137 Kinetics of In Vitro Embryonic Development According to the Follicular Population in Bos Indicus

2018 ◽  
Vol 30 (1) ◽  
pp. 208
Author(s):  
J. G. Soares ◽  
F. M. Morato ◽  
G. F. Rossi ◽  
B. M. Bayeux ◽  
A. S. Oliveira ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Formation Rate ◽  
Bos Indicus ◽  
Prostaglandin F2α ◽  
Lower Percentage ◽  
Embryo Production ◽  
High Category ◽  
Kinetics Of ◽  
In Vitro Embryonic Development

The present study aimed to evaluate the effect of the follicular population from cull Nelore (Bos indicus) on the kinetics of in vitro embryonic development. At random stages of the oestrous cycle (Day –5), a total of 28 cull Nelore cows were synchronized with an intravaginal progesterone device associated with oestradiol benzoate (2.0 mg IM). At the same moment, a dose of prostaglandin F2α (2.0 mg im) was also administered to promote luteolysis and absence of corpus luteum (CL) at the time of ovum pick-up (OPU). Five days later (Day 0), all cows underwent an OPU session and the recovered oocytes were submitted to in vitro embryo production (IVEP). The same procedures were repeated 2 times at 30-day intervals (Day 25 and Day 55). Semen from a single batch of a previously tested bull was used for all IVEP. Blastocyst production and hatching were verified on Days 7, 8, and 9 of the IVEP. Data were analysed by the GLIMMIX procedure of SAS 9.3® (SAS Institute Inc., Cary, NC, USA). Data of the 3 OPU sessions were grouped and the cows were classified into 3 categories according to the follicular population: Low (19.7 ± 0.9 follicles, n = 27), Medium (33.5 ± 0.8 follicles, n = 29), and High (58.7 ± 3.2 follicles, n = 28). The Low category had a lower rate of viable oocytes [(number of viable oocytes/total number of oocytes) × 100; 62.0 v. 69.5%; P = 0.02] and cleavage rate [(number of cleaved/total number of oocytes) × 100; 55.9 v. 66.8%; P = 0.001] than the High category. The blastocyst formation rate [number of blastocysts/total number of oocytes) × 100] on Day 7 and Day 8 was lower for Low and Medium compared with the High category (Day 7: 26.1b v. 29.0b v. 35.1a %; P = 0.001; and Day 8: 29.2b v. 30.2b v. 34.7a %; P = 0.05). No differences were found in blastocysts rate on Day 9 among Low, Medium, and High categories (14.1 v. 15.9 v. 16.2%; P = 0.61). However, Low category had a lower percentage of hatched blastocysts [(number of hatched blastocysts/number of blastocysts) × 100] on Day 7 compared with High category (2.9 v. 12.0%; P = 0.01). These results reported that cull Nelore with High follicular population showed higher rates of embryo production and hatched blastocysts compared with cows with Low follicular population. We concluded that the kinetics of in vitro embryonic development was compromised in cull Nelore (Bos indicus) with low follicular population submitted to OPU-IVEP. This research was supported by Fapesp 2012/50533–2 (GIFT).

287 EFFECT OF CYSTEAMINE DURING IN VITRO MATURATION OF OPU DERIVED BOVINE OOCYTES ON FURTHER IN VITRO EMBRYONIC DEVELOPMENT AND PREGNANCY RATE

2006 ◽  
Vol 18 (2) ◽  
pp. 251
Author(s):  
J. S. Merton ◽  
B. Landman ◽  
E. Mullaart
Keyword(s):  
Embryonic Development ◽  
Pregnancy Rate ◽  
Production Rate ◽  
In Vitro Maturation ◽  
Radical Scavenging ◽  
Protective Role ◽  
Embryo Production ◽  
Bovine Oocytes ◽  
In Vitro Embryonic Development

