Ultrasound-guided Follicle Ablation Research Articles

Fertility response following ablation-induced follicular wave emergence and ovulation induction in anestrous buffaloes

The fertility response following induction of follicular wave emergence by follicle ablation combined withprogesterone based ovulation synchronization in postpartum anestrous buffaloes was assessed. Postpartum anestrousbuffaloes (n=24) with absence of overt estrus signs categorized as treatment group (Group 1; n=15) and controlgroup (Group 2; n=9). Group 1 was subjected to transvaginal ultrasound-guided follicle ablation on a random dayof the estrous cycle (Day=0) and insertion of CIDR. Seven days later, CIDR was removed and prostaglandin F2αanalogue was given intramuscularly. GnRH analogue was injected on day 9.5 and fixed-time AI was done 12 h and24 h later. Ultrasonography was performed to follow ovarian structures on different occasions and on day 38 postovulationfor detecting pregnancy. Group 2 did not receive any treatment however, estrus detection in this groupwas done daily by bull parading and visual monitoring followed by AI if found in standing estrus. In treatmentgroup, the follicular wave emergence occurred by 24 h following ablation in 60% buffaloes and in 40% remaininganimals by 48 h. The estrus exhibition was 100 % in group 1 versus 22.2 % in group 2. Mean interval from CIDRremoval to ovulation was 81.2±3.08 h and from wave emergence to ovulation was 9.92±0.18 days. The first servicepregnancy rate in treatment group was higher than control (66.67% vs 22.2%). The induction of follicular waveemergence through ultrasound guided follicle ablation and progesterone based ovulation synchronization can beused to improve pregnancy rates in anestrous buffaloes.

An attempt to potentiate the ovarian superstimulatory response in cattle by co-treatment with an aromatase inhibitor

Letrozole is used for the treatment of subfertility in women undergoing ovarian superstimulation, but the mechanism of action has not been investigated critically. The objective was to test the hypothesis that treatment with letrozole will potentiate the superstimulatory response following gonadotropin treatment by increasing the number of follicles present at ovarian follicular wave emergence in cattle. In Experiment 1, ovarian follicular wave emergence was synchronized among beef heifers (n = 8) by transvaginal ultrasound-guided follicle ablation. On Day 0 (wave emergence), a letrozole-releasing device (LRD) was placed intravaginally for 5 days, followed again by transvaginal follicle ablation on Day 5. The number of follicles ≥3 mm was recorded by transrectal ultrasonography on Days 0 and 6.5 (i.e., pre- vs. post-LRD treatment). In Experiment 2, non-lactating dairy cows were assigned randomly to one of two groups (n = 15/gp) after follicle ablation-induced synchronization of wave emergence (Day 0), and given either an LRD or sham device for 5 days. Superstimulatory treatment was initiated on Day 0, consisting of 8 doses of 50 mg of porcine FSH im at 12 h intervals, and luteolytic doses of prostaglandin on Days 3 and 3.5. The LRD/sham devices were removed on Day 3.5, GnRH was given im on Day 5, estrus response was determined on Days 5 and 6, and the ovarian response was recorded by ultrasonography on Days 0, 3.5, 5, 6.5, and 12. In Experiment 1, no difference was detected in the number of antral follicles at wave emergence pre- vs. post-LRD treatment (23.2 ± 3.2 vs. 23.5 ± 3.8 follicles; P = 0.67; mean ± SEM). In Experiment 2, the interval from prostaglandin treatment to estrus was longer (50.3 ± 1.1 vs. 40.7 ± 2.0 h; P < 0.001) and less variable (residuals: 3.1 ± 0.5 vs. 6.7 ± 0.9 h; P < 0.01) in the LRD vs. sham group. The proportion of ovulations (number of CL on Day 12 over the number of follicles ≥3 mm on Day 0) did not differ (0.65 ± 0.02 vs. 0.70 ± 0.02; P = 0.15) nor did the number of CL on Day 12 (15.9 ± 2.5 vs. 19.0 ± 2.0; P = 0.32) between the LRD and sham groups. In summary, treatment with letrozole did not increase the number of antral follicles at wave emergence or the superstimulatory response, but increased precision in the interval to estrus and may be useful for artificial insemination at a fixed time in superstimulatory protocols.

175 Use of equine chorionic gonadotrophin in a minimum-handling protocol for oocyte collection in bison

Wood bison (Bison bison athabascae) and plains bison (Bison bison bison) are threatened subspecies native to North America. The creation of a germplasm biobank will connect valuable and inaccessible genetics from geographically distant herds in a biosecure manner. Protocols that are feasible in field conditions are required for cumulus-oocyte complex (COC) collection for the purpose of invitro embryo production (IVP). The efficacy of a single dose of equine chorionic gonadotrophin (ECG) was tested in an effort to develop a minimum-handling ovarian superstimulation protocol for bison. The experimental design enabled comparison between ECG-treated and non-superstimulated bison. Transvaginal ultrasound-guided follicle ablation was performed in mature wood bison (n=24) during May (anovulatory season) to induce follicular wave emergence the following day. Immediately after ablation, the bison were assigned to one of three groups (n=8 per group) and treated intramuscularly with 5000IU of ECG (Folligon, Merck), 2500IU of ECG, or saline (control). Transvaginal COC collection was performed 5 days later. Follicular and COC data were recorded, and only grade 1 and 2 COC were used for IVP. The COC were matured invitro for 25-28h at 38.8°C, fertilised (2×106 spermmL−1), and co-incubated at 38.8°C in 5% O2, 5% CO2, and 90% N2 for 18h. Presumptive zygotes were denuded and cultured at 38.8°C in 5% O2, 5% CO2, and 90% N2. Nominal data were compared among groups using analysis of variance, and proportional data were compared using GLIMMIX. The total number of follicles ≥3mm on the day of COC collection was greater in the 5000-IU ECG group than in the 2500-IU ECG and control groups (37.5±6.9, 17.5±2.0, and 16.9±2.0, respectively; P&amp;lt;0.005). The number of follicles 5-8mm was also greater in the 5000-IU ECG group than in the 2500-IU ECG and control groups (12.5±2.1, 7.6±1.0, and 5.8±0.9, respectively; P&amp;lt;0.01), as was the number of follicles &amp;gt;8mm (21.1±5.4, 3.3±1.2, and 0.9±0.2, respectively; P&amp;lt;0.0005). The proportion of grade 1 and 2 COC/total COC recovered was greater in the 5000-IU ECG group than in the 2500-IU ECG and control groups (84/124 (68%), 39/76 (51%), and 37/75 (49%), respectively; P&amp;lt;0.05). The proportion of cleaved zygotes/number of COC matured, assessed at 56h after fertilisation, was similar among the 5000-IU ECG, 2500-IU ECG, and control groups (42/84 (50%), 27/39 (69%), and 21/37 (57%), respectively; P=0.20). The proportion of embryos of IETS grades 1-3/number of COC matured was also similar among the 5000-IU ECG, 2500-IU ECG, and control groups (15/84 (17.9%), 8/39 (20.5%), and 7/37 (18.9%), respectively; P=0.94), but the bison in the 5000-IU ECG group produced twice as many embryos than those in the other groups. In summary, a single dose of 5000IU of ECG increased the number and size of follicles available for COC collection, more than doubled the number of COC collected for IVP, and resulted in the production of more embryos than the other groups. A single dose of 5000IU of ECG is effective in a minimum-handling protocol for ovarian superstimulation and IVP in bison. This research was supported by NSERC.

