Estudou-se o perfil de proteínas de plasma seminal de touros das raças Nelore (Bos taurus primigerus indicus) e Simental (Bos taurus primigerus taurus), criados a campo, em regime de acasalamento múltiplo e manejo extensivo, na região de Dourados/MS, a fim de analisar a relação entre estas proteínas e características ligadas ao desempenho reprodutivo de touros. Coletaram-se amostras de sêmen no período de inverno (Julho/2001) e verão (Fevereiro/2002), realizando-se inicialmente o exame andrológico nos animais, e em seguida a análise padrão do sêmen. O plasma seminal foi, então, submetido à dosagem de proteínas totais, à dosagem de substâncias reativas ao ácido tiobarbitúrico (TBARS), para avaliação da ocorrência de lipoperoxidação, e à eletroforese bidimensional de proteínas. Para análise do perfil proteico de plasma seminal da cada raça, foram utilizados os seguintes parâmetros: comparação entre os períodos de inverno e verão; comparação de grupos de amostras separados de acordo com a quantificação de características ligadas ao desempenho reprodutivo (morfologia espermática, dosagem de proteína total e dosagem de TBARS); e comparação de grupos de amostras separados de acordo com classificações para avaliação da capacidade reprodutiva, previamente descritas na literatura (Breeding Soundness Evaluations – BSEs). Os touros da raça Simental demonstraram menor adaptação às condições ambientais tropicais, com valores mais altos de defeitos espermáticos maiores, proteína total e dosagem de TBARS no período de verão. Observou-se grande variabilidade no perfil proteico entre as raças Nelore e Simental e, dentro de cada raça, encontrou-se expressiva variação individual. O perfil de proteínas de plasma seminal de touros Nelore apresentou 155 spots, entre os quais 04 se destacaram como possíveis marcadores de características ligadas ao desempenho reprodutivo. Já na raça Simental foram identificados 248 spots, com destaque para 04 spots, que demonstraram maior potencial como marcadores de características de interesse para a reprodução. Os resultados mostram que o perfil de proteínas de plasma seminal está relacionado ao desempenho reprodutivo de touros, fazendo-se necessários maiores estudos sobre as proteínas que se destacaram como marcadores, a fim de identificar as funções que elas exercem no trato reprodutivo e nos eventos ligados à fertilização. / Seminal plasma proteins profiles from Nelore (Bos taurus primigerus indicus) and Simmental (Bos taurus primigerus taurus) bulls, used for multiple-sire breeding under range conditions, at Dourados/MS region, were studied aiming to analyze the relation between these proteins and traits linked to bulls’ reproductive performance. Semen samples were collected in the winter (July/2001) and summer (February/2002). Initially, an andrological examination was performed, followed by standard semen analysis. Seminal plasma was subjected to total protein quantification, thiobarbituric acid reactive substances quantification (TBARS, to evaluate lipid peroxidation), and two-dimensional protein electrophoresis. To analyze seminal plasma protein profile of each breed, the following parameters were used: comparison between winter and summer, comparison among groups of samples divided by quantification of traits linked to reproductive performance (sperm morphology, total protein quantification, TBARS quantification) and comparison among groups of samples divided by breeding soundness evaluations (BSEs), previously reported in literature. Simmental bulls showed lower adaptability to tropical environment conditions, with higher values of major sperm defects, total protein and TBARS in the summer. A great variability was observed in the protein profile between Nelore and Simmental breeds, and, for each breed it was found great individual variability too. Nelore seminal plasma protein profile presented 155 spots, 04 of which were considered potential markers for traits linked to reproductive performance. For Simmental breed, 248 spots were identified, with special interest for 04 spots, which showed higher potential as markers to reproductive characteristics. Results show that seminal plasma protein profile is related to bulls’ reproductive performance. Further studies about those proteins which are potential markers are needed, to determine their function at bulls’ reproductive system and in the events related to fertilization.

