increased from 0.8 days during week 1 to 1.9 days during week 4 post partum. Individual data for all the injected kits are presented in Fig. 1 along with the calculated regression line, given by the equation: 3
HHO half-life=0.0131 × Bwt. +0.739 (r 2 =0.828; df= 79)
(2)
where Bwt. is body weight in g.
5.3. Milk intake Individual values of daily milk intake by the injected mink kits, plotted for each litter during weeks 1 – 4 post partum in Fig. 2. (panel B), show a considerable, between kit within litter, variation in daily milk con-
Fig. 2. Total daily milk production (g day − 1) of mink (Mustela 6ison) dams through weeks 1 – 4 post partum (Panel A), calculated as the sum of individual daily milk intake (g day − 1) measured in the tritium loaded kits (Panel B) plus the estimated milk intake by the two unloaded (control) kits of each litter. Dams no. 239, 250, 449 and 453 (with their total number of kits given in parentheses) were studied for 2 days in weeks 1 (days 3 – 5) and 3 (days 16-18), and dams no. 178, 418, 435 and 486 were studied for 2 days in weeks 2 (days 8–10) and 4 (days 22 – 24) post partum. Dams no. 250 and 418 lost one kit each between the two study periods. For details, see Section 2 P: 000–000.
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ported by Tauson [35], but still within the range of normal kit performance. The half-life of body water turnover increased linearly with increasing body mass of the kits (Fig. 1), and the clustered values obtained at each level of body weight indicated similar rates of milk water intake by kits of equal size. At peak lactation, the biological half-life of 3HHO in the mink kits was comparable to that observed by Coward et al. [9] in suckling rat pups. Furthermore, in the mink, water recycling during lactation appeared to be of minor importance, as in lactating rats [4] and dogs [26], whereas in mice [3] recycling accounts for \ 50% of the daily MWI.
6.2. Milk intake of the young and milk yield of the dam Fig. 3. Ratio of daily milk intake (MI, g day − 1) to body growth (BG, g day − 1) of the mink kits studied during postnatal weeks 1– 4.
dams indicated that the amount of milk water recirculated was somewhat lower in the second measurement period. The calculated values for daily MI per unit of weight gain for each kit (Fig. 3) remained rather stable with a mean value of 4.0 (g g − 1) during the first 3 weeks post partum whereas, in week 4, the data were much more scattered around a somewhat higher mean value of 5.6 (g g − 1), the effect of sex being significant (P = 0.006), but not consistent since there were significant interaction effects between week and sex (P =0.002).
Daily milk intake varied considerably between individual kits within litters as well as between litters. This variation could be explained by differences in kit live weight, which to a great extent could be attributed to sex, but factors such as genetic predisposition, feed intake of the dam, litter size and nursing stimulus [14,18,21,22] might also have influenced milk intake. As expected, the daily milk intake was higher in male kits than in female kits and increased markedly during the 4 week experiment. For any measurement period, the largest milk yield was found in dams with the heaviest litters, and male kits having significantly higher MI than female kits. The tendency towards a stabilization of daily milk intake by the suckling young and of the
5.4. ME supply by milk and ME requirements of kits Based on the assumptions stated above, the ME supplied to the kits by the milk increased from : 450 kJ litter − 1 per day in the first week of lactation to 990 kJ litter − 1 per day in week 4. Simultaneously, the energy requirements of the kits increased from 300 kJ litter − 1 per day in week 1 to 920 kJ litter − 1 per day in week 4 (Fig. 4). Milk production increased at a lower rate from week 3 to week 4 than earlier in lactation (Fig. 4), and the relative importance of MEm for the total ME requirement of the kits increased as lactation progressed, accounting for 3791.5% in week 1 and 4692.2% in week 4.
6. Discussion
6.1. Body weight changes and body water turno6er The rates of feed intake and weight change of the dams observed in the present study (Table 1) were comparable to those seen under normal farm conditions [33,34], and kit mortality rate was low. The recorded kit weight gain was somewhat below the growth rate re-
Fig. 4. Total daily output of metabolizable energy (ME)(kJ dam − 1) in mink milk () compared to the estimated (total) daily energy requirements of the kits during the first 4 weeks of lactation. Hatched columns represent the mean ME requirement for body growth (kJ day − 1) and open columns represent the mean ME requirement for maintenance (kJ day − 1). Values are means with standard errors represented by vertical bars. For details, see text, P: 000 – 000.
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total milk yield of the dam in week 4 of lactation was reflected in a lower average daily weight gain by the kits, in agreement with the decrease in relative growth rate previously recorded after week 3 of lactation [35]. Furthermore, the average ratio of milk intake to body growth rate observed during weeks 1 – 3 (4.0 g g − 1) (Fig. 3) was in agreement with the values obtained in suckling mice (3.5 g g − 1) [18], puppies (Section 4.4) [26] and in hibernating bear cubs (3.7 g g − 1) [12]. The increased milk intake to body growth ratio observed in week 4, may be explained, at least in part, by the increasing requirements for MEm of the rapidly growing mink kits (Fig. 4). The mean daily MI increased from 11 – 28 g kit − 1 per day, corresponding to an average total milk yield of 4200 g for the dams in the 4 week experiment, which is in agreement with the estimated total milk production for dams raising 5 – 8 kits [14]. Information on the milk yield of the lactating mink is sparse and the results presented here represent one of only two attempts to measure milk intake in suckling mink kits by a direct method. In previous studies, milk production in mink has been estimated by factorial methods, but the experimental data needed for correct calculations is still too limited to permit precise estimates. For instance, there is no experimental information on MEm for mink kits in the suckling period, values for kp and kf are lacking, and there is little information about the chemical composition of mink milk at various stages of lactation [28]. Therefore, based on the assumption of a fixed relationship between the rates of daily milk intake and body growth of suckling mink kits, estimated values for MI at peak lactation range from far below, i.e. 4 –14 g day − 1 per kit [13,20] to within fair agreement with our present data, i.e. 24 – 30 g day − 1 per kit [33,39] and 30 – 35 g day − 1 per kit [7]. The lower values can to some extent be attributed to differences in litter size [22], to a lower weight gain by the kits studied, and to the increasing demand for maintenance energy of the rapidly growing young as demonstrated by Knight et al. [18] and by the data in Fig. 4 of the present study.
