207 DEVELOPMENT OF A ROUTINE IN VITRO EMBRYO PRODUCTION SYSTEM USING SINGLY IN VITRO-MATURED, FERTILIZED, AND CULTURED BOVINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 201
Author(s):  
I. G. F. Goovaerts ◽  
J. L. M. R. Leroy ◽  
J. B. P. De Clercq ◽  
S. Andries ◽  
P. E. J. Bols
Keyword(s):  
Production System ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Control Individual ◽  
Embryo Production ◽  
Group Culture ◽  
In Vitro Embryo Production ◽  
Bovine Oocytes ◽  
Hatching Rates

An in vitro embryo production system (IVP), in which a single oocyte can be tracked from the moment of retrieval up to the blastocyst stage, would be a valuable tool for studies linking developmental competence and embryo metabolism to oocyte quality and follicular environment. Unfortunately, to date, data on individual IVP are inconsistent, and in most cases show unsatisfactory blastocyst rates. Earlier studies revealed that individual culture on a cumulus cell (CC) monolayer resulted in comparable numbers of good-quality embryos as obtained after regular group culture (Goovaerts et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 190). Because, in the latter study, single culture was performed after group maturation and fertilization, the aim of this study was to develop and test an individual IVP system using bovine oocytes or zygotes obtained after single maturation and single fertilization. Therefore, 532 grade I COC from slaughterhouse ovaries (3 replicates) were randomly assigned to 1 of 2 treatments: a complete individual IVP protocol, or a routine group IVP as a control. Individual maturation (TCM-199 + 20% serum) and fertilization were performed in 20-μL droplets under oil in 24-well plates. Subsequently, each zygote was cultured in 20 μL of medium (SOF + 5% serum, 90% N2, 5% CO2, 5% O2) on a 6-day-old monolayer of matured CC (5% CO2 in air). Group maturation and fertilization were carried out per 100 COC in 500 μL, whereas group culture was performed per 25 zygotes in 50-μL droplets under oil. Cleavage, blastocyst, and hatching rates were assessed 2, 8, and 10 days postfertilization, respectively. Possible effects of individual and group culture were evaluated with binary logistic regression (SPSS 15.0). No interactions between replicate and treatment could be found (P > 0.05). Cleavage and blastocyst rates were significantly lower after individual IVP, compared with group IVP, whereas the blastocyst rates on cleaved zygotes and the hatching rates did not differ significantly (Table 1). In conclusion, acceptable blastocyst rates (25.1%) could be obtained after individual IVP. The lower blastocyst rates were associated with the lower cleavage rates, and no effect of the individual embryo culture system on embryo development could be found. Table 1.Cleavage, blastocyst, and hatching rates after individual and group in vitro embryo production (IVP)

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.

242 USE OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN IN VITRO PREMATURATION OF BOVINE OOCYTES SUBJECTED TO PARTHENOGENETIC ACTIVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 200
Author(s):  
T. H. C. De Bem ◽  
R. Rochetti ◽  
P. R. L. Pires ◽  
F. F. Bressan ◽  
P. R. Adona ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Polar Body ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Parthenogenetic Activation ◽  
Bovine Oocytes ◽  
Hatching Rates

Prematuration provides an additional time for oocyte capacitation and maturation in an attempt to improve in vitro embryo production (IVP) rates and allows media supplementation during this period for IVP. The aim of this study was to use brain-derived neurotropic factor (BDNF) in prematuration to improve maturation of bovine oocytes subjected to parthenogenetic activation and cultured with different media. Oocytes were subjected to prematuration in TCM-199 medium supplemented with 10 µm butyrolactone I, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin for 24 h in the absence of BDNF (control) or in the presence of 10 ng mL–1 BDNF (BD). Oocytes were then in vitro-matured (IVM) in TCM-199 medium supplemented with 10% FCS, 0.5 µg mL–1 FSH, 5.0 µg mL–1 LH, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin at 38.5�C under 5% CO2 in air. After 19 h oocytes were denuded using hyaluronidase and vortexing for 3 min for the 1st polar body (1PB) selection. Those which extruded the 1PB were maintained in IVM until 26 h, when parthenogenetic activation was performed (5 min in 5 µm ionomycin, followed by 3 h in 2 mm 6-DMAP). Activated oocytes were then transferred to in vitro culture (IVC) for embryo development evaluation. Embryos from both groups were cultured in SOF medium with 2.5% FCS, 0.05 g mL–1 BSA, 0.2 mm pyruvate, and 10 mg mL–1 gentamicin. Cleavage rates on the second day of in vitro culture (D2), embryo production at Days 7 and 8 (D7 and D8), and hatching rate at Day 8 were evaluated. Data regarding 1PB extrusion, cleavage, blastocyst development on D7 and D8, and blastocyst D8 hatching rates of three replicates were analyzed by chi-square test at 5% significance using the BIOESTATS 4.0 software. Control and BD, respectively, did not show differences (P > 0.05) regarding 1PB extrusion (n = 164, 63.81%, and n = 175, 66.79%) or cleavage (n = 117, 71.34%, and n = 138, 78.86%). However, for control and BD, respectively, blastocyst development on D7 (n = 63, 38.41%, and n = 89, 50.86%), D8 (n = 63, 38.41%, and n = 91, 52.00%), and hatching on D8 (n = 22, 34.92%, and n = 39, 43.82%) were all significantly higher for BD when compared with control (P < 0.05). In conclusion, BDNF during prematuration improved in vitro embryo development by increasing blastocyst and hatching rates of parthenogenetic embryos.

