293 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION ON IN VITRO EMBRYO PRODUCTION IN COWS

2006 ◽  
Vol 18 (2) ◽  
pp. 254
Author(s):  
A. F. Ramos ◽  
R. Rumpf ◽  
M. R. Mollo ◽  
J. U. Câmara ◽  
I. Pivato ◽  
...  

In order to achieve the ideal time of ovum pickup (OPU) for in vitro embryo production (IVP) in cows regarding number and quality of oocytes recovered, this study investigated the effect of synchronization of wave emergence with estradiol benzoate (EB) injected 7 days prior to follicular aspiration. In a Latin square design, 12 crossbred beef cows were randomly divided into three groups, with three replicates each. Cows were synchronized with a norgestomet ear implant for 7 days followed by an i.m. prostuglandin F2� (PGF2�) injection and aspiration of all ovarian follicles larger than 3 mm in diameter. After that, follicles from cows in group 2X were aspirated twice a week with 4- and 3-day intervals, and follicles from groups 1X and 1X-EB were aspirated once a week. Cows from group 1X-EB also received an im injection of 2 mg of EB immediately after OPU. Throughout the study cows were kept with an ear norgestomet implant that was replaced every 2 weeks. Ultrasound evaluations of numbers of follicles greater than 3 mm in diameter and size of the largest follicle at the time of OPU were performed. Recovered oocytes were evaluated for quality, and the viable ones (Grades I, II, and III) were in vitro-fertilized on Day 0. Cleavage rate was evaluated on Day 2 and blastocyst production on Day 7. Continuous variables were compared by ANOVA and binomial data were compared by chi-square. For the 2X group, only data from the OPU performed 3 days after the last OPU were used for analysis. Results are presented as percentages or mean � SEM. Size of the largest follicle was greater (P < 0.05) in 1X coes (12.9 � 0.2 mm) than in 1X-EB cows (11.1 � 0.3 mm), which was greater than in 2X (9.6 � 0.4 mm) cows. The 1X cows had more follicles at OPU than 2X cows (17.5 � 0.7 vs. 14.1 � 0.9), whereas the 1X-EB group (15.9 � 0.7) was intermediate and not different from the others. There was no difference in the mean number of recovered oocytes among 2X (9.6 � 0.6), 1X (12.7 � 0.8) and 1X-EB (12.3 � 1.0) cows, and the mean number of viable oocytes among groups (5.8 � 0.5, 7.3 � 0.5, and 7.0 � 0.6) for 2X, 1X, and 1X-EB cows, respectively). The rate of viable oocytes was also similar among groups [58.8% (191/325) for 2X, 58.4% (267/457) for 1X, and 57.2% (231/404) for 1X-EB cows]. Cleavage [68.6% (131/191), 65.2% (174/267), and 68.4% (158/231)] and blastocyst [38.7% (74/191), 43.8% (117/267), and 44.2% (102/231)] rates were also not different among 2X, 1X, and 1X-EB groups, respectively. Although the use of 2 mg of EB in association with a norgestomet implant 7 days prior to OPU altered the follicular wave profile, it was not enough to improve number and quality of the oocytes recovered. Moreover, this study failed to demonstrate a positive effect of OPU earlier after wave emergence, when the effect of dominance should be less pronounced, on IVP in cows. The first author was supported by the fellowship 141077/2004-2 of CNPq, Brazil.

2008 ◽  
Vol 20 (1) ◽  
pp. 182 ◽  
Author(s):  
K. Imai ◽  
Y. Inaba ◽  
H. Yoshioka ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
...  

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.


2021 ◽  
Vol 42 (3) ◽  
pp. 1147-1158
Author(s):  
Maria Fernanda Zamai ◽  
◽  
Fábio Luiz Bim Cavalieri ◽  
Marcia Aparecida Andreazzi ◽  
Fabio Morotti ◽  
...  

