In-straw cryoprotectant dilution of IVP bovine blastocysts vitrified in hand-pulled glass micropipettes

The aim of this study was to determine the influence of two ethylene glycol-based vitrification solutions on in vitro and in vivo survival after in-straw cryoprotectant dilution of vitrified in vitro-produced bovine embryos. Day-7 expanded blastocysts were selected according to diameter (> or = 180 microm) and osmotic characteristics and randomly assigned to one of three groups (i) VSa: vitrification in 40% EG+17.1% SUC+0.1% PVA; (ii) VSb: vitrification in 20% EG+20% DMSO; (iii) control: non-vitrified embryos. Vitrification was performed in hand-pulled glass micropipettes (GMP) and cryoprotectant dilution in 0.25 ml straws after warming in a plastic tube. Embryo viability was assessed by re-expansion and hatching rates after 72 h of IVC and by pregnancy rates after direct transfer of vitrified embryos. No differences in re-expansion rates were observed between vitrified groups after 24 h in culture (VSa=84.5%; VSb=94.8%). However, fewer VSa embryos (55.2%, P<0.05) hatched after 72 h than the VSb (75.8%) and control embryos (80.0%). To evaluate in vivo viability, vitrified embryos (VSa=20; VSb=21) were warmed under field conditions and individually transferred to synchronous recipients. Pregnancy rates (day 60) were similar between groups (VSa=20%; VSb=19%). Greater hatching rates occurred after 72 h of IVC for EG+DMSO than EG+SUC+PVA vitrification solutions. However, using a GMP vitrification container and in-tube warming, both solutions provided similar pregnancy rates after the in-straw cryoprotectant dilution and direct embryo transfer.     

Effect of sugars-addition on the survival of vitrified bovine blastocysts produced in vitro

We investigated the effect of addition of sugars to a vitrification solution on the survival rate of bovine blastocysts produced in vitro. In vitro-matured (IVM) and in vitro-fertilized (IVF) bovine Day-6 to Day-8 bovine blastocysts were classified into 3 developmental stages: early blastocysts, blastocysts and expanded blastocysts. The blastocysts were cryopreserved in 1 of 3 vitrification solutions: 1) 25% glycerol25% ethylene glycol (GE); 2) 20% glycerol20% ethylene glycol3/4 M sucrose (GES); and 3) 20% glycerol20% ethylene glycol3/8 M sucrose3/8 M dextrose (GESD). The basic solution was Dulbecco's PBS supplemented with 20% of fetal calf serum. Embryos were exposed to each vitrification solution in 3 steps, and after loading into 0.25-ml straws, were plunged into liquid nitrogen. After warming in water bath at 20 degrees C, cryoprotectants were diluted in 1/2 M and 1/4 M sucrose each for 5 min. Equilibration and dilution procedure except warming were conducted at room temperature (23 to 27 degrees C). After dilution, the embryos were cultured in Ham's F10 medium0.1 mM beta-mercaptoethanol20% fetal calf serum. Survival rates of embryos at 48 h of incubation of each of the 3 developmental stages (early blastocysts, blastocysts and expanded blastocysts) exposed to the 3 types of the vitrification solutions (GE, GES and GESD) were 23.5, 33.3, 65.8% (early blastocysts, blastocysts and expanded blastocysts respectively) in GE, 55.6, 71.9, 90.5% in GES and 84.6, 83.3, 95.8% in GESD respectively. These results indicate that a mixture of 25% glycerol25% ethylene glycol is not suitable for vitrification of early bovine blastocysts; however, addition of sugars to the solution significantly (P<0.01) improved the survival rate of the vitrified blastocysts, independently of their stage of development.     

