Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures

In this study, three different vitrification systems (open pulled straw: OPS; superfine open pulled straw: SOPS; and Vit-Master technology using SOPS: Vit-Master-SOPS) were compared in order to investigate the influence of cooling rate on in vitro development of vitrified/warmed porcine morulae, early blastocysts, or expanded blastocysts. Embryos were obtained surgically on Day 6 of the estrous cycle (D0 = onset of estrus) from weaned crossbred sows, classified and pooled according their developmental stage. A subset of embryos from each developmental stage was cultured to evaluate the in vitro development of fresh embryos; the remaining embryos were randomly allocated to each vitrification system. After vitrification and warming, embryos were cultured in vitro for 96 h in TCM199 with 10% fetal calf serum at 39 degrees C, in 5% CO(2) in humidified air. During the culture period, embryos were morphologically evaluated for their developmental progression. The developmental stage of embryos at collection affected the survival and hatching rates of vitrified/warmed embryos (P < 0.001). The vitrification system or the interaction of vitrification system and developmental stage had no effect on these parameters (P > 0.05). Vitrified expanded blastocysts showed the best development in vitro (P < 0.001), with survival and hatching rates similar to those of fresh expanded blastocysts. The hatching rate of fresh morula or early blastocyst stage embryos was higher than their vitrified counterparts. In conclusion, under our experimental conditions, cooling rates greater than 20,000 degrees C/min, as occurs when SOPS or Vit-Master-SOPS systems are used, do not enhance the efficiency of in vitro development of vitrified porcine embryos.     

Vitrification of in vitro produced ovine embryos at various developmental stages using two methods

This study was conducted to evaluate the effects of developmental stage of in vitro produced (IVP) ovine embryos and the type of vitrification procedure used on embryo cryotolerance.The IVP embryos were vitrified at five different developmental stages: 4-, 8- and 16-cell, morula, and blastocyst. For each stage, half of the embryos were vitrified in either 30 μl 3.4 M glycerol + 4.6 M ethylene glycol in straw (method 1) or in <0.1 μl 2.7 M ethylene glycol + 2.1 M Me₂SO + 0.5 M sucrose placed on the inner surface of a straw (method 2) of vitrification solution, based on two different procedures. After warming embryo viability was determined by assessing the rates of re-expansion, survival, and blastocyst formation. The quality of surviving embryos was evaluated by their hatching rate and blastocyst cell numbers. In both vitrification methods, embryo survival progressively increased as the developmental stage progressed. In method 1 few of the early cleavage stage embryos (4-, 8- and 16-cell) could reach to the blastocyst stage following warming. There was no significant difference in blastocyst cell numbers (total, ICM, and trophectoderm cells) or hatching rate of blastocysts derived from vitrified embryos at different developmental stages. The number of dead cells in vitrified blastocysts in method 1 was higher than for non-vitrified blastocysts (P < 0.05). The number of apoptotic cells in vitrified blastocysts was higher than for non-vitrified counterparts (P < 0.05). In conclusion, both the developmental stage of IVP ovine embryos and the method of vitrification have a significant effect on the viability and developmental competence of sheep embryos.     

Tolerance to vitrification of rat embryos at various developmental stages

Numerous genetically engineered rat strains have been produced via genome editing. Although freezing of embryos is helpful for the production and storage of these valuable strains, the tolerance to freezing of embryos varies at each developmental stage of the embryo. This study examined the tolerance to freezing of rat embryos at various developmental stages, particularly at the pronuclear stage. Embryos that had developed to the pronuclear, 2-cell, and morula stages were frozen via vitrification using ethylene glycol- and propylene glycol-based solutions. More than 90% of the embryos at all developmental stages survived after warming. The developmental rates to offspring of thawed embryos at the pronuclear, 2-cell, and morula stages were 19%, 41%, and 52%, respectively. Pronuclear stage embryos between the early and late developmental stages were then vitrified. The developmental rates to offspring of the thawed pronuclear stage embryos collected at 24, 28, and 31 h after the induction of ovulation were 17%, 21%, and 23%, respectively. These results indicated that the tolerance to vitrification of rat embryos increased with the development of embryos. The establishment of vitrification method of rat embryos at various developmental stages is helpful for improving the production and storage of valuable rat strains used for biomedical science.     

