scholarly journals 246 FOLLICULAR DEVELOPMENT AND OOCYTE QUALITY AFTER OVUM PICKUP IN DONOR COWS

2005 ◽  
Vol 17 (2) ◽  
pp. 273
Author(s):  
K. Imai ◽  
M. Tagawa ◽  
S. Matoba ◽  
M. Narita ◽  
N. Saito
Keyword(s):  
Formation Rate ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Follicular Development ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
The Mean ◽  
Follicle Aspiration ◽  
Student’S T

The present study was designed to assess the renewal of follicular development and oocyte quality after ovum pickup (OPU) in Holstein dry cows. Cows were kept under the same feeding and environmental conditions. In Experiment 1, follicle aspiration (more than 2 mm) by OPU using a 7.5 MHz linear transducer with needle (cova needle, Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200, ALOKA, Tokyo, Japan) was performed in four cows. After OPU ovaries were observed from Day 4 (Day 0 = the day of OPU) to Day 11 to assess the number of follicles that developed. In Experiment 2, two sessions of OPU (n = 11) were performed with a 7-day interval between to assess the quality of developing follicles and oocytes. Oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. To assess the developmental competence of oocytes, collected cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM-199 supplemented with 5% calf serum (CS) in a microdroplet (volume was adjusted to 5 μL/oocyte) at 38.5°C under atmosphere of 5% CO2 in air. After maturation, the COCs were inseminated with frozen-thawed semen collected from the same ejaculation of a single bull. The fertilization was performed with BO solution as described by Imai et al. (2002 J. Vet. Med. Sci. 64(10), 887–891). The putative zygotes were then cultured in CR1aa supplemented with 5% CS under the same conditions as maturation culture for nine days. Embryo development was assessed by the cleavage rate on Day 2 and the blastocyst formation rate on Days 7 to 9 (the day of insemination = Day 0). Data were analyzed by ANOVA or Student's t-test. In Experiment 1, the mean number of developing follicles (larger than 2 mm in diameter) were increased from Day 4 to Day 11 (Day 4: 19.8 ± 10.0, Day 7: 32.5 ± 9.5; Day 11: 39.5 ± 10.7 (mean ± SD), respectively, P < 0.05). In Experiment 2, the mean number of developing follicles and collected oocytes on the day of OPU were significantly (P < 0.05) different between the first and second sessions (54.2 ± 12.4 and 40.8 ± 12.7, 45.7 ± 20.2 and 27.7 ± 8.7, respectively). The percentage of Grade 1 and 2 oocytes for the first session was significantly lower (P < 0.05) than those for the second session (59.1 ± 8.4 and 69.0 ± 11.8), and no significant differences were found within cleavage and blastocyst rates. The mean numbers of blastocysts obtained per session were 14.2 ± 8.9 and 9.7 ± 6.3 in the first and second sessions, respectively. These results indicate that populations of follicles were increased till Day 11 after OPU, and proportion of normal oocytes were increased in the renewal follicles.

277 FOLLICULAR GROWTH, SUBSEQUENT OVUM PICKUP, AND DOMINANT FOLLICLE REMOVAL IN COWS

2006 ◽  
Vol 18 (2) ◽  
pp. 246
Author(s):  
K. Imai ◽  
M. Tagawa ◽  
S. Matoba ◽  
M. Narita ◽  
K. Kanayama
Keyword(s):  
Formation Rate ◽  
Cumulus Cell ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Dominant Follicle ◽  
Cleavage Rate ◽  
Significant Difference ◽  
The Mean ◽  
Follicle Aspiration ◽  
Removal Group

