Ultrasound-guided Transvaginal Follicular Aspiration Research Articles

PSXVI-16 The quality of cows’ oocyte derived at different timing regimens of transvaginal follicular puncture

Abstract Recovery of oocytes from live animals through ultrasound-guided transvaginal follicular aspiration (Ovum Pick-Up, OPU) is an essential element of modern livestock development. The aim of our work was to study the effect of two different timing regimens on the quality of oocytes, recovered from cycled Simmental heifers (n = 4). Follicle aspirations were performed once a week (1/w) or twice a week (2/w). The same donor animals were used for each timing regimens. 228 and 224 follicles were aspirated at 2/w and 1/w regimen, respectively, and 280 cumulus-oocyte complexes (COCs) were recovered (140 COCs at each of timing regimen), which corresponds to the recovery rate of 61.4±5.9% and 62.1±6.3%. On average, 4.4 oocytes were received from each donor per session. We observed a significant increase by 1.2 times (P < 0.05) of the proportion of OPU-oocytes of good quality, which were characterized by normal morphology, when performing the procedure in the 2/w compared to 1/w regimen (65.7±4.0% vs. 53.6±3.0% of the total number of recovered oocytes, respectively). The proportion of oocytes at the MII stage of meiosis after 24 hours of maturation was similar in both groups: 73.3±5.6% (2/w regimen) and 74.7±2.4% (1/w regimen). Proportion of oocytes with signs of apoptotic changes was also not significantly differed between the groups: 8.0±3.1 and 9.8±3.3%, respectively. We did not observe the effect of the frequency of OPU sessions on the embryo cleavage rate (on average 63.5%) and the blastocysts development rate (on average 16.7%). Our data will be used to optimize the timing regimen of oocyte retrieval from living Simmental donor heifers. The studies were performed under financial support of the RFBR (project No. 20-316-90003) and the Russian Ministry of Science and Higher Education.

Effects of pretreating in vitro matured of native Thai cattle oocyte with docetaxel before vitrification on their viability

The present study was to investigate the efficacy of docetaxel (DT) at reducing microtubule damage during vitrification and maintaining the developmental competence of vitrified-warmed oocytes in native Thai cattle. Native Thai cattle cow oocyte from ultrasound-guided transvaginal follicular aspiration (UTFA) were subjected to in vitro maturation (IVM). Then, the IVM oocytes were pretreating with DT in various concentration (0.0, 1.0, 2.0 and 3.0 µM) and subjected to cryopreservation by Cryotop vitrification method. The survival rate of vitrified-warmed oocyte, cleavage and blastocyst formation rate after in vitro fertilization (IVF), and blastocyst nucleic cell numbers were evaluated in this study. Pretreatment of IVM native Thai cattle oocytes with 0.05 µM DT before vitrification resulted in significantly higher (P < 0.05) rates of oocyte survival and cleavage after IVF, and subsequent blastocyst formation on Days 7-8, hatching and hatched on Days 8-9, compared with oocytes pretreated with 0.5 and 1.0 µM DT before vitrification or those vitrified and the control group. Pretreatment of IVM native Thai cattle oocytes with 0.05, 0.5 and 1.0 µM DT before vitrification without side effects on blastocyst nucleic cell numbers. In summary, pretreatment of IVM native Thai cattle oocytes with 0.05 μM DT before vitrification improved survival of vitrified-warmed oocytes, fertilization and developmental competence.

123 Efficiency of embryo production using ovum pickup oocytes recovered from stimulated and nonstimulated cows

Recovery of oocytes from live animals through ultrasound-guided transvaginal follicular aspiration (ovum pickup, OPU) is an essential element of modern livestock development. To increase the number of OPU oocytes retrieved, hormonal stimulation is broadly applied; however, the results are ambiguous. The aim of the present study was to compare recovery rate, quality of oocytes, and efficiency of invitro production of cattle embryos using OPU oocytes derived from hormonal treated and untreated cows. The study was performed in Simmental heifers at the age of 17 to 23 months. The heifers in the first group (n=7) were previously synchronized using prostaglandin F2α (Estrumate, MSD Animal Health) and gonadotrophin-releasing hormone (GnRH; Receptal, MSD Animal Health) and then underwent superstimulation with Stimofol (Reprobiol SPRL, Belgium) in a total amount of 0.25mg of FSH and 0.05mg of LH in a treatment of 6 injections on 3 consecutive days starting on Day −4 before OPU. The heifers of the second group (n=2) underwent OPU once a week for 5 weeks (10 OPU sessions). Groups of recovered cumulus–oocyte complexes (COCs) were matured in TCM-199 containing 10% fetal calf serum, 1mM Na-pyruvate, 50μg mL−1 gentamycin, 10μg mL−1 FSH, and 10μg mL−1 LH for 24h, fertilized in Fert-TALP (Tyrode’s-albumin-lactate-pyruvate) for 18 to 20h, and cultured in CR1aa medium for 7 days. All steps of IVP were performed at 38.5°C with 5% CO2 and 90% humidity. The cleavage and blastocysts rates were assessed on Day 2 and 7, respectively. In addition, Day-7 blastocysts were fixed, and the total cell number was determined using 4′,6-diamidino-2-phenylindole (DAPI) staining. The data were analysed by ANOVA using SigmaStat software package. As expected, the number of aspirated follicles per session in the first group of cows was higher than that in the second group: 14.6±1.2 vs. 6.8±0.5 (P&amp;lt;0.05). In total, 54 and 42 COCs were collected, which corresponds to recovery rates of 54.5±7.7 and 61.3±4.9% for stimulated and nonstimulated cows, respectively. After the quality evaluation, 36 COCs of SS heifers (67.2±1.9%) and 31 COCs of non-SS heifers (75.3±5.0%) were selected for IVP. We did not observe a significant difference in cleavage rate between two groups: 77.8% in the SS group vs. 71.0% in the non-SS group, whereas the rate of blastocyst production was higher (P&amp;lt;0.05) for SS heifers (25.0%) compared with non-SS animals (16.1%). The total number of cells in Day 7 blastocysts was significantly (P&amp;lt;0.01) higher in the first group (89.8±1.3) compared with the second group (70.6±3.2). Thus, superstimulation of heifers before OPU increases the efficiency of oocyte retrieval by ultrasound-guided transvaginal follicular aspiration as well as the developmental competence of OPU-derived bovine oocytes invitro. These studies were performed under financial support of the Russian Science Foundation (project No. 19-16-00115) and the Ministry of Science and Higher Education of Russia.

