The purpose of the studies reported in this thesis was to provide a better understanding of the effects of purified ovulation-inducing factor (OIF) from llama seminal plasma in reflex ovulators (lama glama) and spontaneous ovulators (Bos taurus). The objective of the first study was to determine if the dose of OIF of llama seminal plasma required to elicit ovulation is physiologically relevant, and to test the hypothesis that CL form and function is affected by OIF in a dose-dependent manner. Llamas were treated with four different doses (500 £gg, 250 £gg, 125 £gg and 60 £gg) based on knowledge that for every ejaculate there is approximately 3 mg of OIF. Results supported the hypothesis that OIF affects ovulation and CL form and function in a dose-dependent manner. The high dose of OIF (500 Ýg) was associated with the highest incidence of ovulation, maximum CL diameter, plasma progesterone concentrations and plasma LH concentrations. The low dose of OIF (60 Ýg) was minimally effective for induction of ovulation and associated with smaller CL diameter and lower plasma concentrations of progesterone and LH. The second study was carried out to test the hypotheses that OIF will induce ovulation and affects CL form and function in cattle (Experiment 1), and that OIF given at different stages of development of the first follicular wave will induce atresia of the dominant follicle and hasten emergence of a new follicular wave (Experiment 2). Heifers were treated on Day 5 (Day 0 = wave emergence; Experiment 1) or on Days 3, 6 and 9 (Experiment 2) with a) 1ml of saline, b) 100 £gg of GnRH, or c) 1.0 mg purified OIF per 100 kg of body weight. Results of Experiment 1 demonstrated that OIF did not induce ovulation in cattle but it did induce atresia of the dominant follicle and earlier emergence of a new follicular wave. Results from the second study suggested that the effect previously demonstrated could be accomplished in sexually mature females after treatment on Day 6 corresponding to the late growing phase of the dominant follicle. In summary, the minimum dose of OIF necessary to induce ovulation in llamas was between 60 £gg and 250 £gg. This dose is physiologically relevant and represents less than 1/6th of what is normally present in a single llama ejaculate. In cattle OIF induced regression of the dominant follicle and early emergence of a new follicular wave in pre-pubertal heifers and had a similar effect in sexually mature heifers after treatment on Day 6 of the estrous cycle.

The purpose of the studies reported in this thesis was to provide a better understanding of the effects of purified ovulation-inducing factor (OIF) from llama seminal plasma in reflex ovulators (lama glama) and spontaneous ovulators (Bos taurus). The objective of the first study was to determine if the dose of OIF of llama seminal plasma required to elicit ovulation is physiologically relevant, and to test the hypothesis that CL form and function is affected by OIF in a dose-dependent manner. Llamas were treated with four different doses (500 £gg, 250 £gg, 125 £gg and 60 £gg) based on knowledge that for every ejaculate there is approximately 3 mg of OIF. Results supported the hypothesis that OIF affects ovulation and CL form and function in a dose-dependent manner. The high dose of OIF (500 Ýg) was associated with the highest incidence of ovulation, maximum CL diameter, plasma progesterone concentrations and plasma LH concentrations. The low dose of OIF (60 Ýg) was minimally effective for induction of ovulation and associated with smaller CL diameter and lower plasma concentrations of progesterone and LH. The second study was carried out to test the hypotheses that OIF will induce ovulation and affects CL form and function in cattle (Experiment 1), and that OIF given at different stages of development of the first follicular wave will induce atresia of the dominant follicle and hasten emergence of a new follicular wave (Experiment 2). Heifers were treated on Day 5 (Day 0 = wave emergence; Experiment 1) or on Days 3, 6 and 9 (Experiment 2) with a) 1ml of saline, b) 100 £gg of GnRH, or c) 1.0 mg purified OIF per 100 kg of body weight. Results of Experiment 1 demonstrated that OIF did not induce ovulation in cattle but it did induce atresia of the dominant follicle and earlier emergence of a new follicular wave. Results from the second study suggested that the effect previously demonstrated could be accomplished in sexually mature females after treatment on Day 6 corresponding to the late growing phase of the dominant follicle. In summary, the minimum dose of OIF necessary to induce ovulation in llamas was between 60 £gg and 250 £gg. This dose is physiologically relevant and represents less than 1/6th of what is normally present in a single llama ejaculate. In cattle OIF induced regression of the dominant follicle and early emergence of a new follicular wave in pre-pubertal heifers and had a similar effect in sexually mature heifers after treatment on Day 6 of the estrous cycle.