Growth factors and growth hormone enhance in vitro embryo production and post-thaw survival of vitrified bovine blastocysts

The objective of this study was to assess the influence of specific growth factors and growth hormone (GH) in the culture medium on in vitro embryo production and post-thaw survival of vitrified blastocysts. In total, 1673 bovine oocytes were used for evaluating the nuclear status of the oocytes after in vitro maturation (n=560) or for in vitro fertilization (IVF, n=1113) and distributed in five treatment groups: (1). medium only control; (2). activin (10 ng/ml); (3). epidermal growth factor (EGF) (10 ng/ml); (4). insulin 5 microg/ml and (5). GH (100 ng/ml). There was an increase (P<0.05 and P<0.01, respectively) in the percentage of oocytes that reached meta phase II, developed to blastocysts and hatched, as well as in the blastocyst cell number in the groups treated with activin, EGF and GH compared to controls. There was no significant difference between insulin and control groups. A total of 465 blastocysts were vitrified in a three-step protocol using ethylene glycol and polyvinylpyrrolidone. After thawing, embryos were cultured in five treatments groups as described above. Groups EGF and GH had higher (P<0.05) survival rates with a mean blastocyst survival of 95.0+/-1.5 and 93.1+/-3.5%, respectively, while mean hatching rate was higher for EGF and activin groups (75.3+/-3.4 and 62.0+/-3.2%, respectively). Thawed control blastocysts had a mean cell count of 52.7+/-3.3%. With the exception of insulin, all growth factors and GH tested showed higher (P<0.01) total cell numbers when compared to controls. In conclusion, addition of growth factors and GH in the culture media has favorable effects on in vitro maturation, in vitro embryo production, and post-thaw survival of vitrified blastocysts.     

Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP)

The present work was designed to study the in vitro and in vivo viability, as assessed by blastocyst formation, pregnancy rate and term delivery of bovine embryos produced under completely defined conditions with or without insulin-like growth factor I (IGF-I) following direct transfer after cryopreservation. Slaughterhouse-derived bovine oocytes were matured for 24h, fertilized with frozen-thawed spermatozoa and cultured in vitro under completely defined conditions with or without exposure to IGF-I (5 ng/ml). Only those embryos classified as excellent or good quality blastocysts were frozen. Each blastocyst was individually loaded into a straw, seeded and pre-cooled to -7 degrees C. After 10 min at -7 degrees C straws were frozen further to -30 degrees C at a rate of 0.3 degrees C/min and then plunged into liquid nitrogen. Synchronized recipient cows received one embryo in the horn ipsilateral to the corpus luteum (CL). Pregnancies were diagnosed by ultrasonography 35-45 days after embryo transfer (ET). IGF-I failed to improve cleavage rate, as well as blastocyst production, when added during in vitro culture (IVC). Pregnancy outcome was not significantly improved in cows that received an IGF-I-treated embryo compared with controls (4/10 versus 3/10, respectively). Five out of six calves delivered to date were born alive and healthy. We have shown that it is possible to obtain healthy live offspring from frozen-thawed embryos produced under chemically defined conditions after direct transfer.     

Gene expression profiles during differentiation and transdifferentiation of bovine myogenic satellite cells

The molecular mechanisms underlying myogenic satellite cells (MSCs) differentiation into myotube-formed cells (MFCs) and transdifferentiation into adipocyte-like cells (ALCs) are unclear. As a step towards understanding the molecular mechanisms underlying MSC differentiation and transdifferentiation, we attempted to identify the genes differentially expressed during differentiation and transdifferentiation using gene microarray analysis (GMA). Thirty oligonucleotide arrays were used with two technical replicates and nine and six biological replicates for MFCs vs. MSCs and ALCs vs. MSCs, respectively, to contrast expression profile differences. GMA identified 1,224 differentially expressed genes by at least 2-fold during differentiation and transdifferentiation of MSCs. To select the highly expressed genes for future functional study, genes with a 4-fold expression difference were selected for validation by real time RT-PCR and approximately 96.9% of the genes were validated. The up-regulation of marker genes for myogenesis (MYL2, MYH3) and adipogenesis (PPAR??, and FABP4) was observed during the differentiation and transdifferentiation of MSCs into MFCs and ALCs, respectively. KOG analysis revealed that the most of the genes up-regulated during differentiation and transdifferentiation of MSCs were related to signal transduction. Again the exact location of 109 differentially expressed genes by 4-fold were analyzed by chromosome mapping. Among those, co-localization of 29 genes up-regulated during transdifferentiation with QTL for marbling score and intramuscular fat percentage supports the involvement of these genes in cellular transdifferentiation. Interestingly, some genes with unknown function were also identified during the process. Functional studies on these genes may unfold the molecular mechanisms controlling MSC differentiation and transdifferentiation.     

