Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Male fertility has for many years been defined in vitro as the ability of sperm to fertilize oocytes and to obtain early cleavage-stage embryos. Spermatozoa comprise of an extraordinary high percentage of polyunsaturated fatty acids in their plasma membrane. Due to an extremely low content of cytoplasm, sperm cells have a particularly low potential to scavenge reactive oxygen species (ROS), and are therefore highly sensitive to oxidative processes, which lead to sperm nucleus DNA damage/fragmentation. Normally, DNA fragmentation occurs in every ejaculate and can be induced by an excessive ROS production of active leukocytes or the spermatozoa themselves. Under distressed conditions, DNA fragmentation may also occur in the testis as a result of oxidative processes in the apoptotic cascade. These DNA fragmentations can be regarded as late signs of programmed cell death (apoptosis). Clinically, DNA fragmentation in spermatozoa results in significantly decreased implantation and pregnancy rates especially in patients with oligo- and/or teratozoospermia. The p-pattern normal sperm morphology has been shown to give poorer fertilization rates in vitro than the g- and n-patterns. In this study there is reported on the significant correlation found between the p-pattern normal sperm morphology and sperm DNA fragmentation as measured with the terminal deoxynucleotidyl transferase-mediated dUDP-biotin end labeling (TUNEL) assay. This finding further explains the lower fertility potential of patients presenting with p-pattern normal sperm morphology. In addition, this study explores the intricate relations between ROS in the semen, DNA fragmentation of the spermatozoa, as measured with the TUNEL assay and the sperm chromatin structure assay (SCSA ), spermatozoa apoptotic status and sperm parameters as measured with a standard semen analysis. Positive correlations were found between ROS and the apoptotic status of the sperm, as well as between sperm with non-fragmented DNA and sperm concentration and percentage motility. The results emphasize the importance of sperm selection especially when the treatment of choice is intracytoplasmic sperm injection (ICSI). An early sign of programmed cell death, also known as apoptosis, is the externalization of phosphatidylserine (PS) from the inner membrane leaflet to the outer leaflet. PS shows a high affinity to Annexin V. Apoptotic spermatozoa are able to fertilize oocytes, but embryo senescence may occur at the time when the paternal genes are activated. In this study there is reported on a novel method whereby spermatozoa can be separated on the basis of their apoptotic status through flow cytometry. Results showed that the normal sperm morphology, according to strict criteria, of the resultant nonapoptotic sperm fraction is significantly higher than that of the apoptotic counterpart. With refinement of this technique, it will be possible in future to use these separated non-apoptotic sperm cells during ICSI for fertilization. From the above it is apparent that the spermatozoon has to play a vital role in the development of the embryo from fertilization to implantation and pregnancy. It is, however, important to note that besides the gametes, there are other critical factors which contribute to a successful in vitro fertilization (IVF) cycle, among these are the in vitro culture conditions. In this regard, this study compared two sequential embryo culture systems. It was found that the more complex medium resulted in better day three embryo quality and a better blastocyst formation rate and pregnancy rate. These findings highlight the importance of a holistic perspective towards the complexity of the factors involved in affecting embryo quality and pregnancy outcome. / AFRIKAANSE OPSOMMING: Manlike fertiliteit is vir baie jare gedefinieer as die in vitro vermoë van ‘n spermsel om ‘n eiersel te bevrug om sodoende embrios te verkry. Die spermsel se plasmamembraan bestaan uit ‘n hoë persentasie poli-onversadigde vetsure. As gevolg van die klein hoeveelhede sitoplasma van die spermsel het dit ‘n beperkte weerstand teen reaktiewe suurstof spesies (ROS) en is gevolglik baie sensitief vir oksidasie. Oksidasie lei tot DNS skade/fragmentasie. DNS fragmentasie kom in spermselle van alle ejakulate voor en is gewoonlik die gevolg van ROS produksie deur die leukosiete in die semen of vanaf die spermselle self. Onder sekere omstandighede kan DNS fragmentasie ook voorkom in