A CIDR-based timed AI protocol can be effectively used for dairy cows with follicular cysts

The present study evaluated whether a controlled internal drug release (CIDR)-based timed AI (TAI) protocol could be used as an efficient tool for the treatment of ovarian follicular cysts in lactating dairy cows. In the first experiment, lactating dairy cows diagnosed with follicular cysts were randomly assigned to two treatments: (1) a single injection of GnRH at diagnosis (Day 0) and AI at estrus (AIE) within 21 days (GnRH group, n=70), or (2) insertion of a CIDR device containing progesterone and an injection of GnRH on Day 0, PGF(2alpha) injection at the time of CIDR removal on Day 7, GnRH injection on Day 9, and TAI 16h after the GnRH injection (CIDR-based TAI group, n=65). Conception rate after the CIDR-based TAI protocol (52.3%) was greater (P<0.05) than that after AIE following a single GnRH injection (26.9%). In the second experiment, lactating dairy cows diagnosed with follicular cysts (Cyst group, n=16) and cows having normal estrous cycles (CYC group, n=15) received the same treatment: a CIDR device containing progesterone and an injection of GnRH on Day 0, PGF(2alpha) injection at the time of CIDR removal on Day 7, and GnRH injection on Day 9. The proportion of cows with follicular wave emergence and the interval from treatment to follicular wave emergence did not differ (P>0.05) between groups. The mean diameters of dominant follicles on Days 4 and 7 as well as preovulatory follicles on Day 9, and the synchrony of ovulation following the second injection of GnRH did not differ (P>0.05) between groups. These data suggest that the CIDR-based TAI protocol results in an acceptable conception rate in dairy cows with follicular cysts.     

Long-term follicular dynamics and biochemical characteristics of dominant follicles in dairy cows subjected to acute heat stress

The objective of this study was to examine the quality of successive dominant follicles (DFs) after induced heat stress. Non-lactating dairy cows expressing estrus at normal intervals were allocated randomly to heat stress (HS; n=8) and control (C; n=8) groups. Cows received GnRH (100 microg, i.m.) on Day 0, a progesterone CIDR-B device on Day 4 and prostaglandin (PGF(2alpha); 25mg, i.m.) on Day 7 upon removal of the CIDR device. The DF and follicles >5mm were aspirated on Day 8, and GnRH (100 microg) injected following aspiration, to initiate a new follicular wave. In this manner, a DF was aspirated every 8 days (one "follicular cycle") for 10 cycles. After the first follicular cycle, HS cows were placed in environmental chambers for 7 days during the second follicular cycle (8h per day at 43.3 degrees C set point and 16h per day at 24 degrees C for 4 days, and 8h per day at 43.3 degrees C set point and 16h per day at 32.2 degrees C set point for 3 days; relative humidity, 40%) and thereafter maintained outdoors with control cows at a mean ambient temperature (18.5 degrees C; range 12.7-26 degrees C). Rectal temperature increased (P<0.001) in HS as compared with C cows (39.28+/-0.01 degrees C versus 38.78+/-0.01 degrees C). Concentrations of estradiol (E(2); 1662+/-189 versus 1493+/-188ng/ml) and progesterone (P(4); 44.7+/-5 versus 54.1+/-5.1ng/ml) in follicular fluid (FF) of DF did not differ between C and HS treatments, respectively. Total FF protein concentration was greater (P<0.05) in HS (99.7+/-2.3mg/ml) than in C (92.7+/-2.3mg/ml). Heat shock protein 90 (Hsp 90) in FF was not altered by heat stress. IGF-II ligand blots were conducted with FF samples (n=79) from four HS and four C cows. There was a predominance of IGFBP-3 in 76 of 79 FF samples, indicating healthy follicular status, and only three FF samples had the lower molecular weight IGFBP-2 indicative of a poor quality follicle. Plasma P(4) and E(2) concentrations did not differ between C and HS groups. The number of class 1 and 3 follicles increased during and just after heat stress, but the number of class 2 follicles did not differ between C and HS cows. Heat stress appeared to induce a decrease in follicular dominance, but GnRH-induced follicular cycles resulted in development of healthy preovulatory follicles in both groups.     

