Set up of a serum-free culture system for bovine embryos: Embryo development and quality before and after transient transfer (original) (raw)
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Theriogenology, 2015
The aim of this study was to develop an in vitro embryo culture medium without either fetal calf serum or BSA, using various growth factors and cytokines (GFs-CYKs; IGF-I, IGF-II, bFGF, LIF, GM-CSF, TGF-b1, and PDGF-BB), and other molecules with surfactant and embryotrophic properties, such as recombinant albumin (RA) and hyaluronan (HA). The first part of the study was dedicated to define the best combination of GFs-CYKs þ RA þ HA for optimal embryonic development. Next, we compared development rates and embryo quality (inner cell mass [ICM]-to-total cell number [TCN] ratio), and postthaw survival and hatching rates using this synthetic medium (T1) and a control medium: synthetic oviduct fluid þ BSA þ ITS (insulin, transferrin, and selenium). The blastocyst rates were significantly higher with T1 than those with the control at 7 and 8 days after fertilization. There was no significant difference in TCN or the ICM/TCN ratio between the two treatments. Survival and hatching rates 48 hours after thawing were similar for both treatments. Finally, nine embryo transfers were conducted using fresh and previously frozen Day-7 blastocysts to evaluate the in vivo viability of embryos produced in this synthetic medium; four gestations were obtained from six fresh embryos and one gestation from three frozen embryos. In conclusion, the fetal calf serum and BSA-free medium, supplemented with GFs-CYKs þ RA þ HA, improved embryo development and gave comparable ICM/TCN ratios and postthaw survival rates to the control with BSA. Fresh and frozen embryos produced in this medium are viable for embryo transfer. This fully synthetic method of embryo culture is a useful means of reducing the risk of disease transmission via embryo transfer.
Evaluation of different culture systems on the in vitro production of bovine embryos
Theriogenology, 2005
The objective of this study was to determine the development potential and quality of in vitro produced bovine embryos cultured individually or in groups. After IVM and IVF, presumptive zygotes were cultured in groups or individually, either in drops or in the modified ''well of the well'' (mWOW) system. In Experiment 1, four culture systems were utilized: T1: drop in group (control); T2: mWOW in groups; T3: mWOW individually; and T4: drop individually. Cleavage and blastocyst rates at Days 6, 7 and 8 and total cell number of Day 6 blastocysts were similar (P > 0.05) for all treatments. However, in Day 7 blastocysts, total cell number was lower (P < 0.05) in embryos cultured individually in a small drop than those cultured in the mWOW. In Experiment 2, blastocysts of T1, T2 and T3 were allocated into two groups, control and vitrified. After warming, the vitrified embryos were cultured for 72 h. At 48 h, the development of the Days 6 and 7 embryos was similar (P > 0.05) for all treatments in the control group. For the vitrified embryos, lower hatching rates (P < 0.05) were observed in the T3 group. In conclusion, embryos cultured in groups in the mWOW system had the same blastocyst rates but better quality (measured by their survival after vitrification) than those cultured individually in the mWOW system. #
Animals
Supplementation of the culture media for in vitro production (IVP) of bovine embryos with fetal bovine serum (FBS) is associated with inconsistent outcomes. The present study sought to replace FBS and BSA by insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). In Experiment 1, absence of FBS from maturation medium (MM) did not affect the rate of in vitro maturation, as assessed by the extrusion of the first polar body. However, when gonadotropins and FBS were removed from the MM, the maturation rate was significantly reduced even in the presence of growth factors. Therefore, gonadotropin-supplemented MM medium was established as the base medium for the defined maturation condition. In Experiment 2, the addition of growth factors to gonadotropin-supplemented MM medium supported similar maturation (~90%) compared to the undefined condition (FBS-carrying). In Experiment 3, the addition of growth factors to embryo culture medium showe...
Theriogenology, 1994
With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 pg/ml insulin, 5 pg/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for ftrst cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastoeoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of f'trst cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The " culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0,05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAiTS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicatc that a serum-free, cellfree culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.
Theriogenology, 2006
The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/ mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest postcryopreservation survival rates, regardless of the presence of serum in the culture medium. #
VIABILITY OF IN-VIVO AND IN-VITRO PRODUCED BOVINE EMBRYOS
Reproduction in Domestic Animals, 1993
The viability of in-vivo and in-vitro produced bovine embryos is compared. Available data indicate that in-vitro produced embryos are more susceptible to freezing and subject to a higher incidence of fetal loss following transfer. A comparison between in-vivo (sheep oviducts) and in-vitro (bovine oviduct epithelial cells) culture conditions revealed that the in-vivo condition was superior in terms of yielding embryos with higher viability following freezing and thawing. The time of first cleavage division is important for the compaction and the viability. It is yet to be established if and how growth factors and proteins are involved in the regulation of early embryonic development. Suffice it to say that specific growth factors are produced and secreted from the bovine oviduct epithelium in-vivo and that addition of certain growth factors may improve the magnitude of embryonic development in-vitro. Much attention has been devoted the in-vitro culture use, but oocyte quality, the in-vitro maturation conditions and the in-vitm fertilization system may be equally important.
