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Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Premature luteal regression occurs on average in 30% of superovulated sheep ewes. This phenomenon occurs early in the cycle before the embryo’s can be collected and is a major contributor to failure in embryo transfer programs. This research was done to understand the physiological mechanisms involved. Chapter two provides a general background of the physiology of natural luteolysis and the maternal recognition of pregnancy. The chapter introduces some new concepts on the topic of cell death and provides a recent literature review on research done on the phenomenon of premature luteal regression. This chapter forms the base of ideas and arguments that follows in the two studies containing new original work in this field. The research contained in this thesis comprises of two in vivo studies. The first study (Chapter 3) compare premature luteal regression to Prostaglandin F2α (PGF2α) induced regression with emphasis on the changes in levels of the steroid hormones progesterone (P4) and estradiol - 17β (E2-17β) and changes in structure and ultra structure. The following conclusions were made: 1. Premature luteal regression is not merely inadequate luteal support, but indeed early luteal regression, since seasonal influences could merely be nutritional influences, and a definitive increase in P4 were recorded in animals exhibiting the phenomena. 2. Nutritional influences could play a role, but the type and quality of nutrients and mechanism involved, is still unclear. 3. PGF2α-induced regression differs from premature luteal regression in that: a) The progression of functional and structural regression in PGF2α -induced regression is slower than in premature luteal regression. b) Regressed corpora lutea do not occur with normal functioning corpora lutea. 4. There is a distinct second E2-17β peak preceding the decline in P4 in animals that exhibits signs of premature luteal regression. A threshold initiating premature luteal regression was not established. The second study (Chapter 4) compares the changes in the ovine β estradiol - 17 β receptor (oERβ) between premature luteal regression and PGF2α induced regression. The study concludes that there could be a potential role for oERβ in premature luteal regression. The findings of these two studies raise some questions about the conventional perception that early release of PGF2α is the cause of premature luteal regression. The thesis concludes in a hypothesis (Chapter 4) explaining the phenomenon. / AFRIKAANSE OPSOMMING: Premature luteale regressie kom gemiddeld in 30% van gesuperovuleerde skaap-ooie voor. Die verskynsel kom vroeg in die siklus voor, voor die embrios gekollekteer kan word, en is een van die belangrikste oorsaake van mislukkings in ‘n embrio-oorplaasingsprogram. Die huidige navorsing poog om die fisiologiese meganismes betrokke by premature luteale regressie te verstaan. Hoofstuk twee verskaf ‘n algemene agtergrond van die fisiologiese aspekte betrokke by natuurlike luteale regressie en maternale herkenning van swangerskap. Die hoofstuk stel nuwe konsepte voor oor sel afsterwing en verskaf ‘n opgedateerde literatuuroorsig met betrekking tot die navorsing wat in die veld oor die verskynsel gedoen is. Die hoofstuk vorm die basis vir die idees en argumente, wat volg in die twee studies en wat oorspronklike nuwe navorsing bevat oor die onderwerp. Die navorsing in die tesis bestaan uit twee in vivo studies. Die eerste studie (Hoofstuk 3) vergelyk premature luteale regressie en prostaglandien F2α (PGF2α) ge-induseerde regressie met ‘n klem op die vlakke van die steröiedhormone progesteroon (P4) en estradiol - 17β (E2-17β) en veranderinge in die mikroskopiese struktuur en ultra struktuur van die corpus luteum. Die studie bevind: 1. Premature luteale regressie is nie slegs onvoldoende luteale funksie nie, maar vroë luteale regressie aangesien seisoenale invloede eitlik voedings invloede kan wees en P4 gestyg het in diere waar die verskynsel voorgekom het. 2. Voeding kan ‘n rol speel maar die tiepe en gehalte van die voedingstowwe en die meganismes betrokke is nie duidelik nie. 3. PGF2α - ge-induseerde regressie verskil van premature regressie in dat: a) Die verloop van funksionele en strukturele regressie is stadiger in PGF2α - ge-induseerde regressie in vergelyking met premature luteale regressie. b) Corpora lutea wat regressie ondergaan het kom nie voor saam met corpora lutea wat normal voorkom nie. 4. Daar die ‘n duidelike tweede piek van E2-17β gaan die afname in P4 vooraf in diere waar premature regressie voorkom. 5. Daar is nie geslaag om ‘n drempel vas te stel waar premature regressie ge-inisieer word nie. Die tweede studie vergelyk die veranderinge in estradiol-17β reseptore (oERβ) in die skaap tydens premature luteale regressie en PGF2α geinduseerde regressie. Die studie bevind dat daar ‘n moontlike rol is vir ERβ in premature luteale regressie. Die bevindinge van die twee studies bevraagteken die konvensionele opvatting dat vroë vrystelling van PGF2α verantwoordelik is vir premature luteale regressie. Die tesis sluit af met ‘n nuwe hipotese om die verskynsel te verduidelik.

