Etiologic agents of bacteremia in the early period after simultaneous pancreas-kidney transplantation.

BACKGROUND: Bacteremia is among the known complications in simultaneous pancreas-kidney transplantation (SPKT). This study evaluated the frequency of microbial isolates and their susceptibility profiles among cultures of clinical samples obtained from blood and from the tips of blood vessel catheters of 26 SPKT recipients suspected of bacteremia in the early posttransplant period. PATIENTS AND METHODS: Data on microbiologic blood cultures of 26 adult patients undergoing SPKT were collected prospectively from 2001 to the end of 2006. The isolation and identification of cultured microorganisms were performed according to standard microbiological procedures and commercially available tests. The susceptibility of the strains to antibacterial agents was established by the Clinical and Laboratory Standards Institute guidelines. RESULTS: All patients were followed prospectively for the first 4 weeks after surgery. Among 66 clinical samples, there were 23 microbial isolates from blood samples of 17 recipients and catheter tips of 12 recipients. The most common isolates were Gram-positive bacteria (73.9%) with domination of staphylococci (64.7%) and MRCNS strains (81.8%). Gram-negative bacteria comprised 17.4% of positive cultures, whereas yeast-like fungi, 8.7% with a predominance of Candida glabrata. CONCLUSION: Our study showed predominately Gram-positive bacteria in 73.9% of isolates. The increased proportion of multi-drug-resistant bacteria and fungi to antimicrobial agents may be due to the frequent use of these agents for prophylaxis of bacterial infections in patients.

Relationships between FSH patterns and follicular dynamics and the temporal associations among hormones in natural and GnRH-induced gonadotropin surges in heifers.

Preovulatory LH and FSH surges and the subsequent periovulatory FSH surge were studied in heifers treated with a single injection of GnRH (100 microg, n = 6) or saline (n = 7). Blood samples were collected every hour from 6 h before treatment until 12 h after the largest follicle reached > or =8.5 mm (expected beginning of follicular deviation). The GnRH-induced preovulatory LH and FSH surges were higher at the peak and shorter in duration than in controls, but the area under the curve was not different between groups. The profiles of the preovulatory LH and FSH surges were similar within each treatment group, suggesting that the two surges involved a common GnRH-dependent mechanism. Concentrations of FSH in controls at the nadir before the preovulatory surge and at the beginning and end of the periovulatory surge were not significantly different among the three nadirs. A relationship between variability in the periovulatory FSH surge and number of 5.0 mm follicles was shown by lower FSH concentrations during 12-48 h after the beginning of the surge in heifers with more follicles (11.0 +/- 1.0 follicles (mean+/-s.e.m.) n = 7) than in heifers with fewer follicles (5.7 +/- 0.4, n = 6). This result was attributed to increased FSH suppression from increased numbers of follicles reaching 5.0 mm. Grouping of heifers into those with longer vs shorter intervals from a 4.5 mm to an 8.5 mm largest follicle did not disclose any relationship between length of the interval and FSH characteristics (e.g. profile of surge, area under curve, FSH concentrations at specific events). The hypothesis of a relationship between variation in the periovulatory FSH surge and variation in follicular dynamics was supported for the number of 5.0 mm follicles but not for the hour the largest follicle reached 8.5 mm. Thus, the expected time of follicle deviation was not altered by the extensive variation in the wave-stimulating FSH surge.

In vivo effects of an intrafollicular injection of insulin-like growth factor 1 on the mechanism of follicle deviation in heifers and mares.

In cattle and mares, free insulin-like growth factor 1 (IGF-1) is higher in the future dominant follicle (F1) than in the future largest subordinate follicle (F2) before deviation in diameter or selection is manifested between the two follicles. The effect of IGF-1 on other follicular-fluid factors and on the destiny of F2 were studied in two experiments in each species, using a total of 40 heifers and 42 mares. An injection of IGF-1 was made into F2 at the expected beginning of deviation (heifers, F1 >or= 8.5 mm; mares, F1 >or= 20.0 mm; Hour 0). In heifers, follicular fluid was taken from F2 at Hours 3, 6, 12, or 24; each heifer was sampled only once. In mares, sequential F2 samples were taken from each mare at Hours 0, 6, and 24 or at Hours 12 and 24. Transvaginal ultrasound guidance was used for treatment and sample collection. In heifers, IGF-1 treatment of F2 stimulated the secretion of estradiol (P < 0.05) between Hours 3 and 6 and androstenedione (P < 0.05) between Hours 3 and 12. In F2 of control heifers, estradiol decreased (P < 0.05) and androstenedione did not change significantly. In mares, IGF-1 treatment of F2 did not affect the concentrations of estradiol during the 24-h posttreatment period; androstenedione decreased (P < 0.04) in the IGF-1 group and increased (P < 0.006) in the controls. Compared with control mares, the IGF-1 group had higher (P < 0.04) activin-A at Hours 12 and 24 and higher (P < 0.0006) inhibin-A at Hour 24. After ablating F1 at Hour 24 in mares, F2 became dominant and ovulated in more mares (P < 0.0002) in the IGF-1 group (12/14) than in the control group (2/14). These results are consistent with reported temporal relationships among follicular factors during deviation in both species and indicate that IGF-1 plays a key role in controlling the temporal relationships; however, no indication was found that IGF-1 stimulated estradiol production in mares during the 24 h after treatment.