Title page, abstract and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / In mice and other mammalian species, deposition of semen into the female reproductive tract elicits a local inflammatory response. Whether a comparable response occurs within the human cervix has not previously been studied. The experiments described in this thesis demonstrate, using cervical tissue biopsies taken before and after intercourse, that exposure to semen elicits an infiltration of leukocytes into the cervical tissue of peri-ovulatory women. Immunohistochemical analysis identified macrophages and dendritic cells as the predominant leukocytes recruited into the cervical epithelium and stroma following intercourse. Cytotoxic / suppressor T lymphocytes and memory T cells were also increased. Comparable responses were not detected following condom-protected intercourse. Quantitative real-time PCR was performed on duplicate tissue biopsies to investigate the molecular regulation of this response. Expression of GM-CSF, a potent stimulator of myeloid cell recruitment, was found to increase by 2.5-fold following unprotected intercourse. Trends towards increased IL-6 and IL-8 mRNA were also observed. Condom-protected intercourse did not activate cytokine expression, further suggesting that exposure to semen, as opposed to mechanical trauma, provides the inflammatory stimulus. In an in vitro model using the immortalised Ect-1 cell line, TGFβ was identified as a candidate active seminal factor. All three TGFβ isoforms were capable of mimicking the stimulatory ability of seminal plasma in Ect-1 cells and were comparable in their capacity to stimulate both GM-CSF and IL-6 expression in a dose-responsive manner. The addition of TGFβ isoform-specific neutralising antibodies inhibited seminal plasma-induced increases in these cytokines. However TGFβ was unable to stimulate IL-8 production. Addition of IFNƴ was found to strongly inhibit TGFβ-stimulated GM-CSF production, and 19-0H PGE₁ was found to increase IL-6 and IL-8, but not GM-CSF production. Responses to seminal plasma constituents were almost exactly replicated in primary cultures of human ectocervical cells. These results identify TGFβ as the major active constituent in human seminal plasma and indicate that other seminal agents, 19-0H PGE₁ and IFNƴ, interact with TGFβ to differentially regulate cervical cytokine expression. Finally, whether human seminal plasma cytokine content was associated with fertility in men was examined. No relationship between seminal plasma TGFβ₁, TGFβ₂, TGFβ₃, IL-8 or bacterial endotoxin content and fertility status was observed. However, there was an increased likelihood of high IFNƴ content in the male partners of couples experiencing infertility, most notable in recurrent miscarriage. The discriminating value of IFNƴ was increased when evaluated as a ratio of total TGFβ content. Inflammatory changes after exposure of the female reproductive tract to seminal plasma are implicated in 'conditioning' the maternal immune response, to facilitate successful embryo implantation and pregnancy. The studies described in this thesis provide a mechanistic basis for the observations linking exposure to semen with pregnancy success in humans and have expanded our knowledge of the cellular and molecular events that occur within the female reproductive tract following intercourse. Seminal plasma can therefore no longer be thought of as merely a transport medium for spermatozoa, rather as a means for communication between the male and female reproductive tissues, potentially required for optimal pregnancy success. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1213820 / Thesis (Ph.D.) -- University of Adelaide, Medical School, 2005

Title page, abstract and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / In mice and other mammalian species, deposition of semen into the female reproductive tract elicits a local inflammatory response. Whether a comparable response occurs within the human cervix has not previously been studied. The experiments described in this thesis demonstrate, using cervical tissue biopsies taken before and after intercourse, that exposure to semen elicits an infiltration of leukocytes into the cervical tissue of peri-ovulatory women. Immunohistochemical analysis identified macrophages and dendritic cells as the predominant leukocytes recruited into the cervical epithelium and stroma following intercourse. Cytotoxic / suppressor T lymphocytes and memory T cells were also increased. Comparable responses were not detected following condom-protected intercourse. Quantitative real-time PCR was performed on duplicate tissue biopsies to investigate the molecular regulation of this response. Expression of GM-CSF, a potent stimulator of myeloid cell