Transvaginal Follicle Aspiration Research Articles

Blastocyst production using a new equine FSH for in vitro maturation

Currently, the majority of the equine ART laboratories collect germinal vesicle (GV)-stage oocytes to perform ICSI, and the gonadotrophins used for in vitro maturation are mainly porcine, bovine, or human. In this study, the effect of two different concentrations of equine FSH (eFSH) in the maturation medium was evaluated. Cumulus-oocytes complexes (COCs) were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to the groups. The control group was our standard maturation medium (BMM - TCM199-Earle's salts, 10% FBS, and additives) with human gonadotrophins added (Lazzari.et.al. Journal of Equine Veterinary Science. 2020; 89:103097); experimental groups were composed of BMM with eFSH added (0.01 and 0.05 IU/mL). After holding, COCs were transferred to maturation medium and cultured for 36h. ICSI was performed, and presumptive zygotes were cultured. The cleavage rate and number of cells were evaluated on D3, blastocyst formation and embryo quality, classified on A-B-C grade scale (adapted from Lewis.et.al. Reproduction, Fertility and Development. 2023; 35: 338-351), were evaluated from D6-10. Differences were analyzed by Fisher's Exact Test (significance level P<0.05). There was no difference (P>0.05) in degenerated, GV-stage, or MI oocytes after maturation for control, 0.01, and 0.05, respectively. The number of oocytes that reached the (meiosis) MII-stage was lower (P<0.05) for 0.01 U/mL (56.5%, 35/62)than the control (73.2%, 52/71), and also tended to be lower (P=0.0985) compared to 0.05 U/mL (71.2%, 47/66). There was no difference in cleavage or cell number among groups (Table 1). Group 0.01 U/mL had a higher (P<0.05) blastocyst rate (45.7%, 16/35) than control and 0.05 U/mL groups (28.8%,15/52; 25.5%, 12/47; respectively). No difference was found in embryo quality among groups. Although the 0.01 U/mL group showed a lower MII rate, it also showed numerically less 2-cell stage embryos, more 4-8 and 8-16 cells stage embryos, and more cleaved embryos. Additionally, the 0.01 U/mL group tended to have more high-quality embryos (grade A) than the control group and showed a higher total blastocyst rate compared to the other groups. Additional studies are required to establish an ideal maturation protocol using this new equine FSH.

Effect of day of estrus cycle at time of transvaginal follicle aspiration for oocyte recovery on rates of in vitro maturation and blastocyst production after intracytoplasmic sperm injection.

To determine the effect of stage of estrus cycle (day after ovulation) at the time of transvaginal ultrasound-guided follicle aspiration (TVA) on parameters related to the success of in vitro equine embryo production. 14 healthy mares were used; 11 completed the study and were included for analysis. Mares underwent TVA of all follicles ≥ 5 mm diameter at each of 3 timepoints: 7 days after ovulation, 14 days after ovulation, and S-DSF (subordinate to a dominant stimulated follicle), during estrus at 24 hours after gonadotropin administration. The 3 treatments were assigned to each mare in random order; mares underwent follicle growth and ovulation between treatments. Recovered oocytes were matured in vitro, subjected to intracytoplasmic sperm injection (ICSI), and cultured to the blastocyst stage in vitro. Total follicle numbers differed significantly between individual mares but did not differ between treatments. The number of follicles of different sizes significantly (P < 0.05) differed between treatments, with mares in the Day 7 treatment having more follicles 5 to 9 mm in diameter and fewer follicles 20 to 24 mm in diameter than mares in the other 2 treatments. After in vitro maturation culture, there were significantly more mature oocytes in the S-DSF treatment than in the other 2 treatments. There were no differences in blastocyst rate after ICSI among treatment groups. Timing of TVA for aspiration of S-DSFs may increase the number of mature oocytes available for ICSI. Understanding of the effects of timing of TVA will help veterinarians to maximize the efficiency of this procedure.

Spontaneous Ovarian Hyperstimulation Syndrome: Looking Beyond the Ovary

We report a case of a 22-year-old single female with spontaneous ovarian hyperstimulation syndrome (s-OHSS) referred for transvaginal aspiration of follicles. Investigations revealed primary hypothyroidism, mild hyperprolactinaemia and unelevated levels of both follicle stimulating hormone (FSH) and estradiol. Supplementation with L-thyroxine lead to euthyroid status and gradual resolution of signs and symptoms of ovarian hyperstimulation syndrome (OHSS) over 4 months.

