Poor reproductive performance is a major challenge for the bovine dairy industry, with implications for profitability and animal welfare. Early pregnancy diagnosis and accurate oestrus detection can improve reproductive performance through efficient herd management. However currently available methods do not allow this. Circulating microRNAs (miRNAs) have been proposed as non-invasive biomarkers of reproductive status in humans. The hypothesis for this work was that differentially expressed miRNAs in plasma will be detectable during early pregnancy / oestrus, which may provide novel potential biomarkers. Using sequencing and PCR-based platforms I successfully identified and validated increases in miR-26a and the miR-26a / miR-205 ratio as early as Day 8 of pregnancy (max. 7.5-fold) in the plasma of pregnant compared to non-pregnant heifers. These miRNAs are known regulators of immunity, angiogenesis and metabolism, however their specific roles in early pregnancy remain to be investigated. I also identified small but significant increases in the levels of miR-125b, let-7f, miR-145 and miR-99a-5p at oestrus, when compared with the luteal phase of the cycle. These miRNAs have been previously shown to regulate the follicular to luteal transition in the bovine ovary. Finally, I provide a validated high-throughput resource which can help identify potential global biomarkers of tissue function, as shown for the liver-enriched miR-802 in the present results. The findings of this work may be useful in the development of diagnostic methods for early pregnancy and oestrus, and pave the way for the functional characterisation of these miRNAs in bovine reproduction.

Two studies were designed to characterize the effects of a non-steroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The specific objective was to test the hypothesis that letrozole will arrest dominant follicle growth resulting in emergence of a new follicular wave at a predictable interval post-treatment. In a first experiment, postpubertal beef heifers were assigned randomly to four treatment groups and given phosphate-buffered saline (controls; n=10), or letrozole at a dose of 500 (n=9), 250 (n=10), or 125 (n=10) µg/kg intravenously 4 days after follicular ablation (~2.5 days after wave emergence). In a second study, postpubertal beef heifers were assigned randomly to four treatment groups. One group received no treatment (control; n=17) and the other groups (n=9-10) were given 85 µg/kg of letrozole per day (250 µg/kg total dose), from Days 1 to 3, Days 3 to 5, or Days 5 to 7 (Day 0 = pre-treatment ovulation,) corresponding to the periods before, during and after selection of the dominant follicle, respectively. Follicular dynamics were monitored ultrasonically and blood samples were collected for endocrine assays. Follicle diameter profiles and plasma LH, FSH, and estradiol concentrations were analyzed. Additionally, during the second trial, CL diameter profiles and plasma progesterone concentrations were measured. In both studies, the diameter profile of the dominant follicle was larger in heifers treated with letrozole than in control heifers (P<0.05) and the intervals to new wave emergence and onset of regression of the extant dominant follicle were longer (P<0.05) in heifers treated with letrozole than in controls, regardless of the dose (high, medium, or low; single vs multiple) and the stage of the follicle wave in which treatments were initiated. Furthermore, during the second experiment, the mean CL diameter was larger in letrozole-treated heifers, although there were no differences in plasma progesterone concentrations between treated and control animals. The effects on dominant follicle and CL diameter profiles appeared to be the result of the significantly increased plasma LH concentrations observed in letrozole-treated animals during both treatment approaches (single vs multiple dose). Incomplete and inconsistent inhibition of estradiol production and the lack of a surge on FSH observed in both experiments may be a result of insufficient circulating levels of letrozole during the treatment period. In summary, a single or multiple dose of letrozole did not induce regression of the extant dominant follicle, nor did it directly affect FSH release. Conversely, letrozole extended the lifespan of the dominant follicle, in association with increased endogenous levels of LH, thereby delaying the next FSH surge and subsequent follicular wave emergence. Results suggest that letrozole has potential as a non-steroidal method for controlling ovarian function in cattle, but further studies are warranted to clarify dosage and timing of treatment to predictably affect follicular wave dynamics in cattle.