198 Superovulatory response in cows undergoing aromatase inhibitor treatment

The aromatase inhibitor letrozole has been used in the treatment of infertility in women by inducing mild ovarian superstimulation or augmenting the ovarian response to FSH treatment. The effect has been attributed to an apparent up-regulation of FSH receptors on granulosa cells as a result of increased androgens (Weil et al. 1999 J. Clin. Endocrinol. Metab. 84, 2951-2956). The objective of this study was to determine whether letrozole will augment the superstimulatory response in FSH-treated cattle. Mature, non-lactating Holstein cows (n=30) were given 2 luteolytic doses of PGF2α 12h apart and scanned daily by transrectal ultrasonography to detect ovulation. Transvaginal ultrasound-guided ablation of follicles ≥5mm was performed in all cows as a group, at 5 to 8 days after ovulation. On the day of ablation, cows were assigned randomly to 2 groups (n=15 per group) and given either an intravaginal letrozole-releasing device or a blank device for 5 days. Coinciding with expected wave emergence (Day 0), cows in both groups were given 8 doses of 50mg of pFSH (Folltropin; Vetoquinol, Lure, France) IM at 12-h intervals, and PGF2α on Days 3 and 3.5. At the time of the second PGF2α, vaginal devices were removed and paint was applied to the tailhead to facilitate detection of oestrus. On Day 5, cows were given gonadotropin-releasing hormone (100mg gonadorelin) IM and artificially inseminated 12 and 24h later. Ova/embryos were collected by transcervical uterine flush on Day 12. The ovaries were examined by ultrasonography on Day 0, 3.5, 5, 6.5, and 12 to record the follicular and luteal response. Nominal data were compared between groups by t-test and by ANOVA for repeated-measures and are expressed as mean±s.e.m. Binomial data were compared by chi-squared test. The number of follicles at wave emergence did not differ between letrozole and control groups (24.6±3.1 and 26.5±3.3 respectively; P=0.68). There was no treatment effect or treatment×day interaction in the number of follicles &amp;gt;8mm on Day 3.5, 5, and 6.5. Letrozole-treated cows exhibited oestrus later than controls (50.3±1.1h v. 40.7±2.0h after first PGF2α; P&amp;lt;0.001) and had less variance in interval to oestrus (residuals, 3.1±0.48h v. 6.7±0.87 h; P&amp;lt;0.01). The number of CL on Day 6.5 was lower in the letrozole group than in the control group (9.1±1.1v. 12.3±1.1; P=0.05). The proportion of ovulations (number of CL on Day 12 over number of follicles ≥3mm on Day 0) was lower in the letrozole group than in the control group (0.65±0.05v. 0.82±0.08; P=0.02). The total number of ova/embryos collected per cow did not differ between letrozole and control groups (5.0±1.9v. 5.4±1.8; P=0.75), nor did the number of transferable embryos differ (3.0±1.2v. 4.3±1.5; P=0.56). In conclusion, treatment with a letrozole-releasing device during ovarian superstimulation did not improve the superovulatory response or embryo collection rate in mature Holstein cows, but letrozole treatment resulted in more synchronous oestrus, which may be used in the design of a fixed-time AI protocol following superstimulatory treatment in cattle. This research was supported by the Alberta Livestock and Meat Agency.

122 TIMED ARTIFICIAL INSEMINATION IN WOOD BISON USING FROZEN-THAWED SEMEN

Recent progress with methods to control ovulation and semen cryopreservation in Wood Bison was the impetus to test the feasibility of timed AI to facilitate reclamation of this threatened species. A 2 × 2 design was used to compare the efficacy of 2 ovulation synchronization techniques and 2 semen cryopreservation protocols. Female Wood Bison were assigned randomly to 2 groups (n = 24/group) in which ovarian synchronization was induced by ultrasound-guided ablation of follicles &gt;5 mm or intramuscular treatment with 2.5 mg of estradiol 17B + 50 mg of progesterone (E+P) in canola oil. A progesterone-releasing intravaginal device (PRID) was placed at the time of follicle ablation (for 5 days) or E+P treatment (for 8 days) in the respective groups. A luteolytic dose of prostaglandin was given at the time of PRID removal, and 2500 IU of hCG was given IM 3 days later. Bison were inseminated 24 and 36 h after hCG treatment using frozen-thawed semen. The semen was collected by electro-ejaculation from 4 Wood Bison bulls, pooled, and divided into aliquots diluted in either egg-yolk extender (EY) or cholesterol-loaded cyclodextrin extender (CLC). Half the bison in each synchronization group were inseminated with either EY- or CLC-extended semen. Bison were examined by ultrasonography every 12 h beginning on the day of hCG treatment for 3 days or until ovulation was detected, whichever occurred first. Pregnancy diagnosis was made by ultrasonography 34–36 days after insemination. Two bison were excluded during the experiment because of handling difficulty; therefore, the total number of bison used was 46. Ovulation rate and interval to ovulation were compared between synchronization groups by chi-square and t-test, respectively. Pregnancy rates were compared among groups by 2-way ANOVA after transforming data to arcsin. The ovulation rate was not different between synchronization groups [combined mean, 37/46 (80%)], nor was the degree of synchrony, as assessed by the residuals (variation from the mean) in the respective groups. However, the diameter (mean ± standard error of the mean) of the dominant follicle at the time of hCG treatment was smaller in the follicle ablation group than in the E+P group (10.5 ± 0.6 v. 13.9 ± 0.6; P &lt; 0.04), and the interval from hCG treatment to ovulation tended to be longer (35.3 ± 1.6 v. 31.8 ± 1.3 h; P ≤ 0.10). Pregnancy rate was not affected by synchronization procedure, but pregnancy was detected only in the EY-inseminated group (9/23 v. 0/23; P &lt; 0.01). Despite that post-thaw sperm motility was similar for EY and CLC semen (41.7 ± 2.9 and 44.6 ± 3.3%; respectively), CLC-treated semen failed to impregnate bison in vivo. We concluded that synchronization and timed insemination with frozen-thawed semen is feasible in Wood Bison. Of the 23 bison inseminated with EY-extended semen, 21 ovulated (91%), and of those that ovulated 9 became pregnant (43%). Both synchronization schemes were effective, but the ablation protocol may be improved by an additional day between ablation and hCG treatment. We thank Vetoquinol Canada and Merck Animal Health for providing hormone treatments.

