Cryotolerance Of Embryos Research Articles

65 Effect of oviductal fluid extracellular vesicle supplementation during invitro culture on development and quality of bovine embryos

Recently, it has been postulated that oviductal extracellular vesicles (oEV) might act as natural nanoshuttles bringing key components (small noncoding RNAs and proteins) of the oviduct into gametes and embryos. Furthermore, co-incubation of frozen-thawed oEV with invitro-produced bovine embryos was reported to increase blastocyst rate and quality (Almiñana et al. 2017 Reproduction 154, 153-168). The objective of this study was to determine the dose-dependent effect of oEV supplementation of embryo culture medium on the invitro development and cryotolerance of embryos. Briefly, oEV were isolated by ultracentrifugation from a pool of oviductal fluids (8 cows/sample) collected at the slaughterhouse at the post-ovulatory stage and ipsilateral to ovulation and stored at −80°C until used. Slaughterhouse-derived bovine oocytes were invitro matured and fertilised with frozen-thawed semen from one bull (4 replicates; 194 presumptive zygotes per group), according to our standard procedures. After IVF, groups of presumptive zygotes (n=20/drop) were cultured under humidified air with 5% CO2, 5% O2 at 38.8°C for 7 days in 30µL of synthetic oviductal fluid-bovine serum albumin supplemented with oEV at different protein concentrations: 0.5, 0.05, or 0.005mgmL−1 and without (control). Cleavage rates were evaluated on Day 2 and blastocyst rates were assessed on Days 6 and 7 (IVF as Day 0). At Day 7, expanded grade 1 blastocysts were evaluated (International Embryo Technology Society classification) and embryos at the expanded grade 1 blastocyst stage were slow frozen in 1.5M ethylene glycol + 0.1M sucrose and stored in liquid nitrogen. For cryotolerance evaluation, embryos were thawed and cultured for 48h in synthetic oviductal fluid-bovine serum albumin + 1% estrous cow serum. Hatching rates were assessed at 48h post-thawing. Data were analysed by a logistic regression mixed model (SAS, SAS Institute Inc.; Glimmix procedure) followed by post-hoc Tukey for multiple comparisons. Differences were considered significant at P<0.05. No differences were observed among the different oEV concentrations tested for cleavage and Day 6 blastocysts. A tendency (P=0.0535) was observed for Day 7 blastocyst rates (19.1±2.8, 29.4±3.3, 16.0±2.6, and 20.6±2.9 for 0.5, 0.05, 0.005mgmL−1, and control, respectively) in favour of the 0.05mgmL−1 group. However, a significant difference (P<0.0288) for Day 7 grade 1 expanded blastocyst rates in favour of the 0.05mgmL−1 group was observed (5.2±1.6, 12.9±2.4, 3.1±1.2, and 9.8±2.2 for 0.5, 0.05, 0.005mgmL−1, and control, respectively). For cryopreserved embryos, hatching rates of frozen-thawed embryos were not significant among experimental groups (81.6±10.2 (n=19), 89.6±6.6 (n=27), 77.2±12.2 (n=10), and 60.2±13.6 (n=23) for 0.5, 0.05, 0.005mgmL−1, and control, respectively). In conclusion, under our experimental conditions, the supplementation of the embryo culture medium with frozen-thawed post-ovulatory oEV at the protein concentration of 0.05mgmL−1 increased the Day 7 grade 1 expanded blastocyst rate. Moreover, we showed a tendency to improve Day 7 blastocyst rates but with no apparent effects on the cryotolerance of embryos. This work was supported by APIS GENE.