6.3. Milk energy output and estimated energy requirement of the suckling young The calculated total daily ME output in the milk was sufficient to cover the ME requirements of the suckling kits. During the first two periods of measurement, the calculated ME output in milk far exceeded the kits’ requirement (Fig. 4), but during the two last measurement periods ME in milk was only 5 – 7% above the estimated energy requirements of the kits. It may be that the measurements on the very small kits were somewhat less accurate than those on larger kits. Alternatively, our estimates of MEm of newborn kits may
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have been too low. Data on rats indicate that MEm per unit of metabolic weight decreases with increasing body weight (A. Chwalibog, personal communication). On the other hand, the kits in the present study had very low locomotor activity, and were kept warm by the dam in the nest-box, so a MEm requirement higher than that of adult animals [6,40] seems unlikely.
6.4. Validity of the water isotope technique for studies on mink In the past two decades, the isotope dilution technique has been applied to rats and mice [3,9,18,31], dogs [26], lambs [9,11], calves [16], and pigs [10]. Detailed discussions of the theoretical and experimental implications of utilizing tracer disappearance rates to measure milk production have appeared in review format [9,11,24,26]. Lately, the stable isotope deuterium oxide has been used successfully in studies of wild animals such as bears [12] and the harp seal [27]. It appears that experimental studies based on the isotope dilution technique lead to more reliable and reproducible estimates of milk production than conventional methods based on ‘test-weighing’ of the dams and/or the offspring [15,22,23]. This is partly due to lack of data on the efficiency of energy utilization for growth, and to the fact that the conventional methods require separation of the young from their mother for several hours, which reduces the suckling stimulus to milk production. In the present study, the tritiated water dilution technique was found feasible for repeated short-term measurements of body water turnover and daily MI in suckling mink kits up to 4 weeks of age with a minimum of interference with the mother–young interaction. The daily milk yield of the mink dams increased markedly as lactation progressed, reaching a maximum of 30 g kit − 1 per day, or more than 200 g dam − 1 per day. The total daily milk energy output by the dam was in excess of 1 MJ day − 1, corresponding well with estimated values for daily requirements of energy for maintenance and body growth of the kits. It should be emphasized that using the single-isotope tracer technique for measurements of daily MI it is assumed, that the dams’ milk is the only source of water intake [9,11]. Since it cannot be excluded that some kits may have started to eat solid food during the final measuring period of the present study, the calculated daily milk intake in week 4 may have been slightly overestimated. Data obtained in previous studies of MI in hand-reared calves [16] and suckling bear cubs [12] indicate that the isotope dilution technique may be improved by simultaneous administration of two different water isotopes, i.e. deuterium oxide to the nursing dam and tritiated water to the suckling young, thereby distinguishing between daily intake of milk water and
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other sources of non-milk water taken up by the suckling young.
Acknowledgements The financial support of The Danish Agricultural and Veterinary Research Council (Grants No. 9400135 and 9502267) and the Danish Fur Breeder’s Association is gratefully acknowledged. The authors thank Inge Andersen, Lise Larsen, Boye Pedersen and Merethe Stubgaard for skilled technical assistance.
References [1] Anonymous. European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. European Treaty Series No. 123, Strasbourg, Council of Europe, 1986. [2] Armitage P, Berry G. Statistical Methods in Medical Research, 3rd. Oxford: Blackwell, 1994:154–63. [3] Baverstock PR, Elhay S. Water balance of small lactating rodents. III. Estimates of milk production and water recycling in lactating Mus musculus under various water regimes. J Math Biol 1981;13:1 – 22. [4] Baverstock PR, Green B. Water recycling in lactation. Science 1975;187:657 – 8. [5] Chwalibog A. Energetics of animal production. Research in Copenhagen, review and suggestions. Acta Agric Scand 1991;41:147 – 60. [6] Chwalibog A, Glem-Hansen N, Henckel S, Thorbek G. Energy metabolism in adult mink in relation to protein-energy levels and environmental temperature. Proceedings VIIIth Symposium on Energy Metabolism in Farm Animals EAAP Publication No. 26, 1980:283 – 286 [7] Clausen TN, Olesen CR, Hansen O, Wamberg S. Nursing sickness in lactating mink (Mustela 6ison) I. Epidemiological and pathological observations. Can J Vet Res 1992;56:89–94. [8] Coleman TG, Manning RD Jr, Norman RA Jr, Guyton AC. Dynamics of water-isotope distribution. Am J Physiol 1972;223:1371– 5. [9] Coward WA, Cole TJ, Gerber H, Roberts SB, Fleet I. Water turnover and the measurement of milk intake. Pflu¨g Arch 1982;393:344 – 7. [10] Cromwell GL, Stahly TS, Edgerton LA, Monegue HJ, Burnell TW, Schenk BC, Schricker BR. Recombinant porcine somatotropin for sows during late gestation and throughout lactation. J Anim Sci 1992;70:1404–16. [11] Dove H, Freer M. The accuracy of tritiated water turnover rate as an estimate of milk intake in lambs. Aust J Agric Res 1979;30:725 – 39. [12] Farley SD, Robbins CT. Lactation, hibernation, and mass dynamics of American black bears and grizzly bears. Can J Zool 1995;73:2216 – 22. [13] Glem-Hansen N. Nutrition of mink. In: Jørgensen G, editor. Mink Production. Hillerød Scientifur, 1985:189–248. [14] Hansen BK. The lactating mink (Mustela 6ison). Genetic and metabolic aspects. Ph.D. Thesis. Copenhagen. Department of Animal Science and Animal Health, The Royal Veterinary and Agricultural University, 1997:1–27. [15] Hanwell A, Linzell JL. A simple technique for measuring the rate of milk secretion in the rat. Comp Biochem Physiol 1972;43A:259 – 70.
