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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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O conhecimento do processo de luteólise dentro da fisiologia reprodutiva se torna importante para que ocorra a compreensão do reconhecimento materno da gestação e seja possível formular estratégias anti-luteolíticas que visem manter esta fase conferindo aumentos na eficiência reprodutiva e produtiva. Durante o processo de luteólise, o tecido luteal sofre mudanças bruscas na capacidade esteroidogênica, vascularização e remodelamento, resultando em substituição da glândula por tecido conjuntivo, sendo a PGF2α o principal determinante na indução espontânea da luteólise em bovinos. Simultaneamente, ocorre um íntimo processo entre as relações anatômicas que compreende o ovário, útero e suas vascularizações. A exposição do útero à interação coordenada entre P4, E2 e ocitocina aliada à expressão de seus receptores são essenciais para a secreção pulsátil de PGF2α pelo endométrio a qual induzirá a luteólise. Nesta regressão do corpo lúteo (CL), estão envolvidos ainda os hormônios (LH, prolactina, cortisol) e várias substâncias vasoativas (óxido nítrico, VEGF, EG-VEGF, bFGF, END-1, AngII). A concentração de PGF2α pode ser representada pela mensuração sanguínea da concentração de seu metabólito 13,14-dihidro-15-ceto- PGF2α (PGFM) sendo a pulsatilidade da secreção de PGF2α durante os períodos pré-luteolítico, luteolítico e pós-luteolítico mensurável pelo aumento na concentração de PGFM. Neste artigo de revisão, será possível compreender e discutir os principais aspectos endócrinos e moleculares que ocorrem com as mudanças morfofuncionais no CL durante o processo de luteólise fisiológica em fêmeas bovinas.

The knowledge of the luteolysis process within reproductive physiology becomes important to occur the understanding of maternal recognition of gestation being possible to formulate anti-luteolytic strategies aimed at maintaining this phase conferring increases in reproductive and productive efficiency. During the luteolysis process, luteal tissue undergoes abrupt changes in steroidogenic capacity, vascularization and remodeling, resulting in replacement of the gland by connective tissue, and PGF2α is the main determinant in the spontaneous induction of luteolysis in cattle. Simultaneously, an intimate process occurs between the anatomical relationships that comprise the ovary, uterus and its vascularizations. The exposure of the uterus to the coordinated interaction between P4, E2 and oxytocin, together with the expression of its receptors, are essential for the pulsating secretion of PGF2α by the endometrium, which will induce luteolysis. In this regression of the corpus luteum (CL), hormones (LH, prolactin, cortisol) and various vasoactive substances (nitric oxide, VEGF, EG-VEGF, bFGF, END-1, AngII) are also involved. The concentration of PGF2α can be represented by the blood measurement of the concentration of its metabolite 13,14-dihydro-15-keto-PGF2α (PGFM),being the pulsatility of PGF2α secretion during the pre-luteolytic, luteolytic and post-luteolytic measured by the increased concentration of PGFM. In this review article, it will be possible to understand and discuss the main endocrine and molecular aspects that occur with the morphofunctional changes in CL during the physiological luteolysis process in bovine females.

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