Progesterone is unequivocally required for the establishment and maintenance of pregnancy in all mammals studied. Its known functions are complex and encompass global changes in gene expression. Therefore, studies were conducted to characterize the effects of progesterone on expression of genes for endometrial factors having roles in conceptus growth, implantation and establishment of pregnancy. The first study characterized the effect of an artificially induced early increase in circulating progesterone on conceptus growth and development and regulation of expression of galectin-15 (LGALS15), a recently identified protein secreted by the ovine uterine luminal epithelium (LE). Exogenous progesterone beginning on Day 1.5 post-mating accelerated conceptus development on Days 9 and 12. On Day 12 the conceptus was functionally and morphologically advanced to produce greater quantities of interferon tau (IFNT) than blastocysts from control ewes. Further, the endometrium responded to early progesterone and IFNT with early expression of cathepsin L (CTSL), radical S-adenosyl methionine domain containing 2 (RSAD2), and LGALS15 within the endometrium. The second study identifed structural changes within the luminal epithelium which could alter the flux of factors into and out of the uterine lumen to maintain appropriate fetal/maternal communication. In this study, progesterone reduced quantities of proteins associated with both tight and adherens junctions during the elongation period. IFNT subsequently increased these proteins after conceptus elongation. The third and fourth studies identified progesterone-regulated genes which have been implicated as having importance to implantation in sheep, mouse, and human. WNT signaling was transiently downregulated by progesterone, while members of several growth factor families are upregulated including insulin-like growth factor binding proteins (IGFBPs) 1 and 3, hepatocyte growth factor (HGF) and fibroblast growth factor 7 (FGF7), which may enhance conceptus growth. Collectively, these studies assess the role of progesterone in altering gene uterine expression to establish a favorable environment for conceptus development. The long-term goals of these studies are to establish biomarkers of receptivity to conceptus development and implantation, enhance our understanding of gene and pathway regulation in early pregnancy loss, and identify genes which may be targeted in therapeutic strategies to improve reproductive success in humans and animals.

Progesterone is unequivocally required for the establishment and maintenance of pregnancy in all mammals studied. Its known functions are complex and encompass global changes in gene expression. Therefore, studies were conducted to characterize the effects of progesterone on expression of genes for endometrial factors having roles in conceptus growth, implantation and establishment of pregnancy. The first study characterized the effect of an artificially induced early increase in circulating progesterone on conceptus growth and development and regulation of expression of galectin-15 (LGALS15), a recently identified protein secreted by the ovine uterine luminal epithelium (LE). Exogenous progesterone beginning on Day 1.5 post-mating accelerated conceptus development on Days 9 and 12. On Day 12 the conceptus was functionally and morphologically advanced to produce greater quantities of interferon tau (IFNT) than blastocysts from control ewes. Further, the endometrium responded to early progesterone and IFNT with early expression of cathepsin L (CTSL), radical S-adenosyl methionine domain containing 2 (RSAD2), and LGALS15 within the endometrium. The second study identifed structural changes within the luminal epithelium which could alter the flux of factors into and out of the uterine lumen to maintain appropriate fetal/maternal communication. In this study, progesterone reduced quantities of proteins associated with both tight and adherens junctions during the elongation period. IFNT subsequently increased these proteins after conceptus elongation. The third and fourth studies identified progesterone-regulated genes which have been implicated as having importance to implantation in sheep, mouse, and human. WNT signaling was transiently downregulated by progesterone, while members of several growth factor families are upregulated including insulin-like growth factor binding proteins (IGFBPs) 1 and 3, hepatocyte growth factor (HGF) and fibroblast growth factor 7 (FGF7), which may enhance conceptus growth. Collectively, these studies assess the role of progesterone in altering gene uterine expression to establish a favorable environment for conceptus development. The long-term goals of these studies are to establish biomarkers of receptivity to conceptus development and implantation, enhance our understanding of gene and pathway regulation in early pregnancy loss, and identify genes which may be targeted in therapeutic strategies to improve reproductive success in humans and animals.

The objective of this study was to determine concentrations of amino acids and polyamines as well as nitric oxide (NO) and polyamine synthesis in the ovine conceptus (embryo/fetal and associated placental membrane). Ewes were hysterectomized on Days 30, 40, 60, 80, 100, 120, or 140 of gestation to obtain allantoic and amniotic fluids, intercotyledonary placenta, placentomes and uterine endometrium for the analyses. Alanine, citrulline plus glutamine accounted for about 80% of total α-amino acids in allantoic fluid during early gestation. Serine (16.5 mM) contributed about 60% of total α-amino acids in allantoic fluid on Day 140 of gestation. Maximal ornithine decarboxylase (ODC) and arginase activities and highest rates of polyamine and NO synthesis occured in all tissues on Day 40 of gestation. In ovine allantoic and amniotic fluids, polyamines were most abundant during early (Days 40-60) and late (Days 100-140) gestation, respectively. Activity of guanosine 5??-triphosphate-cyclohydrolase I (GTP-CH), and concentrations of NOS cofactors, tetrahydrobiopterin (BH4) and NADPH (nicotinamide adenine dinucleotide), peaked on Day 40 of gestation in placental and endometrial tissues. In these tissues, NO synthesis was positively correlated with total NOS activity, GTP-CH activity, and concentrations of BH4 and NADPH. The physiological significance of these changes was manifested by undernutrition-induced intrauterine growth retardation (IUGR). Maternal undernutrition (50% of National Research Council nutrient requirements) reduced concentrations of total α-amino acids in fetal plasma and fluids, and retarded fetal growth at both mid (Day 78) and late (Day 135) gestation. Concentrations of polyamines in fetal fluids were lower in underfed ewes than in control-fed ewes. Realimentation of underfed ewes between Days 78 and 135 of gestation increased concentrations of total α-amino acids and polyamines in fetal plasma and fluids, when compared with non-realimented ewes. Results of these studies demonstrate metabolic coordination among the several integrated pathways to enable high rates of polyamine and NO synthesis in the placenta and endometrium during early pregnancy. Collectively, our findings may have important implications for both IUGR and fetal origins of adult disease.

