Ultrasound-guided transvaginal oocyte collection in prepubertal calves

The present study was designed to develop a technique for oocyte collection using an ultrasound-guided transvaginal approach in prepubertal calves too small to accommodate manual transrectal manipulation. A commercially available, 5 MHz, convex-array ultrasound transducer designed for intravaginal use in women was custom-modified for use in calves. In Experiment 1, calves 10 to 16 wk old (n = 10) were restrained in the standing position in an adjustable squeeze chute with regional anesthesia. In Experiment 2, the follicle aspiration procedure was performed in 6 wk-old calves (n = 20) in dorsal recumbency after tranquilization and caudal epidural anesthesia. Ovarian superstimulation was induced in half of the calves using 750 IU eCG (Experiment 1) or 200 mg Folltropin (Experiment 2). Consistent visualization of both ovaries using the transvaginal approach was accomplished after considerable practice. Two methods of ovarian immobilization were attempted, but both interfered with the ultrasound image and were consequently abandoned. The inability to immobilize the ovary resulted in attempts to spear the intended follicle "free-hand." The contents of follicles >or= 6 mm in diameter were aspirated, filtered, and oocytes were located using a stereomicroscope. Although ovarian superstimulation did not resolve the problem of ovary movement, the number of follicles of adequate size to aspirate was dramatically increased. Extremely sharp needles were found to be very important in the free-hand technique. A total of 232 follicles was aspirated, and 100 oocytes were collected (43%). In summary, a transvaginal ultrasound-guided technique for oocyte collection was developed for young calves in a standing position (10 to 16 wk of age) and dorsal recumbency (6 wk of age). Results demonstrate the potential utility of this approach for deriving oocytes from young calves.     

Changes in the cumulus-oocyte complex of subordinate follicles relative to follicular wave status in cattle

We investigated factors that affect cumulus-oocyte complex (COC) morphology and oocyte developmental competence in subordinate follicles on different days after follicular wave emergence in beef heifers. In Experiment 1, heifers (n = 13) were assigned at random to COC aspiration during the growing/static (Days 1 to 3) or regressing (Day 5) phase of subordinate follicle development (follicular wave emergence = Day 0). Follicular wave emergence was induced by transvaginal ultrasound-guided follicular ablation, ovaries were collected at slaughter, all follicles > or = 2 mm except the dominant follicle were aspirated, and COC were microscopically evaluated for morphology. There was a greater percentage of COC with expanded cumulus layers on Day 5 (42.4%) than on Days 1 to 3 (2.2%). In Experiment 2, heifers (n = 64) at random stages of the estrous cycle had all follicles > or = 5 mm ablated and 4 d later, 2 doses of PGF were injected 12 h apart; heifers were monitored daily by ultrasonography for ovulation (Day 0 = follicular wave emergence). Heifers were assigned to the following time periods for oocyte collection from subordinate follicles: Days 0 and 1 (growing phase), Days 2, 3 and 4 (static phase), and Days 5 and 6 (regressing phase). Ovaries were individually collected at slaughter, and all follicles > or 2 mm except for the dominant follicle were aspirated. The COC were morphologically evaluated and then matured, fertilized and cultured in vitro. Expanded COC were more frequent during the regressing phase (53.4%) than the growing or static phase (14.4 and 17.8%, respectively; P < 0.05). While the proportions of COC with > or = 4 layers of cumulus cells and denuded oocytes were higher (P < 0.05) in the growing and static phases, the production of morulae was highest (P < 0.05) with COC collected from subordinate follicles during the regressing phase. In Experiment 3, heifers (n = 18) were assigned at random to oocyte collection from subordinate follicles 3 and 4 d (static phase) or 5 and 6 d (regressing phase) after follicular wave emergence. The heifers were monitored ultrasonically for ovulation (Day 0 = follicular wave emergence); COC were collected from all follicles (> or = 5 mm) except for the dominant follicle by transvaginal ultrasound-guided follicle aspiration 3 to 6 d later. Recovered oocytes were stained and examined microscopically to evaluate nuclear maturation. A higher proportion of oocytes collected on Days 5 and 6 showed evidence of nuclear maturation (50%) than on Days 3 and 4 (8.3%; P < 0.05). Results support the hypothesis that COC morphology and oocyte developmental competence change during the growing, static and regressing phases of subordinate follicle development.     