35 Ethanolic extracts of Cerrado plants in cryotolerance of invitro-produced bovine embryos

Invitro embryo culture induces excessive production of reactive oxygen species (ROS) that impair the quality of produced blastocysts, making them less cryotolerant. Supplementation of culture media with antioxidants would be an alternative to minimise these effects. The present study evaluated the effect of ethanolic extracts obtained from Brazilian Cerrado plants on the cryotolerance of invitro-produced embryos. Bovine ovaries from a slaughterhouse were used to obtain grade I and II oocytes, which were submitted to maturation, fertilization (Day 0), and invitro culture. After fertilization, zygotes were distributed into five groups: one fresh control group (GCont), cultured in high O2 tension (~21%), and four groups cryopreserved at Day 7: a control group subject to high O2 tension without extract supplementation (GDT); a group (G5%) cultured in low O2 tension (5%) without extract supplementation, and two groups cultured under high O2 tension, one supplemented with 0.01mgmL−1 of the ethanolic cagaita extract (Eugenia dysenterica; GCag) and another with 0.01mgmL−1 of murici extract (Byrsonima crassifolia; GMur). Only the expanded blastocysts (EB) of each group were submitted to the direct-transfer cryopreservation system, with slow freezing with ethylene glycol (Sanches et al. 2016 Theriogenology 85, 1147-1151). A total of 163 thawed embryos were evaluated 12, 24, and 36h post-thawing for re-expansion, hatching, and embryonic degeneration rate. In addition, after 12h of post-thaw culture, only the EB embryos were subjected to ROS measurement, quantified in confocal microscopy using 2′,7′-dichlorodihydrofluorescein diacetate, and the rate of apoptosis was obtained by the terminal deoxynucleotidyl transferase dUTP nick end labelling method. Data were analysed by analysis of variance, and the means were compared by TUKEY. A total of 133 EB were used (GCont: 20; GDT: 22; GCag: 35; GMur: 36; G5%: 20). Results are expressed as means±standard deviation. No differences were observed on re-expansion among treatments and time of culture. However, 24h post-thaw, hatching and degeneration rates differed between groups. The group GCont had a higher (P<0.05) hatching rate (47.5±10.3) than the GDT (14.4±6.7) and G5% (10.5±5.8) groups but was similar (P>0.05) to groups supplemented with extracts GCag (22.2±17.1) and GMur (17.5±17.5). Degeneration rate was higher (P<0.05) at 12h for the direct-transfer group (30.2±11.3) and similar among the others (GCont: 0; GCag: 16.8±12; GMur: 10.8±10.8; G5%: 11±11). Nevertheless, GCag had lower embryonic degeneration rates (20±16.7) than the others cryopreserved groups 24h after thawing (GDT: 37.7±5.5; GMur: 38.9±8.2; G5%: 41.3±14.4). Although ROS levels were similar among all groups (P>0.05), apoptosis rate was higher (P<0.05) in the GDT group (23.4±1.9) than in the GCont groups (10.6±1.3): GCag (10.8±1.1), GMur (10.1±1.1), and G5% (7.8±1.2). Therefore, supplementation of ethanolic extracts (0.01mgmL−1) of cagaita and murici improved the embryo quality by reducing the degeneration and apoptosis rates of cryopreserved embryos when compared with the group without supplementation (GDT). Such extracts may be an alternative to increase the cryotolerance of invitro-produced bovine embryos.

Role of endoplasmic reticulum stress on developmental competency and cryo-tolerance in bovine embryos

Endoplasmic reticulum (ER) stress, a dysfunction in protein folding capacity of the ER, is involved in many physiological responses including mammalian reproductive systems. Studies have shown that ER stress interferes with the developmental process of in vitro oocyte maturation and embryo development; however, little is known about its effects on bovine preimplantation embryonic development. In this study, we examined the effects of ER stress during IVC on developmental competency and cryo-tolerance in bovine embryos. IVF-derived zygotes were cultured in CR1aa medium supplemented with tauroursodeoxycholic acid (TUDCA) and/or tunicamycin (TM), which are ER stress-inhibitory and stress-inducing agents, respectively, for 8 days. TM treatment decreased the blastocyst developmental rate and increased the percentage of apoptotic cells compared to that in the control group (10.2 ± 2.3% vs. 39.75 ± 1.3% and 17.8 ± 1.2% vs. 3.6 ± 1.1%, respectively; P < 0.01). However, the blastocyst developmental rate was increased and the percentage of apoptotic cells was decreased by addition of TUDCA in IVC medium compared to that in the control group (50.9 ± 0.9% vs. 39.75 ± 1.3% and 1.13 ± 1.0% vs. 3.6 ± 1.1%, respectively; P < 0.01). Importantly, in the group treated with TM plus TUDCA, the developmental rate and the percentage of apoptotic cells in blastocysts were similar to that in the control group, indicating that TUDCA ameliorates the adverse effects of TM alone on embryo development. In addition, TUDCA treatment significantly reduced the reactive oxygen species, expression of ER stress (GRP78, ATF4, ATF6, IER1, and sXBP1) and pro-apoptotic (CHOP and BAX) genes, while it increased anti-apoptotic BCL2 gene expression and glutathione levels. Moreover, TUDCA improved blastocyst cryo-tolerance as marked by a significantly increased hatching rate and decreased the number of apoptotic cells recorded at 48 h after a post-warming. Therefore, in concordance with a previous report in mice or pig, we showed that TUDCA supplementation during IVC increases the developmental competency of bovine in vitro-derived embryos. Additionally, we found that the presence of TUDCA in IVC medium improves the cryo-tolerance of bovine embryos. These results suggest that modulation of ER stress during IVC contributes to the production of high-quality bovine embryos in terms of cryo-tolerance.

Effect of addition of l-carnitine to media for oocyte maturation and embryo culture on development and cryotolerance of bovine embryos produced in vitro

