Intrafollicular testosterone concentration and sex ratio in individually cultured bovine embryos

Recent studies have suggested a relationship between bovine follicular fluid testosterone concentration and the likelihood of the oocyte being fertilised by an X- or Y-bearing spermatozoon; however, this theory has been challenged. To further test this hypothesis, follicles were dissected from the ovaries of slaughtered heifers, measured and carefully ruptured. The cumulus–oocyte complex (COC) was removed and the follicular fluid collected and testosterone concentration determined by radioimmunoassay. COCs were matured, fertilised and cultured in an individually identifiable manner; all cleaved embryos (2- to 4-cell stage, n = 164) had their sex determined by PCR. Testosterone concentrations were positively skewed. There was no significant difference between follicular fluid testosterone concentrations in male and female embryos (mean ± s.e.m. 51.5 ± 5.59 and 49.5 ± 7.42 ng mL–1, respectively). Linear, quadratic and cubic logistical regression showed that follicular testosterone concentration could not reliably predict the sex of the embryo with odds ratios of 1.001, 1.013 and 1.066, repectively, and coefficient of determination (R2) values of 0.0003, 0.0126 and 0.0567, respectively. Follicular size and testosterone concentration were not related (R2 = 0.087). Finally, follicular size had no influence on embryo sex determination (P = 0.70). In conclusion, under the conditions of the present study, the likelihood of an oocyte being fertilised by an X- or Y-bearing spermatozoon was not affected by the size of the follicle from which it was derived, nor by the testosterone concentration in the follicular fluid.

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78 NONINVASIVE CELL LINEAGE TRACING IN BOVINE EMBRYOS FROM 2-CELL STAGE UP TO BLASTOCYST STAGE

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab78 ◽

2015 ◽

Vol 27 (1) ◽

pp. 132

Author(s):

L. P. Sepulveda-Rincon ◽

D. Dube ◽

P. Adenot ◽

L. Laffont ◽

S. Ruffini ◽

...

Keyword(s):

Cell Mass ◽

Cell Lineage ◽

Blastocyst Stage ◽

Lineage Tracing ◽

Specific Cell ◽

Bovine Embryos ◽

Cell Stage ◽

Daughter Cells ◽

Significant Difference ◽

Allocation Patterns

The first lineage specification occurs during pre-implantation mammalian development. At the blastocyst stage, 2 cell lineages can be distinguished: the inner cell mass (ICM) and the trophectoderm (TE). The exact timing when embryo cells are skewed to these lineages is not clearly determined in mammalian species. In murine embryos, it has been suggested that the first cleavage plane might be related to the embryonic-abembryonic (Em-Ab) axis at blastocyst stage. Thus, the daughter cells of the 2-cell embryo might already be predisposed to a specific cell lineage further on development. The objective of the present study was to observe how the first cleavage in bovine embryos may be related to cell lineage allocation at the blastocyst stage, using a noninvasive tracing approach. Bovine oocytes were harvested, in vitro matured, and fertilised. At the 2-cell stage, embryos were injected in one blastomere with the membrane tracer DiI. At the blastocyst stage, embryos (n = 346) were classified as orthogonal when the Em-Ab axis was orthogonally divided by the borderline between labelled and non-labelled cells; as deviant if the borderline was overlapping the Em-Ab axis; and as random when the labelled and non-labelled cells were randomly distributed. Total cell count (TCC) and the ICM/TE ratio was allowed by DNA staining with 4′,6-diamidino-2-phenylindole (DAPI) and by immunostaining of the ICM with Sox2 antibody. Analysis of variance was performed by one-way ANOVA employing IBM SPSS v21 (SPSS Inc., Chicago, IL, USA) to determine any difference between the cell lineage allocation patterns, TCC, and the ICM/TE ratio. P-values = 0.05 were considered significant. All values are reported as mean ± standard error of mean. Within 40 repetitions, the blastocyst classification was as follows: orthogonal 14.9% (±2.32, n = 56), deviant 22.2% (±2.58, n = 80), and random 62.9% (±2.64, n = 210). A significant difference was found in the incidence between the random group against the orthogonal and deviant, but not between the latter two. Regarding TCC, a significant difference was observed only between the orthogonal (99.6 ± 11.7 cells, n = 15) and deviant (135 ± 7.3 cells, n = 25) groups, but not with random embryos (116 ± 5.5 cells, n = 42). Finally, no significant difference was found among the groups concerning the ICM/TE ratio (0.43 ± 0.07 for orthogonal, n = 7; 0.54 ± 0.06 for deviant, n = 14; and 0.40 ± 0.03 for random embryos, n = 26). In conclusion, bovine embryos present a marked tendency for a random distribution of the daughter cells derived from the 2-cell blastomeres. However, around 37% of the blastocysts present a patterned cell division, where the daughter cells remain together through pre-implantation development. The effect of these cell lineage allocation patterns on implantation and further embryo development needs to be addressed.The authors acknowledge Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73) and CONACyT Mexico for funding.