Reduction in size of the ovulatory follicle reduces subsequent luteal size and pregnancy rate

We hypothesized that reducing the size of the ovulatory follicle using aspiration and GnRH would reduce the size of the resulting CL, reduce circulating progesterone concentrations, and alter conception rates. Lactating dairy cows (n=52) had synchronized ovulation and AI by treating with GnRH and PGF2alpha as follows: Day -9, GnRH (100 microg); Day -2, PGF2alpha (25 mg); Day 0, GnRH (100 microg); Day 1, AI. Treated cows (aspirated group; n=29) had all follicles > 4 mm in diameter aspirated on Days -5 or -6 in order to start a new follicular wave. Control cows (nonaspirated group: n=23) had no follicle aspiration. The size of follicles and CL were monitored by ultrasonography. The synchronized ovulation rate (ovulation rate to second GnRH injection: 42/52=80.8%) and double ovulation rate of synchronized cows (6/42=14.3%) did not differ (P > 0.05) between groups. Aspiration reduced the size of the ovulatory follicle (P < 0.0001; 11.5 +/- 0.2 vs 14.5 +/- 0.4 mm), and serum estradiol concentrations at second GnRH treatment (P < 0.0002; 2.5 +/- 0.4 vs 5.7 +/- 0.6 pg/mL). The volume of CL was less (P < 0.05) for aspirated than nonaspirated cows on Day 7 (2,862 +/- 228 vs 5,363 +/- 342 mm3) or Day 14 (4,652 +/- 283 vs 6,526 +/- 373 mm3). Similarly, serum progesterone concentrations were less on Day 7 (P < 0.05) and Day 14 (P < 0.10) for aspirated cows. Pregnancy rate per AI for synchronized cows was lower (P < 0.05) for aspirated (3/21=14.3%) than nonaspirated (10/21=47.6%) cows. In conclusion, ovulation of smaller follicles produced lowered fertility possibly because development of smaller CL decreased circulating progesterone concentrations.     

Gonadotropin stimulation regimens for follicular aspiration and in vitro embryo production from calf oocytes

Crossbred beef x dairy calves were randomly allocated at 3 wk of age to different gonadotropin treatment regimens for stimulation of follicle development and induction of oocyte maturation in vivo. Follicular responses were assessed laparoscopically, and oocytes were aspirated for assessment of maturational state or for in vitro fertilization (IVF) and culture to determine developmental capacity. Follicle-stimulating Hormone (FSH), administered in a single subcutaneous injection together with a low dosage of PMSG, was as effective as the same total dosage of FSH administered in 6 injections over a 3-d period. Without accompanying PMSG, this dose of FSH was ineffective in stimulating follicle development. The mean number of preovulatory follicles (> 5mm, with hyperemic appearance) doubled with each successive stimulation at 3-wk intervals, reaching 35 follicles per calf at 9 wk of age. Oocyte yields ranged from 55 to 81% of follicles aspirated, and did not differ significantly among age, FSH regimen and oocyte maturation stimulus. A combination of LH + FSH was more effective in stimulating cumulus cell expansion than LH by itself (73 vs 22% of recovered oocyte-cumulus cell complex (OCC) respectively; P<0.05). Of 33 unselected immature oocytes (cumulus unexpanded) subjected to in vitro maturation (IVM) and IVF, 30% developed to blastocysts during co-culture with bovine oviduct epithelial cells, which was not significantly different from 25% of 36 oocytes from adult ovaries which reached the blastocyst stage under similar conditions. The results indicate that follicle responses of calf ovaries to FSH stimulation increase progressively from 3 to 9 wk of age, and that oocytes recovered laparoscopically from these follicles produce blastocysts in culture at rates similar to oocytes from adult cattle ovaries collected at slaughter. The approach offers promise for embryo production from donor calves of superior genetic merit for embryo transfer, thereby enhancing the rate of genetic gain above that attainable by conventional breeding or by embryo transfer in adult cattle.     

