Hormonal mechanisms regulating follicular wave dynamics I: Comparison of follicle growth profiles under different physiological conditions in heifers.

Follicle diameter deviation has been identified as the pivotal morphological manifestation of follicle selection, however, many of the hormonal mechanisms leading to this key event and regulating variation between individual follicular waves remain undefined. This study compared circulating FSH, LH, and P4 with the follicular dynamics during three different physiological conditions. We hypothesized that these end-points would: 1) be similar for a spontaneous wave 2 vs one induced by follicular aspiration, 2) but would differ between wave 1 and 2, and 3) between conventional (F2 > 7.0 mm at deviation) vs undersized (F2 < 7.0 mm) deviations in either wave. Holstein dairy heifers (N = 24) were studied daily during an interovulatory interval. All heifers were evaluated during wave 1 and randomized 6 days after ovulation into an induced wave 2 and a spontaneous wave 2. Values were normalized to the day of expected diameter deviation (day 0) and compared for day -2 to 0 and 0 to 2. Hypothesis 1 was supported that an induced wave 2 from ablation of follicles of wave 1 and spontaneous wave 2 have similar follicle dynamics. However, the peak FSH surge was more prominent at emergence of an induced wave 2 (P < 0.003). Hypothesis 2 was supported that waves 1 and 2 differ in follicle and hormone dynamics. Circulating P4 was lower and LH was greater (P < 0.01) with no difference in diameter of F1 but with a greater (P < 0.01) diameter of F2 on day 0 in wave 1 (7.3 ±â€¯0.2 mm) than in wave 2 (6.6 ±â€¯0.2 mm). Differences between waves were not found when each follicular wave was categorized into conventional vs undersized deviation and analyzed separately. Hypothesis 3 was supported as there were differences in circulating hormones between conventional and undersized deviations. Growth rate of F2 differed (P < 0.0005) during days -2 to 0 (conventional, 2.6 ±â€¯0.2 mm/2d; undersized, 1.4 ±â€¯0.3 mm/2d). However, circulating FSH and P4 concentration on days -1 and 0 tended to be greater (P < 0.06) in undersized than conventional deviations. In conclusion, the effect of different hormonal conditions on follicle dynamics was observed for F2 and not for F1. Furthermore, understanding the physiology that produces conventional vs undersized deviations is crucial since these categories explained most differences in follicular dynamics and circulating FSH observed in these different physiological conditions. In addition, future studies of wave 2 may be facilitated by using an induced wave 2 since it was similar to a spontaneous wave 2.

Gonadotropin concentrations associated with variations in diameter deviation during follicle selection in Holstein heifers.

Diameter deviation or selection of the future dominant follicle (F1) from the future largest subordinate follicle (F2) during a follicular wave occurs when F1 is 8.5 mm (expected deviation, day 0). Deviation has been classified as conventional (F2 ≥ 7.0 mm), F2-undersized (F2 < 7.0 mm), and F1,F2-switched (F2 larger than F1 on day -1 or 0). Concentrations of gonadotropins were compared within and among deviation classifications in waves 1 and 2 in 48 heifers. A three-way (wave 1 compared with 2, classification, day) analysis indicated no effect of wave 1 compared with 2 on F2 or FSH. An interaction of classification by day for F2 diameter (P < 0.001) and FSH concentration (P < 0.005) was primarily from differences on day -1. Rankings on day -1 from greatest to least for F2 diameter were switched, conventional, and undersized and for FSH concentration were undersized, conventional, and switched. Lower FSH concentration in conventional compared with undersized deviations during the decline in the FSH surge was presumed to represent greater output of FSH inhibitors by larger follicles. The incline in FSH surge 2 began significantly later for undersized than for conventional deviation. Switched deviation was associated significantly with emergence of F2 before F1, lower FSH concentration during the decline in the FSH surge, and earlier occurrence of the post-surge FSH nadir. Results supported the hypothesis that diameter differences among deviation classifications are temporally associated with differences in FSH concentration within each classification. These novel findings may complicate studies on the mechanisms of follicle selection.

Concentrations of follicle stimulating hormone associated with follicle selection, number of follicles, and ipsilateral vs contralateral relationships in mares.

