Experiments were conducted to evaluate the ability of prebiotic and probiotic supplementation of commercial fish feed to improve the physiological, immunological, and growth responses of stressed Nile tilapia. To investigate these objectives, tilapia were divided in two major groups: control fish (fed regular commercial feed) and stressed fish (induced by dietary cortisol supplemented to regular commercial feed). Stressed fish were further divided into three sub-groups: stressed fish fed regular feed, stressed fish fed probiotic-supplemented feed, and stressed fish fed a mixture of prebiotic and probiotic supplemented feed. Fish were maintained and tested over an eight-week long experimental period. A variety of physiological, immunological, and growth parameters were measured over the course of the experimental period. These parameters include: serum cortisol, blood glucose, plasma protein, packed cell volume, hepato-somatic index (HSI), spleen-somatic index (SSI), lysozyme activity, feed conversion ratio (FCR), specific growth rate (SGR), protein efficiency ratio (PER), length gain, weight gain, length gain, and condition factor (K). The results of this study does not support the use of these specific prebiotic and probiotic as functional feed additives in Nile tilapia at the levels tested in this study. Further research is needed to determine which probiotic species are best suited for use in Nile tilapia and which prebiotic, when used in combination, will allow these probiotics to have maximum effect.

A reprodução é custosa para ambos os sexos, mas a magnitude dos gastos e sua relação com o sucesso reprodutivo diferem entre os gêneros. Os custos reprodutivos são divididos em duas categorias: custos de sobrevivência e custos energéticos. Crotalus durissus possui um ciclo reprodutivo sazonal com cópula ocorrendo no outono e a parturição no final no verão. Os machos competem por fêmeas receptivas. A inferência de custos reprodutivos associados à sobrevivência foi realizada por meio de levantamentos das taxas de atividade entre machos e fêmeas. A variação da quantidade de gordura abdominal e dos substratos energéticos do fígado e dos rins foi avaliada para mensurar o custo energético. Os resultados sugerem que para fêmeas a reprodução exige um alto investimento energético evidenciado pelos maiores níveis de gordura abdominal e de lipídios no fígado durante a fase vitelogênica e o padrão de atividade diferenciada entre machos e fêmeas sugere que a estação reprodutiva embute um custo de sobrevivência maior para os machos. / Reproduction is costly for both sexes, but the magnitude of spending and its relation to reproductive success differ between genders. Reproductive costs are divided into two categories: survival costs and energy costs. Crotalus durissus has a seasonal reproductive cycle with mating occurring in the fall and parturition in late summer. The males compete for receptive females. The inference of survival costs was accomplished through surveys of activity rates between males and females. The variation of the amount of abdominal fat and energy substrates in liver and kidneys was evaluated to measure the energy cost. The results suggest that for females reproduction requires a high energy investment - evidenced by the higher levels of abdominal fat and lipids in the liver during vitellogenic phase and activity patterns differentiated between males and females suggests that the reproductive season embeds a higher cost of survival for males.