Histotroph, secretions from luminal (LE), superficial glandular (sGE) and glandular (GE) epithelia and molecules selectively transported into the uterine lumen, are essential for peri-implantation ovine conceptus development and maternal recognition of pregnancy. Among them, several components of histotroph including nutrients, cell matrix proteins and growth factors may activate mTOR (mammalian target of rapamycin; also known as FRAP1) to stimulate hypertrophy, hyperplasia, and/or migration of conceptus trophectoderm cells, as well as expression of IFNT for pregnancy recognition and critical proteins for conceptus development. Therefore, studies were conducted to examine effects of select nutrients (arginine, leucine, glutamine and glucose), IGF2 and SPP1 on mTOR signal transduction pathways and determine their biological effects on proliferation, migration and/or attachment of ovine trophectoderm (oTr) cells and conceptuses (embryo and it extra-embryonic membranes). The first study defined the expression of IGF2, RPS6K, phosphorylated AKT, RPS6K, P38 and ERK1/2 MAPK by the uterus and conceptus during the periimplantation period. In addition, effects of IGF2 on the PI3K signaling pathway were evaluated using oTr cells isolated from Day 15 conceptuses. IGF2 was most abundant in compact stroma of endometrial caruncles and also present in all cells of the conceptus, but particularly abundant in the endoderm and yolk sac. Phosphorylated AKT1, RPS6K, P38 and ERK1/2 proteins were abundant in nuclei of endometrial LE and conceptus trophectoderm. IGF2 activated multiple cell signaling pathways including PDK/AKT/mTOR/RPS6K and MAPKs that are critical to survival, growth and migration of the ovine trophoblast cells. The second study demonstrated the multifunctional effects of secreted phosphoprotein 1 (SPP1) on oTr cells including cell signaling transduction, migration, and adhesion. Novel results of this study indicated that SPP1 binds ?v?3 and ?5?1 integrins to activate PI3K/mTOR/RPS6K, MAPK as well as crosstalk between mTOR and MAPK pathways that are essential for expansion and elongation of conceptuses and attachment of trophectoderm to uterine LE during implantation. The third study identified effects of arginine (Arg), leucine (Leu), glutamine (Gln) and glucose on oTr cells. Arg, Leu and glucose, but not Gln, activated PI3KAKT1 and mTOR-RPS6K-RPS6 signaling pathways. Arg, Leu and glucose increased abundance of p-RPS6K in nuclei and p-RPS6 in cytoplasm of oTr cells. In addition, results of this study demonstrated that Arg and Leu are remarkably stimulatory to cell proliferation and migration. The fourth study determined effects of Arg on signal transduction pathways and oTr cell proliferation, as well as inhibition of oTr cell proliferation by L-NAME (an inhibitor of NOS) or Nor-NOHA (an inhibitor of arginase) on oTr cells. Arg increased p-mTOR, RPS6K and 4EBP1 protein and also increased protein synthesis and reduced protein degradation in oTr cells. Both NO and polyamines enhanced cell proliferation in a dose-dependent manner. The effects of Arg were partially inhibited by both L-NAME and Nor-NOHA. These results indicate that Arg enhances production of polyamines and NO and activates the mTOR-FRAP1-RPS6K-RPS6 signaling pathway to stimulate proliferation of oTr. The fifth study identified differential effects of Arg, Leu, Gln and glucose on gene expression and protein translation in explants cultures of ovine conceptuses. Expression of mRNAs was not affected by treatments with the select nutrients; however, Arg, Leu, Gln and glucose increased abundance of total and phosphorylated forms of mTOR, RPS6K, 4E-BP1 and RPS6. Arg, Leu, Gln and glucose also increased the amounts of NOS and ODC1, but only Arg stimulated a significant increase in abundance of IFNT. Collectively, these studies indicated that IGF2, SPP1 and select nutrients activate mTOR cell-signaling pathways that converge on AKT1 and that are likely critical to mechanism(s) responsible for survival, elongation an development of conceptuses. A more complete understanding of this mechanism will be important to development of strategies to reduce early embryonic losses in ruminants and and in other species including humans.

