Nerve growth factor from seminal plasma origin (spß-NGF) increases CL vascularization and level of mRNA expression of steroidogenic enzymes during the early stage of Corpus Luteum development in llamas.

The objectives of the study were to determine the effect of seminal plasma ß-NGF on Corpus Luteum morphology and function and level of mRNA expression of steroidogenic enzymes. Llamas were assigned (n = 12/per group) to receive an intramuscular dose of: (a) 1 ml phosphate buffered saline (PBS), (b) 5 µg gonadorelin acetate (GnRH), or (c) 1.0 mg of purified llama spß-NGF. Ovaries were examined by transrectal B-mode ultrasonography from treatment to ovulation (Day 0 = treatment). B mode/Power Doppler ultrasonography and blood samples collection were performed at Days 4, 8 and 10 (n = 3 llamas per treatment group/per time point) to determine CL diameter, vascularization and plasma progesterone concentration respectively. Plasma progesterone concentration was analyzed in all llamas at Day 0. Then females were submitted to ovariectomy at Days 4, 8 and 10 (n = 3 llamas/treatment/time), CL was removed to determine vascular area, proportion of luteal cells and CYP11A1/P450scc and STAR expression by RT-PCR. Ovulation was similar between llamas treated with GnRH or spß-NGF and CL diameter did not differ between GnRH or spß-NGF groups by Day 4, 8 or 10. Vascularization area of the CL was higher (P < 0.01) in llamas from the spß-NGF than GnRH-treated group by Day 4 and 8. Plasma progesterone concentration was higher (P < 0.05) in llamas from the spß-NGF compared to females of GnRH group by Day 4 and 8. The proportion of small and large luteal cells did not differ between GnRH or spß-NGF groups by Day 8. CYP11A1/P450scc was upregulated 3 folds at day 4 and 10 by spß-NGF compared to GnRH. STAR transcription was 3 folds higher at day 4 in females treated with spß-NGF. In conclusion, the luteotrophic effect of spß-NGF could be related to an increase of vascularization and up regulation of CYP11A1/P450scc and STAR transcripts enhancing progesterone secretion.

In vitro and in vivo embryo production in cattle superstimulated with FSH for 7 days

Over the past two decades, research efforts have resulted in superstimulation protocols that are user-friendly, but embryo production has increased only marginally. Studies to-date have not adequately answered the question of whether superstimulatory protocols can be used to overcome the follicle wave pattern, increase the number of follicles that enter the wave, or rescue a greater number of small follicles within the wave. Studies which appear to facilitate greater utilization of follicles within the wave are described in this review. The number of large follicles at the time of first AI tended to be greater, and more ovulations and CL occurred with lengthened protocol (7-day) than with the convention 4-day FSH treatment. In addition, there was greater synchrony of ovulations and the mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos were numerically higher in the 7-day group. When used in an in vitro fertilization model, FSH treatment for 7 days resulted in a greater number of follicles for aspiration, a greater proportion of expanded cumulus-oocyte-complexes, and more transferable embryos after in vitro culture. Daily ultrasonography revealed a reduction in the number of small (1-2 mm) antral follicles from the beginning to the end of the superstimulatory treatment that was associated with a progressive shift of follicles to the next size category in both 4-day and 7-day groups. The number of follicles<5 mm decreased during superstimulation suggesting that there was no continuous recruitment of small follicles, and the number of follicles>1 mm at the end of superstimulation did not differ from the number of follicles >1 mm at the beginning of Super stimulation. However, the total number of follicles >3 mm at the end of superstimulation, was greater than the number of follicles >3 mm at the beginning of superstimulation due to growth of the 1-2 mm population into larger size categories during treatment. (AU)

In vitro and in vivo embryo production in cattle superstimulated with FSH for 7 days

