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Developmental effects of intraperitoneal saline injections in neonatal rats
Life Sciences, Vol. 29, pp. 143-149 Printed in the U.S.A.
Pergamon Press
DEVELOPh~NTAL EFFECTS OF INTRAPERITONEAL SALINE INJECTIONS IN NEONATAL RATS
Sandra M. Martin and Gary P. Moberg Department of Animal Science, University of California Davis 95616 (Received in final form May 7, 1981)
Summar X The response to neonatal intraperitonal t i P ) s a l i n e injections was investigated in neonatal male and female rats from split- and whole-litters. The results demonstrate that male or female rats housed under s p l i t - l i t t e r conditions and subjected to daily IP injections during the early neonatal period have the same body weight and plasma corticosteroid and testosterone (males only) levels as littermate controls. On the other hand, treated male and female animals from whole-litters weigh significantly less than controls. Additionally, plasma testosterone and cortisteroids from treated males did not differ from controls at any age tested, but treated females from whale-litters had significantly elevated corticoids at days 100 and IS0 when compared to controls. These results suggest that when doing developmental studies involving IP injections, it would be advisable to house animals under split-litter conditions.
Studies concerned with the effects of various agents an neonatal development often involve intraperitoneal (IP) drug administration, but in most af these investigations the method of drug administration has been considered to have no influence on the developmental changes. This view, however, has been questioned by a study where male rats that were treated with IP saline injections from days 2 to 12 after birth demonstrated significantly increased body weights at 200 days when compared to noninjected controls (I). These investigators raised the interesting possibility that androgens were responsible for these differences, but the o~thors did not actually measure circulating levels of the steroid. Another concern in developmental studies is litter composition. Even when pups from different clams are nat combined in the same litter, a question remains of whether to have treated and control pups in separate litters or whether to combine the groups within the same litter. In spite of this problem, few studies have actually investigated the potential developmental differences resulting from split- vs. whole-litter design. However, one review article has raised the question maintaining that "all animals within a litter be given the same experimental treatment" (2). The purposes of this study were threefold: (a) to test and extend the results of the study concerning the developmental effects of IP saline injections in male rats; (b) to determine if female rats respond to neonatal injections in the same manner as male rats~ and (c) to investigate the potential interaction between control and experimental pups in the same litter. 0024-3205/81/020143-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
144
Saline Injection and Development
Vol. 29, No. 2, 1981
Materials and Methods Sprague-Dawley rats (from the University of California, Davis colony) were used in this experiment. On the second day post partum (day 2), litters were standardized to eight pups, four male and four female. Each litter was housed with its mother in a plastic cage with woad shavings for bedding under controlled temperatures (23ac + 4°C) and light (14:10, L:D) conditions with food and water available ad libitum. Litter~ were weaned on day 21 with four pups of the same sex per cage. On day SO, animals were further separated into pairs and thus housed for the duration of the experiment. To determine the interaction, if any, between control and treated pups in the same litter, split- vs. whole-litter design was employed. Split-litters were composed of four control (two male, two female) and four treated (two male, two female) pups, while whole-litters consisted of either all control or all treated pups. The experiment consisted of a total of 5 split-litters and 12 (6 control and 6 treated) whole-litters. In all cases, treated animals received a single daily IP injection of I00 lJ1 normal saline on days 2 to 12 after birth, while controls were not treated. All animals were weighed weekly. Animals from split-litters were sacrified at 200 days of age, while selected experimental and control whole-litters were sacrificed on days 100, IS0, and 200. Under these experimental conditions, the order of sacrifice within litters or between treatment groups had no effect on corticosteroid values. All animals were sacrificed between 0800 and 1000 hours and trunk blood collected in chilled tubes and maintained on ice until centrifuged. Plasma aliquots were maintained at -20°C until analyzed for hormone concentrat ions. Plasma corticosteroids were determined, with modifications, by a competitive protein-binding method established for rat plasma (3). The assay-binding protein was stripped of endogenous steroids on a sephadex column (4) and diluted to S percent with borate buffer (0.0SM, pH 9.0). To3100 ml stock-binding protein solution (5 percent) was added 0.8 ml (50 tJCi/ml) 1,2,6,7- H(N)-corticosterone (New England Nuclear). On the day of use, the stock solution was dilute~, with borate buffer to a 0.25 percent working solution. Separation of bound and free H-corticosterone was accomplished by the use of small sephadex columns (4). Plasma testosterone (males only) was determined by IRIA (S) using specific antiserum (S-250, I I-BSA) kindly supplied by G. D. Niswender. To calculate the significance of differences between groups, a least squares analysis of variance was used (6). Analysis of variance utilizing a hierarchical model was used in the examination of whole-litter experiments. A cross-classified model was used for split-litters.