The objective of this study was to determine concentrations of amino acids and polyamines as well as nitric oxide (NO) and polyamine synthesis in the ovine conceptus (embryo/fetal and associated placental membrane). Ewes were hysterectomized on Days 30, 40, 60, 80, 100, 120, or 140 of gestation to obtain allantoic and amniotic fluids, intercotyledonary placenta, placentomes and uterine endometrium for the analyses. Alanine, citrulline plus glutamine accounted for about 80% of total α-amino acids in allantoic fluid during early gestation. Serine (16.5 mM) contributed about 60% of total α-amino acids in allantoic fluid on Day 140 of gestation. Maximal ornithine decarboxylase (ODC) and arginase activities and highest rates of polyamine and NO synthesis occured in all tissues on Day 40 of gestation. In ovine allantoic and amniotic fluids, polyamines were most abundant during early (Days 40-60) and late (Days 100-140) gestation, respectively. Activity of guanosine 5??-triphosphate-cyclohydrolase I (GTP-CH), and concentrations of NOS cofactors, tetrahydrobiopterin (BH4) and NADPH (nicotinamide adenine dinucleotide), peaked on Day 40 of gestation in placental and endometrial tissues. In these tissues, NO synthesis was positively correlated with total NOS activity, GTP-CH activity, and concentrations of BH4 and NADPH. The physiological significance of these changes was manifested by undernutrition-induced intrauterine growth retardation (IUGR). Maternal undernutrition (50% of National Research Council nutrient requirements) reduced concentrations of total α-amino acids in fetal plasma and fluids, and retarded fetal growth at both mid (Day 78) and late (Day 135) gestation. Concentrations of polyamines in fetal fluids were lower in underfed ewes than in control-fed ewes. Realimentation of underfed ewes between Days 78 and 135 of gestation increased concentrations of total α-amino acids and polyamines in fetal plasma and fluids, when compared with non-realimented ewes. Results of these studies demonstrate metabolic coordination among the several integrated pathways to enable high rates of polyamine and NO synthesis in the placenta and endometrium during early pregnancy. Collectively, our findings may have important implications for both IUGR and fetal origins of adult disease.

Decidualization involves the differentiation of the endometrial tissue into the decidual tissue of the pregnant uterus in several species including humans and rodents. This differentiation occurs only after the onset of implantation in mice and can be artificially-induced causing the formation of deciduomal tissue. The purpose of this study was to identify a group of differentially expressed genes between the developing decidua and deciduoma and study their expression as it may relate to conceptus influenced changes in endometrial gene expression during decidualization. In this study we artificially induced decidualization by transferring blastocyst-sized ConA-coated agarose beads into the uterus on Day 2.5 of pregnancy as we had previously found this model to be more "physiological". Total RNA was isolated from implantation sites of the uteri of pregnant mice as well as pseudopregnant mice that received beads. This RNA was then used for microarray analysis using Mouse Illumina Beadarray chips. This revealed potential differential mRNA levels of over 1,000 genes between the decidua and bead-induced deciduoma tissues of Day 7.5 pregnant and pseudopregnant mice, respectively. Of these, the mRNA levels of 102 genes were 2-fold greater in the decidual tissue while almost twice as many were 2-fold greater in the deciduoma. The broad functions of the protein encoded by the mRNAs included protein binding (e.g. Copz1, Gjb2, Dctn1, Islr, Nisch, Wwc1, Cdc20, Rxrb, Klhl7, Adam10), calcium transport (e.g. Anxa6, Itga11, Clta, Smoc2, Vdr), hydrolase/peptidase activity (e.g. Klk5, Klk26, Klk24, Tmprss4, Ptpn14, Ddx3x, Atp1a2, Usp25, Smarca1), ligase activity (e.g. Iars, Farsla, Ube2v1, Cbll1, Rnf19, Mccc1), and transcription (e.g. Irf1, Hip1, Bhlha15, Supt6h, Scand1, Myocd, Sp3, Mitf, Papolg). We confirmed the differential mRNA levels of a number of gene transcripts using quantitative RT-PCR. Finally, the level and localization of some of the mRNA's identified by our microaray analysis were examined in the mouse uterus during decidualization in more detail and included: Aldh3a1, Bcmo1, Guca2b, GCC, and Inhbb. Localization of mRNA expression in the Day 7.5 implantation site occurred in the mesometrial region near the lumen (Aldh3a1), luminal and glandular epithelia (Guca2b), and endothelial cells lining the sinusoids (Inhbb). This study provides the identity and expression analysis of steady-state mRNA levels of genes whose expression may be influenced by the conceptus using a physiological model for implantation.

Early embryogenesis is highlighted by the emergence of several embryonic end extraembryonic lineages. One such lineage is the primitive endoderm, which will eventually give rise to the yolk sac. Once believed to be a vestigial structure, the yolk sac is now believed to play a more prominent role in embryogenesis as it provides nutrients to the preimplantation embryo. The endoderm may also interact with the trophectoderm lineage, as they develop in close contact within the embryo. The efficiency of developing primitive endoderm in vitro is considerably low, leading to a lapse in our understanding of its development and function in cattle and other ruminants. The goal of the first study was to establish a protocol for developing primitive endoderm cultures and characterizing these cells. Bovine embryos were produced in vitro, and primitive endoderm outgrowths were created with fibroblast growth factor 2 (FGF2) supplementation. These cells can be produced in culture with 80.3 5.6% efficiency. Furthermore, outgrowths can be maintained in culture for 6-8 weeks before reaching a quiescent state. A true bovine primitive endoderm cell line does not currently exist, however, these cells hold potential in improving the current understanding of early lineage specification in cattle. A second set