Over the past two decades, research efforts have resulted in superstimulation protocols that are user-friendly, but embryo production has increased only marginally. Studies to-date have not adequately answered the question of whether superstimulatory protocols can be used to overcome the follicle wave pattern, increase the number of follicles that enter the wave, or rescue a greater number of small follicles within the wave. Studies which appear to facilitate greater utilization of follicles within the wave are described in this review. The number of large follicles at the time of first AI tended to be greater, and more ovulations and CL occurred with lengthened protocol (7-day) than with the convention 4-day FSH treatment. In addition, there was greater synchrony of ovulations and the mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos were numerically higher in the 7-day group. When used in an in vitro fertilization model, FSH treatment for 7 days resulted in a greater number of follicles for aspiration, a greater proportion of expanded cumulus-oocyte-complexes, and more transferable embryos after in vitro culture. Daily ultrasonography revealed a reduction in the number of small (1-2 mm) antral follicles from the beginning to the end of the superstimulatory treatment that was associated with a progressive shift of follicles to the next size category in both 4-day and 7-day groups. The number of follicles1 mm at the end of superstimulation did not differ from the number of follicles >1 mm at the beginning of Super stimulation. However, the total number of follicles >3 mm at the end of superstimulation, was greater than the number of follicles >3 mm at the beginning of superstimulation due to growth of the 1-2 mm population into larger size categories during treatment.

LH release and ovulatory response after intramuscular, intravenous, and intrauterine administration of ß-nerve growth factor of seminal plasma origin in female llamas.

The objective of the study was to compare the pituitary and ovarian responses after intramuscular, intravenous, or intrauterine administration of ß-nerve growth factor (ß-NGF) of seminal plasma origin (SP-NGF) in llamas. In experiment 1, mature female llamas with a growing follicle of 7 mm or greater were assigned randomly to four groups (n = 7/group) and given 2 mg of purified SP-NGF in a volume of 2 mL by (1) intramuscular administration, (2) intravenous administration, and (3) intrauterine infusion, or (4) intrauterine infusion of 2 mL of PBS (negative control). Because ovulations were not detected after intrauterine infusion in experiment 1, a second experiment was done to determine if a higher dose of SP-NGF given by intrauterine infusion, similar to a natural dose during copulation, will elicit an ovulatory response. In experiment 2, llamas with a growing follicle of 7 mm or greater were assigned randomly to three groups (n = 6/per group) given an intrauterine infusion of (1) 4 mL of raw seminal plasma, (2) 4 mL of PBS containing 20 mg of purified llama SP-NGF, or 3) 4 mL of PBS (negative control). In both experiments, the ovaries were examined daily by transrectal ultrasonography using a B-mode scanner and power Doppler mode to detect ovulation and to monitor CL growth, regression, and vascularization. Blood samples were collected to determine plasma LH and progesterone concentrations. In experiment 1, only llamas treated by intramuscular or intravenous administration of SP-NGF ovulated (7 of 7 and 6 of 7, respectively). Plasma LH concentration did not differ between the intramuscular and intravenous SP-NGF-treated groups, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. In experiment 2, the ovulation rate was 100% for llamas treated by intrauterine infusion of raw seminal plasma or llama SP-NFG, whereas no ovulations were detected in females treated with PBS. Plasma LH concentrations did not differ between groups that ovulated, nor did CL diameter, CL vascularization, or plasma progesterone concentration profiles. We conclude that ß-NGF from llama seminal plasma origin elicits a preovulatory LH surge, followed by ovulation and the development of a functional CL, regardless of the route of administration. However, the dose required to elicit pituitary and ovarian responses is higher when administered by intrauterine infusion than by intramuscular or intravenous routes.

The effect of repeated administrations of llama ovulation-inducing factor (OIF/NGF) during the peri-ovulatory period on corpus luteum development and function in llamas.

The objective of the study was to test the hypothesis that repeated administrations of OIF/NGF during the peri-ovulatory period (pre-ovulatory, ovulatory, early post-ovulatory), will enhance the luteotrophic effect in llamas. Female llamas were examined daily by transrectal ultrasonography in B- and Doppler-mode using a scanner equipped with a 7.5-MHz linear-array transducer to monitor ovarian follicle and luteal dynamics. When a growing follicle ≥7mm was detected, llamas were assigned randomly to one of the three groups and given 1mg of purified OIF/NGF im (intramuscular) (a) pre-ovulation (single dose; n=12), (b) pre-ovulation and at the time of ovulation (2 doses, n=10), or (c) pre-ovulation, at the time of ovulation, and 24h after ovulation (3 doses, n=10). The pre-ovulatory follicle diameter at the time of treatment, ovulation rate and the first day of CL detection did not differ (P=0.3) among groups. However, maximum CL diameter was greatest (P=0.003) in llamas in the 2-dose group, and smallest in the 3-dose group. Accordingly, the 2 dose-group had the largest day-to-day profile for CL diameter (P<0.01), area of CL vascularization (<0.01), and plasma progesterone concentration (P=0.01) compared to the other groups. Interestingly, the luteal response to 3-doses of OIF/NGF during the peri-ovulatory period was not different from a single dose. In conclusion, OIF/NGF isolated from llama seminal plasma is luteotrophic and the effect on CL size and function is affected by the number and timing of treatments during the peri-ovulatory period.

