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SummaryThis study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

Protein-free media are essential for the sanitary cryopreservation of bovine genetic resources. Our aim was to set up an optimized protocol for the vitrification of immature bovine oocytes using protein free media which can provide the highest embryo development rates and embryo quality after subsequent in vitro maturation and fertilization. First, using a protein free NCSU-37 as base medium we compared the efficacy of vitrification on Cryotop device with two different CPA protocols. “Protocol A″ employed a combination of ethylene glycol and propylene glycol as permeating cryoprotectants (pCPA) and equilibration in 4% total pCPA (2% ethylene glycol + 2% propylene glycol). “Protocol B″ employed a combination of ethylene glycol and DMSO and equilibration in 15% total pCPA (7.5% ethylene glycol + 7.5% DMSO). The 2 protocols were equally effective in terms of oocyte survival and subsequent development to the blastocyst stage. However, blastocyst cell numbers were significantly higher with “Protocol A”. TCM-199 and NCSU-37 were equally effective as base media for vitrification. Vitrification with “Protocol A″ reduced the percentage of live oocytes and subsequent development to blastocyst stage but did not affect the hatching and cell numbers of blastocysts when compared to the non-treated group. CPA treatment of “Protocol A″ without cooling did not affect embryo development. Storage of ovaries in PBS at 15 °C for overnight reduced the percentage of surviving oocytes after vitrification but not their subsequent development to the blastocyst stage. In conclusion we established a vitrification protocol for the cryopreservation of immature bovine oocytes employing protein-free media which provided high blastocyst quality without noticeable toxic effects.

The aim of the present study was to clarify whether or not our vitrification procedure at the germinal vesicle (GV)-stage triggers the apoptotic cascade in oocytes and subsequent embryos. Immature porcine cumulus-oocyte complexes were either vitrified and warmed (vitrified group) or subjected to cryoprotectant agents (CPA group) or cultured without any treatment (control). Oocytes of all treatment groups were subjected to in vitro maturation (IVM), fertilization, and embryo culture. Apoptosis was assayed in live oocytes at the end of IVM culture and in cleavage-stage embryos after in vitro fertilization (IVF). We detected similar frequencies of DNA fragmentation, levels of caspase activity, phosphatidylserine externalization, and mRNA levels for pro-apoptotic Bax and CASP3 genes in oocytes at the end of IVM and in early embryos among all groups. However, in the vitrified group, the anti-apoptotic Bcl-XL gene was upregulated in 4-8 cell embryos, which caused an 8-fold significant increase in the Bcl-XL/Bax mRNA ratio compared with the control and CPA groups (P < 0.05). In conclusion, vitrification of porcine oocytes at the GV stage by our method did not trigger the apoptotic cascade in oocytes and subsequent embryos but triggered the upregulation of the anti-apoptotic Bcl-XL gene in embryos.

The present study was conducted to examine the feasibility of in vitro embryo production and transfer technologies for producing piglets of Agu, an Okinawan indigenous pig breed. After collection of oocytes from surgically dissected ovaries, they were subjected to in vitro maturation. After in vitro maturation/fertilization, a total of 616 putative embryos were transferred into four commercial Western pig recipients, one of which became pregnant and farrowed a total of eight Agu piglets. These results demonstrate that in vitro embryo production using ovaries from Agu females is useful for breeding management and conservation of indigenous breeds.

This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = -0.560, p

Vitrification by the Cryotop method is frequently used for bovine oocyte cryopreservation. Nevertheless, vitrified oocytes still have reduced developmental competency compared with fresh counterparts. The objective of this study was to compare the effect of vitrification either at the germinal vesicle (GV) stage or at the metaphase II (MII) stage on epigenetic characteristics of bovine oocytes and subsequently developing embryos. Our results demonstrated that vitrification of oocytes at each meiotic stage significantly reduced blastocyst development after in vitro fertilization (IVF). However, vitrification at the GV stage resulted in higher blastocyst development than did vitrification at the MII stage. Irrespective of the meiotic stage, oocyte vitrification did not affect 5-methylcytosine (5mC) immunostaining intensity in oocyte DNA. However, at both stages, it caused a similar reduction of 5mC levels in DNA of subsequently developing blastocysts. Oocyte vitrification had no effect on the intensity of H3K9me3 and acH3K9 immunostaining in oocytes and subsequent blastocysts. The results suggest that irrespective of meiotic stage, oocyte vitrification alters global methylation in resultant embryos although such alteration in the oocytes was not detected. Oocyte vitrification might not influence histone acetylation and methylation in oocytes and resultant embryos. Vitrification at the immature stage was more advantageous for blastocyst development than at the mature stage.

