Hatched Blastocyst Rates Research Articles

Tauroursodeoxycholic Acid Supplementation in In Vitro Culture of Indicine Bovine Embryos: Molecular and Cellular Effects on the In Vitro Cryotolerance.

During embryo development, the endoplasmic reticulum (ER) acts as an important site for protein biosynthesis; however, in vitro culture (IVC) can negatively affect ER homeostasis. Therefore, the aim of our study was to evaluate the effects of the supplementation of tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, in the IVC of bovine embryos. Two experiments were carried out: Exp. 1: an evaluation of blastocyst rate, hatching kinetics, and gene expression of hatched embryos after being treated with different concentrations of TUDCA (50, 200, or 1000 μM) in the IVC; Exp. 2: an evaluation of the re-expansion, hatching, and gene expression of hatched embryos previously treated with 200 µM of TUDCA at IVC and submitted to vitrification. There was no increase in the blastocyst and hatched blastocyst rates treated with TUDCA in the IVC. However, embryos submitted to vitrification after treatment with 200 µM of TUDCA underwent an increased hatching rate post-warming together with a down-regulation in the expression of ER stress-related genes and the accumulation of lipids. In conclusion, this work showed that the addition of TUDCA during in vitro culture can improve the cryotolerance of the bovine blastocyst through the putative modulation of ER and oxidative stress.

Influence of superovulation on the production of native Vietnamese pig embryos in vivo and embryonic survival after vitrified-thawed

We aimed to evaluate the influence of superovulation on the production of native Vietnamese pig embryos in vivo and embryonic survival after vitrified-thawed. The average number of follicles, corpora lutea, and recovered embryos of the superovulation group was higher than non-superovulation group (13.56 vs. 7.45, 12.32 vs. 6.12, and 10.91 vs. 5.67; P < 0.05, respectively). The average number of follicles of 4th repeated superovulation was lower than the first, second, and third repeated superovulation (10.57 vs. 13.56, 12.91, and 12.32, P < 0.05, respectively). The average number of corpora lutea and recovered embryos of 4th and third repeated superovulation was lower than first and second superovulation (9.57 and 7.58 vs. 12.32 and 11.84, 10.91 and 9.28 vs. 6.86 and 5.42, P < 0.05, respectively). The average number of follicles of the Co Binh Thuan pig was higher compared to Muong Te pig (16.78 vs. 14.58, P > 0.05, respectively) and Kieng Sat pig (16.78 vs. 13.56, P < 0.05, respectively). The average number of corpora lutea and recovered embryos of the Co Binh Thuan pig was higher than Muong Te pig and Kieng Sat pig (16.14 vs. 12.36, and 12.32, 14.34 vs. 10.98 and 10.91, P < 0.05, respectively). There was no difference in survival and hatching blastocyst rates of native Vietnamese pig embryos in vivo after thawing between breeds (P > 0.05). When vitrified-thawed pig embryos in vivo were transferred, 3 out of 5 recipients became pregnant at Day 60 and one recipient miscarried at Day 31 after embryo transfer. In conclusion, superovulation improved the production of native Vietnamese pig embryos in vivo, the highest number of repeated superovulation on the native Vietnamese pig breeds was four times and the breed affected the superovulation parameters evaluated. We successfully created pregnant pigs after transferring vitrified-thawed embryos in vivo of native Vietnamese pigs for the first time.

Cathepsin L regulates oocyte meiosis and preimplantation embryo development.

Early embryonic loss, caused by reduced embryo developmental competence, is the major cause of subfertility in humans and animals. This embryo developmental competence is determined during oocyte maturation and the first embryo divisions. Therefore, it is essential to identify the underlying molecules regulating these critical developmental stages. Cathepsin L (CTSL), a lysosomal cysteine protease, is involved in regulating cell cycle progression, proliferation and invasion of different cell types. However, CTSL role in mammalian embryo development is unknown. Using bovine in vitro maturation and culture systems, we show that CTSL is a key regulator for embryo developmental competence. We employed a specific CTSL detection assay in live cells to show that CTSL activity correlates with meiotic progression and early embryo development. Inhibiting CTSL activity during oocyte maturation or early embryo development significantly impaired oocyte and embryo developmental competence as evidenced by lower cleavage, blastocyst and hatched blastocyst rates. Moreover, enhancing CTSL activity, using recombinant CTSL (rCTSL), during oocyte maturation or early embryo development significantly improved oocyte and embryo developmental competence. Importantly, rCTSL supplementation during oocyte maturation and early embryo development significantly improved the developmental competence of heat-shocked oocytes/embryos which are notoriously known for reduced quality. Altogether, these results provide novel evidence that CTSL plays a pivotal role in regulating oocyte meiosis and early embryonic development.