Effect of frequency of follicle aspiration on oocyte yield and subsequent superovulatory response in cattle

The effect of frequency of transvaginal follicular aspiration on oocyte yield and subsequent superovulatory response was studied in 2 experiments. In Experiment 1, 32 primiparous Hereford x Friesian cows were assigned to 4 treatments (n = 8 per treatment). Oocyte recovery was carried out once a week for 12, 8, 4 or 0 (control) wk. Embryo recovery for all animals was 7 wk after the completion of the aspiration schedules. In Experiment 2, the effects of oocyte recovery once or twice a week (n = 8 per treatment; control n = 18) for 12 wk and response to superovulation 4 wk after the last aspiration were compared using nulliparous purebred Simmental heifers. Increasing the period of once weekly aspirations from 4 to 12 wk (Experiment 1) did not affect the number of follicles observed per session (mean +/- SEM; 10.0 +/- 0.82) or aspirated (7.8 +/- 0.71), but the recovery rate of oocytes from follicles aspirated was greater for donors aspirated for either 4 or 8 wk than for 12 wk (32.3 +/- 3.73 vs 28.4 +/- 2.61 vs 20.1 +/- 2.13 %; P < 0.05). Following the last aspiration and prior to commencing superovulatory procedures, estrus or estrous activity was observed in 7 8 , 8 8 , 7 8 and 6 8 of the animals aspirated over 12, 8, 4 or 0 wk, respectively. Subsequent superovulatory responses and in vivo embryo recoveries were similar for all aspiration treatments and for control animals. Changing the frequency of oocyte recovery from once to twice weekly (Experiment 2) did not affect the numbers of follicles observed (9.1 +/- 0.63 vs 8.3 +/- 0.85), follicles aspirated (5.9 +/- 0.56 vs 6.2 +/- 0.69), oocytes recovered (1.7 +/- 0.27 vs 1.9 +/- 2.0) per session or the oocyte recovery rate (29.4 +/- 2.4 vs 30.4 +/- 2.4 %); nor was there any effect of frequency of aspiration on subsequent superovulatory response and embryo recovery. In conclusion, increasing the period of aspiration from 4 to 12 wk and the frequency from once to twice a week over 12 wk did not reduce the number of follicles observed or aspirated, or number of oocytes recovered per donor per session. Subsequent estrous cyclicity and responses to superovulation were unaffected by the periods or frequencies of oocyte recovery examined here.     

Effects of once- versus twice-weekly transvaginal follicular aspiration on bovine oocyte recovery and embryo development

Ultrasound-guided transvaginal follicular aspiration combined with in vitro maturation/in vitro fertilization (IVM/IVF) and culture was used to obtain bovine preimplantation stage embryos. Evaluated were the effects of aspiration frequency on oocyte recovery and embryo development following IVM/IVF. In Experiment 1, transvaginal follicular aspiration was performed once (n=5) or twice (n=5) weekly in multiparous Angus cows with the aid of a transvaginal sector transducer (5-MHz). In Experiment 2, aspiration was performed on Angus cows once weekly (n=6), twice weekly (n=4), or twice weekly after treatment with FSH (15 mg; n=4). Follicles (>2 mm) were punctured using a 55-cm needle (17g), and oocytes were aspirated through the needle and silastic tubing (2 m) by vacuum suction (75 mmHg). The oocytes were examined for morphology and were in vitro matured and fertilized. Following IVF, all ova were co-cultured in vitro for 7 d on Buffalo Rat liver cells. Oocyte recovery rates per asp?ration session in Experiment 1 were not different between groups aspirated once or twice weekly (6.8+/-2.0 vs 6.3+/-1.1 oocytes/session; x+/-SEM) or in Experiment 2 between groups aspirated once, twice, or twice plus FSH treatment (7.7+/-1.8 vs 9.5+/-1.1 vs 6.2+/-1.1; P>0.10). In vitro development to the blastocyst stage was not different between the once, twice or twice-weekly aspiration plus FSH treatments or control oocytes obtained from cows at slaughter (23.1 vs 26.1 vs 18.0 vs 27.9%; P>0.10). Oocytes from the twice-weekly and twice-weekly plus FSH aspiration groups generated a higher percentage of Grade-1 quality embryos than the once-weekly group (P<0.05). In commercial bovine oocyte aspiration, more transferable embryos can be generated from twice-weekly aspirations than from once-weekly aspiration.     