of studies was performed to examine the effects of maternal obesity on the preimplantation conceptus and endometrium. Exposure to maternal obesity in utero affects offspring development at the postnatal, adolescent, and adult stages of development; however, its impacts on early embryogenesis are not well studied. This work utilized an obese ewe model. Once the obese phenotype was established, ewes were bred. Conceptus and endometrial tissue were collected at D 14 of pregnancy, and samples were processed for RNA-sequencing analysis. There were no differences in pregnancy rate, ovulation rate, or pregnancies/ovulation between obese and lean animals. At an RPKM threshold of 0.2, fold-change 2, and FDR 0.05, 669 and 21 differentially expressed genes (DEGs) were identified between obese- and lean-derived endometrial samples and conceptus samples, respectively. Likewise, 137 DEGs were identified between male and female conceptuses. The PANTHER GO-Slim Biological Process system identified several biological processes affected by obesity in both the endometrium and conceptus tissue. GO terms do not currently exist for "placenta" and "trophoblast", so a literature search was conducted to identify DEGs involved in implantation and placentation. This revealed 125 placentation DEGs in the endometrium, and 4 DEGs in conceptuses between obese and lean groups. A follow-up study was conducted to examine the abundance of transcripts with regulatory roles in embryogenesis. Conceptuses exhibited differential expression of DNA methyltransferase 1 (DNMT1) based on obesity exposure, fibroblast growth factor receptor 2 (FGFR2) in a sex*obesity interaction, and peroxisome proliferator-activated receptor gamma (PPARG) and prostaglandin-endoperoxide synthase 2 (PTGS2) in a sex-specific manner. Collectively these results identify the preimplantation period as a susceptible time to maternal obesity in both conceptus and endometrial tissue. Together, these studies aim to provide a better understanding of the events controlling early embryogenesis, and insight into the implication of insults during this time. These findings will prove to be beneficial in establishing the link between maternal health, endometrial function, and subsequent offspring outcomes, with the hope of promoting a more viable embryo and thus healthier offspring. / Ph. D. / Early embryogenesis in cattle is afflicted with high embryonic loss, costing the dairy and beef industries a fair portion of their profits. The mechanisms behind these losses are not well understood, however, cellular miscommunications during lineage specification are likely to blame. Of particular interest is the endoderm lineage, which gives rise to the embryonic yolk sac. Initially believed to be a transient structure, we now believe the yolk sac is indispensable in embryogenesis as it provides nutrients to the preimplantation embryo. Our current understanding of primitive endoderm and the resulting yolk sac in cattle is severely lacking because few primitive endoderm in vitro models exist. A portion of the following work is focused on developing a protocol for producing primitive endoderm cell lines in vitro. This work improved the rate of producing primitive endoderm cells in vitro and characterized those cells. These cells will be used as a tool to better understand the mechanisms involved in early embryogenesis. Furthermore, they may help identify targets for manipulating early development to lessen the high rate of embryonic loss in cattle. The stage of early embryogenesis may also be particularly susceptible to intrauterine stressors, such as maternal obesity, because of the lineage segregation events occurring at this time. Insults to the earliest lineages can have lasting developmental effects, as these cell types will give rise to the embryo proper, yolk sac, and placenta. The effects of maternal obesity have been extensively studied in the postnatal, adolescent, and adult stages of development, however, insights into the effects on early embryogenesis are missing. The final studies of the following work are focused on the effects of maternal obesity on the preimplantation ovine conceptus and endometrium. This work utilized RNA-sequencing technology as well a qRT-PCR to identify differential gene and transcript expression in conceptus and endometrial samples collected from lean and obese ewes. Following analysis, we identified several crucial biological processes affected by maternal obesity. Of particular interest were those involved in implantation and placentation, indicating developmental programming events during early embryogenesis may be at fault for the abnormal offspring outcomes observed in previous studies. This work highlights the susceptibility of the preimplantation conceptus to maternal obesity and identifies the endometrium as a mediator between maternal nutrition and conceptus development. Additionally, this work identifies alterations in genes involved in placentation in both the conceptus and endometrium, indicating developmental programing events have occurred. As a whole, this work developed a new tool for examining early embryogenesis and the specification events that occur during that time. It also examines the embryonic impacts of maternal obesity during that critical window of development. These findings will prove to be invaluable in factors involved in early embryo development and function in ruminant species.

