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Chapter 2: Materials & Methods
Introduction
Animal Experimentation
This study utilises a small animal model to examine the effects of altered maternal nutrition on the long-term development and health of the offspring. The rat has been used extensively in DOHaD research and the laboratory of Dr Vickers has over 20 years experience in nutritional modelling in the Wistar rat. All animal experiments were undertaken under the guidelines and approval of the Animal Ethics Committee at the University of Auckland. All animal experimental designs were performed with the best principles of laboratory animal experimentation in mind, that is the ‘Three Rs’ of Russell and Burch – Replacement, Reduction and Refinement, first proposed in 1959 (284-286), while maintaining the necessary statistical power as determined from prior independent experimental cohorts.
Replacement We felt that an animal model was required and could not be replaced by any other model for this study, which is informed in part by previous epidemiological observations in human populations, and which seeks to tease apart mechanistic determinants of the effects noted in humans. The Liggins Institute has developed animal models displaying a phenotype that closely resembles that of the human metabolic syndrome. The value of models utilising laboratory animals lies in the ability to manipulate one variable at a time whilst controlling against confounds, thus allowing for an examination of the interaction between the maternal and postnatal nutritional environments.
Reduction All endeavours were made to reduce the numbers of animals used, and to reduce ethical costs. Numbers were based on power equations derived from prior experimental cohorts in our laboratory, utilising the Wistar strain of rats. All work was done under the guidelines of the New Zealand Animal Welfare Act (287-289). Animals had free access to water at all times, and the level of undernutrition in the UN cohorts was at a moderate level. All other animals had food supplied ad libitum. Due to the social nature of rats, all animals were caged as pairs. The temperature and humidity of the housing facility were set at optimal levels and carefully monitored. A registered veterinary surgeon was available at all times should any animal appear unwell.
Refinement This refers to techniques of care, in all aspects, that minimise suffering and distress. Refinement comes with experience and with attention to animal welfare. The laboratory of Dr Vickers has extensive experience in large animal cohorts and as such has implemented a monitoring system that has been refined over numerous experimental cohorts to minimise disturbances to the animals, extraneous stressors and minimisation of handling stress.
All animal experimentation was approved by the University of Auckland Animal Ethics Committee (Application No. R402).
Broad Outline of the Experimental Design
Parental Generation Normal SPF-derived males and female Wistars were raised for breeding. They were placed on various diets during rearing as dictated by experimental protocols detailed below. Animals were acquired at a weaning age (day 22) to allow familiarisation with the handlers prior to mating.
Mating, Pregnancy, Lactation Dietary manipulation occurred during pre-conception, pregnancy and/or lactation. This study encompassed two broad categories of nutritional manipulation that resulted in a total of 6 maternal nutritional groups: Controls, 3 levels of undernutrition, and 2 levels of maternal obesogenic diets.
Offspring Litter size was adjusted at birth to 8 pups (4 male and 4 female) to ensure standardised nutrition until weaning. Weight gain and food consumption were monitored through lactation and during the postnatal period. At weaning the offspring were divided into male and female cohorts and further divided into 2 post-weaning dietary groups to be raised either on standard chow or HF.
Phenotypic measurements Body weights were recorded every three days during the post-weaning period and food intakes recorded at defined postnatal periods representing pre-pubertal, pubertal, mature adulthood and later adulthood. At day 150, body composition was quantified via dual energy x-ray absorptiometry (DEXA). At day 175, animals were killed (decapitation following anaesthesia) and plasma and tissues collected for later analysis. Plasma samples were analysed for leptin, insulin and glucose. Pancreas tissue was immediately dissected and snap frozen for gene expression analysis via qPCR.
Generation of the experimental cohorts
Animals for Breeding
Wistar rats were obtained from the SPF breeding colony at the Vernon Jansen Unit, the animal laboratory facility of the University of Auckland. As described earlier, animals were acquired at weaning age (day 22) to allow familiarisation with the handlers prior to mating and to provide a continuum of experimental handling and conditions throughout the course of the experiments. All breeders were housed under standard conditions with a 12:12 light:dark cycle, constant temperature and humidity and ad libitum access to food and water.