die testis waar dit deel vorm van apoptose. Hierdie tipe DNS skade word gesien as laat tekens van geprogrammeerde seldood (apoptose). In oligo- en/of teratozoospermiese mans lei DNS fragmentasie tot verlaagde implantasie- en swangerskapssyfers. Die p-patroon normale sperm morfologie groep gee laer in vitro bevrugting en swangerskapsyfers as die g- en n-patrone. In hierdie studie doen ons verslag oor die statisties betekenisvolle korrelasie wat gevind is tussen die p-patroon normale sperm morfologie en DNS fragmentasie soos gemeet met die ‘terminal deoxynucleotidyl transferase-mediated dUDP-biotin end labeling’ of te wel TUNEL toets. Hierdie bevinding is ‘n verdere verklaring vir die laer fertiliteits potensiaal van pasiënte wat voordoen met p-patroon sperm morfologie. ‘n Verdere doel van die studie was om die moontlike verband tussen ROS in die semen, spermatozoa DNS fragmentasie, apoptotiese status van die sperms en die motiliteits parameters van die spermatozoa te bepaal. ‘n Positiewe korrelasie is gevind tussen ROS en sperm apoptotiese status. Sperms met ongeframenteerde DNS is ook positief gekorreleer met sperm konsentrasie en motiliteit. Die resultate beklemtoon die belangrikheid van spermseleksie veral in pasiënte waar die keuse van behandeling intrasitoplasmiese sperm inspuiting (ICSI) is. ‘n Vroeë teken van apoptose is die eksternalisering van ‘phosphatidylserine’ (PS) vanaf die interne oppervlakte van die plasmamembraan na die eksterne oppervlak. PS het ‘n hoë affiniteit vir Annexin V. Apoptotiese sperms het die vermoë om ‘n oösiet te bevrug, maar kan lei tot die staking van embrio deling wanneer die vaderlike gene ‘n rol begin speel in embrio ontwikkeling. In hierdie studie het ons ‘n nuwe metode ontwikkel waarvolgens die spermatozoa in die ejakulaat op grond van hul apoptotiese status geskei kan word in apoptotiese en nie-apoptotiese fraksies. Die normale sperm morfologie van die nie-apoptotiese fraksie is betekenisvol beter as dié van die apoptotiese fraksie. Verdere verfyning van die tegniek kan daartoe lei dat dit in die toekoms toegepas kan word om vir nie-apoptotiese sperms te selekteer veral voor die uitvoering van ICSI. Uit die bogenoemde is dit duidelik dat die spermsel ‘n baie belangrike rol in die ontwikkeling van ‘n embrio, vanaf bevrugting tot implantasie en swangerskap, speel. Dit is egter ook belangrik om in gedagte te hou dat daar ander bydraende faktore tot ‘n suksesvolle in vitro swangerskap is, soos laboratorium toestande en embrio kultuursisteem. Om hierdie rede is daar ook twee kultuurmedia in hierdie studie vergelyk. Daar is bevind dat die meer komplekse medium beter kwaliteit embrios op dag drie lewer, asook meer blastosiste en ‘n hoër swangerskapsyfer. Dit is dus duidelik dat dit uiters belangrik is om ‘n holistiese perspektief te hê op die komplekse faktore wat ‘n invloed mag hê op bevrugting, embrio kwaliteit asook die swangerskapsyfer.

Orientador: José Antonio Dell'Aqua Junior / Banca: Marco Antonio Alvarenga / Banca: Silvia Ferrari / Resumo: A indução de múltiplas ovulações nas espécies domésticas é de extrema importância para a melhoria dos resultados e custos de biotécnicas como a transferência de embriões, inseminação com sêmen de baixa fertilidade, transferência de oócitos, entre outras. Este estudo teve como objetivo determinar se a sulpirida poderia induzir multiplas ovulações em éguas. Dezessete éguas foram aleatoriamente utilizadas nos dois grupos experimentais. No grupo de éguas tratadas com sulpirida 1mg/kg (IM) a cada 12 horas, o protocolo foi iniciado apenas em éguas que alcançaram um folículo de 24 mm ± 2mm de diâmetro e um segundo folículo ≥ 20 ± 2 mm de diâmetro. No grupo controle, utilizou-se o mesmo protocolo de início do tratamento porém as éguas foram tratadas com solução salina 0,9% (IM) a cada 12 horas. Em ambos os grupos, se durante as avaliações ultrassonográficas era verificado crescimento folicular concomitante de dois ou mais folículos, o tratamento perdurava até quando o menor folículo atingisse no mínimo 33 mm de diâmetro. Neste momento a ovulação foi induzida com 1500 UI de hCG. A inseminação artificial foi realizada 24 horas após a indução, e os embriões foram coletados no oitavo dia após a primeira ovulação. As variáveis estudadas foram analisadas utilizando o teste t de Student e teste exacto de Fisher. No grupo tratado, 35% das éguas tiveram mais que uma ovulação e uma média de 1,4 ovulações por ciclo enquanto que no grupo controle apenas 11% das éguas tiveram mais que