Equilibrium vitrification of mouse embryos at various developmental stages

Previously, we developed a new method by which 2‐cell mouse embryos can be vitrified in liquid nitrogen in a near‐equilibrium state, and then kept at ?80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight‐cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol‐based solutions, named EFSc because of their composition of ethylene glycol (30–40%, v/v) and FSc solution. The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM‐70 plus 1.5 M sucrose. The extent of equilibrium was assessed by examining how well vitrified embryos survived after being kept at ?80°C. When 8‐cell embryos and morulae were vitrified with EFS35c or EFS40c and then kept at ?80°C, the survival rate was high even after 4 days in storage and remained high after re‐cooling in liquid nitrogen. On the other hand, the survival of vitrified‐expanded blastocysts kept at ?80°C was low. Therefore, 8‐cell embryos and morulae can be vitrified in a near‐equilibrium state using the same method as for 2‐cell embryos. A high proportion of C57BL/6J embryos at the 2‐cell, 8‐cell, and morula stages vitrified with EFS35c developed to term after transportation on dry ice, re‐cooling in liquid nitrogen, and transfer to recipients. In conclusion, the near‐equilibrium vitrification method, which is effective for 2‐cell mouse embryos, is also effective for embryos at the 8‐cell and morula stages. The method would enable handy transportation of vitrified embryos using dry ice. Mol. Reprod. Dev. 79: 785–794, 2012. © 2012 Wiley Periodicals, Inc.     

Vitrification of mouse embryos at various stages by open-pulled straw (OPS) method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure-that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method-that is, embryos were first pretreated in 10%E + 10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E + 10%D for 0.5 min, exposed to EDFS30for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

Sensitivity to chilling of medaka (Oryzias latipes) embryos at various developmental stages

As an essential step toward cryopreservation of fish embryos, we examined the chilling sensitivity of medaka (Oryzias latipes) embryos at various developmental stages. Embryos at the 2-4 cell, 8-16 cell, morula, blastula, and early gastrula stages were suspended in Hanks solution. They were chilled to various temperatures (usually 0 degrees C), kept for various periods (usually 20 min), then cultured for up to 14 d to determine survival (assessed by the ability to hatch). Embryos at the 2-4 cell stage were the most sensitive to chilling to 0 degrees C, but sensitivity decreased as development proceeded. The survival rate of 2-4 cell embryos was affected after 2 min of chilling at 0 degrees C; although the rate decreased gradually as the duration of chilling increased, 38% of them still survived after 40 min of chilling. Embryos at the 2-4 cell stage were sensitive to chilling at 0 or -5 degrees C, but much less sensitive at 5 or 10 degrees C. The survival rate of 2-4 cell embryos subjected to repeated rapid cooling and warming was similar to that of those kept chilled. When early gastrula embryos were preserved at 0 or 5 degrees C, the hatching rate did not decrease after 12 and 24h of chilling, respectively, but then decreased gradually as storage was prolonged; however, 3-10% of the embryos hatched even after storage for 10 d. In conclusion, although later-stage medaka embryos would be suitable for cryopreservation (from the perspective of chilling sensitivity), chilling injury may not be serious in earlier stage embryos.     

Double vitrification of rat embryos at different developmental stages using an identical protocol

The aim of the present investigation was to test the effectiveness of a method of vitrifying rat embryos at different stages of development (from early morula to expanding blastocyst) in a double vitrification procedure. Wistar rat embryos were vitrified and warmed in super-fine open-pulled straws (SOPS). Before being plunged into liquid nitrogen, the embryos were exposed to 40% ethylene glycol+0.75 M sucrose in TCM-199+20% fetal calf serum (FCS) for 20s at 38 degrees C. Subsequent warming and direct rehydration of the embryos was conducted in culture medium (TCM-199+20% FCS) at 38 degrees C. Early morula stage (7-10 blastomeres) embryos (n=358) were vitrified, warmed and cultured in vitro (EM group). Batches of these embryos were then cryopreserved again (revitrified) at the early blastocyst (EB group, n=87), blastocyst (B group, n=93) or expanding blastocyst stage (ExpB group, n=73). After the first (EM group) and repeated (EB, B, and ExpB groups) vitrification procedures, developmental rates of 81, 83, 34 and 76%, respectively were achieved (for EM-EB-ExpB P>0.1; for EM, EB, ExpB-B P<0.005). Our data demonstrate the possibility of using the described identical protocol for the SOPS vitrification of rat early morulae, early blastocysts and expanding blastocysts. The low survival rate of blastocysts subjected to double vitrification requires further investigation.     

Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method

Mouse oocytes and embryos at various developmental stages were exposed directly to an ethylene glycol-based vitrification solution (EFS) for 2 or 5 minutes at 20 degrees C. They were then vitrified at -196 degrees C and were warmed rapidly. At the germinal vesicle stage, the proportion of morphologically normal oocytes was 36 to 39% if they had cumulus cells, whereas in cumulus-removed immature oocytes and in ovulated oocytes it was only 2 to 4%. This low survival was attributed to the harmful action of ethylene glycol. After fertilization, on the other hand, the post-warming survival rate of 1-cell zygotes, as assessed by cleavage to the 2-cell stage, increased markedly (62%). As the developmental stage proceeded, higher proportions of vitrified embryos developed to expanded blastocysts; the rates increased up to 77 and 80% in 2-cell and 4-cell embryos, respectively. For embryos at the 8-cell, morula and early blastocyst stages, the proportion of embryos developed after vitrification (90 to 95%) was not significantly different from that of the untreated embryos (95 to 100%) when the period of exposure to EFS solution was 2 minutes. As the blastocoel began to enlarge, however, survival began to decrease again, with rates of 79 and 57% in blastocysts and expanded blastocysts, respectively. After the cryopreserved 2-cell, 4-cell and 8-cell embryos as well as morulae and blastocysts were transferred to recipients, 43 to 57% of the recipients became pregnant, and 48 to 60% of these various stage embryos developed into live young.     

Expression and localization of TRPC proteins in rat ventricular myocytes at various developmental stages

Growing evidence indicates that transient receptor potential canonical (TRPC) channels play important roles in various Ca²⁺-mediated physiological and pathophysiological processes, including development. Many types of TRPC proteins are expressed in the heart. However, limited data are available comparing the expression and localization among TRPC proteins in the ventricular myocyte at various developmental stages. Our purpose is to investigate the expression and localization profile of TRPC proteins in ventricular myocytes of fetal (18.5 days), neonatal (< 24 h after birth) and adult (8 week old) rats. Western blotting, immunofluorescence and confocal laser scanning microscopy were employed. TRPC1/3-6 proteins were expressed in the rat ventricle throughout the three developmental stages. The expression profile of TRPC1/3/4 in the ventricle followed an upward trend from the fetus to the adult. By contrast, TRPC6 in the ventricle was expressed at the highest level in the fetal group and was sharply down-regulated immediately after birth. TRPC5 expression in the ventricle did not change significantly during the three stages. TRPC1/3/5/6 proteins were localized to the T-tubule and TRPC1/3/4/6 to intercalated disks in adult myocytes. The wide spatiotemporal overlap and dynamic regulation of TRPC expression in ventricular myocytes indicates potential complex combinations and redundancy of native TRPC proteins in the heart and gives important clues for further investigations into the exact subunit compositions and functional properties of native TRPC channels in the heart.     

Sex ratios in mule duck embryos at various stages of incubation

Mule duck hatcheries have long reported varying degrees of unbalance in the sex ratio, with a preponderance of male mules at hatching. The aim of the present study was to assess the distributions of sex ratios at various stages of development in embryos originating from intra- and intergeneric crosses between parental lineages (Muscovy male x Muscovy female, Pekin male x Pekin female, Muscovy male x Pekin female or Mule, and Pekin male x Muscovy female or Hinny). In Experiment I, embryo sexing was performed on Days 1 and 5 of incubation (by multiplex PCR) and at hatching (by vent observation). The sex ratio was not significantly modified during the early stages of embryo development whatever the genetic origin (P>0.05, Days 1 and Day 5) but our results in mule and hinny ducklings confirmed the preponderance of males among normally hatched ducklings originating from the intergeneric lineage (58.9 and 55.4% males in mules and hinnies, respectively; P<0.05 in both cases). Sex ratio (vent sexing) in second grade (cull) ducklings revealed that 68% of these ducklings were females (P<0.05). In Experiment II, the distribution of sex ratio was also performed in mule duck eggs from 6 batches (400,000 eggs/batch) first examined for fertility (candling) on Day 18 of incubation. These results indicate that the percentage of males present in the population of normally hatched ducklings increases when fertility decreases. In addition, this experiment also revealed that 83.7-90.5% of viable male mule embryos develop up to hatching, compared to only 43.0-51.0% of female mule embryos. Given that a deviation in sex ratio during the first stages of incubation is unlikely (Experiment I), it is concluded that the skewed sex ratio of mule ducks at hatching is primarily due to increased late mortality in female mule embryos occurring between egg transfer and hatching. This mortality originated, at least in part, from the intergeneric origin of female mules, and was marked to a greater or lesser extent depending on the initial success of fertilization in a given batch, a possible indication that the initial quality of gametes may selectively exert its influence at the later stages of embryo development.     

Cell proliferation study at various stages of gastrulation in axolotl embryos

A study of cell proliferation in different regions of axolotl embryos has shown a rather uniform distribution of the S phase and mitotic indices in the animal half of the early and midgastrulae. The dorsal blastoporal lip is characterized by a very low S phase index as compared with the other regions of the embryo.     