Acquired immune deficiency syndrome (AIDS) is characterized by a chronic, progressive depletion of T-lymphocytes bearing the CD4 cell surface marker. Human immunodeficiency virus (HIV), the causative agent of AIDS, is a cytolytic retrovirus which infects CD4+ T-lymphocytes resulting in virus-mediated cytopathic effects (CPE). HIV-induced CPE includes fusion of cells to form multinucleated giant cells or syncytia and the death of single cells due to a 'ballooning degeneration' Quantitation of intracellular volume regulation ions, sodium and potassium, was performed using ion-specific fluorescent probes, fluorescent microscopy, digital image analysis, and fluorescent concentration analysis (FCA). There were observable increases in the intracellular free concentrations of both cations in HIV-infected cells. The activity and expression of the Na+/K+/2C1$-$ cotransporter, a diuretic sensitive ion transporter, increased markedly during acute HIV infection of CD4+ lymphocytes. Inhibition of the cotransporter by diuretics inhibits HIV-mediated CPE supporting a critical role for this ion transporter in cell killing by HIV. The activity and expression of the Na+/K+ ATPase (sodium pump), a ouabain-sensitive electrogenic ion transporting enzyme, is reduced early in acute infection and in chronically HIV-infected cells as measured by ouabain binding, western immunoblotting, and binding of $\gamma-\sp{32}$P from ATP. The loss of this critical physiology regulator may be important in CD4+ cell depletion which characterizes HIV infection / acase@tulane.edu

Wild Canis species possess a unique suite of reproductive traits including social monogamy, copulatory lock/tie, and biparental care. Females are seasonally monestrous and experience an obligatory pseudopregnancy after spontaneous ovulation. While these characteristics have been ascribed to coyotes, an integrated profile of behavior and physiology has not yet been described. In this study, temporal correlations between steroid hormone levels and socio-sexual mating behaviors were documented, as were changes in vaginal epithelium. Pseudopregnancy was compared to pregnancy by contrasting hormone (progesterone, estradiol, prolactin and relaxin) profiles of unmated females to patterns obtained in alternate years when they bred. Meanwhile, social interactions between pseudopregnant females and their mates appeared similar to pregnant coyotes, suggesting a proximate role of pseudopregnancy in pair-bond enforcement. Finally, out-of-season stimulation of ovarian hormones and estrous behaviors suggested that reproductive seasonality of the coyote may possess some degree of plasticity, providing an adaptive response mechanism to environmental change.

With the signing of the Kyoto Protocol, New Zealand must reduce its national greenhouse gas emissions. As New Zealand has a large proportion of its national emissions as methane (~31%), and methane (CH4) has a short atmospheric lifetime, it provides a good target for mitigation strategies. The initial aim of this research was to identify high and low CH4-emitting cattle to assess factors that contribute to low CH4 production. Initial studies using the SF6 tracer technique to estimate CH4 production could not identify consistently high and low CH4 emitters. Research was therefore undertaken to confirm whether this was due to high variation in estimated CH4 yields, and to quantify the within- and between-animal variation in CH4 production when using the SF6 technique. This research showed considerable within- (coefficient of variation, CV = 7-10%) and between-animal (CV = 7-18%) variation in CH4 yield (g CH4/kg DMI) over time when using the SF6 technique. This is larger than the within- (CV = 3%) and between-animal (CV = 10%) variation reported for calorimetry. This led to the recommendation that the SF6 technique not be used in identifying animals for high or low CH4 yield. A power analysis was developed based on the measured variances for the SF6 technique. Results from this analysis provide researchers with important information on the number of animals and measurements per animal required when undertaking CH4 experiments. One of the sources of variation with the SF6 technique is the SF6 release from permeation tubes. Estimated CH4 yield increases by approximately 8.5% when going from a release rate of 3 mg SF6/day to a rate of 5 mg SF6/day. Further, an in vitro study indicated that SF6 release from permeation tubes is approximately 8% lower in rumen fluid than in air. While further research is required to confirm these results, they emphasise the need to allow time for the release rate to stabilise in the rumen for 4-5 days prior to undertaking measurements. It also led to the recommendation that release rates used in experiments should be within a narrow range, and balanced across experimental treatments.

