Much has been improved in the basic and applied aspect of female reproduction after understanding of the wave-like developmental pattern of follicles ¡Ý4 mm. However, it is speculated that the understanding of the developmental pattern of small follicles <4 mm may bring about efficient management of ovarian functions for essential reproductive interventions. Present studies were therefore, carried out to characterize the developmental pattern of 1-3 mm follicles in cattle using ultrasonography and to validate a method to histologically characterize the developmental pattern of follicles <1 mm using non-serial data. Transrectal ultrasonography was used once daily (n=18 Hereford-cross heifers) to examine changes in the diameter of follicles ¡Ý1 mm for one interovulatory interval (IOI), as well as every 6 h (n = 9 Hereford-cross cows) from 5 to 13 days after ovulation to encompass emergence of Wave 2. A periodic shift in the peak number (P < 0.05) of 1-3 mm and ¡Ý4 mm follicles and a significant inverse relationship (P < 0.05) between them suggested a wave-like developmental pattern. The number of 1-3 mm follicles detected in anovulatory waves did not differ (P = 0.53) between 2- versus 3-wave IOIs. A difference (P < 0.05) was noticed between anovulatory and ovulatory waves in 3-wave IOIs but not (P = 0.63) in 2-wave IOIs. The future dominant follicle was identified at 1 mm and was found to emerge 6-12 h earlier than the largest subordinate follicle (P < 0.01). Emergence of the future dominant (r = 0.71) and 1st subordinate (r = 0.78) follicles was temporally associated (P < 0.05) with a rise in circulating concentrations of FSH. <p> The developmental pattern of follicles not detectable by ultrasonography (<1 mm) may be assessed histologically by examining the ovaries from different animals on different days (i.e., non-serial method) using follicle diameter or number profiles. A data set (n = 56 heifers) of follicles ¡Ý4 mm was tabulated in a serial (same set of heifers each day; n = 7/day, N = 7) and non-serial (different set of heifers each day; n = 7/day, N = 56) manner for number and diameter profiles around emergence of the first follicular wave. Profiling of serial and non-serial data (serial and non-serial methods) revealed a change in the number of follicles (4-5 mm, 6-8 mm, ¡Ý9 mm) over days (P < 0.01), but the effect of method and the day-by-method interaction were not different (P > 0.28). Similarly, the diameter of the dominant and first 2 subordinate follicles changed over days (P < 0.01), but the effect of method and the day-by-method interaction were not different (P > 0.06), indicating that non-serial data provide wave-like profile of follicles. <p> In conclusion, the hypothesis that follicles 1-3 mm develop in a wave-like manner was supported, and a new non-serial method was validated for the study of follicle dynamics using non-serial (e.g. histologic) data.

Much has been improved in the basic and applied aspect of female reproduction after understanding of the wave-like developmental pattern of follicles ¡Ý4 mm. However, it is speculated that the understanding of the developmental pattern of small follicles <4 mm may bring about efficient management of ovarian functions for essential reproductive interventions. Present studies were therefore, carried out to characterize the developmental pattern of 1-3 mm follicles in cattle using ultrasonography and to validate a method to histologically characterize the developmental pattern of follicles <1 mm using non-serial data. Transrectal ultrasonography was used once daily (n=18 Hereford-cross heifers) to examine changes in the diameter of follicles ¡Ý1 mm for one interovulatory interval (IOI), as well as every 6 h (n = 9 Hereford-cross cows) from 5 to 13 days after ovulation to encompass emergence of Wave 2. A periodic shift in the peak number (P < 0.05) of 1-3 mm and ¡Ý4 mm follicles and a significant inverse relationship (P < 0.05) between them suggested a wave-like developmental pattern. The number of 1-3 mm follicles detected in anovulatory waves did not differ (P = 0.53) between 2- versus 3-wave IOIs. A difference (P < 0.05) was noticed between anovulatory and ovulatory waves in 3-wave IOIs but not (P = 0.63) in 2-wave IOIs. The future dominant follicle was identified at 1 mm and was found to emerge 6-12 h earlier than the largest subordinate follicle (P < 0.01). Emergence of the future dominant (r = 0.71) and 1st subordinate (r = 0.78) follicles was temporally associated (P < 0.05) with a rise in circulating concentrations of FSH. <p> The developmental pattern of follicles not detectable by ultrasonography (<1 mm) may be assessed histologically by examining the ovaries from different animals on different days (i.e., non-serial method) using follicle diameter or number profiles. A data set (n = 56 heifers) of follicles ¡Ý4 mm was tabulated in a serial (same set of heifers