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Effect of caffeine on rat offspring from treated dams
Toxicology Letters, 7 (1980) 71-77 Elsevier/North-Holland Biomedical Press
EFFECT OF CAFFEINE ON RAT OFFSPRING FROM TREATED
H.U. AESCHBACHER,
71
DAMS
H. MILON, A. POOT and H.P. WeRZNER
Experimental Biology Laboratory, 1350 CH-Orbe, Switzerland
Nest16 Products Technical Assistance Co., Ltd.,
(Received June 12th, 1980) (Accepted July 8th, 1980)
SUMMARY
Pregnant Sprague-Dawley rats were given caffeine at 1.0,0.5 and 0.25 g/kg diet during gestation and lactation. At birth, half of the pups from control and treated rats at each dose level were exchanged and cross fostered. Two litters were produced by each animal from each of the experimental groups. Caffeine at dietary concentrations of 0.5 and 0.25 g/kg throughout gestation and lactation had no significant effect on birth weight, litter size or development. There was also no effect at these doses following treatment during either gestation alone, or lactation alone. At 1.0 g/kg there was a slight reduction of birth weight, as well as a trend towards lower weight gain in litters from dams fed the test diet throughout gestation and lactation.
INTRODUCTION
The intake of caffeine, consumed in tea and coffee, as a common ingredient of soft drinks, or in analgesics and OTC drugs has steadily increased worldwide. In recently published epidemiological studies [ 4, 10,161, concern was raised about the possible involvement of caffeine and coffee consumption during pregnancy on spontaneous abortion, premature births, and small-for-dates babies. Hill et al. [9] confirmed data from Nelson and Fofar [13] that OTC drugs are probably consumed by 75% of pregnant women, whereas coffee consumption was much lower at about 45%. Fetal resorptions, teratogenic effects and fetal death have been reported in experiments with rodents [ 2,6, 71. However, Thayer and Kensler [ 151, in an extensive study involving the exposure of 4 generations of mice to caffeine in the drinking water, observed no adverse effects on litter size or on the incidence of fetal abnormalities. Abbreviations:
ANOVA,
analysis of variance; OTC, over-the-counter.
72
Our objectives in the design and conduct of the present “crossoverfoster” study in rats were to investigate possible effects of caffeine when administered during pregnancy and/or lactation, on mortality, birth weight and development of the young. MATERIALS AND METHODS
Sprague-Dawley rats (Charles-River Farms; Saint-Aubin-les-Elbeuf, France) at 11 weeks of age, with initial body weight of approx. 250 g were offered a commercial standard laboratory feed (No. 185) (Nafag A.G., Gossau, Switzerland). This was pelleted at 3-weeks intervals for the controls and, after incorporation of caffeine purum (Fluka, Buchs, Switzerland), for the test groups. The diets were stored at 4°C and were tested for caffeine homogeneity. Tap water was provided ad libitum. Temperature and humidity in the animal rooms were maintained at 22” C and 55% relative humidity, respectively, throughout the study and there were artificial, 12-h day-night light periods. 2 females were mated with 1 male over a 4-day period. Since the influence of caffeine on sperm cells was to be excluded the male and female rats were not treated during the breeding period. Caffeine treatment of females started on the 4th day of the breeding period after removing the males. Following mating, all the females were divided into an untreated control group with 80 animals, and 3 treatment groups of 40 animals each. At the end of gestation the pregnant dams (60 controls or 30 treated animals) were selected randomly and distributed in groups of 15 dams. For the 2nd breeding, only the 15 dams/group which became pregnant at the first mating were remated. Rem~n~g untreated pregnant dams (not used for exchange) were added to the control group, whereas infertile dams were excluded from the study. At birth half of the dams from each group had their pups removed and exchanged either for pups from treated mothers in the case of control dams, or pups from untreated animals in the case of treated dams. The remaining animals per group were maintained with their own pups. EXPERIMENTAL DESIGN (EXAMPLE FOR FIRST LITTER)
Treatment of dams g caffeine/kg diet
Untreated
0.25
0.5
1.0
Number of dams which nursed their own young
15
15
15
15
Number of dams where pups are exchanged after birth between treated and untreated mothers
15 -15 15 P 15 f
15 ,15
73 TABLE I CAFFEINE
INTAKE DURING PREGNANCY
Dose (g caffeine1 kg feed)
1.0 0.5 0.25
AND LACTATION
Mean caffeine enposurea (mg/kg body weight) Pregnancy
Lactation
1st litter
2nd litter
1st litter
2nd litter
62.3 35.3 15.9
62.3 33.2 17.0
120.6 60.3 31.1
107.5 53.6 27.7
aDaily dose calculated from values for food consumption, and concentration of caffeine in diet.