recruitment, was found to increase by 2.5-fold following unprotected intercourse. Trends towards increased IL-6 and IL-8 mRNA were also observed. Condom-protected intercourse did not activate cytokine expression, further suggesting that exposure to semen, as opposed to mechanical trauma, provides the inflammatory stimulus. In an in vitro model using the immortalised Ect-1 cell line, TGFβ was identified as a candidate active seminal factor. All three TGFβ isoforms were capable of mimicking the stimulatory ability of seminal plasma in Ect-1 cells and were comparable in their capacity to stimulate both GM-CSF and IL-6 expression in a dose-responsive manner. The addition of TGFβ isoform-specific neutralising antibodies inhibited seminal plasma-induced increases in these cytokines. However TGFβ was unable to stimulate IL-8 production. Addition of IFNƴ was found to strongly inhibit TGFβ-stimulated GM-CSF production, and 19-0H PGE₁ was found to increase IL-6 and IL-8, but not GM-CSF production. Responses to seminal plasma constituents were almost exactly replicated in primary cultures of human ectocervical cells. These results identify TGFβ as the major active constituent in human seminal plasma and indicate that other seminal agents, 19-0H PGE₁ and IFNƴ, interact with TGFβ to differentially regulate cervical cytokine expression. Finally, whether human seminal plasma cytokine content was associated with fertility in men was examined. No relationship between seminal plasma TGFβ₁, TGFβ₂, TGFβ₃, IL-8 or bacterial endotoxin content and fertility status was observed. However, there was an increased likelihood of high IFNƴ content in the male partners of couples experiencing infertility, most notable in recurrent miscarriage. The discriminating value of IFNƴ was increased when evaluated as a ratio of total TGFβ content. Inflammatory changes after exposure of the female reproductive tract to seminal plasma are implicated in 'conditioning' the maternal immune response, to facilitate successful embryo implantation and pregnancy. The studies described in this thesis provide a mechanistic basis for the observations linking exposure to semen with pregnancy success in humans and have expanded our knowledge of the cellular and molecular events that occur within the female reproductive tract following intercourse. Seminal plasma can therefore no longer be thought of as merely a transport medium for spermatozoa, rather as a means for communication between the male and female reproductive tissues, potentially required for optimal pregnancy success. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1213820 / Thesis (Ph.D.) -- University of Adelaide, Medical School, 2005

Title page, abstract and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / In mice and other mammalian species, deposition of semen into the female reproductive tract elicits a local inflammatory response. Whether a comparable response occurs within the human cervix has not previously been studied. The experiments described in this thesis demonstrate, using cervical tissue biopsies taken before and after intercourse, that exposure to semen elicits an infiltration of leukocytes into the cervical tissue of peri-ovulatory women. Immunohistochemical analysis identified macrophages and dendritic cells as the predominant leukocytes recruited into the cervical epithelium and stroma following intercourse. Cytotoxic / suppressor T lymphocytes and memory T cells were also increased. Comparable responses were not detected following condom-protected intercourse. Quantitative real-time PCR was performed on duplicate tissue biopsies to investigate the molecular regulation of this response. Expression of GM-CSF, a potent stimulator of myeloid cell recruitment, was found to increase by 2.5-fold following unprotected intercourse. Trends towards increased IL-6 and IL-8 mRNA were also observed. Condom-protected intercourse did not activate cytokine expression, further suggesting that exposure to semen, as opposed to mechanical trauma, provides the inflammatory stimulus. In an in vitro model using the immortalised Ect-1 cell line, TGFβ was identified as a candidate active seminal factor. All three TGFβ isoforms were capable of mimicking the stimulatory ability of seminal plasma in Ect-1 cells and were comparable in their capacity to stimulate both GM-CSF and IL-6 expression in a dose-responsive manner. The addition of TGFβ isoform-specific neutralising antibodies inhibited seminal plasma-induced increases in these cytokines. However TGFβ was unable to stimulate IL-8 production. Addition of IFNƴ was found to strongly inhibit TGFβ-stimulated GM-CSF production, and 19-0H PGE₁ was found to increase IL-6 and IL-8, but not GM-CSF production. Responses to seminal plasma constituents were almost exactly