Preovulatory Estradiol Concentrations Research Articles

Associations among the largest follicle, preovulatory estradiol concentrations, and predominant vaginal epithelial cells at the completion of hormonal ovarian stimulation for fixed-time artificial insemination in goats.

The objective of the present study was to investigate the association among the largest follicle (LF), preovulatory estradiol (E2), and predominant vaginal epithelial cell at the completion of hormonal ovarian stimulation for fixed-time artificial insemination (FTAI) in goats. Thirty-seven crossbred Boer does received gonadotropin-releasing hormone (GnRH) and intravaginal progesterone (P4)-releasing devices (day 0). On day 5, P4 devices were removed and does received prostaglandin F2α and equine chorionic gonadotrophin. On day 7, does received GnRH, and FTAI was undertaken. On day 7, does were divided into three groups, i.e. small-sized (3-3.9 mm; n = 5), medium-sized (4-4.9 mm; n = 8), and large-sized (≥5 mm; n = 24) according to the diameter of the ovarian LF; follicular characteristics (number and diameter) were identified, and blood samples and vaginal smears were collected. The average diameters of total antral follicles and LF and the percentage of superficial cell were greatest in large-sized LF does (p < .01). The average diameters of total antral follicle (r = .68) and LF (r = .71), number of preovulatory follicle (r = .58), and plasma E2 concentrations (r = .61) were positively correlated with the percentage of superficial cells (p < .01). The likelihood of a pregnancy outcome after the FTAI increased by 13.71 times in does with a greater average diameter of antral follicle, 14.18 times with emergence of a large preovulatory follicle, and 36.83 times with a higher percentage of vaginal superficial cells (p < .01). It was concluded that there is a relationship between the cell types of the vaginal epithelium, the diameters of the largest ovarian follicles, and the concentration of E2 in goats subjected to FTAI protocols.

Importance of preovulatory estradiol on uterine receptivity and luteal function.

Animals that exhibited estrus had greater pregnancy success compared to animals that did not exhibit estrus before fixed-time AI (FTAI). Estradiol is synthesized in bovine ovarian follicles under gonadotropin regulation and can directly and indirectly regulate the uterine receptivity and luteal function. Estradiol concentrations at FTAI impacted oviductal gene expression and has been reported to play an important role in establishing the timing of uterine receptivity. These changes have been reported to impact uterine pH and sperm transport to the site of fertilization. After fertilization, preovulatory estradiol has been reported to improve embryo survival likely by mediating changes in uterine blood flow, endometrial thickness and changes in histotroph. Cows with greater estradiol concentrations at the time of GnRH-induced ovulation also had a larger dominant follicle size and greater circulating progesterone concentrations on day 7. Therefore, it is impossible to accurately determine the individual benefit of greater estradiol concentrations prior to ovulation and greater progesterone concentrations following ovulation to pregnancy establishment, as these two measurements are confounded. Research has indicated an importance in the occurrence and timing of increasing preovulatory concentrations of estradiol, but increasing estradiol concentrations by supplementation may not be sufficient to increase fertility. Increased production of estradiol by the preovulatory follicle may be required to enhance fertility through the regulation of sperm transport, fertilization, oviductal secretions, the uterine environment, and embryo survival.

Impact of preovulatory estradiol concentrations on subsequent luteal function in beef cattle

It has been hypothesized that circulating concentrations of estradiol during the preovulatory period, can impact subsequent progesterone concentrations. Ovulation was synchronized in nonlactating beef cows (n = 53). Cows that exhibited estrus before gonadotrophin-releasing hormone (GnRH)-induced ovulation (d 0) had greater (p<.01) peak concentrations of estradiol compared with cows that did not express estrus (11.5 ± 0.8 vs. 6.2 ± 0.6 pg/mL), respectively, but there was no difference in ovulatory follicle size (p= .80) or interval from GnRH2 to ovulation (p=.23). Circulating concentrations of progesterone during luteal formation (d 3–7; p=.70 and p=.77) or mid-luteal phase (d 8–14; p=.39 and p=.12) were not affected by elevated periovulatory estradiol or an interaction with day. To investigate the direct influence of estradiol on luteal function, ovulation (d 0) was synchronized in nonlactating beef cows and cows were allocated to three groups (control, n = 5; vehicle injection, n = 4; or an estradiol antagonist (Fulvestrant; ICI 182,780), n = 4. Intrafollicular injection of vehicle (100 µL) or an estradiol antagonist (25 μg Fulvestrant in 100 µL) into the largest follicle occurred on d –2. Concentrations of estradiol increased (p<.0001) from d –2 to 0 but did not differ among groups (p>.50). Furthermore, plasma concentrations of progesterone on d 0 through 20 were not affected by treatment (p=.86). These results indicate that elevated preovulatory estradiol before ovulation was not required to prepare granulosa cells for luteinization or subsequent luteal progesterone secretion but did tend to impact luteal lifespan.