Ovarian follicular development following administration of progesterone or aspiration of ovarian follicles in Holstein cows

The objective of this study was to compare the effects of administration of a single injection of progesterone (P4) and follicle aspiration on Day 7 of the estrous cycle on the timing and synchrony of follicular wave emergence, time of ovulation, and concentrations of P4, estradiol and FSH in Holstein cows. Twenty cows were assigned to 4 groups (n=5 cows per group) in a 2 by 2 factorial arrangement. Cows were treated on Day 7 (Day 0 = estrus) of the estrous cycle with either sham follicular aspiration and an oil vehicle administered intramuscularly (control), aspiration of ovarian follicles (aspiration), 200 mg of P4 im, or aspiration and 200 mg of P4 im (aspiration + P4). On Day 11, PGF(2alpha)(25mg) was administered to all groups. Synchrony of ovulation was less variable in each of the treatment groups compared with the control group (P<0.05), whereas ovulation was delayed in cows in the P4 group (P<0.05). Day of follicular wave emergence was delayed in the cows of the P4 group compared with cows in the aspiration and aspiration + P4 groups (P<0.01), whereas variability in wave emergence was less among both groups of aspirated cows compared with the cows in the control group (P<0.01). More follicles 4 to 7 mm in diameter were detected in the 2 aspiration groups compared with the cows in the control and P4 group (P<0.05). No difference was detected among groups in the maximum concentration of FSH associated with follicular wave emergence. We conclude that both the administration of P4 and the aspiration of follicles on Day 7 of the estrous cycle improves the synchrony of ovulation when luteolysis is induced on Day 11 and results in similar concentrations of FSH at the time of follicular wave emergence, but the timing of wave emergence and the number of follicles post-emergence differ.     

Effect of timing of prostaglandin PGF 2 alpha injection subsequent to embryo collection on the resumption of normal follicular development following superovulatory treatment in cattle

Nonlactating Holstein and Jersey cows (n = 24) were superovulated and ovarian follicular development was monitored by transrectal ultrasound during the period after embryo recovery. Luteolysis was induced by two injections of prostaglandin F(2)alpha (PGF; 25 mg Lutalyse; 12-h interval) at specific times after superovulatory induced estrus (Treatment 1, Day 9; Treatment 2, Day 12; Treatment 3, Day 17; Treatment 4, Day 25; superovulatory estrus = Day 0 of Cycle 1). Follicular development was monitored during Cycle 1 before and after PGF injection and continued through the ensuing estrous cycle (Cycle 2). Superovulation led to more than one embryo collected in 14 cows (mean = 8.71 embryos: positive superovulatory response [PSR] cows), while 10 cows were not successfully superovulated (mean = 0.1 embryo; negative superovulatory response [NSR] cows). These cows differed in terms of number of unovulated follicles detected at embryo collection (4.21 vs 17.2, PSR vs NSR) and plasma progesterone during the superovulatory estrous cycle (32.3 ng/ml PSR vs 8.6 ng/ml NSR). Follicular development during Cycle 1 started sooner in NSR than in PSR cows (day by class by response P<0.03) and was initiated on Days 11 to 12 in NSR cows and on Days 19 to 20 in PSR cows. Interval to estrus after PGF averaged 6.3 d. Cows having short intervals to estrus had follicles at the time of PGF injection. Treatment influenced the length of Cycle 1, but it did not affect the interval to estrus after PGF, the length of Cycle 2, or follicular development during Cycle 2. The results indicate that 1) the timing of PGF injection after embryo collection does not influence subsequent follicular populations, 2) elongated estrous cycles and intervals to estrus after PGF in superovulated cattle are a function of decreased follicular activity, and 3) the presence of numerous corpora lutea and not the superovulatory treatment, per se, seem to attenuate follicular growth.     

Synchronization of ovulation in beef cows (Bos indicus) using GnRH, PGF2alpha and estradiol benzoate

The objective of this study was to evaluate protocols for synchronizing ovulation in beef cattle. In Experiment 1, Nelore cows (Bos indicus) at random stages of the estrous cycle were assigned to 1 of the following treatments: Group GP controls (nonlactating, n=7) received GnRH agonist (Day 0) and PGF2alpha (Day 7); while Groups GPG (nonlactating, n=8) and GPG-L (lactating, n=9) cows were given GnRH (Day 0), PGF2alpha (Day 7) and GnRH again (Day 8, 30 h after PGF2alpha). A new follicular wave was observed 1.79+/-0.34 d after GnRH in 19/24 cows. After PGF2alpha, ovulation occurred in 19/24 cows (6/7 GP, 6/8 GPG, 7/9 GPG-L). Most cows (83.3%) exhibited a dominant follicle just before PGF2alpha, and 17/19 ovulatory follicles were from a new follicular wave. There was a more precise synchrony of ovulation (within 12 h) in cows that received a second dose of GnRH (GPG and GPG-L) than controls (GP, ovulation within 48 h; P<0.01). In Experiment 2, lactating Nelore cows with a visible corpus luteum (CL) by ultrasonography were allocated to 2 treatments: Group GPE (n=10) received GnRH agonist (Day 0), PGF2alpha (Day 7) and estradiol benzoate (EB; Day 8, 24 h after PGF2alpha); while Group EPE (n=11), received EB (Day 0), PGF2alpha (Day 9) and EB (Day 10, 24 h after PGF2alpha). Emergence of a new follicular wave was observed 1.6+/-0.31 d after GnRH (Group GPE). After EB injection (Day 8) ovulation was observed at 45.38+/-2.03 h in 7/10 cows within 12 h. In Group EPE the emergence of a new follicular wave was observed later (4.36+/-0.31 d) than in Group GEP (1.6+/-0.31 d; P<0.001). After the second EB injection (Day 10) ovulation was observed at 44.16+/-2.21 h within 12 (7/11 cows) or 18 h (8/11 cows). All 3 treatments were effective in synchronizing ovulation in beef cows. However, GPE and, particularly, EPE treatments offer a promising alternative to the GPG protocol in timed artificial insemination of beef cattle, due to the low cost of EB compared with GnRH agonists.     

