Unravelling the role of 17ß-estradiol on advancing uterine luteolytic cascade in cattle.

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.

Effects of estradiol treatments on PGF2α release in beef heifers submitted to estrous resynchronization 14 days after timed-AI.

The effects of 17ß-estradiol (E2) or estradiol benzoate (EB) on PGF2α release were studied in bred-non-pregnant and pregnant Nelore beef heifers. The day of timed artificial insemination (TAI) was designated day 0 (D0), and a single treatment was given on D14. All heifers also received an intravaginal P4 device on D14, and were randomly assigned to three groups: Control (C, P4 device only, n = 12); E2 (1 mg E2 + 9 mg P4, n = 10); or EB (1 mg, n = 10). Blood samples were collected hourly for 8 hours after treatment (Hours 0-8) to measure plasma concentrations (pg/mL) of a PGF2α metabolite (PGFM). The P4 device was removed on D22 and pregnancy was diagnosed on D28. Pregnancy rate was not different among groups (C, n = 7/12; E2, n = 5/10; EB, n = 5/10). More (P < 0.05) heifers had a CV-identified prominent PGFM pulse (peak of > 100 pg/mL) in E2 group (6/10) than in EB (1/10) and C (0/12) groups. Hourly concentration of PGFM for Hours 0 to 8 showed significant effects of group and hour and an interaction of group by hour but did not show an interaction of group or hour with pregnancy status. In preliminary post-hoc analyses, PGFM concentrations during Hours 0 to 8 and pulse characteristics were analyzed within each pregnancy status. For the non-pregnant heifers, a group-by-hour interaction was detected tentatively indicating an increase (P < 0.005) in PGFM concentrations in E2 group from Hours 4 to 6 and in EB group at Hours 5 and 6. Maximum PGFM concentration during Hours 0 to 8 did not differ (P > 0.1) between E2 (124 ± 23) and EB (110 ± 30) groups, but was greater (P < 0.05) in each group than in C (32 ± 3). Furthermore, PGFM concentrations of pulses at the peak, amplitude, and area under pulse curve (pg/mL/h) were greater (P < 0.05) in E2 group than in C group whereas the EB group did not differ (P > 0.1) from the other groups. For pregnant heifers, no effects of group, hour, or their interaction were detected in PGFM concentrations during the hourly sessions, except that maximum PGFM concentration was greater (P < 0.05) in E2 than in EB and C groups. In addition, the number of prominent pulses was greater in E2 group than in Control or EB groups. In conclusion, PGFM increased earlier and in greater concentration combined for bred-non-pregnant and pregnant heifers treated 14 days after TAI with 1 mg E2 plus 9 mg P4 than with 1 mg EB. Tentatively, a positive effect for each of E2 and EB on PGFM concentrations was attenuated in pregnant heifers.

Type I interferon receptors and interferon-τ-stimulated genes in peripheral blood mononuclear cells and polymorphonuclear leucocytes during early pregnancy in beef heifers.

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.

Increased pregnancy rate in beef heifers resynchronized with estradiol at 14 days after TAI.