replicated in primary cultures of human ectocervical cells. These results identify TGFβ as the major active constituent in human seminal plasma and indicate that other seminal agents, 19-0H PGE₁ and IFNƴ, interact with TGFβ to differentially regulate cervical cytokine expression. Finally, whether human seminal plasma cytokine content was associated with fertility in men was examined. No relationship between seminal plasma TGFβ₁, TGFβ₂, TGFβ₃, IL-8 or bacterial endotoxin content and fertility status was observed. However, there was an increased likelihood of high IFNƴ content in the male partners of couples experiencing infertility, most notable in recurrent miscarriage. The discriminating value of IFNƴ was increased when evaluated as a ratio of total TGFβ content. Inflammatory changes after exposure of the female reproductive tract to seminal plasma are implicated in 'conditioning' the maternal immune response, to facilitate successful embryo implantation and pregnancy. The studies described in this thesis provide a mechanistic basis for the observations linking exposure to semen with pregnancy success in humans and have expanded our knowledge of the cellular and molecular events that occur within the female reproductive tract following intercourse. Seminal plasma can therefore no longer be thought of as merely a transport medium for spermatozoa, rather as a means for communication between the male and female reproductive tissues, potentially required for optimal pregnancy success. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1213820 / Thesis (Ph.D.) -- University of Adelaide, Medical School, 2005

The use of AI in dogs has been limited by the lack of effective and reliable means of cryopreservation of semen and by the poor correlation between traditional methods of post-thaw assessment of semen quality and fertility. In order to address these problems, the present study focuses upon methods of cold storage and cryopreservation of dog spermatozoa by undertaking comparative evaluations of post-thaw motility and in vitro induction of acrosome reaction. Split-ejaculate protocols were used to compare the effect of storage at +4°C and cryopreservation upon (i) the maintenance of spermatozoa motility and (ii) spontaneous or A23187-induced acrosome reactions during incubation at 39°C (in 5% CO2 in the humidified air) for 60 or 120 min. The assessments of samples were made by Bright-Field, Phase Contrast (PC), Differential Interference Contrast (DIC), Scanning (SEM) and Transmission (TEM) Electron Microscopy. The interaction between the process of glycerolisation and the presence of seminal plasma is one of the key limitors for success in cryopreservation of dog spermatozoa. Interactions between the effects of removal of seminal plasma (by centrifugation), dilution rate, the temperature at which glycerolisation took place and the concentration of glycerol upon survival of spermatozoa at +4°C were studied in a series of split-ejaculate experiments. Spermatozoa were suspended in Tris-fructose-citric acid extender containing 20% (v/v) egg yolk and 8% (v/v) glycerol at +4°C for 48 h. Survival was assessed as the percentage of spermatozoa displaying progressive motility. Survival of spermatozoa was higher (P < 0.05) after glycerolisation at +4°C than at the room temperature. At dilution rates of 1:1 and 1:2 (semen: extender), the survival was higher (P < 0.05) in samples that were centrifuged and glycerolised at +4°C than the samples that were neat and glycerolised at the room temperature. While at the dilution rate of 1:16 it was higher (P < 0.05) in samples that were neat plus glycerolised at +4°C than all samples that were glycerolised at the room temperature. Concentrations of glycerol that were > 2% (v/v) resulted in lower (P < 0.05) survival than at lower concentrations. Following the initial stage of the investigations, the optimisation and validation of a method for in vitro induction of acrosome reactions were required. Suspensions of spermatozoa in TALP medium were incubated in the presence of a logarithmic. series of concentrations of the calcium ionophore, A23187. Induction of acrosome reactions was assessedb y bright-field (using naphthol yellow S/aniline blue stain, NA) and phase contrast (PC) microscopy. Using these methods, it was determined that incubation in the presence of 1 μM/1 A23187 for a period of 30-45 min was optimal for inducing acrosomer eactions in fresh semen. It was also noted that the assessments of acrosome reactions by using NA staining, were highly correlated with PC microscopy. In consequence, the simple procedure of NA staining might be an acceptable alternative to PC microscopy for use in the field. Subsequently, the effect of chilling and glycerolisation upon in vitro induction of acrosome