O presente trabalho teve como objetivo avaliar os efeitos de diferentes condições nutricionais (suplementação e não suplementação) na atividade cíclica reprodutiva (ACR) em ovelhas das raças Santa Inês (SI), Romney Marsh (RM) e Suffolk (SU) mantidas sob pastejo, e submetidas ao efeito macho, durante época de anestro sazonal sob fotoperíodo natural no Estado de São Paulo. Avaliou-se também os efeitos destes tratamentos nas concentrações plasmáticas de melatonina (MEL) e na duração (horas) da secreção noturna deste hormônio. Todos animais foram mantidos sob pastejo, sendo que metade de cada raça recebeu suplementação e a outra não, caracterizando os tratamentos de suplementação e não suplementação, respectivamente. O tratamento de suplementação forneceu de 100 a 110% dos requerimentos protéicos (PB) e de 130 a 140% dos requerimentos energéticos (NDT), enquanto que o tratamento de não suplementação forneceu de 60 a 70% de PB e 100% de NDT. Os tratamentos nutricionais iniciaram 21 dias antes da introdução dos machos, e continuaram por todo o período de permanência dos mesmos (45 dias). Para o efeito macho, foram utilizados três machos (rufiões), impregnados com tinta na região prepucial para detecção do estro, os quais permaneceram isolados das fêmeas por um período de 60 dias. Colheitas de sangue foram realizadas antes (a cada 3 dias) e após (a cada 2 dias) a introdução dos machos, para dosagem de progesterona plasmática, a fim de monitorar a ACR das fêmeas. Foram também realizadas colheitas de sangue para dosagem de MEL plasmática: antes do início do experimento, antes da introdução dos machos e no final do experimento, em período de 24 horas, a cada 2 (noite) ou 4 (dia) horas. Os parâmetros avaliados foram: ganho de peso, ACR após introdução dos machos nos animais que se encontravam em anestro, dias para retorno da ACR, concentrações plasmáticas de MEL, duração da secreção de MEL. Estes parâmetros foram analisados utilizando o procedimento GLM no SAS, seguido pelo teste de ukey para estabelecer a comparação entre médias. O tratamento nutricional teve efeito no ganho de peso (P<0,001), no número de dias para retorno da ACR (P<0,1), nas concentrações de MEL (P<0,1) e na duração da secreção deste hormônio (P<0,1). Animais suplementados apresentaram em média, maior ganho de peso, maiores concentrações de MEL plasmática, e maior duração da secreção deste hormônio. Diferenças raciais foram encontradas para ganho de peso, retorno da ACR e concentrações plasmáticas de MEL. Fêmeas SI, perderam peso, enquanto que as RM mantiveram e as SU ganharam peso durante todo o período experimental, independente do tratamento nutricional. Todas as fêmeas SI retornaram à ACR em média 5 dias após a introdução dos machos, já as fêmeas SU suplementadas não retornaram à ACR, enquanto que as não suplementadas retornaram em 23 dias. Nenhuma ovelha RM retornou à ACR. Assim, conclui-se que para a raça SI só efeito macho é eficiente em ativar o retorno da ACR, e para a raça SU, deve-se aliar esta prática com um bom manejo nutricional, e para a RM, o efeito macho mesmo associado à nutrição não são capazes de amenizar a estacionalidade da ACR. E por fim, a nutrição afeta a duração da secreção de MEL. / This study evaluated the effects of different nutritional conditions (suplementation and no suplementation) in reproductive cyclic activity (RCA) for Santa Inês (SI), Romney Marsh (RM) and Suffolk (SU) breeds, in pasture, submitted to the male effect, during seasonal anoestrus, under natural photoperiod in São Paulo State. It was also evaluated the effects of nutritional treatments for plasmatic melatonin (MEL) concentration and for the duration of MEL secretion. All ewes were mainted in pasture, receiving food supplementation or not, distinguishing the supplementation or not supplementation treatments. The supplementation treatment supplied 100-110% of protein requeriments (CP) and 130-140% of energy requeriments (TDN). The no supplementation treatment supplied 60-70% CP and 100% TDN requeriments. The nutritional treatments were started 21 days before the rams introduction and lasted throughout the permanence period (45 days). To the male effect, it used three vasectomized rams impregnated with ink at the prepucial area in order to facilitate the estrus verification, isolated from the females for 60 days. Blood samples were collected before (every 3 days) and after (every 2 days) the rams introduction to determine de plasmatic progesterone to follow up the ewes reproductive cyclic activity (RCA). Blood samples were also collected to determine the plasmatic MEL dosage: before the beginning of the experiment, before the rams introduction and in the end of the experiment, during 24 hours, every 2 dark hours or 4 light hours. There was evaluated the following parameters: weight gain, RCA after male introduction for the ewes in anoestrus, days to RCA return, MEL concentration and MEL secretion duration. The parameters were analyzed by analisis of variance using the GLM (General Linear Procedure) of the Statistical System (SAS) software. Tukey test was used to compare means. The nutritional treatment resulted efficient for weight gain (P<0,001), days to return RCA (P<0,1), MEL concentration (P<0,1) and the duration of its hormone secretion (P<0,1). Supplemented ewes showed higher weight gain, higher MEL plasmatic concentration and higher hormone secretion duration in average. There was verified breed differences for weight gain, RCA return and MEL plasmatic concentration. SI ewes lost weight in average, RM ewes maintained it and SU ewes gained weight during the whole experimental period not taking into account the nutritional treatment. SI ewes had, in average, their RCA returned 5 days after the rams introduction not taking into account the nutritional treatment. However, SU ewes presented different behaviours due treatments: the ones that received supplementation did not return the RCA while, the non supplemented ones returned the RCA within 23 days. RM ewes did not return RCA no matter which treatment had received. Therefore, one may conclude that for SI ewes the male effect is enough for the RCA return. For SU breed one should combine this practice to good nutritional management. And for RM neither the male effect nor the nutrition are capable to diminish the RCA seasonally. Finally, one may conclude that the nutrition affects the amount and duration of MEL secretion.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

O presente trabalho teve como objetivo avaliar os efeitos de diferentes condições nutricionais (suplementação e não suplementação) na atividade cíclica reprodutiva (ACR) em ovelhas das raças Santa Inês (SI), Romney Marsh (RM) e Suffolk (SU) mantidas sob pastejo, e submetidas ao efeito macho, durante época de anestro sazonal sob fotoperíodo natural no Estado de São Paulo. Avaliou-se também os efeitos destes tratamentos nas concentrações plasmáticas de melatonina (MEL) e na duração (horas) da secreção noturna deste hormônio. Todos animais foram mantidos sob pastejo, sendo que metade de cada raça recebeu suplementação e a outra não, caracterizando os tratamentos de suplementação e não suplementação, respectivamente. O tratamento de suplementação forneceu de 100 a 110% dos requerimentos protéicos (PB) e de 130 a 140% dos requerimentos energéticos (NDT), enquanto que o tratamento de não suplementação forneceu de 60 a 70% de PB e 100% de NDT. Os tratamentos nutricionais iniciaram 21 dias antes da introdução dos machos, e continuaram por todo o período de permanência dos mesmos (45 dias). Para o efeito macho, foram utilizados três machos (rufiões), impregnados com tinta na região prepucial para detecção do estro, os quais permaneceram isolados das fêmeas por um período de 60 dias. Colheitas de sangue foram realizadas antes (a cada 3 dias) e após (a cada 2 dias) a introdução dos machos, para dosagem de progesterona plasmática, a fim de monitorar a ACR das fêmeas. Foram também realizadas colheitas de sangue para dosagem de MEL plasmática: antes do início do experimento, antes da introdução dos machos e no final do experimento, em período de 24 horas, a cada 2 (noite) ou 4 (dia) horas. Os parâmetros avaliados foram: ganho de peso, ACR após introdução dos machos nos animais que se encontravam em anestro, dias para retorno da ACR, concentrações plasmáticas de MEL, duração da secreção de MEL. Estes parâmetros foram analisados utilizando o procedimento GLM no SAS, seguido pelo teste de ukey para estabelecer a comparação entre médias. O tratamento nutricional teve efeito no ganho de peso (P<0,001), no número de dias para retorno da ACR (P<0,1), nas concentrações de MEL (P<0,1) e na duração da secreção deste hormônio (P<0,1). Animais suplementados apresentaram em média, maior ganho de peso, maiores concentrações de MEL plasmática, e maior duração da secreção deste hormônio. Diferenças raciais foram encontradas para ganho de peso, retorno da ACR e concentrações plasmáticas de MEL. Fêmeas SI, perderam peso, enquanto que as RM mantiveram e as SU ganharam peso durante todo o período experimental, independente do tratamento nutricional. Todas as fêmeas SI retornaram à ACR em média 5 dias após a introdução dos machos, já as fêmeas SU suplementadas não retornaram à ACR, enquanto que as não suplementadas retornaram em 23 dias. Nenhuma ovelha RM retornou à ACR. Assim, conclui-se que para a raça SI só efeito macho é eficiente em ativar o retorno da ACR, e para a raça SU, deve-se aliar esta prática com um bom manejo nutricional, e para a RM, o efeito macho mesmo associado à nutrição não são capazes de amenizar a estacionalidade da ACR. E por fim, a nutrição afeta a duração da secreção de MEL. / This study evaluated the effects of different nutritional conditions (suplementation and no suplementation) in reproductive cyclic activity (RCA) for Santa Inês (SI), Romney Marsh (RM) and Suffolk (SU) breeds, in pasture, submitted to the male effect, during seasonal anoestrus, under natural photoperiod in São Paulo State. It was also evaluated the effects of nutritional treatments for plasmatic melatonin (MEL) concentration and for the duration of MEL secretion. All ewes were mainted in pasture, receiving food supplementation or not, distinguishing the supplementation or not supplementation treatments. The supplementation treatment supplied 100-110% of protein requeriments (CP) and 130-140% of energy requeriments (TDN). The no supplementation treatment supplied 60-70% CP and 100% TDN requeriments. The nutritional treatments were started 21 days before the rams introduction and lasted throughout the permanence period (45 days). To the male effect, it used three vasectomized rams impregnated with ink at the prepucial area in order to facilitate the estrus verification, isolated from the females for 60 days. Blood samples were collected before (every 3 days) and after (every 2 days) the rams introduction to determine de plasmatic progesterone to follow up the ewes reproductive cyclic activity (RCA). Blood samples were also collected to determine the plasmatic MEL dosage: before the beginning of the experiment, before the rams introduction and in the end of the experiment, during 24 hours, every 2 dark hours or 4 light hours. There was evaluated the following parameters: weight gain, RCA after male introduction for the ewes in anoestrus, days to RCA return, MEL concentration and MEL secretion duration. The parameters were analyzed by analisis of variance using the GLM (General Linear Procedure) of the Statistical System (SAS) software. Tukey test was used to compare means. The nutritional treatment resulted efficient for weight gain (P<0,001), days to return RCA (P<0,1), MEL concentration (P<0,1) and the duration of its hormone secretion (P<0,1). Supplemented ewes showed higher weight gain, higher MEL plasmatic concentration and higher hormone secretion duration in average. There was verified breed differences for weight gain, RCA return and MEL plasmatic concentration. SI ewes lost weight in average, RM ewes maintained it and SU ewes gained weight during the whole experimental period not taking into account the nutritional treatment. SI ewes had, in average, their RCA returned 5 days after the rams introduction not taking into account the nutritional treatment. However, SU ewes presented different behaviours due treatments: the ones that received supplementation did not return the RCA while, the non supplemented ones returned the RCA within 23 days. RM ewes did not return RCA no matter which treatment had received. Therefore, one may conclude that for SI ewes the male effect is enough for the RCA return. For SU breed one should combine this practice to good nutritional management. And for RM neither the male effect nor the nutrition are capable to diminish the RCA seasonally. Finally, one may conclude that the nutrition affects the amount and duration of MEL secretion.

La saisonnalité de la reproduction consiste en l’alternance entre des périodes d’activité et de repos sexuel. Ce phénomène s’observe chez les caprins de la région subtropicale du Mexique et rend impossible la reproduction à contre saison. La réintroduction d’un mâle dans un groupe de femelles peut rompre l’anoestrus saisonnier et rapidement stimuler leur activité ovarienne. Cependant, l’utilisation de boucs induits à un comportement sexuel intense pendant la période de repos sexuel au moyen d’un traitement lumineux –mâles photo-stimulés- est nécessaire afin d’assurer la qualité de la stimulation fournie par ceux-ci. Le temps de contact entre les sexes est un autre facteur pouvant moduler la réponse des femelles et il a longtemps été suggéré que ce contact devait être maintenu de manière continue (24h/24) pendant plusieurs jours afin que la majorité des femelles ovulent. L’objectif de cette thèse a été de déterminer si une réduction du temps de contact journalier entre les sexes diminue les réponses ovulatoire et reproductrice de chèvres en anoestrus saisonnier exposées aux mâles. / Seasonality of reproduction consists of alternations between periods of sexual activity and sexual rest (anoestrus). This seasonality is observed in goats from subtropical Mexico and makes impossible out-of-season reproduction. The reintroduction of a male in a group of females can break the seasonal anoestrus and quickly stimulate ovarian activity. However, the use of bucks induced to a high sexual activity during the sexual rest by a treatment of long days –photo-stimulated males- is necessary to ensure the quality of stimulation provided by them. The duration of contact between sexes is another factor that modulates the response of females and it has long been suggested that it should be maintained continuously (24h/24) for several days so that the majority of females ovulate. The purpose of this thesis was to determine whether a reduction of daily contact between sexes decrease ovulatory and reproductive responses of goats exposed to males during the seasonal anoestrus. The results indicate that duration of contact between sexes can be drastically reduced from 16 to 1 h per day without reducing the ovulatory and reproductive responses of females.