Effect of in-straw thawing on in vitro- and in vivo-development of vitrified mouse morulae

For the purpose of ascertaining parameters to embryo transfer on some domestic animals, mouse morulae were used as a model to investigate the effect of in-straw thawing on in vitro and in vivo-development of vitrified embryos. Embryos were vitrified in 0.25 ml straws preloaded with dilution solution (0.5 M Sucrose) and thawed in the straw by mixing the vitrification solution (Ethylene glycol + Ficoll 70 + Sucrose) and the dilution solution at 25 degrees C. The embryos were randomly divided into six groups and expelled from the straws after they had been suspended in the in-straw mixture for 3 min, 5 min, 8 min, 12 min, 16 min, and 20 min, respectively, and then they were collected under a microscope for in vitro culture or direct transfer. The in vitro developmental rates of the embryos were 92.3% to 98.4% and hatching rates were 64.1% to 75.6% for the groups of 3 min to 16 min, showing no significant differences with those of nonfrozen controls (100%, 76.2%; P > 0.05). While embryos were suspended in the straw for 20 min, the developmental rate (86.6%) and hatching rate (52.4%) were significant lower than those of the control (100%, 76.2%; P < 0.01). When the 168 frozen-thawed embryos (in-straw thawing for 5 min) and 168 fresh embryos were transferred, respectively, the proportion of live fetuses in the pregnant recipients between them (58.7% vs. 54.5%) showed no significant difference (P > 0.05). The data indicate that vitrification with EFS30 and suspension in the in-straw mixture for 3 min to 16 min, when thawing, did not affect the in vitro developmental rate and hatching rate. Moreover, the in vivo developmental rate between vitrified embryos and fresh embryos did not differ significantly. It can be concluded that this method is fit for nonsurgical embryo transfer in some domestic animals with a suggestion that the operation of embryo transfer should be accomplished within 16 min.     

Development of porcine blastocysts from in vitro- matured and activated haploid and diploid oocytes

The purpose of this study was to determine the developmental capacity of electro-activated porcine oocytes. Follicular oocytes collected from gilts at local slaughterhouses were matured for 48 h and were then subjected to a single square pulse of direct current for 100 rhojusec at 1,500 V/cm for activation. To obtain activated diploid oocytes, some were treated with 5.0 micro/ml cytochalasin B for 4 h immediately after electro-activation. The frequency of activation ranged from 96 to 100%. While 91% of activated oocytes that had not been treated with cytochalasin B had 2 polar bodies and a nucleus (haploids), 92% of the oocytes treated with cytochalasin B had only the first polar body and 2 nuclei (diploids). Haploids and diploids were further cultured in TCM-199 medium that contained 10% (v/v) heat- treated fetal calf serum (FCS) and 0.1 mg/ml sodium pyruvate (mTCM) or in Whittenk medium plus 0.4% (w/v) bovine serum albumin (BSA). The frequency of abnormal oocytes was significantly higher in mTCM (83%) than in Whitten's medium (65%) 96 h after the electro-activation (P < 0.01), suggesting that Whitten's medium supported the development of activated oocytes beyond the morula stage. In all cases, several oocytes developed to the blastocyst stage 144 h after electro- activation (1 to 12%). The frequency was significantly higher in the case of diploids cultured in Whitten's medium (12%) (P < 0.01) than in the case of haploids cultured in Whitten's medium (4%), or in the case of haploids cultured in mTCM (1%). The mean number of nuclei per blastocyst was significantly lower in mTCM (haploids, 15; diploids, 16.1) than in Whitten's medium (haploids, 35.7; diploids, 40.1; P < 0.01), suggesting that the number of nuclei in blastocysts was affected by the culture medium.     

