Effect of bovine oviductal fluid on development and quality of bovine embryos produced in vitro

2017 ◽  
Vol 29 (3) ◽  
pp. 621 ◽  
Author(s):  
Ricaurte Lopera-Vasquez ◽  
Meriem Hamdi ◽  
Veronica Maillo ◽  
Valeriano Lloreda ◽  
Pilar Coy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Cell Mass ◽  
Water Channel ◽  
Calf Serum ◽  
Cell Counting ◽  
Bovine Embryos ◽  
Oviductal Fluid

To evaluate the effect of bovine oviductal fluid (OF) supplementation during in vitro culture of bovine embryos on their development and quality, in vitro-produced zygotes were cultured in synthetic oviductal fluid (SOF; negative control; C–) supplemented with OF or 5% fetal calf serum (positive control; C+). Embryo development was recorded on Days 7–9 after insemination and blastocyst quality was assessed through cryotolerance, differential cell counting of the inner cell mass and trophectoderm, and gene expression. OF was added to the culture medium at concentrations ranging from 0.625% to 25%. The higher OF concentrations (5%, 10% and 25%) had a detrimental effect on embryo development. Lower OF concentrations (1.25% and 0.625%) supported embryo development until Day 9 (27.5%) and produced higher-quality blastocysts, as reflected by their cryotolerance (53.6% and 57.7% survival at 72 h, respectively, vs 25.9% in C+) and total cell number (mean (± s.e.m.) 165.1 ± 4.7 and 156.2 ± 4.2, respectively, vs 127.7 ± 4.9 in C– and 143.1 ± 4.9 in C+). Consistent with these data, upregulation of the water channel aquaporin 3 (AQP3) mRNA was observed in blastocysts supplemented with 1.25% OF compared with C– and C+. Serum supplementation resulted in a reduction in the expression of glucose and lipid metabolism-related genes and downregulation of the epigenetic-related genes DNA methyltransferase 3A (DNMT3A) and insulin-like growth factor 2 receptor (IGF2R). In conclusion, in vitro culture with low concentrations of OF has a positive effect on the development and quality of bovine embryos.

scholarly journals Effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro

Reproduction ◽  
2017 ◽  
Vol 153 (4) ◽  
pp. 461-470 ◽  
Author(s):  
Ricaurte Lopera-Vasquez ◽  
Meriem Hamdi ◽  
Veronica Maillo ◽  
Alfonso Gutierrez-Adan ◽  
Pablo Bermejo-Alvarez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Extracellular Vesicles ◽  
Water Channel ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Analysis System ◽  
Oviductal Fluid

The aim of this study was to evaluate the effect of extracellular vesicles (EV) from oviductal fluid (OF), either from the ampulla or isthmus, on the development and quality of in vitro-cultured bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF + 3 mg/mL BSA) without calf serum (C− group), in the presence of 3 × 105 EV/mL from ampullary or isthmic OF at either 1 × 104 g (10 K) or 1 × 105 g (100 K), and compared with SOF + 5% FCS (C+ group). OF-EV size and concentration were assessed by electron microscopy and nanotracking analysis system. Embryo development was recorded on Days 7–9, and blastocyst quality was assessed through cryotolerance and gene expression analysis. Lower blastocyst yield was observed on Day 7 in the C− and OF-EV groups (12.0–14.3%) compared with C+ (20.6%); however, these differences were compensated at Days 8 and 9 (Day 9: 28.5–30.8%). Importantly, the survival rate of blastocysts produced with isthmic 100 K OF-EV was higher than that of C+ and C− group at 72 h after vitrification and warming (80.1 vs 34.5 and 50.5% respectively, P < 0.05). In terms of gene expression, blastocysts produced in the presence of 100 K isthmic OF-EV upregulated the water channel AQP3 and DNMT3A and SNRPN transcripts compared with the C+, with the expression in C− being intermediate. The lipid receptor LDLR was downregulated in C+ compared with all other groups. In conclusion, the addition of oviductal fluid extracellular vesicles from isthmus, to in vitro culture of bovine embryos in the absence of serum improves the development and quality of the embryos produced.