Effects of antifreeze protein from Lolium perenne L. (LpAFP) in the vitrification of in vitro-produced bovine embryos.

In the present study, the cryoprotective effects of Lolium perenne antifreeze protein (LpAFP) on the vitrification of bovine embryos were evaluated. In vitro-produced blastocysts were divided into two groups: the control group (CG) without the addition of LpAFP and the treatment group (TG) with the addition of 500 ng/ml of LpAFP in the equilibrium and vitrification solution. Vitrification was carried out by transferring the blastocysts to the equilibrium solution [7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO)] for 2 min and then to the vitrification solution (15% EG, 15% DMSO and 0.5M sucrose). The blastocysts were deposited on a cryotop device and submerged in liquid nitrogen. Warming was carried out in three steps in solutions with different sucrose concentrations (1.0, 0.5, and 0.0 M, respectively). Embryos were evaluated for re-expansion/hatching, the total cell count, and ultrastructural analysis. There was no significant difference in the re-expansion rate 24 h after warming; however, there was variation (P < 0.05) in the hatching rate in the TG and the total number of cells 24 h after warming was higher in the TG (114.87 ± 7.24) when compared with the CG (91.81 ± 4.94). The ultrastructural analysis showed changes in organelles related to the vitrification process but, in the TG, there was less damage to mitochondria and rough endoplasmic reticulum compared with the CG. In conclusion, the addition of 500 ng/ml of LpAFP during the vitrification of in vitro-produced bovine embryos improved the hatching rate and total cell number of blastocysts after warming and mitigated intracellular damage.

O-148 Cumulative embryotoxic effect of IVF plastic devices used sequentially in routine clinical practice could slow the embryo development and delay blastulation

Abstract Study question Is there an embryotoxic cumulative effect resulting from IVF plastic consumables (PC) when used sequentially under clinical routine practice? Summary answer Reduced blastulation rates and slow development were detected in some associations of PC evaluated by the Mouse Embryo Assay (MEA) suggesting possible cumulative embryotoxicity. What is known already Recommendations from European (ESHRE) and American (ASRM) societies regarding the use of IVF PC suggest that they should undergo appropriate quality control tests to detect the possible presence of embryotoxins. The main test used by manufacturers to exclude any embryotoxicity is the MEA. This assay is currently performed individually on each PC so that a certificate of conformity is delivered for each product/lot. However, several PC are used during a single IVF cycle, potentially creating an embryotoxic cumulative effect. To our knowledge, the cumulative toxicity of several PC used sequentially on gametes and embryos has not been studied so far. Study design, size, duration The objective of this prospective study was to determine if there is cumulative embryotoxicity when several PC are used together under clinical routine conditions. Ten associations, each containing 13 to 31 PC, were designed, and assessed for embryotoxicity using a MEA methodology. The tested combinations replicated the different steps used in an IVF cycle, including sperm collection and selection, oocyte pick-up, fertilization, embryo culture, embryo transfer, vitrification and thawing and sperm freezing. Participants/materials, setting, methods A defined volume of culture media was used to extract the toxicity of several PC used in every association in triplicate. The MEA tests were performed on each medium extraction with 21 one-cell stage fresh mouse embryos. Blastocyst formation rates after 96 and 120h of culture (Day 5/6), blastocyst good quality rates and hatched blastocyst rates were compared between the tested and control groups. The total cell number per blastocyst was also evaluated. Main results and the role of chance No toxicity was detected in the first two associations comprised of 22 and 23 PC, which mimicked the collection (either in sperm cups or in spermicide-free condoms) and processing of sperm samples. However, the third association comprised of 32 plastic devices replicating the sperm collection in sperm cups, processing, and freezing in high security straws, showed toxicity as the mean blastulation rate at day 5 was reduced to 20.6% compared to 95.6% in the control group (p &amp;lt; 0.05). Surprisingly, the mean blastulation rates observed at day 6 was 55.6% versus 100.0% in the control group (p &amp;lt; 0.05). The blastocysts obtained at day 6 showed a mean number of cells (n = 112.5) significantly reduced in comparison with the control group (n = 167.9) (p &amp;lt; 0.05), suggesting a sublethal presence of toxins which slowed embryo development down and delayed blastulation. More experiments involving toxicity assessment in other associations of PC are currently undergoing and will be presented at the conference. Limitations, reasons for caution Sensitivity could vary depending on the methodologies used for toxicity extraction and MEA. The main cause, if any, of the detected toxicity will need to be pinpointed by analyzing each element of the affected associations individually. Wider implications of the findings Also, professionals should rationalize and minimize the number of plastic consumables used during the IVF procedures to reduce the potential accumulation of toxicity in the embryo culture system. Trial registration number Not applicable

