RESUMO
In cattle, 17ß-estradiol (E2) stimulates prostaglandin F2α (PGF2α) synthesis, which causes luteolysis. Except for the well-established upregulation of oxytocin receptor gene (OXTR), molecular mechanisms of E2-induced PGF2α release in vivo remain unknown. We hypothesized that E2-induced PGF2α release requires de novo transcription of components of the PGF2α synthesis machinery. Beef cows (n = 52) were assigned to remain untreated (Control; n = 10), to receive 50% ethanol infusion intravenously (Placebo; n = 21), or 3 mg E2 in 50% ethanol infusion intravenously (Estradiol; n = 21) on day 15 (D15) after estrus. We collected a single endometrial biopsy per animal at the time of the treatment (0h; Control B0h group), 4 hours (4h; Placebo B4h group and Estradiol B4h group), or 7 hours (7h; Placebo B7h group and Estradiol B7h group) post-treatment. Compared to the Placebo group, the Estradiol group presented significantly greater 13,14-dihydro-15-keto-PGF2α concentrations between 4h and 7h and underwent earlier luteolysis. At 4h, the qPCR analysis showed a lower abundance of ESR1, ESR2 and aldo-keto reductase family 1 member B1 (AKR1B1) genes in the Estradiol B4h group, and a greater abundance of OXTR compared to the Placebo B4h group. Similarly, the E2 treatment significantly reduced the abundance of AKR1B1, and AKR1C4 in the Estradiol B7h group, compared to the placebo group. Overall, E2-induced PGF2α release and luteolysis involved an unexpected and transient downregulation of components of the PGF2α-synthesis cascade, except for OXTR, which was upregulated. Collectively, our data suggest that E2 connects newly-synthesized OXTR to pre-existing cellular machinery to synthesize PGF2α and cause luteal regression.
Assuntos
Dinoprosta , Luteólise , Animais , Bovinos , Corpo Lúteo/fisiologia , Dinoprosta/farmacologia , Endométrio , Estradiol/farmacologia , Feminino , Progesterona , Receptores de Ocitocina/genética , ÚteroRESUMO
Number of pubertal heifers at time of breeding season initiation is a primary determinant to pregnancy success during the breeding season. It was hypothesized that pre-breeding progesterone (P4) supplementation (induction) would increase the number of heifers pubertal at the time of imposing estrous synchronization treatment regimens and P/AI. Yearling, Bos indicus-influenced (n = 577) or Bos indicus (n = 174) heifers were or were not treated with P4 (CIDR and Non-CIDR, respectively) for 10 d starting on D-23 (D0 = TAI). Presence of a CL on D-33 or D-23 was considered to indicate heifers were pubertal. On D-13, there was a PGF analogue administered. On D-9, there was treatment with GnRH analogue, 6d-CIDR and PGF. There were inseminations based on estrus (D-2 to D0) or TAI on D0 for non-estrous animals. There were 5.2 % and 62.9 % purebred and crossbred heifers pubertal, respectively. Proportion of prepubertal crossbred than purebred heifers with CL on D-3 was greater as a result of imposing the pubertal induction regimen (P < 0.05 and P> 0.10, respectively). Regardless of puberty status, proportion of heifers in estrus prior to AI in the CIDR group was similar to the heifers of the Non-CIDR group for crossbreds and purebreds. Similarly, P/AI of CIDR group was similar to the Non-CIDR group for crossbreds and purebreds. In summary, imposing the pubertal induction regimen hastened attainment of puberty in yearling crossbred, but not purebred heifers. Puberty induction did not affect estrous response, neither fertility after imposing an estrous synchronization treatment regimen.
Assuntos
Bovinos/genética , Bovinos/fisiologia , Sincronização do Estro/efeitos dos fármacos , Progesterona/farmacologia , Maturidade Sexual/efeitos dos fármacos , Maturidade Sexual/fisiologia , Envelhecimento , Animais , Feminino , Hibridização Genética , Progestinas/administração & dosagem , Progestinas/farmacologia , Prostaglandinas F Sintéticas/administração & dosagem , Prostaglandinas F Sintéticas/farmacologiaRESUMO
Induction protocols based on progesterone (P4) are used prior to a synchronization program for timed-AI (TAI) to increase number of pubertal heifers and pregnancy per AI (P/AI). Injectable, long-lasting P4 (iP4) is a novel, practical method to supplement P4 in cattle. Here, we aimed to test the effect of an induction protocol based on a single injection of iP4 on P/AI of heifers. Bos indicus (Nellore) heifers were classified as pubertal (PUB; n = 224) or prepubertal (PRE; n = 414) based on two ovarian ultrasonographyc exams conducted 10 d apart. Heifers with a corpus luteum (CL) in any of the exams were considered PUB. Within each puberty status, heifers were assigned to receive nothing (NoiP4) or an induction protocol (iP4). Induction consisted of a single injection of 150 mg of iP4 on D-31, followed by injections of 1 mg of estradiol benzoate (EB) and 150 µg of prostaglandin analogue (PGF) on D-21. On D-9, all heifers received 2 mg of EB + 75 µg of PGF associated to intravaginal P4-device insertion. On D-3, P4-releasing devices were removed and 150 µg of PGF injected. Heifers were inseminated based on estrus on D-1 or were TAI on D0. On D0, all heifers received a dose of GnRH analogue. On D-21, iP4 treatment stimulated a 50% increase in the uterine score (UTS) and a 19% increase in the diameter of the largest follicle of PRE heifers (P < 0.01). On D-9, PRE|iP4 group had a greater proportion (P < 0.01) of CL (63.3%) than PRE|NoiP4 group (11.6%). On D-3, exposure to 6 d P4-releasing device stimulated UTS of PRE|NoiP4 group in a similar fashion than the induction protocol, but it did not have any additional positive effect for PRE|iP4 heifers. P/AI of PRE|iP4 group was similar to that of the PUB groups (44.7 vs 46.9%), but was more than that of PRE|NoiP4 (34.2%). There was an overall 7.7% increment (P = 0.07) on P/AI of iP4 treated heifers (iP4: 46.0% vs. NoiP4: 38.3%). In conclusion, implementation of an induction protocol based on iP4 was efficacious to hasten puberty. Induction stimulated uterine development and follicular growth of prepubertal heifers, ultimately leading to pregnancy success similar to that of pubertal heifers.
