Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts

1996 ◽  
Vol 8 (5) ◽  
pp. 835 ◽  
Author(s):  
T Pinyopummintr ◽  
BD Bavister
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryo Development ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Development In Vitro ◽  
Defined Culture

Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

P–283 Combination between amino acids profile of the spent culture media and morphokinetics parameters of human embryos to determine its viability

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
N Adel ◽  
M Kadah ◽  
S Abdulghafar ◽  
M Elmahdy ◽  
D Ghareeb ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryo Development ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Human Embryos ◽  
Data Set ◽  
Group A ◽  
Amino Acids Profile ◽  
Group D

Abstract Study question How to determine human embryo viability noninvasively before embryo transfer? Summary answer We propose that the combination of the amino acid profile of an individual embryo with its morphokinetics will provide noninvasive tool to determine its viability. What is known already It was already known that human embryos at early cleavage require non-essential amino acids, while at the 8-cell to blastocyst stages, a mixture of non-essential and essential amino acids. Amino acids have important roles during embryo development. Acting as biosynthetic precursors,buffers of intracellular pH in the embryo, antioxidants, energy sources and regulators of metabolic function and signaling pathways. Many studies have used time-lapse to analyze human embryonic development including the process of fertilization and assessment of early events and introducednoninvasive prognostic markers which predict embryo development and correlate it to IVF treatment outcomes. Study design, size, duration This study was a prospective cohort study approved by the Clinical Trial Ethical Committee of Faculty of Medicine, Alexandria University according to ethical standards of scientific research (Serial number: 0303721).Thirty females aged 30.13 ± 4.83 years undergoing ICSI cycle in the Madina Fertility Center, during the period of March 2018 to November 2019.202 MII oocytes were incubated individually in embryoscope. Participants/materials, setting, methods Embryos (n = 161) were divided on Day 5 into two groups –developed embryos “Group D” (embryos that developed to blastocyst) and arrested embryos “Group A” (embryos remain at cleavage stage and fail to develop to blastocys).Developed embryos (Group D) included 99 embryos, and Arrested embryos (Group A) included 62 embryos. For each group, morphokinetic developmental points using embryoscope and the different amino acids concentrations in spent culture medium were analyzed using LC- mass spectro etry. Main results and the role of chance On one hand, the first appearance of pronuclei (TPNa), t2, t4 and CC2 in group D occurred significantly earlier than those of Group A.Analysis of 19 essential and non-essential amino acids in spent culture medium of each embryo in the two studied groups D and A showed a significantly higher concentration of two essential amino acids L-Valine (145.73 ± 150.96) and L-Phenylalanine (61.59 ± 55.78) in Group D than their concentration in Group A ( 104.58 ± 33.58, 44.24 ± 14.61, respectively , p ≤ 0.05).and significantly lower concentration of three non-essential amino acids L-Tyrosine (62.56 ± 41.03) , L-Cysteine (19.48 ± 11.90), and L-Alanine (136.0 ± 389.83) observed in Group D when compared to Group A (69.57 ± 20.78, 22.37 ± 8.59,145.33 ± 165.22, respectively, Limitations, reasons for caution It is important to note, that results were developed on a data set from one clinic with different stimulation protocols, a multicenter data and a correlation with the stimulation protocol used should be involved in future studies, in addition a larger sample size to avoid high standard deviation is recommended Wider implications of the findings: We can conclude that amino acid turnover is independent of the traditional morphological assessment of embryos and it may reflect its viability. The prospective combined use of amino acids profile of individual embryo and its morphokinetic parameters may contribute to introduce a new noninvasivs tool that may improve implantation rate Trial registration number 0303721

Amino acid metabolism of bovine blastocysts derived from parthenogenetically activated or in vitro fertilized oocytes

1998 ◽  
Vol 10 (3) ◽  
pp. 279 ◽  
Author(s):  
Y. G. Jung ◽  
T. Sakata ◽  
E. S. Lee ◽  
Y. Fukui
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Culture Medium ◽  
Acid Metabolism ◽  
Essential Amino Acids ◽  
Fluid Medium ◽  
Vitro Fertilization ◽  
Oviduct Fluid