Development of buffalo embryo derived from ICSI: effects of various somatic-cell co-culture

Context Somatic-cell co-culture of intracytoplasmic sperm injection (ICSI) buffalo embryos has not been reported earlier. Aim This study aimed to determine the effects of buffalo oviductal epithelial-cell, granulosa-cell, and cumulus-cell co-culture on in vitro culture of early embryo development as ICSI and post-activation. Methods Selected oocyte–cumulus complexes were cultured for 19–20 h in 50-μL drop of tissue culture medium (TCM199 + 10% buffalo follicular fluid, hCG 50 IU/mL, 0.02% arbitrary units (AU)/mL follicle-stimulating hormone and 1 μg/mL estradiol-17βE2). Oocytes reaching Metaphase II were subjected to ICSI with immobilised spermatozoa. All ICSI oocytes were activated with calcium ionophore for 5 min, followed by cycloheximide for 5 h. The embryos at 6–8-cell stages were co-cultured. Key results The morula, blastocyst, and hatched blastocyst rates when co-cultured with oviductal epithelial cells were 68.18%, 48.18%, and 30.00% respectively. The morula, blastocyst, and hatched blastocyst rates when co-cultured with cumulus cells were 51.49%, 34.33%, and 16.42% respectively. The morula, blastocyst, and hatched blastocyst rates when co-cultured with granulosa cells were 52.14%, 32.48%, and 13.68% respectively. Conclusions In vitro maturation buffalo oocytes can be fertilised in vitro with ICSI and co-cultured with different types of cells. Oviductal epithelial cell co-culture was shown to be superior in supporting in vitro embryo development in this study. Implications The oviductal epithelial cells are easy to prepare and may be used for co-culture to increase the efficiency of in vitro production of buffalo embryos.

Influence of the method of vitrification and developmental stages of embryos on cryopreservation efficiency of in vitrobovine embryos produced in Vietnam

The study aimed to evaluate the influence of different vitrification methods and developmental stages of bovine embryos produced in vitro during cryopreservation in Vietnam. In vitro bovine embryos were frozen by two methods: (1) Vitrification by cryotop and (2) Vitrification by microdrop. The percentage of embryos obtained after thawing of method (1) was higher than that of (2) (100 vs 81.01%, p&lt;0.05). However, there was no difference in survival, re-expansion and hatching blastocyst rates after thawing between the two methods (1) and (2) (88.43 vs 91.89%; 83.1 vs 84.43%; and 20.34 vs 17.79%, p&gt;0.05; respectively). In this study, the survival, cleavage, blastocyst and hatching blastocyst rates of in vitro bovine zygotes after thawing were 86.83, 63.83, 21.39, and 3.91%, respectively. The authors did not observe any difference in survival and hatching blastocyst rates after thawing while vitrification at morula/compact morula and blastocyst stages (82.67 vs 88.43%, 16.54 vs 20.34%, p&gt;0.05; respectively). The results showed that in vitro bovine embryos were successfully frozen at the zygotes, the morula/compact morula and blastocyst stage by vitrification.

