N-(2-mercaptopropionyl)-glycine enhances in vitro pig embryo production and reduces oxidative stress.

This study evaluated the effects of different concentrations (1, 10, 25, 50, and 100 µM) of the antioxidant N-(2-mercaptopropionyl)-glycine (NMPG), during the culture of in vitro-fertilized porcine oocytes. While the highest concentrations of NMPG (50 and 100 µM) were toxic to the developing embryos during the first two days of culture, 25 µM NMPG achieved cleavage rates that were similar to those achieved by the control but did not sustain blastocyst production by Day 7 of culture. Compared to the control culture medium, the culture medium supplemented with 10 µM NMPG increased (P < 0.05) the rates of blastocyst formation, decreased (P < 0.05) the intracellular levels of reactive oxygen substances, and downregulated (P < 0.05) the expression of the oxidative stress related gene GPX1. In conclusion, these results demonstrated that supplementation of porcine embryo culture medium with 10 µM NMPG can attenuate oxidative stress and increase the yield of in vitro production of blastocysts.

Three-to-5-day weaning-to-estrus intervals do not affect neither efficiency of collection nor in vitro developmental ability of in vivo-derived pig zygotes.

An efficient system to collect large numbers of vital zygotes is a pre-requisite for application of zygote genome-editing technology, including development of efficient models for xenotransplantation using pigs. Owing to the sub-optimal in vitro production of zygotes in pigs, efficient collection of in vivo developed zygotes is required. Timing of ovulation is a key factor to sustain efficiency since the interval between pronuclear formation and the first division is very short in pigs. The weaning-to-estrus interval can, due to its inverse relation with length of estrus and time of ovulation, interfere with ovulation and make it asynchronous, which reduces the probability of obtaining zygotes. This retrospective study compared the effects of three weaning-to-estrus intervals of 3, 4 or 5 days on zygote collection efficiency in a total of 17 trials over a 3-year period including 223 sows. Donor sows in groups of 10-15 animals were super-ovulated with eCG 24 h after weaning and those in estrus at 48-72 h post-eCG were immediately treated with hCG, followed by insemination 6 and 24 h thereafter. Collected structures during laparotomy on Day 2 (Day 0: onset of estrus) were morphologically evaluated and only those with a single cell and two visible polar bodies were considered as zygotes. Zygotes were injected with CRISPR-Cas9 editor mixture and cultured for 6 days to evaluate their developmental ability against non-injected control zygotes. Of all recovered structures (N = 5,468), 67.4%, 30.8% and 1.8% were zygotes, 2-cell embryos and oocytes-degenerated embryos, respectively. The different weaning-to-estrus intervals did not affect either the percentages of collected zygotes (range: 64.1%-70.0%) or the percentages of sows with zygotes at collection time (range: 69.0%-73.3%). The weaning-to-estrus intervals did not affect the in vitro developmental ability of zygotes. After 24 h of culture, 78.1 ±â€¯2.0% and 95.1 ±â€¯0.6 (P < 0.05) of injected (N = 2,345) and non-injected (N = 335) zygotes, respectively, developed to 2-to-4-cell embryo stage. The total efficiency of the system was 64.1 ±â€¯2.2% and 85.8 ±â€¯1.5% (P < 0.05) for injected and non-injected zygotes, respectively. In conclusion, the results indicate that neither the efficiency of collecting in vivo derived porcine zygotes from superovulated sows nor the zygote ability to develop to blastocyst after cytoplasmic genome-editing injection were affected by a weaning-to-estrus interval between 3-to-5 days.

The cytokine platelet factor 4 successfully replaces bovine serum albumin for the in vitro culture of porcine embryos.