Glutathione (GSH) plays an important protective role in relation to reactive oxygen species generated by normal oxidative metabolism in the cell. The presence of cysteamine during in vitro maturation may facilitate the synthesis of GSH by immature oocytes. In a previous study we showed a positive effect of the presence of cysteamine during in vitro maturation of slaughterhouse derived bovine oocytes on subsequent in vitro embryonic development (Merton et al. 2004 Rep. Fert. Dev. 16, 279 abstract). This report shows the results of a field trial with ultrasound guided transvaginal oocyte collection (OPU) derived oocytes, in order to confirm our previous results obtained with slaughterhouse derived oocytes. Immature cumulus–oocyte complexes (COCs) were recovered twice weekly by ovum pick-up (OPU) at two collection centres from 11 cows and 147 pregnant heifers. COCs were matured in vitro in TCM199/FCS/LH/FSH supplemented either with or without cysteamine (0.1 mM). Subsequently, matured oocytes were fertilised with frozen-thawed gradient-separated semen and further cultured for 7 days in SOFaaBSA. At Day 7, Morula grade 1 (IETS) were transferred fresh and early-, mid- and exp-Blast grade 1 and 2 were transferred either fresh or frozen/thawed. The experimental design was a 2 × 2 factorial. Results were analysed by Chi-square analyses. The results show that the presence of cysteamine during in vitro maturation significantly affected embryo production from OPU derived COCs (23.4% and 34.4% Morula + Blastocyst rate at Day 7 for control and cysteamine, respectively; Table 1). This higher embryo production rate was mainly due to an increased number of Blastocysts. Also the proportion of grade 3 embryos was significantly reduced in the cysteamine group (P < 0.01). The number of transferable embryos per session was 1.06 and 1.73 for control and cysteamine, respectively. Pregnancy rate was not significantly affected by the presence of cysteamine during in vitro maturation for both fresh and frozen/thawed embryos (fresh: 40.5% and 44.8%, frozen/thawed: 44.4% and 47.2% for control and cysteamine, respectively). These results show that the presence of cysteamine during in vitro maturation affects further in vitro embryonic development, resulting in a higher embryo production rate. This suggest that an apparently ‘simple’ extra protection of the oocyte, due to the free radical scavenging potency of GSH, can have an enormous effect (63.2% relative increase in transferable embryos) on its in vitro developmental potency. The intrinsic quality of the ‘extra’ produced transferable embryos seems not to be different, since pregnancy rate was not affected. Table 1. Effect of cysteamine during in vitro maturation of OPU-derived bovine oocytes on subsequent in vitro embryonic development

scholarly journals 320EFFECT OF CYSTEAMINE DURING IN VITRO MATURATION ON FURTHER EMBRYONIC DEVELOPMENT AND POSTTHAW SURVIVAL OF IVP BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 279
Author(s):  
J.S. Merton ◽  
M. Gerritsen ◽  
D. Langenbarg ◽  
Z.L. Vermeulen ◽  
T. Otter ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Production Rate ◽  
In Vitro Maturation ◽  
Survival Rates ◽  
Protective Role ◽  
Bovine Embryos ◽  
Embryo Production ◽  
In Vitro Embryonic Development ◽  
In Vitro Survival

The uptake of cysteamine by immature oocytes may facilitate the synthesis of glutathione (GSH) during in vitro maturation, as reported by Matos et al. (1995 Mol. Reprod. Dev. 42 432–436). GSH plays an important protective role in relation to reactive oxygen species generated by normal oxidative metabolism. This study investigated the effects of the presence of cysteamine during in vitro maturation on subsequent in vitro embryonic development and postthaw in vitro survival. Immature Cumulus-Oocyte-Complexes (COCs) were recovered from ovaries 6 to 8h after slaughter. COCs were matured in vitro for 22 to 24h in TCM199/FCS/LH/FSH supplemented either with or without cysteamine (0.1mM), Subsequently, matured oocytes were fertilized with frozen-thawed Percoll-separated semen and further cultured for seven days in SOFaaBSA. Morulae grade 1 (IETS) and blastocysts grades 1 and 2 (IETS) were frozen on Day 7 in 10% Glycerol using a conventional slow freezing procedure (Wagtendonk-de Leeuw et al. 1995 Cryobiology;; 32 157–167). In vitro survival was measured by rates of blastocyst formation and reexpansion at 24h and hatching/ed blastocysts at 72h in SOFaaBSA supplemented with 5% FCS. Results were analyzed by Chi-square analyses. The presence of cysteamine during in vitro maturation significantly affected the embryo production rate (19.4% and 24.0% for control and cysteamine at Day 7, respectively). The higher number of embryos at Day 7 was totally due to an increased number of blastocysts (Table 1); however, the distribution of embryos among the different quality grades was not affected. Addition of cysteamine did not affect the post thaw survival of the frozen/thawed embryos (85% v. 91% reexpansion and 33% v. 34% hatching/ed for control v. cysteamine, respectively). These results show that the presence of cysteamine during in vitro maturation, does affect further in vitro embryonic development, resulting in a higher embryo production rate. Embryo quality, expressed in morphological grades and postthaw survival rates, were not affected. A field trial will be conducted in order to confirm these results with ovum pick up-derived oocytes. Table 1 Effect of cysteamine during in vitro maturation on subsequent in vitro embryonic development of IVP bovine embryos (number of replicates: 5)

scholarly journals Kinetics of a nucleoside release from lactide-caprolactone and lactide-glycolide polymers in vitro.

