Embryo transfer plays a very important role in the cattle industry and its application requires a consistent supply of viable embryos for use in such programmes. One way of achieving this is the development of reliable superovulation regimens, yielding a large number of high quality embryos. Superovulation with pregnant mare serum gonadotrophin (PMSG) induces a second wave of follicles after ovulation because it is eliminated slowly from the peripheral blood, causing high concentrations of oestradiol. This oestradiol may have an adverse effect on fertilization and early embryonic development. Administering PMSG antiserum after ovulation may improve the quality by neutralizing PMSG activity. The object of this study was to examine the role of monoclonal anti- PMSG (Neutra-PMSG; Intervet UK) in a superovulatory regime for cattle based on PMSG with the objective of increasing the number of viable embryos produced. Two experiments were conducted in this study. In experiment 1, cows were superovulated with 2500 iu of PMSG (Folligon; Intervet UK) im on day 101 of their oestrous cycle, whereas in experiment 2, heifers were superovulated with 1000, 2000, 3000 or 4000 iu of PMSG (PMSG1, 2, 3 or 4, respectively) im on day 10 1 of their oestrous cycle. In experiments 1 and 2, animals were given 2 ml of PG (PG3) im 48 h after PMSG injection and oestrus was observed in the same manner as described above. When data were evaluated in respect of the PMSG/APMSG dose level, there were no significant differences (P>0.05) in the numbers of CL and usable number of embryos between APMSG treatment and the appropriate control. Treatment with APMSG in the 3000 iu PMSG dose group reduced (P 0.05) the numbers of LF compared to control (1.3 v 5.5). When data were analysed based on the dose levels of PMSG, the total number of ova/embryos and the number of usable embryos were higher in heifers which received 2000 iu of PMSG compared to those which received 1000, 3000 or 4000 iu (7.1 v 2.1, 6.6 or 5.6 and 6.3 v 2.1, 4.8 or 3.2, respectively) . In conclusion the results indicate that PMSG dose 2000 iu is the favoured dose for superovulating heifers. The administration of Neutra-PMSG 36, 48, 60, 72, 84 or 96 h after PG3 injection, despite reducing LF numbers and preventing the rise in 0E2 after ovulation, had no significant effect on the number of usable embryos recovered.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 81-83). Also available via the Internet.

Call number: LD2668 .T4 1986 S675 / Master of Science / Animal Sciences and Industry

In vitro embryo production has vastly improved over the past decade through the study of the in vivo environment and the metabolic requirements of embryos. In contrast, in vitro oocyte maturation ( IVM ) culture conditions have remained relatively unchanged and are suboptimal. The aim of this thesis was to create improved systems for bovine IVM by studying the metabolic profiles and requirements of intact cumulus oocyte complexes ( COCs ) during IVM and determining the ion and energy substrate composition of bovine follicular fluid ( FF ). Glucose, pyruvate and oxygen consumption of bovine COCs increased 2 - fold over the 24 h IVM period, with glucose being the preferred energy substrate. While initially the majority of glucose consumed by COCs is metabolised via glycolysis ( Llactate production ), a considerable proportion of glucose is used as a substrate for extracellular matrix ( ECM ) synthesis towards the end of IVM. Glucosamine ( an intermediate substrate of hyaluronic acid ) supplementation of IVM media lead to decreased glucose consumption and incorporation into ECM during FSH - stimulated expansion. Biochemical analyses of bovine FF demonstrated that the concentration of some ions and energy substrates varied with follicle size. Although follicular glucose concentrations increased with follicle size, levels were ~ 2 - fold lower than that found in Tissue Culture Medium ( TCM199 ), the most commonly employed medium for bovine IVM. Synthetic Follicular Fluid Medium ( SFFM ) was created, based on the FF data and also contained glucosamine. Two different glucose concentrations were examined, 2.3 mM glucose to represent physiological concentrations and 5.6 mM glucose, the same concentration as is in TCM199. Culturing COCs in different glucose concentrations manipulated the completion of nuclear maturation and this was dependant on concentration, gonadotrophin supplementation and the timing of media changes, demonstrating the importance of this substrate to meiotic competence. Although glucosamine had no effect on oocyte nuclear maturation, supplementation during IVM led to a dose - dependent decrease in blastocyst rates. The detrimental effects of glucosamine manifested during early cleavage and were associated with a 0.6 - fold decrease in protein synthesis levels within the oocyte compared to oocytes cultured in media with no glucosamine, suggesting a detrimental effect on developmental competence. Interestingly oocytes cultured in media containing glucosamine and EGF had significantly higher protein synthesis compared to the control group. The biochemical profiles of COCs during IVM and FF were determined and used to create new media that allowed manipulation of oocyte nuclear maturation but compromised cytoplasmic maturation. Further research is required to optimise SFFM and to investigate the detrimental effects of glucosamine on developmental competence. / Thesis (Ph.D.)--Medical School, 2004.