Associated and independent comparisons between the two largest follicles preceding follicle deviation in cattle.

Follicle diameters and concentrations of follicular fluid factors were studied in the two largest follicles (F1 and F2) using F1 diameters in increments of 0.2 mm (equivalent to 4 h intervals) and extending from 7.4 to 8.4 mm (12 heifers in each of 6 groups). Changes were compared between follicles using the F2 associated with each F1-diameter group. Diameter deviation began in the 8.2-mm group as indicated by a greater (P < 0.05) diameter difference between F1 and F2 in the 8.4-mm group than in the 8.2-mm group. In the 8.0-mm group, estradiol concentrations began to increase (P < 0.05) differentially in F1 versus F2, and free insulin-like growth factor-1 (IGF-1) began to decrease differentially in F2 (P < 0.06). Combined for F1 and the associated F2, activin-A concentrations increased (P < 0.05) between the 7.6- and 8.2-mm groups and then decreased (P < 0.05). Results supported the hypothesis that estradiol and free IGF-1 concentrations simultaneously become higher in F1 than in the associated F2 by the beginning of diameter deviation. Results did not support the hypothesis that a transient elevation in activin-A is present in F1 but not in the associated F2 at the beginning of the estradiol and IGF-1 changes; instead, a mean transient elevation in activin-A occurred at this time only when data for the two follicles were combined. Comparisons between F1 and F2 also were made by independently grouping F2 and using diameter groups at 0.2-mm increments for F2 as well as for F1. In the diameter groups common to F1 and F2 (7.4, 7.6, 7.8, and 8.0 mm) there was a group effect (P < 0.003) for estradiol involving an increase (P < 0.05) beginning at the 7.6-mm group averaged over F1 and F2. For free IGF-1 concentrations, a fluctuation (a significant increase followed by a significant decrease) occurred independently in F1 between the 7.4- to 7.8-mm groups and independently in F2 between the 7.0- to 7.4-mm groups.

Role of low circulating FSH concentrations in controlling the interval to emergence of the subsequent follicular wave in cattle.

The intervals between emergence of follicular waves 1 (first wave of an oestrous cycle) and 2, and between the associated FSH surges (surges 1 and 2), were studied in control (n = 7) and recombinant bovine (rb)FSH-treated (n = 7) heifers. The expected start of the deviation in follicle diameter between the two largest follicles of wave 1 was defined as the day on which the largest follicle reached 8.5 mm (day 0). In the control heifers, circulating concentrations of FSH decreased and oestradiol increased between day 0 and day 1.5 or day 2.0 in a reciprocal relationship. The opposite reciprocal relationship between an FSH increase and an oestradiol decrease occurred during the next 3 days. This temporal result is consistent with a negative systemic effect of oestradiol on FSH at this time. rbFSH was administered in a dosage regimen that was expected to result in a similarity between FSH surge 2 in the rbFSH-treated group and surge 2 in the control group. On average, surge 2 and wave 2 occurred approximately 2 days earlier in the rbFSH-treated group than in the control group, and characteristics of the FSH surge and follicular wave were similar (no significant differences) between groups. These results support the hypothesis that low circulating FSH concentrations after the deviation in follicle diameter control the interval to emergence of the subsequent follicular wave. However, in one of seven rbFSH-treated heifers, the largest follicle from the apparent stimulation of rbFSH reached only 5.7 mm; therefore, the possibility of involvement of additional mechanisms cannot be dismissed.

Activin A, estradiol, and free insulin-like growth factor I in follicular fluid preceding the experimental assumption of follicle dominance in cattle.