Diameter deviation or follicle selection during a follicular wave begins with continuous growth rate of F1 (dominant or selected follicle; DF) and decreasing growth rate of F2 (largest subordinate). Intraovarian patterns based on presence or absence of the DF and CL are DF-CL, DF, CL, and devoid (neither DF nor CL). The DF and CL relationships in a pair of ovaries are ipsilateral (DF-CL pattern and devoid pattern) and contralateral (DF pattern and CL pattern). The effects of patterns and relationships on F1, F2, and FSH during deviation were determined in 21 mares. Diameters of F1 and F2 at expected beginning of deviation (F1, 22.5 mm; day 0) defined deviation classifications of conventional (F2 ≥ 18.5 mm on day 0), F2-undersized (F2 < 18.5 mm), and F1,F2-switched (F2 larger than F1 on day -1 or 0). During the decline in the FSH surge, an interaction (P < 0.001) of deviation classification and day indicated that FSH concentration was greater (P < 0.05) in undersized than in conventional deviations on days -3 and -1 and intermediate in switched deviation. Low FSH during days -3 to -1 in switched deviation began to increase on day -1 during switching in diameter rank between F1 and F2. The number of subordinate follicles per wave that attained a maximum of 11 to <16 mm was greatest in the DF-CL and devoid patterns and in ipsilateral (4.2 ±â€¯0.5 follicles) than in contralateral (2.5 ±â€¯0.7) relationships (P < 0.03). Concentration of FSH was greater (P < 0.04) on days -4 to 5 in ipsilateral (5.3 ±â€¯0.2 ng/mL) than in contralateral (4.2 ±â€¯0.4 ng/mL) relationships consistent with more follicles 11 to < 16 mm. In a survey (N = 63), an ipsilateral vs contralateral relationship was associated with 17 vs 17 conventional deviations and 7 vs 22 nonconventional deviations (P < 0.04), and conventional deviation (54%) was more frequent (P < 0.0001) than undersized (21%) or switched (25%) deviations. Results supported the hypotheses that (1) diameter deviation can be classified in mares into distinct categories based on diameter and rank of the future largest subordinate at expected deviation, (2) the number of subordinate follicles is influenced by ipsilateral vs contralateral relationships of DF and CL, and (3) conventional deviation is more common when the relationship between DF and CL is ipsilateral. Knowledge on follicle diameters for the different deviation classifications should be considered in studying follicle selection especially when terminal follicle sampling is done before expected deviation.

Role of luteal biosynthesis of prostaglandin F2α on function and structure of the corpus luteum during luteolysis in heifers.

The role of endogenous prostaglandin F2α (PGF) in the induction of luteolysis by exogenous PGF was studied by simultaneous inhibition of endogenous PGF with flunixin meglumine (FM). Groups were controls (n = 8), PGF treated (n = 8), and FM + PGF treated (n = 9). Treatments were given 10 d postovulation at hours 0, 8, and 16. The protocol was based on (1) the assumption that luteolytic characteristics of exogenous PGF would be altered if the synthesis of endogenous PGF is simultaneously inhibited and (2) the reports that luteolysis involves a direct effect of uterine PGF on large luteal cells followed by an effect of the large cells on the small cells. At hour 48, progesterone concentration was greater in the controls (7.6 ± 0.8 ng/mL) than that in the FM + PGF group (3.0 ± 0.5 ng/mL) and lower in the PGF group (0.7 ± 0.3 ng/mL) than in the FM + PGF group (interaction, P < 0.0001). The effects of each of the 3 groups on percentage change in CL volume were similar to the effects on progesterone. At hour 48, the percentage of CL tissue with color-Doppler signals of blood flow was similar between the controls (56.2% ± 3.8%) and FM + PGF group (50.0% ± 6.4%) and lowest in the PGF group (15.6% ± 7.2%) (interaction, P < 0.0001). A resurgence in progesterone concentration began at hours 24 or 48 in 6 of 9 heifers in the FM + PGF group compared to 0 of 8 heifers in each of the other groups (P < 0.007). The progesterone resurgence in the FM + PGF group was associated with the maintenance of percentage of CL tissue with blood-flow signals. The experimental hypothesis that an inhibitor of endogenous PGF reduces the luteolytic response to exogenous PGF was supported.