Prolonging bovine sperm-oocyte incubation in modified medium 199 improves embryo development rate and the viability of vitrified blastocysts

This study was conducted to compare the efficacy of four in vitro fertilization (IVF) media: Bracket and Oliphant's medium (BO), modified medium 199 (IVF-M199), modified Tyrode's medium (MTM), and modified KSOM (m-KSOM) on fertilization efficiency and blastocyst formation rate. In addition, we wanted to investigate the benefit of prolonging the IVF period (from 6 to 18 h) using the two most effective IVF media determined in our initial experiment; subsequently, blastocyst viability was assessed following vitrification. A higher incidence of polyspermic fertilization was observed in the MTM (6%) and in BO, in both the 6 and 18 h (7% and 11%, respectively) groups, than in the m-KSOM (1%) or in the IVF-M199 6 or 18 h (1 and 3%, respectively) groups. Cleavage rates were similar in BO, IVF-M199, and MTM 48 h post-fertilization; however, the lowest cleavage rate was observed for m-KSOM. A greater proportion of zygotes developed into 8-cell embryos in IVF-M199 than in other IVF media. Subsequently, a greater proportion of blastocyst formation and hatching was achieved in IVF-M199 (40% and 79%, respectively) or BO (35% and 74%, respectively) than in m-KSOM (18% and 58%, respectively) or MTM (22% and 66%, respectively). Prolonging IVF to 18 h did not alter cleavage rates; however, the highest rate of overall blastocyst formation was achieved in the IVF-M199 18 h (49%), rather than in the BO 18 h (20%) group. Vitrified/thawed blastocysts from IVF-M199 groups re-expanded and developed better, as compared to the BO 18 h group, and hatching rate and total cell number in IVF-M199 18 h group was comparable to the control groups (non-vitrified). Vitrification reduced survival compared to controls. In conclusion, IVF-M199 was successfully used for IVF, compared favorably to BO medium, and offered the advantage of an extended IVF period for up to 18 h that requires only one-half a dose of semen, and resulted in better quality blastocysts that endured vitrification with a hatching rate comparable to that of control groups.     

X chromosome regulation of autosomal gene expression in bovine blastocysts

Although X chromosome inactivation in female mammals evolved to balance the expression of X chromosome and autosomal genes in the two sexes, female embryos pass through developmental stages in which both X chromosomes are active in somatic cells. Bovine blastocysts show higher expression of many X genes in XX than XY embryos, suggesting that X inactivation is not complete. Here, we reanalyzed bovine blastocyst microarray expression data from a network perspective with a focus on interactions between X chromosome and autosomal genes. Whereas male-to-female ratios of expression of autosomal genes were distributed around a mean of 1, X chromosome genes were clearly shifted towards higher expression in females. We generated gene coexpression networks and identified a major module of genes with correlated gene expression that includes female-biased X genes and sexually dimorphic autosomal genes for which the sexual dimorphism is likely driven by the X genes. In this module, expression of X chromosome genes correlates with autosome genes, more than the expression of autosomal genes with each other. Our study identifies correlated patterns of autosomal and X-linked genes that are likely influenced by the sexual imbalance of X gene expression when X inactivation is inefficient.     

Infectivity of bovine viral diarrhea virus associated with in vivo-derived bovine embryos

Early research indicated that bovine viral diarrhea virus (BVDV) would not adhere to zona pellucida-intact (ZP-I), in vivo-derived bovine embryos. However, in a recent study, viral association of BVDV and in vivo-derived embryos was demonstrated. These findings raised questions regarding the infectivity of the embryo-associated virus. The objectives of this study were to evaluate the infectivity of BVDV associated with in vivo-derived bovine embryos through utilization of primary cultures of uterine tubal cells (UTC) as an in vitro model of the uterine environment and to determine if washing procedures, including trypsin treatment, were adequate to remove virus from in vivo-derived embryos. One hundred and nine ZP-I morulae and blastocysts (MB) and 77 non-fertile and degenerated (NFD) ova were collected on day 7 from 34, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to International Embryo Transfer Society (IETS) standards and exposed for 2h to approximately 10(6) cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1. Following exposure, some groups of <10 MB or NFD ova were washed in accordance with IETS standards. In addition, an equivalent number of MB and NFD ova were subjected to IETS standards for trypsin treatment. Subsequently, NFD ova were immediately sonicated and sonicate fluids were assayed for presence of virus, while individual and groups of MB were placed in microdrops containing primary cultures of UTCs and incubated. After 3 days, embryos, media, and UTCs were harvested from each microdrop and assayed for BVDV. Virus was detected in the sonicate fluids of 56 and 43% of the groups of NFD ova that were washed and trypsin-treated, respectively. After 3 days of microdrop culture, virus was not detected in media or sonicate fluids from any individual or groups of MB, regardless of treatment. However, virus was detected in a proportion of UTC that were co-cultured with washed groups of MB (30%), washed individual MB (9%) and trypsin treated individual MB (9%), but no virus was detected in the UTC associated with groups of trypsin-treated embryos. In conclusion, virus associated with developing embryos was infective for permissive cells. Further, the quantity of virus associated with a proportion of individual embryos (both washed and trypsin treated) was sufficient to infect the UTC. In light of these results, an attempt should be made to determine if the quantity of a high-affinity isolate of BVDV associated with an individual embryo would infect recipients via the intrauterine route.     

Gene expression profiles of vitrified in vivo derived 8-cell stage mouse embryos detected by high density oligonucleotide microarrays

Very little is known about the effect of vitrification on gene functions after warming. The goals of our study were to compare the gene expression patterns, and identify those most affected. For this, 8-cell stage embryos were collected from ICR mice and vitrified with solid surface vitrification technique, while maintaining equal numbers of embryos as control. Total RNAs were extracted and two rounds of amplification were employed. Finally three micrograms of contrasting RNA samples were hybridized on the Agilent Mouse 22 K oligonucleotide slides and the results were analyzed with subsequent verification by independent real-time PCR analyses. The two rounds of amplification with 5 ng tRNA input have yielded 15-16 microg of cRNA. The analyses of repeated hybridizations showed 20,183 genes/ESTs as common signatures, and unsupervised analysis identified 628 differentially expressed (P < 0.01) genes. However, with at least 1.5-fold change considerations, 183 genes were differentially expressed (P < 0.01) out of which 107 were upregulated. The independent analysis with real-time PCR and unamplified samples fully confirmed the results of microarray, indicating the linearity of amplification. Furthermore, this novel gene expression study for vitrified embryos identified many new candidate genes with overrepresentation in some important biological processes. Thus, it is possible to conclude that the expression pattern reflected a broad spectrum of consequences of vitrification on embryos, with most effects on metabolism, regulatory role and stress response genes and allowed the identification of new candidate marker genes for cryosurvival.     

Higher survival rate of vitrified and thawed in vitro produced bovine blastocysts following culture in defined medium supplemented with beta-mercaptoethanol

The present study was conducted to compare bovine embryo developmental quality, after culture in different defined culture media, up to blastocyst stage, and subsequently cultured in media supplemented with beta-mercaptoethanol (beta-ME) following blastocyst vitrification and thawing. In part one of this study, presumptive zygotes were randomly allocated into the following media: (1) CR1, (2) KSOM, (3) SOF, and (4) sequential KSOM-SOF. In the second part of the study, blastocysts derived from four different culture media were subjected to a solid surface vitrification (35% (v/v) ethylene glycol+0.5M Sucrose+5% (w/v) Polyvinylpyrrolidone (PVP), and tested for the effect of beta-ME on their post-vitrification survival. Following thawing, blastocysts were cultured with or without beta-ME. Culture medium had no effect on cleavage rates; however, a significantly greater number of zygotes cultured in KSOM, KSOM-SOF, or SOF developed to the 8-cell stage, compared with those cultured in CR1. A greater proportion of the zygotes cultured in SOF or KSOM-SOF reached blastocysts, than did those cultured in CR1 or KSOM. The use of sequential KSOM-SOF significantly increased total cell numbers of Day 7 expanded-blastocysts when compared to those cultured in CR1, KSOM, or SOF. Addition of beta-ME into culture media after vitrification and thawing improved blastocyst survival, hatching rates, and total cell numbers of blastocysts. In conclusion, supplementation of beta-ME into culture medium after vitrification and thawing significantly increased blastocyst survival, hatching rates, and their total cell numbers. These results suggest that vitrified IVF embryos should be thawed and briefly cultured in beta-ME medium prior to embryo transfer.     

