Heat stress-induced apoptosis in porcine in vitro fertilized and parthenogenetic preimplantation-stage embryos

Decades worth of research have consistently shown the adverse effects of elevated temperatures on reproductive parameters of livestock species. The objective of this study was to evaluate the developmental and apoptotic responses of porcine in vitro fertilized (IVF) and parthenogenetically activated (PA) embryos heat stressed at the late 1-cell stage. Embryos were heat stressed (HS) at 42 degrees C for 9 hr starting 22 hr after insemination or artificial activation stimulus. Non heat-stressed (NHS) control embryos were maintained at 39 degrees C for the duration of the experiments. TUNEL staining on Day 5 of development demonstrated that heat stress elicited a significant apoptotic response in IVF embryos (45.6% of HS embryos and 26.7% of NHS embryos were apoptotic; P<0.05), but not in PA embryos (51.1% and 39.9% for HS and NHS embryos, respectively; P>0.1). And, while IVF embryos were highly susceptible to heat-induced developmental perturbations (20.6% and 8.8% development to blastocyst for NHS and HS embryos, respectively; P<0.05), elevated temperatures did not affect blastocyst rates in PA embryos (22.2% for NHS PA embryos and 21.2% for HS PA embryos; P>0.1). These findings indicate that, as in other systems studied, IVF pig embryos are directly affected adversely by heat stress conditions. Parthenogenetic embryos, though, appear to be surprisingly tolerant of the elevated temperatures. The differences between IVF and PA embryos in their response to heat stress warrants further investigation.     

Proteomic analysis of parthenogenetic and in vitro fertilized porcine embryos

Proteomic data from embryos are essential for the completion of whole proteome catalog due to embryo‐specific expression of certain proteins. In this study, using reverse phase LC‐MS/MS combined with 1‐D SDS‐PAGE, we identified 1625 mammalian and 735 Sus scrofa proteins from porcine zygotes that included both cytosolic and membranous proteins. We also found that the global protein profiles of parthenogenetically activated (PA) and in vitro fertilized (IVF) zygotes were similar but differences in expression of individual proteins were also evident. These differences were not due to culture conditions, polyspermy or non‐activation of oocytes, as the same culture method was used in both groups, the frequency of polyspermy was 24.3±3.0% and the rates of oocyte activation did not differ (p>0.05) between PA and IVF embryos. Consistent with proteomic data, fluorescent Hoechst 33 342 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay also revealed that PA embryos were of poor quality as they contained less cells per blastocyst and were more predisposed to apoptosis (p<0.05), although their in vitro development rates were similar. To our knowledge, this is the first report on global peptide sequencing and quantification of protein in PA and IVF embryos by LC‐MS/MS that may be useful as a reference map for future studies.     

Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure

The fertilized mouse egg actively demethylates the paternal genome within a few hours after fertilization, whereas the maternal genome is only passively demethylated by a replication-dependent mechanism after the two-cell stage. This evolutionarily conserved assymetry in the early diploid mammalian embryo may have a role in methylation reprogramming of the two very different sets of sperm and egg chromatin for somatic development and formation of totipotent cells. Immunofluorescence staining with an antibody against 5-methylcytosine (MeC) showed that the incidence of abnormal methylation patterns differs between mouse two-cell embryos from superovulated females, nonsuperovulated matings, and in vitro fertilization (IVF). It also depends on embryo culture conditions and genetic background. In general, there was a good correlation with the number of embryos (from the same experiment) which did not develop in vitro up to the blastocyst stage. Thus, aberrant genome-wide DNA methylation in early embryos may be an important mechanism contributing to the high incidence of developmental failure in mammals. Similar to the situation in abnormally methylated embryos from nuclear transfer, it may cause a high incidence of pregnancy loss and abnormal phenotypes.     