each day; n = 7/day, N = 7) and non-serial (different set of heifers each day; n = 7/day, N = 56) manner for number and diameter profiles around emergence of the first follicular wave. Profiling of serial and non-serial data (serial and non-serial methods) revealed a change in the number of follicles (4-5 mm, 6-8 mm, ¡Ý9 mm) over days (P < 0.01), but the effect of method and the day-by-method interaction were not different (P > 0.28). Similarly, the diameter of the dominant and first 2 subordinate follicles changed over days (P < 0.01), but the effect of method and the day-by-method interaction were not different (P > 0.06), indicating that non-serial data provide wave-like profile of follicles. <p> In conclusion, the hypothesis that follicles 1-3 mm develop in a wave-like manner was supported, and a new non-serial method was validated for the study of follicle dynamics using non-serial (e.g. histologic) data.

A prolonged growth phase of the ovulatory follicle results in follicular aging. Whether follicular aging is detrimental or beneficial to oocyte competence is not fully known. The objective of this thesis is to investigate the effects of follicular aging on oocyte competence and granulosa cell gene expression in cattle. Four sets of experiments were designed to address the objective. The following hypotheses were tested during the course of these studies: 1) oocyte competence will improve by the longer growing phase but will be adversely affected by FSH starvation, 2) follicles that undergo superstimulation will have different gene expression than dominant follicles from a natural cycle, 3) extending the superstimulation protocol by 3 days will allow follicles to mature better and 4) markers of maturity, cellular health and survival will be turned off by FSH starvation. The objective of the first study (Chapter 3) was to determine the effects of extending the length of superstimulation and follicular aging on oocyte competence by in vitro embryo production. Multiple follicles were allowed to grow for 4 (Short FSH) or 7 days (Long FSH) under the treatment of 8 or 14 injections of FSH (at 12-hour intervals), respectively. Multiple follicles in the FSH starvation group were allowed to grow for 7 days but FSH was provided for only the first 4 days of superstimulation. Extending the duration of follicular growth by superstimulation resulted in a greater number of ≥9 mm follicles and in 2.5 more transferable embryos per animal (morulae+blastocysts) at Day 9 of in vitro embryo culture. The FSH starvation resulted in a greater proportion of poor quality oocytes lower cleavage rate and lower embryonic development. Microarray analysis was used to assess the effect of superstimulation (Chapter 4), follicular aging (Chapter 5) and FSH starvation (Chapter 6) on the gene expression profile of superstimulated granulosa cells. Gene expression of granulosa cells from the post-LH preovulatory dominant follicle was compared (Chapter 4) with those from follicles of the same status after a standard 4-day superstimulation (same protocol as Short FSH group from Chapter 3). A total of 190 genes were down-regulated and 280 genes were upregulated in the superstimulated group when compared with the reference (non-superstimulated control). Data analysis showed that superstimulated follicles are still in a growing phase compared to untreated dominant follicles (most of the upregulated genes are related to matrix remodeling due to tissue proliferation) and did not respond to LH properly (down regulation of LH gene markers). Four-day superstimulation also disturbed genes related to angiogenesis and activated oxidative stress response genes. Extending the superstimulation protocol (7 days; same protocol as Long FSH from Chapter 3) allowed more time for follicles to leave the growing stage and properly respond to LH surge (most of the upregulated genes in the Long FSH group are markers of post LH surge) when compared to the standard 4 day superstimulation protocol (Short FSH; reference group) (Chapter 5). Moreover, the follicles from Long FSH show proximity to ovulation. The continuous FSH support during the extended superstimulation protocol is crucial for follicular health since FSH starvation disturbed genes markers of oocyte quality and embryo development (Chapter 6). Granulosa cells that underwent FSH starvation do not respond to LH surge, which could be detrimental to ovulation (Chapter 6). Therefore, follicles from Short FSH are delayed in maturation and differentiation but the oocyte competence is not compromised. Extending superstimulation protocol by 3 d enhanced the ovarian response to FSH treatment and allowed more time for follicles to mature and properly respond to the LH stimulus. A period of FSH starvation after superstimulatory treatment compromised follicular health, ability to respond to LH and ovulate, oocyte quality and the fertilization process.