Open pulled straw vitrification of goat embryos at various stages of development

This investigation addresses the question whether it is possible to apply the open pulled straw (OPS) vitrification method, found to be effective for cryopreserving caprine (Capra aegagrus hircus) blastocysts, to other embryonal stages. Morulae, blastocysts and hatched blastocysts were cryopreserved by way of OPS vitrification and blastocysts and hatched blastocysts by conventional freezing. Morulae were not included with conventional freezing because in our experience the survival rate is very low. To assess the viability of the cryopreserved embryos, they were transferred to synchronized does; in most cases, two embryos per doe. After OPS vitrification, of nine does receiving morulae, not a single one became pregnant; of 11 does receiving blastocysts, nine (82%) became pregnant (all of which kidded and gave birth to, on average, 1.8 kids); and of nine does receiving hatched blastocysts, three (33%) became pregnant (two of which [22%] kidded, giving birth to a single kid each). After conventional freezing, of 10 does receiving blastocysts, five became pregnant (four of which [40%] carried to term and gave birth to a pair of twins each); and of nine does receiving hatched blastocysts, three (33%) became pregnant (and gave birth to a single kid each). Embryo survival (kids born/embryos transferred) after vitrification for morulae, blastocysts, and hatched blastocysts was 0, 70% (16 of 23), and 13% (2 of 16), respectively, and after conventional freezing for blastocysts and hatched blastocysts was 42% (8 of 19) and 19% (3 of 16), respectively. The difference in pregnancy and kidding rate between vitrified and conventionally frozen blastocysts was significant, and so was the difference in pregnancy rate between hatched and nonhatched blastocysts, regardless whether OPS-vitrified or conventionally frozen. The results of the current study indicate that OPS vitrification is a very effective means of cryopreserving caprine blastocysts. Unfortunately, the superiority of OPS vitrification over conventional freezing does not apply to caprine morulae and hatched blastocysts.     

Effect of yolk redistribution at different developmental stages on survivorship of zebrafish embryos

Survival of zebrafish Danio rerio embryos subjected to yolk redistribution during early development (cleavage to segmentation) was found to be dependent on the stage of manipulation and the quantity of redistributed yolk. The findings point to the presence and importance of yolk intrinsic organization during early development.     

Two-dimensional protein maps of xenopus eggs and embryos at different developmental stages.

Protein expression during the early development of Xenopus has been followed by 2D-polyacrylamide gel electrophoresis (PAGE). The analysis of two-dimensional maps of eggs and embryos at different stages of development has allowed the separation of more than 2000 spots. Identification of numerous polypeptides was obtained in four different ways: (1) immuno-blotting; (2) amino terminal sequence after blotting on to PVDF membranes; (3) comigration; and (4) assignment in comparison with proteins separated by 2D techniques on reference maps such as human liver, red blood cells, plasma and cerebrospinal fluid reported in the Swiss 2D-PAGE Data Base. The maps presented in this report are a step toward the study of the protein expression in Xenopus eggs and embryos and may be a powerful working tool since Xenopus embryos are popular models for the study of development.     

Intracellular lectins of Lentinus edodes at various developmental stages of the fungus

A number of lectins varying in polypeptide composition and carbohydrate specificity were isolated from Lentinus edodes at different stages of its morphogenesis: nonpigmented mycelium, brown mycelium film, and fruiting body. Three lectins were identified at the nonpigmented mycelium stage, two of them being dimers consisting of 16 and 45 kDa and 16 and 42 kDa subunits; the third is a tetramer of 16, 39, 42, and 45 kDa subunits. The fractions with lectin activity obtained at the brown mycelium film stage contained polypeptides of 24, 30, and 38 kDa, characteristic of this morphological structure. The fruiting body was shown to contain two lectins of 43 and 55 kDa. All of the isolated lectins expressed the highest affinity towards L,D-melibiose, D-lactose, and D-galactose.     

Sensitivity to gamma radiation of Tribolium confusum eggs at various developmental stages

Eggs of six different age classes were exposed to gamma rays up to 9.000 rads. As regards the effect on hatchability, the eggs can be divided into two groups. The first group—eggs more than 4 days old—is not influenced by treatment, whereas the second group—younger eggs—is highly susceptible to radiation. As regards the post-embryonic development, relative numbers of the post-embryonic stages 40 days after egg-laying and the post-embryonic mortality are recorded.

