Large Follicle Group Research Articles

Evaluation of cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles in jennies.

Reference values of follicular fluid components at different stages of follicular development in jennies are unknown. It is important to estimate such values because the biochemical composition of follicular fluid provides a suitable microenvironment for oocyte development, growth and maturation. The experimental protocol was carried out in the Equine Reproduction Clinic of the FMVZ-UNAM and was approved by an Institutional Sub-committee for the Care and Use of Experimental Animals (SICUAE.MMVZ-2020/4-2). Seven healthy female donkeys “Mexican burro” with average body condition score of 3 on a scale of 1 to 5 (Svendsen et al. The professional Handbook of the Donkey. 2008), were monitored by transrectal ultrasonography through the Veterinary Digital Diagnostic Ultrasound System 5 MHz (Model, DUS 60 VET) with a multi-frequency linear transducer. The ovum pick up (OPU) by transvaginal aspiration (TVA) was performed in a total of 70 preovulatory follicles, 10 for each jenny using a real- time B- mode ultrasound vaginal 6.5 MHz (Sonovet 600) with curvilinear transducer and 12 gauge (12 G) needle. Follicular fluid components were measured and analyzed by Luminex Multiplex Assays at the Universidad Nacional Autónoma de México. The relationship between cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles and follicular size was assessed. The preovulatory follicles are classified in three groups accordingto the diameter: small (27-30 mm), medium (31-35 mm) and large (36-38 mm). Concentrations of cortisol were lower in the large follicles group (18.8 ng/ml) compared with small (721.44 ng/ml) and medium follicles (361 ng/ml). The concentrations of thyroxine were higher in the medium follicle group (49 ng/ml) than in the small (21ng/ml) and large follicle groups (15.9 ng/ml). Finally, the concentration of triiodothyronine was higher in the small follicles group (1043 ng/ml) followed by the medium (545 ng/ml) and the lowest concentration was in the large follicles group (22 ng/ml). It is concluded that thyroxine concentrations are lower than T4 concentrations in preovulatory follicles irrespective of size as reported for human follicular fluid (Wakim. et. al. Fertil Steril 1993; 59(6) 1187–1190). Furthermore, no relationship was found between preovulatory follicular size and thyroxine hormone concentrations. It has been suggested that cortisol and triiodothyronine concentrations are related to preovulatory follicle size. Cortisol concentrations are higher in preovulatory follicles that in subordinate follicles and cortisol is a mediator of apoptosis in granulosa cells, differentiation, development and maturation of tissues, specifically in the oocyte (Acosta & Miyamoto. Anim Reprod Sci 2004; 82:127-140, Scarlett. et al. Dom Anim Endocrinol 2016; 59:11-22). The role of cortisol within the different sizes of preovulatory follicles is unknown.

Does a Large (>24 mm) Follicle Yield a Competent Oocyte/Embryo?

Aim: To evaluate the effect of large follicular size (≥24 mm) at day of oocyte retrieval on oocyte/embryo quality. Patients and Methods: A cohort study was conducted in a single tertiary medical center between July 2018 and May 2019. Before ultrasound-guided follicular aspiration, follicles were measured and divided into 2 groups according to their maximal dimensional size: large: ≥24 mm and normal: <24 mm. Microscopic examination of the follicular aspirates was performed by an embryologist. Each follicle aspirated was evaluated for oocyte maturation, oocyte fertilization, and embryo quality. Results: 428 follicles were measured, including 383 (62.81%) in the normal and 45 (14.06%) in the large follicle groups. Oocytes were achieved during aspiration from 297 (75.5%) and 29 (64.4%) of the normal and large follicle groups, respectively (p = 0.05). No in-between group differences were observed in mature oocyte (MII), fertilization, and top-quality embryo (TQE) rates. Nevertheless, once a zygote (2PN) was achieved, a trend toward a higher TQE rate/2PN was found in the large follicle group (16/19 [84.2%] vs. 115/171 [67.3%]; p = 0.062). Conclusion: While a nonsignificant decrease in oocyte recovery rate was found in follicles ≥24 mm, the zygote and TQE per follicle were comparable.