363 EXTENDING THE IN VIVO MATURATION TIME TO PERMIT FLEXIBLE TIMING OF OOCYTE COLLECTION IN SUPERSTIMULATED WOOD BISON (BISON BISON ATHABASCAE)

Wood bison are a species threatened by endemic brucellosis and tuberculosis. Reproductive technologies are being developed in an effort to ensure the genetic diversity of wild wood bison, and to prevent disease transmission to healthy bison, livestock, and humans. For the purposes of IVF, recent results revealed that cumulus cell expansion was more extensive in in vivo- v. in vitro-matured cumulus-oocyte complexes (COC), and more oocytes reached maturity after 30 v. 24 h of in vivo maturation following hCG treatment (Cervantes et al. 2013 Reprod. Fert. Develop. 25, 283). An experiment was designed to determine the effects of an additional 4 h of in vivo maturation on follicle development, unwanted ovulation, and COC collection efficiency. Wood bison cows (n = 28) underwent transvaginal ultrasound-guided follicle ablation to induce emergence of a new follicular wave (Day 0 = day of wave emergence, 1 day after ablation) during the non-breeding season. Bison were given FSH diluted in hyaluronan IM on Days 0 (300 mg) and 2 (100 mg), and 2500 IU hCG IM on Day 4. Bison were then assigned randomly to 2 groups (n = 14 per group) in which transvaginal oocyte collection was done at either 30 or 34 h after hCG treatment. The number and size of follicles available for aspiration (i.e. = 5 mm) was compared between groups by Student's t-test. Binomial data (COC collection rate and ovulation rate) were compared by chi-square, and the proportion of cows that ovulated was compared using a Fisher's exact test. Ovulation was defined as the sudden disappearance of follicles ≥10 mm from the hCG treatment to the time of COC collection. The numbers of follicles ≥5 mm and ≥10 mm at the time of COC collection were not different between the 30 and 34 h groups (19.0 ± 1.4 v. 17.4 ± 2.4, and 9.5 ± 1.2 v. 7.7 ± 1.8), nor was the average size of follicles = 5 mm (9.9 ± 0.2 v. 9.8 ± 0.2 mm). The number of follicles aspirated was similar between the 30 and 34 h groups (16.4 ± 1.4 v. 13.4 ± 2.1), but the pre-collection ovulation rate was lower in the 30 h group (12/89 [13.5%] v. 47/147, [32.0%]; P = 0.003), as was the proportion of bison that ovulated (3/14 v. 10/14, P = 0.02). The COC collection rate was lower in the 30 v. 34 h group (64.3% v. 78.2%; P = 0.003), but the total number of COC collected per bison was similar (10.6 ± 1.7 v. 10.5 ± 1.5). Although waiting for 34 h before COC collection resulted in a larger proportion of unwanted ovulations, a greater collection efficiency in the 34 h group resulted in a similar number of COC collected per bison. We conclude that the 30 to 34 h in vivo maturation window provides flexibility for the purposes of oocyte collection and immediate in vitro fertilization in wood bison.We thank Bioniche Animal Health for providing FSH (Folltropin-V) and hyaluronan (MAP-5), and Merck Animal Health for hCG (Chorulon).

Differential abundance of IGF1, bile acids, and the genes involved in their signaling in the dominant follicle microenvironment of lactating cows and nulliparous heifers

It is well documented that incidence of fertility problems is high in lactating cows but not in heifers of the same genetic merit. Understanding the metabolic and molecular differences between fertile heifers and relatively infertile lactating cows will help us understand the pathogenesis of infertility in dairy cows. Follicular waves in lactating cows (30–50 days in milk; n = 12) and heifers (n = 10) were synchronized by ultrasound-guided follicle ablation. Follicular fluid and granulosa cells of the dominant follicle were collected by ultrasound-guided aspiration along with blood sampling on Day 6 after synchronization. Dominant and subordinate follicles were larger in lactating cows than in heifers. Metabolic stress in lactating cows was evidenced by lower glucose and higher ß-hydroxy butyric acid compared with heifers. Insulin-like growth factor 1 signaling was reduced in the dominant follicle in lactating cows through reduced insulin-like growth factor 1 concentrations in plasma and follicular fluid of the dominant follicle, and reduced expression of pregnancy-associated plasma protein A (PAPPA) in their granulosa cells. We also found increased levels of total bile acids in the follicular fluid of the dominant follicle of lactating cows compared with heifers. Granulosa cells of the dominant follicle had higher expression of SLC10A2 and GPBAR1 (bile acid transporter and receptor, respectively) in lactating cows. These novel data are indicative of increased bile acid signaling within the dominant follicles of lactating cows compared with heifers. Overall, we demonstrate in the present study the metabolic, endocrine, and molecular differences within the microenvironment of the dominant follicles in lactating cows and heifers. These differences in follicular microenvironment may contribute toward abnormal ovarian function in lactating dairy cows.

183 EFFECTS OF LONG-TERM NUTRITIONAL RESTRICTION ON PREOVULATORY LUTEINIZING HORMONE SURGE, LEPTIN, AND LUTEAL PHASE IN LLAMAS (LAMA GLAMA)

The aim of the study was to determine the effect of long-term energy restriction on preovulatory LH surge and luteal phase in llamas. Mature nonlactating, nonpregnant, female llamas (n = 16) were assigned randomly to the following groups: (1) llamas received Ballica sp. hay ad libitum and 300 g d–1 of commercial concentrate (control group, n = 8, BCS = 3.9) or (2) llamas were fed the same feed but the amount was progressively reduced from 70% to 40% of their maintenance energy requirement (MER) for a period of 87 days, until the final BCS was 2.5 (restricted group, n = 8). After 2 months and 21 days of 40% MER diet, ovarian follicular wave emergence was synchronized by transvaginal ultrasound-guided ablation of follicles ≥5 mm in diameter using a 17-ga needle attached to a 5-MHz convex-array transducer. Ten days after follicle ablation (Day 0), llamas were given an i.m. dose of 50 µg of gonadorelin acetate to induce ovulation. Frequent blood samples were taken every 30 min for 6 h and every 15 min for 1 h for plasma LH and leptin concentration measurements, respectively, immediately after treatment. Llamas were examined daily by transrectal ultrasonography for 20 days to record serial changes in the CL diameter. In addition, blood samples were taken every 2 days from Day 0 to 16 to determine plasma concentrations of leptin and progesterone. Discrete dependent variables were compared between groups by Student t-tests and continuous dependent variables were analysed by analyses of variance with repeated-measures. There was an effect of time on plasma LH concentration (P ≤ 0.001), but not an effect of group (P = 0.1) or interaction (P = 0.6) after gonadorelin acetate treatment. Plasma leptin concentration was lower in llamas from the restricted than that of the control group after gonadorelin treatment [effect of time, group (P ≤ 0.01), and interaction (P ≤ 0.01)]. The maximum diameter of the CL and plasma progesterone concentration were lower in the restricted than in the control group, and both variables were affected by group (P ≤ 0.01, P ≤ 0.02), and day (P ≤ 0.001, P ≤ 0.001, respectively). Plasma leptin concentration was lower in the restricted than that of the control group during the entire experiment. In conclusion, long-term nutritional restriction affected negatively CL diameter, progesterone secretion, and leptin plasma concentration in llamas. The differential response of leptin to gonadorelin acetate treatment could reflect a subfertlity-nutritional induced problem related with steroidogenesis, oocyte quality, ovulation, and early embryo development processes. Research supported by Postdoctoral Fondecyt N° 3110095.

313 EFFECT OF DURATION OF THE GROWING PHASE OF OVULATORY FOLLICLES IN SUPERSTIMULATED HEIFERS ON OOCYTE COMPETENCE AFTER IN VITRO FERTILIZATION

We tested the hypotheses that extending the duration of follicular growth by superstimulation increases oocyte competence, and that FSH starvation at the end of superstimulatory treatment decreases oocyte competence. Heifers were allocated randomly to short FSH duration (n = 8), FSH starvation (n = 8), or long FSH duration (n = 8) groups. Five to 8 days after ovulation, transvaginal ultrasound-guided follicle ablation was done to synchronize follicle wave emergence, and a progesterone-releasing device (CIDR; Pfizer Animal Health, New York, NY, USA) was placed intravaginally. Short FSH and FSH starvation groups were given 8 doses of FSH (Folltropin-V; Bioniche Animal Health Inc., Belleville, ON, Canada) IM, whereas the long FSH group was given 14 doses of FSH at 12-h intervals, starting from the day of wave emergence (Day 0). Prostaglandin F2α (PGF) was administered twice, 12 h apart, on Day 3 in the short FSH group and on Day 6 in the other 2 groups. In all heifers, the CIDR was removed at the time of the second PGF treatment; pLH (Lutropin-V; Bioniche Animal Health Inc.) was given IM 24 h after CIDR removal, and cumulus–oocyte complexes (COC) were collected 24 h after pLH treatment. The COC were matured in vitro (6 h) and fertilized (IVF), and the embryos were cultured for 10 days. At 12 h after pLH, the long FSH group had a greater number of ≥9 mm follicles than the FSH starvation and short FSH groups (25.4 ± 5.3 v. 11.0 ± 2.1 and 10.6 ± 2.3, respectively; P &lt; 0.03). The long FSH group also had more expanded COC than the FSH starvation group (P &lt; 0.001), but did not differ from the short FSH group (93, 54, and 74%, respectively). The FSH starvation group had a greater proportion (P &lt; 0.0001) of partially expanded COC (32%) and poor quality oocytes (70%) than did the long (1 and 33%) and short (4 and 45%) FSH groups; oocyte quality did not differ between long and short FSH groups. At 48 h after IVF, the cleavage rate was lower in the FSH starvation group compared with the short and long FSH groups (35, 54, and 56%, respectively; P = 0.003). After 9 days in culture, embryo development (morula + blastocyst) in the FSH starvation group was lower than that in the long FSH group, (18 v. 37%; P = 0.04), but did not differ from that in the short FSH group (25%). After removal of the data of one heifer in the FSH starvation group that produced 52% of total embryos in that group (outlier), the Day 9 blastocyst rate was lower in the FSH starvation group than in the short and long FSH groups (2% v. 14 and 21%, respectively; P = 0.02). In conclusion, extending the standard superstimulation protocol by 3 days enhanced ovarian response to FSH treatment, but did not improve oocyte competence, whereas a period of FSH starvation after FSH treatment compromised oocyte quality and embryo development.

227 SUPEROVULATION AND EMBRYO COLLECTION IN WOOD BISON DURING THE ANOVULATORY SEASON

Brucellosis and tuberculosis are endemic in Wood Buffalo National Park, the largest reserve of wood bison (Bison bison athabascae) in Canada. Our goal is to produce and preserve disease-free embryos for the purpose of wood bison repopulation. This study was designed to determine if embryo collection is feasible in wood bison during the anovulatory season (May–July) and to test if progesterone priming is required for superovulation. A 2-by-2 design was used to determine the effectiveness of LH (Lutropin) or hCG (Chorulon) for induction of ovulation with or without intravaginal progesterone releasing device (PRID) in 32 wood bison cows. Follicular wave emergence was synchronized among bison by transvaginal ultrasound-guided follicle ablation. Synchronized bison were assigned to 4 groups: PRID+LH (n = 12), PRID+hCG (n = 4), no-PRID+LH (n = 12) and no-PRID+hCG (n = 4). A PRID was inserted on the day of follicular ablation in the respective groups. A single SC dose of 400 mg FSH (Folltropin) in a slow-release formulation was given the day after follicular ablation (i.e. on the expected day of a new follicular wave emergence, Day 0). The PRID was removed on Day 4 and either 25 mg LH or 2000 IU hCG was given IM on Day 5. Artificial insemination was done at 24, 36 and 48 h after LH or hCG treatment. Embryos were collected nonsurgically on Day 13 using commercial bovine equipment. Transrectal ultrasonography was done on Days 0, 5, 6, 7, 8 and 13 to record follicular and ovulatory responses. Count data (mean ± SEM) were analysed by two-way ANOVA and proportions by chi square. The number of ovulatory-sized follicles (≥10 mm) on Day 5 did not differ among groups (P = 0.33; Table 1). Ovulation rate (number of ovulations/number of follicles ≥10 mm) was greater in bison treated with hCG (P &lt; 0.05; Table 1). The number of corpora lutea (CL) on Day 13 was greater in bison treated with hCG without a PRID (P &lt; 0.05; Table 1). No differences in number of ova/embryos and transferable embryos were found among groups (P = 0.36 and P = 0.52, respectively; Table 1). In conclusion, progesterone priming (PRID) had no effect on ovarian superstimulation in wood bison in the anovulatory season. The ovulatory response was satisfactory only in bison treated with hCG. Embryo collection is feasible in wood bison, but the reasons for a low embryo collection rate in all groups remain unclear. Table 1.Response to superovulation and embryo collection in wood bison Funded by Advancing Canadian Agriculture and Agri-Food and Agri-Food Innovation.

207 EFFECT OF LOCATION AND STAGE OF THE DOMINANT FOLLICLE ON OVULATION AND PREGNANCY RATES IN ALPACAS

Gestation in camelids occurs in the left horn regardless of whether ovulation has taken place in the left or right ovary, suggesting uterine embryo migration (Fernandez-Baca S et al. 1970 Biol. Reprod. 3, 243-251). On the other hand, we have previously documented (Ratto MH et al. 2003 Theriogenology 60, 1645-1656) that more than 90% of llamas with the presence of a follicle ≥6 to 7 mm in diameter, regardless of their stage of development, did accept the copula and ovulate after mating. However, it is unknown whether these oocytes are competent to achieving acceptable pregnancy rate. This study was designed to determine the effect of location of the preovulatory dominant follicle (right or left ovary) on ovulation and pregnancy rates and to evaluate the effect of stage of ovarian follicle development at mating (growing, maintenance, and regression) on ovulation rate and embryo survival in alpacas. In Experiment 1, nonlactating alpacas (4-6 years of age) weighing 55 to 75 kg were randomly assigned to 1 of 2 groups according to the location of the dominant follicle detected by ultrasonography: right ovary [right dominant follicle (RDF); n = 96] or left ovary [left dominant follicle (LDF); n = 108]. Ovulation and pregnancy diagnoses were assessed by ultrasonography on Days 2 (Day 0 = mating) and 30, respectively. Ovulation was defined as the sudden disappearance of a large follicle (≥6 mm) that was detected during the ultrasonographic examination 24 to 36 h after mating. Ovulation rates (96.5 and 96.3% for RDF and LDF, respectively) and pregnancy rates (60.2 and 56.7% for RDF and LDF, respectively) rates did not differ (P = 0.9) among groups. In Experiment 2, nonlactating alpacas (4-8 years of age) weighing 60 to 80 kg (n = 4116) were submitted to ultrasound-guided follicle ablation to synchronize follicular wave emergence and, after daily ultrasonography examination, were randomly assigned to the following groups according to the growth phase and diameter of the dominant follicle: early growing (G1; 5-6 mm, n = 27), mid-growing (G2; 7-12 mm, n = 30); static (G3; 7-12 mm, n = 430), or regressing (G4; 12-7 mm, n = 29). All alpacas were mated with a proven male, except 5 alpacas from G1 that rejected the sire. The ovarian response was determined by ultrasound examinations that were carried out on Day 2 (ovulation rate), Day 9 (CL size), and Day 35 (presence of embryonic vesicle or embryo). Ovulation was determined as described in Experiment 1. No differences were detected in ovulation rate among groups (95, 96, 100, and 96%, respectively; P = 0.8) or in CL size (10.3 ± 0.8; 11.7 ± 0.6; 11.1 ± 0.8; and 11.1 ± 0.9 mm, respectively; P = 0.6). Pregnancy rate was highest in G2 (65.5%), intermediate in G1 (52.4%) and G3 (53.3%), and lowest in G4 (42.9%; P &lt; 0.05). Results suggest that location of the dominant follicle has no influence on ovulation and pregnancy rates and that although mating during the regressing phase of the dominant follicle has no effect on ovulation, pregnancy rate may be compromised. This study was supported by DID-UACH, Universidad Austral de Chile.

