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.     

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.     

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.     

Ultrasound image characteristics of ovarian follicles in relation to oocyte competence and follicular status in cattle

Assessment of the quality of the female gamete has become paramount for in vitro procedures. There is a need to identify reliable indicators of oocyte competence and develop a simple, non-invasive method to assess competence. The aim of this study was to investigate the relationships among ultrasonographic attributes of a follicle, its stage of development and the competence of the oocyte that it contains. We tested the hypotheses that follicular echotexture characteristics are related to: (1) the phase of development of the follicle, (2) the presence of the corpus luteum (CL) and/or the dominant follicle in the ovary, and (3) developmental competence of cumulus oocyte complexes (COC) from the same ovary. Crossbred beef cows (n=143), age 4-14 years, were given a luteolytic dose of dinoprost to cause ovulation. Ultrasound-guided ablation of all follicles > or = 4mm was done 8 days later to induce new follicular wave emergence during a luteal phase. Ultrasonographic images of dominant follicles and the three largest subordinate follicles (n=402 follicles; 84 cows) were acquired on Days 2, 3, 5 or 7 of the follicular wave (Day 0: wave emergence), i.e. growing, early-static, late static, and regressing phases of subordinate follicle development, respectively. From a subset of these animals (n=33), ovaries were collected within 30 min of slaughter and COC from subordinate follicles > or = 3mm underwent in vitro maturation, fertilization and culture to the blastocyst stage.Image analysis revealed differences in echotexture between dominant and subordinate follicles among Days 2-7 of the follicular wave. Images of dominant and subordinate follicles at Day 7 of the wave displayed consistently lower grey-scale values (P<0.05) in the peripheral antrum, follicular wall and perifollicular stroma than all other days. Follicle images displayed a consistent pattern of variation in echotexture among follicular phases. Data did not support the hypothesis of a local effect of the CL or dominant follicle on follicular echotexture. Echotexture values of the perifollicular stroma were lower in ovaries that did not produce embryos compared to ovaries that produced embryos. Our results showed that the changes in follicular image attributes are consistent with changes in follicular status. The sensitivity of the technique is not yet sufficient for use in a diagnostic setting, but results provide rationale for further development of image analysis as a tool for evaluating oocyte competence in situ.     

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.     

Endocrine and ovarian responses associated with the first-wave dominant follicle in cattle.

To examine endocrine and biochemical differences between dominant and subordinate follicles and how the dominant follicle affects the hypothalamic-pituitary-ovarian axis in Holstein cows, the ovary bearing the dominant follicle was unilaterally removed on Day 5 (n = 8), 8 (n = 8), or 12 (n = 8) of synchronized estrous cycles. Follicular development was followed daily by ultrasonography from the day of detected estrus (Day 0) until 5 days after ovariectomy. Aromatase activity and steroid concentrations in first-wave dominant and subordinate follicles were measured. Intact dominant and subordinate follicles were cultured in 4 ml Minimum Essential Medium supplemented with 100 microCi 3H-leucine to evaluate de novo protein synthesis. Five days after unilateral ovariectomy, cows were resynchronized and the experiment was repeated. Follicular growth was characterized by the development of single large dominant follicles, which was associated with suppression of other follicles. Concentrations of estradiol-17 beta (E2) in follicular fluid and aromatase activity of follicular walls were higher in dominant follicles (438.9 +/- 45.5 ng/ml; 875.4 +/- 68.2 pg E2/follicle) compared to subordinate follicles (40.6 +/- 69.4 ng/ml; 99.4 +/- 104.2 pg E2/follicle). Aromatase activity in first-wave dominant follicles was higher at Days 5 (1147.1 +/- 118.1 pg E2/follicle) and 8 (1028.2 +/- 118.1 pg E2/follicle) compared to Day 12 (450.7 +/- 118.1 pg E2/follicle). Concentrations of E2 and androstenedione in first-wave dominant follicles were higher at Day 5 (983.2 +/- 78.2 and 89.5 +/- 15.7 ng/ml) compared to Days 8 (225.1 +/- 78.6 and 5.9 +/- 14.8 ng/ml) and 12 (108.5 +/- 78.6 and 13.0 +/- 14.8 ng/ml). Concentrations of progesterone in subordinate follicles increased linearly between Days 5 and 12 of the estrous cycle. Plasma concentrations of FSH increased from 17.9 +/- 1.4 to 32.5 +/- 1.4 ng/ml between 0 and 32 h following unilateral removal of the ovary with the first-wave dominant follicle. Increases in plasma FSH were associated with increased numbers of class 1 (3-4 mm) follicles in cows that were ovariectomized at Day 5 or 8 of the cycle. Unilateral ovariectomy had no effects on plasma concentrations of LH when a CL was present on the remaining ovary. First-wave dominant follicles incorporated more 3H-leucine into macromolecules and secreted high (90,000-120,000) and low (20,000-23,000) molecular weight proteins that were not as evident for subordinate follicles at Days 8 and 12.(ABSTRACT TRUNCATED AT 400 WORDS)     