A presente tese foi dividida em 5 Experimentos. Os objetivos do Experimento 1 foram avaliar a utilização da gonadotrofina coriônica equina (eCG) e/ou do cipionato de estradiol (ECP) na dinâmica folicular e taxa de concepção de vacas holandesas submetidas a inseminação em tempo fixo (IATF). No D0, todos os animais (n = 782) receberam 2 mg de benzoato de estradiol (BE) e um dispositivo intravaginal de progesterona (CIDR). Oito dias depois, o CIDR foi retirado e todos os animais receberam PGF2?. Simultaneamente, os animais foram divididos em 4 grupos: G1) eCG + ECP no Dia 8; G2) eCG no Dia 8 + GnRH após 48h; G3) ECP no Dia 8; G4) GnRH após 48h. Amostras de sangue e exames utlra-sonográficos foram realizados frequentemente em um subgrupo de animais (n = 96). As análises estatísticas de todos os experimentos foram efetuadas com o proc GLIMMIX e proc MIXED do SAS. O uso de eCG e o escore de condição corporal (ECC) dos animais afetaram as concentrações circulantes de progesterona no diestro. Os animais do G2 apresentaram maior taxa de concepção que os do G4 (33,8% vs. 28,9%). Além disso, para animais de menor ECC, ficou evidente o benefício da aplicação de eCG (G2 = 44,4% vs. G4 = 6,1%). No Experimento 2 (n = 26), o objetivo foi comparar o efeito da administração da eCG no dia da remoção do CIDR em animais de menor (2,0-2,5) ou maior (3,0-3,5) ECC. Foram avaliadas algumas características do corpo lúteo (CL) como o volume e histologia, assim como as concentrações plasmáticas de progesterona no diestro. Independentemente da condição corporal dos animais, a eCG aumentou o volume do CL e a concentração plasmática de progesterona no diestro. O ECC afetou negativamente o volume e concentração de progesterona sérica no diestro. Não foi encontrado diferença na proporção de células grandes/pequenas, assim como no fluxo sanguíneo no CL entre os grupos experimentais. No Experimento 3, foi comparado a taxa de concepção em vacas de leite de alta produção (n = 388) após o uso do protocolo G2 do Experimento 1 (Capítulo I), com ou sem adição de ECP no momento da retirada do CIDR (novo ou usado). Não foi verificado efeito da adição do tratamento com ECP e nem do tipo do dispostivo na taxa de concepção. No Experimento 4 (n = 199), apesar no aumento verificado no diâmetro folicular no grupo tratado com GnRH 56h (17,8 mm) comparado com GnRH 48h (16,5 mm); e do atraso no momento da ovulação após a retirada do CIDR (GnRH 56h = 75,3h; GnRH 48h = 79,8h) não foi constatado qualquer diferença na concepção ao se atrasar a aplicação do GnRH de 48h para 56h em vacas inseminadas 16h depois do GnRH. No Experimento 5 (n = 185), a taxa de concepção não diferiu em animais que receberam o GnRH 48h ou 56h (momento da IATF) após a retirada do CIDR, indicando a possibilidade do emprego de um protocolo com apenas 3 manejos em vacas de leite de alta produção. / The current thesys has been divided in 5 Experiments. Objectives of Experiment 1 were to evaluate the effects of equine chorionic gonadotropin (eCG) and/or estradiol cypionate (ECP) on follicular dymanics and conception rate in Holstein cows receiving fixed timed artificial insemination (TAI). On D0, all cows (n = 782) received 2 mg of estradiol benzoate (EB) and one intravaginal progesterone device (CIDR). Eight days later, CIDR was removed and all animals were treated with PGF2?. Simultaneously, animals were divided in 4 groups: G1) eCG + ECP on Day 8; G2) eCG on Day 8 + GnRH 48h later; G3) ECP on Day 8; G4) GnRH 48h later. Blood samples and ultrasound exams were frequently performed in a subset of the animals (n = 96). All the statistical analyses for all experiments were performed with proc GLIMMIX and proc MIXED of SAS. Equine chorionic gonadotropin (eCG) treatment and body condition score (BCS) affected circulating progesterone in the diestrus. Cows in G2 had greater conception rates than cows in G4 (33,8% vs. 28,9%). In addition, in cows with lower BCS, eCG seems to be even more affective (G2 = 44,4% vs. G4 = 6,1%). In Experiment 2 (n = 26), the objective was to compare the effect of eCG the day of CIDR removal in animals with lower (2,0-2,5) or higher (3,0-3,5) BCS. Some variables such as corpus luteum (CL) volume, histology and circulating progesterone concentration in the diestrus were evaluated. Regardless of the body condition of the animals, eCG increased CL volume and circulating progesterone concentration in the diestrus. BCS negatively affected CL volume and circulating progesterone. There were no differences in large/small CL cell ratio, as well as CL blood flow between experimental groups. In Experiment 3, it was compared conception rate in dairy cows (n = 388) after using the same protocol G2 from Experiment 1 (Chapter I), with or without an ECP treatment