The effects of melatonin, glutathione and vitamin E on semen cryopreservation of Mediterranean buffalo

The aim of this study was to investigate the effects of melatonin (MLT), reduced glutathione (GSH) and vitamin E (Vit. E) or their combinations on semen cryopreservation of Mediterranean buffalo. The quality parameters such as viability, abnormality rate, motility, structural integrity and the antioxidant capacity of frozen-thawed sperm were evaluated. The efficiency of frozen-thawed sperms in performing their functions was further analyzed by in vitro fertilization (IVF). In those separately supplemented groups, 0.2 mg/mL MLT, 0.2 mM GSH and 0.4 mg/mL Vit. E had the best effect on antioxidant capacity, kinetics and morphology, respectively. In addition, the cleavage, blastocyst and hatching blastocyst rates of IVF embryos were higher in 0.2 mg/mL MLT, 0.2 mM GSH, 0.2 and 0.4 mg/mL Vit. E groups than the blank control. Among the three combination groups, the kinetics and structure integrity of frozen-thawed sperms, cleavage, blastocyst and hatching blastocyst rates of IVF embryos were higher in 0.4 mg/mL Vit. E plus 0.2 mg/mL MLT group than the blank control group, revealed that this combination had comprehensive protection on frozen-thawed sperm of Mediterranean buffalo. These results support to develop special semen freezing extender containing an optimal choice of MLT, GSH and Vit. E, and to enhance the efficiency of frozen-thawed sperm of Mediterranean buffalo for IVF.

Sperm chromatin protamination influences embryo development in unsexed and sexed bull semen.

Sex selection through sperm sorting offers advantages in regards selection pressure in high-producing livestock. However, the sex-sorting process results in sperm membrane and DNA damage that ultimately decrease fertility. We hypothesized that given the role of protamines in DNA packaging, protamine deficiency could account, at least partially, for the DNA damage observed following sperm sex sorting. To test this, we compared protamine status between unsexed and sexed spermatozoa from two bulls using the fluorochrome chromomycin A3 (CMA3) and flow cytometry. Then, we assessed embryo development following in vitro fertilization (IVF) using the same sperm treatments. Overall, sperm protamination was not different between sexed and unsexed semen. However, one of the two bulls displayed higher rates of protamine deficiency for both unsexed and sexed semen (P < 0.05). Moreover, unsexed semen from this bull yielded lower blastocyst (P < 0.05) and blastocyst hatching rates than unsexed sperm from the other bull. CMA3-positive staining was negatively correlated with cleavage (R2 85.1, P = 0.003) and blastocyst hatching (R2 87.6, P = 0.006) rates in unsexed semen. In conclusion, while the sex-sorting process had no effect on sperm protamine content, we observed a bull effect for sperm protamination, which correlated to embryo development rates following IVF.

Discovery and Preclinical Development of Orally Active Small Molecules that Exhibit Highly Selective Follicle Stimulating Hormone Receptor Agonism.

