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.     

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 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.     

Effect of ultrasonically-guided follicle aspiration on estrous cycle and follicular dynamics in Holstein cows

Two experiments were conducted to test the effect of ultrasonically-guided follicle aspiration on estrous cycle and follicular dynamics in Holstein cows. The objective of the first experiment was to determine if aspiration of all visible follicles would influence the estrous cycle. All visible follicles > or = 5 mm in diameter were punctured and the oocytes aspirated once during the second and fourth cycle, with no aspirations occurring during Cycles 1, 3 and 5 in 4 Holstein cows. Between aspirations the follicles were scanned ultrasonically every 2 to 3 d to monitor follicular waves. Estrus was monitored twice daily and plasma progesterone concentrations were measured daily. The inter-estrus periods were significantly longer when aspiration was performed than in the 3 cycles in which aspiration did not occur (25.0 vs 21.1 d; P < 0.01). To determine the maximum number of follicles harvestable on a continuing basis, a second experiment was conducted to contrast unstimulated cows subjected to ultrasonically-guided oocyte collection twice-weekly with animals from which oocytes were collected once a week after gonadotrophin stimulation. All visible follicles of multiparous Holstein cows were aspirated every 3 or 4 d (unstimulated group, n = 4), and the total number of follicles was compared with the number of follicles in cows that were aspirated weekly after 400 mg FSH were administered over 2 d (stimulated, n = 6). The study continued for 8 consecutive weeks. The weekly mean number of follicles available for aspiration in the unstimulated cows was 15.7 +/- 3.3 (mean +/-SEM), which was not different from the 14.2 +/- 1.9 follicles available for aspiration from the FSH-stimulated cows. Within the unstimulated cows, there was an increased number of follicles available for aspiration over time during the period of study (P < 0.001). The number of follicles available for aspiration from the stimulated cows did not change, but plasma concentrations of progesterone increased over time as persistent luteal tissue developed on the ovaries.     

Utilization of the growth phase of the first follicular wave for bovine oocyte collection improves blastocyst production

Characteristics of the follicle population and oocyte developmental competence at selected stages of follicular development were studied in cows with the aim to increase embryo production derived from oocytes collected by transvaginal aspiration. In Experiment 1, the growth phase before dominant follicle selection and the low dominant phase during dominant follicle regression were compared. Twenty-four cyclic Holstein cows, 4 to 6 yr of age, were divided into 2 groups. Animals were synchronized using two injections of prostaglandin F2alpha at 11 d intervals, and onset of estrus was determined (Day 0). Using ultrasonography, all follicles were counted and classified. Oocytes were aspirated once on Days I through 3 (Group 1, n=5) or Days 15 and 16 (Group 2, n=3) of the estrus cycle. The experiment was carried out in 3 replicates. In Experiment 2, the growth phase of the first follicular wave before dominant follicle selection was characterized in detail. Twelve cows of the same breed and age were divided into 3 groups. Their first estrus was synchronized as in Experiment 1, and each following estrus was induced using one injection of prostaglandin F2alpha administered 4 to 6 d after each aspiration performed. The ovaries were examined, and oocytes were collected repeatedly (total of 5 times per cow) on Days 1 (Group 3, n=4), 2 (Group 4, n=4) or 3 (Group 5, n=4) after estrus at 10 d intervals during a 40 d period. Viable oocytes were matured, fertilized and cultured using the standard methods. In Experiment 1, the mean numbers (+/-SD) of all follicles and of recovered and viable oocytes per donor were higher in Group 1 than in Group 2, but only the mean numbers (+/-SD) of larger follicles and recovered oocytes were statistically significant (8.0 +/- 0.6 and 6.2 +/- 0.6.vs. 3.3 +/- 0.5 and 2.8 +/- 0.2; P< 0.05). In Experiment 2, the percentage of larger follicles out of all visible follicles and the mean numbers (+/-SD) of larger follicles per donor were significantly higher (P<0.05) in Groups 4 (75.7 and 9.1 +/- 2.7) and 5 (66.3 and 8.5 +/- 2.9) when compared to Group 3 (27.9 and 3.8 +/- 0.8). The development rate of fertilized oocytes was significantly higher (P<0.05) in Groups 4 (27.8) and 5 (27.5) than in Group 3 (12.8). It can be concluded that it is possible to improve the efficiency of transvaginal aspiration and in vitro embryo production by utilization of the growth phase of the first follicular wave before dominant follicle selection.     