The cytokine platelet factor 4 (PF4) enhances differentiation and cell viability of different stem cells lines in vitro. This study investigated whether PF4 addition to customary pig embryo semi-defined culture media can improve their developmental outcome (Experiment 1) and ultimately replace the need for bovine serum albumin (BSA, Experiment 2). Experiment 1 added PF4 (100-1000 ng/mL, 0 = control) to NCSU-23 with 0.4 mg/mL BSA culturing 3430 presumptive zygotes. Experiment 2 added PF4 (100-1000 ng/mL, 0 = Control-PVA) to a BSA-free medium (NCSU-23 with 0.3 mg/mL PVA) culturing 3820 presumptive zygotes. Zygote culture in NCSU-23 with 0.4 mg/mL BSA was used as overall control. All groups of Experiment 1 displayed similar rates of day 2-cleavage (range: 65.0 ±â€¯10.9 to 70.0 ±â€¯5.8%); of day 7-blastocyst rates (range: 46.6 ±â€¯10.0 to 56.4 ±â€¯8.2%) and of total day 7-blastocyst efficiency (range: 32.3 ±â€¯8.3 to 37.2 ±â€¯7.3%). Addition of PF4 did not affect total cell numbers of day 7 blastocysts (range: 44.1 ±â€¯23.2 to 50.5 ±â€¯26.4). In Experiment 2, PF4 accelerated embryo development, increasing (P < 0.01) blastocyst yield compared to 0-PF4, and blastocyst formation by day 5 adding PF4 100-500 ng/mL (range: 29.9 ±â€¯7.8 to 31.8 ±â€¯5.5%; P < 0.05) compared with BSA-control (17.2 ±â€¯8.2%) and PF4 1000 ng/mL (15.5 ±â€¯7.9%); showing similar blastocyst rates (range: 42.0 ±â€¯11.5 to 49.3 ±â€¯10.0%), total efficiency (28.0 ±â€¯8.2 to 32.3 ±â€¯7.1%) total cell numbers (range: 42.6 ±â€¯19.3 to 45.7 ±â€¯23.9) as BSA-controls. In conclusion, although PF4 did not show additive improvement under usual semi-defined, BSA-supplemented embryo media, it successfully replaced BSA sustaining porcine blastocyst production in chemically defined conditions.

Porcine blastocyst viability and developmental potential is maintained for 48 h of liquid storage at 25 °C without CO2 gassing.

Short- and medium-term storage of pig embryos has become relevant for commercial application of non-surgical deep uterine embryo transfer (NsDU-ET) in the light of the strict legal and administrative requirements posed by the International Association for Air Transport (IATA) to allow shipment of liquid nitrogen (LN2) containers and the technical drawbacks when using vitrified embryos. Therefore, this study developed an efficient method for the liquid storage of in vivo-derived porcine blastocysts for a moderate duration (48 h) without controlled CO2 gassing. We evaluated two storage temperatures (25 °C and 37 °C) and three HEPES-supplemented media: the chemically defined media TL-PVA and NCSU-PVA and the semi-defined medium NCSU-BSA. We observed no differences in survival, hatching rate or final developmental stage between the two temperatures, but storage at 25 °C was more efficient to preserve zona pellucida (ZP) integrity. Blastocysts were successfully stored for 24 h in a chemically defined medium. Yet, only 48 h storage in NCSU-BSA medium supported blastocyst development. Although all storage conditions resulted in an embryonic developmental delay, blastocysts stored in NCSU-BSA at either tested temperature could hatch and attain the same final developmental stage as control blastocysts when cultured under standard conditions after storage. Moreover, blastocysts stored at 25 °C for 48 h in NCSU-BSA medium could produce pregnancies after surgical transfer. In conclusion, porcine blastocysts maintain their viability and developmental potential after storage in the semi-defined medium NCSU-BSA for at least 48 h at 25 °C.

High pre-freezing sperm dilution improves monospermy without affecting the penetration rate in porcine IVF.