Two studies were designed to characterize the effects of a non-steroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The specific objective was to test the hypothesis that letrozole will arrest dominant follicle growth resulting in emergence of a new follicular wave at a predictable interval post-treatment. In a first experiment, postpubertal beef heifers were assigned randomly to four treatment groups and given phosphate-buffered saline (controls; n=10), or letrozole at a dose of 500 (n=9), 250 (n=10), or 125 (n=10) µg/kg intravenously 4 days after follicular ablation (~2.5 days after wave emergence). In a second study, postpubertal beef heifers were assigned randomly to four treatment groups. One group received no treatment (control; n=17) and the other groups (n=9-10) were given 85 µg/kg of letrozole per day (250 µg/kg total dose), from Days 1 to 3, Days 3 to 5, or Days 5 to 7 (Day 0 = pre-treatment ovulation,) corresponding to the periods before, during and after selection of the dominant follicle, respectively. Follicular dynamics were monitored ultrasonically and blood samples were collected for endocrine assays. Follicle diameter profiles and plasma LH, FSH, and estradiol concentrations were analyzed. Additionally, during the second trial, CL diameter profiles and plasma progesterone concentrations were measured. In both studies, the diameter profile of the dominant follicle was larger in heifers treated with letrozole than in control heifers (P<0.05) and the intervals to new wave emergence and onset of regression of the extant dominant follicle were longer (P<0.05) in heifers treated with letrozole than in controls, regardless of the dose (high, medium, or low; single vs multiple) and the stage of the follicle wave in which treatments were initiated. Furthermore, during the second experiment, the mean CL diameter was larger in letrozole-treated heifers, although there were no differences in plasma progesterone concentrations between treated and control animals. The effects on dominant follicle and CL diameter profiles appeared to be the result of the significantly increased plasma LH concentrations observed in letrozole-treated animals during both treatment approaches (single vs multiple dose). Incomplete and inconsistent inhibition of estradiol production and the lack of a surge on FSH observed in both experiments may be a result of insufficient circulating levels of letrozole during the treatment period. In summary, a single or multiple dose of letrozole did not induce regression of the extant dominant follicle, nor did it directly affect FSH release. Conversely, letrozole extended the lifespan of the dominant follicle, in association with increased endogenous levels of LH, thereby delaying the next FSH surge and subsequent follicular wave emergence. Results suggest that letrozole has potential as a non-steroidal method for controlling ovarian function in cattle, but further studies are warranted to clarify dosage and timing of treatment to predictably affect follicular wave dynamics in cattle.

This study was performed to test the viability of administering a single 40 mg dose of Folltropin-V® (FSH, Bioniche Animal Health) diluted in SRF (MAP-5 50, Sodium Hyaluronate, Bioniche Animal Health) on day 5 of a recipient synchronization protocol to beef cows to evaluate its effect on recipient fertility. All recipients were administered an estradiol 17beta (2.5 mg, IM) and progesterone (50 mg, IM) combination injection on day 0 and a CIDR® (progesterone 1.34 g, Pfizer Animal Health) was inserted. Lutalyse® (dinoprost tromethamine, Pfizer Animal Health, 25 mg, IM) was administered at the time of CIDR removal on day 7, and estradiol 17beta (1 mg, IM) was administered on day 8. On day 16, the presence of at least one corpus luteum (CL), detected via ultrasound, resulted in the recipient receiving an embryo (both fresh and frozen-thawed embryos were used). Embryos were not transferred into cows that did not show the presence of a CL. Dependent variables for which data were collected included circulating progesterone levels at the time of transfer, number of CLs and CL diameter, circumference, and area; measured in millimeters. The study (n=572) consisted of a treatment group (n=268) and a control group (n=304), and included both Bos indicus (Brahman influenced) crossbred (n=115) and Bos taurus (Angus based) cows (n=457). Pregnancy rates for Treated recipients (40.67%A) and Control recipients (52.96%B) differed (P<.05). There was no difference in the mean number of CLs per recipient for Treated (1.14 +/- .03) and Control (1.10 +/- .02) cows, nor was there a difference in progesterone (P4) at the time of transfer for Treated (3.14 +/- .40 ng/mL) and Control (3.23 +/- .18 ng/mL) recipients. Overall, the inclusion of Folltropin-V® diluted in hyaluronan in a FTET synchronization protocol did not improve the fertility of beef recipients.

