Transvaginal Ultrasound-guided Ovum Pick-up Research Articles

Ovum Pick Up and in vitro Embryo Production using Sex Sorted Semen in Cows

Background: Ovum pick-up, a technique for extracting immature oocytes from antral follicles, with in vitro embryo production, is a way to produce large number of embryos from live donors. Methods: By transvaginal ultrasound-guided ovum pick-up, oocytes were collected from large (9-19 mm), medium (4-8 mm) and small (2-3 mm) ovarian follicles of donor cows (n=8). The recovered oocytes were classified as A, B, C, D and E grades. Oocytes were subjected toin vitro maturation, fertilization (with sex sorted semen) and culture. Cleavage and blastocyst rates were recorded on day 4 and day 7 of culture respectively. Result: Total of 278 follicles were aspirated. The follicles aspirated per cow per OPU session was 12.67±1.86. The oocyte recovery rate was 45.31±7.35. Mean percentage of grade A, B, C, D and E oocytes was 15.69±3.91, 24.97±3.02, 37.53±3.45, 19.44±3.76 and 2.38±2.38 respectively. In vitro maturation rate (IVM%) was 91.71±2.38% and fertilization rate was 90.18±1.52%. Cleavage and blastocyst rates observed was 55.1±5.90 and 24.81±2.95% respectively. The mean percentages of different grades of embryos observed was, code 1: 21.13±2.78, code 2: 14.41±3.25, code 3: 19.56±4.11 and code 4: 44.9±5.90%. Transvaginal ovum pick up can be performed repeatedly without hormonal stimulation. Sex sorted semen can be used for in vitro production of female embryos.

Experiences with transvaginal Ovum Pick-Up (OPU) in sows

Transvaginal ultrasound-guided Ovum Pick-Up (OPU) is an established technique in other species. Due to several challenges, there are few publications addressing the procedure in sows. An efficient OPU technique may allow for the collection of numerous oocytes from valuable sows for porcine in vitro embryo production, gene editing and cloning programmes, or cryopreservation. We aimed to improve transvaginal OPU and equipment for this technique in sows. In experiment 1, we conducted 13 OPU sessions on three Landrace x Large White hybrid sows under general anaesthesia, while the second experiment explored OPU in non-sedated animals (N = 6) physically restrained in a commercial claw trimming chute. The experiments resulted in 6.6 ± 5.6 (mean ± SD) and 7.7 ± 8.9 recovered cumulus-oocyte complexes per session, respectively. Post-mortem examination of the pelvic and abdominal cavities of the three sows subjected to repeated OPU sessions did not reveal major acute or chronic pathological lesions. The only sow which was inseminated after the experiment delivered 16 liveborn piglets at term. Salivary cortisol levels increased during the procedure in non-sedated and physically restrained sows but returned to baseline 1 h later (n = 5), indicating a short-term stress response. The described OPU technique and equipment have the potential to retrieve considerable numbers of oocytes by repeated procedures on valuable mature sows. Follow-up studies are needed to optimize the efficiency of the aspiration of high-quality oocytes and to describe the developmental competence of these OPU-derived oocytes. It is also essential to further investigate sow welfare during and after the procedure before recommending porcine transvaginal OPU as a sustainable and welfare-friendly procedure.

Experimentally Induced Endometritis Impairs the Developmental Capacity of Bovine Oocytes†.

Uterine infection is associated with infertility in women and dairy cows, even after the resolution of infection. However, the mechanisms causing this persistent infertility are unclear. Here, we hypothesized that induced endometritis in non-lactating dairy cows would reduce the developmental competence of oocytes. Non-lactating Holstein cows received an intrauterine infusion of endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; n=12) or vehicle control (n=11) on day 2 of the estrous cycle. Bacterial infusion increased expression of endometrial inflammatory mediators, and a mucopurulent discharge in the vagina confirmed the establishment of endometritis. Oocytes were collected by transvaginal ultrasound-guided ovum pickup on days 2, 24, 45, and 66 following infusion and subjected to in vitro fertilization and embryo culture. Bacterial infusion resulted in fewer cleaved oocytes developing to morulae compared to vehicle-infused controls (30.7 versus 45.0%), with the greatest effect observed in oocytes collected on day 24. Development to morula was inversely correlated with endometrial expression of IL6 on day 6. The expression of genes associated with embryo quality did not differ significantly between morulae from bacteria-infused and control cows. Artificial insemination 130days after intrauterine infusion resulted in normal, filamentous embryos that produced interferon tau 16days after conception in both infusion groups. This model of experimentally induced uterine infection successfully resulted in endometritis and a reduction in the proportion of oocytes that developed to morulae following in vitro fertilization. In conclusion, endometritis reduced the capacity of oocytes to develop to morulae.