In Vitro Production of Bovine Embryos : We Need to Stop or Proceed -A Review
Production of embryos in vitro (in vitro Fertilization technology) provides an excellent and cheap source of embryos for carrying out basic research on developmental physiology, farm animal breeding and for commercial application of the emerging biotechniques like cloning and transgenic livestock production. Embryos of high genetic quality can be obtained from oocytes collected from slaughtered house ovaries or from donors of high genetic quality by ultrasound guided follicular aspiration. Despite technological progress in the last two decades, the practical application of in fertilization technology (IVF) is still less than anticipated because of low efficiency and high cost. Recent data indicate high rate of meiotic maturation (> 85% in cattle, >80% in buffalo), fertilization (> 80% in cattle, >70% in buffalo) and cleavage (> 70% in cattle, >50% in buffalo) but low rates of blastocyst formation (~ 30% in cattle, ~25% in buffalo) and calf production (~10 %) and so...
Development of bovine embryos in vitro following oocyte maturation under defined conditions
Reproduction, nutrition, development, 1994
A total of 4,615 immature bovine oocytes were used in a series of experiments aimed at the systematic evaluation of the role of the different components of our in vitro maturation (IVM) medium in imparting developmental competence to the oocytes. The results clearly demonstrate that both tissue culture medium 199 (M199) and synthetic oviduct fluid (SOF) are capable of supporting the IVM of bovine oocytes at high rates in the absence of macromolecular supplements, as evidenced by subsequent development to the blastocyst stage (20 and 25%, respectively). However, both were significantly lower than the control (containing 10% fetal calf serum, 5 micrograms/ml pLH (porcine luteinizing hormone), 1 microgram/ml pFSH (porcine follicle-stimulating hormone), and 1 microgram/ml-17 beta-estradiol, E2) in terms of blastocyst yield. Inclusion of bovine serum albumin (3 mg/ml) was not beneficial and in fact significantly depressed development when added to SOF. It was shown that the advantage of ...
Theriogenology, 1995
a total of 3465 bovine oocytes this study was aimed at improving the efficiency of bovine embryo production under defined and undefined conditions. Following in vitro maturation (IVM) and in vitro fertilization (IVF), oocytes were allocated to various culture treatments using synthetic oviduct fluid (SOF). In our 3 experiments we showed that : 1) the addition of fetal calf serum (FCS 10% v/v) to SOF droplets after 20 to 24 h significantly improved blastocyst yields on Day 6 (21 vs 12%; P<O.Ol), but not at later stages, and resulted in significantly higher Day-8 blastocyst cell numbers (148+61 vs 92+35; PiO.05); 2) the removal of bovine serum albumin (BSA) from the standard SOF medium resulted in significantly reduced blastocyst yields on Days 6,7 and 8, respectively (17 vs 8%: 28 vs 18%; 3 1 vs 2 1%; PiO.05); 3) the presence or absence of cumulus cells surrounding the presumptive zygote in culture in SOF had no effect on cleavage rate, percentage of 5-8 cell embryos or blastocyst yields (Day 6,7 or 8); 4) the culture of presumptive zygotes in SOF in an atmosphere of 5% CO2 in air (20% 02) resulted in significantly reduced development compared with culture in 5% CO;?, 5%02,90% N2 in terms of blastocyst yield on Days 6,7 and 8 and on Day 8 hatching rate, respectivelv (5 vs 22%; 9 vs 33%: 13 vs 48%; 50 vs 8%; P<O.OOl) and 5) embryo d&sity.( 1 embryo per' 1 or 3 pl SOF) or replacing the culture medium every 48 h had no effect when SOF was supplemented with serum; however, under serum-free conditions, changing of the media resulted in a slightly improved Day-6 blastocyst yield such that renewal of serum-free medium mimicked the effect of serum addition.
Theriogenology, 2004
The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF þ 5% FCS for 9 days; (2) KSOM þ 0:1% BSA for 4 days and then KSOM þ 1% BSA to Day 9; (3) SOF þ 5% FCS for 4 days and then KSOM þ 1% BSA to Day 9; and (4) KSOM þ 0:1% BSA for 4 days and then SOF þ 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0:05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM þ BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P < 0:05) were obtained by vitrification at 6 h (71%), 24 h (64%), and 48 h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality Theriogenology 62 (