[16] Holleman DF, White RG, Luick JR. New isotope methods for estimating milk intake and yield. J Dairy Sci 1975;58:1814–21. [17] Houpt TR. Water and electrolytes. In: Swenson MJ, Reece WO, editors. Dukes’ Physiology of Domestic Animals, 11th. Ithaca, NY: Cornell University Press, 1993:9 – 21. [18] Knight CH, Maltz E, Docherty AH. Milk yield and composition in mice: Effects of litter size and lactation number. Comp Biochem Physiol 1986;84A:127 – 33. [19] Knoche HW. Radioisotopic Methods for Biological and Medical Research. Oxford: Oxford University Press, 1991:187 –210. [20] Kostina VS, Bogacheva ES. Milk production of mink females. (In Russian). Krolikovodstvo i Zverods 1976;3(14). Cited from Dairy Science Abstracts 1977;39:480. [21] Kumaresan P, Anderson RR, Turner CW. Effect of litter size upon milk yield and litter weight gains in rats. Proc Soc Exp Biol Med 1967;126:41 – 5. [22] Moon RC. Mammary growth and milk yield as related to litter size. Proc Soc Exp Biol Med 1969;130:1126– 8. [23] Morag M. Estimation of milk yield in the rat. Lab Anim 1970;4:259 – 72. [24] Nagy KA, Costa DP. Water flux in animals: Analysis of potential errors in the tritiated water method. Am J Physiol 1980;238:454 – 65. [25] Oftedal OT. Milk, protein and energy intakes of suckling mammalian young: A comparative study. Diss Abstr Int 1981;42:1246-B. [26] Oftedal OT. Lactation in the dog: Milk composition and intake by puppies. J Nutr 1984;114:803 – 12. [27] Oftedal OT, Bowen WD, Boness DJ. Lactation performance and nutrient deposition in pups of the harp seal, Phoca groenlandica, on ice floes off Southeast Labrador. Physiol Zool 1996;69:635– 57. [28] Olesen CR, Clausen TN, Wamberg S. Compositional changes in mink (Mustela 6ison) milk during lactation. Norw J Agric Sci Suppl 1992;9:308 – 14. [29] Pinson EA. Water exchanges and barriers as studied by the use of hydrogen isotopes. Physiol Rev 1952;32:123 – 34. [30] Pullar JD, Webster AJF. The energy cost of fat and protein deposition in the rat. Br J Nutr 1977;37:355 – 63. [31] Rath EA, Thenen SW. Use of tritiated water for measurement of 24 h milk intake in suckling lean and genetically obese (ob/ob) mice. J Nutr 1979;109:840 – 7. [32] Statistical Analysis System. SAS User’s Guide: Statistics, 1985 Edition, SAS Institute, 1985. Cary, NC. [33] Sørensen HJ. Water turnover in pregnant and lactating female mink. M.Sc. Thesis. Copenhagen, Department of Animal Science and Animal Health, The Royal Veterinary and Agricultural University, 1997:1 – 32. [34] Tauson A-H. Varied energy concentration in mink diets. II. Effects on kit growth performance, female weight changes and water turnover in the lactation period. Acta Agric Scand 1988;38:231 – 42. [35] Tauson A-H. Postnatal development in mink kits. Acta Agric Scand Sect A Anim Sci 1994;44:177 – 84. [36] Tauson A-H. Prolactin profiles of pregnant, lactating and nonmated female mink (Mustela 6ison). J Reprod Fertil Suppl 1997;51:195 – 201. [37] Tauson A-H, Elnif J, Hansen NE. Energy metabolism and nutrient oxidation in the pregnant mink (Mustela 6ison) as a model for other carnivores. J Nutr 1994;124:2609S– 13S. [38] Tauson A-H, Elnif J, Wamberg S. Nitrogen balance in adult female mink (Mustela 6ison) in response to normal feeding and short-term fasting. Br J Nutr 1997;78:83 – 96. [39] Tauson A-H, Sørensen HJ, Wamberg S, Chwalibog A. Energy metabolism, nutrient oxidation and water turnover in the lactating mink (Mustela 6ison). J Nutr 1998 (in press).
S. Wamberg, A.-H. Tauson / Comparati6e Biochemistry and Physiology, Part A 119 (1998) 931–939 [40] Wamberg S. Rates of heat and water loss in female mink (Mustela 6ison) measured by direct calorimetry. Comp Biochem Physiol 1994;107A:451– 8. [41] Wamberg S. Measurements of total body water in adult female mink (Mustela 6ison). In: Frindt A, Brzozowski M, editors. Proceedings from the VIth International Scientific Congress in Fur Animal Production, Warsaw, Polish Society of Animal Produc-
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tion, 1996:203 – 9. [42] Wamberg S, Clausen TN, Olesen CR, Hansen O. Nursing sickness in lactating mink (Mustela 6ison) II. Pathophysiology and changes in body fluid composition. Can J Vet Res 1992;56:95 –101. [43] Wamberg S, Tauson A-H, Elnif J. Effects of feeding and shortterm fasting on water and electrolyte turnover in female mink (Mustela 6ison). Br J Nutr 1996;76:711 – 25.