Made available in DSpace on 2016-12-08T16:24:17Z (GMT). No. of bitstreams: 1 PGCA13MA135.pdf: 267002 bytes, checksum: 8293274ee87b3870ff68763d1a4e7cbe (MD5) Previous issue date: 2013-11-01 / Due to the drastic reduction of the number of animals, the Flemish Red breed is endangered. This study aims to generate knowledge on the follicular development and serum progesterone (P4) profiles in females of the Flemish Red breed, created in Southern Brazil, in order to subsidize the choice of reproductive biotechniques more adequate for the reproduction of the breed. Two groups of non-lactating pubescent females with ages between four and six years old, Flemish Red (FLE, n=7) and Holstein (HOL, n=7), received two doses of Sodium Cloprostenol in order to synchronize their estrus. Starting from the estrus, the ovaries were assessed through the use of transrectal ultrasound every 24 hours for 21 days or until the second ovulation was detected. The diameter of the dominant follicle (DF) and the subordinate follicle (SF) were recorded according to the day of the cycle. The diameter of the DF and the SF were assessed when the follicle emerged, the day and the diameter of the DF in the divergence, the daily growth of the ovulatory follicle and the non-ovulatory dominant follicle (NODF). Blood samples were collected every five days for P4 administration by radioimmunoassay. None of the variables assessed in the FLE group differed significantly of the HOL group or of the values described for other European cattle breeds. In wave emergence DF measured 3.97 ± 0.19 mm for FLE and 4.00 ± 0.35 mm for the HOL, while the SF reached 3.40 ± 0.22 mm in FLE and 3 , 07 ± 0.26 mm in the HOL. The FLE daily growth rate was 1.10 ± 0.04 mm for DF and 0.67 ± 0.06 mm for the SF , whereas the HOL daily growth rate was 1.01 ± 0.05 mm for DF and 0.72 ± 0.09 mm for the SF . Since the emergence DF was larger than the SF and divergence occurred on the third day , with the DF measuring 8.04 ± 0.37 mm and 8.39 ± 0.47 in FLE and HOL, respectively. The maximum diameter of the ovulatory follicle in FLE cows was 13.16 ± 0.33 mm and the average maximum diameter of the non- ovulatory dominant follicle was 13.01 ± 0.48 mm . Holstein cows showed values of 14.20 ± 0.60 and 13.00 mm ± 0.59 mm , respectively, for the same measurements . These values are in accordance with generally accepted for European breeds, situated between 13-20 mm . The P4 concentration was adjusted to a cubic regression , and in FLE group ranged from 0.155 ± 0.016 ng/ml to 6.651 ± 1.868 ng/ml. In HOL group, P4 ranged from 0.300 ± 0.048 ng/ml to 5.957 ± 1.233 ng/ml.The conclusion was that cows of the Flemish Red breed have similar follicle development and P4 profile to the Holstein cows, which suggests it is possible to use reproductive biotechnologies applicable to animals of the Holstein breed in animals of the Flemish Red breed / Em função da drástica redução no número de animais, a raça Flamenga corre risco de extinção. Este estudo teve objetivo de gerar conhecimento sobre o perfil de desenvolvimento folicular e de Progesterona sérica (P4) hormonal em fêmeas da raça Flamenga criadas no Sul do Brasil, visando subsidiar a escolha das biotécnicas reprodutivas mais apropriadas para multiplicação desta raça. Dois grupos de fêmeas púberes, não lactantes com idade variando entre quatro e seis anos, Flamenga (FLA, n=7) e Holandesa (HOL, n=7), tiveram seus estros sincronizados com Cloprostenol. A partir do estro, os ovários foram avaliados a cada 24 horas por ultrassonografia transretal até que a segunda ovulação fosse detectada. Os diâmetros do folículo dominante (FD) e do subordinado (FS) foram registrados em função do dia do ciclo. Foram avaliados o diâmetro do FD e do FS na emergência folicular, o dia e o diâmetro do FD na divergência, o crescimento diário de FD e FS, o diâmetro máximo do folículo ovulatório e do dominante não ovulatório. Amostras de sangue foram coletadas a cada cinco dias para dosagem de progesterona sérica (P4) por radioimunoensaio. Nenhuma das variáveis avaliadas no grupo FLA diferiu significativamente do grupo