First attempts for vitrification of immature oocytes in donkey (Equus asinus): Comparison of two vitrification methods

Most wild donkey breeds are severely threatened by poaching for meat, habitat loss, and competition with livestock for food resources. Moreover, due to the mechanization in agriculture and in transport, most domestic donkey breeds are at risk of extinction. Considering the importance of biodiversity and preservation of genetic resources, the creation of genetic banks for endangered donkey breeds is urgently needed. Cryopreservation of immature jennies oocytes would be an efficient tool to allow storage of female genetics. The aim of the present study was to establish conditions for immature donkey oocyte vitrification, using equine oocytes as a control. Asine and equine immature cumulus-oocyte complexes were collected by transvaginal ultrasound-guided follicular aspiration and flushed to obtain oocytes surrounded by only corona radiata. Oocytes were vitrified after exposure to increasing concentrations of dimethyl sulfoxide, ethylene glycol and sucrose as cryoprotectants in a solution of INRA-Freeze™ medium or TCM199-Hepes supplemented with bovine serum albumin. Oocytes were warmed in decreasing concentrations of sucrose and processed for in vitro maturation. The recovery rate was 48% for jennies oocytes (4.8 oocyte per female) and 42% for mares oocytes (3.5 oocyte per female). When oocytes were exposed to cryoprotectants in INRA-Freeze™ medium none of the jennies re-warmed oocytes matured, whereas 24% of the mares re-warmed oocytes reached metaphase II after in vitro maturation. When oocytes were exposed to cryoprotectants in TCM199-Hepes-BSA medium, 33% of the jennies re-warmed oocytes matured. In conclusion, we developed a method for the vitrification of immature oocytes from jennies that allows in vitro maturation of the vitrified-warmed asine oocytes. Their competence for fertilization and development has to be ascertain.

Comparing transabdominal and transvaginal ultrasound-guided follicular aspiration: A risk assessment formula

ObjectiveWe sought to identify patients at risk of incomplete transvaginal oocyte retrieval, develop a risk assessment formula to identify patients who would benefit from a transabdominal approach, and compare complication and pregnancy rates between these two approaches. Materials and MethodsIn this retrospective case control study in a private in vitro fertilization center, 95 cases of women undergoing transabdominal follicular aspiration for oocyte retrieval (15 transabdominal only and 80 transabdominal and vaginal combined) were compared with 278 controls of women undergoing the transvaginal aspiration only. Transabdominal oocyte retrieval was performed when one or more ovaries could not be retrieved via the transvaginal approach. Main study outcomes included need for transabdominal retrieval, pregnancy rates, and complications. ResultsA risk assessment scoring system was developed as follows: difficulty seeing ovaries on ultrasound (+4), history of pelvic surgery (+3), and body mass index of 30 kg/m2 or greater (+2). With a cutoff score of 4 or greater, the overall sensitivity is 75%, specificity is 80%, positive predictive value is 57%, and negative predictive value is 90%. No statistically significant differences were found for pregnancy rates or complications. ConclusionThe transabdominal approach is an alternative option that would increase the total number of oocytes retrieved with no statistical difference in complication or pregnancy rates. We also developed a scoring system that can serve as a useful screening tool for identifying women at increased risk of transabdominal oocyte retrieval.