Superovulation can reduce the developmental competence of bovine embryos

This study was done to determine if different superovulatory regimens could have an effect on the percentage of embryos produced using IVM/IVF/IVC. Cyclic heifers (n = 22) were superovulated between Days 8 and 12 of the estrous cycle with 4, 6 or 8 constant doses of FSH-P (4 mg each, twice daily) +/- the addition of 1 mg prostaglandin 24 h before slaughter. Ovaries from these superovulated cows and from untreated cows were collected and the follicles dissected. Oocytes were classified according to the appearance of their cumulus and cytoplasm. Individual culture as well as group culture were performed but an individual culture reduced the percentage of oocytes developing into embryos for both untreated and superovulated animals. The results indicated that despite the superovulation regimen the developmental competence of the oocytes collected was lower (0 to 15% embryos) than that of oocytes from untreated animals (20 to 34% embryos). Small follicles ( < or = 2.7 mm) yielded mostly oocytes with an incomplete or partially expanded cumulus investment that never developed into an embryo. Differences in the morphology of the oocytes from medium (2.7 to 8 mm) and large ( > or = 8 mm) follicles were apparent, but equal developmental rates were obtained between all classes of oocytes (12 and 8% embryos, respectively). Follicular atresia was reduced significantly after superovulation (81% nonatretic follicles in treated vs 42% nonatretic follicles in untreated animals); however oocytes from atretic and slightly atretic follicles developed similarly to those from nonatretic follicles. These results suggest that although superovulation increases follicular size and decreases atresia, these conditions are not sufficient to confer developmental competence on the oocytes.     

Developmental competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vitro embryo production

Developmental competence of bovine oocytes collected from follicles of different size categories (in either the growth or the dominant phase of the first follicular wave) was studied, with the aim of improving in vitro embryo production. Estrus and ovulation of 39 cyclic Holstein dairy cows were synchronized by two prostaglandin F2alpha treatments at 11-day intervals and one hCG treatment on the day of onset of estrus (Day 0). Cows with follicles in either the growth (Day 3, n=25) or the dominant phase (Day 7, n=14) were slaughtered, and follicles >5 mm were counted. Three oocyte populations were recovered separately from large (11-15 mm), medium (6-10 mm) and small (2-5 mm) follicles in both follicular phases. All collected cumulus-oocyte complexes (COC), except for markedly atretic oocytes without cumulus cells, were used in experiments. Oocytes were matured, fertilized and cultured by standard methods. There were no significant differences between the growth and the dominant phases for mean numbers of large follicles, usable oocytes and embryos per donor. Generally, those numbers were low, but the development rates of oocytes into blastocysts were high, particularly in the growth phase (60.0%). Mean (+/- S.E.M.) numbers of medium follicles, oocytes and embryos per donor were higher in the growth as compared with the dominant phase; in the usable oocytes and embryos, this difference was significant (9.6 +/- 1.4 and 3.5 +/- 0.6 versus 3.9 +/- 0.6 and 1.1 +/- 0.3; P<0.01). The development rates of oocytes into blastocysts, however, did not differ significantly between the growth and the dominant phases (36.7% versus 27.8%). Mean numbers of usable oocytes and embryos per donor recovered from small follicles in both follicular wave phases were similar. The development rate of oocytes into blastocysts was generally low, but higher (P<0.01) in the growth than in the dominant phase (24.5% versus 11.7%). Comparison between the two phases showed that mean number of all counted follicles and all usable oocytes collected per donor were similar, but the mean number of embryos per donor and the development rate of oocytes into blastocysts were higher in the growth phase than in the dominant phase (8.0 +/- 1.2 versus 3.8 +/- 2.4; P=0.012 and 30.3% versus 14.9%; P<0.01). The interaction between follicle size and the phase of follicular wave affected the efficiency of embryo production. The yield of embryos was primarily influenced by the number of oocytes collected from medium follicles and the developmental competence of oocytes from small follicles. The growth phase was more effective for oocyte collection; the number of oocytes from medium follicles and the developmental competence of oocytes from small follicles decreased in the dominant phase.     

