Scientific African 7 (2020) e00268 
Contents lists available at ScienceDirect 
Scientific African 
journal homepage: www.elsevier.com/locate/sciaf 
Natural cocoa improves birth weight and viability of rabbit 
pups born to hypercholesterolemic mothers 
Richard M. Blay∗ , Benjamin Arko-Boham,  Frederick K. Addai 
Department of Anatomy, College of Health Sciences, University of Ghana Medical School, University of Ghana, P. O. Box KB 143, Korle-Bu 
Campus, Ghana 
a r t i c l e i n f o a b s t r a c t 
Article history: Background: Maternal nutrition affects fe tal growth and development in humans and ani- 
Received 17 October 2019 mals. Low birth weight can be caused by maternal hypercholesterolemia and is associated 
Revised 9 December 2019 with increased risk of cardiovascular and other metabolic diseases later in adult life. Cocoa 
Accepted 21 January 2020 
powder is rich in flavonoids and reduces plasma cholesterol levels. The study investigated 
the effect of hypercholesterolemia and natural cocoa intake during pregnancy on the birth 
Editor: DR. B. Gyampoh weight and viability of rabbit pups.     
Keywords: Methods: Hypercholesterolemic female New Zealand White rabbits were crossed with nor-  
Maternal nutrition mocholesterolemic males and randomly grouped into 2 ( n = 4 each). One group (HCC)   
Hypercholesterolemia received natural cocoa powder in their water in addition to high cholesterol diet during 
Birth weight pregnancy, whereas the other (HC) received only water and high cholesterol diet. Litter 
Natural cocoa size, birth weight and viability of pups were assessed and compared to a control group 
Fetal growth (NC) fed normal rabbit chow ( n = 2). 
Results: Average litter sizes of hypercholesterolemic rabbits (HC and HCC groups) were 
3, whereas that of control group, NC was 5. Mean birth weight of pups was significantly 
lower in offspring of HC (42.73 g, SD 8.47) and HCC (50.0 g, SD 3.54) as compared to NC 
(73.0 g, SD 5.37). Mortality of offspring was highest in HC group (58.3%) as compared to 
50% in HCC. 
Conclusions: Maternal hypercholesterolemia during pregnancy reduces litter size, birth 
weight and viability of offspring in rabbits and these deleterious effects may be minimized 
by regular intake of natural cocoa. Although further studies in humans need to be con- 
ducted, the results of this study suggest the need to monitor maternal cholesterol levels 
during pregnancy and the effects on pregnancy outcomes and health of offspring later in 
life. 
© 2020 The Author(s). Published by Elsevier B.V. on behalf of African Institute of 
Mathematical Sciences / Next Einstein Initiative. 
This is an open access article under the CC BY license. 
( http://creativecommons.org/licenses/by/4.0/ ) ∗ Corresponding author. 
E-mail addresses: rmblay@ug.edu.gh (R.M. Blay), barko-boham@ug.edu.gh (B. Arko-Boham), fkaddai@ug.edu.gh (F.K. Addai). 
https://doi.org/10.1016/j.sciaf.2020.e00268 
2468-2276/© 2020 The Author(s). Published by Elsevier B.V. on behalf of African Institute of Mathematical Sciences / Next Einstein Initiative. This is an 
open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) 
2 R.M. Blay, B. Arko-Boham and F.K. Addai / Scientific African 7 (2020) e00268 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Introduction 
Intra-uterine environment and nutrition can result in changes that predispose the fetus to cardiovascular and metabolic
diseases later in life [1 , 2] as suggested by the fetal programming hypothesis. Maternal malnutrition and over-nutrition in
animal models enhance the development of cardiovascular diseases and metabolic syndrome later in life in the offspring
[3] . Exposure to these conditions constitute a disturbance to which cells of the developing fetus adapt in order to survive,
leading to irreversible changes or genetic programming that later in life increases susceptibility to diseases [4] . 
In humans, first evidence for fetal programming in-utero showed low birth weight increased the risk for hypertension
[5] . Low birth weight is associated with maternal nutrition and women who recorded high carbohydrate intake with low
protein and dairy intake in early pregnancy had babies with lower birth weights suggesting impaired intrauterine growth
[2 , 6] . Moreover, epidemiological studies show that death from coronary heart disease is high in individuals born with low
birth weight [2] . Putting all these together, maternal nutrition especially early pregnancy may result in priming fetuses to
develop disorders that enhances the development of cardiovascular diseases later in life. 
