Unveiling how vitrification affects the porcine blastocyst: clues from a transcriptomic study.

BACKGROUND: Currently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes. To date, vitrification (VIT) is the most efficient method for pig embryo cryopreservation. Despite a high number of embryos survives in vitro after vitrification/warming procedures, the in vivo embryo survival rates after embryo transfer are variable among laboratories. So far, most studies have focused on cryoprotective agents and devices, while the VIT effects on porcine embryonic gene expression remained unclear. The few studies performed were based on vitrified/warmed embryos that were cultured in vitro (IVC) to allow them to re-expand. Thus, the specific alterations of VIT, IVC, and the cumulative effect of both remained unknown. To unveil the VIT-specific embryonic alterations, gene expression in VIT versus (vs.) IVC embryos was analyzed. Additionally, changes derived from both VIT and IVC vs. control embryos (CO) were analyzed to confirm the VIT embryonic alterations. Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA-sequencing: (1) VIT embryos (vitrified/warmed and cultured in vitro), (2) IVC embryos and (3) CO embryos. RESULTS: RNA-sequencing revealed three clearly different mRNA profiles for VIT, IVC and CO embryos. Comparative analysis of mRNA profiles between VIT and IVC identified 321, differentially expressed genes (DEG) (FDR < 0.006). In VIT vs. CO and IVC vs. CO, 1901 and 1519 DEG were found, respectively, with an overlap of 1045 genes. VIT-specific functional alterations were associated to response to osmotic stress, response to hormones, and developmental growth. While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects. CONCLUSIONS: Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs. IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos. The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts. Selected validated genes were pointed as potential biomarkers that may help to improve vitrification.

Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation.

Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryopreserved embryos either 24 h after in vitro culture or after transfer to recipients. Fresh and nonbiopsied embryos were used as controls. Sex, coat color genes, myotony (neuromuscular disorder) diagnosis, and markers of parentage were investigated using PCR on biopsied cells after whole-genome amplification and on remaining embryos. The embryo survival rate after transfer was not affected by the micropipette biopsy (50%, = 8; 43%, = 7; and 50%, = 12, at d 30 for fresh biopsied embryos, vitrified biopsied embryos, and control embryos, respectively) but was significantly reduced by the use of microblade biopsy: 9 ( = 11) vs. 67% ( = 12) for control embryos. Successful sex determination was achieved for 82% ( = 28) of the micropipette biopsies and 100% ( = 50) of the microblade biopsies. Sex determined on biopsied cells was found to correspond completely (100%) with that determined on the remaining embryo ( = 37). More than 90% of the parentage checking markers, coat color, and myotony diagnosis were successfully determined on biopsies obtained with either a micropipette or a microblade. Mendelian incompatibility (7.5 and 5.5%) and embryo genotyping errors (6.6 and 8.6%) were low and not significantly different between the 2 methods. In conclusion, for the first time, pregnancy at Day 30 was obtained after transfer of Welsh pony biopsied and vitrified embryos >300 µm in diameter to recipient pony mares. The biopsied cells collected enabled multigenetic embryo diagnoses to be performed to a high degree of accuracy. The micropipette biopsy is the better method to apply on Welsh pony embryos.

Effect of different culture systems on adipocyte differentiation-related protein (ADRP) in bovine embryos.

Bovine embryos cultured in serum-containing media abnormally accumulate lipid droplets, compared to their in vivo counterparts. The objective of this study was to investigate the effect of different culture systems on the mRNA expression and on the quantification and localisation of adipocyte differentiation-related protein (ADRP), a protein associated with lipid accumulation in bovine blastocysts. Two experiments were independently performed for ADRP mRNA expression analysis. In experiment A, blastocysts were produced in modified synthetic oviduct fluid (mSOF)+10% foetal calf serum (FCS), in coculture (bovine oviduct epithelial cells, Boec) and in ewe oviducts, whereas in experiment B, they were produced in mSOF+10µM docosahexaenoic acid (DHA) and in vivo. Control groups were also performed. ADRP mRNA expression was downregulated in the Boec, ewe oviduct and in vivo groups compared to the 10% FCS or DHA groups, respectively. Moreover, the expression of this protein was downregulated in the Boec group compared to the control group (P<0.05). A third experiment (experiment C) was performed to quantify and localise ADRP protein. Boec, in vivo and control groups were tested. After immunofluorescence staining followed by confocal microscopy analysis, embryonic ADRP was clearly localised around lipid droplets, indicating that ADRP is also a lipid droplet coat protein in bovine embryos. In conclusion, our results demonstrate that bovine embryos at the blastocyst stage expressed ADRP mRNA and protein, and that the embryonic culture system modified this expression.