Effect of repeated eCG treatments and ovum pick-up on ovarian response and oocyte recovery during early pregnancy in suckling beef cows

This study was designed to evaluate in suckling early pregnant beef cows with and without eCG-pre-stimulation: (i) the influence of day gestation (from 40 to 101 days) and the consecutive eCG treatments on the follicular growth induced by means of ultrasound-guided transvaginal follicle ablation (FA; all follicles ≥ 5 mm) and the number and quality oocytes recovered by ovum pick-up (OPU) and (ii) the possible effects of repeated hormonal stimulation and FA/OPU on pregnancy outcome. Twelve suckling early pregnant Angus cows (40 days post fixed-time artificial insemination) were randomly assigned to each of two groups (n=6 group(-1)). Group 1 treatments included: FA (Day 0), eCG (1600 IU; Day 1) and OPU (Day 5). Group 2: as cited Group 1 with no eCG treatment. In both groups, OPU was repeated five times (Days 45, 59, 73, 87 and 101 of gestation). The numbers (mean ± SEM) of class II (5-9 mm; 4.3 ± 0.9) and class III (≥10 mm; 2.5 ± 0.4) follicles visualized per cow per OPU session in eCG-treated cows were greater (P<0.05) than for non-treated cows (0.9 ± 0.1 and 0.9 ± 0.1, respectively). In contrast, the number (mean ± SEM) of class I (<5mm) follicles per cow per OPU session was lower for cows with eCG treatment (2.8 ± 0.4) than for non-treated cows (5.7 ± 0.5). The mean number of aspirated follicles was not significantly different (P<0.05) between eCG-treated cows and non-treated cows at 45 and 59 days of pregnancy. However, the mean number of aspirated follicles was greater (P=0.03) in eCG-treated cows than non-treated cows from 73 day of pregnancy onwards. The numbers (mean ± SEM) of recovered oocytes and viable oocytes/cow/session were greater (P<0.05) for eCG-treated cows (2.2 ± 0.2 and 1.6 ± 0.4, respectively) than for non-treated cows (1.0 ± 0.2 and 0.9 ± 0.2, respectively). No donor pregnancies were lost either during or following OPU procedure. We can conclude that (1) eCG-treated pregnant suckled cows can be a source of oocytes for IVF at least to 100 days of gestation and (2) repeated FA/eCG treatment/OPU procedures did not affect the pregnancy outcome.     

Development of dominant follicles and length of ovarian cycles in post-partum dairy cows

The resumption of ovarian activity after normal calvings was studied in 18 lactating Friesian cows. Since, in 17 cows, first post-partum ovulation occurred without overt oestrous behaviour being detected, the resultant cycles were called 'ovarian cycles'. The mean (+/- s.d.) length of the ovarian cycles was 21.0 +/- 8.7 days. The duration of cycles tended to be normal (18-24 days) or long (greater than or equal to 25 days) when the ovulatory dominant follicles were identified before Day 10 post partum; they were consistently short (9-13 days) when dominant follicles identified after Day 20 post partum ovulated. When such follicles were detected between Days 10 and 20 post partum, long, normal and short ovarian cycles were detected. The number of waves of follicular growth with associated dominant follicles observed during the ovarian cycles tended to be related to cycle length; short cycles had 1 dominant follicle, normal cycles predominantly 2, and long cycles mostly 3 dominant follicles. The mean (+/- s.d.) duration of 13 oestrous cycles studied was 23.1 +/- 2.1 days. Of these cycles, 7 had 3 and 6 had 2 dominant follicles. The oestrous cycles with 3 dominant follicles had a mean (+/- s.d.) duration of 24.0 +/- 1.2 days and the respective dominant non-ovulatory follicles reached maximum sizes on Days 8 and 18, respectively; oestrous cycles with 2 dominant follicles were 22.2 +/- 2.6 days in duration, and the dominant non-ovulatory follicle reached maximum size by Day 8. Ovarian follicular development during the first 45 days of pregnancy was characterized by the growth and regression of successive dominant follicles, each lasting 10-12 days. These results show that the first ovarian cycle was predominantly short when the ovulatory dominant follicle was first detected after Day 20 post partum.     

Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos

The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used as cytoplast donors. The rates of development of the reconstituted embryos to the blastocyst stage were 23.1% (Group I), 15.0% (Group II), 10.9% (Group III), 4.9% (Group IV), and 29.0% (Group V). The results indicate that the developmental potential of follicles from the same living donors were different when different intervals of OPU were adopted and early atretic follicles from the second follicular wave had higher competence to support the early development of cloned bovine embryos.     

Effect of dominant follicle removal before superstimulation on follicular growth, ovulation and embryo production in Holstein cows

This study was to investigate whether removing the dominant follicle 48 h before superstimulation influences follicular growth, ovulation and embryo production in Holstein cows. After synchronization, ovaries were scanned to assess the presence of a dominant follicle by ultrasonography with a real-time linear scanning ultrasound system on Days 4, 6 and 8 of the estrus cycle (Day 0 = day of estrus). Twenty-six Holstein cows with a dominant follicle were divided into 2 groups in which the dominant follicle was either removed (DFR group, n=13) by ultrasound-guided follicular aspiration or left intact (control group, n=13) on Day 8 of the estrus cycle. Superovulation treatment was initiated on Day 10. All donors were superovulated with injections of porcine FSH (Folltropin) twice daily with constant doses (total: 400 mg) over 4 d. On the 6th and 7th injections of Folltropin, 30 mg and 15 mg of PGF2alpha (Lutalyse) were given. Donors were inseminated twice at 12 h and 24 h after the onset of estrus. Embryos were recovered on Day 6 or 7 after AI. During superstimulation, the number of follicles 2 to 5 mm (small), 6 to 9 mm (medium) and > or = 10 mm (large) was determined by ultrasonography on a daily basis. At embryo recovery, the number of corpora lutea (CL) was also determined by ultrasonography and blood samples were collected for analysis of progesterone concentration. Follicular growth during superstimulation was earlier in the DFR group than in the control group. The number of medium and large follicles was greater (P < 0.01) in the DFR group than in the control group on Days 1 to 2 and Days 3 to 4 of superstimulation, respectively. The numbers of CL (9.6+/-1.1 vs 6.1+/-0.9) and progesterone concentration (30.9+/-5.4 vs 18.6+/-3.5 ng/mL) were greater (P < 0.05) in the DFR group than in the control group, respectively. The numbers of total ova (7.7+/-1.3 vs 3.9+/-1.0) and transferable embryos (4.6+/-0.9 vs 2.3+/-0.8) were also greater (P < 0.05) in the DFR group than in the control group, respectively. It is concluded that the removal of the dominant follicle 48 h before superstimulation promoted follicular growth, and increased ovulation and embryo production in Holstein cows.     