Concentrations of a PGF2α metabolite during pregnancy on the days that luteolysis occurs in nonbred heifers.

Concentrations of a metabolite of PGF2α (PGFM) were compared between nonbred (n = 6) and pregnant (n = 8) heifers on days 16, 17, and 18 postovulation. On each day, an 8-h session of hourly blood sampling was done. Averaged over the 8-h sessions, mean concentration of PGFM was less (P < 0.0009) in the pregnant group (45.2 ± 3.2 pg/mL) than that in the nonbred group (65.6 ± 7.9 pg/mL), but the minimal concentration per session was not significantly different between groups. Pulses of PGFM (identified by coefficient of variance) were similar in frequency between groups but were less (P < 0.03) prominent at the peak in the pregnant group (60.0 ± 5.3 pg/mL) than that in the nonbred group (92.8 ± 10.7 pg/mL). These results indicated similarity between groups in frequency and initial development of a PGFM pulse but without later development and a reduction in prominence in the pregnant group. The progesterone response to a PGFM pulse of similar prominence was made before the beginning of luteolysis in individuals in the nonbred group and during the hourly sessions on days 16 to 18 in the pregnant group. Progesterone concentration in the nonbred group decreased (P < 0.05) during 2 h before the PGFM peak (8.8 ± 1.6 to 5.6 ± 1.0 ng/mL) and rebounded (P < 0.05) completely during the 2 h after the peak (5.6 ± 1.0 to 9.6 ± 2.2 ng/mL). A transient progesterone decrease during a similar PGFM pulse and similar initial progesterone concentration did not occur in the pregnant group. Results supported the hypotheses that (1) pregnant heifers have identifiable but less prominent PGFM pulses during the days that luteolysis occurs in nonbred heifers and (2) the corpus luteum locally resists the luteolytic effect of PGF2α in pregnant heifers before the days of onset of luteolysis in nonbred heifers.

Follicles and gonadotropins during waves 2 and 3 in three-wave interovulatory intervals in Bos taurus heifers.

Observations were made on follicle dynamics and gonadotropin concentrations in anovulatory wave 2 and ovulatory wave 3 in three-wave interovulatory intervals (n = 15). Hypotheses were not used owing to inadequate availability of rationale. The future dominant follicles for waves 2 and 3 were designated DF2 and DF3 and the largest future subordinate follicles as SF2 and SF3, respectively. The day of expected diameter deviation (day 0) was defined as the day that DF2 or DF3 was closest to 8.5 mm. The first day that DF2 became smaller (P < 0.05) than DF3 was day 2 (10.7 ± 0.2 mm vs 11.8 ± 0.3 mm). The FSH surges 2 and 3 that stimulated waves 2 and 3 were similar at peak concentration, but the postsurge nadir of surge 2 occurred 1 day earlier than for surge 3. An LH increase was not temporally associated with deviation in wave 2, but an increase (P < 0.05) in LH in wave 3 began on day -1. Diameter of SF2 (6.5 ± 0.2 mm) on day 0 was less (P < 0.005) than for SF3 (7.2 ± 0.2 mm). Mean diameter of subordinate follicles in wave 2 did not differ among days. Diameter of subordinate follicles that attained ≥6 mm in wave 3 was greater (interaction, P < 0.02) by day 3 when in the right ovary (RO, 7.4 ± 0.2 mm) than when in the left ovary (LO, 5.6 ± 0.2 mm). The frequency of a conventional classification of deviation (future SF greater than 7.0 mm on day 0) was less (P < 0.001) for wave 2 (1 of 15 waves) than for wave 3 (8 of 15 waves). Novel observations involving DF2 and DF3 were (1) before deviation, diameter of DF2 vs DF3 and an incline in FSH surge 2 vs surge 3 were similar and (2) after deviation, smaller diameter of DF2 vs DF3 by day 2 was associated with an earlier cessation (nadir) in FSH surge 2 vs surge 3 and an absence of an LH increase during deviation. Novel observations involving subordinate follicles ≥6 mm were (1) before deviation, diameters were similar between waves 2 and 3 in association with the similar incline in FSH surges 2 vs 3 and (2) after deviation, a greater diameter increase of subordinates occurred in RO than in LO for wave 3, but an increase did not occur for either ovary in wave 2. The characteristics of diameter deviation were profoundly different between waves 2 and 3 owing to a smaller SF2 than SF3 at deviation but similar diameter of DF2 and DF3.