at the time of CIDR (new or used) removal. Both ECP treatment and type of CIDR did not significantly affected conception rates. In Experiment 4 (n = 199), despite the fact that follicular diameter was increased in group GnRH 56h (17,8 mm) compared with GnRH 48h (16,5 mm); and of the delayed time of ovulation after CIDR removal (GnRH 56h = 75,3h; GnRH 48h = 79,8h), there were no differences in conception rates after delaying the GnRH treatment from 48h to 56h in cows inseminated 16h after GnRH. In Experiment 5 (n = 185), conception rate did not differ in animals that received GnRH 48h or 56h (at the time of TAI) after CIDR removal, indicating the possibility of using a protocol with only 3 handlings in high producing dairy cows.

A transferência de embriões (TE) é uma biotecnologia amplamente utilizada na produção de equinos no Brasil, e a égua receptora exerce um papel fundamental nos resultados finais nos programas de reprodução. Alguns tratamentos têm sido preconizados com objetivo de exercer ação luteotrófica, melhorando a função luteal, e com isto aumentando os níveis sistêmicos de progesterona, como também o uso de drogas anti-inflamatórias, que evitam a perda da função luteal causada pela liberação de prostaglandina em processos inflamatórios prévios ou por manipulação na passagem transcervical do embrião no momento da TE. O objetivo deste estudo foi investigar os efeitos do anti-inflamatório inibidor seletivo COX 2 (meloxicam) associado ao uso luteotrófico da hCG na gestação, características de corpo lúteo, fertilidade e desenvolvimento embrionário em éguas receptoras. Para isto, 60 éguas receptoras alojadas em central de reprodução foram aleatoriamente divididas em 4 grupos de 15 animais, de acordo com os seguintes tratamentos: aplicação de 5ml solução fisiológica no dia da ovulação (controle); aplicação de 2500 UI hCG no dia da ovulação; aplicação de 2500 UI hCG no dia da ovulação associado a 0,6 mg/kg de meloxicam; aplicação de 2500 UI hCG no momento da transferência do embrião associado a 0,6 mg/kg meloxicam. No dia da TE, 7 dias após a TE e 21 dias após a TE para as éguas que ficaram gestantes, foi avaliada a irrigação do corpo lúteo por ultrassonografia modo doppler colorido por meio de score do percentual de irrigação (0-100%), e a irrigação do útero avaliado por score (1-4). Por meio da ultrassonografia no modo B, a área do CL em mm2 foi avaliada nos mesmos momentos, a área da vesícula em mm2 aos 15 e 21 dias da gestação e o tamanho do embrião em cm aos 29 dias de gestação. Além disso, foi realizada dosagem plasmática de progesterona no dia da TE, 7 dias após a TE e 21 dias para as éguas gestantes. As comparações entre os grupos foram analisadas para efeitos principais de tratamento e tempo, assim como para efeito de interação tratamento x tempo. Não foi observado influencia da aplicação de hCG e/ou meloxicam nos animais tratados comparados ao controle (P>0,05). No entanto, foi observado um aumento numérico na taxa de gestação para os dois grupos de receptoras que receberam anti-inflamatório meloxicam, correspondendo a um índice superior em 13,3 % de gestação para éguas tratadas. Em conclusão, não há influencia da aplicação de hCG associado ou não meloxicam sobre a perfusão sanguínea uterina, do corpo lúteo, no desenvolvimento embrionário e nas concentrações plasmáticas de progesterona em receptoras de embriões. No entanto, existe, pela aplicação de meloxicam, evidência de melhora nos índices de gestação, podendo estar associado ao efeito anti-inflamatório seletivo COX-2, que pode melhorar resultados em programas comerciais de transferência de embriões em equinos. / Embryo transfer (ET) is a biotechnology widely used in equine production in Brazil, and the recipient mare plays a key role in the final results of breeding programs. Several treatments have been advocated both for the purpose of exerting luteotrophic action, improving luteal function, and thereby increasing systemic levels of progesterone, such as the use of anti-inflammatory drugs, which prevent the loss of luteal function caused by the release of prostaglandin in previous inflammatory processes or by manipulation in the transcervical passage of the embryo at the time of ET. The objective of this study was to investigate the effects of the anti-inflammatory COX 2 inhibitor (meloxicam) associated with luteotrophic use of hCG in gestation, corpus luteum characteristics, fertility and embryonic development in recipient mares. For this, 60 receiving mares housed in a reproduction center were randomly divided into 4 groups of 15 animals, according to the following treatments: application of 5ml physiological solution on the day of ovulation (control group); application of 2500 IU hCG on ovulation day; application