In cattle, the two largest follicles of a wave (F1, F2) begin to deviate into a dominant follicle and a subordinate follicle when F1 is a mean of 8.5 mm in diameter. After the beginning of deviation, F1 and F2 are diameter-defined dominant and subordinate follicles. Changes associated with the conversion of F2 into a future dominant follicle were studied by ablating F1 at the expected beginning of deviation (F1, 8.5 mm; Hour 0) and assessing the follicular-fluid factors in F2. Follicles were designated F1C and F2C in controls and F2A in F1-ablated heifers. Follicular-fluid collections were made at Hours 0, 4, 8, or 12 (n = 7 heifers per hour; fluid from F1C, F2C, and F2A; experiment 1) or at Hours 4, 6, 8, 10, or 12 (n = 9 heifers per hour; fluid from F2A; experiment 2). Postablation concentrations of circulating FSH increased (P < 0.05) between Hours 2 and 6. Diameter of F2A increased (P < 0.05) after Hour 8 in both experiments so that the diameter of F2A at Hours 10 or 12 was not different (P > 0.1) from the diameter of F1 at Hour 0. A transient elevation (P < 0.05) in follicular-fluid activin A occurred in F2A at Hour 8 in both experiments. Concentrations of estradiol (P < 0.05) and insulin-like growth factor I (IGF-I; P < 0.1) decreased in F2C by Hour 8. In F2A, the concentrations of both factors began to increase (P < 0.05) after Hours 4 or 8 so that there was no difference (P > 0.1) between F1C and F2A at Hour 12. Concentrations of IGF-I and IGF binding protein 2 (IGFBP-2) in F2A changed in opposite directions at the same hours. No differences between follicles were found for concentrations of progesterone, androstenedione, inhibin A, and inhibin B. The order of events in the conversion of a future subordinate follicle to a future dominant follicle was an increase in systemic FSH, a transient elevation in follicular-fluid activin A, and a simultaneous increase in follicular-fluid estradiol and restoration of an apparent growth-compatible balance of free IGF-I and IGFBP-2.

Follicle selection in cattle: dynamics of follicular fluid factors during development of follicle dominance.

Follicle diameter deviation during follicular waves in cattle begins with a reduction in growth rates of developing subordinate follicles, in contrast to the maintenance of a constant growth rate by a developing dominant follicle. In experiment 1, the temporal changes encompassing deviation in concentrations of follicular fluid factors relative to one another in the three largest follicles (F1, F2, and F3) were studied. Follicular fluid samples were collected when F1 reached diameter ranges of 7.0-7.9, 8.0-8.9, 9.0-9.9, and 10.0-10.9 mm (n = 12 per range). The first increase (P < 0.05) in the difference between F1 and F2 for estradiol occurred at the 8.0- to 8.9-mm range, which was one range earlier than for diameter (P < 0.05). Free insulin-like growth factor (IGF)-1 concentrations in F1 were similar among diameter ranges, but concentrations in F1 were higher (P < 0.05) than in F2 for each range except 7.0-7.9 mm. Concentrations of free IGF-1 in F2 decreased (P < 0.05). No significant differences were detected in concentrations of progesterone, androstenedione, total inhibin, and inhibin-A. Averaged over follicles, inhibin-B decreased (P < 0.05) between the 8.0- to 8.9- and 10.0- to 10.9-mm ranges, and activin-A increased (P < 0.05) between the 7.0- to 7.9- and 9.0- to 9.9-mm ranges. However, no differences were found among follicles. In experiment 2, changes associated with the development of dominance by F2 were studied using ablation of F1 at the beginning of expected deviation (F1, 8.5 mm; Hour 0) as the reference point. Follicular fluid factors were compared at Hour 12 between F2 of a control group (F1 intact; n = 10) and an ablated group (F1 ablated; n = 10). Diameter (P < 0.02), estradiol (P < 0.001), free IGF-1 (P < 0.002), and progesterone (P < 0.003) were greater and IGF-binding protein-2 was lower (P < 0.01) in F2 of the ablated group at Hour 12. No differences were detected in concentrations of androstenedione, total inhibin, and inhibin-A. The results of the two experiments indicated, on a temporal basis, that intrafollicular changes in estradiol and the IGF system, but not in the inhibin/activin system, could account for a reported greater FSH responsiveness by the future dominant follicle than by the future subordinate follicles by the beginning of diameter deviation in cattle.

Follicle selection in monovular species.