Reproductive biotechnologies are emerging as an important element for livestock; however, some strategies must be modified to adapt to different breeding systems, such as the use of follicular synchronization protocols. This study aimed to evaluate follicular synchronization using estradiol benzoate (EB), in the presence of the corpus luteum (CL) from Wagyu oocyte donors in in vitro embryo production (IVEP). Rounds of IVEP were performed in heifers and cows (n=19) that were classified into three groups: G1/CL - animals with CL, G2/WCL - animals without CL, and G3/CL + EB - animals with CL that were subjected to follicular synchronization with EB at D0. The groups G1/CL and G2/WCL were considered the control and undertook the natural process of follicular dynamics. The results showed that the synchronization of the follicular wave with the application of EB in the presence of CL, presented a smaller number of small (6.05 ± 0.55) and large follicles (0.45 ± 0.15), but increased (P < 0.05) the number of medium-sized follicles (16.20 ± 0.90). However, the results of ovum pick up showed that regardless of whether or not EB was applied, and regardless of the presence or absence of CL in the Wagyu donor, there was no difference among the groups (P > 0.05) concerning the number of viable oocytes and the viability rate. It was concluded that follicular synchronization using EB in Wagyu oocyte donors that presented a CL, increased the number of medium-sized follicles. However, there was no improvement in the efficiency of ovum pick up, in vitro embryo production, and pregnancy rate.


2011 ◽  
Vol 23 (1) ◽  
pp. 213
Author(s):  
L. T. Iguma ◽  
J. N. S. Sales ◽  
R. I. T. P. Batista ◽  
M. M. Pereira ◽  
C. C. R. Quintão ◽  
...  

Recent data reveal that approximately 80% of bovine embryos produced in vitro worldwide are from (Brazil J. H. M. Viana, personal communication). Adoption of this biotechnology by Brazilian producers might be attributed to particularities of the Bos indicus subspecies. Zebu breeds provide 2 to 3 times more viable oocytes than Bos taurus breeds per ovum pickup (OPU) session. This work aimed to evaluate the quality of cumulus–oocyte complexes (COC) retrieved from Gir breed cows (B. indicus) by OPU with a synchronized follicular wave and subjected to an in vitro embryo production (IVP) technique. All COC were obtained by OPU of 14 Gir cows performed every 14 days. There were 4 OPU sessions, preceded by synchronization of the follicular waves. Wave synchronization was achieved by an injection of 2 mg of oestradiol benzoate followed by the insertion of a norgestomet implant that stayed in place for 5 days. On Day 5, follicles of 2 to 8 mm were aspirated. The oocytes recovered were distributed according to classification: Group 1, with ≥3 cumulus cell layers and homogeneous or slightly heterogeneous cytoplasm (n = 314); and Group 2, ≤2 cumulus cells layers and slightly or coarsely granulated cytoplasm (n = 236). Oocytes from both groups were followed to in vitro maturation–IVF–fertilization–in vitro culture. Two (Day 2) and 8 (Day 8) days after the beginning of fertilization, the cleavage and blastocyst rates were measured, respectively. The statistical analyses were performed using a chi-square test (P < 0.05). The mean cleavage rates observed were 72.93 and 69.07% (not significant), whereas the mean blastocyst rates were 37.90 and 17.80%, and were significantly different for Groups 1 and 2, respectively. The results suggest that the morphology of oocytes (number of cumulus cell layers and cytoplasm homogeneity) is a strong indicator of the in vitro developmental capacity of oocytes recovered from Gir cows with synchronized follicular waves, although we did not observe compromised cleavage rates. Financial support was provided by Fapesp/Fapemig/CNPq/Embrapa. Epamig/Faz. Calciolândia/Nutricell–Nutrientes Celulares is acknowledged.


2016 ◽  
Vol 28 (2) ◽  
pp. 219
Author(s):  
P. M. S. Rosa ◽  
A. J. R. Camargo ◽  
R. V. Serapião ◽  
L. S. A. Camargo ◽  
C. S. Oliveira