Production of piglets from in vitro-produced blastocysts by ultrasound-guided ovum pick-up from live donors

The objective of this research was to develop a system for piglet production by transvaginal ultrasound-guided ovum pick-up (OPU), in vitro production (IVP) of embryos and embryo transfer. First, to establish a culture system for a small number of oocytes or embryos, we evaluated the effect of different incubation volumes and culture densities on fertilizing ability and developmental competence in vitro. Porcine oocytes derived from slaughterhouse ovaries were matured, fertilized and then cultured in vitro in groups as follows: 50 oocytes in 500 μL medium for IVM, 20 oocytes in 100 μL medium for IVF and 20 embryos in 40 μL medium for IVC (Group I); 20 in 100 μL for IVM, 20 in 100 μL for IVF and 20 in 40 μL for IVC (Group II); and 10 in 100 μL for IVM, 10 in 100 μL for IVF and 10 in 40 μL for IVC (Group III). Percentages of sperm penetration, cleavage and blastocyst formation did not differ among the groups. Second, to increase the collection efficiency of porcine oocytes by transvaginal ultrasound-guided OPU, the effects of aspiration pressure on follicular oocyte collection were assessed. Oocytes were aspirated from ovaries of live sows using 80 or 100 mmHg. The recovered oocytes were divided into four categories according to the surrounding cumulus cells and quality of oocytes. The number of oocytes recovered using 100 mmHg pressure was significantly higher than with 80 mmHg pressure. However, there were no significant differences in the population of oocytes grouped by the morphological criteria, number of blastocysts per session and the total cell number in blastocysts between the two vacuum pressures. Finally, 81 oocytes obtained by OPU from five donor sows were subjected to IVP and 47 transferable embryos (9.4 ± 4.0 [mean ± SD] morulae/blastocysts per session) were obtained at 5 days after IVF. When they were transferred into five recipient gilts (5–16 embryos per recipient), three of five recipients became pregnant and farrowed a total of 12 live piglets. The present results demonstrate that porcine blastocysts can be produced by OPU-IVP and develop to full term after embryo transfer.

Supplementation of cows with linseed: effects on antral follicle count, oocyte quality, and nutritional and plasma metabolite parameters

Linseed is distinguished by its composition of fatty acids, which are rich in unsaturated acids, and by the slow release of oil in the rumen, consequently providing a greater amount of lipids of interest for intestinal absorption. The aim of this study was to evaluate the effect of linseed supplementation on the antral follicle count (AFC) and oocyte quality of Girolando cows, as well as their digestive parameters and plasma metabolite. Twelve Girolando cows were used in the study and were randomly distributed in two experimental groups: control (CTL, n = 6) and linseed (LINS, n = 6). During the 126 days of supplementation, seven transvaginal ultrasound-guided ovum pick-up (OPU) sessions were performed at intervals of 21 days. AFC was performed in each OPU session. The cows fed with linseed showed no significant difference in the dry matter coefficient of digestibility (63.11 and 62.88), crude protein (62.7 and 55.26), neutral detergent fiber (NDF; 75.36 and 77.15), or acid detergent fiber (ADF; 72.45 and 74.77) for LINS and CTL, respectively. On the contrary, a higher level of ethereal extract was observed for LINS (69.31 vs. 40.7). There was no effect (p > 0.05) of lipid supplementation on plasma metabolite between groups and OPU sessions. There was no difference (p > 0.05) in the mean number of antral follicles (CTL: 31.14 ± 1.97; LINS: 25.52 ± 2.68), cumulus oocyte complexes recovered (CTL: 6.45 ± 1.66; LINS: 5.28 ± 1.18), or oocyte quality (CTL: 60.48% ± 8.46; LINS: 64.54% ± 7.77). The supplementation of 800g of linseed in the diet of Girolando cows did not alter the apparent digestibility of nutrients, AFC, or quality of oocytes obtained by OPU.

Plasma anti-Müllerian hormone as a predictive endocrine marker to select Bos taurus (Holstein) and Bos indicus (Nelore) calves for in vitro embryo production