A series of eight experiments was conducted to investigate the requirement for thyroid hormones in neuroendocrine processes which lead to the seasonally anoestrous state in red deer hinds. The first two experiments used thyroidectomized, ovariectomized, oestradiol-treated hinds which received various thyroid hormone replacement treatments (n=5 per group) to investigate the timing and dose-responsiveness of thyroid hormones in bringing about seasonal oestradiol-induced suppression of plasma LH concentration. A significant seasonal decline in mean plasma LH concentration during September (coinciding with the onset of anoestrus in entire cycling hinds in New Zealand) was observed in all thyroidectomized hinds in both experiments regardless of T₄ or T₃ treatment. When oestradiol implants were removed in November or December, mean plasma LH concentrations increased significantly in all but one of hinds in which T₄ had been administered at very low doses by subcutaneous implants, and mean plasma LH concentrations and LH pulse amplitude increased in approximately half of hinds administered T₃ at varying doses by subcutaneous injections over a one-week period in October. These results suggested that thyroid hormones are not required for steroid-dependent reproductive suppression, but could possibly play a role in steroid-independent suppression of LH secretion. Because problems were encountered in delivering appropriate doses of thyroid hormones in both experiments, further confirmation of these findings was required. Therefore in the next experiment the role of thyroid gland secretions was examined in euthyroid (n=5) and thyroidectomized (n=4) ovariectomized hinds treated with oestradiol implants. These implants were removed for about one month on three occasions to examine the effect of thyroidectomy on steroid-independent control of seasonal LH secretion. During the non-breeding season basal and GnRH-induced plasma LH concentrations declined in all hinds in the presence of oestradiol, but returned to breeding season levels when oestradiol was withdrawn in November. In a concurrent experiment, thyroidectomy of ovary-entire hinds (n=7) during the breeding season prevented the cessation of oestrous cyclicity in spring; this was in contrast to oestrous cyclicity in T₄replaced (n=4) or euthyroid control (n=5) hinds which ceased to occur in early September. Collectively, these results indicate that thyroid hormones are required for the termination of the breeding season in cycling red deer hinds and that this action occurs via steroid-independent neuroendocrine pathways. Two experiments were conducted using neurotransmitter receptor agonists and antagonists to identify neural pathways in the brain which mediate LH suppression by oestradiol and by steroid-independent mechanisms, and to test if the thyroid gland is required for activation of these pathways during the non-breeding season. It was concluded from the lack of plasma LH responses to dopaminergic and opioidergic agonists and antagonists in ovariectomized and ovariectomized, thyroidectomized hinds (n=5) that neural pathways involving dopamine-D₂receptors do not mediate oestradiol-induced seasonal suppression of plasma LH concentrations, and neither dopaminergic or opioid neural pathways mediate non-steroidal suppression of plasma LH concentrations. However preliminary evidence was obtained for a stimulatory role of serotonergic neural pathways in controlling LH secretion. Another experiment was conducted to identify when the steroid-independent mechanisms which suppress LH concentrations during the non-breeding season are responsive to thyroid hormones. T₄treatment at the beginning of or during the non breeding season was effective in bringing about suppression of plasma LH concentration in thyroidectomized, ovariectomized hinds (n=5 per group), but this action of thyroid hormones did not occur during the breeding season. These results show that the steroid-independent mechanisms which contribute to seasonal suppression of plasma gonadotrophin concentrations require thyroid hormones to be present only from around the time of the end of the breeding season for their normal expression, and they remain responsive to thyroid hormones after this period. Lastly, the feasibility of achieving out-of-season breeding using thyroidectomized hinds (n=9) was evaluated by comparing oestrous behaviour, ovulation and pregnancy rates to those of euthyroid control hinds (n=7) following synchronization of oestrous cycles. There was a non-significant trend for a greater occurrence of oestrous behaviour and ovulation in thyroidectomized hinds compared with euthyroid controls during the non-breeding season, but the pregnancy rate following out-of season mating with a thyroidectomized stag was low, suggesting that a side effect of thyroidectomy may be impaired fertility. Six out-of-season pregnancies were obtained from eight matings, however because three of these pregnancies occurred in euthyroid control hinds no improvement in out-of-season reproductive performance could be attributed to thyroidectomy. It is likely that if the actions of the thyroid glands are to be exploited as a tool for achieving out-of-season breeding in this species, techniques will have to be developed for specifically blocking or overcoming the effects of thyroid hormones on the reproductive neuroendocrine centres without causing general hypothyroidism and its associated side-effects.

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.