Bovine in vitro embryo production is highly relevant for dairy systems in Brazil, and Gyr dams are commonly used as oocyte donors. The aim of this study was to evaluate the use of prepubertal Gyr heifers as oocyte donors, an alternative to anticipate reproduction of those animals. For that, 11 Gyr [4 prepubertal (PP) donors and 7 adult cows © donors] were used in ovum pickup (OPU) sessions. The PP cows presented an average of 282.5 kg and 26.75 months, and had never displayed oestrous. Non-lactating cows presenting an average of 492 kg and 136 months were selected for C. Five replicates were performed, totaling 27 OPU sessions (C-17, PP-10) and 2–3 sessions per animal. Follicular wave was synchronised by aspiration of follicles larger than 8 mm 96 h before OPU. Cumulus-oocyte complexes (COC) were classified accordingly to their quality in viable (G1, G2, and G3) or non-viable (G4). Viable oocytes were matured and fertilized, and the presumptive zygotes were cultured in SOF medium at 38.5°C and 5% CO2 in air. Cleavage rate was assessed 48 to 72 h post-insemination (hpi) and blastocyst rate at 168 hpi. Mean number of structures was analysed by t-test, and percentage of viable, G1, G2, G3, G4, cleavage, and blastocyst rates were compared among groups by Fisher’s exact test (GraphPadInstat, La Jolla, CA, USA; P = 0.05). Results are followed by standard error values. All procedures were approved by a local ethics committee. We found that despite higher (P < 0.05) numbers for both viable oocytes (PP: 15 ± 2.6; C: 6.11 ± 0.76) and total oocytes (PP: 23.70 ± 2.83; C: 8.82 ± 1.19) in the PP group, the rate of viable oocytes was similar (P > 0.05) among PP and C groups (PP: 61.5 ± 6.51%, C: 66.79 ± 3.79%). Mean numbers of G1, G2, G3, and G4 oocytes were higher (P < 0.05) in the PP group (G1 = 7.1 ± 1.18; G2 = 4.9 ± 1.74; G3 = 3.9 ± 1.09; G4 = 7.8 ± 1.38) than in the C group (G1 = 2.70 ± 0.740; G2 = 2.47 ± 0.44; G3 = 1.11 ± 0.31; G4 = 2.52 ± 0.39). However, the proportion was similar (P > 0.05) among PP and C groups (PP: G1 = 29.5 ± 4.21%; G2 = 19.5 ± 2.85%; G3 = 15.9 ± 13.5%; G4 = 35.1 ± 6.33%; and C: G1 = 27.24 ± 4.44%; G2 = 29.60 ± 5.08%; G3 = 12.34 ± 3.01%, G4 = 30.79 ± 4.93%). Cleavage rate (PP: 91.3 ± 17.94%; C: 74.09 ± 4.65%), mean blastocyst number per OPU session (PP: 3.3 ± 1.29; C: 1.76 ± 0.28), and blastocyst rate (PP: 19.74 ± 7.40%; C: 27.03% ± 4.07%) were similar (P > 0.05) among groups. We conclude that prepubertal heifers presented increased numbers of viable oocytes per OPU session, but blastocyst yield was similar to adult cows. This data suggests that prepubertal Gyr heifers can be used as oocyte donors. Support from FAPERJ and Embrapa is acknowledged.


2015 ◽  
Vol 27 (1) ◽  
pp. 211 ◽  
Author(s):  
H. E. Tribulo ◽  
J. Carcedo ◽  
R. J. Tribulo ◽  
B. Bernal ◽  
J. Garzon ◽  
...  