body weight
At birth, the pups were counted, weighed and checked for viability and gross abnormalities. They were counted again and weighed at days 4 and 14 after birth and the average pup weight was calculated, since this method was found to be more precise than weighing individual pups. The pups were studied until 14 days of age when the experimental diets were offered. Caffeine intake of dams during pregnancy and lactation, calculated in relation to body weight from food intake date (Table I), almost doubled during lactation, due to considerably increased food intake. That of pups was estimated by determining the caffeine content in the milk of the treated dams and estimating the daily milk consumption by pups. For this purpose, an additional 10 female rats were treated in an identical manner during pregnancy and lactation and at each of the dose levels of caffeine used in the main experiment. At day 10 and againat day 14 of lactation, the-dams were separated from their pups overnight and milked the’following morning, using a modified method of Gupta et al. [ 81. The caffeine content in the milk samples was determined by gas chromatography [12]. To estimate the amount of milk absorbed by a pup the following formula was used 153. Milk intake g/day
Body wt. (g) X Calorie requirement (k&/g
body wt./day)
= Gross energy value (kcal/g X Coeff. utilization)
Statistical analysis of litter size and birth weight was calculated by the analysis of variance and addition~y the covariance analysis for birth weight. RESULTS
At birth, litter size in the control and caffeine-treated groups was identical, thus excluding any possible caffeine-induced increase in foetal resorptions (Table II). In the first litter O-4 stillbirths were observed per group (4 in untreated
74 TABLE II WEIGHT AND LITTER SIZE AT BIRTH The results are given as mean v&es + standard deviation of the mean. There was no signi~cant difference of the litter size between all the groups in either litter (each group was compared with each group using the ANOVA). For the birth weight when each group was compared with each other group the analyses of variance and co-variance showed no significant difference between groups except for the highest dose of caffeine of the first litter where a significantly (P < 0.04)a lower birth weight was observed. Treatment (g caffeine/ kg feed) -untreated 0.25 0.50 1.00
Litter
Number of dams/group
Mean litter size (number of pups)
first first first first
57 30 30 30
12.4*3.0 12.8t 2.4 13.0t1.8 12.722.8
..-.... 6.3t0.7 6.1kO.4 6.1t0.5 6.Oi 0.7a
untreated 0.25 0: 50 1.00
second second second second
49 26 22 20
14.0+ 2.5 13.2k4.3 13.7~ 2.1 14.1k2.9
6.2tOo.6 6.2t:l.O 6.2t0.6 6.1kO.5
Birth weight g
TABLE III GROWTH RATE DURING LACTATION
- FIRST LITTER
Results are given as the group mean of the average weight gain per pup i standard deviation. _-_Pup weight gain Pup weight. gain Number of Treatment of dams during the period during first 4 dams/group (g caffeine/kg feed) 4th to 14th day days (g) During During (g) lactation pregnancy untreated untreated untreated untreated 0.25 0.25 0.50 0.50 1.00 1.00
untreated 0.25 0.50 1.00 untreated 0.25 untreated 0.50 untreated 1.00
10 15 15 15 15 15 15 15 15 14
4.81t0.97 4.9420.79 4.4OkO.78 4.37kO.81 4.59kO.55 4.64t0.69 4.341to.75 4.05*0.94 4.5811.05 4.17*1.02
19.47k2.24 18.83t2.94 17.0314.01 17.6022.75 l&04*2.51 18.69t3.89 17.32kl.82 17.72t2.16 17.85t3.00 19.45t4.60
controls) but there were no stillbirths in the second litter. I-Iowever, since births were not always witnessed it is possible that some stillborn pups were cannibalised by their dams. In the first 4 days Z-7 (7 in untreated control group) of the first litter and l-10 (4 in controls) of the 2nd litter died. In
75
