Improved cryopreservation of in vitro-produced bovine embryos using a chemically defined freezing medium

This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in bovine embryo cryopreservation solutions. During the experiment, fetal calf serum (FCS) and bovine serum albumin (BSA) were used as references. A combination of a thermodynamic approach using differential scanning calorimetry and a biological approach using in vitro-produced bovine embryo slow-freezing was used to characterize cryopreservation solutions containing CRYO3, FCS and BSA. The CRYO3 and fetal calf serum (FCS) slow-freezing solutions were made from Dulbecco's phosphate-buffered saline containing 1.5 m ethylene glycol, 0.1 m sucrose and 20% (v.v⁻¹) of CRYO3 or FCS. The bovine serum albumin (BSA) solution was made by adding 0.1 m sucrose to a commercial solution containing 1.5 m ethylene glycol and 4 g L⁻¹ BSA. These solutions were evaluated using three characteristics: the end of melting temperature, the enthalpy of crystallization (thermodynamic approach) and the embryo survival and hatching rates after in vitro culture (biological approach). The CRYO3 and FCS solutions had similar thermodynamic properties. In contrast, the thermodynamic characteristics of the BSA solution were different from those of the FCS and CRYO3 solutions. Nevertheless, the embryo survival and hatching rates obtained with the BSA and FCS solutions were not different. Similar biological properties can thus be obtained with slow freezing solutions that have different physical properties within a defined range. The embryo survival rate after 48 h of in vitro culture obtained with the CRYO3 solution (81.5%) was higher than that obtained with the BSA (42.2%, P = 0.000 12) and FCS solutions (58%, P = 0.016). Similarly, the embryo hatching rate after 72 h of in vitro culture was higher with the CRYO3 solution (61.1%) than with the BSA (31.1%, P = 0.0055) and FCS solutions (36%, P = 0.018). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in in vitro-produced bovine embryo cryopreservation solutions.     

Effects of phenazine ethosulfate during culture of bovine embryos on pregnancy rate, prenatal and postnatal development

The objective was to evaluate the use of phenazine ethosulfate (PES) during culture of embryos on fetal and postnatal calf development. Oocytes collected from abbatoir-derived ovaries were matured and fertilized, and the resulting embryos were cultured in vitro by standard procedures, in a chemically defined medium plus BSA. Day 0 of culture was 18 ± 2 h after the onset of IVF. From 2.5 to 6.5 d, half of the eight-cell embryos served as controls, and the remainder were exposed to 0.3 μM PES, which decreases lipid content of embryos. Good-quality blastocysts exposed to PES (n = 38) or control (n = 35) blastocysts were transferred nonsurgically to synchronized recipients in estrus 6-7.5 d earlier, resulting in 9 calves in each group. These in vitro-produced pregnancies were evaluated weekly between 35 and 98 d postestrus by ultrasonography, and postnatal development of the calves was monitored for 1 month. Based on a limited number of transfers, use of PES during in vitro culture did not affect pregnancy rates compared to the control at 35 or 98 d (P > 0.1). Transfer at 7-7.5 d after estrus resulted in higher 98-d pregnancy rates than at 6-6.5 d (34% vs. 10%; P < 0.05). In vitro-derived fetuses that aborted had retarded fetal and placental development compared to those that went to term, but there was no difference in fetal loss between the PES treatment and controls. One calf in the PES group weighing 36 kg was born dead at 252 d of gestation, and another calf in this group was dead some hours after birth and weighed 22.2 kg when parturition was induced at 310 d of gestation. It is unclear whether these two abnormal calves were caused by the PES treatment, or were due to in vitro procedures in general. In conclusion, the use of PES during in vitro culture had no effect (P > 0.1) on pregnancy rates, conceptus losses between Days 35 and 98 of pregnancy, nor fetal postnatal development in calves born normally.     