Ovarian superstimulation after follicular wave synchronization with GnRH at two different stages of the estrous cycle in cattle

The objective of this study was to evaluate superovulatory programs based on synchronization of follicular waves with GnRH at 2 different stages of the estrous cycle. Sixteen Holstein cows were randomly assigned to 1 of 3 groups and administered GnRH (Cystorelin, 4 ml i.m.) between Days 4 and 7 (Groups 1 and 3) or between Days 15 and 18 (Group 2) of the estrous cycle (estrus = Day 0). Four days after GnRH treatment, > or = 7-mm follicles were punctured in Groups 1 (n = 6) and 2 (n = 6) or were left intact in Group 3 (n = 4). All cows were superstimulated 2 d later (i.e., from Days 6 to 10 after GnRH treatment) with a total of 400 mg NIH-FSH (Folltropin-V) given twice daily in decreasing doses. The GnRH treatment caused a rapid disappearance of large follicles (P < 0.005), rapid decrease in estradiol concentrations (P < 0.003), and increase in the number of recruitable follicles (4 to 6 mm; P < 0.04), indicative of the emergence of a new follicular wave within 3 to 4 d of treatment. Between 4 and 6 d after GnRH treatment, the mean number of 4- to 6-mm follicles decreased (4.7 +/- 1.8 to 1.5 +/- 3.3) in the nonpunctured group but increased (3.9 +/- 1.0 to 7.3 +/- 1.9) in the punctured group of cows (P < 0.05). In response to FSH treatment, the increase in the number of > or = 7-mm follicles was delayed by approximately 2 d in the nonpunctured group (P < 0.006). Moreover, the mean number of > or = 7-mm follicles at estrus was higher (16.9 +/- 1.7 vs 11.5 +/- 3.0; P < 0.1) in the punctured than the nonpunctured group. The increase in progesterone concentration after estrus was delayed in the nonpunctured group (P < 0.1) compared with the punctured follicles. Mean numbers of CL as well as freezable (Grade 1 and 2) and transferable (Grade 1, 2 and 3) embryos were similar (P > 0.1) in punctured and nonpunctured groups. Spontaneous estrus did not occur prior to cloprostenol-induced luteolysis in any group, and stage of the estrous cycle during which GnRH was given did not affect (P > 0.1) hormonal and follicular responses in the punctured groups. In conclusion, GnRH given at different stages of the estrous cycle promotes the emergence of a follicular wave at a predictable time. Puncture of the newly formed dominant follicle increases the number of recruitable follicles (4 to 6 mm) 2 d later and, in response to superstimulation with FSH, causes a greater number and faster entry of recruitable follicles into larger classes (> or = 7 mm) and a faster postovulatory increase in progesterone concentrations.     

Changes in morphological appearance and functional capacity of recruited follicles in cows treated with FSH in the presence or absence of a dominant follicle

This study was designed to determine the effect of the presence of a dominant follicle at the beginning of FSH stimulation on the morphological appearance and functional capacity of recruited follicles during FSH stimulation in cattle. Synchronized nonlactating dairy cows were assigned to 1 of 2 groups and treated with FSH in the presence (n = 5) or absence (n = 6) of a dominant follicle between Days 7 and 12 of the estrous cycle (Day 0 = estrus) to stimulate follicular growth. Dominant follicles were identified by daily ultrasonographic observations, beginning on Day 3 of the estrous cycle. Dominant follicle had an ultrasonographic diameter > or = 10 mm and were in a growing phase, or maintaining a constant diameter (> or = 10 mm) for less than 4 d. Ovaries were collected at slaughter on the morning of the third day following initiation of the FSH stimulation. All follicles > 2 mm were dissected, classified according to diameter (Class 1: 2 to 4.4 mm; Class 2: 4.5 to 7.9 mm; Class 3: > 8 mm), and incubated individually for 90 min in medium M-199 (37 degrees C, 5% CO2). Following incubation, integrity of each follicle was evaluated histologically to assess the level of atresia and biochemically to determine the in vitro release of estradiol (E2) and androstenedione in culture media. On Day 3 of the FSH treatment, mean number of follicles in each class was similar (P > 0.1) between the 2 groups. The percentage of atretic follicles in Classes 1 and 3 on Day 3 of the FSH stimulation did not differ (P > 0.1) between the 2 groups. However, the percentage of atretic follicles in Class 2 was higher (P < 0.005) in cows treated with FSH in presence than in absence of a dominant follicle (60.8 vs 38.2%). The release of E2 in culture media by small Class 1 atretic or healthy follicles, by Class 2 atretic and by Class 3 healthy follicles was not affected (P > 0.1) by the ovarian status. However (P < 0.001), the release of E2 in culture media of Class 2 healthy and Class 3 atretic follicles was less for follicles harvested from cows bearing than from those not bearing a dominant follicle. Within each follicular class, concentrations of androstenedione in the culture media did not differ between the 2 groups (P > 0.1). These results suggest that the presence of a dominant follicle at the beginning of FSH stimulation alters the population of follicles recruited FSH stimulation. This may be associated with the reported decrease of the superovulatory response in cows superovulated in presence of a dominant follicle.     

