The authors have declared that no competing interests exist.
Our study aims to help researchers calculate resources based on a rat breeding colony for experiments on fetal lung development.
The Wistar rat is commonly used in experimental research. In the context of fetal lung studies, the nitrofen rat is a model for pulmonary hypoplasia and congenital diaphragmatic hernia. Data needed to calculate resources for new experiments are not easily available.
We prospectively acquired data on the reproductive performance and fetal outcome of 314 consecutive virgin Wistar dams in our fetal lung research breeding colony.
We define the impact of breeding conditions on rat fertility and evaluate different methods to diagnose early rat gestation. Effects of nitrofen exposure as well as fetal surgery in terms of prenatal mortality and gross anatomical parameters of lung development are quantified in nitrofen-exposed and -unexposed fetuses from embryonic day 19 to 21 in 12h intervals.
A reduced mating interval (1h) provides a great degree of experimental control with feasible pregnancy rates and a large litter size. Nitrofen exposure, as well as fetal surgery, depict high fetal survival rates. Fetal lung findings were very reproducible and could allow for reduction of animals utilized within experimental groups.
Rats reach estrous approximately every fourth day.
A setting in which early diagnosis of pregnancy is desired is before administering nitrofen (NF) to any dam. First reports about the teratogenic potential of this herbicide were published in 1971.
CDH occurs in 1-2/5,000 newborns. At birth, the associated lung hypoplasia leads to pulmonary insufficiency and hypertension, which is fatal in up to 30% of babies in whom the condition is isolated.
This is a prospective analysis of the reproductive performance and fetal outcome of 314 consecutive virgin Wistar dams which were part of our experiments on prenatal lung development. Female rats were ordered at the age of 9-14 weeks, male rats (n=20) at the age of 11 weeks (Elevage Janvier, Le Genest-St-Isle, France). All were first given one week to adjust to their new environment. Animals were accommodated in a separate room of the animalium of the Faculty of Medicine, KU Leuven. This room was only accessible to the staff and the researchers directly involved in animal care (V. B., S. D., T. D.). The top rows of the shelves were avoided for a possible negative effect of direct light.
To determine the reproductive cycle in female rats, vaginal smears were obtained over at least 4 consecutive days (12.00-14.00). These were analyzed by light microscopy (Zeiss Axioplan microscope, Carl Zeiss, Oberkochen, Germany).
Pulmonary hypoplasia and CDH were induced on ED9 (embryonic phase of lung development) by gavage feeding 100mg of NF (dissolved in 1ml of olive oil) (Maybridge, Acros Organics NV, Geel, Belgium) to pregnant rats. (NF was given independently of female weight. It was suggested, that data might be more valid and uniform if NF dosage would be based on actual weight, which could be explored in a future study.)Those animals later underwent fetal surgery, consisting either of TO with a clip or sham surgery with neck dissection only (sham) on ED18, ED18.5 (both pseudoglandular phase), ED19 (pseudoglandular-cannalicular phase) or ED20 (cannalicular phase) as earlier reported in detail.
Harvest was performed at 0.5-day intervals between ED18 and ED21. Following laparotomy, the uterus was assessed for the presence and viability of fetuses and compared to the status at fetal surgery. Fetuses were harvested one by one through hysterotomy under general maternal anesthesia. We recorded the fetal body weight (fBW), the fetal right and total lung weight (fRLW, fTLW) (Acculab, Sartorius group, VIC-303, NY measuring accurately up to 0.001g), as well as the presence or absence of CDH (CDH-) (LCDH, RCDH, bilateral CDH (BCDH)). Fetal left lung weight (fLLW) and fetal lung-body-weight ratio (fLBWR) were calculated from the obtained values. Female weight was obtained before NF-administration, surgery and harvest.
There were two episodes of pinworm infections of other animals housed in the same facility, and our animals received preventive treatment with fenbendazole (FBZ)-medicated pellets (A153F0300 R/M-H 10mm containing 3g/kg Fenbendatat 5%, Ssniff Spezialdiäten; nutritional value identical to regular pellets. Target dose 8-12mg/kg/day for 2 months after diagnosis of the infection
Data are presented as mean +/- standard error of the mean. Maternal weight and parental age is specified as mean +/- standard deviation. Absolute numbers and percentages are given if applicable. Sets on fetal survival rates and biometrical data in the different experimental groups were analyzed by Kruskal-Wallis and Dunn’s multiple comparison test (GraphPadPrism, Version 5.0, San Diego, CA). Remaining data was tested by uni-/ multivariate stepwise regression analysis (JMP, Version 7.0, Cary, NC). P-values <0.05 were considered statistically significant (* <0.05; ** <0.01; *** <0.001). Data on the very early intervention groups are not displayed for easy reading purposes.
