<p>A series of experiments were designed to study alternative estrus synchronization and resynchronization protocols to facilitate the use of artificial insemination in cattle. </p><p>Studies were conducted to study the effects of estradiol cypionate (ECP) on follicular dynamics, time of ovulation, and pregnancy rate to timed-AI (TAI) in CIDR-based protocols. Although administration of 1 mg ECP did not result in synchronous follicular wave emergence, a dose of 0.5 mg ECP synchronized LH release and ovulation. Administration of ECP 24 h after CIDR removal resulted in acceptable pregnancy rate. However, treatment with ECP at CIDR removal resulted in acceptable pregnancy rate only if follicular wave emergence was synchronized with estradiol-17â (E-17â). </p><p>The efficacy of two estradiol preparations (5 mg of E-17â or estadiol valerate; EV) and reduced doses of EV on CL and ovarian follicular dynamics and superovulatory response were examined. When doses of 5 mg were compared, EV treatment resulted in a more variable interval to follicular wave emergence and a lower superovulatory response than E-17â. However, EV at a dose of 1 or 2 mg was efficacious in synchronizing follicle wave emergence in CIDR-treated cattle.</p><p>Pregnancy rates were compared following TAI in cattle given a new or previously used CIDR and injections of estradiol, with or without progesterone, to synchronize follicular wave emergence. Pregnancy rate following TAI did not differ between cattle treated with a new or once-used CIDR, but pregnancy rate was lower in cattle treated with one or two twice-used CIDR. The addition of an injection of progesterone to the estradiol treatment at CIDR insertion did not enhance pregnancy rate. </p><p>The efficacy of progestins (used CIDR and MGA), and E-17â, ECP, GnRH, or progesterone treatment for resynchronization of estrus in cattle not pregnant following TAI were investigated. Progestin treatment resulted in the majority of nonpregnant heifers detected in estrus over a 4-d interval. Conception rates were higher in heifers resynchronized with a once-used CIDR than with MGA. GnRH at CIDR insertion synchronized follicular wave emergence in cows, but did not increase conception rate in heifers. E-17â at CIDR insertion (1.5 mg) and removal (0.5 mg) resulted in decreased pregnancy rate following TAI. In summary, protocols described in this thesis resulted in acceptable pregnancy rates following TAI and resynchronization of previously inseminated heifers with progestins resulted in variable estrus and pregnancy rates.</p>

In the ewe, one to three antral follicles emerge or grow from a pool of small antral follicles (1 to 3 mm in diameter) every 3 to 5 days and reach diameters of ¡Ý5 mm before regression or ovulation. Each follicular wave is triggered by a peak in serum concentrations of FSH. It is not clear what characteristics of an FSH peak cause follicular wave emergence and what aspects of development of a follicular wave are regulated by its preceding FSH peak.<p> In Experiment 1, we found that the amplitude of FSH peaks decreased, while basal serum FSH concentrations increased across the inter-ovulatory interval (P < 0.05). However, there were no associated changes in the growth, static or regression phases of follicular waves or the number and size of follicles in a wave. In Experiment 2, using computer-assisted quantitative echotextural analysis, we found that the numerical pixel value (NPV) for the wall of anovulatory follicles emerging in the third wave of the cycle was significantly higher than for waves 1 and 2 at the time of wave emergence but it decreased as follicles reached maximum follicular diameter (P < 0.05). A tendency for a similar pattern for the wall of follicles in the last wave of the cycle was also observed (P = 0.07).<p> In Experiment 3, treatment with ovine FSH (oFSH) increased the amplitude of an FSH peak by 5 to 6 fold. This treatment increased estradiol production (P < 0.05) but had little effect on other characteristics of the subsequent follicular wave. Daily injections of oFSH (Experiment 4) for four days, resulted in the occurrence of 4 discrete peaks in serum FSH concentrations. Each injection of oFSH resulted in the emergence of a new follicular wave.<p> In Experiment 5, six cyclic ewes received oFSH (0.1 ¦Ìg/kg, sc) every 6 h for 42 h, to try to give a gradual increase in the leading slope of an FSH peak. Serum FSH concentrations increased in oFSH treated ewes (P < 0.05) resulting in an additional peak between two endogenously driven FSH peaks and therefore, did not give the planned gradual leading slope to an FSH peak. Ovine FSH treatment occurred in the early growth phase of wave 1 of the inter-ovulatory interval and increased the growth rate of growing follicles in that wave, compared to control ewes (P < 0.05). This apparently induced dominance in follicles in wave 1, causing them to suppress wave emergence in response to the injected FSH. In Experiment 6, oFSH was infused constantly (1.98 ¦Ìg/ewe/h, iv, n = 6) for 60 h. Infusion of oFSH maintained serum FSH concentrations at a level similar to the zenith of a peak. This resulted in a superstimulatory effect with a peak in the mean number of large follicles on Day 2 after the start of FSH infusion (P < 0.001).<p> A hormonal milieu similar to low serum progesterone concentrations was created by treatment of ewes with prostaglandin and medroxyprogesterone acetate (MAP) sponges (Experiment 7). This treatment delayed regression of the penultimate follicular wave of a cycle. However, the delayed follicular atresia was accompanied by a greater degree of apoptosis in somatic cells of follicles growing in the penultimate wave compared to those in the final wave of the cycle, when collected one day before expected ovulation.