Altas taxas de mortalidade embrionária ocorrem no período de implantação em embriões provenientes de IATF e FIV. Acredita-se que a falha no desenvolvimento do saco vitelino possa estar relacionada com estas perdas embrionárias. O objetivo deste trabalho foi descrever as características macroscópicas, microscópicas e expressão gênica do processo de involução do saco vitelino, visto que os dados da literatura são contraditórios ao descrever os processos de formação e involução do saco vitelino em embriões provenientes de IATF e FIV. Foram utilizados 19 embriões de IATF e 16 de FIV, provenientes da Sociedade Agropecuária Imaculada Conceição-Redenção/PA, Brasil. Observou-se que não houve diferença significativa no tamanho dos embriões nas idades de 25 (p=0,1), 35 (p=0,1), 40 (p=0,4) e 45 (p=0,36) dias, quando comparados os grupos de IATF e FIV. Entretanto entre os embriões de IATF e FIV com 30 dias de gestação, apresentaram diferença estatística (p= 0,03). A curva de crescimento (Crow-Rump) aumentou gradativamente e linearmente em função da idade gestacional. Macroscopicamente, observou-se que aos 25 dias de gestação, o saco vitelino apresentava formato de T e coloração translúcida. Uma porção central se encontrava próxima a região ventral do embrião emitindo duas extremidades que eram finas membranas, estas acompanhavam o alantóide juntamente com os vasos umbilicais. Aos 30 a 35 dias, apresentava coloração amarelada, sua parte central e extremidades bem diminuídas mostrando sinais de um início de regressão. Aos 40 a 45 dias, o seu processo de involução estava caracterizado, com extremidades enoveladas e região central assemelhando-se a um pequeno grão achatado que se encontrava em contato com o âmnio. Microscopicamente, o saco vitelino era formado por três camadas: o endoderma, camada única que reveste a cavidade vitelínica, uma camada intermediária mesenquimal vascular e o mesotélio, camada simples voltada ao exoceloma. O epitélio do saco vitelino formava dobras ou criptas que se projetavam para a luz. Imunohistoquímica notou-se marcação para os anticorpos VEGF, Flt-1 e KDR. Observou-se que não houve diferença significativa na expressão do mRNA do VEGF, KDR e Flt-1 entre os grupos e nas idades gestacionais analisadas, aos 30 dias notou-se uma tendência entre os grupos em relação a expressão do Flt-1. Os fatores de crescimento b FGF, FGFR-1 e FGFR-2, não apresentaram diferenças em suas expressões, somente aos 35 dias de gestação para o mRNA FGFR-1. Conclui-se que o saco vitelino está intensamente relacionado com o desenvolvimento embrionário, na qual sua involução e desaparecimento relacionam-se com o crescimento e \"maturação\" embrionária. / High mortality rates occur during embryonic implantation embryos from IVF and FTAI. It is believed that the failure in the development of the yolk sac may be related to these embryonic losses. The objective of this study was to describe the macroscopic, microscopic and mRNA expression features of the yolk sac involution process, since the literature relates contradictory information regarding the processes of formation and involution of the yolk sac in IVF and TAI embryos. Nineteen embryos were used for FTAI and 16 IVF; the specimes were obtained from the Sociedade Agropecuária Imaculada Conceição-Redenção/PA, Brazil. No significant differences were observed in the size of embryos aged 25 (p = 0.1), 35 (p = 0.1), 40 (p = 0.4) and 45 (p = 0.36) days in the groups of IVF and FTAI. However between FTAI and IVF embryos with 30 days of gestation, a difference (p = 0.03) was detectable. The growth curve (Crow-Rump) increased gradually and linearly with gestational age. Macroscopically, it was observed that after 25 days of gestation, the yolk sac was \"T-shaped\" and transparent colored. A central portion was observed in the vicinity of the ventral ends of the embryo exhibiting two thin membranes, which accompanied the allantois with the umbilical vessels. At 30 to 35 days this portion becomes yellowish and its central part together with the extremities was reduced displaying clearly signs of regression. At 40 to 45 days, the process of involution was presented in a curled fashion and ends with the central region resembling a flat small grain in contact with the amnion. Microscopically, the yolk sac was formed by three layers: the endoderm, single layer lining the vitelline cavity, an intermediate layer and vascular mesenchymal mesothelium, exoceloma focused on single layer. The epithelium of the yolk sac formed folds or crypts, which projected to the light. Immunohistochemistry marking was noted for antibodies VEGF, Flt-1 and KDR. No significant difference were observed in mRNA expression of VEGF, KDR and Flt-1 between groups and the gestational ages analyzed at 30 days there has been a trend among the groups regarding the expression of Flt-1. The growth factors bFGF, FGFR-1 and FGFR-2 showed no differences in their expressions, with the exception of 35 days of gestation for the m-RNA FGFR 1. It was concluded that the yolk sac is strongly related to embryonic development, in which the involution and disappearance are related to growth and \"maturity\" stage.