.
Daily milk intake and body water turnover in suckling mink (Mustela 6ison) kits1 Søren Wamberg a,*, Anne-Helene Tauson b b
a Department of Physiology, Institute of Medical Biology, Odense Uni6ersity, Winsløwparken 19, DK-5000 Odense C., Denmark Di6ision of Nutrition and Production, Department of Animal Science and Animal Health, The Royal Veterinary and Agricultural Uni6ersity, DK-1870 Frederiksberg C., Denmark
Received 19 August 1997; accepted 3 December 1997
Abstract Daily (24 h) milk intake and body water turnover were measured in eight litters of suckling mink (Mustela 6ison) kits (6–9 kits litter − 1) during weeks 1–4 post partum using the tritiated water (3HHO) dilution technique. The biological half-life of body water turnover in the mink kits increased linearly from 0.9 days in week 1 (3 – 5 days post partum) to 1.9 days in week 4 (22 –24 days post partum). The daily milk intake varied markedly among the mink kits within a litter and increased significantly with increasing body mass from (mean9 SEM) 10.990.4 g per kit during week 1 to 27.79 1.0 g per kit during week 4. Throughout the study, male kits were 10% heavier and had a significantly higher milk intake than female kits. The results were corrected for water recycling between the dam and her kits, ranging from 4 to 15% of the daily milk water intake, and the calculated daily milk yield of the 2 year old lactating mink dams increased from 87 9 7 g day − 1 in week 1 to 190 915 g day − 1 in week 4 post partum. The average body growth rate of the mink kits ranged from 2.9 g kit − 1 per day in week 1 to 5.4 g kit − 1 per day in week 4, and the calculated mean intake of mink milk per unit of body weight gain was remarkably stable at 4.0 (g g − 1) during weeks 1–3 post partum, but increased to 5.6 (g g − 1) in week 4 post partum. The amount of metabolizable energy supplied to the kits by the daily milk yield of the dam increased from :450 to :990 kJ day − 1, which corresponded well with the calculated daily energy requirements of the kits. The tritiated water dilution technique was found feasible and reliable for repeated measurements of milk intake in suckling mink kits up to 4 weeks of age. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Body composition; Carnivores; Milk intake; Milk yield; Mink kits; Mustela 6ison; Tritiated water; Water turnover
1. Introduction The mink is a strict carnivore and a seasonal breeder with one annual breeding season. In the Northern hemisphere, the kits are born over a short period of time from late April until mid May. Mink kits are born blind, almost hairless and devoid of thermoregulatory capacity and, since they virtually * Corresponding author. Fax: + 45 66 133479; e-mail: [email protected] 1 Part of this study was presented at the Waltham International Symposium on Pet Nutrition and Health in the 21st Century, Orlanda, Florida, May 26 –29, 1997. 1095-6433/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(98)00007-5
lack mobilisable energy reserves, it is of the utmost importance for their survival that lactation is established immediately after birth [35]. Mink kits are capable of very rapid growth with a maximum relative growth rate of 23% per 24 h, recorded from the first to the second day of life, and an average relative growth rate of 12% during the first 3 weeks of life [35]. During this period, their body weight increases from 8–10 g to 120–150 g and normally, the body weight of the male kits exceeds that of the females by up to 10% [14]. The kits are completely dependent on mother’s milk for nourishment during the first 24–25 days of life; from then until weaning at : 6 weeks, they consume solid feed in addition to milk.
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The lactation period places very high energetic demands on the dams, and usually they are unable to cover their total energy expenditure by feed consumption, resulting in a situation of negative energy balance [36] and weight loss [14,34], due to mobilisation of body fat [36]. In some cases, this leads to nursing sickness, a condition characterised by heavy weight loss, emaciation, severe metabolic disorders and a high mortality rate [7,42]. Despite the vital importance of a high milk yield for kit survival and growth performance, and the challenge of food energy requirements for milk production and sustenance of female health [7,42], direct measurements of milk production in mink have, to our knowledge, been reported in only a single study with few animals at peak lactation [25]. The main objective of the present study was to use the isotopic water dilution technique as described by Coward et al. [9] for measurements of milk consumption by suckling mink kits during the first 4 weeks of life, during which mother’s milk is thought to be the only source of water intake, and to evaluate the applicability of the method to studies in mink. Secondly, data on body growth, and the size and turnover rate of the total body water pool of each individual mink kit, were recorded during the first 4 weeks of life and used to calculate the energy requirement of the kits for comparison with the amount of metabolizable energy (ME) supplied by their daily milk intake (MI).
2. Materials and methods
respectively, but in two cases (dams no. 418 and 250) one kit was lost in the interval between the first study period and the re-study. The kits were not replaced, resulting in average litter sizes of 7.5 and 6.8 kits in the third and fourth experimental periods, respectively (Table 1). All experimental procedures followed the guidelines approved by the member States of the Council of Europe for the use of live animals for scientific research [1]. Energy metabolism parameters and water turnover in the lactating dams and their progeny were studied in consecutive 1 week balance experiments, each including a 22 h respiration experiment by means of indirect calorimetry in an open-air circulation system. The routines applied in the balance experiments have been described in detail by Wamberg et al. [43] and Tauson et al. [38]. The methods used in the respiration experiments have been given in detail by Tauson et al. [37]. Results from this part of the study will be reported elsewhere.