of 2500 IU hCG on the day of ovulation associated with 0.6 mg/kg of meloxicam; application of 2500 IU hCG at the time of embryo transfer associated with 0.6 mg/kg meloxicam. On the day of ET, 7 days after ET and 21 days after ET for pregnant mares, corpus luteum irrigation was evaluated by color Doppler ultrasonography using a score of the percentage of irrigation (0-100%), and irrigation of the uterus evaluated by score (1-4). Using B-mode ultrasonography, corpus luteum area in mm2 was assessed at the same time points, the area of the embryonic vesicle in mm2 at 15 and 29 days of gestation, and the size of the embryo in cm at 29 days of gestation. In addition, plasma progesterone dosage was performed on ET day 7 days after ET in all groups and 21 days after ET for pregnant mares. The comparisons between the groups were analyzed for main effects of treatment and time, as well as for interaction effect treatment x time. No influence of application of hCG and / or meloxican was observed on treated animals compared to control (P>0.05). However, a numerical increase in gestation rate was observed for the two groups of recipients who received anti-inflammatory meloxicam, corresponding to an index higher than 13.3% of gestation for treated mares. In conclusion, there is no influence of the application of hCG associated or not to meloxicam on uterine irrigation, corpus luteum, embryo development and plasma concentrations of progesterone in embryo recipients. However, there is evidence of improvement in gestation rates due to the application of meloxicam, which may be associated with the COX-2 selective anti-inflammatory effect of this drug, improving results in commercial embryo transfer programs in horses.

A aplicação de drogas durante o diestro para melhorar a taxa de prenhez na égua inseminada e na receptora de embrião tem sido o foco de alguns grupos de pesquisa. Estudos com hCG encontraram resultados promissores nas taxas de prenhez e características uterinas de receptoras de embrião no dia da transferência. Com o objetivo de avaliar o efeito da aplicação da hCG em diferentes momentos do ciclo estral sobre as características do trato reprodutivo, vascularização e concentração sérica de progesterona, os animais foram submetidos à aplicação da hCG para induzir ovulação, no dia da ovulação e no 5o dia pós ovulação, além do grupo controle. O presente trabalho foi dividido em estudo preliminar com 4 animais em cada grupo e um estudo principal, com 12 animais por grupo sendo que todos os animais foram submetidos a todos os tratamentos. As características examinadas ao longo dos 15 dias pós ovulação foram tônus, morfoecogenicidade e vascularização do útero; tônus da cérvix; o diâmetro e área do corpo lúteo e do pedículo ovariano e seu RI; RI e vascularização mesometrial. Além disso, amostras de sangue para mensuração sérica de progesterona foram coletadas. Não foi encontrado efeito do tratamento nas características avaliadas com hCG em nenhum dos grupos. As características somente apresentaram variação ao longo do tempo, conforme descrito na literatura. Novos estudos avaliando o efeito da hCG em éguas devem ser realizados para averiguar outras variáveis e possíveis efeitos. / The use of drugs during diestrus in order to improve conception rates in inseminated and recipient mares has been the focus of several research groups. Studies using hCG found promising results regarding pregnancy rates and recipient uterine characteristics on the Day of the transfer. The objective of the study was to evaluate the effect of hCG administration in different moments of the oestrous cycle on reproductive tract characteristics, vascularization and serum progesterone concentration. Therefore, groups consisted of control, hCG to induce ovulation; hCG on day 0 and hCG on day 5 postovulation. The current study was performed as a preliminar study with 4 animals per group, one cycle each animal and a main study with 12 animals per group, each animal receiving all the treatments during consecutive cycles. The evaluations were performed daily from day 0 until day 15 postovulation. Characteristics examined were uterine tone, morphoecogenicity and vascularization; cervical tone; area, diamaterand vascularization of the corpus luteum, ovarian pedicle vascularization and RI; mesometrial vascularization and RI. Besides, blood samples were collected for serum progesterone concentration. No diferences were detected comparing treated and control groups. The characteristics only varied through the days, as described in the literature. Therefore, it can be concluded that hCG administered to induce ovulation, on the day of ovulation and on day 5 postovulation did not alter the characteristics evaluated in the current study.