Piglets born from centrifuged and vitrified early and peri-hatching blastocysts

Cryopreservation of zona-intact porcine embryos has been relatively unsuccessful to date, although some success has been obtained with lipid reduced morulae and early blastocysts. This study adapted some vitrification protocols used successfully with late blastocysts for use with early zona-intact blastocysts, using actin depolymerization, centrifugation, and open-pulled (OPS) straws. Initially, Day 6 peri-hatching blastocysts were collected, cultured for 40 min in 7.5 microg/ml cytochalasin B and vitrified in 6.5 M glycerol and 6% BSA (VS1) in either heat-sealed (HS) or open straws (OS). The post-thaw survival of those stored in HS was 15.4% after 24 and 48 h in vitro; storage in OS significantly improved survival (58.8% for both 24 and 48 h). When similar stage blastocysts were cultured in cytochalasin B and vitrified with 8 M ethylene glycol and 7% polyvinylpyrrolidone (PVP; VS2) in OS, survival was 44.4 and 33.3% for 24 and 48 h, respectively. Day 5 late morulae and early blastocysts were collected, cultured with cytochalasin B, and centrifuged or left intact (control), then vitrified with VS1 in HS or OS, or vitrified in VS2 in OS only. None of the intact control embryos survived thawing and 48 h culture in vitro. Centrifuged early blastocysts vitrified with VS1 showed good post-thaw survival in culture when stored in HS (62.8 and 60.5% for 24 and 48 h, respectively), or OS (75 and 63.6%). When vitrified with VS2 in OS, survival improved (80 and 76.7%). Peri-hatching blastocysts were vitrified in VS1, and early blastocysts were vitrified with VS1 and VS2. All blastocysts were stored in OS. The embryos were recovered and transferred to Day 4 and 5 pseudopregnant recipients (for Day 5 and 6 blastocysts, respectively). Of the five recipients receiving peri-hatching blastocysts, two became pregnant and delivered a total of eight piglets. All three recipients of early blastocysts vitrified in VS1 had a delayed return to estrus; while of the four receiving embryos vitrified with VS2, two were delayed in returning to estrus, and one was confirmed pregnant after 45 days. A litter of five piglets, one male and four female, was produced at 116 days of gestation. To our knowledge, this is the first litter of piglets produced from early blastocysts vitrified without micromanipulation to remove polarized lipid droplets.     

Ultrastructure and cell death of in vivo derived and vitrified porcine blastocysts

Morphological and molecular signs of injury and cell death and subsequent regeneration following vitrification of porcine blastocysts were evaluated by light (LM) and transmission electron microscopy (TEM) as well as TUNEL/propidium iodide (PI) nuclear staining followed by confocal microscopy (CSM). In vivo derived blastocysts were assigned to one of the following four groups: Controls-(1) fixed immediately after collection (C0h) and (2) after 24 hr culture in vitro (C24h) and vitrified embryos-(3) fixed immediately after vitrification and warming (V0h), and (4) after 24 hr of culture upon warming after vitrification (V24h). Observation by LM and TEM showed that the V0h embryos displayed collapse of the blastocoele cavity (BC) and cell swelling, a general distension or shrinkage of mitochondria and massive increase in the amount of vesicles, vacuoles, and secondary lysosomes (SLs). Approximately 2/3 of the V24h embryos had recovered, whereas the remaining 1/3 were degenerated. Recovered embryos displayed almost normal blastocyst morphology, except for a widening of the perivitelline space, accumulation of debris and partial distension of mitochondria, whereas degenerated embryos were disintegrated into a poorly defined mass of cells and debris including cells with abundant degeneration of mitochondria and other organelles. Both recovered and degenerated embryos displayed a persistent abundance of presence of small membrane bounded vesicles, vacuoles, and SLs. Evaluation of TUNEL/PI stained embryos showed only occasional appearance of TUNEL positive nuclei with typical apoptotic morphology in controls (C0h 0.67%, C24h 1.22%) and in the V0h embryos (0.93%). The percentage of apoptotic nuclei in embryos at V24h was significantly higher than in all other groups (2.64%). Vitrified embryos showed significantly increased appearance of DNA fragmented nuclei without typical morphological features of apoptosis (V0h 1.43%, V24h 4.30%) compared with controls (C0h 0.26%, C24h 0.45%). The observed morphological changes and increased DNA fragmentation observed immediately after vitrification and warming probably reflects a direct damaging effect of vitrification. During 24 hr of culture a portion of the embryos was able to regenerate and along with the regenerative process, apoptosis--a possible pathway for elimination of damaged cells--became evident.