Pregnancy, is associated with high levels of cholesterol in maternal circulation and this is a physiological response to
increased demands for normal development of the fetus [7] . However, maternal hypercholesterolemia beyond the pregnancy
related physiological levels, also known as maternal supra-physiological hypercholesterolemia (MSPH) may predispose the 
fetus to early development of atherosclerosis and other cardiovascular diseases [8–10] . Although the placenta is known to
be restrictive to the transfer of lipids and MSPH may not result in increased cholesterol levels in off spring. There is a cor-
relation between maternal and fetal blood cholesterol levels during pregnancy [11 , 12] and this may be due to enhanced
endogenous synthesis of cholesterol in the fetus. Increased levels of cholesterol in fetal circulation, may enhance atheroscle-
rosis by inducing endothelial dysfunction and intima-media thickening [7 , 13 , 14] . 
Maternal hypercholesterolemia restricts fetal growth which has been shown to be associated with low birth weight and
the development of disorders including cardiovascular diseases, later in life [9] . Low birth weight is associated with hyper-
tension and ischemic heart disease as well as overall health outcomes and may be due to restriction of fetal growth and
nutrition [6 , 15 , 16] . Interestingly, there are no established clinical reference values for maternal hypercholesterolemia and
the mechanism of in-utero fetal programming is not fully understood [9 , 10] . Targeting fetal programming and interventions
with phytosterols and antioxidants during pregnancy may reduce the adverse effects of maternal hypercholesterolemia on
offspring [9 , 17 , 18] . Consumption of cocoa is known to reduce oxidative stress and obesity by increasing antioxidant levels
[19] , high blood pressure as well as risk of coronary heart disease and cardiovascular mortality [20] . This study, aimed at in-
vestigating the effect of hypercholesterolemia and natural cocoa intake during pregnancy on the birth weight and viability of
rabbit pups. With limited safe lipid-lowering treatments for pregnant women, finding supplements or dietary interventions
that inhibit the deleterious effects of maternal hypercholesterolemia on offspring may be beneficial. 
Materials and methods 
This study was approved by the Ethical and Protocol Review Committee of the College of Health Sciences, University of
Ghana. 
Animal experiments 
Ten adult New Zealand White female rabbits (age: 6 months, body weight: 1.5–2.8 kg) obtained from the Noguchi Memo-
rial Institute for Medical Research (NMIMR) of the University of Ghana were used for this experiment. They were housed
under standard conditions of local temperature (28 °C) and relative humidity (80%) and exposed to 12-hour light/dark cycle.
Rabbits were randomly assigned by lottery to three groups and housed individually in cages. 
Hypercholesterolemia was established as previously described by Blay et al. [14] . In summary, the rabbits were arbitrarily
numbered from 1 to 10 and the numbers written on pieces of paper. After mixing the pieces of paper, rabbits were placed
in the groups when the numbers were drawn. The first two groups of four rabbits each (HC and HCC) were fed cholesterol-
enriched diet which was prepared by mixing standard chow (Kosher Feedmill Ltd, Accra) with 0.5% (w/w) cholesterol (Hop-
kin and William Ltd, London) and 10% (v/w) coconut oil (open market, Accra). The rabbits were given 24 h unrestricted
access to the cholesterol-enriched diet for two weeks, and after a lipid profile test confirmed hypercholesterolemia, they
were crossed with normocholesterolemic males. The normal cholesterol range for rabbits is 0.14–1.86 mmol/l. Hyperchole-
strolemia was defined as a total plasma cholesterol level higher than twice the upper level of the normal range. One group
of rabbits given cholesterol-enriched diet was given 2% (w/v) of natural cocoa powder (NCP) (GoodFood brand, Kakawa
Enterprise Ltd, Accra) as an aqueous suspension instead of drinking water. This group (HCC) had 24 h access to the NCP
suspension, which they drank voluntarily, after being mated until they littered (28–30 days). The second group of rabbits on
cholesterol-enriched diet (HC) were given 24 h access to filtered tap water. The third group of rabbits ( n = 2), designated
as normal control (NC), were given standard chow without cholesterol enrichment and filtered tap water throughout the
duration of the experiment. 
In the previous paper [14] , we demonstrated alteration of lipid profile and intima-media thickness of aorta in rabbits
born to hypercholesterolemic mothers fed same dose of cholesterol-enriched diet. The present paper focuses on the effect
on birth weight and viability of pups born to experimentally-induced hypercholesterolemic mothers. 
R.M. Blay, B. Arko-Boham and F.K. Addai / Scientific African 7 (2020) e00268 3 
Fig. 1. Mean serum cholesterol just before pregnancy; after hypercholesterolemia had been induced and during pregnancy. NC = Control group, HC = group 
given cholesterol but not cocoa and HCC = group given cholesterol and cocoa. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood samples were obtained by bleeding of the marginal ear vein of the female rabbits 3 weeks into pregnancy and
total cholesterol levels during pregnancy assessed. Total levels of cholesterol were determined after an overnight fast by an
enzymatic colorimetric test in a laboratory at the Medical Biochemistry Department (University of Ghana Medical School),
using a semi-automated clinical analyser, Microlab 300 (Vital Scientific, the Netherlands).The skin over the ear of the rabbits
was anaesthetised using a local anaesthetic cream containing lidocain (Lignocaine 2% Jelly, Purna Pharmaceuticals, Belgium)
after the fur over the ear was shaved and the skin sanitised with alcohol. Blood samples were then drawn and stored in
sterilised test tubes containing heparin. 
Litter size, birth weight and mortality rate of pups 
The number of pups or litter size was recorded for each animal after delivery and each pup weighed. Mortality was
recorded as number of pups that died within 1 week post-delivery. There were no deaths of the pups beyond 1 week
post-delivery. 
Statistical analyses 
The results were analysed using Graphpad Prizm software (5.0). T-test was used to compare the means of two groups
while one-way ANOVA was used to compare the means of three groups. A p-value < 0.05 was considered to be statistically
significant. Bonferroni’s Multiple Comparison Test was done to show actual differences between the three groups. 
Results 
Hypercholesterolemia in female rabbits during pregnancy 
Cholesterol levels in hypercholesterolemic mothers decreased significantly during pregnancy (3 weeks of pregnancy) as
shown in Fig. 1 . Mean cholesterol level of HC during pregnancy was 1.9 (SD 0.18) mmol/l compared to 7.33 (SD 1.93) mmol/l
before pregnancy. An unpaired T-test showed a significant difference ( p < 0.05, t = 5.60 and df = 6) between cholesterol
levels of HC before and during pregnancy. The HCC group had a mean cholesterol level of 2.7 (SD 0.97) mmol/l during
pregnancy and this was significantly lower ( p < 0.05, t = 4.14 and df = 6) than the mean cholesterol level of 7.40 (SD 2.05)
mmol/l before pregnancy. During pregnancy, mean cholesterol levels of rabbits receiving high cholesterol diet showed no
significant difference from control rabbits as shown in Fig. 1 . 
Litter size and mortality rate 
The total number of pups delivered by control group ( n = 2) was 10, giving an average litter size of 5 pups, whereas HC
group ( n = 4) delivered a total of 12 pups (average litter size = 3) ( Table 1 ). The total number of offspring of the HCC group
( n = 4) was 6. Two rabbits from the HCC group did not litter at all and so average litter size (3) was calculated using the
two that littered. Neonatal mortality was highest among HC group (58.3%), whereas the mortality rate of HCC pubs was 50%
( Table 1 ). No death was recorded in the control group. 
Birth weight of pups 
Differences in birthweight of the neonates of the three groups were statistically significant ( p < 0.0 0 01, F = 58.42 and
df = 2) as shown in Fig. 2 . Offspring or pups of the control rabbits (NC) had the highest mean birthweight of 73.0 (SD 5.37)
4 R.M. Blay, B. Arko-Boham and F.K. Addai / Scientific African 7 (2020) e00268 
Table 1 
Litter size and mortality rate of rabbit pups. 
Treatment Group Number of Litter Average litter size Mortality rate (%) 
NC ( n = 2) 10 5 0 
HC ( n = 4) 12 3 58.3 
HCC ( n = 4) 6 3a  50 
a 2 mothers did not litter. 
75
NC
HC
50 HCC
25
0
NC HC HCC
Offspring of rabbits
Fig. 2. Mean birth weight of offspring. NC = Control group, HC = group given cholesterol but not cocoa and HCC = group given cholesterol and cocoa. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Birthweight/gg followed by that of HCC with 50.0 (SD 3.54) g. Pups from the HC group had lowest mean birthweight of 42.73 (SD 3.54) g.
Statistically significant differences existed between NC and HC ( p < 0.001) and NC and HCC ( p < 0.001). Mean birth weight
of HCC was higher than the mean birth weight of HC but was not statistically different ( p > 0.05). 