Submitted by Adriana Martinez (amartinez@unoeste.br) on 2017-06-01T14:24:03Z No. of bitstreams: 1 Fernanda.pdf: 1636861 bytes, checksum: 75751b060fb15b626d5f147c36748a99 (MD5) / Made available in DSpace on 2017-06-01T14:24:03Z (GMT). No. of bitstreams: 1 Fernanda.pdf: 1636861 bytes, checksum: 75751b060fb15b626d5f147c36748a99 (MD5) Previous issue date: 2017-03-14 / The objective of this study was to study the influence of the periods of the year and reproductive phases on body temperature, pregnancy and milk components of cows of the Black and White Dutch breed. Cows (n = 24) of the Black and White Dutch breed aged 5.0 ± 1.3 years were used in the reproductive stages: 1 (1-95 days of pregnancy); 2 (96-190 days of pregnancy); 3 (191-285 days of pregnancy); 4 (puerperium) and 5 (non-pregnant cows), kept on pasture of Urochloa decumbens, corn silage, mineral mixture and water at will. The experiment period was 365 days, dry period (April to September) and rainy (October to March). Infrared thermography of body areas was performed every 28 ± 5 days, followed by rectal thermometry, ultrasonography for the diagnosis of pregnancy, measurement of climatic parameters, CMT examination and milk collection to study its composition. Statistical analysis was performed using the Tukey test at the significance level of 5% and in the Leeds stadium comparison. For temperatures obtained by thermograms there were differences (P <0.05) between periods of the year and reproductive phases. For the flank, lateral udder and perineal temperatures there were differences (P <0.05) between periods of the year. The udder, perineum and rectal temperatures differed for reproductive phases (P <0.05). There were significant correlations (P <0.01) between: reproductive phase with rectal temperature, ocular globe, muzzle, flank and perineum. The CMT correlated with the temperature of the different reproductive phases presented significant differences (P <0.05) in all the reproductive phases during the rainy season, there were positive correlations (P <0.01) between CMT and CCS. It was concluded that the areas of the body examined by thermographic images presented different temperatures, showing physiological variations that aid in the clinical evaluation of each of the examined areas. Infrared thermography images have also been shown to detect changes in skin surface temperature in response to varying degrees of severity of mammary gland infection, as CMT suggests. The periods of the year and reproductive phases influenced the temperature of areas of the body of milk cows. The image processing was practical, and routine thermography examination was recommended as a complement to the clinical examination. / Objetivou-se estudar a influência dos períodos do ano e fases reprodutivas na temperatura de áreas do corpo, prenhez e componentes do leite de vacas da raça Holandês Preto e Branco. Foram utilizadas vacas (n=24) da raça Holandês Preto e Branco com idade de 5,0±1,3 anos, nas fases reprodutivas: 1(1-95 dias de prenhez); 2 (96-190 dias de prenhez); 3 (191-285 dias de prenhez); 4 (puerpério) e 5 (vacas não prenhes), mantidas em pastagem de Urochloa decumbens, silagem de milho, mistura mineral e água à vontade. O período do experimento foi de 365 dias, período seco (abril a setembro) e chuvoso (outubro a março). Termografia de infravermelho de áreas do corpo foi realizada a cada 28±5 dias, seguido de termometria retal, ultrassonografia para o diagnóstico de prenhez, mensuração de parâmetros climáticos, exame de CMT e coleta de leite para estudo da sua composição. A análise estatística utilizou-se do teste Tukey ao nível de significância de 5% e na comparação estádio Leeds. Para as temperaturas obtidas por termogramas houve diferenças (P <0,05) entre períodos do ano e fases reprodutivas. Para as temperaturas do flanco, úbere lateral e períneo houve diferenças (P <0,05) entre períodos do ano. As temperaturas do úbere, períneo e temperatura retal diferiram para fases reprodutivas (P <0,05). Houve correlações significativas (P <0,01) entre: fase reprodutiva com temperatura retal, globo ocular, mufla, flanco e períneo. O CMT correlacionado com a temperatura das diferentes fases reprodutivas apresentou diferenças significativas (P<0,05) em todas as fases reprodutivas durante a estação chuvosa, houve correlações positivas (P<0,01) entre o CMT e a CCS. Conclui-se que as áreas do corpo examinadas por imagens termográficas apresentaram diferentes temperaturas, mostrando variações fisiológicas que auxiliam na avaliação clínica de cada uma das áreas examinadas. As imagens de termografia por infravermelho também se revelaram sensíveis para detectar alterações de temperatura na superfície da pele em resposta a graus variados de gravidade da infecção da glândula mamária, como sugere o CMT. Os períodos do ano e fases reprodutivas influenciaram na temperatura de áreas do corpo de vacas de leite. O processamento das imagens foi prático, sendo recomendado o exame de termografia na rotina como complementar ao exame clínico.