Effect of ovarian superstimulation on COC collection and maturation in alpacas

The objective of the present study was to compare the ovarian follicular response, cumulus-oocyte complex (COC) collection rate, and maturational status of COC collected from alpacas subsequent to treatment with two different superstimulatory protocols. Alpacas (n=7 per group) were treated with: (1) 200mg of FSH im divided bid for 3d, plus a single i.v. dose of 1000IU hCG 24h after the last FSH treatment, or (2) 1200IU of eCG as a single i.m. dose, plus a single i.v. dose of 1000IU of hCG on day 3 after eCG treatment (day 0=start of superstimulatory treatment). At 20-24h post-hCG treatment, the ovaries were surgically exposed and COC were collected by needle aspiration of all follicles > or =6mm. The FSH and eCG treatment groups did not differ with respect to the number of follicles > or =6mm at the time of COC collection (20.0+/-7.5 versus 27.0+/-3.3; P=0.5), the number of COC collected (26.2+/-8.4 versus 23.3+/-3.7; P=0.7), or the collection rate per follicle aspirated (89% versus 87%; P=0.7). No differences were detected between FSH- and eCG-treated alpacas in the number of expanded COC collected per alpaca (11.5+/-2.9 versus 8.8+/-2.8; P=0.54), the number of expanded COC in metaphase II (8.5+/-1.9 versus 6.0+/-2.1; P=0.1), or the number of compact COC with > or =3 layers of cumulus cells (12.5+/-4.3 versus 14.3+/-2.6; P=0.72). A greater proportion (P<0.05) of compact COC collected after FSH treatment matured in vitro to the metaphase II stage than after eCG treatment. Eight expanded alpaca COC were fertilized in vitro with llama sperm, three of which were fixed and stained 18h after exposure to sperm and five were cultured in vitro. Two of the three stained oocytes were in the pronuclear stage, and all five of the cultured oocytes developed to the two-cell and morula stages at 2 and 7 days, respectively, after in vitro fertilization. In summary, FSH and eCG treatments were equally effective for ovarian superstimulation and oocyte collection. Cumulus-oocyte complexes were collected from more than 80% of follicles aspirated during laparotomy. Nearly one third of the COC collected after superstimulation were in metaphase II, and more than 70% of the remaining COC progressed to metaphase II after in vitro maturation for 26h, bringing the mean number of oocytes available for in vitro fertilization to 16 per alpaca. Preliminary results support the hypothesis that alpaca oocytes obtained after superstimulation in the absence of progesterone are developmentally competent since morulae developed from all five COC fertilized and cultured in vitro.     

Effects of ultrasound-guided transvaginal follicular aspiration on oocyte recovery and hormonal profiles before and after GnRH treatment

Endocrine changes and recovered oocytes were evaluated during 16 wk of ultrasound-guided transvaginal follicular aspiration (TVFA) and prior to and following administration of GnRH at the cessation of aspiration. Nonlactating previously aspirated (PAC, n = 4) and non-aspirated, (AC, n = 4) Holstein cows were subjected to 16 wk of twice-weekly aspiration. Four control cows (OAC) were aspirated 1 time only at the final TVFA session (wk 16). Jugular blood samples were collected from all cows during aspiration, before and after the final TVFA session, and during an 18-d period following cessation of aspiration. Ovarian activity was monitored in all cows after cessation of aspiration for 18 d. The PAC and AC cows averaged 3.4 +/- 1.2 (+/- SE) and 6.8 +/- 1.2 oocytes per session, respectively. Progesterone concentrations during TVFA did not differ between the PAC and AC (0.8 +/- 0.1 and 0.9 +/- 0.1 ng/mL, respectively). Progesterone concentration in OAC was 4.5 +/- 0.2 ng/mL before TVFA, while the PAC and AC averaged 0.5 +/- 0.2 and 0.3 +/- 0.2 ng/mL, respectively, at 16 wk. At Week 16 LH was 1.0 +/- 0.2 ng/mL and it increased to 7.5 +/- 0.1 ng/mL after GnRH treatment. The LH concentration before the final aspiration session was higher at peak amplitude in PAC than in AC groups and peak length was longer in OAC than in AC cows (P < 0.07). Between 18 and 24 h after the last aspiration there were more LH peaks and greater peak frequencies in PAC than in OAC cows (P < 0.07), and the interval between peaks was longer in PAC and AC cows (P < 0.10) than in OAC cows. Mean FSH concentrations were lower (P < 0.01) for OAC than for PAC and AC groups at 20 and 24 h after the last aspiration. Follicle numbers after GnRH varied most among treatment groups for follicles < 9 mm, with the PAC, AC and OAC averaging 5.1 +/- 1.0, 5.1 +/- 1.0, and 3.8 +/- 1.0 follicles/d, respectively. Progesterone concentrations increased to 1.1 +/- 0.3 ng/mL in PAC cows and 2.5 +/- 0.3 and 3.4 +/- 0.3 ng/mL in AC and OAC groups, respectively, during the 18-d period. These results suggest that long-term TVFA affects progesterone, LH and FSH profiles and ovarian dynamics in cows.     