In Vitro Embryo Production and Transfer of Bubaline Embryos using Oocytes Derived from Transvaginal Ultrasound-Guided Follicular Aspiration (TUFA)

Transvaginal ultrasound-guided follicular aspiration (TUFA) has become a popular tool for embryo production in vitro due to its high degree of repeatability in terms of recovering oocytes from live animals. In Study 1, the quantity and quality of oocytes from Bulgarian Murrah buffalo cows (n=10) of varying ages (Group 1, 8-12; and Group 2, 13-17 years) were assessed. Group 1 buffalo donor cows yielded significantly higher (P&lt;0.05) number of oocytes vs Group 2 buffalo donor cows (71 vs 29 oocytes, respectively), though in terms of oocyte quality, no difference was observed. In Study 2, oocytes collected (n=100) in Study 1 were matured, fertilized in vitro and the resulting zygotes were cultured which developed to blastocyst stage embryos. The maturation, fertilization and blastocyst development rates obtained were 53.0%, 40.0% and 32.5%, respectively. In Study 3, the viability of resulting blastocyst stage embryos was determined by transferring to recipient cows. Of 10 recipients 1 got pregnant and delivered a 35 kg male calf after 310 days gestation period. Overall, the results of the studies conducted demonstrated the potential of TUFA technology in the in vitro production of embryos which eventually could be used in the production of live offspring.DOI: http://dx.doi.org/10.3126/ijasbt.v2i2.10369Int J Appl Sci Biotechnol, Vol. 2(2): 180-184

Effect of potential oocyte transport protocols on blastocyst rates after intracytoplasmic sperm injection in the horse

Intracytoplasmic sperm injection (ICSI) is used to produce foals from otherwise infertile mares and from stallions with limited sperm stores, but requires expensive equipment and is technically demanding. Methods to transport oocytes to ICSI laboratories would allow collection of oocytes by the referring veterinarian and enable greater application of this technique. This study was conducted to evaluate protocols that could be used to transport immature and maturing oocytes for ICSI. In vitro experiment. Oocytes were recovered by transvaginal ultrasound-guided follicular aspiration either from dominant follicles 24 h after deslorelin administration (dominant stimulated follicle [DSF]), or from subordinate (immature) follicles at the same time. To mimic transport, DSF oocytes were incubated overnight under differing conditions before ICSI; immature oocytes were placed in varying conditions overnight before in vitro maturation, followed by ICSI. The rate of blastocyst production was compared among treatments. Blastocysts were produced in all groups. Dominant stimulated follicle oocytes held in sealed tubes in pre-equilibrated control maturation medium maintained at 37°C yielded blastocyst development equal to that obtained for control incubated oocytes (70%). Dominant stimulated follicle oocytes held similarly in a warm passive device yielded poor blastocyst development (10%). Immature oocytes held for one or 2 nights in modified M199 medium, or for one night in commercial embryo holding solution, in air at room temperature, yielded 35-37% blastocyst development per injected oocyte. A commercially available medium can be used for shipping immature oocytes at room temperature with good resulting blastocyst rates. Better blastocyst rates per oocyte are obtained from DSF oocytes; however, these require maintenance at 37°C and as they are already maturing at the time of collection, are more sensitive to delays. This new, practical information supporting transport of both immature and DSF oocytes for ICSI may allow wider use of this procedure.

Ovarian superstimulation and oocyte collection in wood bison (Bison bison athabascae) during the ovulatory season

The objective of the study was to establish an effective ovarian superstimulatory protocol and subsequently obtain oocytes from bison by transvaginal ultrasound-guided follicular aspiration. Two experiments involving 22 wood bison were done during the breeding season (September to December). In experiment 1, the bison were given a luteolytic dose of prostaglandin (Day 0) and underwent follicular ablation (Day 8) to induce ovarian synchrony. Synchronized bison were then assigned randomly to two groups (n = 11 per group) and given either 200 mg FSH diluted in saline sc, or 200 mg FSH diluted in a proprietary slow-release formulation (SRF) im on Days 9 and 11. Prostaglandin was given to both groups on Day 11 followed by 25 mg LH on Day 13. Oocytes were collected by transvaginal ultrasound-guided aspiration of follicles ≥5 mm on Day 14. In experiment 2, bison were synchronized as in experiment 1 and assigned randomly to one of two groups (n = 11 per group) and given either a single dose of 2500 IU eCG im on Day 9, or 200 mg FSH sc on Days 9 and 11. Prostaglandin was given to both groups on Day 11, and LH (25 mg) was given on Day 13. Oocyte collection was done as described in experiment 1. Cumulus-oocyte-complexes (COC) were classified according to morphologic characteristics. In experiment 1, more follicles ≥5 mm were detected on Day 14 in bison treated with FSH versus eCG (12.2 ± 1.73 vs. 5.8 ± 0.52; P < 0.05), and more COC were collected from FSH-treated animals (7.2 ± 1.41 vs. 3.4 ± 0.62; P < 0.05). In experiment 2, the FSH-saline and FSH-SRF groups had a similar number (mean value ± standard error of the mean) of follicles ≥5 mm on Day 14 (12.4 ± 1.49 vs. 13.8 ± 1.24, respectively) and a similar number of COC were collected (6.5 ± 1.13 vs. 6.3 ± 0.96, respectively). The proportion of COC collected per follicle aspirated and the percentage of compact, expanded, and denuded oocytes did not differ between groups in either experiment 1 or 2. In summary, a two-dose regimen of FSH diluted in saline and given sc or in a SRF and given im induced a similar ovarian response in wood bison, whereas a single dose of eCG resulted in a significantly lower ovarian response. Overall, COC were collected from 55% of follicles after transvaginal, ultrasound-guided needle aspiration in wood bison.