Closed pulled straw vitrification of in vitro-produced and in vivo-produced bovine embryos

The objective of this study was to evaluate the efficiency of the closed pulled straw (CPS) method for cryopreserving in vitro-produced and in vivo-produced bovine (Bos taurus) embryos. Based on the open pulled straw (OPS) protocol, the top end of a CPS was closed by tweezers (heated in a flame) to prevent the cryoprotectant medium containing embryos from contacting the liquid nitrogen. Bovine in vitro or in vivo morulae and early blastocyst embryos were frozen by slow cryopreservation, OPS vitrification, or CPS vitrification. Morphology of postthawed embryos was evaluated, and normal embryos were used for successive culture for 72 h. There were no significant differences between OPS and CPS freezing groups in postthawed in vitro-produced embryos with respect to rates of morphologically normal embryos (mean ± SD, 87.9 ± 5.2% vs. 85.4 ± 4.9%), survival at 24 h (58.0 ± 6.8% vs. 56.3 ± 4.4%), and survival at 72 h (35.2 ± 6.0% vs. 34.9 ± 6.7%). However, both OPS and CPS vitrification resulted in higher postthaw rates of morphologically normal embryo and survival at 24 and 72 h than those of the slow-freezing method (P < 0.05). Similar results were obtained for in vivo-derived embryos. We concluded that CPS vitrification was a feasible method to cryopreserve both in vitro-derived and in vivo-derived bovine embryos. This method not only eliminated the risk of embryo contamination by preventing contact with liquid nitrogen but also retained the advantages of the OPS vitrification method.     

New device for the vitrification and in-straw warming of in vitro produced bovine embryos

Two experiments were designed to test the use of a new device designed to vitrify and in-straw warm in vitro produced (IVP) embryos, which can potentially be used for their direct transfer to recipient females in field conditions. In experiment 1, IVP embryos from both prepubertal and adult animals were vitrified on cryotops and warmed in steps (1, 0.5 and 0 M sucrose; protocol W3) or directly in 0.5 M (protocol W1/0.5) or 0 M sucrose (protocol W1/0). Similar survival rates were recorded 24 h after warming for calf embryos irrespective of the warming procedure (W3: 79.2%, W1/0.5: 62.5%, W1/0: 66.7%). For cow embryos, survival rates at 24 h post-warming were significantly higher when embryos were warmed using the W3 (85.7%) or W1/0.5 (89.1%) protocols compared to the W1/0 protocol (70.5%). In experiment 2, IVP embryos were vitrified on the new designed device followed by their in-straw cryoprotectant (0.5 M sucrose) dilution/warming and different warming temperatures (45, 50, 60 and 70 °C) were tested. When warming solution passed through the new vitrification/warming device at 45 °C, 61.5% of blastocysts were fully re-expanded or hatched at 24 h post-warming, being not significantly different to the control (65%). Other warming temperatures triggered significantly lower survival rates at 24 h post-warming. No significant differences were detected in total cell numbers and blastocyst apoptosis indices in response to vitrification followed by warming at 45 °C respect to the control. Our findings indicate that the new device allows vitrification and in-straw warming of IVP bovine embryos, being a useful option for their direct transfer in field conditions.     

Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitro-produced bovine embryos

Embryo quality of in vitro-produced bovine blastocysts was assessed at several steps of a vitrification procedure in which glycerol and ethylene glycol were used as cryoprotectants (3-step equilibration with cryoprotectants followed by vitrification, dilution of the cryoprotectants in 0.85 M galactose then in embryo transfer freezing medium [ETF], and finally co-culture for periods). To visualize cell membrane alterations, double staining was performed using a cell permeant fluorochrome (bisbenzimide--BIS) and a nonpermeant one (propidium iodide--PI). In Experiment 1, the effect of the vitrification procedure on the hatching rate and total cell number was assessed 72 h after treatment. Hatching rate and the number of stained nuclei were decreased in comparison with untreated embryos when blastocysts were exposed to the whole procedure with or without vitrification (respectively 42 and 53% vs 76% for hatching and 128 +/- 17 and 141 +/- 17 vs 226 +/- 13 for stained nuclei). In Experiment 2, the effect of cryoprotectants and their dilution was evaluated on membrane permeability and total cell numbers at various steps of the vitrification procedure. Blastocysts exposed only to cryoprotectant solutions and stained immediately after dilution in galactose showed no modification. After dilution in ETF, the total number of stained nuclei decreased, and the number of blastomeres showing membrane permeabilization (PI-stained) increased (respectively, 74 +/- 5 vs 110 +/- 5 and 32 +/- 2% vs 0.1 +/- 1.8%). In Experiment 3, we demonstrated that the total number of stained nuclei after ethanol fixation (membrane permeabilization) was higher when embryos treated up to dilution in ETF were stained with PI than when the same embryos were stained with BIS. This suggests that, for unknown reasons, some nuclei of the treated embryos were not stained with BIS. Membrane permeabilization and inability of BIS to stain some nuclei were the most obvious alterations probably induced by osmotic shock at dilution. This hypothesis is supported by the fact that the introduction of a further dilution step in 0.42 M galactose (Experiment 4) before dilution in ETF decreased the proportion of cells permeant to PI and increased the hatching rate after 72 h of co-culture. In conclusion, double staining with BIS and PI allowed for discrimination between different types of cellular injuries after the various steps of our vitrification protocol. It represents a useful tool for adjusting equilibration and dilution conditions during a cryopreservation procedure.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