Ovulation-inducing factor (OIF/NGF) from seminal plasma origin enhances Corpus Luteum function in llamas regardless the preovulatory follicle diameter.

Ovulation-inducing factor (OIF) is a protein present in llama seminal plasma that has recently been identified as ß-Nerve Growth Factor (NGF) and it induces not only a high rate of ovulation but also appears to have luteotrophic properties in this species. A 2-by-2 experimental design was used to determine the effect of treatments (OIF/NGF vs GnRH) and categories of preovulatory follicle diameter (7-10 vs >10mm) on ovulation rate, CL diameter and function in llamas. Llamas (n=32 llamas per group) were randomly assigned to receive an intramuscular dose of: (a) 1mg purified OIF/NGF in the presence of a follicle of 7-10mm in diameter; (b) 50 µg of GnRH in the presence of a follicle of 7-10mm in diameter; (c) 1mg purified OIF/NGF in the presence of a follicle >10mm in diameter; (d) 50 µg of GnRH in the presence of a follicle >10mm in diameter. Llamas were examined by ultrasonography every 12h from treatment to Day 2 (Day 0=treatment) to detect ovulation, and again on Day 8 to determine CL diameter. Ovulation rates did not differ among groups. There was an effect of preovulatory follicle size on Corpus Luteum diameter at Day 8 (P<0.001), however plasma progesterone concentration (n=15/per group) was higher (P<0.05) in the OIF/NGF - than that of the GnRH - treated group by the same day. We conclude that OIF/NGF treatment enhances CL function regardless preovulatory follicle size at the time of treatment.

Luteotrophic effect of ovulation-inducing factor/nerve growth factor present in the seminal plasma of llamas.

The hypothesis that ovulation-inducing factor/nerve growth factor (OIF/NGF) isolated from llama seminal plasma exerts a luteotrophic effect was tested by examining changes in circulating concentrations of LH and progesterone, and the vascular perfusion of the ovulatory follicle and developing CL. Female llamas with a growing follicle of 8 mm or greater in diameter were assigned randomly to one of three groups (n = 10 llamas per group) and given a single intramuscular dose of PBS (1 mL), GnRH (50 µg), or purified OIF/NGF (1.0 mg). Cineloops of ultrasonographic images of the ovary containing the dominant follicle were recorded in brightness and power Doppler modalities. Llamas were examined every 4 hours from the day of treatment (Day 0) until ovulation, and every other day thereafter to Day 16. Still frames were extracted from cineloops for computer-assisted analysis of the vascular area of the preovulatory follicle from treatment to ovulation and of the growing and regressing phases of subsequent CL development. Blood samples were collected for the measurement of plasma LH and progesterone concentrations. The diameter of the dominant follicle at the time of treatment did not differ among groups (P = 0.48). No ovulations were detected in the PBS group but were detected in all llamas given GnRH or OIF/NGF (0/10, 10/10, and 10/10, respectively; P < 0.0001). No difference was detected between the GnRH and OIF/NGF groups in the interval from treatment to ovulation (32.0 ± 1.9 and 30.4 ± 5.7 hours, respectively; P = 0.41) or in maximum CL diameter (13.1 ± 0.4 and 13.5 ± 0.3 mm, respectively; P = 0.44). The preovulatory follicle of llamas treated with OIF/NGF had a greater vascular area at 4 hours after treatment than that of the GnRH group (P < 0.001). Similarly, the luteal tissue of llamas treated with purified OIF/NGF had a greater vascular area than that of the GnRH group on Day 6 after treatment (P < 0.001). The preovulatory surge in plasma LH concentration began, and peaked 1 to 2 hours later in the OIF/NGF group than in the GnRH group (P < 0.05). Plasma progesterone concentration was higher on Day 6 in the OIF/NGF group than in the GnRH group (P < 0.001). Results support the hypothesis that OIF/NGF exerts a luteotrophic effect by altering the secretion pattern of LH and enhancing tissue vascularization during the periovulatory period and early stages of CL development.