reactions by A23187 was assessed. Acrosome reactions were studied as these have been described in the literature as providing accurate bioassay of spermatozoal functionality in vivo. Acrosome reactions were assessed by using DIC microscopy. The acrosomal integrity was impaired after chilling, which accelerated the A23187-induced acrosome reaction such that a lower concentration (0.1 μM/1) of A23187 was also effective to induce the reaction within 60 min of incubation. However, the presence of 2% glycerol (v/v, final) in standard Tris extender, containing 20% egg yolk, did not significantly affect the sequence of acrosome reaction. The optimal freezing regimen (from +4°C to -120°C) was determined by using a programmable biological freezer in a series of experiments, in which various cooling rates were combined in a Latin square design. Semen was diluted in standard Tris extender containing 20% egg yolk and 2% glycerol (v/v, final) and packed in 0.25 ml French paiettes (straws). The optimal cooling regimen was -0.5°C/min from +4°C to -9°C, -40°C/min to -20°C, -100°C/min to -120°C, followed by direct immersion of the straws in liquid nitrogen. Changes in temperatures within an individual straw were continuously measured and these data were found to be highly correlated with the eventual post-thaw motility of frozen-thawed spermatozoa. Although freezing and thawing resulted in major acrosomal deterioration, there were no significant differences between freezing regimens on the basis of in vitro induction of acrosome reactions, as assessed by DIC microscopy. Finally, ultrastructural studies, using SEM and TEM, upon chilled (as 'ready to freeze') and frozen-thawed spermatozoa subjected to A23187-induced acrosome reaction demonstrated that freeze-thawing provoked the acrosome reaction such that, with TEM (i) the plasma membrane was usually damaged or missing, (ii) the acrosomal changes (including the loss of acrosomal content, as seen by decondensation and swelling) except vesiculation of the acrosomal membranes, exceeded to the equatorial segment and (iii) a further damage occurred to the post-acrosomal region. In summary, these results show that semen should be; (i) centrifuged for dilutions of < 1:8, (ii) diluted at 1:8 in Trisfructose-citric acid extender containing 20% egg yolk, (iii) glycerolised at +4°C at a final concentration of 2% glycerol (v/v), (iv) cooled at -0.5°C/min from +4°C to -9°C, at -40°C/min to -20°C and at -100°C/min to -120°C, followed by direct immersion of the straws in liquid nitrogen for cryopreservation and (i) introduced to 1 μM/1 A23187 in TALP, (ii) incubated for at least 30-45 min for induction of acrosome reaction in vitro and, thereby, demonstrated that optimisation of cryopreservation and in vitro induction of acrosome reaction of dog spermatozoa are possible.

Internal fertilization occurs in birds and eutherian mammals. Foetal development, however, is either extra- respectively intra-corpore (egg vs uterus). In these animal classes, the female genital tract stores ejaculated spermatozoa into a restricted oviductal segment; the functional pre-ovulatory sperm reservoir, where they survive until ovulation/s occur. Paradoxically, this immunologically foreign sperm suspension in seminal fluid/plasma, often microbiologically contaminated, ought to be promptly eliminated by the female local immune defence which, instead, tolerates its presence. The female immune tolerance is presumably signalled via a biochemical interplay of spermatozoa, as well as the peptides and proteins of the extracellular seminal fluid, with female epithelial and immune cells. Such interplay can result in gene expression shifts in the sperm reservoir in relation to variations in fertility. To further aid our understanding of the underlying mechanisms, this thesis studied the proteome of the seminal fluid (using 2D SDS-PAGE and mass spectrometry) including cytokine content (using Luminex and/or ELISA) of healthy, sexually mature and fertile boars and cocks. As well, gene expression changes (using cDNA microarray) in the oviductal sperm reservoirs of sexually-mature females, mated or artificially infused with homologous sperm-free seminal fluid/plasma were studied. Pigs were of commercial, fertility-selected modern breeds (Landrace), while chicken belonged to the ancestor Red Junglefowl (RJF, low egg laying-capacity), a selected egg-layer White Leghorn (WL) and of their Advanced Intercross Line (AIL). Ejaculates were manually collected as single sample in cocks or as the sperm-rich fraction [SRF] and the post- SRF fraction in boars to harvest seminal fluid/plasma for proteome/cytokine and infusion-studies. Oviducts were retrieved for gene-expression analyses via microarray immediately post-mortem (chicken) or at surgery (pig), 24 