Effect of unilateral ovariectomy, gonadotropin stimulation and immunization against a synthetic peptide of the inhibin alpha-subunit on follicular development and oocyte recovery in cattle

Nulliparous Holstein cows were randomly distributed among 4 treatment groups to test the effects of treatments, including unilateral ovariectomy, anti-inhibin immunization and gonadotropin stimulation on ovarian follicle population and oocyte recovery. The Control treatment consisted of intact cows (I-Control). Unilaterally ovariectomized cows were included in the 3 remaining treatments consisting of ovariectomy alone (U-Control), cows immunized against a synthetic peptide of the alpha(c)-subunit of bovine inhibin (alpha(c)I; U-IH), and cows stimulated with FSH (Super-Ov; 75 units/female/week) and also immunized with alpha(c)I as in the previous treatment (U-IH/FSH). Oocytes were collected by transvaginal ultrasound-guided aspiration on a weekly basis from cows in each treatment for 5 consecutive weeks. Intact Control cows had a greater (P<0.05) number of follicles > or = 3 mm per female (4.7) than the U-Control and U-IH cows (2.6 and 2.9, respectively), and had a similar number of follicles as the U-IH/FSH treatment group (3.5). The numbers of follicles aspirated (2.7 to 3.6) and oocytes recovered/cow (1.6 to 2.6) were similar for cows in the I-Control, U-IH and U-IH/FSH treatment groups. Cows in the U-Control treatment group had a lower (P<0.05) number of aspirated follicles (2.0) and recovered oocytes (1.1) than the I-Control cows. Cows in the U-IH/FSH and U-IH treatments had follicles with larger (P<0.01) diameters (8.7 and 8.2 mm, respectively) than cows in the I-Control (6.6 mm) and U-Control (5.7 mm) treatments. In conclusion, unilateral ovariectomy did not result in compensatory increase of follicle number or size in the intact ovary; cows in the U-IH/FSH treatment group had a greater number of follicles aspirated than the U-Control cows. In addition, the anti-alpha(c)I immunization may have played a role in increasing the number and diameter of the follicles. None of the treatments evaluated in this study improved oocyte retrieval over that of the intact, nontreated cows.     

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.     

Superstimulation of ovarian follicular growth with FSH oocyte recovery, and embryo production from Zebu (Bos indicus) calves: effects of treatment with a GnRH agonist or antagonist

The capacity of heifer calves of a late sexually maturing Zebu (Bos indicus) genotype to respond to superstimulation with FSH at a young age and in vitro oocyte development were examined. Some calves were treated with a GnRH agonist (deslorelin) or antagonist (cetrorelix) to determine whether altering plasma concentrations of LH would influence follicular responses to FSH and oocyte developmental competency. Brahman calves (3-mo-old; 140 +/- 3 kg) were randomly assigned to 3 groups: control (n = 10); deslorelin treatment from Day -8 to 3 (n = 10); and cetrorelix treatment from Day -3 to 2 (n = 10). All calves were stimulated with FSH from Day 0 to 2, and were ovariectomized on Day 3 to determine follicular responses to FSH and to recover oocytes for in vitro procedures. Before treatment with FSH, heifers receiving deslorelin had greater (P < 0.001) plasma LH (0.30 +/- 0.01 ng/ml) than control heifers (0.17 +/- 0.02 ng/ml), while plasma LH was reduced (P < 0.05) in heifers treated with cetrorelix (0.13 +/- 0.01 ng/ml). Control heifers had a surge release of LH during treatment with FSH, but this did not occur in heifers treated with deslorelin or cetrorelix. All heifers had large numbers of follicles > or = 2 mm (approximately 60 follicles) after superstimulation with FSH, and there were no differences (P > 0.10) between groups. Total numbers of oocytes recovered and cultured also did not differ (P > 0.05) for control heifers and heifers treated with deslorelin or cetrorelix. Fertilization and cleavage rates were similar for the 3 groups, and developmental rates to blastocysts were also similar. Zebu heifers respond well to superstimulation with FSH at a young age, and their oocytes are developmentally competent.     