Ultrasound-guided follicle aspiration: the collection of bovine cumulus-oocyte complexes from ovaries of slaughtered or live cows

To increase the collection efficiency of bovine cumulus-oocyte-complexes (COCs) by transvaginal aspiration, the effects of aspiration pressure and needle diameter on bovine follicular oocyte collection were assessed. Oocytes were aspirated from ovaries of slaughtered cows using 2 different diameter needles (18- or 21-gauge) with 4 different aspiration pressures (40, 80, 120 or 160 mmHg) and of live cows using 18-gauge needles with 40 or 80 mmHg, or using 21-gauge needles with 80 or 120 mmHg. The recovered oocytes were divided into 4 categories according to the surrounding cumulus cells and quality of oocytes: 1) 4 or more layers, 2) between 1 and 3 layers, 3) completely or partially denuded and 4) all others, including expanded cumulus cells and degenerated oocytes. The highest oocyte recovery rates from Categories 1 and 2 were obtained using 18-gauge needles with 40 mmHg pressure and 21-gauge needles with 120 mmHg pressure, respectively, from the ovaries of slaughtered cows. When oocytes were collected from live cows, the highest recovery rates for Categories 1 and 2 were obtained using an 18-gauge needle and 40 mmHg pressure, and 21-gauge needle and 80 mmHg, respectively. In addition, the proportion of oocytes in each category were compared between ovaries from slaughtered and live cows. The proportion of Category 1 oocytes collected from live cows was lower than from slaughtered cows when 18-gauge needles at 80 mmHg (P<0.05). The results show that the combination of aspiration pressure and needle diameter is crucial for COC collection, and they suggest that optimal aspiration conditions for ovaries of slaughtered cows are not necessarily applicable to live cows.     

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.     

Transvaginal ultrasound guided follicular aspiration of bovine oocytes

A transvaginal ultrasound guided follicular aspiration technique was developed for the repeated collection of bovine oocytes from natural cycling cows. In addition, the feasibility of using this method for collecting immature oocytes for in vitro embryo production was also evaluated. Puncturing of visible follicles for ovum pick-up was performed in 21 cows over a three month period. All visible follicles larger than 3 mm were punctured and aspirated three times during the estrous cycle on Day 3 or 4, Day 9 or 10 and Day 15 or 16. The mean (+/- SEM) estrous cycle length after repeated follicle puncture was 22.2 +/- 0.3 days. The mean total number of punctured follicles per estrous cycle was 12.6 +/- 0.3. The largest (P<0.05) number of follicles punctured (5.1 +/- 0.3) for ovum pick-up was on Day 3 or 4 of the estrous cycle. The overall recovery rate of 541 punctured follicles was 55%. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm. Following in vitro maturation and fertilization (IVM/IVF), 104 oocytes were transferred to sheep oviducts. Six days later, 75 ova/embryos were recovered, after flushing the oviduct of the sheep, of which 24% developed into transferable morulae and blastocysts. In this study, a reliable nonsurgical, follicular aspiration procedure was used for the repeated collection of immature oocytes which could be used successfully for in vitro production of embryos. This procedure offers a competitive alternative to conventional superovulation/embryo collection procedures.     

