scholarly journals 312 BLASTOCYST DEVELOPMENT OF MALE AND FEMALE BOVINE EMBRYOS PRODUCED BY IVF WITH FLOW CYTOMETRICALLY-SORTED SPERM

2005 ◽  
Vol 17 (2) ◽  
pp. 306
Author(s):  
M. Zhang ◽  
K.H. Lu ◽  
G.E. Seidel Jr
Keyword(s):  
Y Chromosome ◽  
Calf Serum ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Male And Female ◽  
Significant Difference

Several studies have demonstrated that male bovine embryos produced in vitro develop faster than female embryos produced in vitro, which results in more male than female blastocysts. The objective of this study was to compare the rate of blastocyst development of male and female bovine embryos derived from sexed sperm and cultured in a chemically defined medium + fatty acid-free (FAF)-BSA. Bovine oocytes (n = 1364) were fertilized with two types of frozen-thawed sperm (X- or Y-chromosome-bearing sperm sorted at 90% accuracy). Oocytes, aspirated from slaughterhouse ovaries, were matured in TCM199 supplemented with 10% fetal calf serum plus hormone additives (15 ng FSH, 1 mg LH, 1 mg 17 β-estradiol mL−1) for 22–24 h at 39°C, 5% CO2 in air with maximum humidity. Semen from one bull was sorted by flow cytometry into X- and Y-chromosome bearing sperm and frozen for later use with IVF. The procedures for IVF and IVC have been previously described (Lu KH, et al. 1999 Theriogenology 52, 1393–1405). Presumptive zygotes were removed from culture and placed in chemically defined medium (CDM-1 [Zhang M et al. 2003 Theriogenology 60, 1657–1663]) 6–7 h after insemination and cultured for 65–66 h. Embryos which had cleaved by 72 h post insemination were further cultured 96 h in CDM-2 (Zhang M et al. 2003 Theriogenology 60, 1657–1663) containing 0.12 IU insulin mL−1. Cleavage and blastocyst rates per oocyte inseminated were recorded on Day 3 and Days 7–8 after insemination, respectively. Data were analyzed by ANOVA procedures with replicates and treatments in the model. There was no significant difference in cleavage rate or blastocyst rate between X and Y sperm treatments. These results indicate that embryos produced with Y sperm do not reach the blastocyst stage in significantly higher proportions than embryos produced with X sperm in this chemically defined medium + FAF-BSA. Apparently, this IVC system leads to a more synchronous development of male and female embryos than other methods of producing bovine embryos in vitro. Table 1. Cleavage and blastocyst rates with X and Y sperm This research was supported by XY, Inc., Fort Collins, CO, USA.

Developmental kinetics and gene expression in male and female bovine embryos produced in vitro with sex-sorted spermatozoa

2010 ◽  
Vol 22 (2) ◽  
pp. 426 ◽  
Author(s):  
Pablo Bermejo-Álvarez ◽  
Patrick Lonergan ◽  
Detlef Rath ◽  
Alfonso Gutiérrez-Adan ◽  
Dimitrios Rizos
Keyword(s):  
Gene Expression ◽  
Mrna Abundance ◽  
Bovine Embryos ◽  
Same Sex ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Female Embryo ◽  
Male And Female ◽  
Kinetics Of

Using bovine embryos generated in vitro from IVF with X-sorted, Y-sorted and unsorted spermatozoa, we compared the kinetics of male and female embryo development and gene expression between male and female blastocysts. Bovine in vitro-matured oocytes (n = 8858) were fertilised with spermatozoa from each of three different bulls (X-sorted, Y-sorted or unsorted spermatozoa depending on the experiment). The cleavage rate was assessed 24, 27, 30, 33, 36, 40, 44 and 48 h post insemination (h.p.i.) and blastocyst development was recorded on Days 6–9. The relative mRNA abundance of nine genes (GSTM3, DNTM3A, PGRMC1, TP53, BAX, COX2, IGF2R, AKR1B1 and PLAC8) was analysed in male and female Day 7 blastocysts produced with sorted and unsorted spermatozoa from one bull. Cumulative cleavage rate and blastocyst yield were significantly higher in the unsorted group compared with the X- or Y-sorted group from the same bull (P ≤ 0.05). Although differences existed between bulls in terms of cleavage rate, no differences were observed in cleavage rate between X- and Y-sorted spermatozoa within a bull. The blastocyst yield was significantly higher only for Bull 3 when the Y-sorted spermatozoa were used (27.1+2.8 v. 19.1+1.4 for Y- and X-sorted spermatozoa, respectively; P < 0.05). There were no differences in the mRNA abundance of the nine genes analysed between embryos of the same sex produced with sorted or unsorted spermatozoa. However, significant differences in polyA mRNA abundance were observed between male and female blastocysts for three genes (GSTM3, DNMT3A and PGRMC1; P ≤ 0.05). In conclusion, the use of sorted rather than unsorted spermatozoa in IVF significantly delays the onset of first cleavage. Differences were noted between bulls, but not between X- and Y-sorted spermatozoa, and although no differences were found in terms of the mRNA abundance of the nine genes tested between sorted and unsorted spermatozoa, sex-related differences were found in the case of three genes.