Waves of follicle development during the estrous cycle in sheep

The pattern of ovarian follicle development in maiden cyclic lambs was characterized using the definition of a follicle wave as the changes in the number of follicles among the days of the estrous cycle, as originally defined in cattle by Rajakoski in 1960. We also examined the steroid content relationships among follicles on Days 5 (Wave 1) and 14 (Waves 2 and 3) of the estrous cycle. In Experiment 1, the ovaries of 20 cyclic lambs (40 to 45 kg) were examined daily using transrectal ultrasonography for 1 or 2 estrous cycles (n = 31 cycles). The number of small (2 and 3 mm in diameter), medium (4 and 5 mm) and large (> or = 6 mm) follicles were aligned with the beginning and end of the average length estrous cycle and then compared among days. Identified follicles were defined as those that grew to > or = 4 mm and remained at > or = 3 mm for > or = 3 d. The number of identified follicles emerging (retrospectively identified at 2 or 3 mm) per ewe per day was also aligned with the average length estrous cycle. In Experiment 2, ewe lambs were ovariectomized on Day 5 (n = 6) or 14 (n = 5) of the estrous cycle, then follicle diameters and follicular fluid concentrations of estradiol and progesterone were compared among follicles. Data were analyzed by repeated measures ANOVA and compared among days using Fisher's LSD. In Experiment 1, either 2 (n = 10 cycles), 3 (n = 20 cycles) or 4 (n = 1 cycle) periods of emergence of identified follicles occurred during individual cycles, with estrous cycle lengths of 15.6 +/- 1.6, 16.1 +/- 1.1 and 17 d respectively. In animals with 2 or 3 periods of emergence of identified follicles, the total number of small, medium and large follicles differed (P < 0.05) among days of the estrous cycle showing a wave-like pattern. In Experiment 2, a single follicle collected on each of Days 5 and 14 of the cycle (6.2 +/- 0.2 and 3.9 +/- 0.2 mm in diameter) had a higher (P < 0.05) concentration of follicular fluid estradiol (36.2 +/- 4.4 and 50.9 +/- 21.6 ng/mL) than other follicles collected on the same day (next largest follicle: 4.3 +/- 0.3 and 3.5 +/- 0.4 mm; 4.3 +/- 0.9 and 18.2 +/- 6.7 ng/mL estradiol). The results showed that 1) there was a synchronous emergence of follicles associated with fluctuations in the number and size of follicles during the estrous cycle; 2) within a wave there was a hierarchy among follicles for diameter and steroid content; 3) ovarian follicle growth in ewe lambs occurred in 2 or 3 organized waves during the estrous cycle.     

Simultaneous injection of PGF2alpha and GnRH into diestrous dairy cows delays return to estrus

Simultaneous injections of prostaglandin F2alpha (PGF) and gonadotropin releasing hormone (GnRH) or saline were given to 32 diestrous dairy cows to test the ability of GnRH to improve estrous and ovulation synchrony beyond that of PGF alone. Cows were randomly assigned to receive PGF on Day 8 or Day 10 of the estrous cycle (estrus = Day 0), and all cows were further assigned to simultaneous injection of GnRH or saline. Corpus luteum (CL) regression, return to estrus and follicular activity were monitored by plasma progesterone assay, twice-daily estrous detection and ultrasonographic examination, respectively. Plasma progesterone concentrations declined to <1.0 ng/ml at 24 hours after PGF in all cows and were not affected by GnRH. Gonadotropin releasing hormone inducted premature ovulation or delayed return to estrus in 7 of 8 cows treated with PGF/GnRH on Day 8 and 3 of 8 cows treated with PGF/GnRH on Day 10. Further, cows with premature GnRH-induced ovulations failed to develop and maintain a fully functional CL, and all returned to estrus 7 to 13 days after the induced ovulation. These data indicate that GnRH administered simultaneously with a luteolytic dose of PGF disrupts follicular dynamics and induces premature ovulation or delays normal return to estrus and, therefore, does not improve the synchrony of estrus and ovulation achieved with PGF alone.     