Kinetics of pronuclear development and the effects of vector type and timing of injection on the efficiency of gene transfer into rhesus macaque embryos

A series of experiments was performed to determine the dynamics of pronuclear development as well as the efficiency of either adenovirus-associated (AAV) or lentivirus-derived vectors to introduce a green fluorescent protein (GFP) reporter gene into rhesus macaque (Macaca mulatta) embryos. Assessment of pronuclear development at various times after fertilization revealed that the appearance of pronuclei was determined by the presence of the first and the timing of the second polar body. The dynamics of pronuclear formation was a significant determinant of whether an oocyte reached the blastocyst stage, however, when the percentage of blastocysts were based on the number of zygotes, the timing of the appearance of polar bodies did not appear to have any effect on subsequent development. Injection of different AAV-derived vectors showed that the serotype of the vector did not affect development or the proportion of transgenic embryos. Moreover, all putative transgenic embryos proved to be expression mosaics. Injection of embryos with lentiviral vectors showed that timing of injection (before or after fertilization) had no effect on subsequent transgene expression, but that the type of reporter gene determined post-injection development and rate of transgenesis. The transfer of embryos following injection of a lentiviral vector into three recipients resulted in one pregnancy which was lost during the second trimester. Analysis of fetal tissues showed ubiquitous presence of the transgene and GFP expression in all tissues examined. These results show that lentivirus-derived vectors can efficiently transform rhesus embryos and are suitable for the generation of transgenic rhesus monkeys.     

小鼠电激活孤雌胚胎早期体内外发育能力的比较

目的探讨小鼠电激活孤雌胚胎的早期体内、外发育能力。方法 利用不同电脉冲参数和激活液对小鼠卵母细胞进行活化,观察激活后的小鼠孤雌胚体外发育状况和移植后的发育能力。结果非电解质激活液优于电解质液,脉冲强度、脉冲宽度和脉冲次数3个参数各自处于某一范围内时,他们之间存在某种相关性,降低其中1个参数可通过升高另外2个参数得到补偿,经筛选较适宜的电脉冲参数为：1.0 kV/cm、40μs、2 p,或者1.5kV/cm、30/μs、2 p,分别为74.65%和71.19%,体外囊胚发育率分别为43.40%和47.62%。电激活孤雌胚体外发育时序比正常胚胎慢,但囊胚细胞数与对照组差异不显著。它们经胚胎移植后,其中的一部分能够着床,但着床率仅为3.6%,极显著低于对照组（67%,P〈0.01）。结论电刺激能够较好地模拟正常受精过程激活小鼠卵母细胞,但激活后的多数小鼠孤雌胚胎着床能力较低,不能够顺利着床。     

Relationship between age of blastocyst formation and interferon-τ secretion by in vitro–derived bovine embryos

This study was designed to examine the relationship between the speed at which bovine embryos reach the blastocyst stage, their cell number, and interferon-τ production. A total of 800 oocytes were fertilized by frozen-thawed semen. On day 2, 44 hr after exposure to sperm, 78, 320, and 296 embryos were at the two-, four-, and eight-cell stages, respectively, with an overall cleavage rate of 86.8%. Within these three groups 15 (19.2%), 106 (33.1%), and 158 (53.4%) embryos proceeded to the blastocyst stage. Of these 46.7%, 65.1%, and 63.3% hatched in the three groups, respectively. Blastocysts began to appear at day 7, but a few did not form until as late as day 13. Expanded blastocysts (n = 279) were cultured individually for 48 hr in 50-μl droplets of medium, fixed for cell counts, and the concentration of interferon-τ in the medium was determined. Blastocysts originating from two-cell embryos had significantly fewer cells (46.5 ± 23.3) than either four-cell- (97.2 ± 13.5) or eight-cell-derived embryos (113.8 ± 13.6; P < 0.05). Hatching was accompanied by an increase in cell number (129.8 ± 15.5 versus 41.9 ± 14.4; P < 0.01). Blastocysts derived from embryos that had reached the eight- or four-cell stage 44 hr after insemination produced significantly more interferon than embryos derived from two-cell embryos (941.7 ± 92.1, 930.1 ± 163.1, versus 232.8 ± 70.1 pM). In contrast, hatching, ovary batch, the speed of early cleavage, cell number, and quality grade had no effect on interferon-τ secretion. The embryo's age at blastocyst formation was not related to the number of its cells but did have a significant effect (P < 0.001) on interferon-τ production, with mean concentrations in the medium of 294.8 ± 57.9, 563.3 ± 82.0, 1126.3 ± 133.6, 1778.5 ± 297.2, 512.9 ± 82.0, 315.0 ± 157.5, and 157.5 pM among blastocysts appearing from days 7 to 13, respectively. These data suggest that blastocysts that form at days 7 and 8 produce less interferon-τ than those that form on days 9 or 10. Since early-forming blastocysts are generally considered more developmentally competent than those which form late, there may be a negative relationship between early interferon-τ production and competence. Mol. Reprod. Dev. 49:254–260, 1998. © 1998 Wiley-Liss, Inc.     