Altas taxas de mortalidade embrionária ocorrem no período de implantação em embriões provenientes de IATF e FIV. Acredita-se que a falha no desenvolvimento do saco vitelino possa estar relacionada com estas perdas embrionárias. O objetivo deste trabalho foi descrever as características macroscópicas, microscópicas e expressão gênica do processo de involução do saco vitelino, visto que os dados da literatura são contraditórios ao descrever os processos de formação e involução do saco vitelino em embriões provenientes de IATF e FIV. Foram utilizados 19 embriões de IATF e 16 de FIV, provenientes da Sociedade Agropecuária Imaculada Conceição-Redenção/PA, Brasil. Observou-se que não houve diferença significativa no tamanho dos embriões nas idades de 25 (p=0,1), 35 (p=0,1), 40 (p=0,4) e 45 (p=0,36) dias, quando comparados os grupos de IATF e FIV. Entretanto entre os embriões de IATF e FIV com 30 dias de gestação, apresentaram diferença estatística (p= 0,03). A curva de crescimento (Crow-Rump) aumentou gradativamente e linearmente em função da idade gestacional. Macroscopicamente, observou-se que aos 25 dias de gestação, o saco vitelino apresentava formato de T e coloração translúcida. Uma porção central se encontrava próxima a região ventral do embrião emitindo duas extremidades que eram finas membranas, estas acompanhavam o alantóide juntamente com os vasos umbilicais. Aos 30 a 35 dias, apresentava coloração amarelada, sua parte central e extremidades bem diminuídas mostrando sinais de um início de regressão. Aos 40 a 45 dias, o seu processo de involução estava caracterizado, com extremidades enoveladas e região central assemelhando-se a um pequeno grão achatado que se encontrava em contato com o âmnio. Microscopicamente, o saco vitelino era formado por três camadas: o endoderma, camada única que reveste a cavidade vitelínica, uma camada intermediária mesenquimal vascular e o mesotélio, camada simples voltada ao exoceloma. O epitélio do saco vitelino formava dobras ou criptas que se projetavam para a luz. Imunohistoquímica notou-se marcação para os anticorpos VEGF, Flt-1 e KDR. Observou-se que não houve diferença significativa na expressão do mRNA do VEGF, KDR e Flt-1 entre os grupos e nas idades gestacionais analisadas, aos 30 dias notou-se uma tendência entre os grupos em relação a expressão do Flt-1. Os fatores de crescimento b FGF, FGFR-1 e FGFR-2, não apresentaram diferenças em suas expressões, somente aos 35 dias de gestação para o mRNA FGFR-1. Conclui-se que o saco vitelino está intensamente relacionado com o desenvolvimento embrionário, na qual sua involução e desaparecimento relacionam-se com o crescimento e \"maturação\" embrionária. / High mortality rates occur during embryonic implantation embryos from IVF and FTAI. It is believed that the failure in the development of the yolk sac may be related to these embryonic losses. The objective of this study was to describe the macroscopic, microscopic and mRNA expression features of the yolk sac involution process, since the literature relates contradictory information regarding the processes of formation and involution of the yolk sac in IVF and TAI embryos. Nineteen embryos were used for FTAI and 16 IVF; the specimes were obtained from the Sociedade Agropecuária Imaculada Conceição-Redenção/PA, Brazil. No significant differences were observed in the size of embryos aged 25 (p = 0.1), 35 (p = 0.1), 40 (p = 0.4) and 45 (p = 0.36) days in the groups of IVF and FTAI. However between FTAI and IVF embryos with 30 days of gestation, a difference (p = 0.03) was detectable. The growth curve (Crow-Rump) increased gradually and linearly with gestational age. Macroscopically, it was observed that after 25 days of gestation, the yolk sac was \"T-shaped\" and transparent colored. A central portion was observed in the vicinity of the ventral ends of the embryo exhibiting two thin membranes, which accompanied the allantois with the umbilical vessels. At 30 to 35 days this portion becomes yellowish and its central part together with the extremities was reduced displaying clearly signs of regression. At 40 to 45 days, the process of involution was presented in a curled fashion and ends with the central region resembling a flat small grain in contact with the amnion. Microscopically, the yolk sac was formed by three layers: the endoderm, single layer lining the vitelline cavity, an intermediate layer and vascular mesenchymal mesothelium, exoceloma focused on single layer. The epithelium of the yolk sac formed folds or crypts, which projected to the light. Immunohistochemistry marking was noted for antibodies VEGF, Flt-1 and KDR. No significant difference were observed in mRNA expression of VEGF, KDR and Flt-1 between groups and the gestational ages analyzed at 30 days there has been a trend among the groups regarding the expression of Flt-1. The growth factors bFGF, FGFR-1 and FGFR-2 showed no differences in their expressions, with the exception of 35 days of gestation for the m-RNA FGFR 1. It was concluded that the yolk sac is strongly related to embryonic development, in which the involution and disappearance are related to growth and \"maturity\" stage.