Synchronisation of ovarian follicular waves in the dromedary camel ( Camelus dromedarius)

This study was designed to compare the efficacy of various treatments intended to synchronise follicular wave cycles in dromedary camels by removing the existing follicle of unknown size and replacing it with a follicle capable of ovulating at a known time. Camels were randomly assigned to one of five groups and treated with either (1) 5 mg oestradiol benzoate (i.m.) and 100 mg progesterone (i.m.; E/P, n = 15), (2) 20 μg GnRH analogue, buserelin (i.m.; GnRH, n = 15), (3) 20 μg buserelin (i.m.) on Day 0 ( T = 0) and 500 μg prostaglandin on Day T + 7 (GnRH/PG n = 15), (4) transvaginal ultrasound—guided follicle ablation of all follicles ≥0.5 cm (ABL, n = 15) or (5) 5 ml saline (i.m; Controls n = 15). All camels were subsequently injected with 20 μg buserelin 14 days after the first treatment was given. The ovarian response was monitored daily by transrectal ultrasonography and the intervals from treatment to follicular wave emergence and also the day on which the new dominant follicle reached 1.3 cm was recorded. Amongst the treatment groups the mean interval from treatment to new follicle wave emergence and treatment to time taken for the new dominant follicle to reach 1.3 cm in diameter was shortest in the ABL group (2.3 ± 0.5 days and 8.8 ± 1.1 days respectively, P = 0.044) and longest in the E/P group (6.4 ± 0.8 days and 12.2 ± 1.0 days respectively, P < 0.001) whereas the GnRH and GnRH/PG groups were intermediate (3.0 ± 0.5 days and 11.1 ± 0.8 days GnRH; and 4.5 ± 0.5 days and 10.7 ± 0.7 days GnRH/PG). A total of 11/15 camels in both the GnRH and GnRH/PG groups had dominant follicles between 1.3 and 1.9 cm 14 days post treatment, of which 21 of the 22 follicles ovulated after GnRH injection on T + 14. The ABL, E/P and control groups however, showed greater variability in follicle size with less camels having dominant follicles between 1.3 and 1.9 cm than the GnRH and GnRH/PG groups and more in the ≥2.0 cm or follicle regressing groups, therefore fewer of these camels ovulated (ABL n = 7; E/P n = 9; Control n = 6) after GnRH injection on Day T + 14. In conclusion, two GnRH injections 14 days apart or two GnRH injections 14 days apart and PG on Day 7 after the first GnRH were the most effective methods to synchronise ovulation rate in dromedary camels at a fixed time interval of 14 days after treatment.

Induction of ovarian follicular wave emergence in wapiti ( Cervus elaphus)

Two experiments were done to test the effects of treatments designed to electively induce ovarian follicular wave emergence in wapiti for the purpose of group synchronization. In Experiment 1, hinds were assigned randomly to three groups and given saline im (controls; n = 5), 5 mg of estadiol-17ß im ( n = 4), or 5 mg estradiol-17ß plus 100 mg progesterone im ( n = 5). In Experiment 2, hinds were assigned randomly to two groups and given no treatment (controls; n = 6), or transvaginal ultrasound-guided follicle ablation ( n = 7). In both experiments, ovarian follicular dynamics were monitored by daily transrectal ultrasonography from Day 0 (day of treatment) to Day 9. In Experiment 1, blood samples were collected at each examination for measurement of serum concentrations of progesterone and FSH. Both experiments were conducted during the late anestrous period (July and August). The mean (±S.E.M.) day of wave emergence did not differ between the control and estradiol alone groups, but tended to be later in the estradiol plus progesterone group Day 4.0 ± 0.7, Day 3.5 ± 0.3, and Day 5.2 ± 0.2, respectively; P = 0.06). The interval from treatment to wave emergence was less variable in the estradiol plus progesterone group ( P < 0.05) and tended to be less variable in the estradiol-alone group ( P = 0.07) than in the control group. The day of wave emergence was more variable ( P < 0.05) and tended to be later ( P = 0.10) in the control group compared to the ablation group (Day 2.5 ± 0.8 versus Day 1.4 ± 0.2). All three treatments were effective in synchronizing ovarian follicular wave emergence among a group of wapiti hinds. Follicle ablation may be an alternative method for synchronization of follicular waves in estrus synchronization and superstimulatory protocols.

Ovulation synchronization following commercial application of ultrasound-guided follicle ablation during the estrous cycle in mares

A regimen of progesterone plus estradiol (P&E) was used as a standard for ovarian synchronization to test the efficacy and evaluate the commercial application of ultrasound-guided follicle ablation as a non-steroidal alternative for ovulation synchronization in mares. Recipient mares at a private embryo transfer facility were at unknown stages of the estrous cycle at the start of the experiment on Day 1 when they were randomly assigned to an ablation group (n=18-21 mares) or to a P&E group (n=20-21 mares). In the ablation group, mares were lightly sedated and all follicles > or = 10 mm were removed by transvaginal ultrasound-guided follicle aspiration. In the P&E group, a combination of progesterone (150 mg) plus estradiol (10mg) prepared in safflower oil was given daily (im) for 10 d. Two doses of prostaglandin F(2alpha) (PGF, 10mg/dose, im) were given 12 h apart on Day 5 in the ablation group, or a single dose on Day 10 in the P&E group. Human chorionic gonadotropin (hCG, 2500 IU/mare, im) was given at a fixed time, 6 and 10 d after PGF treatment in the ablation and P&E groups, respectively, with the expectation of a follicle > or = 30 mm at the time of treatment. In both the ablation and P&E groups, transrectal ultrasonography was done at the start of the study (Day 1) and again on the day of hCG treatment and daily thereafter to determine the presence of a CL, measure diameter of the largest follicle and detect ovulation. The mean interval from the start of the study and from PGF treatment to ovulation was shorter (P<0.0001) in the ablation group (13.7 and 9.7 d, respectively) compared to the P&E group (22.3 and 13.2 d, respectively). Following fixed-day treatment with hCG after PGF treatment, the degree of ovulation synchronization was not different (P>0.05) between the ablation and P&E groups within a 2-d (56 and 70%) or 4-d (83% and 90%) period. Although ultrasound-guided follicle ablation may not be practical in all circumstances, it excluded the conventional 10-d regimen of progesterone and estradiol and was considered an efficacious and feasible, non-steroidal alternative for ovulation synchronization in mares during the estrous cycle.