3. Experimental techniques The isotopic water dilution technique was used to calculate the daily milk intake of the mink kits. Following an intraperitoneal injection of tritiated water (3HHO), the decline in specific activity of plasma water
Table 1 Mink litter dataa Lactation week
2.1. Animals Eight lactating mink dams of the wild colour type, each 2 years old and raising litters of 6 – 9 kits (Fig. 2) were used in this study. The animals were apparently healthy when transferred to the laboratory : 1 week prior to delivery. They were confined to individual metabolism cages equipped with plywood nest-boxes (21 cm long, 19 cm wide and 24 cm high) in a controlled environment (room temperature: 18 – 20°C; relative humidity: 40–60% and a natural spring daylight cycle; 55°N, 12°E). Each morning the females were given a weighed portion of a conventional wet mink diet (DM: 303 g kg − 1; crude protein (N×6.25): 167 g kg − 1; with a calculated metabolizable energy content of 5.60 MJ kg − 1). They had free access to tap water from a closed water bottle system throughout the study. All the kits were born between 5 – 8 May 1996. The day after delivery was designated day 1 of lactation. The kits were reared by the dam throughout the 6 week nursing period. Litter size averaged 7.8 and 7.0 kits for the females studied in week 1 and week 2 post partum,
Week 1
Week 2
No. of littersb 4 4 No. of kits per 7.8 90.53d 7.0 90.4 litterc Dam’s feed in95 9 6 169 914 take, (g d−1) Dam weight 1081 9 7 1133 9 26 (g) Litter weight 141 911 250 934 (g) Litter weight 21.7 9 2.1 31.4 9 4.1 gain (g d−1) Kit weight gain 2.8 90.3 4.4 9 0.3 (g d−1)
Week 3
Week 4
4 7.5 9 0.6
4 6.89 0.5
219 9 11
270 9 13
1064 9 11
1093 9 32
584 926
762 9 89
45.1 92.1
36.0 9 3.4
6.1 9 0.4
5.4 9 0.6
Number of mink (Mustela 6ison) litters studied, average number of kits per litter, dam’s feed intake and body weight, litter weight, and weight gain of litters and individual mink kits during lactation weeks 1 – 4. b The four litters studied in week 1 and week 2 were re-studied in weeks 3 and 4, respectively. For details, see Section 2, P: 000. c For details on number of kits per litter, see Fig. 2(upper panel) and Section 2, P: 000 – 000. d Values are mean 9SEM. a
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was multiplied by the total body water pool, as described by Coward et al. [9]. In order to minimize the influence of experimental procedures on mother – young interaction [15], the study was conducted as follows: Four dams (dams no. 239, 250, 449 and 453) were studied with their litters for a 2 day period in week 1 (days 3–5 post partum) and re-studied 2 weeks later (days 16–18 post partum) and the remaining four dams (dams no. 178, 418, 435 and 486) were studied in week 2 (days 8–10 post partum) and re-studied in week 4 (days 22–24 post partum) as shown in Fig. 2 (upper panel). For each experimental period, the changes in body weight of all dams and their kits were recorded (Table 1). In the morning of the first day of each experimental period, all kits in the litter were weighed, ranked by weight, and marked with blue ink for identification. The kits were then given an intraperitoneal injection of an accurately weighed amount (0.2 – 0.5 ml) of sterile isotonic saline (0.154 M), containing 370 kBq of 3HHO per ml, except for two kits per litter who served as controls for the calculation of water recirculating between the dam and her kits due to the uptake of urinary and faecal water by the dam [4].
3.1. Blood sampling At the end of the 2 h equilibration and the 48 h experimental period, respectively, the mink kits were placed on a thermostat-controlled heating pad for 10 min before blood sampling by the following procedure: The unanaesthetized animal was fixed with one hand and the tail tip was cleaned with antiseptic tissue. The skin of the outer tail tip was cut off using a pair of sharp surgical scissors and : 60 ml of venous blood was carefully collected in a heparinized micro-haematocrit capillary tube by repeated gently sweepings of the tail. Haemostasis was ensured by compressing the tail tip with gauze containing chloramine dusting powder (20 mg g − 1) before the kit was returned to the nest box. After centrifugation (12000 RPM for 15 min) the haematocrit value was recorded and the capillary tube cut to separate the plasma, which was transferred directly to a calibrated 25 ml micropipette (Vitrex®, Hounisen, Risskov, Denmark) and prepared for radioactivity counting as described below.
3.2. Body composition At the end of the study (24 days post partum) two kits from each litter were randomly selected for blood sampling by heart puncture using a 5 ml heparinized syringe under light ketamine (Ketaminol® Vet, 50 mg ml − 1; 40 mg kg − 1 Lwt.) and xylazine (Narcoxyl® Vet, 20 mg ml − 1; 2 mg kg − 1 Lwt.) anaesthesia. The kits were then killed by exsanguination, the gastrointestinal
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tract excised and emptied, and the carcass and organs weighed, cut into small pieces (B 0.5×0.5× 0.5 cm) using a poultry shears, and homogenised in a Waring® household blender (Waring, Winsted, CT).
3.3. Analytical procedures The total body water of the mink kits was determined by dessication to constant weight at 100°C, whereas plasma water was measured by dessication at 80°C of 1000 ml samples of mink plasma, obtained from eight adult female mink and 16 kits at the end of the study. Carcass fat and protein were determined by petroleum ether (b.p. 40–60°) extraction using a Soxhlet apparatus [41] and the micro-Kjeldahl technique [38], respectively. All samples were assayed in duplicate and the chemicals used were analytical grade, purchased from E. Merck, Darmstadt, FRG. Tritiated water (185 MBq ml − 1; code TRS3) was obtained from Amersham International, Buckinghamshire, UK, and the pharmaceuticals, Ketaminol® Vet. and Narcoxyl® Vet., were obtained from Veterinaria AG, Zu¨rich, Switzerland.