An orally active follicle stimulating hormone receptor allosteric agonist would provide a preferred treatment for over 16 million infertile women of reproductive age in low complexity methods (ovulation induction-intrauterine insemination) or in high complexity methods (controlled ovarian stimulation-in vitro fertilization). We present two oral follicle stimulating hormone receptor allosteric agonist compounds that have the desired pharmacology, drug metabolism, pharmacokinetics, and safety profile for clinical use. These molecules provide a single agent suitable for ovulation induction-intrauterine insemination or controlled ovarian stimulation-in vitro fertilization that is more convenient for patients and achieves similar preclinical efficacy as rec-hFSH. TOP5668, TOP5300 were evaluated in vitro in Chinese hamster ovary cells transfected with individual glycoprotein receptors measuring cAMP (FSHR, LH/CGR, thyroid stimulating hormone receptor). TOP5668 was found to have solely follicle stimulating hormone receptor allosteric agonist activity while TOP5300 was found to have mixed follicle stimulating hormone receptor allosteric agonist and LHR-AA activity. Both compounds stimulated concentration-dependent increases in estradiol production from cultured rat granulosa cells in the presence or absence of low dose rec-hFSH, while only TOP5300 stimulated testosterone production from rat primary Leydig cells. In pooled human granulosa cells obtained from patients undergoing controlled ovarian stimulation-in vitro fertilization, TOP5300 stimulated 7-fold greater maximal estradiol response than rec-hFSH and TOP5668 was 10-fold more potent than TOP5300. Both TOP5300 and TOP5668 stimulated follicular development in immature rat to the same efficacy as recombinant follicle stimulating hormone. In mice treated with TOP5300, in the presence of low dose of follicle stimulating hormone, there were no differences in oocyte number, fertilization rate, and hatched blastocyst rate in mice with TOP5300 and low dose follicle stimulating hormone vs. reference proteins pregnant mare serum gonadotropin or high dose rec-hFSH. ADME/PK and safety profiles were favorable. In addition, there was no appreciable activity on thyroid hormones by TOP5300 in 14-days toxicological study in rat or dog. The selected lead compound, TOP5300 stimulated a more robust increase in estradiol production from granulosa-lutein cells from women with polycystic ovarian syndrome patient compared to rec-hFSH. Conclusions: Two novel oral FSHR allosteric agonist, TOP5668 and TOP5300, were found to mimic the biological activity of rec hFSH in preclinical studies. Both compounds led to folliculogenesis and superovulation in rat and mice. Specifically, TOP5300 led to a similar number of ovulated oocytes that fertilized and developed into hatched blastocysts in mice when compared to rec-hFSH. The safety profile demonstrated lack of toxicity.

117 Supplementation of IVF medium with nerve growth factor improved bovine embryonic cleavage rates during summer months

Nerve growth factor-β (NGF), a protein originally associated with regulation of neuron development, has been found to play a role in the reproductive system of mammals. Previous research showed that administration of NGF to cows resulted in enhanced conceptus development. Although these effects were speculated to be a result of improved corpus luteum function, whether NGF could act directly on the embryo remained undetermined. Therefore, the direct effects of NGF on fertilization and embryo development warrant investigation to see whether it can be used as a novel tool to improve cleavage and blastocyst rates when producing embryos via IVF during periods of suboptimal oocyte quality, such as with heat stress. The objective of this study was to explore how supplementation of NGF, purified from bull seminal plasma, during IVF may directly affect embryo development in oocytes harvested in the summer. Abattoir-derived bovine ovaries were used for recovery of cumulus-oocyte complexes (COCs) over eight replicates through May and June. On Day −1, COCs were collected and matured for 20h in oocyte maturation medium incubated at 38.5°C. On Day 0, matured oocytes were added to a solution of IVF-Tyrode's albumin lactate pyruvate (TALP) and either phosphate-buffered saline (PBS; control) or 100ngmL−1 NGF. Pooled frozen-thawed semen from two different bulls per replicate were added to the IVF solutions and incubated with COCs for 20h at 38.5°C in a humidified atmosphere of 5% CO2. On Day 1, zygotes were washed in HEPES-TALP, and cumulus cells were removed using 1% hyaluronidase. The zygotes were plated in synthetic oviductal fluid (SOF-BE2) culture medium and incubated at 38.5°C in a tri-gas chamber (5% CO2, 5% O2, and balanced N2). Cleavage rates were recorded at 24 and 48h, calculated by dividing the number of cleaved embryos by the total zygote count. Embryos were incubated until Day 8, when the rate of blastocysts was assessed. This study found that the treatment of IVF medium with NGF increased the cleavage rate of embryos after 48h (Control: 59%; NGF: 66%; P=0.04) and the hatched blastocyst percentage per oocyte on Day 8 (Control: 6.7%; NGF: 9.4%; P=0.01). The treatment did not affect the percentage of blastocysts per cleaved embryos (Control: 21%; NGF: 22%; P=0.16) or the hatched blastocyst rate at Day 8 (Control: 53%; NGF: 55%; P=0.67). These results show that NGF can act directly on the oocyte during fertilization to alter subsequent development, specifically through increased embryonic cleavage rates. Further studies are needed to assess different dosages of NGF in order to mitigate the detrimental effects of heat stress on oocyte competence for use in IVF. Follow-up studies using a whole-animal model are needed to understand the clinical relevance of these findings in the ability of embryos to promote maternal recognition of pregnancy.

