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Most mammary development is postnatal. Mammary growth that occurs before puberty is diminutive in amount but consequential for future milk production, especially in dairy heifers. With advanced knowledge on fundamental aspects that govern prepubertal mammary development, scientists and farmers alike can ensure that heifers perform their best once they become cows. The Hippo pathway has been identified as an evolutionarily conserved pathway that regulates organ size in many animal species; it might contribute to mammary growth in dairy heifers. This pathway is mediated by yes-associated protein (YAP) and through downstream gene transcription activation, results in cell proliferation. Because YAP has never been identified in bovine mammary tissue, questions examined in this body of work mainly focused on the abundance and localization of YAP in mammary tissue of prepubertal heifers. The first trial investigated effects of in vivo estradiol administration on YAP abundance and localization in prepubertal bovine mammary epithelial and myoepithelial cells. While YAP was present in nuclei and cytoplasm of both cell types, it was also discovered that estrogen did not influence YAP abundance or location. The second research trial focused on determining the effects of in vivo estradiol blockade on YAP abundance and localization in prepubertal bovine mammary epithelial and myoepithelial cells. Similar to the first experiment, results indicate that YAP abundance and localization was not influenced by estrogen blockade. Despite not being responsive to in vivo estradiol administration (experiment 1) or estradiol blockade (experiment 2) under the conditions of our experiments, YAP was present in nearly all mammary epithelial cells and myoepithelial cells of the 21 total prepubertal heifers examined. Its presence hints at an underlying biological function but that function was not ascertained here. It will be up to the next researcher to deduce what YAP contributes to mammary growth in prepubertal dairy heifers. / MSLFS / Replacement dairy heifers are the future of any dairy herd and the successful growth and development of their mammary glands, during early stages of life, can have long term impacts on future milk production. Today, many aspects of mammary growth remain unclear. The studies described herein investigated the effects of the hormone estradiol on a biologically conserved pathway, the Hippo Pathway, which is implicated in organ size across many animal species. The first study investigated the effects of administering estradiol to heifers on Hippo pathway activity, which was thought to be implicated with mammary growth. It was discovered that estradiol administration did not affect pathway activity and therefore may not be the hormone implicated in causing growth and development through Hippo pathway signaling. The second experiment investigated the effect of blocking estradiol’s mammogenic actions on the mammary gland and whether the Hippo pathway was impacted. Results indicated that estradiol blockade did not affect pathway activity. It is possible that another hormone may be linked to mammary growth and development through Hippo pathway signaling. Overall this work contributes to the understanding of biological mechanisms involved in mammary growth and development in dairy heifers.

Intramammary infections (IMI) are common in non-lactating heifer and dry cow mammary glands and occur during periods of appreciable mammary growth and development. The presence of these infections is expected to negatively impact mammary growth and development but has yet to be investigated. The works reported here investigated how IMI affects mammary tissue structure, cellularity, and the expression of integral mammogenic hormone receptors implicated in mammary growth. Non-pregnant non-lactating cows (n = 19) were administered estradiol and progesterone to stimulate mammary growth and 2 quarters of each cow were subsequently infused with either saline (n = 19) or Staphylococcus aureus (n = 19). Intramammary infusion of Staphylococcus aureus increased the number of immune cells present in gland secretions and also increased the proportion of neutrophils comprising these secretion somatic cells. Mammary tissues from quarters infused with Staphylococcus aureus contained more immune cells, less mammary epithelial tissue area, and greater tissue areas of intralobular stromal tissue than saline quarters. Staphylococcus aureus quarters also contained more apoptotic mammary epithelial cells and a lower proportion of apoptotic cells in the intralobular stroma compartment than saline infused quarters; this signified that Staphylococcus aureus quarters had less epithelial growth and experienced an expansion and/or lack of regression of stromal tissues. The number of cells expressing estrogen receptor α (ESR1) and progesterone receptor (PGR), as well as staining characteristics of ESR1 and PGR positive nuclei was also examined in these tissues. No appreciable differences were observed in any of the examined ESR1 and PGR measures between Staphylococcus aureus and saline mammary glands, but myoepithelial cells from Staphylococcus aureus glands had a greater nuclear staining area than saline quarters, indicating that these cells were affected by IMI. The results of these investigations indicate that IMI, in mammary glands that are concurrently stimulated to grow and develop, limits the growth of mammary epithelium and impairs regression of the stromal tissue, both of which are necessary for successful lactational performance. / PHD / Successful growth and development of the dairy cow udder (mammary gland) is important and has long-term impacts on milk production. Most mammary growth occurs during the first pregnancy but, at this same time, a bacterial infection can be present within the mammary gland and is expected to hinder normal growth and development. The studies conducted here sought to examine how a bacterial infection, within a cow’s udder, affects mammary gland growth and development. Overall, it was observed that a bacterial infection in the mammary gland reduced the amount of functional tissue that would eventually produce milk while simultaneously increasing the amount of connective tissue. Infected mammary glands also had a greater number of dying mammary cells, reducing the number of cells that would eventually produce milk. Estrogen and progesterone are known to be integral in supporting mammary growth, so an examination of the number of cells being able to receive signals from estrogen and progesterone was also undertaken; presence of an infection did not alter the number of cells able to receive estrogen and progesterone’s signal. This work furthered our understanding of how bacterial infections affect mammary tissue and alter normal developmental processes.

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