Mitochondrial DNA Copy Number in Cleavage Stage Human Embryos—Impact on Infertility Outcome

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between mitochondrial DNA (mtDNA) content of cleavage-stage preimplantation embryos and their developmental potential. A total of 69 couples underwent IVF treatment (averaged women age: 36.5, SD 4.9) and produced a total of 314 embryos. A single blastomere was biopsied from each embryo at the cleavage stage (day-3 post-fertilization) subjected to low-pass next generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number amount was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 6.3 ± 7.5 versus 7.1 ± 5.8, p < 0.004; U Mann–Whitney test), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (6.6 ± 4.8 vs. 8.5 ± 13.6, p 0.09), sex (6.6 ± 4.1 vs. 6.2 ± 6.8, p 0.16), maternal age (6.9 ± 7.8 vs. 6.7 ± 4.5, p 0.14) or its ability to implant (7.4 ± 6.6 vs. 5.1 ± 4.6, p 0.18). The mtDNA content cannot serve as a useful biomarker at this point in development. However, further studies investigating both quantitative and qualitative aspects of mtDNA are still required to fully evaluate the relationship between mitochondrial DNA and human reproduction.     

Concentration and composition of free amino acids and osmolalities of porcine oviductal and uterine fluid and their effects on development of porcine IVF embryos

The concentration of free amino acids and the osmolalities in porcine oviductal (OF) and uterine fluids (UFs) on day 3 (D3) and day 5 (D5) were measured by HPLC and Vapor Pressure Osmometer, respectively. Based on these measurements we designed new media based on PZM3 by modifying the amino acid composition and osmolality. The effectiveness of the modified PZM3 on the development of porcine IVF embryos was then investigated. A total of 24 free amino acids were measured, including 20 protein and 4 nonprotein amino acids (beta-alanine, taurine, ornithine, and citrulline). There was no significant difference in the total concentration of amino acids among D3OF (13.06 +/- 3.63 mmol/L), D3UF (10.54 +/- 5.16 mmol/L), or D5UF (10.23 +/- 6.69 mmol/L). But the total concentration of amino acids in D5OF (5.89 +/- 1.47 mmol/L) was significantly lower than the three fluids above. Some individual amino acids varied significantly depending on where they were collected and from which day. The blastocyst rates of porcine IVF embryos were not improved when embryos were cultured in PZM3 with amino acids at D3OF (PZM3-D3OF, 20.3 +/- 7.9%) or D5UF (PZM3-D5UF, 14.3 +/- 10.7%) concentrations or in PZM3-D3OF for the first 48 (20.5 +/- 15.1), 72 (25.6 +/- 10.4), and 96 (18.7 +/- 10.0) hr and then transferred into PZM3-D5UF compared with PZM3 with Sigma amino acid solution (PZM3-SAA) (30.8 +/- 9.1%). However, when IVF embryos were cultured in PZM3-D5UF, the average nuclear number per blastocyst (57.6 +/- 8.3) was increased compared to PZM3-SAA (40.5 +/- 3.5). The osmolalities in D3OF, D3UF, D5OF, and D5UF were 318 +/- 8, 320 +/- 32, 321, and 293 +/- 8 mOsM, respectively. When the IVF embryos were cultured in PZM3-SAA and PZM3-D3OF at a variety of osmolalities (150-360 mOsM), higher blastocyst rates were obtained at 270-300 mOsM in the PZM3-SAA group (24.6-33.9%) and 270-290 mOsM in PZM3-D3OF group (22.4-24.2%). The blastocyst rate gradually decreased when the osmolality was increased or decreased in both groups. When the embryos were cultured in PZM3-SAA at 330 mOsM for the first 72 hr and then transferred to 250 mOsM (33.3 +/- 3.4%), the blastocyst rate was higher than original PZM3 (21.2 +/- 2.2%) (288 mOsM).     

Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.     

Effects of oxygen tension and humidity on the preimplantation development of mouse embryos produced by in vitro fertilization: analysis using a non-humidifying incubator with time-lapse cinematography

To examine the effects of oxygen tension and humidity on early embryonic development, the preimplantation development of mouse embryos produced by in vitro fertilization was assessed by time-lapse cinematography to evaluate morphokinetic development with higher precision. Zygotes were produced from spermatozoa and oocytes from ICR mice and cultured in KSOM under low or high oxygen tension in a non-humidified incubator with time-lapse cinematography (CCM-iBIS). The developmental rates of embryos to the 4-cell and blastocyst stages under lower oxygen tension in CCM-iBIS were significantly higher than those under higher oxygen tension in CCM-iBIS. Ninety-six hours after insemination, a large number of embryos cultured under low oxygen tension developed to the hatching blastocyst stage. Embryonic development was more synchronized under lower oxygen tension. Non-humidified cultures did not affect embryonic development. On average, mouse embryos cultured at lower oxygen tension reached 2-cell at 18 h, 3-cell at 39 h, 4-cell at 40 h, initiation of compaction at 58 h, morula at 69 h, and blastocyst at 82 h after insemination. In conclusion, lower oxygen tension better supports preimplantation development of mouse embryos fertilized in vitro, and non-humidified culture conditions do not influence the embryonic development in vitro.     

Developmental,qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro

The objective of this study was to compare bovine and ovine oocytes in terms of (1) developmental rates following maturation, fertilization, and culture in vitro, (2) the quality of blastocysts produced in vitro, assessed in terms of their ability to undergo cryopreservation, and (3) the ultrastructural morphology of these blastocysts. In vitro blastocysts were produced following oocyte maturation/fertilization and culture of presumptive zygotes in synthetic oviduct fluid. In vivo blastocysts were used as a control from both species. In Experiment 1, the cleavage rate of bovine oocytes was significantly higher than that of ovine oocytes (78.3% vs. 58.0%, respectively, P < 0.001). The overall blastocyst yield was similar for both species (28.7% vs. 29.0%). However, when corrected for cleavage rate, significantly more ovine oocytes reached the blastocyst stage at all time-points (36.6% vs. 50.0% on day 8, for bovine and ovine, respectively, P < 0.001). Following vitrification, there was no difference in survival between in vivo produced bovine and ovine blastocysts (72 hr: 85.7% vs. 75.0%). However, IVP ovine blastocysts survived at significantly higher rates than IVP bovine blastocysts at all time points (72 hr: 47.4% vs. 18.1%, P < 0.001). At the ultrastructural level, compared with their in vivo counterparts, IVP blastocysts were characterized by a lack of desmosomal junctions, a reduction in the microvilli population, an increase in the average number of lipid droplets and increased debris in the perivitelline space and intercellular cavities. These differences were more marked in bovine IVP blastocysts, which also displayed electron-lucent mitochondria and large intercellular cavities. These observations may in part explain the species differences observed in terms of cryotolerance. In conclusion, the quality of ovine blastocysts was significantly higher than their bovine counterparts produced under identical in vitro conditions suggesting inherent species differences between these two groups affecting embryo quality.     