The high incidence of polyspermy is still an unresolved problem for the production of in vitro-produced porcine embryos. In this work, we modified the usual sperm processing sequence for in vitro fertilization (IVF), and the spermatozoa from four boars were frozen directly at a low sperm concentration of 20 × 106 sperm/mL (high pre-freezing sperm dilution group; F20), thawed and processed for IVF in three replicates. Spermatozoa from the same boars frozen at a conventional concentration (1000 × 106 sperm/mL) were used as the control group. The post-thaw sperm quality evaluation demonstrated that despite there being no differences in the percentage of motile spermatozoa between groups, the proportion of live spermatozoa with intact acrosomes was significantly higher in the F20 group than in the control. The in vitro penetration rate was also similar between groups; however, the co-incubation of oocytes with F20 sperm increased monospermy, IVF efficiency, cleavage rate and the efficiency of blastocyst formation compared with the results for oocytes co-incubated with control spermatozoa. These results indicate, for the first time, that a high pre-freezing sperm dilution increases monospermy without affecting penetration rates, thereby increasing blastocyst formation.

Exogenous ascorbic acid enhances vitrification survival of porcine in vitro-developed blastocysts but fails to improve the in vitro embryo production outcomes.

In this study, the effects of addition of the antioxidant ascorbic acid (AsA) were evaluated during porcine in vitro embryo production (IVP) and vitrification. In experiment 1, the effects of AsA supplementation during IVM, IVF and IVC were evaluated, using a total of 2744 oocytes in six replicates. The IVM, IVF and embryo IVC media were supplemented or not (control) with 50 µg/mL AsA in all possible combinations. No significant effects of AsA were detected in any of the maturation, fertilization or embryo development parameters assessed. In experiment 2, we evaluated the effects of adding AsA to vitrification-warming media on the post-warming survival and quality of blastocysts. Day-6 in vitro-produced blastocysts (N = 588) from six replicates were randomly divided in two groups, with vitrification and warming media either supplemented with 50 µg/mL AsA (VW + group) or un-supplemented (VW- control). Addition of AsA increased (P < 0.05) blastocyst survival rate after vitrification compared with that of VW- control embryos. Vitrification and warming increased (P < 0.05) the production of oxygen species (ROS) and reduced (P < 0.05) the glutathione levels in blastocysts. Although VW + blastocysts displayed higher (P < 0.05) ROS levels than those of fresh control blastocysts, the levels were lower (P < 0.05) than those found in VW- control blastocysts. In conclusion, under the experimental conditions, supplementation of IVM/IVF/IVC media with AsA did not improve the embryo production in vitro. By contrast, the addition of AsA to chemically defined vitrification and warming media increased the survival of in vitro-produced porcine blastocysts by decreasing ROS production.

Simple storage (CO2-free) of porcine morulae for up to three days maintains the in vitro viability and developmental competence.

The advancement of porcine embryo transfer (ET) technology is constrained by regulatory hurdles (liquid nitrogen transportation) or, more importantly, the technical obstacles of using vitrified embryos in combination with nonsurgical deep uterine ET technology. Maintaining embryos in culture during transport and prior ET collides with the need of CO2 gassing and the best choice of culture medium. In this work, we describe storage conditions for short-term embryo CO2-free storage that allowed for a majority of in vivo-derived porcine morulae to survive after 3 days of storage in a liquid state, and to develop to the blastocyst stage unhatched, a sanitary prerequisite for ET. The storage conditions included NCSU-23 medium supplemented with bovine serum albumin, where bicarbonate was partially replaced by HEPES to avoid the need for CO2 gassing, and a temperature of 37 °C. These conditions were able to maintain the functionality of the stored embryos (hatching capacity after exposure to conventional culture conditions) and their developmental competence after ET (normal fetuses by day 38 of pregnancy). Use of this strategy of CO2-free storage should allow the shipment of fresh embryos worldwide in the absence of liquid nitrogen.

Peroxidized mineral oil increases the oxidant status of culture media and inhibits in vitro porcine embryo development.