This study evaluated the association between plasma anti-Müllerian hormone (AMH) concentrations and in vitro embryo production in Bos indicus (Nelore; experiment 1) and Bos taurus (Holstein; experiment 2) calves superstimulated or not with 140 mg of porcine follicle-stimulating hormone (pFSH; 4 decreasing doses twice daily). Oocytes were recovered from calves aged 2 to 4 mo after receiving gonadotropin stimulation (Nelore, n = 15; Holstein, n = 12) or not (Nelore, n = 15; Holstein, n = 12). Cycling heifers formed a positive control group (n = 15 for Nelore [aged 18–24 mo], n = 10 for Holstein [aged 14–16 mo]). All the calves underwent laparoscopic ovum pickup, and cycling heifers underwent a regular transvaginal ultrasound-guided ovum pickup for oocyte recovery. Immediately before oocyte retrieval, blood samples were taken for subsequent AMH determination (ng/mL). Regardless of the genetic group, calves that received pFSH (3.6 ± 1.1 in Nelore and 4.6 ± 1.2 in Holstein) or did not receive pFSH (3.2 ± 1.0 in Nelore and 2.5 ± 0.8 in Holstein) had greater plasma AMH concentrations (P = 0.01 in Nelore and P = 0.003 in Holstein) than cycling heifers (1.1 ± 0.2 in Nelore and 0.6 ± 0.07 in Holstein). AMH concentrations in calves with or without pFSH were similar in both genetic groups (3.6 ± 1.1 vs 3.2 ± 1.0 in Nelore; 4.6 ± 1.2 vs 2.5 ± 0.8 in Holstein). In calves, positive correlations were observed between plasma AMH concentrations and the numbers of follicles >2 mm (r = 0.86, P < 0.0001 in Nelore; r = 0.78, P < 0.0001 in Holstein), cumulus-oocyte complexes (COCs) retrieved (r = 0.91, P < 0.0001 in Nelore; r = 0.82, P < 0.0001 in Holstein), COCs cultured (r = 0.71, P < 0.0001 in Nelore; r = 0.79, P < 0.0001 in Holstein), and blastocysts produced (r = 0.62, P = 0.0003 in Nelore; r = 0.58, P = 0.009 in Holstein), and these results were independent of pFSH treatment. In conclusion, calves had greater plasma AMH concentrations than cycling heifers. In addition, treatment with pFSH did not influence AMH concentrations in calves, regardless of the genetic group. More importantly, plasma AMH concentrations were positively correlated with the antral follicle population and the number of COCs retrieved, COCs cultured, and blastocysts produced in B indicus and B taurus calves. Therefore, AMH is a promising tool for selecting oocyte donor calves to maximize results during in vitro embryo production.

Minimizing the Risk of Infection and Bleeding at Trans-Vaginal Ultrasound-Guided Ovum Pick-up: Results of a Prospective Web-Based World-Wide Survey.

The objective of this study was to identify clinical practices worldwide, which would help in recognizing women at risk of excessive bleeding or of developing pelvic infection following trans-vaginal ovum pick-up (TV-OPU), measures taken to minimize risks and their management. A prospective, web-based questionnaire with distinct questions related to the practice of TV-OPU. A total of 155 units from 55 countries performing 97,200 IVF cycles annually responded to this web-based survey. A majority (65%) responded that they would routinely carry out full blood count, while 35% performed coagulation profile. Less than a third agreed screening women for vaginal infections. About a third used both sterile water and antiseptic to minimize ascending infection, and 52% used antibiotics for prophylaxis. Doppler ultrasound was routinely used by 20% of clinicians. 73% of the clinicians preferred conservative management as their first line management for patients diagnosed with intra-abdominal bleeding. The study has identified a wide variation in the practices of minimizing infection and bleeding complications. The dearth of good quality evidence may be responsible for the lack of published guidelines, and therefore a lack of consensus on the optimum practice for minimizing the risk of infection and bleeding during TV-OPU.

Acid–Base Parameters and Steroid Concentrations in Pre‐Ovulatory Follicles and Plasma of Lactating Dairy Cows with Spontaneous and Synchronized Oestrus or Follicular Cyst

The study aimed to compare the acid-base balance and steroid concentrations between follicular fluids (FF) of pre-ovulatory follicles derived from a spontaneous oestrus (SO), synchronized or induced oestrus (IO) and follicular cysts (CYS) and between FF and blood in dairy cows. Forty-two dairy cows were included in this study. The animals were allocated to three groups: SO (n=23); IO (n=11) using GnRH at day 0 and day 9 and PGF2 α at day 7; and animals with CYS (n=10). The follicular fluids (FF) were aspirated from the cyst/pre-ovulatory follicles (∅≥15mm) after SO and after second GnRH dose in IO by transvaginal ultrasound-guided ovum pick-up technique. Blood samples (BL) were collected in heparinized vacutainer tubes. The oxygen tension (pO2) in FF of IO was higher (p<0.05) than in SO and CYS groups. There were negative correlations (p<0.001, r=-0.89) between FF and blood pO2. The carbon dioxide tension (pCO2) and lactate level in FF of CYS group were higher (p<0.05) than in SO and IO groups. There were negative correlations (p<0.01, r=-0.73) between blood and FF pO2. Oestradiol-17β concentration in pre-ovulatory follicles and plasma of the SO group was higher (p<0.001) than in IO and CYS groups. Progesterone concentration in pre-ovulatory follicles and plasma of the SO and IO groups was lower (p<0.01) than in CYS group. Plasma androstenedione concentration in SO and IO groups was higher (p<0.05) than in CYS group. In conclusion, acid-base parameters, E2 and P4 levels in the follicular fluid of both IO and CYS groups were deviated greatly from the physiological level (disturbances of intrafollicular/intracystic environment), which may affect the quality of both the oocyte and the granulosa cells.