Can caprine arthritis encephalitis virus (CAEV) be transmitted by in vitro fertilization with experimentally infected sperm?

For each of the five fertilization trials of the experiment, frozen semen was prepared for in vitro capacitation at a concentration of 1 × 10(7) spz/ml and divided into three groups. One group was used as a control, while the two others were inoculated with 100 µl/ml of either culture medium from non-infected cells (placebo group) or cell culture medium containing virus at a concentration of 10(5) TCID(50)/ml (infected group). A total of 789 oocytes were used for IVF. For each of the five trials a group of oocytes were used as a non-infected control and were found to be caprine arthritis-encephalitis virus (CAEV) free. The other oocytes were divided in two equal batches. Oocytes in the first batch were in vitro fertilized with CAEV infected sperm (infected group) and the second batch were fertilized with CAEV non-infected sperm (placebo and control groups). After IVF, the zygotes of each group were washed 12 times. The CAEV genome was not detected (using RT-PCR) in the washing media of either the control or placebo groups from each trial. In contrast, the first three washing media from the infected group were consistently found to be positive for the CAEV genome (5/5), whereas subsequent washing media were CAEV-free (P < 0.05). Zygotes obtained using all semen groups tested negative for both the provirus and genome of CAEV. These results clearly show that the first four washes were sufficient to remove viral particles from CAEV infected fertilization media and that CAEV-free embryos can be produced by IVF using spermatozoa infected in vitro by CAEV.

Sex and PRNP genotype determination in preimplantation caprine embryos.

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo.

Effect of coculture with oviduct epithelial cells on viability after transfer of vitrified in vitro produced goat embryos.

This study evaluates the effect of coculture with goat oviduct epithelial cells (GOEC) on the pregnancy rate, embryo survival rate and offspring development after direct transfer of vitrified/thawed caprine in vitro produced (IVP) embryos. Oocytes were recovered from slaughterhouse goat ovaries, matured and inseminated with frozen/thawed capacitated semen, and presumptive zygotes were randomly cultured in synthetic oviduct fluid (SOF) (n=352) or GOEC (n=314). The percentage of cleaved embryos reaching the blastocyst stage was 28% and 20% in SOF and GOEC, respectively (P<0.05). Overall, 26 blastocysts of SOF were transferred freshly in pairs to recipient goats, whereas 58 of SOF and 36 of GOEC were vitrified and transferred directly in pairs to recipient goats after thawing without removal of cryoprotectants or morphological evaluation. The kidding rate was 92% for SOF fresh, 14% for SOF vitrified (P<0.001) and 56% for GOEC vitrified (P<0.05); the difference was also significant between vitrified groups (P<0.01). The embryo survival rate was 62% for SOF fresh, 9% for SOF vitrified (P<0.001) and 33% for GOEC vitrified (P<0.05) with a significant difference between vitrified groups (P<0.01). The results showed that the coculture of IVP goat embryos with GOEC significantly improves the pregnancy and embryo survival rates and leads to the birth of healthy offspring. However, further research using more defined GOEC coculture is required to confirm its capacity to increase the success rate of IVP embryo technology in goat.

Improved vitrification method allowing direct transfer of goat embryos.

The aim of this study was to design a vitrification method suited to field embryo transfer experiments in goat. In a first experiment, a standard vitrification protocol, previously designed for sheep embryos was compared to slow freezing of goat embryos. No significant difference was observed on kidding rate (48% versus 69%, respectively), nor on embryo survival rate (35% versus 45%). Second experiment: all embryos were vitrified. After warming, embryos were either transferred directly (direct transfer), or after in vitro dilution of the cryoprotectants (conventional transfer). The kidding rate was not affected by the transfer method (38% versus 23%, respectively). However, embryo survival rate tended to be higher after direct transfer (26% versus 14%). Third experiment: OPS vitrification was compared to standard vitrification. The kidding rate was not affected (22% versus 39%, respectively), but the embryo survival rate was lower after OPS (14% versus 28%). Fourth experiment: 0.4M sucrose was added with cryoprotectants in vitrification. The kidding rate after direct transfer was significantly enhanced after addition of sucrose (56% versus 27%, respectively), whereas embryo survival rate was not significantly affected (32% versus 18%). Fifth experiment: vitrification with sucrose supplementation was compared to slow freezing. No significant difference was observed after direct transfer on kidding rate (52% versus 31%, respectively), but embryo survival rate tended to be higher after vitrification (34% versus 21%). In conclusion, our results indicate that addition of 0.4M sucrose in association with direct transfer improves significantly the viability of goat vitrified embryos.