A transferência de embriões (TE) é uma biotecnologia amplamente utilizada na produção de equinos no Brasil, e a égua receptora exerce um papel fundamental nos resultados finais nos programas de reprodução. Alguns tratamentos têm sido preconizados com objetivo de exercer ação luteotrófica, melhorando a função luteal, e com isto aumentando os níveis sistêmicos de progesterona, como também o uso de drogas anti-inflamatórias, que evitam a perda da função luteal causada pela liberação de prostaglandina em processos inflamatórios prévios ou por manipulação na passagem transcervical do embrião no momento da TE. O objetivo deste estudo foi investigar os efeitos do anti-inflamatório inibidor seletivo COX 2 (meloxicam) associado ao uso luteotrófico da hCG na gestação, características de corpo lúteo, fertilidade e desenvolvimento embrionário em éguas receptoras. Para isto, 60 éguas receptoras alojadas em central de reprodução foram aleatoriamente divididas em 4 grupos de 15 animais, de acordo com os seguintes tratamentos: aplicação de 5ml solução fisiológica no dia da ovulação (controle); aplicação de 2500 UI hCG no dia da ovulação; aplicação de 2500 UI hCG no dia da ovulação associado a 0,6 mg/kg de meloxicam; aplicação de 2500 UI hCG no momento da transferência do embrião associado a 0,6 mg/kg meloxicam. No dia da TE, 7 dias após a TE e 21 dias após a TE para as éguas que ficaram gestantes, foi avaliada a irrigação do corpo lúteo por ultrassonografia modo doppler colorido por meio de score do percentual de irrigação (0-100%), e a irrigação do útero avaliado por score (1-4). Por meio da ultrassonografia no modo B, a área do CL em mm2 foi avaliada nos mesmos momentos, a área da vesícula em mm2 aos 15 e 21 dias da gestação e o tamanho do embrião em cm aos 29 dias de gestação. Além disso, foi realizada dosagem plasmática de progesterona no dia da TE, 7 dias após a TE e 21 dias para as éguas gestantes. As comparações entre os grupos foram analisadas para efeitos principais de tratamento e tempo, assim como para efeito de interação tratamento x tempo. Não foi observado influencia da aplicação de hCG e/ou meloxicam nos animais tratados comparados ao controle (P>0,05). No entanto, foi observado um aumento numérico na taxa de gestação para os dois grupos de receptoras que receberam anti-inflamatório meloxicam, correspondendo a um índice superior em 13,3 % de gestação para éguas tratadas. Em conclusão, não há influencia da aplicação de hCG associado ou não meloxicam sobre a perfusão sanguínea uterina, do corpo lúteo, no desenvolvimento embrionário e nas concentrações plasmáticas de progesterona em receptoras de embriões. No entanto, existe, pela aplicação de meloxicam, evidência de melhora nos índices de gestação, podendo estar associado ao efeito anti-inflamatório seletivo COX-2, que pode melhorar resultados em programas comerciais de transferência de embriões em equinos. / Embryo transfer (ET) is a biotechnology widely used in equine production in Brazil, and the recipient mare plays a key role in the final results of breeding programs. Several treatments have been advocated both for the purpose of exerting luteotrophic action, improving luteal function, and thereby increasing systemic levels of progesterone, such as the use of anti-inflammatory drugs, which prevent the loss of luteal function caused by the release of prostaglandin in previous inflammatory processes or by manipulation in the transcervical passage of the embryo at the time of ET. The objective of this study was to investigate the effects of the anti-inflammatory COX 2 inhibitor (meloxicam) associated with luteotrophic use of hCG in gestation, corpus luteum characteristics, fertility and embryonic development in recipient mares. For this, 60 receiving mares housed in a reproduction center were randomly divided into 4 groups of 15 animals, according to the following treatments: application of 5ml physiological solution on the day of ovulation (control group); application of 2500 IU hCG on ovulation day; application of 2500 IU hCG on the day of ovulation associated with 0.6 mg/kg of meloxicam; application of 2500 IU hCG at the time of embryo transfer associated with 0.6 mg/kg meloxicam. On the day of ET, 7 days after ET and 21 days after ET for pregnant mares, corpus luteum irrigation was evaluated by color Doppler ultrasonography using a score of the percentage of irrigation (0-100%), and irrigation of the uterus evaluated by score (1-4). Using B-mode ultrasonography, corpus luteum area in mm2 was assessed at the same time points, the area of the embryonic vesicle in mm2 at 15 and 29 days of gestation, and the size