Effects of follicular atresia and size on the developmental competence of bovine oocytes: a study using the well-in-drop culture system

The effect of granulosa cell (GC) apoptosis and follicle size on the competence of bovine oocytes were studied using a well-in-drop (WID) oocyte/embryo culture system, which allows identification of follicular origin. Hatching rates of blastocysts did not differ (P>0.05) between oocytes cultured in the WID system (13%) and those cultured in the conventional group system (16%). Hatching rates of blastocysts were higher (P<0.05) in early atretic (17%) than in non-atretic (8%) and late atretic follicles (10%) of the same size (4-8mm), and in 6-8mm (22%) than in 4-5mm follicles (15%) at the early atretic stage. More oocytes (P<0.05) from late atretic (17%) than from non-atreteic (7%) or early atretic follicles (9%) of the same size (4-8mm) were arrested at Grade 1 cumulus expansion (only cells in the peripheral two layers began to expand). Similarly, more oocytes from 2 to 3mm follicles (30%) than from 6 to 8mm follicles (21%) at the same (late) atretic stage had Grade 1 cumulus expansion (P<0.05). Hatching blastocyst percentages of oocytes with Grade 3 (all layers of the cumulus except corona radiate cells expanded) or Grade 4 (full) cumulus expansion were higher in early atretic (20%) than in non-atretic (13%) or late atretic follicles (12%). Hatching blastocyst percentages of oocytes from follicles at the early atretic stage increased as cumulus expanded from Grade 2 (9%) to Grade 4 (27%). Regardless of the degree of follicle atresia, 72-76% of the floating cells in the follicular fluid (FF) were undergoing apoptosis. The floating cell density in FF was highly (r=0.6-0.7) correlated with oocyte developmental potency. In conclusion, the WID culture system was as efficient as group culture and allowed identification of follicular origin. Furthermore, the developmental potential of oocytes was affected by GC apoptosis, follicle size and cumulus expansion, and the floating cell density in FF could be used as a simple and non-invasive marker of oocyte quality.     

The effect of a GnRH analogue on the dynamics of follicular development and synchronization of estrus in lactating cyclic dairy cows

A GnRH analogue was used to synchronize ovarian follicular development prior to an injection of PGF(2alpha) for the synchronization of estrus in lactating Holstein cows. On Day 12 (estrus = Day 0) of the experimental cycle, cows (n = 8) were injected with 8 mug Buserelin (BUS group), followed by 25 mg PGF(2alpha) 7 d later (Day 19). Control cows (n = 7) received PGF(2alpha) on Day 12 (PGF group). Ovaries were scanned daily via ultrasonography, and plasma progesterone and estradiol concentrations were determined. Sizes of all visible follicles were recorded. Follicles were classified as small (3 to 5 mm), medium (6 to 9 mm), or large (>/= 10 mm). Between Days 12 and 16 of the cycle, the number of large follicles in PGF cows remained unchanged (1.2), whereas in the BUS group, the number of large follicles decreased from 1.3 on Day 12 to 0.5 on Day 15. Only 4 of 7 PGF cows ovulated a second-wave dominant follicle. In the BUS group, 7 of 8 cows ovulated a GnRH analogue induced dominant follicle that was first identified on Day 15. During the follicular phase (last 5 d prior to estrus), plasma progesterone declined in association with CL regression in both groups, and estradiol concentrations increased, reaching higher (P<.0.05) preovulatory peak concentration in BUS cows than in PGF cows (14.0 +/- 1.0 vs 10.4 +/- 1.1 pg/ml). The number of medium-size follicles was smaller and the number of small-size follicles tended to be higher in BUS cows than in the PGF-treated group. On the day of estrus, the size of the ovulatory follicle (16.1 vs 13.3 mm) and the size difference between the ovulatory and second largest follicle (11.4 vs 6.2 mm) were both larger in BUS cows than in PGF-treated cows, suggesting a more potent dominance effect of the ovulatory follicle in the BUS cows. This study suggests that a GnRH analogue can alter follicular development prior to synchronization of estrus with an injection of PGF(2alpha) in lactating dairy cows.     