Bovine muscle 20S proteasome. II: Contribution of the 20S proteasome to meat tenderization as revealed by an ultrastructural approach.

The role of the 20S proteasome proteolytic effects was revisited using an ultrastructural approach with the aim to explain some particular structural changes identified in type I muscles and in high pH meat. In both types of meat, major changes observed after ageing are an increase in the thickness of the Z-line followed by the appearance of an amorphous protein structure spreading out over the I-band. This was followed by a total degradation of this amorphous structure and of the Z-line. Partial transversal fragmentation of the myofibrils within the I-band can also be detected. The data reported clearly demonstrate that the 20S proteasome was able to mimic these sequential structural changes, a feature never obtained with either calpains or cathepsins. It is the first time that a direct implication of this complex in postmortem muscle is postulated.

In vitro comparisons of two cryopreservation techniques for equine embryos: slow-cooling and open pulled straw (OPS) vitrification.

Vitrification using open pulled straw (OPS) has provided encouraging results with embryos from other species. The aim of this study was to compare the survival of 6.5- and 6.75-day-old equine embryos after OPS vitrification and slow-cooling. Eighteen embryos were frozen using a slow-cooling method. Embryos were placed in modified PBS with increasing glycerol concentration (2.5%, 5%, 7.5% and 10% (v/v) 5 min each). Embryos were loaded into 0.25 ml straws then placed in a programmable freezer and subsequently plunged into liquid nitrogen. After thawing, cryoprotectant was removed by five steps with decreasing glycerol and sucrose concentrations. Twenty embryos were vitrified using the OPS method. Embryos were exposed to 7.5% dimethyl-sulfoxide (DMSO)+7.5% ethylene glycol (EG) for 3 min and in 18% DMSO+18% EG+0.4M sucrose for 1 min, loaded in OPS and plunged into liquid nitrogen. After warming, embryos were placed in decreasing sucrose concentrations. All embryos were cultured in synthetic oviduct fluid (SOF) medium for 3h and evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining. The percentage of cells entering in S-phase (%SC) was evaluated by incorporation of BrdU. No significant differences were observed for mean diameter, morphological grade and percentage of degenerate embryos after 3h of culture for slow-cooling and OPS methods. The percentage of dead cells per embryo was similar for the two procedures (42+/-6 versus 46+/-9). The percentage of cells entering in S-phase did not differ significantly between the two procedures (27+/-5 versus 26+/-6). OPS vitrification may be as efficient as slow-cooling for the cryopreservation of equine embryos. However, these results should be confirmed by the transfer of OPS vitrified embryos to recipient mares.

Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation.

In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before, and cleavage rate reached after IVF, following short (1.5 to 4 h) and long (6 to 8 h) storage. Temperatures in the storage container decreased from 37-C to 32 degrees and 27.5 degrees C during the short and long interval, respectively. The cumulus-oocytes complexes (COCs) were classified as having a compact cumulus, completely or partially surrounding the oocyte (compact); those having only a corona radiata surrounding the oocyte (corona); those having a completely or partially expanded cumulus, showing a cellular or sparsely cellular, gelatinous cloud around the oocyte (expanded); and those that were completely denuded of both cumulus and corona cells (denuded). All COCs, except the denuded ones, which were discarded, were matured in vitro for 30 h at 38.5 degrees C in 5% CO2. The recovery rate of oocytes was significantly higher after long vs short storage (48 vs 35%; P < 0.01), but the distribution of the collected COCs into the 4 classes was not affected by the storage time. After in vitro maturation nuclear maturity was not affected by the storage time, but oocytes with intact cytoplasmic membranes were more frequently found after short than after long storage (54 vs 34%; P = 0.07), and fully matured oocytes were more often seen with intact membrane (P < 0.01). Moreover, oocytes with intact membranes in metaphase II (MII) were associated with short storage intervals and the corona COC class, while damaged membranes and incomplete maturation were associated with the long storage and the compact COC class.

Preliminary observations in in vitro development of equine embryo after ICSI.