Vitrification reduces the developmental competence of porcine immature oocytes. We investigated the effects of modifying various factors on the viability and development of oocytes after vitrification. These factors included: 1) exposure to the vitrification solution, 2) macromolecule addition (bovine serum albumin (BSA) or polyvinyl pyrrolidone (PVP)), 3) treatment with cytochalasin B, 4) equilibration temperature, and 5) vitrification method (microdrop or Cryotop). Oocytes were equilibrated and vitrified using medium containing ethylene glycol and propylene glycol. After warming, oocytes were subjected to in vitro maturation, stimulated parthenogenetically, and cultured in vitro. Survival rate, nuclear maturation, cleavage, development to the blastocyst stage and their quality were compared between the vitrified groups and the non-vitrified control group. It was found that 1) exposure to the vitrification solution for longer than 30 s was detrimental to embryo development; 2) replacement of BSA with PVP improved embryo development; 3) cytochalasin B treatment reduced the survival rates, but did not affect the blastocyst development rates, 4) equilibration at room temperature (25 °C) was the most beneficial, and 5) the microdrop method improved survival rates. With these adjustments, we were able to establish a simplified and defined cryopreservation system for porcine immature oocytes with improved efficacy.

Our aim was to establish an efficient culture system to produce embryos by SCNT of the endangered Vietnamese Ỉ pig. Reducing the serum concentration from 10.0% to 0.2% during culture efficiently synchronized Ỉ pig fibroblasts used as donor cells at the G0/G1 stage. Oocyte maturation in a defined porcine oocyte medium (POM) supplemented with EGF and gonadotrophins resulted in higher cleavage and blastocyst rates compared with a non-defined POM containing pig follicular fluid (but without EGF) and both the defined and non-defined variants of NCSU-37. For embryo culture PZM3 and PZM5 media were superior to NCSU-37, in terms of the percentage of cleaved embryos. Addition of serum to PZM3 medium on Day 5 of culture (Day 0 = SCNT) improved blastocyst development. When SCNT embryos were transferred at the blastocyst stage, 7 of 11 recipients became pregnant. However, live offspring were not obtained. In conclusion, we established a system for the production of Ỉ pig embryos by SCNT and achieved blastocyst production rate at 26.4% by improving culture systems for donor cells, oocytes and embryos culture. Transfer of embryos resulted in pregnancies; however, live offspring were not obtained.

Oocyte cryopreservation is a potent approach to keep female germplasm safe from epidemic diseases. In the last decade, we developed simple, cheap, and robust vitrification protocols which enable quick cryopreservation of immature porcine oocytes and zygotes in large numbers. In this chapter, we describe vitrification procedures for porcine oocytes and zygotes where they are vitrified in 1-2 μL aliquots of a defined (protein-free) vitrification medium and dropped either on a metal surface pre-cooled from the bottom with liquid nitrogen (solid surface vitrification) or directly into liquid nitrogen. Vitrified microdrops can be stored in cryo-vials in liquid nitrogen. Low concentrations of permeating cryoprotectants during equilibration and proper temperatures during equilibration and warming are crucial for achieving high survival rates. The device used for cooling does not seem to affect system efficacy as vitrification of oocytes or zygotes either on Cryotop

Oocyte cryopreservation is a potent approach to keep female germplasm safe from epidemic diseases. In the last decade, we developed simple, cheap, and robust vitrification protocols which enable quick cryopreservation of immature porcine oocytes and zygotes in large numbers. In this chapter, we describe vitrification procedures for porcine oocytes and zygotes where they are vitrified in 1-2 μL aliquots of a defined (protein-free) vitrification medium and dropped either on a metal surface pre-cooled from the bottom with liquid nitrogen (solid surface vitrification) or directly into liquid nitrogen. Vitrified microdrops can be stored in cryo-vials in liquid nitrogen. Low concentrations of permeating cryoprotectants during equilibration and proper temperatures during equilibration and warming are crucial for achieving high survival rates. The device used for cooling does not seem to affect system efficacy as vitrification of oocytes or zygotes either on Cryotop® sheets or in microdrops were equally effective.