of the embryo in cm at 29 days of gestation. In addition, plasma progesterone dosage was performed on ET day 7 days after ET in all groups and 21 days after ET for pregnant mares. The comparisons between the groups were analyzed for main effects of treatment and time, as well as for interaction effect treatment x time. No influence of application of hCG and / or meloxican was observed on treated animals compared to control (P>0.05). However, a numerical increase in gestation rate was observed for the two groups of recipients who received anti-inflammatory meloxicam, corresponding to an index higher than 13.3% of gestation for treated mares. In conclusion, there is no influence of the application of hCG associated or not to meloxicam on uterine irrigation, corpus luteum, embryo development and plasma concentrations of progesterone in embryo recipients. However, there is evidence of improvement in gestation rates due to the application of meloxicam, which may be associated with the COX-2 selective anti-inflammatory effect of this drug, improving results in commercial embryo transfer programs in horses.

Transrectal ovarian ultrasonography and hormone measurements were used to study ovarian antral follicular dynamics and development of luteal structures during the middle portion of the breeding season in non-prolific cross-bred Western white-faced ewes and prolific Finn sheep. Studies were also done on ovarian activity in Western white-faced ewes during the transition to seasonal anoestrus and at the onset of the breeding season. Lastly, two experiments were carried out to examine ovulatory responses and subsequent luteal function in Western white-faced ewes treated with luteolysin (PgF 2á) and progestogen (medroxyprogesterone acetate-MAP) during the luteal phase of the oestrous cycle and after ovulation induction with gonadotrophin-releasing hormone (GnRH) in mid-anoestrus. The results of the present experiments showed that the growth of ovine antral follicles reaching ovulatory sizes of >=5 mm in diameter occurred in a wave-like pattern throughout the oestrous cycle in both breeds of sheep under study. There were typically 3 or 4 waves of follicle production throughout the 17-day interovulatory period. Ovarian follicular emergence, or beginning of growth from the pool of 3-mm follicles, appeared to be primarily controlled by changes in circulating concentrations of follicle-stimulating hormone (FSH). In cyclic ewes, the largest ovarian follicles acquired the ability to secrete oestradiol from the day of emergence and a peak of oestradiol secretion occurred about the time they reached their maximum diameter. The high ovulation rate in prolific Finn sheep appeared to be achieved mainly by the ovulation of follicles emerging in the last two waves of the interovulatory interval. Interestingly, prolific Finn ewes produced more but smaller corpora lutea (CL) and had lower serum concentrations of progesterone during the luteal phase of the oestrous cycle as compared to non-prolific Western white-faced ewes. During the transition into seasonal anoestrus in Western white-faced ewes, FSH secretion resembled that during the breeding season but the pattern of emergence of sequential follicular waves was dissociated from FSH and oestradiol secretion. Prior to the first ovulation of the breeding season, there was a distinct elevation in circulating concentrations of progesterone produced by luteinized unovulated follicles and/or interstitial tissue of unknown origin. This increase in serum levels of progesterone, heralding the resumption of ovulatory cycles, did not alter the rhythmic pattern FSH secretion or follicular wave emergence. Treatment of non-prolific Western white-faced ewes with PgF2á and MAP applied late in the oestrous cycle changed follicular dynamics and increased ovulation rate to resemble that in prolific Finn sheep. Effects of MAP on the recruitment and growth of ovulatory follicles in Western white-faced ewes did not have a clear gonadotrophic dependancy, suggesting a possible local regulation of ovarian activity by progestins in ewes. Following the induction of ovulation with GnRH in anoestrous Western white-faced ewes, an array of ovarian responses were detected with ultrasonography, including failure of ovulation of large antral follicles, normal (fall-lifespan) and short-lived CL post-ovulation, and luteinized cystic-like follicles. The normal luteinization of ovulated follicles appeared to be related to the amplitude of episodic elevations in daily serum FSH concentrations before induction of ovulation and characteristics of the preovulatory LH surge.