2000 ◽  
Vol 47 (1) ◽  
pp. 59-64
Author(s):  
T Kryczka ◽  
P Grieb ◽  
M Bero ◽  
J Kasperczyk ◽  
P Dobrzynski
Keyword(s):  
Formation Rate ◽  
Local Treatment ◽  
Extracellular Fluid ◽  
Brain Extracellular Fluid ◽  
Artificial Cerebrospinal Fluid ◽  
Fluid Formation ◽  
Further Development ◽  
Kinetics Of ◽  
The Brain

We assessed the rate of release of a model nucleoside (adenosine, 5%, w/w) from nine different lactide-glycolide or lactide-caprolactone polymers. The polymer discs were eluted every second day with an artificial cerebrospinal fluid at the elution rate roughly approximating the brain extracellular fluid formation rate. Adenosine in eluate samples was assayed by HPLC. Three polymers exhibited a relatively constant release of adenosine for over four weeks, resulting in micromolar concentrations of nucleoside in the eluate. This points to the necessity of further development of polymers of this types as intracerebral nucleoside delivery systems for local treatment of brain tumors.

scholarly journals Hydrosalpingeal fluid inhibits in-vitro embryonic development in a murine model

1997 ◽  
Vol 12 (12) ◽  
pp. 2724-2728 ◽  
Author(s):  
S. A. Beyler ◽  
K. P. James ◽  
M. A. Fritz ◽  
W. R. Meyer
Keyword(s):  
Embryonic Development ◽  
Murine Model ◽  
In Vitro Embryonic Development

106 COMPARISON OF IN VITRO EMBRYO PRODUCTION IN PANAMA USING HOLSTEIN OOCYTES WITH BOS INDICUS SEXED SEMEN FROM DONORS IN DIFFERENT LOCATIONS: FIELD DATA

2016 ◽  
Vol 28 (2) ◽  
pp. 183
Author(s):  
S. J. R. Rodriguez ◽  
Y. E. Ramirez ◽  
E. Gomes ◽  
L. F. Nasser ◽  
J. H. F. Pontes ◽  
...  
Keyword(s):  
Embryo Development ◽  
Bos Indicus ◽  
Embryo Production ◽  
University Of Illinois ◽  
Oocyte Collection ◽  
Sexed Semen ◽  
The Usa ◽  
In Vitro Embryo Production ◽  
The University

The objective of this work was to compare in vitro embryo production of Bos taurus × Bos indicus cross embryos using oocytes from Holstein donors under different production and environment systems. This study also examined the possibility for in vitro production using oocytes imported and transported fresh between the USA and Panama. All animals were mature Holstein cows going through a normal lactation. The first group of donors was from the University of Illinois dairy herd and went through 3 ovum pickup sessions. The second group of donors were Holstein cows already adapted to Panama and went through 10 ovum pickup sessions. The Panamanian herd of Holstein donors were born and raised in Panama in an area of mountains, on average 1300 m above sea level. This environment does not have the typical hot and humid tropical weather seen in other regions of Panama. Both groups of donors were aspirated without stimulation during the years 2013 and 2014. Oocytes recovered from donors in Illinois were imported fresh under a special sanitary research protocol between Panama and the University of Illinois. The transport of fresh oocytes from the USA to Panama was done using a portable incubator set at 39°C (Minutube of America). Oocytes were matured during transport in 5-mL tubes (~30–35 oocytes per tube) containing 400 µL of maturation media (TCM-199) that had been equilibrated with 5% CO2. Oocytes recovered from donors in Panama were matured using the same media. For both groups, oocytes were inseminated 24 h after ovum pickup using sexed semen from the same bull. All embryo production procedures followed the protocols of the In vitro Brasil™ commercial system. At 72 h postinsemination, cleavage was evaluated. On Day 7 after insemination, embryo development to the blastocyst stage (early to expanded) was recorded. Data were analysed using Chi-squared. As shown in Table 1, there was no effect of oocyte collection location on embryo development. These results indicate that it is possible to produce a viable in vitro-produced embryo using fresh oocytes collected and transported from different countries. This work opens the possibility to access superior genetics and improve herds in countries seeking to increase their production systems and potentially improve their quality of life. Table 1.Effect of oocyte collection location on embryo development This project was supported by Programa de Competitividad ProCom Senacyt, Panama.