The interrelationship between the occurrence of oversized follicles and the peripheral and intra-follicular concentrations of E2, P4, FSH, and LH in female dromedary camels

The present study aimed to clarify the phenomenon of presence of larger than normal follicles (OVGF) in female dromedary camels. Females with OVGF (n=125) were examined by manual palpation and ultrasonography. Accordingly, the OVGF were subdivided into those with thin walls and clear hypoechogenic content (OVGF-TH, n=18) and those with thick walls and fibrous trabeculae (OVGF-TK, n=107). Transvaginal follicle aspiration was performed in females with OVGF and from a control group with growing follicles (1-2 cm in diameter, GF group, n=5). Serum was collected at the same time of follicle aspiration and analyzed for Follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P4) and estradiol-17β profiles (E2). The follicular fluid (FF) was analyzed for E2 and P4. The results showed that mean E2 concentration in FF and serum were lower in OVGF-TH and OVGHTK groups than in the GF group (P &lt; 0.05). Difference between OVGF-TH and OVGH-TK groups was not significant. P4 in FF did not significantly differ among groups. Positive correlation was found between E2 in FF and E2 in serum (r = 0.495, r = 0.03). Mean FSH concentration in serum was higher in OVGF-TH and OVGH-TK groups than in the GF group (P = 0.03). Mean LH concentration was non-significantly (P=0.1) greater in OVGF-TH and OVGH-TK groups than in the GF group. In conclusion, female dromedary camels with OVGF had endocrine characteristics differed from camels with no OVGF. It seems that the high FSH and/or LH concentration(s) stimulated the continuing growth of the developing follicles to reach these large sizes, suggesting that the phenomenon of OVGF in camels is a pathological finding.

The effect of seminal plasma \u03b2-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells

BackgroundNerve growth factor (β-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified β-Nerve Growth Factor (β-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas.MethodsExperiment I: Female llamas (n = 64) were randomly assigned to receive an intramuscular administration of: a) 50 μg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama β-NGF (n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas (n = 16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20 h after treatment (Time 0 = administration of treatment, n = 8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas (n = 24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20 h (n = 4 replicates) and progesterone secretion at 48 h (n = 4 replicates) after LH or β-NGF treatment.ResultsExperiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified β-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified β-NGF with respect to the control group. All treatments except β-NGF (20 h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20 h), or β-NGF at 10 or 20 h after treatment. VEGF was up regulated only in those llamas submitted to mating (10 h) or treated with purified β-NGF (10 and 20 h). Experiment II: Only β-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20 h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20 h of in vitro culture from granulosa cells treated with β-NGF whereas LH treatment increases VEGF mRNA abundance only at 20 h of in vitro culture. In addition, there was a significant increase on progesterone secretion from llama granulosa cells 48 h after LH or β-NGF treatment.ConclusionsSystemic administration of purified β-NGF from llama seminal fluid induced a rapid shift from estradiol to progesterone production in the preovulatory follicle. Differences in gene expression patterns of steroidogenic enzymes between GnRH and mated or β-NGF-treated llamas suggest local effects of seminal components on the preovulatory follicle.

51 Effect of Cryoprotectant Exposure Time on Development of Vitrified-Warmed Immature Equine Oocytes

Effective methods for cryopreservation of equine oocytes have not yet been established. Vitrification involves use of high cryoprotectant (CPA) concentrations, which can be cytotoxic. Thus, it is critical to determine a CPA concentration and exposure time able to protect the cell during cooling but with a minimal toxicity. Using a rapid non-equilibrating system, we fixed the time in the first, lower CPA concentration solution (V1) at 40 s, based on the time to maximal shrinkage. We then evaluated different exposure times in the final vitrification solution (V2). Cumulus-oocyte complexes (COC) were collected from slaughterhouse-derived ovaries and held overnight in commercial embryo holding medium. Fetal bovine serum was used as the base medium (BM). In experiment 1, COC were held in BM, incubated in V1 (2% propylene glycol + 2% ethylene glycol) for 40 s followed by incubation in V2 (17.5% propylene glycol + 17.5% ethylene glycol + 0.3 M trehalose) for 0, 45, 75, or 110 s, and then loaded in groups of 6 to 10 oocytes on a 75-µm steel mesh and plunged into liquid nitrogen. Warming was performed in decreasing trehalose concentrations in BM: 0.4 M (60-70 s), 0.2 M (5 min), 0.1 M (5 min), 0.05 M (5 min), and 0 M. After warming, oocytes were cultured for in vitro maturation (IVM) and evaluated after staining with Hoechst 33258. Differences between treatments were analysed by Fisher’s exact test. The maturation (metaphase II, MII) rate of the Control (non-vitrified oocytes; 38.8%, 31/80) was similar to that of the 75-s treatment (34.8%, 16/46; P = 0.71), and higher (P &lt; 0.05) than those of the 0, 45, and 110 s treatments (0.0%, 0/10; 11.4%, 4/35; and 3.6%, 1/28; respectively). In experiment 2, timings in V2 focusing around 75 s were evaluated. The COC were collected and vitrified as for experiment 1, except that time in V2 was 50, 60, 70, 80, 90, or 100 s. The vitrified COC were then shipped to the intracytoplasmic sperm injection (ICSI) laboratory. After warming and IVM, oocytes were subjected to ICSI and embryo culture. Control oocytes were recovered by transvaginal follicle aspiration. The MII rate of the Control (60%, 33/55) was similar (P &gt; 0.05) to that of the 60- and 70-s treatments (38.9%, 7/18, and 35.3%, 6/17, respectively), and higher (P &lt; 0.05) than those of the 50-, 80-, 90-, and 100-s treatments (5.6 to 31.6%). The cleavage rates were 94% (31/33) for the Control and 71 to 100% for vitrified oocytes (P &gt; 0.05). No blastocyst was produced from vitrified oocytes compared with 8/33 (24.2%) for Control. This work demonstrates that a rapid, non-equilibrating vitrification technique using a 40-s initial exposure and 70- to 80-s final exposure to CPA is associated with maintenance of meiotic competence of immature equine oocytes; however, further work is required to optimize embryonic development with this method. Research supported by the Clinical Equine ICSI Program and the Link Equine Research Fund, Texas A&amp;M University.