HOL ou dos valores descritos para outras raças taurinas. Na emergência da onda os FD mediram 3,97 ± 0,19 mm para vacas Flamengas e de 4,00 ± 0,35 mm para as Holandesas, enquanto o FS chegou a 3,40 ± 0,22 mm nas Flamengas e 3,07 ± 0,26 mm nas Holandesas. O crescimento folicular diário das Flamengas foi de 1,10 ± 0,04 mm para o FD e de 0,67 ± 0,06 mm para o FS; já nas Holandesas o crescimento diário foi de 1,01 ± 0,05 mm para o FD e de 0,72 ± 0,09 mm para o FS. Desde a emergência o FD foi maior que o FS e a divergência ocorreu no terceiro dia, com o FD medindo 8,04 ± 0,37 mm e 8,39 ± 0,47, em Flamengas e Holandesas, respectivamente. O diâmetro máximo do folículo ovulatório em vacas Flamengas foi de 13,16 ± 0,33 mm e o valor médio do diâmetro máximo do folículo dominante não ovulatório foi de 13,01 ± 0,48 mm. Vacas Holandesas apresentaram valores de 14,20 ± 0,60 mm e 13,00 ± 0,59 mm, respectivamente, para as mesmas medidas. Estes valores estão de acordo com os normalmente aceitos para raças taurinas, situados entre 13 20 mm. A concentração de progesterona sérica ajustou-se a um modelo cúbico de regressão e, em vacas Flamengas variou de 0,155 ± 0,016 ng/ml até 6,651 ng/ml ± 1,868 ng/ml. Para vacas Holandesas os limites da concentração da progesterona oscilaram entre 0,300 ± 0,048 ng/ml e 5,957 ± 1,233 ng/ml. Conclui-se que vacas da raça Flamenga possuem desenvolvimento folicular e perfil de P4 semelhantes ao de vacas Holandesas, sugerindo ser possível a utilização das biotecnologias reprodutivas aplicáveis em animais da raça Holandesa em animais da raça Flamenga

O cavalo Crioulo é originário de raças de cavalos espanhóis trazidos há quatro séculos e reproduziu-se livremente no sul do continente da América.A seleção natural que ocorreu durante séculos fez o cavalo Crioulo ser conhecido por sua rusticidade e resistência, o que pode ter influenciado positivamente a fertilidade e longevidade desta raça, mas informações sobre fertilidade e performance reprodutiva da égua Crioula sob manejo natural ou controlado ainda são escassas. Os objetivos do presente estudo foram: (1) elucidar a dinâmica folicular das éguas Crioulas; (2) caracterizar o desenvolvimento e a regressão do corpo lúteo; e (3)estudar as mudanças de tônus e de ecotextura uterina ultrassonográfica durante dois ciclos estrais consecutivos. No experimento, 26 intervalos interovulatórios (IIO) foram estudados em 13 éguas. O ciclo estral natural foi caracterizado por: (i) o comprimento do IIO foi de 21 dias; (ii) os folículos dominante e subordinado emergem juntos no dia 5 e a taxa de crescimento é lenta até o momento do divergência; (iii) o divergência folicular ocorreu quando o folículo dominante atingiu o diâmetro de 22mm; (iv) o diâmetro pré-ovulatório foi de 43 mm; (v) o corpo lúteo atingiu o tamanho máximo 1,5 dias depois da ovulação e no dia 15 já não pode ser mais visualizado por ultrassonografia transretal; e (vi) a forte correlação de repetibilidade demonstrou que o tônus uterino e cervical podem ser usados para determinar a fase do ciclo estral. Os resultados apresentados neste trabalho possuem grande importância para melhor compreensão das características reprodutivas naturais do ciclo estral da égua Crioula. / The Criollo horse originates from breeds of spanish horses that were brought four centuries ago and reproduced freely in the southern part of the American continent. The natural selection that occurred over the centuries made the Criollo horse known for its roughness and resistance, which may have influenced positively the fertility and longevity of this breed, but information regarding fertility and reproductive performance of Criollo breed mare under natural or controlled management conditions is scarce. The objectives of the present study were to: (1) elucidate the follicular dynamics of Criollo breed mares; (2) characterize growth and regression of the corpus luteum; and (3) study the tone and ultrasonographic uterine echotexture changes during two consecutive estrous cycles. In this trial, 26 interovulatory intervals (IOI) were evaluated in 13 mares. The spontaneous estrous cycles