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    Pre-implantation maternal uterine effects on embryo growth and development : an investigation using models of maternal constraint in sheep : a thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science, Massey University, Turitea, Palmerston North, New Zealand
    (Massey University, 2017) Fermin, Lisanne Monique
    Prenatal development and growth are critical to survival of the fetus and neonate. Recent evidence suggests that a critical period for determining growth is the pre-implantation period of pregnancy during which differentiation, organogenesis and development of the embryo occur and the embryo is considerably vulnerable to uterine environmental factors. The objectives of the present study were to examine the effects of restrictive uterine environments on embryo development using two sheep models of maternal constraint: litter size and dam size, and to identify embryonic and maternally-driven mechanisms that regulate development of the peri-implantation sheep embryo. Morphometric analysis (embryo length, width and heart bulge width) of the embryos in peri-implantation single and twin embryos was inconclusive; as the transcriptomics analysis of whole embryos using RNA-seq to examine differential gene expression that may be responsible for differential regulation of growth. In a dam size model, large-breed Suffolk embryos gestated in small-breed Cheviot ewes (constrained environment) were smaller than Suffolk embryos gestated in Suffolk ewes (control) at day 19 of pregnancy, confirming previous findings that maternal constraint is evident in early pregnancy when limitations of space are not of consequence. Progesterone administered in the post-ovulatory period, day 0 to 6, alleviates this apparent constraint such that suffolk embryos gestated in Cheviot ewes that received progesterone are larger than those gestated in Cheviot ewes that did not. Further, differential gene expression analysis of maternal uterine tissues showed that at day 6 and day 19 endometrial genes that encode for histotroph secretion and uterine receptivity are altered by post-ovulatory progesterone administration. Timing of administration of progesterone is critical not only to embryo growth but also to embryo survival. There were lower pregnancy rates in the ewes that received progesterone from day 0 than those that received progesterone from day 2. The results of this thesis indicate that progesterone exerts its effects by regulation of genes that encode for uterine structural and secretory activity to advance the uterus. This likely forces the asynchronous embryo to accelerate its growth in order to adapt to its environment. These findings contribute to the knowledge of the regulatory mechanisms controlling early embryo growth and present a platform within the livestock industry and human reproductive technology practice to manipulate embryo growth to improve survival of offspring.
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    Maternal exercise during pregnancy affects the rat musculoskeletal system and indices of energy metabolism : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Manawatū, New Zealand
    (Massey University, 2013) Rosa, Brielle Vastola
    The developmental origins of health and disease hypothesis postulates that environmental cues perceived by the developing organism during early life program long-term health outcomes. A series of studies were undertaken to examine the developmental programming effects of maternal exercise during pregnancy on offspring musculoskeletal health and energy metabolism using a rat model. Firstly, an exercise that did not cause a potentially confounding stress response in the exercising animal was identified. Secondly, pregnant dams then performed this exercise and its effects on fetal growth and the maternal stress response were quantified. Finally, the offspring of dams that exercised throughout pregnancy were allowed to grow to maturity, and the effects of maternal exercise on their musculoskeletal health and energy metabolism were assessed. Throughout these experiments, body composition was assessed by dual- energy X-ray absorptiometry, and tibial parameters were measured using peripheral quantitative computed tomography. Maternal stress was quantified by measurement of faecal corticoid metabolites. Serum concentrations of the fully and undercarboxylated forms of the bone-derived hormone osteocalcin, and expression of genes related to osteocalcin carboxylation, were measured to explore their role in the response of offspring bone and energy metabolism to maternal exercise. Two exercise types, rising to an erect bipedal stance and tower climbing, were initially tested in non-pregnant rats. Both rapidly caused changes in the tibias of exercised animals without inducing stress. In pregnant rats, both exercises increased fetal growth relative to controls, and neither caused a physiological stress response in the dams. Since rising to an erect bipedal stance had the greater effect on fetal growth, it was selected for use in the final study in which the offspring were grown to maturity. Maternal exercise throughout pregnancy was associated with sex-dependent changes in the bone and body composition of the mature offspring. Male offspring of exercised dams had increased adiposity and serum undercarboxylated osteocalcin concentrations, while offspring of both genders had lower volumetric bone mineral density at the tibial diaphysis, relative to controls. These results suggest that maternal exercise has long- term effects on the musculoskeletal system and energy metabolism, and that undercarboxylated osteocalcin may play a role in these effects.