Efficient harvesting methods for early‐stage snake and turtle embryos

Reptile development is an intriguing research target for understating the unique morphogenesis of reptiles as well as the evolution of vertebrates. However, there are numerous difficulties associated with studying development in reptiles. The number of available reptile eggs is usually quite limited. In addition, the reptile embryo is tightly adhered to the eggshell, making it a challenge to isolate reptile embryos intact. Furthermore, there have been few reports describing efficient procedures for isolating intact embryos especially prior to pharyngula stage. Thus, the aim of this review is to present efficient procedures for obtaining early‐stage reptilian embryos intact. We first describe the method for isolating early‐stage embryos of the Japanese striped snake. This is the first detailed method for obtaining embryos prior to oviposition in oviparous snake species. Second, we describe an efficient strategy for isolating early‐stage embryos of the soft‐shelled turtle.     

Nuclear lamin antigen and messenger RNA expression in bovine in vitro produced and nuclear transfer embryos

The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10). Lamin A/C was detected in 9/10 immature oocytes, 10/10 zygotes, 8/10 2-cell embryos, 4/10 morulae, 10/10 blastocysts but absent during the maternal embryonic transition. Lamin B was ubiquitously expressed during IVF preimplantation development but was only detected in 4/10 GV oocytes. Messenger RNA expression confirms that the major lamins, A/C and B1 are expressed throughout preimplantation development and transcribed by the embryo proper. Lamin A/C and B expression were observed (15 min, 30 min, 60 min, 120 min) following somatic cell nuclear transfer using adult fibroblasts and at the 2-cell, 8-cell, 16-32-cell, morula and blastocyst stage (n = 5). Altered expression levels and localization of nuclear lamins A/C and B was determined in nuclear transfer embryos during the first 2 hr post fusion, coincidental with only partial nuclear envelope breakdown as well as during the initial cleavage divisions, but was restored by the morula stage. This mechanical and molecular disruption of the nuclear lamina provides key evidence for incomplete nuclear remodeling and reprogramming following somatic cell nuclear transfer.     

Total protein content and protein synthesis within pre-elongation stage bovine embryos

Protein content was measured in zona-free bovine oocytes and pre-elongation stage embryos, following in vitro maturation, fertilisation, and then culture in Synthetic Oviduct Fluid medium supplemented with amino acids and 8 mg ml^-1 bovine serum albumin (BSA). Values (ng embryo^-1) of 122 ± 7.8, 137 ± 8.6, 111 ± 8.8, 115 ± 10.4, 139 ± 9.0 and 152 ± 10.1 were obtained for zona-free mature oocytes, 2-cell (day 2), 8-cell (day 3), compact morula (day 6), blastocyst (day 7), and expanded blastocyst (day 8) stage embryos, respectively. The protein content of day 7 zona-enclosed blastocysts was 337 ± 58.0 ng embryo^-1. These values suggest that prior to compaction and blastulation, the early cleavage stage bovine embryo has a higher rate of protein degradation than that of synthesis. Net growth is observed only after initiation of compaction. The protein content of day 7 blastocysts was measured in embryos following in vitro production and culture in the same media supplemented with either 0.5% w/v polyvinyl alcohol (PVA), 8 mg ml^-1 BSA, 8 mg ml^-1 BSA and further supplemented with 10% fetal calf serum (FCS) from the beginning of culture (FCS-D1), 8 mg ml^-1 BSA and 10% FCS from the fourth day of culture (day 5 of development) or from in vivo-derived day 7 blastocysts. Protein content was significantly (P< 0.05) lower in PVA-cultured embryos than other treatments. To determine if this difference in PVA-cultured embryos was due to a difference in the rate of protein synthesis, comparisons were made between day 7 embryos derived from BSA-culture and either PVA-culture, FCS-D1 culture or in vivo-derived embryos. Despite differences in diameter, no significant difference was observed in the incorporation of L-[2,3,4,5,6-³H]-phenylalanine into the TCA-precipitable fraction in any of the three comparisons made. However, incubation in the presence of FITC-labelled BSA or β-casein and examination under either fluorescence or confocal microscopy revealed that protein in the extra-embryonic environment was actively taken up by the trophectoderm of day 7 blastocysts, most likely by endocytosis. These results suggest that exogenous protein is an important nutritive source, probably maintaining intracellular amino acid pools. Results obtained from the production of embryos in protein-free medium should be viewed with the knowledge that such embryos differ metabolically from those embryos grown in the presence of protein, including in vivo-derived embryos. Mol. Reprod. Dev. 50:139–145, 1998. © 1998 Wiley-Liss, Inc.     