Color-Doppler signals of blood flow in the corpus luteum and vascular perfusion index for ovarian and uterine arteries during expansion of the allantochorion in Bos taurus heifers.

Hemodynamics of the CL and each main uterine artery during expansion of the allantochorion from the ipsilateral side (CL side) to the contralateral side were studied in heifers (n = 8 nonbred, 9 pregnant). Progesterone concentration, vascular perfusion index for each uterine artery (by spectral ultrasonography), extent of blood flow in the CL (percentage of CL tissue with color-Doppler signals of blood flow), and intrauterine location of the expanding allantochorion (by gray-scale ultrasonic imaging) were determined daily from Days 14-60 (Day 0 = ovulation). In the pregnant group, but not in the nonbred group, the percentage of CL tissue with blood-flow signals increased (P < 0.003) from Days 16 (66.7 ± 4.2%) to 23 (79.4 ± 2.5) and then more slowly increased (P < 0.02) from Days 24 (76.7 ± 2.9%) to 50 (85.0 ± 2.0%). The volume of CL increased (P < 0.0001) progressively from Days 26 (6.1 ± 0.4 cm3) to 60 (7.3 ± 0.7 cm3). The vascular perfusion index in the ipsilateral uterine artery did not change in the nonbred group and progressively increased in the pregnant group beginning on Day 17 in approximate temporal association with the increase in luteal blood-flow signals. Functional increases in the CL during early pregnancy were attributed to the greater uterine arterial blood flow on the ipsilateral or CL side and the reported prominent anastomosis from a branch of the uterine artery to the ovarian artery that supplies the CL ovary. After an initial significant decrease in vascular perfusion index in each uterine artery in the pregnant group, the index began to increase on Day 17 in the ipsilateral artery and on Day 18 in the contralateral artery. The perfusion continued in the ipsilateral artery but discontinued in the contralateral artery on Day 21. Perfusion and diameter of the uterine artery were greater for the ipsilateral side until the vesicle entered the contralateral horn on Day 33 and increased for the contralateral horn between vesicle entry and filling of the horn on Day 44. During Days 35-60, the perfusion index (P < 0.04) and artery diameter (P < 0.001) were lower in the contralateral than in the ipsilateral artery. Results supported the hypotheses that (1) CL function increases during early pregnancy in temporal association with an increase in blood flow in the ipsilateral uterine artery and (2) blood flow in each of the ipsilateral and contralateral uterine arteries increases as the allantochorion expands in each uterine horn.

An intraovarian mechanism that enhances the effect of an FSH surge on recovery of subordinate follicles in heifers.

The effect of the future dominant follicle (DF), corpus luteum (CL), and side (left ovary [LO] and right ovary [RO]) on FSH-induced recovery (increase in diameter) of regressing subordinate follicles was studied in heifers. The DF of wave 2 and the largest subordinate follicle remained intact (controls, n = 14 heifers) or were ablated (n = 14 heifers) on a mean of 13 d postovulation when the DF was ∼10 mm (hour 0). Concentration of FSH (P < 0.0004) and diameter of subordinate follicles (P < 0.0002) decreased between hours -48 to 0 combined for the control and ablation groups. Thereafter, follicle diameter continued to decrease in the controls. Concentration of FSH increased (P < 0.05) and diameter of subordinates began to increase at hour 12 in the ablation group. Follicle-stimulating hormone increased to hour 24 and then returned to the hour 0 concentration by hour 72, completing the induced FSH surge. Concentration of LH began to increase at hour 0 in each group and at a similar rate between groups. Follicle recovery in the ablation group was compared among 8 subgroups as defined by the 2 sides and 4 intraovarian patterns (DF-CL pattern, both structures in same ovary; DF pattern, DF alone; CL pattern, CL alone; and devoid pattern, both structures absent). Follicle diameter increased (P < 0.05) between hours 24 and 48, and diameter at hours 24, 48, 72, and 96 involved a 3-way interaction (P < 0.0001) of pattern, side, and hour. The interaction was similar when diameter of the DF that originated from a recovered subordinate was either included or excluded in the analysis. Diameter of subordinate follicles in the ablation group at hour 96 was greater (P < 0.05) in the DF-CL/RO and DF/RO subgroups than that in the devoid/LO, devoid/RO, and CL/LO subgroups. The DF-CL/LO and CL/RO subgroups were intermediate. For follicles that decreased in diameter before hour 0, a greater (P < 0.05) percentage increased after hour 0 when the ovary contained a DF and was in the RO (DF-CL/RO and DF/RO subgroups) than for the remaining subgroups even after excluding the DF that originated from a subordinate. Results supported the hypotheses that (1) an induced FSH surge can stimulate the recovery of regressing subordinate follicles and (2) recovery of regressing subordinate follicles by FSH involves an intraovarian mechanism. Our interpretation is that the intraovarian mechanism that enhances the stimulatory effect of FSH on recovery of subordinate follicles was effective only in RO and only when it contained a DF.