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142 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN KSOM, CR1AA, OR KSOM/CR1aa

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab142 ◽

2005 ◽

Vol 17 (2) ◽

pp. 221 ◽

Cited By ~ 1

Author(s):

M.R.B. Mello ◽

C.E. Ferguson ◽

A.S. Lima ◽

M.B. Wheeler

Keyword(s):

Embryo Culture ◽

Cumulus Cells ◽

Bovine Embryos ◽

In Vitro Production ◽

Cell Stage ◽

In Vitro Development ◽

Significant Difference ◽

Developmental Rates ◽

Vitro Development

In vitro embryo culture is an important step of in vitro production of bovine embryos. It has been shown that IVF-derived bovine embryos cultured in KSOM or CR1aa have high development rates. In our laboratory, we have observed that 8-cell embryos are morphologically superior when embryos are cultured in KSOM whereas blastocysts are morphologically superior when embryos are cultured in CR1aa. Based on these observations, we hypothesized that development of IVF-derived bovine embryos can be improved by sequential use of these media (KSOM and CR1aa). The aim of this experiment was to compare the in vitro development of bovine embryos cultured in KSOM, CR1aa or KSOM/CR1aa supplemented with BSA at Day 0 and BSA and FBS at Day 3. In order to accomplish the sequential culture, fertilized oocytes where cultured in KSOM to the 8-cell stage and then transferred to CR1aa for further development. Oocytes were purchased from Bomed (Madison, WI, USA), and after 22 hours of maturation were fertilized with frozen-thawed semen for 5 hours at 39°C in 5% CO2. After fertilization, the presumptive zygotes were denuded from cumulus cells by votexing and were randomly allotted to one of 3 treatments: (1) cultured only in KSOM (n = 110), (2) cultured only in CR1aa (n = 102), and (3) cultured in KSOM in the first 3 days and then in CR1aa from Day 3 to Day 9 (n = 110). The embryo culture was carried out in 50-μL droplets of medium that were placed in an airtight modular incubator filled with 5% CO2, 5% O2 and 90% N2. The embryos were evaluated on Days 6 to 9 post insemination. All embryo developmental rates were calculated from presumptive zygotes. The Day 6 morula rates were 52%, 40%, and 47% for KSOM, CR1aa, and KSOM/CR1aa, respectively. The Day 7 blastocyst rates for KSOM (40%), CR1aa (25%), and KSOM/CR1aa (30%) were not significantly different; however, Day 9 hatched blastocyst rates were significantly higher (P < 0.05) for KSOM (22%) compared to CR1aa (9%) but not different from KSOM/CR1aa (14%). Regarding embryo quality, Day 7 transferable embryos rates (Grade 1 and Grade 2) were 35%, 25%, and 30%, respectively for KSOM, CR1aa, and KSOM/CR1aa; however, no significant difference was observed. These results indicate that IVF-derived bovine embryos can develop in KSOM, CR1aa, or KSOM/CR1aa with no significant difference among morula, blastocyst and hatched blastocyst rates. However, the combination of KSOM and CR1aa during in vitro culture did not decrease the morula and blastocyst rates.

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218 GENE AND PROTEIN EXPRESSION OF ANTIOXIDANT ENZYMES IN BOVINE EMBRYOS EXPOSED TO HEAT SHOCK

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab218 ◽

2009 ◽

Vol 21 (1) ◽

pp. 207

Author(s):

M. Sakatani ◽

K. Yamanaka ◽

M. Takahashi

Keyword(s):