Development of bovine embryos in vitro in coculture with murine mesenchymal stem cells and embryonic fibroblasts

Despite the progress on development of new culture media, in vitro-produced embryos still display lower quality when compared to the in vivo-produced counterparts. Coculture has been reconsidered as an alternative to improve embryo quality. Mesenchymal stem cells (MSC) and murine embryonic fibroblasts (MEF) have been extensively used as feeder layers due to their capacity to release growth factors. In the present study we investigated the effect of these feeder layers in oocyte maturation and/or embryo development under in vitro conditions. Oocytes were matured in control (CTRL) conditions or in coculture with MSC or MEF. In vitro fertilization and embryo culture until fourth day were performed in CTRL condition for all groups. Embryos from fourth day on were then cultured until the eighth day in CTRL or in coculture system. No significant differences for metaphase II stage and apoptosis in oocytes were found among the groups. There was also no difference among the groups when we evaluated blastocyst formation on the seventh and eighth day, with exception of a higher hatched blastocyst rate in the group maturated and cultivated in CTRL condition when compared to the group matured and cocultured with MSC. Also no difference was observed in the number of cells in the whole embryos, in the inner cell mass, in the trophoblast and at apoptotic stage on the eighth day. We conclude that coculture with MSC or MEF during maturation and/or embryo development do not enhance the in vitro production of bovine embryos.

Treatment with chemical delipidation forskolin prior to cryopreservation improves the survival rates of swamp buffalo (Bubalus bubalis) and bovine (Bos indicus) in vitro produced embryos

The cryopreservation of embryos is a technology developed for long-term genetic preservation. However, high sensitivity to low temperatures due to a large number of intracellular lipids within ruminant embryos compromises the success of this technique. The aim of this study was to examine the effects of using of lipolytic chemical agent forskolin, during in vitro producing of buffalo and bovine embryos on lipid contents, cryotolerance and subsequent developmental competence of these embryos. Buffalo and bovine oocytes were collected by the aspiration technique from follicles and submitted for in vitro fertilisation; the embryos were later divided into four experiments. Experiment 1, buffalo and bovine embryos were pre-treated in the presence and absence of 10 μM forskolin for 24 h. Lipid contents were determined by Nile red staining and confocal microscopy. We found that 10 μM forskolin was capable to reduce lipid contents within developing embryos in both of species (P < 0.01). Lipid contents within Day 2 embryos exhibited greater fluorescence intensity than did Day 7 embryos in both animal species. The purpose of Experiment 2 was to investigate the adverse effects of 10 μM forskolin on embryo development. In Experiments 3 and 4, Day 2 (4- to 8-cell stage) and Day 7 (blastocyst stage) embryos were pre-treated with 10 μM forskolin for 24 h and further cryopreserved with a controlled-rate freezing technique. The successful cryopreservation was determined by post-thawed embryonic development in vitro. The results showed that the blastocyst rate of the 4–8 cell stage in the forskolin-treated group had increased in both species, while the hatching and hatched blastocyst rates of forskolin-treated day 7 bovine embryos were significantly higher than those of the non-treated group (52.1% vs. 39.4%; P < 0.05). However, delipidation with forskolin did not affect the developmental rate of the day 7 buffalo embryos (P = 0.73). Our studies showed that delipidation by forskolin treatment increased the survival rate of cryopreservation in buffalo and bovine in vitro produced embryos.

