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Item Effect of heavier live weight of ewe lambs at breeding on reproductive performance, mammary gland development and subsequent live weight : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science, Massey University, Palmerston North, New Zealand(Massey University, 2021) Haslin, EmmanuelleBreeding ewe lambs at seven to eight months of age can increase farm profitability and ewe lifetime performance. In New Zealand, 30 to 40% of ewe lambs are bred each year with a minimum recommended pre-breeding live weight of 40 kg. Ewe lamb reproductive performance increases with breeding live weight, therefore, some farmers aim to breed ewe lambs heavier than 40 kg. Increasing ewe-lamb growth rates prior to puberty, to achieve a heavier breeding live weight could, however, impair ewe lamb mammary gland development and lactational performance. Currently, little is known about the impact of breeding heavier ewe lambs on their subsequent performance, live weight, and efficiency. The aim of this thesis was to investigate the effect of breeding heavier ewe lambs on their reproductive performance, mammary gland development and live weight over their first three breeding seasons. Ewe lambs were managed from weaning to breeding and achieved an average live weight of 47.9 ± 0.38 or 44.9 ± 0.49 kg at breeding. A growth rate of 150 g/d prior to the first breeding did not affect ewe lamb mammary gland development to the weaning of their second litter, as measured using ultrasonography. Positive relationships were found between ewe lamb mammary ultrasound measures at one year of age and the growth of their progeny to weaning. The associations between ultrasound measurements and growth of the progeny indicate that ultrasound scanning has the potential to be used as a selection technique for heavier lamb live weight at weaning. Although the live weight difference between treatments was limited to three kilograms, compared with lighter ewes, heavier ewe lambs at their first breeding showed greater fertility rate, litter size and lambing percentage but did not differ in the second and third breeding seasons. Over the first three breeding seasons, heavier ewe lambs had greater lamb production than their lighter counterparts. Ewe lamb live weight treatment had no effect on progeny performance to weaning, nor ewe efficiency over the three-year period. A positive association was found between ewe lamb breeding live weight and their mature live weight at 39 months of age. Farmers should aim to breed their ewe lambs at heavier live weights to maximise their reproductive performance as a ewe lamb and, if well managed, they can achieve increased ewe performance over the first three breeding seasons, although there would be no impact on efficiency. Before firm recommendations can be made to farmers, lifetime performance and longevity of the heavier ewe lambs at breeding needs to be examined.Item A study of a strain of albino mice with regard to suitabilty for investigations of the role of the adrenal cortex in mammary gland growth : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in the University of New Zealand(Massey University, 1956) Munford, R EThe experiments reported in this thesis were carried out in the hope that they might assist in the clarification of the role of the endocrine system in the regulation of mammary gland development. A large part of the work was concerned with the elucidation of the general effects of adrenal insufficiency in mice, and with the maintenance of adrenalectomised mice by the injection of cortisol acetate. It is hoped that the results obtained with these mice will be of some assistance in future studies of the endocrine control of the growth of the mammary glands in mice, where it is dersired to exclude any influence mediated by, or originating from the animal's own adrenal cortex. This study w_as prompted by Dr. D.S. Flux, to whom the author is indebted for guidance, encouragement, and patient instruction. The advice of Professor I.L. Campbell, in whose department the work was carried out, and the assistance of Mr D.J. Myers are gratefully acknowledged. The author also wishes to thank the staff of the College Library for their considerable efforts in obtaining a large number of .journals on loan from other institutions, and Mr. K.A. Rose for his work in connection with the reproduction of the illustrations. Thanks are also due to the following for gifts of material to Dr. D.S. Flux: Organon Ltd., England for oestrone (through the courtesy of Mr. G.B. Davis of The Dental and Medical Supply Co. Ltd.) A.M. Satterthwaite and Co. Ltd. for cortisol (through the courtesy of Mr. F.A. Hacking). [From Preface]Item Prognostic significance of tumour-associated inflammation related markers in canine mammary gland tumours : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterniary Science at Massey University, Manawatū, New Zealand(Massey University, 2020) Ariyarathna, HarshaCanine mammary gland tumours (CMGTs) are a major cause of illness and premature death in female dogs, especially in countries like Sri Lanka where early de-sexing is not a routine veterinary