An experiment was designed to evaluate in vitro embryo production following the use of frozen-thawed conventional or Y-sexed semen from a single Brangus and a single Braford bull of proven fertility. Semen was obtained by splitting the same ejaculate to be frozen directly or sex-sorted and then frozen. Oocytes were obtained from 69 ultrasound-guided follicle aspiration (ovum pickup) sessions performed at random stages of the oestrous cycle without superstimulation in 24 Brangus and 10 Braford cows and heifers. Viable oocytes (n = 1120) were matured in TCM-199 medium with NaHCO3 and supplemented with 1% fetal bovine serum. Frozen-thawed sperm from the Brangus and Braford bulls were selected with Percoll for IVF, capacitated in Fert Medium, and used at a final concentration of sperm per milliliter for conventional (non-sexed) semen and 2 × 106 sperm mL–1 for Y-sexed semen. After 16 h (sexed) or 18 h (conventional) of co-incubation with oocytes in Fert Medium, presumptive zygotes were denuded and cultured in SOF supplemented with 0.4% BSA under oil at 37°C, 5% CO2, and saturated humidity for 7 days. The total number of oocytes matured and fertilized from the Brangus donors was 538 and 318 for conventional and sexed semen, respectively. The total numbers of oocytes matured and fertilized from the Braford donors were 139 and 125 for conventional and sexed semen, respectively. Data were compared by ANOVA for mixed models, using breed and type of semen as fixed variables and cow (i.d.) as a random variable. Cleavage and blastocyst rates were first transformed by square root and then analysed by ANOVA for mixed models. Mean (± s.e.m.) number of total viable oocytes collected, cleaved zygotes, and blastocysts produced per ovum pickup session did not differ (P = 0.18) between breeds (Brangus: 17.1 ± 1.6, 10.0 ± 0.9, and 6.2 ± 0.7 v. Braford: 13.9 ± 2.8, 7.6 ± 1.5, and 4.0 ± 0.8), and there was no breed × semen interaction on the mean number of cleaved zygotes and blastocysts produced. However, the mean (± s.e.m.) number of cleaved zygotes and blastocysts produced was significantly higher (P < 0.05) when the oocytes were fertilized with conventional semen (10.7 ± 1.2 and 6.5 ± 0.8) than with sexed semen (7.7 ± 0.7 and 4.3 ± 0.6). The mean cleavage rate was also significantly higher (P < 0.05) when the oocytes were fertilized with conventional semen (76.8 ± 3.9) than with sexed semen (54.1 ± 4.2). Blastocyst rate tended to be higher (P = 0.1) with conventional semen (40.5 ± 3.3) than with sexed semen (33.6 ± 4.2). Although in vitro production may be the preferred alternative for the production of embryos of a known sex, the number of blastocysts produced might be reduced as compared with the use of non-sexed semen from the same bull.


Author(s):  
M.H. Pitroda ◽  
K.P. Khillare ◽  
M.B. Amle ◽  
M.D. Meshram ◽  
A.B. Mali ◽  
...  

Background: In vitro embryo production in buffaloes has gained much importance in this current scenario due to ever increasing population and high demand of milk and meat. Slaughter house derived bubaline ovaries are a cheap and abundant source of cumulus oocyte complexes.Methods: Oocytes from the buffalo ovarian follicles were recovered by aspiration technique as it facilitates quick recovery. Total 155 ovaries were used in the present study. Surface follicles were measured using vernier calliper and categorized into three groups viz. less than 3 mm, 3-5 mm and greater than 5 mm based on follicular diameter and oocytes were processed for IVM, IVF and IVC using conventional non sorted semen.Result: Overall percentage of small, medium and large follicles in the ovaries were recorded as 16.29 ± 0.94%, 8.14±0.60%, 5.35 ± 0.76%, respectively. Overall recovery rate of COCs was 38%. The percentage of these oocytes were 16.74% (A), 15.25% (B), 25.26% (C), 18.33% (D) and 29.87% (E) respectively. Maturation rate of oocytes were 81.96 ± 2.70%. Fertilization rate was 74.98 ± 3.87%, Cleavage rate % was 40.84±2.51% and Blastocyst percentage was 21.57±1.75% respectively. Application of in vitro embryo production technique using slaughter house ovaries can salvage the genetic potential of bubaline species.


2010 ◽  
Vol 22 (1) ◽  
pp. 290 ◽  
Author(s):  
R. M. Ferreira ◽  
H. Ayres ◽  
M. L. Ferraz ◽  
A. B. Araújo ◽  
M. R. Chiaratti ◽  
...  

The association of ovum pickup (OPU) and in vitro embryo production (IVP) has been widely used to improve bovine reproduction. However, previous reports have indicated the occurrence of low fertility associated with summer heat stress. In the present work, we hypothesized that different categories of Holstein cattle [heifers (H), high-producing cows in peak lactation (PL), and repeat-breeders (RB)] would respond differently to OPU and IVP during the summer, because of their different metabolisms. This experiment was conducted on 2 commercial dairy farms in southeast Brazil in summer 2009. Cattle (n = 36/category) started a protocol to synchronize follicular wave emergence: 2 mg of estradiol benzoate (Sincrodiol®, OuroFino, Minas Gerais, Brazil) + 50 mg of progesterone (OuroFino) + 150 μg of D-cloprostenol (Sincrocio®, OuroFino) i.m. + a norgestomet ear implant (Crestar®, Intervet, São Paulo, Brazil) on Day 0, implant removal and OPU on Day 5. Respiration rate (RR), rectal temperature (RT), and cutaneous temperature (CT) were recorded on Day 0. Semen from a single Holstein bull previously tested was used in IVP, and oocytes from slaughterhouse were submitted to IVP as a quality control. Statistical analyses were done using PROC GLIMMIX of SAS (SAS Institute, Cary, NC, USA). The average and maximum environmental temperature and humidity were 30 and 39.8°C and 61 and 88%. Heifers were on average 15.7 months old; PL and RB cows had 112.8 ± 5.4a v. 422.8 ± 27.6b days in milk, milk production of 32.8 ± 0.9a v. 21.7 ± 1.1b kg, and number of insemination of 0.9 ± 0.1a v. 6.8 ± 0.3b; P < 0.0001; mean ± SE). Heifers and cows had different RR (H = 11.2 ± 0.3a, PL = 18.2 ± 0.6b, RB= 18.5 ± 0.4b breaths/min; P < 0.0001), RT (H = 38.7 ± 0.1a, PL = 39.56 ± 0.2b, RB = 39.32 ± 0.1b; P < 0.0001), and CT (H = 31.3 ± 0.2a, PL = 33.2 ± 0.3b, RB = 32.9 ± 0.3b; P < 0.0001). At OPU, heifers had greater number of follicles than PL cows, but they were similar to RB cows (H= 18.5 ± 1.9a, PL = 12.4 ± 1.1b, RB = 17.2 ± 2.0a; P = 0.04). Heifers had also greater number of oocytes (H = 9.6 ± 1.6a, PL = 5.0 ± 0.9b, RB = 8.8 ± 13ab; P = 0.03) and viable oocytes (H = 7.6 ± 1.5a, PL = 3.6 ± 0.8b, RB = 6.8 ± 1.2ab; P = 0.05) recovered from OPU than PL cows and similar to RB cows. However, at IVP, heifers had greater rates than both other categories (cleavage at Day 3: H = 47.8%a, PL = 31.1%b, RB = 35.4%b, P = 0.008; blastocyst at Day 7: H = 21.0%a, PL = 4.1%b, RB = 3.8%b, P < 0.0001) and more grade I embryos (H = 1.3 ± 0.4a, PL = 0.3 ± 0.2b, RB = 0.5 ± 0.2b, P = 0.04). The quality control had 80.7% cleavage and 45.4% blastocyst rates. The differences found among heifers and cows are probably related to their metabolism under heat stress, compromising oocyte number and quality. Also, although RB had similar number of viable oocytes than heifers, these oocytes are probably compromised, leading to poorer results at IVP, as observed. Fazendas Santa Rita e Campestre, Vida Reprodutiva, LMMD, SAMVET, VITROGEN, OuroFino Saúde Animal, FAPESP (proc09/00938-3).


2016 ◽  
Vol 28 (2) ◽  
pp. 209
Author(s):  
M. Nkadimeng ◽  
E. van Marle-Koster ◽  
K. P. M. Lekola ◽  
M. L. Mphaphathi ◽  
M. M. Seshoka ◽  
...  

