Effects of Ovum Pick-up Frequency and FSH Stimulation: A Retrospective Study on Seven Years of Beef Cattle In Vitro Embryo Production

The aim of this retrospective study was to compare the number of follicles, cumulus oocyte complexes (COCs) and cultured In Vitro Produced (IVP) embryos obtained from 1396 non‐stimulated Ovum Pick‐up (OPU) sessions on 81 donor animals in a twice weekly OPU scheme. Results were obtained from 640 sessions following FSH‐LH superstimulation, on 112 donors subjected to OPU once every 2 weeks. The stimulation protocol started with the insertion of an ear implant containing 3 mg norgestomet (Crestar, Intervet, Belgium) 8 days before puncture (day ?8). The dominant follicle was ablated by ultrasound‐guided follicle puncture on day ?6. On day ?3 and day ?2, cows were injected with FSH (Ovagen, ICP) twice daily (8 am to 8 pm ), i.e. a total dose of 160 μg FSH and 40 μg LG per donor per stimulation cycle. Animals were punctured 48 h after the last FSH injection (day 0). Progesterone implants were removed the next day. Stimulated donor cows were treated with this protocol at 14‐day intervals. Follicles were visualized with a Dynamic Imaging ultrasound scanner, equipped with a 6.5 MHz sectorial probe. Follicles were punctured with 55 cm long, 18 gauge needles at an aspiration pressure corresponding to a flow rate of 15 ml/min. Cumulus oocyte complexes were recovered and processed in a routine IVF set‐up. Results demonstrate that, expressed per session, FSH stimulation prior to OPU increases production efficiency with significantly more follicles punctured and oocytes retrieved. However, when overall results during comparable 2‐week periods are considered (four non‐stimulated sessions vs one stimulated), more follicles are punctured and more oocytes are retrieved using the non‐stimulated protocol. No significant differences in the number of cultured embryos could be detected, indicating that FSH/LH stimulation prior to OPU might have a positive effect on in vitro oocyte developmental competence as more embryos are cultured with less, presumably better‐quality, oocytes.     

The in vitro developmental competence of oocytes from juvenile calves is related to follicular diameter

This study investigated the relationship between follicle size (FS) and developmental competence of calf oocytes. Cumulus-oocyte-complexes (COCs) from follicles>8 (L-COCs; n=19), 4-8 (M-COCs; n=54), and 2-3 mm (S-COCs; n=155) were recovered from non-stimulated 1-4 months old dairy calves post mortem and ex vivo (laparoscopy), and in parallel from slaughtered adult cows from follicles of identical size categories [>8 (n=91); 4-8 (n=138); 2-3 mm (n=193)]. Morphologically intact COCs were subjected to in vitro maturation, fertilization, and embryo culture. Cleavage rate (CR; 46 h post-insemination=p.i.), rate of morulae/blastocysts (M/Bl; day 7 p.i.), and blastocysts (Bl; day 9 p.i.) were recorded. FS had no effect on the CR in calves. However, calf L-COCs yielded the highest rates of M/Bl and Bl compared with the two other size categories (P<0.05). In contrast, calf S- and M-COCs gave similar rates of M/Bl, whereas the proportion of Bl was lowest for S-COCs (P<0.05). This was almost identical to findings in cows, except that the CR was highest for L-COCs and M/Bl yields were lowest for S-COCs (P<0.05). There were no differences between calf and cows with regard to CR for the respective FS categories. L-COCs from calves and cows yielded similar rates of M/Bl and Bl, whereas calf S- and M-COCs yielded lower rates of Bl than S- and M-COCs from cows and a lower rate of M/Bl when S-and M-COCs were analyzed as one group (P<0.05). Whereas the CR was similar in calves and cows, calf COCs yielded lower rates of M/Bl and Bl (P<0.05). In conclusion, the results show that the developmental competence of calf oocytes is higher in those derived from follicles larger than 8 mm, and thus are almost equally as competent as cow oocytes derived from follicles of identical size. This suggests that calf oocytes acquire developmental competence within the large follicle, potentially due to a process similar to prematuration of the oocyte in the adult cow. It is proposed that procedures that facilitate prematuration, such as "coasting" following a preceding superstimulation, might increase the developmental competence of calf oocytes.     

