Orthopaedics Section DOI : 10.7860/JCDR/2017/29004.10874
Year : 2017 | Month : Nov | Volume : 11 | Issue : 11 Page : RC05 - RC08

Chronic Effects of Maternal Malnutrition during Lactation: Severe Influences on Cortical Bone Mass of Wistar Rats Femur

Guilherme Schultz1, Fabiano Medeiros2, Mauricio Arcoverde3, Max Alves Cavalcante4, Lucas Pires5, Monique Babinski6, Marcio Antonio Babinski7

1 Resident, Department of Orthopaedics and Traumatology, Salgado Filho Municipal Hospital, Rio de Janeiro, Brazil.
2 Resident, Department of Orthopaedics and Traumatology, Salgado Filho Municipal Hospital, Rio de Janeiro, Brazil.
3 Resident, Department of Orthopaedics and Traumatology, Salgado Filho Municipal Hospital, Rio de Janeiro, Brazil.
4 Tutor, Department of Orthopaedics and Traumatology, Salgado Filho Municipal Hospital, Rio de Janeiro, Brazil.
5 Postgraduate Student, Medical Sciences Post-Graduation Program, Fluminense Federal University, Niterói, Rio de Janeiro, Brazil.
6 Nurse, Medical Sciences Post Graduation Program, Fluminense Federal University, Niterói, Rio de Janeiro, Brazil.
7 Postgraduate Advisor, Medical Sciences Post-Graduation Program, Fluminense Federal University, Niterói, Rio de Janeiro, Brazil.

NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Marcio Antonio Babinski, Av. Prof. Hernani Mello, 101, CEP 24.210-150, Niterói, Rio De Janeiro, Brazil.
E-mail: mababinski@gmail.com


Malnutrition is a world health problem and has high incidences in developing countries. It is known that the nutritional status of the mother during gestation and lactation affects the growth and development of its child. Maternal malnutrition during breastfeeding has been shown to cause alterations of thyroid function and bone tissue.


The present study aimed to evaluate the chronic effects in bone tissue of energy-protein restriction in rats during lactation period through a histological and morphometric analysis of the cortical mass of the femur.

Materials and Methods

At parturition, Wistar rat dams were divided in three groups: (1) Control group - which received 23% protein diet without restrictions; (2) Protein-Energy restriction group - which received 8% protein diet; (3) Energy restriction group - which received 23% protein diet in limited amounts, according to the ingestion of the divond group. Each group had 12 pups. After weaning, all pups were separated from its dams and received free access to 23% protein diet until 180 days and then euthanized. Afterwards, the femur of these specimens were excised and fixed in 4% formaldehyde solution and decalcified by EDTA. 10 μm thick transverse divtions on the middle third of the femur diaphysis were performed. Then, the tissue was stained with Haematoxylin/Eosin and analysed under a microscope.


The cortical area of the control, energy restriction and protein-energy restriction groups were, respectively: 6.54±0.23 μm, 5.51±0.29 μm and 6.18±0.19 μm, while the medullary cavity showed the following results: 3.65±0.16 μm, 4.1±0.20 μm and 3.53±0.12 μm. Reduction of the cortical area and an increase of medullary zone were observed on the protein-energy restriction group.


Maternal malnutrition during lactation has negative effects on femur growth even after normalization of the diet in adult aged Wistar rats.

How to cite this article

Guilherme Schultz, Fabiano Medeiros, Mauricio Arcoverde, Max Alves Cavalcante, Lucas Pires, Monique Babinski, Marcio Antonio Babinski, .CHRONIC EFFECTS OF MATERNAL MALNUTRITION DURING LACTATION: SEVERE INFLUENCES ON CORTICAL BONE MASS OF WISTAR RATS FEMUR.Journal of Clinical and Diagnostic Research [serial online]2017 Nov[cited:2019 Aug 24] 11 RC05 - RC08
 Available from


According to recent data, roughly 805 million people are estimated to suffer from malnutrition [1]. Furthermore, in developing countries low birth weight and associated diseases (such as coronary heart diseases) usually arise from a poor nutritional intake of the mother (before and during pregnancy and lactation) [2]. Malnutrition remains as a common world health issue and it contributes to impaired childhood growth and mortality [2].