90 OPEN PULLED STRAW VITRIFICATION OF IN VITRO PORCINE BLASTOCYTS IN A CHEMICALLY DEFINED MEDIUM

2011 ◽  
Vol 23 (1) ◽  
pp. 150
Author(s):  
J. Sanchez-Osorio ◽  
C. Cuello ◽  
J. Gomis ◽  
C. Maside ◽  
M. A. Gil ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Formation Rate ◽  
Calf Serum ◽  
Defined Medium ◽  
Basic Medium ◽  
Chemically Defined Medium ◽  
Cleavage Rate ◽  
Number Of Cells ◽  
Serum Components

The aim of this study was to design a chemically defined medium for the vitrification of in vitro produced porcine blastocysts avoiding the use of serum or serum components. Cumulus–oocyte complexes were matured in vitro in NCSU-23 for 44 h and were inseminated with frozen–thawed spermatozoa. Presumptive zygotes were cultured for 16 h to assess in vitro fertilization (IVF) parameters (N = 200) or for 6 days (N = 600) in order to obtain blastocysts. For chemically delipidation, 10 μM forskolin was added to the culture medium on Day 5 of in vitro culture. On Day 2, embryos were evaluated for cleavage rate. On Day 6, embryos were assessed for blastocyst formation; only those blastocysts showing excellent morphological appearance were selected for vitrification. Blastocysts were vitrified using as basic medium TCM-199 HEPES supplemented with 20% of newborn calf serum (NBCS; n = 65), with 0.1% of polyvinyl alcohol (PVA; n = 64) or without additives (WA; n = 65). The OPS-vitrification and warming were performed as described by (Sanchez-Osorio et al. 2010 Theriogenology 73, 300–308) using 16% of Etylenglycol and 16% of dimetyl sulfoxide as final concentrations of cryoprotectants. Vitrified blastocysts were warmed and cultured in vitro for 24 h to assess their viability. Blastocysts that totally reformed their blastocoel cavity showing a normal or excellent morphology were considered viable. In addition, after in vitro culture vitrified-warmed viable embryos were fixed in 4% paraformaldehyde in PBS medium and stained with Hoechst 33342 in order to assess the total number of cells. Data were analysed by using the MIXED procedure of SPSS. The threshold for significance was set at P < 0.05. Results are expressed as least squares means ± SEM. The maturation, penetration, and monospermy rates were 98.5 ± 1.2%, 85.3 ± 3.6%, and 48.8 ± 5%, respectively. The efficiency of IVF (defined as the ratio of monospermic oocytes to the total number of inseminated oocytes) was 41.0 ± 4.9%. The values of cleavage rate at Day 2 and blastocysts formation rate were 67.8 ± 1.4% and 37.3 ± 1.6%, respectively. After vitrification and warming, similar survival rates were observed for NBCS (33.8 ± 5.9) PVA (40.6 ± 6.0), and WA (30.8 ± 5.9) groups. No significant differences were found for the total number of cells (ranged from 35.4 ± 6.8 to 50.8 ± 8.3) among vitrification groups. In conclusion, in vitro derived porcine blastocysts can be vitrified in the absence of serum and serum components. Furthermore, PVA is a suitable substitute for serum in vitrification solutions with no detrimental effect on the viability of in vitro produced pig blastocysts. This study was supported by the Seneca foundation of Murcia (GERM 04543/07).

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

149 EFFECTS OF FSH ADDED IN DEFINED MEDIUM MATURATION ON THE PRE-IMPLANTATION DEVELOPMENT AND EXPRESSION OF Bax, Bcl-2, AND CONNEXIN 43 GENES IN BOVINE IVP BLASTOCYSTS

2010 ◽  
Vol 22 (1) ◽  
pp. 233
Author(s):  
L. V. M. Gulart ◽  
L. Gabriel ◽  
L. P. Salles ◽  
G. R. Gamas ◽  
D. K. Souza ◽  
...  
Keyword(s):  
Internal Control ◽  
Connexin 43 ◽  
Calf Serum ◽  
Defined Medium ◽  
Culture Conditions ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Blastocyst Rate

FSH at low concentrations affect embryo production. In vitro culture conditions also affect embryo production and embryonic expression of genes and alter oocyte competence to produce embryos. The search for better and less variable culture conditions simulating those in vivo has led to the development of several systems of oocyte in vitro maturation culture. To compare the efficiency of the systems of MIV we utilized 4 groups: (1) TCM-199 control; (2) α-minimal essential medium (MEM); 3) α-MEM + 1 ng of FSH; 4) α-MEM+ 10 ng of FSH. The medium of Group 1 is non-defined by the presence of fetal calf serum (10%). Groups 2, 3, and 4 are defined and polyvinyl alcohol (1%) was used as a macromolecule. Porcine FSH (1 IU mg-1) was used at 1 and 10 ng mL-1 and at 100 ng in defined and non-defined medium, respectively. Bovine ovaries were collected at an abbatoir. Oocytes (n = 1718) with homogeneous cytoplasm and with more than 3 layers of granulosa cells were used. Mature oocytes from the 4 treatments (11 replicates of each treatment) were inseminated with frozen-thawed, motile sperm separated by Percoll, using Sperm TALP HEPES medium. Presumptive zygotes with up to 2 or 3 layers of cumulus cells were cultured in 50-mL drops of SOF medium, supplemented with 10% FCS and 1 mg mL-1 BSA under mineral oil in a humid 5% CO2 atmosphere at 38.5°C after. Cleavage rate was evaluated 72 h post-insemination (hpi), and blastocyst rate was evaluated 168-192 hpi. Cleavage and blastocyst rates were calculated on the basis of number of presumptive zygotes. The expression of the following genes (Bax, Bcl-2, and conexin 43) was evaluated in blastocysts by RT-PCR. One-way ANOVA was used to compare blastocyst number. There was no difference in the proportion of embryos with more than 8 blastomeres in all groups tested, indicating that the rate of development during the first 72 hpi was similar for oocytes matured in chemically defined medium and for oocytes matured in medium containing serum. Bax is a pro-apoptotic marker and Bcl-2 an antiapoptotic marker. Connexin 43 (Cx43) may be a marker of embryo competence. Glyceraldehyde 3-phosphate dehydrogenase was used as internal control. The Bax gene was not expressed in any group. The Bcl-2 and Cx43 genes were expressed, mainly in the α-MEM 10. Although no differences were observed in blastocyst rate among the groups (30% to 40%), the strong expression of Bcl-2 and of Cx43 on the group containing 10 ng mL-1 of FSH may indicate that FSH could improve embryo quality under defined conditions. The authors thank FAP-DF, CNPq, FUNPE, FINATEC, CAPES, and Biovitro Tecnologia de Embrioes Ltda, for laboratory assistance and grants, and Frigorifico Ponte Alta, Brasília-DF, for supplying bovine ovaries.

Production of in vitro bovine embryos supplemented with l-carnitine in different oxygen tensions and the relation to nitric oxide

Zygote ◽  
2020 ◽  
Vol 28 (5) ◽  
pp. 403-408
Author(s):  
Daniela Moraes Pereira ◽  
Christopher Junior Tavares Cardoso ◽  
Wilian Aparecido Leite da Silva ◽  
Mirela Brochado Souza-Cáceres ◽  
Mariana Santos ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryo Development ◽  
Calf Serum ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Low Oxygen ◽  
Cleavage Rate ◽  
Treatment Groups ◽  
Oxygen Tensions