In vitro embryo production after microinjection and ovarian dynamics following transvaginal follicular oocyte aspiration

Ultrasound-guided transvaginal follicular aspiration of oocytes from live cows combined with IVM, IVF and in vitro culture (IVC) is a procedure for producing preimplantation-stage bovine embryos and a source of oocytes for pronuclear microinjection of DNA for producing transgenic cattle. This experiment was designed to compare in vitro embryo development rates between oocytes derived from transvaginal follicular aspiration and those obtained from cows at slaughter. Nine cows were subject to a twice-weekly aspiration. Oocytes were aspirated with a 5 MHz ultrasound transducer packaged in a vaginal probe equipped with a dorsal-mounted needle guide (16-ga). All visible follicles (>2 mm) were punctured with a 17-ga, 55-cm needle at each aspiration session and the contents removed under vacuum suction. Oocytes underwent IVM/IVF/IVC. Microinjection of DNA was performed during the pronuclear stage of development, and the zygotes were co-cultured on Buffalo Rat Liver (BRL) cells in modified M199 at 39 degrees C in 5% CO2 and air. After 7 d in culture, embryos were removed and scored for development. A Chi-square analysis was used to compare transvaginal follicular-derived oocytes (microinjected and not) and slaughterhouse-derived, matured in transit oocytes (SHDMT; microinjected and not). Nonmicroinjected embryos resulting from IVF of transvaginal aspiration-derived oocytes developed to blastocysts at a higher rate than SHDMT oocytes (40.0 vs 30.8%; P < 0.05). There was no difference in development rates between the microinjected groups (aspiration = 15.9% vs SHDMT = 12.8%). Higher proportions of the embryos generated from the aspirated oocytes were of excellent or good quality following culture (P < 0.05). In the present experiments the effects of microinjection may overshadow some effects of ova source, but transvaginal follicular aspiration may provide a more consistent, synchronous population of oocytes than those derived from commercial slaughter house sources for use with in vitro systems.     

Comparison of repeated transvaginal ovum pick up in heifers by ultrasonographic and endoscopic instruments

Endoscopically and ultrasonographically guided ovum pick up were studied in 15 heifers once per week. Althogether 60 sessions were carried out. The number of aspirated follicles per animal (13.0 vs 10.3) did not differ significantly between both methods. Similar recovery rates (39 % vs 44 %) were achieved after repeated sessions. As a consequence the number of recovered oocytes per animal (5.2 vs 4.7) were not significantly different. Inter-animal- and intra-animal-variations were more important for the results than the applied methods. In contrast to these findings the quality of recovered cumulus-oocyte-complexes (COC) was significantly influenced by the methods. The COC were divided into 4 categories a) oocytes with compact cumulus, b) oocytes with rest of compacted cumulus, c) oocytes with expanded cumulus and d) oocytes without cumulus. There was a higher denudation rate of COC when using the endoscopic aspiration (62.0 % vs 6.6 %) because of turbulent current in this aspiration system. Advantages and disadvantages of both methods are described and discussed. Generally, the ultra sonographic method is the less traumatic procedure for the vagina, fornix and for abdominal organs. The other method is less traumatic for the ovaries.     

The relationship of follicle atresia to follicle size, oocyte recovery rate on aspiration, and oocyte morphology in the mare

Oocytes were collected by aspiration of follicles from horse ovaries obtained at surgery or post-mortem. The oocytes were classified according to morphology of the ooplasm and cumulus. The size of the corresponding follicles was measured, and sections of the follicles were fixed and examined histologically to determine the stage of viability or atresia. In Part 1, 11 pairs of ovaries were examined and all follicles were sectioned; in Part 2, 9 pairs of ovaries were examined and only those follicles from which oocytes were recovered were sectioned. The number of follicles examined per pair of ovaries in Part 1 (average +/- SD) was 12.9 +/- 4.1. The proportion of follicles that were viable increased with increasing follicular size (P < 0.01); the percentage of viable follicles was 21, 42 and 83% for follicles < 10 mm, 10 to 19 mm, and >/= 20 mm in diameter, respectively. The overall oocyte recovery rate on aspiration of follicles was 46%. There was no significant difference in the oocyte recovery rate between viable and atretic follicles. A significantly higher proportion of oocytes recovered from viable follicles had granular ooplasm (64 vs 39%; (P < 0.05); whereas significantly more oocytes from atretic follicles had a misshapen or dense ooplasm (23 vs 6%; P < 0.05), or an expanded or pyknotic cumulus (24 vs 6%; P < 0.05). The most common cumulus morphology (63% of oocytes from viable follicles and 48% of oocytes from atretic follicles) was presence of only the corona radiata. Only 11% of oocytes from viable follicles and 9% of oocytes from atretic follicles had a complete cumulus present.     

