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.     

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.     

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.     

Calculated follicle deviation using segmented regression for modeling diameter differences in cattle

Segmented linear regression alone or in combination with simple linear regression was evaluated as an objective method to calculate the beginning of follicle deviation by modeling the sequential (Experiment 1) and non-sequential or single-point (Experiment 2) differences in diameter between the future dominant (F1) and largest subordinate (F2) follicles of Wave 1 in cattle. The segmented regression consisted of Segment 1 representing the common growth phase, Segment 2 representing the period of dominance, and a Join Point connecting the two segments and representing the end of the common growth phase and the beginning of deviation. The model was fit to the diameter differences for each heifer in Experiment 1 (n=15) and the group of heifers in Experiment 2 (n=40). The optimal Join Point value that corresponded to the maximum R(2) was designated the calculated hour (Experiment 1) or diameter of F1 (Experiment 2) at the beginning of deviation. In Experiment 1, simple linear regression was used to calculate the corresponding diameter of F1 at the beginning of deviation. Observed deviation was determined by inspection of the diameter profiles of F1 and F2 for comparison to calculated deviation. In Experiment 1, the observed method determined the beginning of deviation in 80% of the heifers, whereas, the regression method calculated deviation in 93% of the heifers including two of the three heifers in which observed deviation was not discernable (no significant difference between methods). The mean hours of deviation after wave emergence (Hour 0) and diameters of F1 at the corresponding hours were not significantly different between the observed (62 h and 8.4 mm) and calculated (61 h and 8.8 mm) methods. In Experiment 2, the diameter of F1 at the beginning of calculated deviation was 8.2 mm. The results indicated that the segmented regression model can provide an objective and more accurate alternative to estimate follicle deviation, especially when observed deviation is obscured by the complexity of follicle development in some waves.     

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.     

Selection of the dominant follicle in cattle: establishment of follicle deviation in less than 8 hours through depression of FSH concentrations

Deviation in follicle diameter in cattle is characterized by continued growth of the largest follicle of a follicular wave and a reduction or cessation of growth of the smaller follicles. Deviation begins when the largest follicle reaches about 8.5 mm. Two experiments were done to test the hypothesis that the deviation mechanism is established in < 8 h, as indicated by the temporal relationships between follicle removal and an increase in FSH concentrations (Experiment 1) and between a decrease in FSH concentrations and follicle inhibition (Experiment 2). In Experiment 1, the role of the first follicle to reach 8.5 mm was studied by follicle ablation (Hour 0). The combined mean FSH concentrations for the control group (n = 8) and ablation group before ablation (n = 7) progressively decreased (P < 0.02) over two 8-h intervals before the largest follicle reached > or = 8.5 mm (Hour-16, 1.77 +/- 0.11 ng/mL; Hour 0, 1.49 +/- 0.08 ng/mL). In controls, the concentrations continued to decrease (P < 0.02) until Hour 10 (1.21 +/- 0.09 ng/mL). Ablation of the largest follicle at > or = 8.5 mm resulted in increased (P < 0.02) circulating FSH concentrations between Hours 5 (1.34 +/- 0.04 ng/mL) and 8 (1.61 +/- 0.09 ng/mL). Growth rate of the second-largest follicle between Hours 0 and 8 was greater (P < 0.05) in the ablation group than in the controls, and the second largest follicle became dominant in 7 of 7 heifers following ablation of the largest follicle. In Experiment 2, a minimal single injection of a depressant of FSH concentrations (4.4 mL of steroid-reduced follicular fluid) was given when the largest follicle was a mean of 8.4 mm (Hour 0; controls, n = 4; treated, n = 4). An interaction of group and hour (P < 0.005) for FSH concentrations was attributable to an FSH decrease (P < 0.002) by Hour 6 and an increase (P < 0.002) between Hours 9 and 12 in the treated group. The growth rate of the largest follicle between Hours 0 and 12 was less (P < 0.05) in the treated group (0.2 +/- 0.2 mm/12 h) than in the control group (1.2 +/- 0.4 mm/12 h). The reduced diameter was recorded within 6 h after suppression of FSH concentrations, supporting the hypothesis. Our preferred interpretation is that when the largest follicle reaches a critical diameter of about > or = 8.5 mm, FSH concentrations continue to decrease and become lower than required by the smaller follicles but not the largest follicle. The results further indicate that a close temporal coupling between a change in FSH concentrations and the follicular response could establish the deviation mechanism in < 8 h or before the second largest follicle reaches a similar critical diameter.     