Assuntos
Sincronização do Estro , Progesterona , Animais , Bovinos , Dinoprosta , Feminino , Hormônio Liberador de Gonadotropina , Inseminação Artificial/veterinária , Gravidez , Taxa de Gravidez , Maturidade SexualRESUMO
This study characterised the expression of interferon (IFN)-τ-stimulated genes (ISGs) and Type I IFN receptors in circulating polymorphonuclear cells (PMNs) of beef heifers and compared it with expression in peripheral blood mononuclear cells (PBMCs) up to Day 20 of gestation. Nelore heifers (n=26) were subjected to fixed-time AI (FTAI) on Day 0. PMNs and PBMCs were isolated on Days 0, 10, 14, 16, 18 and 20 after FTAI. The abundance of target transcripts (ubiquitin-like protein (ISG15), 2'-5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance 1 (MX1), myxovirus resistance 2 (MX2), IFN receptor I (IFNAR1) and IFN receptor 2 (IFNAR2)) was determined using real-time quantitative polymerase chain reaction and compared between pregnant (n=8) and non-pregnant (n=9) females. In both PBMCs and PMNs, ISG15 and OAS1 expression was greater in pregnant than non-pregnant heifers on Days 18 and 20. There were no significant differences in the expression of ISGs between PBMCs and PMNs. A time effect on expression was found for IFNAR1 in PBMCs and IFNAR2 in PMNs, with decreased expression of both genes on Days 18 and 20. When the expression of these genes was compared between cell types only in pregnant heifers, IFNAR2 expression in PMNs had an earlier decrease when compared to its expression in PBMCs, starting from Day 18. In conclusion, PMNs do not respond earlier to the conceptus stimulus, and ISG15 and OAS1 expression in both PMNs and PBMCs can be used as a suitable marker for pregnancy diagnosis on Days 18 and 20. In addition, gestational status did not affect IFNAR1 and IFNAR2 expression, but IFNAR2 showed a distinct response between PMNs and PBMCs of pregnant heifers.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Neutrófilos/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Ubiquitinas/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , Animais , Bovinos , Feminino , Proteínas de Resistência a Myxovirus/genética , Gravidez , Progesterona/sangue , Receptor de Interferon alfa e beta/genética , Ubiquitinas/genéticaRESUMO
Long-acting injectable progesterone (iP4) supplementation during early diestrus is a strategy to increase conception rates in cow-calf beef operations. However, iP4 treatment causes early functional and structural regression of the corpus luteum (CL) in a proportion of iP4-treated animals, resulting in pregnancy loss. The hypothesis evaluated was that iP4 accelerates downregulation of sex-steroid receptors (PGR, ESR1, ESR2) during early to mid-diestrus and the upregulation of genes controlling PGF2α secretion (OXTR, PTGS2, AKR1B1) during late diestrus in the endometrium. Ovulations of cyclic, multiparous Nelore (Bos indicus) cows were synchronized, and cows were divided to receive placebo or 300 mg iP4 3 d postovulation (D3). Growth and vascularization of luteal tissue were evaluated by ultrasonography. Blood samples were collected from 3 d postovulation to 3 d after luteolysis, and P4 plasma concentrations were measured by radioimmunoassay. On days 3, 5, 7, 9, 11, 13, and 16 luminal endometrial samples were taken using a cytologic brush. Transcript abundance was measured by qPCR. Structural luteolysis occurred 3 d earlier in cows receiving iP4 compared to the control group. Analyzing only cows that received iP4, those that presented early luteolysis (ie, ≤ D16) showed a decrease in CL area and P4 concentration after D5, compared to the control group. Cows that presented early luteolysis showed a reduced abundance of transcripts on D5 for the ESR2 gene and a greater abundance of transcripts for OXTR and ESR1 on D16, compared to cows that did not present early luteolysis. The iP4-induced early luteolysis can be explained by two nonexclusive possibilities: the activation of uterine mechanisms that trigger early secretion of endometrial PGF pulses and the formation of a subfunctional CL that is prone to early regression.