239 Increased Preovulatory Estradiol Improves Pregnancy Success of Embryo Transfer in Beef Cows

Abstract Preovulatory estradiol concentrations and expression of estrus have been associated with greater pregnancy rates in beef cattle following AI, thus an experiment was designed to determine if supplemental estradiol (E2) at GnRH-induced ovulation would improve pregnancy retention in postpartum beef cows after embryo transfer. Cows were synchronized with the 7-d CO-Synch + CIDR® protocol. On d0 (48h post-prostaglandin) cows were grouped by estrual status (Estrual; n=198, nonestrual; n=406). Nonestrual cows were administered GnRH and randomly assigned to either no treatment (Control; n=204) or administration of 0.1 mg (IM) estradiol 17-β (Estradiol; n=202). In a preliminary study, 0.1 mg (IM) estradiol 17-β increased plasma estradiol (17.11±3.4 pg/mL; P &amp;lt; 0.01) and remained elevated above baseline for approximately 8h compared to control (1.70±0.58 pg/mL). All cows received an in vivo produced embryo on d7. Embryos were matched by grade, stage, and flush across treatments. Pregnancy was classified on d30 by either plasma pregnancy-associated glycoproteins analysis (Year 1) or ultrasonography (Year 2). Plasma estradiol concentrations (d-2, d0h0, d0h2) were analyzed by PROC MIXED as repeated measures in SAS(9.4), while GLIMMIX procedures were used to analyze differences in pregnancy rates with treatment, group, year, and their interactions as fixed effects. There was a treatment by time interaction (P &amp;lt; 0.001) in E2 concentration. There was no difference in plasma E2 concentrations on d0h0 (P &amp;gt;0.30) between treatments. At d0h2, Estradiol cows (15.7±0.25 pg/mL) had greater plasma E2 compared to Estrual (4.0±0.13 pg/mL) or Control (3.4±0.25 pg/mL) cows (P &amp;lt; 0.001), and Estrual cows had greater plasma E2 than Control cows (P = 0.02). Control cows had decreased (26%) pregnancy rates compared to both Estradiol (37%; P = 0.03) and Estrual (40%; P = 0.01) cows. Pregnancy rates did not differ (P = 0.51) between the Estrual and Estradiol cows. In conclusion, elevated preovulatory concentrations of estradiol are critical for improved pregnancy retention following embryo transfer.

Effect of equine chorionic gonadotropin (eCG) administration and proestrus length on ovarian response, uterine functionality and pregnancy rate in beef heifers inseminated at a fixed-time

The objective of the present study was to evaluate the effect of equine chorionic gonadotropin (eCG) administration associated to different proestrus lengths for Fixed-time AI (FTAI) in beef heifers. In Experiment 1, pre-pubertal heifers (n = 46) received a 6-day estradiol/progesterone-based treatment (J-Synch protocol), and were then allocated into four experimental groups in a 2 × 2 factorial design, to receive or not receive eCG (300 IU) at the time of intravaginal progesterone device removal, and to receive GnRH at 48 h or 72 h after device removal (to induce shortened and prolonged proestrus length, respectively). Endometrial samples were obtained 6 d after ovulation from the cranial portion of the uterine horn. The eCG administration induced greater serum estradiol-17β concentrations before ovulation (P < 0.05) and greater proportion of heifers bearing a competent corpus luteum after ovulation (P = 0.054). Delaying GnRH administration from 48 h to 72 h induced a longer interval from device removal to ovulation (i.e., prolonged proestrus; P < 0.05), larger diameter of the ovulatory follicle, and greater progesterone concentrations on Day 10–11 after ovulation. Heifers in eCG + GnRH72h group had more uterine receptors in luminal epithelium than those in eCG + GnRH48h group (PR and ERα), and than those in No eCG + GnRH72h group (PR) (P < 0.05). No effect of eCG or GnRH treatments was found in endometrial gene expression of progesterone and estrogen receptors. In Experiment 2, a total of 2,598 heifers received the J-Synch protocol associated or not with eCG administration at device removal, followed by FTAI/GnRH at 60 or 72 h after device removal (i.e., prolonged proestrus protocol). Heifers that received eCG had greater P/AI than those not receiving eCG (P < 0.05) and there was an interaction between eCG treatment and time of FTAI. The lowest P/AI was found in those heifers that received FTAI/GnRH at 72 h without eCG treatment at device removal (P < 0.05), and no differences were found between the other experimental groups. In conclusion, prolonging the length of proestrus in J-Synch protocol improves ovulatory follicular diameter and luteal function; and the administration of eCG at device removal improves preovulatory estradiol concentrations and luteal function. Finally, P/AI was enhanced by eCG treatment and the improvement was more evident when FTAI/GnRH was performed at 72 h after device removal.

Role of preovulatory concentrations of estradiol on timing of conception and regulation of the uterine environment in beef cattle

ABSTRACTThe ability to induce ovulation with an injection of GnRH facilitated the development of fixed-time artificial insemination (AI) protocols. However, animals that exhibit estrus prior to fixed-time AI have greater pregnancy success. Thus, the objectives of the present experiments were to determine the impact of estrus expression prior to fixed-time AI on timing of conception and to characterize the role of preovulatory estradiol in regulating changes in expression of uterine genes. In experiment 1, data were collected from 4,499 beef cows inseminated by fixed-time AI in 31 different herds. Animals that did not conceive to AI but exhibited estrus before timed-AI were more likely to conceive during cycle 1 after AI, and overall conceived earlier in the breeding season compared to animals that did not exhibit estrus. In experiment 2, beef cows were synchronized using a fixed-time AI protocol. Uterine horn biopsies and blood samples were collected on Day 0, 5, 10, or 16. Concentrations of estradiol on Day 0 did not influence expression of progesterone receptor, ER beta, or oxytocin receptor. Increased concentrations of estradiol on Day 0 increased expression of ER alpha from Days 0 to 5 of the estrous cycle. Furthermore, cows with increased concentrations of estradiol on Day 0 had increased expression of inhibin beta A, and uterine milk protein precursor. Thus, animals that do not exhibit estrus prior to fixed-time AI had decreased breeding season pregnancy success and conceived later in the breeding season, and preovulatory concentrations of estradiol likely play a major role in this establishment of pregnancy, not only directly by regulating uterine gene expression, but also indirectly throughout the subsequent estrous cycle.