Antral follicle count reliably predicts number of morphologically healthy oocytes and follicles in ovaries of young adult cattle

Methods to predict numbers of healthy oocytes in the ovaries of young adults could have important diagnostic relevance in family planning and animal agriculture. We have observed that peak antral follicle count (AFC) determined by serial ovarian ultrasonography during follicular waves is very highly reproducible within individual young adult cattle, despite 7-fold variation among animals. Herein, we tested the hypothesis that AFC is positively associated with the number of morphologically healthy oocytes and follicles in ovaries and with serum concentrations of anti-Müllerian hormone (AMH), an indirect marker for number of healthy follicles and oocytes in ovaries. In the present study, age-matched young adult cattle (12-18 mo old) were subjected to serial ultrasonography to identify animals with a consistently high (> or =25 follicles that were > or =3 mm in diameter) or low (< or =15 follicles) AFC during follicular waves. Differences in serum AMH concentrations, ovary weight, and number of morphologically healthy and atretic follicles and oocytes were determined. The phenotypic classifications of cattle based on AFC during follicular waves or AMH concentrations both predict reliably the relative number of morphologically healthy follicles and oocytes in ovaries of age-matched young adult cattle.     

Association between numbers of ovarian follicles in the first follicle wave and superovulatory response in ewes

The aim of this study was to examine the variability in the number of ovarian follicles in sheep and to determine if the average number of follicles per day influences the response to superovulation and resulting embryo quality. Ewes (n = 83) were synchronized and the number of follicles (≥2 mm diameter) in the ovaries were counted daily between Days 0 and 4 of the oestrous cycle using transrectal ultrasonography. Fourteen to 21 days later, 47 ewes were randomly chosen from the group and were treated with an intravaginal progestagen pessary for 12 days and superovulated with 1500 IU eCG administered as a single injection 10 days after sponge insertion. Ewes were mated and reproductive tracts were recovered after slaughter on Day 6 of pregnancy. The number of corpora lutea was counted, uterine horns were flushed and the morphology and developmental stage of the recovered oocytes/embryos was assessed. The mean daily number (±S.D.) (≥2 mm diameter) of follicles per ewe was 8.5 ± 2.8 (ranging between 3 and 16). After superovulation animals with few follicles (Low group: <8 follicles/day; n = 21) had fewer (P < 0.005) corpora lutea, total structures (unfertilized oocytes and embryos), good quality and total embryos compared to animals with many follicles (High group: ≥8 follicles/day; n = 23). No difference was found in the proportion of good quality embryos (relative to the total number; Low 0.68 ± 0.11 versus High 0.79 ± 0.08; P = 0.21) between the two groups, or the recovery rate, the number of unfertilized oocytes or the number of poor quality embryos per animal. We conclude that ewes with a higher number of follicles (≥8) during the first follicular wave had a better superovulatory response (in terms of corpora lutea and high quality embryos) 2–3 weeks later; however, there was no relationship between the number of follicles and the proportion of good quality embryos per animal.     