Assuntos
Bovinos/fisiologia , Corpo Lúteo/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Progesterona/farmacologia , Animais , Corpo Lúteo/fisiologia , Endométrio/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Progesterona/administração & dosagem , Fatores de TempoRESUMO
Progesterone (P4) supplementation in early diestrus advances changes in the endometrial transcriptome, stimulating embryonic development. However, it also induces early onset of luteolysis. Occurrence of luteolysis before D16 postmating can be detrimental to fertility. A potential counteracting role of the elongating conceptus on early luteolysis is understood poorly. The aim of the study was to evaluate the effect of artificial insemination (AI; ie, pregnancy) on the temporal dynamics of luteolysis of cows supplemented with P4. Nonsuckled beef cows were inseminated at 12 h after estrus (D0: ovulation) or were not inseminated (no-AI). On D3, the AI cows were assigned to receive a single dose of 150 mg of injectable long-acting P4 via intramuscular injection (AI + iP4; n = 23), and the no-AI cows were assigned to receive iP4 (iP4; n = 21) or saline (control, n = 22). Corpus luteum (CL) development and regression were determined by ultrasonography (US) between D3 and D21. Plasma P4 concentrations were measured on D3 and every other day from D9 to D21. Pregnancy status was determined by US (D28âD32). iP4 supplementation reduced luteal development (D5-D10) compared to the control group and increased incidence of luteolysis between D14 and D15. On D15, the proportion of cows that underwent luteolysis and plasma P4 concentrations differed between the iP4 group (47.6; 2.10 ± 0.47) and the control group (13.6; 4.40 ± 0.46) and was intermediate in the AI + iP4 group, respectively (26.1%; 3.70 ± 0.45 ng/mL; P < 0.05). The AI effects were due to the pregnant cows (n = 7). Considering nonpregnant cows only, the proportion of early luteolysis in the AI + iP4 group (37.5%) was similar to the iP4 group. Pregnancy was not established in cows having a shortened luteal lifespan. Indeed, interval to luteolysis in the AI + iP4 group (15.50 ± 0.66 d) was similar to the iP4 group (16.38 ± 0.46 d), but less than the control group (17.38 ± 0.40 d; P = 0.05). In conclusion, the effect of AI on extending luteal lifespan occurred exclusively in cows that maintained pregnancy.
Assuntos
Bovinos/fisiologia , Diestro , Inseminação Artificial/veterinária , Luteólise , Progesterona/administração & dosagem , Animais , Brasil , Diestro/sangue , Feminino , Injeções Intramusculares/veterinária , Inseminação Artificial/métodos , Gravidez , Progesterona/sangue , Carne Vermelha , Fatores de TempoRESUMO
The aims were to characterize follicular dynamics in response to supplemental progesterone (P4) and to investigate the relationships between follicular growth and onset of luteolysis in P4-treated cows, submitted or not to artificial insemination (AI). Nonsuckled beef cows detected in estrus were assigned to receive AI or to remain non-AI. Three days after ovulation (ie, D3), AI cows were injected with 150 mg of long-acting P4 (AI + injectable P4 formulation [iP4]; n = 22), and the non-AI cows were assigned to receive 150 mg iP4 (n = 19) or saline (control, n = 19). Between D3 and D21, growth dynamics of the dominant follicles (DFs) was monitored by ultrasonography. Plasma P4 concentrations were measured every other day from D9 to D19. Pregnancy status (ie, P: pregnant and NP: nonpregnant) was examined by ultrasound on D28 to D32. Injectable P4 formulation supplementation decreased average maximum diameter of first-wave DF (DF1). Neither day of emergence of DF2 or DF3 nor the proportion of two- or three-wave cycles were altered by supplemental P4. Daily mean diameter of DF2 and DF3 was also similar between control and iP4 groups. Consistently, daily mean diameter of DF1 in iP4-treated cows was smaller for cows that underwent luteolysis by D15 compared to a later onset. Progesterone concentrations between D9 and D19 decreased earliest in the iP4 group, latest in the control group and was intermediate for the NP-AI + iP4 group. In addition, three-wave cycles presented a delayed decrease on plasma P4 concentrations than two-wave cycles. Further analysis revealed that on two-wave cycles, P4 concentrations on D15 were lowest in the iP4 and NP-AI + iP4 animals compared to the control and P-AI + iP4 groups. Conversely, for three-wave cycles, on D15, P-AI + iP4, NP-AI + iP4, and controls had greater P4 concentrations than the iP4 group. In summary, our data indicate that impairment of first follicular growth was associated with P4-induced shortened luteal lifespan (D14-D15) and that three-wave cycles after AI can be more supportive for pregnancy maintenance in P4-treated cows. We speculate that such conditions play a critical role in the embryonic ability to inhibit iP4-induced early luteolysis reported in part I of this series.
Assuntos
Bovinos , Diestro , Luteólise/efeitos dos fármacos , Progesterona/administração & dosagem , Animais , Esquema de Medicação , Feminino , Inseminação Artificial/veterinária , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologiaRESUMO
BACKGROUND: Insufficient production of anti-luteolytic signals by the pre-attachment embryo is considered a major cause of pregnancy failure in cattle. We tested the hypothesis that transfer of multiple blastocysts (n = 5/recipient) and progesterone (P4) supplementation amplify anti-luteolytic signaling and reduce embryonic losses in beef cattle. Cows detected in estrus (D0; n = 104) were assigned randomly to receive 150 mg of injectable long-acting P4 (iP4) or vehicle (non-iP4) on D4 and transcervical transfer of none or five, grade 1, not-frozen, in vitro-produced blastocysts, on D7. Luteal development and time of structural luteolysis were monitored by ultrasonography. Plasma P4 concentrations were determined on D4, D5 and D7, and daily between D14 and D20. Conceptus signaling was monitored by transcript abundance of interferon-stimulated gene 15 (ISG15) in peripheral blood mononuclear cells isolated on D14, D16, D18 and D20. Early embryonic mortality (EEM) was defined as the absence of ISG15 mRNA upregulation over time and/or luteal regression up to D20. Late embryonic mortality (LEM) was defined as the absence of a conceptus with a heartbeat on pregnancy diagnosis at D30 (PD30) after observing upregulation of ISG15 mRNA and extension of luteal lifespan. Pregnant cows presented conceptuses with heartbeat at PD30. RESULTS: On D5, iP4-treated cows had P4 concentrations 2.07-fold greater than non-iP4 treated (P < 0.001). On D7, P4 concentrations were similar. Pregnant and LEM animals showed a progressive increase in the abundance of ISG15 from D14 to D20. iP4-treated cows detected pregnant at PD30 had 1.53-fold greater abundance of ISG15 mRNA between D14 and D20 than non-iP4 treated cows (P = 0.05). iP4 doubled the frequency of EEM while it did not affect LEM. At PD30, embryonic survival was 37.0% vs. 55.6% for iP4-treated vs. control cows. Majority of pregnant cows (71%) presented only a single viable embryo. CONCLUSIONS: A substantial proportion of cows had EEM (31%) and LEM (20%) even after transferring multiple blastocysts. This argues that mortality was due to poor uterine receptivity that could not be reversed by supplemental P4 or overcome by transferring multiple blastocysts. Further, a given uterine environment was not necessarily adequate to all embryos.