Effects of preovulatory estradiol on uterine environment and conceptus survival from fertilization to maternal recognition of pregnancy.

Preovulatory estradiol is known to impact embryo quality and survival. The objective of this study was to determine the effects of preovulatory estradiol on the uterine environment and conceptus survival through maternal recognition of pregnancy. Beef cows/heifers were AIed following induced ovulation. Cows were grouped into high and low preovulatory estradiol. Conceptuses were collected on day 16 nonsurgically (Rep 1; n=20), or following slaughter (Rep 2; n=29). Blood was collected to determine plasma glucose concentrations, and uterine luminal fluid (ULF) was analyzed for protein, glucose, and interferon tau (IFNT) concentrations. Total cellular RNA was extracted from caruncular (CAR) and intercaruncular (INCAR) endometrial tissue. There was no effect of preovulatory estradiol on conceptus recovery rate (P=0.38) or on apoptosis rate in the trophectoderm (P=0.64). Cows in which a conceptus was recovered had greater concentrations of protein in the ULF (P=0.04). Animals with elevated preovulatory estradiol had greater endometrial abundance of SLC2A1 (P=0.05) and SLC5A1 (P=0.04) in both INCAR and CAR tissue. Presence of a conceptus also tended to increase (P=0.10) abundance of SLC5A1 in INCAR. In CAR tissue, cows with a conceptus had decreased SLC2A4 abundance (P=0.05). In summary, conceptus recovery rates, apoptosis in the trophectoderm, IFNT, glucose, and protein concentration in ULF did not differ between cows that did or did not have an increase in preovulatory estradiol concentrations. Thus, there is no indication of increased conceptus survival to day 16 of pregnancy based on estradiol concentrations.

Dynamic remodeling of endometrial extracellular matrix regulates embryo receptivity in cattle.

We aimed to evaluate in the bovine endometrium whether (1) key genes involved in endometrial extracellular matrix (ECM) remodeling are regulated by the endocrine peri-ovulatory milieu; and (2) specific endometrial ECM-related transcriptome can be linked to pregnancy outcome. In Experiment 1, pre-ovulatory follicle growth of cows was manipulated to obtain two groups with specific endocrine peri-ovulatory profiles: the Large Follicle-Large CL group (LF-LCL) served as a paradigm for greater receptivity and fertility and showed greater plasma pre-ovulatory estradiol and post-ovulatory progesterone concentrations when compared to the Small Follicle-Small CL group (SF-SCL). Endometrium was collected on days 4 and 7 of the estrous cycle. Histology revealed a greater abundance of total collagen content in SF-SCL on day 4 endometrium. In Experiment 2, cows were artificially inseminated and, six days later, endometrial biopsies were collected. Cows were retrospectively divided into pregnant and non-pregnant (P vs. NP) groups after diagnosis on day 30. In both experiments, expression of genes related to ECM remodeling in the endometrium was studied by RNAseq and qPCR. Gene ontology analysis showed an inhibition in the expression of ECM-related genes in the high receptivity groups (LF-LCL and P). Specifically, there was down-regulation of TGFB2, ADAMTS2, 5 and 14, TIMP3 and COL1A2, COL3A1, COL7A1 and COL3A3 in the LF-LCL and P groups. In summary, the overlapping set of genes differently expressed in both fertility models: (1) suggests that disregulation of ECM remodeling can impair receptivity and (2) can be used as markers to predict pregnancy outcome in cattle.

The periovulatory endocrine milieu affects the uterine redox environment in beef cows.