We aimed to evaluate the treatment with estradiol benzoate (EB) or 17ß-estradiol (E2) associated with progesterone (P4) for resynchronization of ovulation 14 days after timed artificial insemination (TAI). In Experiment 1 (Exp. 1), Nelore heifers were submitted to TAI (D0). On D14, the animals received an intravaginal P4 device and were randomly assigned to one of three groups: control (no treatment; n = 17); EB (1  mg EB; n = 17); and E2+P4 (1 mg E2 + 9 mg P4; n = 18). Ultrasonography evaluations were performed daily from D14 to D22 to map follicular and luteal dynamics. On D22, the P4 devices were removed and non-pregnant (NP) animals were determined using corpus luteum blood flow Doppler ultrasonography. In Exp. 2, 1295 beef heifers were resynchronized and randomly allocated to the same experimental groups as described in Exp. 1. On D22, the largest follicle (LF) was measured in NP and a second TAI was performed on D24. In a subset of heifers (n = 337), an estrus detection patch was used between D22 and D24 to monitor estrus expression and the LF was measured at D24. Confirmatory diagnosis of pregnancy was performed between D37-67 and D43-67 after first and second TAI, respectively. In Exp 1, the proportion of heifers with a synchronized follicular wave emergence (from 3 to 5 days after treatment) was greater (P < 0.05) in the EB group (93.8%) than in the control (62.5%) and E2+P4 (64.7%) groups. Structural luteolysis occurred earlier (P < 0.05) in the EB and E2+P4 groups than in the controls. The pregnancy rate after first TAI did not differ (P > 0.1) among the groups at D22 and at confirmatory diagnosis in both experiments. In Exp 2, the potential pregnancy loss between D22 and D37-67 was similar (P > 0.1) in the control (19% [36/185]), EB (15% [28/182]) and E2+P4 (15% [28/184]) groups. The LF diameter (mm) on D22 was greater (P < 0.05) in the control group (11.9 ± 0.1) than in EB (11.3 ± 0.1) and E2+P4 (11.5 ± 0.1). No difference (P > 0.1) was observed in the proportion of heifers detected in estrus, but LF growth rate (mm/day) between D22 and D24 was greater (P < 0.05) in EB group (0.9 ± 0.08) than in control (0.6 ± 0.07) and E2+P4 (0.7 ± 0.09) groups. The pregnancy rate for the second TAI was greater (P < 0.05) in the EB group (47% [94/200]) than in the control (37% [76/203]), but did not differ (P > 0.1) from the E2+P4 group (43% [93/214]). In conclusion, the treatment with 1 mg EB or 1 mg E2 + 9 mg P4 at 14 days post-TAI anticipates luteolysis in NP heifers but does not compromise pregnancy. The EB treatment induces a new synchronized follicle wave emergence and increases the pregnancy rate of resynchronized NP heifers.

Supplemental progesterone induces temporal changes in luteal development and endometrial transcription in beef cattle.

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.

Supplementation with long-acting progesterone in early diestrus in beef cattle: I. effect of artificial insemination on onset of luteolysis.

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 (D28‒D32). 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.

Supplementation with long-acting progesterone in early diestrus in beef cattle: II. Relationships between follicle growth dynamics and luteolysis.

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.

Impact of estradiol cypionate prior to TAI and progesterone supplementation at initial diestrus on ovarian and fertility responses in beef cows.

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.

Cytobrush: A tool for sequential evaluation of gene expression in bovine endometrium.

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.

Improved fertility in suckled beef cows ovulating large follicles or supplemented with long-acting progesterone after timed-AI.

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.

The Receptive Endometrial Transcriptomic Signature Indicates an Earlier Shift from Proliferation to Metabolism at Early Diestrus in the Cow.

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.

Modulation of periovulatory endocrine profiles in beef cows: consequences for endometrial glucose transporters and uterine fluid glucose levels.

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.

Impact of probing the reproductive tract during early pregnancy on fertility of beef cows.

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.

Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows.

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.

Corpus luteum development and function after supplementation of long-acting progesterone during the early luteal phase in beef cattle.

Strategic supplementation of P4 may be used to increase conception rates in cattle, but timing of supplementation in relation to ovulation, mass of supplementary P4 and formulation of the P4-containing supplement has not been determined for beef cattle. Effects of supplementation of long-acting progesterone (P4) on Days 2 or 3 post-ovulation on development, function and regression of corpus luteum (CL) were studied in beef cattle. Cows were synchronized with an oestradiol/P4-based protocol and treated with 150 or 300 mg of long-acting P4 on Day 2 or 3 post-ovulation (6-7 cows/group). Colour-doppler ultrasound scanning and blood sample collection were performed from Day 2-21.5. Plasma P4 concentrations were greater (p < 0.05) from Day 2.5-5.5 in the Day 2-treated groups and from Day 3.5-5.5 in the Day 3-treated cows than in the control group. CL area and blood flow during Day 2-8.5 did not differ (p > 0.05) among groups, suggesting no effect of P4 treatment on luteal development. The frequency of cows that began luteolysis before Day 15 was greater (p < 0.04) in cows treated with 300 mg than in the controls, but there were no differences between non-treated and 150 mg-treated cows. The interval from pre-treatment ovulation to functional and structural luteolysis was shorter (p < 0.01) in the combined P4-treated groups than in the control cows. In conclusion, was showed for the first time that long-acting P4 supplementation on Day 2 or 3 post-ovulation increases P4 concentrations for ≥3 day, has no effect on luteal development, but anticipates the beginning of luteolysis in beef cattle.