h after mating or genital infusion. In pigs, the protein-rich seminal plasma showed the highest amounts of cytokines [interferon-γ, interferon gamma-induced protein 10 (IP-10/CXCL10), macrophage derived chemokine (MDC/CCL22), growth-regulated oncogene (GRO/CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemo-attractant protein-1 (MCP-1/ CCL2), interleukin (IL)-6, IL-8/CXCL8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-β1-3) in the larger, protein-rich and sperm-poor post-SRF, indicating its main immune signalling influence. Chicken showed also a plethora of seminal fluid proteins with serum albumin and ovotransferrin being conserved through selection/evolution. However, they showed fewer cytokines than pigs, as the anti-inflammatory/immune-modulatory TGF-β2 or the pro-inflammatory CXCL10. The RJF contained fewer immune system process proteins and lacked TGF-β2 compared to WL and AIL, suggesting selection for increased fertility could be associated with higher expression of immune-regulating peptides/proteins. The oviductal sperm reservoir reacted in vivo to semen exposure. In chicken, mating significantly changed the expression of immune-modulatory and pH-regulatory genes in AIL. Moreover, modern fertile pigs (Landrace) and chicken (WL), albeit being taxonomically distant, shared gene functions for preservation of viable sperm in the oviduct. Mating or SP/SF-infusion were able to change the expression of comparable genes involved in pH-regulation (SLC16A2, SLC4A9, SLC13A1, SLC35F1, ATP8B3, ATP13A3) or immune-modulation (IFIT5, IFI16, MMP27, ADAMTS3, MMP3, MMP12). The results of the thesis demonstrate that both mating and components of the sperm-free seminal fluid/plasma elicit gene expression changes in the pre-ovulatory female sperm reservoir of chickens and pigs, some conserved over domestication and fertility-selection.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Em altas latitudes tem-se observado uma variaÃÃo na composiÃÃo bioquÃmica do plasma seminal (PS) de caprinos por influÃncia da estacionalidade. Em baixas latitudes essa variaÃÃo tem sido atribuÃda unicamente ao baixo valor nutricional das dietas na Ãpoca seca. Objetivou-se por meio deste estudo avaliar a ocorrÃncia de variaÃÃo na composiÃÃo bioquÃmica do PS de caprinos SPRD criados intensivamente no Estado do CearÃ, Brasil. O experimento foi conduzido nas instalaÃÃes do Dep. de Zootecnia da Universidade Federal do Cearà â UFC. Foram utilizados 20 bodes SPRD, alimentados segundo o NRC/caprinos (1981). Os ejaculados foram coletados semanalmente em vagina artificial e letroejaculador e em seguida avaliados, mensurados, centrifugados e separados o PS (sobrenadante) das cÃlulas espermÃticas (precipitado), sendo acondicionados em tubos eppendorffs a -18ÂC, durante um ano (Set/2005 a Ago/2006). Para realizaÃÃo das anÃlises as amostra semanais passaram a constituir um pool individual mensal, onde foram avaliadas as concentraÃÃes de Ca, P, Mg, PTt, AC e frutose. Foi observada variaÃÃo significativa (p<0,05) na composiÃÃo bioquÃmica do PS entre as Ãpocas do ano, registrando-se os nÃveis mais elevados dos parÃmetros bioquÃmicos na Ãpoca chuvosa. A variaÃÃo individual foi significativa (p<0,01), sà havendo interaÃÃo significativa, animal/Ãpoca para a frutose (p<0,05). O estudo das correlaÃÃes mostrou associaÃÃes significativas (p<0,0001) entre PTt, AC e frutose, entre os volumes de ejaculado e de PS, com os componentes, e de temperatura, umidade, precipitaÃÃo e ITU, com os parÃmetros bioquÃmicos do PS. Concluindo-se que a frutose à um bom parÃmetro bioquÃmico para o estudo da qualidade seminal, entretanto, mais estudos sÃo necessÃrios para identificar as causas desta variaÃÃo, e compreender melhor o papel da frutose, acido cÃtrico e proteÃnas totais no metabolismo espermÃtico. / Goat seminal plasma (SP) biochemical composition observed to very in high latitudes due to seasonality. In low latitudes this variation has been attributed only to the low nutritional value of diets in the dry period. The aims of this study were to evaluate the occurrence of variation in the SP biochemical composition of SPRD goats breed intensively in the State of CearÃ, Brazil. The experiment was carried out in the facilities of Dep. of Zootecnia of the Federal University of Cearà - UFC. Twenty male SPRD goats were used, they were fed according to NRC/goats (1981). Ejaculated were collected weekly in artificial vagina and eletroejaculador, and evaluated, measure, centrifuged and separated the SP (sobreswing) from the cells spermatic (precipitate), being stored in eppendorff tubes at -18ÂC, during one year (Sep/2005 the Aug/2006). For analyses purpose weekly sample were mixed to constitute a monthly individual pool, from which were evaluated the concentrations of Ca, P, Mg, PTt, CA and fructose. Significant variation was observed (p <0,05) in the biochemical composition of the SP among the periods of the year, registering the highest levels of biochemical parameters in the rainy period. The individual variation was significant (p <0,01), only having significant interaction animal/period to the fructose (p <0,05). The study of correlations showed significant associations (p <0,0001) among PTt, CA and fructose, among the ejaculate volume and of SP with the components, and of temperature, humidity, precipitation and ITU with the biochemical parameters of the SP. It is concluded that fructose is a good biochemical parameter for the study of seminal quality. However, more studies are necessary to identify the causes of this variation, and to better understand the role of the fructose, citric acid and total proteins in the metabolism spermatic.

The purpose of the studies reported in this thesis was to provide further evidence in support of the hypothesis that ovulation-inducing factor (OIF) is a component of seminal plasma which is conserved amongst mammals. Based on studies conducted in vivo, the results indicate that males ejaculate a substance during copulation which is responsible for the ovulatory and luteotrophic effect in female camelids. In our lab we have developed an <i>in vivo</i> llama bioassay to study the presence and biological effects of OIF in seminal plasma from different species.<p> The objective of the first experiment within the first study was to determine if llama seminal plasma would stimulate ovulation in prepubertal mice. Mice were treated with a single 0.1 mL intraperitoneal dose of 1) phosphate-buffered saline (negative control), 2) 5 µg gonadotropin-releasing hormone (GnRH), 3) 5 IU of human chorionic gonadotropin (hCG) or 4) llama seminal plasma. Results indicate that prepubertal mice treated with GnRH, hCG or llama seminal plasma stimulated similar proportions of mice to ovulate, which were all higher than the proportion of mice that ovulated after saline treatment. The number of oocytes observed under a stereomicroscope was also higher in all treatment groups than in mice treated with saline. However, the number of oocytes observed was lower in mice treated with seminal plasma than those treated with GnRH, both of which were similar to the number of oocytes observed in hCG treated mice.<p> In a second part of this study the corollary that OIF is present in the seminal plasma of horses and pigs was examined. Seminal plasma from horses or pigs was administered intramuscularly to female llamas and ovulation was monitored using transrectal ultrasonography. Llamas were treated with an intramuscular dose of 1) phosphate buffered saline (negative control), 2) llama seminal plasma (positive control), 3) equine seminal plasma or 4) porcine seminal plasma. Ovulations were detected in llamas treated with seminal plasma while none were observed in saline-treated llamas. The proportion of llamas that ovulated when treated with equine seminal plasma was higher than llamas treated with saline. The proportion of llamas that ovulated after porcine seminal plasma tended to differ from negative control groups, but did not reach statistical significance. The proportion of llamas that ovulated after equine or porcine seminal plasma treatment was lower than animals treated with llama seminal plasma which indicates that either OIF is not present in equal concentration among mammals, or that OIF is not structurally the same across mammals.<p> The second study was carried out to test the hypothesis that OIF stimulates LH secretion at the level of the anterior pituitary gland. The second objective was to determine if the degree of LH release was related to the dose of OIF treatment. Anterior pituitary cells (2 x 10^6 cells/ well) from either llamas (reflex ovulator) or cattle (spontaneous ovulator) were incubated for 2 hours with either media containing no treatment (control), GnRH or OIF. In all experiments, GnRH and OIF stimulated more LH secretion than control groups. An effect of dose was evident in the llama pituitary cell culture where mean LH concentrations were greater in wells treated with a higher dose of OIF in comparison to wells treated with a lower dose, both of which were higher than in wells with no treatment. Although OIF stimulated LH release in bovine cell cultures, an apparent dose response was not detected. Results indicate that the preovulatory LH surge observed after OIF treatment in camelids may be the result of OIF directly stimulating LH release from gonadotrope cells within the anterior pituitary gland. In conclusion these results illustrate that the presence and the response to OIF is conserved among species that share no relation or common reproductive strategy.