<p> In conclusion, trends in basal serum concentrations of FSH and peaks in serum FSH concentrations, across the estrous cycle, are associated with changes in the image attributes of follicles emerging later in the estrous cycle, perhaps reflecting a greater readiness of those follicles for ovulation and formation of CL. The ovine ovary can respond to discrete peaks in serum FSH concentrations with the emergence of new follicular waves on a daily basis. This led us to conclude that follicular dominance is not evident in the ewe and peaks in serum FSH concentrations are likely to be driven by some endogenous rhythm that is unrelated to ovarian follicular secretory products. However, direct dominance can be induced by giving supplemented FSH during the growth phase of a follicle. Extended exposure of ovine ovaries to the serum concentrations of FSH found at the zenith of a peak overrides the mechanisms that recruit follicles into a wave and induces a superovulatory response in cyclic ewes. Finally, an increase in the incidence of apoptosis occurs in antral follicles in sheep that have an extended lifespan, prior to any morphological changes detectable by ultrasonography. This would seem to cause decreased follicular viability and lowered fertility of the oocytes that the follicles contain.

<p>A series of experiments were designed to study alternative estrus synchronization and resynchronization protocols to facilitate the use of artificial insemination in cattle. </p><p>Studies were conducted to study the effects of estradiol cypionate (ECP) on follicular dynamics, time of ovulation, and pregnancy rate to timed-AI (TAI) in CIDR-based protocols. Although administration of 1 mg ECP did not result in synchronous follicular wave emergence, a dose of 0.5 mg ECP synchronized LH release and ovulation. Administration of ECP 24 h after CIDR removal resulted in acceptable pregnancy rate. However, treatment with ECP at CIDR removal resulted in acceptable pregnancy rate only if follicular wave emergence was synchronized with estradiol-17â (E-17â). </p><p>The efficacy of two estradiol preparations (5 mg of E-17â or estadiol valerate; EV) and reduced doses of EV on CL and ovarian follicular dynamics and superovulatory response were examined. When doses of 5 mg were compared, EV treatment resulted in a more variable interval to follicular wave emergence and a lower superovulatory response than E-17â. However, EV at a dose of 1 or 2 mg was efficacious in synchronizing follicle wave emergence in CIDR-treated cattle.</p><p>Pregnancy rates were compared following TAI in cattle given a new or previously used CIDR and injections of estradiol, with or without progesterone, to synchronize follicular wave emergence. Pregnancy rate following TAI did not differ between cattle treated with a new or once-used CIDR, but pregnancy rate was lower in cattle treated with one or two twice-used CIDR. The addition of an injection of progesterone to the estradiol treatment at CIDR insertion did not enhance pregnancy rate. </p><p>The efficacy of progestins (used CIDR and MGA), and E-17â, ECP, GnRH, or progesterone treatment for resynchronization of estrus in cattle not pregnant following TAI were investigated. Progestin treatment resulted in the majority of nonpregnant heifers detected in estrus over a 4-d interval. Conception rates were higher in heifers resynchronized with a once-used CIDR than with MGA. GnRH at CIDR insertion synchronized follicular wave emergence in cows, but did not increase conception rate in heifers. E-17â at CIDR insertion (1.5 mg) and removal (0.5 mg) resulted in decreased pregnancy rate following TAI. In summary, protocols described in this thesis resulted in acceptable pregnancy rates following TAI and resynchronization of previously inseminated heifers with progestins resulted in variable estrus and pregnancy rates.</p>

In the ewe, one to three antral follicles emerge or grow from a pool of small antral follicles (1 to 3 mm in diameter) every 3 to 5 days and reach diameters of ¡Ý5 mm before regression or ovulation. Each follicular wave is triggered by a peak in serum concentrations of FSH. It is not clear what characteristics of an FSH peak cause follicular wave emergence and what aspects of development of a follicular wave are regulated by its preceding FSH peak.<p> In Experiment 1, we found that the amplitude of FSH peaks decreased, while basal serum FSH concentrations increased across the inter-ovulatory interval (P < 0.05). However, there were no associated changes in the growth, static or regression phases of follicular waves or the number and size of follicles in a wave. In Experiment 2, using computer-assisted quantitative echotextural analysis, we found that the numerical pixel value (NPV) for the wall of anovulatory follicles emerging in the third wave of the cycle was significantly higher than for waves 1 and 2 at the time of wave emergence but it decreased as follicles reached maximum follicular diameter (P < 0.05). A tendency for a similar pattern for the wall of follicles in the last wave of the cycle was also observed (P = 0.07).<p> In Experiment 3, treatment with ovine FSH (oFSH) increased the amplitude of an FSH peak by 5 to 6 fold. This treatment increased estradiol production (P < 0.05) but had little effect on other characteristics of the subsequent follicular wave. Daily injections of oFSH (Experiment 4) for four days, resulted in the occurrence of 4 discrete peaks in serum FSH concentrations. Each injection of oFSH resulted in the emergence of a new follicular wave.