Exposure of in vitro-produced bovine embryos to foot-and-mouth disease virus

The aim of this study was to investigate whether foot and mouth disease virus (FMDV) interacts with in vitro produced (IVP) bovine embryos. One milliliter of a suspension of FMDV (2 x 10(7) TCID50/mL) was added to several batches of these embryos 7 d after in vitro fertilization, by which time they had either developed to the morula/blastocyst stage (n = 256) or degenerated (n = 260). Six experiments were performed in which developed or degenerated batches of embryos were incubated with FMDV for periods of 1 h (3), 2 h (2) or 4h (1). After this, the embryos were washed 10 times according to the International Embryo Transfer Society (IETS), then pooled and ground up to form a suspension, and assayed on cell cultures for FMDV. The cell cultures were observed daily for cytopathic effects for 3 d post exposure. In addition to the cell culture method, the polymerase chain reaction (PCR) technique was used to assay for the presence of the virus in the washing fluids. Assays for FMDV were also conducted on the first and second wash and on the pooled sample constituting the eight, ninth and tenth wash. With the exception of the second wash from a batch of embryos exposed to FMDV for 2 h, all samples of the first and second wash produced FMDV cytopathic effects, but none occurred with the pooled samples of the 8th, 9th and 10th wash. FMDV was also isolated from all but 1 of the batches of embryos after 1 h of incubation, from 1 of 4 batches after 2 h of incubation and from all batches after 4 h incubation. By contrast, the presence of virus could not be demonstrated by PCR based on the technique used here. These results show that 7 d old IVP bovine embryos can retain FMDV after washing, unlike in vivo-derived embryos, which do not appear to carry risks of FMDV transmission when washed according to IETS recommendations. Stricter controls are, therefore, necessary when using IVP embryos from cattle in a non-FMD-free zone in domestic or international trade.     

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.     

Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

The objective of this study was to determine the effect of fetal calf serum (FCS) on the quality of in vitro produced bovine embryos. Cumulus oocyte-complexes (COCs, n = 2 449) recovered by ovum pick-up from Bos taurus indicus donors were randomly assigned to experimental groups. Sperm selected by Percoll gradient was used for in vitro fertilization (insemination = Day 0). In Experiment 1 (n = 1 745 COCs), zygotes were cultured in vitro in Synthetic Oviduct Fluid + 4 mg/mL of bovine serum albumin (BSA), or BSA + 2% FCS (BSA+FCS). In Experiment 2 (n = 704 COCs), the COCs were cultured in SOF + BSA, BSA + 2% FCS, or BSA + 2% FCS on D4 (BSA + FCSD4). In Experiment 1, blastocyst yield (51%) and Quality I blastocysts (41%) at Day 7 were higher (P < 0.05) in the BSA + FCS treatment than in BSA (42 and 30%, respectively). In Experiment 2, blastocyst yield was higher (P < 0.05) in the BSA+FCS (47%) treatment. Quality I blastocyst yield was higher (P < 0.05) for BSA + FCS (34%) and BSA+FCSD4 (32%) compared to the BSA treatment (20%). A total of 820 embryos were transferred, with no significant differences among groups in pregnancy rates. In conclusion, in vitro culture in SOFaaci + BSA + FCS enhanced blastocyst yield and Quality I blastocysts; adding FCS to the culture medium increased the efficiency of IVP of bovine embryos.     