scholarly journals 165RE-EXPANSION AND QUALITY OF SPLIT BOVINE EMBRYOS IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 205 ◽  
Author(s):  
P. Kesseler ◽  
E. Mahabir ◽  
M. Köster ◽  
M. Gilles ◽  
K. Wimmers ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Counting ◽  
Bovine Embryos ◽  
Becton Dickinson ◽  
Group A ◽  
Group B ◽  
Number Of Cells

Transferring split bovine embryos results in a higher number of calves born per embryo. In addition to generating genetically identical progeny, biopsies can be made for molecular biological analyses. We aimed to determine the effect of splitting ratio on the in vitro development of Day 7 (164 to 168h post insemination) IVP bovine embryos. The inner cell mass (ICM) and trophoblast cells were split in three ratios (50:50, 60:40 and 70:30) with a Beaver microblade (Becton Dickinson, N.J., USA.) fixed to a micromanipulator under an inverse microscope at 100 X (Leica, Bensheim, Germany). Split blastocysts were cultured singly in 50μL drops of CR1aa medium at 39°C under 5% CO2 in a moisture-saturated atmosphere. After 1 and 2h culture, the morphology was assessed by judging the shape of the embryos and re-development of the blastocoel. On Day 8 (after 22h culture), the shape of the blastocysts, development of the ICM, blastocoel, proportion of degenerated cells and embryos and re-expanded blastocysts were recorded. Embryos were stained with propidium iodide and Hoechst 33258 for cell counting. The re-expansion status of Group A (50, 60 and 70%) and B (50, 40 and 30%) embryos after 1 and 2h and their quality after 22h culture (1: excellent=&lt;10% degenerated cells, well-defined ICM; 2: fair=&lt;20% degenerated cells) are shown in Table 1. With regards to Group A split blastocysts, a higher (P&lt;0.05) percentage of embryos that re-expanded after 1 and 2h and which yielded Quality 1 and 2 embryos suitable for embryo transfer was observed with the 60% and 70% than with demi-embryos. There were significant differences (P&lt;0.05) between all split blastocysts in Group B after 1h culture. The 30% split embryos showed the lowest re-expansion rate and quality of embryos after 2h and 22h culture, respectively. No differences (P&lt;0.05) were seen in the ratio of the ICM to the total number of cells in both Group A and B. This study showed that the ratio in which blastocysts were split had a significant effect on re-expansion and quality but not on the number of cells. Table 1

scholarly journals Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture

2005 ◽  
Vol 17 (8) ◽  
pp. 799 ◽  
Author(s):  
Natalie I. Alexopoulos ◽  
Gábor Vajta ◽  
Poul Maddox-Hyttel ◽  
Andrew J. French ◽  
Alan O. Trounson
Keyword(s):  
In Vitro Culture ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Collagen Gel ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Term Survival

Attempts to support survival of mammalian embryos after hatching have met with limited success, although some mouse studies have reported growth at the post-implantation stage. The aim of the present research was to establish and characterise an in vitro culture system that could support extended growth and differentiation of bovine embryos. Abattoir-derived oocytes were matured and fertilised in vitro. Presumptive zygotes were cultured in modified synthetic oviduct fluid (SOFaaci) medium supplemented with 5% cow serum (CS). On Day 9, single hatched blastocysts (n = 160) were randomly allocated to SOFaaci supplemented with either 5% bovine serum albumin, 5% CS, 5% fetal calf serum (FCS) or SOF only and cultured on a collagen gel substrate for up to 45 days. Embryos were evaluated at various time-points until complete disaggregation or the total disappearance of embryonic cells. Blastocyst viability post hatching was severely compromised in protein-free SOFaaci medium. Addition of FCS generated increased embryonic growth for the longest time period (Day 45) when compared to the other groups. Long-term survival of embryonic cells was observed stereomicroscopically by the proliferation and development of three-dimensional tubular structures to 85% confluence in culture. Haematoxylin and eosin staining of morphological structures obtained from all treatment groups revealed embryos displaying trophoblast, inner cell mass and hypoblast development to varying degrees. Regardless of treatment, extended in vitro culture did not result in development comparable with that described for in vivo embryos. In the present work, however, there was evidence of extended culture of bovine embryos beyond that achieved previously. However, further research is required to identify the exact requirements for extended in vitro culture for bovine embryos.