<p>In all vertebrate species, reproduction is a hormonally controlled process, important for growth and maturation of gonads and germ cells. Production of functional germ cells is of outmost importance to secure the survival of a species. Fish comprises 50% of the known vertebrates and are found in aquatic habitats all over the world. Even though fish have evolved a wide variety of morphological and physiological characteristics, due to large differences in the living environment, the growth an maturation of germ cells follows the same pattern in all species. In this thesis the focus has been directed on oocyte growth and development in Arctic char (Salvelinus alpinus), and if stress might inflict disturbances on the reproductive systems.</p><p>All sexually mature female egg laying vertebrates produces yolky eggs surrounded by an eggshell. Production of yolk and egg shell is under estrogenic control and it is known that production of egg components can be induced in male and juvenile fish by estrogenic substances. Many manmade chemicals have been found to interfere with hormonally controlled processes. Therefore production of the egg yolk precursor, vitellogenin (VTG), and the egg shell components, vitelline envelope proteins (VEP), have been used as biomarkers for estrogenic effect. Exposure to endocrine disrupting substances (EDS) does not only give rise to hormonal effects on the organism, but in addition it also gives rise to an increase in stress hormone, cortisol (F), levels. </p><p>It is evident that a wide variety of substances may affect Arctic char oocyte growth and maturation. VTG and VEP production is found to be under dose dependent estrogenic control, but the production was directly affected by F. Under natural condition it has been found that F increases towards ovulation. Even though both VTG and VTG is under estrogenic control, these studies showed that stress lead to a decrease of VTG while the VEP production increased. These effects was only observed on protein levels indicating that a post transcriptional down regulation of VTG production is mediated by F in Arctic char.</p><p>In order for an egg to become fertilizatible, it must undergo a maturation phase. This maturation phase is primarily induced by gonadotropins, which in turn induce the production of species specific maturation inducing substances (MIS). To investigate oocyte development in Arctic char a characterization of its MIS receptor was made. The MIS receptor is localized on the oocyte surface and displays a single class of high affinity and low capacity binding sites. The binding moieties displays association and dissociation kinetics typical of steroid membrane receptors.</p><p>Even though high specificity for Arctic char MIS was observed, it was found that some EDS bind to the Arctic char oocyte membrane receptor. This suggest that certain EDS might affect oocyte maturation and thereby might alter the reproductive success. Furthermore, it was found that F did not bind to the MIS receptor in Arctic char. It is therefore suggested that oocytes are more sensitive to stress during the growth phase than during maturation</p>

<p>The crayfish plague refractory crayfish, <i>Pacifastacus leniusculus</i>, which can harbour the fungal parasite within melanotic sheath, are found to constitutively express the gene encoding for prophenoloxidase (proPO) after mimicking parasite attack. In contrast, the susceptible crayfish, <i>Astacus astacus</i>, responds to the parasite by increased levels of proPO transcript, particularly in the semigranular haemocytes. The upregulation of proPO could confer a temporary resistance towards the fungal infection, suggesting that additional factors are involved in maintaining the balance between host and parasite. The resistant crayfish may have adapted to the parasite by increasing the transcript level of immune genes. The parasite can be considered as a symbiont since it does not harm the host rather than it activates the immune gene and possibly preventing other pathogens to become established.</p><p>Two serine proteinase genes encoding a subtilisin-like (<i>AaSP1</i>) and a trypsin (<i>AaSP2</i>) enzyme were isolated from the crayfish plague fungus, <i>Aphanomyces astaci</i>. These proteinases are prepropeptides and generate mature proteins of 39 kDa and 29 kDa, respectively. Characterisation of <i>AaSP1</i> suggests that the enzyme may be involved in intracellular control mechanisms rather than playing a role in pathogenesis. The <i>AaSP2 </i>transcript was not controlled by catabolic repression, but was induced by crayfish plasma, implying a role in pathogenesis toward the crayfish host. </p><p>Physiology and genetics of five <i>Aphanomyces</i> strains, which were isolated from moribund crayfish, were characterised with regard to their pathogen diversity. These strains are not virulent against crayfish. Some physiological properties of these strains differed from <i>A. astaci</i>, such as growth rate, germination and production of chitinase. Genetic analysis clearly indicated that they are not related to <i>A. astaci</i> and their name are proposed to be <i>Aphanomyces repetans</i>.