Improve the developmental competence of porcine oocytes from small antral follicles by pre-maturation culture method

The oocytes from small antral follicle have low developmental potential to reach blastocyst due to incomplete cytoplasmic maturation during in vitro maturation (IVM). Thus, we developed an in vitro culture system for porcine oocytes derived from small antral follicles with l-ascorbic acid supplement during pre-maturation (pre-IVM) to support their development to blastocyst stage. Besides that, how l-ascorbic acid effect on the developmental competence of porcine oocytes with a special focus on histone modifications will be elucidated.The in vitro culture process consisted of two steps. The first step is 22 h of pre-IVM and the second step is 42 h of IVM. We utilized dibutyryl-cyclicAMP (dbcAMP) with L-ascorbic supplement during pre-IVM. Based on the result of this procedure, we proposed that the best culture condition in which hormone human chorionic gonadotropin (hCG) be added during the last 7 h of pre-IVM and continued culture to complete IVM. We observed that, in this culture system, the meiotic competence of porcine oocytes derived from small follicles was as high as those derived from large follicles after undergoing IVM. In addition, our study suggested that l-ascorbic acid supplementation at 100 μg/mL sharply enhanced the developmental potential of porcine oocytes. Interestingly, oocytes from small antral follicles treated with l-ascorbic acid could obtain the blastocyst quantity and quality as high as that of large antral follicles. The treated groups showed a significantly higher number of blastomeres compared to those in non-treated groups in both small and large follicle groups. Besides that, = The increasing levels of acetylation of histone H3 at lysine 9 (H3K9) and methylation of histone H3 at lysine 4 (H3K4) in blastocyst derived from small and large antral follicle under the present of l-ascrobic acid lead to a significant positive effect on the developmental competence and improvement in quality of porcine embryos.

Effect of roscovitine on developmental competence of small follicle-derived buffalo oocytes.

Background & objectives:The lower recovery of competent oocytes in buffalo species limits the commercialization of in vitro embryo production technology in field condition. In this context, pre-maturation of small follicle (SF)-derived oocytes with meiotic inhibition may be a promising alternative to obtain more number of competent oocytes. Thus, the present study was conducted with an objective to enhance the developmental potential of less competent SF-derived buffalo oocytes.Methods:All the visible follicles (used for aspiration) from buffalo ovaries were divided into two categories: large follicle (LF) (follicles having diameter ≥6 mm) and SF (follicles of diameter <6 mm). The competence of LF and SF oocytes was observed in terms of brilliant cresyl blue (BCB) staining, cleavage rate, blastocyst rate and relative gene expression of oocyte and blastocyst competence markers. Thereafter, less competent SF oocytes were treated with 0, 12.5, 25, 50 and 100 mM doses of roscovitine (cyclin-dependent kinase inhibitor) to enhance their developmental potential.Results:Based on parameters studied, LF oocytes were found to be more competent than SF oocytes. Pre-maturation incubation of SF oocytes with roscovitine reversibly arrested oocyte maturation for 24 h to ensure the proper maturation of less competent oocytes. A significantly higher number of BCB-positive oocytes were noted in roscovitine-treated group than SF group. Cleavage and blastocyst rates were also higher in roscovitine-treated group. The relative messenger RNA expression of oocyte (GDF9, BMP15, GREM1, EGFR, PTGS2 and HAS2) as well as blastocyst (INF-τ, GLUT1 and POU5F1) competence markers was significantly greater in roscovitine-treated group relative to SF group. Again, on comparison with LF group, these parameters depicted a lower value in the treatment group.Interpretation & conclusions:The findings of this study has revealed that pre-maturation incubation of SF-derived oocytes with 25 μM roscovitine can improve its developmental competence and thus can be utilized to get maximum number of competent oocytes for better commercialization of in vitro embryo production technology in buffalo.

Association between three glycosidases activity [±-mannosidase (±-oe'), ²--acetyloglucosaminidase (NAGASE) and ²-galactosidase (²-GAL)] andin vitro fertilization of bovine oocytes collected from different-sized follicles