The objective of this study was to perform intracytoplasmic sperm injection (ICSI) on in vitro matured equine oocytes and to improve in vitro embryonic development on Vero cells after activation of the microinjected oocytes with calcium ionophore. After maturation (23 or 40 h, 38.5 degrees C, 5% CO2), the cumulus-oocyte complexes were denuded, centrifuged and all oocytes exhibiting the first polar body were microinjected. ICSI was performed using fresh semen from three fertile stallions. Microinjected oocytes were activated with calcium ionophore A23187 (10 min, 10 microM) and cultured individually for 7 days on Vero cells in microdrops. In seven trials, 353 cumulus-oocyte complexes were matured and 103 oocytes were microinjected. Eight oocytes were sham microinjected. After ICSI, 85 oocytes (82.5%) survived the sperm injection procedure. Among the 76 successfully microinjected oocytes, 52 (68%) were fertilized (two pronuclei, syngamy stage and cleaved ova). Sham microinjected oocytes were not activated. After in vitro culture, 35 ova (46%) were cleaved 2 days after ICSI and early embryonic development was obtained (three embryos of 23 cells, 50 cells and more than 80 cells) 5 to 7 days after ICSI.

Relationships between post-mortem pH changes and some traits of sensory quality in veal.

The aim of this study was to evaluate the relationships between the rate and extent of post-mortem pH changes and the colour, the cooking loss and the eating quality of veal. The experiment used 12 calves aged 18 weeks. Variations in ultimate pH were induced by adrenalin administration (0.1-0.4 mg/kg liveweight) to six of the animals. Measurements were made on the Longissimus thoracis muscle. pH and osmotic pressure were measured at 0.5 h, 4 h and 29 h after slaughter. Pigment content, drip loss and cooking loss were measured at 29 h after slaughter, and colour was measured at 2 days and 9 days after slaughter. Cooking loss, tenderness, juiciness and flavour of roasts were assessed at 9 days after slaughter. Correlations between colour traits and pH values were higher with ultimate pH than with pH at 0.5 h or 4 h after slaughter. Lightness, redness and reflectance decreased when the ultimate pH increased. Drip loss was correlated with the rate of pH fall (r = -0.80, P < 0.01 with pH at 4 h), while cooking loss was correlated with ultimate pH (r = -0.94, P < 0.01). Ultimate pH and the sensory quality traits were linearity and positively correlated (r = 0.83) for tenderness, 0.81 for juiciness and 0.71 for flavour, respectively).

Post mortem evolution of myofilament spacing and extracellular space in veal muscle.

The post mortem evolution of water distribution was studied in muscle tissue from veal calves in two experiments. Myofilament spacing, extracellular space and rate and extent of pH fall were determined in Psoas major muscle in Experiment 1. Extracellular space and rate and extent of pH fall were determined in Longissimus dorsi, Psoas major and Trapezius muscles in Experiment 2. In Experiment 2, the variability of ultimate pH was increased by using adrenalin injections. The myofilament spacing decreased after slaughter when pH reached values around 5·9. In both experiments, the extracellular space began to increase soon after slaughter in close relation with the pH changes. The size of the ultimate extracellular space was significantly correlated with the rate of pH fall, but not with the ultimate pH.

The role of ultimate pH in proteolysis and calpain/calpastatin activity in bovine muscle.

Of a total of three Friesian cows, two of which had been treated with adrenalin before slaughter, Mm longissimus (LO), supraspinatus (SS), triceps brachii (TB) and rectus abdominis (RA) were sampled at different times post mortem (pm). pH, calpain/calpastatin activities and degradation of myofibrillar proteins, as evidenced by SDS-PAGE, were assessed. Contraction characteristics were measured by determining myofibrillar ATPase activities. Adrenalin treatment resulted in a high ultimate pH (6.48 +/- 0.40) and a faster decline pm of calpain I activity. The effect was similar in all four investigated muscles (72.4 +/- 5.4% decline at 24 h pm). The decline in calpain I activity in the control muscles was muscle-dependent and ranged from 22.8-74.3% at 24 h pm. Differences in ultimate pH did not lead to distinct rates of breakdown of proteins with molecular weights lower than that of myosin heavy chain. Calpastatin levels were muscle-dependent and correlated with myofibrillar ATPase activity (r = -0.99). In a second experiment Mm rectus abdominis (RA) and psoas major (PM) of adrenalin-treated (n = 6) and control (n = 6) Friesian-Holstein calves were sampled at 1 and 29 h pm for assessment of calpain activities. At seven days pm the M longissimus (LO) was sampled for tenderness evaluation. pH values were measured at 30 min, 4 h and 29 h pm. Adrenalin treatment resulted in a higher ultimate pH in the three muscles. Higher ultimate pH resulted in lower calpain activities in the RA at 29 h pm (P less than or equal to 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)