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    Characterisation of the physical environment of embryos throughout in vitro culture : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
    (Massey University, 2011) Blomfield, Sophia Elizabeth
    Characterisation of the physical environment embryos are exposed to throughout in vitro culture for treatments involving in vitro fertilisation (IVF) has been limited due to measurement difficulties (since the position of an embryo is the point of interest) and the lack of a theoretical framework. Temperature, oxygen concentrations and pH are all important factors in an oocyte’s and an embryo’s environment which, if away from desired levels, may impact on embryo viability. The development of mathematical models provides a structured approach which helps to overcome measurement difficulties. The IVF process was broken down into 70 discrete back-to-back steps, from oocyte aspiration to embryo transfer, which could be modelled. Models of heat transfer were developed for a Petri dish, 4-well dish, Pasteur pipette (un-pulled and pulled), plastic pipette tip (two sizes), denuding pipette and transfer catheter. Models of oxygen and carbon dioxide mass transfer were developed for the Petri dish, in which oocytes and embryos spend the majority of their time in culture. The models were solved by the finite element method in the software package COMSOL Multiphysics 3.3a used in conjunction with MATLAB R2006a. Models were then validated against experimental data. There is considerable variation in the embryology culture process, with respect to the number and timing of steps, both between and within laboratories. Of all the factors in an embryo’s environment embryology practice has the greatest impact on temperature. Embryos are cultured in dishes in incubators which maintain the required gaseous and thermal environment. While paraffin oil, which overlays culture media in a dish, successfully buffers embryos from great changes in oxygen and pH when dishes are removed from an incubator, maintenance of embryo temperature is dependent on numerous factors including the setting of the surface temperature of microscope stages, whether the lid is on or off the dish, the embryo position across the floor of the dish, the dish’s foot height, the time out of incubator and the depth of liquid in the dish. For a period of 5 minutes out of an incubator in a standard Petri dish set up, the pH an embryo is exposed to will not likely rise from pH 7.33, as in an incubator, to above pH 7.38. However, the temperature an embryo is exposed to may change by ± < 0.5 °C or may change by ± 1 to 3 °C, depending on embryology practice. Importantly, an increase of 1 °C in embryo temperature may adversely affect embryo viability while a decrease of 1 °C will likely have little impact. Transfer of an embryo in a pipette is the step identified which subjects embryos to the greatest rate and magnitude of temperature change. While temperature in a dish may change by 1 to 3 °C during 5 minutes out of an incubator, the temperatures within a pulled glass Pasteur pipette can fall by > 10 °C in 10 seconds. Use of plastic pipette tips instead of glass pipettes is beneficial for maintaining embryo temperature as the temperature will fall by approximately 3 °C in 10 seconds, 7 °C less than in the glass pipette under the same conditions. This work identified many simple practical steps, such as the use of plastic pipette tips instead of glass, which minimise temperature changes embryos are exposed to throughout the culture process. Applying the Model of mass transfer of O2 in a Petri dish disproved the belief that equilibration of gas in the dish occurs significantly faster without a lid. The model of O2 transport in a Petri dish demonstrated that it takes ˜1 hour to reach 67 % and ˜4 hours to reach 95 % full equilibration of oxygen between atmospheric and 5 vol % O2 at 37 C. Modelling mass transport of CO2 provided a means to predict pH changes within a media drop in a Petri dish. In equilibration from atmospheric to 6 vol % CO2, the pH reached within 0.1 unit of the final value in ˜1.5 hours. An important finding of this work was that sufficient equilibration of gas may be achieved in ˜2 hours and therefore the pre-equilibration time for dishes (currently overnight) may be shortened, reducing the degradation of amino acids, which occurs at 37 °C, to ammonium (embryo toxic). There is considerable variation in embryology practice. This work successfully utilised engineering knowledge and mathematical modelling to describe the physical environment of temperature, oxygen and pH that oocytes and embryos may be exposed to throughout an open embryo culture system, used by the majority of IVF clinics worldwide. The findings here provide a basis for establishing best practice. Further work is needed to quantify the effects on the embryo of fluctuation in the embryo’s environment but this work demonstrates that mathematical modelling of the embryo’s environment in IVF is a viable tool for improving laboratory practice.