3.4. Measurements of tritiated water The plasma samples were stored for 24 h at 4°C before measuring the radioactivity by liquid scintillation counting, using disposable minivials containing 3 ml of scintillation fluid (Ecoscint-A, National Diagnostics, Manville, NJ) and the Packard Liquid Scintillation Analyzer, model Tri-Carb 2100TR (Packard Instrument, Meriden, CT). Standards were prepared by dilution (1:100) with distilled water of the 3HHO-solution injected and corrections were made for the DM content of plasma, 72.09 1.0 g l − 1 in adult mink and 59.592.1 g l − 1 in mink kits. Corrections for chemical quenching were made by the external standard channels ratio method [19]. The efficiency for tritium counting was in the range of 51–57%.
4. Calculations
4.1. Total body water For each experimental period, it was assumed that the body water pool of the injected kits remained a constant fraction of their body mass during the 48 h observation period. Hence, the change in total body water was calculated from the change in body weight multiplied by a factor relevant to the age of the kits, which was taken from a linear relation between 0.82 (g water g − 1 body weight), the value found in 3 –5 day old mink kits [35], and 0.74 (g g − 1), the value obtained
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in 4 week old mink kits at the end of this study. The biological half-life (T1/2) of total body water turnover in the mink kits was calculated from a semi-logarithmic plot of the corrected plasma activities against time for each experimental 2 day period using the formula, T1/2 =ln 2/k, where k (day − 1) is the rate constant for disappearance of tritium from the body water pool.
4.2. Milk intake For each kit, the daily (24 h) milk water intake (MWI) was calculated from the rate of disappearance of tritiated water injected in the kit by the equation given by Coward et al. [9]: MWI =(Q1 − Q2)× log (C1/C2)/log (Q1/Q2) (ml day − 1)
(1)
where Q1 and Q2 are the volumes of total body water and C1 and C2 are the specific activities of tritium in the body water pool at two different points in time (t1 and t2) after injection of 3HHO into the young. In small mammals, hydrogen isotopes equilibrate in B2 h [8,29]. Thus, for each litter in each experimental period, the tritium-radioactivities of samples of plasma water of the injected kits, obtained at 2 and 48 h after equilibration, were used to calculate the individual rates of body water turnover. In each case, the fraction of isotopic water being recycled between the dam and the kits [4] was accounted for by subtraction of the mean plasma water radioactivity of the two non-injected kits from the corresponding (individual) values of the 3 HHO-injected kits. In a previous study [28], the chemical composition of mink milk was found to vary little during the first 3–4 weeks of lactation, and based on these data, the average chemical composition of mink milk during postnatal weeks 1–4 of the present study was assumed to be: water 78%, protein 7.5%, fat 8.5% and carbohydrate 5%. Furthermore, taking the amount of water produced by complete oxidation of 1 MJ of protein, fat and carbohydrate to be 23.9, 28.7 and 33.4 ml, respectively [17] and assuming a DM metabolisability of 85%, the intake of 100 g of mink milk would provide a total amount of 91 (=78 +13) ml of water. Therefore, for each individual mink kit, the total (preformed+ metabolic) water intake, measured over the 2 day interval, was multiplied by 1/0.91 and by 24/46 in order to calculate the actual MI in g day − 1.
4.3. Total milk and milk energy yields For each period, the daily milk yield produced by each dam was calculated as the sum of the daily milk intake of all tritium-loaded kits in the litter plus the milk intake of the non-injected control kits, calculated
as the average daily milk intake (g kg − 1 body wt.) of the injected kits multiplied by the mean body weight of the non-injected kits during the 2 day observation period (Fig. 2). Using the average data for mink milk composition [28] given above to calculate the gross energy (GE) content in milk, and assuming 85% metabolisability of GE, the amount of metabolizable energy supplied by the intake of 1 g of mink milk would be : 5.2 kJ g − 1. Based on this value, the total daily milk energy output was calculated for each dam and compared to the estimated value for total daily energy requirements of the corresponding litter of suckling kits. It was assumed that the DM content of the body followed the linear relationship mentioned above (Section 4.1), and that all DM retention occurred as protein and fat. Thus, using a value of 53 kJ for each g of protein or fat deposited [30], the energy deposition associated with body growth in the kits corresponded to a calculated efficiency of utilization of ME for deposition of protein (kp) of 0.45 and for fat deposition (kf) of 0.75, efficiencies which are in good agreement with experimental findings in other monogastric species [5]. Estimates of the ME requirement for maintenance (MEm) of newborn mink kits are lacking and therefore, the value of 525 kJ/kg0.75, which is in close agreement with findings in adult male [6] and female [40] mink in the thermoneutral zone, was tentatively taken to represent the MEm requirement of the mink kits during the 4 week experimental period (Fig. 4).
4.4. Statistics All results and literature data are given as means with their standard errors, unless otherwise stated. The equation of the linear regression line (Fig. 1) was calculated by the least squares method [2]. Data on body weight, daily weight gain, milk intake and milk intake per g daily weight gain were evaluated with two way variance analysis according to the GLM procedure in SAS [32]. The fixed effects of kit age (week 1, 2, 3 or 4) and sex (male or female) and the interaction between week and sex were tested for the dependent variables.
5. Results
5.1. Animal performance and kit weight gain The rates of feed intake by the dams and the body weights of the nursing dams and their litters (Table 1) demonstrated that the animals were apparently normal and thrived well throughout the 4 week experimental period. Kit losses were low since only two out of a total of 60 kits were lost during the experiment.