Estrus synchronization (ES) is a valuable technology that can help beef cow-calf producers to overcome infertility caused by prolonged anestrus. Protocols for ES that use progesterone (P4) supplementation are of particular value to cows with prolonged postpartum anestrus as P4 triggers them to begin cycling and allows them to have fertility similar to that of cycling cows. Supplementation of P4 intravaginally with the use of a controlled internal drug release device (CIDR) improves cycle induction when compared to oral administration of P4. Vaginitis has been reported as a side effect to CIDR use in cattle, which raises concerns about its downstream effects on fertility. More specifically, the effects of CIDR use on the vaginal environment requires exploration, as no studies have explored the changes in vaginal microbiome in response to CIDR based ES protocols. In cattle, the vaginal microbiome has not been widely explored. On the contrary, the human vaginal microbiome is a well-defined environment, rich in bacteria from the genus Lactobacillus, which are responsible for promoting an environment of acidic pH. The dominance of Lactobacillus in the human vagina, however, fluctuates according to steroid hormone concentrations, and disruptions in the vaginal environment will cause depletion of Lactobacillus species, increase in vaginal pH and decreased fertility. Based on this data in humans, our objectives were to describe incidence of vaginitis caused by the CIDR in beef cows, as well as the vaginal microbiome changes in response to CIDR based protocols, and explore their relation to fertility. We found high incidences of vaginitis caused by CIDR use, yet CIDR-induced vaginitis did not negatively affect pregnancy outcomes. However, at CIDR withdrawal, there was decreased bacterial diversity, increased vaginal pH, increased bacterial abundance, and increased vaginal inflammation when compared to what was observed prior to CIDR insertion. Furthermore, abundance of bacteria, vaginal inflammation, and bacterial diversity, but not vaginal pH, were restored to normal values by the day of timed artificial insemination. This important finding suggests that although the vaginal microbiome was disrupted by the use of CIDR, the vaginal microbiome is resilient and capable of restoring its natural conditions without intervention. Finally, cows that ultimately became pregnant were found to have had increased bacterial diversity and decreased vaginal pH prior to protocol initiation, suggesting that the vaginal microbiome may play a role in individual cow fertility. In conclusion, our results indicate that for beef cows a more diverse vaginal microbiome with decreased vaginal pH presents greater resilience of the microbiome towards disruptions caused by an ES protocol, which is translated in greater pregnancy success. / Doctor of Philosophy / According to the Food and Agriculture Organization, the world population is expected to grow by 51% by the year of 2100. The efficiency of food production must therefore be optimized, given the finite availability of farmable land. In beef production, cow fertility is of great importance, since it will ultimately determine the number of animals available for slaughter. The main reproductive issue that cow-calf producers face is that after calving, cows will undergo a period known as postpartum anestrus, in which cows fail to ovulate. Artificial insemination (AI) can help to optimize beef production efficiency, since it allows for the dispersal of semen from valuable bulls to facilitate the genetic enhancement of herds. The use of estrus synchronization (ES) protocols allows for induction and synchronization of ovulation, which allows AI to be performed at the same time for large groups of cows. Progesterone is often used in ES protocols and is the hormone responsible for inducing cyclicity in postpartum cows. Progesterone can be administered either orally or intravaginally via the use of a controlled internal drug release (CIDR). While the CIDR seems to be more effective at inducing cyclicity of cattle compared to oral progesterone administration, vaginal inflammation as response to the CIDR has been reported in cattle. Little is known about the downstream effects of this inflammation on the normal vaginal microbiota and fertility in cattle. In humans, the vaginal microbiome is predominated by a single genus of bacteria (Lactobacillus), that has an essential role in producing lactic acid, which results in the human vagina being remarkably acidic. In humans, depletion of this bacteria, a condition called bacterial vaginosis (BV), allows for other types of bacteria to grow, which results in an increased vaginal pH and decreased fertility. The bovine vaginal microbiome composition and pH in response to the hormones administered during ES protocols and its relation to fertility have not been widely explored. Our objectives were to document the incidence of vaginitis caused by the CIDR in beef cows and evaluate its effects on the vaginal microbiome changes and fertility. We found high incidences of vaginitis caused by the CIDR, yet no effects of CIDR-induced vaginitis were seen on pregnancy success to the protocol. However, decreased bacterial diversity, followed by increases in vaginal pH, abundance of bacteria and vaginal inflammation are all detected at CIDR withdrawal when compared to before CIDR insertion. Furthermore, abundance of bacteria, vaginal inflammation, and bacterial diversity, but not vaginal pH, were restored to normal values by the day of timed AI, indicating that although the vaginal microbiome was disrupted using CIDR, the vaginal microbiome can restore to natural conditions, and indicate resilience of the vaginal microbiome. Finally, cows that became pregnant to the protocol presented increased bacterial diversity and decreased vaginal pH prior to the protocol. In conclusion, our results indicate that for beef cows a more diverse vaginal microbiome with decreased vaginal pH presents greater resilience of the microbiome towards disruptions caused by an ES protocol, which is translated in greater pregnancy success.