Ultrasound biomicroscopy: a non-invasive approach for in vivo evaluation of oocytes and small antral follicles in mammals.

The use of ultrasonography has changed our understanding of the ovarian function in live animals. However, most of the studies that have used ultrasonography to image the ovary have provided data only of structures >1mm in diameter. The recent availability of high-resolution ultrasound technology with high-frequency transducers (25-70 MHz), offers the potential to examine the developmental dynamics of small antral follicles and the cumulus-oocyte complex (COC) in vivo. In this review we provide data from a series of studies performed by Veterinary Biomedical Sciences Laboratory describing the advantages and disadvantages, as well as image characteristics, of ultrasound biomicroscopy (UBM) to study ovarian biology in mammals. Data and images of small ovarian structures in rabbits, cattle, mice and humans are shown. The UBM technique allowed visualisation of small antral follicles ranging in size from 300 to 700 µm in all species examined, as well as COC within follicles in rabbits, cattle and humans. Furthermore, UBM permitted clear distinction of the follicular wall from the surrounding ovarian stroma in cattle and humans. At present, the limited depth of penetration of UBM restricts the use of this technique to an experimental setting. In that regard, further studies using UBM will probably result in a greater understanding of the pattern and control of early antral folliculogenesis and oogenesis.

Effects of nutritional restriction on metabolic, endocrine, and ovarian function in llamas (Lama glama).

The objectives of the study were to determine the effects of nutritional restriction on ovarian function in llamas. Mature female llamas were assigned randomly to a Control group, fed 100% of maintenance energy requirements (MER) (n=8), or a Restricted group (n=8) fed from 70% to 40% of MER until a body condition score of 2.5 was attained. Blood samples were taken every-other-day to determine plasma concentrations of LH, estradiol, leptin and metabolic markers, and follicular dynamics were monitored daily by ultrasonography for 30 days (Experiment 1). Llamas were then treated with GnRH to compare the ovulatory response and corpus luteus (CL) development between groups (Experiment 2). Blood samples were taken to measure LH, leptin, progesterone and metabolic markers and ovarian structures were assessed as in Experiment 1. Llamas in the Restricted group had lower body mass and body condition scores than those in the Control group (P<0.001). Plasma concentrations of cholesterol, non-esterified fatty acids, triglycerides, and urea were higher in the Restricted group (P<0.05) than in the Control group. The day-to-day diameter profiles of the dominant follicles were smaller (P<0.05) in the Restricted group than in the Control group but plasma estradiol concentration did not differ. The ovulation rate and LH secretion in response to GnRH did not differ. Day-to-day profiles of CL diameter, plasma progesterone and leptin concentrations were smaller (P<0.01) in the Restricted group. In conclusion, nutritional restriction in llamas was associated with suppressed follicle and CL development, and lower plasma concentrations of progesterone and leptin.

In vivo imaging of cumulus-oocyte-complexes and small ovarian follicles in cattle using ultrasonic biomicroscopy.

The objectives of this study were to: (1) evaluate the feasibility of ultrasonic biomicroscopy (UBM) for imaging ovarian follicles in vivo in cows and heifers, (2) compare the transvaginal to the transrectal approach, (3) compare the number of follicles detected by UBM to conventional ultrasonography (US), and (4) identify cumulus-oocyte-complexes (COC) within follicles by UBM. Mature cows (n=5) and peri-pubertal heifers (n=5) were examined once using conventional B-mode US (Aloka 900) with a 7.5 MHz transrectal, and a 5 MHz transvaginal transducers. A second series of examinations was performed using UBM (Visualsonics Vevo 660) equipped with a 30 MHz transrectal and a 40 MHz transvaginal transducer. A three- to four-fold increase in the number of small follicles (<3 mm) was detected using the transvaginal approach with UBM compared to conventional US in both heifers (32.4 ± 4.24 compared to 7.2 ± 1.4; P<0.0001) and cows (35.0 ± 13.8 compared to 10.7 ± 7.5; P=0.0013). However, fewer follicles (all sizes combined) were detected using the transrectal approach with UBM compared to conventional US in both heifers (8.6 ± 5.6 compared to 17.6 ± 4.9; P=0.02) and cows (5.3 ± 6.1 compared to 20.3 ± 7; P=0.04). In heifers, COC-like structures were identified in 39 of 202 (19.3%) follicles examined. In conclusion, UBM using a transvaginal approach is feasible and may be used for in vivo assessment of early antral follicles as small as 0.4 mm, and COC within follicles.