Follicular dynamics and superovulatory response in Holstein cows treated with FSH-P in different endocrine states

The effect of follicular and/or endocrine environments on superovulatory response was tested. Eighteen nonlactating Holstein cows were superovulated with 32 mg FSH-P given in decreasing doses at 12-h intervals plus two injections of prostaglandin F2-alpha (25 mg each) on the third day of treatment. Cows were assigned randomly to treatments: T1, superovulatory treatment initiated on estrous cycle Day 10.5; T2, CIDR (intravaginal device containing 1.9 g of progesterone) inserted from Days 3 to 9 and superovulation initiated on Day 6.5; T3, identical to T2 but Buserelin (GnRH agonist) was injected (8 mug, i.m.) on Day 3 at the time of CIDR insertion. Embryos were recovered on Day 7 after the superovulatory estrus. Cows were examined daily by ultrasonography and blood was collected for progesterone and estradiol determinations. Mean diameter of the dominant follicle (frequency of first-wave dominant follicle) at the beginning of FSH injections was 13.7 mm (4 6 ), 11.2 mm (6 6 ) and 8.7 mm (6 6 ) (P<0.01) for T1, T2 and T3, respectively. Following initiation of superovulation, follicles moved into larger follicle classes (Class I, <3 mm; Class II, 3 to 4 mm; Class III, 5 to 9 mm; Class IV >9 mm) earliest in T1 (P<0.01). Cumulative follicular diameter and plasma concentrations of estradiol at Day 4 of superovulation were higher (P<0.01) in T1 (200 mm, 82 pg/ml) compared with T2 (123 mm, 24 pg/ml) and T3 (130 mm, 18 pg/ml). Proportion of cows in estrus prior to 12 h vs 12 to 24 h differed (P<0.05) between groups (T1: 5 vs 1; T2: 2 vs 4; T3: 1 vs 5). Mean number of follicles on the last day of superovulation treatment, number of CL and number of embryos plus unfertilized ova recovered were 17.5, 12.2 and 13.3; 13.8, 10 and 8.2 (P<0.1) and 8.7, 4.5 and 2.3 (P<0.05) for T1, T2 and T3, respectively. The developmental stage of the dominant follicle was associated with not only the number of ovulations, but also the size and periestrous concentrations of plasma estradiol associated with the recruited follicles.     

Effect of an implant containing the GnRH agonist deslorelin on secretion of LH, ovarian activity and milk yield of postpartum dairy cows

Prevention of high plasma progesterone concentrations in the early postpartum period may improve fertility. Our objective was to determine whether a Deslorelin implant (DESL; 2100 microg, s.c.) would reduce secretion of LH and alter follicle dynamics, plasma concentrations of progesterone, estradiol and PGF2alpha metabolite (PGFM) in postpartum dairy cows. Cows received DESL on Day 7 postpartum (Day 7, n=8) or were untreated (Control, n=9). All cows were injected with GnRH (100 microg, i.m.) on Day 14 to assess LH response. A protocol for synchronization of ovulation with timed AI was initiated on Day 60 (GnRH [Day 60], CIDR [Day 60 to Day 67], PGF2alpha [Day 67, 25 mg and Day 68, 15 mg], GnRH [Day 69] , AI [Day 70]). The LH response to injection of GnRH on Day 14 was blocked in animals treated with DESL. Numbers of Class 1 (<6 mm) follicles were unaffected (P > 0.05) whereas numbers of Class 2 (6 to 9 mm) (P < 0.01) and Class 3 (>9 mm) follicles were less (P < 0.01) in DESL cows between Day 7 and Day 21. From Day 22 to Day 60, DESL-treated cows had more of Class 1 follicles and less Class 2 (P < 0.01) and Class 3 (P < 0.01) follicles, and lower plasma concentrations of progesterone and estradiol (P < 0.01). Concentrations of PGFM between Day 7 and Day 42 were not affected by treatment (P > 0.05). All cows ovulated in response to GnRH on Day 69. Subsequent luteal phase increases in plasma progesterone concentrations (Day 70 to Day 84) did not differ. The use of the DESL implant associated with PGF2alpha given 14 days later suppressed ovarian activity and caused plasma progesterone concentrations to remain < 1 ng/mL between Day 22 and Day 51. The DESL implant did not affect milk production.     