The uptake and synthesis of 19 amino acids by fresh or frozen–thawed bovine blastocysts produced by parthenogenesis (PT) or in vitro fertilization (IVF) were compared in the present study. Fresh blastocysts, 180 h after IVF or PT activation, and frozen–thawed blastocysts, 168 h old and cultured for 12 h post-thawing, were cultured in synthetic oviduct fluid medium (SOFM) containing polyvinyl alcohol (PVA) with both essential and non-essential amino acids (EAA and NEAA, respectively) (Medium 1: M1) or SOFM containing PVA with only EAA (Medium 2: M2). In Experiment 1, when fresh or frozen–thawed PT blastocysts were cultured in M1, the uptake of glutamate (in fresh only), aspartate and arginine, and the synthesis of glutamine and alanine were significantly enhanced. In the culture with M2, serine, asparagine, glutamate, glutamine, glycine, arginine and alanine were significantly taken up. It was found that the glutamine concentrations was significantly higher (P < 0.001) in the culture medium drops containing embryos than in the drops without embryos. In Experiment 2, when PT blastocysts were cultured in M1, the uptake of aspartate and synthesis of alanine were greater (P < 0.01) than those by IVF blastocysts. When M2 was used, a significant (P < 0.01) production of serine, asparagine, glutamate, glutamine and alanine, and the uptake of arginine by PT blastocysts were observed. In Experiment 3, when IVF blastocysts were cultured in M1, fresh blastocysts depleted more aspartate and glutamate, and produced more glutamine and alanine than frozen–thawed blastocysts. When cultured in M2, frozen–thawed blastocysts depleted more threonine (P < 0.01) than fresh blastocysts. These results indicate that the uptake and synthesis of amino acids were different in fresh or frozen–thawed bovine blastocysts derived from PT or IVF. These differences in amino acid metabolism may be related to the viability of the blastocysts.

144 USE OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT AND SURVIVAL FOLLOWING VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
J. Block ◽  
L. Bonilla ◽  
P. J. Hansen
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Fresh Embryos ◽  
Hatching Rates

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos. Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.

133 EFFECTS OF ACTIVINA ON mRNA EXPRESSION IN IN VITRO FERTILIZED BOVINE EMBRYOS CULTURED FROM CHEMICALLY DEFINED TWO-STEP CULTURE MEDIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 147
Author(s):  
J. E. Park ◽  
G. Jang ◽  
H. J. Oh ◽  
S. G. Hong ◽  
I. S. Yang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Early Stage ◽  
Activin A ◽  
Developmental Competence ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Defined Culture

During the preimplantation stage, embryo development occurs in a maternal environment within the oviducts and uterine horns. It has been speculated that both the embryo itself and the maternal reproductive tract provide paracrine factors that influence embryo development (Jones et al. 2006 Reproduction 132(5), 799–810). Activins are known for FSH releasers, and several previous studies have reported that activin subunits and activin receptors mRNA were expressed in oocytes, zygotes, and oviduct (Yoshioka et al. 1998 Reprod. Fertil. Dev. 10(3), 293–298; Gandolfi et al. 1995 Mol. Reprod. Dev. 40(3), 286–291). The purposes of the present study were Experiment 1) to evaluate the effects of activin A on developmental competence of bovine embryos derived from two-step defined culture medium (Lim et al. 2007 Theriogenology 67(2), 293–302) and Experiment 2) to analyze the effects of activin A on transcriptional level of the genes in IVF embryos. Cumulus–oocyte complexs were harvested from ovaries obtained from a local slaughter house, matured, and fertilized in vitro. In vitro fertilized zygotes cultured in media supplemented with activin A in the early stage at the concentrations of 0, 10, or 100 ng mL–1 or in the later stage medium at the concentrations of 0, 10, or 100 ng mL–1. Data were analyzed using the Statistical Analysis System (SAS) program. In Exp. 1, although the development competence of embryos that cultured with activin A in the early stage medium was not significantly different, development to blastocysts on day 8 in the later stage medium with 100 ng mL–1 activin A was significantly higher than the control group [22.4% (54/264) v. 34.7% (76/233); P < 0.05]. Hatching rate of blastocyst on day 8 was significantly higher in the presence of 100 ng mL–1 activin A in the later stage culture medium compared with the control group [9.3% (5/54) v. 22.4% (17/76); P < 0.05]. In Exp. 2, the relative expression of 3 genes (Na/KATPase, E-cad, Glut-1) related to blastocyst hatching and implantation was analyzed. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitative reverse transcription-polymerase chain reaction. The expression level of the Na/K ATPase, E-cad, and Glut-1 gene were higher in the presence of activin A in the culture medium compared with the control group. In conclusion, this study suggests that activin A during the later stage of in vitro bovine embryo development can enhance the developmental competence of preimplantation embryos, increase the hatching rate, and affect expression level of genes related to hatching and implantation in defined culture medium. This study was financially supported by KOSEF (grant ? M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.

Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following transfer

1998 ◽  
Vol 49 (6) ◽  
pp. 1239-1249 ◽  
Author(s):  
J.G Thompson ◽  
N.W Allen ◽  
L.T McGowan ◽  
A.C.S Bell ◽  
M.G Lambert ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Bovine Embryo ◽  
Development In Vitro

189 EFFECT OF PROGESTERONE AND EPIDERMAL GROWTH FACTOR ON IN VITRO-PRODUCED EIGHT-CELL BOVINE EMBRYOS IN A SERUM-FREE CULTURE MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 211 ◽  
Author(s):  
B. Merlo ◽  
E. Iacono ◽  
G. Mari
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Culture Medium ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Serum Free ◽  
Epidermal Growth ◽  
Blastocyst Rate

The role of progesterone (P4) and epidermal growth factor (EGF) in early bovine embryo development is still not clear. P4 has been administered at different times of embryo development, and a direct effect on IVF-derived bovine 8-cell embryos has been noted even if there was an interference due to the P4 vehicle (Ferguson et al. 2005 Reprod. Fertil. Dev. 17, 219 abst). EGF has been added to the culture medium from the presumptive zygote stage at different concentrations, resulting in improved blastocyst rates compared to that in control medium (Mtango et al. 2003 Theriogenology 59, 1393–1402; Sirisathien et al. 2003 Anim. Reprod. Sci. 77, 21–32), and gave results similar to those with 5% or 10% FCS (Palasz et al. 2000 Anim. Reprod. Sci. 58, 229–240). The objective if this experiment was to determine the effect of P4 and EGF on development of in vitro-produced bovine embryos when administered alone or in combination at the 8-cell stage in the absence of serum. In vitro-produced bovine 8-cell embryos were randomly allotted to treatments: (1) control, SOFaaBSA medium (BSA, 16 mg mL−1; n = 198); (2) P4, SOFaaBSA + P4 (15 ng mL−1 in ethanol; n = 198); (3) EGF, SOFaaBSA + EGF (25 ng mL−1; n = 200); (4) P4 + EGF, SOFaaBSA + P4 (15 ng mL−1 in ethanol) + EGF (25 ng mL−1; n = 201); and (5) FBS, SOFaaBSA + FBS (5%; n = 197). In order to minimize the toxic effect of ethanol, it was allowed to evaporate from the culture dish and then medium was added. All in vitro procedures were carried out at 38.5°C in a humidified atmosphere of 5% CO2 in air; presumptive zygotes were cultured in SOFaaBSA until 8-cell stage. Embryo development was evaluated on Day 6 and on Day 8 after IVF (Day 0), and rates calculated from 8-cell embryos. The study was done in 4 replicates and chi-square test was used for statistical analysis (Statistica for Windows; Stat Soft Inc., Tulsa, OK, USA); significance was assessed at P &lt; 0.05. Results are reported in Table 1. No differences were found in the number of morulae between P4 and control, between P4 + EGF and FBS, and between P4 + EGF and EGF (P &gt; 0.05), whereas the combination P4 + EGF was better than P4 alone (P &lt; 0.05). Blastocyst rate was not different (P &gt; 0.05) among EGF, P4 + EGF, and FBS groups. P4 achieved an higher (P &lt; 0.05) blastocyst rate than control but it was lower (P &lt; 0.05) than that of P4 + EGF or FBS. In conclusion, P4 alone improves embryo development from the 8-cell embryo to the blastocyst stage in a serum-free culture system, and EGF alone achieves a blastocyst rate not significantly different from that of FBS; furthermore, the combination of P4 and EGF can be considered the most suitable as an alternative to FBS because similar results were obtained in terms of both morulae and blastocysts. Table 1.Eight-cell bovine embryo development in SOFaaBSA medium in presence of P4, EGF, P4+EGF, or FBS

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

scholarly journals Nutritional quality evaluation of Whitemouth croaker (Micropogonias furnieri) protein isolate

2014 ◽  
Vol 44 (2) ◽  
pp. 134-143
Author(s):  
William Renzo Cortez-Vega ◽  
Irene Rodrigues Freitas ◽  
Sandriane Pizato ◽  
Carlos Prentice
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nutritional Quality ◽  
Troponin I ◽  
Essential Amino Acids ◽  
Protein Isolate ◽  
Content Type ◽  
Sds Page ◽  
Apparent Bioavailability