Follicle deviation is proposed to be the eminent event in follicle selection in monovular species. At deviation, the largest follicle establishes dominance apparently before the second-largest follicle can reach a similar diameter. In cattle, based on diameters of the two follicles at the beginning of deviation, the mechanism becomes established in <8 h. An FSH:follicle-coupling hypothesis has been supported as the essence of follicle selection. According to the hypothesis, the growing follicles cause the FSH decline from the peak of the wave-stimulating FSH surge until deviation, even though the follicles continue to require FSH (two-way functional coupling involving multiple follicles). During multiple-follicle coupling, inhibin is the primary FSH suppressant. Near the beginning of deviation, the largest follicle secretes increased estradiol, and apparently both estradiol and inhibin contribute to the continuing FSH decline; only the more-developed largest follicle is able to utilize the low FSH concentrations (single-follicle coupling). Deviation is encompassed by a transient elevation in LH in heifers and by a component, often distinct, of the long ovulatory LH surge in mares. In heifers, receptors for LH appear in the granulosa cells of the future dominant follicle about 8 h before the beginning of deviation. The LH stimulates the production of estradiol and insulin-like growth factor-1. These intrafollicular factors and perhaps others account for the responsiveness of the largest follicle to the low concentrations of FSH. The smaller follicles have not reached a similar developmental stage and because of their continued and close dependency on FSH become susceptible to the low concentrations. Thereby, follicle selection is established.

Follicle selection in cattle: follicle deviation and codominance within sequential waves.

Follicle deviation during bovine follicular waves is characterized by continued growth of a developing dominant follicle and reduction or cessation of growth of subordinate follicles. Characteristics of follicle deviation for waves with a single dominant follicle were compared between wave 1 (begins near ovulation; n = 15) and wave 2 (n = 15). Follicles were defined as F1 (largest), F2, and F3, according to maximum diameter. No mean differences were found between waves for follicle diameters at expected deviation (F1, > or =8.5 mm; Hour 0) or observed deviation or in the interval from follicle emergence at 4.0 mm to deviation. For both waves, circulating FSH continued to decrease (P < 0.05) after Hour 0, estradiol began to increase (P < 0.05) at Hour 0, and immunoreactive inhibin began to decrease (P < 0.05) before Hour 0. A transient elevation in circulating LH reached maximum concentration at Hour 0 (P < 0.01) in both waves and was more prominent (P < 0.0001) for wave 1. Waves with codominant follicles (both follicles >10 mm) were more common (P < 0.02) for wave 1 (35%) than for wave 2 (4%). Codominants (n = 6) were associated with more (P < 0.05) follicles > or=4 mm and a greater concentration (P < 0.04) of circulating estradiol at Hours -48 to -8 than were single dominant follicles (n = 15). A mean transient increase in FSH and LH occurred in the codominant group at Hour -24 and may have interfered with deviation of F2. In codominant waves, deviation of F3 occurred near Hour 0 (F1, approximately 8.5 mm). A second deviation involving F2 occurred in four of six waves a mean of 50 h after the F3 deviation and may have resulted from a greater suppression (P < 0.05) of FSH in the codominant group after Hour 0. In conclusion, follicle or hormone differences were similar for waves 1 and 2, indicating that the deviation mechanisms were the same for both waves. Waves that developed codominant follicles differed in hormone as well as follicle dynamics.

Effect of LH on circulating oestradiol and follicular fluid factor concentrations during follicle deviation in cattle.

Progesterone was used to reduce LH concentrations starting at the time when the largest follicle was > or = 5.7 mm in diameter or well before the expected start of follicle deviation (largest follicle > or = 8.5 mm in diameter). Plasma concentrations of LH, FSH and oestradiol were determined at 4 h intervals in control and progesterone-treated heifers (n = 8 per group). Concentrations of LH were lower (P < 0.05) in the progesterone-treated group, reflecting an absence of the transient increase in LH concentrations that encompasses follicle deviation. An increase in oestradiol and a continued decrease in FSH occurred at the start of follicle deviation in the control cows but not in the treated heifers. In a second experiment, follicular fluid of the two largest follicles of control and progesterone-treated heifers was sampled at the expected start of deviation (n = 8--10 per group). The concentrations of oestradiol, but not androstenedione and free insulin-like growth factor I (IGF-I), in follicular fluid were higher (P < 0.001) in the largest follicle than in the second largest follicle. Progesterone treatment reduced (P < or = 0.02) the concentrations of all three factors in follicular fluid and increased (P < 0.05) the concentrations of insulin-like growth factor binding protein 2 (IGFBP-2). These results confirm that oestradiol contributes to the continued decrease in FSH concentrations after the start of follicle deviation. Prevention of the transient LH increase, the oestradiol increase and the continued FSH decrease did not significantly alter the mean time or follicle diameters characteristic of expected follicle deviation. However, in some treated individuals (three of eight), the observed follicle deviation was delayed. In addition, these results indicate that the secretion of oestradiol into the circulation and the increase in oestradiol and IGF-I and decrease in IGFBP-2 concentrations in the follicular fluid at the start of deviation are functions of the transient increase in LH concentrations that encompasses follicle deviation.