Transvaginal ultrasound guided follicular aspiration of bovine oocytes

A transvaginal ultrasound guided follicular aspiration technique was developed for the repeated collection of bovine oocytes from natural cycling cows. In addition, the feasibility of using this method for collecting immature oocytes for in vitro embryo production was also evaluated. Puncturing of visible follicles for ovum pick-up was performed in 21 cows over a three month period. All visible follicles larger than 3 mm were punctured and aspirated three times during the estrous cycle on Day 3 or 4, Day 9 or 10 and Day 15 or 16. The mean (+/- SEM) estrous cycle length after repeated follicle puncture was 22.2 +/- 0.3 days. The mean total number of punctured follicles per estrous cycle was 12.6 +/- 0.3. The largest (P<0.05) number of follicles punctured (5.1 +/- 0.3) for ovum pick-up was on Day 3 or 4 of the estrous cycle. The overall recovery rate of 541 punctured follicles was 55%. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm. Following in vitro maturation and fertilization (IVM/IVF), 104 oocytes were transferred to sheep oviducts. Six days later, 75 ova/embryos were recovered, after flushing the oviduct of the sheep, of which 24% developed into transferable morulae and blastocysts. In this study, a reliable nonsurgical, follicular aspiration procedure was used for the repeated collection of immature oocytes which could be used successfully for in vitro production of embryos. This procedure offers a competitive alternative to conventional superovulation/embryo collection procedures.     

Ovarian follicular activity and hormonal profile during estrous cycle in cows: the development of 2 versus 3 waves

Most estrous cycles in cows consist of 2 or 3 waves of follicular activity. Waves of ovarian follicular development comprise the growth of dominant follicles some of which become ovulatory and the others are anovulatory. Ovarian follicular activity in cows during estrous cycle was studied with a special reference to follicular waves and the circulating concentrations of estradiol and progesterone. Transrectal ultrasound examination was carried out during 14 interovulatory intervals in 7 cows. Ovarian follicular activity was recorded together with assessment of serum estradiol and progesterone concentrations. Three-wave versus two-wave interovulatory intervals was observed in 71.4% of cows. The 3-wave interovulatory intervals differed from 2-wave intervals in: 1) earlier emergence of the dominant follicles, 2) longer in length, and 3) shorter interval from emergence to ovulation. There was a progressive increase in follicular size and estradiol production during growth phase of each wave. A drop in estradiol concentration was observed during the static phase of dominant anovulatory follicles. The size of the ovulatory follicle was always greater and produced higher estradiol compared with the anovulatory follicle. In conclusion, there was a predominance of 3-wave follicular activity that was associated with an increase in length of interovulatory intervals. A dominant anovulatory follicle during its static phase may initiate the emergence of a subsequent wave. Follicular size and estradiol concentration may have an important role in controlling follicular development and in determining whether an estrous cycle will have 2 or 3-waves.     

The Expression Pattern of microRNAs in Granulosa Cells of Subordinate and Dominant Follicles during the Early Luteal Phase of the Bovine Estrous Cycle

This study aimed to investigate the miRNA expression patterns in granulosa cells of subordinate (SF) and dominant follicle (DF) during the early luteal phase of the bovine estrous cycle. For this, miRNA enriched total RNA isolated from granulosa cells of SF and DF obtained from heifers slaughtered at day 3 and day 7 of the estrous cycle was used for miRNAs deep sequencing. The results revealed that including 17 candidate novel miRNAs, several known miRNAs (n = 291–318) were detected in SF and DF at days 3 and 7 of the estrous cycle of which 244 miRNAs were common to all follicle groups. The let-7 families, bta-miR-10b, bta-miR-26a, bta-miR-99b and bta-miR-27b were among abundantly expressed miRNAs in both SF and DF at both days of the estrous cycle. Further analysis revealed that the expression patterns of 16 miRNAs including bta-miR-449a, bta-miR-449c and bta-miR-222 were differentially expressed between the granulosa cells of SF and DF at day 3 of the estrous cycle. However, at day 7 of the estrous cycle, 108 miRNAs including bta-miR-409a, bta-miR-383 and bta-miR-184 were differentially expressed between the two groups of granulosa cell revealing the presence of distinct miRNA expression profile changes between the two follicular stages at day 7 than day 3 of the estrous cycle. In addition, unlike the SF, marked temporal miRNA expression dynamics was observed in DF groups between day 3 and 7 of the estrous cycle. Target gene prediction and pathway analysis revealed that major signaling associated with follicular development including Wnt signaling, TGF-beta signaling, oocyte meiosis and GnRH signaling were affected by differentially expressed miRNAs. Thus, this study highlights the miRNA expression patterns of granulosa cells in subordinate and dominant follicles that could be associated with follicular recruitment, selection and dominance during the early luteal phase of the bovine estrous cycle.     