Fertility Preservation in Female Patients with Breast Cancer - a Current Overview.

Many premenopausal patients who develop breast cancer have not yet completed their family planning, so measures of fertility protection to preserve their fertile potential would be beneficial. Polychemotherapy causes irreversible damage to the ovarian follicles – irrespective of whether in a neoadjuvant or adjuvant setting – and this can sometimes result in permanent infertility. Depending on which cytostatic agents are used and on the age-related ovarian reserve of the woman, gonadotoxic risk must be classified as low, moderate or high. Options of fertility preservation include: a) cryopreservation of fertilised or unfertilised oocytes. After ovarian hyperstimulation, mature oocytes are retrieved by transvaginal follicle aspiration, after which they are cryopreserved, either unfertilised or on completion of IVF or ICSI treatment. During b) cryopreservation of ovarian tissue, about 50% of the ovarian cortex of one ovary is resected with the aid of a laparoscopic procedure and cryopreserved. The application of c) GnRH agonists as a medicinal therapy option is an attempt at endocrine ovarian suppression in order to protect oocytes, granulosa cells and theca cells from the cytotoxic effect of chemotherapy.

Characterization of blood flow and the effects of exogenous estradiol benzoate on residual follicles formed after ultrasound-guided transvaginal follicle aspiration in cattle.

BackgroundRemoval of the follicular content by ultrasound-guided transvaginal follicle aspiration (TVFA) may fail to induce immediate atresia and loss of function, resulting in the occurrence of residual follicles (RF). The aims of this study were to characterize the blood flow in RF and to determine the effects of the treatment with estradiol benzoate on RF fate. Lactating, cyclic Holstein-Gir crossbred cows were used. In Experiment 1, follicular wave emergence (D0) was synchronized in cows (n = 10) and follicular growth was then monitored by transrectal ultrasonography from D0 to D8, followed by TVFA of the largest follicle present on the ovaries 24 h later. Color Doppler ultrasound imaging was used to examine blood flow on the follicular wall, which was recorded immediately before and every 12 h after TVFA, up to 72 h. In experiment 2, cows (n = 22) were randomly allocated to receive either 2 mL of saline i.m. (Control group, n = 11) or 2 mL estradiol benzoate i.m. (EB group, n = 11) immediately after TVFA. Ovaries were scanned every 12 h to confirm the presence and to measure the diameter of RF. The contents of the RF, if present, were collected 72 h after the first TVFA, using the same aspiration procedures. Follicular fluid from original follicles and RF were stored at -20 °C until hormonal assays.ResultsIn Experiment 1, there was no reduction (P > 0.05) of blood flow in the remaining follicle walls after TVFA and maximum blood flow values were observed at 49.5 ± 19.7 h post-TVFA. In Experiment 2, formation of RF after TVFA was proportionally similar between Controls (5/9) and EB (5/10) cows. Also, RF diameter did not differ between groups (P > 0.05). Nonetheless, the content of RF from cows in the EB group had lower (P = 0.0004) estradiol (E2) concentration and lower (P = 0.0005) E2:P4 ratio compared with Controls.ConclusionsIn conclusion, 1) the persistence of vascularization in the remaining follicle wall may contribute to the formation of RF after follicle aspiration, and 2) the treatment with estradiol benzoate does not prevent formation of RF, but does reduce their estradiol production.