practice. Therefore, there is a need for prognostic markers which can better predict the behaviour of a CMGT. In human breast cancers (HBCs), some markers of tumour-associated inflammation (TAI) have been shown to better predict prognosis than many conventional prognostic markers. This thesis investigated whether TAI prognostic markers adopted from human breast cancers are similarly prognostic for CMGTs. The prognostic markers investigated in this thesis included tumour stromal mast cell density determined by toluidine blue staining, gene expression of chemokines: CCL5, CXCL12, CXCL10, and chemokine receptors: CXCR3, CXCR4, CXCR7, CCR4, CCR9 and gene expression and immunostaining of two immune checkpoint molecules: programme death ligand-1 (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) . Similar to HBCs, all markers except CXCL10 and CCR4 were prognostic of the disease outcome determined by disease-specific survival times of the dogs with mammary neoplasms. Of them, stromal mast cell density, CCL5 gene expression and PD-L1 immunostaining were prognostic independent of tumour size, tumour histological grade, and lympho-vascular invasion observed in histological sections. In conclusion, this thesis identified that similar to HBCs, TAI related prognostic markers are useful to better predict the behaviour of CMGTs while stromal mast cell density has the potential to be adopted for routine laboratory prognostic determination. In addition to identification of prognostic markers, surveys of CMGTs in Sri Lanka and New Zealand conducted for sample collection gathered large amounts of information that allowed a comparison of CMGTs between the two countries. These studies allowed a determination of the characteristics of dogs with CMGTs, as well as allowing histological characterisation of the tumours within the two countries.Item Effect of mechanical stress o the integrity, signalling mechanisms and function of bovine mammary epithelial cells : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Manawatū, Palmerston North, New Zealand(Massey University, 2014) Biet, JulianeMammary gland engorgement due to milk accumulation in late lactation leads to changes in cell morphology and has been recognised as a potential key initiator of involution and remodelling of the mammary gland. The physical distension of mammary epithelial cells (MEC), due to udder filling, is likely to result in mechanical tension on cell-cell and cell-matrix interactions. Cell-cell and cellmatrix junctions provide tissue integrity, promote cell polarity, guarantee sufficient communication between cells to ensure synchronised milk secretion and support cell survival. Their disruption may be one of the early initiators of the mammary gland remodelling process. As a consequence, the primary goal of this study was to determine the potential effects of MEC stretch on changes in cell sensing within the mechanical micro-environment in the initiation of bovine MEC involution. During this investigation, particular emphasis was put on three potential mechanosensors: tight junctions (TJ), focal adhesions (FA) and primary cilia (PC), and their regulation in the early stages of involution using in vivo and in vitro experimental approaches. Static, biaxial in vitro cell stretch and acute physical distension in vivo resulted in changes in TJ protein expression levels implying a potential disruption of cell-cell communication as well as communication with the cell‟s cytoskeleton. Furthermore, down-regulation of Akt and pAkt following different periods of mechanical strain applied in vitro and decreased levels of pAkt following acute physical distension in vivo indicated a disruption of β1-integrin-FAK survival signalling through the PI3K-Akt pathway downstream of FA interactions. Increased numbers of ciliated MEC following extended periods of non-milking indicated a dedifferentiation of MEC. Furthermore, increased levels of STAT6 transcription (part of PC signalling following mechanical stimulation) factor indicates the initiation of macrophage accumulation and promotion of tissue remodelling of the bovine mammary gland. In conclusion, this study supports the hypothesis that local factors play an important role during bovine mammary gland involution and that mechanical stimulation may play a part in the initiation of this process.Item Mammogenesis in the mouse : a study of the responses of the immature mammary gland to minimal oestrogenic stimulation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University(Massey University, 1979) Aye, Khin MaungThe response of the mammary glands of immature ovariectomized mice of the NOS strain to minimal levels of oestradiol monobenzoate was investigated in two experiments using both objective and subjective measurements as indices of response. Uterus weight, thickness of the uterine wall and vaginal opening were used as additional measures of the effectiveness of the oestrogenic stimulation. In the first experiment single injections of OMB at four levels (0.01, 0.03, 0.09 and 0.27µg) were used and mice were killed at four intervals after the injection (1,2,4 and 8 days). A significant dose response relationship was observed for mammary gland area to OMB which was essentially linear. Different stages of the response were observed both with respect to the morphology (in whole mounts) and the micro-anatomy (in serial histological sections) of the duct system. The sampling errors of a histometric estimate of volume of glandular tissue were investigated and the results used to design a stratified sampling system for the second experiment. In the second experiment dual injections at one of three levels (0.04, 0.1 and 0.2µg total), given at one of three spacings (2, 4 and 8 days) were used and mice were killed at one of three intervals after the second injection (2, 6 and 14 days). The response of the mammary gland to log-dose of OMB was essentially linear for the estimate of volume of glandular tissue, but no response to increasing level of OMB was seen with mammary gland area. The detailed observations of the morphological and histological changes have been discussed in relation to the results reported in other studies. The following stages have been proposed as the sequence of events, which can extend over a period greater than a week, following discrete doses of oestrogen at minimally effective levels: (1) Increase in width of principal ducts, thickening of the epithelial wall and the appearance of a non-specific secretion: (2) Formation of peripheral 'clubs' accompanied by mitotic activity along the length of the principal ducts; (3) Extension of the principal ducts from the peripheral clubs and formation of small end buds at discrete points along the principal ducts. (4) Extension of the small end buds to form higher order duct branches.Item The role of the mammary fat pad during mammogenesis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, New Zealand(Massey University, 1996) Hovey, Russell Charles; Hovey, Russell CharlesDevelopment of the female mammary gland involves the proliferation and morphogenesis of epithelial cells within a matrix of adipose and connective tissue which constitutes the mammary fat pad. The objective of this research was to investigate the mechanisms by which this stromal environment locally regulates postnatal mammogenesis. Initial experiments showed that the mouse mammary fat pad liberates a diffusible activity in vitro which stimulates the growth of mouse mammary epithelial cells and enhances their proliferative response to insulin-like growth factor-I, epidermal growth factor and insulin. This effect was specific to these mitogens, and of a variety of cell lines tested was most pronounced for mouse mammary epithelial cells. Subsequent investigations indicated that these responses were likely induced by unsaturated fatty acids, particularly linoleic acid, from mammary adipocytes. Such responses may be effected by increased intracellular signalling via the actions of protein kinase C. The mitogenic capacity of the mouse mammary fat pad was also evaluated across several physiological states. Mammary fat pad-stimulated proliferation during the estrus cycle was increased at estrus concomitant with a phase of ductal elongation in vivo. In certain medium treatments there was evidence for epithelial upregulation of the mitogenic effect of the mammary fat pad, where intact mammary tissue was more stimulatory than mammary fat pad cleared of endogenous epithelium. Further experiments demonstrated that while the mitogenic effect of the mammary fat pad was unaltered by ovariectomy, ovarian function was required for this effect to be increased by exogenous progesterone. The effect of estrogen was independent of ovarian function but was altered by the local epithelial-stromal interaction, where it increased the mitogenic effect of epithelium-free mammary fat pad and decreased that of intact mammary tissue. Mitogenic stimulation by mammary tissues also declined during virginal development to be least in mature virgin and mid-pregnant states. Stimulation by intact mammary tissue increased during lactation, while that from epithelium-free mammary fat pad remained constant in the presence of steroid hormones and increased in the presence of growth factors. Further experiments investigated the stromal regulation of epithelial growth within the ruminant mammary gland. Differences between the ruminant and rodent mammary fat pad were emphasised in vitro where ovine mammary fat pad stimulated the growth of mouse mammary epithelial cells but did not markedly potentiate their growth factor-responsiveness. A subsequent study examined the expression of stroma-derived growth factors within the ruminant mammary gland during postnatal development, and their regulation by several physiological influences. The level of insulin-like growth factor (IGF)-I mRNA in the ovine mammary fat pad was elevated prior to puberty and during late gestation, while IGF-II mRNA was upregulated in mammary parenchyma of virgin ewes in a transcript-specific manner. Abundance of IGF-I mRNA in mammary tissues of prepubertal ewe lambs tended to be increased by exogenous estrogen whereas IGF-II mRNA levels were reduced. Messenger RNA for keratinocyte growth factor (KGF) was detected within the ovine mammary fat pad throughout development as 2.4 and 1.5 