Behzad M. Toosi

Ovarian antral follicular dynamics in sheep revisited: comparison among estrous cycles with three or four follicular waves.

In this study, the characteristics of ovarian follicular waves and patterns of serum concentrations of follicle-stimulating hormone (FSH), estradiol, and progesterone were compared between cycles with three (n=9) or four (n=10) follicular waves in Western White Face (WWF) ewes (Ovis aries). Transrectal ultrasonography and blood sampling were performed daily during one cycle. Estrous cycles were 17.11+/-0.3 and 17.20+/-0.2 d long in cycles with three and four waves, respectively (P>0.05). The first interwave interval and the interval from the emergence of the final wave to the day of ovulation were longer in cycles with three waves compared with those in cycles with four waves (P<0.05). The growth phase (5.1+/-0.5 vs. 3.1+/-0.4 d) and life span (5.67+/-0.3 vs. 4.3+/-0.3 d) of the largest follicle growing in the last or ovulatory wave was longer in cycles with three waves compared with that in cycles with four waves (P<0.05). The maximum diameter of the largest follicle was greater in the first wave and the ovulatory wave compared with that in other waves of the cycle (P<0.05). The regression phase of the largest follicle growing in the first wave was longer in cycles with three waves compared with that in cycles with four waves (4.44+/-0.4 vs. 3.4+/-0.4 d; P<0.05). The length of the life span, regression phase, and, although not significant in every case, FSH peak concentration and amplitude decreased across the cycle (P<0.05). We concluded that estrous cycles with three or four follicular waves were confined within the same length of cycle in WWF ewes. In this study, there were no apparent endocrine or follicular characteristics that could explain the regulation of the different number of follicular waves (three vs. four) during cycles of similar length.

Markers of ovarian antral follicular development in sheep: comparison of follicles destined to ovulate from the final or penultimate follicular wave of the estrous cycle.

Treatment of non-prolific western white-faced ewes with prostaglandin F(2α) (PGF(2α)) and medroxyprogesterone acetate (MAP) increases the ovulation rate as a result of ovulations from the penultimate wave in addition to the final wave of the cycle. The objective of the current study was to evaluate the expression of markers of vascularization/angiogenesis, a marker of intercellular communication, and cellular proliferation and apoptosis in follicles from the penultimate and final waves. On day 8 of the estrous cycle, 15 ewes were administered a single injection of PGF(2α) and an intravaginal MAP sponge, which remained in place for 6 days. Two days after sponge removal, ovaries which contained follicles from the penultimate and final waves were collected and processed for immunohistochemistry followed by image analysis, and for quantitative real-time RT-PCR. Expression of factor VIII (marker of vascularization), proliferating cell nuclear antigen, and GJA1 (Cx43; marker of gap junctional communication) was greater (P<0.05) in follicles from the final wave compared with follicles from the penultimate wave. For theca cells, mRNA expression for vascular endothelial growth factor (VEGF) was greater (P<0.05) and tended to be greater (P≤0.1 and ≥0.05) for GJA1 and endothelial nitric oxide synthase in follicles from the final wave compared with follicles from the penultimate wave. For granulosa cells, the mRNA expression for GJA1 was greater (P<0.05) and tended to be greater (P≤0.1 and ≥0.05) for VEGF in follicles from the final wave compared with follicles from the penultimate wave. In conclusion, extension of the lifespan of follicles in the penultimate wave reduces follicular viability in the ewe.

LH pulse frequency and the emergence and growth of ovarian antral follicular waves in the ewe during the luteal phase of the estrous cycle

BackgroundIn the ewe, ovarian antral follicles emerge or grow from a pool of 2–3 mm follicles in a wave like pattern, reaching greater than or equal to 5 mm in diameter before regression or ovulation. There are 3 or 4 such follicular waves during each estrous cycle. Each wave is preceded by a peak in serum FSH concentrations. The role of pulsatile LH in ovarian antral follicular emergence and growth is unclear; therefore, the purpose of the present study was to further define this role.MethodsEwes (n = 7) were given 200 ng of GnRH (IV) every hour for 96 h from Day 7 of the estrous cycle, to increase LH pulse frequency. Controls (n = 6) received saline. In a second study, ewes (n = 6) received subcutaneous progesterone-releasing implants for 10 days starting on Day 4 of the cycle, to decrease LH pulse frequency. Controls (n = 6) underwent sham surgery. Daily transrectal ovarian ultrasonography and blood sampling was performed on all ewes from the day of estrus to the day of ovulation at the end of the cycle of the study. At appropriate times, additional blood samples were taken every 12 minutes for 6 h and 36 min or 6 h in studies 1 and 2 respectively.ResultsThe largest follicle of the follicular wave growing when GnRH treatment started, grew to a larger diameter than the equivalent wave in control ewes (P < 0.05). Mean serum estradiol and progesterone concentrations were higher but mean serum FSH concentrations were lower during GnRH treatment compared to control ewes (P < 0.05). The increased serum concentrations of estradiol and progesterone, in GnRH treated ewes, suppressed a peak in serum concentrations of FSH, causing a follicular wave to be missed. Treatment with progesterone decreased the frequency of LH pulses but did not have any influence on serum FSH concentrations or follicular waves.ConclusionWe concluded that waves of ovarian follicular growth can occur at LH pulse frequencies lower than those seen in the luteal phase of the estrous cycle but frequencies seen in the follicular phase, when applied during the mid-luteal phase, in the presence of progesterone, do enhance follicular growth to resemble an ovulatory follicle, blocking the emergence of the next wave.