In vivo and in vitro maturation of oocytes collected from superstimulated wood bison (Bison bison athabascae) during the anovulatory and ovulatory seasons

Experiments were done to compare the in vivo and in vitro maturational characteristics of cumulus-oocyte complexes (COC) collected from live wood bison. In Experiment 1 (anovulatory season), follicular ablation was done to synchronize follicle wave emergence among bison on Day −1, and FSH was given on Days 0 and 2. Bison were then assigned to 5 groups (n=5/group) in which COC were collected by transvaginal follicle aspiration on Day 4 and either fixed immediately with no maturation (control), matured in vitro for 24 or 30h, or collected on Day 5 after in vivo maturation for 24 or 30h (i.e., after hCG treatment). In Experiment 2 (ovulatory season), bison were treated as described for Experiment 1, but PGF2α (cloprostenol) was given to control the luteal phase on Days −9 and 3. In both experiments, cumulus cell expansion was more extensive following in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30h groups. Nuclear maturation occurred more rapidly in vitro; 60–70% of oocytes were at the MII stage 24h after in vitro maturation while only 25–27% of oocytes had reached the MII stage after 24h of in vivo maturation. In conclusion, nuclear maturation occurred more rapidly during in vitro vs. in vivo maturation, but was associated with less cumulus expansion than in vivo maturation. In vivo oocyte maturation was more complete at 30 vs. 24h after hCG treatment. Season had no effect on the maturational capacity of wood bison oocytes.

43 MASS VITRIFICATION OF GERMINAL-VESICLE STAGE EQUINE OOCYTES

Oocyte cryopreservation has the potential to preserve female genetics. In addition, equine oocytes are not readily available in some areas, and vitrification could be used to accumulate oocytes at remote locations to provide material for research. To preserve large numbers of oocytes, a method for rapid vitrification of multiple oocytes is needed. First, we determined whether immature equine oocytes could be held overnight before vitrification, and we tested the use of a mesh+capillary-action media-removal vitrification platform. Oocytes were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to either immediate vitrification or overnight holding (24 to 27 h in 40% M199-Earle’s salts, 40% M199-Hanks’ salts, 20% fetal bovine serum, and 0.3 mM pyruvate) then vitrification. Oocytes were vitrified using different times (1 or 4 min) in vitrification solution and first warming solution: 1v1w, 1v4w, 4v1w, and 4v4w. The base solution was MH (80% M199-Hanks’ salts and 20% fetal bovine serum). Cryoprotectant concentration (vol/vol) was increased in 3 steps until reaching 7.5% dimethyl sulfoxide and 7.5% ethylene glycol. The oocytes were then held in vitrification solution (MH with 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose) for either 1 or 4 min, according to treatment, and 3 to 10 oocytes were transferred to a 75-μm sterile stainless steel mesh. The mesh was placed on sterile paper to absorb excess medium, then plunged in LN. The oocytes were warmed in MH solution with 1.25 M sucrose for either 1 or 4 min, then placed in 0.62 M and 0.31 M sucrose solutions for 5 min each and undetermined time in MH. After warming, oocytes were cultured for maturation (in vitro maturation) in M199-Earle’s salts, 5 mU mL–1 FSH, and 10% fetal bovine serum. After 30 to 36 h, the oocytes were denuded and stained with Hoechst 33258. Data were analysed by Fisher’s exact test. There were no significant differences (P &gt; 0.05) in rates of meiotic resumption among timing treatments (35, 24, 26, and 39% for 1v1w, 1v4w, 4v1w, and 4v4w, respectively), nor between immediately vitrified (17/55, 31%) and overnight held-vitrified groups (18/56, 32%). In the second experiment, all oocytes were held overnight. They were vitrified and warmed using only the 1v1w and 4v4w schedules, then subjected to in vitro maturation, intracytoplasmic sperm injection, and embryo culture. The MII rate of the control group (27/37, 73%) was higher (P &lt; 0.05) than that for 1v1w (12/33, 36%) or 4v4w treatments (10/35, 29%). The cleavage rate for control (25/27, 93%) was higher than that for 1v1w (5/9, 56%) but not than that for 4v4w (6/9, 67%). Blastocyst rates were 19% (5/27), 11% (1/9), and 0% (0/9) for control, 1v1w, and 4v4w, respectively (P &gt; 0.05). These results indicate that blastocysts may be produced from equine immature oocytes vitrified en masse; however, both the maturation and blastocyst production rates were relatively low. Additional studies are required to improve the efficiency of this technique. This work was supported by the Clinical Equine ICSI Program, Texas A&amp;M University.