DNA Fragmentation in Canine Immature Grade 1 Cumulus‐Oocyte Complexes

In this work, we studied the incidence of DNA fragmentation, interpreted as apoptotic changes and assessed by the TUNEL assay, in cumulus cells and oocytes of immature Grade 1 cumulus‐oocyte complexes (COCs) obtained from healthy bitches (n = 27) of three age groups: young (1–3 years; n = 13), adult (4–6 years; n = 8) and elderly (7–10 years; n = 6). Age affected (p < 0.05) Grade 1 COCs recovery rates, with young animals yielding more (p < 0.01) Grade 1 COCs than the other two age groups. Conversely, no differences were observed in the incidence of DNA fragmentation (TUNEL‐positive) in cumulus cells or oocytes between the three age groups. Overall, more than 80% of Grade 1 COCs presented <15% of TUNEL‐positive cumulus cells and enclosed TUNEL‐negative (intact DNA) oocytes. Despite a higher proportion of TUNEL‐negative oocytes being found in the germinal vesicle stage, most of the oocytes with nuclear material compatible with meiosis resumption (MR) or with non‐identifiable nuclear material (ND) did not present DNA fragmentation. No correlation was observed between DNA fragmentations in oocytes and in cumulus cells. We concluded that the morphological parameters used to classify canine Grade 1 COCs are reliable to select a homogeneous population of COCs with low incidence of DNA fragmentation. Furthermore, these results indicate that DNA fragmentation can only explain a minor proportion of the incidence of MR and degeneration in canine oocytes at collection.     

Effect of Days Post-Partum, Breed and Ovum Pick-Up Scheme on Bovine Oocyte Recovery and Embryo Development

The objective of this study was to investigate (i) the effect of two different ovum pick-up (OPU) schemes (once vs twice weekly aspirations) on oocyte recovery rate, quality and subsequent in vitro embryo development, (ii) the influence of days post-partum on oocyte recovery and (iii) possible differences in OPU results from two different herds. In group A, OPU was performed twice weekly in two Holstein Friesian (HF) and three Danish Red and White (DRW) cows from a private herd. In the research herd, two groups of eight HF cows were investigated: group B (OPU once weekly) and group C (OPU twice weekly). The collected oocytes were subsequently submitted to in vitro embryo production. More oocytes were recovered from the private herd when compared with the research herd. In the research herd, the twice weekly scheme aspirated more oocytes than the once weekly scheme. The quality of the retrieved oocytes was significantly different between groups B and C but not between groups A and C, and HF cows yielded higher quality oocytes than DRW cows (p = 0.029). Oocytes from group C showed higher level of embryonic development than group B oocytes. No differences in blastocyst rates were observed between groups A and C. Session affected the number of retrieved oocytes and subsequent developmental rates, with these being lower in the first compared with the last sessions. Finally, there was no significant effect of days post-partum in the number and quality of the retrieved oocytes, likely because of the small group size and high variation between sessions.     

Postpartum Variations of Plasma IGF and IGFBPs,Oocyte Production and Quality in Dairy Cows: Relationships With Parity and Subsequent Fertility

The aim of this study was to determine whether postpartum variations of plasma IGF‐1 and IGFBP concentrations, oocyte production and quality were related to parity and subsequent conception rate in Holstein dairy cows. Holstein dairy cows [10 primiparous (PP) and 22 multiparous (MP)] were allotted in six batches and sampled once weekly between calving and oestrous synchronization treatment started at 71.2 ± 2.0 days postpartum. During the 3 weeks before treatment, ovum pick‐up (OPU) was performed twice weekly. Oocytes were scored on a 4‐point scale, and oocytes from OPU1, 3 and 5 were fertilized in vitro. Seventeen cows became pregnant after first and second AI and were considered as fertile (F), while the others were considered to be subfertile (SF). Logistic regression was carried out to investigate the relationships between repeated measurements and fertility including parity and batch effects in the models. Likelihood of fertility significantly increased when plasma urea and IGFBP‐3 concentrations decreased and was higher in PP compared with MP cows. There was a trend for fertility to increase when plasma IGF‐1 concentrations increased (p = 0.07). In vitro cleavage and development rates were similar between SF and F cows (46.4% and 28.3% in SF vs 55.0% and 22.1% in F). Parity had an effect on plasma IGF‐1 concentrations (PP: 61.65 ± 2.67 vs MP: 41.63 ± 5.81 ng/ml, p < 0.001), mean number of follicles aspirated per session (PP: 5.7 ± 1.3 vs MP: 9.5 ± 0.8, p < 0.05) and fertility (PP: 8/10 = 80% vs MP: 9/22 = 41%, p < 0.05) but not on the number of oocytes recovered per session nor their quality. In conclusion, postpartum plasma urea and IGFBP‐3 concentrations, but not oocyte production and quality before breeding, were related to subsequent conception rate in our experimental design. Parity had a significant effect on energy status, follicular growth and fertility and needs to be considered when investigating relationships between nutrition and reproduction.     