Discussion 
Maternal hypercholesterolemia during pregnancy and low birth weight (LBW) predispose offspring to developing car- 
diovascular diseases later in life. Cocoa consumption is known to be protective against cardiovascular diseases. This study
shows maternal hypercholesterolemia during pregnancy reduced significantly birth weight and viability of rabbit pups and
consumption of natural cocoa minimized this effect. 
Studies in humans show that low birth weight increases rate of illness and reduces significantly the probability of survival
within the first year [16 , 21 , 22] . LBW is also associated with coronary heart disease, stroke, hypertension, type 2 diabetes,
obesity and hyperlipidaemia later in life [23 , 24] . The present study showed that maternal hypercholesterolemia is associated
with low birth weight and increased mortality in rabbit pups. Mortality rate was highest in the group with the lowest birth
weight, whiles no death was recorded in the control group that had the highest birth weight. Low birth weight in this
study may have been the result of exposure to high levels of cholesterol in circulation resulting in an abnormal intra-uterine
environment, leading to restricted growth and hence reduced weight at birth [25–28] . 
The mechanism by which maternal hypercholesterolemia restricts fetal growth is yet to be fully understood but increased
inflammation and oxidative stress that lead to impaired vascular function may be implicated [29–32] . Ingestion of cocoa by
hypercholesterolemic mothers increased birth weight slightly although the change was not statistically significant. Regular 
intake of natural cocoa also decreased the mortality from 58.3% to 50%. Altering the dosage of natural cocoa may enhance
the beneficial effects on birth weight and viability [33] . Reduced litter size in this study may be attributed to inhibited in-
trauterine development [34] . Further investigations is required to elicit the mechanisms through which hypercholesterolemia
reduces growth and survival, however, evidence from previous studies suggest natural cocoa through its antioxidant activity
may reduce oxidative stress and inflammation and hence preserve vascular function and growth during development [35] . 
An interesting observation of this study was that although pregnant rabbits continued to receive high cholesterol diet,
maternal cholesterol levels significantly reduced during pregnancy. In humans, cholesterol levels during pregnancy has 
shown conflicting results with some studies indicating lower levels during pregnancy, while other studies showed higher
levels [36] . Maternal cholesterol levels may reduce during pregnancy due to the high demand for cholesterol for develop-
ment of the fetus [37] and differ during the course of pregnancy [38 , 39] . In the early stages, cholesterol levels are high due
to increased synthesis, but in later trimesters there is an increased break down of lipid storage for the growing fetus [37] .
Another possible explanation why maternal cholesterol levels were reduced is that, there may have been increased choles-
terol transport into fetal circulation [40] . Cholesterol is an essential component of cell membranes and is therefore in high
demand during processes like cell proliferation, cell differentiation and cell-to-cell communication as well as metabolism
R.M. Blay, B. Arko-Boham and F.K. Addai / Scientific African 7 (2020) e00268 5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
during fetal development [38] . Although the transfer of cholesterol into the fetus is physiologically necessary to enhance
growth, high cholesterol levels in the mothers at the beginning of pregnancy may have led to higher levels of cholesterol in
fetal circulation. Increased cholesterol transfer beyond physiologically normal levels may therefore have restricted growth,
resulting in low birth weight and decreased survival of pups. 
Natural cocoa intake minimized to an extent the effects of high cholesterol during pregnancy. The beneficial effects of
cocoa may be dose-dependent and as such may account for the reason why a significant reduction in birth weight was
not observed in this study [33 , 41] . Cocoa may reduce the deleterious effects of maternal hypercholesterolemia on birth
weight and viability when consumed at the appropriate dose. Further studies will be required to determine the right dose.
The mechanism by which cocoa protects against pathological changes also needs further investigations, but experimental
evidence supports the fact that improvement of vascular function and antioxidant activity are benefits of cocoa intake [42] .
Consumption of cocoa increases serum high density lipoprotein (HDL) and decreases oxidation of low density lipoprotein
(LDL) and these effects are associated with decreased cardiovascular diseases [43] . 
Conclusions 
Maternal hypercholesterolemia during pregnancy reduces litter size, birth weight and viability of rabbit pups and inges-
tion of unsweetened natural cocoa may improve birth weight and viability. The results of this study provide evidence for
further study both in animals and humans to understand cholesterol metabolism in pregnancy and to monitor the effect of
increased maternal cholesterol on offspring. 
Declaration of Competing Interest 
The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper. 
Acknowledgments 
We acknowledge the College of Health Sciences, University of Ghana for providing financial support for this study and
technicians from Departments of Anatomy, Medical Biochemistry and Pharmacology, University of Ghana for their assistance.
Funding 
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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