It is known that breastfeeding has an extremely positive effect on the child and mother’s health. Exclusive breastfeeding is recommended on the first six months of the child. Mothers who do not breastfeed have an increase of ovarian cancer risk [3]. Factors such as low maternal age, low educational attainment and low socioeconomic position may influence on the nutritional status of the mother during pregnancy and lactation [4].

Studies have shown that the nutritional status of the mother during gestation and lactation is essential to normal growth and development in humans [5], and in animals [6-10]. Studies regarding the effects of protein-energy restriction during lactation period are scarce, although they showed that there are alterations in thyroid function [9,11,12] and on the skeletal system of Wistar rats [7,13].

Bone tissue is sensitive to protein malnutrition due to the fact that its non-mineral composition is essentially constituted by protein, thus, there is a relation between an adequate protein absorption and osseous metabolism [8]. Furthermore, it is known that pre-natal and neonatal malnourishment will cause disturbances during bone growth [7,8,13,14].

The present study aimed to investigate the effects caused by protein and energy restriction during lactation period on cortical dimensions of the femur in the adult offspring of Wistar rats.

Materials and Methods

The present study was approved by the Animal Care and Use Committee of the State University of Rio de Janeiro, and is in accordance with the American Physiological Society guidelines [15]. The study was conducted between January 2015 and January 2017. The present study was also performed in compliance with the general guidelines of the Brazilian College for Animal Experimentation (COBEA). Furthermore, the same protocols and design were used in previous studies by the same research group [7,8,13].


Six three-month-old female virgin Wistar rats were obtained from the Biomedical Center of the State University of Rio de Janeiro and were housed at 25±1°C and on a 12 h light/dark cycle (lights on from 07:00 a.m. to 07:00 p.m.) throughout the experiment. The rats were house with one male at a proportion of 2:1. After mating, each female was placed in an individual cage with free access to water and food until delivery. Afterwards the pregnant rats were divided into three different groups. Each female gave birth to six pups, totaling 12 per group. All pups showed good health condition. There was no statistical significant difference in body weight or linear growth.

Experimental Procedures and Diets

The pregnant Wistar rats were separated at delivery into three groups: 1) control group (C) – with free access to a standard laboratory diet (in grams per 100g) containing 23% protein, 68% carbohydrate, 5% lipid, 4% salts and 0.4% vitamins, and a total of 17,038.7 (kJ/kg) energy; 2) Protein-Energy-Restricted group (PER) – with free access to an isoenergetic, protein-restricted diet containing 8% protein, and; 3) Energy-Restricted group (ER) – fed with a standard laboratory diet in restricted quantities (calculated based on average ingestion of the PER group).

The protein-restricted diet had cornstarch instead of the full protein content of the C group diet [Table/Fig-1]. Vitamin and mineral mixtures [Table/Fig-2,3] were formulated to equal those found in the control diet and to meet the American Institute of Nutrition AIN- 93G recommendation for rodent diets [16]. The protein-energy restriction started at birth (immediately postpartum), which was defined as day 0 of lactation, and ended at weaning (d21= 21 days postpartum).

Composition of the normal and hypoproteic diets.

Total energy – kcal4070.44070.4
Protein %23.08.0
Carbohydrates %66.081.0
Lipids %11.011.0
Protein g230.080.0
Carbohydrates g676.0826.0
Lipids g50.050.0
Vitamin mix g4.04.0
Mineral mix g40.040.0

*According to recommendations from the American Institute of Nutrition Rodents Diets, AIN-93G [16]

Vitamin mix composition

Vitamins*g/kg of mix
Nicotinic acid3.000
Ca Panthotenate1.600
Pyridoxine (B6)0.700
Thiamin (B1)0.600
Folic Acid0.200
Cyanocobalamin (B12)2.500
Vitamin E (500 UI/g)15.00
Vitamin A (500.000 UI/g)0.800
Vitamin D (400.000 UI/g)0.250
Vitamin K0.075

*According to recommendations from the American Institute of Nutrition Rodents Diets, AIN-93G [16].

Mineral mix composition.

Minerals*g/kg of mix
Calcium carbonate, 40.04% Ca357.00
Potassium phosphate, 22.76% P; 28,73% K196.00
Potassium citrate, 36.16% K70.78
Sodium chloride, 39.34% Na; 60,66% Cl74.00
Potassium sulfate, 44.87% K; 18,39% S46.60
Magnesium oxide, 60.32% Mg24.00
Ferric citrate, 16.5% Fé6.06
Zinc carbonate, 52.14% Zn1.65
Manganous carbonate, 47.79% Mn0.63
Cupric carbonate, 57.47% Cu0.30
Potassium iodate, 59.3% I0.01
Sodium selenate, 41.79% Se0.010
Ammonium paramolybdate, 54.34% Mo0.008

*According the recommendations from the American Institute of Nutrition Rodents Diets, AIN-93G [16].

To evaluate the nutritional state, the body weight of the pups was monitored during the 21 days throughout the experiment. After weaning (d22–d180), male pups of the same group were housed in packs of three animals per cage and given unlimited access to food and water until adult period (day 180). Then, the pups were anaesthetized with lethal thiopental anaesthesia (0.15 mL/100 g/body weight) and fixed with a 10% formalin solution (perfused through the left ventricle with buffered saline). The right femurs of the specimens were excised, dissected, weighted and analysed.

Then, the bones were fixed in a 10% formaldehyde solution for 24 hours. After that the bones were decalcified with Ethylenediaminetetraacetic acid. Histological processing was done as routinely to embed the samples in paraffin blocks. Then, 10 μm thick transversal sections were performed at the middle third of the femur diaphysis and stained with Haematoxylin/Eosin. Microscopic analysis was made through the Scion-image® software.

Morphometric Parameters

Measurements of the circumference of the femur’s external surface and the internal surface of the medullary cavity were taken with the results presented in micrometers (μm) [Table/Fig-4]. Afterwards, the software calculated the cortical bone mass through the previously measured structures.

*Haematoxylin and eosin stain of the measured parameters.

Black arrow = Medular area;*= Cortical area. The total area of the femur is the sum of both areas

Statistical Analysis

Data was reported as mean±Standard Deviation (SD). Statistical significance of experimental observations was determined by the ANOVA one-way analysis of variance, followed by Newman-Keuls test to compare the three experimental groups. The level of significance was set at p<0.05. All statistical analysis was done using GraphPad Prism six software (GraphPad Inc., CA, USA).


ER and PER groups presented a significant reduction of cortical mass, weight and length when compared to the control group (p < 0.05 was considered significant). Histomorphometric results of the cortical area from the C, ER, and PER groups were, respectively: 6.54±0.23 μm; 5.51±0.29 μm and 6.18±0.19 μm, while the measures from the medullary cavity were: 3.65±0.16 μm; 4.1±0.20 μm and 3.53±0.12 μm, respectively [Table/Fig-5].

Histomorphometric results of 10 femurs (180 days) from the three groups. Values are presented as mean±SD.

TA (μm)10.19±9.61± (**)9.71± (*)
CA (μm)6.54±0.235.51±0.29 (**)6.18±0.19 (*)
MA (μm)3.65±0.164.1±0.20 (**)3.53±0.12 (*)

* = C vs ER (p≤0.01) **= C vs PER (p≤0.01) ns= no statistical sigfinicance

*Legend: TA= Total area body weight; CA= cortical area; MA= medullary area.


Bones and joints are subject to an ample variety of forces that gives origin to tensions, compressions, shears, torsions and angulations. Those forces result in internal and compressive loads in bone tissue [17].

Bone strength is determined by architectural variables like geometry, porosity, cortical thickness and trabecular structure. Furthermore, it is also determined by the intrinsic properties of the bone tissue [17,18]. The bones are vital tissues and are susceptible to a great number of congenital defects, malnourishment, hormonal and metabolic imbalances, inflammatory and degenerative diseases, as well as pathological or traumatic fractures [17,19].

Bony tissue is critically affected by protein-energy malnutrition [7,13,20-24]. Malnutrition brings biochemical and morphological alterations that leads to an insufficient growth [25]. A study reported that restricted growth during foetal period and insufficient development during childhood are associated with bone mass reduction and higher fracture risks. Moreover, bone consolidation, wound healing, infection recoveries and muscle mass gain are also in risk [23].

Many studies have examined the effects of nutritional deficiencies on bone growth of offsprings in different periods, such as gestation [26], lactation [7,8,13,27,28], and during the post-weaning phase [29]. The growth delay varies in accordance to the gravity and duration of the nutritional deficiency, which may be associated with body size and composition abnormalities in adult age, as well as the bone length and mineral content [21,29-31].