SummaryThe aim of this study was to evaluate the production of bovine embryos in vitro when supplemented with l-carnitine for 24 h beginning on day 5 (d 5) under two different oxygen tensions (20% or 5%) and the relationship of nitric oxide (NO) in in vitro culture (IVC) medium to embryo development. Cumulus–oocyte complexes (COC; n = 837) were matured in vitro for 24 h and fertilization was performed for 18 h. Zygotes were cultured in vitro for 9 days after in vitro fertilization in synthetic oviductal fluid (SOF) medium with 5% fetal calf serum. At d 5 the plates were assigned to one of four treatment groups: high (20%) or low (5%) O2 tension either with or without the addition of 3.03 mM l-carnitine (High-Cont, High-Lcar, Low-Cont, Low-Lcar). The concentration of NO in the culture medium was evaluated on d 5, d 6 and d 9. On d 7, parts of the embryos were submitted for evaluation of intracellular lipid droplets. The cleavage rate was similar (P > 0.05) between high and low O2 tension and the blastocyst rate was similar in all conditions evaluated. The hatching rate was higher (P < 0.05) for Low-Cont. The NO concentration was higher at d 9 under low O2 tension (P < 0.1). The addition of 3.03 mM l-carnitine between d 5 and d 6 of IVC was not efficient in reducing cytoplasmic lipid content of bovine embryos. Additionally, IVC at a low oxygen tension without l-carnitine promoted better conditions for embryo development. A higher concentration of NO in medium was observed under low O2 tension.

Efficient one-step direct transfer to recipients of thawed bovine embryos cultured in vitro and frozen in chemically defined medium

2020 ◽  
Vol 146 ◽  
pp. 39-47 ◽  
Author(s):  
Enrique Gómez ◽  
Susana Carrocera ◽  
David Martín ◽  
Juan José Pérez-Jánez ◽  
Javier Prendes ◽  
...  
Keyword(s):  
Defined Medium ◽  
Direct Transfer ◽  
Chemically Defined Medium ◽  
Bovine Embryos ◽  
One Step

179 EFFECT OF SERUM DURING CULTURE ON DAY 14 ELONGATED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 191 ◽  
Author(s):  
J. Angulo ◽  
G. T. Gentry ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Expression Levels ◽  
The Mean

It has been reported that the addition of serum to embryo culture media alters gene expression and triggers the development of large offspring syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and in vivo-derived (IVD) embryos and to determine the effects of serum on the length of elongated embryos. Abattoir-derived oocytes were obtained from a commercial provider and fertilized at 24 h of maturation with semen from a bull previously used for IVF. At 18 h post-insemination (hpi), embryos were denuded and groups of 15 presumptive zygotes were cultured in 30-μL drops of modified SOF medium with amino acids and 6 mg mL–1 of BSA (mSOFaa). At 72 hpi, cleavage rate was assessed and embryos were randomly allocated into 2 treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed and recorded. Blastocysts (n = 5 to 10) from each treatment were transferred into synchronized recipients, and Day 14 embryos were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at –80°C in a minimal volume of PBS + 0.1% polyvinyl alcohol. Messenger RNA was isolated using a Dynabeads mRNA Direct Kit™ (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA). Quantitative PCR was performed to determine the transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R, and GAPDH for each sample. The GAPDH was used as a reference gene, and gene expression was calculated as a ratio of expression levels between each gene of interest and GAPDH. Expression levels for each gene were determined from standard curves generated by serial dilutions of PCR amplicons starting with 0.4 pg/reaction. Blastocyst development rates were higher in embryos cultured with serum compared with the nonserum treatment (14.9 and 7.4% respectively; chi-square, P < 0.001). Lengths of elongated embryos from the serum (3395.3 ± 414.7 μm) and nonserum (2784 ± 741.8 μm) culture treatments differed from the IVD (6297.7 ± 677.2 μm) treatment (mean ± SE; ANOVA, P < 0.0052). There were no differences in the mean expression levels for COX6A, IFNT1a, PLAC8, and IGF2R across treatment groups, but in the serum treatment, 3 out 11 overexpressed IFNT1a, 4 out of 11 overexpressed IGF2R, and 2 out of 11 overexpressed PLAC8, defined as being 2 standard deviations above the mean of the IVD treatment for each respective gene. In the in vitro-produced nonserum and IVD treatments, overexpression by this definition was not observed. Although mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R, and PLAC8 was observed in some embryos cultured with serum, but not in embryos cultured without serum or IVD embryos.

208 EFFECT OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR ON BLASTOCYST DEVELOPMENT AND POST-TRANSFER SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 183 ◽  
Author(s):  
B. Loureiro ◽  
L. Bonilla ◽  
G. Entrican ◽  
P. J. Hansen
Keyword(s):  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cho Cell ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Cell Supernatant