</p><p>The crayfish <i>P. leniusculus </i>was found to be susceptible to white spot syndrome virus infection. The virus has a significant effect to the population of crayfish haemocyte. The number and proportion of granular cell from virus-infected crayfish were higher than in controls, indicating granular cells are more resistant to and may interact by some means with the virus.</p><p>Two morphotypes of the crayfish parasite <i>Psorospermium haeckeli</i> obtained from different crayfish hosts of different geographical origin were analysed for ribosomal ITS DNA in order to compare their genetic diversity. The sequence difference between them was found largely in ITS 1 and ITS 2 regions, which was variable in length and showed 66% and 58% sequence similarity. Thus, different morphotypes of <i>P. haeckeli</i> are genetically diverse.</p>

The crayfish plague refractory crayfish, Pacifastacus leniusculus, which can harbour the fungal parasite within melanotic sheath, are found to constitutively express the gene encoding for prophenoloxidase (proPO) after mimicking parasite attack. In contrast, the susceptible crayfish, Astacus astacus, responds to the parasite by increased levels of proPO transcript, particularly in the semigranular haemocytes. The upregulation of proPO could confer a temporary resistance towards the fungal infection, suggesting that additional factors are involved in maintaining the balance between host and parasite. The resistant crayfish may have adapted to the parasite by increasing the transcript level of immune genes. The parasite can be considered as a symbiont since it does not harm the host rather than it activates the immune gene and possibly preventing other pathogens to become established. Two serine proteinase genes encoding a subtilisin-like (AaSP1) and a trypsin (AaSP2) enzyme were isolated from the crayfish plague fungus, Aphanomyces astaci. These proteinases are prepropeptides and generate mature proteins of 39 kDa and 29 kDa, respectively. Characterisation of AaSP1 suggests that the enzyme may be involved in intracellular control mechanisms rather than playing a role in pathogenesis. The AaSP2 transcript was not controlled by catabolic repression, but was induced by crayfish plasma, implying a role in pathogenesis toward the crayfish host. Physiology and genetics of five Aphanomyces strains, which were isolated from moribund crayfish, were characterised with regard to their pathogen diversity. These strains are not virulent against crayfish. Some physiological properties of these strains differed from A. astaci, such as growth rate, germination and production of chitinase. Genetic analysis clearly indicated that they are not related to A. astaci and their name are proposed to be Aphanomyces repetans. The crayfish P. leniusculus was found to be susceptible to white spot syndrome virus infection. The virus has a significant effect to the population of crayfish haemocyte. The number and proportion of granular cell from virus-infected crayfish were higher than in controls, indicating granular cells are more resistant to and may interact by some means with the virus. Two morphotypes of the crayfish parasite Psorospermium haeckeli obtained from different crayfish hosts of different geographical origin were analysed for ribosomal ITS DNA in order to compare their genetic diversity. The sequence difference between them was found largely in ITS 1 and ITS 2 regions, which was variable in length and showed 66% and 58% sequence similarity. Thus, different morphotypes of P. haeckeli are genetically diverse.

In all vertebrate species, reproduction is a hormonally controlled process, important for growth and maturation of gonads and germ cells. Production of functional germ cells is of outmost importance to secure the survival of a species. Fish comprises 50% of the known vertebrates and are found in aquatic habitats all over the world. Even though fish have evolved a wide variety of morphological and physiological characteristics, due to large differences in the living environment, the growth an maturation of germ cells follows the same pattern in all species. In this thesis the focus has been directed on oocyte growth and development in Arctic char (Salvelinus alpinus), and if stress might inflict disturbances on the reproductive systems. All sexually mature female egg laying vertebrates produces yolky eggs surrounded by an eggshell. Production of yolk and egg shell is under estrogenic control and it is known that production of egg components can be induced in male and juvenile fish by estrogenic substances. Many manmade chemicals have been found to interfere with hormonally controlled processes. Therefore production of the egg yolk precursor, vitellogenin (VTG), and the egg shell components, vitelline envelope proteins (VEP), have been used as biomarkers for estrogenic effect. Exposure to endocrine disrupting substances (EDS) does not only give rise to hormonal effects on the