Effect of addition of hyaluronan to embryo culture medium on survival of bovine embryos in vitro following vitrification and establishment of pregnancy after transfer to recipients

Two experiments were conducted to determine whether addition of hyaluronan to culture medium could improve survival of bovine embryos after vitrification or following embryo transfer. In Experiment 1, embryos were produced in vitro and cultured for 7 days in modified synthetic oviductal fluid (SOF) containing one of four concentrations of hyaluronan (0, 0.1, 0.5, or 1 mg/mL), with or without 4 mg/mL of bovine serum albumin (BSA). On Day 7 after insemination, blastocysts and expanded blastocysts were vitrified using open-pulled straws. At a concentration of 1 mg/mL, hyaluronan increased (P < 0.05) the percentage of oocytes that were blastocysts and re-expansion rate at 24 h after warming. At 0.5 mg/mL, hyaluronan tended (P < 0.10) to increase re-expansion rate at 48 h after warming and increased (P < 0.05) embryo hatching rate at 24 and 72 h. Treatment with BSA caused a slight reduction in cleavage rate (P < 0.05), but only for cultures containing hyaluronan (BSA × hyaluronan, P = 0.10), an increase in the percentage of oocytes that became blastocysts (P < 0.001), and a reduction in re-expansion rates (P < 0.001) and hatching rates (P < 0.05 or P < 0.01) at all times examined. In Experiment 2, embryos were produced in vitro and cultured in modified SOF containing 4 mg/mL BSA, with or without 1 mg/mL hyaluronan. At 159-162 h after insemination, grade 1 morula, blastocysts and expanded blastocysts were harvested for embryo transfer. Harvested embryos were transferred individually to lactating Holstein recipients with a palpable corpus luteum on Day 7 after presumptive ovulation. There was an interaction (P < 0.05) between hyaluronan and embryo stage on pregnancy rate. Recipients that received morula and blastocyst stage embryos treated with hyaluronan had a higher pregnancy rate than recipients that received control embryos of the same stage. There was no effect of hyaluronan on pregnancy rates of recipients that received expanded blastocysts. In conclusion, addition of hyaluronan to embryo culture enhanced blastocyst yield, improved survival following vitrification, and enhanced the post-transfer survival of fresh morula and blastocyst stage embryos.     

Influence of sire breed (Bos indicus versus Bos taurus) and interval from slaughter to oocyte aspiration on heat stress tolerance of in vitro-produced bovine embryos

Based on in vitro experiments, Bos indicus embryos were more resistant to heat stress (HS) than Bos taurus embryos. To increase knowledge regarding differences between Bos indicus and Bos taurus in resistance to HS, the primary objective of this study was to determine if tolerance to HS is due to the breed, origin of the oocyte, sperm, or both. Additionally, the influence of the interval between ovary acquisition (in the abattoir) and oocyte aspiration in the laboratory, on early embryo development was ascertained. Oocytes were collected from Nelore and Holstein cows in an abattoir; 4.0 or 6.5 h later, oocytes were aspired in the laboratory, and then matured and fertilized using semen from Nelore (N), Gir (GIR), or Holstein (H) bulls. Ninety-six h post insemination (hpi), embryos with ≥ 16 cells were divided in two groups: control and HS. In the control group, embryos were cultured at 39°C, whereas in the HS group, embryos were subjected to 41°C for 12 h, and then returned to 39°C. Rates of cleavage, and formation of morula and blastocysts were higher (P < 0.05) for oocytes aspirated at 4.0 versus 6.5 h after ovaries were acquired. Heat stress decreased rates of blastocyst formation for all breeds (N × N; H × H; and H × GIR) and in both time intervals (4.0 and 6.5 h). However, N × N had higher cleavage rate (P < 0.05) in both time intervals when compared with H × H and H × GIR. In addition, Nelore oocytes fertilized with Nelore semen (N × N) had higher blastocyst yields (P < 0.05) in the control and HS group, when compared with the other two breeds (H × H and H × GIR). We concluded that the breed of origin of the oocyte was more important than that of the sperm for development of thermotolerance, because bull breed did not influence embryo development after HS, and in vitro early embryonic development was impaired by increasing (from 4 to 6.5 h) the interval between ovary acquisition and oocyte aspiration.     

Morphokinetic changes and apoptotic cell death in vitrified and non-vitrified in vitro-produced ovine embryos

This article addresses morphokinetic changes and the extent of apoptosis in vitrified and non-vitrified in vitro-derived ovine blastocysts. Cumulus-oocyte complexes were collected after ovarian scarification obtain after slaughter and in vitro maturation was performed in TCM 199 medium supplemented with Earle’s Salt, 10 % of FBS, and 5 µg/mL of LH/FSH at 38 °C for 24 h. After maturation, the oocytes were co-incubated with thawed ram semen (IVF) for 19 h.Embryo development was monitored with the aid of the Primo Vision Time-Lapse (TL) system. Twenty-five out of thirty-one ovine blastocysts that were vitrified using the Cryotop system at the early blastulation stage of development subsequently re-expanded. Both the vitrified (n = 25) and non-vitrified (control group: n = 28) blastocysts were examined for detection of apoptosis (TUNEL assay) and total blastomere counts at the time they attained the expanded blastocyst stage. Blastocyst formation occurred earlier in non-vitrified than in vitrified ovine embryos (147:49 ± 20:23 compared with 156:46 ± 19:24; hours:minutes post-insemination; mean ± SD; P < 0.05). The average number of blastocyst collapses was greater (2.45 ± 1.64 compared with 1.45 ± 1.64), but the number of weak contractions was less for vitrified than non-vitrified ovine blastocysts (P < 0.05). The mean number of blastomeres was greater (131.8 ± 38.6 compared with 91.5 ± 18.3; P < 0.05) while the number of TUNEL-positive cells (4.4 ± 1.6 compared with 6.3 ± 2.3) and apoptotic index (3.4 ± 1.2 % compared with 6.9 ± 2.6 %) were less (P < 0.05) in non-vitrified compared with vitrified blastocysts. Vitrification of ovine embryos was associated with a delayed blastocyst formation, greater numbers of apoptotic cells, significant reduction in the number of blastomeres, and higher/lower incidence of blastocyst collapse/weak contractions.     

Errors in development of fetuses and placentas from in vitro-produced bovine embryos

In vitro systems for oocyte maturation, fertilization and embryo culture [in vitro production (IVP)] have the potential for more wide-spread use in creative breeding programs for dairy and beef cattle. However, one negative consequence of both IVP and somatic cell nuclear transfer (SCNT) in cattle and other species is that embryos, fetuses, placentas, and offspring can differ significantly in morphology and developmental competence compared with those from embryos produced in vivo. Fetuses and placentas derived from IVP and SCNT embryos may fall within the normal range of development, may have obvious abnormalities such as increased fetal and placental weights, or may have subtle abnormalities such as aberrant development of fetal skeletal muscle, placental blood vessels, and altered metabolism. Failures in physiologic and/or genetic mechanisms essential for proper fetal growth and survival outside of the uterus contribute significantly to pregnancy and neonatal losses. Oversized fetuses are at increased risk of death during parturition and the adverse consequences of severe dystocia may compromise the dam. Collectively, these abnormalities have been referred to as 'large offspring syndrome' or 'large calf syndrome'. Abnormal phenotypes resulting from IVP and SCNT embryos are stochastic in occurrence and they have not been consistently linked to aberrant expression of single genes or specific pathophysiology. Thus, reliable methods of early diagnosis of the condition are not yet available. The objective of this paper is to examine abnormal development of fetuses and placentas resulting from embryos produced using in vitro systems. The term 'abnormal offspring syndrome (AOS)' is introduced and a classification system of developmental outcomes is proposed to facilitate research efforts on the mechanisms of the various abnormal phenotypes. We also discuss potential genetic and physiologic mechanisms that may contribute to abnormal phenotypes following transfer of IVP and SCNT embryos.     

Culture of bovine embryos in intermediate host oviducts with emphasis on the isolated mouse oviduct

The oviduct provides the optimal environment for the transport of sperm and oocyte at the earliest stages of mammalian embryo development. During the early postfertilization period, several major developmental events occur in the embryo including (i) the first cleavage division, (ii) activation of the embryonic genome, (iii) compaction of the morula, and (iv) formation of the blastocyst. Most of these events are initiated in the oviduct. The absence of assistance from the oviduct may compromise the developmental ability of the cattle embryo under in vitro culture conditions. The oviducts of several mammalian species, including rabbits, cow, sheep (in situ), and mice (organ culture), can sustain early bovine embryos and yield blastocysts of better quality compared with those of culture conditions in vitro, leading to normal pregnancy rates in recipient animals. This review focuses on the use of oviducts in vitro or in vivo as intermediate hosts for postfertilization culture environment of bovine in vitro-produced zygotes with emphasis on the mouse model.     