Apoptosis in porcine cumulus‐oocyte complexes: Relationship with their morphology and the developmental competence

The aim of this study was to assess the presence and distribution of apoptosis in porcine cumulus‐oocyte complexes (COCs) and its relations with COC morphology and developmental competence. The COCs were obtained from slaughterhouse ovaries, classified into A1 (top category), A2, B1, B2, C, and D based on their morphology. A1, A2, and B1 were matured and fertilized in vitro, and blastocyst rate was compared among them. Before and after in vitro maturation (IVM), annexin‐V staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to assess early and late apoptosis, respectively. There was a significant increase in both annexin‐V (+) oocytes and TUNEL (+) cumulus cells as morphology further deteriorated. There were no statistical differences regarding annexin‐V (+) oocytes within immature and post‐IVM COCs, but TUNEL (+) oocytes were only observed in post‐IVM COCs. Early and late apoptosis was detected in cumulus cells of all categories of immature and post‐IVM COCs. However, the difference was only significant for annexin‐V (+). There were no significant differences in embryo development. Therefore, apoptosis increases as the morphological features of the immature COCs decrease. In conclusion, the selection of COCs from Categories A1, A2, and B1 may be used as a selection criterion for in vitro development.     

Effects of hyaluronic acid in culture and cytochalasin B treatment before freezing on survival of cryopreserved bovine embryos produced in vitro

Summary One limitation to the widespread use of in vitro-produced embryos in cattle is their poor survival following cryopreservation. Two approaches for enhancing survival of in vitro-produced bovine embryos following cryopreservation were evaluated: culture in the presence of hyaluronic acid and alterations in the cytoskeleton through cytochalasin B treatment. The experiment was a 2×2 factorial design to test main effects of hyaluronic acid added to culture at day 5 after insemination (+or−) and cryopreservation treatment (control or cytochalasin B). Embryos used for cryopreservation were blastocysts and expanded blastocysts harvested on day 7 after insemination. Cytochalasin B increased the percent of embryos that re-expanded (P<0.0001) and that hatched following thawing (P<0.05). The hatching percent was 29.6% for embryos treated with cytochalasin B versus 9.1% for control embryos. There was no significant effect of hyaluronic acid on survival although there was a tendency for embryos cultured with hyaluronic acid to have higher percent hatching if not treated with cytochalasin B (12.7% for hyaluronic acid versus 4.5% for control; hyaluronic acid x cytochalasin B interaction; P=0.09). In conclusion, cytochalasin B treatment before freezing improved cryosurvival of bovine embryos produced in vitro. Such a treatment could be incorporated into methods for cryopreservation of bovine embryos provided post-transfer survival is adequate. In contrast, culture with hyaluronic acid was of minimal benefit—the increased cryosurvival in the absence of cytochalasin B was not sufficient to allow an adequate number of embryos to survive.