Ultrasound-guided Transvaginal Follicular Aspiration and Development of Vitrified-thawed Thai Indigenous Beef Cattle (Bos indicus) Oocytes after in vitro Fertilization

Ultrasound-guided Transvaginal Follicular Aspiration and Development of Vitrified-thawed Thai Indigenous Beef Cattle (Bos indicus) Oocytes after in vitro Fertilization

Normal Calves Produced After Transfer of Embryos Cultured in a Chemically Defined Medium Supplemented with Epidermal Growth Factor and Insulin-like Growth Factor I Following Ovum Pick Up and &lt;i&gt;In Vitro&lt;/i&gt; Fertilization in Japanese Black Cows

The objective of this study was to examine whether high concentrations of epidermal growth factor (EGF) and/or insulin-like growth factor I (IGF-I) would have a beneficial effect on bovine embryo development in vitro and to obtain normal calves by using an ovum pick up method and embryo culture in a chemically defined medium. When compared with controls, EGF (100 or 200 ng/ml) or IGF-I (50 or 100 ng/ml) significantly increased the rate of embryos that developed into blastocysts during an 8-day culture after the in vitro fertilization of oocytes obtained from ovaries from a slaughterhouse. IGF-I induced a dose-dependent increase in cell number in both the inner cell mass and the trophectoderm, whereas EGF stimulated proliferation only in the inner cell mass. A combination of EGF (100 ng/ml) and IGF-I (50 ng/ml) produced an additive effect, and embryos developed into blastocysts at a comparatively high rate (27.9%) compared with controls (12.0%). A similar rate of development was achieved using a combination of EGF and IGF-I in the culture of embryos following ovum pick up by ultrasound-guided transvaginal follicular aspiration and in vitro fertilization, and 5 blastocysts that developed after the culture were transferred into uteri; two embryos implanted, and normal calves were born. These results suggest that the combined use of EGF and IGF-I makes bovine embryo culture in a chemically defined medium a practical and useful procedure for producing blastocysts, and its application to embryo culture following ovum pick up and in vitro fertilization could be useful for producing normal calves.

125 FOLLICULAR FLUID AND GRANULOSA CELL RECOVERY FROM BOVINE FOLLICLES OF DIFFERENT DIAMETERS USING AN ADAPTED TRANSVAGINAL-GUIDED FOLLICULAR ASPIRATION SYSTEM

Most studies of granulosa cell (GC) function have been performed in vitro, using follicular fluid (FF) and GC recovered from slaughterhouse ovaries. This approach does not consider the reproductive status and follicular developmental stage of the donor, limiting data interpretation and usefulness. The aim of this study was to evaluate the efficiency of an adapted ultrasound-guided transvaginal follicular aspiration (TVFA) procedure to recover FF and mural GC from live cows. A preliminary in vitro trial was performed to calculate fluid losses using a conventional TVFA circuit. A known volume of PBS, expected to be the volume of FF in follicles ranging from 4 to 12 mm in diameter, was aspirated using a 20 G needle connected to a Teflon circuit 80 cm long, with a 1.0-mm internal diameter, connected to a 1.5-mL tube. Losses of the expected volume of FF were 12.7 ± 1.1, 19.9 ± 2.5, 54.4 ± 4.0, 87.6 ± 4.3 and 100%, for follicles of 12, 10, 8, 6 and 4 mm in diameter (0.90, 0.52, 0.27, 0.11 and 0.03 mL, respectively). When an adapted system for small-volume recoveries was used, there was a decrease (P &lt; 0.05) in fluid losses for follicles of 8, 6 and 4 mm. An experiment was performed in vivo, using the adapted system, to evaluate FF and GC recovery from follicles of 4 and 5 (n = 16), 6 (n = 19), 7 (n = 7), 8 (n = 13), 9 (n = 15), 10 (n = 24), 11 (n = 11), 12 (n = 15), 13 (n = 13), 14 (n = 6) and 16 (n = 9) mm in diameter. Follicular wave emergence was synchronized with 2 mg of oestradiol benzoate and an intravaginal progesterone device and follicles that reached the desired diameter were aspirated using an ultrasound machine equipped with a 7.5-MHz probe and disposable 20 G needle. The recovered FF volume was measured and centrifuged at 600 × g for 10 min. The GC pellet was vortexed with 0.1% hyaluronidase (5 min) and washed twice in PBS and the number of cells was determined using a Neubauer chamber. Ribonucleic acid was extracted using an RNAeasy Microkit and quantified in NanoDrop. The efficiency of FF recovery was estimated by the difference between the recovered and expected volumes for each follicle diameter (4/3πr3), which were compared by ANOVA. From all the follicles aspirated, the recovery of FF and GC was not successful in 2 (1.3%). Overall, FF recovery efficiency was 84.7%. The recovered volume ranged from 0.03 to 3.80 mL and increased with follicular diameter (y = 0.011x2 – 0.012x + 0.043; R2 = 0.99). Losses of FF were significant (P &lt; 0.05) for follicles larger than 12 mm. The mean (±s.e.m.) number of mural GC recovered was 716,708 ± 68,536, providing 6.8 ± 0.7 samples of 100 000 cells with 14.8 ± 0.7 ng of RNA μL–1 for each punctured follicle. A high coefficient of variation (57.4%) was observed in cell recovery. There was no difference in the number of cells recovered from follicles of different diameters, but sample contamination with blood was more frequent (75%) in follicles larger than 10 mm. In conclusion, an adapted TVFA system can be used successfully on an individual basis and from follicles of different diameters for in vivo recovery of FF and GC for further endocrine and gene expression analyses. This research was supported by CNPq and Fapemig (CVZ APQ 02863/09).