uma ovulação e uma média de 1,1 ovulações por ciclo. No grupo tratado com sulpirida o tempo de início do tratamento até a primeira ovulação foi de 3,9 ± 0,9 dias enquanto que no grupo controle foi de 4,9 ± 1,0 (P<0,001). A administração da Sulpirida durante o desenvolvimento folicular, estimulou a atividade ovariana e não contribuiu para um aumento significativo nas taxas de duplas ou triplas ... / Abstract: The induction of multiple ovulations in domestic species is of extreme importance to provide improvements of results and costs of biotechnologies such as embryo transfer, artificial insemination with poor quality semen, oocyte transfer among others. This study aimed to determine if sulpiride could induce multiple ovulations in mares. Seventeen mares randomly passed the two experimental groups. In the group of mares treated with sulpiride 1mg/kg (IM) every 12 hours, the protocol was initiated only in mares that have achieved a follicle of 24 mm ± 2 mm in diameter and a second follicle ≥ 20 ± 2 mm in diameter. In the control group, were used the same protocol, but the mares were treated with 0.9% saline solution (IM) every 12 hours. During the ultrasound evaluation if was observed the follicular growth the treatment even when the lower follicle reached at least 33 mm in diameter. At this time the ovulation was induced with 1500 IU of hCG. Artificial insemination was performed 24 hours after ovulation induction, and the embryos were collected on the eighth day after the first ovulation. The variables were analyzed using Student's t Fisher's exact test and test. In group treated, 35% of the mares had more than one ovulation and ovulation an average of 1.4 per cycle while in control group only 11% of the mares had more than one ovulation and ovulation an average of 1.1 per cycle. In the group treated with sulpiride the time of the treatment beginning to the first ovulation was 3,9 ± 0.9 days whereas the control group was 4.9 ± 1.0 (P<0,001). Administration of Sulpiride during follicular development stimulated ovarian activity but did not contribute to a significant increase in the rates of double or triple ovulations, however some mares had a higher incidence of multiple ovulations when subjected to the proposed protocol, suggesting that further studies should be conducted for clarification of these results / Mestre

Abstract not available

Made available in DSpace on 2015-03-03T11:52:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-11-25Bitstream added on 2015-03-03T12:06:49Z : No. of bitstreams: 1 000809261.pdf: 277300 bytes, checksum: fd4b97c5598b7219cc2c5e30b2f73157 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo do presente estudo foi avaliar se aplicação de dose reduzida de bST (333mg), associada ao protocolo de superovulação de doadoras não lactantes, aumenta a produção de embriões e a manutenção de gestação após transferência para receptoras em lactação. Um total de 267 doadoras foram submetidas ao protocolo de superovulação, e subdivididas em 3 grupos: CONT (grupo controle = não tratados com bST), bST/IA (grupo recebeu uma dose de bST no dia da IA) e bST/InIA (grupo recebeu duas doses de bST, uma no início de protocolo e outra no dia da IA). A coleta não cirúrgica dos embriões foi realizada 6,5 dias após a inseminação. Os embriões frescos foram transferidos para receptoras lactantes previamente sincronizadas. Os diagnósticos de gestação foram realizados no d30 e d60. A concentração sérica de IGF1 nas doadoras no dia da coleta de embriões foi maior (p<0,01) para os grupos bST/IA (306,4 ± 13,0 ng/mL) e bST/InIA (307,3 ± 12,4 ng/mL) em relação ao CONT (195,8 ±13,3 ng/mL). A aplicação de bST no momento da IA aumentou a proporção de embriões transferíveis por coleta (69,9%), em relação ao controle (54,7%) e aos animais que receberam duas doses de bST (55,8%), resultando em 3,4±0,6 embriões por coleta no grupo controle, 4,7±0,6 embriões por coleta no grupo tratado com duas doses e 6,0±0,6 no grupo tratado com uma dose na IA. O tratamento com bST na IA aumentou o número de blastocistos recuperados por coleta (3,0±0,4) em relação ao controle (1,6±0,4), enquanto o grupo que recebeu duas aplicações de bST tendeu de aumentar o numero de blastocistos (2,6 ±0,4) em relação ao controle. A prenhez aos 60 dias foi 25,6%, 30% e 28,6% para os grupos CONT, bST/IA e bST/InIA respectivamente. Foi detectada interação entre tratamento e o estágio de desenvolvimento do embrião na prenhez por TE, em que blastocistos dos grupos tratados com bST ... / The objective of this study was evaluate if a low-dose of bST (333mg), associated with the superovulation protocol of non-lactating donors, would increases the embryo production and pregnancy maintenance after transfer to lactating recipients. For this, 267 donors were subjected to the superovulation protocol and subdivided into 3 groups: CONT (control group = no bST), bST/IA (group received one dose of bST at AI) and bST/INIA (group received two doses of bST, one at the beginning protocol and another at AI). Non-surgical collection of embryos occurred 6.5 days after insemination. The fresh embryos were transferred to previously synchronized lactating recipients. The pregnancy diagnoses were performed at d30 and d60. The The donors IGF1 serum concentration at embryo recovery day was higher (p <0.01) for bST/IA groups (306.4 ± 13.0 ng / mL) and bST/INIA (307.3 ± 12 4 ng / ml) compared to the CONT (195.8 ± 13.3 ng / ml). The bST application at AI increased proportion of transferable embryos per collection (69.9%), when compared with the control (54.7%) and with the animals that received two doses of somatotropin (55.8%) resulting 3.4 ± 0.6 embryos per collection in the control group, 4.7 ± 0.6 embryos per collection in two doses group and 6.0 ± 0.6 in the one dose at IA group. Treatment with bST at AI increased the number of blastocysts recovered by collection (3.0 ± 0.4) compared to control (1.6 ± 0.4), while the group that received two bST applications tended to increase number of blastocysts (2.6 ± 0.4) compared to the control. Pregnancy at 60 days was 25.6%, 30% and 28.6% for the CONT group, bST/AI and bST/INIA respectively. Was detected interaction between treatment and embryo development stage in pregnancy by ET, when blastocysts in the bST treated groups had higher P/ET at 60 days compared to the control group blastocysts, while morulae of the treated groups had lower P/ET that morulae ...

Made available in DSpace on 2015-01-26T13:21:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-06-26Bitstream added on 2015-01-26T13:30:36Z : No. of bitstreams: 1 000793045.pdf: 198695 bytes, checksum: adc16ea3cdb610acdec5679f22a1a6f8 (MD5) / A indução de múltiplas ovulações nas espécies domésticas é de extrema importância para a melhoria dos resultados e custos de biotécnicas como a transferência de embriões, inseminação com sêmen de baixa fertilidade, transferência de oócitos, entre outras. Este estudo teve como objetivo determinar se a sulpirida poderia induzir multiplas ovulações em éguas. Dezessete éguas foram aleatoriamente utilizadas nos dois grupos experimentais. No grupo de éguas tratadas com sulpirida 1mg/kg (IM) a cada 12 horas, o protocolo foi iniciado apenas em éguas que alcançaram um folículo de 24 mm ± 2mm de diâmetro e um segundo folículo ≥ 20 ± 2 mm de diâmetro. No grupo controle, utilizou-se o mesmo protocolo de início do tratamento porém as éguas foram tratadas com solução salina 0,9% (IM) a cada 12 horas. Em ambos os grupos, se durante as avaliações ultrassonográficas era verificado crescimento folicular concomitante de dois ou mais folículos, o tratamento perdurava até quando o menor folículo atingisse no mínimo 33 mm de diâmetro. Neste momento a ovulação foi induzida com 1500 UI de hCG. A inseminação artificial foi realizada 24 horas após a indução, e os embriões foram coletados no oitavo dia após a primeira ovulação. As variáveis estudadas foram analisadas utilizando o teste t de Student e teste exacto de Fisher. No grupo tratado, 35% das éguas tiveram mais que uma ovulação e uma média de 1,4 ovulações por ciclo enquanto que no grupo controle apenas 11% das éguas tiveram mais que uma ovulação e uma média de 1,1 ovulações por ciclo. No grupo tratado com sulpirida o tempo de início do tratamento até a primeira ovulação foi de 3,9 ± 0,9 dias enquanto que no grupo controle foi de 4,9 ± 1,0 (P<0,001). A administração da Sulpirida durante o desenvolvimento folicular, estimulou a atividade ovariana e não contribuiu para um aumento significativo nas taxas de duplas ou triplas ... / The induction of multiple ovulations in domestic species is of extreme importance to provide improvements of results and costs of biotechnologies such as embryo transfer, artificial insemination with poor quality semen, oocyte transfer among others. This study aimed to determine if sulpiride could induce multiple ovulations in mares. Seventeen mares randomly passed the two experimental groups. In the group of mares treated with sulpiride 1mg/kg (IM) every 