Different forms of disrupted growth have been reported in the literature, and it seems that each form is dependent of the type, duration and intensity of the malnutrition, with special regards to the period that it was performed. Furthermore, femur growth in rats can be influenced by sex, animal species and nutritional state [25-30].

In the present study, the femur was significantly affected by malnutrition during lactation period, and the morphological effects remained until adult life of the animals, even after receiving unlimited amounts of a diet with protein and energy after weaning. An under development of the femur was observed in adult aged offspring (180 days) whose mothers belonged to the ER and PER group during lactation. These morphological alterations were accompanied by significant differences regarding body weight gain of its pups during lactation (21 days).

Body weight alterations can be partly explained by a reduction of ingestion from the PER group dams, as observed in our experimental model. It is thus possible that PER diet may induce behavioural changes, such as humor of the breastfeeding mother. Moreover, it was observed that the dams from both restriction groups ingested smaller amounts of food, and this pattern was repeated throughout the experiment. We believe that this biological phenomenon could lead to a diminished ingestion of magnesium and other minerals essential to the behavior [32,33].

Body weight reduction of the ER and PER groups as well as their osseous mass during adult life may be related to quantitative and qualitative changes of milk composition, as a study showed a reduction of milk components (protein, lipids) available to its offspring [9].

Microscopic analysis showed reduced growth of femoral dimensions on the ER and PER groups. In a previous study [13], we observed that the femur was proportionally underdeveloped and displayed alterations of the anatomical and mechanical axis, as well as the angle between them. Furthermore, the study also presented significant alterations of the head and anatomical neck diameters, together with alterations of the cervico-diaphyseal angle in rats from ER and PER groups, in comparison to the control group.

Geometrical alterations of the femur’s diameter and cortical thickness (as presented in this study) may affect the regular bipedal stance, thus, its torsion and heavier loads resistances are reduced [17,34,35]. There is also a reduction of energy absorption capacity, which leads to fractures [17,36] and a deficit in bone consolidation [17,37,38].

Malnourished rats develop an imbalance, in which bone resorption is increased and bone absorption is decreased. This fact led us to believe that our experiment simulated osteoporosis type II, which could not be reversed with nutritional reposition. The observed alterations may increase the risk of pathological fractures [23].

The first week of life is a critical period regarding the nutritional state, and malnutrition during this interval can cause metabolic incapacity of bone recover up to 100 days in rats [20], and until six months of life in humans [39], or yet, it can influence the metabolism through the rest of the life [5,16,25].

According to the results of this study, catch-up growth is not possible after puberty in malnourished rats dams, in accordance with other studies [13,20,24]. Although, there are other studies that had opposite results [21,22,29]. There was no catch-up growth in rats even after free access to a protein-energy diet even after adult age (180 days). Thus, the importance of a protein-energy adequate diet to the breastfeeding mother during gestational and lactation period is highlighted.

The small number of studied animals, the lack of more detailed morphometric parameters and a less refined measurement instrument limited the study from the methodological point of view.


A poor ingestion of proteins and calories during lactations provokes irreversible morphological changes on the cortical area of the femur in rat’s offspring, which resulted in an incomplete growth during adult life even after regular protein diet ingestion. Diet regulation did not reversed the effects caused in bone mass by maternal malnutrition, thus, proving the “metabolical imprinting theory”.

Further studies are needed in order to quantify the alterations and enlighten the mechanisms through which they occur in association with the morphology of these structures. Moreover, more studies should focus on chronic effects of maternal malnutrition in their offspring, so that one may study more effective methods to deal with potential complications that affect patients.

* = C vs ER (p≤0.01) **= C vs PER (p≤0.01) ns= no statistical sigfinicance*Legend: TA= Total area body weight; CA= cortical area; MA= medullary area.