Submitted by DARCIO ZANGIROLAMI FILHO null (darcio.z@hotmail.com) on 2018-03-26T18:21:29Z No. of bitstreams: 1 TESE ZANGIROLAMI FILHO, DARCIO 26.03.pdf: 1014256 bytes, checksum: b38519b6ca6817296b518a39076e5200 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-03-27T10:56:11Z (GMT) No. of bitstreams: 1 zangirolamifilho_d_dr_jabo.pdf: 1014256 bytes, checksum: b38519b6ca6817296b518a39076e5200 (MD5) / Made available in DSpace on 2018-03-27T10:56:12Z (GMT). No. of bitstreams: 1 zangirolamifilho_d_dr_jabo.pdf: 1014256 bytes, checksum: b38519b6ca6817296b518a39076e5200 (MD5) Previous issue date: 2018-02-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As técnicas de fertilização in vitro e transferência de embrião são amplamente utilizadas como multiplicadores genéticos em fêmeas de alto valor zootécnico. Para sua realização é mandatório o uso de anestesia epidural, porém sabe-se que a punção repetida do espaço epidural pode causar alterações inflamatórias e fibrose do canal, aos quais reduzem a eficácia da técnica. Objetivou-se, por meio deste estudo desenvolver uma técnica cirúrgica de implantação de cateter epidural com portal de acesso subcutâneo e avaliar a patência deste dispositivo para a realização de aspiração folicular em vacas, por um período de 510 dias. Para a realização deste estudo foram utilizadas doze vacas, com idade entre 3-5 anos e peso entre 308-518 kg, que foram alocadas em dois grupos, grupo A, animais da raça Nelore e B animais da raça Holandesa. Após contenção em tronco, foi realizada sedação com 0,04 mg/kg de acepromazina 1% associada a 0,01 mg/kg de xilazina 2% pela via intramuscular e bloqueio anestésico local da região sacrococcígea e sacral direita. Procedeu-se uma incisão de pele semicircular de dez centímetros (incisão-I), abrangendo o espaço sacrococcígeo e intercoccígeo. Após divulsão da tela subcutânea, a pele foi rebatida lateralmente. Através de uma agulha de Tuohy (16G) posicionada na articulação sacrococcígea ou intercoccígea, introduziu-se dez centímetros do cateter (17G) em sentido cranial, no canal epidural. Procedeu-se uma segunda incisão (incisão-II), de quatro centímetros, no terço médio da região sacral direita. O cateter epidural foi transposto, da incisão-I para a incisão-II, através do espaço subcutâneo utilizando um guia metálico, para ser conectado ao portal de acesso, o qual foi sepultado no espaço subcutâneo da região sacral (incisão-II). As incisões foram suturadas com pontos simples separados. Após 15 dias da implantação, a patência do cateter e do portal foi comprovada por meio da administração de 3 mL de cloridrato de lidocaína 2%. Decorridos 30 dias, reaplicou-se o anestésico local, através do portal de acesso para se proceder a aspiração folicular transvaginal. Este procedimento foi repetido a cada 30 dias, durante 240 dias (8 ciclos). Na sequência, foi realizado descanso de 9 meses, seguido de nova aspiração folicular aos 510 dias após a implantação. A técnica cirúrgica de implantação do cateter e portal foi de média complexidade de execução. As anestesias foram eficazes para a realização da aspiração folicular durante os oito ciclos propostos. Não foram constatadas quaisquer evidências de rejeição do portal e tampouco obstrução total ou parcial dos cateteres. A ferida cirúrgica para a implantação do cateter epidural e do portal de acesso cicatrizou por primeira intenção em todos os animais. A implantação do portal e do cateter permitiu administrações múltiplas de anestésico local, otimizando a aspiração folicular, cuja patência abrangeu 510 dias desde a implantação. / In vitro fertilization and embryo transfer are often used as genetic multipliers in cows of high zootechnical value. Epidural anesthesia is mandatory preceding these procedures, but repeated puncture of the epidural space may cause inflammation and fibrosis, which reduce the effectiveness of the technique in time. The objective of this study was to describe a surgical technique for implantation of an epidural catheter with subcutaneous access portal, and to evaluate the patency this