The use of oils with undetected alterations is a long-recognized problem for in vitro embryo production systems. Since peroxides in oils have been associated with reduced embryo production outcomes, our goals were (1) to evaluate the effects of a batch of mineral oil (MO) that was suspected to be altered on the in vitro production of pig embryos and (2) to determine oil peroxide values throughout culture and the transfer of oxidant agents from oil to culture media. Sunflower oil, which has a completely different chemical composition than MO but a higher oxidative status, and unaltered MO were used as controls. Oocyte maturation, fertilization and embryo development were affected differently depending on the oil overlay used. While the suspected MO was not able to sustain in vitro maturation and fertilization, the oocytes incubated in the presence of sunflower oil were matured and fertilized similarly to those of the unaltered MO group. Moreover, the cleavage rate of presumed zygotes cultured under the suspected MO was severely reduced compared with those cultured under the other oils, and none of the cleaved embryos developed to the blastocyst stage. Although the cleavage rates in the sunflower oil and unaltered MO groups were similar, embryos cultured under sunflower oil also failed to develop to the blastocyst stage. Our results revealed that the suspected MO and sunflower oil had similar levels of peroxides and that these levels were much higher than those of the unaltered MO. The total oxidant status was higher in media incubated under peroxidized oils than in fresh media or media incubated without an oil overlay or under unaltered MO, indicating that oxidant agents were transferred to the incubation media. However, unlike the sunflower oil group, the culture media incubated under the suspected MO had high levels of total oxidant status and low levels of hydrogen peroxide and reactive oxygen species, suggesting the presence of other unknown oxidant agents in that oil. These results indicate that a peroxidized MO overlay dramatically decreases embryo production outcomes. This decrease could be associated with the higher peroxide values of the oil but cannot be explained by the levels of hydrogen peroxide and reactive oxygen species transferred from the oil to the culture media. It is likely that different oxidant agent(s) and/or other toxic compounds present in the peroxidized MO are responsible for its damaging effects on oocytes and embryos.

Effects of meiotic inhibitors and gonadotrophins on porcine oocytes in vitro maturation, fertilization and development.

This study evaluated the effect of three reversible meiotic inhibitors (MINs) and their interaction with gonadotrophins (Gns) on the meiotic maturation and developmental competence of porcine oocytes. In experiment 1, the oocytes were matured for 22 hr in the presence or absence of dbcAMP (1 mM), cycloheximide (7 µM) or cilostamide (20 µM) with or without Gns, and for an additional 22 hr in the absence of MINs and Gns. At 22 hr of maturation, regardless of the presence of Gns, a higher proportion (p < .001) of oocytes cultured in the presence of MINs were effectively arrested at the germinal vesicle stage compared with the oocytes cultured without MINs. At 44 hr of maturation, the proportion of oocytes that reached MII was higher (p < .05) in groups with Gns compared with groups without Gns. In experiment 2, oocytes that were matured as in experiment 1 were inseminated and cultured for 7 days to evaluate fertilization parameters and blastocyst formation. Only oocytes from the dbcAMP + Gns group had higher (p < .05) efficiency of fertilization compared with the other treatment groups. The presence of dbcAMP during maturation also increased (p < .05) blastocyst formation and efficiency of blastocyst formation in both the presence and absence of Gns. These results indicate that the interaction of Gns with the tested MINs improved meiotic progression. In addition, regardless of supplementation with Gns, the presence of dbcAMP during the first maturation period increased and even doubled the capacity of oocytes to develop to the blastocyst stage.

Factors of importance when selecting sows as embryo donors.

The improvement in porcine embryo preservation and non-surgical embryo transfer (ET) procedures achieved in recent years represents essential progress for the practical use of ET in the pig industry. This study aimed to evaluate the effects of parity, weaning-to-estrus interval (WEI) and season on reproductive and embryonic parameters at day 6 after insemination of donor sows superovulated after weaning. The selection of donor sows was based on their reproductive history, body condition and parity. The effects of parity at weaning (2 to 3, 4 to 5 or 6 to 7 litters), season (fall, winter and spring), and WEI (estrus within 3 to 4 days), and their interactions on the number of corpus luteum, cysts in sows with cysts, number and quality of viable and transferable embryos, embryo developmental stage and recovery and fertilization rates were evaluated using linear mixed effects models. The analyses showed a lack of significant effects of parity, season, WEI or their interactions on any of the reproductive and embryonic parameters examined. In conclusion, these results demonstrate that fertilization rates and numbers of viable and transferable embryos collected at day 6 of the cycle from superovulated donor sows are not affected by their parity, regardless of the time of the year (from fall to spring) and WEI (3 or 4 days).

Surgical embryo collection but not nonsurgical embryo transfer compromises postintervention prolificacy in sows.

Recent advances in nonsurgical deep uterine (NsDU) embryo transfer (ET) technology allow the noninvasive transfer of porcine embryos into recipients, overcoming the most important impediment for commercial ET in this species. Although many factors in the porcine ET-field have been recently evaluated, many others remain to be explored. We investigated here the future reproductive performance of donors and recipients after artificial insemination subsequent to the default surgical embryo recovery approach and to the NsDU-ET procedure, respectively. Although surgical embryo collection did not influence subsequent farrowing rates (90.5%), litter size decreased severely (8.9 ± 0.8 piglets) compared to presurgery (10.8 ± 0.3 piglets) and control group (10.7 ± 0.3 piglets). In contrast, NsDU-ETs did neither affect fertility nor prolificacy of recipients in the cycle subsequent to ET, regardless of whether they were pregnant after NsDU-ET or not. These results indicate that while the surgical embryo collection procedure compromises the reproductive future of donor sows, the NsDU-ET approach does not affect the reproductive potential of the recipients after reintroduction to the breeding stock of the farm. Further research is thus needed to improve surgical embryo collection.

Generation of human organs in pigs via interspecies blastocyst complementation.

More than eighteen years have passed since the first derivation of human embryonic stem cells (ESCs), but their clinical use is still met with several challenges, such as ethical concerns regarding the need of human embryos, tissue rejection after transplantation and tumour formation. The generation of human induced pluripotent stem cells (iPSCs) enables the access to patient-derived pluripotent stem cells (PSCs) and opens the door for personalized medicine as tissues/organs can potentially be generated from the same genetic background as the patient recipients, thus avoiding immune rejections or complication of immunosuppression strategies. In this regard, successful replacement, or augmentation, of the function of damaged tissue by patient-derived differentiated stem cells provides a promising cell replacement therapy for many devastating human diseases. Although human iPSCs can proliferate unlimitedly in culture and harbour the potential to generate all cell types in the adult body, currently, the functionality of differentiated cells is limited. An alternative strategy to realize the full potential of human iPSC for regenerative medicine is the in vivo tissue generation in large animal species via interspecies blastocyst complementation. As this technology is still in its infancy and there remains more questions than answers, thus in this review, we mainly focus the discussion on the conceptual framework, the emerging technologies and recent advances involved with interspecies blastocyst complementation, and will refer the readers to other more in-depth reviews on dynamic pluripotent stem cell states, genome editing and interspecies chimeras. Likewise, other emerging alternatives to combat the growing shortage of human organs, such as xenotransplantation or tissue engineering, topics that has been extensively reviewed, will not be covered here.

Non-viable sperm in the ejaculate: Lethal escorts for contemporary viable sperm.

Non-viable sperm ("dead sperm") are present in variable numbers in mammalian ejaculates and their number increase substantially when semen is stored, particularly cryopreserved. This review comparatively highlights, with experimental data in porcine, the role-played by non-viable sperm in the outcome of semen used in assisted reproductive technologies. As well, the review discusses our current understanding of their origin and the pathways involved when their large numbers negative influence the functional lifespan of contemporary viable sperm to eventually cause irreversible dysfunction that reduces their fertility potential and their ability to develop healthy embryos. Finally, it highlights procedures currently available to mitigate these harmful effects.

The Recipients' Parity Does Not Influence Their Reproductive Performance Following Non-Surgical Deep Uterine Porcine Embryo Transfer.

With the development of the non-surgical deep uterine (NsDU) embryo transfer (ET) technology, the commercial applicability of ET in pigs is now possible. There are, nevertheless, many factors that influence NsDU-ET effectiveness that need to be addressed. The aim of this study was to evaluate the effects of the weaned recipients' parity on fertility and prolificacy following NsDU-ET. The recipients (n = 120) were selected based on their reproductive history and body condition and grouped into three categories according to their parity: primiparous sows, sows of parity 2 and sows of parities from 3 to 5. Thirty fresh embryos (morulae and unhatched blastocysts) were non-surgically transferred into one uterine horn of each recipient. It was possible to insert the NsDU-ET catheter through the cervix along a uterine horn in 98.3% of the recipients. The parity had no influence on the difficulty grade of the insertions or on the percentage of correct insertions. The cervix and uterine wall were not perforated during the insertions, and vaginal discharge was not observed after transfer in any of the recipients. There were no differences in the pregnancy rates (74.8%), farrowing rates (71.2%) or litter sizes (9.6 ± 3.3) between groups. Also, there were no differences between groups regarding to the piglets' birthweights or piglet production efficiency. In conclusion, these results demonstrate that weaned sows from parity 1 to 5 are appropriate to be used as recipients in NsDU-ET programs, which increase the possibilities for the utilization of ET in the recipient farms.

Effects of two combinations of cryoprotectants on the in vitro developmental capacity of vitrified immature porcine oocytes.

This study evaluated two cryoprotectant (CPA) combinations, ethylene glycol (EG) + DMSO and EG + propylene glycol (PG), used for the vitrification of germinal vesicle (GV) porcine oocytes. In experiment 1, the equilibration of GV with the two CPA combinations increased (P < 0.05) the percentage of oocytes that degenerated after IVM (18.1 ± 2.3% and 19.4 ± 2.6% for EG + DMSO and EG + PG groups, respectively) compared with control oocytes (7.6 ± 1.3%). However, CPAs did not affect the fertilization or developmental parameters of the embryos. In experiment 2, the percentages of live vitrified-warmed GV oocytes at 2 hours after warming (EG + DMSO: 67.0 ± 2.3% and EG + PG: 57.6 ± 2.3%) were lower than those of fresh control GV oocytes (97.3 ± 0.7%). The percentage of degenerated oocytes after IVM was higher (P < 0.001) in vitrified-warmed oocytes (EG + DMSO: 59.8 ± 2.3% and EG + PG: 56.2 ± 2.6%) than in the control (1.6 ± 1.3). Fertilization efficiency was higher (P < 0.05) in the EG + PG (39.6 ± 2.4%) and control (42.0 ± 2.2%) groups than in the EG + DMSO (26.3 ± 7.7%) group. The cleavage and blastocyst formation rates of the EG + DMSO (25.9 ± 3.5% and 6.6 ± 2.5%, respectively) and EG + PG (20.2 ± 5.4% and 4.7 ± 1.6%, respectively) vitrification groups were lower (P < 0.001) than those observed in the control oocytes (53.4 ± 2.7% and 31.9 ± 1.7%, respectively). In conclusion, in the absence of vitrification, the toxic effects of both CPA combinations on the GV oocytes were minimal. Vitrification resulted in important losses in viability at each step of the in vitro embryo production procedure. However, the surviving oocytes were able to mature and be fertilized, although the fertilization efficiency in the EG + DMSO group was lower. Blastocysts formation was similar for both CPA combinations.

The effects of superovulation of donor sows on ovarian response and embryo development after nonsurgical deep-uterine embryo transfer.

This study aimed to evaluate the effectiveness of superovulation protocols in improving the efficiency of embryo donors for porcine nonsurgical deep-uterine (NsDU) embryo transfer (ET) programs. After weaning (24 hours), purebred Duroc sows (2-6 parity) were treated with 1000 IU (n = 27) or 1500 IU (n = 27) of eCG. Only sows with clear signs of estrus 4 to 72 hours after eCG administration were treated with 750 IU hCG at the onset of estrus. Nonhormonally treated postweaning estrus sows (n = 36) were used as a control. Sows were inseminated and subjected to laparotomy on Days 5 to 6 (Day 0 = onset of estrus). Three sows (11.1%) treated with the highest dosage of eCG presented with polycystic ovaries without signs of ovulation. The remaining sows from nonsuperovulated and superovulated groups were all pregnant, with no differences in fertilization rates among groups. The number of CLs and viable embryos was higher (P < 0.05) in the superovulated groups compared with the controls and increased (P < 0.05) with increasing doses of eCG. There were no differences among groups in the number of oocytes and/or degenerated embryos. The number of transferable embryos (morulae and unhatched blastocysts) obtained in pregnant sows was higher (P < 0.05) in the superovulated groups than in the control group. In all groups, there was a significant correlation between the number of CLs and the number of viable and transferable embryos, but the number of CLs and the number of oocytes and/or degenerated embryos were not correlated. A total of 46 NsDU ETs were performed in nonhormonally treated recipient sows, with embryos (30 embryos per transfer) recovered from the 1000-IU eCG, 1500-IU eCG, and control groups. In total, pregnancy and farrowing rates were 75.1% and 73.2%, respectively, with a litter size of 9.4 ± 0.6 piglets born, of which 8.8 ± 0.5 were born alive. There were no differences for any of the reproductive parameters evaluated among groups. In conclusion, our results demonstrated the efficiency of eCG superovulation treatments in decreasing the donor-to-recipient ratio. Compared with nonsuperovulated sows, the number of transferable embryos was increased in superovulated sows without affecting their quality and in vivo capacity to develop to term after transfer. The results from this study also demonstrate the effectiveness of the NsDU ET procedure used, making possible the commercial use of ET technology by the pig industry.

Forskolin improves the cryosurvival of in vivo-derived porcine embryos at very early stages using two vitrification methods.

This study was aimed to determine the effect of forskolin on the viability of in vivo-derived porcine embryos vitrified by the superfine open pulled straw (SOPS) or solid surface vitrification (SSV) methods at the 2-cell, 4-cell, and blastocyst stages. Zygotes, 2- to 4-cell embryos, and morulae were obtained from superovulated sows. After collection, embryos were cultured for 24h with 0 or 10 µM forskolin and then vitrified using the SOPS and SSV method, or not vitrified (fresh controls). Fresh and vitrified-warmed 2-cells, 4-cells, and blastocysts were cultured for additional 96 h, 72 h and 24 h, respectively. At the end of the culture, embryos were evaluated for progression to the blastocyst stage and total cell number. The vitrification method did not affect any of the parameters evaluated for any embryo stage. Forskolin increased (P<0.01) the blastocyst formation and the final developmental stage of vitrified 2- and 4-cell embryos. However, these embryos exhibited lower (P<0.003) blastocyst formation rates than their fresh counterparts. The total cell number and hatching rate were similar in both groups (vitrified and fresh) of 2- and 4-cell embryos. Vitrified blastocysts exhibited viabilities, final developmental stages, hatching rates, and total cell numbers that were similar to those of their fresh counterparts, regardless of the addition of forskolin. In conclusion, the SOPS and SSV methods are suitable for the cryopreservation of in vivo-derived 2- to 4-cell porcine embryos. Pre-treatment with forskolin for 24h before vitrification improves the cryotolerance of 2- and 4-cell porcine embryos.

Effects of lipid polarisation on survival of in vivo-derived porcine zygotes vitrified by the superfine open pulled-straw method.

This study aimed to evaluate the post-warming in vitro viability of intact porcine zygotes vitrified using the superfine open pulled-straw (SOPS) method and to investigate whether cryotolerance is increased by lipid polarisation before vitrification. In vivo-derived zygotes (n=317) were either untreated before SOPS vitrification or subjected to one of the following pre-treatments: (1) centrifugation (20 min, 15000 g) or (2) equilibration in high-osmolality medium (6 min, 400 mOsm kg(-1)) followed by centrifugation. Vitrified-warmed and non-vitrified fresh zygotes were cultured in vitro for 120 h. There were no differences in the blastocyst formation rates between the vitrification groups (from 35.4±5.3% to 48.2±5.6%), but fresh zygotes exhibited higher (P<0.001) blastocyst formation rates (87.5±5.3%) than did vitrified-warmed zygotes. The total blastocyst cell number was similar among all groups (from 34.9±2.8 to 44.1±2.8). In conclusion, SOPS vitrification is a promising method for the cryopreservation of untreated in vivo-derived porcine zygotes. Neither lipid polarisation by centrifugation nor exposure to a high-osmolality medium followed by centrifugation affected the post-warming in vitro viability of zygotes. Our study also demonstrated that the donor is an important factor in determining the success of vitrification for in vivo-derived porcine zygotes.

The in vitro and in vivo developmental capacity of selected porcine monospermic zygotes.

In this study, we evaluated the in vitro and in vivo developmental capacity of selected monospermic zygotes produced in vitro. Cumulus-oocyte complexes were matured in vitro and inseminated with frozen-thawed spermatozoa. Thirteen hours after insemination, presumptive zygotes were centrifuged at 15,000 ×g for 20 minutes to polarize the lipids in the cytoplasm and permit the visualization of pronuclei. Then, the oocytes were individually classified as bipronuclear (2PN) or polypronuclear (three or more pronuclei, PPN). To examine embryo development, 102 selected zygotes were cultured for 7 days. There were no differences in cleavage rate (93.0% and 88.9% for 2PN and PPN zygotes, respectively). However, the blastocyst formation rate was higher (P < 0.003) in 2PN (80.7%) zygotes than in PPN (53.3%) zygotes. The control (noncentrifuged, nonselected zygotes) group showed lower (P < 0.003) cleavage rate and blastocyst formation than the 2PN and PPN zygotes. In a second experiment, 2PN zygotes and control zygotes were transferred (30 zygotes per transfer) by laparoscopy into the oviducts of recipient gilts (10 recipients per group) on the first day of standing estrus. The farrowing rates were 70% and 40% for transfers made with 2PN and control zygotes, respectively. The average number of piglets born per recipient farrowed did not differ between groups (4.9 ± 0.6 and 4.5 ± 1.2, respectively), but the efficiency (number of live piglets per total transferred embryos) was higher (P < 0.01) for 2PN zygotes than for the control group (9.3% and 4.0%, respectively). These results demonstrate the effectiveness of centrifugation for the selection of monospermic zygotes as a procedure to improve in vitro embryo production in pigs. In addition, the results indicate that the laparoscopic technique described here is a simple and effective procedure for transferring embryos into one oviduct.

Effect of MEM vitamins and forskolin on embryo development and vitrification tolerance of in vitro-produced pig embryos.

The aims of this study were (1) to determine the effect of in vitro maturation (IVM) medium supplementation with MEM vitamins on in vitro embryo development and sensitivity to vitrification of Day 6 blastocysts and (2) to evaluate whether the addition of forskolin to in vitro culture (IVC) medium enhances blastocyst survival following Super Open Pulled Straw (SOPS) vitrification. Cumulus-oocyte complexes (COCs; n=4000) were matured with 0.0% or 0.05% (v/v) MEM vitamins. After 44h of IVM, the oocytes were in vitro fertilized, and presumptive zygotes were cultured. At Day 5 of IVC, embryos from both experimental groups were cultured for 24h with 0 or 10µM forskolin, achieving a 2×2 factorial design. The blastocyst formation rate was assessed on Day 6, and subsets of samples from the four experimental groups were vitrified (n=469) or kept fresh (n=546). Fresh and vitrified-warmed blastocysts were cultured for 24h prior to embryo survival and total blastocyst cell number assessment. The MEM vitamins increased (P<0.001) the blastocyst formation rate at Day 6, but they did not affect embryo survival after vitrification. In contrast, the addition of forskolin to the culture medium enhanced (P<0.05) the blastocyst vitrification tolerance. The total blastocyst cell number was similar among the groups. In conclusion, supplementation with 0.05% MEM vitamins improved the blastocyst formation rate, and the addition of 10µM forskolin to the culture medium increased survival in Day 6 in vitro-produced blastocysts after SOPS vitrification.