kb mRNA transcripts which were expressed by stromal adipocytes and fibroblasts, respectively. The level of KGF mRNA in mammary tissues of prepubertal lambs was increased by ovariectomy and decreased by estrogen, while KGF mRNA expression in cultures of mammary fibroblasts was suppressed by dexamethasone. Messenger RNA for hepatocyte growth factor, a paracrine mitogen and morphogen for mammary epithelial cells, was expressed in the ovine mammary fat pad and by cultured mammary fibroblasts. The abundance of basic fibroblast growth factor (bFGF) mRNA was highest within the ovine mammary fat pad, while in vitro results suggest bFGF may be a paracrine/autocrine mitogen for multiple cell types within the mammary gland. Basic FGF gene expression in mammary tissues of prepubertal ewes was reduced by estrogen treatment. For each of these growth factors there was evidence suggesting that their expression within the mammary fat pad was upregulated by the adjacent mammary epithelium. In conclusion, these findings indicate that the mammary fat pad may stimulate the proliferation of mammary epithelial cells during postnatal mammogenesis by a variety of influences. Such mechanisms may involve the direct stimulation of epithelial growth or the modulation of epithelial responsiveness to other mitogens. These effects may function to mediate the actions of certain mammogenic hormones. Furthermore, strong evidence indicates that mammary growth may be locally regulated by the interaction between epithelial and stromal cells.Item Nitric oxide production in the mammary gland : a thesis submitted in partial fulfilment for the degree of Doctor of Philosophy at Massey University, New Zealand(Massey University, 2001) Turner, Sally-AnneAlthough the effects of nitric oxide (NO) have been widely studied in many different cell and tissue types, very little is known of the role it plays in the mammary gland. Thus, the production of NO by mammary gland was investigated in a series of experiments. NO is a free radical gas which is produced by a wide variety of cells by the action of the enzyme nitric oxide synthase (NOS) on arginine. This results in the formation of citrulline and NO. The study first examined several methods for their suitability for the detection of NO or NOS. Evaluation of the methods revealed that the indirect measurement of NO production by the detection of nitrate and nitrite (NOx), the spontaneously produced metabolites of NO, was the most valid and reliable. The measurement of NOx was carried out in culture medium using a fluorescent-based assay, which was developed by the modification of published methods, during the course of this study. Comma-D cells (murine mammary epithelial cell line) were used to investigate the production of NOx by the inducible form of NOS (iNOS) following treatment with cytokines and cytotoxins. The cell's response was characterised and showed that mammary epithelial cells produce NOx in a dose dependent manner in response to interferon-γ (IFN-γ). Lipopolysaccharide (LPS), a component of bacterial cell walls, was employed to examine the response of the mammary epithelial cells to cytotoxins and it was found that the treated cells produced more NOx than the untreated ones, however, no dose response was apparent. The specific iNOS inhibitor, aminoguanidine (AG) and general NOS inhibitor Nω-nitro-L-arginine (L-NNA) were both used to confirm that the NOx measured in the medium was produced by NOS. The production of NOx by the mammary gland was also examined in cultured explants of mammary tissue taken from pregnant (D 12-14 of pregnancy) and lactating (D 12-14 postpartum or D 17-18 postpartum) rats. A significant difference was found in the basal production of NOx between the different developmental stages. The method of euthanasia of the rats also affected the amount of NOx produced. The inclusion of prolactin (PRL) also increased the production of NOx from both Comma-D cells and explants of mammary tissue. Xanthine oxidase (XO), an enzyme responsible for the conversion of NOx to NO under anaerobic conditions, does not interfere with the determination of NO production using the NOx assay. The measurement of NOx was carried out in the milk of cows following the intramammary infusion of Streptococcus uberis or interleukin-1β (IL-1β). By comparing the milk NOx concentration with the somatic cell count (SCC) and electrical conductivity (EC) of the milk, it was concluded that the source of the NOx in the milk could not be attributed entirely to the epithelium or the somatic cells. The experiments in this Thesis clearly show that the mammary gland is capable of the production of NO in response to a variety of situations and that the regulation of the production is very complex. The work also identifies some new areas of research, which if completed would further enhance the understanding of the role NO plays in the mammary gland.Item DNA synthesis in mammary epithelial cells of Swiss mice during lacation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand(Massey University, 2003) Croke-Auldist, Danielle EProliferation of extra secretory epithelial cells in mammary glands during lactation could potentially