<p dir="ltr">Nulliparous replacement beef heifers represent an opportunity to improve both genetic potential and lifetime production within the herd. However, advances in reproductive efficiency and synchrony in the herd require heifers to conceive earlier than multiparous cows in order to account for the extended postpartum interval following first-time parturition. Further, those heifers that achieve early calving dates continue to calve early throughout their lifetime. This early calving results in increased weaning weights and better synchrony in subsequent years. One obstacle producers face when breeding nulliparous heifers is the ability to identify which animals have reached puberty at the initiation of the breeding season. This results in the variation observed in synchronization success within a group of heifers. In Chapter 2 our laboratory formulated a study to analyze the efficacy of the 7-d CO-synch + CIDR protocol when utilizing short-term exposure to a progestin (melengestrol acetate, MGA) as a pre-synchronization protocol. Our hypothesis was that the heifers may respond more efficiently to an orally fed progestin as the increase in serum progesterone is less pronounced, when compared to the CIDR. This first study resulted in a protocol application error, in which MGA was fed an extra day (8 vs. 7). This additional day of treatment with MGA following the administration of a prostaglandin eliminated our laboratory’s opportunity to collect meaningful data from this first attempt. Thus, it was pertinent to restart the experiment in an attempt to obtain data for analysis. In order to avoid confounding data, half of the heifers in each treatment received a new treatment, while the other half remained on the treatment assigned during the first attempt. After completing the second round of experiments it was observed that at the first pregnancy check (study day 30), the heifers that were originally assigned to MGA, but were switched to a CIDR for the second attempt numerically outperformed their counterparts in the other three formulated treatment groups. In an attempt to replicate these results, a second study was formulated (Chapter 3), to mimic the timeline from the preliminary study. This resulted in a pre-synchronization treatment protocol prior to the initiation of the breeding season synchronization protocol. Previous studies that have been conducted with similar protocols were designed to provide heifers with a pre-synchronization period that would aid in the attainment of puberty prior to their first attempt at breeding through exposure to progesterone. The purpose of the Chapter 3 experiment was to evaluate the effects of feeding melengestrol acetate (MGA®) as a pre-synchronization for 10 days immediately prior to estrous synchronization and fixed-timed artificial insemination (FTAI). The ten days were chosen as this was a novel protocol that was the result of the preliminary study and the subsequent restart. Ninety-three crossbred heifers (395.67 ± 5.37 kg) were blocked by BW, genetics, and reproductive tract scores and allotted to 3 treatments. The 3 treatment groups were: 7-day CO-synch + CIDR without pre-synchronization (CON, n=31); 25 mg PGF2α (Lutalyse®) followed immediately by MGA feeding for 7 d prior to the start of the 7-day CO-synch + CIDR program (PRE, n=31); and 25 mg PGF2α followed immediately by MGA feeding for 7d followed 10 d later by the start of the 7-day CO-synch + CIDR program (PRE+10, n=31). The 7-day Co-synch + CIDR protocol in all three treatments was initiated on d 0 by administering a 2 cc IM injection of GnRH (Cystorelin®) and placing a CIDR into the vagina. The CIDRs were removed 7 days later and accompanied by 25 mg IM injection of PGF2α. An injection of GnRH occurred 60-66 hours following PGF2α at FTAI with frozen semen from a single bull. Ten days after FTAI, heifers were exposed to a bull. Estrotect® patches were applied throughout the study to assess estrous behavior. Ovaries were visualized by transrectal ultrasonography 24 hours post-FTAI to determine whether ovulation occurred. Pregnancy was determined on days 40, 64, and 109 post-FTAI via transrectal ultrasonography. Blood was collected via jugular venipuncture (d -19 and -12, MGA initiation and termination, days 0, 7, and 9) and serum progesterone determined. Performance data were analyzed using the MIXED procedure and conception data were analyzed using the GLIMMIX procedure of SAS. Heifers in the PRE+10 treatment group had higher levels of progesterone (P=0.04) at d 0 compared to PRE heifers. At d 7 (CIDR removal) there was a tendency (P=0.07) for PRE+10 heifers to have higher levels of progesterone than PRE, but did not differ by d 9 (FTAI, P=0.36). FTAI conception rates in heifers in the PRE treatment group (63%) tended (P = 0.09) to be higher compared to the controls (35%), but not different from PRE+10 (43%), with no differences in season-long pregnancy rates (P > 0.15). Pre-synchronization with MGA immediately prior to FTAI synchronization appears to increase conception rates early in the breeding season in beef heifers. In Chapter 4, our laboratory analyzed the efficacy of supplementation strategies post-insemination. Unlike the first two experiments, the third study focused on reproductive failure that occurs after insemination. The overarching goal remained the same, increasing the reproductive efficiency within our nulliparous heifer herd. A common practice for beef producers in the United States is to use estrous synchronization, and immediately turn heifers out to lush spring pasture immediately following FTAI. The fresh forage is high in water content which lowers dry matter intake (DMI) and creates a negative energy balance. Ultimately, this reduced energy intake can result in a reduction in reproductive performance. In the Chapter 4 our laboratory formulated a supplementation strategy utilizing the SmartFeed™ technology to deliver soybean hulls to our treatment group following insemination for 45 days. The utilization of the SmartFeed® technology provided the opportunity to analyze the efficacy of the supplementation strategy using each individual animal as an experimental unit. Sixty-two nulliparous crossbred heifers were fed in drylot to obtain moderate body condition prior to breeding at d 0 via FTAI and trained to utilize the SmartFeed™ system.Heifers were blocked by weight and body condition score to either the soybean hull supplementation group (SOY) or the non-supplemented control group (CON). . Heifers in the treatment group received their supplementation by entering the SmartFeed™ system, allowing for RFID controlled release of 2.27 kg. per head each day for 45 days. Scales located beneath each feed pan sent real time weight data for regulation and analysis on individual animal feeding behavior. On study d 6, a subset of the nulliparous crossbred heifers (n = 12; n = 6/ treatment) were transported by trailer from the Feldun Purdue Agricultural Center to Purdue West Lafayette main campus (approximately 217.74 km.). The subset of heifers had embryos flushed and evaluated for embryo quality and number of live/dead cells. Ultrasonography was utilized to monitor ovarian activity throughout the duration of the study and to determine pregnancy status 30 days after FTAI and 30 days after the 45-day breeding season. Though conception rates were not statistically (P=0.17) different (SOY 16/25; 64% vs. CON 11/25; 44%) when comparing treatment groups, the numerical differences suggest there is potential in pursuing a similar supplementation strategy following breeding in nulliparous beef heifers. The supplementation of SBH resulted in greater weight gain over time (P = 0.04), potentially explaining the numeric improvement in conception rate. The two pre-synchronization studies from Chapters 2 and 3 resulted in numerical improvements in conception rate as a result of exposing replacement heifers to a source of progestin prior to the initiation of their synchronization protocol. The implementation of progesterone priming mitigates the occurrence of short cycles and immature oocyte maturation at the time of ovulation. Based on results from previous studies conducted by our laboratory, the source of progestin greatly determines the timing and concentration of P4 circulating in replacement heifers immediately following treatment. Additionally, the 7 & 7 estrous synchronization protocol has grown in popularity and implementation. This protocol when broken down is very similar to the pre-synchronization protocol our laboratory utilized for the first two studies. Several studies have been conducted to analyze the efficacy of the 7 & 7 and the conception data was comparable to the MGA derived protocol. Thus, one potential direction for the future would be to formulate a research study that compares the two protocols. Since the conception rates were similar, it may be hypothesized that the MGA protocol could be more widely accepted as there is a reduction number of times animals are handled.</p>