<p> In Experiment 5, six cyclic ewes received oFSH (0.1 ¦Ìg/kg, sc) every 6 h for 42 h, to try to give a gradual increase in the leading slope of an FSH peak. Serum FSH concentrations increased in oFSH treated ewes (P < 0.05) resulting in an additional peak between two endogenously driven FSH peaks and therefore, did not give the planned gradual leading slope to an FSH peak. Ovine FSH treatment occurred in the early growth phase of wave 1 of the inter-ovulatory interval and increased the growth rate of growing follicles in that wave, compared to control ewes (P < 0.05). This apparently induced dominance in follicles in wave 1, causing them to suppress wave emergence in response to the injected FSH. In Experiment 6, oFSH was infused constantly (1.98 ¦Ìg/ewe/h, iv, n = 6) for 60 h. Infusion of oFSH maintained serum FSH concentrations at a level similar to the zenith of a peak. This resulted in a superstimulatory effect with a peak in the mean number of large follicles on Day 2 after the start of FSH infusion (P < 0.001).<p> A hormonal milieu similar to low serum progesterone concentrations was created by treatment of ewes with prostaglandin and medroxyprogesterone acetate (MAP) sponges (Experiment 7). This treatment delayed regression of the penultimate follicular wave of a cycle. However, the delayed follicular atresia was accompanied by a greater degree of apoptosis in somatic cells of follicles growing in the penultimate wave compared to those in the final wave of the cycle, when collected one day before expected ovulation.<p> In conclusion, trends in basal serum concentrations of FSH and peaks in serum FSH concentrations, across the estrous cycle, are associated with changes in the image attributes of follicles emerging later in the estrous cycle, perhaps reflecting a greater readiness of those follicles for ovulation and formation of CL. The ovine ovary can respond to discrete peaks in serum FSH concentrations with the emergence of new follicular waves on a daily basis. This led us to conclude that follicular dominance is not evident in the ewe and peaks in serum FSH concentrations are likely to be driven by some endogenous rhythm that is unrelated to ovarian follicular secretory products. However, direct dominance can be induced by giving supplemented FSH during the growth phase of a follicle. Extended exposure of ovine ovaries to the serum concentrations of FSH found at the zenith of a peak overrides the mechanisms that recruit follicles into a wave and induces a superovulatory response in cyclic ewes. Finally, an increase in the incidence of apoptosis occurs in antral follicles in sheep that have an extended lifespan, prior to any morphological changes detectable by ultrasonography. This would seem to cause decreased follicular viability and lowered fertility of the oocytes that the follicles contain.

For this thesis our objectives were to establish an efficient method of ovarian synchronization and superstimulation in bison, and determine the effects of gonadotropin treatments on oocyte collection efficiency and quality in bison. In the first study we conducted two experiments to develop an efficient protocol for synchronization of follicular wave emergence during the anovulatory season. In Experiment 1, we compared the synchronizing effect of follicular ablation (n = 9) and treatment with 2 mg estradiol (E-) 17β in oil (n = 10), while in Experiment 2, we compared follicular ablation (n = 9) and treatment with 2 mg E-17β + 100 mg progesterone (P4; n = 10). Results showed that the degree of synchrony did not differ between ablation and hormone treatment groups in either Experiment, but follicular wave emergence was more synchronous in both treatment groups compared to the untreated control phase. The second study was conducted to develop an efficient method for ovarian superstimulation and oocyte collection during the anovulatory and ovulatory seasons. During the anovulatory season, one experiment was conducted in two replicates to compare the superstimulatory effect of 2500 IU of eCG (n = 10) given intramuscularly vs two doses of 200 mg of pFSH each (n = 10) given subcutaneously. Additionally, the effect of 25 mg of pLH given 24 hours prior oocyte collection on oocyte quality and collection rate was evaluated for each superstimulatory treatment. Results showed that treatment with pFSH induced a higher superstimulatory response and more cumulus oocyte complexes (COC) collected than did eCG during the anovulatory season. Furthermore, treatment with pLH increased the proportion of expanded COC that were collected with ultrasound-guided follicular aspiration. Two experiments were conducted during the ovulatory season, to develop an efficient protocol for superstimulation and oocyte collection. In Experiment 1, we compared the effect of two intramuscular doses of 200 mg of pFSH in saline (n = 11) vs two intramuscular doses of 200 mg of pFSH in a proprietary slow release formulation (SRF; n = 11). In Experiment 2, we compared the effect of a single dose of 2500 IU eCG intramuscularly vs two doses of 200 mg of pFSH administered subcutaneously. Results showed that a 2-dose regime of pFSH, diluted in either saline or a slow-release formulation induced a similar superstimulatory ovarian response in wood bison, while bison given a single-dose of 2500 IU eCG had a significantly lower ovarian response. In summary, synchronization of follicle wave emergence can be effectively accomplished in wood bison during the anovulatory season and follicular ablation, E-17β and E-17β + P4 treatments all shortened, and decreased the variability in the interval to follicular wave emergence. In addition, oocyte collection by transvaginal ultrasound-guided follicle aspiration from superstimulated bison was feasible and practical. Finally, treatment with pFSH was more effective than eCG to induce ovarian superstimulation for ultrasound-guided follicle aspiration in wood bison during both the anovulatory and ovulatory seasons.