We studied the role of three glycosidases (α- mannosidase - α-ΜΑΝ, β-N-acetyloglucosaminidase - NAGASE and β-galactosidase - β-GAL) in follicular fluid (FF) and in fertilization medium (FM) of bovine oocytes. Oocytes were allocated into 3 groups according to the follicular size (controls - CF: 2-8 mm, small follicle group - SF: 2-5 mm, large follicle group - LF: >5-8 mm). Bovine embryos were produced in vitro either in groups (experiment 1, n = 2099 oocytes) or individually (experiment 2, n = 79 oocytes). In both experiments, the activity of all glycosidases in the FF of large follicles was significantly lower than in the FF of small follicles group. In the FM of LF-group oocytes, α- MAN and NAGASE were significantly higher compared to SF- and CF-group oocytes (experiment 1) and β-GAL was significantly higher in SF- compared to CF-group oocytes (experiment 2). Cleavage rate was similar among all groups in both experiments; however significantly higher blastocyst formation was noted in CF-group compared to LF- (days 7, 8, 9) and SF- (days 8, 9) groups (experiment 1). In follicular fluid of small follicle group, β-GAL was associated positively with degenerating oocytes’ number and negatively with blastocyst rate at days 7, 8 (P = 0.065) and 9 (experiment 1). In fertilization medium of control group, α-MAN related negatively to cleavage rate (P < 0.05) and β-GAL to blastocyst rate at day 8 (P = 0.089) or day 9 (P = 0.072) (experiment 1). During fertilization, in experiment 1 all oocytes consumed β-GAL and only control or small follicle oocytes consumed α-MAN; in experiment 1, only large follicle oocytes released NAGASE, whereas all oocytes released all three glycosidases in experiment 2. In conclusion, glycosidases affect the developmental competence of oocytes collected from different sized follicles during in vitro fertilization, performed either in groups or individually; their role in follicular fluid is different from that in fertilization medium.

Improved fertility in suckled beef cows ovulating large follicles or supplemented with long-acting progesterone after timed-AI

We aimed to evaluate the effects and the interaction of size of the preovulatory follicle (POF) and long-acting progesterone (P4) supplementation after timed-AI on CL function and pregnancy success in beef cows. In experiment 1, ovulations of beef cows were synchronized starting on Day −10, and cows were split to receive sodium cloprostenol (large follicle group; LF; n = 31) or nothing (small follicle group; SF; n = 35). Ovulations were induced on Day 0, and cows were inseminated. Ovulated cows were assigned to receive placebo (LF/control group, n = 14; and SF/control group, n = 9) or 150 mg of long-acting P4 on Day 4.5 (LF/P4 group, n = 13; and SF/P4 group, n = 12). Diameter of POF, blood flow in POF wall, ovulation rate, and size and vascularization of CL were greater (P < 0.05) in LF group. In experiments 2 (unknown cyclic status) and 4 (noncycling), ovulations were synchronized, and beef cows received placebo or 150 mg of long-acting P4 on Day 4 after timed-artificial insemination. In experiment 2, pregnancy/AI (P/AI) did not differ (P > 0.1) between P4-treated (53.2%; 209/393) and control cows (56.2%; 219/390), but P/AI was greater in cows with a CL < 0.9 cm2 on Day 4 that were P4-treated (57.9%, 22/38) versus placebo-treated (40.4%, 21/52; P < 0.05). In Experiment 4, P/AI was greater (P < 0.05) in P4-treated cows (55.6%, 105/189 vs. 46.0%, 86/187). In Experiment 3, cyclic–suckled beef cows were treated as described in Experiment 1 to generate animals with small (SF; n = 111) or large POF (LF; n = 109), and subdivided to receive placebo or P4 on Day 4. POF size, ovulation rate, CL area, and P/AI were greater (P < 0.007) in the LF group. Pregnancy/AI in ovulated cows were lower (P = 0.05) in the SF/control group (41.5%, 17/41) compared to LF/control group (62%, 31/50) and were similar for the SF/P4 group (55.6%, 25/45) and LF/P4 group (57%, 28/49) compared to others. In summary, smaller and less vascularized POF results in less functional CL and reduces ovulatory rate and P/AI in cyclic beef cows; the long-acting P4 injection on Day 4 after timed-artificial insemination may attenuate the negative effects of small POF/CL; and postovulatory P4 supplementation improved fertility in anestrous beef cattle.

11 FERTILITY RESPONSE IN SUCKLED BEEF COWS SUPPLEMENTED WITH LONG-ACTING PROGESTERONE AFTER TIMED ARTIFICIAL INSEMINATION