Evaluation of the ultrasound image attributes of developing ovarian follicles in the four follicular waves of the interovulatory interval in ewes

Computer-assisted quantitative echotextural analysis was applied to ultrasound images of antral follicles in the follicular waves of an interovulatory interval in sheep. The ewe has three or four waves per cycle. Seven healthy, cyclic Western White Face ewes (Ovis aris) underwent daily, transrectal, ovarian ultrasonography for an interovulatory interval. Follicles in the third wave of the ovulatory interval had a longer static phase than that of those in Waves 1 and 2 (P<0.05). The numeric pixel value for the wall of anovulatory follicles emerging in the third wave of the cycle was significantly higher than that for Waves 1 and 2 at the time of emergence (156.7+/-8.09, 101.6+/-3.72, and 116.5+/-13.93, respectively), and it decreased as follicles in Wave 3 reached maximum follicular diameter (P<0.05). The numeric pixel value of the antrum in the ovulatory follicles decreased as follicular diameter increased to > or =5mm in diameter (P<0.05). The pixel heterogeneity of the follicular antrum in Wave 1 increased from the end of the growth phase to the end of the regression phase for follicles in that wave (P<0.05). The total area for the wall and antrum of the follicles studied were correlated with follicular diameter in all follicular waves (r=0.938, P<0.01 and r=0.941, P<0.01 for the wall and antrum, respectively). Changes in image attributes of the follicular wall and antrum indicate potential morphologic and functional differences among antral follicles emerging at different stages of the interovulatory interval in cyclic ewes.

Superovulation and embryo transfer in wood bison (Bison bison athabascae)

Two experiments were done to develop an effective superovulatory treatment protocol in wood bison for the purpose of embryo collection and transfer. In experiment 1, donor bison were assigned randomly to four treatment groups (N = 5 per group) to examine the effects of method of synchronization (follicular ablation vs. estradiol-progesterone treatment) and ovarian follicular superstimulation (single slow-release vs. split dose of FSH). Recipient bison were synchronized with donor bison by either follicular ablation (N = 8) or estradiol-progesterone treatment (N = 9). In experiment 2, bison were assigned randomly to four treatment groups (N = 5 per group) to examine the ovarian response to two versus four doses of FSH, and the effect of progesterone (ovarian superstimulation with or without an intravaginal progesterone-releasing device). Donor bison were inseminated with fresh chilled wood bison semen 12 and 24 hours after treatment with GnRH (experiment 1) or LH (experiment 2). The ovarian response was assessed using ultrasonography. In experiment 1, the number of large follicles (≥ 7 mm) increased in response to both FSH treatments, but the diameter of the largest follicle detected 4 and 5 days after the start of ovarian superstimulation was greater in bison treated with a single dose of FSH than in those treated with two doses (P < 0.05). A total of 10 ova and/or embryos were collected. One blastocyst was transferred to each of five recipient bison resulting in the birth of two live wood bison calves. In experiment 2, two doses of FSH resulted in a greater number of large follicles (≥ 9 mm) on Days 4, 5, and 6 (P < 0.05) after beginning of superstimulation (Day 0), and more ovulations than four doses of FSH (11.2 ± 2.4 vs. 6.4 ± 0.8; P < 0.05). Embryo collection was performed on only five donors, and a total of 19 ova and/or embryos were recovered. In summary, fewer FSH treatments were as good or better than multiple treatments, consistent with the notion that minimizing handling stress improves the superovulatory response in bison. Follicular ablation and estradiol plus progesterone treatment were effective for inducing ovarian synchronization in embryo donor and recipient bison, and an intravaginal progesterone-releasing device during superstimulatory treatment did not influence the superovulatory response or embryo collection. Delaying ovulation-inducing treatment (GnRH or LH) to 5 days after superstimulatory treatment resulted in a greater number of ovulations and improved embryo collection efficiency (experiment 2). Embryo collection and transfer resulted in live offspring from wild wood bison.