Maturation competence of swamp buffalo oocytes obtained by ovum pick-up and from slaughterhouse ovaries

This study was designed with the final goal of improving in vitro embryo production in the Thai swamp buffalo (Bubalus bubalis carabensis). Oocytes were collected by ovum pick-up (OPU) from six non-lactating multiparous swamp buffalo twice per week for 10 consecutive sessions followed by once-weekly collection for 10 consecutive sessions without hormone stimulation. In addition, oocytes were collected from slaughterhouse ovaries that were classified as follows: ovaries from non-pregnant cows with a visible corpus luteum (NPCL); pregnant cows with a corpus luteum (P); and non-pregnant cows without a corpus luteum (NP). Follicles in each group of ovaries were categorized as small (2-4 mm), medium-sized (5-8 mm) or large follicles (≥ 9 mm). The quality of the oocytes was assessed by their capacity to undergo in vitro maturation. The total number of observed follicles per session (all sizes combined) was larger in the once-weekly OPU group compared with the twice-weekly OPU group. In particular, the numbers of small and large follicles were higher in the once-weekly OPU group (5.2 ± 0.7 and 0.9 ± 0.2, respectively) than in the twice-weekly OPU group (3.9 ± 0.5 and 0.5 ± 0.1). The number of medium-sized follicles did not differ between the groups. The percentages of oocytes with an abnormal spindle morphology were not different between oocytes from the twice-weekly (30.0%) and the once-weekly (28.6%) OPU groups. A higher percentage of oocytes obtained in vitro (49.5%) exhibited nuclear abnormalities compared with those obtained in vivo (≤34.8%) after in vitro maturation. In conclusion, oocytes can be successfully collected by OPU in the swamp buffalo, without hormonal pretreatment, and per week more good-quality oocytes can be collected by twice-weekly OPU. In addition, oocytes collected from slaughterhouse ovaries can be used with the reproductive status of the cow having no influence on the maturation competence of oocytes.     

Supplementation with cumulus cell masses improves the in vitro meiotic competence of porcine cumulus–oocytes complexes derived from small follicles

The present study was conducted to examine the supplemented effect of cumulus cell masses (CCMs) derived from middle follicle (MF; 3–6 mm diameter) on the morphology and the meiotic or developmental competence of oocytes from small follicles (SF; 1–2 mm diameter). The number of cumulus cells surrounding oocytes just after collection was also lower in cumulus–oocyte complexes (COCs) from SF than MF. The ooplasmic diameter of oocytes was significantly smaller in SF‐derived oocytes than MF‐derived ones before and after in vitro maturation (IVM), whereas the diameter significantly increased during the culture. Co‐culture of SF‐derived COCs with MF‐derived CCMs during IVM significantly improved the meiotic competence of the oocytes to the metaphase‐II stage. Furthermore, the ooplasmic diameter of SF‐derived COCs during IVM was increased to the similar size of MF‐derived those in the presence of MF‐derived CCMs. The abilities of oocytes to be penetrated, to form male pronuclear formation and to cleave or develop to the blastocyst stage were not affected by the co‐culture with CCMs. Electrophoretic analysis of CCM secretions clearly showed the presence of more protein(s) approximately 27.6 kDa in the conditioned medium when supplemented with MF‐derived CCMs. In conclusion, we demonstrate that supplementation with MF‐derived CCMs improves the ooplasmic diameter and meiotic competence of SF‐derived oocytes.     

Differential expression of insulin‐like growth factor family members in immature cumulus–oocyte complexes from dairy cows with different genotypes

It has been evident the improvement of in vitro embryo production (IVEP) in dairy cows. Nevertheless, it is known that differences in the number and quality of oocytes between taurine and zebu females impact the efficiency and economic viability of IVEP. As the insulin‐like growth factor (IGF) system is related to follicular and oocyte development, we aimed to quantify mRNA abundance of IGF system members and pregnancy‐associated plasma protein‐A (PAPPA) in the cumulus–oocyte complexes (COCs) of Gir, 1/2 Holstein × 1/2 Gir and Holstein cows. Four pools of 30 immature COCs from Gir, 1/2 Holstein × 1/2 Gir and Holstein cows were obtained by ovum pickup (OPU), and the oocytes and cumulus cells (CC) were mechanically separated and stored at ?80°C. Total RNA was extracted from pools of 30 oocytes and their respective CC. Expression of target genes was assessed by real‐time RT‐PCR. In oocytes, the abundance of IGFR1 mRNA was higher (p < .05) in Gir cows compared with the other breeds. In contrast, in CC, mRNA encoding IGF2 (p < .05), IGFR2 (p < .05) and IGFBP4 (p < .01) was higher in Holstein donors compared with Gir and 1/2 Holstein × 1/2 Gir cows. Additionally, the abundance of PAPPA mRNA was higher in oocytes (p < .001) and CC (p < .01) in Gir and 1/2 Holstein × 1/2 Gir cows compared with the Holstein donors. In conclusion, the higher abundance of PAPPA mRNA in the oocytes and CC from Gir and cross‐breed donors combined with the low expression of IGFBP4 in the CC suggests an enhancement of the bioavailability of IGF‐free when compared with Holstein COCs.     

Bovine OPU‐Derived Oocytes can be Matured In Vitro for 16–28 h with Similar Developmental Capacity

The aim of this study was to determine the optimal maturation culture period of ovum pick up (OPU)‐derived cumulus oocytes complexes (COCs) in relation to their developmental capacity. Embryo production, embryo cryotolerance, post‐transfer embryonic survival and calf characteristics such as gestation length, birthweight and sex ratio were investigated. This retrospective study covers the analyses of ovum pick up –in vitro production and calving results from a commercial programme that took place between March 1994 and September 2004. Donors were both heifers (of which approximately 90% pregnant) and cows (of which approximately 10% pregnant). Embryo production analyses were based on 7800 OPU sessions conducted from January 1995 until January 1999. Analyses of calving rate were based on 13 468 embryo transfers performed during January 1995 until May 2002. Analyses on calf characteristics were based on 2162 calves born between March 1994 and September 2004. The in vitro maturation culture period ranged from 16 to 28 h. The mean production rate of transferable embryos was 16.5% (1.2 embryos per OPU session). Length of maturation culture period did not affect the production of transferable embryos. Mean calving rate was 40.9% and 38.7% for fresh and frozen/thawed embryos, respectively. Calving rate was not affected by the maturation culture period. Mean birthweight, gestation length and proportion of male calves were 46 kg, 281.9 days and 52.8%, respectively. Maturation culture period did not affect these variables. In conclusion, this study shows that the in vitro maturation culture period within the range of 16–28 h does not affect in vitro embryo production, embryo cryotolerance, post‐transfer embryonic survival and calf characteristics, suggesting that all COC batches collected by OPU on the same day, can be fertilized in one IVF session without a significant loss in the production from oocyte to calf.     

The Anti‐Müllerian Hormone Profile is Linked with the In Vitro Embryo Production Capacity and Embryo Viability after Transfer but Cannot Predict Pregnancy Outcome

The current study investigated the possibility of using the AMH concentration as a predictor of the ability of Korean Hanwoo cows to produce cumulus‐oocyte complexes, embryos that survive after transfer as well as the pregnancy outcome of surrogates. Eight sessions of ovum pick‐up (OPU) were performed with 19 donor cows at an interval of 3–4 days. Antral follicle count (AFC), oocyte quality and in vitro embryo development were recorded for each cow. Embryos produced from cows with different AMH profiles were transferred into recipients (n = 96). Cows in the high (≥0.25 ng/ml) and intermediate (0.1≥ to <0.25 ng/ml) AMH groups had a significantly higher AFC per OPU session (20.40 ± 1.36 and 16.91 ± 1.52, respectively; mean ± standard deviation) than cows in the low AMH group (<0.1 ng/ml; 12.19 ± 2.14). In addition, more cumulus‐oocyte complexes per donor were recovered in the high (11.46 ± 1.22) and intermediate (7.38 ± 0.83) AMH groups than in the low AMH group (4.77 ± 0.44). The percentage of oocytes reached blastocyst stage was significantly higher in the intermediate (47.0%) and high (38.5%) AMH groups than in the low AMH group (32.3%). The number of embryos produced per cow was higher in the high (3.9 ± 0.2) and intermediate (6.9 ± 0.6) AMH groups than in the low AMH group (2.2 ± 0.3). The percentage of embryos that gave birth to viable calves when transferred into recipients was higher for those derived from cows in the intermediate AMH group (50.7%) than for those derived from cows in the low (35.7%) and high (36.4%) AMH groups. In conclusion, a single measurement of AMH concentration predicted the in vitro embryo production potential of donor Korean native cows before OPU and is linked with embryo viability after transfer into recipients.     