Corpus luteum (CL) and progesterone (P4) secretion are affected by preovulatory follicle (POF) size. Increased circulating P4 during early diestrus has a positive effect on embryo development in beef cattle. However, the combined effects of the POF size and P4 supplementation during early diestrus on fertility of beef cows are not known. The objective was to evaluate the effects of POF size and supplementation of long-acting P4 after timed-AI on pregnancy rates (P/AI). Suckled Nelore cows (n = 596) were evaluated twice by transrectal Doppler ultrasonography (US) 10 days apart to detect the cyclic status. In Study 1, anestrous cows (absence of CL on both exams) received an intravaginal P4 device and an oestradiol benzoate (EB) injection on Day –10 (day of the second US). Devices were removed and sodium cloprostenol [prostaglandin F2α (PGF2α)], oestradiol cypionate, and eCG were given on Day –2. Cows were timed-AI on Day 0 and assigned to receive placebo (control group, n = 187) or 150 mg of long-acting P4 on Day 4 (P4 group, n = 189). In Study 2, cyclic cows (presence of CL) received a PGF2α injection on Day –20 (first US). Cows with a new CL on Day –10 received an intravaginal P4 device and an injection of EB and were split to receive an injection of PGF2α [large follicle (LF); n = 109] or not [small follicle (SF); n = 111]. Devices were removed and PGF2α was injected on Day –2. Ovulation was induced with buserelin acetate, and cows were timed-AI on Day 0 and split to receive placebo (LF/control group, n = 55, and SF/control group, n = 55) or 150 mg of long-acting P4 on Day 4 (LF/P4 group, n = 56, and SF/P4 group, n = 54). Ultrasonographic scanning was done on Days 0, 4, and between 35 and 40 to detect the POF and CL sizes and P/AI, respectively. Data were analysed using PROC GLIMMIX (SAS Institute Inc., Cary, NC). In anestrous cows, P/AI was reduced in POF with &lt;11 mm. The P/AI was greater in the P4-treated group than in the control group for all cows (55.6% v. 46.0%; P = 0.05) and for ovulated cows (59%, 105/178 v. 49%, 86/173; P = 0.08). For cyclic cows, POF size (mm) on Day 0 (13.5 ± 0.3 v. 11.2 ± 0.2), ovulation rate (90% v. 77%), and CL area (cm2) on Day 4 (1.46 ± 0.05 v. 1.25 ± 0.05) were greater (P &lt; 0.007) in the LF group than in the SF group. There was a main effect of follicle group on P/AI (54%, LF group v. 38%, SF group; P &lt; 0.01). Moreover, P/AI were greater (P &lt; 0.05) in the LF/control (56%) and LF/P4 (52%) groups than in the SF/control group (31%), whereas no difference was detected between the SF/P4 group (45%) and the other groups. Among cows that ovulated, P/AI was lower (P = 0.05) in the SF/control group (41%, 17/41) compared with the LF/control group (62%, 31/50) and were similar for the SF/P4 group (56%, 25/45) and LF/P4 group (57%, 28/49) compared with others. We suggest that P4-stimulated embryotrophic effects improved fertility in anestrous beef cows supplemented with long-acting P4 on Day 4 after timed-AI. Also, the presence of a functional CL during follicle growth results in smaller POF and CL and reduces the ovulatory and P/AI rates in cyclic cows. Post-AI P4 supplementation may attenuate the negative effects of small POF/CL. Research was supported by CNPq, FAPESP, Ouro Fino Agronegócio, and Innovare.

11 STRATEGIES TO MODULATE THE PERI-OVULATORY ENDOCRINE MILIEU BY CONTROLLING PROGESTERONE CONCENTRATIONS PRE- AND POST-INSEMINATION IN BEEF COWS