The objective of this study was to develop a reliable method for intracytoplasmic sperm injection (ICSI) in bovine oocytes. Oocytes recovered from abattoir-derived ovaries were centrifuged for 5 min at 6000xg to facilitate sperm injection. Sperm were pre-treated in vitro with 5mM dithiothreitol (DTT), and diluted (approximately 1:5) with 5% polyvinylpyrrolidone (PVP) in 0.9% saline. After sperm injection, various activation procedures were compared. Initially, 3 h after activation with 5muM Ionomycin (A23187), oocytes with second polar bodies were selected and treated with 1.9mM 6-dimethylaminopurine (DMAP). The cleavage rate of sperm-injected oocytes treated with Ionomycin and DMAP was higher than with Ionomycin alone (62.1 vs. 27.3%, p < 0.05). In sham-injected control oocytes treated with Ionomycin and DMAP, the cleavage rate was approximately six times higher than that of oocytes treated with Ionomycin alone (44.3 vs. 7.4%, p < 0.001). Upon examination 16 h after ICSI, pronuclear formation was observed in 33 of 47 (70.2%) DMAP-treated oocytes. Two pronuclei were present in 18 of 33 (54.6%), while one and three pronuclei were seen in 8 of 33 (24.2%) and 7 of 33 (21.2%), respectively. In sham-injected DMAP-treated control oocytes, 6 of 15 (40.0%) had one pronucleus while 9 of 15 (60.0%) had two pronuclei. Since a single Ca2+ stimulation provided insufficient activation and DMAP treatment could result in triploidy, activation by multiple Ca2+ stimulations following ICSI was tested. Three Ca2+ stimulations were given at 30-min intervals. Pronuclear formation was observed in 16 of 41 (39.0%) oocytes at 16 h after sperm injection, with one and two pronuclei found in 4 of 16 (25.0%) and 12 of 16 (75.0%), respectively. Although one pronucleus was formed in 3 of 33 (9.1%) sham-injected control oocytes treated with three Ca2+ stimulations, two pronuclei were not seen in any of these oocytes. Due to the low rate of pronuclear formation after 5muM Ionomycin, 50muM

Thesis (M.S.)--University of Wisconsin--Madison, 1966. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

No description available.

Call number: LD2668 .T4 1985 B74 / Master of Science

Thesis (M.S. in animal science)--Washington State University, May 2007. / Includes bibliographical references.

Made available in DSpace on 2015-03-03T11:52:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-26Bitstream added on 2015-03-03T12:06:08Z : No. of bitstreams: 1 000809964.pdf: 1108463 bytes, checksum: 43fa89dc624ca99b382ecaa200b42518 (MD5) / A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende- se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2). As caveolinas -1 foram localizadas nos vilos fetais e maternos, mas sua marcação mais forte foi observada no estroma endometrial. As caveolinas -2 tiveram marcação positiva no trofoblasto e membrana corioalantoide, e, especificamente em célula trofoblástica gigante binucleada. Sendo assim, os resultados mostram que a proteína CAV-1 teve uma maior expressão em relação a proteína CAV-2 e que as proteínas CAV-1 e -2 são parte da composição das cavéolas, sendo . / The transgenic application of green fluorecent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these are animals that present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytocis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples will be cutted and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS) at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 ( -1 CAV and CAV- 2). The caveolinas -1 were found in fetal and maternal villi , but its strongest staining was observed in the endometrial stroma . The caveolinas -2 had positive staining in trophoblast and chorioallantoic membrane , and specifically in giant trophoblastic binucleated cell . therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and ...