Limitations of ultrasound guided follicular aspiration for analysis of ovarian follicular fluid in dairy cattle

The aim of this study was to evaluate the applicability of ovum pick-up equipment for follicular fluid collection from various follicular structures (experiment 1) and for recovery of follicular fluid for acid-base balance analysis (experiment 2). An ultrasound scanner equipped with a 5-MHz convex transducer was used for transvaginal ultrasound-guided follicular aspiration. A 17-gauge, 60-cm aspiration needle was connected with a shortened aspiration line. The fluid was aspirated manually into a 2 ml plastic syringe at a speed of approximately 0.2 ml/s. The success of aspiration was higher in ovarian cysts (100%) and single follicles larger than 13 mm (76.7%) compared to single follicles smaller than 12 mm (20%,p&lt; 0.001). The success of aspiration of multiple follicles on day 4 (diameter of 7–9 mm) was higher (90.9%) compared to follicles on day 2 (diameter of 4-6 mm) (66.7%,p&lt; 0.05) in experiment 1. The fluid from ovarian cysts &gt; 25 mm in diameter was aspirated in a two-step procedure (samples 1 and 2) for the determination of pH, HCO3,BE, pCO2and pO2(experiment 2). The indicators were compared between samples 1 and 2. Higher pO2as well as pH and lower pCO2in sample 1 compared to sample 2 showed insufficient anaerobic conditions during the first phase of the puncture in experiment 2. Our study brings for the first time the finding that the ovum pick-up equipment used in our experiments is suitable for the collection of follicular fluid only from larger follicular structures. The sampling of follicular fluid for acid-base balance assays requires the development of a special new device to prevent samples from coming into contact with air during aspiration.

Collection of Oocytes Through Transvaginal Ovum Pick‐up for In Vitro Embryo Production in Nanyang Yellow Cattle

The objectives of this study were to use ultrasound-guided transvaginal follicular aspiration to develop a technique for preservation of Nanyang Yellow cattle germ plasm. In Nanyang Yellow heifers, aspiration twice per week yielded a higher mean numbers of aspirated follicles, recovered oocytes and viable oocytes per session than once-weekly aspiration. Intriguingly, twice-weekly aspiration from Holstein heifers yielded a higher mean numbers of aspirated follicles, recovered oocytes and viable oocytes per session than in Nanyang Yellow heifers. Moreover, the oocyte recovery rate was significantly higher in Holstein heifers than in Nanyang Yellow heifers. Importantly, similar rates of embryos undergoing cleavage division were observed in Nanyang Yellow and Holstein, as was the number of viable day 8 blastocysts. These observations demonstrate that twice-weekly aspiration harvested the similar proportions of viable oocytes from Nanyang Yellow and Holstein follicles, and that twice-weekly aspiration yields a higher number of viable oocytes than once-weekly aspiration in Nanyang Yellow heifers. The results also suggest that the combination of ultrasound-guided transvaginal follicular aspiration and in vitro production is an effective and practical strategy for establishment of a germ plasm preservation programme for Nanyang Yellow cattle.

159 TECHNIQUES FOR OVUM PICK-UP IN GONADOTROPIN-TREATED ALPACAS

The objective of the present study was to evaluate the quantity and quality of oocytes collected when using 2 methods for ovum pick-up and 2 different regimens for ovarian stimulation in live alpaca donors. Thirty-four non-pregnant female alpacas of 3 to 5 years of age maintained at 4100 m elevation in southern Peru were randomly distributed into 4 experimental groups. Groups 1 (n = 8) and 3 (n = 9) received an intravaginal device containing 0.78 mg of progesterone (Cue-Mate�, Bioniche Animal Health, Belleville, Ontario, Canada) plus an i.m. injection of 1 mg of estradiol benzoate on Day 0; the intravaginal device was removed on Day 7. Groups 2 (n = 7) and 4 (n = 10) received an i.m. injection of 3.1 mg of LH (Lutropin�, Bioniche Animal Health) on Day 0. Females received 700 IU of eCG (Pregnecol�, Bioniche Animal Health) i.m. on Day 7 (Groups 1 and 3) or Day 2 (Groups 2 and 4). In all groups, oocyte collection was done 2 days after the injection of eCG. Groups 1 and 2 were subjected to ventral laparotomy aspirating the oocytes from follicles &gt;3 mm in diameter using a 10-mL hypodermic syringe containing 1 mL of aspiration media (Ringer's lactate solution plus 10% bovine serum) and connected to an 18 G � 1 inch aspiration needle. After collection, the follicular fluid was searched and the COC were graded. Groups 3 and 4 were subjected to ovum pick-up by transvaginal recovery using an ultrasound scanner (Parus 240�, Pie Medical, Maastricht, the Netherlands) equipped with a vaginal probe of 7.5 MHz (MEVA�, Pie Medical) and a 17G � 55 cm aspiration needle introduced through a needle guide. Follicles &gt;3 mm in diameter were aspirated into 50-mL centrifuge tubes containing 5 mL of aspiration media with 75 IU mL–1 of heparin. The aspirated fluid was filtered and rinsed using an embryo filter (EmCon�, Immunosystems, Menomonie, WI), and COC were searched and graded under a microscope based on the intactness of the cumulus cell layers. Data were analyzed by ANOVA. There were no differences (P &gt; 0.05) between groups in the mean number of follicles aspirated per donor (11.0, 13.8, 9.4, and 9.1 for Groups 1 to 4 respectively), and in the mean number of COC recovered per donor (7.6, 7.0, 6.0, and 6.1 respectively for Groups 1 to 4). The proportions of good quality COC were significantly (P &lt; 0.01) different between surgical (81.0 and 79.5% for Groups 1 and 2) and transvaginal/ultrasound-guided (7.4% for Group 3) methods of collection; however, they were similar to the proportion in Group 4 (64.9%) retrievals. The results show that in the absence of an intravaginal device, a similar quantity and quality of alpaca oocytes can be collected when using a surgical approach or minimally invasive ultrasound-guided transvaginal follicular aspiration.