Antioxidant Enzymes ◽

Heat Shock ◽

Protein Expression ◽

Early Stage ◽

Bovine Embryos ◽

Cell Stage ◽

Expression Levels ◽

Gene And Protein Expression ◽

Significant Difference

In a previous study, we reported that 8-cell-stage embryos exposed to a temperature of 41°C for 6 h had significantly increased embryonic mortality and intracellular reactive oxygen species (ROS). There have been some reports that ROS regulates the expression of genes encoding antioxidant enzymes in culture cells. In this study, we investigated the gene and protein expression of antioxidant enzymes in bovine 8-cell-stage embryos exposed to heat shock. In vitro-produced bovine embryos were used for the experiment. Embryos were cultured with CR1aa + 5% FCS at 38.5°C in 5% CO2 and 5% O2. On Day 2 after fertilization, 8-cell-stage embryos were exposed to heat shock at 41°C in 5% CO2 and 5% O2 for 6 h (HS). Eight-cell-stage embryos cultured at 38.5°C in 5% CO2 and 5% O2 were sampled at the same collection time as controls. After HS, 20 embryos were immediately collected for gene expression analysis. Expression of heat shock protein 70 (HSP70), CuZn-containing superoxide dismutase (SOD), catalase (CAT), and glutathione peroxide (GPx) genes was examined by real-time polymerase chain reaction. Twenty embryos were also collected after 3 h of HS (3 h) and at 18 h after HS (18 h) to evaluate the expression of proteins. Expression of HSP70, SOD, and CAT proteins was examined by Western blotting. Both the gene and protein expression levels of HS groups were normalized to those of the controls to obtain the relative expression levels. All results were analyzed by Student’s t-test. Expression of the HSP70 gene significantly increased in HS embryos (P < 0.05). Expression of the SOD and CAT genes tended to increase in HS embryos (P < 0.07), but there were no significant differences in expression of the GPx gene. There was no significant difference in protein expression in all the antioxidant enzymes in 3-h-sampled embryos. Expression of the HSP70 protein increased significantly in heat-shocked embryos sampled at 18 h (P < 0.05). These results indicate that expression of antioxidant enzymes was not greatly affected in 8-cell-stage embryos exposed to HS. Thus, these results suggest the possibility that the early-stage embryos were stressed and damaged from heat shock because of their poor antioxidative potency. Table 1.Gene and protein expression of embryos This work was supported by KAKENHI [16780209, Grant-in-Aid for Young Scientists (B)].

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145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab145 ◽

2010 ◽

Vol 22 (1) ◽

pp. 231

Author(s):

C. Y. Choe ◽

S. R. Cho ◽

J. K. Son ◽

S. H. Choi ◽

C. Y. Cho ◽

...

Keyword(s):

Oxygen Consumption ◽

Embryo Quality ◽

Blastocyst Stage ◽

Bovine Embryo ◽

Bovine Embryos ◽

Cell Stage ◽

Significant Difference ◽

Morula Stage

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.

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Quantitative Breast Density in Contrast-Enhanced Mammography

Journal of Clinical Medicine ◽

10.3390/jcm10153309 ◽

2021 ◽

Vol 10 (15) ◽

pp. 3309

Author(s):

Gisella Gennaro ◽

Melissa L. Hill ◽

Elisabetta Bezzon ◽

Francesca Caumo

Keyword(s):

Breast Density ◽

Potential Role ◽

Multiple Correlation Coefficient ◽

Low Energy ◽

Coefficient Of Determination ◽

Contrast Enhanced ◽

Increased Risk ◽

Significant Difference ◽

Automatic Software ◽

The Relationship

Contrast-enhanced mammography (CEM) demonstrates a potential role in personalized screening models, in particular for women at increased risk and women with dense breasts. In this study, volumetric breast density (VBD) measured in CEM images was compared with VBD obtained from digital mammography (DM) or tomosynthesis (DBT) images. A total of 150 women who underwent CEM between March 2019 and December 2020, having at least a DM/DBT study performed before/after CEM, were included. Low-energy CEM (LE-CEM) and DM/DBT images were processed with automatic software to obtain the VBD. VBDs from the paired datasets were compared by Wilcoxon tests. A multivariate regression model was applied to analyze the relationship between VBD differences and multiple independent variables certainly or potentially affecting VBD. Median VBD was comparable for LE-CEM and DM/DBT (12.73% vs. 12.39%), not evidencing any statistically significant difference (p = 0.5855). VBD differences between LE-CEM and DM were associated with significant differences of glandular volume, breast thickness, compression force and pressure, contact area, and nipple-to-posterior-edge distance, i.e., variables reflecting differences in breast positioning (coefficient of determination 0.6023; multiple correlation coefficient 0.7761). Volumetric breast density was obtained from low-energy contrast-enhanced spectral mammography and was not significantly different from volumetric breast density measured from standard mammograms.

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Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

Scientific Reports ◽

10.1038/s41598-021-91158-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Karina Cañón-Beltrán ◽

Yulia N. Cajas ◽

Serafín Peréz-Cerezales ◽

Claudia L. V. Leal ◽

Ekaitz Agirregoitia ◽

...

Keyword(s):

Embryonic Development ◽

Signaling Pathway ◽

Akt Signaling ◽

Mitochondrial Activity ◽

Bovine Embryos ◽

Akt Signaling Pathway ◽

Cell Stage ◽

Development In Vitro

AbstractIn vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

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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

Reproduction Fertility and Development ◽

10.1071/rd05048 ◽

2005 ◽

Vol 17 (8) ◽

pp. 751 ◽

Cited By ~ 7

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.