Maternal influence on oocyte collection and embryo development after transvaginal puncture and in vitro fertilisation in dairy cows

2001 ◽  
Vol 26 (2) ◽  
pp. 433-435
Author(s):  
S. Chastant-Maillard ◽  
H. Quinton ◽  
C. Douar ◽  
J. Marchal ◽  
C. Richard ◽  
...  
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Formation Rate ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Cell Counting ◽  
In Vitro Fertilisation ◽  
Oocyte Collection

AbstractThe aim of the study was to evaluate differences between cows in the “quality” of their oocytes defined as their ability to support embryonic development. Ten cows from the same herd, all primiparous and non-pregnant were submitted for oocyte collection by Ovum-Pick Up (OPU). The oocytes were matured in vitro and fertilised with semen from the same bull. In vitro embryo development, both quantitatively (percentages showing cleavage and forming blastocysts) and qualitatively (differential cell counting in blastocysts) was determined at the blastocyst stage (Day 7). The number of oocytes collected, the number of blastocysts obtained and the blastocyst formation rate varied between cows (P<0.001). The mean percentage of inner cell mass cells tended to higher for embryos derived from one cow. These results provide evidence that the quality of the oocytes was influenced by their maternal origin. Follicular growth also varied between cows.

140 PRODUCTION OF RECONSTRUCTED IN VITRO PRODUCED BOVINE EMBRYOS BY INNER CELL MASS AND TROPHECTODERM AGGREGATION IN VITRO

2011 ◽  
Vol 23 (1) ◽  
pp. 174
Author(s):  
I. P. Emanuelli ◽  
E. Razza ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
In Vitro Culture ◽  
Fetal Calf Serum ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Calf Serum ◽  
Cell Aggregation ◽  
Bovine Embryos ◽  
Advanced Stages

The efficiency of embryonic chimerism tends to decrease when embryos in advanced stages of development, such as morulae and blastocysts, are used. To perform the inner cell mass (ICM) transfer to a trophectoderm (TE) receptor, it is essential to use embryos at an advanced stage and blastocoel presence. This method of embryo reconstruction has been performed only by the micromanipulator microinjection method (Zheng et al. 2005 Zygote 1, 73–71; Loi et al. 2007 Trends Biotechnol. 25, 195–200; Roth et al. 1989 Biol. Reprod. 41, 675–682; and Murakami et al. 2006 Cloning Stem Cells 8, 51–60). This study aimed to validate a manual procedure to reconstruct embryos using the method of ICM and TE approximation in the presence of phytohemagglutinin. Bos indicus ovaries from the abattoir were used to obtain 230 cumulus–oocyte complexes (COC; quality I and II). The COC were matured in 90-μL drops of TCM-199 bicarbonate supplemented with 10% fetal calf serum (FCS) and incubated in vitro for 22 to 24 h. Fertilization occurred in TALP-IVF medium, and the COC were incubated for 18 h. Presumptive zygotes were transferred to SOF medium to in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. In vitro produced (IVP) embryos 8.5 days after fertilization were used for the experiment. Ninety-three hatching or hatched blastocysts were put into 3-μL microdrops of protein-free HEPES-buffered SOF (HSOF) medium to hold the embryos on the dish bottom and to allow handmade sections of ICM and TE. The section was performed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under a stereomicroscope (35× magnification). Seventy half-structures from 35 different blastocysts were obtained to form pairs (ICM+TE). Each pair was transferred to drops with 500 μg mL–1 of phytohemagglutinin-L (3 min) before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) to in vitro culture (38.5°C; 5% O2 and 5% CO2). The aggregation rate was 25.7% (9/35) and all the reconstructed blastocysts by aggregation (24 h) re-expanded after 48 h of culture. The technique of handmade ICM and TE section and posterior aggregation in the presence of an agglutinating agent was feasible for the structural and functional reconstruction and re-expansion of the blastocyst produced. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).

