Lactation in tiie Dog: Mill< Composition and Intal^e by Puppies OLAV T. OFTEDAL' Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853 ABSTRACT The composition and intake of milk by mother-reared puppies was studied to compare protein and energy intakes of puppies with estimated require- ments. Milk samples were obtained from five beagle bitches over the period of 7-37 days postpartum. Dog milk contained on average 22.7% dry matter, 9.47% fat, 7.53% protein, 3.81% sugar and 146 kcal gross energy per 100 g. Protein comprised 31% of milk energy. Nonprotein nitrogen averaged 0.054%, equivalent to 4.4% of total nitrogen. Milk intakes of puppies in the five litters were estimated from water kinetics following administration of deuterium oxide (D2O). D2O dilution indicated that body water comprised 72-73% of puppy body weight, and fractional turnover rate of body water averaged 0.15-0.17% per day in weeks 3 and 4 postpartum. Milk intakes were calculated as 160 ? 5.4 g (mean ? SEM) at 19 days and 175 ? 5.3 g at 26 days, equivalent to 17.0 and 14.6% of body weight, respectively. Daily milk yields of the bitches averaged 964 g at 19 days and 1054 g at 26 days. Dry matter intakes of the puppies were equivalent to 3.9 and 3.3% of body weight at 19 and 26 days, respec- tively. Gross energy intakes averaged 223-224 kcal/kg?'^ per day, and protein intakes averaged 0.33-0.36 g per gram body weight gain at these ages. Estimates of the energy requirements of young puppies by the National Research Council appear to be too hi?i. J. Nutr. 114: 803-812, 1984. INDEXING KEY WORDS dogs ? lactation ? milk composition ? milk yield ? water kinetics Milk composition and yield vary greatly age or less are reported to consume the among diverse mammalian species (1-3). equivalentof 10-14% of body weight per day Estimation of the nutrient requirements of (7, 9). The effects of maternal nutrition (9, both mother and suckling young requires 10), maternal size (2), breed (6), and litter size quantitative information on lactation per- and mass on lactation performance in dogs formance Dogs are know^n to produce a need clarification. rather concentrated milk containing 21-26% The following study was undertaken to total solids, 8-12% fat and 7-10% protein measure milk composition and milk yield at (4-7), although lower levels of fat and protein peak lactation in well-nourished dogs of the have recently been reported (8). Little in- beagle breed. This study is part of a larger formation is available on milk yields in dogs, project in which lactation performance is Several litters of various breeds have been compared among several species, and nutri- studied by weighing puppies before and after ent intakes of suckling young are related to suckling (6, 7). A German shepherd was esti- body size, growth rates and estimated re- mated to produce 1.7 kg milk per day at the quirements (3, 11). Milk production was lactation peak at 3 weeks postpartum, where- as bitches of smaller breeds produced less ? 1984 American institute of Nutrition. Received for publication milk but were only Studied in the first 9 days ^TJunewss ,,,.,? u x, , /e>\ c> 11* 'A 1 r Current address: Department of Zoological Research, National postpartum (0). Suckling puppies 4 weeks or zoological Park, Smithsonian Institution, Washington, DC 20008. 803 804 OFTEDAL measured from the dilution and turnover of deuterium oxide (D2O) administered to puppies. Hydrogen isotopes have been shown to yield valid estimates of milk production if corrections are made for changes in body water pool size and for isotope recycling via maternal milk (11-15). Peak lactation was assumed to occur in week 3 or 4 postpartum since puppies do not initiate feeding on semisolid food until the emergence of deciduous dentition at 21 to 35 days postpartum (16-18). Milk alone will support normal growth up to 4 weeks post- partum; thereafter, withholding of supple- mental food may result in a reduced growth rate (19). MATERIALS AND METHODS Experimental animals. Five bitches were studied at the long-established beagle re- search colony at Cornell University (20). The bitches were 1.5-3.7 years of age, had postpartum weights of 9.3-15.2 kg (mean = 12.7 kg) and were producing their first or second litters. One week prior to the ex- pected parturition date each dog was removed from the main colony to an isolated whelping room. They were individually housed in 1.2- x 1.1-m cages (horizonte dimensions) with 0.7- X 0.7-m heated whelping boards, and were fed a commer- cial dry dog food (Wayne Dry Dog Food, Allied Mills, Inc., Chicago, XL) containing about 26% crude protein, 9% fat, 4% crude fiber and 2.75 kcal metabolizable energy (ME) per gram.* Water was provided ad libitum in elevated stainless-steel bowls that were too high for the puppies to drink from. At birth litter size ranged from 5 to 10 pup- pies; 5 to 7 puppies per litter survived beyond 1 week postpartum. The puppies were first offered supplemental feed [dry dog food (Wayne Dry Dog Food) soaked in evaporated milk and water] at 29-30 days postpartum. Puppies were weighed to the nearest gram at least three times per week over the course of the study. Milk sampling and analysis. Milk samples were collected at weekly intervals from 7-37 days postpartum. Additional samples were also taken during weeks 2 and 5 post- partum. Bitches were removed from their litters for 2-3 hours prior to milking. Oxy- tocin (5 lu) was administered by intramus- cular injection, and one or two teats evacu- ated as completely as possible by gentle manual expression. An average of 21 ml ( ? 6.3 SD) was obtained in 10-15 minutes. Samples were frozen in sealed vials until analyzed. Milk samples were thawed quickly, ho- mogenized in a Potter-Elvehjem tissue grinder and subsampled. The weekly sam- ples were assayed in duplicate for major constituents. Total solids were determined by oven drying, total nitrogen (TN) and nonprotein nitrogen (NPN) by a Kjeldahl procedure, fat by the Roese-Gottlieb method (21) and sugar by the phenol-sulfuric acid colorimetric method, as previously described (21). Sample size did not permit NPN deter- mination on eight samples. In such cases an additional sample collected from the same bitch within 2 to 3 days was substituted. Both TN and NPN were measured on these additional samples. Protein was calculated as 6.38 X (TPN - NPN). Gross energy was estimated from an equation developed by Perrin (22), as previously presented (21). Milk intake estimation procedure. Milk intake was estimated from water kinetics of 25 puppies in the five litters. D2O (99.8% purity) was administered by stomach tube to puppies at 15-16 days and 22-23 days postpartum at a rate of 2.3 g/kg body weight. One young in each litter was not given D2O so that correction could be made for isotope recycling. Two hours were al- lowed for isotope equilibration prior to collection of about 2 ml of blood by jugular puncture; in young puppies hydrogen iso- topes equilibrate in 1.5 hours (23). Each puppy was bled at 2- to 3-day intervals such that four samples were collected during each weekly study period for determination of water turnover. The second isotope administration immediately followed the final bleeding of the preceding period such that residual isotope levels could be mea- sured. Blood water was isolated by heat distillation and assayed for deuterium con- centration by infrared spectrophotometry (24) using matched barium fluoride cells (0.11-mm path length) in a double beam 'Nutritional information supplied by manufacturer. LACTATION IN THE DOG 805 grating infrared spectrophotometer (Model 521, Perkin-Elmer Corp., Norwalk, CT). Assayed deuterium levels were corrected for body weight changes in computations of fractional turnover rate (k) and body water fraction (F)(ll). Isotope recycling via mater- nal ingestion of the excreta of suckling young, followed by transfer of isotope in milk water from mother to young (12), necessitated an additional correction. The accumulation of deuterium in an unin- jected, control puppy in each litter was monitored. On the assumption that these levels are representative of recycled isotope in littermates, the deuterium levels in con- trol puppies were subtracted from the deuterium levels of their littermates prior to regression of corrected log D2O concentra- tion against time after administration. Daily water loss, water gain and water intake were computed as previously de- scribed (21). The proportions of milk constituents catabolized to produce meta- bolic water were estimated by iterative calculations detailed elsewhere (11). Sta- tistical analyses were performed using programs of the Statisticid Package for the Social Sciences (SPSS) on a Honeywell computer at the Smithsonian Institution. Mean values are presented as mean ? SEM unless otherwise indicated. RESULTS Milk composition. Over the period of 7 to 37 days postpartum (table 1) there were no significant differences in total solids, fat, protein or gross energy content among sampling times [P > 0.05, analysis of vari- ance (ANOVA)]. Sugar content did differ among sampling times (P < 0.05, ANOVA), the mean value rising from 3.47% at 7 to 9 days postpartum to 4.13% at 29 to 30 days postpartum. The mean values for all sam- pling times were: 22.7 ? 0.41% total solids, 9.47 ? 0.386% fat, 7.53 ? 0.123% protein, 3.81 ? 0.079% sugar and 146 ? 3.6 kcal gross energy per 100 g. If converted to a dry matter basis dog milk was found to contain 41.4 ? 0.87% fat, 33.4 ? 0.60% protein, 17.0 ? 0.049% sugar and 641 ? 3.9 kcal/ 100 g dry matter. Fat, protein and sugar provided58.7 ? 0.86%, 30.5 ? 0.66% and 10.5 ? 0.33% of total gross energy, respec- tively. There were no significant differences in milk composition among the five bitches, whether compared on a whole-milk, dry matter or gross energy basis (F > 0.05, ANOVA). NPN ranged from 0.045 to 0.068% (mean = 0.054 ? 0.0012%), equivalent to 3.4-5.2% of total nitrogen. NPN did not differ significantly among bitches or sam- pling times (P > 0.05, ANOVA). Water and milk intakes. Water and milk intakes were calculated for the midpoints of each study period, i.e., for 19 and 26 days postpartum (table 2). Growth rates and esti- mated body weights at these ages derive from regressions of puppy weight on post- natal age. These regressions were highly linear (mean r^ = 0.990). Regressions of the logarithm of corrected deuterium concen- tration on time after isotope administration were also highly linear (mean r* = 0.996). Deuterium content in body water of unin- jected control puppies reached levels equiva- lent to 11.5 ? 0.45% and 10.6 + 0.30% of the levels in injected littermates by the final TABLE 1 Composition of dog milk''' Time of milk sampling, days postpartum Constituent' 7-9 15-16 22-23 29-30 36-37 Total Total solids, % 23.5 ? 1.31' 22.9 ? 1.41" 22.1 ? 0.51' 22,3 ? 0.64' 22.6 ? 0.72' 22.7 ? 0.41 Fat, % 10.90 ? 1.370" 9.67 ? 1.171" 8.73 ? 0.230' 8.89 ? 0.410' 9.16 ? 0.482' 9.47 ? 0.386 Protein, % 7.17 ? 0.092" 7.59 ? 0.275' 7.72 ? 0.390' 7.49 ? 0.303' 7.70 ? 0.281' 7.53 ? 0.123 Sugar, % 3.47 ? 0.087" 3.61 ? 0.111'"' 4.04 ? 0.178''" 4.13 ? 0.149" 3.81 ? 0.186'''" 3.81 ? 0.079 Gross energy. kcal/100 g 155 ? 12.3' 147 ? 11.9' 141 ? 3.4' 142 ? 4.5' 144 ? 5.0' 146 ? 3.6 'Mean ? SEM; n = 5 for each time period; n = 25 for total. 'Means with the same superscript in a row do not differ by more than the shortest significant range at the 0.05 significance level (Duncan's multiple-range test). 'Percentage values represent gram per 100 g. 806 OFTEDAL blood sampling in the first and second study periods, respectively. Correction for isotope recycling was therefore warranted. Fractional turnover rate of body water (k) declined from 0.168 ? 0.0036 per day for the first study period to 0.152 ? 0.0034 per day for the second (P < 0.001, paired i-test). By contrast body water fraction (F) did not differ significantly (P > 0.05, paired i-test) between the two periods (table 2). Calcu- lated water losses, water gains and water intakes at 19 and 26 days postpartum are presented in table 2. Water intake was equivalent to 15.1 and 13.9% of body weight at 19 and 26 days, respectively. Water intake derives from both preformed milk water and metabolic water from the catabolism of milk solids. Body composition data presented by Sheng and Huggins (25) indicate weight gain comprises 12% protein and 13% fat in the period of 16-33 days postpartum. On the basis of these values, combined with milk composition, growth rate and water intake data, one can calcu- late that the amounts of fat and protein catabolized are equivalent to 69 and 72% of ingested fat and 63 and 67% of ingested protein at 19 and 26 days, respectively. It was assumed that 100% of ingested sugar was catabolized. Ingestion of 100 g milk will then yield 77.3 g preformed and 11.7 g meta- bolic water at 19 days and 77.3 g preformed and 11.7 g metabolic water at 26 days. Milk intake was estimated as water intake X 1.129 at 19 days and water intake x 1.124 at 26 days (table 3). Individual milk intakes of puppies ranged from 126 to 239 g/day at 19 days and from 134 to 229 g/day at 26 days. TABLE 2 Body water turnover in suckling puppies Time after parturition, day Measure 19 26 Body wt, g 942 ? 