The objective of these experiments was to determine the effect of l-carnitine during oocyte maturation or embryo culture on embryo development and cryosurvival. For Experiments 1–3, embryos were produced in vitro using abattoir-derived cumulus-oocyte complexes (COCs). At d 7 after insemination, embryo development was assessed, and blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Post-thaw cryosurvival was determined by re-expansion and hatching rates at 24, 48 and 72 h post-thaw. In Experiment 1, COCs were matured with or without 3.03 mM l-carnitine. There was no effect of l-carnitine supplementation during maturation on embryo development or post-thaw cryosurvival. In experiment 2, presumptive zygotes were cultured in medium supplemented with or without 5% (v/v) fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5 and 3.03 mM. There was no effect of l-carnitine treatment on embryo development, but embryos treated with l-carnitine had increased (P ≤ 0.05) post-thaw re-expansion rates, irrespective of concentration. In experiment 3, presumptive zygotes were cultured with or without 0.75 mM l-carnitine from d 1 to d 4, from d 4 to d 7 or for the entire culture period. There was no effect of l-carnitine during culture on embryo development or post-thaw cryosurvival, regardless of the timing of addition. In Experiment 4, COCs were harvested by ovum pick-up from virgin dairy heifers (n = 24) and subjected to in-vitro embryo production with presumptive zygotes cultured with or without 0.75 mM l-carnitine. At d 7 after insemination, morula and blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Lactating Holstein cows (n = 102) were used as recipients and synchronized for timed embryo transfer. At d 7 after anticipated ovulation, a single embryo was thawed and transferred to the ipsilateral uterine horn of each recipient with a corpus luteum. Pregnancy was diagnosed at d 33, 44 and 72 of gestation. l-carnitine had no effect on the percentage of cows pregnant per embryo transfer (P/ET) after transfer of a frozen-thawed embryo. In conclusion, media supplementation with l-carnitine during in vitro embryo production can improve post-thaw cryotolerance as assessed in vitro but had no effect on P/ET after transfer of frozen-thawed embryos.

Effect of sex on cryotolerance of bovine embryos produced in vitro

Male and female embryos are known to be different in developmental kinetics, metabolism, gene expression, and epigenetic patterns. Therefore, the objective of this study was to clarify whether the morphological criteria used to select embryos for cryopreservation lead to a deviation in the male:female ratio, and whether vitrification effects vary according to embryo sex. Initially, five sires were tested to evaluate the effect of the bull on embryo development, sex ratio, speed of development, and response to cryopreservation. Results showed that bulls affected (P < 0.05) embryo production, response to cryopreservation, and sex ratio. Then, one bull was selected, and used to produce embryos in vitro to characterize the responses of male and female embryos to vitrification. Results suggested that male and female embryos have the same morphological responses to vitrification, as no differences (P > 0.05) were observed between the two sexes in post-warming survival and re-expansion rates. However, their molecular responses as evaluated by gene expression (FOSL1, HSPB1, CASP3, CASP8, HSPA5, HSPA1A, G6PD, and PGK1) analysis indicated an effect of sex on vitrification; vitrified female embryos exhibited higher mRNA levels of HSPA1A, CASP3, and G6PD compared to their male counterparts. In conclusion, bulls affected embryo production, speed of development, sex ratio, and response to cryopreservation. Male and female embryos differed in their molecular responses to vitrification; and also, deviations in the male:female ratio when selecting embryos for cryopreservation were confirmed.

Intraoviductal concentrations of steroid hormones during in vitro culture changed phospholipid profiles and cryotolerance of bovine embryos.

The objective of this study was to evaluate the effect of progesterone (P4), estradiol (E2), and cortisol (CO) at intraoviductal concentrations on bovine embryo development and quality in vitro. After fertilization of in vitro matured oocytes, zygotes were cultured for 8 days in synthetic oviductal fluid, supplemented with 55 ng/ml P4, 120 pg/ml E2, 40 ng/ml CO, or their combination (ALL). Control embryos were cultured with vehicle (0.1% ethanol). Exposure to steroids did not affect the embryo developmental rate nor the mean number of cells per blastocyst. However, at 24 hr after vitrification-warming, exposure to P4 improved the proportion of embryos that re-expanded and were viable while exposure to CO decreased the proportion of viable embryos. By intact cell MALDI-TOF mass spectrometry, a total of 242 phospholipid masses of 400-1000 m/z were detected from individual fresh blastocysts. Exposure to ALL induced the highest and most specific changes in embryo phospholipids, followed by P4, E2, and CO. In particular, the m/z 546.3 and 546.4 attributed to lysophosphatidylcholines were found less abundant after exposure to P4. In conclusion, exposure of bovine embryos to intraoviductal concentrations of steroid hormones did not affect in vitro development but changed blastocyst quality in terms of cryotolerance and phospholipid profiles.