Supplementing in vitro embryo production media by NPPC and sildenafil affect the cytoplasmic lipid content and gene expression of bovine cumulus-oocyte complexes and embryos

In our study, we added natriuretic peptide type C (NPPC) and/or sildenafil during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs) followed by in vitro culture (IVC) of embryos with or without sildenafil. We evaluated the effects on the lipid content (LC) of oocytes and embryos and also verified the expression of 96 transcripts related to competence in matured COCs and 96 transcripts related to embryo quality in blastocysts. After IVM, LC was decreased in oocytes by NPPC while sildenafil did not affect LC in oocytes. The genes involved in lipid metabolism and lipid accumulation (DGAT1, PLIN2and PLIN3) were not affected in COCs after treatment during IVM, although the expression of PTX3 (a cumulus cells expansion biomarker) was increased and the hatched blastocyst rate was increased by NPPC during IVM. During IVM, sildenafil increased the mRNA relative abundance of HSF1 and PAF1 and decreased REST in blastocysts. The use of sildenafil in IVC increased the LC of blastocysts. The mRNA abundance in blastocysts produced during IVC with sildenafil was changed for ATF4, XBP1, DNMT3A, DNMT3B, COX2, and SOX2. Although NPPC reduced the LC of oocytes after IVM and upregulated markers for cumulus expansion, embryo production was not affected and the produced blastocysts were able to regain their LC after IVC. Finally, the use of sildenafil during IVC increased the cytoplasmic LC of embryos but did not affect embryo quality, as measured by analysis of 96 transcripts related to embryo quality.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

Vitrification of bovine matured oocytes and blastocysts in a paper container.

In the present study, we aimed to determine the applicability of a paper container for the vitrification of invitro matured (IVM) bovine oocytes. In experiment 1, IVM oocytes were exposed to vitrification solution (20% dimethylsulfoxide (DMSO), 20% ethylene glycol (EG), and 5mol/L sucrose), using a two-step method, for 30s; loaded onto either a paper container or Cryotop; and stored in liquid nitrogen. No significant difference (P<0.05) in the survival and blastocyst formation rates after invitro vitrification was observed between the paper container and Cryotop. In experiment 2, IVM oocytes were exposed to either a two- or three-step vitrification solution. The three-step vitrification solution was not significantly different from the two-step solution in terms of oocyte survival, cleavage and blastocyst rates. In experiment 3, invitro produced blastocysts were graded according to the manual of the International Embryo Transfer Society (grades 1 and 2) and vitrified using the two- and three-step methods. For grade 2 blastocysts, the three-step method showed significantly higher (P<0.05) survival and hatched blastocyst rates than the two-step method, whereas for grade 1 blastocysts, no significant difference was observed. In conclusion, the paper device and three-step technique are suitable for oocytes and embryo vitrification.

Hyperspectral microscopy can detect metabolic heterogeneity within bovine post-compaction embryos incubated under two oxygen concentrations (7% versus 20%).

Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity? Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy. Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment. The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8; embryo developmental competence) or 5 days (D5; metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40-50 embryos per replicate used for the developmental competency experiment, 10-20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21-22 embryos used for the hyperspectral microscopy study. In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy. Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen (P < 0.05), demonstrating that atmospheric oxygen was a viable model for studying mild metabolic stress. The metabolic profiles of D5 embryos was determined and although metabolic heterogeneity was evident within the cleavage stage (i.e. arrested) embryos exposed to fluorophores, there were no detectable difference in fluorescence intensity and pattern localisation in morula exposed to the two different oxygen concentrations (P > 0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P > 0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P < 0.05), with no change in the redox ratio. Hyperspectral autofluorescence imaging (in 18-spectral channels) of the D5 morula revealed highly significant differences in four features of the metabolic profiles of morula exposed to the two different oxygen concentrations (P < 0.001). These four features were weighted and their linear combination revealed clear discrimination between the two treatment groups. Metabolic profiles were assessed at a single time point (morula), and as such further investigation is required to determine if differences in hyperspectral signatures can be detected in pre-compaction embryos and oocytes, using both cattle and subsequently human models. Furthermore, embryo transfers should be performed to determine the relationship between metabolic profiles and pregnancy success. Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer. This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. The authors declare there are no conflict of interest.