Temporal gene expression in bovine corpora lutea after treatment with PGF2alpha based on serial biopsies in vivo.

There is growing evidence to indicate that PGF(2alpha)-induced luteolysis involves altered gene expression in the corpus luteum. Concentrations of mRNA encoding nine different gene products were quantified at three time points from corpora lutea in situ. Serial luteal biopsies (2.1-5.5 mg per biopsy) were collected using an ultrasound-guided transvaginal method and mRNA concentrations were quantified with standard curve quantitative competitive RT-PCR. In the first experiment, three luteal biopsies were collected from three heifers and analysed in multiple assays to evaluate the repeatability of the methods. Concentrations of mRNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PGF(2alpha) receptor (FP receptor) and LH receptor were found to be highly repeatable between assays, between multiple biopsies and between animals (coefficients of variation 1.3-17.3%). In the second experiment, heifers on days 9-11 after ovulation were assigned randomly to receive saline only (n = 6), saline with biopsies taken at t = 0, 0.5 and 4.0 h after injection (n = 6), PGF(2alpha) only (n = 6) or PGF(2alpha) with biopsies taken at t = 0, 0.5 and 4.0 h after treatment (n = 7). Biopsy alone did not change corpus luteum diameter, serum progesterone concentrations or days to next ovulation within the saline- or PGF(2alpha)-treated groups. Concentrations of mRNA for steroidogenic acute regulatory protein, FP receptor, 3beta-hydroxysteroid dehydrogenase, cytosolic phospholipase A(2) and LH receptor were decreased at 4.0 h after PGF(2alpha) injection. In contrast, PGF(2alpha) increased mRNA concentrations for prostaglandin G/H synthase-2, monocyte chemoattractant protein-1 and c-fos but the time course differed for induction of these mRNAs. Concentrations of mRNA for GAPDH did not change after PGF(2alpha) treatment. In conclusion, the techniques allowed analysis of multiple, specific mRNAs in an individual corpus luteum at multiple time points without altering subsequent luteal function. Use of these techniques confirmed that luteolysis involves both up- and downregulation of specific mRNA by PGF(2alpha).

Follicular-fluid factors and granulosa-cell gene expression associated with follicle deviation in cattle.

Intrafollicular changes in the largest follicle (F1) and second-largest (F2) follicle were examined in relation to follicle diameter deviation. Deviation is characterized by continued growth of the largest follicle and the cessation of growth of the smaller follicles. Granulosa cells and follicular fluid were obtained from slaughterhouse ovaries (n = 95 pairs, experiment 1), and follicular fluid was collected in vivo (n = 28 heifers, experiment 2). Several ranges in the diameter of F1 were used to represent the progressive growth of the follicle. The diameter range with the first significant increase in the difference between F1 and F2 was determined for each end point and was used as an indicator of the sequence of events associated with diameter deviation. An increased difference for diameter and for estradiol concentration occurred (P: < 0.05) simultaneously at the 8.5- to 8.9-mm range in both experiments. In experiment 1, the increased difference between F1 and F2 in LH receptor (LHr) mRNA expression occurred (P: < 0.05) at the 8.0- and 8.4-mm range. In F2 of experiment 2, there was a progressive decrease (P: < 0.05) in free insulin-like growth factor (IGF)-1 and a progressive increase (P: < 0.05) in IGF binding protein (BP)-2 across the follicle-diameter ranges (7.5-11.2 mm). No differences were detected between F1 and F2 for 3beta-hydroxysteroid dehydrogenase mRNA expression in experiment 1 and testosterone, total inhibin, and dimeric inhibin-A concentrations in experiment 2. The results indicated that the acquisition of granulosa cell LHrs by F1, as indicated by increased LHr mRNA expression, occurred one diameter range before an increased difference between F1 and F2 for diameter or estradiol concentrations. On a temporal basis, it is concluded that LHr acquisition plays a role in the establishment of diameter deviation. In addition, the reduced growth of F2 may have involved the reduced bioavailability of IGF-1 in association with elevated IGFBPs.

Follicle selection in cattle: role of luteinizing hormone.