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 and endocrine dynamics during experimental follicle deviation in mares

Deviation during a follicular wave in mares begins when the largest follicle (F1) reaches a mean diameter of 22.5 mm and is characterized by continued growth of F1 to become the dominant follicle and regression of F2 to become the largest subordinate follicle. In the present study, F1 was ablated at the expected beginning of deviation (Hour 0) to provide a reference point for characterizing the intrafollicular changes preceding experimental deviation between F2 and F3. Diameters and concentrations of follicular fluid factors in F2 and F3 were determined in F1-ablated mares at Hours 0, 12, 24, 48, or 72 (n = 8 mares/group). Circulating FSH concentrations were greater (P < 0.05) in the Hour 72 ablation group than in controls 12 h after ablation and then progressively decreased. The diameters of F2 and F3 increased (P < 0.05) during Hours 0 to 24. Thereafter, F2 continued to increase but F3 did not, indicating that experimental deviation began at Hour 24. The diameter of F2 and circulating FSH concentration at Hour 24 were similar (P > 0.1) to the diameter of F1 and FSH concentration at Hour 0, respectively. A differential change between F2 and F3 was not detected in follicular fluid concentrations of estradiol, inhibin-A, and activin-A by the beginning of experimental deviation. However, estradiol was higher in F2 at Hours 0 and 12 and inhibin-A was higher in F2 throughout the experiment, and both factors could have been involved in experimental deviation. Free insulin-like growth factor-1 (IGF-1) increased (P < 0.05) in F2 beginning at Hour 12 and was higher (P < 0.05) in F2 than in F3 by the beginning of experimental deviation. Temporally, this result indicated that intrafollicular IGF-1 was involved in conversion of F2 from a destined subordinate follicle to a dominant follicle.     

Echotextural changes in the follicular wall during follicle deviation in mares

The echotextural changes occurring in the follicular wall in association with deviation in diameters were studied in 8 pony mares. Echotextural changes could be useful as a reference point for future studies of the follicle-selection phenomenon. Follicles were examined daily by transrectal ultrasound from 3 d before to 3 d after the beginning of deviation (Day 0). The following echotextural end points were recorded based on a scoring or percentage system: 1) thickness of granulosa, 2) echogenicity of granulosa, 3) prominence of an anechoic layer located beneath the granulosa, and 4) extent of involvement of the circumference of the follicle by the anechoic layer. Significant main effects indicated increasing values over Days -3 to 3 and a higher mean value for the dominant-follicle status for each end point. The interaction approached significance for thickness of granulosa and was significant for each of the other 3 end points. The interaction seemed due primarily to lower values in the subordinate follicle on Days 2 and 3. The first day of an overall echotextural difference between the 2 largest follicles (15.9 +/- 0.5 d after ovulation) or between days within the largest follicle (16.1 +/- 0.5 d) was about 1 d earlier (P < 0.05) than the beginning of diameter deviation (17.4 +/- 0.8). The effects of follicle diameter versus future follicle status were compared for Days -3 to 0 by grouping follicles for each status into 2 diameter categories (16.0 to 18.9 mm and 19.0 to 22.5 mm). Increasing echotextural values for the granulosa (thickness and echogenicity) were more attributable to follicle growth, whereas the increasing values for the anechoic layer were more attributable to status as a future dominant follicle. Results indicated that development of an anechoic layer beneath the granulosa distinguished the future dominant follicle from the largest future subordinate follicle before the beginning of diameter deviation.     

Follicular deviation and acquisition of ovulatory capacity in bovine follicles.