the 4-14 day period very few pups died (an average of 0.5 pups/litter). Deaths were evenly distributed between groups throughout the study. The evaluations showed that birth weight was not influenced by caffeine at the lower dietary concentrations. At 1 g caffeine/kg diet the birth weight was significantly reduced but only at a low probability (P < 0.04) and only in the first litter (Table II). As regards development of litters during lactation, there were no evident abnormalities. Since a significant correlation was observed between litter size and weight at birth a larger litter size resulted in lower birth weight of pups in the litter irrespective of caffeine treatment of the dams. In this regard, using the ANOVA which is not strictly correct in this context, little effect on pup weight was obtained during development, but with the covariance analysis the effect disappeared. It appeared that there were trends and minimal signs of adverse effects only in those pups that were exposed to caffeine at the highest dose level during both intrauterine and postnatal life (Tables III, IV). The concentrations of caffeine in the rat milk are given in Table V. We checked, by weighing their food intake, that the overnight separation needed for these determinations did not affect the quantity of caffeine ingested by the dams. In a separate experiment, an acute dose of 14 and 57 mg caffeine/kg body weight was given by gavage to nursing dams and samples of milk were taken at different time intervals. The results obtained showed that no accumulation of caffeine occurred. TABLE IV GROWTH RATE DURING LACTATION - SECOND LITTER Treatment of dams (g caffeine/kg feed) During pregnant y
During lactation
untreated untreated untreated untreated 0.25 0.25 0.50 0.50 1.00 1.00
untreated 0.25 0.50 1.00 untreated 0.25 untreated 0.50 untreated 1.00
For legend see Table III.
Number of dams/group
Pup weight gain during first 4 days (g)
19 10 10 9 10 14 10 12 9 10
4.9oi1.02 5.17*1.11 4.82tO.86 4.19*0.43 4.8520.79 5.05i1.28 5.43k1.34 4.9OkO.65 5.19*0.58 4.41io.99
Pup weight gain during the period 4th to 14th day (9) 17.6222.29 19.68i1.93 17.69k2.29 19.22i2.26 18.24* 2.97 18.79i3.54 19.67k3.00 18.68* 2.54 i8.94* 2.49 15.9Ok2.14
76 TABLE V CAFFEINE CONTENT IN RAT MILK The values represent the means + S.E.M. of 8 lactating females per group. Litter
Day of lactation
Treatment g caffeine/ kg feed
fig caffeine/ ml milk
10th day
1.0 0.5 0.25
3.5kO.5 not measured not measured
14th day
1.0 0.5 0.25
5.9* 2.4 2.OkO.5 0.7+0.2
10th day
1.0 0.5 0.25
7.0t2.5 2.2k1.2 1.8kO.4
14th day
1.0 0.5 0.25
5.6kO.7 3.Ok1.3 not measured
-____
First
Second
DISCUSSION
Although there is evidence of the importance of maternal nutrition as a determinant of the course and outcome of pregnancy, it has been extremely difficult to establish exactly the correlating factors, particularly between maternal nutrition and birth weight [ 31. Philips and Johnson [ 141 found that 9 different dietary and related factors accounted for 86% of the variability of birth weight in offspring from a large group of pregnant women. Mau [ll] ,also confirmed earlier observations, that smoking during pregnancy results in significantly lower birth weights. Heavy smoking, however, is often associated with excessive coffee drinking. This emphasizes the need for controlled experimental work. Our results indicate that in rats there is no effect of caffeine on birth weight or pup mortality below concentrations of 1 g/kg diet. This corresponds to a dose of about 60 mg/kg/day during gestation. It has been concluded [ 11 that the mean human caffeine consumption figure for any age group does not exceed 3 mg/kg/day. The concentration of caffeine in the rat milk was linearly dose-dependent. Prom the estimation of the quantities of milk ingested daily by the pups, their caffeine intake during the lactation period was estimated to be 2% of that of their nursing mothers as expressed per kg body wt. This low caffeine intake accounts for the fact that the pups were not significantly affected by caffeine ingested during lactation.