Results
At no age tested (7 to 196 days) did IP saline injections result in statistical differences in the weights of either male or female rats housed in split-litters when compared to their littermate controls (Fig. I). Whole-litter design combined with neonatal IP saline injections did result, however, in reduced weight for both the treated males and the treated females (Fig. 2). Statistical analysis demonstrated these differences to be significant on days 7, 14, 21, 112, 119, 126, and 133 for the males and on days 14, 126, and 140 to 196 far the females (Table I). It should be noted that the 200-day weights for control males from split-litters were slightly larger than those from control males from whole-litters. Statistical anlaysis of the weights of whole-litter animals revealed variability within the litters, which may have been responsible for the observed phenomenon.
Vol. 29, No. 2, 1981
700
Saline Injection and Development
145
SPLIT-LITTERS Soline Treated .... Control
600
MALE 500 FIG. I ul
E
o
Results of saline treatment on postnatal days 2 to 12 on body weight in male and female rots from split-litters. Each value represents the mean + S.E.M. of at least 7 animals°
400
t"1-
_~ 500 i,i
FEMALE
200
IOO
O 1" O
I 50
1 I OO
I 150
J 200
AGE (days) Plasma concentrations of neither corticosteroids nor testosterone were different for treated vs. control male rats from either split- (day 200) or whole-litters (days 100, 150, 200) when compared to controls (Table 2). While the female treated rats from split- or whole-litters demonstrated no differences in plasma cortieosteroid concentrations from their respective controls at day 200, on days 100 and 150 the whale-litter treated females had significantly elevated plasma carticoids with respect to controls (Table 2). Split-litters had plasma hormones determined on day 200 only. Discussion
These data demonstrate that neonatal saline injections combined with split-litter design result in no difference in weight gain, whereas injected animals housed separately from noninjected animals (whole-litter design) exhibit reduced body weights when compared to their respective controls. Other studies involving whole-litter design, with handling as the neonatal treatment, have reported varying results an weight gain. In some studies, treated
146
Saline Injection and Development
Vol. 29, No. 2, 1981
WHOLE-LITTERS ..... Saline Treated ........... C o n t r o l
600
500
LE
-
FIG. 2
f
E 400 o ~
.'
i
-r
LD hi
300 -
.
~
Results of saline treatment on postnatal days 2 to 12 on male and female rats from wholelitters. Each value represents the mean + S.E.M. of at least 7 animals.
.. .
.
.
FEMALE
_..'}:
"1 .Jl"
200
I00
-
/ /
O
/ O
I 50
I I00 AGE
I 150
I 200
(days)
animals weighed more than nontreated (7,8,9,10), while in others either no differences were observed (I I) or the treated animals weighed less than the nontreated controls
(12). With results similar to the present study, another investigation (13) reported weight depression at 30 days in treated animals as a result of vehicle injection on days 12 to 14, 22 to 24, or 22 to 29; however, the differences were transient and were not observed by day 50 of age. The different time course of weight changes reported here probably reflects the fact that the present experiment involves treatment in the early neonatal period (from day 2 to 12). As discussed in the introduction, neonatal saline injections have been reported to result in increased weight in treated animals at 200 days of age, but in this case it is not clear whether split- or whole-litters were utilized. The mechanism by which early neonatal injections lead to depressed body weights in whole- but not split-litters is not evident. The results of the present study suggest that androgens do not participate, since the phenomenum is observed in both males and females, and plasma testosterone concentratians in treated and control adult males do
Vol. 29, No. 2, 1981
Saline Injection and Development
147
not differ in animals from split- or whole-litters. The results of the plasma corticosteroid determinations are difficult to interpret. In spite of difference in body weight, corticoid levels do not differ between treated and control males from whole litters, yet treated females from whole litters have significantly elevated levels. Since cortlcoids are known to depress growth (14,15), they may have participated in the body weight depression observed in the females. Such an explanation, however, seems unlikely in view of the fact that the same elevation of plasma corticoids is not seen in males with decreased body weights. Recent studies have reported that the maternal response to animals returned to the nest can affect pup weight gain (16,17,18). This information strongly suggests that the phenomena observed in the present investigation is a result of the maternal response elicited by the treated pups when they were returned to the nest. It follows then that the injected pups elicited a response from the mother that was also experienced by the control pups in the split-litters, resulting in similar weights for all pups within a litter. On the other hand, the control pups from the whole-litters were not subjected to treated pup induced altered maternal behavior and, consequently, present body weights different from comparable treated animals.
TABLE I Analysis of Variance of the Effects of Neonatal Saline Treatment on Body Weight in Male and Female Rats from Whole-Litters MALE Age(days)
F(df/df)
112
5.8(y4(I/36) 12.374(I/36) 9.8o2(I/36) 4.381(I/21)
7 14 21
FEMALE p <
F(df/df)
.025 .005
p <
NS 5.067 (I/41 )
.05
.005
NS
.05
NS
I19
8.077(I/21)
.01
126
7.112(1/21)
.025
133
5.328(I/20)
.05
NS 5.791 (I/23)
.025 NS
140
NS
5.211(1/23)
.05
147
NS
4.786(1/14)
.05
154
NS
5.169(1/14)
.05
161
NS
6.916(I/14)
.025
168
NS
5.666(I/14)
.05
175
NS
4.981(1/14)
.05
182
NS
.05
189
NS
5.998(I/14) 6.297(I/14)
.05
196
NS
7.54 (I/14)
.025
I SO
16.5+_1.2 (4)
10.0+_2.3 (8)
Saline Treated
p <.001 compared to respective controls
p < .025
eNumber of subjects
CTestosterone in ng/mI dMean + S.E.M.
bcorticosteroids in !Jg/100 ml
aAge in days
7.7+_2.3(8)
6.2+_1.8(3)
5.6+0.7(8)
CS
2.4+_0.3(10)
1.5+_0.2
1.5+0.4
Tc
Control
Female
1.7+_0.6(8)
Saline Treated
1000
1.5+0.2d(6) e
CS b
Control
Male
SUBJECTS
0.8+-0.2
1.5+0.2
T
WHOLE-LITTERS
8.3_+1.9(7)
10.~+_2.d(5)
6.8+_I .5(I 5)
2.8+0.6~8)
CS
200
1.0Z0. I
0.9+0.2
T
5.2+_1.7_(10)
6.7_+1.4(8)
3.9+-0.5(9)
4.6+0.8(10)
CS
200
0.9+_0.I
0.9+0.2
T
SPLIT-LITTERS
The Effects of Neonatal Saline Treatment on Plasma Corticosteroid and Testosterone Concentrations at 100, IS0~ end 200 Days of Age in Rats from Split- and Whole-Litters
TABLE II
OO
h~
O
< o
f~
<
r~
O
c~ ¢-t
2.
~°
Oo
T.
Vol. 29, No. 2, 1981
Saline Injection and Development
149
In conclusion, it appears from these data that studies investigating the developmental effects of neonatal treatment involving injections must include both treated and nontreated animals in the same litter. This split-litter design would allow the nonspecific maternal response to pup treatment to become "background," and, therefore, the specific developmental results of treatment would more easily be de l ineated.
Acknowledgements The authors wish to thank Dr. M. S. Barkley of the Animal Physiology Department, University of California, Davis for assaying plasma testosterone, and Dr. E. J. Pollak and C. A. Piper of the Animal Science Department, University of California, Davis for their assistance with the statistical analysis.
References I. 2.
R. ERDOSOVA, B. JAKOUBEK, and M. KRAUS, Experientia 37 62-63 (1975). V.H. DENENBERG, Laborator X Techniques in Neuropsychology and Neurobiology. Ed. by R.D. Myers, pp. 127-147, Academic Press, New York (1977). 3. E.H.D. CAMERON, and J.J. SCARISBRICK, J. Steroid Biochem. 4 577-584 (1973). 4. H.M. BASSETT, and N.T. HINKS, Endocrinol. 44 387-403 (1969). 5. M.S. BARKLEY, and B.D. GOLDMAN, Hormones and Behov. 8 208-218 (1977). 6. W.R. HARVEY, USDA, ARS H-4 1-157 (1975). 7. J. ALTMAN, G.D. DAS, and W.J. ANDERSON, Dev. Psychobiol. I 10-20 (1968). 8. B.M. CINES, and M. WINICK, Dev. Psychobiol. 12 381-389 (1979). 9. S. LEVINE, Dev. Psychobiol. I 6;7-70 (1968). I0. R.E. MCMICHAEL, J. Comp.-Physiol. Psych. 62 433-436; (1966). II. R. ADER, J. Camp. Physiol. Psychol. 60 233-238 (1965). 12. G. SIECK, and J.A. RAMALEY, Physiol. Behav. 15 487-489 (1975). 13. R.J. LORENZ, B.J. BRANCH, and A.N. TAYLOR, Proc. Sac. Exp. Biol. Med. 145 528-532 (I 974). 14. E. HOWARD, J. Neurochem. 12 181-191 (1965). 5. S. SCHAPIRO, Gen. Camp. Endocrinol. 10 214-228 (1968). 6;. P.A. RUSSELL, Psychol. Bull. 75 192-202 (1971). 7. W.D. SMOTHERMAN, S.P. MENDOZA, and S. LEVINE, Dev. Psychobiol. 10 365-371 (I 977). 8. R. VlLLESCAS, R.W. BELL, I_. WRIGHT, and M. KUFNER, Dev. Psychobiol. 10 323-329 (I 977).
Pergamon Press
DEVELOPh~NTAL EFFECTS OF INTRAPERITONEAL SALINE INJECTIONS IN NEONATAL RATS
Sandra M. Martin and Gary P. Moberg Department of Animal Science, University of California Davis 95616 (Received in final form May 7, 1981)
Summar X The response to neonatal intraperitonal t i P ) s a l i n e injections was investigated in neonatal male and female rats from split- and whole-litters. The results demonstrate that male or female rats housed under s p l i t - l i t t e r conditions and subjected to daily IP injections during the early neonatal period have the same body weight and plasma corticosteroid and testosterone (males only) levels as littermate controls. On the other hand, treated male and female animals from whole-litters weigh significantly less than controls. Additionally, plasma testosterone and cortisteroids from treated males did not differ from controls at any age tested, but treated females from whale-litters had significantly elevated corticoids at days 100 and IS0 when compared to controls. These results suggest that when doing developmental studies involving IP injections, it would be advisable to house animals under split-litter conditions.
Studies concerned with the effects of various agents an neonatal development often involve intraperitoneal (IP) drug administration, but in most af these investigations the method of drug administration has been considered to have no influence on the developmental changes. This view, however, has been questioned by a study where male rats that were treated with IP saline injections from days 2 to 12 after birth demonstrated significantly increased body weights at 200 days when compared to noninjected controls (I). These investigators raised the interesting possibility that androgens were responsible for these differences, but the o~thors did not actually measure circulating levels of the steroid. Another concern in developmental studies is litter composition. Even when pups from different clams are nat combined in the same litter, a question remains of whether to have treated and control pups in separate litters or whether to combine the groups within the same litter. In spite of this problem, few studies have actually investigated the potential developmental differences resulting from split- vs. whole-litter design. However, one review article has raised the question maintaining that "all animals within a litter be given the same experimental treatment" (2). The purposes of this study were threefold: (a) to test and extend the results of the study concerning the developmental effects of IP saline injections in male rats; (b) to determine if female rats respond to neonatal injections in the same manner as male rats~ and (c) to investigate the potential interaction between control and experimental pups in the same litter. 0024-3205/81/020143-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
144
Saline Injection and Development
Vol. 29, No. 2, 1981
Materials and Methods Sprague-Dawley rats (from the University of California, Davis colony) were used in this experiment. On the second day post partum (day 2), litters were standardized to eight pups, four male and four female. Each litter was housed with its mother in a plastic cage with woad shavings for bedding under controlled temperatures (23ac + 4°C) and light (14:10, L:D) conditions with food and water available ad libitum. Litter~ were weaned on day 21 with four pups of the same sex per cage. On day SO, animals were further separated into pairs and thus housed for the duration of the experiment. To determine the interaction, if any, between control and treated pups in the same litter, split- vs. whole-litter design was employed. Split-litters were composed of four control (two male, two female) and four treated (two male, two female) pups, while whole-litters consisted of either all control or all treated pups. The experiment consisted of a total of 5 split-litters and 12 (6 control and 6 treated) whole-litters. In all cases, treated animals received a single daily IP injection of I00 lJ1 normal saline on days 2 to 12 after birth, while controls were not treated. All animals were weighed weekly. Animals from split-litters were sacrified at 200 days of age, while selected experimental and control whole-litters were sacrificed on days 100, IS0, and 200. Under these experimental conditions, the order of sacrifice within litters or between treatment groups had no effect on corticosteroid values. All animals were sacrificed between 0800 and 1000 hours and trunk blood collected in chilled tubes and maintained on ice until centrifuged. Plasma aliquots were maintained at -20°C until analyzed for hormone concentrat ions. Plasma corticosteroids were determined, with modifications, by a competitive protein-binding method established for rat plasma (3). The assay-binding protein was stripped of endogenous steroids on a sephadex column (4) and diluted to S percent with borate buffer (0.0SM, pH 9.0). To3100 ml stock-binding protein solution (5 percent) was added 0.8 ml (50 tJCi/ml) 1,2,6,7- H(N)-corticosterone (New England Nuclear). On the day of use, the stock solution was dilute~, with borate buffer to a 0.25 percent working solution. Separation of bound and free H-corticosterone was accomplished by the use of small sephadex columns (4). Plasma testosterone (males only) was determined by IRIA (S) using specific antiserum (S-250, I I-BSA) kindly supplied by G. D. Niswender. To calculate the significance of differences between groups, a least squares analysis of variance was used (6). Analysis of variance utilizing a hierarchical model was used in the examination of whole-litter experiments. A cross-classified model was used for split-litters.
Results
At no age tested (7 to 196 days) did IP saline injections result in statistical differences in the weights of either male or female rats housed in split-litters when compared to their littermate controls (Fig. I). Whole-litter design combined with neonatal IP saline injections did result, however, in reduced weight for both the treated males and the treated females (Fig. 2). Statistical analysis demonstrated these differences to be significant on days 7, 14, 21, 112, 119, 126, and 133 for the males and on days 14, 126, and 140 to 196 far the females (Table I). It should be noted that the 200-day weights for control males from split-litters were slightly larger than those from control males from whole-litters. Statistical anlaysis of the weights of whole-litter animals revealed variability within the litters, which may have been responsible for the observed phenomenon.
Vol. 29, No. 2, 1981
700
Saline Injection and Development
145
SPLIT-LITTERS Soline Treated .... Control
600
MALE 500 FIG. I ul
E
o
Results of saline treatment on postnatal days 2 to 12 on body weight in male and female rots from split-litters. Each value represents the mean + S.E.M. of at least 7 animals°
400
t"1-
_~ 500 i,i
FEMALE
200
IOO
O 1" O
I 50
1 I OO
I 150
J 200
AGE (days) Plasma concentrations of neither corticosteroids nor testosterone were different for treated vs. control male rats from either split- (day 200) or whole-litters (days 100, 150, 200) when compared to controls (Table 2). While the female treated rats from split- or whole-litters demonstrated no differences in plasma cortieosteroid concentrations from their respective controls at day 200, on days 100 and 150 the whale-litter treated females had significantly elevated plasma carticoids with respect to controls (Table 2). Split-litters had plasma hormones determined on day 200 only. Discussion
These data demonstrate that neonatal saline injections combined with split-litter design result in no difference in weight gain, whereas injected animals housed separately from noninjected animals (whole-litter design) exhibit reduced body weights when compared to their respective controls. Other studies involving whole-litter design, with handling as the neonatal treatment, have reported varying results an weight gain. In some studies, treated
146
Saline Injection and Development
Vol. 29, No. 2, 1981
WHOLE-LITTERS ..... Saline Treated ........... C o n t r o l
600
500
LE
-
FIG. 2
f
E 400 o ~
.'
i
-r
LD hi
300 -
.
~
Results of saline treatment on postnatal days 2 to 12 on male and female rats from wholelitters. Each value represents the mean + S.E.M. of at least 7 animals.
.. .
.
.