RESUMO
Supplementation with compounds rich in linoleic acid, including sunflower seed supplementation, promotes increase in conception rates in cows. We aimed to evaluate whether the sunflower seed (linoleic acid source) supplementation in beef donor females alters the plasma concentrations of cholesterol, triglycerides, HDL and LDL, increases the number and quality of oocytes, increases the cleavage rates and determines an improvement in number and quality of in vitro produced blastocysts. Thus, Nelore females were divided into two groups of 15 animals to receive supplementation with or without sunflower seed for 57 days. Females underwent follicular aspiration and the oocytes were subjected to in vitro embryo production. There was no difference (p > .1) between control group and group supplemented with sunflower seed on the number of displayed follicles; number of aspired oocytes; recovery rate; cleavage rate; number of embryos; number of blastocysts; embryos number of grades I and II; plasma concentrations of total cholesterol, triglycerides; HDL and LDL. Therefore, sunflower seed supplementation in oocyte donors did not increase the number and quality of oocytes, cleavage rates and the number and quality of blastocysts produced in vitro.
Assuntos
Bovinos/fisiologia , Suplementos Nutricionais , Helianthus , Oócitos/fisiologia , Animais , Blastocisto , Bovinos/embriologia , Colesterol/sangue , Feminino , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos , Ácido Linoleico , Recuperação de Oócitos/veterinária , Sementes , Triglicerídeos/sangueRESUMO
The growth profiles of the future dominant follicle (DF) and subordinate follicle (SF) and the gene expression of the granulosa cells during luteolysis induction in Bos indicus cows were evaluated. Forty cows were synchronized with a progesterone and estradiol based protocol. After synchronization, cows with a corpus luteum (CL) were evaluated by ultrasonography every 12â¯h, beginning at eight days post ovulation. Cows identified with a follicle of at least 6.0â¯mm in diameter in the second wave were split into two groups (BD-before follicular deviation and AD-after follicular deviation. In the BD group cows received 500⯵g of cloprostenol (a synthetic analogue of prostaglandin F2α) when the DF reached a mean diameter of 7.0â¯mm (6.5-7.5â¯mm). In the AD group, cows received 500⯵g of cloprostenol when the DF reached a mean diameter of 8.0â¯mm (7.5-8.5â¯mm). Cows in both groups were submitted to aspiration of the DF at 96 and 72â¯h after prostaglandin was given. Follicular aspirations were performed to quantify IGF1R, LHR and PAPPA transcripts in the granulosa cells. The diameter of the DF at the moment of prostaglandin administration (Pâ¯=â¯0.001) and the growth rate of the SF (Pâ¯=â¯0.05) were greater in the AD group. There was greater abundance of LHR transcripts in BD cows (Pâ¯=â¯0.04). The remaining variables tested were similar between the experimental groups (Pâ¯>â¯0.05). In conclusion, the induction of luteolysis before follicular deviation does not interfere with dominant follicle dynamics. However, it causes granulosa cell LHR down regulation.
Assuntos
Cloprostenol/farmacologia , Luteólise/efeitos dos fármacos , Folículo Ovariano/fisiologia , Animais , Bovinos , Estradiol , Sincronização do Estro , Feminino , Folículo Ovariano/efeitos dos fármacos , Progesterona/farmacologia , RNA Mensageiro , Receptores do LH , Receptores de Somatomedina/metabolismoRESUMO
In cattle, early diestrus progesterone (P4) supplementation modulates endometrial function to exert pro- and anti-pregnancy establishment effects; specifically, P4 stimulates conceptus growth, but also induces early onset of luteolysis. This paradoxical effect is frequently related to the inconsistent fertility outcomes that result from P4 supplementation experiments. Aim was to investigate the impact of exogenous estradiol (E2) treatment at the end of timed fixed AI (TAI) on frequency of early luteolysis and pregnancy of beef cows supplemented with P4. Ovulations (D0 of study) of suckled multiparous (n = 643) and primiparous (n = 193) Nelore cows (Bos indicus) were synchronized with an E2/P4-based protocol for TAI and assigned to receive 1.0 mg of estradiol cypionate (CP) or nothing (NoCP) on D-2 and 150 mg of injectable long-acting P4 (iP4) or Placebo (NoiP4) on D4 on a 2 × 2 factorial arrangement. On D15, the iP4 supplementation increased (P < 0.05) the frequency of early luteolysis (NoCP + iP4: 26.0%; [13/50] vs. NoCP: 8.0% [4/50]), but CP prevented this effect (CP + iP4: 8.3% [4/48] and CP: 6.4% [3/47]). The CP improved pregnancy/AI (P/AI) of multiparous (CP: 51.6% [165/320] and NoCP: 35.0% [113/323]; P < 0.001) and primiparous cows (CP: 40.4% [40/99] and NoCP: 24.5% [23/94], P < 0.05), regardless of iP4 treatment. The iP4 supplementation affected P/AI of CP and NoCP treated cows according to follicle size at TAI. For the CP treated cows, the iP4 supplementation improved P/AI of sub-populations of cows with follicles <12.35 mm (42.0% [34/81] vs. 53.1% [34/64]), while for NoCP treated cows, the improvements occurred in subpopulations of cows with follicles ≥12.35 mm (46.1% [35/76] vs. 58.7% [37/63]). In conclusion, strategies associating E2 and P4 supplementation decrease the incidence of early onset of luteolysis and improve P/AI of suckled beef cows with smaller follicles.