Effect of suckling on embryo production by repeated ovum pick-up before and after timed artificial insemination in early postpartum Japanese black cows

We investigated whether suckling would affect embryo production of cows bred by timed artificial insemination (TAI) following an ovulation synchronization protocol combined with ovum pick-up and progesterone releasing intravaginal device (OPU-PRID-TAI protocol). The number of oocytes and transferable embryos collected by repeated OPU, performed before and after TAI, were recorded. A total of 14 Japanese Black cows were divided into weaned (n=7) and suckled groups (n=7). All 14 cows were treated with OPU on day 0 (the first day of treatment) and then with a PRID for 9 days. Prostaglandin F(2alpha) analog was administered on day 7, GnRH analog was administered on day 10 (36 h after removal of the PRID) and TAI was performed 12 h later. Ovulation was confirmed by palpation per rectum the following day. After TAI, additional OPU sessions were performed on days 18, 25 and 32. The synchronized ovulation rates of the weaned and suckled groups were 100 and 85.7%, and the conception rates were 71.4 and 42.9%, respectively. Immature oocytes were fertilized and cultured in vitro. The numbers of oocytes collected and blastocysts generated were similar between the individual OPU sessions in both groups. However, the total numbers of oocytes collected, cultured oocytes, cleavage embryos and blastocysts as well as the proportions of cleavage embryos and blastocysts to cultured oocytes were all significantly (P<0.05) greater in the weaned group compared with the suckled group. These results suggest that the OPU-PRID-TAI protocol has the potential to produce a significant number of good-quality embryos in vitro after repeated OPU in early postpartum weaned Japanese Black cows. To collect more oocytes and produce more embryos, we suggest that calves be removed from cows scheduled for treatment using this protocol.     

A three‐dimensional alginate system for in vitro culture of cumulus‐denuded feline oocytes

In the case of high valuable individuals with very precious genetic material, widening the genetic pool including gametes with poor morphological characteristics, as cumulus‐denuded oocytes (CDOs), could be an option. To improve the in vitro culture of low‐competence feline CDOs, an enriched three‐dimensional (3D) system in association with competent cumulus–oocyte complexes (COCs) was developed. For this purpose, domestic cat CDOs were cultured with or without companion COCs in the 3D barium alginate microcapsules. The overall viability and the meiotic progression of feline CDOs cocultured with COCs or cultured separately in 3D or in 2D (traditional microdrops) system were compared. The 3D system was able to support viability and meiotic resumption of the feline oocytes, as well as the 2D microdrops. In 3D microcapsules, the presence of COCs resulted in a higher viability of CDOs (91.1%, p < .05), than that obtained without COCs or in 2D microdrops (71.2% and 67.3%, respectively), but the percentages of meiotic resumption were similar of those of CDOs cultured separately (55.4% vs. 40.4%, p > .05). It is notable that the presence of CDOs seemed to enhance the meiotic progression of the associated COCs. In conclusion, the 3D barium alginate microcapsules are a suitable system for feline oocytes in vitro culture, but more specific enriched conditions should be developed to improve the CDOs full competence in vitro.     

Influence of co‐culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus‐enclosed porcine oocytes in a defined system

Co‐culture of cumulus‐oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte‐secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β‐mercaptoethanol. Cumulus‐oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co‐culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus‐enclosed porcine oocytes in a defined system.     