VEGFA164_B mRNA Abundance Has a Positive Relationship to AMH, BCL2 and the Ratio of E2:A4 in Mural Granulosa Cells of Estrogen Active and Inactive Follicles Prior to Ovulation.

Previously, we reported differential expression of Vascular Endothelial Growth Factor A (VEGFA) isoforms in somatic cells from bovine females of differing fertility. Thus, we sought to determine how expression of VEGFA pro and antiangiogenic isoforms were regulated after synchronization protocols that stimulated normal or persistent follicle development. Furthermore, we examined relationships between VEGFA isoforms, steroid hormones and genes that regulate follicular development in mural granulosa cells and cumulus oocyte complexes (COC). Our hypothesis was that dominant estrogen active (EA) follicles would have greater mRNA abundance of VEGFA angiogenic isoforms and genes that stimulate follicle development; while estrogen inactive (EI) follicles would contain greater mRNA expression of VEGFA antiangiogenic isoforms. Thirty-five composite beef cows [75% MARC III (1/4 Angus, 1/4 Hereford, 1/4 Pinzgauer, 1/4 Red Poll) and 25% Red Angus] were treated with either 1) a modified Co-Synch protocol (CIDR) or 2) injected with prostaglandin and offered Melengesterol Acetate (MGA) for 14 days. Dominant follicle aspirations were performed 18, 36, and 60 hours after the last PGF2a injection utilizing transvaginal ultrasound guided ovum pickup. Follicular fluid steroid hormone concentrations were determined via RIA (E2 and P4) or ELISA (A4). Follicles were then classified as EA (E2:P4 > 1) or as EI (E2:P4 = 1). There was no effect of time on gene expression or steroid hormone levels, thus animals were grouped based on treatment and follicle E2-activity. CIDR and MGA EI follicles had a greater (P = 0.02) concentrations of progesterone compared to CIDR EA follicles. Although A4 concentration was similar among groups, CIDR EA follicles had greater E2:A4 ratio compared to EI follicles suggesting reduced efficiency in conversion of A4 to E2.To determine if the reduced E2:A4 ratio in EI follicles alters the mRNA abundance of VEGFA isoforms in the COC or mural granulosa cells quantitative, real-time RT-PCR was performed. There was no difference in VEGFA_164 gene expression among groups. However, abundance of VEGFA_164B mRNA was 7-fold and 2-fold greater (P < 0.01) in the mural granulosa cells of MGA EI and CIDR EI, respectively, compared to CIDR EA follicles. Furthermore, there was a trend (P = 0.06) for an increased ratio of VEGFA_164/ VEGFA_164B in mural granulosa of CIDR EA follicles. Follicles classified as MGA EI had greater (7-fold; P = 0.02) mRNA abundance of AMH compared to CIDR EA follicles in granulosa cells. Furthermore, BCL2 mRNA expression tended to be increased (P = 0.07) in MGA EI follicles. In contrast to the mural granulosa cells, VEGFA_164B mRNA abundance was not different in COC. In mural granulosa cells, there was a positive correlation between VEGFA_164B and BCL2; VEGFA_164B and AMH; VEGFA_164B and the ratio of E2:P4; and the ratio of VEGFA_164/VEGFA_164B and BCL2.Taken together these data suggest a tendency for increased levels of anti-apoptotic factors and reduced efficiency of conversion of A4 to E2 in granulosa cells of EI follicles. Furthermore, there is a positive correlation between the antiangiogenic VEGFA isoform and BCL2 and AMH which play a role in regulating follicle development. USDA is an equal opportunity provider and employer.

Ultrasonographic-guided retrieval of cumulus oocyte complexes after super-stimulation in dromedary camel (Camelus dromedarius)