Direct effect of PGF2α pulses on PRL pulses, based on inhibition of PRL or PGF2α secretion in heifers.

The relationships between PRL and PGF(2α) and their effect on luteolysis were studied. Heifers were treated with a dopamine-receptor agonist (bromocriptine; Bc) and a Cox-1 and -2 inhibitor (flunixin meglumine [FM]) to inhibit PRL and PGF(2α), respectively. The Bc was given (Hour 0) when ongoing luteolysis was indicated by a 12.5% reduction in CL area (cm(2)) from the area on Day 14 postovulation, and FM was given at Hours 0, 4, and 8. Blood samples were collected every 8-h beginning on Day 14 until Hour 48 and hourly for Hours 0 to 12. Three groups of heifers in ongoing luteolysis were used: control (n = 7), Bc (n = 7), and FM (n = 4). Treatment with Bc decreased (P < 0.003) the PRL concentrations averaged over Hours 1 to 12. During the greatest decrease in PRL (Hours 2-6), LH concentrations were increased. Progesterone concentrations averaged over hours were greater (P < 0.05) in the Bc group than in the controls. In the FM group, no PGFM pulses were detected, and PRL concentrations were reduced. Concentrations of PGFM were not reduced in the Bc group, despite the reduction in PRL. Results supported the hypothesis that a decrease (12.5%) in CL area (cm(2)) is more efficient in targeting ongoing luteolysis (63%) than using any day from Days 14 to ≥ 19 (efficiency/day, 10-24%). The hypothesis that PRL has a role in luteolysis was supported but was confounded by the known positive effect of LH on progesterone. The hypothesis was supported that the synchrony of PGFM and PRL pulses represents a positive effect of PGF(2α) on PRL, rather than an effect of PRL on PGF(2α).

Role of LH in luteolysis and growth of the ovulatory follicle and estradiol regulation of LH secretion in heifers.

The role of LH in luteolysis and development of the ovulatory follicle and the involvement of GnRH receptors in estradiol (E2) stimulation of LH secretion were studied in heifers. A pulse of PGF(2α), as indicated by a metabolite, was induced by E2 treatment on Day 15 (Day 0 = ovulation) and LH concentration was reduced with a GnRH-receptor antagonist (acyline) on Days 15, 16, and 17. Blood samples were collected every 6 h on Days 14-17 and hourly for 10 h beginning at the Day-15 treatments. Four groups were used (n = 6): control, acyline, E2, and E2/acyline. The number of LH pulses/heifer during the 10 h posttreatment was greater (P < 0.0002) in the E2 group (2.3 ± 0.4, mean ± SEM) than in the acyline group (0.2 ± 0.2) and was intermediate in the E2/acyline group (1.4 ± 0.2). Concentrations of progesterone in samples collected every 6 h on Day 15 showed a group-by-hour interaction (P < 0.02); concentrations decreased in the acyline group but not in the control group. The 12 heifers in the combined acyline and E2/acyline groups had three follicular waves compared to two waves in 10 of 12 heifers in the combined control and E2 groups. Results (1) supported the hypothesis that LH delays the progesterone decrease associated with luteolysis, (2) supported the hypothesis that LH has a positive effect on the continued development and growth of the selected ovulatory follicle, and (3) indicated that E2 stimulates LH production through an intracellular pathway that involves GnRH receptors on the gonadotropes and a pathway that does not involve the receptors.

Induction of PGFM pulses and luteolysis by sequential estradiol-17ß treatments in heifers.