77
ACKNOWLEDGEMENT
We are grateful to Dr. L. Vuataz and Dr. R. Munoz-Box for carrying out the s~tistic~ evaluations and Ms. R. Acheson for editorial assistance.
REFERENCES 1 Abrams, FASEB/SCGGS, Hearing Sept. 26 (1977). 2 H, Bartel and M. Gnacikowska, Histological studies on the effe& of Caffeine on the fetal development of mouse extremities, Folia Morphol., 31 (I972) 193. 3 L+Bergner and M.W. Susser,Low birth weight and prenatal nutrition: an interpretative review, Pediatrics, 46 (1970) 946. 4 I. Borlee, M.F. Lechat, A. Bouckaert and C. Mllion, Le caW, facteur de risque pendant la grossesse? Louvain Med., 97 (1978) 279. 6 R.L. Bornschein, D.A. Fox and IA Michaelson, Estimation of daily exposure in neonatal rats receiving lead via dam’s milk, Toxicol. Appl. Pharmacol., 40 (1977) 577. 6 T. Fujii, II. Sasaki and H. ~~~~~~ Teratogenicity of caffeine in mice related to its mode of administration, Jap. J. Pharmacol., Kyoto, 19 (1969) 134. 7 T. Fujii and II. Niihimura, Adverse effects of prolonged administration of caffeine on rat fetus, Toxicol. Appl. Pharmacol., 22 (1972) 449; 8 B.N. Gupta, G.H. Conner and R.F. Langham, A device for collecting milk from guinea pigs, Am. J. Vet. Res., 31 (1970) 567. 9 R.M. Hill, J.P. Craig, M.D. Chaney, L.N. Tennyson and .M.B. MCCulley, Utilisation of Over-The-Counter Drugs during pregnancy, Clin. Obstet Gynecol. 20 (1977) 381. 10 G. Mau, Nahrungs-und Genussmittelkonsum in der Schwangerschaft und seine Auswirkung auf perinatale Sterblichkeit. Friihgeburtlichkeit und andere perinatale Faktoren, Mschr. Kinderklin., 122 (1974) 593. 11 G. Mau, Rauchen und Schwangerschaft, Med. Welt, 26 (1975) 28. 12 H. Milon and J.A. Antonio& Caffeine de@rmination in rat plasma: a comparative study of ~cromethod~~ J. C~orn~~~., 162 (1979) 228. 13 M.M. Nelson and J.O. Fofar, Association between drugs admin@zered during pregnancy and congenital abnormalities’ofthe fetus, Br. Med. J., l(1971) 523. 14 C. Philips and N.E. Johnson, The impact of quality of diet and other factors on birth weight of infants, Amer. J. Clin. Nutr., 30 (1977) 216. 15 P.S. Thayer and C.J. Kensler, Exposure of four generations of mice to caffeine in drinking water, Toxicol. Appl. Pharmacol., 25 [1973) 169. 16 P.S. Weathersbee, L.K. Olsen and J.R. Lodge, Caffeine and pregnancy, Postgrad. Med., 62 (1977) 64.