70 Does the addition of docosahexaenoic acid to in vitro systems during culture improve the quality of bovine embryos?

The aims of this study were to decrease the apoptotic index and increase cryotolerance of bovine embryos produced in vitro with the addition of 1 µM docosahexaenoic acid (DHA). On Day 1, presumed zygotes were cultivated with 1µM DHA (Sigma, St. Louis, MO, USA; n=437), and without the agonist (control group; n=450). The cleavage rate (%) was evaluated on Day 2 and the development of blastocysts on Day 7. Embryos before and after vitrification were fixed for the TUNEL trial. After vitrification, the embryos were heated and re-cultivated to evaluate the hatching rate at 12, 24, 36, 48, 60, and 72h. A sample of re-cultivated embryos at 12h of DHA (n=5), and without the agonist (control group; n=6), was frozen for mass spectrometry (MALDI-MS). Statistical analyses of deviance were carried out considering generalized mixed linear models, and the effect of the collection day (block) was considered as random. For the count variables, the Poisson distribution and the log link function were considered. In the cases of variables represented by rates, binomial distribution and the logit link function were used. In the study of cryotolerance, ANOVA of the hatching rate for each one of the times evaluated was carried out. In cases of significance of the effect of treatments, the Dunnett test was applied to compare treatments. Multivariate and univariate statistical models were used for analysis of MALDI-MS. All analyses were made using the GLIMMIX procedure of the SAS software (SAS Institute Inc., Cary, NC, USA). The cleavage rate was not different between the groups (P&amp;gt;0.05) and the production of blastocysts was lower in the DHA group (P&amp;lt;0.05). The number of cells per embryo was higher (P&amp;lt;0.05) by the addition of 1μM DHA in blastocysts pre- and post-vitrification. The rate of total and internal cell mass apoptosis in fresh embryos (11.73 and 15.98%) increased compared with the control group (9.62% and 11.03%, respectively). The proportion of internal cell mass in fresh embryos decreased in the DHA group (39.93%) compared with the control group (57.00%). Hatching rates at 48, 60, and 72h after devitrification in the group treated with 1μM DHA were not different (P&amp;gt;0.05) compared with the control group. Phosphatidylcholine [phosphatidylcholine (32: 0)+H] was more abundant (P&amp;lt;0.05) in embryos cultured with DHA, and thus was considered as a negative apoptosis biomarker. In conclusion, the use of 1μM DHA in vitrification of bovine embryos impairs embryonic quality and development under the conditions of the present study. Research was supported by CAPES, FAPEMIG, PPGCV/UFLA, EVUFMG, CENATTE EMBRIÕES.

164 Evaluation of the lipid content of in vitro-matured bovine cumulus - oocyte complexes in the presence of natriuretic peptides A, B, and C types

One of the difficulties still observed in in vitro production (IVP) of bovine embryos is the lower cryotolerance of such embryos, which has been related to their increased lipid accumulation during culture in the presence of fetal bovine serum (FBS). Previous studies have indicated that the cyclic guanosine monophosphate (cGMP) pathway may be involved in the lipid metabolism of bovine cumulus-oocyte complexes (COC). Synthesis of cGMP can be caused by activation of membrane guanylate cyclase (mGC), also called natriuretic peptide receptors (NPR1 and NPR2), which are activated by natriuretic peptides (NP) A, B, and C types (NPPA, NPPB, and NPPC). The objective of this study was to investigate the influence of supplementation with NP during in vitro maturation (IVM) on lipid content and nuclear maturation of bovine COC. Pools of 25 COC were submitted to IVM in TCM-199 with 0.2mM sodium pyruvate, 10μg mL−1 gentamicide, 0.5μg mL−1 FSH, 10% FBS, and NPPA (10−5 M), NPPB (10−7 M), or NPPC (10−5 M). The control group was matured without NP. After 24h, cumulus cells (CC) were removed and oocytes (OO) were fixed and permeabilized in 4% paraformaldehyde+0.5% Triton for 20min, stained with 10μg mL−1 Hoechst 33342 for 15min and 1μg mL−1 Nile Red for 30min. Then, the OO were placed in 13μL of ProLong (Thermo Fisher Scientific, Waltham, MA, USA) on glass slides, covered with a coverslip, and submitted to epifluorescence microscopy to evaluate nuclear maturation (emission 445-450nm and excitation 475-490nm) and lipid content (emission 590nm and excitation 516-560nm). Data for 4 replicates/group were tested for normality of results and homogeneity of variance, and then submitted to ANOVA, followed by Tukey test using a GraphPad Prism software (GraphPad Inc., San Diego, CA, USA), with a significance level of 5%. Nuclear maturation was influenced only by one of the NP added to IVM medium, NPPC, which reduced maturation rate (68.3% metaphase II, MII) compared with the control (82.7% MII; P&amp;lt;0.05). Maturation rates of NPPA (79.5% MII) and NPPB (85.1% MII) did not differ from the control (P&amp;gt;0.05). Activation of mGC by NPPB generated oocytes with lower lipid content (34.02±1.2 IF/μm2) compared with control (36.98±0.7 IF/μm2; P&amp;lt;0.05). Neither NPPA (36.5±1.4 IF/μm2) nor NPPC (39.3±1.4 IF/μm2) was able to reduce the lipid content in oocytes matured in vitro relative to control (P&amp;gt;0.05). Different NP may have different effects on maturation and lipid content in in vitro matured bovine oocytes; NPPB may be favourable for reducing the lipid content of matured bovine oocytes in vitro.