device during eight follicular aspiration cycles in cows. Twelve cows, aged between 3-5 years, weighting 308-518 kg were used in this study. They were allocated in two groups, (A) Nellore and (B) Holstein. Following physical retrain in a hydraulic stock and sedation with 0.04 mg/kg of acepromazine 1% associated with 0.01 mg/Kg of xylazine 2%, administered intramuscularly, local anesthetic block of the sacrococcygeal and right sacral region was made. Then, a 10-cm semicircular followed by skin incision (I-incision), was performed over the sacrococcygeal space, after divulsion of the subcutaneous tissue. Through a Tuohy needle (16G) positioned within the sacrococcygeal joint, ten centimeters of the catheter (17G) were cranially inserted in the epidural space. A second incision (incision-II), four centimeters long, was made at the middle third of the right sacral region. The epidural catheter was transposed from the I-incision to the II-incision through the subcutaneous by using a metallic guide, and promptly connected to the access portal, which was accommodated in the subcutaneous space of the sacral region. Both incisions were sutured with simple interrupted pattern. After 15 days of implantation, the patency of the catheter and portal was confirmed by administration of 3 mL of lidocaine 2%. After 30 days, the local anesthetic was reapplied through the access portal to perform transvaginal follicular aspiration. This procedure was repeated every 30 days for 240 days (8 cycles). Following, 9 months interval a new follicular aspiration was performed on the 510th day following implantation. The surgical technique for the catheter and portal implantation was of medium complexity. Anesthesia was effective for follicular aspiration along the eight cycles. No evidence of portal rejection and total or partial catheter obstruction were noticed. The surgical wounds promptly closed by primary intention. The implantation of the portal and the catheter allowed multiple administrations of local anesthetic optimizing follicular aspiration until the 510th day of implantation. / 2014/11630-8

Nous avons caractérisé cinq protéines bovines qui sont potentiellement impliquées dans la reproduction.Un travail de clonage a été initié qui permettra à terme de purifier les GPA2 et GPB5 recombinantes puis naturelles pour étudier leurs structures. GPA2 et GPB5 sont considérés comme les ancêtres moléculaires des sous-unités α et β des hormones glycoprotéiques. Nous avons montré la relative fragilité thermique de la structure quaternaire de la FSH bovine par rapport aux FSH ovine et humaine et nous avons étudié les propriétés enzymatiques de la PDI (Protein Disulfide Isomerase) en préalable à l’étude de l’activité PDI de GPA2/GPB5. Nous avons aussi purifié la phosphatidyl-ethanolamine-binding protein (PEBP) et l’ubiquitine testiculaires par chromatographie hydrophobe à très haute concentration de sulfate d’ammonium. A partir de la PEBP purifiée, on a produit des anticorps spécifiques chez le lapin qui nous ont permis d’être les premiers à développer un dosage ELISA fiable pour cette protéine. / We characterized five bovine proteins that are potentially involved in reproduction. We started with the cloning of gpa2 and gpb5 cDNAs in order to eventually purify recombinant and natural GPA2 and GPB5 to study their possible quaternary structure. GPA2 and GPB5 are the evolutionary ancestors of Glycoprotein hormones α and β subunits respectively. Meanwhile, we have shown the relative quaternary structure fragility of bovine FSH compared to human and sheep FSH. We also studied the effect of endocrine disruptors on PDI (Protein Disulfide Isomerase) before addressing GPA2/GPB5 PDI activity of GPA2/GPB5 once purified.We succeeded to purify the phosphatidyl-ethanolamine-binding protein (PEBP) and ubiquitin from bovine testis by hydrophobic interaction chromatography at very high ammonium sulfate concentration and we produced specific antibodies (anti-PEBP) in rabbits that allowed us to be the first to develop a reliable Elisa assay for this protein.