25.4 1199 ? 33.3 Wt gain, g/day 36.8 ? 1.28 36.8 ? 1.28 Fractional turnover, (k) 0.168 ? 0.0036 0.152 ? 0.0034 Wt fraction,' (F) 0.726 ? 0.0068 0.716 ? 0.0084 Water loss, g/day 115 ? 4.2 130 ? 4.1 Water gain, g/day 26.6 ? 0.80 26.1 ? 0.68 Water intake, g/clay 142 ? 4.8 156 ?4.7 'Grams body water per gram body weight. Milk intakes of puppies were compared among litters and between the two post- natal ages by two-way ANOVA. Whether expressed as a daily amount, as a percentage of body weight per day, or per gram body weight gain, milk intake was significantly influenced by both litter and age effects (table 3). Although the absolute amount of milk consumed per day at 26 da)^ (175 ? 5.3 g) was greater (P < 0.01) than that at 19 days (160 ? 5.4 g), this amount repre- sented a smaller percentage of body weight (14.6% at 26 days vs. 17.0% at 19 days, P < 0.001). Since the same estimate of growth rate was used for both age categories, milk intake per gram body weight gain was of course greater at 26 days (table 3). The mean milk intakes in four litters were rela- tively similar (147-156 g/day at 19 days; 159-178 g/day at 26 days), but the puppies in the litter of bitch BR 82 consumed appre- ciably more milk on average (203 g/day at 19 days and 221 g/day at 26 days). Puppies in the larger litters tended to be smaller and grow at a reduced rate but did not appear to ingest substantially less milk than the pup- pies in smaller litters at these ages (table 3). Total milk output of lactating bitches (milk intake per puppy X litter size) was estimated as 964 ? 57.6 g/day (n = 5) at 19 days and 1054 ? 57.7 g/day at 26 days (table 3). These estimates are not signifi- cantly different (P > 0.05, paired i-test). These yields correspond to 7.6% of maternal weight or 143 g/kg" ''^ at 19 days and 8.3% or 157 g/kg? '' at 26 days. Nutrient intakes. By combining mean milk composition (table 1) and mean milk intake (table 3) data, the intake of various constituents can be calculated. At 19 days suckling puppies ingested 36.3 g dry matter (3.86% of body weight), 15.2 g fat, 12.0 g protein, 6.1 g sugar and 234 kcal (224 kcal/kg" ''^). At 26 days intakes were 39.7 g dry matter (3.31% of body weight), 16.6 g fat, 13.2 g protein, 6.7 g sugar and 256 kcal (223 kcal/kg"''^). For each gram of body weight gain, puppies ingested 0.33 g protein and 6.4 kcal at 19 days and 0.36 g protein and 7.0 kcal at 26 days. DISCUSSION Milk composition. Milk collected from beagle bitches contained on average 22.7% LACTATION IN THE DOG 807 TABLE 3 Milk intakes of suckling puppies^ Litter Milk intake of puppies Bitch Daily Per gram MUk output designation size intake As % wt wt gain of bitch g % g g 19 days postpartum BR82 5 203 18.7 4.51 1015 BR50 5 152 14.8 4.02 760 CA46 6 154 16.7 4.13 924 CD 18 7 156 17.3 4.54 1092 CA45 7 147 17.3 4.55 1029 All litters 160 ? 5.4 26 17.0 ? 0.30 days postpartum 4.37 ? 0.074 964 ? 57.6 BR82 5 221 15.7 4.91 1105 BR50 5 166 12.8 4.39 830 CA46 6 178 15.1 4.80 1068 CD 18 7 165 14.4 4.82 1155 CA45 7 159 14.8 4.92 1113 All litters 175 ? 5.3 14.6 ? 0.22 4.79 ? 0.071 1054 ? 57.7 Analysis of variance Litter effect F(4,44) P < 0.001 P < 0.001 P < 0.001 ? Age effect F(l,44) P < 0.01 P < 0.001 P < 0.001 ? 'Milk intakes are litter means. Values after ? are SEM. total solids, 9.5% fat, 7.5% protein and 3.8% sugar. Mean values from prior studies on dog milk are tabulated for comparison (table 4). This list includes 19th century results of questionable analytical accuracy as well as studies involving only a few sam- ples from one or two dogs. Samples collected very early or late in lactation have been excluded as not representative of established lactation. Despite variation in sampling and analytical procedures, most reports fall within the ranges of 21-26% total solids, 8-12% fat, 7-10% protein and 3-4% sugar (table 4). The results reported herein are consistent with these values. By contrast the recent data of L?nnerdal and colleagues (8) indicate much lower fat (4.8%) and protein (5.2%) levels in beagle milk collected 11-40 days postpartum. This discrepancy may stem from inappropriate application of rapid spectrophotometric methods. Color development in the sulfuric acid-phosphoric acid-vanillin reaction employed in the determination of fat depends on the degree of unsaturation of the lipids (34). Binding of Coomassie brilliant blue G250 dye to pro- tein is likewise a function of the amino acid composition of the protein (35, 36). These methods are valid only if standardized to the particular mix of lipid and protein constitu- ents found in dog milk. It appears that this was not done. The variation among the remaining studies may be a function of sampling or analytical bias or may represent real differ- ences among dogs. Dog breeds vary tre- mendously in body size and conformation, but no correlation to the gross composition of milk could be determined by Russe (6) who studied breeds ranging in size from dachshunds to Saint Bernards. Beagle milk collected by Luick and colleagues (5) con- tained more total solids, fat and protein, but less sugar (table 4) than was found in the present study. By contrast the recent data of Mundt and colleagues (7) are very similar to 808 OFTEDAL TABLE 4 Published data on the composition of dog milk at midlactation Females Days after No. of Total Source milked birth samples solids Fat Protein Sugar % % % % Ssubotin 1866 (26) 3 12-38? 12' 20.8 8.8 8.8" 2.7 Tolmatscheff 1867 (27) 1 35 2 11.8 8.2^ 3.2 Abderhalden 1898 (28) 2 5-11 9' 11.5 7.0" 3.3 Abderhalden 1899 (29) 1 10-14 3 11.6 7.3* 3.1 Dijkstra 1910 (30) 1 6-20 2 6.5 6.8' 2.6 Grimmer 1915 (31) 1 5-26 22' 20.8 8.5 7.2* Daggs 1931 (10) 3 21-35 8' 23.9 12.4 8.0? 3.2 Deniges 1935 (32) 2 ? 2 24.2 10.6 9.3' 2.7 Anderson et al. 1940 (4) 1 18-30 2-4' 22.6 8.3 7.5' 3.7 Luick et al. 1960 (5) 3 15-36 30 26.0 12.3 9.8' 3.3 Russe 1961 (6) 7 5-35 32-63' 22.0 11.1 6.9' 3.3 Lauer et al. 1969 (33) 2 30 2 26.4 12.3 8.2' 2.9 Mundt et al. 1981 (7) 7 7-28 18-28' 22.0 9.7 7.3' 3.6 L?nnerdal et al. 1981 (8) 16 11-40 62-71' ? 4.8 5.2' 4.5 Present study 5 7-37 25 22.7 9.5 7.5* 3.8 'Early and/or late lactation samples excluded. "Protein determined by precipitation and weighing. 'Assay method not specified. "Protein calculated as protein nitrogen (Kjeldahl) x 6.38. 'Protein calculated as total nitrogen (Kjeldahl) x 6.38. "Protein measured by dye-binding procedure. the present results although four breeds as well as mongrels were included. Breed dif- ferences in the composition of dog milk must be minor if they exist at all. An elevation in total solids and protein at both the beginning and the end of lactation has been noted in prior reports (4, 6, 31). In the present study there were no substantial changes in milk composition from 7 to 37 days postpartum, a week after supplemental feeding of puppies commenced. Marked compositional changes are apparently asso- ciated with mammary involution at about 39-49 days (4, 6). Weaning is of course influenced by feeding and management practices, and perhaps by breed as well (6). L?nnerdal et al. (8) have reported a rise in fat and protein contents of beagle milk from 0 to 40 days, and a subsequent decline in fat content. These trends are at odds with other findings and could reflect sensitivity of the spectrophotometric assay procedures to qualitative as well as quantitative changes in milk constituents. Canine caseins and whey proteins certainly differ in amino acid composition (37) such that varying propor- tions during lactation (6) will aiffect protein content as measured by dye-binding. The NPN content of dog milk averaged 0.054%. The data of Grimmer (31) and Russe (6) indicate mean NPN values of 0.068 and 0.112%, respectively. In the present study NPN accounted for only 4.4% of total nitrogen, as compared to 5.7 (31) and 9.3% (6). Protein estimates based on total nitro- gen (e.g., see refs. 4, 5, 7, 33) will over- estimate true protein by an amount equal to NPN X 6.38, i.e., by about 0.3 to 0.7 percentage points. In 100 g milk, 1.88 ? 