Day Of Follicular Wave Emergence Research Articles

Early resynchronization of follicular wave emergence among Nelore cattle using injectable and intravaginal progesterone for three timed artificial inseminations

Follicular dynamics and pregnancy per AI (P/AI) of cattle submitted to resynchronization 13 d after TAI using a progesterone (P4) insert and supplementary injectable progesterone (iP4) were determined. There was synchronization of ovulation timing imposed (Day 0 = expected estrus; Experiment 1). On Day 13, animals were assigned to: control (only P4 insert; 15 cows and 13 heifers) and iP4 (P4 insert+100 mg iP4; 13 cows and 12 heifers) groups, and submitted to daily ovarian ultrasonography and blood collection (Day 13 to Day 22). In Experiment 2, 760 suckled cows and 498 heifers were submitted to a TAI on Day 0 and assigned into control and iP4 groups on Day 13. In animals with luteolysis on Day 22, there was a second TAI on Day 24, and on Day 37 were subjected to the opposite treatments as imposed in first resynchronization procedure. On Day 37, there was pregnancy diagnosis in animals with functional CL. The third TAI was performed on Day 48. Day of follicular wave emergence did not differ between groups and parities. Dominant follicles were larger in cows than heifers, and in animals of the control group on Day 24. Greater P4 concentrations were detected on Day 14 and Day 15 in the iP4-treated animals. Luteolysis occurred earlier in cows than heifers. Overall P/AI percentage as a result of second and third TAIs, regardless of parity, was greater in iP4-treated animals. In conclusion, females treated with a supplementary iP4 had a greater plasma P4 concentration and P/AI, but there was no effect of iP4 treatment on synchrony of timing of follicular wave.

Repeatability of antral follicle counts and anti-Müllerian hormone and their associations determined at an unknown stage of follicular growth and an expected day of follicular wave emergence in dairy cows

The objectives were to determine (1) the repeatability of antral follicle counts (AFC) and anti-Müllerian hormone (AMH) concentrations at an unknown stage of follicular growth (FG) and at an expected day of follicular wave emergence (FWE) in dairy cows, and (2) the association between AFC and AMH at the above two stages. Transrectal ultrasound imaging of the ovaries and blood sample collection were performed on 91 lactating Holstein cows (33 primiparous, 58 multiparous) to determine AFC and serum concentrations of AMH at an unknown stage of FG (mean ± SEM; 14 ± 0.5 d postpartum) and an expected day of FWE (mean ± SEM; 73 ± 0.5 d postpartum, approximately 36 h after the 2nd GnRH of Ovsynch protocol). The AFC ranged from 10 to 53 (mean ± SEM; 26.1 ± 1.0) and 6 to 45 (mean ± SEM; 23.4 ± 1.0) at an unknown stage of FG and expected day of FWE, respectively (P = 0.02). Serum concentrations (pg/mL) of AMH ranged from 13.9 to 528.8 (mean ± SEM; 187.3 ± 13.1) and 38.2 to 774.1 (mean ± SEM; 218.7 ± 14.5) at an unknown stage of FG and expected day of FWE, respectively (P < 0.01). The repeatability estimates for AFC and serum AMH concentrations between an unknown stage of FG and the expected day of FWE were 0.37 and 0.73 (P < 0.01), respectively. The correlation between AFC and AMH was moderate at an unknown stage of FG (r = 0.54; P < 0.01) and at an expected day of FWE (r = 0.59; P < 0.01). In summary, both AFC and AMH were repeatable when measured at an unknown stage of FG and an expected day of FWE, but the repeatability was greater for AMH. Thus, if cows are tested at an unknown stage of FG, serum AMH may be a more reliable phenotype than AFC to test association with reproductive outcomes. Furthermore, AFC and AMH were moderately correlated at the two stages examined.

Study of two strategies to induce follicular wave emergence for assisted reproductive treatments (ART)-a preliminary trial.

This study aimed to induce follicular wave emergence (FWE) using pharmacological (recombinant hCG administration) or mechanical (aspiration of dominant follicle) interventions in infertile women. Sixteen infertile women (≤35 years) with indications for in vitro fertilization due to tubal and/or male factor infertility were randomized into three groups: control (n = 6), pharmacological (n = 5) and mechanical (n = 5) groups. Women in both experimental groups underwent serial transvaginal sonograms (TVS) from menstrual cycle day 10 until identification of a dominant follicle ≥15 mm. Women in the pharmacological group received 250 μg of recombinant-hCG to induce ovulation, and resumed serial TVS 2 days later. In the mechanical group, dominant and subordinate follicles ≥10 mm were aspirated, and daily TVS was resumed on the following day. An increased pool of follicles ≥5 and ≤9 mm after interventions characterized FWE. Women in the control group underwent ovulation induction (OI) with 150 IU/day of recombinant follicle-stimulating hormone started on menstrual cycle day 3 (D3). OI was started on the day of FWE in the experimental groups. Endometrial asynchrony with development of the embryo was expected in the experimental groups. Therefore, all viable embryos were cryopreserved and transferred in an endometrial-stimulated cycle. The number of follicles ≥5 and ≤9 mm increased after the interventions in both experimental groups (p < .001), indicating induction of FWE. OI outcomes were similar among the groups. The pharmacological and mechanical interventions are efficient in inducing FWE; outcomes of OI synchronized with FWE should be further investigated.