For the undernutrition cohorts, all females were fed a standard rat Chow ad libitum (Diet 2018, Harlan-Teklad, please refer to 2.2.2) from weaning until the time of mating. For the maternal high fat (HF) model, females were either fed the standard rat Chow ad libitum until mating or fed a moderate high fat diet (D12451, research Diets, please refer to 2.2.2) ad libitum from weaning until mating. A total of 76 females were used for breeding (56 maintained on the Chow diet and a further 20 fed the HF diet post-weaning).
Males for breeding purposes (n=20) were fed the standard rat Chow from weaning until the time of mating and housed under identical conditions to that of the females. Male background was normalised as much as possible across the maternal nutritional groups to minimise paternal/litter influences on experimental outcome.
All animal work was approved by the animal ethics committee at the University of Auckland.
Diet Composition
An outline of the two diets is given in Tables 2.1 and 2.2, and detailed compositional analysis is available via:
Chow diet, D2018: www.harlan.com
HF diet, D12451: www.researchdiets.com/pdf/Data%20Sheets/D12451.pdf
The HF diet utilised has been well cited in the literature and reflects an “open source” diet, thus ensuring consistency across batches. The standard control chow diet was routinely tested for compositional deviations and no significant differences were observed across batches during the course of these studies.
Animals of the parental generation were weighed twice weekly from weaning until the time of mating (day 100). Females were DEXA scanned prior to mating to examine the effect of pre-conceptional HF nutrition on body composition.
Mating
Mating commenced when the animals had reached 100 days of age. Females were probed using an EC40 Estrus Cycle Monitor (FST 22500-1 Rat, Fine Science Tools Inc., Canada) to detect the day of oestrus, at which time they were placed into a cage overnight with a selected male. Mating was confirmed the next morning by microscopic examination of material obtained by vaginal lavage. Presence of sperm and a vaginal mucus plug were indicative of successful mating. There were a few females where oestrus proved difficult to determine, even after a week of daily probing, and these were placed in cages with a selected male and left together for seven days to cover 1 oestrus cycle.
Female age range at mating was 103 – 119 days old, with the median at day 108.
Pregnant females were housed as singletons. Food intake and body weight were measured daily. Of the 56 females raised on chow, 44 were successfully mated (78.6% success rate). Of note, of the 20 females raised on the HF diet, only 8 were successfully mated (40% success rate). Following a two week mating period males and any females that did not become pregnant were euthanased, with tissues and plasma collected for pools.
Chapter 1: Introduction
Part A Epidemiological evidence for developmental programming
A1.1 Introduction: David Barker & the Hertfordshire cohort
A2 Non-nutritional factors influencing birth weight
A3 Conclusions:
Part B: Animal Models of Developmental Programming
B1.1 Introduction.
B2 Developmental Programming of the Rodent Pancreas
Part C: Insulin, Leptin & the Adipoinsular Axis
C1 Insulin
C2 Glucose-stimulated insulin secretion
C3 Leptin
C4 Leptin Receptors
C5 Leptin Receptor Signalling
C6 The Adipoinsular Axis
Part D: Obesity & Type 2 diabetes
D1 Insulin resistance and obesity
D2 Insulin resistance and the development of T2DM
D3 Subcutaneous versus visceral fat deposition
D4 Insulin resistance and T2DM
D5 An underlying susceptibility in some people?
Chapter 2: Materials & Methods
2.1 Introduction
2.2 Generation of the experimental cohorts
2.3 Evaluation of offspring and postnatal development
2.4 Laboratory Work
Chapter 3: Maternal undernutrition during critical windows of development results in differential and gender specific effects on postnatal adiposity and related metabolic profiles in adult rat offspring
3.1 Introduction
3.2 Materials and Methods
3.3 Results
3.4 Discussion
Chapter 4: Maternal nutritional history predicts obesity in adult offspring independent of postnatal diet
4.1 Background
4.2 Materials and Methods
4.3 Results
4.4 Discussion
Chapter 5: Gene Expression
5.1 Introduction
5.2 Materials and Methods
5.3 Results
5.4 Discussion
Chapter 6: Discussion
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