Influence of intrauterine infections and follicular development on the response to GnRH administration in postpartum dairy cows

The effects of intrauterine infections and prior follicular development on the response to gonadotropin releasing hormone (GnRH) administration in postpartum dairy cows were studied. Fifty lactating Holstein cows were assigned at random to one of two groups after calving. Group I (control) consisted of 25 cows given a single intramuscular injection of saline on Day 15 postpartum. Group II (treated) consisted of 25 herdmates given a single i.m. injection of 100 mug of GnRH on Day 15 postpartum. Palpation per rectum and real-time ultrasonography were used to monitor ovarian activity, and endometrial swabs were cultured to determine the presence of uterine infection. Blood samples were collected for progesterone (P(4)) and luteinizing hormone (LH) analysis. Fourteen cows (control, n = 5; treated, n = 9) did not ovulate during the first 60 d postpartum. Ovaries in these cows contained 4 to 8-mm size follicles and both P(4) and LH remained at basal concentrations. Fourteen other cows (control, n = 6; treated, n = 8) ovulated by Day 15 postpartum. Follicles >/= 10 mm were demonstrable in the ovaries of these cows before or by Day 12 postpartum. GnRH treatment had no effect on the lifespan of the existing corpus luteum in these cows. In the remaining cows, 7 of 14 Control and all 8 Treated cows ovulated within 3 d of treatment. All cows ovulating within this period were free of uterine infection and the ovaries contained follicles 相似文献   

The ability of subordinate follicles of the second follicular wave to become dominant is lost by day 15 of the estrous cycle in cattle

Generally, unilateral ovariectomy before a critical period in the latter part of the estrous cycle induces a transitory increase in plasma FSH, which causes subordinate follicles to develop and maintain ovulation rates characteristic of the species. A limiting period for subordinate follicles to assume dominance and from which ovulation occurs has not been shown for cattle. Growth and/or regression of subordinate follicles were characterized following removal of the dominant follicle at different days of the luteal phase of the estrous cycle in cattle in this study. In the mid-luteal phase (Day 13 or 15), the ovary with the dominant follicle of the second wave was ablated via unilateral ovariectomy; the corpus luteum also was removed. In the late luteal phase (Day 17 or 19), the dominant follicle was ablated with an ultrasonically guided 20 gauge needle. When the dominant follicle was removed on Day 13, the largest subordinate follicle of the second wave of follicular development became dominant and ovulation occurred from this follicle in 4 of 4 animals. However, when the dominant follicle was removed on Day 15, 17 or 19, a new wave of follicular development was induced in 14 of 15 animals. Moreover, the recovered subordinate follicle of the second wave of follicular development had similar growth characteristics to naturally occurring dominant follicles. In conclusion, the subordinate follicle in the second follicular wave in cattle retained the ability to become dominant, but this ability was lost by Day 15 of the estrous cycle. However, cattle then were able to maintain ovulation by developing a new wave of follicular growth.     

The effect of a GnRH analogue on the dynamics of follicular development and synchronization of estrus in lactating cyclic dairy cows

A GnRH analogue was used to synchronize ovarian follicular development prior to an injection of PGF(2alpha) for the synchronization of estrus in lactating Holstein cows. On Day 12 (estrus = Day 0) of the experimental cycle, cows (n = 8) were injected with 8 mug Buserelin (BUS group), followed by 25 mg PGF(2alpha) 7 d later (Day 19). Control cows (n = 7) received PGF(2alpha) on Day 12 (PGF group). Ovaries were scanned daily via ultrasonography, and plasma progesterone and estradiol concentrations were determined. Sizes of all visible follicles were recorded. Follicles were classified as small (3 to 5 mm), medium (6 to 9 mm), or large (>/= 10 mm). Between Days 12 and 16 of the cycle, the number of large follicles in PGF cows remained unchanged (1.2), whereas in the BUS group, the number of large follicles decreased from 1.3 on Day 12 to 0.5 on Day 15. Only 4 of 7 PGF cows ovulated a second-wave dominant follicle. In the BUS group, 7 of 8 cows ovulated a GnRH analogue induced dominant follicle that was first identified on Day 15. During the follicular phase (last 5 d prior to estrus), plasma progesterone declined in association with CL regression in both groups, and estradiol concentrations increased, reaching higher (P<.0.05) preovulatory peak concentration in BUS cows than in PGF cows (14.0 +/- 1.0 vs 10.4 +/- 1.1 pg/ml). The number of medium-size follicles was smaller and the number of small-size follicles tended to be higher in BUS cows than in the PGF-treated group. On the day of estrus, the size of the ovulatory follicle (16.1 vs 13.3 mm) and the size difference between the ovulatory and second largest follicle (11.4 vs 6.2 mm) were both larger in BUS cows than in PGF-treated cows, suggesting a more potent dominance effect of the ovulatory follicle in the BUS cows. This study suggests that a GnRH analogue can alter follicular development prior to synchronization of estrus with an injection of PGF(2alpha) in lactating dairy cows.     

