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.

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.

scholarly journals Post-hatching development of bovine embryos in vitro: the effects of tunnel preparation and gender

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 123-134 ◽  
Author(s):  
Grazieli Marinheiro Machado ◽  
Ester Siqueira Caixeta ◽  
Carolina Madeira Lucci ◽  
Rodolfo Rumpf ◽  
Maurício Machaim Franco ◽  
...  
Keyword(s):  
Gene Expression ◽  
Morphological Characteristics ◽  
Tunnel Construction ◽  
Agarose Gel ◽  
Male And Female ◽  
Embryo Size ◽  
And Gender ◽  
Phosphate Buffered Saline ◽  
Kinetics Of

SummaryThe objective of this study was to compare morphological characteristics, kinetics of development, and gene expression of male and female IVP embryos that were cultured until day (D)15 (fertilization = D0), using either phosphate-buffered saline (PBS) or Milli-Q water (MQW) to dilute the agarose gel used for tunnel construction. On D11, embryos (n = 286) were placed in agarose gel tunnels diluted in PBS and MQW. Embryos were evaluated for morphology, and embryo size was recorded on D11, D12.5, D14 and D15. Then, embryos were sexed and used for gene expression analyses (G6PD, GLUT1, GLUT3, PGK1, PLAC8, KRT8, HSF1 and IFNT). The percentage of elongated embryos at D15 was higher (p < 0.05) in the PBS (54%) than in the MQW (42%) gel. However, embryos produced in MQW were bigger (p < 0.05) and had a lower expression of GLUT1 (p = 0.08) than those cultured in PBS. There was a higher proportion of male than female embryos at D15 in both treatments, MQW (65% vs. 35%; p < 0.05) and PBS (67% vs. 33%; p < 0.05); however, embryo size was not significantly different between genders. Moreover, D15 female embryos had greater expression of G6PD (p = 0.05) and KRT8 (p = 0.03) than male embryos. In conclusion, the diluent used for tunnel construction affected embryo development in the post-hatching development (PHD) system, and the use of MQW was the most indicative measure for the evaluation of embryo quality. Male and female embryos cultured from D11 to D15, either in an MQW or PBS agarose gel, demonstrated similar development but different gene expression.

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.

168 CORRELATION OF DEVELOPMENT KINETICS AND SEX OF IN VITRO-PRODUCED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 196
Author(s):  
A. R. Buzzo ◽  
A. R. Pupulim ◽  
J. Mazucheli ◽  
F. V. Meirelles ◽  
I. P. Emanuelli
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Proteinase K ◽  
Bovine Embryos ◽  
Male And Female ◽  
Stage Of Development ◽  
Males And Females ◽  
Kinetics Of

Approaches to improve the culture medium for in vitro production (IVP) of bovine embryos have been continuous because of the high commercial demand and a portion of this attempts the production of female cattle (dairy cows and stud cattle). However, in some embryonic in vitro culture systems, the development kinetics is faster in male than in female embryos (Avery 1992 Mol. Reprod. Dev. 32, 265–70; Xu 1992 Mol. Reprod. Dev. 31, 249–50). The aim of this work was to relate the kinetics of blastocyst expansion with the production rates of male and female embryos. Cumulus–oocyte complexes (n = 917; classes I and II) of cows from a slaughterhouse were matured with TCM-199 bicarbonate and 10% FCS (38.5°C, 5% CO2) for 24 h and fertilized with frozen-thawed semen in TALP-IVF medium for 18 h. Presumptive zygotes were culture in SOF medium supplemented with 10% FSB (5% O2, 38.5°C). Seven days after IVF, embryos were divided in 2 groups according to their kinetic stage of development: nonexpanded blastocysts (n = 175), or hatched and expanded blastocysts (n = 146). Hence, embryos were individually frozen in LN and stored in cryotubes. After thawing, Proteinase K (16 mg mL–1) was added to each tube and the tubes were incubated for 60 min at 37°C. Proteinase was denatured at 98°C for 10 min and the contents of each tube were divided into 2 samples (A and B) and subjected to the PCR technique. Two pairs of primers for the specific sequence of the Y chromosome were used to amplify the sequence of 210 and 250 bp for the male bovine and 1 pair of primers was used for the autosomal bovine sequence with a 280-bp fragment. Female embryos with a 280-bp product were observed in sample A and none were observed in sample B. The presence of 2 amplicons (280 and 210 bp) in sample A and 1 amplicon of 250 bp in sample B indicated that the embryo was male. A chi-square test was used to evaluate homogeneity. An analysis of the percentage of males and females between the experimental groups was performed by logistic regression and significance was considered when P < 0.05. There was no difference in the proportions of males and females in the nonexpanded blastocyst group (49.71 and 50.29%; P > 0.05). In the hatched and expanded blastocyst group, the proportion of males (65.75%) was statistically different from the proportion of females (34.25%); that is, the chance of the embryo being male was twice as high (P < 0.0038). These results suggest that there is a difference in the kinetics of embryo development between male and female embryos and that blastocyst expansion can point that out. In vitro culture media with FCS support the development of expanded male blastocysts. Further research in culture medium modifications (FCS, the energy source, amino acids and others) are needed to respond to the trend in the production of sex-defined embryos.