Heat stress during IVF is associated with reduced fertility in cattle oocytes. It may, however, enhance thermo-tolerance or cause detrimental effects on a variety of cell types or organisms, depending on the duration and intensity of the thermal challenge. The aim of this study was to evaluate the developmental potential of cumulus-oocyte complexes (COC) matured for 18 or 24 h and incubated at 39°C or 41°C. A total of 1000 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The COC were randomly allocated (100/treatment) into 2 maturation times (18 or 24 h) and cultured in M199 + FSH-LH-estradiol medium under oil at 100% humidity and 5% CO2 at 39°C or 41°C. Post maturation, oocytes were subjected to normal subsequent embryo conditions. The Bracket and Oliphant medium was used for IVF. All matured oocytes were fertilised for 6 h with frozen-thawed Nguni bull semen at a concentration of 265 × 106. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate 48 h post IVF. After Day 7 of culture, blastocyst were stained (Hoechst 33323) for nuclei cell count. Statistical analyses was performed using Genstat® software of SAS (SAS Institute, Cary, NC, USA; P < 0.05). Oocytes that were matured for 18 h in 41°C resulted in more 8-cell embryos (41%) compared with those incubated at 39°C (21.6%). However, no difference was observed for cleavage rate at both maturation times and incubation temperatures (41 or 39°C). There was more morula formation from oocytes matured for 18 h (19.6%) and 24 h (19.0%) at 41°C compared to 39°C (8.4%) group. The results further showed more blastocyst formation during 18 h at 41°C (15.2%) than at 39°C (7.4%) and during 24 h at 41°C (11.2%), 39°C (11.4%). However there was no difference in the nuclei cell number during 18 h at 41°C (45.2), 24 h (45.8), and 18 h at 39°C (43.4) of maturation. Thus, there was a significant difference in the nuclei cell numbers at 24 h on 39°C (n = 133.2) and 41°C (n = 45.8). In conclusion, oocytes that were matured for 18 and 24 h at 41°C or for 18 h at 39°C developed further to blastocyst stage on in vitro embryo production, however, with low nuclei cell numbers due to accelerated maturation temperature or shortened maturation period.


2011 ◽  
Vol 23 (1) ◽  
pp. 205
Author(s):  
K. Imai ◽  
M. Ohtake ◽  
Y. Aikawa ◽  
S. Sugimura ◽  
M. Hirayama ◽  
...  

We previously reported that superstimulatory (SS) treatment-induced follicular wave synchronization after ovum pickup (OPU) was effective in enhancing the quality of obtained oocytes and blastocysts derived from in vitro maturation (IVM) and fertilization (IVF; Imai et al. 2010 Reprod. Fertil. Dev. 22, 296). The present study was designed to examine the efficiency of embryo production by 4 sessions of OPU-IVF using a series of the SS treatment-induced follicular wave synchronizations. For the SS protocols, 3 consecutive SS (3CSS) and 2 separated SS (2SSS) were used. In the 3CSS group, the first OPU was performed on random days of the oestrous cycle (Day 0) and all follicles larger than 2 mm in diameter were aspirated. On Day 5, follicles larger than 8 mm in diameter were aspirated and a CIDR (InterAg, Hamilton, New Zealand) was inserted. The cows then received 20 armour units of FSH (Kawasaki-Seiyaku, Kawasaki, Japan) in twice-daily decreasing doses by IM injection from Day 7 to 10. Cloprostenol (PGF; 0.75 mg, Fujita-Pharm, Tokyo, Japan) was administered on the morning of Day 9. The second OPU was performed 48 h after PGF administration on Day 11; the CIDR was removed from the cows just before OPU. After the second OPU, donors were treated consecutively with the SS protocol mentioned above for the third and fourth OPU sessions. In the 2SSS group, donors received 2 sets of the SS treatment mentioned above, with an interval of 11 days between the second and the third OPU session. Four OPU sessions were performed every 11 days on all cows. In this study, 8 Japanese Black cows were divided into the 3CSS and 2SSS groups, and the treatment for each group was reversed after a 65-day interval as crossover trials. After OPU, Grade 1 and 2 oocytes were used for IVM and IVF, and putative zygotes were cultured as described by (Imai et al. 2006 J. Reprod. Dev. 52, S19–S29 suppl.). A part of the zygotes were cultured in a micro-well system. Data were analysed by Student’s t-test and chi-square test. There were differences (P < 0.05) in the mean (±SD) number of follicles, collected oocytes, and cultured oocytes in the 3CSS (35.0 ± 8.6 and 24.4 ± 11.2, respectively) and 2SSS (30.8 ± 10.5 and 20.2 ± 9.0, respectively) groups. There were no differences in mean percentage of blastocyst formation and Grade 1 blastocyst rates between the 3CSS (38.5 and 55.8%, respectively) and 2SSS (34.8 and 54.8%, respectively) groups. However, the mean number of blastocysts produced per OPU session was significantly (P < 0.05) higher in the 3CSS group (8.1 ± 6.3) compared with the 2SSS group (5.8 ± 4.4). These results indicate that a series of 3 consecutive SS treatments had greater efficiency in producing OPU-IVF embryos. This work was supported in part by the Research and Development Program for New Bio-industry Initiatives.


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