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    PCR probes for ammonia hyper-producing bacteria in the rumen of New Zealand ruminants : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Nutritional Science at Massey University, Albany campus, New Zealand
    (Massey University, 2000) Manickavasagar, Shanmuganathan
    Competitive PCR (cPCR) primers were developed to detect and enumerate 5 hyper ammonia-producing (HAP) bacteria previously isolated from New Zealand ruminants, and 3 previously described HAP bacteria, Clostridium aminophilum, C. sticklandii and Peptostreptococcus anaerobius. Primers were designed by aligning 16S ribosomal RNA gene sequences and identifying unique site for each bacterium. Primers were matched as closely as possible in terms of length, G+C content and Tm to either the universal eubacterial forward (fdl*) or reverse (rdl*) primers to anchor the PCR at either the 5' or 3' end of the 16S rRNA gene. Primer specificity was tested in amplification reactions with DNA extracted from 35 bacterial isolates, mostly from the rumen. The primers designed for isolates C2 and D5 produced amplified PCR products only with their respective target DNAs. Primers developed for isolates S1, D4 and P. anaerobius also amplified DNA from closely related species, P. asaccharolyticus, Fusobacterium necrophorum and isolate D1, respectively, in addition to their respective target DNAs. Internal controls were developed for each of the chosen primers by creating deletions in the amplified target DNA using restriction endonuclease digestions and religating the terminal fragments. The deleted internal control fragments were reamplified and cloned into the PCR cloning vector pGEM-T. Cloned internal control DNAs were coamplified with known amounts of their respective target DNAs to generate standard curves so that unknown samples could be quantitated. DNAs extracted from rumen samples from sheep fed a diet of chaffed lucerne and infused with either monensin or buffer were probed for HAP bacteria using the cPCR probes. The results showed that isolates C2, D5, S1 and C. sticklandii and C. aminophilum were below the detectable limits of the cPCR technique and their population could not be enumerated. The absence of any PCR amplifiable DNA of these organisms in the rumen samples was confirmed by conventional PCR in the absence of internal control DNAs, by additional purification of rumen DNAs followed by reamplification, and by preamplifying rumen DNA with the universal eubacterial primers fdl* and rdl* prior to PCR with primers specific to each organism. However the D4/F. neewphorum and D1/P. anaerobius probes showed detectable populations in the samples. In vivo the D1/P. anaerobius population in the rumen ranged from 3 to 7x108 cells ml-1. Monensin showed no inhibitory effect on the D1/P.anaerobius population, which maintained steady levels throughout the sampling period. D4/F.necrophorum populations ranged from 3x108 to 1.4x109 bacteria ml-1". Monensin had little effect over the first 48hr compared to control sheep but after 72hr D4/F.necrophorum populations increased and finally reached 1.4 x109 bacteria ml-1 at 96 hrs.
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    Isolation of 5' regulatory sequences for ruminant ATP citrate lyase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1998) Sanders, Rebecca Jane
    ATP citrate lyase is an essential enzyme in the pathway for conversion of glucose to fatty acids in mammalian tissues. The enzyme catalyses the cleavage of cytosolic citrate to acetyl CoA and oxaloacetate in an ATP-dependent reaction. The sequence of the cDNAs for both rat and human ATP citrate lyase have been published and have 96.3% identity at the amino acid level. This high level of identity may also extend to other mammals, including ruminants. The ruminant presents a unique system in which to study the regulation of ATP citrate lyase as levels of expression of the enzyme change during the development of a functional rumen. An analysis of the 5'-regulatory region of ruminant ATP citrate lyase will be important in determining factors that contribute to the developmental regulation of this enzyme in ruminants. In order to analyse the 5'-regulatory region of ruminant ATP citrate lyase, a probe was constructed with which to screen an amplified bovine genomic library. The probe was produced by cloning a 282 bp PCR product containing rat ATP citrate lyase exon II sequence, amplified from rat genomic DNA. This clone was sequenced to verify that it contained rat ATP citrate lyase exon II sequence. This probe was then used for northern and Southern blotting, and for screening an amplified bovine genomic library. Northern blotting of rat and lamb total RNA showed that the probe hybridised with rat RNA, but not with lamb RNA. Conditions for hybridisation were not optimised, as hybridisation between the probe and rat RNA was not as specific as expected. The quality of RNA used for preparing the northern blots could have also affected the specificity of hybridisation. Southern blotting experiments were also inconclusive, as the hybridisation signals seen were not specific. However the probe was shown to hybridise to rat and human genomic DNA. Screening of the bovine genomic library was unsuccessful, but once conditions for hybridisation are optimised, then the probe could be used to rescreen the amplified bovine genomic library, and isolate a clone containing the 5'-regulatory sequences for bovine ATP citrate lyase.
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    Water and electrolyte transfers in ruminants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 1969) Simpson, Heather Vivian
    At the present time, the economy of New Zealand is largely dependent upon the health end well-being of the ruminant animal. The national loss due to primary water and electrolyte disturbances, and to those secondary to other diseases, is of considerable importance. Deficiencies in our knowledge of water and electrolyte metabolism in the ruminant have become apparent, even of the principal cations sodium and potassium. The specialized form of nutrition in the ruminant has entailed some changes in the water and electrolyte economy. In adapting to a diet of plant material rich in cellulose, they have developed a large forestomach, the reticulo-rumen, where a symbiotic population of bacteria and protozoa is maintained and exploited. Microbial fermentation breaks down plant cellulose and converts carbohydrate to volatile fatty acids, principally acetic, propionic and butyric acids, which are rapidly absorbed by the host for use as an energy source. Microbial protein and certain vitamins are also made available further down the gastro-intestinal tract. The development of the reticulo-rumen has resulted in an increase in the content and daily turnover of gut water and electrolytes. A major source of this content of the reticulo-rumen liquor, which provides a well-buffered medium for the microbes, is the copious salivary flow. The rumen of the sheep may contain 500-800 m-equiv of Na+, approximately half that in the extracellular fluid. The daily digestive cycle of salivary secretion and later reabsorption may involve double this amount of Na+.
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    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 Charles
    Development 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.