For this thesis our objectives were to establish an efficient method of ovarian synchronization and superstimulation in bison, and determine the effects of gonadotropin treatments on oocyte collection efficiency and quality in bison. In the first study we conducted two experiments to develop an efficient protocol for synchronization of follicular wave emergence during the anovulatory season. In Experiment 1, we compared the synchronizing effect of follicular ablation (n = 9) and treatment with 2 mg estradiol (E-) 17β in oil (n = 10), while in Experiment 2, we compared follicular ablation (n = 9) and treatment with 2 mg E-17β + 100 mg progesterone (P4; n = 10). Results showed that the degree of synchrony did not differ between ablation and hormone treatment groups in either Experiment, but follicular wave emergence was more synchronous in both treatment groups compared to the untreated control phase. The second study was conducted to develop an efficient method for ovarian superstimulation and oocyte collection during the anovulatory and ovulatory seasons. During the anovulatory season, one experiment was conducted in two replicates to compare the superstimulatory effect of 2500 IU of eCG (n = 10) given intramuscularly vs two doses of 200 mg of pFSH each (n = 10) given subcutaneously. Additionally, the effect of 25 mg of pLH given 24 hours prior oocyte collection on oocyte quality and collection rate was evaluated for each superstimulatory treatment. Results showed that treatment with pFSH induced a higher superstimulatory response and more cumulus oocyte complexes (COC) collected than did eCG during the anovulatory season. Furthermore, treatment with pLH increased the proportion of expanded COC that were collected with ultrasound-guided follicular aspiration. Two experiments were conducted during the ovulatory season, to develop an efficient protocol for superstimulation and oocyte collection. In Experiment 1, we compared the effect of two intramuscular doses of 200 mg of pFSH in saline (n = 11) vs two intramuscular doses of 200 mg of pFSH in a proprietary slow release formulation (SRF; n = 11). In Experiment 2, we compared the effect of a single dose of 2500 IU eCG intramuscularly vs two doses of 200 mg of pFSH administered subcutaneously. Results showed that a 2-dose regime of pFSH, diluted in either saline or a slow-release formulation induced a similar superstimulatory ovarian response in wood bison, while bison given a single-dose of 2500 IU eCG had a significantly lower ovarian response. In summary, synchronization of follicle wave emergence can be effectively accomplished in wood bison during the anovulatory season and follicular ablation, E-17β and E-17β + P4 treatments all shortened, and decreased the variability in the interval to follicular wave emergence. In addition, oocyte collection by transvaginal ultrasound-guided follicle aspiration from superstimulated bison was feasible and practical. Finally, treatment with pFSH was more effective than eCG to induce ovarian superstimulation for ultrasound-guided follicle aspiration in wood bison during both the anovulatory and ovulatory seasons.