BackgroundIn cattle, recent studies have shown positive associations between pre-ovulatory concentrations of estradiol (E2), progesterone (P4) at early diestrus and fertility. However, information on cellular and molecular mechanisms through which sex steroids regulate uterine function to support early pregnancy is lacking. Based on endometrial transcriptome data, objective was to compare function of the redox system in the bovine uterus in response to different periovulatory endocrine milieus.MethodsWe employed an animal model to control growth of the pre-ovulatory follicle and subsequent corpus luteum (CL). The large follicle-large CL group (LF-LCL, N = 42) presented greater levels of E2 on the day of GnRH treatment (D0; 2.94 vs. 1.27 pg/mL; P = 0.0007) and P4 at slaughter on D7 (3.71 vs. 2.62 ng/mL, P = 0.01), compared with the small follicle-small CL group (SF-SCL, N = 41). Endometrium and uterine washings (N = 9, per group) were collected for analyses of variables associated with the uterine redox system.ResultsThe SF-SCL group had lower endometrial catalase (0.5 vs. 0.79 U/mg protein, P < 0.001) and glutathione peroxidase (GPx; 2.0 vs. 2.43 nmol β-nicotinamide adenine dinucleotide phosphate reduced/min/mg protein, P = 0.04) activity, as well as higher lipid peroxidation (28.5 vs. 17.43 nmol malondialdehyde/mg of protein, P < 0.001) and superoxide dismutase (SOD) activity (44.77 vs. 37.76 U; P = 0.04). There were no differences in the endometrial reactive species (RS) or glutathione (GSH) concentrations between the groups. The uterine washing samples showed no differences in the concentrations of RS or GSH or in total SOD activity (P > 0.1). Additionally, catalase, GPx4, SOD1 and SOD2 gene expression was lower in the SF-SCL group than in the LF-LCL group.ConclusionsWe concluded that the intrauterine environment of cows from the LF-LCL group exhibited higher antioxidant activity than that of the cows from the SF-SCL group. We speculate that uterine receptivity and fertility are associated with an optimal redox environment, such as that present in the animals in the LF-LCL group.

Relationship of follicle size and concentrations of estradiol among cows exhibiting or not exhibiting estrus during a fixed-time AI protocol

Cows exhibiting estrus near fixed-time artificial insemination (AI) had greater pregnancy success than cows not showing estrus. The objective of this study was to determine the relationship between follicle size and peak estradiol concentration between cows that did or did not exhibit estrus during a fixed-time AI protocol. Ovulation was synchronized in beef cows by applying the CO-Synch protocol [GnRH (100 μg) on day-9, prostaglandin F2α (PGF2α; 25 mg) on day-2, and a second injection of GnRH 48 h after PGF2α (day 0)] to both suckled (experiment 1) and nonsuckled (experiment 2) cows. Follicle size (day 0) and ovulation (day 2) was determined by ultrasonography. Blood samples were collected every 3 or 4 h beginning at the time of PGF2α injection (0 h). Estrus was detected by visual observation with the aid of estrus-detection patches, and cows that ovulated were classified as exhibited estrus (n = 46) or did not exhibit estrus (n = 63). In both suckled and nonsuckled cows, there was a positive relationship between all cows (P < 0.05) and among those that exhibited estrus (P < 0.05) between follicle size and peak estradiol concentration, but no linear relationship (P > 0.50) between follicle size and peak estradiol concentration was observed among cows not exhibiting estrus. Cows that exhibited estrus had greater (P < 0.01) peak estradiol concentrations than cows that did not exhibit estrus. Suckled cows exhibiting standing estrus had greater (P < 0.001) preovulatory concentrations of estradiol beginning 6 h (replicate 1) or 4 h (replicate 2) after the injection of PGF2α on day-2 compared with cows not exhibiting standing estrus. Nonsuckled cows exhibiting standing estrus had greater (P < 0.001) preovulatory concentrations of estradiol beginning at the injection of PGF2α on day-2 compared with cows not exhibiting standing estrus. Furthermore, cows that exhibited estrus had an increased (P < 0.01) rate in the rise in concentrations of estradiol following the PGF2α to peak estradiol than cows not exhibiting estrus. In summary, follicle diameter had a positive relationship with peak concentrations of estradiol, but only among cows that exhibited standing estrus, and estradiol increased earlier in cows that exhibited estrus compared with cows that did not.

Comparison of follicular dynamics and hormone concentrations between the 7-day and 5-day CO-Synch + CIDR program in primiparous beef cows

The objectives were to compare follicular dynamics, preovulatory estradiol concentrations, and progesterone concentrations between the 7-day (7CO, n = 15) and 5-day (5CO, n = 13) CO-Synch + controlled internal drug release device (CIDR) program in primiparous suckled beef cows. On Day −7 (7CO) or Day −5 (5CO), GnRH (100 μg) was administered (GnRH-1) and a CIDR was inserted. On Day 0, hour 0, CIDR was removed and cows received PGF2α (25 mg) at hours 0 and 12. Animals were administered GnRH (100 μg, GnRH-2) at either hour 60 (7CO) or 72 (5CO). Follicular growth and ovulation to both GnRH-1 and GnRH-2 were evaluated using ultrasonography. Concentrations of estradiol were determined in blood samples taken at hours 0, 36, 60, and 72 (5CO). Blood samples were collected on Days 5, 8, and 14 for progesterone quantification. Ovulation rate to GnRH-1 did not differ between the 7CO (11/15) and 5CO (8/13) treatments, and for all dependent variables the statistical model included treatment, ovulation to GnRH-1, and their interaction. Diameter (mm) of the ovulatory follicle did not differ between treatments (13.4 ± 0.3) but was greater (P < 0.05) in cows that responded to GnRH-1 (13.8 ± 0.3) than those did not (12.6 ± 0.6). Maximum estradiol concentrations tended (P = 0.06) to be greater in the 5CO (7.3 ± 0.5 pg/mL) than 7CO (6.1 ± 0.7 pg/mL) treatment and tended to be greater (P = 0.08) in cows that responded to GnRH-1 (7.1 ± 0.5 pg/mL) than those did not (5.6 ± 0.9 pg/mL). Three cows in the 7CO treatment failed to develop a CL after GnRH-2. There was a treatment by response to GnRH-1 interaction (P < 0.05) for progesterone concentrations. In cows that did not respond to GnRH-1 in the 7CO treatment, progesterone concentrations were less (P < 0.05) than in those that responded to GnRH-1 in the 7CO treatment and tended (P = 0.09) to be less than in cows in the 5CO treatment that did not respond to GnRH-1. In conclusion, these findings demonstrate that failure to respond to GnRH-1 is detrimental to estradiol and progesterone concentrations with a 7-day interval between GnRH-1 and PGF2α but of little consequence when this interval is shortened to 5 days.

Impact of preovulatory estradiol concentrations on conceptus development and uterine gene expression

This experiment was conducted to determine the effect of altering preovulatory estradiol concentrations, through manipulation of length of proestrus, on peripheral progesterone concentrations, conceptus development, interferon tau (IFNT) production and uterine gene expression in cattle. Approximately 6 days after a time-synchronized ovulation, all antral follicles (≥5mm) were ablated from the ovaries in beef heifers. To manipulate preovulatory estradiol concentrations, the length of proestrus prior to the GnRH-induced LH surge was altered between treatments. Heifers were administered PGF2α either −2.5 days (2.5 days of proestrus; HiE2; n=5) or −1.5 days (1.5 days of proestrus; LoE2; n=5) prior to GnRH (Day 0 of the experiment; 6.75 days after follicle ablation). Follicular dynamics and estradiol concentrations were evaluated during proestrus and progesterone concentrations were analyzed in the subsequent estrous cycle. On Day 7, embryos were transferred into all heifers using standard procedures. On Day 15.5 heifers were slaughtered, the reproductive tract was flushed to collect the conceptus and uterine flush media, and the uterine tissue was processed for subsequent analyses. Peripheral progesterone concentrations, conceptus development and IFNT production were similar between treatments. However, amount of nuclear progesterone receptor in the deep glandular epithelium and mRNA concentrations for estradiol receptor alpha (ESR1) in the uterine endometrium were less in the LoE2 than HiE2 treatment. These changes in uterine characteristics in heifers with lower preovulatory estradiol concentrations were not related to aspects of conceptus development monitored, however, it is speculated that the alterations in mRNA and receptor protein detected may contribute to pregnancy failure subsequent to day 15.5 of gestation.

Agonist and antagonist coast

The use of GnRH-a in ovarian stimulation permitted stronger stimulation resulting in an increased incidence of OHSS. The first Cochrane review comparing GnRH agonist and GnRH antagonist protocols for ovarian stimulation showed no significant difference in OHSS rate between the two protocols, however, a recent Cochrane review showed a highly significant decrease in the incidence in OHSS rate in the antagonist protocol. Coasting is a commonly used procedure for preventions of OHSS. The optimum time to start coasting is when the lead follicle reaches 16 mm in diameter and hCG should be given when E2 level drops below 3000 pg/ml. Coasting may act by diminishing the functioning granulosa cell cohort. Administration of daily GnRH antagonist in high risk patients for OHSS who were down-regulated by GnRH-a resulted in rapid drop of E2 and decrease in incidence of OHSS. A series of patients who developed early OHSS were treated by daily GnRH antagonist injections, all embryos were cryopreserved. No progression to severe OHSS was observed.

The Effect of Duration of the Preovulatory Period on Expression of ISG-15, SPP1, and Mx1 mRNA in Endometrial Tissues Collected on Day 17.5 in Pregnant and Cyclic Beef Cattle.

The aim of this study was to determine the impact of the length of the preovulatory period and consequent influences on preovulatory plasma estradiol concentrations, on expression of mRNA for ISG-15, SPP1 and Mx1 by endometrium collected from beef cows slaughtered 17.5 d after GnRH-induced ovulation. Twenty-one non-lactating cows in 2 replicates (Rep 1, n = 10; Rep 2, n = 11) were pre-synchronized to a common day of estrus and ultrasonographic-guided follicle aspiration was performed 6.2 ± 0.4 d after estrus (d -7 of the experiment). Cows were assigned to receive prostaglandin F2α on either d -3 (high estradiol group, n = 9; HiE), or d -2 (low estradiol group, n = 12; LoE). All cows received GnRH (100 µg) on d 0 (h 0), resulting in a preovulatory period of either 3 (HiE) or 2 (LoE) days. On d 7, cows received no further treatment (Cyclic = C) or were implanted with an embryo (Pregnant = P) resulting in 4 treatments (HiE-P, n = 5; HiE-C, n = 4; LoE-P, n = 8; LoE-C, n = 4). Ovarian ultrasonography was performed on d -7, -3, 0, 2 and 6 to monitor emergence and maximum diameter of the preovulatory follicle and formation of a new corpus luteum after GnRH. Blood samples collected at 12 h intervals from h -72 to h -12 and at h -6 and 0 were used to determine preovulatory concentrations of estradiol. Progesterone concentrations were assessed in samples collected on d -3, -2, 0, 2, 4, 6, 9, 11, 13 and daily to d 17.5. All cows were slaughtered on d 17.5 and endometrium was collected and snap frozen for later analyses. Number of animals in the HiE-P and LoE-P treatments reflects those from which embryos were recovered and for the HiE-C and LoE-C treatments, those cows with luteal phase progesterone concentrations at slaughter. Gene expression was determined using RT-qPCR for ISG-15, Mx1, SPP1 and RPL19. Statistical analyses were conducted using mixed procedures of SAS with appropriate models that included treatment, pregnant/cyclic, Rep and interactions for gene expression, with the addition of d as a repeated measure for hormone concentrations. Estradiol concentrations differed between the HiE and LoE treatments (treatment x h, P <.05), being greater (P<.05) in the HiE than LoE treatment at h 48 (3.2 ± 0.32 and 1.6 ± 0.26 pg/ml, respectively). Concentrations of progesterone from d 0 to d 17 did not differ between the HiE and LoE treatment. Relative amount of mRNA for Mx1 was greater in P than C females, but did not differ between the HiE-P (10.3 ± 2.9) and LoE-P (9.4 ± 2.8) treatments. ISG-15 mRNA was undetectable in 5/8 cows in the C treatments and did not differ between the HiE-P and LoE-P treatments (4.8 ± 1.1 and 5.9 ± 1.5, respectively). For SPP1, amount of mRNA did not differ between the HiE-C and HiE-P treatments, but was was greater (P < 0.05) in the LoE-P (2.8 ± 0.3) than LoE-C treatment (1.3 ± 0.03). In Rep 1, relative amount of SPP1 was greater (P < 0.05) in the HiE-P than LoE-P treatment (4.2 ± 0.5 and 2.5 ± 0.4, respectively) whereas in Rep 2 the inverse tended (P = 0.06) to occur (treatment x Rep, P < 0.05). In conclusion, altered concentrations of estradiol during the preovulatory period did not influence amounts of endometrial mRNA for ISG-15 and Mx1 in pregnant cows, and had variable effects on amounts of mRNA for SPP1. (poster)

Influence of the length of proestrus on fertility and endocrine function in female cattle

Previous research from our laboratory in beef cattle suggests that ovarian follicle maturity and subsequent fertility is influenced by length of proestrus across a range of follicle sizes. To test this hypothesis an animal model was used in which ovulation from similar sized follicles was induced following either a long (LPE; ∼2.25 days) or short (SPE; ∼1.25 days) proestrus (interval from PGF 2α administration to a GnRH-induced LH surge). Specific objectives were to compare pregnancy rates and luteal phase concentrations of progesterone (Experiment 1) and to characterize preovulatory concentrations of estradiol, the GnRH-induced LH surge, and concentrations of progesterone in the subsequent estrous cycle (Experiment 2) between the LPE and SPE treatments. In Experiment 1, ovulation from follicles that were previously synchronized using follicular aspiration was induced with GnRH (Day 0) after either 2.25 days (LPE; n = 40) or 1.25 days (SPE; n = 38) of proestrus. Lactating and non-lactating cows were inseminated 12 h following GnRH administration. Ovulatory follicle diameter was similar between treatments. Pregnancy rates to AI were greater ( P < 0.01) in the LPE (50.0%) compared to the SPE (2.6%) treatment. The proportion of cows having a short luteal phase in the subsequent estrous cycle was greater ( P < 0.01) in the SPE than LPE treatment. In cows with a luteal phase of normal length, timed-AI pregnancy rates and concentrations of progesterone in the subsequent luteal phase were greater ( P < 0.05) in the LPE than SPE treatment. In Experiment 2, a similar experimental approach was taken with non-lactating beef cows and ovulation was induced following either 2.2 days (LPE; n = 8) or 1.2 days (SPE; n = 8) of proestrus. Ovulatory follicle diameter was similar between treatments. Concentrations of estradiol during the proestrus period were greater ( P < 0.05) in the LPE than SPE treatment from Days −1.9 to Day 0 (GnRH administration). Concentration of LH during the GnRH-induced LH surge and concentrations of progesterone in the subsequent estrous cycle did not differ between treatments although there was a tendency ( P = 0.10) for increased incidence of short luteal phases in the SPE treatment. In conclusion, decreasing the length of proestrus before induction of ovulation of a large follicle resulted in lesser pregnancy rates and an increased incidence of short luteal phases. The impact of a shortened proestrus on concentrations of progesterone in cows with luteal phases of normal length varied among experiments. Decreased circulating concentrations of estradiol during the preovulatory period and/or reduced concentrations of progesterone during the subsequent estrous cycle in cows that do not experience a shortened luteal phase may represent the mechanism responsible for reduced fertility with the SPE treatment.

The effect of strain of Holstein–Friesian cow on size of ovarian structures, periovulatory circulating steroid concentrations, and embryo quality following superovulation

When managed under grass-based systems of production, the New Zealand (NZ) strain of Holstein–Friesian cow has superior reproductive performance compared to the North American (NA) strain despite having similar solids-corrected milk (SCM) yields. This study compared the ontogeny of early pregnancy events in NZ and NA cows. Ten NZ and 10 NA cows were submitted to a superovulation protocol on three occasions. Blood samples were collected daily from every cow from days −3 to +7 relative to a synchronized oestrus during each superovulation protocol. Pre-ovulatory oestradiol concentrations, follicle diameter, post-ovulatory progesterone concentrations, corpus luteum (CL) diameter, and circulating insulin-like growth factor-I concentrations did not differ between the two strains. Uteri were non-surgically flushed 7 days post-AI, embryos were isolated and graded. The proportion of transferable embryos recovered was higher ( P < 0.01) in the NZ cows compared with the NA cows. A greater ( P = 0.01) proportion of the recovered structures were at the blastocyst stage in the NZ cows. Peak SCM yield and body condition score (BCS) at the time of peak SCM yield were not different between strains. However, during the experimental period the NA cows maintained significantly higher daily SCM yields, whereas the NZ cows replenished significantly greater levels of BCS. The results indicate that differences in periovulatory steroid concentrations and size of ovarian structures do not explain the differences in embryo quality between the two strains. However, strain differences in nutrient partitioning from the time of peak SCM yield through late lactation may provide the key signals responsible for superior embryo quality in NZ cows.

Association Between Preovulatory Concentrations of Estradiol and Expression of Uterine Milk Protein Precursor, Inhibin Beta A, and Proenkephalin.

Eliminating the preovulatory surge of estradiol decreased embryo survival following embryo transfer, uterine weight, uterine protein, RNA to DNA ratio, and the rate of protein synthesis in sheep. Furthermore, cows that did not exhibit standing estrus (decreased preovulatory concentrations of estradiol) around the time of gonadotropin-releasing hormone (GnRH)-induced ovulation had decreased pregnancy success compared to cows that did have elevated preovulatory concentrations of estradiol. Thus, induction of ovulation alone may not be sufficient for maximum pregnancy success. Uterine environment, as influenced by endometrial protein expression, plays a central role in early embryo development and survival. Therefore, the objective of the present experiment was to characterize changes in expression of uterine milk protein precursor, inhibin beta A, and proenkephalin between cows with high (highE2; n = 6) and low (lowE2; n = 5) concentrations of estradiol at time of GnRH induced ovulation (day 0). Beef cows were treated with 100 μg GnRH on day -9, 25 mg PG on day -2, and 100 μg GnRH on day 0. Uterine horn biopsies (collected ipsilateral to the ovulatory follicle) and blood samples were collected on days 0 and 16. Total cellular RNA was extracted from all biopsies and relative mRNA levels for uterine milk protein precursor, inhibin beta A, proenkephalin, and polyubiqutin were determined by real-time RT-PCR and corrected for polyubiqutin. Circulating concentrations of estradiol on day 0 were greater (P < 0.001) in highE2 cows (8.33 ± 1.13 pg/mL) compared to lowE2 cows (2.72 ± 1.24 pg/mL). Relative expression of uterine milk protein precursor was greater (P = 0.04) in highE2 cows compared to lowE2 cows on day 0 (12.51 ± 3.07 vs. 2.10 ± 3.37, respectively). However, expression decreased (P = 0.002) from day 0 to day 16 in highE2 cows, and there was no difference (P = 0.95) in uterine milk protein precursor expression between highE2 and lowE2 cows on day 16 (1.11 ± 0.21 vs. 1.13 ± 0.34, respectively). Relative expression of inhibin beta A was greater (P = 0.03) in highE2 cows compared to lowE2 cows on day 0 (8.81 ± 1.64 vs. 2.82 ± 1.80, respectively), and expression decreased (P = 0.002) in highE2 cows from day 0 to 16. On day 16, there was no difference (P = 0.74) in inhibin beta A expression between highE2 and lowE2 cows (0.28 ± 0.03 vs. 0.29 ± 0.05, respectively). Expression of proenkephalin was not detectable in either highE2 or lowE2 cows on day 0. However, relative expression of proenkephalin on day 16 was greater (P = 0.02) in highE2 cows (10.40 ± 1.95) compared to lowE2 cows (2.70 ± 2.49). In summary, preovulatory concentrations of estradiol regulated uterine expression of uterine milk protein precursor, inhibin beta A, and proenkephalin during the subsequent estrous cycle. Reduced fertility among cows with decreased concentrations of estradiol prior to GnRH-induced ovulation may be associated with effects of changes in uterine protein expression on uterine environment.

Effect of preovulatory concentrations of estradiol and initiation of standing estrus on uterine pH in beef cows

Previous studies have indicated that initiation of standing estrus within 24 h of fixed-time AI influenced pregnancy rates. Furthermore, uterine environment at time of insemination can influence sperm transport. We hypothesized that preovulatory concentrations of estradiol would influence uterine pH at time of insemination. The objective of this study was to determine the influence of elevated preovulatory concentrations of estradiol on uterine pH following a fixed-time AI protocol. Cows were synchronized with the CO-Synch ( n = 57) protocol, and 29 cows were treated with an injection of estradiol cypionate (ECP; 1 mg) 36 h before the second injection of GnRH. Cows that exhibited standing estrus or were treated with ECP had increased ( P < 0.05) concentrations of estradiol compared to cows not in estrus and not administered ECP, respectively. There was an ECP by standing estrus interaction on uterine pH ( P = 0.01). Control cows that exhibited estrus had a reduced uterine pH (6.72 ± 0.10; P = 0.05) compared to control cows not exhibiting estrus (7.0 ± 0.06). Cows treated with ECP and detected in standing estrus had a greater uterine pH (7.0 ± 0.07) compared to control cows in estrus ( P = 0.02) and ECP cows not in estrus (6.81 ± 0.09; P = 0.06). The interval between the initiation of standing estrus and when pH was determined also influenced uterine pH. Cows that initiated standing estrus within 4 h of pH determination had a lower uterine pH (6.74 ± 0.12) compared to cows that initiated estrus 4–8 h (7.09 ± 0.08; P = 0.07) or 8–12 h (7.10 ± 0.15; P = 0.03) after pH determination. In summary, elevated concentrations of estradiol influenced standing estrus but only influenced uterine pH when pH was determined within 4 h of the initiation of standing estrus.