12 hours, the protocol was initiated only in mares that have achieved a follicle of 24 mm ± 2 mm in diameter and a second follicle ≥ 20 ± 2 mm in diameter. In the control group, were used the same protocol, but the mares were treated with 0.9% saline solution (IM) every 12 hours. During the ultrasound evaluation if was observed the follicular growth the treatment even when the lower follicle reached at least 33 mm in diameter. At this time the ovulation was induced with 1500 IU of hCG. Artificial insemination was performed 24 hours after ovulation induction, and the embryos were collected on the eighth day after the first ovulation. The variables were analyzed using Student's t Fisher's exact test and test. In group treated, 35% of the mares had more than one ovulation and ovulation an average of 1.4 per cycle while in control group only 11% of the mares had more than one ovulation and ovulation an average of 1.1 per cycle. In the group treated with sulpiride the time of the treatment beginning to the first ovulation was 3,9 ± 0.9 days whereas the control group was 4.9 ± 1.0 (P<0,001). Administration of Sulpiride during follicular development stimulated ovarian activity but did not contribute to a significant increase in the rates of double or triple ovulations, however some mares had a higher incidence of multiple ovulations when subjected to the proposed protocol, suggesting that further studies should be conducted for clarification of these results

Made available in DSpace on 2016-07-01T13:10:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-06-22. Added 1 bitstream(s) on 2016-07-01T13:14:08Z : No. of bitstreams: 1 000866261.pdf: 2011639 bytes, checksum: de26a1680120fdc75d49c236f0f98bb9 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Com o objetivo de investigar os efeitos da administração de 2,5 mg de benzoato de estradiol (BE) seguido de 1500 mg de progesterona de longa ação (P4 LA) sobre a dinâmica molecular endometrial, características morfológicas do útero e concentrações plasmáticas de estrógeno e progesterona em éguas receptoras acíclicas, foram avaliados: a expressão proteica e as alterações na abundância relativa dos transcritos para os receptores de estrógeno (ER) e progesterona (PR) no endométrio por meio da RT-qPCR e imunohistoquímica; as características morfológicas do útero por ultrassonografia e palpação retal; e as concentrações plasmáticas do estrógeno e progesterona por meio de radioimunoensaio. A aplicação de 2,5 mg, 5 mg ou 10 mg de BE foram eficazes em causar edema uterino máximo 24 h após a administração. A dose de 2,5 mg de BE provocou picos de concentração de estradiol similares aos de éguas tratadas com 5 mg, 10 mg e aos de éguas cíclicas, sem elevar a concentração de estrógeno conjugado como observado com as outras doses de BE. Ainda, a concentração de P4 foi reduzida após a aplicação da P4 LA quando 10 mg de BE foram utilizados. A administração dos tratamentos hormonais alterou a abundância relativa dos transcritos para ERα, ERβ e PR de forma similar às alterações encontradas em éguas cíclicas quando o final do estro e início do diestro foram comparados. Em conclusão, o uso de 2,5 mg de BE seguido de 1500 mg de P4 LA provoca alterações similares na morfologia uterina, abundância relativa dos transcritos para ERα, ERβ e PR no endométrio, e de concentrações circulantes de estrógeno e progesterona às encontradas em éguas cíclicas / To investigate the effects of the administration of 2.5 mg of estradiol benzoate (EB) followed by 1500 mg of long acting progesterone (LA P4) on endometrial molecular dynamics, uterine morphological characteristics, as well as estrogen and progesterone plasma concentrations in non-cyclic recipient mares, we evaluated: protein expression and changes in relative abundance of transcripts for estrogen (ER) and progesterone receptors (PR) in the endometrium by RT-qPCR and immunohistochemistry; uterine morphological characteristics by ultrasonography and rectal palpation; and estrogen and progesterone plasma concentrations by radioimmunoassay. The administration of 2.5 mg, 5 mg or 10 mg of EB were effective at promoting maximum uterine edema 24 h after their injection. The dose of 2.5 mg of EB caused similar estradiol concentration peaks to those observed in mares treated with 5 mg or10 mg of EB and to those observed in cyclic mares, which was not followed by increased concentrations of estrogen conjugate as observed for the other doses. Moreover, P4 concentration was reduced 24 h after LA P4 administration when the dose of 10 mg of EB was injected. The administration of the hormonal protocols altered the relative abundance of transcripts for ERα, ERβ and PR in non-cyclic mares as observed in cyclic ones when late estrus and early diestrus were compared. In conclusion, the use of 2.5 mg of EB followed by 1500 mg of LA P4 causes similar uterine morphology changes, relative abundance of transcripts for ERα, ERβ and PR, and estrogen and progesterone circulating concentrations to those found in cyclic mares