The effects of sequential induction of PGFM pulses by estradiol-17ß (E2) on prominence of PGFM pulses and progesterone (P4) concentration were studied in heifers. Three treatments of vehicle (n = 12) or E2 (n = 12) at doses of 0.05 or 0.1 mg were given at 12-h intervals beginning on Day 15 postovulation. Blood samples were collected every 12 h from Days 13-24 and hourly for 12 h after the first and third treatments. On Day 15, all heifers were in preluteolysis and on Day 16 were in preluteolysis in the vehicle-treated heifers (n = 11) and either preluteolysis (n = 4) or luteolysis (n = 8) in the E2-treated heifers. Peak concentration of induced PGFM pulses during preluteolysis on Day 15 was greater (P < 0.04) than for pulses during preluteolysis on Day 16. The interval from ovulation to the beginning of luteolysis was shorter (P < 0.04) in the E2-treated heifers than in the vehicle-treated heifers. An E2-induced PGFM pulse was less prominent (P < 0.008) in heifers in temporal association with a transient resurgence in P4 than in heifers with a progressive P4 decrease. The hypothesis that repeated E2 exposure stimulates increasing prominence of PGFM pulses was not supported. Instead, repeated exposure reduced the prominence of PGFM pulses, in contrast to the stimulation from the first E2 treatment. Reduced prominence of a PGF(2α) pulse during luteolysis can lead to a transient resurgence in P4 concentration.

Effect of dose of estradiol-17ß on prominence of an induced 13,14-dihydro-15-keto-PGF(2α) (PGFM) pulse and relationship of prominence to progesterone, LH, and luteal blood flow in heifers.

Various doses of estradiol-17ß (E(2)) were used in heifers to induce a pulse of 13,14-dihydro-15-keto-prostaglandin F(2α) (PGFM). The effect of E(2) concentration on the prominence of PGFM pulses and the relationship between prominence and intrapulse concentration of progesterone (P(4)), LH, and luteal blood flow were studied. A single dose of 0 (vehicle), 0.01, 0.05, or 0.1 mg of E(2) was given (n = six/group) 14 d after ovulation. Blood samples were collected, and luteal blood flow was evaluated hourly for 10 h after the treatment. The 0.05-mg dose increased and the 0.1-mg dose further increased the prominence of the induced PGFM pulse, compared with the 0.0-mg dose and the 0.01-mg dose. The PGFM pulses were subdivided into three different prominence categories (<50, 50 to 150, and >150 pg/mL at the peak). In the 50 to 150 category, P(4) concentration increased (P < 0.05) between -2 h and 0 h (0 h = peak of PGFM pulse). In the >150 category, P(4) decreased (P < 0.05) between -1 h and 0 h, LH increased (P < 0.05) at 1 h, and luteal blood flow apparently decreased (P < 0.05) at 2 h of the PGFM pulse. The novel results supported the following hypotheses: (1) an increase in E(2) concentration increases the prominence of a PGFM pulse, and (2) greater prominence of a PGFM pulse is associated with a greater transient intrapulse depression of P(4) at the peak of the PGFM pulse. In addition, the extent of the effect of prostaglandin F(2α) on the increase in LH and changes in blood flow within the hours of a PGFM pulse was related positively to the prominence of the PGFM pulse.

Effects of inhibition of prostaglandin F2α biosynthesis during preluteolysis and luteolysis in heifers.

Flunixin meglumine (FM; 2.5 mg/kg) was given to heifers at three 8-h intervals, 16 d after ovulation (first treatment = Hour 0) to inhibit the synthesis of prostaglandin F(2α) (PGF), based on plasma concentrations of a PGF metabolite (PGFM). Blood samples were collected at 8-h intervals from 15 to 18 d in a vehicle (control) and FM group (n = 16/group). Hourly samples were collected from Hours -2 to 28 in 10 heifers in each group. Heifers that were in preluteolysis or luteolysis at Hour 0 based on plasma progesterone (P4) concentrations at 8-h intervals were partitioned into subgroups. Concentration of PGFM was reduced (P < 0.05) by FM treatment in each subgroup. For the preluteolytic subgroup, the first decrease (P < 0.05) in P4 concentration after Hour 0 occurred at Hours 24 and 40 in the vehicle and FM groups, respectively. Plasma P4 concentrations 32 and 40 h after the beginning of luteolysis in the luteolytic subgroup were greater (P < 0.05) in the FM group. Concentration at the peak of a PGFM pulse in the FM group was greater (P < 0.05) in the luteolytic than in the preluteolytic subgroup. The peak of a PGFM pulse occurred more frequently (P < 0.001) at the same hour as the peak of an LH fluctuation than at the ending nadir of an LH fluctuation. In conclusion, a reduction in prominence of PGFM pulses during luteolysis delayed completion of luteolysis, and treatment with FM inhibited PGFM production more during preluteolysis than during luteolysis.