An estimated 12% of couples worldwide are infertile. The contributing factor is approximately equal between men and women with nearly 25% diagnosed as idiopathic. Despite the increasing numbers of couples seeking assistance from infertility clinics, few molecular mechanisms have been identified for treatment. Autophagy is an evolutionarily conserved cellular process for bulk degradation and recycling of cytosolic components through the lysosome to maintain homeostasis. Several studies have observed increased levels of autophagy during ovarian folliculogenesis and gonadal steroidogenesis; however, no genetic studies to determine the significance of autophagy exist. To investigate the function of autophagy in the ovary and testis, a directed genetic knockout approach was used to independently knockout two key autophagy genes, Becn1 and Atg7. Chapter 2 reports that deficiency of Becn1 results in 56% fewer primordial follicles at postnatal day 1. In addition, Atg7 knockout mice do not have identifiable primordial follicles, suggesting that autophagy is necessary for survival of female germ cells during embryogenesis. Chapter 3 presents that Becn1 is necessary to sustain pregnancy and the deficiency of Becn1 in granulosa cells is a novel genetic model to study preterm labor due to impaired corpora lutea function. The results indicate that Becn1 is necessary for lipid droplet formation and subsequent progesterone production in luteal cells. In contrast, Atg7 is not necessary and deficiency results in overproduction of progesterone throughout pregnancy, suggesting that the defect in Becn1 conditional knockout mice is additional to autophagy. Chapter 4 presents that Sertoli cell expression of Becn1 is required for spermatogenesis after 8 weeks of age. Beyond 9-weeks-old, Becn1 conditional knockout mice are unable to sire a litter due to a failure of spermatogenesis and a Sertoli-cell-only phenotype in a majority of the seminiferous tubules. Atg7 was also identified as a necessary factor for spermatogenesis beyond 26-weeks-old. Together the data presented in Chapter 4 suggests that autophagy is necessary for adult Sertoli cell function. Primarily, this dissertation presents data from the first functional studies on autophagy in the reproductive tract. The results demonstrate an understanding of the functional significance for Becn1 and Atg7 in both the ovary and testis.

[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Apoptosis is a morphologically distinct form of cell death that is involved in the regulation of normal and aberrant cell systems. The complexities of the apoptotic cell death pathway arise from variation in both the cellular specialisation and initial stimulus. The corpus luteum (CL) is an endocrine gland that whilst critical to the maintenance of pregnancy in the rat, regresses at the completion of each oestrous cycle and pregnancy. This regression is facilitated through apoptosis; though, the stimulus and factors involved in the apoptotic pathway are poorly understood. Previous studies suggest that CL regression is not initiated through failure of luteotrophic support, but rather the active production of a luteolytic factor, of which tumour necrosis factor -alpha (TNFα) is one possible candidate. Several publications have reported the participation of the immune system in ovarian events. There is evidence that TNFα expression within the ovary is coordinated between cells of the immune system and the hormonal regulation of the CL. This study has focussed on the role of TNFα in CL apoptosis and the factors involved in this apoptotic pathway. TNFα-induced cell death is governed by the presence of the two TNFα receptors (TNFR) and several second messenger systems that include; the sphingolipids, mitogen-activated protein (MAP) kinases, nitric oxide (NO), nuclear factor-kappaB (NF-κB) and the caspases. These factors and their interactions were assessed in the rat CL during pregnancy and post-partum, and in vitro. Apoptosis was measured through the analysis of DNA fragmentation using DNA 3’ end labelling and single cell electrophoresis (COMET assay). Assessment of mRNA and protein expression was through Real-time RT-PCR and Western blot analysis; proteins were localised within the CL by immunocytochemistry. In addition, specific measurement of sphingolipid expression and nitric oxide (NO) production was by high performance liquid chromatography (HPLC) and NO assay respectively. Following parturition, TNFα mRNA and protein expression increased corresponding to the onset of CL apoptosis and increased expression of the chemotactic factor monocyte chemoattractant protein -1 (MCP-1). Furthermore, CL apoptosis was induced by treatment with recombinant TNFα in a time- and dose-dependent manner. A similar effect was observed in isolated luteal cells. Simultaneously, the functional regression of the CL was assessed by measurement of both progesterone synthesis and steroidogenic acute regulatory (StAR) protein expression. StAR mRNA and protein expression declined toward parturition in vivo. Immunocytochemical studies revealed the presence of TNFα receptors 1 (TNFR1) and 2 (TNFR2) in luteal cells. Furthermore, TNFR mRNA