scholarly journals Difference in Developmental Kinetics of Y-Specific Monoclonal Antibody Sorted Male and Female In Vitro Produced Bovine Embryos

2019 ◽  
Vol 21 (1) ◽  
pp. 244 ◽  
Author(s):  
Tabinda Sidrat ◽  
Rami Kong ◽  
Abdul Khan ◽  
Muhammad Idrees ◽  
Lianguang Xu ◽  
...  
Keyword(s):  
Oxygen Species ◽  
Bovine Embryos ◽  
Male And Female ◽  
Endogenous Factors ◽  
Epigenetic Events ◽  
Mitochondrial Distribution ◽  
Growth Differences ◽  
Kinetics Of ◽  
Implantation Period

Sex-related growth differences between male and female embryos remain an attractive subject for reproductive biologists. This study aimed to investigate the endogenous factors that play a crucial role in the pace of early development between male and female bovine embryos. Using sex pre-selected semen by Y-specific monoclonal antibodies for the production of bovine embryos, we characterized the critical endogenous factors that are responsible for creating the development differences, especially during the pre-implantation period between male and female embryos. Our results showed that at day seven, (57.8%) Y-sperm sorted in vitro cultured embryos reached the expanded blastocyst (BL) stage, whereas the X-sperm sorted group were only 25%. Y-BLs showed higher mRNA abundance of pluripotency and developmental competency regulators, such as Oct4 and IGF1-R. Interestingly, Y-sperm sorted BLs had a homogeneous mitochondrial distribution pattern, higher mitochondrial membrane potential (∆Ѱm), efficient OXPHOS (oxidative phosphorylation) system and well-encountered production of ROS (reactive oxygen species) level. Moreover, Y-blastocysts (BLs) showed less utilization of glucose metabolism relative to the X-BLs group. Importantly, both sexes showed differences in the timing of epigenetic events. All these factors directly or indirectly orchestrate the whole embryonic progression and may help in the faster and better quality yield of BL in the Y-sperm sorted group compared to the X counterpart group.

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos

2001 ◽  
Vol 55 (5) ◽  
pp. 1117-1126 ◽  
Author(s):  
A. Gutiérrez-Adán ◽  
P. Lonergan ◽  
D. Rizos ◽  
F.A. Ward ◽  
M.P. Boland ◽  
...  
Keyword(s):  
Sex Ratio ◽  
In Vitro Culture ◽  
Culture System ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
In Vitro Culture System ◽  
Kinetics Of

125 EFFECT OF BOVINE OVIDUCTAL FLUID ON DEVELOPMENT AND QUALITY OF IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 154
Author(s):  
R. Lopera ◽  
M. Hamdi ◽  
V. Maillo ◽  
C. Nunez ◽  
P. Coy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Count ◽  
Embryo Culture ◽  
Cell Number ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Low Concentrations ◽  
Fluid Supplementation