The timing of ovulation and insemination schedules in superstimulated cattle

The development of treatments that control follicular wave dynamics during the bovine estrous cycle has resulted in interesting possibilities for the precise control of follicular wave emergence and the time of ovulation. For superstimulation, follicular wave emergence can be controlled by ultrasound-guided follicle ablation with FSH treatments initiated 1 or 2 d later, or injection of estradiol combined with progesterone at the time of insertion of a progestogen releasing device and FSH treatments beginning 4 d later. These are the most widely used protocols for superstimulation of donor cows because they offer the convenience of being able to initiate treatments quickly and at a self-appointed time, without reducing the number of transferable embryos. However, these protocols still require precise estrus detection of donors following superstimulation in order to conduct AI at the most appropriate time. Recent studies have been designed to develop superstimulation protocols that involve fixed-time AI of donors, without regard to estrus detection. Results presented herein indicate that delaying the removal of a progestogen releasing device, combined with the administration of GnRH or porcine LH (pLH) 12 or 24 h later results in predictable, synchronous ovulations, permitting fixed-time AI without reducing the numbers or quality of embryos. These protocols facilitate the application of on-farm embryo transfer programs because they are practical, easy to administer by farm personnel, and more importantly, they eliminate the need for detecting estrus.

366 THE BOVINE MODEL FOR REPRODUCTIVE AGING: SUPEROVULATORY RESPONSE

Two experiments were done to test the hypotheses that aging in cattle is associated with a reduction in the number of follicles recruited into an ovarian follicular wave, and a reduction in the ovarian response following gonadotropin treatment for superovulation. Old cows (13 to 16 years old; n = 6 to 9) and their young daughters (3 to 6 years old; n = 7 to 9) were treated to induce an ovarian superstimulatory response twice in each of two consecutive years (i.e. 31 and 33 superstimulatory cycles, respectively). In Experiment 1, follicular wave emergence was synchronized using a single treatment of estradiol (5 mg) combined with progesterone (100 mg) at the time of CIDR-B (Bioniche Animal Health, Belleville, Ontario, Canada) insertion. Superstimulatory treatment consisted of a total dose of 50 mg NIH-FSH-P1 (Folltropin-V; Bioniche Animal Health) per 100 kg body weight divided into twice daily intramuscular injections over 4 days. CIDR devices were removed at the time of the last FSH treatment and 500 �g cloprostenol (Estrumate; Schering-Plough Animal Health, Montreal, Quebec, Canada) was administered twice along with last two FSH treatments to induce luteolysis. Ovulation was induced using 25 mg porcine LH (Lutropin-V; Bioniche Animal Health) administered 24 h after the last FSH treatment. Ovulations were detected by ultrasonography at 24, 48, and 72 h after LH treatment. In Experiment 2, follicular wave emergence was synchronized with ultrasound-guided follicle ablation in the presence of a functional corpus luteum. Superstimulatory treatment was given as in Experiment 1. Ultrasound-guided follicle aspirations were performed 48 to 72 h after the last FSH for the purpose of another experiment. In both experiments, the ovarian follicular response was monitored daily by ultrasonography. There were no differences (P &gt; 0.3) in mean body weight and dose of FSH treatment between old cows and their daughters. Fewer 2-5-mm follicles (P &lt; 0.01) were detected at the expected time of wave emergence in old cows than in their daughters. In Experiment 1, the difference in mean number of ovulations in old and young cows (32 � 4 vs. 40 � 3) did not reach significance (P = 0.11), but fewer follicles e6 mm (P &lt; 0.01) were detected on the day before ovulation in old cows than in their daughters. The number of follicles e6 mm detected just before ovulation (Experiment 1) or follicular aspiration (Experiment 2) in successive superstimulatory cycles was correlated within animals (r &gt; 0.8; P &lt; 0.0001). Similarly, the number of ovulations in successive superstimulatory cycles (Experiment 1) was correlated within animals (r = 0.6; P = 0.04). In conclusion, aging was associated with fewer 2-1 mm follicles at the time of wave emergence, a lower follicular response, and a tendency for a lower ovulatory response subsequent to gonadotropin treatment. The follicular and ovulatory response to superstimulatory treatment was repeatable within individuals.

Ovarian follicular wave synchronization and pregnancy rate after fixed-time natural mating in llamas

The study was designed to compare the efficacy of treatments intended to induce follicular wave synchronization among llamas (Experiment 1), and to determine the effect of these treatments on pregnancy rates after fixed-time natural mating (Experiment 2). In Experiment 1, llamas were treated with: (1) saline (control, n=20); (2) estradiol and progesterone (E/P, n=20); (3) LH (LH, n=20); or (4) transvaginal ultrasound-guided follicle ablation (FA, n=20). The ovarian response was monitored daily by transrectal ultrasonography. The intervals from treatment to follicular wave emergence and to the day on which the new dominant follicle reached ≥7 mm, respectively, did not differ between the LH (2.1±0.3 days and 5.2±0.5 days, respectively) and FA groups (2.3±0.3 days and 5.0±0.5 days), but both were shorter ( P<0.05) and less variable ( P<0.01) than in the control group (5.5±1.0 days and 8.4±2.0 days), while the E/P group (4.5±0.8 days and 7.7±0.5 days) was intermediate. In Experiment 2, llamas at unknown stages of follicular development were assigned randomly to control, E/P, and LH groups ( n=30 per group). A single, fixed-time natural mating was permitted 10–12 days after treatment. Ovulation rates did not differ among groups (control, 93%; E/P, 90%; LH, 90%; P=0.99), but the pregnancy rate was higher ( P<0.05) for synchronized llamas (LH and E/P groups combined, 41/54) than for non-synchronized llamas (control group, 15/28). In conclusion, LH and FA treatments were most effective for inducing follicular wave synchronization, while E/P treatment was intermediate. Synchronization treatments did not influence ovulation rate subsequent to fixed-time natural mating, but a higher pregnancy rate in synchronized than non-synchronized llamas warrants critical evaluation of the effects of follicular status on the developmental competence of the contained oocyte.