The experimental protocol was approved by the Ethics Committee for Animal Experimentation of the Faculty of Medicine, KU Leuven. Rats were treated according to current guidelines on animal well-being.
Characteristics of the adult rat population and environmental factors in relation to rat fertility are summarized in
Independent variable | Type of effect | 60% cut-off | Univariate analysis | Multivariate analysis (R2=0.13) |
Female weight (grams) | ↓ | 300 | <0.0001 | 0.0249 |
Female age (weeks) | ↓ | 18.5 | 0.0009 | n.s. |
Male age (weeks) | ↓ | 35 | 0.0006 | n.s. |
Number of mating attempt | n.s. | |||
Previous pseudopregnancy | ↓ | 0.0004 | 0.0147 | |
FBZ exposure | n.s. | |||
Clidox-S exposure | ↓ | 0.0347 | n.s. | |
Day of the week/ Year | n.s. |
FBZ= fenbendazole, n.s. = not significant.
Pregnancy rate was 74% after the first mating attempt. 11, respectively, 3 female rats underwent a second or third mating attempt resulting in a subsequent pregnancy in 64% and 67% (n.s.,
We compared three methods to predict successful mating: (1) the presence of sperm in the vaginal smear, (2) the presence of a vaginal plug after mating and (3) a maternal weight gain ≥15% at ED9 of a potential pregnancy. All 3 parameters were strongly correlated with the occurrence of an actual pregnancy (p<0.001). The presence of only sperm fragments rather than complete sperm or a mixture of any amount was most indicative of a subsequent pregnancy (98.4% versus 87%, p<0.001). Positive and negative predictive values of these methods are listed in
Vaginal plug | Vaginal sperm | Weight gain ≥15% | |
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92.7% | 91% | 94.4% |
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78.5% | 67.8% | 45.5% |
= positive predictive value,
= negative predictive value.
Of 227 pregnant dams 200 had received NF and 27 did not. Mean litter size was 13 in NF+ (range 1-20) as well as NF- (range 3-16) groups. Litter size was smaller in females with previous pseudopregnancies (mean litter size of 7 versus 13; p=0.0025). FBZ and Clidox-S exposure did not have an impact on the total litter size.
The mean rate of spontaneous abortions of any gestational age (GA) was 8.5% (0-83%) in NF+ fetuses compared to 3.9% (0-33%; n.s.) in NF- fetuses. Of the 1961 NF+ fetuses, 37% had LCDH, 7% RCDH and 3% BCDH. NF+ pups without CDH had a lower lung and body weight compared to NF- pups (p<0.001). The fLBWR, a measure of pulmonary hypoplasia, was lower in NF+ fetuses with CDH than those without (p<0.05). The effect on fLBWR of the combination of CDH and NF-exposure was more pronounced later in pregnancy, suggesting the progressive nature of the disease. In the absence of CDH, NF-exposure is not associated with a decreased fLBWR near term (
ED19 | ED19.5 | ED20 | ED20.5 | ED21 | |||||||||||
NF- | CDH- | LCDH | NF- | CDH- | LCDH | NF- | CDH- | LCDH | NF- | CDH- | LCDH | NF- | CDH- | LCDH | |
(n=55) | (n=43) | (n=31) | (n=39) | (n=61) | (n=39) | (n=52) | (n=70) | (n=40) | (n=39) | (n=81) | (n=57) | (n=43) | (n=173) | (n=116) | |
fBW (mg) | 2,496±27 | 1,987±25 | 2,034±31 | 2,549±57 | 2,236±34 | 2,187±44 | 4,144±57 | 2,934±36 | 2,957±60 | 4,392±68 | 3,275±44 | 3,311±50 | 5,926±55 | 4,197±33 | 4,081±90 |
fTLW (mg) | 82±1.6 | 54±1.1 | 50±1.4 | 89±2.6 | 62±1.4 | 54±1.3 | 136±2.1 | 91±1.4 | 70±1.9 | 137±3 | 96±2 | 74±1.4 | 168±3 | 119±2.6 | 71±2.6 |
fLLW (mg) | 28±0.5 | 19±0.5 | 17±0.5 | 30±0.9 | 22±0.5 | 18±0.5 | 45±0.8 | 32±0.6 | 23±0.6 | 46±1.1 | 34±0.7 | 23±0.5 | 56±1.1 | 41±0.9 | 22±0.9 |
fLBWR (%) | 3.3±0.05 | 2.7±0.05 | 2.5±0.06 | 3.5±0.05 | 2.8±0.05 | 2.5±0.05 | 3.3±0.04 | 3.1±0.04 | 2.4±0.06 | 3.1±0.04 | 2.9±0.04 | 2.3±0.05 | 2.8±0.06 | 2.8±0.05 | 1.7±0.05 |
NF- vs. NF- at different GA or NF+ at same GA: | |||||||||||||||
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LCDH versus CDH-: | |||||||||||||||
fBW | n.s. | n.s. | n.s. | n.s. | n.s. | ||||||||||
fTLW | n.s. |
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n.s. = not significant; = p<0.05; = p<0.01; = p<0.001. ED= embryonic day, fBW= fetal body weight, fTLW= fetal total lung weight, fLLW= fetal left lung weight, fLBWR= fetal lung-body-weight ratio.