Purpose – The purpose of this study was to isolate Whitemouth croaker protein by alkaline solubilization process and evaluate their nutritional quality to evaluate the bioavailability of essential amino acids. Design/methodology/approach – The proximate composition, essential amino acid composition, in vitro digestibility, apparent bioavailability, chemical score of amino acids and SDS-PAGE were determined for the isolated croaker proteins. Findings – The isolated protein showed a high level of protein 92.21 percent and low amount of lipids 0.57 percent. The protein is rich in lysine and leucine, 108.73 and 96.75 mg/g protein, respectively. The protein isolate had high digestibility, 94.32 percent, which indicates proper utilization of this protein source, while the tryptophan had lower bioavailability (12.58 mg amino acid/mg protein). The high chemical scores were found for the amino acids lysine, methionine+cysteine (6.79 and 5.14). SDS-PAGE of proteins extracted showed appearance of the heavy chain of myosin (220 kDa), actin (50 kDa) and other fractions, with molecular weight between 20 and 50 kDa, such as troponin I, C and T. Originality/value – The products obtained from croaker muscle can be incorporated as a high value supplements in human diets. The isolated protein exhibited a high content of essential amino acids and digestibility, indicating that the protein has a high nutritional quality.

scholarly journals 133 INSULIN, TRANSFERRIN AND SELENIUM WITH OR WITHOUT BSA IN A SERUM-FREE CULTURE SYSTEM FOR BOVINE EMBRYO, AND ITS SUITABILITY FOR EMBRYOS CULTURED IN SMALL GROUPS

2005 ◽  
Vol 17 (2) ◽  
pp. 217 ◽  
Author(s):  
C. Daniaux ◽  
B. Verhaeghe ◽  
I. Donnay
Keyword(s):  
Small Groups ◽  
Culture Medium ◽  
Culture Media ◽  
Quality Parameters ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Serum Free ◽  
Abnormal Accumulation ◽  
Development In Vitro

Serum in embryo culture medium may be a potential cause of abnormal accumulation of lipid droplets, which is correlated to a higher sensitivity to cryopreservation. Moreover, serum may introduce pathogens. With the aim of developing a serum-free culture medium, we first (Experiment 1) investigated the effect of adding ITS (5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium) as a serum substitute in SOF medium on embryos cultured in large groups (20 embryos per culture drop of 20 μL) and we then (Experiment 2) analyzed the effect of adding BSA. In this second experiment, our serum-free culture media were also tested on embryos cultured in small numbers (5 embryos per drop of 20 μL) in order to mimic ovum pickup (OPU) conditions. Embryos were obtained from slaughterhouse oocytes, matured in vitro for 24 h in a serum-free enriched 199 medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) containing ITS, and fertilized for 18 h. In experiment 1, embryos were cultured in SOF (Holm et al. 1999 Theriogenology 52, 683–700) supplemented with 0.1 mg/mL polyvinylpyrrolidane (PVP) without (SOF) or with ITS (SOF-ITS), or with 5% FCS (SOF-FCS). Cavitation occurred earlier in presence of serum (Table). Adding ITS to SOF increased blastocyst rates at Day 7 and Day 8 post-insemination (p.i.) and also the hatching rate. In experiment 2, embryos were cultured in SOF-FCS, SOF-ITS, or SOF-ITS supplemented with 4 mg/mL fatty acid free BSA (SOF-ITS-BSA). Within each condition, no differences were observed for blastocyst and hatching rates between embryos cultured in large or in small groups. Adding BSA to SOF-ITS increased blastocyst rate at Day 6 p.i. and also the hatching rate. At Days 7 and 8 p.i., blastocyst rates were higher in SOF-FCS than in SOF-ITS and tended to be higher than in SOF-ITS-BSA, especially for embryos cultured in small groups. Cell numbers of the resulting embryos were unaffected. These results indicate that: (1) ITS as supplement to SOF medium promotes embryo development in vitro. (2) BSA as protein supplement to SOF-ITS medium accelerates blastulation and improves hatching rate. (3) SOF-ITS and SOF-ITS-BSA are two serum-free culture media that can sustain development of embryos, also when cultured in small number, even though SOF-FCS tended to afford better rates of development. Further studies will include evaluation of other quality parameters including resistance to cryopreservation. This work was supported by the Ministery of Agriculture of the Region wallonne de Belgique.

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