The progesterone (P4) produced by the corpus luteum (CL) is essential for maintenance of pregnancy. On the other hand, the interferon tau (IFNT) produced by the embryo during elongation process, besides being the primary signal for recognition, also is responsible for maintenance of the CL during early pregnancy. The presence of oxytocin receptors (OXTR) in endometrium during expected time of luteolysis is determinant for trigger the uterine release of prostaglandin F2∝ (PGF), which is in charge of CL regression. The IFNT avoid the luteolysis by suppressing the OXTR appearance. However, during second month, accessory CLs are able to regress, indicating that the PGF release occurs with the advancing of the pregnancy and the mechanisms that initiated luteolysis are recovered. Therefore, failures in maintenance of the CL can cause luteolysis and pregnancy loss during this period of 30 to 60 days, which is one of the most important problems in reproductive efficiency in cattle, specially when in vitro produced (IVP) embryos are transferred. Two experiments were designed to study this factors, focused on point when uterus recover its PGF release during pregnancy and to identify possible differences between those mechanisms on pregnancies from IVP or artificial insemination (AI) embryos. The first study evaluated circulating PGF metabolite (PGFM) after an oxytocin challenge throughout first two months of pregnancy in lactating Holstein cows. Treatment with oxytocin did not affected PGFM concentration in d11 pregnant (P) and non-pregnant (NP), on d18 had a little increase in P cows, while increased 2-fold in NP cows. Oxytocin-induced PGFM in P cows on day 25 was greater than d18 P, however was lower than P cows on d53 and d60. Days 32, 39 and 46 of pregnancy had intermediate response. The second study evaluated the oxytocin-induced PGFM in Nelore cows pregnant from AI or IVP embryos on days 17 and 31, and its association with factors that can impact in success of the pregnancy, such as P4 levels, conceptus length on d18 and size of the embryo on d32. Also, OXTR and interferon-stimulated gene 15 (ISG15) gene expression were evaluated and located in uterine endometrium. Embryo size on d32 and P4 on d31, were higher in AI than IVP. Cows from IVP on d17 presented lower oxytocin-induced PGFM than AI in the same day, however, d31 for both groups had higher PGF release after oxytocin. On d31 there was similar PGFM increase in synchronized non-inseminated group (NI). The OXTR are highly suppressed on pregnant cows on d18, especially in IVP group, but were high expressed in NI cows and on d32 for both groups, AI being higher than IVP at this day. The ISG15 had irrelevant expression on NI and d32 groups, while had extremely high expression in d18 pregnant cows for both groups. Concluding, the CL in early pregnancy is maintained by PGF release suppression, while during second month there is oxytocin-induced PGF release, suggesting that other mechanisms are responsible for maintaining CL after d25. In addition, these results demonstrate there are signaling differences between IVP and AI pregnancies, impacting the molecular and endocrine environment that influences PGF release during these time points. / A progesterona (P4) produzida pelo corpo lúteo (CL) é essencial para a manutenção da gestação. Por sua vez, o interferon tau (IFNT) produzido pelo embrião durante o processo de alongamento, além de ser o sinal primário para reconhecimento e manutenção da gestação também é responsável pela manutenção do CL durante a gestação inicial. A presença de receptores de ocitocina (OXTR) no endométrio no momento esperado da luteólise é determinante para liberação uterina de prostaglandina F2∝ (PGF), a qual é responsável pela regressão do CL. O IFNT evita a ocorrência da luteólise por meio da supressão da expressão de OXTR no endométrio. Entretanto, durante o segundo mês de gestação, CLs acessórios, principalmente contralaterais são capazes de regredir, indicando que ocorre liberação de PGF pelo útero conforme a gestação avança, e os mecanismos que iniciam a luteólise são restabelecidos. Portanto, falhas na manutenção do CL podem causar luteólise e perdas gestacionais de 30 para 60 dias, um dos importantes problemas de eficiência reprodutiva em bovinos, principalmente quando embriões produzidos in vitro (PIV) são transferidos. Dois estudos foram delineados para estudar estes fatores, com foco em determinar o momento em que o útero gravídico retoma a liberação de PGF, e identificar prováveis diferenças entre estes mecanismos em gestações de embriões PIV ou de inseminação artificial (IA). O primeiro estudo avaliou a concentração circulante do metabólito de PGF (PGFM) após desafio com ocitocina durante os primeiros dois meses de gestação em vacas Holandesas lactantes. O tratamento com ocitocina não afetou a concentração de PGFM em vacas de d11 prenhes (P) e não-prenhes (NP), no d18 apresentou um ligeiro aumento em vacas P, enquanto aumentou cerca de duas vezes em relação ao nível basal em vacas NP. O aumento de PGFM induzido por ocitocina em vacas P no dia 25 foi maior que P em d18, entretanto foi menor que vacas P nos dias 53 e 60. Os dias 32, 39 e 46 da gestação tiveram resposta intermediária. O segundo estudo avaliou a PGFM circulante em resposta a ocitocina em vacas Nelore prenhes de embriões PIV ou IA, nos dias 17 e 31 de gestação, e sua associação com fatores que podem impactar no sucesso da prenhes, como P4 circulante, tamanho de concepto no d18, e o tamanho de embrião no d32. Além disso, foi avaliada e localizada a expressão de OXTR e do gene estimulado por interferon 15 (ISG15) no endométrio uterino. O tamanho de embrião no dia 32 e a P4 circulante no dia 31 foram maiores no grupo IA. Vacas do grupo PIV d17 apresentaram menor resposta a ocitocina na concentração de PGFM do que as de IA no mesmo dia, contudo no dia 31 ambos os grupos tiveram maior resposta do que PIV d17. As vacas do d31 dos dois grupos tiveram aumento na PGFM similar às vacas não-inseminadas (NI). Os OXTR foram altamente suprimidosnas vacas prenhes do d18, especialmente no grupo PIV, mas com alta expressão em vacas NI e no dia 32 para os dois grupos, sendo a IA com maior expressão que a PIV neste dia. O gene ISG15 apresentou expressão irrelevante em NI e d32 para IA e PIV, mas apresentou expressão extremamente alta no d18 nos dois grupos prenhes. Conclui- se que o CL na gestação inicial é mantido pela supressão da liberação de PGF, enquanto que no segundo mês, ocitocina induz liberação de PGF, sugerindo que outros mecanismos regem a manutenção do CL a partir do dia 25. Além disso, nossos resultados demonstram que há diferenças entre a sinalização de gestações provenientes de embriões PIV e IA, que impactam no ambiente molecular e endócrino, influenciando a liberação de PGF nestes momentos.