178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 197 ◽  
Author(s):  
B. S. Song ◽  
J. S. Kim ◽  
D. B. Koo ◽  
J. S. Park ◽  
K. K. Lee ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Developmental Rate ◽  
Treated Group ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Frozen Semen ◽  
Oviductal Fluid

The microenvironment of the follopian tube, in which the oviductal fluid contains a variety of cytokines and growth factors, affects pre-implantation development of fertilized embryos in mammals. Prostaglandin I2 (PGI2, prostacyclin) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. PGI2 also enhances the implantation rate of mouse embryos. In this study, the effect of PGI2 analog on the development of bovine embryos was examined. Bovine cumulus oocytes complexes (COCs) were matured in TCM-199 medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL hCG, and 10 ng/mL epidermal growth factor (EGF) at 39�C, 5% CO2 in air for 20-22 h. Following in vitro maturation, COCs were fertilized in Fert-TALP medium containing 0.6% BSA using frozen semen. Also, oocytes matured in vitro were enucleated, individually reconstructed with bESF cells, fused, and then activated by treatment with 5 �M ionomycin for 5 min and 2 mM 6-DMAP for 4 h. In vitro-fertilized (IVF) and nuclear-transferred (NT) eggs were cultured in 50 ��L drops of CR1-aa medium supplemented with 0.3% BSA in the absence or presence of 1 �M PGI2 analog at 39�C, 5% CO2 in air, respectively. At 3 days of culture, cleaved embryos were further cultured in the same culture media supplemented with 10% FBS for 4 days. Allocations of blastocysts to inner cell mass (ICM) and trophoblast (TE) cells were investigated to assess embryo quality. All experiments were repeated more than three times. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA) and numbers of nuclei in blastocysts were expressed as mean � SE. No difference was detected in the cleaved rate of the eggs between the treated- and nontreated groups. IVF zygotes treated with PGI2 analog represented a higher developmental rate (33%, 122/418) to the blastocyst stage than nontreated controls (24%, 107/456) (P < 0.05). Among IVF-derived blastocysts, interestingly, the proportion (46%, 84/181) of expanded blastocysts was significantly higher in the PGI2 analog-treated group compared with that in the nontreated group (28%, 46/164). The number of nuclei in (165 � 6.1, n = 15) in blastocysts in the PGI2 analog-treated group was higher than that (146.12 � 5.7, n = 18) in the nontreated group (P < 0.05). No difference was detected in the ratio of ICM to total cells between PGI2 analog-treated (42.0 � 3.0%) and nontreated groups (41.9 � 2.9%). Like the IVF embryos, NT embryos in the PGI2 analog-treated group showed a higher in vitro developmental rate (33.6%, 43/128) than the nontreated embryos (24.2%, 32/132) (P < 0.05). Our results indicate that PGI2 analog improves the kinetics of embryo development in cattle.

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 146-157 ◽  
Author(s):  
Daniela Martins Paschoal ◽  
Mateus José Sudano ◽  
Midyan Daroz Guastali ◽  
Rosiára Rosária Dias Maziero ◽  
Letícia Ferrari Crocomo ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Similar Rate ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Vitrification Solution ◽  
Oviductal Fluid

SummaryThe objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

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.

137 EFFECTS OF X-SORTED SPERM IN QUALITY OF BOVINE BLASTOCYST DERIVED FROM IN VIVO-MATURED OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 182
Author(s):  
K. Imai ◽  
M. Ohtaku ◽  
Y. Aikawa ◽  
H. Matsuda ◽  
S. Kobayashi ◽  
...  
Keyword(s):  
Prognostic Factors ◽  
In Vitro Culture ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Early Embryo ◽  
Lag Phase ◽  
Culture Dish