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Fig. 1. Half-life (days) of tritiated water in suckling mink kits measured over a 2 day period plotted as a function of body mass (g). The following equation for the linear regression line was calculated by the least squares method: y= 0.0131× +0.739 (r 2 = 0.828; df = 79). For details, see Sections 2 and 4 P: 000–000.
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sumption. Furthermore, the mean daily MI increased remarkably from 10.9 9 0.4 g per kit per day in week 1 (3–5 days post partum) to 27.79 1.0 g per kit per day in week 4 (22–24 days post partum). MI reflected differences in body weight between sexes by being significantly greater in male than in female kits (PB 0.001). In Fig. 2 (panel A), the individual data for MI were summed for all kits to give the total daily milk yield for each dam. Hence, the calculated milk yield of the lactating dams increased from 879 7 g day − 1 in week 1 to 1909 15 g day − 1 in week 4, indicating a total milk production of 4200 g by an 1100 g mink dam during the first 4 weeks of lactation. In all litters studied, water recirculation accounted for, on average, 9.0% (range: 4.3–14.9%) of the daily MWI by the kits, although the data from individual
The body mass of the lactating dams decreased from a starting level of 1081 and 1133 g (periods 1 and 2, respectively) to 1064 and 1093 g (periods 3 and 4), but this change did not reach statistical significance. Kit body weights increased from an average of 18.49 0.6 g in the first experimental period to 1159 2.8 g in the last period, when the kits were 22 – 24 days old. Throughout the experiment, sexual dimorphism was considerable, male kits being the heavier (P B0.001). The average rate of weight gain of the kits was 2.8 g day − 1 in week 1 and reached 6.1 and 5.4 g day − 1 in weeks 3 and 4, respectively (Table 1), but the rate of weight gain was not different between sexes (P = 0.33).
5.2. Kit body water turno6er The mean biological half-life of total body water turnover in the mink kits increased from 0.8 days during week 1 to 1.9 days during week 4 post partum. Individual data for all the injected kits are presented in Fig. 1 along with the calculated regression line, given by the equation: 3
HHO half-life=0.0131 × Bwt. +0.739 (r 2 =0.828; df= 79)
(2)
where Bwt. is body weight in g.
5.3. Milk intake Individual values of daily milk intake by the injected mink kits, plotted for each litter during weeks 1 – 4 post partum in Fig. 2. (panel B), show a considerable, between kit within litter, variation in daily milk con-
Fig. 2. Total daily milk production (g day − 1) of mink (Mustela 6ison) dams through weeks 1 – 4 post partum (Panel A), calculated as the sum of individual daily milk intake (g day − 1) measured in the tritium loaded kits (Panel B) plus the estimated milk intake by the two unloaded (control) kits of each litter. Dams no. 239, 250, 449 and 453 (with their total number of kits given in parentheses) were studied for 2 days in weeks 1 (days 3 – 5) and 3 (days 16-18), and dams no. 178, 418, 435 and 486 were studied for 2 days in weeks 2 (days 8–10) and 4 (days 22 – 24) post partum. Dams no. 250 and 418 lost one kit each between the two study periods. For details, see Section 2 P: 000–000.
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ported by Tauson [35], but still within the range of normal kit performance. The half-life of body water turnover increased linearly with increasing body mass of the kits (Fig. 1), and the clustered values obtained at each level of body weight indicated similar rates of milk water intake by kits of equal size. At peak lactation, the biological half-life of 3HHO in the mink kits was comparable to that observed by Coward et al. [9] in suckling rat pups. Furthermore, in the mink, water recycling during lactation appeared to be of minor importance, as in lactating rats [4] and dogs [26], whereas in mice [3] recycling accounts for \ 50% of the daily MWI.
6.2. Milk intake of the young and milk yield of the dam Fig. 3. Ratio of daily milk intake (MI, g day − 1) to body growth (BG, g day − 1) of the mink kits studied during postnatal weeks 1– 4.
dams indicated that the amount of milk water recirculated was somewhat lower in the second measurement period. The calculated values for daily MI per unit of weight gain for each kit (Fig. 3) remained rather stable with a mean value of 4.0 (g g − 1) during the first 3 weeks post partum whereas, in week 4, the data were much more scattered around a somewhat higher mean value of 5.6 (g g − 1), the effect of sex being significant (P = 0.006), but not consistent since there were significant interaction effects between week and sex (P =0.002).
Daily milk intake varied considerably between individual kits within litters as well as between litters. This variation could be explained by differences in kit live weight, which to a great extent could be attributed to sex, but factors such as genetic predisposition, feed intake of the dam, litter size and nursing stimulus [14,18,21,22] might also have influenced milk intake. As expected, the daily milk intake was higher in male kits than in female kits and increased markedly during the 4 week experiment. For any measurement period, the largest milk yield was found in dams with the heaviest litters, and male kits having significantly higher MI than female kits. The tendency towards a stabilization of daily milk intake by the suckling young and of the
5.4. ME supply by milk and ME requirements of kits Based on the assumptions stated above, the ME supplied to the kits by the milk increased from : 450 kJ litter − 1 per day in the first week of lactation to 990 kJ litter − 1 per day in week 4. Simultaneously, the energy requirements of the kits increased from 300 kJ litter − 1 per day in week 1 to 920 kJ litter − 1 per day in week 4 (Fig. 4). Milk production increased at a lower rate from week 3 to week 4 than earlier in lactation (Fig. 4), and the relative importance of MEm for the total ME requirement of the kits increased as lactation progressed, accounting for 3791.5% in week 1 and 4692.2% in week 4.