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that has been implicated in preimplantation embryo development. Granulocyte-macrophage-CSF improves the proportion of bovine embryos that become blastocysts in vitro (Moraes and Hansen 1997 Biol. Reprod. 57, 1060–1065) and increases blastocyst cell numbers in mice (Robertson et al. 2001 Biol. Reprod. 64, 1206–1215). The long-term goal of the present research was to evaluate the effects of GM-CSF on post-transfer survival of bovine embryos. The experiments used recombinant ovine GM-CSF produced in transfected Chinese hamster ovary (CHO) cells or an equivalent volume of cytokine-free CHO cell supernatant (control). The objective of the first study was to evaluate the effects of GM-CSF on post-transfer survival. Embryos were cultured with 10 ng mL–1 of either GM-CSF or cytokine-free CHO cell supernatant added to culture medium at Day 1 after insemination. Embryos were transferred at Day 7 to lactating dairy cows according to a timed embryo transfer protocol. Pregnancy was evaluated at approximately Day 45 of gestation. There was no significant difference in the proportion of embryos becoming blastocysts at Day 7 after insemination (34.8 v. 37.5% for the control and GM-CSF; SEM = 2.4%). There was also no difference in pregnancy rates between cows receiving control embryos (6/24; 25%) and cows receiving embryos treated with GM-CSF (8/35; 23%). A second study determined the effects of various concentrations of GM-CSF on the development of in vitro-produced embryos to the blastocyst stage. Embryos were cultured in 5% (v/v) oxygen (low oxygen) or atmospheric oxygen (21%, w/v; high oxygen) in the presence of 0, 1, 10, or 100 ng mL–1 of GM-CSF or an equivalent volume of cytokine-free CHO cell supernatant (control). The GM-CSF was added on either Day 1 or Day 5 after insemination. Cleavage rate was accessed on Day 3 after insemination. Stage of development was recorded at Day 7 and Day 8 after insemination. There was no effect of GM-CSF on cleavage rate. Addition of GM-CSF at Day 5 to embryos cultured in low or high oxygen increased the percentage of oocytes that became blastocysts at Day 7 (P < 0.01) and Day 8 (P < 0.01), but addition at Day 1 did not have a significant effect on blastocyst development. The greatest effects of GM-CSF occurred at a concentration of 10 ng mL–1. At this concentration, least squares means for the percentage of oocytes that became blastocysts at Day 7 were 13.9 v. 21.6% (control v. GM-CSF) when GM-CSF was added at Day 5, and 19.5 v. 21.5% when GM-CSF was added at Day 1. The percentage of blastocysts at Day 8 was 20.9 v. 28.7% when GM-CSF was added at Day 5, and 26.7 v. 27.5% when GM-CSF was added at Day 1. In conclusion, GM-CSF can affect the competence of embryos to develop to the blastocyst stage, but at the concentrations and times given, there was no evidence that GM-CSF enhanced embryo survival after transfer.

Effects of activin A and follistatin on developmental kinetics of bovine embryos: cinematographic analysis in a chemically defined medium

Reproduction ◽  
2000 ◽  
pp. 119-125 ◽  
Author(s):  
K Yoshioka ◽  
C Suzuki ◽  
S Iwamura
Keyword(s):  
Defined Medium ◽  
Activin A ◽  
Lag Phase ◽  
Chemically Defined Medium ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oviduct Fluid ◽  
Kinetics Of ◽  
Number Of Cells

The effects of recombinant human activin A and follistatin on the developmental kinetics of bovine presumptive zygotes matured and fertilized in vitro using time-lapse cinematography were investigated. The presumptive zygotes were cultured for 9 days in a chemically defined medium (modified synthetic oviduct fluid, control) and modified synthetic oviduct fluid supplemented with activin A or follistatin. Development under cine-recording conditions was similar to that in an incubator. Addition of activin A to modified synthetic oviduct fluid increased, while addition of follistatin decreased, the percentage of zygotes that developed to morulae and blastocysts. Follistatin significantly prolonged the timing of development to the 9-16-cell stage compared with the control and activin A media. Activin A significantly shortened the duration of the third cell cycle compared with the control, but follistatin significantly prolonged the fourth cell cycle compared with the control and activin A. Developmental arrest ('lag-phase') during the 4-8-cell stage was observed in 95% of embryos developed to more than the 9-16-cell stage in all treatments. The greater the number of cells at the onset of the lag-phase, the earlier the onset of the phase and the shorter the duration of the phase, the further embryos were able to develop by day 9 in all treatments. The number of cells at the onset of the lag-phase in the medium containing activin A was significantly higher than it was in control or follistatin-containing media. Moreover, activin A significantly shortened the duration of the lag-phase compared with follistatin. The present results indicate that activin A may enhance in vitro development of bovine embryos by improving developmental kinetics, especially by increasing the number of cells at the onset of the lag-phase and shortening the duration of this phase.

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