The Phosphodiesterase Inhibitor,Isobutyl-1-Methylxanthine Prevents the Sudden Drop in Cyclic Adenosine Monophosphate Concentration and Modulates Glucose Metabolism of Equine Cumulus–Oocyte Complexes Matured in Vitro

Spontaneous nuclear maturation of mammalian oocytes can occur when physically removed from the ovarian follicle during in vitro oocyte maturation (IVM), largely because of a decrease in cyclic adenosine monophosphate (cAMP) concentration. Modulation of oocyte cAMP during IVM by using phosphodiesterase inhibitors has been shown to maintain elevated oocyte cAMP concentrations and control meiotic resumption of bovine and ovine oocytes. This study determined the effect of inclusion of isobutyl-1-methylxanthine (IBMX) during collection and the first 12 hours of incubation of equine oocytes on cAMP concentration and glucose metabolism of cumulus–oocyte complexes (COCs). Abattoir-derived COCs were collected in aspiration medium with (Asp-IBMX) or without (Asp) IBMX. Cumulus–oocyte complexes were then incubated for 12 hours in IVM medium with (Mat-IBMX) or without (Mat) IBMX, followed by additional 24 hours in Mat medium. The cAMP concentration, glucose consumption, lactate production, and metaphase II rates of the COCs were assessed. Cumulus–oocyte complexes aspirated into Asp-IBMX (62.2 ± 2.6 fmol per COC) medium had higher cAMP concentration than Asp (31.8 ± 2.8 fmol per COC) control group (P < .05). Likewise, at 12 hours of IVM, Mat-IBMX group (33.2 ± 2.1 fmol per COC) had higher cAMP concentration than the Mat group (7.68 ± 0.5 fmol per COC; P < .05). Glucose consumption and lactate production were lower during the first 12 hours of incubation in COCs cultured in Mat-IBMX (P < .05). Isobutyl-1-methylxanthine prevented the rapid drop in cAMP concentration and altered metabolism of glucose by the COC. Preventing the sudden drop in cAMP prevents the premature nuclear maturation of in vitro–matured oocytes causing poor developmental competence.     

Influence of Superovulatory Protocols on In Vitro Production of Nellore (Bos indicus) Embryos

There are indications in the literature that delaying the period between ovarian superestimulation and ovum pick up (OPU) would induce follicles to a condition of initial atresia, which could be beneficial to oocyte development. In this work, we compared three protocols for OPU and in vitro production (IVP) of embryos, in Nellore cattle. Nellore cows (n = 18) were randomly allocated in three groups: Group 1 (OPU), Group 2 [Follicle stimulating hormone (FSH) and OPU] and Group 3 (FSH deprivation and OPU). Three OPUs were performed, and the animals were switched to a different group each time (crossover), in such a way that at the end of the experiment all cows received the 3 protocols. At random stage of the oestrous cycle (D‐2), all follicles ≥ 6 mm were aspirated to induce a new follicular wave 2 days afterwards (D0). In Group 1, OPU was performed on D2 and oocytes were processed to IVP. In Group 2, starting on D0, cows were superstimulated (FSH, Folltropin^®, 30 mg administered daily, i.m., during three consecutive days, total dose = 180 mg), and 6 h after the last FSH dose, they received exogenous luteinizing hormone (LH) (12.5 mg, i.m., Lutropin^®, D3). The OPU was performed 6 h after LH administration, i.e. 12 h after the last dose of FSH. Animals in Group 3 received the same treatment as those in Group 2, except that LH was administered 42 h after the last dose of FSH, and OPU occurred 6 h later. Therefore, in this group, follicles were deprived of FSH at 48 h. Both cleavage and blastocyst rates were similar (p > 0.05, anova ) among oocytes from Groups 1, 2 and 3, respectively: 77.4% (144/185) and 42.70% (79/185); 75.54% (105/139) and 31.65% (44/139); 63.52% (101/159) and 33.33% (53/159). However, hatched blastocyst rate was higher (p < 0.01) in Group 1 (30.27%, 56/185) when compared with Group 2 (11.51%, 16/139) or 3 (15.72%, 25/159). It is concluded that, contrary to previous work on European breeds (Bos taurus), ovarian superstimulation associated with deprivation of FSH and OPU (Group 3) did not increase IVP of Nellore embryos (Bos indicus). On the contrary, the highest hatched blastocyst rates were observed in oocytes from non‐superstimulated cows.     

Modulation of Glycolysis and the Pentose Phosphate Pathway Influences Porcine Oocyte In Vitro Maturation

Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus–oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose‐dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6‐AN) and the physiological (NADPH) inhibitors of PPP induced a dose‐dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.     