Assuntos
Diestro , Estradiol/análogos & derivados , Inseminação Artificial/veterinária , Ovário/efeitos dos fármacos , Progesterona/farmacologia , Animais , Bovinos , Anticoncepcionais Femininos/administração & dosagem , Anticoncepcionais Femininos/farmacologia , Preparações de Ação Retardada , Estradiol/administração & dosagem , Estradiol/farmacologia , Feminino , Inseminação Artificial/métodos , Ovário/fisiologia , Gravidez , Taxa de Gravidez , Progesterona/administração & dosagemRESUMO
Aims were to (i) compare specific transcript abundance between endometrial samples collected by transcervical biopsy and cytobrush and (ii) measure the abundance of endometrial transcripts involved in PGF2α synthesis in samples collected by cytobrush. In Experiment 1, endometrial samples were taken transcervically by cytobrush and biopsy 10 days after ovulation. Compared to biopsy samples, abundance of transcripts for MSTN, AKR1C4 and PGR was similar, VIM, FLT1 and PTGES was lower (p < .05) and KRT18 and CD3D was greater in cytobrush samples (p < .05). Thus, there was an enrichment of epithelial and immune cells in the cytobrush samples. In Experiment 2, endometrial samples were collected by cytobrush on days 10, 13, 16 and 19 after ovulation. Abundance of PGR2 mRNA was maximum on day 10 then decreased (p < .05). Abundance of ESR1 decreased gradually from day 10 to day 16 then increased again on day 19. The greatest abundance of OXTR was noted on day 19. The sequential alterations in abundance of these transcripts are consistent with the release of PGF2α associated with luteolysis. In summary, cytobrush sampling provides representative, physiologically relevant samples of the luminal epithelium in cattle.
Assuntos
Técnicas de Diagnóstico Obstétrico e Ginecológico/veterinária , Dinoprosta/biossíntese , Endométrio/metabolismo , Expressão Gênica , Animais , Biópsia , Bovinos , Técnicas de Diagnóstico Obstétrico e Ginecológico/instrumentação , Endométrio/citologia , Feminino , RNA Mensageiro/análiseRESUMO
Load-bearing reinforcing elements in a continuous matrix allow for improved mechanical properties and can reduce the weight of structural composites. As the mechanical performance of composite systems are heavily affected by the interfacial properties, tailoring the interactions between matrices and reinforcing elements is a crucial problem. Recently, several studies using bio-inspired model systems suggested that interfacial mechanical interlocking is an efficient mechanism for energy dissipation in platelet-reinforced composites. While cheap and effective solutions are available at the macroscale, the modification of surface topography in micron-sized reinforcing elements still represents a challenging task. Here, we report a simple method to create nanoasperities with tailored sizes and densities on the surface of alumina platelets and investigate their micromechanical effect on the energy dissipation mechanisms of nacre-like materials. Composites reinforced with roughened platelets exhibit improved mechanical properties for both organic ductile epoxy and inorganic brittle cement matrices. Mechanical interlocking increases the modulus of toughness (area under the stress-strain curve) by 110% and 56% in epoxy and cement matrices, respectively, as compared to those reinforced with flat platelets. This interlocking mechanism can potentially lead to a significant reduction in the weight of mechanical components while retaining the structural performance required in the application field.