248 IN VITRO EMBRYO PRODUCTION USING IN VIVO-MATURED OOCYTES COLLECTED TRANSVAGINALLY FROM WOOD BISON (BISON BISON ATHABASCAE)

Reproductive technologies are being developed to help conserve the genetic diversity of wood bison, a threatened species. To date, the efficiency of in vitro embryo production in bison is very low and appears to be related to inadequate in vitro conditions for oocyte maturation. Recently, we have attempted to circumvent the problem by inducing oocyte maturation in vivo and found that more than one-third of superstimulated oocytes collected 30 h after administration of hCG were at metaphase II (Cervantes et al. 2013 Reprod. Fertil. Dev. 25, 283; Cervantes et al. 2014 Reprod. Fertil. Dev. 26, 199). We hypothesise that additional maturation time in vitro, after in vivo maturation, will allow the remaining oocytes to reach the MII stage, and thus improve in vitro embryo production in wood bison. The objective of this study was to determine the effect of an additional 4 h of in vitro maturation on the developmental competence of oocytes collected 30 h after hCG treatment. Wood bison cows (n = 24) were superstimulated by the administration of 300 mg of FSH (Folltropin-V) diluted in 0.05% hyaluronan on the day of follicular wave emergence and 100 mg of FSH in hyaluronan 2 days later. Bison were administered 2500 IU of hCG (Chorulon) IM 2 days after the last dose of FSH. Transvaginal ultrasound-guided follicle aspiration was performed 30 h after hCG treatment to collect cumulus-oocyte complexes (COC). Expanded COC (with no evidence of degeneration) were selected and assigned randomly to 2 groups (n = 38 COC/group) in which IVF was done immediately, or after 4 h of in vitro maturation in TCM 199 with 5% calf serum, 5 μg mL–1 pLH, 0.5 μg mL–1 pFSH, and 0.05 μg mL–1 gentamicin, at 38.5°C, 5% CO2 and high humidity. In vitro fertilization (Day 0) was done with frozen-thawed wood bison semen (dose 5 × 106 sperm mL–1) in Brackett-Oliphant medium at 38.5°C, 5% CO2, and high humidity. Presumptive zygotes were cultured in CR1aa plus 5% calf serum, at 38.5°C and in 5% CO2, 5% O2, and 90% N2 and high humidity. Cleavage was recorded on Day 3, and blastocyst formation was recorded on Days 7 and 8. Cleavage and blastocyst rates (calculated from the total number of oocytes submitted to IVF) were compared between groups by chi-square analysis. No difference was detected between groups (immediate fertilization v. after an additional 4 h in vitro) in cleavage rate on Day 3 (55.3 v. 60.5%, respectively, P = 0.82), or blastocyst rate on Day 7 (13.2 v. 23.7%, respectively, P = 0.37). However, the blastocyst rate on Day 8 was higher in the COC group exposed to an additional 4 h of in vitro maturation (18.4 v. 44.7%, respectively, P = 0.03). Results support the hypothesis that an additional short period of in vitro maturation improves the developmental competence of oocytes collected after 30 h of in vivo maturation.We thank Bioniche Animal Health for providing FSH (Folltropin-V) and hyaluronan (MAP-5), and Merck Animal Health for hCG (Chorulon).

Evidence that the dominant follicle of the first wave is more active than that of the second wave in terms of its growth rate, blood flow supply and steroidogenic capacity in cows

To clarify the different characteristics of the dominant follicle (DF), the DF in first ovarian follicular wave (DF-1) after spontaneous ovulation and DF in second follicular wave (DF-2) and after induced ovulation of the first-wave DF by GnRH were examined in non-lactating Holstein cows. Follicular maturation of DF-1 and DF-2 were induced by PGF2α and GnRH treatment on Day 6 and 8 (Day 0=Day of follicular wave emergence), respectively. Follicular growth and blood flow (BF) in the follicular wall of DF-1 and DF-2 were examined. To analyze sex steroids in follicular fluid (FF) and amount of mRNA in granulosa cells, DF-1 and DF-2 were aspirated on Day 8 or 9 in different estrous cycle. Diameter in DF-1 was larger than DF-2 on Day 8 and 9. From Day 8 to 9, BF area (BFA) and percentage of the follicular wall with BF, which represents the degree of distribution of BF, increased in DF-1 but not in DF-2. BFA per length of follicle circumference with BF, which represents the thickness of BF, was not different between DF-1 and DF-2. Concentrations of 17β-estradiol (E2) in plasma, E2 and androstendione in FF and amounts of LH receptor mRNA were greater in the DF-1 on Day 8. Gene expression for steroidogenesis, prostaglandin synthesis and angiogenesis did not differ between DF-1 and DF-2. These results indicated that DF-1 were more active than DF-2 in growth, BF supply and steroidogenesis. The greater BFA observed in the DF-1 may be derived from as a result of the greater vascularity in the follicular wall.

Effect of purified llama ovulation-inducing factor (OIF) on ovarian function in cattle

Two experiments were designed to determine the effect of purified ovulation inducing factor (OIF) on ovarian function in cattle. In Experiment 1, prepubertal heifers (n = 11 per group) were treated on Day 5 (Day 0 = day of follicular wave emergence) of the follicular wave with an intramuscular dose of saline (1 mL), GnRH (100 μg), or purified OIF (1 mg/100 kg body weight). Ovulation occurred in 9/11 heifers treated with GnRH, and 1/11 heifers in each of the OIF- and saline-treated groups (P < 0.05). Compared to saline-treated controls, OIF treatment was associated with a smaller dominant follicle diameter (P < 0.01), a rise in plasma FSH concentration (P < 0.1), and earlier emergence of the next follicular wave (P < 0.05). In Experiment 2, sexually mature heifers were given either GnRH or purified OIF on Days 3, 6 or 9 of the first follicular wave (i.e., early growing, early static, or late static phase of the dominant follicle; n = 5 per group per day), or were untreated (n = 10). In heifers treated with OIF on Day 6, the dominant follicle diameter profile tended to be smaller than in controls, and was associated with a rise (P < 0.05) in plasma FSH concentrations. A similar rise in FSH was detected after OIF treatment on Day 9. Compared to untreated controls, treatment with OIF and GnRH was associated with a larger CL diameter (Days 3 and 6 groups; P < 0.05) and a greater concentration of plasma progesterone (Days 6 and 9 groups; P < 0.05). Treatment with purified OIF did not induce ovulation in heifers, but hastened new follicular wave emergence in prepubertal heifers, influenced follicular dynamics in a phase-specific manner in mature heifers, and was luteotrophic.

321 OVARIAN SUPERSTIMULATION AND OOCYTE COLLECTION IN WOOD BISON, A THREATENED CANADIAN SPECIES

The wood bison (Bison bison athabascae) is a threatened species, and recovery is constrained by endemic tuberculosis and brucellosis in wild herds in and around Wood Buffalo National Park, Alberta, Canada. The Committee on the Status of Endangered Wildlife in Canada has recommended the eradication of affected herds and repopulation with healthy bison obtained through the use of reproductive technologies. As part of the conservation effort, the specific objective of this study was to develop an effective ovarian superstimulatory protocol in bison that will permit collection of a large number of oocytes for the ultimate purpose of in vitro embryo production. Ovarian function was synchronized among female wood bison during the ovulatory season (December) by giving a luteolytic dose of prostaglandin followed 8 days later by transvaginal ultrasound-guided follicular ablation. On the day after follicular ablation (expected day of follicular wave emergence, Day 0), bison were assigned randomly to 2 groups (n = 11/group) and given either a single intramuscular dose of 2500 IU of eCG or subcutaneous doses of 200 mg of FSH given on Days 0 and 2. A luteolytic dose of prostaglandin was given on Day 2, and 25 mg of LH was given intramuscularly on Day 4. Cumulus–oocyte complexes (COC) were collected 24 h after LH treatment by transvaginal ultrasound-guided follicle aspiration using an 18 G × 2’ aspiration needle attached to a flexible tubing and a regulated vacuum pump (flow rate, 22 mL min–1). The COC were classified morphologically from 1 (excellent) to 4 (poor) based on attributes of the ooplasm and the number of surrounding cumulus cells. The number of COC in grade 1 to 3 was combined (i.e. grades acceptable for IVF) for purposes of statistical interpretation. Ovarian response and oocyte quality were compared between groups by t-tests, and COC collection rate was compared by chi-square analysis. The number (mean ± SEM) of follicles ≥5 mm was greater (P &lt; 0.05) in bison treated with FSH than in those treated with eCG (12.2 ± 1.7 v. 5.8 ± 0.5), resulting in a greater number of follicles aspirated (11.2 ± 1.8 v. 5.6 ± 0.5; P &lt; 0.05), more COC collected (7.2 ± 1.4 v. 3.4 ± 0.6; P &lt; 0.05), and more COC of acceptable quality (6.1 ± 1.4 v. 2.5 ± 0.5). The COC collection rate (i.e. number of COC collected per number of follicles aspirated), however, did not differ between FSH- and eCG-treated groups [79/123 (64%) v. 37/62 (60%)]. The results document the successful collection of viable oocytes from superstimulated wood bison via tranvaginal ultrasound-guided follicle aspiration. The number of large follicles, follicles aspirated, and COC collected were more than twice as high in bison treated with FSH than in those treated with eCG. This work was supported by grants from Advancing Canadian Agriculture and Agri-Food Fund and Agri-Food Innovation Fund. The authors thank Bioniche Animal Health Canada for providing Folltropin, Lutropin, and Pregnecol.

Effect of estradiol benzoate used at the start of a progestagen treatment on superovulatory response and embryo yield in lactating and non-lactating llamas

Two experiments were conducted to determine the effect of estradiol benzoate (EB) and intravaginal progestagen treatment on ovarian follicular dynamics and superstimulatory response in eCG-treated llamas. The purpose of Experiment 1 was to evaluate the effect of EB and progestagen treatment starting at different phases of dominant follicle (DF1) development on regression pattern and subsequent follicle wave emergence (WE2) in lactating and non-lactating llamas. Early lactating ( n = 24, 30 ± 4 days postpartum) and non-lactating ( n = 24) females were assigned in equal numbers ( n = 8) to one of three groups according to the phase of DF1 (growing, static or regressing) determined by ultrasonography from day −3 to day 0. At day 0, llamas received an intravaginal sponge (MPA, 150 mg) and 5 mg of MPA (i.m.). Half of the females ( n = 4) of each group were injected with 2 mg (i.m.) of EB and half were not (control group). A 2 × 2 × 3 (lactational status, EB treatment and follicular phases) factorial design was used. Each sponge was removed 8 days later. Ovaries were monitored from day 0 to day 12. Daily blood samples were taken to determine 17β-estradiol (E 2) profiles from day 0 to day 8. The DF1 regression pattern was not affected ( P > 0.05) by the phase of follicle wave at the start of the treatment or any interactions among main effects. Follicle wave emergence in EB-treated llamas was delayed ( P < 0.05) by 2.3 days compared with non-treated llamas. Following EB treatment, plasma concentrations of E 2 were greater ( P < 0.05) from day 1 to day 5 in the treated than in non-treated females, but not from day 6 onward ( P > 0.05). Experiment 2 was designed to evaluate the effect of this treatment on the ovarian superovulatory response and embryo yield following eCG treatment administered on day of follicular wave emergence as determined in the Experiment 1. The same lactating ( n = 18, 61 ± 4 days postpartum) and non-lactating ( n = 18) llamas at random stages of follicle wave were treated as those in Experiment 1 and received 1200 IU of eCG at the time of WE2 (EB-treated = day 6.5 and non-treated = day 4.5). Llamas were mated 5 days after sponge withdrawal. A second mating was allowed 24 h later. Embryos were collected between 7 and 8 days after the first mating and blood samples were taken to determine progesterone (P 4) concentrations. The mean number of follicles on day of mating and the number of CL on day of embryo collection were not affected by lactational status, EB treatment or their interactions ( P > 0.05). Ovulation rate and mean (±SEM) number of recovered embryos for EB treatment group (67.5% and 2.4 ± 0.4) were greater ( P < 0.05) than for no EB treatment (51.1% and 1.1 ± 0.4). Plasma P 4 concentrations and number of CL per llama were correlated ( r = 0.49; P = 0.014). In conclusion, progestagen plus EB treatment facilitates the prediction of the emergence of a new follicular wave approximately 6 days after treatment and resulted in a higher ovulation rate and embryo production in ovarian superstimulated llamas regardless of lactational status.

Ovarian Follicular Dynamics and Hormonal Changes in Goats during Early Pregnancy

The present study was conducted to characterize follicular development and its hormonal control during early pregnancy in goats. The ovaries of goats (n=8) were scanned daily for follicles (> or = 2 mm in diameter) and corpora lutea by transrectal ultrasound with blood sampling from the jugular vein for monitoring the hormonal changes during the first thirty-five days after mating. During early pregnancy, three (37.5%), four (50%) and one (12.5%) goat showed nine, eight and seven waves of follicular development, respectively. The corpora lutea were detected as early as Day 3.61 ± 0.45 (7.47 ± 0.43 mm) of pregnancy (Day 0=day of mating) and attained their maximal cross-sectional diameter (10.64 ± 0.37 mm) on Day 25.7 ± 0.8 of pregnancy, respectively. A transient rise in FSH levels was temporally associated with the day of follicular wave emergence (up to three days prior to wave emergence). The plasma LH and estradiol levels were negatively correlated with the progesterone concentration. The rise in plasma immunoreactive (ir) inhibin levels was negatively correlated with the FSH concentration and positively correlated with the number of large-sized follicles. Alternatively, the mean plasma ir-inhibin levels showed a noticeable decline with the progression of pregnancy. The present results demonstrated that follicular development during early pregnancy shows a wave-like pattern, with seven to nine waves developing until Day 35 after breeding, and that the number of follicular waves can be predicted by the number of FSH peaks. The current study also demonstrated that the role of inhibin as an FSH regulator is maintained throughout early pregnancy.

27 FOLLICULAR RESPONSE TO DIFFERENT eCG DOSES IN AN OVSYNCH + PROGESTERONE PROTOCOL IN BEEF HEIFERS

An experiment was designed to evaluate the effect of different doses of eCG on ovarian follicular dynamics in heifers treated with a Ovsynch plus progesterone protocol. Twenty-five cyclic yearling Black Angus heifers (373.0 ± 35.7 kg), in 2 replicates, received an injection of 100 μg of GnRH (Ovurelin, Bomac Laboratories Ltd., Auckland, New Zealand) i.m. and an intravaginal progesterone device (1.38 g of progesterone; Eazi-Breed CIDR, Pfizer Animal Health, New Zealand) on Day 0 (beginning of the experiment), followed by 500 μg of cloprostenol (PGF, Estrumate, Intervet/Schering-Plough Animal Health, Auckland, New Zealand) i.m. on Day 7, and a second 100 μg of GnRH injection given i.m. on Day 9 (56 h after PGF). At the time of PGF treatment, heifers were randomly assigned to 5 treatment groups to receive 0, 300, 500, 700, or 1000 IU of eCG (Folligon, Intervet/Schering-Plough Animal Health) i.m. Heifers were monitored by ultrasonography (Aloka 900-SSD equipped with a 7.5-MHz linear-array transducer; Aloka, Tokyo, Japan) daily from Day 0 to 9 (GnRH), and then every 12 h until ovulation. Data were analyzed by one-way ANOVA or Kruskall-Wallis test, and means or ranks were compared with LSD or Wilcoxon rank sum tests, respectively. Because a replicate effect was observed (P &lt; 0.05) in the size of the dominant follicle at the second GnRH and prior to ovulation, replicate effect was included in the analysis. The luteal area at PGF treatment was significantly greater (P &lt; 0.01) in heifers that ovulated (750.0 ± 97 mm2) in response to the first GnRH treatment than in those that did not (301.6 ± 42.7 mm2). The diameter of the dominant follicle at the time of PGF treatment was also greater (P &lt; 0.05) in ovulating (11.2 ± 0.4 mm) than in nonovulating (9.7 ± 0.5 mm) heifers. The interval from the first GnRH treatment to the emergence of the next follicular wave was longer (P = 0.50) and more variable in heifers that did not ovulate (2.9 ± 0.4 d; n = 27) than in those that ovulated (1.9 ± 0.2 d; n = 23). There was no effect (P &lt; 0.37) of eCG on the interval from PGF to ovulation (86 ± 1.9 h). The number of ovulations after the second GnRH was higher (P = 0.01) in the group of heifers treated with 1000 IU of eCG (1.8 ± 0.4) than in the other groups (1.0 ± 0.0; 1.1 ± 0.1; 1.2 ± 0.1; 1.0 ± 0.1). There was an effect of day of follicular wave emergence on the number of ovulations (P &lt; 0.01). Heifers with a wave emerging 1 to 3 days after the first GnRH (n = 37), had one ovulation (1.0 ± 0.0), whereas heifers with a wave emerging on Day 4 (3 out of 4 heifers) and Days 5 to 7 (n = 9), ovulated 2 or more follicles. In summary, the multiple ovulation effect occurred when eCG was given to heifers with a follicular wave emerging on or after Day 4, and was potentiated when heifers received 1000 IU of eCG. Although the dose of eCG given at the time of PGF treatment in an Ovsynch program has a significant effect on follicular development, the time of emergence of the dominant follicle appeared to be more important in the ovulation of preovulatory follicle/s after the eCG and the second GnRH treatment.

The temporal relationship between patterns of LH and FSH secretion, and development of ovulatory-sized follicles during the mid- to late-luteal phase of sheep

The aim of the present study was to investigate the temporal relationship between the secretory pattern of serum LH and FSH concentrations and waves of ovarian antral follicles during the luteal phase of the estrous cycle in sheep. The growth pattern of ovarian antral follicles and CL were monitored by transrectal ultrasonography and gonadotropin concentrations were measured in blood samples collected every 12 min for 6 h/d from 7 to 14 d after ovulation. There were two follicular waves (penultimate and final waves of the cycle) emerging and growing during the period of intensive blood sampling. Mean and basal LH concentrations and LH pulse frequency increased ( P < 0.001) with decreasing progesterone concentration at the end of the cycle. Mean and basal FSH concentrations reached a peak ( P < 0.01) on the day of follicular wave emergence before declining to a nadir by 2 d after emergence. None of the parameters of pulsatile LH secretion varied significantly with either the emergence of the final follicular wave or with the end of the growth phase of the largest follicle of the penultimate wave of the cycle. However, mean and basal LH concentrations did increase ( P < 0.05) after the end of the growth phase of the largest follicle of the final follicular wave of the cycle. Furthermore, the end of the growth phase of the largest follicle of the final wave coincided with functional luteolysis. In summary, there was no abrupt or short-term change in pulsatile LH secretion in association with the emergence or growth of the largest follicle of a wave. We concluded that the emergence and growth of ovarian antral follicles in follicular waves do not require changes in LH secretion, but may involve changes in sensitivity of ovarian follicles to serum LH concentrations.

268 BOVINE AND EQUINE RECOMBINANT FOLLICLE-STIMULATING HORMONE PRODUCED IN TWO HETEROLOGOUS HOSTS: ESCHERICHIA COLI AND TRYPANOSOMA BRUCEI

Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein hormone consisting of � and � subunits produced by different genes. Following release from the anterior pituitary, FSH is responsible for development of ovarian follicles from the time of emergence of a follicular wave until divergence of the dominant follicle. Treatment of cattle and horses with recombinant FSH would be valuable for induction of superovulation in an effort to harvest multiple embryos for use in embryo transfer. Glycosylation of the subunits may be essential for proper function and receptor recognition, thereby complicating the production of recombinant FSH. Therefore, a eukaryotic, rather than a prokaryotic, host would be most beneficial for generation of recombinant protein. To explore recombinant protein synthesis, we utilized the ancient protozoan Trypanosoma brucei. Perhaps best known for its parasitic life style, T. brucei has been the focus of extensive biochemical and genetic research, with the nonpathogenic insect stage of the parasite serving as a model eukaryote for the study of many fundamental eukaryotic pathways, including glycosyl phosphatidylinositol (GPI) anchor biosynthesis and RNA editing. The ease of molecular manipulation of this protozoan, and the organism's robust glycosylation machinery, has led us to explore the possibility of using the non-pathogenic life stage for recombinant protein production. The objective of this study was to produce recombinant bovine and equine FSH in both prokaryotic and eukaryotic expression systems. Protein has been produced in Escherichia coli using the pQE30 expression system. Subunit genes were ligated into pQE30 and expressed in M15 E. coli cells. These cells were found to produce detectable amounts of the individual subunit protein after a three hour induction. To express recombinant protein in T. brucei, subunit genes were cloned into pLEW2T7(6His), a stably integrated vector designed to allow inducible expression of proteins fused to a C-terminal six-histidine tag. Clones were subsequently transformed into T. brucei and are awaiting protein expression. All protein purification will be performed via nickel affinity chromatography and protein concentration will be measured using radioimmunoassay. Bioactivity will be analyzed using a rat ovarian weight assay prior to use in cattle and horses. The purified protein will be administered as intramuscular injections twice daily for four days beginning on the day of follicular wave emergence. Producing recombinant FSH will be useful for the embryo transfer and food safety industries by increasing consistency of biologic activity, decreasing production costs, and, perhaps most importantly, decreasing the opportunity for zoonotic transmission of prions such as bovine spongiform encephalopathy.

Follicular and Hormonal Dynamics during the Estrous Cycle in Goats

Transrectal ultrasonography of ovaries was performed daily in 6 goats for 3 consecutive estrous cycles. Blood samples collected daily were measured for concentrations of FSH, inhibin A, and estradiol-17beta. Follicular and hormonal data were analyzed for associations between the follicular waves and hormonal concentrations. During the interovulatory intervals, follicular growth and regression occurred in a wave like pattern (2-5 waves), and the predominant patterns were three and four follicular waves. In addition, there was no significant difference among the diameters of dominant follicles during the growth phase of the follicular waves. The number of 3 mm follicles peaked on days 0, 7, and 11 in interovulatory intervals that had three follicular waves and on days -1, 5, 11, and 15 in those that had four follicular waves. Plasma concentrations of FSH increased around the day of follicular wave emergence and declined with the growth of follicles. Circulating FSH increased again concomitant with regression of dominant follicles in the anovulatory wave, whereas FSH levels remained low in the ovulatory wave. Inhibin A was negatively correlated with FSH, while it was positively correlated with estradiol-17beta, suggesting that inhibin A is a product of healthy growing follicles and that it contributes to the suppression of FSH secretion. In conclusion, the growth of ovarian follicles in goats exhibits a wave-like pattern, and follicular dominance is less apparent in goats. Moreover, inhibin A may be a key hormone for regulation of the follicular wave through suppression of FSH secretion in goats.

Effect of estradiol valerate on ovarian follicle dynamics and superovulatory response in progestin-treated cattle

Three experiments evaluated the effects of estradiol valerate (EV) on ovarian follicular and CL dynamics, intervals to estrus and ovulation, and superovulatory response in cattle. Experiment 1 compared the efficacy of two norgestomet ear implants (Crestar and Syncro-Mate B; SMB) for 9 d (with PGF at implant removal), combined with either 5 mg estradiol-17β and 100 mg progesterone (EP) or 5 mg EV and 3 mg norgestomet (EN) im at the time of implant insertion on CL diameter and follicular wave dynamics. Ovaries were monitored by ultrasonography. There was no effect of norgestomet implant. Diameter of the CL decreased following EN treatment ( P < 0.01). Mean (± S.D.) day of follicular wave emergence (FWE) was earlier ( P < 0.0001) and less variable ( P < 0.0001) in EP- (3.6 ± 0.5 d) than in EN- (5.7 ± 1.5 d) treated heifers. Intervals from implant removal to estrus ( P < 0.001) and ovulation ( P < 0.01) were shorter in EN- (45.7 ± 11.7 and 74.3 ± 12.6 h, respectively) than in EP- (56.4 ± 14.1 and 83.3 ± 17.0 h, respectively) treated heifers. Experiment 2 compared the efficacy of EP versus EN in synchronizing FWE for superovulation in SMB-implanted cows. At random stages of the estrous cycle, Holstein cows ( n = 78) received two SMB implants (Day 0) and were randomly assigned to receive EN on Day 0 or EP on Day 1. Folltropin-V treatments were initiated on the evening of Day 5, with PGF in the morning and evening of Day 8, when SMB were removed. Cows were inseminated after the onset of estrus and embryos were recovered 7 d later. Non-lactating cows had more CL (16.7 ± 11.3 versus 8.3 ± 4.9) and total ova/embryos (14.7 ± 9.5 versus 7.9 ± 4.6) than lactating cows ( P < 0.05). EP-treated cows tended ( P = 0.09) to yield more transferable embryos (5.6 ± 5.2) than EN-treated cows (4.0 ± 3.7). Experiment 3 compared the effect of dose of EV on ovarian follicle and CL growth profiles and synchrony of estrus and ovulation in CIDR-treated beef cows ( n = 43). At random stages of the estrous cycle (Day 0), cows received a CIDR and no further treatment (Control), or an injection of 1, 2, or 5 mg im of EV. On Day 7, CIDR were removed and cows received PGF. Follicular wave emergence occurred within 7 d in 7/10 Control cows and 31/32 EV-treated cows ( P < 0.05). In responding cows, interval from treatment to FWE was longer ( P < 0.05) in those treated with 5 mg EV (4.8 ± 1.2 d) than in those treated with 1 mg (3.2 ± 0.9 d) or 2 mg (3.4 ± 0.8 d) EV, while Control cows were intermediate (3.8 ± 2.0 d). Diameter of the dominant follicle was smaller ( P < 0.05) at CIDR removal and tended ( P = 0.08) to be smaller just prior to ovulation in the 5 mg EV group (8.5 ± 2.2 and 13.2 ± 0.6 mm, respectively) than in the Control (11.8 ± 4.6 and 15.5 ± 2.9 mm, respectively) or 1 mg EV (11.7 ± 2.5 and 15.1 ± 2.2 mm, respectively) groups, with the 2 mg EV group (10.7 ± 1.5 and 14.3 ± 1.7 mm, respectively) intermediate. Diameter of the dominant follicle at CIDR removal was less variable ( P < 0.01) in the 2 and 5 mg EV groups than in the Control group, and intermediate in the 1 mg EV group. In summary, treatment with 5 mg EV resulted in a longer and more variable interval to follicular wave emergence than treatment with 5 mg estradiol-17β, which affected preovulatory dominant follicle size following progestin removal, and may have also affected superstimulatory response in Holstein cows. Additionally, 5 mg EV appeared to induce luteolysis in heifers, reducing the interval to ovulation following norgestomet removal. Conversely, intervals to, and synchrony of, follicular wave emergence, estrus and ovulation following treatment with 1 or 2 mg EV suggested that reduced doses of EV may be more useful for the synchronization of follicular wave emergence in progestogen-treated cattle.

Effects of estradiol cypionate (ECP) on ovarian follicular dynamics, synchrony of ovulation, and fertility in CIDR-based, fixed-time AI programs in beef heifers

Estradiol cypionate (ECP) was used in beef heifers receiving a controlled internal drug release (CIDR; insertion=Day 0) device for fixed-time AI (FTAI) in four experiments. In Experiment 1, heifers ( n=24) received 1 mg ECP or 1 mg ECP plus 50 mg commercial progesterone (CP) preparation i.m. on Day 0. Eight or 9 days later, CIDR were removed, PGF was administered and heifers were allocated to receive 0.5 mg ECP i.m. concurrently (ECP0) or 24 h later (ECP24). There was no effect of treatment ( P=0.6) on mean (±S.E.M.) day of follicular wave emergence (3.9±0.4 days). Interval from CIDR removal to ovulation was affected ( P<0.05) only by duration of CIDR treatment (88.3±3.8 h versus 76.4±4.1 h; 8 days versus 9 days, respectively). In Experiment 2, 58 heifers received 100 mg progesterone and either 5 mg estradiol-17β or 1 mg ECP i.m. (E-17β and ECP groups, respectively) on Day 0. Seven (E-17β group) or 9 days (ECP group) later, CIDR were removed, PGF was administered and heifers received ECP (as in Experiment 1) or 1 mg EB 24 h after CIDR removal, with FTAI 58–60 h after CIDR removal. Follicular wave emergence was later ( P<0.02) and more variable ( P<0.002) in heifers given ECP than in those given E-17β (4.1±0.4 days versus 3.3±0.1 days), but pregnancy rate was unaffected (overall, 69%; P=0.2). In Experiment 3, 30 heifers received a CIDR device and 5 mg E-17β, with or without 100 mg progesterone (P) i.m. on Day 0. On Day 7, CIDR were removed and heifers received ECP as described in Experiment 1 or no estradiol (Control). Intervals from CIDR removal to ovulation were shorter ( P<0.05) in ECP0 (81.6±5.0 h) and ECP24 (86.4±3.5 h) groups than in the Control group (98.4±5.6 h). In Experiment 4, heifers ( n=300) received a CIDR device, E-17β, P, and PGF (as in Experiment 3) and after CIDR removal were allocated to three groups (as in Experiment 2), with FTAI 54–56 h (ECP0) or 56–58 h (ECP24 and EB24) after CIDR removal. Pregnancy rate did not differ among groups (overall, 63.6%, P=0.96). In summary, although 1 mg ECP (with or without progesterone) was less efficacious than 5 mg E-17β plus 100 mg progesterone for synchronizing follicular wave emergence, 0.5 mg ECP (at CIDR removal or 24 h later) induced a synchronous ovulation with an acceptable pregnancy rate to fixed-time AI.