0.062 g of sohds was not accounted for by the summation of fat, protein and sugar. Part of the residual is due to ash and part to NPN constituents. If published data on the ash content of dog milk in the period of 5 to 35 days postpartum (4, 7, 10, 28, 30-33) are considered collec- tively, a mean ash content of 1.15% can be calculated (n = 57). Assuming the NPN constituents of dog milk to be similar in proportion to those of cow's milk (38), NPN X 5.34 = 0.29% gives an approximation of the combined weight of NPN constituents (11). The remaining 0.44 g (= 1.88 - 1.15 - 0.29) represents minor organic and inorganic compounds not included in the various analj^ical fractions (38) as well as LACTATION IN THE DOG 809 analytical error. On this basis it would appear that any such error was small. Water and milk intakes. Suckling beagle puppies were estimated to consume 142 g water, equivalent to 160 g milk, at 19 days postpartum, and 156 g water, equivalent to 175 g milk, at 26 days postpartum (table 2). These values are only as accurate as the estimates of body water fraction (F) and water turnover rate (k) on which the calcu- lations are based. Isotope dilution proce- dures have been reported by Sheng and Huggins (23, 25) to overestimate body water content in growing beagles when compared to values obtained by direct dessication. Dilution of tritiated water indicated body water percentages of 75-92% at 8-21 days (23), whereas direct dessication values were 65-74% at the same sampling times (25). These results have been contested with al- legations that the analytical procedures must have been in error (39). Neither the mean values obtained in the present study (72-73%) nor the values reported by Romsos and colleagues (9) for 4-week-old puppies administered tritiated water (74-76%) ap- pear to be greatly in excess of expected values. Widdowson (40) reported that 3-week-old puppies contain 68% water. It appears that isotope dilution may over- estimate body water in puppies by a few percentage points but not to the degree reported by Sheng and Huggins (23). The decline in water turnover rate from 0.168 in the first week of study to 0.152 in the second may reflect in part that puppies were not allowed supplemental water or feed prior to 29 to 30 days postpartum. Maternal r?gurgitation of feed to puppies was not observed; disgorging of food has been reported as early as 21-24 days post- partum by Martins (41). Any ingestion of water from sources other than mother's milk would lead to overestimation of milk intake. Fractional turnover rates of 0.168 and 0.152 correspond to body water half-lives of 4.1 and 4.6 days. Four-week-old beagle puppies suckling bitches fed canned, semipurified diets of high or low carbohydrate content had somewhat longer half-lives of 5.0 and 6.8 days, respectively (9). These data were not corrected for either isotope recycling or changing size of the body water pool, how- ever. Baverstock and Green (12) calculate that at least 47% of the isotope lost by dingo puppies is ingested by the mother; some of this is recycled to the young in milk. Isotope recycling was not measured directly in the present study although accumulation of D2O in uninjected control puppies indicated that it did occur. The milk intakes determined herein are much higher than the 81 ? 13 ml/day and 59 ? 13 ml/day reported by Romsos et al. (9) for beagle puppies suckling bitches fed two semipurified diets. Aside from the methodological problems mentioned above, these puppies exhibited abnormally low growth rates, gaining on average only 13 and 11 g/day in the two groups. Normal growth rates for beagle puppies are 35-40 g/day (17, 19, 42, 43). Records for the Cornell dog colony for 1975-1976 indicate average growth rates of 34.9 ? 0.99 g/day for puppies in litters of five (n = 25 pup- pies), 29.9 ? 1.23 g/day for puppies in litters of six (n = 30) and 27.2 ? 1.08 g/day for puppies in litters of seven (n = 35). The growth rates observed in the present study (36.8 ? 1.28 g/day) for puppies suckling bitches fed a commercial dry dog food are somewhat above the colony norms, but within the normal range for beagle puppies. The semipurified diets used by Romsos et al. (9) apparently did not support normal milk yields. The milk intake of 26-day-old puppies was equivalent to 14.6% body weight, a decline from the 17.0% of body weight consumed at 19 days. Mundt and colleagues (7) reported that milk intakes of puppies in three litters of various breeds averaged 10.0-13.5% of body weight in the first 4 weeks postpartum. These data derive from weights taken before and after puppies were allowed to suckle The experimental regi- men of separation and periodic, controlled access of young to mother may cause a reduction in secretion rates, may interfere with normal maternal nursing behavior, or may result in the accumulation of amounts