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.     

Relationship between follicle size and ultrasound-guided transvaginal oocyte recovery.

We evaluated the relationship between follicle size and oocyte recovery (OR) using ultrasound-guided follicle aspiration. Thirty Holstein cows were subjected to OR without gonadotrophic therapy. Oocytes were recovered two to four times from each cow in a total of 67 aspiration sessions. Ovarian follicles with diameters < or =4 mm and >4 mm were aspirated in separated groups. Recovered oocytes from each group were kept separate and submitted to in vitro maturation, fertilization, and culture to the blastocyst stage. A total of 430 follicles were aspirated, of which 154 (35.8%) were from follicles >4 mm and 276 (64.2%) were from follicles < or =4 mm. Seventy-seven oocytes (50%) were recovered from follicles >4 mm and 200 (72.2%) were from follicles < or =4 mm. Nineteen blastocysts were obtained from follicles >4 mm, whereas 45 blastocysts were obtained from follicles < or =4 mm. Recovery rate was greater (P<0.01) in follicles < or =4 mm. Oocyte quality, cleavage rate and blastocyst development did not differ between different follicle sizes. Routine aspiration of small follicles (< or =4 mm) could increase the number of oocytes available for in vitro development.     

Effect of genetic merit, milk yield, body condition and lactation number on in vitro oocyte development in dairy cows

The effects of milk yield, body condition score (BCS) and lactation number on the number of oocytes recovered and blastocysts formed were studied following in vitro maturation, fertilization and culture of bovine oocytes collected from 48 high and 46 medium genetic merit dairy cows in their first and third lactation. The cows were slaughtered between 125 and 229 d post partum. Ovaries were recovered, and 2- to 10-mm follicles were aspirated. Cleavage rate and number of blastocysts were determined at 44 h and 7 d after insemination, respectively. Oocytes from high genetic merit cows formed fewer blastocysts and had lower cleavage and blastocyst formation rates than those from medium genetic merit cows (0.36 +/- 0.19, 70.4 and 6.8% vs 0.85 +/- 0.22, 77.4 and 11.4%, respectively). The effect of milk production was tested by grouping cows in their third lactation into high and low groups. There was no difference in number of oocytes recovered and subsequent development into blastocysts between the cows in the high milk production group (4559 to 5114 kg, n = 20) and cows in the low yield (3162 to 3972 kg, n = 20) group (6.9 +/- 1.34 vs 8.9 +/- 1.32, respectively). The effect of BCS was tested by grouping cows in their first or third lactation into high and low groups. Cleavage and blastocyst formation rates were greater for oocytes from cows with a high BCS (3.3 to 4.0, n = 20) than a low BCS (1.5 to 2.5, n = 20) (75.7 vs 61.9% and 9.9 vs 3.0%, respectively). Cows in the first lactation yielded fewer oocytes (5.7 +/- 1.24) than cows in the third lactation (7.8 +/- 0.79). Thus, the quality of oocytes probably contributes to reduced fertility, often evident in high genetic merit dairy cows.     

Ultrasound-guided follicle aspiration: effect of the frequency of a linear transvaginal probe on the collection of bovine oocytes

The effect of the frequency of an ultrasonic linear transvaginal probe on the collection of bovine oocytes by transvaginal ultrasound-guided follicle aspiration was investigated. Probes with different frequencies (7.5 or 5.0 MHz) were applied to examine the clarity of follicles on the monitor using ovaries of slaughtered cows in Experiment 1. The follicles were visualized on the monitor and divided into small (3- to 5-mm diameter) and large (6- to 10-mm) groups. They were also divided into 2 groups according to the clarity of their outline (clear or obscure). The number of small follicles visualized with a clear outline was greater (P < 0.01) with the 7.5 MHz probe than with the 5.0 MHz probe (9.0 vs 3.2). Oocyte aspiration from live cows was performed using the 7.5 or 5.0 MHz probe in Experiments 2 and 3. The recovered oocytes were divided into 3 categories: cumulus-oocyte-complexes (COCs), denuded oocytes and all others. In Experiment 2, the number of oocytes collected per donor cow was assessed, and in Experiment 3 the number of oocytes per aspirated follicle was examined by aspirating a constant number of follicles per aspiration session. The numbers of oocytes and COCs per donor cow obtained with the 7.5 MHz probe (11.2 and 9.0, respectively) were greater (P < 0.01) than those obtained with the 5.0 MHz probe (4.3 and 3.5). This difference between probes was due to the greater clarity of the follicle images obtained with the 7.5 MHz probe.     