26 Season affects cryotolerance of in vitro-produced buffalo embryos

Buffaloes are tendentially short-day breeders, and seasonality is one of the main factors affecting the feasibility of ovum pickup and in vitro embryo production technology in this species. An improvement of oocyte developmental competence during decreasing daylight months was previously reported in Italian Mediterranean buffalo (Di Francesco et al. 2011 Anim. Reprod. Sci. 123, 48-53). The aim of this work was to evaluate whether season also affects embryo quality and cryotolerance. Abattoir-derived buffalo cumulus-oocyte complexes were collected during the breeding season, characterised by decreasing daylight length (n=349 over 6 replicates), and the non-breeding season, characterised by increasing daylight length (n=770 over 12 replicates). Buffalo cumulus-oocyte complexes were in vitro matured, fertilized, and cultured according to standard procedures (Di Francesco et al. 2011 Anim. Reprod. Sci. 123, 48-53). The embryos obtained by the end of culture (i.e. on Day 7 post-IVF) were scored for quality and developmental stage, and the percentages of total transferable embryos (tight morulae and blastocysts) were recorded. Embryos (n=107 and 110 in the breeding and non-breeding seasons, respectively) were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, and 0.5M sucrose (Boccia et al. 2013 Ital. J. Anim. Sci. 12, 492-496). Warming was carried out by plunging the cryotop strip into a 0.25M sucrose solution and transferring the embryos into 0.15M sucrose for 5min. Embryos were then washed and cultured in SOF for 24h to evaluate post-culture viability. The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, and development rate (i.e. the percentage of embryos that resumed their development and reached a more advanced developmental stage) after 24h post-warming culture. Data were analysed by Student’s t-test. Both cleavage (82.8±4.3v. 73.1±1.7 in the breeding and non-breeding seasons, respectively; P&amp;lt;0.05) and blastocyst (32.9±3.5v. 18.3±1.7 in the breeding and non-breeding seasons, respectively; P&amp;lt;0.01) rates increased during the breeding season, confirming previous observations. Due to the different efficiency, a higher number of replicates was required during the non-breeding season to obtain an equal number of embryos. In addition, a seasonal effect was recorded on embryo quality, indicated by poorer cryotolerance of in vitro-produced buffalo embryos during the non-breeding season. Indeed, both survival (94.6±2.7% and 74.0±5.5% in the breeding and non-breeding seasons, respectively; P&amp;lt;0.01) and development (67.3±7.6% and 40.0±7.2% in the breeding and non-breeding seasons, respectively; P&amp;lt;0.01) rates of vitrified blastocysts decreased after 24h post-warming culture in the non-breeding season. These findings suggest that the reduced developmental competence of buffalo oocytes during the non-breeding season may also lead to lower blastocyst quality. This is in contrast to the evidence in cattle that embryo quality is mainly determined by culture conditions, whereas blastocyst production depends on oocyte quality.

31 Effect of fetal calf serum on production and cryotolerance of in vitro bovine embryos from Ecuadorian Creole heifers

In the Ecuadorian Andes there is a Creole bovine biotype whose population is disappearing. In vitro embryo production and cryopreservation is an important biotechnology that allows the conservation of animals threatened with extinction. The objective of this study was to determine the in vitro production and cryopreservation of embryos from creole heifers raised in the highlands of Ecuador. Immature cumulus-oocyte complexes were retrieved by ovum pickup from 10 Creole heifers (OPU) and from abattoir ovaries (control). The experiment was completed within 8 replicates. Cumulus-oocyte complexes were cultured in a maturation medium (TCM-199 supplemented with 10% fetal bovine serum, 100µg mL−1 of sodium pyruvate, 0.75mg mL−1 of l-glutamine, 4µg mL−1 of FSH-p, 100µM cysteamine, and 250µg mL−1 of gentamicin) following IVF (SOF medium supplemented with 10µg mL−1 heparin) and in vitro culture (citrate SOF medium). After denudation (Day 1 after IVF), presumptive embryos from each oocyte source (OPU and control) were split into 2 groups: with (FCS+) and without (FCS−) FCS (2.5%), which was added on Day 5 after IVF. On Day 7, embryos were evaluated, and those with quality 1 were vitrified. After warming, embryo re-expansion at 2h and embryo re-expansion and hatching at 24 and 48h were evaluated. Data were analysed by logistic regression in SAS software (SAS Institute Inc., Cary, NC, USA). Results of embryo rate at Day 7 and rates of vitrified, re-expanded, and hatched embryos are shown in Table 1. Regardless of the oocyte source, the addition of 2.5% FCS decreased embryo re-expansion at 2h and reduced embryo hatching at 48h in the OPU group. In conclusion, FCS did not improve embryo production and adversely affected the cryotolerance of embryos produced in vitro from Ecuadorian creole heifers. Table 1.Production and cryotolerance of in vitro bovine embryos

Bovine in vitro embryo production protocol: does it really influence embryo cryotolerance?