Repeated ultrasound-guided transvaginal oocyte retrieval from cyclic Murrah buffaloes (Bubalus bubalis): oocyte recovery and quality

The present study was undertaken to explore the potential of the Murrah breed of buffaloes as donors of oocytes and to find out the recovery rate and oocyte quality in cyclic Murrah buffaloes subjected to oocyte recovery once a week. Murrah buffaloes (n = 5) were synchronized for estrus by a single prostaglandin injection schedule. The animals were subjected to transvaginal oocyte retrieval (TVOR) once weekly for 6 weeks, starting from Day 7 of the oestrous cycle (Day 0 = day of oestrus). TVOR was performed using an ultrasound machine with a 5 MHz transvaginal transducer, single lumen 19-gauge, 60 cm long needle and a constant vacuum pressure of 50 mmHg. The number and size of follicles in each ovary was determined before puncture. The follicles were characterized on the basis of their diameter as small (3-5 mm), medium (6-9 mm) and large (> or = 10 mm). The oocytes recovered were classified as grade A, cumulus-oocytes complexes (COCs) with > or = 5 layers of cumulus cells; grade B, those with two to four layers; grade C, partially denuded oocytes; and grade D, completely denuded oocytes. The mean (+/- S.E.M) number of small, medium and large follicles, and the number of total follicles observed per animal per session, which was 2.2 +/- 0.3, 0.6 +/- 0.2, 0.9 +/- 0.1 and 3.7 +/- 0.3, respectively, did not differ between animals or between puncture sessions. Small follicles constituted a major proportion (59%) of the total observed follicles. A mean (+/- S.E.M) number of 3.0 +/- 0.3 follicles were punctured and 2.0 +/- 0.3 oocytes recovered per animal per session, with a recovery rate of 68%. Out of the total 61 oocytes recovered, 36 (59%) were of grades A + B whereas 25 (41%) were of grades C + D. In conclusion, this study describes the potential of cyclic Murrah buffaloes as donors of oocytes collected by repeated TVOR once a week, without any adverse effects on follicular growth and oocyte recovery. It also describes an efficient system for carrying out TVOR in buffaloes.     

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 responses and oocyte recovery in prepubertal swamp buffalo (Bubalus bubalis) calves after FSH or PMSG treatment

Stimulation of follicular growth was examined using two different gonadotropin treatments in 10 prepubertal swamp buffalo calves (8 to 12 mo old). Each calf received an ear implant consisting of 3 mg norgestromet and 5 mg estradiol valerate during hormonal treatment. Five calves were additionally administered FSH (24 mg, im) and, 2 mo later, PMSG (3,000 IU). The remaining 5 calves were first treated with PMSG followed by FSH. Ovarian responses to treatments were examined by laparotomy, 72 h after ear implant removal, and by the number of follicles (diameter > or = 0.8 cm) and corpora hemorrhagica present. Ovaries had more significant response to FSH than PMSG treatment (13.9+/-8.6 vs 5.9+/-3.3 follicles; P<0.01). Although the recovery rate tended to be lower for FSH treated (64%) than PMSG-treated (82%) animals, more oocytes/animal were harvested in the PMSG treatment (8.3+/-5.0 vs 4.6+/-3.2, respectively). The immature oocytes (n = 38) were cultured for 24 to 25 h in maturation medium (TCM-199 NaHCO3+10% fetal calf serum [FCS] in 5%CO2 in air at 39 degrees C). Oocyte maturation was assessed after fixation and staining with aceto orcein. The in vitro maturation rate was 52.6% (20/38). This study shows the possibility of harvesting oocytes from prepubertal swamp buffalo calves and maturing the oocyte in vitro.     

Collection of oocytes from cattle via follicular aspiration aided by ultrasound with or without gonadotropin pretreatment and in different reproductive stages