Although fertilization and early embryonic development take place in the oviduct, the consequences of tubal fluid supplementation during in vitro embryo culture have not been explored. The aim of this study was to evaluate the effect of bovine oviducal fluid (bOF) supplementation during in vitro embryo culture of bovine embryos on their development and quality. The bOF was aspirated from oviducts of slaughtered heifers in the early luteal phase. In vitro-produced zygotes were cultured in SOF (C–; n = 927) or SOF + 5% FCS (C+; n = 872) or in SOF + bOF (n ~900/group) at different concentrations (0.62, 1.25, 2.5, 5, 10, or 25%) in 10 replicates. Blastocysts on Days 7/8 were used for quality evaluation through (a) differential cell count, (b) survival after vitrification/warming, and (c) gene expression (qRT-PCR). One-way ANOVA (development and quality) and t-test (cell count) were used for statistical analysis. The bOF concentrations over 5% were detrimental for blastocysts development (<7% at Day 7) and were discarded. Embryos cultured in absence of FCS exhibited a delay in the kinetics of blastocyst development; at Day 7, the groups cultured without FCS (bOF 0.62–2.5% and C–) had fewer blastocysts (range:12.0 ± 1.7 to 17.4 ± 1.5%) compared with C+ group (22.9 ± 1.2%). However, blastocyst yield at Day 9 was similar in 0.62 and 1.25 bOF groups (27.5 ± 1.7% and 27.5 ± 1.2%, respectively) compared with C+ (27.7 ± 1.0%) and significantly higher than 2.5 bOF (22.7 ± 1.5%) and C– (21.5 ± 1.4%; P < 0.05). In terms of blastocyst quality, 48 h after vitrification/warming, embryos from bOF 1.25%, 0.62%, and C– groups survived significantly higher than C+ (61.3 ± 2.1; 61.6 ± 4.1; 59.3 ± 3.2; and 30.3 ± 2.5, respectively; P < 0.05). This difference was even higher at 72 h (53.6 ± 1.7; 57.7 ± 3.8; 56.1 ± 2.9; and 25.9 ± 2.3%, respectively; P < 0.05). Total cell number of the embryos cultured in bOF 1.25 and 0.62% groups were significantly higher than C+ and C– (165.1 ± 4.7 and 156.2 ± 4.2 v. 143.1 ± 4.9 and 127.7 ± 4.9, respectively), which was associated with an increased TE cell number in 1.25 and 0.62% bOF groups (119.9 ± 3.7 and 127.0 ± 4.5, respectively). Culture with 2.5% bOF had no effect on either blastocyst yield or quality. Gene expression analysis was performed in 1.25% bOF, C–, and C+ groups. The result suggested a higher glucose (SCL2A1) and lipid (CYP51 and FADS1) metabolism in those groups cultured without serum. Gene DNMT3A and the imprinted gene IGF2R were also significantly up-regulated in bOF and C– compared with C+ (P < 0.05). Interestingly, AQP3, a gene positively correlated with survival after vitrification, was significantly up-regulated in bOF compared with C– and C+ (P < 0.05). In conclusion, in vitro culture with low concentrations of bOF has a positive effect in development and quality of bovine embryos cultured in absence of FCS. Funded by the Spanish Ministry of Science and Innovation (AGL2012–37510).

135 RECOMBINANT BOVINE SOMATOTROPIN ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 181
Author(s):  
L. Berté ◽  
L. Vasconcelos ◽  
L. Hatamoto-Zervoudakis ◽  
W. Yamazaki ◽  
L. Yamazaki ◽  
...  
Keyword(s):  
Bovine Embryos ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Recombinant Bovine Somatotropin ◽  
Group 4 ◽  
Group 3 ◽  
Group 2 ◽  
Vitro Production

Bovine growth hormone (bGH) has been used to improve the results for in vitro production of bovine embryos. Inclusion of bGH in the maturation medium increases both rate of cleavage and frequency of blastocyst development. Thus, the purpose of the present study was to evaluate the effect of recombinant bovine somatotropin (rBST) on cleavage and blastocyst development of bovine embryos when included during in vitro maturation (IVM) only (Group 1), during both IVM and in vitro culture (IVC; Group 2), during IVC only (Group 3), or not included during either IVM or IVC (Group 4). Specifically, in Group 1, oocytes were matured in TCM 199 (Earle's salts) supplemented with 10% FCS, LH, FSH, oestradiol, and amikacin (IVM medium), plus 100 ng mL–1 of rBST and cultured in SOFaaci supplemented with essential amino acids, tri-sodium citrate, myo-inositol, and 5% FBS. In group 2, oocytes were matured in IVM medium containing 100 ng mL–1 of rBST and cultured in SOFaaci supplemented with essential amino acid, tri-sodium citrate, myo-inositol, 5% FBS; on Day 5, rBST (50 ng mL–1) was added. In Group 3, oocytes were matured in IVM medium without rBST; on Day 5, rBST (50 ng mL–1) was added. Group 4 was the control, without rBST supplementation. The treatment groups were analysed using the SAS® (SAS Institute Inc., Cary, NC, USA) in a completely randomised design (P < 0.05). Somatotropin has receptors in cumulus cells and in the zona pellucida acting directly in the oocyte; however, the increase in cleavage rate seen in previous studies after rBST treatment was not observed in the present study. Supplementation of culture medium with rBST during Day 2 to 6 of IVC has been shown to increase the number of trophoblast and subsequent pregnancy rate following transfer. However, in the present study, addition of 50 and 100 ng mL–1 of rBST to the maturation or culture medium did not affect the cleavage rate of embryos and blastocyst production. Table 1.Analysis of the meaning and percentages related to cleavage and production of embryos

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