The protocol of in vitro production (IVP) of bovine embryos is one of the critical factors determining embryo viability after cryopreservation. In this study were used two differents protocols to produce IVP bovine embryos, with variations in protein source, oocyte/zygote density per media volume, with the aim to determine the in vitro and in vivo embryo survival after vitrification using hand-pulled glass micropipettes. Expanded blastocysts (D7) were morphologically selected by size ( ? 180 ?m) and osmotic behavior before they were randomly allocated to sub-groups by protocol: non-vitrified embryos (control; C) and vitrified embryos (V). For the evaluation of the in vitro survival, control embryos and a group of warmed vitrified embryos were in vitro -cultured (IVC) for 72 h. Re-expansion rates of warmed embryos at 24 h of IVC were 94.8% and 93.2% for Protocols 1 and 2, respectively. Hatching rates at 72 h of IVC of embryos from Protocol 1 (C=80% and V=75.8%) tended to be higher (P=0.0561, Chi 2 test) than those from Protocol 2 (C=67.2% and V=59.3%). For the evaluation of in vivo survival, 21 vitrified embryos per protocol were singly non-surgically transferred to synchronized recipients (n=42) after the in-straw cryoprotectant dilution, resulting in 4 (19%) pregnancies per group on Day 60 of gestation. In conclusion, despite a lower variation on in vitro embryo development between both IVP protocols, the use of different protocols under the same laboratory conditions did not afect the in vitro and in vivo embryo viability after vitrification into hand-pulled glass micropipettes.

Correction: The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation.

[This corrects the article DOI: 10.1371/journal.pone.0191023.].

The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation.

This study aimed to determine the influence of cyclic guanosine 3’5’-monophosphate (cGMP) and cGMP-dependent kinase (PKG) during in vitro maturation (IVM) on lipolysis-related parameters in bovine cumulus-oocyte complexes (COCs), and on embryo development and cryosurvival. COCs were matured with cGMP/PKG modulators and assessed for metaphase II rates (MII), cGMP levels, lipid content in oocytes (OO), transcript abundance for genes involved in lipolysis (ATGL) and lipid droplets (PLIN2) in cumulus cells (CC) and OO, and presence of phosphorylated (active) hormone sensitive lipase (HSLser563) in OO. Embryo development, lipid contents and survival to vitrification were also assessed. Phosphodiesterase 5 inhibition (PDE5; cGMP-hydrolyzing enzyme) with 10-5M sildenafil (SDF) during 24 h IVM increased cGMP in COCs (56.9 vs 9.5 fMol/COC in untreated controls, p<0.05) and did not affect on maturation rate (84.3±6.4% MII). Fetal calf serum (FCS) in IVM medium decreased cGMP in COCs compared to bovine serum albumin (BSA) + SDF (19.6 vs 66.5 fMol/COC, respectively, p<0.05). FCS increased lipid content in OO (40.1 FI, p<0.05) compared to BSA (34.6 FI), while SDF decreased (29.8 and 29.6 FI, with BSA or FCS, respectively p<0.05). PKG inhibitor (KT5823) reversed this effect (38.9 FI, p<0.05). ATGL and PLIN2 transcripts were detected in CC and OO, but were affected by cGMP and PKG only in CC. HSLser563 was detected in OO matured with or without modulators. Reduced lipid content in embryos were observed only when SDF was added during IVM and IVC (27.6 FI) compared to its use in either or none of the culture periods (34.2 FI, p<0.05). Survival to vitrification was unaffected by SDF. In conclusion, cGMP and PKG are involved in lipolysis in OO and possibly in CC and embryos; serum negatively affects this pathway, contributing to lipid accumulation, and cGMP modulation may reduce lipid contents in oocytes and embryos, but without improving embryo cryotolerance.

79 Effect of Polydatin on Development and Cryotolerance of In Vitro-Produced Bovine Embryos

In vitro-produced (IVP) cattle embryos have high reactive oxygen species levels resulting in poor development and cryotolerance. Polydatin, a naturally occurring antioxidant, improves embryonic metabolism when added to maturation media; however, it has not been evaluated at other stages of embryo production. We hypothesised that embryos cultured with polydatin during maturation and fertilization would have increased development and cryotolerance. Therefore, IVP embryos were produced in 8 treatment groups supplemented with 1 µM polydatin during in vitro maturation, fertilization, and culture, or a combination of the different production stages, and each assigned a letter (Table 1). Embryos were produced in 7 replicates by oocytes (n = 3320) aspirated from abattoir ovaries, matured for 23 h in TCM-199 plus 10% fetal bovine serum and gonadotropins, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130) in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content or 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial activity. Ten images per embryo were acquired using confocal microscopy at 5 µM step size at 40× magnification, and fluorescence was measured by Image Pro software (Media Cybernetics, Rockville, MD, USA). Remaining blastocysts were slow frozen following a 20-min equilibration in conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose in holding medium) with 1 mm l-ascorbic acid. Embryos were thawed and assessed for re-expansion at 48 h. Blastocyst rate, Nile Red, Mitotracker, and re-expansion data were analysed by one-way ANOVA and means separated by least significant difference. Results indicate that treatment B had a higher blastocyst rate than treatment H (P &lt; 0.01), lower lipid content than all other treatment groups (P &lt; 0.01 or 0.05), and higher level of mitochondrial polarity than treatments A, D, E, and G (P &lt; 0.01 or 0.05), suggesting enhanced metabolic activity. Additionally, this treatment enhanced cryotolerance compared with treatment H (P &lt; 0.01). These results suggest that adding polydatin to maturation media has the most effect on embryo developmental competence and cryotolerance. Table 1.Effect of polydatin addition during in vitro maturation (IVM), fertilization (IVF), and culture (IVC) on blastocyst rate, lipid content, Mitotracker, and cryotolerance (± SEM)