Comparison of oocyte developmental competence and follicular steroid content of nulliparous heifers and cows at different stages of lactation

Reduced reproductive performance and lower conception rates of lactating cows are closely associated with genetic progress for high milk production. In contrast, the fertility of nulliparous Holstein heifers has remained fairly stable over the years and appears to be markedly higher than that of mature lactating cows. Possible differences in oocyte quality and follicular steroid levels, which could be associated with the low fertility of high-lactating cows, were examined in 13-month-old heifers, cows around the time of first AI (60-95 d post-partum, yielding 49+/-2.4 kg/d) and cows at mid-lactation (120-225 d post-partum, yielding 37+/-2.1 kg/d). Estrus was synchronized by two doses of PGF2alpha and follicles (5-8 mm) were aspirated on days 4, 8, 11 and 15 of the cycle by an ultrasound-guided procedure. Oocytes were morphologically examined, matured in vitro, chemically activated and cultured for 8d. Cleavage rate and the proportion of developing parthenogenetic blastocysts were determined on days 3 and 8 post-activation, respectively. On day 17, heifers and cows received additional PGF2alpha and follicular fluids from preovulatory follicles were collected on day 19 perior to the expected estrus. Follicular-fluid volumes were similar in cows and heifers, as were estradiol, progesterone and androstenedione concentrations in the follicular fluid. Percentages of high-grade oocytes, proportions of cleaved oocytes and developed blastocysts did not differ between the groups. Results suggest that the fertility gap between nulliparous heifers and high-lactating cows is not directly related to steroid content in the preovulatory follicular fluid or oocyte developmental competence.     

In vivo oocyte recovery and in vitro embryo production from bovine donors aspirated at different frequencies or following FSH treatment

The effects of frequency of follicular aspiration and treatment of donor cattle with FSH on in vivo oocyte recovery and in vitro embryo production were studied. Simmental heifers (n = 24) formed 8 replicates of 3 treatments in which oocyte donors were aspirated 1) once a week, 2) twice a week, or 3) once a week following treatment with FSH for 3 d prior to aspiration. Oocytes were graded, washed, matured for 20 to 24 h and then inseminated with frozen/thawed semen from a single sire, followed by co-culture on granulosa cell layers. Embryo development was observed until Day 7 after insemination. Significantly fewer follicles per heifer per week were counted (14.7+/-2.3 vs. 27.4+/-3.1 vs. 23.1+/-2.8) and aspirated (12.0+/-2.0 vs. 21.8+/-2.7 vs. 20.1+/-2.6) in heifers on the once-weekly than twice-weekly aspiration treatment (P<0.01) or on the once-weekly aspiration after FSH treatment (P<0.05). There were no significant differences between treatments in the total number of oocytes recovered per week (5.6+/-1.2 vs. 8.9+/-1.5 vs. 6.1+/-1.2), but significantly more oocytes per heifer per week recovered from animals treated with FSH were graded Category 1 (2.8+/-0.4), i.e., >4 layers good cumulus with a clear, even cytoplasm, than from animals aspirated once (0.9+/-0.2; P<0.01) or twice a week (1.5+/-0.3; P<0.05). The number of transferable morulae plus blastocysts produced per heifer per week was higher from animals aspirated twice a week (2.4+/-0.4; P<0.05) or once a week following FSH treatment (2.1+/-0.4; P<0.05) than from animals aspirated once a week without FSH treatment (1.0+/-0.3). In conclusion, FSH treatment of bovine oocyte donors aspirated once a week enabled a similar number of transferable embryos to be produced per donor week as aspiration twice a week without FSH treatment. These 2 treatments produced twice as many transferable embryos per donor week as aspiration once a week without FSH treatment.     

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.