Establishing an in vitro production program for buffalo embryos (Bubalus bubalis) in Colombia

Objective. Evaluate the results of the standardization of the in vitro production program of buffalo embryos, using oocytes obtained by ultrasound guided oocyte puncture during the 2012 breeding season in Colombia. Materials and methods. Fifty seven buffalo females were selected for ultrasound guided transvaginal aspiration of follicles, oocytes were identified within follicular fluid, classified and transported to the laboratory and matured in vitro for 18 to 20 hours. Frozen semen of seven Mediterranean bulls were used, motile sperm was obtained using the Percoll technique and oocytes were inseminated with 2 million sperm/ml. Presumptive zygotes were cultured for 6 days, grade 1 embryos obtained were frozen using ethylene glycol. Embryos were transferred to females on day 5 during natural cycle. Results. 97 aspirations were performed on the 57 animals, in 8.2% of the aspirations no oocytes were found. 8 oocytes/aspiration were obtained. Of the 783 oocytes, 92% were classified as viable (721/783) and were fertilized. The cleavage and blastocyst rate were 23% and 19% respectively. 37 embryos were transferred and 11 pregnancies were obtained, confirmed by rectal palpation 60 days after transfer, with a pregnancy rate of 29.7%. Conclusions. The results presented here are comparable with others in literature and shows the feasibility of producing in vitro embryos and pregnancies after the standardization of current protocols, with normal and sexed semen and transfer during natural cycle in buffalo.Key words: Buffaloes, embryo, fertilization in vitro, reproduction (Source: MeSH).

171 IN VIVO AND IN VITRO MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS–OOCYTE COMPLEXES DURING THE OVULATORY SEASON

Technologies are being developed to conserve the genetic diversity of wood bison. Knowledge of the characteristics of in vivo and in vitro maturation of the cumulus–oocyte complex (COC) are needed in wood bison to design efficient in vitro embryo production protocols. The objectives were to (1) determine the optimal interval after hCG treatment for in vivo maturation of COC in superstimulated wood bison, and (2) compare the characteristics of COC after in vitro and in vivo maturation. Ovarian synchronization was induced in 25 bison during October and November by giving a luteolytic dose of prostaglandin followed 8 days later by follicular ablation (Day –1). Ovarian superstimulation was induced with FSH (Folltropin-V) given i.m. on Day 0 (300 mg) and Day 2 (100 mg). A second luteolytic dose of prostaglandin was given on Day 3. Bison were assigned randomly to 5 groups (n = 5/group). The COC were collected by transvaginal follicle aspiration on Day 4 and were either assessed immediately (0 h, control), or matured in vitro for 24 or 30 h (in vitro maturation), or collected on Day 5 (in vivo maturation), 24 or 30 h after bison were given 2000 IU of hCG i.m. on Day 4. In vitro maturation was done in TCM-199 with 5% calf serum, 5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 0.05 μg mL–1 gentamicin, at 38.5°C and in a 5% CO2 humidified atmosphere. Nuclear maturation was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII) with anti-lamin AC/DAPI staining. Groups were compared by analysis of variance and Fisher's exact test (Table 1). A mean (±s.e.m.) of 7.3 ± 1.7 COC were collected per bison, with no difference among groups. The COC in the control (0 h) group were at the nonexpanded GV stage. Cumulus cells were more expanded after in vivo than in vitro maturation, and the percentage of fully expanded COC was the highest in the 30-h in vivo maturation group (87%; P &lt; 0.05). The greatest number of oocytes reached MII stage after 24 h of in vitro maturation, and 30 h of in vivo maturation. In conclusion, nuclear maturation occurred more quickly in vitro compared with in vivo, but the degree and incidence of cumulus expansion was greater after in vivo maturation. The competence of oocytes to undergo fertilization and develop into embryos remains to be investigated. Table 1.Cumulus expansion and nuclear maturation of wood bison oocytes

A viable foal obtained by equine somatic cell nuclear transfer using oocytes recovered from immature follicles of live mares

The presence of heterogenous mitochondria from the host ooplast affects the acceptance of offspring obtained by somatic cell nuclear transfer. This might be avoided by obtaining oocytes from selected females, but is then complicated by low numbers of available oocytes. We examined the efficiency of equine somatic cell nuclear transfer using oocytes recovered by transvaginal aspiration of immature follicles from 11 mares. Use of metaphase I oocytes as cytoplasts and of scriptaid (a histone deacetylase inhibitor) treatment during oocyte activation were evaluated to determine if these approaches would increase blastocyst production. In experiment 1, blastocyst development was 0/14 for metaphase I oocytes and 4/103 (4%) for metaphase II oocytes. Three blastocysts were transferred to recipient mares, resulting in two pregnancies and one live foal, which died shortly after birth. In experiment 2, blastocyst development was 2/47 (4%) for control oocytes and 1/83 (1%) for scriptaid-treated oocytes. No foals were born from two blastocysts transferred in the control group. The blastocyst from the scriptaid treatment resulted in birth of a live foal. In conclusion, this is apparently the first report of production of a viable cloned foal from oocytes collected from immature follicles of live mares, supporting the possibility of cloning using oocytes from selected mares.