249 INTRACYTOPLASMIC SPERM INJECTION OF ELAND (TAUROTRAGUS ORYX) AND BONGO (TRAGELAPHUS EURYCERUS)ANTELOPE OOCYTES

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique applicable in cases of limited male gamete availability. Moreover, it bypasses barriers of the oocyte, thus avoiding poorly understood species-specific capacitation events affecting sperm–egg interaction. In the present study, we evaluated the application of conventional and piezo drill-assisted ICSI and whether subsequent chemical activation is required for initiating embryonic development in eland (Taurotragus oryx) and bongo (Tragelaphus eurycerus) oocytes. Oocytes were collected using transvaginal ultrasound-guided follicular aspiration after gonadotropin-induced ovarian stimulation and incubated in modified TCM-199 medium (Gomez et al. 2000 Reprod. Fertil. Dev. 12, 423) containing 10% FBS. After 3 to 24 h, the cumulus cell layers were removed either by repeated mouth-pipetting and/or by using hyaluronidase. Oocytes with an extruded first polar body were used for ICSI and the other oocytes were returned to culture and evaluated every six hours Piezo drill-assisted (Kimura and Yanagimachi 1995 Biol. Reprod. 52, 709) or conventional (Gomez et al.) ICSI were done as described previously using glass pipettes with internal tip diameters of 9–10 µm. We used frozen–thawed or freshly collected spermatozoa that were kept in HEPES-buffered Tyrode's medium (Gomez et al.) for up to 24 h. Four to 6 h after ICSI, 3 activation treatments were examined: (1) none; (2) 7% ethanol, 5 min; or (3) calcium ionophore (5 µM, 5 min) followed by DMAP (2 mM, 4 h). Then we cultured oocytes in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 at 38.5°C in one of 3 media: SOF, α-MEM, or CR1aa containing essential and nonessential amino acids and FBS. Fifty-three of 70 (76%) eland oocytes survived after piezo-ICSI, and 13 of 16 (81%) survived after conventional ICSI. For bongo oocytes, 27 of 30 (90%) survived piezo-ICSI and all (n = 8) survived after conventional ICSI. Table 1 outlines cleavage data on Day 2. Generally, embryonic development was arrested at about 10 cells. In summary, eland and bongo oocytes can survive both conventional and piezo drill-assisted ICSI. Activation treatments do not appear to be a prerequisite for initiating cleavage after ICSI in eland and bongo antelope oocytes. Table 1.Cleavage of eland and bongo antelope oocytes after conventional or piezo-ICSI and three activation treatments

Aspiration of oocytes from transitional, cycling, and pregnant mares

The aim of this study was to compare the efficacy of three approaches for recovering equine oocytes via transvaginal ultrasound-guided follicular aspiration. Fourteen mares were used as oocyte donors during the spring transition period and physiologic breeding season, and 11 mares were bred for use as oocyte donors during early gestation. In all mares, large (>20 mm) and small (10–20 mm) follicles were aspirated in eight rounds every 10–11 days. In each of the four rounds during the transition period, half the mares received 12.5 mg eFSH once daily for 4 days prior to aspiration. For each of the four rounds during the cycling season, half the mares received 12.5 mg eFSH twice daily for 3 days prior to aspiration. Pregnant mares were aspirated on days 25, 40 and 55 of gestation and received no eFSH. There were more large (>20 mm) follicles in cycling controls (2.25 ± 0.27) and cycling FSH-treated (2.64 ± 0.27) mares than in transitional FSH-treated mares (1.18 ± 0.27). The number of oocytes recovered from small (10–20 mm) follicles varied by mare ( P < 0.05) and averaged 1.08 ± 0.22 per aspiration for transitional mares and 1.23 ± 0.22 per aspiration for cycling mares ( P > 0.1). The number of oocytes per aspiration from large follicles was greater in cycling FSH-treated mares (0.46 ± 0.09) than in transitional control mares (0.11 ± 0.09). In pregnant mares, more large follicles were present at day 25 than at any other time, and the number of oocytes per aspiration from large follicles was greater at day 25 (0.73 ± 0.16) than at day 55 (0.04 ± 0.18). When compared across all seasons and treatments, the day 25 pregnant mares yielded the greatest number of oocytes per aspiration (2.91 ± 0.66 per mare).

416 EFFECTS OF THE CORPUS LUTEUM WITHIN THE OVARY ON THE FOLLICULAR DYNAMICS AFTER FOLLICULAR ASPIRATION AND ON THE DEVELOPMENTAL COMPETENCE OF ASPIRATED OOCYTES

The use of ultrasound-guided transvaginal follicular aspiration combined with in vitro embryo production is increasing in the commercial cattle breeding industry. However, frequent puncture of follicles can induce the dysfunction of corpora lutea (CL) (Petyim et al. 2001 J. Vet. Med. A 48, 449–463), resulting in irregular estrous intervals or absence of estrus. This study was conducted to compare follicular and oocyte characteristics in ovaries ipsilateral vs. contralateral to the CL after follicular aspiration. The experiment was performed using a crossover design with each Japanese Black cow (n = 7) assigned to each of the 3 groups. The treatment period was 30 days for each group, and successive treatments were separated by a 2-month rest period. Hence the study spanned a period of 7 months (3 treatment periods and 2 rest periods). To verify ovulation prior to each treatment, the stage of the estrous cycle in each cow was synchronized with an ovulation synchronization (Ovsynch) program. Follicular aspirations were performed 6 times (every 3 or 4 days) between Day 3 and Day 20 after GnRH administration (Day = 0) of the Ovsynch program. All cows in each group ovulated the follicle before the initiation of follicular aspiration treatment. Follicles (e3 mm in diameter) from the ovary ipsilateral (Group 1) or contralateral (Group 2) to the CL were aspirated a using B-mode ultrasound scanner equipped with a 7.5 MHz probe. As a control (Group 3), the follicles from both ovaries were aspirated, irrespective of the presence of CL. In all groups, follicular development in each cow was monitored daily from Days 0 to 31. The cumulus–oocyte complexes (COCs) collected from each cow in Group 1 (n = 158), Group 2 (n = 140), and Group 3 (n = 277) were subjected to IVM/IVF/IVC. The numbers of embryos cleaved and developed to the blastocyst stage were recorded 3 days and 8 days after IVF, respectively. The mean profiles of total visible follicles (e3 mm in diameter) in ovaries with follicular aspiration in Groups 1 and 2 were similar to those in Group 3, irrespective of the presence of CL. The average numbers of visible follicles in the ovaries with follicular aspiration 7 days after the end of follicular aspiration treatment were not different from those in the ovaries without follicular aspiration (P &amp;gt; 0.05; one-way ANOVA). Moreover, there were no significant differences among the groups with respect to the percentages of cleavage (57.5–65.7%) and development to the blastocyst stage (23.6–32.1%) of collected COCs (P &amp;gt; 0.05; chi-square analysis). These results indicate that the presence of CL within the ovaries does not affect the ovarian follicular dynamics after follicular aspiration and the developmental competence of collected COCs.

Impact of Nutrition on Oocyte Quality: Cumulative Effects of Body Composition and Diet Leading to Hyperinsulinemia in Cattle1

The present study sought to assess the combined effects of body composition and diet (level of feeding) on the postfertilization developmental potential of oocytes recovered from heifers using ultrasound-guided transvaginal follicular aspiration and to relate oocyte quality to the metabolic status of these animals. By collecting oocytes on repeated occasions spanning several weeks, it was possible to assess the cumulative effects of changes in nutritional status on oocyte quality over this period. Twenty-four heifers of low and moderate body condition were placed on one of two levels of feeding (equivalent to once or twice the maintenance requirements of these animals). Oocytes were recovered at two defined time points within each of three successive estrous cycles and were matured, fertilized, and cultured to the blastocyst stage in vitro. The results show that the effect of feeding level on oocyte quality is dependent on the body condition of the animal, with the high level of feeding being beneficial to oocytes from animals of low body condition but detrimental to oocytes from animals of moderately high body condition. Furthermore, the effects of high levels of feeding on oocyte quality were cumulative, with blastocyst yields for relatively fat heifers on twice the maintenance requirement deteriorating with time relative to yields for relatively thin heifers on the same level of feeding. Finally, a significant proportion of the moderately fat animals on the high level of feeding were hyperinsulinemic, and we show, to our knowledge for the first time in ruminants, that this condition is associated with impaired oocyte quality.

238 THE EFFECT OF TRANSVAGINAL FOLLICULAR ASPIRATION AND THE ROLE OF PROGESTERONE IN FOLLICULAR CYSTS OF DAIRY CATTLE

Ovarian follicular cyst (CYS) is a common condition affecting 5-25% of postpartum dairy cows, increasing the number of days open, calving intervals, cow costs, and culling rates, and with an effect on fertility rates. Current hormonal treatment has variable responses. Debate exists regarding progesterone (P4) influence on CYS formation and the effect of the aspirated CYS in resumption of cyclicity. The objectives of this descriptive study were to investigate the effect of ultrasound-guided transvaginal follicular aspiration on the resumption of follicular dynamics, to determine the fate of the aspirated CYS, and to find the P4 relationship. Resumption of ovarian cyclicity was determined by ovulation, formation of a corpus luteum (CL), and a P4 rise over 3.2 nmol/L. Ten (10) postpartum (35-60 days in milk (DIM)) lactating Holstein cows with CYS were selected. They were monitored every other day by rectal palpation, ultrasonography, and P4 concentration for 30 days post-aspiration. Change in size of the aspirated CYS and emergence of a new follicular wave were recorded and images were saved electronically. Results were analyzed using Pearson correlations, linear regression, and one-way ANOVA. Of these cows, 5/10, 3/10, and 1/10 resumed cyclicity after the 1st, 2nd, and 3rd aspirations, respectively. Two (2/10) cows did not resume cyclicity during the observation period. Size of aspirated CYS was 34.6 � 0.2 mm (mean � SEM). Seven (7/10) cows had intermediate (0.98 � 0.3 nmol/L) and 3/10 cows had high (8.6 � 3.6 nmol/L) P4 concentration at aspiration. Mean times from aspiration to emergence of a new follicle, to ovulation, and to detection of a new CL were 3.6 � 1.4, 10.5 � 2.0, and 15.2 � 3.0 days, respectively. New cysts grew in 4/10 cows after the first aspiration and one aspirated CYS refilled twice. Dominant follicles present at first aspiration (DFPF) had the tendency to undergo cyst turnover (4/9) or to ovulate (2/9) at a larger size (25.7 � 3.2 mm). The CYS size was correlated (r = 0.58; P = 0.05) with circulating P4 concentration. These data, together with a significant (P = 0.05) drop in P4 concentration following aspiration (from 3.26 � 1.5 to 2.16 � 0.1 nmol/L), suggest that the CYS may be the source of P4. Persistence of the aspirated cyst was associated (r = 0.87) with increased days to ovulation. The significant association (r = 0.96; P = 0.01) between P4 at ovulation (2.29 � 0.6 nmol/L) and the size of the ovulatory follicle (18.5 � 2.0 mm) may explain the tendency of existing follicles to persist (cyst turnover) or to ovulate at a larger size. In summary, follicular aspiration was successful in inducing resumption of cyclicity and shortening days to ovulation in half of the cases with one aspiration. Persistence of the aspirated CYS increased days to ovulation. P4 may have a follicular origin and an influence on the size of the cyst and the ovulatory follicle. DFPF tend to undergo cyst turnover or to ovulate at a larger size. However, resumption of normal cyclicity may also be influenced by other factors, such as stress, disease, and energy balance.

272OVUM RECOVERY AND BLASTOCYST DEVELOPMENT FOLLOWING INTRACYTOPLASMIC SPERM INJECTION IN CHIMPANZEES

In the present study, we report on oocyte collection, intracytoplasmic sperm injection and early embryogenesis in chimpanzees. Eight adult female chimpanzees, 11–27 years of age, received a single s.c. injection of 3.75mg GnRH (Leuplin, Takeda Co. Ltd., Osaka, Japan) 1 to 3 days after the beginning of menstruation. Daily i.m. injections of hMG (Humegon, Nippon Organon K.K., Tokyo, Japan) were initiated the following day. The dose of hMG was altered from 75 to 300IU according to serum estradiol levels. When at least one follicle of 17mm or more in diameter was observed, 10000IU of hCG (Pregnyl, Nippon Organon K.K.) were administered by i.m injection. Oocytes were recovered by ultrasound-guided transvaginal follicular aspiration 30.5 to 35.5h after hCG injection. Mature oocytes were denuded of cumulus cells by treatment with 0.1% hyaluronidase, and injected with a frozen-thawed or fresh spermatozoan using a Piezo-driven micromanipulator. Zygotes were cultured in Quinn’s Advantage Fertilization Medium (Cooper Surgical, Inc., Trumbull, CT, USA) with 10 serum protein substitute (SPS) at 37°C in a 5% CO2 atmosphere until the pronucleus stage. The medium was replaced by Quinn’s Advantage Cleavage Medium with 10 SPS from the pronuclear to 8-cell stage, and Quinn’s Advantage Blastcyst Medium with 10 SPS, thereafter. Mild ovarian hyperstimulation syndrome (OHSS) occurred in one female chimpanzee with estradiol levels of 7520pgmL−1. No oocytes were collected from 2 chimpanzees in which large follicles were observed. Thirty-five mature oocytes, one immature oocyte and 6 degenerate/fragmented oocytes were retrieved from 6 chimpanzees, including the one with OHSS. Among 35 mature oocytes injected with spermatozoa, 26 oocytes (74%) produced two pronuclei;; 23 zygotes (66%) cleaved to the 2-cell stage, 22 (63%) to the 4-cell stage, 14 (40%) to the 8-cell stage, and 9 (26%) to the morula stage. Seven zygotes (20%) developed to the blastocyst stage by 120h. There were no differences in fertilization rate or early embryogenesis between frozen and fresh spermatozoa. Results indicate that techniques used for human-assisted reproduction may be applicable to the chimpanzee to help preserve this endangered species.