Master of Science / Department of Animal Sciences and Industry / Jeffrey S. Stevenson / We hypothesized that pregnancy outcomes may be improved by inducing luteal regression, ovulation, or both before a control CO-Synch + CIDR program (100 mcg GnRH i.m. [GnRH-1] and insertion of a progesterone-impregnated intravaginal controlled internal drug release [CIDR] insert on d -10, 25 mg PGF2alpha (PG) i.m. and CIDR insert removal on d -3, and 100 mcg GnRH i.m. [GnRH-2] and timed AI [TAI] on d 0) in suckled beef cows. This hypothesis was tested in 2 experiments, in which cows were treated with either PG or PG + GnRH before initiating a control CO-Synch + CIDR program to increase the proportion of cows starting the program in a low (< 1 ng/mL; Exp. 1) or high (≥ 1 ng/mL; Exp. 2) progesterone status, respectively. Blood was collected before each injection for later progesterone analyses. In Exp. 1, cows at 9 locations (n = 1,537) were assigned to either: (1) control or (2) PrePG (same as control with a PG injection on d -13). The PrePG cows had larger (P < 0.05) follicles on d -10 and more (P < 0.05) ovulated after GnRH-1 than controls (60.6 vs. 36.5%). Incidence of estrus between d -3 and 0 was greater (P < 0.05) for treated multiparous cows than multiparous controls and treated and control primiparous cows (74.1 vs. 64.3, 58.6, and 59.1%, respectively). In Exp. 2, cows at 4 locations (n = 803) were assigned to: (1) control (same as Exp. 1) or (2) PrePGG (same as control with PG injection on d -20 and GnRH injection on d -17. Cows with BCS > 5.0 or ≥ 70 d postpartum at TAI were more (P < 0.05) likely to become pregnant than thinner cows or those with fewer days postpartum. Treated cows in both experiments were more (P < 0.05) likely than controls to have luteolysis after initial PG injections and reduced (P < 0.05) serum progesterone. In both experiments, pregnancy rates at d 35 did not differ between treatment and control; however, cows classified as anestrous before d -10, but with elevated progesterone on d -10, had increased (P < 0.05) pregnancy outcomes than remaining anestrous cows with low progesterone concentrations. In summary, luteal regression and ovulation were enhanced by treatments before the 7 d CO-Synch + CIDR program; however, pregnancy per TAI was not improved.

O objetivo do presente estudo foi o de determinar em qual fase da onda de crescimento folicular são obtidas maiores taxas de recuperação e competência de oócitos obtidos por aspiração folicular (OPU) em novilhas cruzadas (Bos indicus x Bos taurus; Capítulo 1) ou em novilhas bubalinas (Bubalus bubalis), zebuínas (Nelore; Bos indicus) e taurinas (Holandês; Bos taurus; Capítulo 2). No Capítulo 1, 30 novilhas cruzadas foram submetidas à OPU aos 2 (D2), 4 (D4) e 6 (D6) dias após a emergência da onda de crescimento folicular (n=10/ grupo), em delineamento &quot;cross-over&quot; realizado em 3 réplicas. Os oócitos recuperados foram selecionados e os viáveis submetidos aos procedimentos de produção in vitro. Os dados foram analisados por ANOVA, utilizando o PROC MIXED, considerando os efeitos fixos de momento de OPU, de réplica e da interação entre eles, e o efeito aleatório de doadora. Foram produzidos maior taxa de blastocistos aos 6 dias pós-FIV, bem como maior número de blastocistos aos 9 dias pós-FIV e maior número de núcleos dos embriões eclodidos quando procedeu-se com aspiração no D2. Contudo, a OPU realizada no D6 também influenciou positivamente a taxa de blastocistos 6 dias pós FIV e não diferiu dos outros momentos com relação ao número de núcleos dos embriões eclodidos. No Capítulo 2, foram realizados 2 experimentos (1 e 2). No Experimento 1, foi avaliado o efeito de 50 mg de progesterona injetável na sincronização de emergência folicular em novilhas Nelore, Holandesas e bubalinas submetidas a protocolo de sincronização à base de progestágeno associado a 2 mg de benzoato de estradiol (n=10/ grupo). Os dados foram analisados por regressão logística, utilizando o PROC GLIMMIX. O momento da emergência folicular não foi afetado pela administração de progesterona injetável, ocorrendo entre 4,2 a 4,5 dias após o início do protocolo de sincronização para os três grupos genéticos. No Experimento 2, foram avaliados os efeitos de momento de sincronização da emergência folicular para a aspiração folicular (1, 3 ou 5 dias após a emergência) e de grupo genético (Nelore, Holandês e búfalo) na produção de embriões in vitro. Um total de 27 novilhas (9 de cada grupo genético, subdivididas nos 3 momentos de aspiração folicular) foram submetidas a 6 sessões de OPU, em delineamento &quot;cross-over&quot;. Os oócitos recuperados foram selecionados e os viáveis submetidos aos procedimentos de produção in vitro. Os dados foram analisados por ANOVA, utilizando o PROC MIXED, considerando os efeitos fixos de momento de OPU, de grupo genético, de réplica e da interação entre eles, e o efeito aleatório de doadora. Os resultados demonstraram