Inhibition of apoptosis by caspase inhibitor Z-VAD-FMK improves cryotolerance of in vitro derived bovine embryos

The aim of this work was to evaluate whether the treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) during cryopreservation and post-warming in vitro culture improves cryotolerance of bovine in vitro produced (IVP) embryos. Abattoir derived bovine oocytes were in vitro matured, fertilized and cultured according to standard procedure. On Day 7, embryo yields were assessed and blastocysts randomly divided in 2 groups: vitrification and post-warming culture in the absence (n = 184) or presence (n = 156) of 20 μM Z-VAD-FMK. Resistance to cryopreservation was evaluated post-warming culture by assessing the survival rate and hatching rate. Differential staining combined with in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) technique was performed to evaluate total cells number, cell allocation into inner cell mass (ICM) and trophectoderm (TE) lineages, as well as the DNA fragmentation rate of vitrified blastocysts, while immunohystochemical staining was used to assess the level of cleaved-caspase 3. It was demonstrated that inhibition of caspase activity by Z-VAD-FMK increases embryo cryotolerance, as indicated by higher survival (76.1 vs 51.1%; P < 0.01) and hatching rates (26.5 vs 17.6%; P < 0.05) after 48 h of post-warming culture. Furthermore, Z-VAD-FMK decreased both the average number (4.7 ± 0.3 vs 7.7 ± 0.5; P < 0.01) and the percentage (3.4 ± 0.2 vs 6.1 ± 0.5; P < 0.01) of DNA fragmented cells in blastocysts compared to the control. No differences were recorded in the average number of ICM, TE and total cells between groups. The level of cleaved-caspase-3, the downstream effector of apoptosis, and its relative percentage on total area of blastocysts was reduced (P < 0.01) in the presence of Z-VAD-FMK both at thawing (1.29 ± 0.17 vs 3.24 ± 0.46) and after 48 h post-warming culture (1.46 ± 0.17 vs 5.06 ± 0.41). In conclusion, the addition of 20 μM Z-VAD-FMK during vitrification/warming and post-warming culture partially inhibits cryopreservation-induced apoptosis by reducing the level of active caspase 3, suggesting a potential use as an additive to ameliorate the efficiency of embryo cryopreservation in cattle, critical for a further diffusion of IVEP technology in the field. Further studies are though needed to evaluate the effect of Z-VAD-FMK on post-transfer embryo development before considering a commercial application.

Effect of charcoal:dextran stripped fetal bovine serum on in vitro development of bovine embryos

This study investigated the ability of charcoal:dextran stripped fetal bovine serum (CDS FBS) and heat-inactivated fetal bovine serum (HI FBS) to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentage of embryos that formed a blastocyst was significantly (P<0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84±0.78% vs. 36.85±0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly (P<0.05) increased mitochondrial activity, as identified by MitoTracker Green, as well as a reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. Quantitative reverse transcription PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly (P<0.05) increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and the anti-apoptotic gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (P<0.05) higher in the CDS FBS-supplemented group than in the HI FBS-supplemented group, whereas that of the pro-apoptotic gene BCL2-associated X protein was significantly lower. Taken together, these data suggest that supplementation of medium with CDS FBS improves the in vitro developmental competence and cryo-tolerance of bovine embryos.