The circulating concentrations of LH were reduced by administration of 50 mg of progesterone every 8 h for 72 h, beginning when the largest follicle was 6.0 mm (experiment 1; n = 10). Progesterone treatment prevented the transient increase in LH that accompanied deviation (partitioning into dominant and subordinate categories) in control heifers (n = 10). The reduced LH concentrations were associated with reduced growth of the largest follicle, beginning a mean of 31 h after deviation, but did not alter the time of deviation or the growth and regression of the second-largest follicle. In experiment 2, 0 mg (controls) or 50 mg of progesterone was given every 8 h for three injections, beginning when the largest follicle was 7.0 mm (predeviation group) or 9.0 mm (postdeviation group; n = 8 for each of the four groups). Blood samples from the jugular vein and follicular-fluid samples from the two largest follicles were taken 8 h after the last treatment when the largest follicle was a mean of 8.7 mm in the predeviation group and 10.8 mm in the postdeviation group. In the controls, follicular-fluid concentrations of estradiol and free insulin-like growth factor (IGF)-1 in the largest follicle and IGF binding protein (IGFBP)-2 in the second-largest follicle were higher (P: < 0.05) in the postdeviation group than in the predeviation group. Progesterone treatment lowered (P: < 0.006) the circulating LH concentrations to a similar extent in both groups. In the predeviation group, progesterone treatment did not have a significant effect on any of the characteristics of the largest follicle. In the postdeviation group, the largest follicle of the progesterone-treated heifers had significant reductions in diameter and in follicular-fluid concentrations of estradiol and free IGF-1. Follicular-fluid concentrations of immunoreactive inhibin were not different for any of the comparisons. The results supported the hypothesis that LH has a positive effect on diameter of the largest follicle but not until after the beginning of diameter deviation. In addition, the results indicated that LH is involved in the production of estradiol by the largest follicle and that free IGF-1 concentrations increase in the largest follicle during deviation.

Selection of the dominant follicle in cattle: role of estradiol.

Involvement of estradiol in the deviation in growth rates between the two largest follicles of a wave was studied in 39 heifers. In experiment 1, the largest follicle remained intact in a control group and was ablated in five estradiol-treated groups when the largest follicle reached 8.5 mm or larger (expected beginning of deviation; Hour 0). The ablation groups were given a single injection of 0, 0.004, 0.02, 0.1, or 0.5 mg of estradiol. Blood samples were taken from a jugular vein every hour at Hours 0 to 16. By Hour 8, FSH concentrations were greater (P < 0.05) in the ablation group that received 0 mg of estradiol than in the controls. Among the estradiol groups, that receiving 0.02 mg had the lowest detectable increase in estradiol. In this group, FSH concentrations were not suppressed below the control concentrations, but the increase in FSH concentrations following ablation of the largest follicle was delayed for 2 or 3 h. This delay in the increase of FSH concentrations corresponded to the hours that estradiol was maximal. In experiment 2, blood samples were taken every 4 h from the caudal vena cava cranial to the junction with the ovarian veins in heifers with the largest follicle intact (controls) or ablated at 8.5 mm or larger (Hour 0). Averaged over Hours 4 to 48, estradiol concentrations were higher (P < 0.04) in the controls than in the ablation group. During Hours 0 to 12, estradiol concentrations increased (P < 0.05) in the controls, whereas FSH concentrations decreased (P < 0.05). In the ablation group, estradiol concentrations were lower than in the controls by Hour 4, and FSH concentrations increased (P < 0.05) between Hours 4 and 12. These results support the hypothesis that the largest follicle releases increased estradiol into the blood at the beginning of follicular deviation, and that the released estradiol is involved in the continuing depression of FSH concentrations to below the requirement of the smaller follicles.

Selection of the dominant follicle in cattle: role of two-way functional coupling between follicle-stimulating hormone and the follicles.