Ultrasound-guided follicular aspiration was performed on 29 Holstein-Friesian cows/heifers twice weekly at 3- to 4-d intervals over a period of 2 consecutive estrous cycles (total 42 d). For visualization of the ovaries and guidance of the aspiration needle, a 6.5 MHz fingertip probe on a 62 cm probe carrier was inserted into the vagina. The disposable aspiration needle was connected to a permanent rinse tubing system, thus ensuring minimum death of oocytes in the aspiration processs. After penetration of the vaginal wall, the needle was inserted into a follicle of the rectally fixed ovary. Cumulus oocyte complexes (COC) were aspirated at a pressure of 100 mm Hg. In the first experiment, the effect of an additional gonadotropin treatment 4 d prior to aspiration was investigated in 8 lactating cows. Following FSH-treatment, the number of aspirated follicles was higher (P < 0.05) than in the nontreated animals (10.6 +/- 0.7 vs 8.9 +/- 0.5). The number of recovered COC (7.0 +/- 0.6 vs 5.8 +/- 0.5), the recovery rate (COC per aspirated follicle) (66.6% vs 65.4%), the percentage of viable COC (56.8% vs 52.1%), the cleavage rate upon in vitro maturation and in vitro fertilization (56.7% vs 59.8%) as well as the rate of morula/blastocyst formation (3.8% vs 2.9%) were similar in both groups. In the second experiment, follicles were aspirated in 4 lactating cows, 6 dry cows, 4 pregnant cows (first 35 d of pregnancy), and 4 heifers. The average number of aspirated follicles and recovered COC was higher (P < 0.05) in the first 2 groups (10.6 +/- 0.6 and 9.3 +/- 0.7 follicles; 7.2 +/- 0.5 and 6.9 +/- 0.7 oocytes) than in trie 2 other treatment groups (7.3 +/- 0.5 and 8.1 +/- 0.5 follicles; 5.0 +/- 0.4 and 5.7 +/- 0.5 oocytes). The percentage of viable COC was higher (P < 0.05; 68.3%) in lactating animals than in all the other groups (49.7, 52.5 and 57.4%, respectively). Similarly, upon in vitro fertilization, cleavage rate was higher (P < 0.05; 63.4%) in lactating cows than in the other groups (43.7, 50.5, 55.1%, respectively). A total of 21.5, 22.7, 11.9 and 13.5%, respectively, in the 4 groups of the in vitro fertilized oocytes reached the morula and blastocyst stages. After transfer of a total of 48 embryos 22 pregnancies (45.8%) were established as detected on Day 65. We conclude that 1) repeated aspiration of viable COC at short intervals is possible, 2) additional FSH-treatment does not increase oocyte yields, and 3) viable blastocysts can be produced from cattle at various reproductive phases irrespective of the reproductive phase.     

Differential requirement for pulsatile LH during the follicular phase and exposure to the preovulatory LH surge for oocyte fertilization and embryo development in cattle

The requirement for pulsatile LH and the LH surge for the acquisition of oocyte fertilizing potential and embryo developmental competency was examined in Zebu heifers. Follicular growth was superstimulated using the GnRH agonist-LH protocol in which pulsatile LH and the preovulatory LH surge are blocked. In experiment 1, heifers were assigned on Day 7 of the estrous cycle to receive: group 1A (n = 5), 1.5 mg norgestomet (NOR) implant; group 1B (n = 5), GnRH agonist implant. Follicular growth was superstimulated with 2x daily injections of FSH from Day 10 (a.m.) to Day 13 (p.m.), with PGF2alpha injection on Day 12 (a.m.). Heifers were ovariectomized on Day 15 (a.m.) and oocytes were placed immediately into fertilization, without 24 h maturation. Respective cleavage and blastocyst development rates were: group 1A, 0/64 oocytes (0%) and 0/64 (0%); group 1B, 34/70 oocytes (48.6%) and 2/70 (2.9%). In experiment 2, heifers were assigned on Day 7 of the estrous cycle to receive: group 2A (n = 10), 1.5 mg NOR implant; group 2B (n = 10), GnRH agonist implant; group 2C (n = 10), GnRH agonist implant. Follicular growth was superstimulated as in experiment 1 above. Heifers in groups 2A and 2B received an injection of 25 mg LH on Day 14 (p.m.) and all heifers were ovariectomized on Day 15 (a.m.); oocytes were placed immediately into fertilization without 24 h maturation. Cleavage rates were similar for heifers in group 2A (84/175 oocytes, 48.0%), group 2B (61/112 oocytes, 54.5%) and group 2C (69/163, 42.3%). Blastocyst development rates were similar for heifers in group 2A (22/175 oocytes, 12.6%) and group 2B (25/112 oocytes, 22.3%) and lower (P < 0.05) for heifers in group 2C (9/163 oocytes, 5.5%). Oocytes obtained from heifers treated with GnRH agonist, without injection of exogenous LH, underwent cleavage indicating that neither pulsatile LH nor the preovulatory LH surge are obligatory for nuclear maturation in cattle oocytes. Exposure to a surge-like increase in plasma LH increased embryo developmental competency indicating that the preovulatory LH surge promotes cytoplasmic maturation. The findings have important implications for controlling the in vivo maturation of oocytes before in vitro procedures including nuclear transfer.     