Impact of hydrosalpinx fluid on early human embryos

ABSTRACTThis study aimed to investigate the impact of hydrosalpinx fluid (HF) on early human embryonic development. A total of 33 patients who underwent laparoscopic surgery for hydrosalpinx were selected, and the HF specimens obtained from these patients were subjected to bacterial culture, Chlamydia antigen detection, biochemical analysis, and cytokine detection. Meanwhile, human embryos derived from three pronuclei (3PN) were cultured in various HF concentrations. There was no significant difference in the chemical components and physical characteristics between colorless and colored HF specimens, apart from the glucose concentration which was significantly higher in colorless HF. K+ and HCO3− were significantly increased (P < 0.05 and P < 0.01), and Ca2+, Mg2+, and glucose were significantly decreased (P < 0.05, P = 0.006, and P = 0.007) in the two HF specimens, compared to blastocyst culture medium (G-2 medium); no phosphates were detected in the HF specimens. Compared to colorless HF, the concentrations of tumor necrosis factor α (TNF-α) and interleukin 2 (IL-2) in the colored HF specimens were significantly increased (P < 0.05). There were no differences in the Chlamydia antigen-positive rate between the HF groups (62.5% vs. 70.6%), and no bacterial growth occurred in the HF specimens. There were no significant differences in the development of the 3PN embryos between the two HF groups (P > 0.05). High-concentration HF (75%) significantly affected the rates of blastulation, blastocyst hatching, and high-quality blastocyst formation (P < 0.05). HF is related to chlamydial infection. Embryonic development may be significantly affected only in high-concentration HF, possibly due to the deficiency of essential elements required for embryonic development. TNF-α and IL-2 concentrations were found to vary between the clear and colored HF specimens; however, TNF-α and IL-2 in HF do not appear to exert adverse effects on embryonic development.

Effect of corifollitropin alfa supplemented with or without LH on ovarian stimulation and embryo viability in rabbit

There is increasing interest in using rabbits for research as a laboratory model as well as for industrial production of meat, wool and fur. Superovulation in animals is used to produce a maximum number of transferable embryos per donor, in order to either support genetic improvement programs, ex situ conservation or to optimize other biotechnologies. Over time, the use of this biotechnology has shown variable outcomes as a consequence of several factors, such as the origin of exogenous hormone, posology and the effect of gonadotropins used simultaneously, the donor and the environment. The aim of this study was to compare the efficacy of a single injection of corifollitropin alfa (CTP), alone or supplemented with LH, versus a FSH standard protocol of five equal doses administered twice daily to superovulate rabbit does (20 per group and 29 control females). We determined: 1) the impact of this stimulation on in vitro development and mRNA expression at blastocyst stage and 2) in vivo embryo development and viability rate at birth of transferred embryos. Our outcomes showed that the ovulation rate was similar among the different ovarian stimulation groups, reaching more than fourfold the ovulation rate of a control doe. While rates of embryos developing to the blastocyst stage after 48 h of in vitro culture were similar between groups, the hatched blastocyst rate was higher for superovulated embryos from CTP group. Moreover, no significant differences among mRNA expression of OCT4, SOX2 and NANOG genes were detected. Nevertheless, embryos from ovarian stimulated does with CTP + LH showed significantly higher implantation rates and survival at birth among the different ovarian stimulation groups and similar to those in the control group. In conclusion, the results of this study suggest that a single injection of long acting corifollitropin alfa can be effectively used in rabbits to elicit a more than fourfold increase in ovulation rate compared to control animals. In addition, the LH supplementation allows us to obtain similar in vivo embryo development results as in the control group.

A novel embryo culture media supplement that improves pregnancy rates in mice.

The preimplantation embryo in vivo is exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture media in vitro The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were divided into one of four supplemented culture media treatment groups: (1) control (media only); (2) 12.5 nM IGF2; (3) 10 µg/mL uPA and 5 µg/mL plasminogen; or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls (P < 0.02). Following B6BcF1 embryo transfer, IGF2 + U + P treatment increased implantation sites at day 8 of pregnancy compared with controls (P < 0.05). Replication in the CBAB6F2 mouse strain showed significant improvements in pregnancy rates at days 8 and 18 but not in blastocyst development. No adverse effects were seen on gestational age, litter size or birthweight, or the reproductive capacity of offspring of IGF2 + U + P treated embryos. For embryos susceptible to detrimental effects of in vitro culture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain.