We investigated the influence of maternal salt restriction during mating or gestation on birth rate and offspring growth in Dahl salt-sensitive rats (DS). DS were divided into 5 groups: DS fed a low-salt (0.3% NaCl, w/w) (DS-low) or high-salt (4% NaCl, w/w) diet (DS-high) during mating and DS-high or DS-low during gestation, and DS fed regular chow (0.75% NaCl, w/w) (DS-regular) throughout mating and gestation. During the unspecified periods, the rats were given regular chow. DS-low during mating delivered fewer infants than high-salt mothers (
For the last several decades, increased salt intake has been blamed for hypertension and related diseases, and a world-wide campaign on salt reduction commenced. Since the 1980s many investigators have reported serious problems in adult physiological and psychological health that are related to excessive salt intake. Despite such trends towards salt reduction and to our surprise, a recent meta-analysis including 25 previous studies on salt intake and cardiovascular events demonstrated that excessively low-salt intake is associated with an increase in cardiovascular events rather than benefits [
Some researchers reported that, particularly during pregnancy, low-salt intake is associated with adverse effects, that is, low birth weight and insulin resistance in offspring in adulthood [
Accordingly, an increasing amount of evidence has suggested that fetal programming during gestation may play an important role in the development of metabolism-related disorders during adulthood [
To our knowledge, however, few studies have investigated the influence that maternal low-salt intake has on the process of fetal growth. There is a necessity to separately examine the influence of low-salt diet on fertilization and on fetal growth. Moreover, it is of interest to disclose the influence of low-salt intake in mothers on the growth of babies after weaning.
Thus, in the present study, we hypothesized that low-salt intake during mating (early pregnancy period) or pregnancy period influences fertilization and fetal growth and causes a decrease in the number of newborn pups. To test this hypothesis, we investigated the effects of a low-salt diet during mating or pregnancy on birth and growth rates of offspring in Dahl salt-sensitive (Dahl S) rats. Moreover, we examined the influence of low-salt intake on the growth of babies after weaning.
We obtained 4-week-old male and female Dahl salt-sensitive (Dahl S) rats from Sankyo Laboratories, Inc., Tokyo, Japan. The rats were fed regular chow (0.75% NaCl, w/w) (CEL Rodent Diet CE-7, CLEA Japan, Inc., Tokyo, Japan). At 10 weeks of age, the rats were allowed to mate for 1 week (mating period (early pregnancy period)). Next, the mated female rats were placed in separate cages for the pregnancy period. Three weeks after mating, the mothers began delivering pups. We monitored the number of mothers that delivered pups, the number of pups delivered, and the growth of pups until weaning.
Dahl S rats were divided into 5 groups (Figure
Protocol of the study. This graph shows each type of salt diet and the time period in which it was fed to the Dahl S rats. 0.3% NaCl, a low-salt (0.3% NaCl, w/w) diet; 0.75% NaCl, regular (0.75% NaCl, w/w) diet; and 4% NaCl, a high-salt (4% NaCl, w/w) diet.
Dahl S rats were fed the regular (0.75% NaCl, w/w) chow during the periods unspecified. Water was available
All statistical analyses were performed using STATISTICA software (StatSoft, Tulsa, OK). Values were expressed as means ± SD. Differences were assessed by
We followed the guidelines for experimental animal handling, and our study was approved by the Animal Care Committee of the Kyoritsu Women’s University. The experiment was conducted in accordance with the National Institutes of Health (NIH) guidelines.
Dahl S rats fed a low-salt diet during the mating (early pregnancy) period delivered less pups compared with those fed a high-salt diet (Table
Number of mothers that delivered pups.
Group | Diet | Mother | Statistics | |||||
---|---|---|---|---|---|---|---|---|
Mating | Pregnant | Total | Delivered | Babies/mother* |
|
|
Versus | |
(NaCl, w/w) | (NaCl, w/w) | (rats) | (rats) | (rats) | ||||
MHPR | 4.0% | 0.75% | 7 | 7 |
|
|||
MLPR | 0.3% | 0.75% | 7 | 4 |
|
3.82 | 0.050 | MHPR |
|
||||||||
MRPR | 0.75% | 0.75% | 8 | 7 |
|
0.94 | 0.332 | MHPR |
1.76 | 0.184 | MLPR | ||||||
1.45 | 0.228 | MRPH | ||||||
2.33 | 0.127 | MRPL | ||||||
|
||||||||
MRPH | 0.75% | 4.0% | 11 | 11 |
|
|||
MRPL | 0.75% | 0.3% | 11 | 6 |
|
6.87 | 0.011 | MRPH |
MHPR: mothers fed the high-salt diet during mating period and the regular chow during pregnancy period; MLPR: mothers fed the low-salt diet during mating period and the regular chow during pregnancy period; MRPR: mothers fed the regular chow during mating period and during pregnancy period; MRPH: mothers fed the regular chow during mating period and the high-salt diet pregnant period; MRPL: mothers fed the regular chow during mating period and the low-salt diet during pregnant period. Total: the total number of mothers; delivered: the number of mothers that gave birth to pups. The differences in delivered mothers were assessed by
*: number of rats delivered per mother.
Similarly, Dahl S rats fed the low-salt diet during the pregnancy period delivered less pups compared with those fed the high-salt diet. There were no differences in the birth rates between Dahl S rats that were fed the high-salt rats and those on regular chow.
The number of babies delivered from one mother was equal among the 5 mother groups (Table
The pups that were born in each group and the offspring that survived during the lactation period are shown in Table
Mortality rates of the newborn pups until weaning.
Group | Diet (NaCl, w/w) | Rats born |
Survival rate during lactation | **Statistics | ||||
---|---|---|---|---|---|---|---|---|
Matching | Pregnant | Alive (%) | Dead (%) |
|
|
Versus | ||
MHPR | 4% | 0.75% | 52 (7) | 35 (67) | 17 (33) | |||
MLPR | 0.3% | 0.75% | 35 (4) | 19 (54) | 16 (46) | 1.51 | 0.21 | MHPR |
|
||||||||
MRPR | 0.75% | 0.75% | 59 (7) | 39 (66) | 20 (34) | 0.02 | 0.89 | MHPR |
1.30 | 0.25 | MLPR | ||||||
26.82 | 0.00 | MRPH | ||||||
0.05 | 0.82 | MRPL | ||||||
|
||||||||
MRPH | 0.75% | 4.0% | 113 (11) | 107 (95) | 5 (5) | |||
MRPL | 0.75% | 0.3% | 53 (6) | 34 (64) | 19 (36) | 19.88 | 0.00 | MRPH |
MHPR: mothers fed the high-salt diet during mating period and the regular chow during pregnancy period; MLPR: mothers fed the low-salt diet during mating period and the regular chow during pregnancy period; MRPR: mothers fed the regular chow during mating period and during pregnant period; MRPH: mothers fed the regular chow during mating period and the high-salt diet pregnancy period; MRPL: mothers fed the regular chow during mating period and the low-salt diet during pregnancy period. Rats born: the total number of newborn pups; alive: the number of rats that were alive until weaning; dead: the number of rats that died before weaning. *: mothers that delivered babies. **The differences in alive pups were assessed by
In contrast, only 64% pups from mothers that were fed the low-salt diet during the pregnancy period survived the lactation period, while the survival rate was 95% for the pups whose mothers were fed the high-salt diet (
Using the data from the pups of mothers that were fed the low- and high-salt diets during the pregnancy period, we determined the Kaplan-Meier survival curve in order to increase our understanding of the time course before death (Figure
Cumulative proportion of offspring survival (Kaplan-Meier analysis). Survival prognosis was evaluated from their birth of the rat pups to weaning. Blue circles refer to the death (complete) of pups from high-salt intake mothers; red circles refer to the death (complete) of pups from a low-salt intake mothers. Plus (+), censor. The difference was analyzed by Cox’s
We assessed pup growth during the lactation period (Table
Body weights of pups during lactation.
Day 2 | Day 9 | Day 16 | Day 23 | Day 30 | |
---|---|---|---|---|---|
LS-male |
|
|
|
|
|
HS-male |
|
|
|
|
|
|
|||||
LS-female |
|
|
|
|
|
HS-female |
|
|
|
|
|
LS: offspring of Dahl S rats fed a low-salt diet during the pregnancy period; HS: offspring of Dahl S rats fed a high-salt diet during the pregnancy period. The differences were assessed by ANOVA and followed by post hoc analysis.
In the present study, we attempted to examine the role that salt intake has on the health of mothers and their children. We demonstrated that mothers on a low-salt (0.3% NaCl) diet during the mating pregnancy period produced lower number of pups than those on a high-salt (4% NaCl) diet. Since 7 out of 8 mothers that were fed regular chow throughout the mating and pregnancy periods produced babies, the low-birth rate of pups from mothers that were fed the low-salt diet during the mating period may be due to the failure to achieve fertilization. However, the mating season usually lasts 6-7 days in rats, and this means that time point of fertilization varied in mothers tested. The separation on the influence on fertilization and pregnancy process is technically difficult in the present experimental setting.
Similarly, the mothers that were fed the low-salt diet during the pregnancy period produced less pups than those that were fed the high-salt diet. As shown in mothers that were fed 0.75% NaCl throughout the mating and pregnancy periods, 7 out of 8 mothers produced pups, suggesting that the low-birth rate of pups from mothers that were fed the low-salt diet during the pregnancy period was due to a failure to maintain the pregnancy. In the present study, we were not able to conclude that fertilization is affected by the low-salt diet; however, it may be concluded that low-salt diet during the mating period and following pregnancy decreases birth rate.
In addition, we demonstrated that there were no differences in the number of babies born per pregnant mother among the experimental groups. These findings suggested that ovulation may not be affected by a low-salt diet.
The mechanism underlying why fewer pregnant mothers on a low-salt diet delivered pups is unclear. We speculate that low-salt intake enhanced the activity of the renin angiotensin system (RAS), and this may affect the intrauterine growth of pups. Moreover, some studies suggest that the overexpression of RAS transgenic mice results in a placenta-fetus imbalance and leads to preeclampsia [
We demonstrated in the present study that rat pups born to mothers that were on a low-salt diet during a pregnancy period were more likely to die before weaning than those born to mothers that were fed a high-salt diet. However, it was also demonstrated that there were no differences in survival rate among the pups from mothers fed 0.75% NaCl or a low-salt diets during a pregnancy period. The 0.75% NaCl chow did not influence the birth rate of pups, but it lowered the survival rate of pups from mothers in comparison with pups from mothers fed a high-salt diet during a pregnancy period. This was demonstrated in Kaplan-Meier survival analysis. Since 95% of the pups from mothers on a high-salt diet during a pregnancy period survived until weaning, it was suggested that the regular (0.75% NaCl) diet was less likely to prevent the death of pups during a lactation period. The sensitivity of salt intake to benefits for mothers and pups differed.
In the present study we investigated Dahl S rats for the implications of mating or gestational salt intake in the birth and growth of the offspring. It is unclear whether salt sensitivity contributes to the relationship of mothers that were on a low-salt diet and their offspring. The future direction of research regarding salt intake will entail evaluating whether these findings are also true in other genetic rat strains of spontaneous hypertension.
We investigated the influence of salt intake on gestation and baby growth using Dahl S rats. Dahl S rats are a genetic model for salt-induced hypertension in humans [
The role of salt intake in the development of hypertension in humans has been the main theme over several decades, and this led to a world-wide campaign of salt reduction. However, the extent of salt reduction is controversial. Recently, a meta-analysis involving 25 studies reported that excessive salt intake is associated with an increased incidence of cardiovascular diseases [
In conclusion, low-salt intake during mating and pregnancy is associated with low-birth rates and high death rates among the surviving offspring. We urge additional investigations into the effects of paragestational salt reduction in humans as the level of salt intake that is required for optimal health varies depending on the subjects’ age or stage of growth. Furthermore, the current finding provides evidence that supports the recommendation that the amount of salt consumed during pregnancy should be considered carefully based on data regarding the relationship between mothers and their offspring.
Dahl salt-sensitive rats
Spontaneously hypertensive rats
Renin angiotensin system
Standard deviation
One-way factorial analysis of variance.
The authors report no conflict of interests regarding the publication of this paper.
The authors acknowledge Grants-in-Aid from the Center for Interdisciplinary Studies of Science and Culture, Kyoritsu Women’s University & Junior College. The authors alone are responsible for the content and writing of the paper.