Characteristics of bovine estrous cycles during repeated transvaginal, ultrasound-guided puncturing of follicles for ovum pick-up

Repeated transvaginal ultrasound guided puncturing of visible follicles was performed for ovum pick-up (OPU) during Periods A and B, each of which lasted 3 mo. During Period A, 10 cows (A) were used in the study. Period B commenced 1 mo after Period A and two groups of animals were used. The first group (B1) consisted of 9 of 10 cows from Group A. The second experimental group of animals in Period B consisted of 11 cows (B2) which had not been submitted to previous puncture. During the study, all visible follicles larger than 3 mm were punctured and aspirated three times, on Day 3 or 4, Day 9 or 10 and Day 15 or 16 of the estrous cycle. The mean estrous cycle length (+/- SEM) after repeated follicle puncture did not differ among the three groups and was 22.3 +/- 0.4, 22.5 +/- 0.4 and 22.1 +/- 0.3 d for groups A, B1 and B2, respectively. The mean total number (+/- SEM) of punctured follicles per estrous cycle in Group A (13.1 +/- 0.5) was significantly larger than in Groups B1 (11.2 +/- 0.4) and B2 (11.6 +/- 0.4). The largest number of follicles punctured for ovum pick-up in all three groups was always on Day 3 or 4 of the estrous cycle: 4.9 +/- 0.3 follicles; the mean (+/- SEM) number of punctured follicles on Day 9 or 10 and Day 15 or 16 was significantly (P<0.05) lower: 3.4 +/- 0.2 and 3.9 +/- 0.2, respectively. In Period A, primarily 3- to 5-mm follicles were punctured per estrous cycle, while 6- to 10-mm follicles were predominantly punctured in Period B (P<0.05). Recovery rate of oocytes on Day 3 or 4, Day 9 or 10 and Day 15 or 16 were 53, 50 and 52%, respectively. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm.     

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.     

Effects of repeated ultrasound-guided transvaginal follicular aspiration on bovine oocyte recovery and subsequent follicular development

We wished to compare cumulus oocyte complex (COC) recovery and follicle development after single and repeated ultrasound-guided transvaginal follicle aspiration (aspiration). Aspirations were performed in Holstein-Friesian heifers every once weekly (every 7 d; n = 12) or twice weekly (every 3 or 4 d; n = 6) starting on Days 3 or 4 of the estrous cycle (estrus = Day 0) and continuing for 4 wk. During each session, all visible follicles > 2 mm were aspirated using an 7.5 MHz transducer to guide an 18 ga x 60 cm single lumen needle and applying 50 mm Hg vacuum which generated 25 mL/min. The COC's harvested from each follicle were counted and classified into 4 categories. Post-aspiration follicle wave emergence was traced by daily ultrasound examinations. A total of 1410 follicles were aspirated during 96 sessions, yielding 632 (45%)oocytes. There was no difference in average COC/follicle recovered between the single vs the repeated aspiration treatment. However, ovaries of heifers subjected to two aspirations per week yielded more follicles (17.2 +/- 5.7 vs 12.4 +/- 6.1; P < 0.01) and COC's (7.7 +/- 4.5 vs 5.4 +/- 3.7; P < 0.01) per session than those subjected to a single aspiration. Ovaries of heifers subjected to twice weekly aspirations at 4-d intervals resulted in a higher recovery rate (51.1 vs 38.6%), yielded more COC's (9.3 +/- 4.7 vs 6.2 +/- 3.8) and a higher number of viable COC's recovered per session (7.6 +/- 3.8 vs 5.2 +/- 3.3) than those aspirated every 3 d, all P < 0.01. Aspiration-induced follicle waves were indicated by an increased number of follicle > or = 4 mm seen within 2 d of the procedure. We conclude that follicle aspiration appears to induce and synchronize follicle waves, and when it is done twice a week it is associated with higher number of harvestable follicles and more oocytes recovered than when done once a week. These results can be attributed to the aspiration of a newly recruited pull of follicles 3 or 4 d after the first aspiration and before the establishment of follicular dominance and regression of subordinate follicles.     