Complexities of follicle deviation during selection of a dominant follicle in Bos taurus heifers.

Follicle deviation during a follicular wave is a continuation in growth rate of the dominant follicle (F1) and decreased growth rate of the largest subordinate follicle (F2). The reliability of using an F1 of 8.5 mm to represent the beginning of expected deviation for experimental purposes during waves 1 and 2 (n = 26 per wave) was studied daily in heifers. Each wave was subgrouped as follows: standard subgroup (F1 larger than F2 for 2 days preceding deviation and F2 > 7.0 mm on the day of deviation), undersized subgroup (F2 did not attain 7.0 mm by the day of deviation), and switched subgroup (F2 larger than F1 at least once on the 2 days before or on the day of deviation). For each wave, mean differences in diameter between F1 and F2 changed abruptly at expected deviation in the standard subgroup but began 1 day before expected deviation in the undersized and switched subgroups. Concentrations of FSH in the wave-stimulating FSH surge and an increase in LH centered on expected deviation did not differ among subgroups. Results for each wave indicated that (1) expected deviation (F1, 8.5 mm) was a reliable representation of actual deviation in the standard subgroup but not in the undersized and switched subgroups; (2) concentrations of the gonadotropins normalized to expected deviation were similar among the three subgroups, indicating that the day of deviation was related to diameter of F1 and not F2; and (3) defining an expected day of deviation for experimental use should consider both diameter of F1 and the characteristics of deviation.

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.

Simultaneous detection and identification of the Xanthomonas species complex associated with tomato bacterial spot using species-specific primers and multiplex PCR.

AIMS: To establish protocols for the simultaneous detection and identification of Xanthomonas species causing tomato bacterial spot. METHODS AND RESULTS: We verified the specificity and sensitivity of the previously reported sets of primers designed for strains of the four species of Brazilian tomato bacterial spot xanthomonads, consisting of 30 of Xanthomonas euvesicatoria, 30 of X. vesicatoria, 50 of X. perforans and 50 of X. gardneri. Furthermore, we tested a multiplex PCR protocol for the purpose of concurrent species identification. The possibility of direct detection of the pathogens in diseased leaf samples was also verified. The primers were highly specific, amplifying only target DNA. The sensitivity of the primers in conventional PCR was 50 pg µl(-1) for purified DNA and ranged from 5 × 10(2) to 5 × 10(4) CFU ml(-1) when bacterial suspensions were analysed. The multiplex PCR was suitable for the detection of all four species and showed similar sensitivity to conventional PCR when tested on purified DNA. When using bacterial suspensions, its sensitivity was similar to conventional PCR only when a biological amplification step (Bio-PCR) was included. Both methods were able to detect the pathogens in symptomatic tomato leaves. CONCLUSIONS: Brazilian Xanthomonas strains causing tomato bacterial spot can be differentiated and identified at species level by a PCR-based method and by a multiplex PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This protocol may be a feasible alternative tool for the identification and detection of these pathogens in plant material and may be used for routine diagnostic purposes in plant pathology laboratories.