Recently, we reported on a promising system for selecting healthy IVF embryos in cattle using kinetics of early embryo development and oxygen consumption of blastocyst [Sugimura et al. 2012 PLoS ONE 7, e36627]. The present study was conducted to examine the differences in embryo quality of bovine blastocysts obtained after IVF of in vivo-matured oocytes with X-sorted and unsorted sperm. Holstein dry cows (n = 8) were reared under the same feeding and environmental conditions. Two ovum pickup (OPU) sessions were conducted in each cow to fertilize with or without X-sorted sperm. In vivo-matured oocytes were collected by OPU just before ovulation after superstimulation treatment. The oocytes were inseminated with 5 × 106 sperm mL–1 of each sperm, and presumptive zygotes were cultured in CR1aa supplemented with 5% newborn calf serum and 0.25 mg mL–1 of linolenic acid albumin at 38.5 C in 5% CO2, 5% O2, and 90% N2 for 168 h. Embryo kinetics were observed individually using a microwell culture dish (Dai-Nippon Print) and time-lapse cinematography (CCM-1.4MZS; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken every 15 min during the in vitro culture period and images were analysed by CCM-1.4 software (Astec). By assessing the quality of blastocysts, a combination of identified prognostic factors were used: (1) timing of the first cleavage (less than 27 h post-insemination); (2) two blastomeres at the end of the first cleavage; (3) absence of fragments at the end of the first cleavage; and (4) six or more blastomeres at the onset of the lag-phase. Data were analysed by ANOVA. In total, 34.1 ± 18.4 oocytes per session per donor were collected by OPU, and 23.7 ± 13.4 oocytes had an expanded cumulus cell. Oocyte recovery rates were recorded at 77.1 ± 15.1%. After IVF and in vitro culture, 10.6 ± 7.7 blastocysts per session per donor were produced in this study. There was no significantly difference in cleavage rates and blastocyst formation rates between X-sorted sperm and unsorted sperm (87.1 ± 10.8 and 82.6 ± 12.1% and 38.4 ± 23.6 and 57.1 ± 23.4%, respectively). However, blastocysts derived from X-sorted sperm showed significantly (P < 0.05) lower quality in the prognostic factor (1) and combined (1) to (4) than that in unsorted sperm (35.3 v. 54.0 and 14.7 v. 42.9%, respectively). Pregnancy rates were higher for the blastocysts that had a high score in the prognostic factors (1) to (4) compared to those that had a low score (75.0%, n = 8 v. 36.4%, n = 22). These results suggest that quality of blastocysts, based on the prognostic factors studied, derived from X-sorted sperm is lower than that from unsorted sperm. Supported by the Research and Development projects for application in promoting new policy of agriculture, forestry and fisheries (22016).

scholarly journals Effect of cysteamine supplementation during in vitro culture of early stage bovine embryos on blastocyst rate and quality

2012 ◽  
Vol 81 (3) ◽  
pp. 229-234 ◽  
Author(s):  
Martina Lojkic ◽  
Iva Getz ◽  
Marko Samardžija ◽  
Mario Matkovic ◽  
Goran Bacic ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Oviductal Fluid

The aim of this study was to evaluate whether the addition of cysteamine to the in vitro culture media enhances the yield, hatching rate, total cell number and inner cell mass/total cell number ratio of bovine embryos. A total of 933 bovine oocytes collected from ovaries of 60 slaughtered donors were subjected to in vitro maturation and in vitro fertilization. Following fertilization, embryos were cultured in synthetic oviductal fluid without glucose. After 24 h embryos were transferred into synthetic oviductal fluid with 1.5 mM glucose and 0 (control), 50, 100 and 200 µM of cysteamine. After 48 h, the embryos were transferred into synthetic oviductal fluid with glucose but without cysteamine and cultured until Day 9. The number of cleaved embryos on Day 2, the total number of blastocysts on Day 7 and the number of hatched blastocysts on Day 9 were calculated. Differential staining of inner cell mass and trophectoderm cells of blastocysts were performed on Day 7 and Day 9 of in vitro culture. Supplementation of in vitro culture media with 100 µM cysteamine increased the blastocyst yield (P < 0.05) without affecting the hatching rate. Furthermore, the embryos cultured in the presence of 100 µM cysteamine had significantly higher number of inner cell mass cells (P < 0.05) and the proportion of inner cell mass cells (P < 0.05) compared with the controls. The results of the present study demonstrated that the addition of 100 µM cysteamine to the in vitro culture media improved blastocyst production rate and enhance embryo quality, which could lead to the improvement of the in vitro culture system for bovine embryos.

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