EFFECT OF CAFFEINE ON RAT OFFSPRING FROM TREATED
H.U. AESCHBACHER,
71
DAMS
H. MILON, A. POOT and H.P. WeRZNER
Experimental Biology Laboratory, 1350 CH-Orbe, Switzerland
Nest16 Products Technical Assistance Co., Ltd.,
(Received June 12th, 1980) (Accepted July 8th, 1980)
SUMMARY
Pregnant Sprague-Dawley rats were given caffeine at 1.0,0.5 and 0.25 g/kg diet during gestation and lactation. At birth, half of the pups from control and treated rats at each dose level were exchanged and cross fostered. Two litters were produced by each animal from each of the experimental groups. Caffeine at dietary concentrations of 0.5 and 0.25 g/kg throughout gestation and lactation had no significant effect on birth weight, litter size or development. There was also no effect at these doses following treatment during either gestation alone, or lactation alone. At 1.0 g/kg there was a slight reduction of birth weight, as well as a trend towards lower weight gain in litters from dams fed the test diet throughout gestation and lactation.
INTRODUCTION
The intake of caffeine, consumed in tea and coffee, as a common ingredient of soft drinks, or in analgesics and OTC drugs has steadily increased worldwide. In recently published epidemiological studies [ 4, 10,161, concern was raised about the possible involvement of caffeine and coffee consumption during pregnancy on spontaneous abortion, premature births, and small-for-dates babies. Hill et al. [9] confirmed data from Nelson and Fofar [13] that OTC drugs are probably consumed by 75% of pregnant women, whereas coffee consumption was much lower at about 45%. Fetal resorptions, teratogenic effects and fetal death have been reported in experiments with rodents [ 2,6, 71. However, Thayer and Kensler [ 151, in an extensive study involving the exposure of 4 generations of mice to caffeine in the drinking water, observed no adverse effects on litter size or on the incidence of fetal abnormalities. Abbreviations:
ANOVA,
analysis of variance; OTC, over-the-counter.
72
Our objectives in the design and conduct of the present “crossoverfoster” study in rats were to investigate possible effects of caffeine when administered during pregnancy and/or lactation, on mortality, birth weight and development of the young. MATERIALS AND METHODS
Sprague-Dawley rats (Charles-River Farms; Saint-Aubin-les-Elbeuf, France) at 11 weeks of age, with initial body weight of approx. 250 g were offered a commercial standard laboratory feed (No. 185) (Nafag A.G., Gossau, Switzerland). This was pelleted at 3-weeks intervals for the controls and, after incorporation of caffeine purum (Fluka, Buchs, Switzerland), for the test groups. The diets were stored at 4°C and were tested for caffeine homogeneity. Tap water was provided ad libitum. Temperature and humidity in the animal rooms were maintained at 22” C and 55% relative humidity, respectively, throughout the study and there were artificial, 12-h day-night light periods. 2 females were mated with 1 male over a 4-day period. Since the influence of caffeine on sperm cells was to be excluded the male and female rats were not treated during the breeding period. Caffeine treatment of females started on the 4th day of the breeding period after removing the males. Following mating, all the females were divided into an untreated control group with 80 animals, and 3 treatment groups of 40 animals each. At the end of gestation the pregnant dams (60 controls or 30 treated animals) were selected randomly and distributed in groups of 15 dams. For the 2nd breeding, only the 15 dams/group which became pregnant at the first mating were remated. Rem~n~g untreated pregnant dams (not used for exchange) were added to the control group, whereas infertile dams were excluded from the study. At birth half of the dams from each group had their pups removed and exchanged either for pups from treated mothers in the case of control dams, or pups from untreated animals in the case of treated dams. The remaining animals per group were maintained with their own pups. EXPERIMENTAL DESIGN (EXAMPLE FOR FIRST LITTER)
Treatment of dams g caffeine/kg diet
Untreated
0.25
0.5
1.0
Number of dams which nursed their own young
15
15
15
15
Number of dams where pups are exchanged after birth between treated and untreated mothers
15 -15 15 P 15 f
15 ,15
73 TABLE I CAFFEINE
INTAKE DURING PREGNANCY
Dose (g caffeine1 kg feed)
1.0 0.5 0.25
AND LACTATION
Mean caffeine enposurea (mg/kg body weight) Pregnancy
Lactation
1st litter
2nd litter
1st litter
2nd litter
62.3 35.3 15.9
62.3 33.2 17.0
120.6 60.3 31.1
107.5 53.6 27.7
aDaily dose calculated from values for food consumption, and concentration of caffeine in diet.
body weight
At birth, the pups were counted, weighed and checked for viability and gross abnormalities. They were counted again and weighed at days 4 and 14 after birth and the average pup weight was calculated, since this method was found to be more precise than weighing individual pups. The pups were studied until 14 days of age when the experimental diets were offered. Caffeine intake of dams during pregnancy and lactation, calculated in relation to body weight from food intake date (Table I), almost doubled during lactation, due to considerably increased food intake. That of pups was estimated by determining the caffeine content in the milk of the treated dams and estimating the daily milk consumption by pups. For this purpose, an additional 10 female rats were treated in an identical manner during pregnancy and lactation and at each of the dose levels of caffeine used in the main experiment. At day 10 and againat day 14 of lactation, the-dams were separated from their pups overnight and milked the’following morning, using a modified method of Gupta et al. [ 81. The caffeine content in the milk samples was determined by gas chromatography [12]. To estimate the amount of milk absorbed by a pup the following formula was used 153. Milk intake g/day
Body wt. (g) X Calorie requirement (k&/g
body wt./day)
= Gross energy value (kcal/g X Coeff. utilization)
Statistical analysis of litter size and birth weight was calculated by the analysis of variance and addition~y the covariance analysis for birth weight. RESULTS
At birth, litter size in the control and caffeine-treated groups was identical, thus excluding any possible caffeine-induced increase in foetal resorptions (Table II). In the first litter O-4 stillbirths were observed per group (4 in untreated
74 TABLE II WEIGHT AND LITTER SIZE AT BIRTH The results are given as mean v&es + standard deviation of the mean. There was no signi~cant difference of the litter size between all the groups in either litter (each group was compared with each group using the ANOVA). For the birth weight when each group was compared with each other group the analyses of variance and co-variance showed no significant difference between groups except for the highest dose of caffeine of the first litter where a significantly (P < 0.04)a lower birth weight was observed. Treatment (g caffeine/ kg feed) -untreated 0.25 0.50 1.00
Litter
Number of dams/group
Mean litter size (number of pups)
first first first first
57 30 30 30
12.4*3.0 12.8t 2.4 13.0t1.8 12.722.8
..-.... 6.3t0.7 6.1kO.4 6.1t0.5 6.Oi 0.7a
untreated 0.25 0: 50 1.00
second second second second
49 26 22 20
14.0+ 2.5 13.2k4.3 13.7~ 2.1 14.1k2.9
6.2tOo.6 6.2t:l.O 6.2t0.6 6.1kO.5
Birth weight g
TABLE III GROWTH RATE DURING LACTATION
- FIRST LITTER
Results are given as the group mean of the average weight gain per pup i standard deviation. _-_Pup weight gain Pup weight. gain Number of Treatment of dams during the period during first 4 dams/group (g caffeine/kg feed) 4th to 14th day days (g) During During (g) lactation pregnancy untreated untreated untreated untreated 0.25 0.25 0.50 0.50 1.00 1.00
untreated 0.25 0.50 1.00 untreated 0.25 untreated 0.50 untreated 1.00
10 15 15 15 15 15 15 15 15 14
4.81t0.97 4.9420.79 4.4OkO.78 4.37kO.81 4.59kO.55 4.64t0.69 4.341to.75 4.05*0.94 4.5811.05 4.17*1.02
19.47k2.24 18.83t2.94 17.0314.01 17.6022.75 l&04*2.51 18.69t3.89 17.32kl.82 17.72t2.16 17.85t3.00 19.45t4.60
controls) but there were no stillbirths in the second litter. I-Iowever, since births were not always witnessed it is possible that some stillborn pups were cannibalised by their dams. In the first 4 days Z-7 (7 in untreated control group) of the first litter and l-10 (4 in controls) of the 2nd litter died. In
75