The functional coupling between the declining portion of the FSH surge and the growing follicles of a wave was studied by treating heifers with a minimal dose of estradiol to decrease FSH concentrations without an associated change in LH concentrations. Estradiol treatment when the largest follicle reached >/= 6.0 mm (Hour 0) resulted in depression of both FSH concentrations and diameter of the largest follicle by Hour 8. The smaller follicles were also inhibited. These results supported the hypothesis that FSH continues to be needed by the growing follicles even when the FSH concentrations are decreasing during the declining portion of the FSH surge. Estradiol treatment when the largest follicle was >/= 8.5 mm (expected time of follicular deviation) also resulted in a transient decrease in both FSH concentrations and diameter of the largest follicle, but the diameters of the smaller follicles were not affected. These results supported the hypothesis that the low concentrations of FSH at the expected time of deviation, although inadequate for the smaller follicles, were required for continued growth of the largest follicle. In another study, ablation (Hour 0) of the largest follicle was done at >/= 7.5 mm vs. >/= 8.5 mm. The mean FSH concentrations for the 8.5-mm groups were greater for the ablation group than for the control group at Hours 8 and 12, but there was no difference between the 7.5-mm groups at any hour. These results supported the hypothesis that by the time the largest follicle reaches the expected beginning of deviation it has developed a greater capacity for suppressing FSH. It is postulated that the essence of the selection of a dominant follicle is a close two-way functional coupling between changing FSH concentrations and follicular growth.

Follicular and hormonal response to experimental suppression of FSH during follicle deviation in cattle.

A near steroid-free fraction of bovine follicular fluid was used to suppress FSH concentrations at the expected time of follicle deviation or when the largest follicle of Wave 1 reached > or = 8.0 mm (actual mean diameter, 8.4 mm; Hour 0). It was hypothesized that the low concentrations of FSH associated with deviation are inadequate for the smaller follicles but are needed for continued growth of the largest follicle. Control heifers (n=8) received 10 mL of saline, and treated heifers (n=16) received either 8.8 mL or 13.3 mL of the follicular-fluid fraction at Hours 0, 12, and 24. Between Hours -48 and 0, FSH concentrations decreased (P<0.05) and diameters of the 4 largest follicles increased (Hour effect, P<0.0001) similarly between groups. Concentrations of LH in the controls increased (P<0.05) between Hours -24 and -12 and decreased (P<0.05) between Hours 8 and 36, demonstrating a transient LH surge encompassing the expected beginning of deviation. In the treated group, a comparable increase in LH occurred before deviation but a decrease did not occur until after Hour 48. By Hour 4.5, the FSH concentrations in the treated group decreased (P<0.05) to below the concentrations in the controls. Suppressed diameter (P<0.001) of the largest follicle was detected at the first post-treatment examination (Hour 12; 7.5 h after FSH suppression) and was accompanied by reduced (P<0.04) systemic estradiol concentrations. The mean growth rates of the 3 smaller follicles in both the treated and control groups began to decrease at Hours -12 to 24 and were not different between groups during Hours 0 to 36. Concentrations of FSH in the treated group returned to control concentrations by Hour 24 (hour of last treatment). A rebound (P<0.05) in concentrations of FSH to >100% above control concentrations occurred by Hour 48 and was accompanied by resumed growth of the largest follicle in 75% of the heifers between Hours 48 and 72. The results demonstrated that the low concentrations of FSH associated with deviation can be further reduced by treatment with a nonsteroidal factor of follicular origin. Transient reduction of FSH concentrations to below the already low control concentrations inhibited the largest follicle but did not further inhibit the smaller follicles. These results support the hypothesis that the low FSH concentrations associated with follicle deviation are below the minimal requirements of the smaller or subordinate follicles but are needed for continued growth of the largest or dominant follicle in cattle.

Transvaginal, ultrasound-guided biopsy of the corpus luteum in cattle.

An ultrasound-guided transvaginal technique for corpus luteum biopsy was developed and tested in cattle. The biopsy needle set consisted of an inner needle (o.d. 1 mm) with a 20-mm long specimen notch, an outer cannula (o.d. 1.2 mm) with a cutting edge, and an automated spring-loaded handle with trigger. The biopsy needle set was inserted into the channel guide of the handle of a convex-array transvaginal ultrasound probe. The transducer was positioned in the vaginal fornix, and the ovary was manipulated transrectally against the vaginal wall and transducer face. During monitoring on the ultrasound screen, the inner needle was pushed through the vaginal wall into the corpus luteum, and the cutting cannula was fired, cutting and trapping luteal tissue in the specimen notch. Three luteal biopsies at each of Hours 0 and 4 were taken 10 d after ovulation in 6 heifers; 6 other heifers served as controls. A biopsy core was obtained in 36 of 39 attempts (92%). The tissue specimens seemed normal based on gross evaluation. The effect of biopsy on luteal function was assessed by daily ultrasound monitoring of luteal area, by assay of progesterone concentrations in blood samples obtained daily, and by the length of the interval from biopsy to ovulation. No significant differences were found for post-biopsy function for any of the 3 end points. The results indicated repeated transvaginal, ultrasound-guided biopsy of the corpus luteum in cattle is a practical procedure and may be useful for experimental and diagnostic purposes.