were characterized by: (i) the length of the IOI was 21 days; (ii) dominant and subordinate follicles emerge together at day 5 and their growth rate were slower until the deviation moment; (iii) deviation occurred when the dominant follicle has achieved 22 mm; (iv) diameter of the preovulatory follicle was 43 mm; (v) corpus luteum has achieved its maximum diameter 1.5 days after ovulation and at day 15 it can no longer be visualized by transrectal ultrasonography; and (vi) the strong correlation for repeatability demonstrated that the uterine and cervical tonus can be used to determine the phase of the estrous cycle. The results presented are of fundamental importance for a better understanding of the reproductive physiological patterns of the estrous cycle in the Criollo mare.

O cavalo Crioulo é originário de raças de cavalos espanhóis trazidos há quatro séculos e reproduziu-se livremente no sul do continente da América.A seleção natural que ocorreu durante séculos fez o cavalo Crioulo ser conhecido por sua rusticidade e resistência, o que pode ter influenciado positivamente a fertilidade e longevidade desta raça, mas informações sobre fertilidade e performance reprodutiva da égua Crioula sob manejo natural ou controlado ainda são escassas. Os objetivos do presente estudo foram: (1) elucidar a dinâmica folicular das éguas Crioulas; (2) caracterizar o desenvolvimento e a regressão do corpo lúteo; e (3)estudar as mudanças de tônus e de ecotextura uterina ultrassonográfica durante dois ciclos estrais consecutivos. No experimento, 26 intervalos interovulatórios (IIO) foram estudados em 13 éguas. O ciclo estral natural foi caracterizado por: (i) o comprimento do IIO foi de 21 dias; (ii) os folículos dominante e subordinado emergem juntos no dia 5 e a taxa de crescimento é lenta até o momento do divergência; (iii) o divergência folicular ocorreu quando o folículo dominante atingiu o diâmetro de 22mm; (iv) o diâmetro pré-ovulatório foi de 43 mm; (v) o corpo lúteo atingiu o tamanho máximo 1,5 dias depois da ovulação e no dia 15 já não pode ser mais visualizado por ultrassonografia transretal; e (vi) a forte correlação de repetibilidade demonstrou que o tônus uterino e cervical podem ser usados para determinar a fase do ciclo estral. Os resultados apresentados neste trabalho possuem grande importância para melhor compreensão das características reprodutivas naturais do ciclo estral da égua Crioula. / The Criollo horse originates from breeds of spanish horses that were brought four centuries ago and reproduced freely in the southern part of the American continent. The natural selection that occurred over the centuries made the Criollo horse known for its roughness and resistance, which may have influenced positively the fertility and longevity of this breed, but information regarding fertility and reproductive performance of Criollo breed mare under natural or controlled management conditions is scarce. The objectives of the present study were to: (1) elucidate the follicular dynamics of Criollo breed mares; (2) characterize growth and regression of the corpus luteum; and (3) study the tone and ultrasonographic uterine echotexture changes during two consecutive estrous cycles. In this trial, 26 interovulatory intervals (IOI) were evaluated in 13 mares. The spontaneous estrous cycles were characterized by: (i) the length of the IOI was 21 days; (ii) dominant and subordinate follicles emerge together at day 5 and their growth rate were slower until the deviation moment; (iii) deviation occurred when the dominant follicle has achieved 22 mm; (iv) diameter of the preovulatory follicle was 43 mm; (v) corpus luteum has achieved its maximum diameter 1.5 days after ovulation and at day 15 it can no longer be visualized by transrectal ultrasonography; and (vi) the strong correlation for repeatability demonstrated that the uterine and cervical tonus can be used to determine the phase of the estrous cycle. The results presented are of fundamental importance for a better understanding of the reproductive physiological patterns of the estrous cycle in the Criollo mare.