The survival rates after fetal surgery on ED18.5 were 96% in untouched, 76% in sham-operated and 66% in TO fetuses (TO/Sham vs. control p<0.001). Later surgery resulted in higher survival rates: Intervention at ED19 resulted in 97%, 87% and 83% surviving fetuses and at ED20 this was 94%, 88% and 92%, respectively (TO18.5 vs. TO19/20 p<0.01). The few animals which underwent interventions at ED18 had a higher mortality rate, so we abandoned this practice (unpublished experience of our group).
We report in great detail outcomes of our large scale breeding and experimental surgery program for CDH in the NF-Wistar rat. With very consistent conditions in terms of personnel, housing and experimental circumstances, we document fetal lung development along 12h intervals near term. GA was well-defined by restricting mating to a 1h period over the entire experimental phase of three years.
Herein we obtained an overall pregnancy rate of 74% at first attempt, a fetal loss rate (before interventions) of 8.5% for NF+ fetuses which did not significantly differ from NF- fetuses (3.9%) and an average litter size of 13. Humphreys (1976) observed at maximum a 97.4% pregnancy rate when leaving virgin Sheffield-Wistar dams at 12 weeks age with males until detection of a plug or obvious pregnancy resulting in an average of 9.3 pups born per mated female. An alternative strain or age resulted in a lower pregnancy rate.
In order to document lung development, we aimed to obtain normative data in healthy and NF+ fetuses (
In NF- pups, the lung grows faster than the body, peaking at ED19.5. In nitrofen-exposed CDH- fetuses this effect can as well be observed with a peak in fLBWR at ED20. In LCDH fetuses the fLBWR is highest at ED19 and decreases thereafter. The latter is known as the progressive nature of lung hypoplasia towards term and observed in animal studies as well as the clinical setting. To obtain severe disturbances in lung development, pups must not only be NF+ but also have CDH. At term LCDH pups have a fLBWR of 60% compared to that of NF-exposed littermates without CDH. Strictly speaking the fLBWR of LCDH fetuses of 1.7% at ED21 does not meet the pathologic criteria of pulmonary hypoplasia used clinically (fLBWR ≤1.5% until 27wks, ≤1.2% 28wks until term).
The occurrence of CDH in our study was 47% in total, 37% of fetuses had left-sided lesions. Our total rate was comparable to the 42% of Kluth,
One of the main goals of our group is to reverse pulmonary hypoplasia prenatally. Fetal TO is one option
In laboratory animal research based on breeding colonies it is difficult to standardize certain conditions. For instance the age of animals in the colony will increase and as a consequence their weight which negatively affects fertility. An alternative would be to order time-mated dams, however, this reduces control over other experimental factors. A strategy to limit the potential extent of such unwanted effects should be to perform experiments in the shortest time frame possible.
We provide data that may help to calculate numbers needed in a breeding colony for experiments on fetal lung development. Limiting the mating interval, which is a requirement for this type of research, still provides reasonably high pregnancy rates and a large litter size. Exposure to NF as well as fetal surgery is associated with high fetal survival rates. In addition, lung findings were very reproducible and eventually will reduce the number of animals within experimental subgroups.
This work was supported by the European Commission in its 6th Framework (EuroSTEC; LSHC-CT-2006-037409) and the Flemish Government (Instituut voor Wetenschap en Technologie; IWT/ 070715). VB was provided a grant within the Marie Curie Early Stage Research Training Programme (MEST CT2005 019707) and was further supported by a Marie Curie Reintegration Grant (PERG07-GA-2010-268330; FP7-PEOPLE-2010-RG). JD was the recipient of a “Fundamental Clinical Researcher” grant of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (1.8.012.07.N.02).