O presente estudo objetivou promover incremento no desenvolvimento do concepto e aumentar a taxa de prenhez de búfalas lactantes por meio da administração de P4 injetável (P4i) três ou seis dias após a IATF. Para tanto, foram conduzidos três experimentos. No Experimento 1, foi aferido o padrão de liberação de P4 por meio da administração de P4i em 8 búfalas ovariectomizadas (delineamento crossover) nas doses 300 ou 600 mg (grupo P300 ou P600, respectivamente). Foram realizadas colheitas de sangue, para posteriores dosagens de P4, nos seguintes períodos: -24, 0, 6, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216 e 240 h da administração da P4i. No Experimento 2, 24 búfalas receberam a aplicação de 10 µg im de GnRH em dia aleatório do ciclo estral (D0). No D7, os animais receberam 0,53 mg im de PGF2α. Após 48 h (D9), foram administrados 10 µg im de GnRH e, 16 h mais tarde, todas as búfalas foram submetidas à IATF (D10). No D13, os animais foram avaliados por ultrassonografia e as fêmeas com ovulação positiva foram distribuídas em 3 grupos: Controle (C; n=8); P4D13 (n=8), que receberam 300mg de P4i no D13 e P4D16 (n=8), que receberam 300 mg de P4i no D16. No D26, as búfalas foram abatidas e os genitais removidos para a realização das seguintes avaliações: diâmetro (DCLa) e peso (PCL) do CL, presença de conceptos íntegros (pCI), íntegros e fragmentados (pCT) e comprimento do concepto (CC). Os CLs e endométrios foram seccionados, fixados e corados para aferir o percentual de células luteínicas pequenas e grandes (SLC e LLC) e o número (GEn), a área (AGEn) e o perímetro (PGEn) das glândulas do endométrio. No Experimento 3, 337 búfalas foram submetidas ao protocolo Ovsynch e, assim como no Experimento 2, as fêmeas ovuladas foram distribuídas em 3 grupos (C, n=81; P4D13, n=84 e P4D16, n=85). Foram avaliadas a funcionalidade (fluxo sanguíneo central FSC; e periférico FSP; escore de 0 a 4, em que 0 corresponde à ausência de fluxo e 4 o máximo fluxo) e o diâmetro do CL (DCL) nos D17, D21 e D25 por meio de ultrassonografia em modo color Doppler. Além disso, foram avaliadas por ultrassonografia as taxas de concepção aos 30 (DG30) e 60 (DG60) dias após a IATF e as perdas gestacionais (PG). Os dados foram analisados utilizando o procedimento GLIMMIX do SAS 9.3. Diferenças com P≤0,05 foram consideradas significativas e aquelas com 0,05<P≤0,10 foram consideradas tendência. As concentrações de P4 foram maiores no P600 em relação ao P300 em todos os pontos das colheitas de sangue (Ptrat<0,01, Ptempo=0,04, Ptrat*tempo=0,18). Verificou-se que as concentrações de P4 permaneceram acima de 1 ng/mL por aproximadamente 3 dias (entre as horas 6 e 72) no grupo P300, o que foi utilizado como critério para a dose de P4i utilizada nos Experimentos 2 e 3. Não houve diferenças entre os grupos para as variáveis avaliadas no Experimento 2: DCLa (P=0,39), PCL (P=0,13), pCI (P=0,85), pCT (P=0,41), CC (P=0,19), SLC (P=0,31), LLC (P=0,31), GEn (P=0,28), AGEn (P=0,72) e PGEn (P=0,91). No Experimento 3, houve interação tratamento*tempo (Ptrat*tempo) para as variáveis FSC (P<0,01), FSP (P<0,01) e DCL (P=0,07). Verificou-se redução do fluxo sanguíneo central e periférico e do diâmetro do CL no P4D13 em relação aos grupos C e P4D16 conforme os momentos de avaliação. Não houve diferença entre os grupos C, P4D13 e P4D16 para o DG30 (56,8 vs. 46,4 vs. 61,2 %; P=0,13) e para a PG (0,0 vs. 10,3 vs. 5,8 %; P=0,73). No entanto, houve menor taxa de concepção no DG60 para o P4D13 em comparação aos C e P4D16 (41,7b vs. 56,8a vs. 57,7a %; P=0,07). Conclui-se que o tratamento com 300 mg de P4i administrados três ou seis dias após a IATF não foi eficiente para aumentar o comprimento do concepto e a taxa de prenhez de búfalas lactantes submetidas à IATF. Ainda, o tratamento com P4i três dias após a IATF reduziu o fluxo sanguíneo central e periférico do CL, o diâmetro do CL e a taxa de prenhez. / The present study aimed to promote improvements on the conceptus development and to increase the pregnancy rate in lactating buffaloes through the administration of injectable P4 (P4i) three or six days after TAI. For this, three experiments were performed. In Experiment 1, was measured the pattern of P4 release by the P4i administration in 8 ovariectomized buffaloes (crossover design) at doses 300 or 600 mg (P300 or P600 group, respectively). Blood samples were collected for subsequent P4 dosages in the following periods: -24, 0, 6, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216 and 240 h of P4i administration. In Experiment 2, 24 buffaloes received the application of 10 µg im GnRH at random day of the estrous cycle (D0). On D7, the animals received 0.53 mg im PGF2α. After 48 h (D9), were administered 10 µg im GnRH and, 16 h later, all buffaloes underwent TAI (D10). In D13, the animals were evaluated by ultrasonography and females with positive ovulation were allocated to one of three groups: Control (C, n=8); P4D13 (n=8) that received 300 mg P4i on D13 and P4D16 (n=8), that received 300 mg P4i on D16. On D26, buffaloes were slaughtered and genitals removed to perform the following assessments: CL diameter (CLDs) and CL weight (CLW), presence of integrate conceptus (pIC), integrate and fragmented (pCT) and the conceptus length (CLe). The CLs and endometrium were sectioned, fixed and stained to assess the percentage of small and large lutein cells (SLC and LLC) and the number (GEn), the area (AGEn) and the perimeter (PGEn) of endometrial glands. In Experiment 3, 337 buffaloes were submitted to the Ovsynch protocol and, as well as in Experiment 2, the ovulated females were divided to one of three groups (C, n=81; P4D13, n=84 and P4D16, n=85). The functionality (central blood flow - CBF, and peripheral blood flow - PBF; score from 0 to 4, where 0 corresponds to absence of flow and 4 the maximum flow) and the CL diameter (CLD) were evaluated on D17, D21 and D25 by ultrasonography in color Doppler mode. Furthermore, the conception rates at 30 (CR30) and 60 (CR60) days after TAI, and pregnancy loss (PL) were evaluated by ultrasonography. Data were analyzed using the GLIMMIX procedure of SAS 9.3. Differences were considered significant with P≤0.05 and those with 0.05<P≤0.10 were considered tendencies. The P4 concentrations were higher in the P600 compared to P300 in all points of blood sampling (Ptreat<0.01, Ptime=0.04, Ptreat*time=0.18). It was found that the P4 concentrations remained above 1.0 ng/ml for approximately 3 days (between hours 6 and 72) in the P300 group, which was used as standard for P4i dose used in Experiments 2 and 3. There were no differences between the groups for the variables evaluated in Experiment 2: CLDs (P=0.39), CLW (P=0.13), pCI (P=0.85), pCT (P=0.41), CoLe (P=0.19), SLC (P=0.31), LLC (P=0.31), GEn (P=0.28), AGEn (P=0.72) and PGEn (P=0.91). In Experiment 3, there was interaction treatment*time (Ptreat*time) for the CBF (P<0.01), PBF (P<0.01) and CLD (P=0.07). There was a reduction of the central blood flow, peripheral blood flow and CL diameter for P4D13 compared to C and P4D16 groups according to the evaluation moments. There was no difference between C, P4D13 and P4D16 groups for the CR30 (56.8 vs. 46.4 vs. 61.2 %; P=0.13) and for the PL (0.0 vs. 10.3 vs. 5.8 %; P=0.73). However, there was lower conception rate in CR60 for P4D13 compared to C and P4D16 (41.7b vs. 56.8a vs. 57.7a %; P=0.07). It was concluded that treatment with 300 mg P4i administered three or six days after TAI was not efficient to increase the conceptus length and the pregnancy rate in lactating buffaloes submitted to TAI. Furthermore, the treatment with P4i three days after TAI reduced the central and the peripheral CL blood flow, the CL diameter and the pregnancy rate.

O presente estudo objetivou promover incremento no desenvolvimento do concepto e aumentar a taxa de prenhez de búfalas lactantes por meio da administração de P4 injetável (P4i) três ou seis dias após a IATF. Para tanto, foram conduzidos três experimentos. No Experimento 1, foi aferido o padrão de liberação de P4 por meio da administração de P4i em 8 búfalas ovariectomizadas (delineamento crossover) nas doses 300 ou 600 mg (grupo P300 ou P600, respectivamente). Foram realizadas colheitas de sangue, para posteriores dosagens de P4, nos seguintes períodos: -24, 0, 6, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216 e 240 h da administração da P4i. No Experimento 2, 24 búfalas receberam a aplicação de 10 µg im de GnRH em dia aleatório do ciclo estral (D0). No D7, os animais receberam 0,53 mg im de PGF2α. Após 48 h (D9), foram administrados 10 µg im de GnRH e, 16 h mais tarde, todas as búfalas foram submetidas à IATF (D10). No D13, os animais foram avaliados por ultrassonografia e as fêmeas com ovulação positiva foram distribuídas em 3 grupos: Controle (C; n=8); P4D13 (n=8), que receberam 300mg de P4i no D13 e P4D16 (n=8), que receberam 300 mg de P4i no D16. No D26, as búfalas foram abatidas e os genitais removidos para a realização das seguintes avaliações: diâmetro (DCLa) e peso (PCL) do CL, presença de conceptos íntegros (pCI), íntegros e fragmentados (pCT) e comprimento do concepto (CC). Os CLs e endométrios foram seccionados, fixados e corados para aferir o percentual de células luteínicas pequenas e grandes (SLC e LLC) e o número (GEn), a área (AGEn) e o perímetro (PGEn) das glândulas do endométrio. No Experimento 3, 337 búfalas foram submetidas ao protocolo Ovsynch e, assim como no Experimento 2, as fêmeas ovuladas foram distribuídas em 3 grupos (C, n=81; P4D13, n=84 e P4D16, n=85). Foram avaliadas a funcionalidade (fluxo sanguíneo central FSC; e periférico FSP; escore de 0 a 4, em que 0 corresponde à ausência de fluxo e 4 o máximo fluxo) e o diâmetro do CL (DCL) nos D17, D21 e D25 por meio de ultrassonografia em modo color Doppler. Além disso, foram avaliadas por ultrassonografia as taxas de concepção aos 30 (DG30) e 60 (DG60) dias após a IATF e as perdas gestacionais (PG). Os dados foram analisados utilizando o procedimento GLIMMIX do SAS 9.3. Diferenças com P≤0,05 foram consideradas significativas e aquelas com 0,05<P≤0,10 foram consideradas tendência. As concentrações de P4 foram maiores no P600 em relação ao P300 em todos os pontos das colheitas de sangue (Ptrat<0,01, Ptempo=0,04, Ptrat*tempo=0,18). Verificou-se que as concentrações