Selection of dominant follicles in cattle is associated with a deviation in growth rate between the dominant and largest subordinate follicle of a wave (diameter deviation). To determine whether acquisition of ovulatory capacity is temporally associated with diameter deviation, cows were challenged with purified LH at known times after a GnRH-induced LH surge (experiment 1) or at known follicular diameters (experiments 2 and 3). A 4-mg dose of LH induced ovulation in all cows when the largest follicle was > or =12 mm (16 of 16), in 17% (1 of 6) when it was 11 mm, and no ovulation when it was < or =10 mm (0 of 19). To determine the effect of LH dose on ovulatory capacity, follicular dynamics were monitored every 12 h, and cows received either 4 or 24 mg of LH when the largest follicle first achieved 10 mm in diameter (experiment 2). The proportion of cows ovulating was greater (P < 0.05) for the 24-mg (9 of 13; 69.2%) compared with the 4-mg (1 of 13; 7.7%) LH dose. To determine the effect of a higher LH dose on follicles near diameter deviation, follicular dynamics were monitored every 8 h, and cows received 40 mg of LH when the largest follicle first achieved 7.0, 8.5, or 10.0 mm (experiment 3). No cows with a follicle of 7 mm (0 of 9) or 8.5 mm (0 of 9) ovulated, compared with 80% (8 of 10) of cows with 10-mm follicles. Thus, follicles acquired ovulatory capacity at about 10 mm, corresponding to about 1 day after the start of follicular deviation, but they required a greater LH dose to induce ovulation compared with larger follicles. We speculate that acquisition of ovulatory capacity may involve an increased expression of LH receptors on granulosa cells of the dominant follicle and that this change may also be important for further growth of the dominant follicle.     

Short intervals between ultrasonographically guided follicle aspiration improve oocyte quality but do not prevent establishment of dominant follicles in the Gir breed (Bos indicus) of cattle

The aim of the present study was to evaluate oocyte recovery and embryo yield using two different ovarian follicular aspiration schedules in donor cows of the Gir breed. Pluriparous, non-lactating Gir cows (n = 14) were randomly allocated to one of two groups, one of which had aspirations of ovarian follicular contents conducted once a week (TVFA-1x) and the other twice weekly (TVFA-2x), for nine consecutive weeks. Before follicle aspiration, follicular population was recorded in three classes according to size (> 6 mm, 6-9 mm and > 9 mm). The cumulus-oocyte complexes (COCs) recovered were identified, morphologically classified and in vitro matured, fertilized with Gir sperm and cultured in CR2 medium for 7 days. There was no difference (P > 0.05) in the size of the largest follicle, number of follicles identified or follicular content aspirations between TVFA-1x and TVFA-2x groups. Large follicles (> 9 mm) were observed for all the aspiration intervals considered (3, 4 or 7 days). More oocytes were recovered per session in TVFA-1x as compared with TVFA-2x (8.9 +/- 0.8 versus 7.0 +/- 0.7, P < 0.01), resulting in a greater recovery rate in this group (74.3% versus 58.7%, P < 0.01). More COCs of Grade I were recovered from TVFA-2x (22.6% versus 13.3%, P < 0.01). There was no difference in cleavage rate between groups, but the percentage of embryos that reached the blastocyst stage was greater in TVFA-2x as compared with the TVFA-1x (31.8% versus 21.6%, P < 0.01). The greater in vitro performance qualities of TVFA-2x oocytes compensates for the greater oocyte recovery rate in TVFA-1x, demonstrating a greater embryo production potential. Despite showing uncommon follicular dynamics characteristics when subjected to follicular aspiration, Gir cows can be successfully used as oocyte donors for in vitro embryo production.     

Differential blood flow changes between the future dominant and subordinate follicles precede diameter changes during follicle selection in mares

Diameter deviation during a follicular wave is characterized by the continued growth of the developing dominant follicle and reduced growth and regression of the subordinate follicles. This study considered the hypothesis that reduced blood flow in the future largest subordinate follicle precedes the beginning of diameter deviation. The hypothesis was tested by quantifying the daily changes in blood-flow velocities and blood-flow area within the wall of follicles before and during diameter deviation in mares (n = 7). The blood-flow end points were quantified daily by transrectal color Doppler ultrasonography. Follicles were identified retrospectively by rank as F1 (largest) and F2 according to the maximum attained diameter. Follicles were grouped into nine F1 diameter ranges of 3.0 mm each (equivalent to 1 day's growth) centered on 6.5, 9.5, 12.5, 15.5, 18.5, 21.5, 24.5, 27.5, and 30.5 mm. Diameter deviation began in the 24.5-mm group, as indicated by a smaller (P < 0.05) difference between F1 and F2 in the 24.5-mm group than in the 27.5-mm group. Based on a similar approach, peak systolic velocity and time-averaged maximum velocity of blood flow began to deviate between F1 and F2 in the 18.5-mm group (P < 0.04) and blood flow area began to deviate in the 21.5-mm group (P < 0.009). Thus, differential blood flow area between F1 and F2 began an average of 3.0 mm (equivalent to 1 day) and differential blood-flow velocities began an average of 6.0 mm before the beginning of diameter deviation. The results demonstrated that deviation between F1 and F2 in the blood flow of the follicle walls occurred 1 or 2 days before deviation in follicle diameter during follicle selection in mares.     