Ovarian Dynamics and Their Associations with Peripheral Concentrations of Gonadotropins, Ovarian Steroids, and Inhibin During the Estrous Cycle in Goats1

Ovarian changes determined by daily transrectal ultrasound and its relationship with FSH, LH, estradiol-17beta, progesterone, and inhibin were investigated in six goats for three consecutive interovulatory intervals. Estrous cycles were synchronized using two injections of prostaglandin F2alpha analogue 11 days apart. All follicles 3 mm or greater in diameter and corpora lutea were measured daily. A follicular wave was defined as one or more follicles growing to 5 mm or greater in diameter. The day that the follicles reached 3 mm in diameter was defined as the day of wave emergence, and the first wave after ovulation was defined as wave 1. During the interovulatory interval (mean +/- SEM, 21.3 +/- 0.4 days; n = 18), follicular waves emerged at 0.3 +/- 0.5, 6.5 +/- 0.2, and 12.1 +/- 0.4 days for wave 1, wave 2, and wave 3, respectively, in goats with three waves of follicular development and at -0.6 +/- 0.3, 4.7 +/- 0.2, 9.4 +/- 0.5, and 13.4 +/- 0.5 days for wave 1, wave 2, wave 3, and wave 4, respectively, in goats with four waves of follicular development (Day 0 = the day of ovulation). The mean diameter of the largest follicle of the ovulatory wave was significantly larger than those of the largest follicles of the other waves. Corpora lutea could be identified ultrasonically at Day 3 postovulation and attained 12.1 +/- 0.3 mm in diameter on Day 8. Transient increases in plasma concentrations of FSH were detected around the day of follicular wave emergence. The level of FSH was negatively correlated with that of inhibin. These results demonstrated that follicular waves occurred in goats and that the predominant follicular wave pattern was four waves with ovulation from wave 4. These results also suggested that the emergence of follicular waves was closely associated with increased secretion of FSH.

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 (P 4) 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 P 4, 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 P 4 im, or aspiration and 200 mg of P 4 im (aspiration + P 4). On Day 11, PGF 2α(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 P 4 group (P<0.05). Day of follicular wave emergence was delayed in the cows of the P 4 group compared with cows in the aspiration and aspiration + P 4 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 P 4 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 P 4 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.

Ovarian follicular dynamics during anoestrus in ewes.

The aim of the present study was to document ovarian antral follicle dynamics throughout seasonal anoestrus in sheep. Daily transrectal ultrasonography was performed during four 17 day scanning periods from March to July in Western White-faced crossbred ewes. Blood samples were collected each day with ultrasonographic scanning for measurement of serum concentrations of FSH, oestradiol and progesterone. Blood samples were also taken every 15 min for 6 h, mid-way through each period of ultrasonographic examination, to determine the patterns of secretion of gonadotrophic hormones. Hormonal data were then related to observed changes in follicular populations and the patterns of antral ovarian follicle turnover. Ultrasonography showed that the ovaries of anoestrous ewes remained active and that the largest ovarian antral follicles grew to a periovulatory size (> or = 5 mm in diameter) at all stages of anoestrus. The total number of all ovarian follicles > or = 3 mm in diameter was lower during early anoestrus compared with at mid-anoestrus because of a significantly smaller number of small (3 mm) and medium (4 mm) ovarian follicles. The largest ovarian follicles (attaining > or = 5 mm in diameter before regression) exhibited a wave-like pattern of growth; an average of three waves of follicular development were recorded in sheep during each of the four 17 day scanning periods in anoestrus, with follicular waves emerging approximately every 5 days. This rhythmic pattern of follicular emergence was found to be associated with the occurrence of fluctuations in serum FSH concentrations. The growth rate of the largest follicles of the wave increased significantly from early to late anoestrus in sheep. In addition, ovarian follicles not growing beyond 3 mm in diameter showed organized patterns of growth and regression; their numbers tended to be lower (P = 0.09) at 3 days before and on the day of follicular wave emergence. Some ewes were seen to maintain synthesis of progesterone throughout anoestrus. This submaximal progesterone secretion tended to occur at irregular intervals and was not coupled with changes in concentrations or patterns of gonadotrophin release, ovulations or detectable morphological luteinization of ovarian antral follicles. It was concluded that the growth of ovarian antral follicles to an ovulatory size was maintained throughout anoestrus in ewes, with a transient shift in the number of small and medium-sized follicles during mid-anoestrus, and that the periodic emergence of waves of large follicles (> or = 5 mm in diameter) occurred in synchrony with an endogenous rhythm of FSH secretion.