Assuntos
Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Nácar/química , Nanocompostos/química , Nanocompostos/ultraestrutura , Dióxido de Silício/química , Animais , Materiais Biomiméticos/síntese química , Módulo de Elasticidade , Dureza , Manufaturas/análise , Teste de Materiais , Tamanho da Partícula , Estresse Mecânico , Propriedades de SuperfícieRESUMO
Studying the multitude of molecular networks and pathways that are potentially involved in a complex trait such as fertility requires an equally complex and broad strategy. Here, we used Next-Generation Sequencing for the characterization of the transcriptional signature of the bovine endometrial tissue. Periovulatory endocrine environments were manipulated to generate two distinctly different fertility phenotypes. Cycling, non-lactating, multiparous Nelore cows were manipulated to ovulate larger (> 13 mm; LF group; high fertility phenotype) or smaller (< 12 mm; SF group) follicles. As a result, greater proestrus estrogen concentrations, corpora lutea and early diestrus progesterone concentrations were also observed in LF group in comparison to SF group. Endometrial cell proliferation was estimated by the protein marker MKI67 on tissues collected 4 (D4) and 7 (D7) days after induction of ovulation. Total RNA extracts from D7 were sequenced and compared according to the transcriptional profile of each experimental group (LF versus SF). Functional enrichment analysis revealed that LF and SF endometria were asynchronous in regards to their phenotype manifestation. Major findings indicated an LF endometrium that was switching phenotypes earlier than the SF one. More specifically, a proliferating SF endometrium was observed on D7, whereas the LF tissue, which expressed a proliferative phenotype earlier at D4, seemed to have already shifted towards a biosynthetically and metabolically active endometrium on D7. Data on MKI67 support the transcriptomic results. RNA-Seq-derived transcriptional profile of the endometrial tissue indicated a temporal effect of the periovulatory endocrine environment, suggesting that the moment of the endometrial exposure to the ovarian steroids, E2 and P4, regulates the timing of phenotype manifestation. Gene expression profiling revealed molecules that may be targeted to elucidate ovarian steroid-dependent mechanisms that regulate endometrial tissue receptivity. Data was deposited in the SRA database from NCBI (SRA Experiment SRP051330) and are associated with the Bio-Project (PRJNA270391). An overview of the gene expression data has been deposited in NCBI's Gene Expression Omnibus (GEO) and is accessible through GEO Series accession number GSE65450. Further assessment of the data in combination with other data sets exploring the transcriptional profile of the endometrial tissue during early diestrus may potentially identify novel molecular mechanisms and/or markers of the uterine receptivity.
RESUMO
We aimed to evaluate the effects and the interaction of size of the preovulatory follicle (POF) and long-acting progesterone (P4) supplementation after timed-AI on CL function and pregnancy success in beef cows. In experiment 1, ovulations of beef cows were synchronized starting on Day -10, and cows were split to receive sodium cloprostenol (large follicle group; LF; n = 31) or nothing (small follicle group; SF; n = 35). Ovulations were induced on Day 0, and cows were inseminated. Ovulated cows were assigned to receive placebo (LF/control group, n = 14; and SF/control group, n = 9) or 150 mg of long-acting P4 on Day 4.5 (LF/P4 group, n = 13; and SF/P4 group, n = 12). Diameter of POF, blood flow in POF wall, ovulation rate, and size and vascularization of CL were greater (P < 0.05) in LF group. In experiments 2 (unknown cyclic status) and 4 (noncycling), ovulations were synchronized, and beef cows received placebo or 150 mg of long-acting P4 on Day 4 after timed-artificial insemination. In experiment 2, pregnancy/AI (P/AI) did not differ (P > 0.1) between P4-treated (53.2%; 209/393) and control cows (56.2%; 219/390), but P/AI was greater in cows with a CL < 0.9 cm(2) on Day 4 that were P4-treated (57.9%, 22/38) versus placebo-treated (40.4%, 21/52; P < 0.05). In Experiment 4, P/AI was greater (P < 0.05) in P4-treated cows (55.6%, 105/189 vs. 46.0%, 86/187). In Experiment 3, cyclic-suckled beef cows were treated as described in Experiment 1 to generate animals with small (SF; n = 111) or large POF (LF; n = 109), and subdivided to receive placebo or P4 on Day 4. POF size, ovulation rate, CL area, and P/AI were greater (P < 0.007) in the LF group. Pregnancy/AI in ovulated cows were lower (P = 0.05) in the SF/control group (41.5%, 17/41) compared to LF/control group (62%, 31/50) and were similar for the SF/P4 group (55.6%, 25/45) and LF/P4 group (57%, 28/49) compared to others. In summary, smaller and less vascularized POF results in less functional CL and reduces ovulatory rate and P/AI in cyclic beef cows; the long-acting P4 injection on Day 4 after timed-artificial insemination may attenuate the negative effects of small POF/CL; and postovulatory P4 supplementation improved fertility in anestrous beef cattle.
Assuntos
Fertilidade/efeitos dos fármacos , Inseminação Artificial/veterinária , Folículo Ovariano/fisiologia , Ovulação/fisiologia , Progesterona/farmacologia , Animais , Animais Lactentes , Bovinos , Tamanho Celular , Ciclo Estral/fisiologia , Sincronização do Estro/métodos , Feminino , Fármacos para a Fertilidade Feminina/administração & dosagem , Lactação/fisiologia , Gravidez , Progesterona/administração & dosagemRESUMO
In this study, endometrial samples were collected in 14 Nelore cows on days zero (ovulation), five, nine, thirteen and nineteen of the estrous cycle (biopsy group), and in 15 females these collections weren't performed (control group). Biopsies were done on the uterine horn endometrium contralateral to the ovary with corpus luteum. Blood samples were taken at -24, -16, -8, 0 +8, +16 and +24 hours in relation to progesterone drop (<1ng/mL, zero moment) and evaluated for 13, 14-dihydro-15-keto prostaglandin F2-alpha (PGFM) by radioimmunoassay (RIA). Plasma progesterone concentration was determined by RIA every 24 hours. Data were analyzed by ANOVA using the PROC GLM and MIXED of the SAS. The mean value for PGFM during the entire period evaluated was greater in the biopsy group. The mean concentration of PGFM at moment zero was not different between the groups; the mean concentration of PGFM was higher in the biopsy group before and after the drop in progesterone level. The maximum mean concentration observed was not different between the groups; however, the mean minimum concentration was different with high values in the biopsy group. Although the PGFM concentrations were higher in the biopsy group, the biopsy and control groups had similar length of estrous cycle showing that repeated endometrial biopsy on the side contralateral to the ovary with corpus luteum does not affect luteolysis and the length of the estrous cycle.