100 COMPARISON OF PREGNANCY RATES OF FRESH IN VITRO-PRODUCED BOS INDICUS EMBRYOS PRODUCED IN THE SAME LABORATORY BUT COLLECTED AND TRANSFERRED IN PANAMA OR THE DOMINICAN REPUBLIC

2014 ◽  
Vol 26 (1) ◽  
pp. 164
Author(s):  
L. F. Nasser ◽  
S. C. Feliú ◽  
E. Rodríguez ◽  
K. Mojica ◽  
E. G. Oliveira ◽  
...  
Keyword(s):  
Dominican Republic ◽  
Bos Indicus ◽  
Disease Status ◽  
Pregnancy Rates ◽  
Bovine Embryo ◽  
Department Of Agriculture ◽  
Embryo Production ◽  
Essential Variable ◽  
Laboratory Facility

Because of Panama's stricter sanitary status, a specialised protocol was developed with the Department of Agriculture in the Dominican Republic to legalize the exchange of biological materials (oocytes/embryos). This protocol allows the team of specialised technicians, currently working in Born® Animal Biotechnology's Panamanian facility, to operate using the same in vitro bovine embryo production system (IVP, In vitro Brasil®) to service Dominican producers. Because the donors are not located at a specific centre with controlled sanitary management, a special protocol was developed in which blood tests were done to certify that the entirety of the herd at each client's farm was free of infectious bovine rhinotracheitis, DBVD, leptospirosis, leucosis, brucellosis, and tuberculosis. As timing during IVP is an essential variable that can have detrimental effects on the final results, precautions were taken to ensure that the oocytes arrived at the Panamanian laboratory facility within 24 h of aspiration. A portable incubator was used to transport oocytes and embryos during the import and export portions of the procedure. A comparison of pregnancy rates based on oocyte source and recipient transfers from September 2012 until May 2013 was analysed with ?2 (Table 1). The number of embryos produced in Panama was significantly higher than in the Dominican Republic, which was likely due to the larger number of donors and oocytes from the Panama herd. However, pregnancy rate was higher in the Dominican Republic likely because of the health status of the Dominican recipients, which were free of the diseases mentioned above. Recipients were the same type and breed and under similar management conditions in both countries. The disease status aspect will be examined with greater numbers of animals in the future. The data suggest that the present IVP and recipient management protocols could serve as a model for other Central American and Caribbean countries under similar management systems. Table 1.In vitro embryo production and pregnancy rates of Bos indicus embryos transferred in Panama or the Dominican Republic (September 2012 through May 2013)

169 THE RELATIONSHIP BETWEEN OOCYTE RECOVERY AND EMBRYO PRODUCTION IN BOS INDICUS

2012 ◽  
Vol 24 (1) ◽  
pp. 196
Author(s):  
M. P. Palhão ◽  
E. R. Oliveira ◽  
M. M. Gioso ◽  
B. C. Carvalho ◽  
L. G. B. Siqueira ◽  
...  
Keyword(s):  
Body Condition Score ◽  
Bos Indicus ◽  
Pregnancy Rates ◽  
Embryo Production ◽  
Exact Test ◽  
Oocyte Recovery ◽  
Conversion Rates ◽  
Condition Score ◽  
The Relationship