Recent evidence indicates that progesterone (P4) secretion from corpus luteum (CL) is affected by preovulatory follicle (POF) size and that increased circulating P4 during early diestrus has a positive effect on embryo development and pregnancy rates in cattle. Despite the embryotrophic effects, early administration of P4 may impair CL growth and anticipates luteolysis. Owing to develop favourable peri-ovulatory endocrine milieus to pregnancy success, we evaluated in beef cows (1) the effect of CL presence during dominant follicle growth on follicular vascularization, size and ovulation rate and (2) the effects of POF size and supplementation of long-acting P4 post-AI on CL development and regression. Sixty-six Nelore cows received an intravaginal P4 device and an injection of oestradiol benzoate on Day 10 (5–10 days post-oestrus), and were split to receive sodium cloprostenol [prostaglandin F2α (PGF2); large follicle (LF); n = 31) or not [small follicle (SF); n = 35]. Devices were removed and PGF2 was injected on Day 1.75. Ovulation was induced with a gonadotropin-releasing hormone (GnRH; buserelin acetate) on Day 0. Cows were inseminated twice on Days 0.5 and 1. Cows that ovulated were assigned to receive placebo (LF/control group, n = 14; and SF/control group, n = 9) or 150 mg of long-acting P4 on D4.5 (LF/P4 group, n = 13; and SF/P4 group, n = 12). Colour-Doppler ultrasound scanning was done on Days 10, 6, 2, 0.5, 1, 1.5, 4.5, 7.5, and 15.5. Structural luteolysis was detected by 25% decrease in CL area and 50% in luteal blood flow of subsequent scanning. Data were analysed for the main effects of POF size and supplementation and their interaction using the PROC MIXED procedure of SAS (SAS Institue Inc., Cary, NC, USA). Follicle diameter (mm) on Day 2 (11.4 ± 0.4 v. 8.4 ± 0.3) and POF (14.1 ± 0.3 v. 12.5 ± 0.3) were greater (P &lt; 0.05) in LF group. Percentage of luteal blood flow in follicle wall was greater (P &lt; 0.05) on Day 0.5 (33.9 ± 3.4 v. 22.8 ± 3.6) and 1 (48.9 ± 4.8 v. 28.3 ± 4.3) in the LF group. Ovulation rate was lower (P &lt; 0.05) in the SF group (60%, 21/35) than in the LF group (90%, 28/31), suggesting that a functional CL during dominant follicle growth is less favourable to pregnancy success due to 30% reduction in fertilizing potential of non-ovulated follicles. For luteal area (cm2), an effect (P &lt; 0.05) of POF size was detected on Day 4.5 (1.7 ± 0.1 v. 1.3 ± 0.1), 7.5 (2.8 ± 0.2 v. 2.2 ± 0.1), and 15.5 (2.5 ± 0.1 v. 2.2 ± 0.2) in the LF and SF groups, respectively. Luteal blood flow was greater (P &lt; 0.05) in the LF groups (40.4 ± 1.7 and 47.6 ± 3.4) than in the SF groups (35.7 ± 1.5 and 37.9 ± 4.1) on Day 4.5 and 15.5, respectively. This indicates that larger and more vascularized POF may be associated with greater CL vascularization in addition to greater CL size. The frequency of cows with early luteolysis did not differ (P &gt; 0.1) between P4-treated (5/25) and placebo-treated (1/23) cows. We suggest that the P4 supplementation did not severely induce early luteolysis in inseminated cows and that the P4-embryotrophic effects may down-regulate the mechanisms involved in the earlier luteolytic process caused by greater exposure of uterus to P4. Support for this study was provided by the São Paulo Research Foundation (FAPESP, Sao Paulo, Brazil) and Ourofino (Cravinhos, SP, Brazil).

78 THE PERIOVULATORY ENDOCRINE MILIEU AFFECTS THE OXIDATIVE STRESS CONTROL ON ENDOMETRIUM OF COWS

Acknowledging that the local oxidative profile (pro or antioxidant) may influence the control of very specific mechanisms such as intracellular signalling as well as the unspecific damage caused by oxidative stress, it is proposed that the balance between reducing and oxidative activities of the uterine environment (i.e. the REDOX profile) is regulated by exposure to ovarian steroids. The aim of this study was to verify the effect of the periovulatory endocrine milieu on concentrations of reactive oxygen species (ROS), activity of antioxidant enzymes, and lipid peroxidation profile on the endometrial tissue of cows in early diestrus. Eighty-three cyclic, nonlactating Nelore cows received an intravaginal progesterone (P4) device and an injection of oestradiol benzoate (E2) on Day –10. Animals were divided into 2 groups to receive sodium cloprostenol (PGF; large follicle group; LFG; N = 42) or not (small follicle group; SFG; N = 41) on Day –10. Progesterone devices were removed and prostaglandin F2α was injected between Day –2.5 and Day –1.75 in cows from LFG, and between Day –1.5 and Day –1.25 in cows from SFG. Ovulation was induced with GnRH on Day 0. Progesterone and oestradiol plasma concentrations were quantified. Animals were slaughtered on Day 7, when endometrial tissue was collected. A subgroup of ovulated cows (n = 9 per group) was selected to assess the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Furthermore, the content of reduced glutathione (GSH) and ROS by spectrofluorometry and lipid peroxidation by assessment of malondialdehyde concentrations were determined. Statistical comparisons between groups were performed by ANOVA (Statistica 4.5; StatSoft Inc., Tulsa, OK, USA). Maximum diameter of the preovulatory follicle (mean ± s.e.m.; 13.36 ± 0.29 v. 11.11 ± 0.33 mm, respectively; P &lt; 0.01), plasma concentrations of P4 on Day 7 (3.71 ± 0.25 v. 2.62 ± 0.26 ng mL–1; P &lt; 0.01) and E2 on Day 0 (2.94 ± 0.28 v. 1.27 ± 0.2 pg mL–1; P &lt; 0.01) were greater in LFG compared with SFG. The SFG showed lower enzymatic activity for CAT (0.79 ± 0.09 v. 0.5 ± 0.07 U mg–1 of protein; P &lt; 0.01) and GPx (2.43 ± 0.39 v. 2.0 ± 0.35 nmol of NADPH/min/mg of protein; P &lt; 0.05) than LFG. Additionally, lipid peroxidation was increased in SFG (177.68 ± 46.46 v. 255.71 ± 40.64 nmol of MDA/g of tissue; P &lt; 0.01), similarly to SOD activity (37.76 ± 3.95 v. 44.77 ± 7.66 IU; P &lt; 0.05). Concentrations of ROS and GSH were not different between groups. In conclusion, the periovulatory endocrine environment alters antioxidant mechanisms in the endometrium of cows in early diestrus. Specifically, it is proposed that the greater antioxidant activity observed on the LFG provided an environment that was less prone to lipid peroxidation than that from the SFG. The increase in SOD activity suggests a compensatory mechanism triggered by an environment potentially more oxidative observed on the SFG. The REDOX profile observed on the experimental groups may be associated with higher fertility observed in cows with higher E2 and P4 concentration during the proestrus and early diestrus, respectively. We acknowledge support from CNPq, FAPESP; Ourofino, CCPS-USP.