was isolated from CL throughout pregnancy and post-partum. Subsequently, the role of the sphingolipids ceramide and sphingosine was examined during CL apoptosis in vitro. Ceramide and sphingosine were found to be potent apoptotic agents when administered in vitro (50µM). The downstream signal transduction of TNFα and ceramide was assessed through MAP kinase expression. Both TNFα and ceramide increased expression of the pro-apoptotic p38 MAP kinase with no change to the non-apoptotic extracellular signal-related kinase (ERK1&2). Despite previous reports of c-Jun NH2 terminal kinase (JNK) involvement in the cell death pathway, JNK expression was not evident in the rat CL. The caspases are a family of cysteine proteases central to the regulation and execution of apoptosis. General inhibition of the caspase cascade in vitro was effective in preventing apoptosis regardless of the apoptotic stimulus (TNFα, ceramide and sphingosine), suggesting that this pathway is central to CL apoptosis. Specific inhibition of several caspases produced a varying effect; inhibition of caspases 3, 6 and 8 significantly reduced the level of TNFα-induced apoptosis, thus supporting their classification as either regulatory or effector caspases. NO is endowed with the unique ability to initiate and to block apoptosis and this dichotomy extends to the cytotoxic actions of TNFα. Inhibition of NO production by treating CL with L-NAME prevented the onset of apoptosis, whilst NO production increased in response to increasing levels of apoptosis following trophic withdrawal. However, this effect was not seen during TNFα-induced apoptosis, suggesting that the actions of NO are independent of TNFα. The data presented within this study examine multiple elements of the TNFα cell death pathway in a single system. The results suggest that these elements are involved in TNFα signal transduction and furthermore, in rat CL apoptosis. It can be said that TNFα plays an active role in CL regression through the activation of the caspases, the sphingolipids and the MAP kinases.

Orientador: Eunice Oba / Resumo: Estratégias que busquem resultados reprodutivos superiores em búfalos devem ser pesquisadas. Para tanto, a gonadotrofina coriônica equina (eCG) vem sendo estudada apresentando resultados satisfatórios em búfalas, porém os mecanismos envolvidos na ação da eCG não foram totalmente esclarecidos. A termografia digital por infravermelho apresenta importância no estudo das variações de temperaturas da vulva durante o ciclo estral, podendo auxiliar na detecção do estro nesta espécie. O objetivo do presente estudo foi avaliar as variações morfológicas e de vascularização dos folículos e corpo lúteo (CL) e nas concentrações plasmáticas de progesterona (P4) e cortisol durante protocolo hormonal para a sincronização da ovulação com e sem eCG. Também objetivou-se avaliar a influência da fase reprodutiva e do clima sobre a temperatura da superfície da vulva em búfalas Murrah. Os animais foram aleatoriamente divididos em dois grupos, grupo com eCG (eCG, n=20) e grupo sem eCG (controle, n=20). No primeiro dia do protocolo hormonal (Dia 0), as vacas receberam um dispositivo intravaginal de P4 e administração de benzoato de estradiol. No dia 9, removeu-se o dispositivo de P4, administrou-se PGF2α em todas as búfalas e eCG nos animais do grupo eCG. No dia 11, foi administrado GnRH. A partir do dia 9, foram realizadas ultrassonografias doppler no período da tarde e termografia digital pela manhã e à tarde, diariamente até o dia 16 e depois nos dias 20, 24, 28 e 32, visando avaliar o desenvolvim... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Strategies that seek better reproductive results in buffaloes should be researched. Thus, equine chorionic gonadotrophin (eCG) was studied presenting satisfactory results when used in buffalo during the breeding season and anestrous period, but the mechanisms involved in the reproductive improvement observed with the eCG implementation were not fully understood. The superficial digital thermography may be important in the study of temperature variations of the vulva inherent to the estrous cycle, and may help in the detection of estrus in this species. The objective of the present study was to evaluate the morphological and irrigation alterations in the follicles and corpus luteum (CL) and the changes in plasma concentrations of progesterone (P4) during hormonal protocol for ovulation synchronization with and without eCG. As well the aim of this study was to evaluate the influence of the reproductive phase and the climate on the superficial vulvar temperature in Murrah buffaloes. The animals were randomly divided into two groups, group with eCG (eCG, n = 20) and group without eCG (control, n = 20). On the first day of the hormonal protocol (Day 0), cows received an intravaginal P4 device and estradiol benzoate administration. On day 9, the P4 device was removed, PGF2α was administered in all buffaloes and eCG in eCG group. On day 11, GnRH was administred. From day 9, Doppler ultrasonography was daily performed in the afternoon and digital thermography in the morning and after... (Complete abstract click electronic access below) / Doutor