organism, but in addition it also gives rise to an increase in stress hormone, cortisol (F), levels. It is evident that a wide variety of substances may affect Arctic char oocyte growth and maturation. VTG and VEP production is found to be under dose dependent estrogenic control, but the production was directly affected by F. Under natural condition it has been found that F increases towards ovulation. Even though both VTG and VTG is under estrogenic control, these studies showed that stress lead to a decrease of VTG while the VEP production increased. These effects was only observed on protein levels indicating that a post transcriptional down regulation of VTG production is mediated by F in Arctic char. In order for an egg to become fertilizatible, it must undergo a maturation phase. This maturation phase is primarily induced by gonadotropins, which in turn induce the production of species specific maturation inducing substances (MIS). To investigate oocyte development in Arctic char a characterization of its MIS receptor was made. The MIS receptor is localized on the oocyte surface and displays a single class of high affinity and low capacity binding sites. The binding moieties displays association and dissociation kinetics typical of steroid membrane receptors. Even though high specificity for Arctic char MIS was observed, it was found that some EDS bind to the Arctic char oocyte membrane receptor. This suggest that certain EDS might affect oocyte maturation and thereby might alter the reproductive success. Furthermore, it was found that F did not bind to the MIS receptor in Arctic char. It is therefore suggested that oocytes are more sensitive to stress during the growth phase than during maturation

The mammalian embryo is encased in a glycoproteinaceous covering, the zona pellucida (ZP/zona) during preimplantation development. Prior to implantation into the recipient maternal endometrium, the blastocyst has to hatch out of this zona. This is a critical and an important event for the successful establishment of pregnancy. Hatching in mammals is characterised by the expansion of the blastocyst, followed by the nicking of the zona and extrusion of the blastocyst by repeated contraction-expansion cycles, thereby leaving the empty zona behind. In species such as the mouse, cow and primates, the empty zona is left behind in the uterine lumen. However, in the hamsters, the features associated with hatching are characteristic for this species. Firstly, the blastocyst remains predominantly in a deflated state. Secondly, the zona undergoes focal rupture which is followed by the complete dissolution of the zona. Third, trophectodermal projections (TEPs) present in the blastocysts, aid the hatching of the blastocyst. Hence, this study was aimed to identify the molecular players involved in hamster blastocyst hatching and to study their embryo-endometrial expression. Earlier work in the laboratory has demonstrated the involvement of cysteine protease-like factors in hamster blastocyst hatching (Mishra and Seshagiri, 2000a). Broad spectrum cysteine protease inhibitors, E-64 and PHMB, completely blocked the hatching of blastocysts. To identify the class of cysteine proteases involved in this phenomenon, class-specific inhibitors were used in this study. Calpain and caspase inhibitors, calpastatin and Z-VAD- FMK, respectively, did not block hamster blastocyst hatching (Fig 2.2). Cathepsin (cts)-specific inhibitors, cystatin-C and peptidyl diazomethane (PPDM) blocked hatching of embryos in a dose-, time- and embryo-stage dependant manner (Figs 2.5, 2.6, 2.10 and 2.11). Continuous exposure of 1.0 µM cystatin to expanded or deflated blastocysts completely blocked hatching (Fig 2.3Aii, iii), without effecting their viability (Fig 2.3Bii and iii). Deflated blastocysts exposed transiently to 1.0 µM cystatin, for 12 or 6 h failed to hatch, but could overcome the inhibition and exhibited hatching, when transferred to fresh, inhibitor-free medium (Fig 2.6). Effect of the inhibitor was less pronounced in the deflated blastocysts when compared to expanded blastocysts (Figs 2.5 and 2.6). The viability of the cystatin-treated embryos was not affected as assessed by vital-dye staining and also their ability to attach and exhibit trophoblast (TB) proliferation on serum-coated dishes was not compromised. The area of the TB outgrowth of cystatin-treated embryos was similar to that of the untreated embryos (Fig 2.9). The inhibitory effect of PPDM, an irreversible inhibitor of cts, on blastocyst hatching was demonstrated. Expanded and deflated blastocysts exhibited a dose-dependent inhibition in hatching, following the exposure to 0.5 and 1.0 µM of PPDM (Figs 2.10A and 2.11A). When treated with the inhibitor for 6 or 12 h, there was a transient inhibition in hatching, as blastocysts could overcome the inhibition and exhibit hatching following transfer to inhibitor-free, fresh medium. Inhibitor-treated hatched blastocysts, when transferred to serum-coated dishes, attached and exhibited TB outgrowth, similar to untreated