the 4-14 day period very few pups died (an average of 0.5 pups/litter). Deaths were evenly distributed between groups throughout the study. The evaluations showed that birth weight was not influenced by caffeine at the lower dietary concentrations. At 1 g caffeine/kg diet the birth weight was significantly reduced but only at a low probability (P < 0.04) and only in the first litter (Table II). As regards development of litters during lactation, there were no evident abnormalities. Since a significant correlation was observed between litter size and weight at birth a larger litter size resulted in lower birth weight of pups in the litter irrespective of caffeine treatment of the dams. In this regard, using the ANOVA which is not strictly correct in this context, little effect on pup weight was obtained during development, but with the covariance analysis the effect disappeared. It appeared that there were trends and minimal signs of adverse effects only in those pups that were exposed to caffeine at the highest dose level during both intrauterine and postnatal life (Tables III, IV). The concentrations of caffeine in the rat milk are given in Table V. We checked, by weighing their food intake, that the overnight separation needed for these determinations did not affect the quantity of caffeine ingested by the dams. In a separate experiment, an acute dose of 14 and 57 mg caffeine/kg body weight was given by gavage to nursing dams and samples of milk were taken at different time intervals. The results obtained showed that no accumulation of caffeine occurred. TABLE IV GROWTH RATE DURING LACTATION - SECOND LITTER Treatment of dams (g caffeine/kg feed) During pregnant y
During lactation
untreated untreated untreated untreated 0.25 0.25 0.50 0.50 1.00 1.00
untreated 0.25 0.50 1.00 untreated 0.25 untreated 0.50 untreated 1.00
For legend see Table III.
Number of dams/group
Pup weight gain during first 4 days (g)
19 10 10 9 10 14 10 12 9 10
4.9oi1.02 5.17*1.11 4.82tO.86 4.19*0.43 4.8520.79 5.05i1.28 5.43k1.34 4.9OkO.65 5.19*0.58 4.41io.99
Pup weight gain during the period 4th to 14th day (9) 17.6222.29 19.68i1.93 17.69k2.29 19.22i2.26 18.24* 2.97 18.79i3.54 19.67k3.00 18.68* 2.54 i8.94* 2.49 15.9Ok2.14
76 TABLE V CAFFEINE CONTENT IN RAT MILK The values represent the means + S.E.M. of 8 lactating females per group. Litter
Day of lactation
Treatment g caffeine/ kg feed
fig caffeine/ ml milk
10th day
1.0 0.5 0.25
3.5kO.5 not measured not measured
14th day
1.0 0.5 0.25
5.9* 2.4 2.OkO.5 0.7+0.2
10th day
1.0 0.5 0.25
7.0t2.5 2.2k1.2 1.8kO.4
14th day
1.0 0.5 0.25
5.6kO.7 3.Ok1.3 not measured
-____
First
Second
DISCUSSION
Although there is evidence of the importance of maternal nutrition as a determinant of the course and outcome of pregnancy, it has been extremely difficult to establish exactly the correlating factors, particularly between maternal nutrition and birth weight [ 31. Philips and Johnson [ 141 found that 9 different dietary and related factors accounted for 86% of the variability of birth weight in offspring from a large group of pregnant women. Mau [ll] ,also confirmed earlier observations, that smoking during pregnancy results in significantly lower birth weights. Heavy smoking, however, is often associated with excessive coffee drinking. This emphasizes the need for controlled experimental work. Our results indicate that in rats there is no effect of caffeine on birth weight or pup mortality below concentrations of 1 g/kg diet. This corresponds to a dose of about 60 mg/kg/day during gestation. It has been concluded [ 11 that the mean human caffeine consumption figure for any age group does not exceed 3 mg/kg/day. The concentration of caffeine in the rat milk was linearly dose-dependent. Prom the estimation of the quantities of milk ingested daily by the pups, their caffeine intake during the lactation period was estimated to be 2% of that of their nursing mothers as expressed per kg body wt. This low caffeine intake accounts for the fact that the pups were not significantly affected by caffeine ingested during lactation.
77
ACKNOWLEDGEMENT
We are grateful to Dr. L. Vuataz and Dr. R. Munoz-Box for carrying out the s~tistic~ evaluations and Ms. R. Acheson for editorial assistance.
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