No presente estudo, foram coletadas amostras endometriais de 14 vacas Nelore nos dias zero (dia da ovulação), cinco, nove, 13 e 19 do ciclo estral (grupo controle), e em 15 fêmeas essas coletas não foram realizadas (grupo controle). As biópsias foram realizadas no corno uterino contralateral ao ovário contendo o corpo lúteo. Amostras plasmáticas foram coletadas nos momentos -24, -16, -8, 0 +8, +16 e +24 horas em relação à queda de progesterona (<1ng/mL, momento zero) e avaliadas quanto à concentração de 13, 14-di-hidro-15-ceto prostaglandina F2-alpha (PGFM) por radioimunoensaio (RIA). As concentrações plasmáticas de progesterona foram avaliadas a cada 24 horas também por RIA. Os dados foram analisados por ANOVA empregando-se PROC GLM e MIXED do SAS. O valor médio de PGFM durante todo o período avaliado foi maior no grupo biópsia. A concentração média de PGFM no momento zero não diferiu entre os grupos, e foi maior no grupo biópsia antes e após a queda de progesterona. A concentração máxima observada não foi diferente entre os grupos, porém a concentração mínima diferiu com maiores valores observados no grupo biópsia. Embora as concentrações de PGFM fossem maior no grupo biópsia, ambos os grupos apresentaram o mesmo comprimento do ciclo estral, demonstrando que a coleta repetitiva de biópsias endometriais no corno uterino contraletral ao ovário contendo o corpo lúteo não afeta a luteólise e o comprimento do ciclo estral.
Assuntos
Animais , Bovinos , Membrana Celular , Corpo Lúteo , Dinoprosta , Endométrio , Progesterona , Biópsia/veterinária , Colo do Útero , Ciclo EstralRESUMO
This study aimed to characterize the endometrial transcriptome and functional pathways overrepresented in the endometrium of cows treated to ovulate larger (≥13 mm) versus smaller (≤12 mm) follicles. Nelore cows were presynchronized prior to receiving cloprostenol (large follicle [LF] group) or not (small follicle [SF] group), along with a progesterone (P4) device on Day (D) -10. Devices were withdrawn and cloprostenol administered 42-60 h (LF) or 30-36 h (SF) before GnRH agonist treatment (D0). Tissues were collected on D4 (experiment [Exp.] 1; n = 24) or D7 (Exp. 2; n = 60). Endometrial transcriptome was obtained by RNA-Seq, whereas proliferation and apoptosis were assessed by immunohistochemistry. Overall, LF cows developed larger follicles and corpora lutea, and produced greater amounts of estradiol (D-1, Exp. 1, SF: 0.7 ± 0.2; LF: 2.4 ± 0.2 pg/ml; D-1, Exp. 2, SF: 0.5 ± 0.1; LF: 2.3 ± 0.6 pg/ml) and P4 (D4, Exp. 1, SF: 0.8 ± 0.1; LF: 1.4 ± 0.2 ng/ml; D7, Exp. 2, SF: 2.5 ± 0.4; LF: 3.7 ± 0.4 ng/ml). Functional enrichment indicated that biosynthetic and metabolic processes were enriched in LF endometrium, whereas SF endometrium transcriptome was biased toward cell proliferation. Data also suggested reorganization of the extracellular matrix toward a proliferation-permissive phenotype in SF endometrium. LF endometrium showed an earlier onset of proliferative activity, whereas SF endometrium expressed a delayed increase in glandular epithelium proliferation. In conclusion, the periovulatory endocrine milieu regulates bovine endometrial transcriptome and seems to determine the transition from a proliferation-permissive to a biosynthetic and metabolically active endometrial phenotype, which may be associated with the preparation of an optimally receptive uterine environment.
Assuntos
Diestro/fisiologia , Endométrio/metabolismo , Transcriptoma/genética , Animais , Apoptose , Caspase 3/fisiologia , Bovinos , Proliferação de Células , Cloprostenol/farmacologia , Biologia Computacional , Endométrio/crescimento & desenvolvimento , Ativação Enzimática/fisiologia , Matriz Extracelular/metabolismo , Feminino , Luteolíticos/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/ultraestrutura , GravidezRESUMO
In beef cattle, proestrus estradiol and subsequent progesterone (P4) concentrations can regulate the endometrial characteristics and thereby determine maternal receptivity toward the embryo. However, the underlying mechanisms linking periovulatory endocrine profiles to receptivity, which is crucial to obtain pregnancy, need to be elucidated. We hypothesized that the size of the preovulatory follicle (POF) and subsequent circulating P4 concentrations, during early diestrus, modulate endometrial levels of glucose transporter transcripts and proteins, and subsequently affect the luminal glucose availability in the uterus. Therefore, follicle growth of Nelore cows was manipulated, and cows were assigned to 2 experimental groups: (1) large follicle and large corpus luteum (LF-LCL) group with a large POF and corpus luteum (CL); and (2) small follicle and small corpus luteum (SF-SCL) group with a small POF and CL. At day 7 post gonadotropin-releasing hormone induced ovulation (gonadotropin-releasing hormone treatment = day 0), animals were slaughtered (n = 18 per group), and uterine tissues and washings were collected for characterization of glucose transporters and glucose levels, respectively. The diameter of POF was larger (P < 0.05) in the LF-LCL cows compared with their SF-SCL counterparts (12.8 ± 0.4 vs 11.1 ± 0.4 mm). Furthermore, CL size (17.49 ± 0.88 vs 14.48 ± 0.52 mm) and circulating P4 concentrations at day 7 (4.5 ± 1.0 vs 3.3 ± 1.1 ng/mL, P < 0.05) were significantly higher in the LF-LCL cows compared with the SF-SCL cows. No differences (P > 0.05) were detected in gene expression patterns of SLC2A1, SLC2A3, SLC2A4, SLC2A5, SLC5A1, ATP1A2, ATP1B2, and SLC37A4. However, the protein abundance of endometrial SLC2A1was increased in the LF-LCL group compared with the SF-SCL group (P < 0.05). SLC2A1 and SLC2A4 protein products were mainly identified at the endometrial luminal and glandular epithelium membranes as well as in the endometrial stroma. Glucose concentrations in uterine washings were similar between groups. In conclusion, we provided information on the potential link between endocrine profiles and glucose transport pathways in the bovine endometrium. More specifically, our data reveal that the size of the POF, and subsequent P4 concentrations, do not functionally affect the main endometrial glucose transporter pathways or uterine fluid glucose concentrations during diestrus.