Ovarian estradiol modulates the stimulatory effect of ovulation-inducing factor (OIF) on pituitary LH secretion in llamas.

This study was designed to: 1) characterize the effect of ovulation-inducing factor (OIF) on pituitary LH secretion in ovariectomized (OVX) llamas; and 2) determine the effect of OIF on LH secretion in OVX llamas pretreated with estradiol-17ß (E-17ß) or estradiol benzoate (EB). In Experiment 1, intact and OVX llamas (n = 5 or 6 per group) were assigned to a two by two factorial design: 1) Intact llamas treated with 1 mL of phosphate buffered saline (PBS); 2) Intact llamas treated with 1 mg of purified OIF; 3) OVX llamas treated with 1 mL of PBS; or 4) OVX llamas treated with 1 mg of purified OIF. In Experiment 2, intact and OVX llamas (n = 5 or 6 per group) were randomly assigned to the following groups: 1) Intact llamas treated with 1 mg of purified OIF; 2) OVX llamas treated with 1.0 mL of PBS; 3) OVX llamas treated with 1.0 mg of purified OIF; 4) OVX llamas primed with E-17ß, followed by 1.0 mg of purified OIF. Experiment 3 was similar as described for Experiment 2, except that priming was done with EB. In Experiment 1, animal category by treatment and animal category by treatment by time interactions tended (P = 0.08) to affect LH concentration. The effect of OIF on LH released was partly restored (P < 0.05), to the values observed for the intact OIF-treated females, when OVX llamas were primed with E-17ß or BE (Experiments 2 and 3). We concluded that peripheral estradiol concentrations in llamas partially modulates the effect of OIF on pituitary LH secretion; however, other ovarian factor(s) could also participate in this modulatory action.

Is an ovulation-inducing factor (OIF) present in the seminal plasma of rabbits?

The objectives of this study were (1) to determine the effect of rabbit seminal plasma on LH secretion and ovulation using the llama animal model as an in vivo ovulation bioassay and (2) to determine the effect of llama or rabbit seminal plasma on ovulation induction in the rabbit model. In Experiment 1, llamas with a growing follicle ≥8mm in diameter were assigned randomly to one of three groups (n=5 per group) and given an intramuscular dose of 1mL of: (a) llama seminal plasma, (b) rabbit seminal plasma, or (c) phosphate buffered saline (PBS; negative control). Blood samples for LH measurement were taken every 15 min from 1.5 h before to 8 h after treatment (Day 0: starting of treatment). Llamas were examined by ultrasonography every 12h from treatment to ovulation, and then every other day until Day 16 after treatment to evaluate corpus luteum (CL) development. Blood samples for progesterone measurement were taken every other day from Day 0 to Day 16. Ovulation was detected in 4 of 5, 5 of 5, and 0 of 0 llamas treated with llama or rabbit seminal plasma and PBS, respectively (P<0.001). After treatment, plasma LH concentration increased and decreased (P<0.01) in the llama and rabbit seminal plasma group but not in the PBS-treated group. No differences were observed on CL development (P≥0.3) and progesterone secretion (P>0.05) between both seminal plasma treated groups. In Experiment 2, receptive female rabbits (n=5-7 per group) were given an intramuscular dose of: (a) 0.5, (b) 1.0 and (c) 2.0mL of either rabbit or llama seminal plasma, (d) 0.5mL PBS (negative control), or (e) 25µg of gonadoreline acetate (GnRH; positive control). Does were submitted to laparotomy 24-36 h after treatment to determine the ovulatory response and the presence of antral and hemorrhagic anovulatory follicles. Ovulation sites (7.0±0.6) were only detected in GnRH-treated does (P<0.01). There was an increase (P<0.01), in the total number of follicles (antral plus hemorraghic follicles) in those females treated with 1mL of rabbit seminal plasma and there was a tendency (P=0.08) for more hemorrhagic anovulatory follicles in does treated with 1.0 and 2.0mL of either rabbit or llama seminal plasma. Results document the presence of OIF in the seminal plasma of rabbits. The differential ovulatory response between species, however, requires further investigation.

In vitro fertilization and development of cumulus oocytes complexes collected by ultrasound-guided follicle aspiration in superstimulated llamas.

The objective was to evaluate the developmental competence of cumulus-oocyte complexes (COC) collected by follicular aspiration in llamas treated with FSH or eCG. Llamas were assigned randomly to two groups (n = 16 per group) and treated, at the time of ovarian follicular wave emergence, with either: 1) 25 mg of FSH im, twice daily for 4 d; or 2) 1000 IU of eCG as a single i.m. dose. The start of gonadotropin treatment was considered Day 0. Both groups were given 5 mg of Armour Standard LH im on Day 6, and COC were collected by follicle aspiration on Day 7. Expanded COC collected from FSH- (n = 157) and eCG-treated llamas (n = 151) were fertilized in vitro using epididymal sperm, and presumptive zygotes were in vitro cultured in SOF medium for 8 d. The FSH and eCG treatment groups did not differ with respect to: the number of follicles ≥7 mm (16.0 ± 2.7 vs 14.0 ± 1.9, respectively; P = 0.5); the number of COC collected (11.5 ± 1.9 vs 9.7 ± 1.2; P = 0.4); the number of expanded COC (9.8 ± 1.4 vs 9.4 ± 1.2; P = 0.8); or the percentage of presumptive zygotes which developed into 2 to 8 cell stage embryos (65.3 vs 63.1), morulas (46.2 vs 42.5), or blastocysts (23.1 vs 20.5; P > 0.05). In conclusion, FSH and eCG treatments were equally effective for recovery of a high number of expanded COC which were used directly for in vitro fertilization. Furthermore, rate of embryo development was not significantly affected by the gonadotropin treatment used.

Ovarian response and embryo production in llamas treated with equine chorionic gonadotropin alone or with a progestin-releasing vaginal sponge at the time of follicular wave emergence.

The objective of the study was to compare the ovulatory response and embryo production in llamas (Lama glama) treated with a single dose of equine chorionic gonadotropin (eCG) alone or combined with intravaginal medroxyprogesterone acetate (MPA) at the time of follicular wave emergence. Llamas with a growing follicle >or=7 mm in diameter were assigned to one of the following groups: (1) Control (n=28): Nonstimulated llamas were mated and embryos were collected 7 d after mating. (2) eCG (n=32): Llamas were given 5mg luteinizing hormone (LH) (Day 0) to induce ovulation, 1000 IU eCG on Day 2, a luteolytic dose of prostaglandin F(2alpha) on Day 6, mating on Day 7, and embryo collection on Day 14. (3) eCG+MPA (n=34): Llamas were treated as those in the eCG group, but a sponge containing 60 mg MPA was placed intravaginally from Days 2 to 6. Llamas that did not respond to synchronization or superstimulation were excluded, leaving data from n=26, 26, and 27 in the control, eCG, and eCG+MPA groups, respectively, for statistical analysis. The mean (+/-SD) number of follicles>7 mm at the time of mating was greatest in the eCG group, intermediate in the eCG+MPA group, and lowest in the control group (16.6+/-5.3, 12.9+/-3.7, and 1.0+/-0.0, respectively, P<0.001). The number of corpora lutea was similar between eCG and eCG+MPA groups (10.1+/-2.9 and 8.6+/-3.7, respectively); both were higher (P<0.001) than in controls (0.9+/-0.3). The number of embryos did not differ significantly between the eCG and eCG+MPA groups (4.8+/-2.8 and 3.5+/-3.0, respectively), but both were higher (P<0.001) than in the controls (0.7+/-0.4). In conclusion, eCG, with or without MPA effectively induced a superovulatory response and multiple embryo production in llamas.

Alternative approaches to setting up donor cows for superstimulation.

Protocols that controlled follicular wave emergence and ovulation have had a great impact on the application of on-farm embryo transfer, as they permitted the initiation of superstimulatory treatments at a self-appointed time. However, the most commonly used approach for synchronization of follicular wave emergence involved estradiol, which cannot be used in many countries. Therefore, alternative treatments are required. Mechanical removal of the dominant follicle by ultrasound-guided follicle aspiration was effective, but required the use of specialized equipment and trained technical staff, which made it difficult to utilize in the field. Exogenous GnRH or pLH have also been used to induce ovulation of a dominant follicle, synchronizing follicular wave emergence, but their efficacy was dependent on the stage of the dominant follicle at treatment; thus, the emergence of the ensuing follicular wave may be too variable for superstimulation. An alternative approach could be initiating treatments at the time of emergence of the first follicular wave, but the need to synchronize ovulation may be a disadvantage in groups of donors at random stages of the estrous cycle. The final alternative may be to use FSH or eCG to initiate a new wave, without regard to the presence of a dominant follicle, followed by superstimulatory treatment at a predetermined time. All alternatives need to be thoroughly investigated in order to confirm their utility in the superstimulation of donor cows, regardless of the stage of the estrous cycle and without compromising ova/embryo production.

Developing Anti-HER2/neu Single-Chain Fv Fragments from Phage Display Libraries.

One of the major failures of conventional cancer therapy has been the inability to achieve a sufficient differential in toxicity between normal and neoplastic tissues. Since hybridoma technology was established (1), monoclonal antibodies (MAbs) have been raised against a variety of tumor-associated antigens to overcome the lack of specificity. Unfortunately, MAbs have for the most part, failed to fulfill the expectations of being the desired "magic bullets" capable of directing cytotoxic effects strictly to malignant cells. The notable exception has been in the treatment of B-cell lymphomas that have exhibited an exciting response rate when treated with unconjugated (2) and radiolabeled (3) MAbs. However, similar progress has not been observed in the treatment of ovarian carcinoma and other solid malignancies. These tumors contain disordered vasculature and lack draining lymphatics, resulting in elevated interstitial pressure. In this environment, the diffusion of a relatively large, 150 kDa IgG molecule is limited to 1 mm in 2-3 d (4). In contrast, smaller molecules such as 50 kDa Fab fragments exhibit faster movement through the tumor interstitium, on the order of 1 to 2 mm per day.

Transvaginal collection and ultrastructure of llama (Lama glama) oocytes.

Ultrasound-guided transvaginal follicle aspiration has been described as a noninvasive and repeatable procedure for oocyte collection in several species, but its use has not been described for any of the members of the family, Camelidae. A study was designed to determine the feasibility of an ultrasound-guided transvaginal approach for oocyte collection in llamas. Fifteen non-pregnant, adult female llamas (10 non-stimulated and 5 superstimulated) were examined by transrectal ultrasonography with a 7.5-MHz linear-array transducer to determine the number and diameter of follicles available for aspiration. After caudal epidural anesthesia was induced, the 7.5-MHz linear-array transducer was fastened to a long rigid handle and inserted intravaginally. The free hand was placed into the rectum to manipulate the ovaries, one at a time, in position against the vaginal wall over the face of the transducer. A 20-gauge, 55-cm-long, single-lumen needle was advanced through the vaginal fornix and into follicles > or = 3 mm in diameter. Follicular contents were aspirated using a regulated vacuum pump (flow rate = 33 mL/min; approximately 150 mm Hg) into a tube containing 3 mL of phosphate buffered saline and 0.2% BSA. Fluid was filtered (75 microm mesh), and oocytes were located and morphologically evaluated using a stereomicroscope. Overall, 134 follicles were aspirated, and 76 oocytes were collected (collection rate = 57%). Thirty-two oocytes (42%) were surrounded by multiple layers of compacted granulosa cells and had homogenous dark ooplasm; 13 oocytes (17%) were surrounded by the corona radiata layer only and had heavily granulated ooplasm; 9 oocytes (12%) were denuded and had homogenous dark ooplasm; and 22 oocytes (29%) were denuded and displayed signs of ooplasm degeneration. The ultrastructure of llama oocytes was similar to that of cattle except for conspicuous accumulation of large lipid droplets in the cytoplasm. Twenty-four hours after follicle aspiration, the ovaries were examined by transrectal ultrasonography and intrafollicular hematomas were detected in 3 llamas (9 of 48 follicles aspirated). Results demonstrate the potential utility of a transvaginal ultrasound-guided technique for oocyte collection and in vitro embryo production in llamas. Oocytes of llamas bear an ultrustructural resemblance to those of cattle, but are distinguished by a predominance of cytoplasmic lipid.