Morphology and developmental competence of bovine oocytes relative to follicular status

In cattle, follicle dimension has been used as the main criterion for selection of oocytes for in vitro embryo production. However, follicles with similar diameters may be in very different physiologic phases. The aim of this study was to investigate whether morphology and developmental competence of cumulus-oocyte complexes (COCs) are related to the phase of development of the follicle, and presence of the corpus luteum (CL) or the dominant follicle in the ovary from which the COCs were collected. Cows (n = 143) were given a luteolytic dose of PGF(2alpha) and 8 days later underwent transvaginal ultrasound guided ablation of follicles > or =4mm to induce emergence of a new follicular wave. Cows (n = 10-20 per replicate) were slaughtered on Day 2, 3, 5 or 7 (Day 0 = follicular wave emergence), equivalent to the growing, early static, late static, and regressing phases of subordinate follicle development. COCs were collected from subordinate follicles > or =3mm, were classified as denuded, degenerated or healthy, and underwent IVM-IVF-IVC. The proportion of oocytes that developed to the blastocyst stage was higher (P<0.05) in those collected on Day 5 after wave emergence (23%) than on Day 2 (12%), 3 (13%) or 7 (16%). Data did not support the hypothesis of a local effect of the CL or dominant follicle. We conclude that a positive relationship exists between early follicular regression and oocyte competence. Moreover, morphologic characteristics of oocyte quality used in this study were not predictive in identifying competent oocytes.     

Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes

Follicular size, follicular atresia, and oocyte morphology were investigated for the possible relation of these characteristics to the developmental competence of bovine oocytes. Ovaries from a local slaughterhouse were dissected to obtain a heterogeneous population of follicles. Half of each follicle was fixed for histological analysis, and the oocytes were detached carefully and cultured individually. Before in vitro maturation, the oocytes were grouped into six different classes based on the morphology of the cumulus and the ooplasm: classes 1 and 2 represent oocytes with a homogeneous ooplasm plus a compact and complete cumulus, and classes 3–6 represent oocytes with a granulated ooplasm and an incomplete and/or expanded cumulus. Oocytes from class 3 (beginning of expansion in outer cumulus layers and slight granulations in the ooplasm) developed past the 16-cell stage significantly (P<0.05) more than oocytes with a compact and complete cumulus (classes 1 and 2) and oocytes from classes 4–6 (incomplete and/or expanded cumulus) after 5 days of in vitro culture. Oocytes from follicles measuring 3 mm or less did not develop past the 16-cell stage, whereas follicles of 3–5 mm and 5 mm or larger developed at similar rates (17% and 21% morulae, respectively). The state of the follicle did not affect whether an embryo reached at least the 16-cell stage, as comparable rates were obtained in all three groups of follicles: nonatretic (20%), intermediate (14%), and slightly atretic (16%). We concluded that oocytes acquire developmental competence late in the follicular phase, possibly when the first signs of atresia have appeared, and that oocytes with beginning signs of degeneration (class 3) will develop significantly more than all other classes. Class 3 oocytes originated from follicles that were generally atretic and therefore in later phases of follicular growth, suggesting that these oocytes, having been subjected longer to the follicular microenvironment, are more differentiated (possibly at the cytoplasmic level) than other classes of oocytes. © 1995 Wiley-Liss, Inc.     