O plasma seminal é o constituintes não celular do sêmen suíno e contém uma série de componentes orgânicos e inorgânico que desempenham ações variadas tanto no trato reprodutivo masculino como no feminino. No entanto, este fluido de constituição complexa, exerce ações ambiguas sobre os espermatozoides suínos, pois pode atuar ao mesmo tempo de forma benéfica ou deletéria sobre a viabilidade destas células. Nesse sentido, alguns estudos sugerem que este não é o melhor meio para a conservação de espermatozoides. Desta forma o objetivo deste trabalho foi avaliar os efeitos do plasma seminal sobre a integridade das membranas plasmática e acrossomal e o potencial de membrana mitocondrial do espermatozoide suíno armazenado sob refrigeração a 17°C por 72 horas. Para tanto, foram obtidos 4 ejaculados de 6 cachaços. Em seguida o sêmen in natura foi avaliado quanto às características da motilidade pelo sistema computadorizado de análise do sêmen, morfologia espermática por contraste de interferência diferencial e concentração espermática. Após essa primeira avaliação, os ejaculados foram acondicionados em tubos cônicos de 50 mL para serem divididos em três tratamentos, a saber: não centrifugado (NC), centrifugado e com o plasma seminal retirado pós-centrifugação (CS) e centrifugado resuspendido (CR). A força de centrifugação utilizada foi de 500xg por 10 minutos. Todos os tratamentos foram submetidos à diluição em meio BTS para que se obtenha uma concentração de 30 x 106 espermatozoides por mililitro (mL). Em seguida, as amostras permaneceram por 90 minutos em temperatura ambiente e protegidas da luz antes de serem armazenadas. As doses com os diferentes tratamentos foram acondicionadas à temperatura de 17°C e foram avaliadas nos intervalos 0 (90 min pós-diluição), 24, 48 e 72 horas para os seguintes parâmetros: características da motilidade (CASA), integridade das membranas plasmática e acrossomal, estabilidade da membrana plasmática e peroxidação das membranas espermáticas (citometria de fluxo). Os tratamentos foram submetidos à análise de variância (PROC GLM), empregando-se o programa SAS (1998). Quando o principal efeito foi significativo, as médias foram comparadas pelo teste de Tukey-kramer ao nível de 5% de significância. Os resultados do presente estudo mostram que a ausência do plasma seminal foi deletéria para algumas características de motilidade, o mesmo ocorreu para a integridade das membranas plasmática e acrossomal uma vez que houve diminuição na percentagem de celulás espermáticas com membrana plasmatica integra e acrossomo integro no tratamento sem plasma seminal. A peroxidação lipídica das membranas e a manutenção da estabilidade da membrana plasmática não foram influenciadas pelo tratamento. Assim, conclui-se que a presença do plasma seminal em doses inseminantes refrigeradas por 72 h é importante para a manutenção das características de motilidade e para a integridade das membranas plasmáticas e acrossomal / The present study aimed to evaluate the effects of seminal plasma, from the rich fraction of the ejaculate, on kinetics, plasma and acrosome membrane integrity, lipid peroxidation and capacitation of extended liquid boar semen stored under refrigeration at 17° C for 72 hours. For this purpose, four ejaculates from each of six boars were used. Shortly after collection and raw semen evaluation, ejaculates were extended in BTS medium and then assigned to one of three treatments, as follows: non-washed seminal plasma (NWSP), washed-seminal plasma (WSP) centrifuged with own seminal plasma suspended (CWSSP). All treatments were evaluated for sperm motility parameters by the sperm class analyzer (SCA). Plasma and acrosome membrane integrity, lipid peroxidation and sperm capacitation were evaluated by flow cytometry at 0, 24, 48 and 72 hours post dilution. The mean percentage of sperm motility (total and progressive) were lower (p<0.05) in the WSP treatment. There was an increase (p<0.05) in the percentage of sperm with damaged acrosome and damage plasma membrane in the WSP treatment. Membrane lipid peroxidation did not differ (p>0.05) irrespective of treatment nor did sperm capacitation, which was similar (p>0.05) among treatments. Our results show that seminal plasma from the sperm rich fraction is important to maintain adequate structural and functional characteristics of extended liquid boar and should be present in seminal doses throughout the period of store

Made available in DSpace on 2014-08-20T14:37:55Z (GMT). No. of bitstreams: 1 dissertacao_carine_dahl_ corcini.pdf: 419232 bytes, checksum: 05d064069666c9b2d7ee37c1f565b4c2 (MD5) Previous issue date: 2008-04-18 / Artificial insemination (AI) in swine with frozen semen presents lower reproductive performance than AI with cooled semen. Such inefficiency can be attributed to many factors, especially to the semen extenders and individual boar effects, since boars may have different protein content in their seminal plasma. The objective of this study was to evaluate the protein content of distinct portions of the ejaculate of boars, prior to freezing, and the association between the presence of the proteins in the seminal plasma and parameters of semen quality post-thawing, to identify biochemical markers potentially associated with semen freezability in different boars and portions of the ejaculate. The sperm-rich portion of the ejaculate was fractioned in two samples, which were frozen in an extender containing 5% dimethylacetamide as a penetrating cryoprotectant. The protein content of the seminal plasma was evaluated through unidimensional electrophoresis and associated with sperm motility and membrane integrity, pre-freezing and post-thawing, and to sperm morphology and acrossome integrity post-thawing. Inter-boar analyses were conducted, adjusting for individual effects, as well as intra-boar analyses, which were stratified by boar. The first portion of the ejaculate, consisting of the first collected 10 mL, presented better post-thawing semen quality than the second portion. Nine proteins present in the seminal plasma presented significant association with parameters of semen quality (P < 0.05), in both inter and intra-boar analyses. Among them, two proteins, having molecular weight of 28 and 100 kDa were associated, respectively, with post-thawing sperm motility and both pre-freezing and post-thawing sperm membrane integrity. Thus, such proteins are potential biochemical markers for boar semen quality. / A inseminação artificial (IA) em suínos com sêmen congelado apresenta índices de desempenho reprodutivo insatisfatórios, em comparação com a IA com sêmen resfriado. Entre outros fatores, esta ineficiência pode ser atribuída aos diluentes e à variação entre os machos doadores de sêmen, que podem apresentar diferente conteúdo de proteínas no plasma seminal. O objetivo deste trabalho foi avaliar o conteúdo protéico de diferentes porções do ejaculado de suínos, antes destas serem submetidas ao congelamento, e a associação entre a presença de proteínas identificadas no plasma seminal e indicadores de qualidade seminal pósdescongelamento, visando a identificação de marcadores bioquímicos associados com a congelabilidade do sêmen de diferentes machos e frações do ejaculado. A porção do ejaculado rica em espermatozóides foi fracionada em duas amostras, as quais foram congeladas em diluente contendo 5% de dimetilacetamida como crioprotetor interno. O conteúdo protéico do plasma seminal foi avaliado por eletroforese unidimensional e associado com motilidade e integridade da membrana espermática pré e pós-congelamento, e morfologia espermática e integridade de acrossoma pós-congelamento. Foram conduzidas análises inter-machos, nas quais o efeito individual foi ajustado, e intra-machos, nas quais as análises foram estratificadas para cada macho. A primeira porção do ejaculado, consistindo dos primeiros 10 mL coletados, apresentou melhores indicadores de qualidade seminal pós-descongelamento, em comparação com a segunda porção do ejaculado. Nove proteínas presentes no plasma seminal apresentaram associação significativa com parâmetros de qualidade do sêmen congelado, tanto na análise inter-machos, como na intra-machos. Dentre estas nove proteínas, duas dela, que possuem peso molecular de 28 e 100 kDa foram associadas, respectivamente, com a motilidade pós-descongelamento e com a integridade da membrana espermática précongelamento e pós-descongelamento (P < 0,05), sendo, portanto, potenciais candidatas a marcadores bioquímicos para qualidade de sêmen de suínos.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

El presente trabajo tuvo por objetivo evaluar el efecto de la aplicación por vía intramuscular (IM) de plasma seminal sobre la supervivencia embrionaria en alpacas luego que fueron servidas por monta natural. El estudio fue realizado durante los meses de enero a marzo del 2013, en el departamento de Puno. El plasma seminal se obtuvo de muestras de semen de alpaca, colectadas y diluidas en proporción 1:1 en buffer fosfato salino (PBS), para luego ser centrifugadas y conservadas en congelación hasta su posterior uso. Alpacas hembras vacías (n = 117), fueron seleccionadas de acuerdo a la presencia de un folículo dominante ≥ de 7 mm detectado por ecografía tras rectal; las cuales fueron servidas por monta natural y distribuidas aleatoriamente en 3 grupos de comparación: T1(n=40) a las cuales se les administró 1 ml de plasma seminal diluido en PBS, vía IM; T2(n=39) a las que cuales se les administró 1 ml de un análogo de GnRH (0.0042 mg de acetato de buserelina), vía IM y T3(n=38) sin administración de producto alguno. Posteriormente se evaluaron mediante ecografía tras rectal el día 25 post monta para determinar la gestación y el día 62 para determinar la tasa de supervivencia embrionaria. Todos los animales en estudio fueron alimentados con pastos naturales y recibieron las mismas condiciones de manejo. Los resultados obtenidos muestran una tasa de concepción al día 25 post monta del 67.5%, 51.3% y 55.3% y una tasa de supervivencia embrionaria al día 62 del 92.6%, 80% y 85.7% para los grupos T1, T2 y T3 respectivamente. Los resultados obtenidos sugieren que la aplicación intramuscular de plasma seminal inmediatamente después de la monta determina un incremento porcentual de la supervivencia embrionaria en comparación a la aplicación de GnRH o la monta natural, sin embargo dicha diferencia no es estadísticamente significativa (P˂0.05).

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.

<p>Prostasomes are submicron-sized, membrane-bound organelles produced by the epithelial cells of the prostate and normally found in the secretion in the gland ducts. Their physiological role is in the promotion of sperm-function in human reproduction. This thesis contains four papers dealing with the production of prostasomes and some possible applications in clinical urology of the prostasome. </p><p>Paper I and II provided an ultrastructural description of the synthesis, storage and secretion of prostasomes in benign as well as in malignant tissue. Most notable were the extracellular appearances of prostasomes in metastatic lesions whereby the prostasomes become exposed to the immune system of the patient. This supported findings in earlier studies in which patients with advanced prostate cancer had elevated levels of anti-prostasome antibodies. The results of paper III reinforced the view of the prostate-unique origin of the prostasome. In particular, there were no indications in SDS-PAGE patterns or flow-cytometric studies of material from seminal vesicle secretion that it contained components that could be associated with a production of prostasomes. </p><p>Some possible clinical functions of the prostasomes were investigated in paper IV. Exposure of prostasomes to the immune system through mechanical and thermal trauma to the prostate did not induce an evident formation of anti-prostasome autoantibodies. Furthermore, the serum levels of anti-prostasome antibodies registered by assays with preparations of prostasomes from seminal plasma as antigen did not correlate with existing prostate cancer. Seminal prostasomes seemed not to function as substitute markers for prostate cancer in the test kit used. A possible explanation could be underestimated differences in antigen properties between seminal or prostate gland-derived prostasomes and prostasomes from tumor tissue.</p>

Prostasomes are submicron-sized, membrane-bound organelles produced by the epithelial cells of the prostate and normally found in the secretion in the gland ducts. Their physiological role is in the promotion of sperm-function in human reproduction. This thesis contains four papers dealing with the production of prostasomes and some possible applications in clinical urology of the prostasome. Paper I and II provided an ultrastructural description of the synthesis, storage and secretion of prostasomes in benign as well as in malignant tissue. Most notable were the extracellular appearances of prostasomes in metastatic lesions whereby the prostasomes become exposed to the immune system of the patient. This supported findings in earlier studies in which patients with advanced prostate cancer had elevated levels of anti-prostasome antibodies. The results of paper III reinforced the view of the prostate-unique origin of the prostasome. In particular, there were no indications in SDS-PAGE patterns or flow-cytometric studies of material from seminal vesicle secretion that it contained components that could be associated with a production of prostasomes. Some possible clinical functions of the prostasomes were investigated in paper IV. Exposure of prostasomes to the immune system through mechanical and thermal trauma to the prostate did not induce an evident formation of anti-prostasome autoantibodies. Furthermore, the serum levels of anti-prostasome antibodies registered by assays with preparations of prostasomes from seminal plasma as antigen did not correlate with existing prostate cancer. Seminal prostasomes seemed not to function as substitute markers for prostate cancer in the test kit used. A possible explanation could be underestimated differences in antigen properties between seminal or prostate gland-derived prostasomes and prostasomes from tumor tissue.

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.