Effects of the estrous cycle on the efficacy of oocyte collection andin vitroembryo production in Duroc-breed

Collection efficacy and in vitro embryo developmental ability of oocytes obtained from Duroc-breed ovary donors at different stages of the estrous cycle (days 6, 12 and 16 after estrus) were performed. The numbers of collected oocytes did not differ significantly among the different estrous cycle groups (total 72-90 oocytes per gilt). However, the blastocyst rates of oocytes collected on days 12 and 16 (9.2% and 19.4%, respectively) were significantly higher than those on day 6 (1.1%). More oocytes were obtained on day 16 from small follicles (<2 mm in diameter; 85.3 oocytes per gilt) than from medium-sized (≥2-<6 mm) and large (≥6 mm) follicles (17.5 and 12.8 oocytes, respectively). The blastocyst rates in both the medium-sized and large follicle groups (20.0% and 19.2%, respectively) were significantly higher than that in the small follicle group (6.3%). The blastocyst cell numbers in both the medium-sized and large follicle groups (39.4 and 43.3 cells, respectively) were significantly higher than that in the small follicle group (30.6 cells). The results suggest that oocyte collection from cycling Duroc pigs can be carried out efficiently from the late luteal to follicular stage. Those oocytes collected from medium-sized and large follicles show better embryo development.

266 COMPARISON OF OOCYTES FROM LARGE-SIZED (≥8-mm) and MEDIUM-SIZED (3- to 7-mm) FOLLICLES FOR IN VITRO EMBRYO PRODUCTION IN PIGS

Despite recent efforts to improve culture systems, the developmental competence of in vitro-matured (IVM) porcine oocytes is still inferior compared with those that have been in vivo matured. In pigs, cumulus–oocyte complexes (COC) are usually aspirated from 3- to 7-mm follicles and matured for 42 to 44 h in vitro. In this study, we compared oocytes obtained from large-sized (≥8-mm) and medium-sized (3- to 7-mm) follicles in terms of nuclear maturation, intracellular reduced glutathione levels, gene expression, and embryo development after IVM. In the control group, COC (n = 521) were aspirated from 3- to 7-mm follicles and matured for 22 h with hormones (eCG/hCG) and subsequently matured in vitro for 20 to 22 h without hormones at 39°C, 5% CO2. In the large follicle (LF) group, COC (n = 256) were obtained from follicles larger than 7 mm and were subjected to IVM reduced for 18 h. The maturation medium was TCM-199 supplemented with 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng mL–1 of epidermal growth factor, 75 µg mL–1 of kanamycin, 1 µg mL–1 of insulin, and 10% (vol/vol) porcine follicular fluid without hormones. Nuclear status and reduced glutathione content in oocytes were investigated by Hoechst 33342 staining and CellTracker Blue (CMF2HC; Invitrogen, Carlsbad, CA, USA), respectively. The abundance of messenger RNA of genes reflecting the developmental competence of oocytes was analysed in cumulus cells by real-time PCR using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as the reference. In addition, oocytes were subjected to parthenogenetic activation to assess in vitro embryo developmental competence. Data were analysed by Student’s t-test using SPSS version 17.0 (IBM Corporation, Armonk, NY, USA) and presented as means. All experiments were replicated at least three times. The average frequency of ovaries having ≥8-mm follicles was 2.3% (44/1953 in 11 replicates). Before IVM, the nuclear-stage oocytes from ≥8-mm follicles were as follows: germinal vesicle stage = 15.2%; metaphase I (MI) stage = 55.4%; anaphase I and telophase I (AI + TI) stages = 15.8%; and metaphase II (MII) stage = 13.6%. After 6 h of IVM, 4.2% of oocytes were at the germinal vesicle stage and frequencies of the MI, AI + TI, and MII stages were 43.6, 9.4, and 42.8%, respectively. After 18 h, IVM frequencies of the MI and MII stages were 13.0 and 87.0%, respectively. Oocytes of the LF group showed a significant (P &lt; 0.001) increase in intracellular reduced glutathione level (1.41 v. 1.00) compared with the control (42- to 44-h matured oocytes). Cumulus cells in the LF group showed lower (P &lt; 0.1) messenger RNA expression of COX-2 (cyclooxygenase-2) and TNFAIP6 (tumor necrosis factor, α-induced protein 6), and higher (P &lt; 0.1) expression of PCNA (proliferating cell nuclear antigen) and Nrf2 (NF-E2-related factor 2) compared with the control. After parthenogenetic activation, the oocytes from the LF group had significantly (P &lt; 0.05) higher blastocyst rates and total cell numbers in blastocysts than did the control group (90.1% and 73.6 v. 50.5% and 55.3, respectively). In conclusion, oocytes from preovulatory LF require only 18 h to complete maturation in vitro, and their developmental competence is higher than those obtained from MF. Although limited, oocytes from ≥8-mm follicles offer an alternative source of material for the production of transgenic pigs by somatic cell nuclear transfer. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008121), Rural Development Administration, Republic of Korea.

177 EFFECTS OF MANIPULATION OF DOMINANT FOLLICLE GROWTH ON SIZE AND FUNCTION OF CORPUS LUTEUM IN BEEF CATTLE

Recent evidence indicates that the progesterone (P4) secretion by corpus luteum (CL) during early diestrus is affected by the size of ovulatory follicle and has a significant impact on embryo development and conception rates. Therefore, strategies to promote the growth of the dominant follicle and/or to stimulate the early development of the CL to increase P4 secretion become an alternative to improve conception rates in the beef cattle industry. Our aim was to study the effect of manipulations of the follicle growth on the diameter of the preovulatory follicle (POF) and subsequent size and function of the CL. Cyclic and non-lactating Nelore cows, pre-synchronized by 2 injections of prostaglandin F2α (PGF) 14 d apart, were manipulated to ovulate large or small follicles according to 3 experiments. In Experiment 1 (Exp. 1; n = 23), animals received a second-use intravaginal P4-releasing device along with an injection of oestradiol benzoate on Day –10 (Day 0 = GnRH injection). Cows were split to receive (large follicle group; LF) or not (small follicle group; SF) a PGF injection on Day –10. Progesterone devices were removed on Day –2.5 in the LF group and on Day –1.5 in the SF group. The PGF was injected at the removal of the P4 device. In Experiment 2 (Exp. 2; n = 38), cows in the LF group had the P4 device removed on Day –2.25 or Day –2, whereas in Experiment 3 (Exp. 3; n = 23), the device (first-use) was removed on Day –1.75 in the LF group and on Day –1.25 in the SF group; the other manipulations were similar to Exp. 1. Data analyses were done only on cows that had a functional CL on Day –10 (P4 &gt; 1 ng mL–1) and that ovulated within 24 and 48 h post-GnRH (Exp. 1, n = 14; Exp. 2, n = 14; Exp. 3, n = 12). The three experiments were successful in inducing POF with different sizes, as indicated by the greater diameter of the POF in the LF group compared with SF in Exp. 1 (12.9 ± 0.5 mm v. 10.7 ± 0.6 mm; P &lt; 0.03), Exp. 2 (14.1 ± 0.6 mm v. 11.7 ± 0.4 mm; P &lt; 0.006), and Exp. 3 (13.8 ± 0.6 mm v. 11.7 ± 0.8 mm; P &lt; 0.06). To evaluate the effect of POF size on size and function of the CL, a factorial analysis was performed by SAS software to test the effect of group, day, and their interaction. For CL volume, an effect of group was detected in Exp. 1 (P &lt; 0.02) and in Exp. 3 (P &lt; 0.06), but not in Exp. 2. The group effect represented greater average CL volume from Day 3 to Day 7 in LF (2.42 ± 0.27 and 2.5 ± 0.39 cm3) than in the SF group (1.39 ± 0.18 and 1.2 ± 0.15 cm3) for Exp. 1 and 3, respectively. For P4 concentrations, a group effect was detected only in Exp. 3 (P &lt; 0.007), as indicated by greater average P4 concentrations from Day 3 to Day 7 in LF (2.31 ± 0.31 ng mL–1) than in the SF group (1.37 ± 0.19 ng mL–1). A day effect was detected in all experiments (P &lt; 0.0001), as indicated by a progressive increase of CL volume and P4 concentrations from Day 3 to Day 7. Manipulation of follicle growth performed in Exp. 3 was the most efficient to modify the function and size of the CL. In conclusion, control of POF size by manipulation of P4 concentrations during growth of the dominant follicle alters the size and function of CL postovulation. CNPq, FAPESP, Ourofino, and PUSP-P.