Effects of repeated transvaginal aspiration of immature follicles on mare health and ovarian status

Transvaginal ultrasound-guided follicle aspiration (TVA) is performed clinically but there is little information available on complications associated with this procedure. It is possible that TVA is associated with damage to the ovary and may induce peritonitis or peritoneal adhesions. This study was conducted to determine the effect of repeated TVA on mare health and ovarian status. Thirty-two mares were used for oocyte recovery via repeated TVA over a 3 year period; different mares were used each year. In Year 1, ovarian status was monitored in 11 mares by transrectal palpation and ultrasonography. In Year 2, 6 of 11 mares underwent abdominocentesis and were examined by laparoscopy after one TVA and again after multiple TVAs. In Year 3, 10 mares underwent multiple TVAs with either a 15 or a 12 gauge needle and the ovaries were removed for examination. Four hundred and twenty-seven aspiration sessions (390 via TVA and 37 via needle placement through the flank) and 3202 follicle punctures (3161 TVA and 41 flank) were performed. One mare developed an ovarian abscess. Transient rectal bleeding was evident after 16% of TVA sessions. No adhesions were found on laparoscopic or gross examination of ovaries and there were minimal changes on histological evaluation. Follicle aspiration carries a small possibility (< 0.5%) of ovarian abscess formation. There is a possibility of rectal abrasion or puncture but little gross or histological damage to the ovary. These results provide a basis for using prophylactic administration of antibiotics after TVA and for advising mare owners of the rare but potential complications associated with the procedure.

Occurrence and characteristics of residual follicles formed after transvaginal ultrasound-guided follicle aspiration in cattle

Ultrasound-guided transvaginal follicle aspiration is used to recover cumulus-oocyte complexes (for IVF) and to synchronize follicular wave emergence (ablation of dominant follicle). Although aspirated follicles are generally supposed to undergo immediate atresia, there are indications that they may remain active. The objective was to evaluate the occurrence and characteristics of residual follicles (RF) after transvaginal follicle aspiration in cattle. Ovarian follicular wave emergence was synchronized in Holstein cows (N = 13) in the presence (groups 1 and 3) or absence (groups 2 and 4) of norgestomet implants. The largest follicle was aspirated at a diameter of 8 mm (groups 1 and 2) or 12 mm (groups 3 and 4). Ovarian follicles were visualized (transrectal ultrasonography) every 12 h after wave emergence. Follicular fluid samples were collected from the largest follicle and from the ensuing RF and concentrations of estradiol and progesterone were determined. After aspiration, 73.2% (52/71) of the follicles refilled with fluid, and a new antrum was detected 12 to 24 h later. Norgestomet did not affect (P > 0.05) RF occurrence or diameter, but in RF from group 4, concentrations of estradiol decreased (−530.7 ± 133.9 ng/mL; P < 0.01) whereas progesterone increased (+429.6 ± 171.7 ng/mL; P < 0.05) relative to preaspiration. In RF, there were three steroidogenesis patterns: (1) high estradiol concentration and high estradiol:progesterone ratio (estradiol-active RF); (2) low estradiol, but high progesterone concentrations (luteinized RF); and (3) low estradiol and low progesterone concentrations (inactive RF). Estradiol-active RF were more likely (P < 0.05) from follicles with high estradiol concentrations (regardless of diameter). In conclusion, fluid-filled structures (RF) with variable steroid production patterns are frequently formed after ultrasound-guided follicle aspiration. The occurrence and features of these RF depended on the diameter and status of these follicles before aspiration.