increase milk production, with flow-on benefits such as improved weaning weights of young or increased exports from the dairy cow industry. The primary objective of the research reported in this thesis was to increase proliferation of mammary epithelial cells during lactation so that the mechanisms associated with this phenomenon could be studied. Induction of increased proliferation in mammary glands was attempted by applying challenges to mice which were used as a laboratory model for agriculturally important species such as cows and pigs. The experiments reported in this thesis also included refinement of methodologies developed to study proliferation of secretory cells in mammary glands during lactation. The first was to improve techniques for describing the chemical composition of mammary glands collected during lactation. This was achieved by collecting and analysing the composition of mouse milk at 3 stages of lactation (Chapter 3). While milk protein and milk fat remained constant throughout, the concentration of lactose increased with time. These data were critically important for correcting the weights of mammary glands for milk content. A second investigation was carried out to compare different methods of calculating the milk production of mice (Chapter 3). Three methods were evaluated with the best based on calculating the maintenance energy requirements of the metabolic weight of the litter which was added to the energy required for measured litter growth. The total energy required was then converted to a quantity of milk. The third methodology developed during the course of the work in this thesis was sample preparation for analysis of lactating mammary cells using flow cytometry. One approach to increasing proliferation of mammary epithelial cells during lactation was to increase the suckling intensity of the mice. This challenge was accomplished by either increasing litter size (Chapter 6) or by increasing the ratio of pups per gland by taping over 5 of the 10 glands (Chapter 5), Suckling intensity was increased to 2 pups per gland but the effect was to accelerate mammary gland development in terms of cell number and milk synthesis status. Once a suckling intensity of >1 pup per gland was reached, there was no additive effect on the size of mammary glands or milk production at mid lactation. Mammary glands appeared to have a limit on their size and output which is reached at a suckling intensity of 1 pup per gland. Manipulation of suckling intensity did not produce a suitable model of elevated proliferation of mammary epithelial cells during lactation. Another approach tested was to use exogenous steroids as these had previously caused increased proliferation in mammary glands (Nagasawa and Yanai, 1978; Knight and Peaker, 1982d). The work reported herein showed that the response of mammary glands of mice to administration of steroids was dependent on stage of lactation and the dose (Chapter 4). In mid lactation, mammary glands were unresponsive for the parameters measured but in late lactation, incorporation of [3H] thymidine into DNA increased and milk production decreased in response to higher doses of estrogen. The high estrogen dose did not however yield a suitable model for the study because the elevated incorporation of [3H] thymidine was associated with early involution of mammary glands rather than proliferation leading to a net increase of epithelial cells. The most promising method of analysis came from histological studies of lactating glands of mice labelled for DNA synthesis. Labelling indices of epithelial cells were >1.5 times greater on the edges of glands on D1 of lactation compared to the inner zones of glands. This within mouse variation was much greater than any between mouse variation arising from the suckling intensity and steroid experiments. An attractive feature is that tissues are derived from the same gland and have therefore been exposed to the same factors such as systemic mitogens and nutrition. In addition, the differences in labelling indices were measured in glands of mice suckling litters of 10 pups which is an easily repeatable treatment compared to some of the more complicated treatments tested during the course of this thesis. Dissection of mammary glands into outer and inner zones could provide useful tissue for the study of local factors involved with increased DNA synthesis of epithelial cells during lactation. Histological studies also revealed that following labelling of mammary epithelial cells for DNA synthesis on the day after parturition, the proportion of cells labelled decrease at a constant rate over the next 23 days (Chapter 8). This project has increased the knowledge of manipulations of mouse mammary glands during lactation. It was found that growth of mouse mammary glands during lactation is difficult to increase experimentally and may have limited application as a model system to study regulation of growth of mammary glands during lactation. However, the work completed in this thesis will allow similar work to continue, with a high chance of success of investigating factors involved in mitosis of epithelial cells in lactating mammary glands.