51 CASPASE-3 INHIBITOR Z-VAD-FMK ENHANCES CRYOTOLERANCE OF IN VITRO-PRODUCED BOVINE PRE-IMPLANTATION EMBRYOS

In vitro-produced (IVP) bovine embryos are still less viable and resistant to cryopreservation than their in vivo counterparts. Cryopreservation induces cell degeneration through the apoptotic pathway in bovine oocytes and embryos (Men et al. 2003 Cryobiology 47, 73–81). Apoptosis can be prevented by inhibition of caspase activity, leading to improved cryosurvival in mammalian cells (Stroh et al. 2002 FASEB J. 16, 1651–3). Interestingly, cryotolerance of porcine embryos was improved by inhibiting apoptosis using a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) during vitrification and subsequent culture (Men et al. 2006 Theriogenology 66, 2008–16). Aim of this work was to evaluate whether cryotolerance of bovine IVP embryos may be improved by using Z-VAD-FMK during cryopreservation and post-warming in vitro culture. Abattoir-derived bovine oocytes (n = 753, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–55). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium at 39°C with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and blastocysts (except the hatched blastocysts) were randomly divided in 2 groups: vitrification and post-warming culture in presence (n = 60) or absence (n = 54) of 20 µM Z-VAD-FMK. Vitrification was carried out by Cryotop in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose (Rubessa et al. 2011 Theriogenology 76, 1347–55). Blastocysts were warmed in decreasing sucrose solutions (0.25 M for 1 min and 0.15 M for 5 min) and cultured for 2 days. Resistance to cryopreservation was evaluated by assessing the survival rate, based on morphological criteria and hatching rate after 48 h culture. Furthermore, TUNEL staining was used to evaluate the total cell (TC) number and the apoptotic rate of vitrified blastocysts after 48-h post-warming culture. Differences between groups in survival and hatching rates after 48-h post-warming culture were analysed by Chi-squared test, whereas differences in TC number and in number and percentage of apoptotic cells were analysed by Student’s t-test. Inhibition of caspase activity induced by Z-VAD-FMK increased embryo cryotolerance, as indicated by higher survival (92.6 v. 55.0%; P &lt; 0.01) and hatching rates (40.7 v. 23.3%; P &lt; 0.05) after 48 h of post-warming culture. Furthermore, Z-VAD-FMK decreased both the average number (7.1 ± 0.6 v. 4.2 ± 0.3; P &lt; 0.01) and the percentage (6.3 ± 0.6 v. 3.0 ± 0.2; P &lt; 0.01) of apoptotic cells in blastocysts. No differences were recorded in TC number between groups (on average, 128.90 ± 1.6). These results suggest that addition of 20 µM Z-VAD-FMK during vitrification/warming and post-warming culture significantly inhibits apoptosis (DNA fragmentation) and improves the cryotolerance of IVP bovine embryos.

Effects of dry matter and energy intake on quality of oocytes and embryos in ruminants.

The success of herd fertility involves the development of healthy follicles, viable oocytes and embryos capable of establishing and maintaining a pregnancy. Herein we discuss how nutrition interacts with reproduction throughout follicle development and pregnancy establishment, focusing on dry matter and energy intake. High feed intake, especially associated with moderate to high body condition, before and through superstimulation protocols, natural or induced single-ovulations or before ovum pick-up has detrimental effects on the quality of oocytes or embryos. Feed restriction or high energy supply can be used strategically to obtain either more or better quality oocytes or embryos. Altering diets that provide different concentrations of circulating insulin may improve ovarian status, oocyte quality, embryo development and pregnancy establishment and maintenance. Some sources of fat can positively affect reproductive performance, such as polyunsaturated fatty acids, improving embryo quality and pregnancy. In contrast, fat supplementation in the diet may compromise embryo cryotolerance. Finally, nutrition can alter concentrations of circulating or intrafollicular hormones and metabolites and the expression of genes in cattle oocytes and embryos. For an adequate feeding program to benefit reproductive performance, factors such as genetic group, source of energy, metabolic status, physiological status and level of feed intake must be taken into account.

1147 Effect of the timing of addition of trans-10, cis-12 conjuaged linoleic acid and L-carnitine during culture on development and cryotolerance of bovine embryos produced in vitro

1147 Effect of the timing of addition of trans-10, cis-12 conjuaged linoleic acid and L-carnitine during culture on development and cryotolerance of bovine embryos produced in vitro

Crocetin improves the quality of in vitro–produced bovine embryos: Implications for blastocyst development, cryotolerance, and apoptosis

The aim of this work was to assess the effect of supplementation of bovine culture medium with the natural antioxidant crocetin on in vitro blastocyst development and quality. This was evaluated as cryotolerance, apoptosis index, and total cells number and allocation. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid medium, supplemented with 0, 1, 2.5, and 5 μM crocetin (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and the blastocysts were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose. Finally, blastocysts produced on Day 8 in the absence (control) and presence of 1 μM crocetin were used for terminal deoxynucleotidyl transferase–mediated dUTP nick end labelling and differential staining to evaluate, respectively, the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm (TE) lineages (experiment 2). Embryo development was higher in the 1 μM crocetin group compared to the control, both in terms of total embryo output (37.7 ± 4.2%, 52.9 ± 6.3%, 40.9 ± 7.6%, and 42.4 ± 8.7%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.01) and grade 1 and 2 blastocysts (33.6 ± 4.9%, 46.1 ± 7.3%, 37.8 ± 7.9%, and 39.4 ± 7.9%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). Moreover, the percentage of fast-developing embryos increased in 1 μM crocetin group compared to the control (23.4 ± 4.7%, 32.7 ± 6.6%, 27.2 ± 6.6%, and 30.1 ± 7.2%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). In addition, the enrichment of culture medium with 1 μM crocetin improved embryo cryotolerance compared to the control, as indicated by higher hatching rates recorded after 48 hours postwarming culture (46.5% vs. 60.4%; P < 0.05). Furthermore, 1 μM crocetin decreased both the average number (9.9 ± 0.4 vs. 7.1 ± 0.3) and the percentage of apoptotic cells (7.1 ± 0.4 vs. 4.2 ± 0.2) in blastocysts compared to the control (P < 0.01). However, no differences were recorded in the average number of ICM, TE, and total cells between 1 μM crocetin and control groups. In conclusion, the enrichment of bovine culture medium with 1 μM crocetin increased both blastocyst yield and quality, as indicated by the improved chronology of embryo development, increased resistance to cryopreservation, and reduced incidence of apoptosis.