Luteal function induced by transvaginal ultrasonic-guided follicular aspiration in mares

Ultrasonic-guided transvaginal follicle aspiration was performed in 58 crossbreed mares in order to determine whether aspiration of various dominant follicle diameters resulted in luteal tissue capable of producing progesterone (P 4). The mares were randomly assigned to three groups according to follicular diameter (25–29 mm; 30–35 mm and >35 mm). Mares that had ovulations naturally served as controls. The serum progesterone (P 4) concentrations in the aspirated mares were greater ( P < 0.0001; r 2 = 0.6687; CV = 21.52) in mares with natural ovulation compared to mares with aspirated follicles regardless of groups. Serum P 4 concentration in aspired mares with follicular diameter of 25–29 mm declined 0.365 ng/ml/day ( P = 0.0065) from the day of aspiration (D0) up to D8. In mares with follicle diameter of 30–35 mm, serum P 4 concentration increased (0.258 ng/ml/day; P = 0.001), as well as in the mares with follicles >35 mm diameter (0.481 ng/ml/day; P < 0.0001), and in mares with natural ovulation (1.236 ng/ml/day; P < 0.0001). Out of the 25 mares with follicular aspirations that formed Corpora hemorragica (P 4 >1 ng/ml), 23 (92%) had greater (>2 ng/ml) serum P 4 concentrations on Day 8 after aspiration. Of these 23 mares, 75% were in the 25–29 mm group, 9/10 (90%) in the 30–35 mm group, and 11/11 (100%) of the mares in the >35 mm follicular diameter group had luteinization (P 4 >2 ng/ml). These results suggest that a functional Corpus luteum can be induced in mares using follicular aspiration and that a minimum 35 mm follicular diameter is needed to reach a progesterone serum concentration compatible with that of a Corpus luteum produced by natural ovulation.

263 IN VITRO FERTILIZATION AND EMBRYO DEVELOPMENT OF CUMULUS - OOCYTE COMPLEXES COLLECTED BY ULTRASOUND-GUIDED FOLLICULAR ASPIRATION IN LLAMAS TREATED WITH GONADOTROPIN

We have previously documented that both FSH and eCG are equally effective in inducing ovarian superstimulation in llamas, resulting in the recovery of a high number of expanded COC suitable for in vitro fertilization (Ratto et al. 2005 Theriogenology 63, 2445-2457). The objective of the study was to evaluate the ovarian response, morphology, and competence of COC collected by ultrasound-guided follicular aspiration in llamas treated with FSH or eCG. Llamas were assigned randomly into 2 groups (n = 16 per group) and treated for 48 h after follicle ablation with (1)25 mg of FSH (Folltropin, Bioniche Animal Health Canada Inc., Belleville, Canada) i.m. twice daily for 4 d; or (2) 1000IU of eCG (Novormon, Bioniche Animal Health Canada) as a single i.m. dose. The starting of gonadotropin treatment was considered Day 0. Both groups were given an i.m. dose of 5 mg of Armour Standard LH (Lutropin, Bioniche Animal Health Canada) on Day 6, and COC were collected by transvaginal ultrasound follicle aspiration of all follicles ≥7 mm on Day 7. The ovarian response was assessed by transrectal ultrasonography using a 7.5-MHz linear-array transducer (Aloka SSD-500, Clinics, Santiago, Chile) immediately before oocyte collection at 24 to 26 h after LH treatment in both groups. The COC were classified as expanded, compact, denuded, or degenerated. Expanded COC collected from FSH- (n = 147) and eCG-treated llamas (n = 141) were fertilized in vitro using epididymal sperm as previously described (Ratto et al. 2006 Anim. Reprod. Sci. 97, 246-257). Gametes were co-incubated at 38.5°C in air with 5% CO2 and high humidity for 18 h. After in vitro fertilization, presumptive zygotes were co-culture in SOF medium supplemented with 0.6% of BSA with llama granulosa cells at 39°C, 5% CO2, 5% O2, and 90% N2 for 7 days. Embryo development was evaluated on Days 2, 5, and 7 of in vitro culture (Day 0 = IVF). Data were analyzed by Student’s t-test or Fisher’s exact test and presented as mean ± SEM. The FSH and eCG treatment groups did not differ with respect to the number of follicles ≥7 mm at the time of COC collection (16.0 ± 2.7 v. 14.0 ± 1.9; P = 0.5), the number of COC collected (11.5 ± 1.9 v. 9.7 ± 1.2; P = 0.4), or the collection rate per follicle aspirated (77.0 v. 71.5%; P = 0.2). No difference was detected between FSH and eCG-treated llamas in the number of expanded COCs (9.8 ± 1.4 v. 9.4 ± 1.2; P = 0.8). The percentage of presumptive zygotes to develop into 2 to 8 cells on Day 2 (65.3 v. 63.1), morulas on Day 5 (46.2 v. 42.5), and blastocyst stage on Day 7 (23.1 v. 20.5) did not differ (P &gt; 0.05) between FSH and eCG-treated llamas, respectively. In conclusion, FSH and eCG treatments were equally effective for ovarian superstimulation and oocyte collection. The recovery of a high number of expanded COC can be used directly for in vitro fertilization and their competence is not affected by gonadotropin treatment. The study was supported by Convenio Desempeño en Investigacion (2007-DGI-CDA-04), Universidad Catolica de Temuco.