---

Résumé Des oeufs de Tribolium confusum agés de 1 à 6 jours ont été irradiés à l'aide de rayons gamma. Les doses employécs variaient de 1,500 à 9,000 rad. L'éclosion des oeufs n'est pas ou est fortement influencée suivant que l'irradiation a lieu après ou avant le quatrième jour. Par contre le développement post-embryonnaire se différencie plus fortement en fonction des doses appliquées. On observe un certain retard dans l'évolution des descendants et une mortalité post-embryonnaire variant non seulement suivant l'âge des oeufs mais également suivant les doses appliquées. Ces arrière-effets sont donnés au diagramme I.

---

---

Research supported by the Institute for the Encouragement of Scientific Research in Industry and Agriculture (I.W.O.N.L.).     

Transitional changes in arachidonic acid metabolism by bovine embryos at different developmental stages

In order to determine the profile of arachidonic acid (AA) metabolites synthesized by bovine embryos during early developmental stages, embryos collected from superovulated beef cattle (days 6 through 17) were incubated with AA and its metabolites were analyzed by high performance liquid chromatography and radioimmunoassay (RIA). Embryos harvested and cultured before day 12 of the estrous cycle metabolized AA primarily to prostaglandin E2 (PGE2), whereas, those harvested on day 13 of the cycle metabolized AA to both PGE2 and PGF2 alpha. Furthermore, embryos collected after day 15 of the cycle metabolized AA to PGI2 in addition to PGE2 and PGF2 alpha. In view of the luteotropic properties that have been attributed to PGE2 and the vasodilatory effect of PGI2, this transitional change in prostaglandin synthesis during early stages of embryonic development may be a part of the mechanism by which the embryo exerts a luteotropic effect leading to maternal recognition of pregnancy and by which the conceptus begins preparing for subsequent implantation.     

Biochemical characterization of developmental stages of cycad somatic embryos

The effect of two light intensities (25 μmol m⁻²s⁻¹ and 50 μmol m⁻²s⁻¹) on four developmental stages ofCeratozamia mexicana somatic embryos growing on semisolid plant growth medium at 25°C was measured. Growth parameters included fresh weight, morphology, and invertase and peroxidase activity. Under low light conditions, fresh weight was greater in stages 1 and 2 than in stages 3 and 4. In addition, there was a high frequency of hyperhydricity and polyembryogenesis in stages 1 and 2, whereas stages 3 and 4 were nonhyperhydric and unbranched. Stages 2–4 were green. Under high light conditions, embryos had lower fresh weights and less hyperhydricity, and stages 2–4 were green. Under low light conditions, peroxidase activity was less, although stage 1 embryos under both light conditions showed the highest activity. Stage 1 embryos required three to four months to develop to stage 2 under high light conditions and two to three months under low light conditions. Invertase activity under low light conditions was minimal in stage 2. All embryos had low invertase activity under high light intensity, and stages 2–4 had high levels of glucose. Embryo development from stage 2 to the next and for each subsequent stage under high light conditions required three to four months, and under low light conditions required four to five months. Higher light intensity therefore promotes the speedy recovery of plants.

---

Resumen  El efecto de dos intensidades de luz (25 μmol m⁻²s⁻¹ y 50 μmol m⁻²s⁻¹) fue registrado en cuatro estados de desarrollo de embriones somáticos deCeratozamia mexicana cultivados en medio semisólido, 25°C. Los parámétras de crecimiento incluyeron peso fresco, peso seco, morfología, y actividad peroxidasa e invertasa. Bajo condiciones de baja iluminación, el peso fresco de los estados 1 y 2 fue mayor que en los estados 3 y 4. Además, hubo una alta frecuencia de hiperhidratación y poliembriogénesis en estados 1 y 2, mientras que los estados 3 y 4 no resultaron hiperhidratados ni ramificados. Los estados 2–4 fueron verdes. Bajo alta iluminación, los embriones tuvieron un menor peso fresco y menos hiperhidratación. En baja iluminación la actividad peroxidasa fue menor, aunque en los embriones en estado 1 en ambas condiciones de iluminación mostraron la más alta actividad. Los embriones en estado 1 requirieron tres o cuatro meses para desarrollarse hasta el estado 2 bajo condiciones de alta iluminación; y dos o tres meses en baja iluminación. La actividad invertasa en condiciones de baja iluminación fue minima en el estado 2. Todos los embriones tuvieron altos nivelés de glucosa. El desarrollo de los embriones de estado 2 al siguiente y a los subsecuentes, bajo alta iluminación, requirió tres o cuatro meses, y bajo condiciones de baja iluminación requirió cuatro o cinco meses. Una alta intensidad luminosa parece promover la velocidad de recuperación de plantas.