This study assessed the effects of cryoprotectant concentration during equilibration on the efficiency of bovine blastocyst vitrification and the expression of selected developmentally important genes. In vitro produced bovine blastocysts were equilibrated in either 7.5% ethylene glycol (EG) + 7.5% DMSO (Va group) or in 2% EG + 2% DMSO (Vb group) then vitrified on Cryotop® sheets in 16.5% EG + 16.5% DMSO + 0.5M sucrose. After warming, embryos were cultured for 48 hr. Re-expansion, hatching, and the numbers of total and membrane damaged cells were compared among vitrified groups and a control. There was no significant difference between the vitrified groups in survival, cell numbers and the extent of membrane damage. Vitrification increased the number of membrane-damaged cells in both groups, however, in a greater extent in the Vb group. Vitrification increased (p < .05) the expression of the HSP70 gene in Va but not in Vb embryos. The expression of IGF2R, SNRPN, HDAC1, DNMT3B, BAX, OCT4, and IFN-t genes were the same in control and vitrified groups. In conclusion, the concentration of cryoprotectants during equilibration did not affect survival rates; however, normal cell numbers could be maintained only by equilibration in 15% cryoprotectants which was associated with increased HSP70 expression.

The aim of this study was to examine whether a morphological approach is efficient for selecting high‐quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen‐thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105 sperms/ml). Under conditions of moderate polyspermy, 4‐cell embryos selected at 48 hr after IVF (single selection) and 8‐cell embryos selected at 79 hr after IVF from the collected 4‐cell embryos (double selection) showed high developmental competence. Likewise, 4‐ and 8‐cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4‐ and 8‐cell embryos produced under conditions of high polyspermy was significantly (p

The Vietnamese Ban pig is a precious genetic resource that needs to be preserved. In vitro embryo production from in vitro matured (IVM) oocytes is an important tool for the utilization of cryopreserved porcine sperm. The aim of this study was to compare two media for the IVM of Ban pig oocytes. Immature oocytes were subjected to IVM either in a non-defined (TCM-199 + pig follicular fluid) or in a defined base medium (POM + epidermal growth factor). At the end of IVM, the oocytes were in vitro fertilized (IVF) with frozen Ban sperm. Ten hours after IVF, the oocytes were either subjected to orcein staining to check fertilization and maturation status or cultured in vitro for 7 days. There was no difference between the two IVM media in terms of percentages of oocyte maturation and blastocyst production. However, the percentage of male pronuclear formation after IVF and the total cell numbers in blastocysts were higher with the defined system. Zygotes obtained by the two IVM systems survived vitrification at similar rates. In conclusion, the two IVM systems were both effective for the production of Ban pig embryos; however, better embryo quality was achieved with the defined one.

The purpose of this study was to examine whether freeze-dried germinal vesicles (GV) can be matured in vitro after being injected into enucleated fresh oocytes in pigs as an alternative method for conservation of genetic resources. Although no reduction of the size of GV (p = .094), resveratrol treatment significantly enhanced the survival rates following GV transfer (GVT) (p < .001). Supplementation with 100 or 200 mmol/L trehalose in freeze-drying medium significantly increased the proportions of GVs with intact nuclear membrane and DNA integrity compared with the control group. Following transfer of freeze-dried GVs into enucleated fresh oocytes, the proportion of reconstructed oocytes reached the metaphase-II stage (2.4% ± 1.4%) was significantly lower (p < .05) than that of the in vitro matured control group (83.2% ± 2.5%), it was comparable with the GVT control group (7.4% ± 2.7%). The rates of freeze-dried GVs with intact nuclear membrane and DNA stored at -20°C for 5 days were significantly higher (p < .05) than those at 4°C and room temperature. The rates of intact nuclear membrane and DNA in the freeze-dried GV stored for 15 or 30 days at -20, 4°C and RT were not significantly different. In conclusion, matured oocytes were produced derived from freeze-dried GVs.

We investigated whether high-quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis-shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p

We compared the efficacy of the microdrop and minimum volume cooling (MVC) methods for the vitrification of in vitro-produced porcine zygotes and blastocysts after equilibration in low concentrations of cryoprotectant agents. Zygotes and blastocysts were equilibrated in 2% (v/v) ethylene glycol and 2% (v/v) propylene glycol for 13-15 min. Then, they were vitrified in a medium comprised of 17.5% ethylene glycol, 17.5% propylene glycol, 0.3 M sucrose, and 50 mg/ml polyvinylpyrrolidone either by either dropping them directly into liquid nitrogen (microdrop method) or placing them on Cryotop sheets in a minimum volume of medium and plunging into liquid nitrogen (MVC method). Both zygotes and blastocysts were successfully vitrified. For the vitrification of zygotes, the MVC and microdrop methods were equally effective; however, for blastocyst vitrification, MVC was superior. For both methods, the vitrification of zygotes produced higher-quality embryos than the vitrification of blastocysts.

Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 μM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.

We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus-oocyte complexes were collected from antral follicles of 7-8 mo old female cyclic Ban pigs and vitrified in micro-drops. Oocyte morphology, lipid content, post-warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post-warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo-tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.

Oocyte vitrification is an important approach for invitro gene banking of female germplasm; however, in pigs, it hampers embryo development. In cattle, vitrification at the MII stage was reported to alter epigenetic status in oocytes and even in subsequently developing embryos (Chen et al. 2016 Theriogenology 86, 868-878). The present study investigated the effect of vitrification at the immature stage of porcine oocytes on DNA methylation status and its relevance to subsequent embryo development. Immature cumulus–oocyte complexes were vitrified in microdrops and warmed (vitrified group) or treated with cryoprotectant agents (17.5% ethylene glycol + 17.5% propylene glycol, CPA group) by our method (Appeltant et al. 2018 Cryobiology 85, 87-94). Then they were subjected to IVM, parthenogenetic activation (PA), and embryo culture. From each batch, a group of oocytes was processed without treatment (control group). Oocyte survival and polar body extrusion were recorded after IVM. Cleavage and blastocyst developmental rates were recorded on Day 2 and 6 of culture, respectively (Day 0=PA). In each replication, DNA methylation was assayed in representative oocytes at the MII stage after IVM and in embryos at the 2- to 4-cell stage on Day 2 by immunostaining with 5-methylcytosine (5mC). Relative fluorescent intensity of 5mC in the chromatin was compared among groups. The experiment was replicated 3 times. Data were analysed by ANOVA. After IVM, there was no significant difference among the control, CPA, and vitrified groups in terms of the percentage of live oocytes (99.3, 96.4, and 94.0%, respectively) or polar body extrusion (88.6, 86.9, and 79.6%, respectively). After PA of oocytes with a polar body, there was no difference between the control and CPA groups in the percentage of cleavage (84.1 and 80.7%, respectively) or blastocyst development of cleaved embryos (63.3 and 79.3%, respectively). However, in the vitrified group, cleavage and blastocyst development rates (46.6 and 33.5%, respectively) were reduced (P