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Human oocyte vitrification with corona radiata, in autologous follicular fluid supplemented with ethylene glycol, preserves conventional IVF potential: birth of four healthy babies

Reproduction Fertility and Development ◽

10.1071/rd13161 ◽

2014 ◽

Vol 26 (7) ◽

pp. 1001 ◽

Cited By ~ 2

Author(s):

Xian-Hong Tong ◽

Li-Min Wu ◽

Ren-Tao Jin ◽

Hong-Bing Luan ◽

Yu-Sheng Liu

Keyword(s):

Ethylene Glycol ◽

Embryo Transfer ◽

Follicular Fluid ◽

Control Group ◽

Human Oocyte ◽

Oocyte Vitrification ◽

Human Oocytes ◽

Corona Radiata ◽

Fertilisation Rate ◽

Significant Difference

The aim of this study was to examine the effects of vitrification with autologous follicular fluid (AFF) supplemented with ethylene glycol (EG) and sucrose on human oocytes with corona radiata. A total of 182 human oocytes with corona radiata from fifteen infertile patients were vitrified using either equilibration solutions (ES) and vitrification solution (VS) consisting of AFF, EG and sucrose (AFF group, n = 67) or commercial ES and VS (control group, n = 115). All oocytes were thawed in the next cycle, with surviving oocytes being inseminated by conventional IVF. The clinical outcome of vitrified–warmed oocytes by both vitrification methods was analysed retrospectively. In the AFF group, six patients received embryo transfer, with three couples taking four healthy babies home. In the control group, nine patients received embryo transfer, with four couples taking five healthy babies home. There was no significant difference in the survival rate (91.0 vs 92.2%), two pronuclei (2PN) fertilisation rate (73.8 vs 73.6%), cleavage rate (100 vs 100%), top-quality embryo rate (62.2 vs 59.2%), clinical pregnancy rate (50.0 vs 44.4%), implantation rate (33.3 vs 25%) or take-home baby rate (50.0 vs 44.4%) between the AFF group and the control group, respectively. These results show that AFF supplemented with EG and sucrose is an efficient, cost-effective cryoprotectant for human oocyte cryopreservation. A corona radiata on vitrified–warmed oocytes retains the oocytes’ fertilisation capability in conventional IVF.

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Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.639249 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yasumitsu Masuda ◽

Ryo Hasebe ◽

Yasushi Kuromi ◽

Masayoshi Kobayashi ◽

Kanako Urataki ◽

...

Keyword(s):

Embryo Transfer ◽

Inner Cell Mass ◽

Cell Mass ◽

Three Dimensional ◽

Preimplantation Embryos ◽

Infrared Light ◽

Bovine Embryo ◽

Bovine Embryos ◽

Cell Stage ◽

Developmental Potential

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

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The effects of brain-derived neurotrophic factor and metformin on in vitro developmental competence of bovine oocytes

Zygote ◽

10.1017/s0967199409005255 ◽

2009 ◽

Vol 17 (3) ◽

pp. 187-193 ◽

Cited By ~ 9

Author(s):

So Gun Hong ◽

Goo Jang ◽

Hyun Ju Oh ◽

Ok Jae Koo ◽

Jung Eun Park ◽

...

Keyword(s):

Tyrosine Kinase ◽

Embryo Development ◽

Neurotrophic Factor ◽

Brain Derived Neurotrophic Factor ◽

Early Embryo ◽

Developmental Competence ◽

Blastocyst Development ◽

Cell Stage ◽

Significant Difference

SummaryBrain-derived neurotrophic factor (BDNF) signalling via tyrosine kinase B receptors may play an important role in ovarian development and function. It has been reported that metformin elevates the activity of Tyrosine kinase receptors and may amplify BDNF signalling. The objective of this study was to investigate the effect of BDNF during in vitro maturation (IVM) and/or in vitro culture (IVC) (Experiment 1), and to evaluate the collaborative effect of BDNF and metformin treatment on the developmental competence of bovine in vitro fertilized (IVF) embryos (Experiment 2). In Experiment 1, BDNF, which was added to our previously established IVM systems, significantly increased the proportions of MII oocytes at both 10 ng/ml (86.7%) and 100 ng/ml (85.4%) compared with the control (64.0%). However, there was no statistically significant difference in blastocyst development between the control or BDNF-supplemented groups. In Experiment 2, in order to investigate the effect of BDNF (10 ng/ml) and/or metformin (10−5 M) per se, TCM-199 without serum and hormones was used as the control IVM medium. The BDNF (48.3%) and BDNF plus metformin (56.5%) significantly enhanced the proportions of MII oocytes compared with the control (34.4%). Although, BDNF or metformin alone had no effect in embryo development, BDNF plus metformin significantly improved early embryo development to the 8–16-cell stage compared with the control (16.5 vs. 5.5%). In conclusion, the combination of BDNF and metformin may have a collaborative effect during the IVM period. These results could further contribute to the establishment of a more efficient bovine in vitro embryo production system.

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