African matrilineages in American Creole cattle: evidence of two independent continental sources

In order to clarify the historical origin and phylogeographic affinities of Creole cattle matrilineages throughout the American continent, we analysed published D-loop mtDNA sequences (n = 454) from Creole, Iberian and African cattle breeds. The Western European T3 haplogroup was the most common in American Creole cattle (63.6%), followed by the African T1 (32.4%) and the Near Eastern T2 haplogroups (4%). None of the sequences were found in Bos indicus types. Within the African T1 haplogroup there were two subclades, T1a and T1*, whose geographic distribution in America was clearly disjointed. T1a is a highly divergent clade originally reported for Creole cattle from Brazil and the Lesser Antilles, but whose geographic distribution in Africa remains unknown. In contrast, lineages attributable to T1* are restricted in America to the region colonized by the Spaniards. We propose a new hypothesis for the origins of Creole cattle that summarizes all previously published historical and genetic data. While the African T1* fraction in Creole cattle may have arrived in America through the Iberian breeds, the divergent T1a lineages may have been introduced by Portuguese and other European crowns from some unknown, not-yet-sampled African location. Additional molecular studies will be required for pinpointing the specific African regional source.     

Testosterone stimulates the primary to secondary follicle transition in bovine follicles in vitro

The mechanisms controlling the initiation and early stages of follicular growth are poorly understood. Our laboratory developed a serum-free culture system that supports spontaneous and wholesale activation of primordial follicles in pieces of cortex dissected from the ovaries of fetal calves and fetal baboons. However, very few follicles activated in vitro progressed to the secondary stage. To determine whether androgens can promote the primary to secondary follicle transition, pieces of fetal bovine ovarian cortex were cultured in serum-free medium in the absence or presence of testosterone (T, 10(-7) and 10(-6) M) or estradiol (E(2), 10(-6) M) for 10 days. Cortical pieces were then fixed and embedded in plastic for serial sectioning and morphometric analysis; fresh cortical pieces fixed on Day 0 served as uncultured controls. Freshly isolated cortical pieces contained mostly primordial follicles, whereas after 10 days in vitro, most primordial follicles had activated, differentiating into primary follicles as expected. Neither T nor E(2) affected the number of primordial and primary follicles compared with controls (P > 0.05). However, T (10(-7) and 10(-6) M) increased the number of secondary follicles (P < 0.05), whereas E(2) had no effect, suggesting that the effect of T was not due to conversion of T to E(2). In the second experiment, the optimal concentration of T for preantral follicle growth was determined. A range of lower doses of T (10(-10)-10(-7) M) increased the number of secondary follicles in cultured cortical pieces in a dose-dependent manner, with 10(-7) M T being the most effective (P < 0.05). In the third experiment, addition of a specific androgen receptor blocker, flutamide, inhibited the stimulatory effects of T on the primary to secondary follicle transition (P < 0.05), suggesting a receptor-mediated action of T. Localization of androgen receptors by immunohistochemistry revealed immunostaining for the androgen receptor in ovarian stromal cells and increasing immunoreactivity in follicle cells as follicular development progressed from primordial and primary to secondary to antral follicles, suggesting the involvement of the androgen receptor in bovine folliculogenesis. In summary, our results show that T promotes the growth of bovine follicles activated in vitro and suggest that its stimulatory effect is mediated through androgen receptors in the stroma and/or follicular cells.     