Repeated ovum pick-up in Italian Mediterranean buffalo cows

The potential of the ovum pick-up technique, used over a long period, was evaluated in 6 Italian Mediterranean buffalo cows that had more than 500 d open. The cows were submitted to ovum pick-up twice weekly for 2 mo. An additional 2-mo cycle of ovum pick-up was performed in 3 of the buffalo. The ovum pick-up sampling did not affect the resumption of reproductive activity of these animals. In fact, all the buffalo conceived, on average, 47.5 +/- 27.5 d after the last ovum pick-up. An average of 5.48 follicles was punctured, and 2.71 oocytes were collected per session. However, only 53.5% of these oocytes were suitable for in vitro embryo production. The number of punctured follicles differed between individual cows. There were no differences in the number of collected oocytes or in the recovery rates. The number of punctured follicles, the number of collected oocytes and the recovery rate were similar in the first and second months; the quality of the oocytes was, however, better in the second than in the first month (P < 0.05). The increasing interval between 2 consecutive ovum pick-up sampling (intersession interval) caused an increase of the percentage of large follicles. Moreover, the increase of the intersession interval from 4 to 5 d decreased the quality of the collected oocytes (P < 0.05). The efficiency of in vitro production of embryos to expanded blastocysts was 16.7%.     

Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation

Developmental competence of oocytes is compromised if they originate from atretic follicles. Apoptosis is the underlying process of atresia. Apoptotic changes in follicular cells are thought to influence the outcome of IVF. The aim of this study was to investigate apoptosis in different compartments of single bovine follicles (follicular wall, granulosa and cumulus cells (CC)) in relation to COC morphology, and to determine whether the addition, in vitro, of exogenous follicular cells from atretic follicles to maturing cumulus oocyte complexes (COCs) influenced the development of oocytes.Antral follicles were dissected from bovine ovaries and opened to obtain COCs and free floating granulosa cells (GC). The COCs were classified according to morphology. Apoptosis was determined in cumulus and granulosa cells and in homogenates of the remaining follicular wall.For every morphological class of COCs, a large variability of apoptotic expression was found in all follicle compartments. Follicular wall apoptosis was not correlated to COC morphology or to the percentage of apoptotic granulosa or cumulus cells. In grade 1 (best morphology) COCs, the degree of apoptosis in granulosa cells was comparable to cumulus cell apoptosis (P<0.01). The overall expression of apoptosis in granulosa cells of follicles containing grade 3 COCs (median+/-median absolute deviation: 37.8+/-13.8%) was significantly higher (P<0.05) than in follicles with grade 1 (22.7+/-10.4%) or grade 2 COCs (20.0+/-17.0%). About 48.3% of grade 3 COCs possessed strongly apoptotic cumulus cells compared to 27.8 and 28.2% of grade 1 or grade 2 COCs, respectively. Nonapoptotic cumulus complexes were observed in grades 1 and 2 COCs only.Adding exogenous follicular cells from atretic follicles to bovine COCs (grades 1 and 2) during in vitro maturation (IVM) had no impact on fertilization, blastocyst formation or hatching after IVF. This is of particular practical relevance to embryo production after ovum pick up (OPU), as during this process, good quality COCs are cultured together with simultaneously collected slightly atretic COCs.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.     

Bovine follicular development and its effect on the in vitro competence of oocytes

The current knowledge is reviewed concerning correlations between follicular development in the cow and the competence of matured oocytes to develop into an embryo following IVF and IVC. At the follicular size of 3 mm, some oocytes become competent and the proportion of competent oocytes does not increase during development up to 7 mm. The proportion of competent oocytes increases greatly in follicles > 8 mm in both untreated and gonadotropin-stimulated cows. The competence of in vitro-matured oocytes from these large follicles is lower than the competence of in vivo-matured oocytes. These observations lead to the following concept. Oocytes have acquired an intrinsic capacity to develop into an embryo after IVM-IVF-IVC at the follicular stage of 3 mm, but require an additional "prematuration" to express this competence. In vivo, this prematuration occurs during preovulatory development before the occurrence of the LH surge. In follicles of 3-7 mm, a low level of atresia appears to improve the in vitro competence of oocytes which may act via a prematuration-like effect. A thorough understanding, however, of the effect of atresia and other factors on the competence of this highly heterogeneous oocyte population is still missing. Two routes to improve the embryo yield in ovum pick-up (OPU) practice are discussed.