The present study was designed to assess the recruitment of follicles after ovum pickup (OPU) and dominant follicle (DF) removal on the follicular wave after OPU in Holstein dry cows. Cows were reared under the same feeding and environmental conditions. In Experiment 1, follicle aspiration (>2 mm in diameter) by OPU using a 7.5-MHz linear transducer with needle (COVA needle; Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200; ALOKA, Tokyo, Japan) was performed in four cows. Then, ovaries were observed after OPU from Day 1 (Day 0 = the day of OPU) to Day 11 to assess the number of follicles developed. In Experiment 2, two sessions of OPU were performed with a 7 day interval between sessions, with or without dominant follicle removal, to assess the quality of developing follicles and oocytes. In the DF removal group, >8-mm follicles were aspirated at Day 5 after the first OPU session, and the same cows without DF removal were designated as a control (n = 4, crossover trial). Oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. To assess the developmental competence of oocytes, Grades 1 and 2 cumulus-oocyte complexes (COCs) were collected, matured, fertilized, and cultured as described by Imai et al. (2002 J. Vet. Med. Sci. 64(10), 887-891). Embryo development was assessed by the cleavage rate on Day 2 and the blastocyst formation rate on Days 7 to 9 (the day of insemination = Day 0). Data were analyzed by ANOVA or Student t-test. In Experiment 1, a dominant follicle (>8 mm in diameter) was developed during Days 3 to 5 after OPU in each donor. The mean number of developing follicles (>2 mm in diameter) were increased from Day 1 to Day 9 (Day 1: 7.5 � 2.1, Day 3: 19.0 � 1.2, Day 5: 23.3 � 9.0, Day 7: 30.3 � 11.0, Day 9: 42.0 � 15.8 and Day 11: 41.0 � 16.7 (mean � SD), P < 0.05). In Experiment 2, there was no difference in the mean number of developing follicles on the day of OPU and collected oocytes between DF removal and control groups (follicles: 47.8 � 23.0 and 39.3 � 6.2; oocytes: 27.0 � 11.6 and 26.5 � 5.4, respectively). The number of Grades 1 and 2 oocytes for the DF removal group was significantly higher (P < 0.05) than that for the control (83.6 � 1.5 and 63.2 � 14.2, respectively), and no significant difference was found within cleavage (60.0 � 37.2, 53.6 � 23.2) and blastocyst rates (34.1 � 33.9, 34.4 � 16.8). These results indicate that populations of follicles were increased till Day 9 after OPU, and the DF removal was effective at increasing oocyte quality in the developing follicles.

206 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON IN VITRO EMBRYO PRODUCTION

2008 ◽  
Vol 20 (1) ◽  
pp. 182 ◽  
Author(s):  
K. Imai ◽  
Y. Inaba ◽  
H. Yoshioka ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cumulus Cell ◽  
Oocyte Quality ◽  
Annual Conference ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Follicular Wave ◽  
The Mean ◽  
In Vitro Embryo Production

We previously reported that follicular wave synchronization, by removal of the dominant follicle on Day 5 after ovum pickup (OPU), was effective in increasing oocyte quality in the developing follicles (Imai et al. 2006 32th Annual Conference of the IETS, poster presentation no. 277). The current study was designed to examine the effect of superstimulatory treatment to induce subsequent follicular wave synchronization on embryo production by OPU and IVM-IVF-IVC in Holstein dry cows. Cows were reared under the same feeding and environmental conditions, and 2 OPU sessions were conducted in each cow. In the first session, OPU was performed in 8 cows on arbitrary days of the estrous cycle by using a 7.5-MHz linear transducer with needle (Cova needle, Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200, Aloka, Tokyo, Japan). Follicles larger than 8 mm in diameter were then aspirated and a CIDR was inserted on Day 5 (the day of first OPU session = Day 0). Cows then received 30 mg of FSH (Antrin-R10; Kawasaki Mitaka Pharmaceutical Co., Tokyo, Japan) twice a day from Days 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 mg) by i.m. injection. Cloprostenol (PGF; Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan; 0.75 mg) was administered in the morning of Day 9 (third day of superstimulation). The second OPU session was performed 48 h after PGF administration (Day 11), and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Collected oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. Grades 1 and 2 COC were matured, fertilized, and cultured as described by Imai et al. [2006 J. Reprod. Dev. 52(Suppl.), S19–S29]. Embryo development was assessed by the cleavage rate on Day 2 and by the blastocyst formation rate on Days 7 to 8 (the day of insemination = Day 0). Data were analyzed by Student's t-test. There were no differences in the mean (� SD) number of aspirated follicles or collected oocytes between the first (32.5 � 6.8 and 26.0 � 12.7, respectively) and second (29.3 � 10.4 and 19.0 � 9.4, respectively) OPU sessions (P > 0.1). The percentage of Grade 1 and 2 oocytes for the second OPU session (90.5 � 13.8%) was significantly higher (P < 0.01) than for the first OPU session (63.1 � 6.3%), and significant differences were found for cleavage (79.4 � 14.1, 61.8 � 25.1, P < 0.01) and blastocyst rates (68.1 � 16.7, 24.2 � 22.3, P < 0.001) between sessions. The mean numbers of blastocysts obtained per session were 4.3 � 2.9 and 12.8 � 8.7 in the first and second sessions, respectively (P < 0.01). These results indicate that superstimulatory treatment and subsequent follicular wave synchronization were effective on in vitro embryo production by increasing the oocyte quality.

scholarly journals 274SIMILARITY OF EMBRYOS PRODUCED BY OVUM PICK-UP AND IN VITRO FERTILIZATION IN IDENTICAL TWIN CATTLE

2004 ◽  
Vol 16 (2) ◽  
pp. 257 ◽  
Author(s):  
K. Imai ◽  
S. Kobayashi ◽  
S. Matoba ◽  
M. Tagawa ◽  
N. Saito
Keyword(s):  
Calf Serum ◽  
Cumulus Cell ◽  
Follicular Development ◽  
Subsequent Development ◽  
Oocyte Quality ◽  
Identical Twin ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Significant Difference

The present study was designed to assess the similarity of follicular development, oocyte quality, and their subsequent development on ovum pick-up (OPU)-IVF in identical twin cattle. Four pairs of identical twin Japanese black cows (A, B pairs at 5 years old and C, D pairs at 3 years old) were kept under the same feeding and environmental conditions. OPU was performed for these cows once a week for seven continuous weeks. OPU was done by using a 7.5-MHz linear transducer with needle (17 G, 530-mm length) connected to an ultrasound scanner (SSD-1200, ALOKA, Tokyo, Japan). Oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. To assess the development, collected COCs were cultured for 20h in TCM-199 supplemented with 5% calf serum (CS) in a microdroplet (volume was adjusted to 5μL/oocyte) at 38.5°C under atmosphere of 5% CO2 in air. After maturation, the COCs were inseminated with frozen-thawed semen collected from the same ejaculation of a single bull. The fertilization was performed with BO solution as described by Imai et al (J. Vet. Med. Sci., 2002, 64(10), 887–891). The zygotes were then cultured in CR1aa supplemented with 5% CS under the same condition of maturation for nine days. Embryo development was assessed by the cleavage rate on Day 2 and the blastocyst production rate on Days 7 to 9 (insemination day=Day 0). Blastocysts were classified according to the IETS creteria. Data were analyzed by ANOVA. A total 56 sessions of OPU were performed in this study. The overall mean number of developing follicles (larger than 2mm in diameter), collected oocytes, and produced blastocysts were 30.3±9.2, 20.1±9.2 and 6.3±3.8 (mean±SD) per session, respectively. The mean number of developing follicles on the day of OPU were significantly different between B and D pairs (38.6±7.5 and 21.9±6.5, P&lt;0.01); however, no significant difference was found within each twin. In oocyte quality, C and D pairs were significantly higher grade than the A pair. The percentages of cleaved oocytes and embryos developed to the blastocyst stage (34±16, 27±10, 41±17 and 39±24) showed no differences among 4 pairs and within each twin. However, the percentage of Grade 1 blastocyst of B pair was significantly lower (P&lt;0.01) than that of other pairs, and C pair was significantly higher (P&lt;0.05) than that of A and D pairs (67±25, 41±22, 93±10 and 71±25; A, B, C and D pairs, respectively). There was no significant difference within twins. These results show little statistical variation between cows of the same genetic background in the production of embryos in vitro.

19 Invitro embryonic development from oocytes collected by ovum pickup of superstimulated females and nonstimulated slaughterhouse ovaries of alpaca (Vicugna pacos)

2021 ◽  
Vol 33 (2) ◽  
pp. 117
Author(s):  
L. Landeo ◽  
M. Zuñiga ◽  
T. R. Gastelu ◽  
J. A. Ruiz
Keyword(s):  
Embryonic Development ◽  
Statistical Significance ◽  
Calf Serum ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Hormonal Stimulation ◽  
Array Transducer ◽  
The Mean

The objective of this study was to evaluate the embryonic development of alpaca oocytes collected by ovum pickup from superstimulated females (OPU, Group 1) and from slaughterhouse ovaries of 8 non-superstimulated females (SHO, Group 2) using a conventional aspiration technique (20G needle and a 3-mL syringe). A total of 8 nonpregnant alpacas, 3 to 4 years old, were superstimulated with a single dose of 200IU of equine chorionic gonadotrophin (eCG, Day=0). Three days later, alpacas were examined by transrectal ultrasonography with a 7.5-MHz linear-array transducer to determine the number and diameter of follicles available for aspiration. A total of 101 follicles were aspirated, recovering 67 oocytes (66.3%) by OPU using an endocavity transducer attached to a 21G needle adapted for alpacas. The follicular fluid was aspirated using a regulated vacuum pump (40 mmHg) into a tube containing 5mL of phosphate-buffered saline (PBS), 0.2% bovine serum albumin (BSA), and 10IUmL−1 heparin, at 37°C. In the SHO group we used 16 ovaries maintained at 28°C. The recovery of oocytes was carried out within 3h of ovary collection. We aspirated 155 follicles from SHO and recovered 117 oocytes (75.5%). After collection, all oocytes recovered were morphologically classified into categories (I and II) and cultured for 26h in an incubator (5% CO2 in air at 38.5°C), in TCM-199 supplemented with 0.2mmol sodium pyruvate, 50µgmL−1 gentamicin sulphate, 0.02IUmL−1 FSH, 1µgmL−1 oestradiol 17β, and 10% fetal calf serum (FCS). After maturation, oocytes were invitro fertilized with epididymal spermatozoa recovered from postmortem males and co-cultured for 18 to 20h. After this period, all cleaved oocytes were incubated (5% CO2 in air, 38.5°C) for 6 days in synthetic oviductal fluid-serum medium. Number and morphological quality of oocytes collected, invitro cleaved, and embryos ratea were registered and compared between groups. Statistical significance was determined using Kruskal–Wallis test. The mean and standard error were calculated from average of the percentages obtained in each repetition. Results indicated that the mean number of oocytes collected per ovary was higher (P&lt;0.05) using SHO (7.8±2.4) than OPU (4.5±3.0). Also, the number of oocytes classified as category I, was higher in the SHO compared with OPU group (56% vs. 30% respectively; P&lt;0.05); however, category II oocytes were the same (16% vs. 15%, respectively). There was no difference in early development (cleavage) rate between OPU (57±2.0) and SHO (49±1.5) groups. However, there was difference in the rate of development (P&lt;0.05) between OPU and SHO groups to reach the morula stage (56±2.0 vs. 42±1.7, respectively) and early blastocyst stage (55±2.0 vs. 34±1.4, respectively). In conclusion, oocyte quality could be affected by hormonal stimulation or by the quality of follicles aspirated by OPU. In contrast, oocytes recovered from live animals by OPU have greater capability of embryonic development invitro than oocytes recovered from slaughterhouse ovaries.

165 THE EFFECTS OF L-CARNITINE AND LINOLEIC ACID ALBUMIN SUPPLEMENTATION ON THE DEVELOPMENT AND CRYOSURVIVAL OF BOVINE IN VITRO-MATURED/IN VITRO-FERTILIZED EMBRYOS IN IN VITRO CULTURE MEDIUM

2012 ◽  
Vol 24 (1) ◽  
pp. 194
Author(s):  
S. Miyashita ◽  
Y. Inaba ◽  
T. Somfai ◽  
M. Geshi ◽  
T. Nagai ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
In Vitro Culture ◽  
Culture Medium ◽  
Formation Rate ◽  
Calf Serum ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Formation ◽  
Cleavage Rate

The objective of this study was to investigate the effects of the supplementation of a lipid metabolism inducer, L-carnitine (LC) and a membrane stabilizer, linoleic acid albumin (LAA), on the developmental competence and cryosurvival of bovine in vitro-matured/in vitro-fertilized embryos in in vitro culture medium. Cumulus–oocyte complexes collected from the ovaries of slaughtered cattle were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH at 38.5°C in an atmosphere of 5% CO2 in air. After IVF (Day 0), presumptive zygotes were cultured in CRlaa containing 5% CS at 38.5°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days. The culture medium was supplemented with 0.6 mg mL–1 of LC (LC group; n = 180) or with 0.25 mg mL–1 of LAA (LAA group; n = 180) or with both LC and LAA (LC + LAA group; n = 180) or without LC and LAA (control; n = 178). The cleavage rates were recorded on Day 2 and the blastocyst formation rates were recorded on Day 7 to 9. Expanded blastocysts harvested on Day 7 and 8 (LAA group: n = 31; LC group: n = 29; LC + LAA group: n = 25; control group: n = 33) were used for freezing in modified PBS supplemented with 1.5 M ethylene glycol, 0.1 M sucrose and 20% CS. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1 mM β-mercaptoethanol at 38.5°C under 5% CO2 in air for 72 h. The rates of re-expansion, hatching and formation of hatched blastocysts were determined at 24, 48 and 72 h after thawing, respectively. The rates of cleavage and blastocyst formation were expressed as mean ± s.e.m. and analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by chi-square test. The cleavage rate in the control group (69.1 ± 2.5%) was significantly lower than that in the LAA (81.8 ± 3.8%) and LC + LAA groups (77.9 ± 1.4%) but did not differ from that in the LC group (73.8 ± 2.4%). The blastocyst formation rate in the control group (21.7 ± 2.8%) was significantly lower (P < 0.05) than those in the LAA and LC + LAA groups (33.5 ± 2.8% and 31.4 ± 2.4%, respectively), but it did not differ significantly from that of the LC group (32.1 ± 3.3%) despite a strong tendency (P = 0.06). There were no significant differences among the control, LC, LAA and the LC + LAA groups in post-thaw re-expansion rates (66.7, 75.9, 67.7 and 76.0%, respectively), hatching rates (48.5, 69.0, 58.1 and 64.0%, respectively) and rates of formation of hatched blastocysts (51.5, 62.1, 61.3 and 64.0%, respectively). These results indicate that the addition of LC and LAA to the medium for in vitro culture of in vitro-matured/in vitro-fertilized bovine embryos improved their ability to develop to the blastocyst stage; however, the effects on the freezing tolerance were not verified.

82 Invitro embryo production outcomes in adult goats is affected by season

2021 ◽  
Vol 33 (2) ◽  
pp. 149
Author(s):  
P. V. Pereira ◽  
L. Correia ◽  
R. Batista ◽  
V. Freitas ◽  
Y. Locatelli ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Formation Rate ◽  
Calf Serum ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Seasonal Effects ◽  
Hatching Rate ◽  
Initial Number ◽  
Cleavage Rate ◽  
Blastocyst Rate

In seasonal breeders, reproductive seasonality can have a substantial influence on the efficiency of assisted reproductive technologies. This study assessed seasonal effects on cleavage and blastocyst rates, as well as on quality of invitro-produced (IVP) goat embryos over 18 months. In total, 2348 (autumn: 811, spring: 404, summer: 639, and, winter: 494) cumulus–oocyte complexes (COC) were recovered from slaughterhouse ovaries during 49 replicates (autumn: 17, spring: 7, summer: 15, and, winter: 10) and matured in TCM-199 with 10ng mL−1 epidermal growth factor and 100µM cysteamine for 22h at 38.8°C (5% CO2 in air). Matured oocytes were fertilized with frozen/thawed semen in synthetic oviductal fluid (SOF) with 10% oestrus sheep serum. Sperm and oocytes were co-incubated for 16h at 38.8°C in a humidified atmosphere of 5% CO2 in air. Presumptive zygotes were cultured in SOF medium supplemented with bovine serum albumin (3mg mL−1) for 8 days at 38.8°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. At Day 2, 10% fetal calf serum was added to the culture droplets. The embryos produced were fixed and stained with Hoechst to count their total number of cells, under an epifluorescence microscope. The results of cleavage and blastocyst rates, including hatching rate, from each routine of IVEP were considered as replicates. These data were tested for normality by the Shapiro-Wilk test, before being subjected to ANOVA, followed by Tukey HSD test. The odds ratio (OR) among seasons (autumn: breeding; spring: anoestrus) were calculated. Values of P&lt;0.05 were considered as significant, and the data reported are mean±s.e.m. Cleavage rate was lower (P&lt;0.05) in spring (51±7.1%) than in either autumn (72±2.1%) or summer (71±2.0%) while winter (66±4.1%) had an intermediate value, being similar (P&gt; 0.05) to all others. Indeed, greater possibility of cleavage was observed in autumn (OR: 2.43) and summer (OR: 2.39) compared with spring. The blastocyst rate from cleaved embryos was greater (P &lt;0.05) in autumn (73±2.7%) than in spring (55±2.6%; OR: 2.18). As a result, the blastocyst formation rate from the initial number of COC entering IVM was greater (P&lt;0.05) in autumn (52±2.5%) than in spring: 28±4.7%, summer: 45±2.3%, and winter: 42±2.1%; indeed, the spring season resulted in the lowest rate (P&lt;0.05), compared with other seasons. Moreover, the OR in the blastocyst rate from initial number of COC was greater (P&lt;0.05) in autumn compared with all other seasons and lower in spring compared with winter (OR: 0.54) and summer (OR: 0.48). There were no differences (P&gt; 0.05) in the embryo hatching (mean: 66±2.0%) and blastocyst cell number (mean: 193±2.0 cells). In conclusion, the breeding season (autumn) leads to improved oocyte developmental competence, resulting in greater cleavage and blastocyst yield, whereas embryo quality remained similar throughout the year. Further studies at the molecular level might indicate the mechanisms involved and provide clues to alleviate the negative effect of season.

P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Á Martíne. Moro ◽  
I Lamas-Toranzo ◽  
L González-Brusi ◽  
A Pérez-Gómez ◽  
P Bermejo-Álvarez
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Developmental Competence ◽  
Success Rates ◽  
Developmental Potential ◽  
Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p &gt; 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 260
Author(s):  
M. Bertoldo ◽  
P. K. Holyoake ◽  
G. Evans ◽  
C. G. Grupen
Keyword(s):  
Cell Morphology ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Oocyte Developmental Competence ◽  
Before And After ◽  
Follicle Size

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had >2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P < 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P < 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P < 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P < 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P > 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P < 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P < 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.

143 EFFECT OF FSH OR EPIDERMAL-GROWTH-FACTOR-LIKE PEPTIDE SUPPLEMENTATION TO MATURATION MEDIUM ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES DERIVED FROM FULLY DEVELOPED FOLLICLES INDUCED BY SUPER-STIMULATION

2017 ◽  
Vol 29 (1) ◽  
pp. 180
Author(s):  
T. Yamanouchi ◽  
S. Sugimura ◽  
H. Matsuda ◽  
M. Ohtake ◽  
Y. Goto ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Formation Rate ◽  
Calf Serum ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Maturation Medium ◽  
Epidermal Growth ◽  
Bovine Oocytes

Bovine oocytes obtained by ovum-pick-up (OPU) following follicle growth treatment (FGT) have improved quality and competence (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). However, the effect of the presence of FSH or epidermal growth factor (EGF) like peptide during in vitro maturation (IVM) on the developmental competence of FGT oocytes has not been well known. This study was undertaken to examine the developmental competence of FGT oocytes following IVM in the presence of FSH (recombinant human FSH) or EGF-like peptide (amphiregulin; Areg) and IVF. Japanese Black cows (n = 17) were used as donors. Five days after arbitrary OPU (opu group), follicles ≥8 mm in diameter were aspirated again, a controlled internal drug release (CIDR) was inserted into the vagina, and then pFSH was injected twice a day from the evening of Day 6 to the morning of Day 10 with decreasing doses (total of 20 AU; 4, 4, 3, 3, 2, 2, 1, 1 AU/day). On the evening of Day 8, PGF2α (0.5 mg of cloprostenol) was administered. On Day 11, oocytes were aspirated from follicles with ≥5 mm in diameter of the treated donors by OPU (fgt group). The cumulus-oocyte complexes (COC) were cultured in the absence (opu-cont and fgt-cont groups) or presence of 0.1 IU mL−1 FSH (opu-fsh and fgt-fsh groups) or 100 ng mL−1 Areg (opu-areg and fgt-areg groups) in IVM medium (mTCM199 containing 5 mg mL−1 BSA) for 20 to 22 h (1 COC/5 µL, total of 162–171 COC per group), and then co-cultured with 3 × 106 sperm/mL for 6 h. The presumptive zygotes were continued to culture in mCR1aa supplemented with 5% newborn calf serum for 216 h (1 zygote/5 µL) using micro-well culture dishes (Dai-Nippon-Print). When repeating this opu-fgt session in the same cow, an interval at least for 50 days was kept, and the session was performed 28 times. Statistical analysis was carried out by Mann-Whitney’s U-test (between opu and fgt groups) or Steel-Dwass test after Kruskal-Wallis test (among all groups). The number of follicles ≥5 mm increased in the fgt than opu group (17.8 v. 2.9; P < 0.01). The number of COC collected was not different between the opu and fgt groups (23.1 v. 19.6; P > 0.05). The blastocyst formation rate was higher in the fgt than opu group (36.9 v. 23.1%; P < 0.01). Within 6 groups, the blastocyst formation rate was higher in the fgt-fsh (43.3%; P < 0.01) and fgt-areg (39.5%; P < 0.05) groups than the opu-cont (16.3%) group. The rate in the fgt-fsh group was also higher than that in the opu-fsh group (43.3 v. 18.7%; P < 0.01). These results suggested that FGT improved the developmental competence of bovine oocytes, probably through improving the ability of the COC to react against FSH/Areg.

132 The Developmental Competence of Bovine Oocytes Exposed to Progesterone and Luteotrophic Hormones During the Second Step of Two-Step Maturation

2018 ◽  
Vol 30 (1) ◽  
pp. 206
Author(s):  
G. Singina ◽  
I. Lebedeva ◽  
T. Taradajnic ◽  
E. Shedova ◽  
A. Lopukhov ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Calf Serum ◽  
Developmental Competence ◽  
Second Step ◽  
Russian Science ◽  
Cleavage Rate ◽  
System 1 ◽  
And Control ◽  
Bovine Oocytes

Data on effects of progesterone (P4) during in vitro maturation of bovine oocytes on their capacity for embryonic development are contradictory. Our study was aimed at characterising effects of P4 and 2 luteotropic hormones, prolactin (PRL) and LH, on bovine oocyte developmental competence during the second step of two-step maturation (from metaphase (M)I to MII). Slaughterhouse-derived cumulus-enclosed oocytes (CEO) were matured for 12 or 24 h [one-step (OS) Control] in TCM-199 containing 10% fetal calf serum (FCS), 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH at 38.5°C and 5% CO2. The CEO cultured for 12 h were transferred to the following culture systems: (1) TCM-199 containing 10% FCS (Control 1) or (2) a monolayer of granulosa cells (GC) precultured for 12 h in TCM-199 containing 10% FCS (Control 2); then, the oocytes were matured for next 12 h. In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (25 and 50 ng mL−1) or ovine LH (5 μg mL−1). All treatments were repeated 5 to 6 times using 138 to 196 oocytes per group. Following IVM, all oocytes underwent IVF as described previously (Singina et al. 2014 Reprod. Fertil. Dev. 26, 154). Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured to Day 7. Embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst formation. Apoptosis was detected by the TUNEL method using 26 to 47 blastocysts per group (from 4 to 5 separate experiments). For each system, arcsine-transformed data were analysed by one-way ANOVA. In OS Control, the cleavage and blastocyst rates were 68.9 ± 4.4% and 22.0 ± 2.4%, respectively. Regardless of the system or medium of two-step culture, the cleavage rate did not differ from that for OS Control, varying between 57.6 and 68.4%. In the absence of GC (System 1), the blastocyst yield in the P4 group (30.4 ± 0.8%) was greater (P < 0.05) than in OS Control and Control 1 (20.2 ± 2.7%) as well as in the groups treated with LH (19.1 ± 3.0%) and 25 ng mL−1 PRL (20.1 ± 2.7%). In the presence of GC, P4 raised the yield from 16.7 ± 2.3% (Control 2) to 27.7 ± 2.4% (P < 0.05). Furthermore, in System 2, the blastocyst rate in groups treated with P4 and 50 ng mL−1 PRL (25.0 ± 2.8%) was higher (P < 0.05) than in the LH group (13.9 ± 2.6%). Meanwhile, the proportion of apoptotic nuclei (2.3-6.9%) was not associated with the system of oocyte maturation or effects of hormones studied. Our data indicate that P4 (50 ng mL−1) can enhance the developmental competence of bovine oocytes during the second step of two-step maturation regardless of the presence of granulosa cells, whereas the similar effect of PRL (50 ng mL−1) is less pronounced and depends on the granulosa-conditioned environment. This research was supported by the Russian Science Foundation (project 16-16-10069).

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