In Experiment 1, studies were conducted to apply the transvaginal ultrasound guided ovum pick-up (OPU) technique in dromedary camels after their ovarian super-stimulation and in vivo oocyte maturation. In Experiment 2, the developmental potential of two commonly used oocyte types, i.e., in vivo matured oocytes collected by OPU and abattoir derived in vitro-matured oocytes was compared after their chemical activation. In Experiment 3, developmental competence of oocytes collected from super-stimulated camels by OPU, matured either in vivo or in vitro, was compared after their chemical activation. Mature female dromedary camels super-stimulated with a combination of eCG and pFSH were given an injection of 20 μg of the GnRH analogue, buserelin 24, 26, or 28 h before the scheduled OPU. For collection of cumulus oocyte complexes (COCs) the transducer was guided through the vulva into the cranial most portion of the vagina and 17-gauge, 55 cm single-lumen needle was placed in the needle guide of the ultrasound probe and advanced through the vaginal fornix and into the follicle. Follicular fluid was aspirated using a regulated vacuum pump into tubes containing embryo-flushing media. Aspirates were searched for COCs using a stereomicroscope, and they were then denuded of cumulus cells by hyaluronidase and repeated pipetting. The oocytes were classified as mature (with a visible polar body), immature (with no visible polar body), activated (with divided or fragmented ooplasm) and others (degenerated and abnormal). Overall an average of 12.12 ± 7.9 COCs were aspirated per animal with an oocyte recovery rate from the aspirated follicles of about 77%. The majority (> 90%) of the collected COCs by OPU were with loose and expanded cumulus cells. The proportion of matured oocytes obtained at 28–29 h (91.2 ± 4.1) and 26–27 h (82.1 ± 3.4) were higher (P < 0.005) when compared with those obtained at 24–25 h (40.4 ± 16.3) after GnRH administration. In Experiment 2, a higher proportion (P < 0.05) of in vivo matured oocytes cleaved (84.6 ± 2.1 vs. 60.9 ± 6.6) and developed to blastocyst stages (52.4 ± 4.1 vs. 30.5 ± 3.3) when compared with in vitro matured oocytes collected from slaughterhouse ovaries. In Experiment 3, no difference was observed between the developmental competences of oocytes, collected from super stimulated camels, matured in vitro with those collected after their in vivo maturation. In conclusion, about 80 –90% mature oocytes can be collected by ultrasound guided transvaginal ovum pick-up from super-stimulated dromedary camels 26 –28 h after GnRH administration. The developmental response, to chemical activation, of in vivo matured oocytes collected by ultrasound guided transvaginal OPU is better than in vitro matured oocytes obtained from slaughterhouse ovaries. However, no difference was observed in the developmental competence of oocytes collected by OPU whether they were matured in vivo or in vitro.

Production of the First Cloned Camel by Somatic Cell Nuclear Transfer1

In this study, we demonstrate the use of somatic cell nuclear transfer to produce the first cloned camelid, a dromedary camel (Camelus dromedarius) belonging to the family Camelidae. Donor karyoplasts were obtained from adult skin fibroblasts, cumulus cells, or fetal fibroblasts, and in vivo-matured oocytes, obtained from preovulatory follicles of superstimulated female camels by transvaginal ultrasound guided ovum pick-up, were used as cytoplasts. Reconstructed embryos were cultured in vitro for 7 days up to the hatching/hatched blastocyst stage before they were transferred to synchronized recipients on Day 6 after ovulation. Pregnancies were achieved from the embryos reconstructed from all cell types, and a healthy calf, named Injaz, was born from the pregnancy by an embryo reconstructed with cumulus cells. Genotype analyses, using 25 dromedary camel microsatellite markers, confirmed that the cloned calf was derived from the donor cell line and the ovarian tissue. In conclusion, the present study reports, for the first time, establishment of pregnancies and birth of the first cloned camelid, a dromedary camel (C. dromedarius), by use of somatic cell nuclear transfer. This has opened doors for the amelioration and preservation of genetically valuable animals like high milk producers, racing champions, and males of high genetic merit in camelids. We also demonstrated, for the first time, that adult and fetal fibroblasts can be cultured, expanded, and frozen without losing their ability to support the development of nuclear transfer embryos, a technology that may potentially be used to modify fibroblast genome by homologous recombination so as to generate genetically altered cloned animals.

Repeated Ovum Pick-Up and In-vitro Embryo Production in Buffalo

Transvaginal ultrasound-guided ovum pick-up (OPU) was performed on 40 Nili-Ravi and Murrah buffaloes for consecutive 3 times each, at an interval of 3 days. A total of 936 follicles were aspirated, 643 oocytes were retrieved and 456 oocytes of them were usable. The recovery rate was 68.77% (643/935) and the percentage of usable oocytes was 70.92% (456/643), with an average of 3.80±0.23 oocytes usable per buffalo. The number of usable oocytes for the first, second and third OPU was 3.82±0.50;3.82±0.35; 4.05±0.43, respectively, and the difference among them was not significant (P<0.05). After in-vitro maturation (IVM), in-vitro fertilization (IVF) and co-incubation with single layer of granular cells, the cleavage rate was 56.36% - 257/456, blastocyst formation rate was 20.39% (93/456) The result proved the feasibility of consecutive OPU at an interval of 3d and produce buffalo embryo via OPU-IVF. Further studies are needed to improve the efficiency of OPU-IVF.