Orientador: José Luiz Moraes Vasconcelos / Banca:José Buratini Junior / Banca: Heraldo César Gonçalves / Resumo: O objetivo do presente estudo foi avaliar se aplicação de dose reduzida de bST (333mg), associada ao protocolo de superovulação de doadoras não lactantes, aumenta a produção de embriões e a manutenção de gestação após transferência para receptoras em lactação. Um total de 267 doadoras foram submetidas ao protocolo de superovulação, e subdivididas em 3 grupos: CONT (grupo controle = não tratados com bST), bST/IA (grupo recebeu uma dose de bST no dia da IA) e bST/InIA (grupo recebeu duas doses de bST, uma no início de protocolo e outra no dia da IA). A coleta não cirúrgica dos embriões foi realizada 6,5 dias após a inseminação. Os embriões frescos foram transferidos para receptoras lactantes previamente sincronizadas. Os diagnósticos de gestação foram realizados no d30 e d60. A concentração sérica de IGF1 nas doadoras no dia da coleta de embriões foi maior (p<0,01) para os grupos bST/IA (306,4 ± 13,0 ng/mL) e bST/InIA (307,3 ± 12,4 ng/mL) em relação ao CONT (195,8 ±13,3 ng/mL). A aplicação de bST no momento da IA aumentou a proporção de embriões transferíveis por coleta (69,9%), em relação ao controle (54,7%) e aos animais que receberam duas doses de bST (55,8%), resultando em 3,4±0,6 embriões por coleta no grupo controle, 4,7±0,6 embriões por coleta no grupo tratado com duas doses e 6,0±0,6 no grupo tratado com uma dose na IA. O tratamento com bST na IA aumentou o número de blastocistos recuperados por coleta (3,0±0,4) em relação ao controle (1,6±0,4), enquanto o grupo que recebeu duas aplicações de bST tendeu de aumentar o numero de blastocistos (2,6 ±0,4) em relação ao controle. A prenhez aos 60 dias foi 25,6%, 30% e 28,6% para os grupos CONT, bST/IA e bST/InIA respectivamente. Foi detectada interação entre tratamento e o estágio de desenvolvimento do embrião na prenhez por TE, em que blastocistos dos grupos tratados com bST ... / Abstract: The objective of this study was evaluate if a low-dose of bST (333mg), associated with the superovulation protocol of non-lactating donors, would increases the embryo production and pregnancy maintenance after transfer to lactating recipients. For this, 267 donors were subjected to the superovulation protocol and subdivided into 3 groups: CONT (control group = no bST), bST/IA (group received one dose of bST at AI) and bST/INIA (group received two doses of bST, one at the beginning protocol and another at AI). Non-surgical collection of embryos occurred 6.5 days after insemination. The fresh embryos were transferred to previously synchronized lactating recipients. The pregnancy diagnoses were performed at d30 and d60. The The donors IGF1 serum concentration at embryo recovery day was higher (p <0.01) for bST/IA groups (306.4 ± 13.0 ng / mL) and bST/INIA (307.3 ± 12 4 ng / ml) compared to the CONT (195.8 ± 13.3 ng / ml). The bST application at AI increased proportion of transferable embryos per collection (69.9%), when compared with the control (54.7%) and with the animals that received two doses of somatotropin (55.8%) resulting 3.4 ± 0.6 embryos per collection in the control group, 4.7 ± 0.6 embryos per collection in two doses group and 6.0 ± 0.6 in the one dose at IA group. Treatment with bST at AI increased the number of blastocysts recovered by collection (3.0 ± 0.4) compared to control (1.6 ± 0.4), while the group that received two bST applications tended to increase number of blastocysts (2.6 ± 0.4) compared to the control. Pregnancy at 60 days was 25.6%, 30% and 28.6% for the CONT group, bST/AI and bST/INIA respectively. Was detected interaction between treatment and embryo development stage in pregnancy by ET, when blastocysts in the bST treated groups had higher P/ET at 60 days compared to the control group blastocysts, while morulae of the treated groups had lower P/ET that morulae ... / Mestre