Sampling follicular fluid without altering follicular status in cattle: oestradiol concentrations early in a follicular wave.

A transvaginal, ultrasound-guided in situ technique was developed for sampling follicular fluid from 6.0-12.5 mm follicles in cattle, with minimal interference with the subsequent development of the sampled follicle. Each follicle was sampled only once and the status of the follicle (future dominant or subordinate) was determined retrospectively. A sample (20 microliters) was successfully obtained from 77% of 132 targeted follicles. Reasons for considering an attempt unsuccessful were as follows: (1) ovaries difficult to manipulate in two heifers (eight follicles), (2) samples contaminated with blood (11 samples), and (3) follicles sampled but the antrum subsequently developed an apparent blood clot (11 follicles). Only the 102 successful collections were used in the statistical analyses for evaluating oestradiol concentrations in the follicular fluid and the follicle growth profiles before and after sampling. The follicles were sampled once on days 1, 2, 3, or 4 after the emerging dominant follicle was 4 mm in diameter. On day 1, there were no differences in the oestradiol concentrations among follicles that later became the dominant follicle, largest subordinate follicle, or smaller subordinate follicles (means for diameters, 6.7-6.9 mm; means for oestradiol, 30-42 ng ml-1). The mean diameter of the dominant follicle increased linearly over the 4 days. Concentrations of oestradiol in the dominant follicle increased curvilinearly, resulting from a slower increase between days 1 (mean, 42 ng ml-1) and 2 (110 ng ml-1) than between days 2 and 3 (313 ng ml-1) and 3 and 4 (554 ng ml-1). Neither mean diameter nor mean oestradiol concentration of the largest subordinate follicle increased after day 2. Data were available from 19 follicular waves in which both the dominant and largest subordinate follicles were sampled on the same day. Oestradiol concentrations were not higher in the dominant follicle than in the largest subordinate follicle until the day after the two follicles began to deviate in growth rates (mean day of deviation, 2.5 +/- 0.2 days after emergence). These observations indicate that the future dominant follicle cannot be identified reliably by either its diameter or oestradiol production before the deviation in growth rates between the two largest follicles.

Associations between emergence of follicular waves and fluctuations in FSH concentrations during the estrous cycle in ewes.

Folliculogenesis was studied daily in 16 interovulatory intervals in 5 Polypay ewes from mid February through April using transrectal ultrasonic imaging. The 3-mm follicles attaining > or = 5 mm on Days--1 (ovulation=Day 0) to 11 showed significant peak numbers on Days 0, 5 and 10. The number of 3- and 4-mm follicles that did not reach > 4 mm was not significant, indicating that these follicles did not manifest a wave pattern. A follicular wave was defined as one or more follicles growing to > or = 5 mm; the day the follicles were 3 mm was the day of wave emergence, and the first wave after ovulation was Wave 1. Waves 1, 2 and 3 emerged on Days -1 to 2,4 to 7 and 8 to 10, respectively. Four interovulatory intervals in April were short (9 to 14 d), indicating the end of the ovulatory season. In the remaining 12 intervals, the ovulatory wave was Wave 3 in one interval, Wave 4 in 8 intervals, and Wave 5 or 6 in 3 intervals. The ovulatory wave followed the rhythmic pattern of Waves 1 to 3 by emerging on Days 11 to 14 in 50% of the intervals. In the remaining intervals, either the ovulatory wave was Wave 4 but did not emerge until Day 16 or other waves intervened between Wave 3 and the ovulatory wave. The longest intervals (22, 24 and 24 d) had >4 waves. Based on a cycle-detection program, peak values of FSH fluctuations were temporally associated with the emergence of waves as indicated by the following: 1) tendency (P < 0.08) for an increase in FSH concentrations between 3 and 2 days before emergence of a wave; 2) close agreement between mean number of waves per interval (mean +/- SEM, 4.1 +/- 0.3) and mean number of identified FSH fluctuations (4.5 +/- 0.3); 3) close agreement in length of interwave intervals (4.0 +/- 0.3) and interpeak (FSH) intervals (3.6 +/- 0.2); 4) positive correlation (r(2)=0.8) for number of the 2 events (follicular waves and FSH fluctuations) within intervals; and 5) a closer (P < 0.01) temporal relationship between the 2 events than would have been expected if the events were independent. The results support a relationship between transient increases in FSH concentrations and emergence of follicular waves throughout the interovulatory interval in Polypay ewes, with the 2 events occurring approximately every 4 d.