6. Discussion
6.1. Body weight changes and body water turno6er The rates of feed intake and weight change of the dams observed in the present study (Table 1) were comparable to those seen under normal farm conditions [33,34], and kit mortality rate was low. The recorded kit weight gain was somewhat below the growth rate re-
Fig. 4. Total daily output of metabolizable energy (ME)(kJ dam − 1) in mink milk () compared to the estimated (total) daily energy requirements of the kits during the first 4 weeks of lactation. Hatched columns represent the mean ME requirement for body growth (kJ day − 1) and open columns represent the mean ME requirement for maintenance (kJ day − 1). Values are means with standard errors represented by vertical bars. For details, see text, P: 000 – 000.
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total milk yield of the dam in week 4 of lactation was reflected in a lower average daily weight gain by the kits, in agreement with the decrease in relative growth rate previously recorded after week 3 of lactation [35]. Furthermore, the average ratio of milk intake to body growth rate observed during weeks 1 – 3 (4.0 g g − 1) (Fig. 3) was in agreement with the values obtained in suckling mice (3.5 g g − 1) [18], puppies (Section 4.4) [26] and in hibernating bear cubs (3.7 g g − 1) [12]. The increased milk intake to body growth ratio observed in week 4, may be explained, at least in part, by the increasing requirements for MEm of the rapidly growing mink kits (Fig. 4). The mean daily MI increased from 11 – 28 g kit − 1 per day, corresponding to an average total milk yield of 4200 g for the dams in the 4 week experiment, which is in agreement with the estimated total milk production for dams raising 5 – 8 kits [14]. Information on the milk yield of the lactating mink is sparse and the results presented here represent one of only two attempts to measure milk intake in suckling mink kits by a direct method. In previous studies, milk production in mink has been estimated by factorial methods, but the experimental data needed for correct calculations is still too limited to permit precise estimates. For instance, there is no experimental information on MEm for mink kits in the suckling period, values for kp and kf are lacking, and there is little information about the chemical composition of mink milk at various stages of lactation [28]. Therefore, based on the assumption of a fixed relationship between the rates of daily milk intake and body growth of suckling mink kits, estimated values for MI at peak lactation range from far below, i.e. 4 –14 g day − 1 per kit [13,20] to within fair agreement with our present data, i.e. 24 – 30 g day − 1 per kit [33,39] and 30 – 35 g day − 1 per kit [7]. The lower values can to some extent be attributed to differences in litter size [22], to a lower weight gain by the kits studied, and to the increasing demand for maintenance energy of the rapidly growing young as demonstrated by Knight et al. [18] and by the data in Fig. 4 of the present study.
6.3. Milk energy output and estimated energy requirement of the suckling young The calculated total daily ME output in the milk was sufficient to cover the ME requirements of the suckling kits. During the first two periods of measurement, the calculated ME output in milk far exceeded the kits’ requirement (Fig. 4), but during the two last measurement periods ME in milk was only 5 – 7% above the estimated energy requirements of the kits. It may be that the measurements on the very small kits were somewhat less accurate than those on larger kits. Alternatively, our estimates of MEm of newborn kits may
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have been too low. Data on rats indicate that MEm per unit of metabolic weight decreases with increasing body weight (A. Chwalibog, personal communication). On the other hand, the kits in the present study had very low locomotor activity, and were kept warm by the dam in the nest-box, so a MEm requirement higher than that of adult animals [6,40] seems unlikely.
6.4. Validity of the water isotope technique for studies on mink In the past two decades, the isotope dilution technique has been applied to rats and mice [3,9,18,31], dogs [26], lambs [9,11], calves [16], and pigs [10]. Detailed discussions of the theoretical and experimental implications of utilizing tracer disappearance rates to measure milk production have appeared in review format [9,11,24,26]. Lately, the stable isotope deuterium oxide has been used successfully in studies of wild animals such as bears [12] and the harp seal [27]. It appears that experimental studies based on the isotope dilution technique lead to more reliable and reproducible estimates of milk production than conventional methods based on ‘test-weighing’ of the dams and/or the offspring [15,22,23]. This is partly due to lack of data on the efficiency of energy utilization for growth, and to the fact that the conventional methods require separation of the young from their mother for several hours, which reduces the suckling stimulus to milk production. In the present study, the tritiated water dilution technique was found feasible for repeated short-term measurements of body water turnover and daily MI in suckling mink kits up to 4 weeks of age with a minimum of interference with the mother–young interaction. The daily milk yield of the mink dams increased markedly as lactation progressed, reaching a maximum of 30 g kit − 1 per day, or more than 200 g dam − 1 per day. The total daily milk energy output by the dam was in excess of 1 MJ day − 1, corresponding well with estimated values for daily requirements of energy for maintenance and body growth of the kits. It should be emphasized that using the single-isotope tracer technique for measurements of daily MI it is assumed, that the dams’ milk is the only source of water intake [9,11]. Since it cannot be excluded that some kits may have started to eat solid food during the final measuring period of the present study, the calculated daily milk intake in week 4 may have been slightly overestimated. Data obtained in previous studies of MI in hand-reared calves [16] and suckling bear cubs [12] indicate that the isotope dilution technique may be improved by simultaneous administration of two different water isotopes, i.e. deuterium oxide to the nursing dam and tritiated water to the suckling young, thereby distinguishing between daily intake of milk water and
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other sources of non-milk water taken up by the suckling young.
Acknowledgements The financial support of The Danish Agricultural and Veterinary Research Council (Grants No. 9400135 and 9502267) and the Danish Fur Breeder’s Association is gratefully acknowledged. The authors thank Inge Andersen, Lise Larsen, Boye Pedersen and Merethe Stubgaard for skilled technical assistance.
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