Gap junctions in ovarian follicles of Drosophila melanogaster: inhibition and promotion of dye-coupling between oocyte and follicle cells

The analysis of chimeras has shown that communication between germ-line and soma cells plays an important role during Drosophila oogenesis. We have therefore investigated the intercellular exchange of the fluorescent tracer molecule, Lucifer yellow, pressure-injected into the oocyte of vitellogenic follicles of Drosophila. The dye reached the nurse cells via cytoplasmic bridges and entered, via gap junctions, the somatic follicle cells covering the oocyte. The percentage of follicles showing dye-coupling between oocyte and follicle cells was found to increase with the developmental stage up to stage 11, but depended also on the status of oogenesis, i.e., the stage-spectrum, in the respective ovary. During late stage 10B and stage 11, dye-coupling was restricted to the follicle cells covering the anterior pole of the oocyte. No dye-coupling was observed from stage 12 onwards. During prolonged incubation in vitro, the dye was found to move from the follicle cells back into the oocyte; this process was suppressable with dinitrophenol. Dyecoupling was inhibited when prolonged in vitro incubation preceded the dye-injection. Moreover, dye-coupling was inhibited with acidic pH, low [K⁺], high intracellular [Ca²⁺], octanol, dinitrophenol, and NaN₃, but not with retinoic acid, basic pH, or high extracellular [Ca²⁺]. Dyecoupling was stimulated with a juvenile hormone analogue and with 20-hydroxyecdysone. Thus, gap junctions between oocyte and follicle cells may play an important role in intercellular communication during oogenesis. We discuss the significance of our findings with regard to the electrophysiological properties of the follicles, and to the coordinated activities of the different cell types during follicle development and during the establishment of polarity in the follicle.     

Development of a persistent ovarian follicle and associated elevated concentrations of 17beta-estradiol preceding ovulation does not alter the pregnancy rate after embryo transfer in cattle

It was hypothesized that prolonged elevation in 17beta-estradiol (E(2)) preceding ovulation as a result of a persistent ovarian follicle would have a detrimental effect on pregnancy rate after Day 7 (behavioral estrus = Day 0) of the estrous cycle. Cows were either treated with exogenous progesterone (P(4)) for 10 d or remained untreated (CON; n = 76). Cows were treated with 1 of 2 doses of P(4) from Day 6 to 16 which was intended to result in either elevated E(2) (EE(2); n = 76) or normal E(2) (NE(2); n = 76) concentration in the circulation. At the initiation of P(4) treatment, cows received prostaglandin F(2alpha) (PGF(2alpha)) to eliminate the endogenous source of P(4). On Day 16, the exogenous source of P(4) was removed from treated cows, while cows in the CON group received PGF(2alpha). A single embryo was transferred into each cow 7 days after observation of behavioral estrus. Blood samples were taken on alternating days during the treatment period to determine concentrations of P(4) and E(2). The pregnancy rate was determined by ultrasonographic examination 25 to 32 d after embryo transfer. There was a treatment-by-day interaction (P < 0.0001) on E(2) concentrations in the plasma during the 10-d treatment period. Cows in the EE(2) group had a higher concentration of E(2) by Day 8 (6.1 +/- 0.5 pg/ml) and this concentration remained elevated until PRID removal compared with that of cows in the NE(2) (2 +/- 0.2 pg/ml) and CON (2.0 +/- 0.3 pg/ml) groups, which had concentrations of E(2) similar to those at the initiation of treatment. Pregnancy rates after embryo transfer did not differ (P = 0.56; X(2) = 1.1) among cows in the EE(2) (30.7%), NE(2) (36.2%) and CON (32.9%) groups. Prolonged elevation of E(2) concentrations associated with the development of a persistent ovarian follicle preceding ovulation did not affect the pregnancy rate to embryo transfer after Day 7 of the estrous cycle in cows.