[1]de Onis M, Blössner M, Borghi E, Prevalence and trends of stunting among pre-school children, 1990-2020 Public Health Nutr 2012 15(1):142-48.  [Google Scholar]

[2]Barker DJ, The developmental origins of adult disease J Am Coll Nutr 2004 23(6 Suppl):588S-95S.  [Google Scholar]

[3]Department of Health. Choosing a better diet: a food and health action plan. London, 2004. Available in: http://www.dh.gov.uk/prod_consum_dh/groups/dh_digitalassets/@dh/@en/documents/digitalasset/dh_4105709.pdf. [Access Date: 10th March 2017]  [Google Scholar]

[4]Bolling K, Grant C, Hamlyn B, Thornton A, Infant feeding 2005. A survey conducted on behalf of The Information Centre for Health and Social Care and the UK health departments by BMRB Social Research 2007 LondonThe Information Centre  [Google Scholar]

[5]Barker DJ, In utero programming of cardiovascular disease Theriogenology 2000 53(2):555-74.  [Google Scholar]

[6]Andrade FM, Judice LF, Cardoso GP, Cisne R, Ramos CF, Babinski MA, Maternal malnutrition during lactation in Wistar rats: effects on elastic fibers of the extracellular matrix in the trachea of offspring J Bras Pneumol 2012 38(5):588-94.  [Google Scholar]

[7]Fernandes RMP, Abreu AV, Schanaider A, Soares Jr ER, Peçanha GCA, Babinski MA, Effects of protein and energy restricted diet during lactation leads to persistent morphological changes on tibia growth in the weaned pups Int J Morphol 2007 25(3):565-71.  [Google Scholar]

[8]Fernandes RMP, Abreu AV, Silva RB, Martinez GL, Babinski MA, Ramos CF, Maternal malnutrition during lactation reduces skull growth in weaned rat pups: experimental and morphometric investigation Anat Sci Int 2008 83(3):123-30.  [Google Scholar]

[9]Passos MCF, Ramos CF, Moura EG, Short and long term effects of malnutrition in rats during lactation on the body weight of offspring Nutr Res 2000 20(11):1603-12.  [Google Scholar]

[10]Teixeira C, Passos MCF, Ramos CF, Dutra S, Moura EG, Leptin serum concentration, food intake and body weight in rats whose mothers were exposed to malnutrition during lactation J Nutr Biochem 2002 13(8):493-98.  [Google Scholar]

[11]Passos MCF, Ramos CF, Dutra SCP, Moura EG, Transfer of iodine through the milk in protein-restricted lactating rats J Nutr Biochem 2001 12(5):300-03.  [Google Scholar]

[12]Ramos CF, Lima AP, Teixeira CV, Brito PD, Moura EG, Thyroid function in post-weaning rats whose dams were fed a low-protein diet during suckling Braz J Med Biol Res 1997 30(1):133-37.  [Google Scholar]

[13]Babinski MSD, Ramos CF, Fernandes RMP, Cardoso GP, Babinski MA, Maternal malnutrition diet during lactation period leads to incomplete catch-up growth in femur of the pups at adulthood Int J Morphol 2016 34(1):71-77.  [Google Scholar]

[14]Monteiro ACT, Paes ST, Santos JA, Lira KDS, Moraes SRA, Effects of physical exercise during pregnancy and protein malnutrition during pregnancy and lactation on the development and growth of the offspring’s femur J Pediatr 2010 86(3):233-38.  [Google Scholar]

[15]Fischbeck KL, Rasmussen KM, Effect of repeated reproductive cycles on maternal nutritional status, lactational performance and litter growth in ad libitum fed and chronically food-restricted rats J Nutr 1987 117:1967-75.  [Google Scholar]

[16]Reeves PG, Nielsen FH, Fahey GC, AIN-93 Purified diets for laboratory rodents: final report of the American Institute of Nutrition Ad Hoc Writing Commitee on the reformulation of the AIN-76 rodent diet J Nutr 1993 123(11):1939-51.  [Google Scholar]

[17]Herbert S, Barros-Filho TEP, Xavier R, Pardini J.A G, Ortopedia e traumatologia– Principios e prática 2009 4th edPorto AlegreArtmed  [Google Scholar]

[18]Boersma B, Wit JM, Catch-up growth Endocr Rev 1997 18:646-61.  [Google Scholar]

[19]Cooper C, Javaid MK, Taylor P, Walker-Bone K, Dennison E, Arden N, The fetal origins of osteoporotic fracture Calcif Tissue Int 2002 70(5):391-94.  [Google Scholar]

[20]Boyer PM, Compagnucci GE, Oliveira MI, Bozzini C, Roig MC, Compagnucci CV, Bone status in an animal model of chronic sub-optimal nutrition: A morphometric, densitometric and mechanical study Br J Nutr 2005 93(5):663-69.  [Google Scholar]

[21]De-Sousa E, Fernandes RMP, Mathias MB, Rodrigues MR, Ambram AJ, Babinski MA, Morphometric study of the proximal femur extremity in Brazilians Int J Morphol 2010 28(3):835-40.  [Google Scholar]

[22]Houdjik ME, Engelbregt MT, Popp-Snijders C, Delemarre van der Wall HA, Long-term effects of early postnatal food restriction on growth hormone secretion in rats JPEN J Parenter Enteral Nutr 2003 27(4):260-67.  [Google Scholar]

[23]Oreffo RO, Lashbrooke B, Roach HI, Clarke NM, Cooper C, Maternal protein deficiency affects mesenchymal stem cell activity in the developing offspring Bone 2003 33(1):100-07.  [Google Scholar]

[24]Rodriguez PN, Friedman SM, Boyer PM, de Portela ML, Influence of dietary calcium concentration on body size and bone composition in rats during recovery from malnutrition J Am College Nutr 1998 17(1):86-91.  [Google Scholar]

[25]Mehta G, Roach HI, Langley-Evans S, Taylor P, Reading I, Oreffo RO, Intrauterine exposure to a maternal low protein diet reduces adult bone mass and alters growth plate morphology in rats Calcif Tissue Int 2002 71(6):493-98.  [Google Scholar]

[26]Fetoui H, Mahjoubi-Samet A, Guermazi F, Zeghal N, Maternal low-protein diet affects bone mass and mineral metabolism in suckling rats J Anim Physiol and Animal Nut 2008 92(4):448-55.  [Google Scholar]

[27]Jones DC, Bernstein M, German RZ, Catch-up and targeted growth following variable duration protein restriction: effects on bone and body mass J Morphol 2011 272(4):485-96.  [Google Scholar]

[28]Medeiros DM, Plattner A, Jennings D, Stoecker B, Bone morphology, strength and density are compromised in iron-deficient rats and exacerbated by calcium restriction J Nutr 2002 132(10):3135-41.  [Google Scholar]

[29]Alippi RM, Meta MD, Oliveira MI, Bozzini C, Schneider P, Meta IF, Effect of protein-energy malnutrition in early life on the dimensions and bone quality of the adult rat mandible Arch Oral Biol 2002 47(1):47-53.  [Google Scholar]

[30]Caddell JL, Scheppner R, The postmortem diagnosis of magnesium defici-ency: studies in an animal model for the human infant J Forensic Sci 1978 23(2):335-44.  [Google Scholar]

[31]Benardot D, Nutrición para deportistas de alto nível 2001 BarcelonaHispano-Europea  [Google Scholar]

[32]Leggon RE, Lindsey RW, Panjabi MM, Strength reduction and the effects of treatment of long bones with diaphyseal defects involving 50% of the cortex J Orthop Res 1988 6:540-46.  [Google Scholar]

[33]McBroom RJ, Cheal EJ, Hayes WC, Strength reduction from metastatic cortical defects in long bones J Orthop Res 1988 6:369-78.  [Google Scholar]

[34]Burstein AH, Zika JM, Helpie KG, Klein L, Contribution of collagen an mineral to the elastic-plastic properties of bone J Bone Joint Surg Am 1975 57(7):956-61.  [Google Scholar]

[35]Edgerton BC, An KN, Morrey BF, Torsional strength reduction due to cortical defects in bone J Orthop Res 1990 8(6):851-55.  [Google Scholar]

[36]Engelbregt MJ, van Weissenbruch MM, Lips P, van Lingen A, Roos JC, Body composition and bone measurements in intra-uterine growth retarded and early postnatally undernourished male and female rats at the age of six months: comparision with puberty Bone 2004 34(1):180-86.  [Google Scholar]

[37]Cesani MF, Ordem B, Zucchi M, Mune MC, Oyhenart EE, Pucciarelli HM, Effect of undernutrition on the cranial growth of the rat. An intergenerational study Cells Tissues Organs 2003 174(3):129-35.  [Google Scholar]

[38]Lucas A, Programming by early nutrition: an experimental approach J Nutr 1998 128(2 Suppl):401S-6S.  [Google Scholar]

[39]Bayne K, Revised guide for the care and use of laboratory animals available. American physiological society Physiologist 1996 39(4):199:208-11.  [Google Scholar]