O cavalo Crioulo é originário de raças de cavalos espanhóis trazidos há quatro séculos e reproduziu-se livremente no sul do continente da América.A seleção natural que ocorreu durante séculos fez o cavalo Crioulo ser conhecido por sua rusticidade e resistência, o que pode ter influenciado positivamente a fertilidade e longevidade desta raça, mas informações sobre fertilidade e performance reprodutiva da égua Crioula sob manejo natural ou controlado ainda são escassas. Os objetivos do presente estudo foram: (1) elucidar a dinâmica folicular das éguas Crioulas; (2) caracterizar o desenvolvimento e a regressão do corpo lúteo; e (3)estudar as mudanças de tônus e de ecotextura uterina ultrassonográfica durante dois ciclos estrais consecutivos. No experimento, 26 intervalos interovulatórios (IIO) foram estudados em 13 éguas. O ciclo estral natural foi caracterizado por: (i) o comprimento do IIO foi de 21 dias; (ii) os folículos dominante e subordinado emergem juntos no dia 5 e a taxa de crescimento é lenta até o momento do divergência; (iii) o divergência folicular ocorreu quando o folículo dominante atingiu o diâmetro de 22mm; (iv) o diâmetro pré-ovulatório foi de 43 mm; (v) o corpo lúteo atingiu o tamanho máximo 1,5 dias depois da ovulação e no dia 15 já não pode ser mais visualizado por ultrassonografia transretal; e (vi) a forte correlação de repetibilidade demonstrou que o tônus uterino e cervical podem ser usados para determinar a fase do ciclo estral. Os resultados apresentados neste trabalho possuem grande importância para melhor compreensão das características reprodutivas naturais do ciclo estral da égua Crioula. / The Criollo horse originates from breeds of spanish horses that were brought four centuries ago and reproduced freely in the southern part of the American continent. The natural selection that occurred over the centuries made the Criollo horse known for its roughness and resistance, which may have influenced positively the fertility and longevity of this breed, but information regarding fertility and reproductive performance of Criollo breed mare under natural or controlled management conditions is scarce. The objectives of the present study were to: (1) elucidate the follicular dynamics of Criollo breed mares; (2) characterize growth and regression of the corpus luteum; and (3) study the tone and ultrasonographic uterine echotexture changes during two consecutive estrous cycles. In this trial, 26 interovulatory intervals (IOI) were evaluated in 13 mares. The spontaneous estrous cycles were characterized by: (i) the length of the IOI was 21 days; (ii) dominant and subordinate follicles emerge together at day 5 and their growth rate were slower until the deviation moment; (iii) deviation occurred when the dominant follicle has achieved 22 mm; (iv) diameter of the preovulatory follicle was 43 mm; (v) corpus luteum has achieved its maximum diameter 1.5 days after ovulation and at day 15 it can no longer be visualized by transrectal ultrasonography; and (vi) the strong correlation for repeatability demonstrated that the uterine and cervical tonus can be used to determine the phase of the estrous cycle. The results presented are of fundamental importance for a better understanding of the reproductive physiological patterns of the estrous cycle in the Criollo mare.