Effect of an implant containing the GnRH agonist deslorelin on secretion of LH, ovarian activity and milk yield of postpartum dairy cows

Prevention of high plasma progesterone concentrations in the early postpartum period may improve fertility. Our objective was to determine whether a Deslorelin implant (DESL; 2100 microg, s.c.) would reduce secretion of LH and alter follicle dynamics, plasma concentrations of progesterone, estradiol and PGF2alpha metabolite (PGFM) in postpartum dairy cows. Cows received DESL on Day 7 postpartum (Day 7, n=8) or were untreated (Control, n=9). All cows were injected with GnRH (100 microg, i.m.) on Day 14 to assess LH response. A protocol for synchronization of ovulation with timed AI was initiated on Day 60 (GnRH [Day 60], CIDR [Day 60 to Day 67], PGF2alpha [Day 67, 25 mg and Day 68, 15 mg], GnRH [Day 69] , AI [Day 70]). The LH response to injection of GnRH on Day 14 was blocked in animals treated with DESL. Numbers of Class 1 (<6 mm) follicles were unaffected (P > 0.05) whereas numbers of Class 2 (6 to 9 mm) (P < 0.01) and Class 3 (>9 mm) follicles were less (P < 0.01) in DESL cows between Day 7 and Day 21. From Day 22 to Day 60, DESL-treated cows had more of Class 1 follicles and less Class 2 (P < 0.01) and Class 3 (P < 0.01) follicles, and lower plasma concentrations of progesterone and estradiol (P < 0.01). Concentrations of PGFM between Day 7 and Day 42 were not affected by treatment (P > 0.05). All cows ovulated in response to GnRH on Day 69. Subsequent luteal phase increases in plasma progesterone concentrations (Day 70 to Day 84) did not differ. The use of the DESL implant associated with PGF2alpha given 14 days later suppressed ovarian activity and caused plasma progesterone concentrations to remain < 1 ng/mL between Day 22 and Day 51. The DESL implant did not affect milk production.     

Follicular and endocrinological turnover associated with GnRH induced follicular wave synchronization in Indian crossbred cows

The goal of this study was to record the hormonal and follicular turnover in Jersey crossbred cows when subjected for follicular wave synchronization using GnRH. Six healthy, non-lactating and regularly cycling Jersey crossbred cows (5-6 y) were used for the study. In the control group, the follicular wave pattern was ultrasonographically investigated in 18 cycles (3 cycles/cow). In the treatment group, GnRH analogue (buserelin acetate 10 μg im) was administered on Day 6 of the cycle and follicular wave pattern was studied in 12 cycles (2 cycles/animal). Follicular population was categorized based on their diameter Class I, ≤5 mm; Class II, >5-<9 mm; Class III, ≥9 mm) and the number of follicles in each category was determined on Day 6, Day 8 and Day 10. Plasma FSH and progesterone concentrations were estimated in both control and treatment groups. Out of 18 estrous cycles studied, 14 cycles (77.8%), three cycles (16.7%) and one cycle (5.6%) exhibited three-, two- and four-follicular waves per cycle, respectively. It was evident that the DF of Wave I established its dominance and was in the growing phase by Day 6 of the estrous cycle in all the normally cycling crossbred cows. The DF ovulated in all the animals (100%) in the mean interval of 27.7 ± 0.2 h after GnRH administration. A synchronized homogenous group of follicles emerged two days after GnRH injection (Day of 8.0 ± 0.0) in all the animals (100%). The combination of LH surge induced ovulation of DF (abrupt termination of Wave I) and FSH surge stimulated homogenous recruitment of Class I follicles, led to a synchronized emergence of follicular wave. All the GnRH treated cows had three follicular waves because of early emergence and short period of dominance of Wave II DF.