198 EXOGENOUS CONTROL OF FOLLICULAR WAVE EMERGENCE IN WOOD BISON (BISON BISON ATHABASCAE)

Two methods for synchronizing ovarian follicular development in both wood bison (Bison bison athabascae) and plains bison (Bison bison bison) were tested as part of a project to conserve wood bison through the application of advanced reproductive technologies. A secondary objective was to test the effect of a long-acting neurolept tranquilizer, pipothiazine palmitate, on ovarian function in bison. Female wood bison (4 years old; n = 14) and plains bison (2-8 years old, n = 10), previously conditioned to daily examination in a chute, were divided randomly into 3 groups in which 1) ovarian follicles ≥5 mm were ablated by ultrasound-guided transvaginal follicle aspiration, 2) 5 mg of estradiol-17β in canola oil was given i.m. or 3) no treatment was given (control). The experiment was conducted in 3 replicates so that each animal rotated through each of the groups. Half of the bison, blocked by subspecies and treatment group, were given a single dose (150 mg) of pipothiazine palmitate i.m. The ovaries were examined daily by transrectal ultrasonography beginning 4 days before treatment and continuing until the dominant follicle of a new wave reached a diameter of 10 mm. No effect of treatment with pipothiazine palmitate was detected for any end point. In addition, no differences were detected between wood and plains bison for any end point. Consequently the pipothiazine palmitate and subspecies treatment groups were collapsed for further analyses. The interval and variation in the interval to new follicular wave emergence were compared by ANOVA. Wave emergence was detected on Day 1.0 ± 0.2 (mean ± SEM; Day 0 = day of treatment) in the follicle ablation group, and was earlier (P &lt; 0.05) than in both the estradiol (Day 3.3 ± 0.3) and control (Day 4.0 ± 0.4) groups. The interval to follicle wave emergence was least variable in the follicle ablation group (P &lt; 0.05) and tended to be less variable in the estradiol group (P = 0.09) than in the control group (residuals, 0.1 ± 0.04, 1.0 ± 0.2, and 1.6 ± 0.3 days, respectively). Ovulations occurred subsequent to estradiol administration in 10 of 23 (43%) bison. In conclusion, ovarian follicular wave emergence can be synchronized in bison during the anovulatory season. Follicular ablation consistently shortened and decreased the variability in the interval to new wave emergence. The synchronizing effect of estradiol was confounded by the induction of ovulation. Progesterone will be added in future studies to control the ovulatory effect of estradiol. Pipothiazine palmitate had no discernable effect on ovarian function and maybe useful in reducing the effects of handling stress on untrained animals. Supported by a grants from the Advancing Canadian Agriculture and Agri-Food Fund, the Agri-Food Innovation Fund, Parks Canada, the World Wildlife Fund, and the Northwest Territories.

(135) Ultrasound-guided biopsy in hepatic tumors: A study based on 247 patients

A new device was developed to hold linear transducers for transvaginal follicle aspiration. Efficacy of follicle aspiration was compared using a linear 6 MHz and a convex 5 MHz transducer. Fifty-five cows were submitted to follicle aspiration at random days of the estrous cycle. Aspirations were conducted with linear (n=28) and convex (n=38) transducers with 18 G needles at a negative pressure corresponding to 13 ml H2O/min. A greater number of follicles were aspirated using convex than to linear probe (12.4 versus 7.8, respectively, P<0.05). Mean number of oocytes and recovery rates were similar for convex (5.4 and 48.6%) and linear (4.6 and 59.3%) transducers. Limited space between the linear transducer and needle guide restricted access to some portions of the ovary, reducing the number of follicles aspirated using a linear transducer. The newly developed adaptor allowed greater stability, holding the ovaries firmly against the linear transducer. This diminished mobility permitted a similar number of oocytes to be recovered with both transducers. In conclusion, this new adaptor provided a low cost alternative for routine follicle aspiration and oocyte recovery in cattle.

Different intervals of ovum pick‐up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos

The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used as cytoplast donors. The rates of development of the reconstituted embryos to the blastocyst stage were 23.1% (Group I), 15.0% (Group II), 10.9% (Group III), 4.9% (Group IV), and 29.0% (Group V). The results indicate that the developmental potential of follicles from the same living donors were different when different intervals of OPU were adopted and early atretic follicles from the second follicular wave had higher competence to support the early development of cloned bovine embryos.