Virus screening of a bovine in vitro embryo production system

1993 ◽  
Vol 132 (26) ◽  
pp. 660-660 ◽  
Author(s):  
B. Avery ◽  
T. Greve ◽  
L. Ronsholt ◽  
A. Botner
Keyword(s):  
Production System ◽  
Embryo Production ◽  
In Vitro Embryo Production ◽  
Virus Screening

329 SUPPRESSION OF BOVINE OOCYTE IVM BY SERUM AND FSH DEPRIVATION OR BY α-AMANITIN: EFFECT ON FERTILIZATION AND EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 272
Author(s):  
K. Kananen-Anttila ◽  
M. Eronen ◽  
J. Matilainen ◽  
M. Kallio ◽  
J. Peippo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Embryo Cleavage ◽  
Bovine Oocytes ◽  
Sperm Aster

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μ­m; serum+FSH-free: 12 ± 0.5 μ­m, P < 0.05; α-amanitin: 10 ± 0.6 μ­m, P < 0.001) and sperm asters (control: 86 ± 4 μ­m; serum+FSH-free: 82 ± 4 μ­m, P < 0.01; α-amanitin: 49 ± 7 μm, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription. Table 1. Average percentages ± (n) for nuclear maturation, fertilization (min two pronuclei), embryo cleavage, and blastocyst development

Effects of cumulus cell density during in vitro maturation on the developmental competence of bovine oocytes

1998 ◽  
Vol 49 (8) ◽  
pp. 1451-1463 ◽  
Author(s):  
S Hashimoto ◽  
K Saeki ◽  
Y Nagao ◽  
N Minami ◽  
M Yamada ◽  
...  
Keyword(s):  
Cell Density ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Bovine Oocytes

229 EVALUATION OF OOCYTE QUALITY RECOVERED FROM Gir BREED COWS WITH SYNCHRONIZED FOLLICULAR WAVE ON IN VITRO EMBRYO PRODUCTION

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 ◽  
...  
Keyword(s):  
Cumulus Cell ◽  
Personal Communication ◽  
Fertilization In Vitro ◽  
Embryo Production ◽  
Follicular Waves ◽  
Follicular Wave ◽  
Cell Layers ◽  
The Mean ◽  
In Vitro Embryo Production

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.

210 CO-CULTURE WITH AUTOLOGOUS CUMULUS CELLS SUPPORTS THE INDIVIDUAL DEVELOPMENT OF SINGLY IN VITRO-MATURED AND FERTILIZED BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 204
Author(s):  
I. G. F. Goovaerts ◽  
J. L. M. R. Leroy ◽  
E. Merckx ◽  
S. Andries ◽  
P. E. J. Bols
Keyword(s):  
Somatic Cells ◽  
Cumulus Cells ◽  
Individual Development ◽  
Control Individual ◽  
In Vitro Production ◽  
Group Culture ◽  
The Individual ◽  
Vitro Production ◽  
Hatching Rates