316 DEVELOPMENTAL COMPETENCE OF OOCYTES SELECTED BY THE BRILLIANT CRESYL BLUE STAINING IN PREPUBERTAL AND ADULT CATTLE

Transvaginal ultrasound guided ovum pickup (OPU) can be carried out repeatedly not only in adult cows but also in prepubertal calves. Moreover, recently the brilliant cresyl blue (BCB) staining has been used successfully to select oocytes homologous stained with BCB as an indirect measure of oocyte growth for in vitro fertilization (IVF) in several farm animals. The aim of this study was to evaluate the effectiveness of BCB staining on the selection of developmentally competent oocytes, collected from cows and calves by OPU, before in vitro maturation by assessing the embryonic development to the blastocyst stage after IVM/IVF. OPU was performed once weekly for a period of 7 weeks on Japanese Black prepubertal calves (9 months old, n = 4) and adult cows (n = 4) without gonadotropin stimulation. Calf and cow oocytes with homogeneous cytoplasm were exposed to 26 �M BCB for 90 min and classified according to their cytoplasmic coloration: blue coloration (BCB+) or colorless (BCB-). Classified oocytes were then matured for 20 h in TCM-199 supplemented with 5% calf serum (CS). Matured oocytes were inseminated with Percoll-separated spermatozoa (3 � 106 mL) for 6 h in BO solution supplemented with 5 mM hypotaurine and 2 U mL-1 heparin. Presumptive zygotes were cultured in CR1aa supplemented with 5% CS for 8 days. Data were analyzed by Student&amp;apos;s t-test. The mean (&amp;plusmn; SEM) percentage of oocytes classified as BCB&amp;plus; in calves was significantly lower than that in cows (34.4 &amp;plusmn; 2.9&amp;percnt; and 69.2 &amp;plusmn; 2.1&amp;percnt;, respectively; P &amp;lt; 0.01). In cows, BCB&amp;plus; oocytes showed significantly higher cleavage and blastocyst formation percentages (72.5&amp;percnt; and 42.4&amp;percnt;, respectively) than those of BCB&amp;minus; oocytes (47.0&amp;percnt; and 13.0&amp;percnt;, respectively). In contrast, in calves there were no significant differences in cleavage and blastocyst formation percentages between BCB&amp;plus; oocytes (56.9&amp;percnt; and 25.3&amp;percnt;, respectively) and BCB&amp;minus; oocytes (65.4&amp;percnt; and 22.4&amp;percnt;, respectively). The mean (&amp;plusmn; SEM) numbers of usable oocytes and blastocysts obtained per calf (19.0 &amp;plusmn; 1.5 and 4.5 &amp;plusmn; 0.6, respectively) were similar to those obtained per cow (16.4 &amp;plusmn; 1.1 and 5.2 &amp;plusmn; 0.6, respectively). No significant difference was observed in the numbers between calves and cows. These results indicate that the selection of developmentally competent oocytes before IVM/IVF, using the BCB staining, was effective for cow oocytes but not for calf oocytes, and that blastocysts could be produced by OPU-IVF of oocytes from 9-month-old prepubertal calves at an efficiency equivalent to that achieved from adult cows.

58 EFFICIENCY OF OVUM PICKUP AND EMBRYO PRODUCTION IN VITRO IN CLONED CATTLE

The reproductive performance of cloned cattle was investigated by assessing the efficiency of transvaginal ultrasound-guided ovum pickup (OPU) and embryo production in vitro. Fetal fibroblasts from the endangered species, German Blackpied Cattle, had been used for nuclear transfer to produce three live cloned offspring (Lucas-Hahn et al. 2002 Theriogenology 57, 433). In the three cloned animals at 12–20 months of age, OPU was performed once per week and the total number of collected oocytes was recorded. In the case of Blondie, the procedure was terminated due to too small ovaries associated with insufficient function. Oocytes suitable for IVF were matured in vitro for 24 h and fertilized in vitro with the semen of a fertile bull. Oocytes derived from abbatoir ovaries were processed in parallel as controls. Embryos were in vitro-cultured in SOFaaBSA medium. Cleavage and developmental rates up to the morula/blastocyst stage were recorded in all groups. Statistical significance was tested using ANOVA and the Student-Newman-Keuls test. The results are presented in Table 1. Embryos from clones had lower cleavage and blastocyst rates compared to those derived from abattoir oocytes. However, results may have been confounded by potential OPU effects. Some of the blastocysts produced from Blacky (n = 5) and Paula (n = 2) were transferred to recipients. Two pregnancies resulted from the Paula transfers. The two male calves were delivered normally. After the completion of this experiment, all three cloned animals were artificially inseminated, became pregnant, delivered healthy calves, and are pregnant again at present. Further studies are needed to explore the fertility of cattle derived from somatic cloning. Table 1. OPU and in vitro embryo production in cloned cattle

246IMPROVEMENT OF THE DEVELOPMENTAL CAPACITY OF OOCYTES FROM PREPUBERTAL CATTLE BY INTRAOVARIAN IGF-I APPLICATION

The developmental potential of oocytes from prepubertal cattle is lower compared to their adult counterparts. Differences between oocytes from calves and cows have been found with regard to size, ultrastructural characteristics and metabolism. GH and IGF-I receptor mRNA have been identified in the cumulus cells and in oocytes (Izadyar F et al. 1997 Mol. Reprod. Dev. 47, 175–180; Armstrong D. et al., 2002 Reproduction 123 (6), 789–797). It is known that IGF-I and GH affect mRNA expression of glucose transporter 1 (Glut1) as well as glucose uptake by embryos. The goal of this study was to improve the developmental capacity of oocytes from prepubertal cattle and to determine whether an induced increase of GH and IGF-I levels affects the mRNA expression pattern of Glut1, UBF and eIF1A. Holstein calves (n=30), 6–7 months old, were randomly divided into three groups. One group received a single s.c. injection of 500mg of somatotrophin (rbST Posilac, Monsanto Company, St. Louis, MO, USA) and during the subsequent 2 weeks their follicles were aspirated 4 times via transvaginal ultrasound-guided ovum pick-up (OPU). The second group received an intraovarian injection of 6μg rhIGF-I (R&amp;amp;D Systems, Wiesbaden-Nordenstadt, Germany). The third group served as control and received an intraovarian injection of 0.6mL of 10mM acetic acid. All animals were i.m. injected with 60mg FSH (Folltropin®; Vetrepharm Inc., Ontario, Canada) 48h prior to aspiration. The treatments were repeated with the same animal groups at the ages of 9–10, 11–12 and 14–15 months. Five adult cows were i.m. injected each with 100mg FSH. For mRNA expression analysis (RT-PCR), embryos were collected with 2–4 cells, 8–16 cells and as blastocysts. The relative abundance of mRNA for Glut 1 and eIF1A was higher (P&amp;lt;0.05) in 8–16 cell embryos from IGF-I-treated calves and cows than in control and rbST treated calves. The in vitro embryo production (IVP) results are shown in the Table 1. Our data show that IGF-I increased mRNA expression of Glut1 and eIF1A which may improve the developmental capacity of embryos produced from calves. Table 1 OPU/IVP-Results for calves (6–15 months old) in comparison with cows

The effects of twisting and type of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up in cattle.

The effects of twisting and type (single- or double-lumen) of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up (US-guided OPU) were investigated in cattle. The first study using slaughterhouse ovaries revealed that twisting of the needle during follicle aspiration improved the oocyte recovery rate without deleterious effects on the attachment of cumulus layers. Vacuum pressure affected the oocyte recovery and cumulus attachment, regardless of the needle type. The needle type did not affect the oocyte recovery or cumulus attachment with an optimized vacuum pressure. In the second study, US-guided OPU was performed in live cows using two types of needles with a vacuum pressure of 75 mmHg. The needle type did not affect the oocyte recovery or cumulus attachment of the recovered oocytes. The results revealed that twisting of the needle is effective in follicle aspiration, and suggested that a single-lumen needle is as useful as a double-lumen needle for US-guided OPU in cattle.

Embryo production using defined oocyte maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH-stimulated Simmental heifers

To determine whether differences in ovarian follicle populations and endocrine status at ovum pick-up (OPU) influenced the quality and developmental competence of oocyte-cumulus complexes (OCC’s) collected from follicle stimulating hormone (FSH)-stimulated donors, 24 Simmental heifers had their ovarian follicles aspirated via transvaginal ultrasound-guided OPU at both 15 (OPU1) and 21 (OPU2) days following a synchronised oestrus, on four consecutive occasions at 15-week intervals. More OCC’s were collected during OPU1 than OPU2 (means±S.E.M.=7.2±0.47 versus 5.7±0.44; P=0.01), but the respective percentages that were of good quality (categories 1 and 2) did not differ significantly (55±3% versus 47±3%). The incidence of zygote cleavage following OCC maturation (Medium 199; protein-free), in vitro fertilization (mTALP; including 0.6% (w/v) albumin) and culture (modified SOF; protein-free) was not significantly different (mean±S.E.M.=81±2% and 71±7% for OPU1 and OPU2, respectively). Corresponding blastocyst yields from good quality OCC’s (24±3% and 26±4%) also did not differ. Although the same 3-day FSH regimen was used immediately prior to each OPU session, plasma FSH concentrations were consistently lower at OPU1 than OPU2 (1.3±0.28 ng/ml versus 2.5±0.45 ng/ml; P<0.05). In contrast, plasma progesterone concentrations were higher at OPU1 (6.6±0.48 ng/ml versus 3.9±0.53 ng/ml; P<0.001), with concentrations at OPU2 being consistent with the presence of luteal tissues, including both persistent corpora lutea and luteinised follicle remnants following OPU1. Failure of the significant differences in follicular and endocrine status between OPU1 and OPU2 to alter the developmental competence of OCC’s suggests that, probably as a result of its stabilising influence on nutritionally-sensitive intraovarian regulators of oocyte competence, the constant feeding regimen had a more profound effect on oocyte quality than observed shifts in the peripheral concentrations of some reproductive hormones. Finally, the study demonstrates that it is possible to generate acceptable numbers of in vitro blastocyst-stage embryos from high genetic merit heifers using strategies which restrict reliance on protein to the in vitro fertilization stage of the production process.

Effects of EGF and IGF-I on porcine oocyte fertilization in vitro in the absence of follicular fluid or hormones

This study evaluated the association between plasma anti-Müllerian hormone (AMH) concentrations and in vitro embryo production in Bos indicus (Nelore; experiment 1) and Bos taurus (Holstein; experiment 2) calves superstimulated or not with 140 mg of porcine follicle-stimulating hormone (pFSH; 4 decreasing doses twice daily). Oocytes were recovered from calves aged 2 to 4 mo after receiving gonadotropin stimulation (Nelore, n = 15; Holstein, n = 12) or not (Nelore, n = 15; Holstein, n = 12). Cycling heifers formed a positive control group (n = 15 for Nelore [aged 18–24 mo], n = 10 for Holstein [aged 14–16 mo]). All the calves underwent laparoscopic ovum pickup, and cycling heifers underwent a regular transvaginal ultrasound-guided ovum pickup for oocyte recovery. Immediately before oocyte retrieval, blood samples were taken for subsequent AMH determination (ng/mL). Regardless of the genetic group, calves that received pFSH (3.6 ± 1.1 in Nelore and 4.6 ± 1.2 in Holstein) or did not receive pFSH (3.2 ± 1.0 in Nelore and 2.5 ± 0.8 in Holstein) had greater plasma AMH concentrations (P = 0.01 in Nelore and P = 0.003 in Holstein) than cycling heifers (1.1 ± 0.2 in Nelore and 0.6 ± 0.07 in Holstein). AMH concentrations in calves with or without pFSH were similar in both genetic groups (3.6 ± 1.1 vs 3.2 ± 1.0 in Nelore; 4.6 ± 1.2 vs 2.5 ± 0.8 in Holstein). In calves, positive correlations were observed between plasma AMH concentrations and the numbers of follicles >2 mm (r = 0.86, P < 0.0001 in Nelore; r = 0.78, P < 0.0001 in Holstein), cumulus-oocyte complexes (COCs) retrieved (r = 0.91, P < 0.0001 in Nelore; r = 0.82, P < 0.0001 in Holstein), COCs cultured (r = 0.71, P < 0.0001 in Nelore; r = 0.79, P < 0.0001 in Holstein), and blastocysts produced (r = 0.62, P = 0.0003 in Nelore; r = 0.58, P = 0.009 in Holstein), and these results were independent of pFSH treatment. In conclusion, calves had greater plasma AMH concentrations than cycling heifers. In addition, treatment with pFSH did not influence AMH concentrations in calves, regardless of the genetic group. More importantly, plasma AMH concentrations were positively correlated with the antral follicle population and the number of COCs retrieved, COCs cultured, and blastocysts produced in B indicus and B taurus calves. Therefore, AMH is a promising tool for selecting oocyte donor calves to maximize results during in vitro embryo production.

Repeated transvaginal ultrasound-guided ovum pick-up in egg-treated cows

Repeated transvaginal ultrasound-guided ovum pick-up in egg-treated cows

Patients' experience of transvaginal follicle aspiration under local anesthesia.

The development of vaginal ultrasound transducers has facilitated ovum pick-up (OPU) by providing higher precision and less trauma than are found with laparoscopy and other ultrasound-assisted techniques. In order to evaluate the patients' acceptance, 65 patients were asked to answer a questionnaire about their experience with the procedure. The punctures were executed with specially designed needles introduced through a needle guide attached to a vaginal transducer with a frequency of 7 MHz (Brüel & Kjaer, Denmark). The anesthesia consisted of a light premedication and a paracervical block. If needed, a small dose of sedative was given intravenously intraoperatively. The patients classified their pain experience within the day of the puncture on a four-grade scale. Their statements were compared to the physician's opinion of the discomfort caused. Oocytes were retrieved from all patients. Fifty-one patients answered the questionnaire, of whom five found the OPU painful. In two cases the physician underestimated the patients' pain experience. The mean time required for the procedure was 16 min/OPU. We conclude that transvaginal ultrasound-guided OPU is well accepted by most patients under local anesthesia and that it is a rapid and accurate procedure.