Characteristics of peaks in serum concentrations of follicle-stimulating hormone and estradiol, and follicular wave dynamics during the interovulatory interval in cyclic ewes.

There are three or four ovarian follicular waves in the interovulatory interval of cyclic ewes. Each follicular wave is preceded by a transient peak in serum follicle-stimulating hormone (FSH) concentrations. Serum concentrations of estradiol also increase concurrent with the growth of follicle(s) in each wave. In the current study, we investigated the patterns of follicular wave development and characteristics of FSH and estradiol peaks in all follicular waves of the interovulatory interval and after induction of a supraphysiologic FSH peak in cyclic ewes (Ovis aris). In Experiment 1, 19 ewes underwent daily ovarian ultrasonography and blood sampling for a complete interovulatory interval. In Experiment 2, seven ewes received two administrations of ovine FSH (oFSH), 8h apart (1 microg/kg; sc), at the expected time of the endogenous FSH peak preceding the second follicular wave of the interovulatory interval. In Experiment 1, the amplitude of the FSH peaks decreased (up to 50%), whereas basal serum FSH concentrations increased across the interovulatory interval (P<0.05). Maximum follicular diameter was greater (P<0.05) for Wave 1 and the Ovulatory wave (6.0+/-0.3 and 6.1+/-0.2 mm, respectively) than for Waves 2 and 3 (5.3+/-0.1 and 5.4+/-0.3 mm, respectively). Life span was greater for follicles in Wave 1 compared with other waves (P<0.05). Treatment with oFSH increased the amplitude of an FSH peak by 5- to 6-fold. This treatment increased estradiol production (P<0.05) but had little effect on other characteristics of the subsequent follicular wave. We concluded that changes in the amplitude and duration of the peaks in serum concentrations of FSH that precede follicular waves across the interovulatory interval do not influence the characteristics of the follicular waves that follow.

Pulsed GnRH secretion and the FSH secretory peaks that initiate ovarian antral follicular wave emergence in anestrous ewes.

In ewes, immunization against GnRH blocks LH pulses but mean serum FSH concentrations are only partly reduced; the fate of the FSH peaks that precede ovarian follicular waves has not been studied. In this study, we used immunization against GnRH to examine the need for pulsed GnRH secretion in the genesis of FSH peaks in the anestrous ewe. Six anestrous ewes were given a GnRH immunogen on Day 0 and a booster injection on Day 28. Control ewes (n=6) received adjuvant only. Transrectal ultrasonography was performed daily for 2 days prior to and 10 days following both the primary (Days -2 to 10) and booster (Days 26-38) injections and for a 13-day period beginning 26 days after booster injection (Days 54-66). Blood samples were collected daily. Intensive bleeding (every 12min for 7h) was performed on Days 9, 37, and 65 of the experimental period to characterize the pulsatile pattern of LH secretion. GnRH antibody titers were increased and LH pulses were abolished immediately after booster immunization (P<0.05). The number of FSH peaks, FSH peak concentration and amplitude and basal FSH concentrations were only decreased in immunized ewes in the period of observations starting 26 days after booster immunization (P<0.05); however, some peaks were still seen. The number of follicular waves was decreased in the period around booster immunization and no follicular waves were seen during the period starting 26 days after booster immunization in immunized ewes (P<0.05). In summary, in anestrous ewes, when pulsed LH secretion was abolished by immunization against GnRH, the peaks in serum concentrations of FSH that trigger ovarian follicular waves continued for a period of time. We concluded that although blocking the effects of GnRH gradually causes a diminution of FSH secretion, there is no acute requirement for GnRH in the regulation of FSH peaks. The existence of FSH peaks in the absence of follicular waves, and pulsed LH secretion, suggests that some endogenous rhythm may drive the occurrence of FSH peaks, independent of both changes in negative feedback by secretory products from ovarian antral follicles and GnRH.

Effects of pipothiazine palmitate on handling stress and on the characteristics of semen collected by electroejaculation in bison (Bison bison) bulls