Orientador: Cezinande de Meira / Coorientador: José Paes de Oliveira Filho / Banca: Fabiana Ferreira de Ssouza / Banca: Fernanda da Cruz Landim / Banca: Paula de Carvalho Papa / Banca: Guilherme Pugliesi / Resumo: Com o objetivo de investigar os efeitos da administração de 2,5 mg de benzoato de estradiol (BE) seguido de 1500 mg de progesterona de longa ação (P4 LA) sobre a dinâmica molecular endometrial, características morfológicas do útero e concentrações plasmáticas de estrógeno e progesterona em éguas receptoras acíclicas, foram avaliados: a expressão proteica e as alterações na abundância relativa dos transcritos para os receptores de estrógeno (ER) e progesterona (PR) no endométrio por meio da RT-qPCR e imunohistoquímica; as características morfológicas do útero por ultrassonografia e palpação retal; e as concentrações plasmáticas do estrógeno e progesterona por meio de radioimunoensaio. A aplicação de 2,5 mg, 5 mg ou 10 mg de BE foram eficazes em causar edema uterino máximo 24 h após a administração. A dose de 2,5 mg de BE provocou picos de concentração de estradiol similares aos de éguas tratadas com 5 mg, 10 mg e aos de éguas cíclicas, sem elevar a concentração de estrógeno conjugado como observado com as outras doses de BE. Ainda, a concentração de P4 foi reduzida após a aplicação da P4 LA quando 10 mg de BE foram utilizados. A administração dos tratamentos hormonais alterou a abundância relativa dos transcritos para ERα, ERβ e PR de forma similar às alterações encontradas em éguas cíclicas quando o final do estro e início do diestro foram comparados. Em conclusão, o uso de 2,5 mg de BE seguido de 1500 mg de P4 LA provoca alterações similares na morfologia uterina, abundância relativa dos transcritos para ERα, ERβ e PR no endométrio, e de concentrações circulantes de estrógeno e progesterona às encontradas em éguas cíclicas / Abstract: To investigate the effects of the administration of 2.5 mg of estradiol benzoate (EB) followed by 1500 mg of long acting progesterone (LA P4) on endometrial molecular dynamics, uterine morphological characteristics, as well as estrogen and progesterone plasma concentrations in non-cyclic recipient mares, we evaluated: protein expression and changes in relative abundance of transcripts for estrogen (ER) and progesterone receptors (PR) in the endometrium by RT-qPCR and immunohistochemistry; uterine morphological characteristics by ultrasonography and rectal palpation; and estrogen and progesterone plasma concentrations by radioimmunoassay. The administration of 2.5 mg, 5 mg or 10 mg of EB were effective at promoting maximum uterine edema 24 h after their injection. The dose of 2.5 mg of EB caused similar estradiol concentration peaks to those observed in mares treated with 5 mg or10 mg of EB and to those observed in cyclic mares, which was not followed by increased concentrations of estrogen conjugate as observed for the other doses. Moreover, P4 concentration was reduced 24 h after LA P4 administration when the dose of 10 mg of EB was injected. The administration of the hormonal protocols altered the relative abundance of transcripts for ERα, ERβ and PR in non-cyclic mares as observed in cyclic ones when late estrus and early diestrus were compared. In conclusion, the use of 2.5 mg of EB followed by 1500 mg of LA P4 causes similar uterine morphology changes, relative abundance of transcripts for ERα, ERβ and PR, and estrogen and progesterone circulating concentrations to those found in cyclic mares / Doutor

Orientador: Gisele Zoccal Mingoti / Banca: Joaquim Mansano Garcia / Banca: Felipe Perecin / Banca: Lindsay Unno Gimenes / Banca: Fernanda da Cruz Landim / Resumo: O presente estudo teve como objetivos avaliar os efeitos de estimuladores da lipólise (Forskolin: Forsk) e inibidores da lipogênese (Ácido Linoléico: LA) durante o cultivo in vitro (CIV) sobre o acúmulo de lipídios intracitoplasmáticos e criotolerância do embrião bovino, bem como avaliar o efeito do tratamento dos embriões com Forsk sobre a taxa de concepção após transferência para receptoras. Oócitos (n=1242) foram maturados in vitro (MIV) durante 22h a 38,5°C e 5% de CO2 em ar em meio TCM-199 bicarbonato com 10% SFB e hormônios. Após fecundação, os prováveis zigotos foram cultivados em meio SOF (grupo Controle) suplementado com: 100 μM LA durante todo o período do CIV (grupo LA); ou 5 μM Forsk a partir do dia 6 do CIV (grupo Forsk); ou com associação de LA e Forsk, como acima descrito (grupo LA+Forsk). O CIV foi conduzido a 38,5°C e 5% de CO2 em ar, por 7 dias. O desenvolvimento embrionário foi avaliado