de P4 permaneceram acima de 1 ng/mL por aproximadamente 3 dias (entre as horas 6 e 72) no grupo P300, o que foi utilizado como critério para a dose de P4i utilizada nos Experimentos 2 e 3. Não houve diferenças entre os grupos para as variáveis avaliadas no Experimento 2: DCLa (P=0,39), PCL (P=0,13), pCI (P=0,85), pCT (P=0,41), CC (P=0,19), SLC (P=0,31), LLC (P=0,31), GEn (P=0,28), AGEn (P=0,72) e PGEn (P=0,91). No Experimento 3, houve interação tratamento*tempo (Ptrat*tempo) para as variáveis FSC (P<0,01), FSP (P<0,01) e DCL (P=0,07). Verificou-se redução do fluxo sanguíneo central e periférico e do diâmetro do CL no P4D13 em relação aos grupos C e P4D16 conforme os momentos de avaliação. Não houve diferença entre os grupos C, P4D13 e P4D16 para o DG30 (56,8 vs. 46,4 vs. 61,2 %; P=0,13) e para a PG (0,0 vs. 10,3 vs. 5,8 %; P=0,73). No entanto, houve menor taxa de concepção no DG60 para o P4D13 em comparação aos C e P4D16 (41,7b vs. 56,8a vs. 57,7a %; P=0,07). Conclui-se que o tratamento com 300 mg de P4i administrados três ou seis dias após a IATF não foi eficiente para aumentar o comprimento do concepto e a taxa de prenhez de búfalas lactantes submetidas à IATF. Ainda, o tratamento com P4i três dias após a IATF reduziu o fluxo sanguíneo central e periférico do CL, o diâmetro do CL e a taxa de prenhez. / The present study aimed to promote improvements on the conceptus development and to increase the pregnancy rate in lactating buffaloes through the administration of injectable P4 (P4i) three or six days after TAI. For this, three experiments were performed. In Experiment 1, was measured the pattern of P4 release by the P4i administration in 8 ovariectomized buffaloes (crossover design) at doses 300 or 600 mg (P300 or P600 group, respectively). Blood samples were collected for subsequent P4 dosages in the following periods: -24, 0, 6, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216 and 240 h of P4i administration. In Experiment 2, 24 buffaloes received the application of 10 µg im GnRH at random day of the estrous cycle (D0). On D7, the animals received 0.53 mg im PGF2α. After 48 h (D9), were administered 10 µg im GnRH and, 16 h later, all buffaloes underwent TAI (D10). In D13, the animals were evaluated by ultrasonography and females with positive ovulation were allocated to one of three groups: Control (C, n=8); P4D13 (n=8) that received 300 mg P4i on D13 and P4D16 (n=8), that received 300 mg P4i on D16. On D26, buffaloes were slaughtered and genitals removed to perform the following assessments: CL diameter (CLDs) and CL weight (CLW), presence of integrate conceptus (pIC), integrate and fragmented (pCT) and the conceptus length (CLe). The CLs and endometrium were sectioned, fixed and stained to assess the percentage of small and large lutein cells (SLC and LLC) and the number (GEn), the area (AGEn) and the perimeter (PGEn) of endometrial glands. In Experiment 3, 337 buffaloes were submitted to the Ovsynch protocol and, as well as in Experiment 2, the ovulated females were divided to one of three groups (C, n=81; P4D13, n=84 and P4D16, n=85). The functionality (central blood flow - CBF, and peripheral blood flow - PBF; score from 0 to 4, where 0 corresponds to absence of flow and 4 the maximum flow) and the CL diameter (CLD) were evaluated on D17, D21 and D25 by ultrasonography in color Doppler mode. Furthermore, the conception rates at 30 (CR30) and 60 (CR60) days after TAI, and pregnancy loss (PL) were evaluated by ultrasonography. Data were analyzed using the GLIMMIX procedure of SAS 9.3. Differences were considered significant with P≤0.05 and those with 0.05<P≤0.10 were considered tendencies. The P4 concentrations were higher in the P600 compared to P300 in all points of blood sampling (Ptreat<0.01, Ptime=0.04, Ptreat*time=0.18). It was found that the P4 concentrations remained above 1.0 ng/ml for approximately 3 days (between hours 6 and 72) in the P300 group, which was used as standard for P4i dose used in Experiments 2 and 3. There were no differences between the groups for the variables evaluated in Experiment 2: CLDs (P=0.39), CLW (P=0.13), pCI (P=0.85), pCT (P=0.41), CoLe (P=0.19), SLC (P=0.31), LLC (P=0.31), GEn (P=0.28), AGEn (P=0.72) and PGEn (P=0.91). In Experiment 3, there was interaction treatment*time (Ptreat*time) for the CBF (P<0.01), PBF (P<0.01) and CLD (P=0.07). There was a reduction of the central blood flow, peripheral blood flow and CL diameter for P4D13 compared to C and P4D16 groups according to the evaluation moments. There was no difference between C, P4D13 and P4D16 groups for the CR30 (56.8 vs. 46.4 vs. 61.2 %; P=0.13) and for the PL (0.0 vs. 10.3 vs. 5.8 %; P=0.73). However, there was lower conception rate in CR60 for P4D13 compared to C and P4D16 (41.7b vs. 56.8a vs. 57.7a %; P=0.07). It was concluded that treatment with 300 mg P4i administered three or six days after TAI was not efficient to increase the conceptus length and the pregnancy rate in lactating buffaloes submitted to TAI. Furthermore, the treatment with P4i three days after TAI reduced the central and the peripheral CL blood flow, the CL diameter and the pregnancy rate.