of milk that young are unable to consume in relatively short suckling bouts (3). Hence weight differential procedures tend to underestimate milk consumption rates. Mundt et al. (7) note that puppies consumed more than 95% of daily milk intake in four or five of the six suckling bouts permitted 810 OFTEDAL per day; i.e., one or two suckling bouts per day were relatively unsuccessful. These puppies reportedly consumed 1.7-3.0 g milk per gram body weight gain in weeks 3 and 4 (7). By contrast puppies in the present study were calculated to consume 4.4 g milk per gram gain during week 3 and 4.8 g milk per gram gain during week 4. On this basis it appears that the weight differential pro- cedure underestimated milk intake by about one-third. Peak milk yields of beagle bitches appear to be about 1 kg/day in week 4 postpartum. Milk yield is undoubtedly influenced by body size (2, 3). Using a weight differential procedure. Russe (6) estimated that at 6 days postpartum a German shepherd pro- duced 915 g as compared to 102 g and 184 g for two dachshunds. Only the German shepherd was studied throughout lactation; peak yield (about 1.7 kg/day) was observed at 22-27 days (6). Assuming a body weight of 30 kg, milk production of this dog was about 130 g/kg"''^ as compared to 157 g/kg"'* for beagle bitches at 26 days post- partum in the present study. Bias associated with the weight differential method may explain some or all of this 20% difference. Nutrient intakes in relation to estimated requirements. The Subcommittee on Dog Nutrition of the National Research Council (NRG) (44), following Payne (45), listed the daily ME requirement of 3- and 6-week-old puppies as 274 kcal/kg"^'. In the present study puppies ingested only 244 kcal gross energy per kilogram"'^ at 26 days. If conver- sion factors of 4.0 kcal ME per gram protein or sugar and 9.0 kcal ME per gram fat are adopted, dog milk can be calculated to contain 131 kcal ME/100 g. The ME intakes would then be 219 kcal/k^''^ at 19 days and 200 kcal/kg* ?'^ at 26 days. It appears that the National Research Council (44) overesti- mated the metabolizable energy needs of young puppies by 25-35%. Recently Mundt et al. (7) estimated the daily maintenance requirements of puppies as 72 kcal gross energy per kilogram"^^ from a regression of gross energy intake of suck- ling puppies on growth rate. As the milk intakes of the puppies were probably under- estimated by a substantial amount (see above), this estimate cannot be considered reliable. Weaned puppies have been estimated by the Subcommittee on Dog Nutrition, NRG (44) to require 22% protein in the dry matter of a diet containing 3.5-4.0 kcal ME per gram dry matter. Dog milk contains about 131 kcal ME/100 g or 5.77 kcal ME per gram dry matter, a value about 50% above the NRG diet. If dog milk is to meet NRG requirements it should contain 1.5 X 22 = 33% protein on a dry matter basis. The protein content of dog milk was indeed found to be 33.4% of dry matter. Puppies ingested 0.33 g protein per gram body weight gain at 19 days and 0.36 g protein per gram body weight gain at 26 days. Payne (45) assumed weight gain in puppies to contain 17% protein, whereas carcass analyses by Sheng and Huggins (25) indicate protein content to remain at about 10-12% of body weight from birth to 6 weeks postpartum. At peak lactation pup- pies apparently incorporate only one-third to one-half of ingested protein into tissue. ACKNOWLEDGMENTS I would like to thank Dr. Richard G. Warner for authorizing use of the dog colony at Gornell University, Dan Shattuck and staff of the colony for care of the ani- mals and assistance with milk and blood collection procedures, and Dr. Gharles Roberts and David Dance of the Smith- sonian Institution for performing statistical analyses. Milk analyses were conducted in the laboratory of Dr. John Sherbon. Drs. Maiden G. Nesheim and Harold F. Hintz offered constructive criticism and support. LITERATURE CITED 1. Jenness, R. & Sloan, R. E. (1970) The composi- tion of milks of various species: a review. Dairy Sei. Abstr. 32, 599-612. 2. Linzell, J. L. (1972) Milk yield, energy loss in milk, and mammary gland weight in different species. Dairy Sei. Abstr. 34, 351-360. 3. Oftedal, O. T. (1984) Milk composition, milk yield and energy output at peak lactation. 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