Assuntos
Líquidos Corporais/química , Bovinos/fisiologia , Endométrio/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glucose/química , Ovulação/fisiologia , Animais , Bovinos/sangue , Corpo Lúteo/fisiologia , Feminino , Regulação da Expressão Gênica/fisiologia , Folículo Ovariano/fisiologia , Ovulação/sangue , Gravidez , ProgesteronaRESUMO
This short communication reports the impact of endometrial biopsies, uterine flushings and follicular fluid aspiration procedures at day 6 post artificial insemination (AI) on pregnancy rates. In Experiment 1, cows were timed AI (TAI) and assigned to the following treatment groups: control (n = 37), uterine flushing (n = 35) and endometrial biopsy (n = 38). On day 30 post AI, pregnancy rates were 40.5%, 33% and 28.5%, respectively (p > 0.1). Pregnancy rate on day 60 was lower (p < 0.004) in flushed cows than in the controls. In Experiment 2, oestrus was detected and cows were assigned to flushing (n = 32) or biopsy (n = 33) treatments 6 days after AI, which resulted in pregnancy rates of 31% and 36%, respectively (p > 0.1). In Experiment 3, cows were, 6 days after TAI, randomly assigned to the following treatments: control (n = 84) or aspiration of the largest follicle (n = 73). Pregnancy rates on day 30 post AI were 63.5% for the control group and 53% for the aspirated group (p > 0.1). In conclusion, uterine flushing and endometrial biopsy negatively affect pregnancy rates, but neither procedure can be considered to be incompatible with pregnancy maintenance. Follicular aspiration during pregnancy does not interact with pregnancy success. The amount and quality of samples obtained are compatible with the use of cellular and molecular analysis of uterine variables from cows that failed or succeeded on maintaining pregnancy.
Assuntos
Biópsia/efeitos adversos , Fertilidade , Irrigação Terapêutica/efeitos adversos , Útero/fisiologia , Animais , Bovinos , Desenvolvimento Embrionário , Endométrio/irrigação sanguínea , Endométrio/patologia , Feminino , Líquido Folicular , Inseminação Artificial/veterinária , Folículo Ovariano , Gravidez , Taxa de Gravidez , Sucção , Fatores de Tempo , Ultrassonografia Pré-Natal/veterináriaRESUMO
In beef cattle, the ability to conceive has been associated positively with size of the preovulatory follicle (POF). Proestrus estradiol and subsequent progesterone concentrations can regulate the endometrium to affect receptivity and fertility. The aim of the present study was to verify the effect of the size of the POF on luteal and endometrial gene expression during subsequent early diestrus in beef cattle. Eighty-three multiparous, nonlactating, presynchronized Nelore cows received a progesterone-releasing device and estradiol benzoate on Day-10 (D-10). Animals received cloprostenol (large follicle-large CL group; LF-LCL; N = 42) or not (small follicle-small CL group; SF-SCL; N = 41) on D-10. Progesterone devices were withdrawn and cloprostenol administered 42 to 60 hours (LF-LCL) or 30 to 36 hours (SF-SCL) before GnRH treatment (D0). Tissues were collected at slaughter on D7. The LF-LCL group had larger (P < 0.0001) POF (13.24 ± 0.33 mm vs. 10.76 ± 0.29 mm), greater (P < 0.0007) estradiol concentrations on D0 (2.94 ± 0.28 pg/mL vs. 1.27 ± 0.20 pg/mL), and greater (P < 0.01) progesterone concentrations on D7 (3.71 ± 0.25 ng/mL vs. 2.62 ± 0.26 ng/mL) compared with the SF-SCL group. Luteal gene expression of vascular endothelial growth factor A, kinase insert domain receptor, fms-related tyrosine kinase 1, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 7 was similar between groups. Endometrial gene expression of oxytocin receptor and peptidase inhibitor 3, skin-derived was reduced, and estrogen receptor alpha 2, aldo-keto reductase family 1, member C4, and lipoprotein lipase expression was increased in LF-LCL versus SF-SCL. Results support the hypothesis that the size of the POF alters the periovulatory endocrine milieu (i.e., proestrus estradiol and diestrus progesterone concentrations) and acts on the uterus to alter endometrial gene expression. It is proposed that the uterine environment and receptivity might also be modulated. Additionally, it is suggested that increased progesterone secretion of cows ovulating larger follicles is likely due to increased CL size rather than increased luteal expression of steroidogenic genes.