Age, FSH Dose and Follicular Aspiration Frequency Affect Oocyte Yield from Juvenile Donor Lambs

Experiments were conducted to determine the effects of lamb age, frequency of follicular aspirations, and hormone stimulation by fixed or variable FSH dose, on the number of collected oocytes and their maturational competence. In trial 1, the characteristics of follicular population (number and diameter of follicles) were studied in 40 lambs which were slaughtered at the age of 30 days (S1), 42 days (S2), 60 days (S3) and 5–6 months (S4), each n = 10. In trial 2, 27 lambs were divided into four groups. group MF lambs (n = 6) had follicular aspiration (OPU) in four monthly intervals commencing from the age of 8–9 weeks (sessions MF1, MF2, MF3 and MF4). In groups SF2, SF3 and SF4 (each n = 6), OPU was conducted once during the 12–13, 16–17 and 20–21 week of age, respectively. Ovarian stimulation was conducted with fixed FSH dose (3.52 mg/animal). In trial 3, 10 lambs (group MV) were treated as those of group MF apart from the FSH dose, which was administered according to the body weight in a dose of 0.27 mg/kg. The number and the size of follicles, the number and the quality of collected oocytes and the maturational competence of the oocytes were compared between and within groups. In trial 1, the total number and the number of small follicles were greater in groups S1 and S2 compared with those of S3 and S4 (p < 0.01). Similarly, the follicular population was greater in group MF1 than in group SF3 (p < 0.01). In sessions MF2, MF3, MV2, MV3 and MV4, more oocytes were collected in comparison with those from the respective once‐aspirated age mates (groups SF2, SF3 and SF4). In total, more (p = 0.02) oocytes per donor were collected from group MV (15.2 ± 5.5) than from group MF (9.0 ± 3.2). An absolute maturational failure was observed in oocytes collected from groups SF2 and SF3. Maturational competence varied between 16.7% and 58.3% (p = 0.017) among sessions of group MF, but it was more uniform among sessions of group MV (range 12.5–42.9%, p > 0.05). Our results indicate that firstly, the number and the quality of harvested oocytes from juvenile lambs can be much improved if follicular stimulation regime is adjusted to the body weight. Secondly, in terms of follicular population and oocyte quality, 3 and 4‐month‐old lambs are naturally bad oocyte donors, but this characteristic can be reversed by a previous follicular ablation.     

Cumulus cell expansion and first polar body extrusion during in vitro oocyte maturation in relation to morphological and morphometric characteristics of the dromedary camel ovary

The morphological and morphometric characteristics of the ovary are fundamental properties for in vitro oocyte maturation. Nuclear maturation, including first polar body (1PB) extrusion, cytoplasmic maturation and cumulus cell (CC) expansion are the criteria for in vitro maturation (IVM) of oocyte. This study was designed to determine the effect of morphological and morphometric features of the ovary on CC expansion and 1PB extrusion during IVM of oocyte in the adult female dromedary camel. The weight, volume and three dimensions of ovaries from slaughtered dromedary camels and oocytes inside zona diameter and zona pellucida thickness were measured. The follicles were classified in regard to the size and oocytes according to their ooplasm appearance and CC compactness. Aspirated cumulus oocyte complexes (COCs) were incubated for 48 hr (with a 6‐hr interval) in Hams‐F10, and CC expansion and 1PB extrusion were assessed. Significant differences were seen in the shape, weight, volume and three dimensions of the ovaries between ≤4‐year‐old and >4‐year‐old dromedary camel (p < .5). Approximately, 95.82% of follicles were 2–4 mm in diameter. The mean (±SD) of inside zona diameter of the oocyte and zona pellucida thickness was 132.22 ± 13.8 and 14.64 ± 2.24 μm, respectively, in >4‐year‐old dromedary camel. The CC expansion and 1PB extrusion were seen in 86% and 21.88% of COCs, respectively. Age and sexual conditions of dromedary camel influence the morphological and morphometric characteristics of the ovary. Most COCs retrieved from 2–6 mm follicles are cultivable. The most slaughterhouse‐derived COCs retrieved from 2–6 mm follicles of non‐pregnant dromedary camels are excellent and good and yielding a most favourable diameter to achieve the developmental competence for IVM in an optimal time of 24–30 hr; the optimal time for CC expansion is 24–30 hr in this species. However, the CC expansion is a prerequisite process, but not sufficient for IVM.     

Effect of Sericin Supplementation During In Vitro Maturation on the Maturation,Fertilization and Development of Porcine Oocytes

This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus‐oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA‐fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.