que novilhas Nelore apresentaram melhor eficiência para a produção de embriões in vitro do que as novilhas Holandesas ou bubalinas, e que a aspiração folicular realizada em diferentes momentos da onda folicular sincronizada não influenciou a produção in vitro. / The objective of the present study was to determine in which phase of follicular wave higher recovery rates and better oocyte competence can be obtained by ovum pickup (OPU) in crossbred (Bos indicus x Bos taurus; Chapter 1) or buffalo (Bubalus bubalis), Nelore (Bos indicus) and Holstein heifers (Bos taurus; Chapter 2). In Chapter 1, 30 crossbred heifers were submitted to OPU on days 2 (D2), 4 (D4) or 6 (D6) after follicular wave emergence (n=10/ group), in a cross-over design performed in 3 replicates. Oocytes recovered were selected and those viable were submitted to in vitro embryo procedures. Data were analyzed by ANOVA, using PROC MIXED, in which time of OPU, replicate and interaction between these factors were considered as fixed effects, and donor was considered as a random effect. Higher blastocyst rate (6 days after IVF), number of blastocysts (9 days after IVF), and number of nuclei of hatched embryos were produced when OPU was done on D2. However, OPU on D6 also produced a higher blastocyst rate (6 days after IVF), and did not differ from other times of OPU concerning number of nuclei of hatched embryos. In Chapter 2, two experiments were performed. In Experiment 1, the effect of 50 mg of injectable progesterone was evaluated in the synchronization of follicular wave emergence of Nelore, Holstein, and buffalo heifers submitted to a progestin plus 2 mg of estradiol benzoate protocol (n=10/ group). Data were analyzed by logistic regression, by PROC GLIMMIX. The time of follicular wave emergence was not affected by administration of injectable progesterone. Besides, follicular wave emergence occurred between 4.2 and 4.5 days after the beginning of synchronization protocol for all genetic groups. In Experiment 2, the effects of time of OPU relative to follicular wave emergence (1, 3 or 5 days) and of genetic group (Nelore, Holstein, and buffalo) on in vitro embryo production were evaluated. A total of 27 heifers (9 of each genetic group, assigned in the 3 times of OPU) were submitted to 6 OPU sessions, in a cross-over design. Oocytes recovered were selected and those viable were submitted to in vitro embryo procedures. Data were analyzed by ANOVA, using PROC MIXED, in which time of OPU, genetic group, replicate and interaction between these factors were considered as fixed effects, and donor was considered as a random effect. Results demonstrated that Nelore heifers showed a better efficiency for in vitro embryo production than Holstein or buffalo heifers. Additionally, OPU performed at different times of synchronized follicular wave did not influence in vitro embryo production.

Três experimentos foram realizados para testar a hipótese de que a resposta superestimulatória de doadoras Nelore (Bos indicus) com tratamentos iniciados próximo à ovulação durante a primeira onda de crescimento folicular seria maior ou comparável àquela decorrente de tratamentos convencionais. Os animais foram aleatoriamente alocados em três grupos. As doadoras dos Grupos 1 - Onda 1 s/P4 e 2 - Onda 1 c/P4 foram superestimuladas na primeira onda de crescimento folicular, e as do Grupo 3 - SincBE+P4/P4 quatro dias após a sincronização da emergência folicular com estradiol e progesterona. Os animais receberam dispositivo intravaginal de Progesterona (CIDR) associado a 50mg de Progesterona e 2,5mg de Benzoato de Estradiol (IM) no Dia 0. Os animais dos Grupos 1 e 2 receberam PGF2&alpha; no Dia 5 e 12,5mg Armour de LH (Lutropin) 24 horas após a remoção do CIDR (Dia 9), e no Dia 11 iniciou-se o tratamento superovulatório. As doadoras do Grupo 2 receberam um novo CIDR juntamente com a primeira dose de FSH (Dia 11). Todos os animais foram superestimulados com 133mg NIH-FSH-P1 de Folltropin diluídos em 10ml, em duas aplicações diárias de 1ml, por cinco dias. No último dia de tratamento, junto com o FSH foram aplicadas doses luteolíticas de PGF2&alpha; nos Grupos 2 e 3 o CIDR foi removido na ultima aplicação. Todas as doadoras receberam 25mg de LH 24 horas após a ultima dose de FSH e foram inseminadas 12 e 24 horas após o LH. Os embriões foram coletados e avaliados pelo mesmo veterinário sete dias após a inseminação. No primeiro experimento, as quantidades de embriões transferíveis não foi significativamente diferente entre os grupos 2 e 3 (8,0 ± 1,8 x 6,6 ± 2,0), e ambas foram maiores que as do 1 (0,2 ± 0,2; P<0,05). Como não houve diferença entre os Grupos 2 e 3 nos parâmetros analisados, o Grupo 3 foi excluído dos outros dois experimentos. No segundo experimento, as doadoras receberam os mesmos tratamentos dos Grupos 1 e 2, mas foram abatidas 12 horas após receberem 25mg de LH (Dia 16) e tiveram seus ovários removidos e levados para o laboratório, para