FEMALE
_..'}:
"1 .Jl"
200
I00
-
/ /
O
/ O
I 50
I I00 AGE
I 150
I 200
(days)
animals weighed more than nontreated (7,8,9,10), while in others either no differences were observed (I I) or the treated animals weighed less than the nontreated controls
(12). With results similar to the present study, another investigation (13) reported weight depression at 30 days in treated animals as a result of vehicle injection on days 12 to 14, 22 to 24, or 22 to 29; however, the differences were transient and were not observed by day 50 of age. The different time course of weight changes reported here probably reflects the fact that the present experiment involves treatment in the early neonatal period (from day 2 to 12). As discussed in the introduction, neonatal saline injections have been reported to result in increased weight in treated animals at 200 days of age, but in this case it is not clear whether split- or whole-litters were utilized. The mechanism by which early neonatal injections lead to depressed body weights in whole- but not split-litters is not evident. The results of the present study suggest that androgens do not participate, since the phenomenum is observed in both males and females, and plasma testosterone concentratians in treated and control adult males do
Vol. 29, No. 2, 1981
Saline Injection and Development
147
not differ in animals from split- or whole-litters. The results of the plasma corticosteroid determinations are difficult to interpret. In spite of difference in body weight, corticoid levels do not differ between treated and control males from whole litters, yet treated females from whole litters have significantly elevated levels. Since cortlcoids are known to depress growth (14,15), they may have participated in the body weight depression observed in the females. Such an explanation, however, seems unlikely in view of the fact that the same elevation of plasma corticoids is not seen in males with decreased body weights. Recent studies have reported that the maternal response to animals returned to the nest can affect pup weight gain (16,17,18). This information strongly suggests that the phenomena observed in the present investigation is a result of the maternal response elicited by the treated pups when they were returned to the nest. It follows then that the injected pups elicited a response from the mother that was also experienced by the control pups in the split-litters, resulting in similar weights for all pups within a litter. On the other hand, the control pups from the whole-litters were not subjected to treated pup induced altered maternal behavior and, consequently, present body weights different from comparable treated animals.
TABLE I Analysis of Variance of the Effects of Neonatal Saline Treatment on Body Weight in Male and Female Rats from Whole-Litters MALE Age(days)
F(df/df)
112
5.8(y4(I/36) 12.374(I/36) 9.8o2(I/36) 4.381(I/21)
7 14 21
FEMALE p <
F(df/df)
.025 .005
p <
NS 5.067 (I/41 )
.05
.005
NS
.05
NS
I19
8.077(I/21)
.01
126
7.112(1/21)
.025
133
5.328(I/20)
.05
NS 5.791 (I/23)
.025 NS
140
NS
5.211(1/23)
.05
147
NS
4.786(1/14)
.05
154
NS
5.169(1/14)
.05
161
NS
6.916(I/14)
.025
168
NS
5.666(I/14)
.05
175
NS
4.981(1/14)
.05
182
NS
.05
189
NS
5.998(I/14) 6.297(I/14)
.05
196
NS
7.54 (I/14)
.025
I SO
16.5+_1.2 (4)
10.0+_2.3 (8)
Saline Treated
p <.001 compared to respective controls
p < .025
eNumber of subjects
CTestosterone in ng/mI dMean + S.E.M.
bcorticosteroids in !Jg/100 ml
aAge in days
7.7+_2.3(8)
6.2+_1.8(3)
5.6+0.7(8)
CS
2.4+_0.3(10)
1.5+_0.2
1.5+0.4
Tc
Control
Female
1.7+_0.6(8)
Saline Treated
1000
1.5+0.2d(6) e
CS b
Control
Male
SUBJECTS
0.8+-0.2
1.5+0.2
T
WHOLE-LITTERS
8.3_+1.9(7)
10.~+_2.d(5)
6.8+_I .5(I 5)
2.8+0.6~8)
CS
200
1.0Z0. I
0.9+0.2
T
5.2+_1.7_(10)
6.7_+1.4(8)
3.9+-0.5(9)
4.6+0.8(10)
CS
200
0.9+_0.I
0.9+0.2
T
SPLIT-LITTERS
The Effects of Neonatal Saline Treatment on Plasma Corticosteroid and Testosterone Concentrations at 100, IS0~ end 200 Days of Age in Rats from Split- and Whole-Litters
TABLE II
OO
h~
O
< o
f~
<
r~
O
c~ ¢-t
2.
~°
Oo
T.
Vol. 29, No. 2, 1981
Saline Injection and Development
149
In conclusion, it appears from these data that studies investigating the developmental effects of neonatal treatment involving injections must include both treated and nontreated animals in the same litter. This split-litter design would allow the nonspecific maternal response to pup treatment to become "background," and, therefore, the specific developmental results of treatment would more easily be de l ineated.
Acknowledgements The authors wish to thank Dr. M. S. Barkley of the Animal Physiology Department, University of California, Davis for assaying plasma testosterone, and Dr. E. J. Pollak and C. A. Piper of the Animal Science Department, University of California, Davis for their assistance with the statistical analysis.
References I. 2.
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