Ovarian response to gonadotropin treatment initiated relative to wave emergence in ultrasonographically monitored ewes.

Follicular recruitment and luteal response to superovulatory treatment initiated relative to the status of the first wave of the ovine estrous cycle (Wave 1) were studied. All ewes (n = 25) received an intravaginal progestagen sponge to synchronize estrous cycles, and ewes were monitored daily by transrectal ultrasonography. Multiple-dose FSH treatment (total dose = 100 mg NIH-FSH-P1) was initiated on the day of ovulation (Day 0 group) in 16 ewes. In the remaining 9 ewes, FSH treatment was started 3 d after emergence of the largest follicle of Wave 1 (Day 3 group). Ewes received PGF(2alpha) with the last 2 FSH treatments to induce luteolysis. Daily blood samples were taken to determine progesterone profiles and to evaluate the luteal response subsequent to superovulation. The ovulation rate was determined by ultrasonography and correlated with direct observation of the ovaries during laparotomy 5 to 6 d after superovulatory estrus when the uterus was flushed to collect embryos. Results confirmed that follicular recruitment was suppressed by the presence of a large, growing follicle. In the Day 0 and Day 3 groups, respectively, mean numbers (+/- SEM) of large follicles (>/= 4 mm) recruited were 6.4 +/- 0.6 and 2.7 +/- 0.7 (P < 0.01) at 48 h after the onset of treatment, and 6.7 +/- 0.5 and 5.1 +/- 0.6 (P = 0.08) at 72 h after the onset of treatment. Ovulation rates were 5.6 +/- 0.8 and 3.3 +/- 0.8 in the respective groups (P < 0.05). The number of transferable embryos was 1.8 +/- 0.5 and 0.3 +/- 0.2 in the respective groups (P < 0.05). Short luteal phases (

Endocrine and ultrasound evaluation of the response to PGF 2alpha and GnRH given at different stages of the luteal phase in cyclic ewes.

To investigate the effects of prostaglandin (PGF 2alpha) plus GnRH at different stages of the luteal phase 13 ewes received PGF 2alpha on Day 9 of the synchronized cycle, followed 36 h later by GnRH. This control regimen resulted in ovulation and normal corpus luteum (CL) function. In the next cycle, the ewes were treated simultaneously with PGF 2alpha and GnRH either on Day 4 (early, n = 7) or Day 9 (late, n = 6). Ovarian activity was monitored daily by ultrasonography, and blood samples were obtained to monitor hormonal patterns. Size of the largest follicle present when GnRH was administered was similar in all groups, but the preceding growth rate was greatest for the early group. In the 36 h after injection of PGF 2alpha, serum progesterone (P4) had declined to basal levels in the control cycles when GnRH was administered, but P4 concentrations were higher in the early group and were highest in the late group when the GnRH was administered with PGF 2alpha. The LH surges induced by GnRH were highest in the control cycles, and were lower in the 2 treated groups. In the early group, 6 of 7 ewes demonstrated ovulation within 48 h of GnRH, resulting in the formation of normal CL. In the late group, ovulation was delayed for about 5 d in 4 of 6 ewes, and subsequent luteal function was normal; no ovulation was detected in the other 2 ewes of this group, but the follicles became luteinized, resulting in a normal P4 profile in one and subnormal in the other. These results suggest that follicles present during the early luteal phase are capable of ovulating and forming fully functional CL in response to exogenous GnRH. In contrast, follicles present during the late luteal phase fail to ovulate in response to GnRH while P4 levels are high, even though the LH stimulus is adequate; however, these follicles persist and subsequently ovulate after P4 levels have decreased. Therefore, the endocrine milieu to which a follicle was exposed may be more important than its size in determining its ability to undergo ovulation and development into a normal CL.