no dia 7 do CIV (D7), quando os blastocistos foram corados com Sudan Black B 1% para determinação do conteúdo lipídico intracitoplasmático. Os embriões foram avaliados em microscopia de luz e as imagens obtidas foram analisadas por Q-Capture Pro Image Software. O grupo Controle foi escolhido como calibrador e a média de cada grupo foi dividida pela média do calibrador para calcular a intensidade relativa de pixels, expressa em unidades arbitrárias. Os blastocistos expandidos foram vitrificados (Vitri Ingá®), aquecidos e cultivados por 24h em SOF para determinação das taxas d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of the present study was to evaluate the effects of supplementation of in vitro culture (IVC) medium with drugs that stimulates the lipolysis (Forskolin: Forsk) and inhibit the lipogenesis (Linoleic Acid LA) on the intracytoplasmic lipid content and cryotolerance of bovine embryos, as well as to evaluate the effect of treatment of embryos with Forsk on the pregnancy rates after transfer to synchronized recipients. Oocytes (n=1242) were matured in vitro for 22h at 38.5°C and 5% CO2 in air in medium TCM199 with 10% FCS and hormones. After fertilization, presumptive zygotes were cultured in SOF medium (Control group) supplemented with: 100 μM LA throughout the culture period (LA group); or 5 μM Forsk from the 6th day to the end of the culture (Forsk group); or with the association of LA and Forsk, as described above (LA+Forsk group). The IVC was conducted at 38.5°C and 5% CO2 in air, for 7 days. Embryonic development was assessed on day 7 of culture (D7), when blastocysts were stained with 1% Sudan Black B to determine the intracytoplasmic lipid content. The embryos were evaluated by light microscopy and the images were analyzed by Q-Capture Pro Image software. The Control group was chosen as a calibrator and the measured value of each treatment was divided by the mean of the calibrator to generate the relative expression levels of pixels, expressed in arbitrary units. The expanded blastocysts were vitrified (Vitri Ingá®), warmed and cultured for 24h in SOF to eval... (Complete abstract click electronic access below) / Doutor

Indiana University-Purdue University Indianapolis (IUPUI) / One’s personal claims regarding personhood will influence his moral belief regarding embryo adoption. In Chapter One, I consider the personhood of the human embryo. If the human embryo is a person, we are morally obligated to permit the practice of embryo adoption as an ethical means to save human persons. However, for those who do not claim that an embryo is a person at conception, embryo adoption is not a necessary practice because we have no moral obligation to protect them. There are still others who claim that personhood is gained at some point during gestation when certain mental capacities develop. I offer my own claim that consciousness and sentience as well as the potential to be self-conscious mark the beginning of personhood. Embryo adoption raises several questions surrounding the institution of marriage. Due to its untraditional method of procreation, embryo adoption calls into question the role of procreation within marriage. In Chapter Two, I explore the nature of the marriage relationship by offering Lisa Cahill’s definition of marriage which involves both a spiritual and physical dimension, and then I describe the concept of marriage from different perspectives including a social, religious, and a personal perspective. From a personal perspective, I explore the relationship between marriage and friendship. Finally, I describe how the concept of marriage is understood today and explore the advantages to being married as opposed to the advantages of being single. Embryo adoption changes the way we customarily think about procreation within a family because in embryo adoption, couples are seeking an embryo from another union to be implanted into the woman. This prompts some philosophers to argue that embryo adoption violates the marriage relationship. In Chapter Three, I further consider the impact of embryo adoption on the family as an extension of the marital relationship as well as the impact of embryo adoption on the traditional roles of motherhood and fatherhood. I examine motherhood by looking at how some philosophers define motherhood and when these philosophers claim a woman becomes a mother. After considering these issues regarding motherhood, I examine the same issues surrounding fatherhood. Peg Brand, PhD., Chair