Pattern and manipulation of follicular development in Bos indicus cattle

Bos indicus cattle are widespread in tropical regions due to their adaptation to these environments. Although data on reproductive performance have indicated both inferior and superior results for B. indicus cattle, there is little doubt that B. indicus cattle are superior than Bos taurus cattle when they are both kept in tropical or subtropical environments, where stressors like hot temperatures, humidity, ectoparasites and low quality forages are greater. Reproductive endocrinology and oestrus behaviour of the B. indicus cattle have been studied for over 30 years; however, the application of technologies such as real time ultrasonography and Heat-Watch systems has expanded our knowledge on the ovarian follicular-wave dynamics during the oestrous cycle and the time of ovulation. Ovarian follicular dynamics in B. indicus cattle is characterised by the occurrence of two, three or sometimes four waves of follicular development. While dominance is similar to that in B. taurus cattle, maximum diameters of the dominant follicle and CL are smaller than those reported in B. taurus and are probably due to a lower capacity for LH secretion than in B. taurus. Duration of oestrus is approximately 10 h and the interval from oestrus to ovulation is about 27 h. However, the variability in response to prostaglandin F2α (PGF) treatments and the difficulty for oestrus detection in B. indicus cattle have limited the widespread application of artificial insemination (AI) and emphasizes the need for treatments that control follicular development and ovulation. Follicular-wave development in B. indicus cattle can be controlled mechanically by ultrasound-guided follicle ablation, or hormonally by treatments with GnRH or oestradiol and progestogen/progesterone in combination. Treatments with GnRH plus PGF and a second GnRH (synchronization protocol known as Ovsynch) or oestradiol benzoate (known as GPE) have resulted in acceptable pregnancy rates after fixed-time AI (FTAI) in cycling cows, but results were lower in heifers and cows in postpartum anoestrus. Alternatively, treatments with oestradiol and progestogen/progesterone releasing devices resulted in synchronous emergence of a new follicular wave, and a second oestradiol or GnRH treatment after device removal resulted in synchronous ovulation and acceptable pregnancy rates to FTAI. Furthermore, oestradiol and progesterone treatments combined with eCG (given at the time of device removal) increased pregnancy rates in suckled B. indicus cows and may be useful for the treatment of cows in postpartum anoestrus. In summary, exogenous control of luteal and follicular development facilitates the application of assisted reproductive technologies in B. indicus cattle by offering the possibility of planning AI programs without the necessity of oestrus detection and without sacrificing the overall results.

The control of follicular wave development for self-appointed embryo transfer programs in cattle

Our expanding knowledge of the control of follicular wave dynamics during the bovine estrous cycle has resulted in renewed enthusiasm for the prospects of precisely controlling the follicular and luteal dynamics and finely controlling the time of ovulation. Follicular wave development can be controlled mechanically by ultrasound-guided follicle ablation or hormonally by treatments with GnRH or estradiol and progestogen/progesterone in combination. Treatment of cattle with GnRH in combination with prostaglandin F 2α (PGF) 7 d later and a second GnRH 48 h after PGF (known as Ovsynch) has resulted in acceptable pregnancy rates after fixed-time AI in lactating dairy cows and in recipients in which embryos were transferred without estrus detection. Alternatively, treatments with estradiol and progestogen/progesterone-releasing devices resulted in synchronous emergence of a new follicular wave and, when a second estradiol treatment was given 24 h after device removal, synchronous ovulation and high pregnancy rates to fixe-time AI. Self-appointed embryo transfer (without estrus detection) using estradiol and progesterone treatments have resulted in pregnancy rates comparable with those obtained with recipients transferred 7 d after estrus. Furthermore, estradiol and progesterone treatments combined with PGF and eCG (given 1 d after the expected time of wave emergence) have resulted in high rates of recipients selected for transfer (84.6%) and an overall pregnancy rate of 48.7% (recipients pregnant/recipients treated). Estradiol and progestogen/progesterone treatments have also been widely used for self-appointed superstimulation protocols with equivalent embryo production to that of donor cows superstimulated using the traditional approach beginning 8 to 12 d after estrus. In summary, exogenous control of luteal and follicular development facilitates the application of assisted reproductive technologies in cattle by offering the possibility of planning the superstimulation of donors and synchronization of recipients at a self-appointed time, without the necessity of estrus detection and without sacrificing results.

Ovarian follicular wave synchronization and superstimulation in prepubertal calves

Two experiments were designed to artificially alter the follicular wave pattern in calves to determine if the mechanisms controlling the well-ordered pattern of follicular growth in adults are extant in prepubertal animals as well. Experiment 1 was designed to test the hypothesis that follicle ablation in a random group of calves will induce synchronous emergence of a new follicular wave which is not different from a spontaneous wave. Experiment 2 was designed to test the hypothesis that ovarian superstimulatory response in calves is enhanced when treatment is initiated before rather than after the time of selection of the dominant follicle. In Experiment 1, 6-month-old calves were assigned randomly to an ablation group (n = 10) and a control group (no ablation, n = 10). Follicle ablation was accomplished by transvaginal ultrasound-guided needle aspiration of all follicles ≥ 4 mm in diameter. Blood samples were taken and ovarian changes were monitored daily. A rise (P < 0.01) in mean plasma FSH concentration was detected 24 h after follicle ablation (1.51 ng/ml in the ablation group and 0.93 ng/ml in the control group). Wave emergence was detected earlier (P < 0.01) and with less variation (P < 0.0001) in the ablation group than the control group (1.2 ± 0.1 vs 4.0 ± 0.7 d). Characteristics of the induced wave were not different from those of the spontaneous wave. In Experiment 2, 7-month-old calves were assigned randomly to a pre-selection group in which superstimulation treatment was initiated at the time of wave emergence (1 d after follicle ablation, n = 11), or to a post-selection group in which superstimulation treatment was initiated after selection of a dominant follicle (4 d after follicle ablation, n = 11). Superstimulation treatment consisted of 30 mg of FSH im twice daily for 3 d. Ultrasound-guided transvaginal follicle ablation was used to synchronize follicle wave emergence at the outset of the experiment. The mean diameter of the largest follicle at the start of superstimulation treatment was 3.2 versus 8.5 mm in the pre- and post-selection groups, respectively (P < 0.001). The day after the last treatment, the number of follicles ≥ 3 mm in diameter was greater (P < 0.002) in the pre-selection group than in the post-selection group (19.3 ± 1.7 versus 11.3 ± 1.3). In summary, ultrasound-guided follicle ablation resulted in synchronous wave emergence in a random group of calves, and superstimulation treatment initiated at the time of wave emergence (pre-selection group) resulted in the growth of more follicles than treatment initiated later (post-selection group). Mechanisms involved in the control of follicle recruitment, selection, and suppression are extant in calves, similar to those found in adults.