classificação. Os oócitos foram aspirados e avaliados quanto à maturação pela expansão do COC. Os animais que não receberam dispositivo de P4 durante o tratamento superovulatório apresentaram maior número de oócitos não maturados (6,4 ± 2,7 x 1,2 ± 0,9; P< 0,05). O terceiro experimento foi semelhante ao segundo, com a diferença de que os animais foram inseminados 12 horas após o tratamento com 25mg de LH, e tiveram as estruturas coletadas 7 dias após a IA. Os resultados desse experimento confirmaram os do primeiro, no qual animais sem suplementação exógena de P4 durante o tratamento superovulatório apresentaram menor número de embriões transferíveis que o grupo com P4 (0,0 ± 0,0 x 3,9 ± 1,1; P<0,05). Os resultados obtidos retificam a hipótese, demonstrando que a superovulação durante a primeira onda de crescimento folicular em novilhas Nelore é eficiente somente com suplementação exógena de P4 / Three experiments were design to test the hypothesis that superovulatory (SPO) response of Nelore (Bos indicus) donors to treatments administered during the first follicular wave, initiated at the expected time of ovulation, will elicited a higher or a comparable response to traditional protocols. In the first experiment, cows were randomly allocated into 1 of 3 treatment groups. Cows in Group Wave 1 without P4 e Group Wave 1 with P4 were superstimulated during the first follicular wave and cows in the Control Group were superstimulated after synchronized follicular wave emergence using estradiol and progesterone. All cows received a CIDR device and an injection of 50mg of P4 and 2.5mg EB on Day 0. Cows in Groups Wave 1 with P4 and Wave 1 without P4 were also treated with PGF2&alpha; on Day 5 and 12,5mg Armour of pLH (Lutropin) 24 h after CIDR removal (Day 9), to synchronize ovulation. The SPO treatments were initiated on Day 11; donor cows in Group Wave 1 with P4 also received a new CIDR device at the time of the first FSH injection. All donors in three groups were superstimulated with a total dose of 133mg NIH-FSH-P1 of Folltropin-V, divided into twice daily injections of the same dosage (13,3mg diluted in 1ml) over 5 days. On the last day of FSH treatment, all animals received PGF2&alpha; after each FSH injection and cows in Group Wave 1 with P4 and Control Group had their CIDR removed at the time of the last FSH injection. All cows received 25 mg of pLH 24h after the last FSH treatment and were AI 12 and 24h later. Ova/embryo collection and evaluation was done 7d after, aIl by the same veterinarian. Results indicate that there was no difference in the numbers of transferable embryos in CIDR treated donor cows when SPO treatments were initiated at the time of emergence of either the first follicular wave Group Wave 1 with P4 (8.0 ± 1.8) or following synchronization of follicular wave emergence with BE + P4, control Group (6.6 ± 2.0), but both were greater than when SPO treatments were initiated at the first follicular wave without the use of a CIDR device, Wave 1 without P4 (0.2 ± 0.2; P<0,05). Since there were no differences between Groups Wave 1 with P4 and control on all the end points, this group was dropped from the others experiments. A second experiment was performed in order to evaluate the quality of oocyte recovered after animals were treated under the same protocol of Groups Wave 1. The SPO treatments were the same described previously, however 12h after the injection of 25mg of pLH animals were slaughtered and their ovaries were taken to an IVF lab. Follicles were aspirated and the oocyte was evaluated and those with an expanded COC were considered mature. Animals in Group Wave 1 without P4 showed a greater (P<0,05) number of immature oocyte (6.4 ± 2.7) than Group Wave 1 with P4 (1.2 ± 0.9). The third experiment was performed to confirm the results of the first experiment. The treatments were similar as the second experiment, however, animals were AI 12 and 24h after the 25mg of pLH and had ova/embryos collection and evaluation 7 d after by the same veterinarian. Results of this experiment confirmed the first experiment presenting a greater (P<0,05) number of transferable embryos in Group Wave 1 with P4 (3.9 ± 1,1) when compared to Group Wave 1 without P4 (0.0 ± 0.0). The results did not support the hypothesis, showing that exogenous P4 is necessary in order to superovulate Nelore during the first follicular wave