The ability to produce embryos singly in vitro (in vitro production, IVP) would be a useful tool for many purposes. Without the interfering effects of other developing or degenerating oocytes or embryos, such an individual IVP system is the tool of choice for studies on oocyte quality and oocyte–embryo metabolism. Unfortunately, individual IVP in most cases leads to unsatisfactorily low blastocyst rates. Earlier work showed that individual culture of zygotes on a cumulus cell (CC) monolayer resulted in comparable numbers of good-quality embryos, as obtained following regular group culture (Goovaerts et al. 2009 Theriogenology 71, 729–738). However, co-culture with somatic cells is often criticised because of the undefined culture conditions and for sanitary reasons. In the cited study, CC for monolayer production were obtained from a different batch of ovaries. Our specific aim was to use CC from the zygote itself (autologous CC). Grade I COC (n = 660) were collected from slaughterhouse ovaries and randomly assigned to 2 treatments (5 replicates): a completely individual ‘single-oocyte’ IVP protocol, or routine group IVP as a control. Individual maturation (TCM-199 + 20% serum) and fertilization were performed in 20-μL droplets under oil in 24-well plates. Subsequently, each zygote was stripped and cultured in 20 μL of medium (SOF + 5% serum, 90% N2, 5% CO2, 5% O2), to which the autologous stripped CC were added. Group maturation and fertilization were carried out per 100 COC in 500 μL, whereas group culture was performed per 25 zygotes in 50-μL droplets under oil. Cleavage, blastocyst, and hatching rates were determined 2, 8, and 10 days post-fertilization, respectively. Possible effects of the individual and group cultures were evaluated with binary logistic regression (SPSS 15.0, SPSS Inc., Chicago, IL). No interactions between replicate and treatment were found (P > 0.05). Although a blastocyst rate of 15.1% was obtained using single IVP, the general efficacy of the single-embryo production system was lower when compared with group culture (Table 1). In conclusion, although developmental competence was impaired using individual IVP, co-culture with autologous cumulus cells can be useful in specific experimental setups in which the influence of other oocytes or embryos or heterologous somatic cells is unacceptable. Table 1.Cleavage, blastocyst, and hatching rates after individual and group in vitro production (IVP)

143 Effect of corpus luteum on recovery rate, quality, and in vitro developmental competence of oocytes in Bos indicus dairy cows

2019 ◽  
Vol 31 (1) ◽  
pp. 196
Author(s):  
M. Saad ◽  
Z. Sarwar ◽  
M. Saleem ◽  
U. Arshad ◽  
M. Shahzad ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Corpus Luteum ◽  
Recovery Rate ◽  
Bos Indicus ◽  
Developmental Competence ◽  
Serum Concentrations ◽  
Embryo Production ◽  
The Mean ◽  
In Vitro Embryo Production

Assisted reproductive technologies have modernized the dairy industry for the rapid multiplication of superior genetic traits. However, the exploitation of genetics through in vitro embryo production in farm animals has been struggling during the last 3 decades. The presence or absence of a corpus luteum (CL) on ovaries from which cumulus-oocyte complexes were recovered has been shown to affect the quality of oocytes and in vitro embryo production outcome in Bos taurus cows. Similar information is lacking in Bos indicus cows. Therefore, the objectives of the present study were to determine the effect of presence or absence of a CL on recovery rate, quality, and in vitro developmental competence of oocytes in Bos indicus dairy cows. This study was conducted from December 2017 to April 2018. The ovaries were harvested from a local abattoir (Bos indicus; 5- to 8-year-old cows having mixed parity with clinically normal reproductive tracts). These ovaries (n=750) were divided into 2 groups [(1) CYCLIC (n=318) and (2) ACYCLIC (n=432)] based on the presence or absence of mature CL having follicles on either left or right ovaries of slaughtered cows. Blood samples from the jugular vein were collected at slaughter. Serum concentrations of progesterone of CYCLIC and ACYCLIC cows were measured using radioimmunoassay. Mean number of recovered oocytes per ovary and serum concentration of progesterone were compared using PROC TTEST model. However, quality, maturation, cleavage, 8-cell, 16-cell, and morula rates were analysed by PROC FREQ model of Chi-squared. All the data was analysed using SAS (SAS Institute Inc., Cary, NC, USA). Results revealed that the mean serum concentrations of progesterone (4.21±0.4v. 0.5±0.2ng mL−1; P&lt;0.05) were higher in CYCLIC as compared with ACYCLIC cows, respectively. The mean number of oocytes recovered per ovary (6.5±4.5v. 4.0±4.4; P&lt;0.05) was higher in CYCLIC than in ACYCLIC cows. The oocytes with grade I+II quality (55.3v. 47.6%; P&lt;0.05) were higher, whereas oocytes with grade III+IV quality (44.5v. 52.4%; P&lt;0.05) were lower, in CYCLIC as compared with ACYCLIC cows, respectively. Furthermore, cleavage rate (70.9v. 52.8%; P&lt;0.05) was higher in CYCLIC than in ACYCLIC cows, respectively. Similarly, the 8-cell (38.5v. 20.8%; P&lt;0.05) and 16-cell (20.0v. 10.9%; P&lt;0.05) stage embryos were greater in CYCLIC as compared with ACYCLIC cows, respectively. In conclusion, the presence of CL has a beneficial effect on recovery rate, quality, and in vitro embryo production outcomes in Bos indicus dairy cows. It is implied that for ovum pickup, cyclic cows should be preferred.

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