Plasma concentrations of inhibin a and follicle-stimulating hormone differ between cows with two or three waves of ovarian follicular development in a single estrous cycle

Patterns of ovarian follicle development were monitored daily in Holstein-Friesian cows that had two (n = 4) or three (n = 4) waves of ovarian follicle development during a single estrous cycle. The plasma from daily blood samples was used in assays for inhibin A, FSH, progesterone, and estradiol-17beta. Mean cycle lengths for cows with two and three waves were 21.8 and 25.3 days, respectively (P < 0.02). Although the average number of follicles >3-mm diameter on each pair of ovaries was similar for two- and three-wave cows on Days 2, 3, and 4 (Day 0 = day of ovulation; 8.6 vs. 9.6 follicles), there were more follicles >6-mm diameter on the ovaries of cows with two waves on Days 3 and 4. This difference was associated with a shorter interval from wave emergence to peak concentrations of inhibin A during the first wave in two-wave cows (2.0 vs. 3.8 days; P = 0.03) and with higher peak concentrations (474 vs. 332 pg/ml; P = 0.03). Differences in peak FSH concentrations were not significant (1.7 vs. 1.3 ng/ml; P = 0.10) and were inversely related to inhibin A concentrations. The peak concentrations of inhibin A and FSH in the second nonovulatory wave in the three-wave cows were similar to the low concentrations measured in the first wave (292 vs. 332 pg/ml of inhibin A, 1.3 vs. 1.3 ng/ml of FSH; P > 0.20). Average peak concentrations of inhibin A and FSH were similar during the ovulatory wave for cows with either two or three waves in a cycle (432 vs. 464 pg/ml of inhibin A, 2.3 vs. 2.1 ng/ml of FSH; P > 0.3). The lower concentrations of FSH during the emergence of the first follicular wave in cows with three-wave cycles may have reduced the rate of development of some of the follicles and reduced the concentrations of inhibin A. This pattern of lower concentrations of FSH and inhibin A was repeated in the second nonovulatory wave but not in the ovulatory wave. Subtle differences in the concentrations of these two hormones may underlie the mechanism that influences the number of waves of ovarian follicle development that occur during the bovine estrous cycle.     

Reproductive responses following postpartum suppression of ovarian follicular development with a deslorelin implant during summer heat stress in lactating dairy cows

The objective was to evaluate pregnancy rate to a timed artificial insemination (TAI) protocol in the autumn for cows treated with a non-degradable GnRH agonist implant (Deslorelin [DESL], 5mg) during the summer heat stress period compared with non-treated controls (CON). Cows were randomly assigned to receive or not a DESL implant within 1-4 days postpartum (dpp) twice weekly, from 25 June through 8 August 2001. All cows in DESL implant and CON treatments were injected with PGF(2alpha) 7 days after enrollment. Ultrasonography (US) monitored numbers of ovarian follicles and corpus luteum (CL) at approximately 10, 30, 35/36, 45/44, 56/55 and 66/63dpp, while DESL implants were in situ and concurrently CON, respectively. DESL implants were removed at two specific days, 28 August and 4 September. Cows had DESL implant in situ for a range of 28-67 days, depending on date of enrollment and implant removal. Within 61-100dpp, 31 days after implant removal, DESL implant and CON cows were initiated in a Presynch-Ovsynch and TAI protocol. Pregnancy was evaluated by US and palpation per rectum at 28 and 46 days after TAI, respectively. Plasma concentrations of progesterone were analyzed for sets of blood samples collected during the Presynch-Ovsynch and at TAI day followed 8 days later. Cows in the DESL-implant treatment had more (P<0.01) Class 1 (3-5mm) follicles, less (P<0.01) Class 2 (6-9mm), Class 3 (> or =10mm) follicles and CL compared with CON cows. Proportion of cows having initiated estrous cycles after calving was less (P<0.01) in the DESL-implant treatment (52.2%, 58/111) compared with CON (93.7%, 104/111) at the beginning of Ovsynch. Pregnancy rate to TAI was less (P<0.01) in the DESL implant (27.5%, 33/120) compared with CON (53.9%, 69/128). Pregnancy rate to TAI was less (P<0.01) in DESL-implanted cows that had initiated estrous cycles after calving (30.6%, 19/62) compared with CON (53.7%, 65/121) cows having initiated estrous cycles after calving. Furthermore, pregnancy rate was less (P<0.01) for cows having ovulations that had initiated estrous cycles after TAI in the DESL implant (39.1%, 18/46) compared with CON (62.1%, 54/87) treatments. Pregnancy losses from day 28 to day 46 of pregnancy did not differ between DESL implant (15.1%, 5/33) and CON (13.0%, 9/69) treatments. The DESL implant induced a delay in initiation of a new wave of follicular development during the postpartum-heat stressed period. The lesser pregnancy rate in the DESL-implant treatment group may be due to a pool of heat stress damaged follicles that were depleted in the control group with re-occurring follicle waves.