New Trends For Estrus Synchronization Using A Combination Of Gonadotropins, Prostaglandin And Estradiol Cypionate In Dairy Cows

This study was conducted to through the light on a recent method for estrus synchronization in lactating dairy cows using PGF2α, GnRH and estradiol cypionate (ECP). Four treatments were performed on sixty dairy cows. In treatment 1 (PP), cows received two injections of PGF2α on days 0 and 11 (n=14); while in treatment 2 (PGP), cows were injected twice PGF2α on days 0 and 11 and 100 ug of GnRH on day 3 after the 1st injection (n=14). In treatment 3 (PGPE-0), cows were treated with PGP and 1 mg of ECP at the same time of 2nd PGF2α injection (n=16). In treatment 4 (PGPE-1), cows were treated with PGP and 1 mg of ECP one day after 2nd PGF2α injection (n=16). The cows were examined rectally and ultrasonographically by B-mode Ultrasound System [Pie-Medical Scanner-240] with a 6-8 MHz linear rectal transducer. Every cow was blood sampled at selected intervals for progesterone and estradiol assay and inseminated at estrus. The results showed a higher percentage of GnRH-treated cows (PGP) ovulated after 1st PGF2α injection than the non treated (PP) cows (64.3% vs 50%; P<0.05). The GnRH-treated cows tended to have a larger mature follicle present at 2nd PGF2α injection. Subsequently, the percentage of cows that ovulated after 2nd PGF2α injection was 71.4% vs 50% for GnRH-treated cows than none treated ones, respectively. Two days after 2nd PGF2α injection, cows treated with PGP had a higher peak preovulatory concentrations of estradiol compared to PP treated cows (3.2±0.48 vs 2.6±0.45 pg/ml). Additionally, cows treated with ECP (PGPE-0 and PGPE-1) had a higher peak preovulatory concentrations of estradiol (6.3±0.43 and 6.99±0.63 pg/ml; P<0.01); and a higher percentage ovulation (75.0% and 87.5%) than the other treated groups (PP or PGP). Thus, a higher percentage of ECP (especially PGPE-1 group) treated cows were observed in standing estrus and ovulated after 2nd PGF2α injection than the other treated groups. Submission rates (number of inseminated/totalX100), differed statistically across treatments, especially those treated with ECP, as well as; the conception and pregnancy rates were observed higher with the ECP treated cows than the other treatments. In conclusion, PGP protocol increased the number of cows that ovulated after 1st PGF2α injection and produced more mature follicles at the time of 2nd PGF2α injection. The combination of ECP and PGP (PGPE-1) enhanced the expression of estrus and increased ovulation percentage, thus it is potentially a new method to routinely synchronize estrus and ovulation in dairy cows.

Stress-Induced versus Preovulatory and Pregnancy Hormonal Levels in Modulating Cytokine Production following Whole Blood Stimulation

Estradiol, progesterone, prolactin and cortisol concentrations are substantially increased during pregnancy. Also, cortisol and prolactin levels are elevated during stress. In the present study, we exposed peripheral blood to estradiol, progesterone, prolactin and cortisol alone or in combination for 24 h before stimulation with T-dependent (phytohemagglutinin, PHA) and independent activators (lipopolysaccharide, LPS) to study their immunomodulatory role in interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and IL-10 production in a whole blood model. This should be similar to in vivo exposure conditions such as long-term stress, preovulatory or pregnancy periods. The present study showed that the stress-induced and preovulatory levels of prolactin and estradiol, respectively, increased the production of IFN-γ and IL-12 levels (and IL-10 in the case of estradiol) in PHA + LPS-stimulated whole blood, and inhibited a hydrocortisone (100 nmol/l) suppressive effect on IFN-γ, IL-12 and IL-10 productions. In LPS-stimulated whole blood, however, prolactin enhanced only IL-10 production levels in a non-concentration-dependent manner. Higher prolactin levels as in pregnancy did not modulate any of the cytokines, but pregnancy estradiol concentrations only induced higher IL-10 levels in PHA + LPS-stimulated whole blood. All progesterone levels tested revealed no effect on any of the cytokines following whole blood stimulation. Our results indicate that (1) a long exposure time of prolactin and estradiol to whole blood modulates the production of cytokines in a concentration- and stimulus-dependent manner; (2) stress-induced levels of prolactin and preovulatory estradiol concentrations can regulate cortisol-induced cytokine suppression, and (3) even though the cytokine pattern is different, pregnancy estradiol and cortisol levels decreased the IFN-γ/IL-10 ratio, thereby keeping the anti-inflammatory IL-10 levels favored during pregnancy, which could be useful in regulating inflammatory-mediated autoimmune diseases.