embryos (Figs 2.13 and 2.14). A PPDM-interacting protease was localised to the cytoplasm of the embryonic cells in the hamster blastocyst, suggesting that the embryo is the source of the zona lysin. Two forms of the enzyme, a probable variant zymogen of molecular mass 65 k and an active form of molecular mass 32 k were detected in the blastocysts (Fig 2.15). In vitro susceptibility of hamster zona to cathepsins is significantly different from that of other species zonae such as the mouse, rat, monkey and human zonae (Table 2.2). All these lines of evidence unequivocally demonstrate the involvement of cathepsins in hamster blastocyst hatching, which is in sharp contrast to what is observed in the mouse, where serine proteases such as strypsin/hepsin, ISP-1 and -2 are reported to play an important role in blastocyst hatching. However, since extensive inhibitor studies were not performed using embryos from other species, it is possible that cysteine proteases maybe involved in the hatching of blastocysts from other species. Having shown the role of cathepsins in hamster zona dissolution, expression of the cathepsins in preimplantation embryos was investigated. Hamster specific cts–L, -B and –P were amplified from day 14 placenta using mouse primers and the amplicons were found to be highly homologous to the cts of other species (Fig 3.2). Hamster and mouse preimplantation embryos i.e., 8-cell, morula and blastocyst were found to express cts–L, -B and –P transcripts (Figs 3.6 and 3.10). Cts-P, present only in the TBs of the placenta (Fig 3.4), for the first time, was also shown to be present in the preimplantation embryos. The immunoreactive cts-L and -P proteins were detected in blastomeres of 8-cell embryo, in the inner cell mass (ICM) and trophectoderm (TE) of the blastocyst (Figs 3.7 and 3.8). These cathepsins could probably correspond to the PPDM- interacting enzymes of molecular mass 32 and 65 kDa, described above. (fig) Fig 5.1. Overview of the expression and the role of embryo-endometrial cathepsins in blastocyst hatching in the golden hamster. Cathepsins ( ) produced by the inner cell mass ( ) or the trophectoderm ( ) of the blastocyst or the endometrial cells ( ) act on the zona matrix ( ), bringing about its lysis. The cathepsins are secreted into the peri-vitelline space or are carried by trophectodermal projections (TEPs, yellow projections) to the zona. Also shown are endogenous inhibitors and growth factors that can regulate these cathepsins. A striking observation made in this study was the detection of the immunoreactive signals for cathepsins in the zona matrix of blastocysts. Since hamster blastocysts possess extracellular projections (TEPs), it is possible for these projections to participate in the transport of cathepsins from TE cells to the zona; as the localisation of the proteases to these projections was demonstrated (Fig 3.9). Also, since the actin-based projections are highly undulating structures, they might potentiate the mechanical rupture of the zona during hatching, apart from acting as carriers for the proteases. Hence, during hatching of the hamster blastocyst, cathepsins, expressed in the ICM and the TE, might be secreted transiently into the peri-vitelline space, whereby they can act on the ZP. Alternatively, in the absence of any apoptotic cells in the embryo that can release the cell contents (Fig 3.13), the cathepsins may be deposited by TEPs in specific pockets of the zona matrix, thereby causing focal zona lysis. In vivo, the hatching of the blastocysts is brought about by both embryonic and maternal proteases. Cts–L and -B transcripts were detected in the maternal endometrium during different stages of the reproductive cycle and early pregnancy (Fig 4.1 and 4.3). Immunoreactive cts-L protein was detected in the uterine luminal epithelium and the stromal cells (Fig 4.5). In the uterus, the PPDM-interacting 32 kDa form was in abundance compared to the 65 kDa form (Fig 4.6). Hence, uterine cathepsins might play a major role in the remodelling of the extracellular matrix during estrous cycle and pregnancy. However, the role of these cathepsins in causing zona dissolution during blastocyst hatching, along with embryonic proteases cannot be ruled out. Reports of recurrent miscarriages in women with low serum cystatin levels imply a role for cysteine proteases in early pregnancy events like blastocyst development, hatching and implantation. Hence, these studies, described in the thesis, could form a basis to investigate the role of cathepsins in early human development. Taken together, the results demonstrate the involvement of embryo-derived cathepsins in hamster blastocyst hatching. These cathepsins may be secreted into the peri-vitelline space or transported to the zona matrix by TEPs (Fig 5.1). Additionally, in vivo, endometrial cathepsins might aid the embryonic zona lysins in the complete zona dissolution. The regulation of these proteases by growth factors, cytokines and their specific inhibitors needs to be explored. (For figure pl see the original document)