The ovarian follicular population has been used as a parameter to evaluate fertility and also the potential of donors undergoing assisted reproductive procedures in both human medicine and animal practice. There is a high correlation between follicular population and oocyte recovery by ovum pickup (OPU), but the relationship between oocyte recovery, embryo production and pregnancy rates may not be fully understood. The aim of the present study was to evaluate whether the conversion rate of oocytes to embryos and further pregnancies could be positively related to the number of cumulus–oocyte complexes (COC) recovered after OPU in cattle. For this purpose, records of 626 OPU sections from 251 nonlactating Gyr cows (dairy Zebu breed) were analysed. The animals had a good body condition score, were kept in a good feeding pasture (Brachiaria spp.) and were supplemented with corn silage and a mixture of corn, soybeans and vitamin and minerals, according to their nutritional requirements. For each ovarian aspiration, the ovarian follicular wave was previously synchronized with an auricular implant (Norgestomet-Crestar®), IM injections of 2 mg of oestradiol benzoate (Gonadiol®) and 0.25 mg of D-cloprostenol (Sincrocio®). The OPU procedures were performed using an ultrasound device (Aquila Vet, Esaote, São Paulo, Brazil) equipped with a vaginal sector 7.5-MHz probe, disposable 20 G needles and a vacuum pressure of 80 mmHg. The cows were ranked in quartiles regarding the total number of COC recovered. To reduce bias related to the eventual fluctuation of OPU results, for the present analysis the authors used only the recorded OPU session of each cow with the highest number of COC recovered. Viable COC were fertilized with sex-sorted (X) semen of Gyr bulls previously tested for in vitro embryo production. Conversion rates (%) of the total and viable oocytes to embryos, viable oocytes to pregnancy and embryo to pregnancy were evaluated for each quartile. Differences between the first and fourth quartiles were accessed by Fisher's exact test. In the 251 OPU, 4246 total and 3173 viable COC were recovered, resulting in the production of 1001 embryos (31.5%) and 453 pregnancies (45.3%). The cows ranked in the first, second, third and fourth quartiles produced >30 (41.6 ± 10.6), 21 to 30 (25.2 ± 3.0), 12 to 20 (15.9 ± 2.6) and <12 (6.7 ± 3.1) total oocytes. The average viable oocyte (29.1 ± 11.0, 18.1 ± 5.3, 11.1 ± 3.7 and 4.5 ± 2.7, respectively) and embryo production (8.6 ± 5.7, 5.2 ± 3.6, 3.8 ± 2.8 and 1.8 ± 1.8, respectively) were different (P < 0.0001) among all quartiles. Pregnancy rates, however, did not differ (46.0, 44.9, 43.9 and 45.6%, respectively; P > 0.05). Interestingly, the conversion rates (viable oocytes to embryos and viable oocytes to pregnancies) were higher (P < 0.0001 and 0.002) in cows from the last quartile (51.1 and 31.9%) compared with those from the first quartile (23.7 and 14.7%). In conclusion, the number of COC recovered by OPU (and consequently the ovarian follicular count) can further predict the total number of embryos and pregnancies produced, but it is not directly related to the oocyte development potential. Biotran and Fapemig Project CVZ APQ 01654/09 and BPD 0007/10.

257 DIFFERENCES BETWEEN BOS TAURUS AND BOS INDICUS EMBRYOS: TRANSCRIPTS AMOUNTS

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
S. Wohlres-Viana ◽  
M. M. Pereira ◽  
A. P. Oliveira ◽  
J. H. M. Viana ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Production System ◽  
Production Systems ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Peroxiredoxin 1 ◽  
Vitro Production

The Zebu breeds (Bos indicus) are different from European breeds (Bos taurus) in some aspects of their reproductive physiology, including follicle recruitment, number of follicular waves, and oocyte ultrastructure. On the other hand, embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. The aim of this study was to evaluate the effect of breed (Gyr v. Holstein) within embryo production system (in vivo and in vitro), as well as effect of production systems within breeds on relative abundance of transcripts related to formation, survival, and subsequent development of blastocysts, such as those involved in water and small solutes transport (Aquaporins 3 and 11), blastocoel formation (Na+/K+-ATPase a1 and |52), and cellular stress response (Peroxiredoxin 1). For in vivo embryo production, donors were superstimulated with FSH and inseminated, and embryos were recovered 7 days after AI. For in vitro embryo production, oocytes recovered by ovum pickup were in vitro matured and fertilized and then cultured for 7 days in culture medium under 5% CO2 at 38.5°C. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA, USA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA, USA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA, USA). The cDNA were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed by REST© software. To evaluate breed effect within the production systems, 2 comparisons were performed: (1) in vivo: Gyr v. Holstein and (2) in vitro: Gyr v. Holstein, considering Holstein data as 1.00. To evaluate production system effect within breeds, 2 comparisons were performed: (1) Gyr: in vivo v. in vitro and (2) Holstein: in vivo v. in vitro, considering in vivo produced embryo data as 1.00. The results are shown as mean ± SEM. For in vivo comparison between breeds, Aquaporin 3 (1.66 ± 0.77), Na+/K+-ATPase a1 (1.61 ± 0.56), and Peroxiredoxin 1 (1.61 ± 0.66) were up-regulated (P < 0.05) in Gyr embryos when compared with Holstein embryos, whereas for in vitro comparison, no differences (P > 0.05) were found. For comparisons between production systems within breeds, only Peroxiredoxin 1 (0.31 ± 0.39) was down-regulated (P < 0.01) in in vitro produced Gyr embryos when compared with in vivo counterparts. No differences (P > 0.05) were found between production systems for the Holstein breed. In conclusion, these data suggest that there is a difference on gene expression between Bos taurus and Bos indicus blastocysts, but such difference between breeds can be attenuated by the in vitro production system, indicating an embryo adaptation to the in vitro culture conditions. The data also suggest that the in vitro production system can influence the amount of transcripts in Gyr embryos. Other genes should be evaluated for a better understanding of these differences. Financial support was provided by CNPq and FAPEMIG.

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.

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