The purpose of the studies reported in this thesis was to provide further evidence in support of the hypothesis that ovulation-inducing factor (OIF) is a component of seminal plasma which is conserved amongst mammals. Based on studies conducted in vivo, the results indicate that males ejaculate a substance during copulation which is responsible for the ovulatory and luteotrophic effect in female camelids. In our lab we have developed an <i>in vivo</i> llama bioassay to study the presence and biological effects of OIF in seminal plasma from different species.<p> The objective of the first experiment within the first study was to determine if llama seminal plasma would stimulate ovulation in prepubertal mice. Mice were treated with a single 0.1 mL intraperitoneal dose of 1) phosphate-buffered saline (negative control), 2) 5 µg gonadotropin-releasing hormone (GnRH), 3) 5 IU of human chorionic gonadotropin (hCG) or 4) llama seminal plasma. Results indicate that prepubertal mice treated with GnRH, hCG or llama seminal plasma stimulated similar proportions of mice to ovulate, which were all higher than the proportion of mice that ovulated after saline treatment. The number of oocytes observed under a stereomicroscope was also higher in all treatment groups than in mice treated with saline. However, the number of oocytes observed was lower in mice treated with seminal plasma than those treated with GnRH, both of which were similar to the number of oocytes observed in hCG treated mice.<p> In a second part of this study the corollary that OIF is present in the seminal plasma of horses and pigs was examined. Seminal plasma from horses or pigs was administered intramuscularly to female llamas and ovulation was monitored using transrectal ultrasonography. Llamas were treated with an intramuscular dose of 1) phosphate buffered saline (negative control), 2) llama seminal plasma (positive control), 3) equine seminal plasma or 4) porcine seminal plasma. Ovulations were detected in llamas treated with seminal plasma while none were observed in saline-treated llamas. The proportion of llamas that ovulated when treated with equine seminal plasma was higher than llamas treated with saline. The proportion of llamas that ovulated after porcine seminal plasma tended to differ from negative control groups, but did not reach statistical significance. The proportion of llamas that ovulated after equine or porcine seminal plasma treatment was lower than animals treated with llama seminal plasma which indicates that either OIF is not present in equal concentration among mammals, or that OIF is not structurally the same across mammals.<p> The second study was carried out to test the hypothesis that OIF stimulates LH secretion at the level of the anterior pituitary gland. The second objective was to determine if the degree of LH release was related to the dose of OIF treatment. Anterior pituitary cells (2 x 10^6 cells/ well) from either llamas (reflex ovulator) or cattle (spontaneous ovulator) were incubated for 2 hours with either media containing no treatment (control), GnRH or OIF. In all experiments, GnRH and OIF stimulated more LH secretion than control groups. An effect of dose was evident in the llama pituitary cell culture where mean LH concentrations were greater in wells treated with a higher dose of OIF in comparison to wells treated with a lower dose, both of which were higher than in wells with no treatment. Although OIF stimulated LH release in bovine cell cultures, an apparent dose response was not detected. Results indicate that the preovulatory LH surge observed after OIF treatment in camelids may be the result of OIF directly stimulating LH release from gonadotrope cells within the anterior pituitary gland. In conclusion these results illustrate that the presence and the response to OIF is conserved among species that share no relation or common reproductive strategy.