O objetivo do presente estudo foi o de determinar em qual fase da onda de crescimento folicular são obtidas maiores taxas de recuperação e competência de oócitos obtidos por aspiração folicular (OPU) em novilhas cruzadas (Bos indicus x Bos taurus; Capítulo 1) ou em novilhas bubalinas (Bubalus bubalis), zebuínas (Nelore; Bos indicus) e taurinas (Holandês; Bos taurus; Capítulo 2). No Capítulo 1, 30 novilhas cruzadas foram submetidas à OPU aos 2 (D2), 4 (D4) e 6 (D6) dias após a emergência da onda de crescimento folicular (n=10/ grupo), em delineamento &quot;cross-over&quot; realizado em 3 réplicas. Os oócitos recuperados foram selecionados e os viáveis submetidos aos procedimentos de produção in vitro. Os dados foram analisados por ANOVA, utilizando o PROC MIXED, considerando os efeitos fixos de momento de OPU, de réplica e da interação entre eles, e o efeito aleatório de doadora. Foram produzidos maior taxa de blastocistos aos 6 dias pós-FIV, bem como maior número de blastocistos aos 9 dias pós-FIV e maior número de núcleos dos embriões eclodidos quando procedeu-se com aspiração no D2. Contudo, a OPU realizada no D6 também influenciou positivamente a taxa de blastocistos 6 dias pós FIV e não diferiu dos outros momentos com relação ao número de núcleos dos embriões eclodidos. No Capítulo 2, foram realizados 2 experimentos (1 e 2). No Experimento 1, foi avaliado o efeito de 50 mg de progesterona injetável na sincronização de emergência folicular em novilhas Nelore, Holandesas e bubalinas submetidas a protocolo de sincronização à base de progestágeno associado a 2 mg de benzoato de estradiol (n=10/ grupo). Os dados foram analisados por regressão logística, utilizando o PROC GLIMMIX. O momento da emergência folicular não foi afetado pela administração de progesterona injetável, ocorrendo entre 4,2 a 4,5 dias após o início do protocolo de sincronização para os três grupos genéticos. No Experimento 2, foram avaliados os efeitos de momento de sincronização da emergência folicular para a aspiração folicular (1, 3 ou 5 dias após a emergência) e de grupo genético (Nelore, Holandês e búfalo) na produção de embriões in vitro. Um total de 27 novilhas (9 de cada grupo genético, subdivididas nos 3 momentos de aspiração folicular) foram submetidas a 6 sessões de OPU, em delineamento &quot;cross-over&quot;. Os oócitos recuperados foram selecionados e os viáveis submetidos aos procedimentos de produção in vitro. Os dados foram analisados por ANOVA, utilizando o PROC MIXED, considerando os efeitos fixos de momento de OPU, de grupo genético, de réplica e da interação entre eles, e o efeito aleatório de doadora. Os resultados demonstraram que novilhas Nelore apresentaram melhor eficiência para a produção de embriões in vitro do que as novilhas Holandesas ou bubalinas, e que a aspiração folicular realizada em diferentes momentos da onda folicular sincronizada não influenciou a produção in vitro. / The objective of the present study was to determine in which phase of follicular wave higher recovery rates and better oocyte competence can be obtained by ovum pickup (OPU) in crossbred (Bos indicus x Bos taurus; Chapter 1) or buffalo (Bubalus bubalis), Nelore (Bos indicus) and Holstein heifers (Bos taurus; Chapter 2). In Chapter 1, 30 crossbred heifers were submitted to OPU on days 2 (D2), 4 (D4) or 6 (D6) after follicular wave emergence (n=10/ group), in a cross-over design performed in 3 replicates. Oocytes recovered were selected and those viable were submitted to in vitro embryo procedures. Data were analyzed by ANOVA, using PROC MIXED, in which time of OPU, replicate and interaction between these factors were considered as fixed effects, and donor was considered as a random effect. Higher blastocyst rate (6 days after IVF), number of blastocysts (9 days after IVF), and number of nuclei of hatched embryos were produced when OPU was done on D2. However, OPU on D6 also produced a higher blastocyst rate (6 days after IVF), and did not differ from other times of OPU concerning number of nuclei of hatched embryos. In Chapter 2, two experiments were performed. In Experiment 1, the effect of 50 mg of injectable progesterone was evaluated in the synchronization of follicular wave emergence of Nelore, Holstein, and buffalo heifers submitted to a progestin plus 2 mg of estradiol benzoate protocol (n=10/ group). Data were analyzed by logistic regression, by PROC GLIMMIX. The time of follicular wave emergence was not affected by administration of injectable progesterone. Besides, follicular wave emergence occurred between 4.2 and 4.5 days after the beginning of synchronization protocol for all genetic groups. In Experiment 2, the effects of time of OPU relative to follicular wave emergence (1, 3 or 5 days) and of genetic group (Nelore, Holstein, and buffalo) on in vitro embryo production were evaluated. A total of 27 heifers (9 of each genetic group, assigned in the 3 times of OPU) were submitted to 6 OPU sessions, in a cross-over design. Oocytes recovered were selected and those viable were submitted to in vitro embryo procedures. Data were analyzed by ANOVA, using PROC MIXED, in which time of OPU, genetic group, replicate and interaction between these factors were considered as fixed effects, and donor was considered as a random effect. Results demonstrated that Nelore heifers showed a better efficiency for in vitro embryo production than Holstein or buffalo heifers. Additionally, OPU performed at different times of synchronized follicular wave did not influence in vitro embryo production.