Early follicle development alters the relationship between antral follicle counts and inhibin B and follicle-stimulating hormone levels on cycle day 3

To verify whether, during the luteal-follicular transition, increased inhibin B production by abnormally large follicles modifies the expected relationship between the number of follicles and peripheral inhibin B and FSH levels. Prospective study. Teaching hospital, France. A total of 192 normo-ovulatory women. Serum inhibin B and FSH levels and numbers and sizes of antral follicles (3-12 mm) were measured on cycle day 3. The strength of hormonal-follicular correlations was assessed in two groups formed according to the presence (large follicle group; n = 73) or absence (small follicle group; n = 119) of one follicle measuring >7 mm. Serum inhibin B and FSH levels were correlated with antral follicle counts (r = 0.32 and r = -0.44, respectively). These relationships were significantly weaker in the large follicle group (r = 0.24 and r = -0.28, respectively) than in the small follicle group (r = 0.51 and r = -0.55, respectively). Antral follicle size influences serum inhibin B and FSH levels and alters their expected relationship with the number of antral follicles on day 3. These results contribute to clarifying the controversial role of serum inhibin B and FSH levels in the prediction of ovarian follicular status.

Effects of VEGF and bFGF on proliferation and production of steroids and nitric oxide in porcine granulosa cells.

Ovarian angiogenesis, which is currently considered to be of crucial importance in controlling the growth of developing follicles, is a physiological process driven by a variety of angiogenic factors. Among these, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been recognized as key players in promoting cell growth and differentiation. Porcine granulosa cells from small (<3 mm), medium (3-5 mm) and large (>5 mm) follicles were seeded at different densities in DMEM:Ham's F12 (1:1) with or without different concentrations of VEGF or bFGF. After 48 h of culture, media were assayed for oestradiol (E2) 17beta, progesterone (P4), nitric oxide (NO) and VEGF levels; in addition, cell proliferation was evaluated by 3H-thymidine incorporation assay. Both bFGF and VEGF effects on E2 and P4 production by cultured granulosa cells resulted to be dependent on follicle size. The bFGF was always ineffective in modulating cell proliferation, while VEGF exerted an inhibitory effect on the proliferation in the small follicle group and a stimulatory one in the medium and large follicle groups. The bFGF consistently reduced NO levels in culture media. The VEGF appeared to be ineffective in modifying NO production in the small follicle group, while it was stimulatory in the medium follicle group and inhibitory in the large follicle group. Basal VEGF production was higher in cells from the large follicle as compared with the small and medium follicle groups, and it was unaffected by bFGF. These results suggest that VEGF plays a modulatory role in granulosa cell functional activity and it is possibly involved in the regulation of follicle growth; on the contrary, bFGF does not appear to represent a significant regulatory factor in our cellular model, except for an inhibitory action on the production of NO, whose anti-angiogenic properties need to be further substantiated.

Relationship between human oocyte maturation and different follicular sizes.

The relationship between the follicular size in the human ovary and oocytes capable of resuming meiosis in vitro was examined in each phase of the menstrual cycle. Intact healthy oocytes with corona cells obtained from small (3-4 mm diameter), medium (5-8 mm diameter) and large (9-15 mm diameter) antral follicles were cultured at 37 degrees C for 43 h. At the end of the culture period the denuded oocytes were examined morphologically to determine whether the resumption of meiosis had occurred. In the follicular phase, the percentage of oocytes resuming meiosis (polar body extruded: PB) in the large-follicle group was significantly higher than that in the small-follicle group (P less than 0.05). The incidence of oocyte maturation including only nuclear maturity seemed to increase as follicles increase in size. However, in the luteal phase the incidence was fairly constant, irrespective of the follicular size. These results suggest that the capacity of human oocyte maturation is closely correlated with follicular maturation.