Effect of container,vitrification volume and warming solution on cryosurvival of in vitro-produced bovine embryos

The aim of the present research was to develop a low cost and easy to perform vitrification method for in vitro-produced cattle embryos. Effect of container material was evaluated (plastic straw compared to glass capillary, experiment 1), two volume sample (1 compared to 0.5 μL, experiment 2) and warming solution composition medium (Tissue Culture Medium 199 (TCM-199) compared to phosphate buffered saline (PBS), experiment 3) as modifications of the open pulled straw (OPS) system in order to reduce embryo damage caused by exposure to cold.     

Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes

Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.     

Successful vitrification of bovine blastocysts,derived by in vitro maturation and fertilization

Bovine blastocysts were produced through maturation, fertilization, and development in vitro. For vitrification, solutions designated EFS, GFS, and PFS were prepared; these were 40% ethylene glycol, 40% glycerol, and 40% propylene glycol, respectively, diluted in modified phosphate-buffered saline (PBS) containing 30% Ficoll + 0.5 M sucrose. The embryos were exposed to the solutions in one step at room temperature, kept in the solutions for various times, vitrified in liquid nitrogen, and warmed rapidly. When the embryos were vitrified in EFS solution after 1 or 2 min exposure, the postwarming survival rate, assessed by the reexpansion of the blastocoel, was 74–77%. However, when the exposure time was extended to 3 min or longer, this rate dropped to 7–0%. This reduction was attributed to the toxicity of ethylene glycol. Of the embryos vitrified in GFS solution, 53% survived when they were cooled after 1 min exposure; as the duration of the exposure increased, the survival rate increased, reaching a peak (72%) at 4 min. The rate then decreased gradually with exposure time. In PFS solution, embryos surviving after vitrification were recovered only with 1 min exposure (33%), reflecting the high toxicity of propylene glycol. After vitrification in EFS or GFS solution, two embryos were nonsurgically transferred into each of 14 recipient animals. Of the 14 recipients, ten (71%) became pregnant; two resulted in early stillbirths, four recipients delivered twins (four alive and four stillborn), and two delivered single live calves, demonstrating the effectiveness of this simple vitrification method for the cryopreservation of in-vitro-produced bovine blastocysts. © 1993 Wiley-Liss, Inc.     

Effects of bovine Herpesvirus Type 5 on development of in vitro-produced bovine embryos

Bovine (Bos indicus) herpesviruses have been associated with reproductive disease. Type 1, the most studied species, is best known for its reproductive and respiratory effects. Type 5 (BoHV-5) has been detected in bull semen and aborted fetuses but not in oocytes and embryos. This study consisted of three experiments that evaluated (1) BoHV-5-infected oocytes matured in medium with fetal bovine serum (BoHV-FBS) or polyvinyl alcohol (BoHV-PVA) and fertilized by noninfected sperm; (2) noninfected oocytes fertilized by BoHV-5-infected sperm; and (3) infection of presumptive zygotes by BoHV-5. Each treatment involved nine drops of 15 to 20 oocytes. Infection with BoHV-5 was detected by polymerase chain reaction and in situ hybridization assay, and fertilization capacity and embryonic development were assessed using in vitro culture. Experimentally induced infection was obtained in all experiments, and vertical transmission of BoHV-5 by gametes was confirmed. The cleavage rate was reduced (P = 0.0201) in BoHV-FBS (80.4 ± 8.9%; mean ± SD) compared with that of noninfected oocytes (89.9 ± 6.5%); neither differed from BoHV-PVA (87.3 ± 7.1%), and the resulting embryo production rate was not significantly different among groups. Rates of cleavage (87.5 ± 7.5% vs. 92.2 ± 5.5%, control vs. infected) and development of embryos (41.7 ± 9.9% vs. 44.3 ± 7.7% to morula/blastocyst/expanded blastocyst [M/B/EB] and 39.6 ± 10.3% vs. 40.8 ± 9.2% to blastocyst/expanded blastocyst/hatching blastocyst [B/EB/HB] stages) were not compromised by infected sperm (P = 0.1462, P = 0.5402, and P = 0.8074, respectively). However, presumptive zygotes directly infected 1 d after fertilization produced a lower number (P = 0.0140 to M/B/EB and P = 0.002 to B/EB/HB stages) of in vitro-produced embryos (31.6 ± 4.6 vs. 25.0 ± 5.5 and 31.6 ± 4.6 vs. 20.2 ± 5.4; control vs. infected). In conclusion, BoHV-5 infected gametes and was transmissible to the embryo during in vitro development. As zygotes infected 1 d after fertilization had compromised development, BoHV-5 has the potential to be a pathogen with economic consequences.