Handling North American bison can pose risk to the handler and evoke stress in the animal. Moreover, this induced stress might affect qualities of semen collected by electroejaculation. The objective of this study was to investigate if a long acting neuroleptic tranquilizer (LAN) would reduce the stress of bison and thereby improve the quality of electroejaculated semen. Eight experimental replicates were conducted between May and November. In each replicate, the same six bison bulls were randomly assigned into LAN-treated (n=3) and non-treated control (n=3) groups. Pipothiazine palmitate (Piportil L4) was administered intramuscularly as a single dose of 100 mg in replicates 1-4 or 200 mg in replicates 5-8. Within each replicate, semen was collected by electroejaculation at 4, 6, 11 and 13 days post treatment. Behavioral parameters, sperm morphology and motility parameters were analyzed. A blood sample was collected before each electroejaculation and serum concentrations of testosterone, cortisol and corticosterone were determined. Treatment bulls with 100 mg of Piportil L4 reduced the restraint time and the struggling of bison bulls during handling compared to the control group (P<0.05). Semen motility parameters and serum concentrations of testosterone, cortisol and corticosterone were not significantly affected when 100mg of the LAN was administered (P>0.05). However, giving 200 mg of Piportil L4 reduced the restraint time of bison bulls and the duration of semen collection (P<0.05). Also, this treatment improved total and progressive sperm motilities when compared to the respective controls (P<0.05). Interestingly, serum concentration of corticosterone, as an endocrine stress indicator, was decreased after administration of 200mg of Pipothiazine palmitate, while testosterone concentrations were increased compared to those values in untreated control bulls (corticosterone: 0.10±0.01 compared with 0.15±0.02 ng/mL; testosterone: 9.11±1.68 compared with 5.33±0.74 ng/mL; P<0.05). In conclusion, this study demonstrated that a treatment dose of 200mg of Piportil L4 can decrease the behavioral and endocrine stress responses in bison bulls, which indirectly increasing testosterone concentrations and improving semen quality.

Effects of the Rate and Duration of Physiological Increases in Serum FSH Concentrations on Emergence of Follicular Waves in Cyclic Ewes

Large antral follicles grow in waves in the ewe, with each wave triggered by a peak in serum FSH concentrations. In this study, our objectives were to determine if the slope of the rise in the FSH peak affects the ability of the peak to trigger wave emergence (experiment 1), and whether increasing serum FSH concentrations and holding them at peak concentrations would provide a stimulus for constant emergence of large antral follicles (experiment 2). In experiment 1, cyclic ewes received ovine FSH (n = 6; 0.1 μg/kg, s.c.) or vehicle (n = 6; control) every 6 h for 42 h. This treatment created a peak in serum FSH concentrations (P < 0.05) during the early growth phase of the first follicular wave of the interovulatory interval and enhanced the growth of follicles in that wave (P < 0.05), but did not trigger emergence of a follicular wave. In experiment 2, cyclic ewes were infused constantly with oFSH (1.98 μg/h; n = 6) or vehicle (control; n = 6) for 60 h starting at the time of the second endogenously driven FSH peak of the interovulatory interval. Infusion of oFSH resulted in a super-stimulatory effect, with a peak in the mean number of large follicles (≥5 mm) on Day 2 after the start of FSH infusion (13 ± 1.2 large follicles per ewe, 1.8 ± 0.2 in control ewes; P < 0.001). In conclusion, exposing early growing antral follicles in a wave to a gradual increase in serum concentrations of FSH enhanced their growth, but did not trigger the expected new follicular wave, and infusion of a dose of oFSH within the physiological range caused a super-ovulatory response in cyclic ewes.

Pulsatile LH secretion and ovarian follicular wave emergence and growth in anestrous ewes

The objective of this study was to determine if pulsatile LH secretion was needed for ovarian follicular wave emergence and growth in the anestrous ewe. In Experiment 1, ewes were either large or small (10 x 0.47 or 5 x 0.47 cm, respectively; n = 5/group) sc implants releasing estradiol-17 beta for 10 d (Day 0 = day of implant insertion), to suppress pulsed LH secretion, but not FSH secretion. Five sham-operated control ewes received no implants. In Experiment 2, 12 ewes received large estradiol-releasing implants for 12 d (Day 0 = day of implant insertion); six were given GnRH (200 ng IV) every 4 h for the last 6 d that the implants were in place (to reinitiate pulsed LH secretion) whereas six Control ewes were given saline. Ovarian ultrasonography and blood sampling were done daily; blood samples were also taken every 12 min for 6 h on Days 5 and 9, and on Days 6 and 12 of the treatment period in Experiments 1 and 2, respectively. Treatment with estradiol blocked pulsatile LH secretion (P < 0.001). In Experiment 1, implant treatment halted follicular wave emergence between Days 2 and 10. In Experiment 2, follicular waves were suppressed during treatment with estradiol, but resumed following GnRH treatment. In both experiments, the range of peaks in serum FSH concentrations that preceded and triggered follicular wave emergence was almost the same as control ewes and those given estradiol implants alone or with GnRH; mean concentrations did not differ (P < 0.05). We concluded that some level of pulsatile LH secretion was required for the emergence of follicular waves that were triggered by peaks in serum FSH concentrations in the anestrous ewe.