Previous research revealed that vitrification at the immature (the germinal vesicle, GV) stage triggers premature meiotic resumption in cumulus-enclosed porcine oocytes and causes a damage in gap junctions (Appeltant et al. 2017 Reprod. Fertil. Dev. 29, 2419-2429). However, the correlation between the two phenomena was not investigated yet. The present research was conducted to clarify whether premature meiotic resumption is caused by gap junction disruption and to assess the importance of cumulus cells for the survival of porcine oocytes vitrified at the GV stage. Cumulus–oocyte complexes (COCs) were collected from 3- to 6-mm antral follicles of slaughtered gilts. Immediately after collection, approximately half of them were denuded mechanically (DOs). In each replicate, groups of COCs and DOs were processed without vitrification (control groups). Treatment groups of COCs and DOs were vitrified on Cryotop sheets in a combination of 17.5% propylene glycol and 17.5% ethylene glycol and warmed in 0.4 M sucrose. The oocytes were then cultured for 22 h in a chemically defined porcine oocyte medium (POM) supplemented with 10 ng mL−1 epidermal growth factor, 10 IU mL−1 equine chorionic gonadotrophin, 10 IU mL−1 human chorionic gonadotrophin, and 1 mM dibutyryl cAMP. After culture, COCs were denuded and oocyte survival was assessed by morphological evaluation of membrane integrity under a stereo microscope. Then, live oocytes were fixed and stained with 1% orcein and nuclear status was evaluated under a phase-contrast microscope. The experiment was replicated 5 times. Data were analysed by ANOVA followed by Tukey’s multiple comparisons test. After vitrification and culture, the survival rate in the COC group was higher (P

We evaluated the effects of polyethylene glycol (PEG) and Supercool X-1000 (SC) as supplements during the vitrification of immature cumulus-enclosed porcine oocytes in a solution based on 17.5% ethylene glycol + 17.5% propylene glycol. After warming, the oocytes were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, equilibration and vitrification solutions were supplemented with or without 2% (w/v) PEG (PEG+ and PEG-, respectively). The survival rate, cleavage and blastocyst development were similar between PEG+ and PEG- groups; however, all values were lower than those in the non-vitrified control. In Experiment 2, vitrification solution was supplemented with or without 1% (v/v) SC (SC+ and SC-, respectively). The percentages of survival and blastocyst development were similar between SC+ and SC- groups but lower than those in the non-vitrified control. The percentage of cleavage in the SC- group was significantly lower than the control and the SC+ groups, which were in turn similar to one another. In both experiments, the cell numbers in blastocysts were not significantly different among the non-vitrified and vitrified groups. In conclusion, PEG did not improve oocyte survival and embryo development, whereas SC improved the ability of surviving oocytes to cleave but not to develop into blastocysts.

Historically, the conservation or preservation of mammalian genetic resources, especially farm animals, has been conducted under in situ conditions by maintaining living individuals as "livestock." However, systems for laboratory in vitro embryo production using gametes such as spermatozoa and oocytes are now available, in addition to ex situ preservation methods for mammalian genetic resources. One of these methods is the cryopreservation of gametes, embryos, and gonadal tissues. In pigs, freezing of sperm is the most reliable and well-established method for this purpose. On the other hand, cryopreservation of female gametes (oocytes) and gonadal tissues-usually by vitrification-has been associated with very low efficacies. Recently, in our laboratory, some research themes related to this issue have been pursued. We have been focusing on advances in porcine in vitro embryo production systems, and here, we introduce recent data on the vitrification of porcine immature oocytes and gonadal tissues followed by their xenografting into host mice to produce gametes.

This study investigated re-expansion dynamics during culture of bovine blastocysts cryopreserved either by slow-freezing or vitrification. Also, the extent and localization of membrane damage and DNA fragmentation in re-expanded embryos were studied. Frozen-thawed embryos showed a significantly lower re-expansion rate during 24 h of post-thawing culture compared to vitrified embryos. Vitrified embryos reached the maximum level of re-expansion rate by 12 h of culture whereas frozen embryos showed a gradual increase in re-expansion rate by 24 h of culture. When assayed by Hoechst/propidium iodide staining there was no difference in the numbers and ratio of membrane damaged cells between re-expanded frozen and vitrified embryos; however, the extent of membrane damage in blastomeres was significantly higher in both groups compared with non-cryopreserved embryos (control). TUNEL assay combined with differential ICM and TE staining revealed a significantly higher number and ratio of TE cells showing DNA-fragmentation in frozen-thawed re-expanded blastocysts compared to vitrified ones; however, vitrification also resulted in an increased extent of DNA fragmentation in TE cells compared with control blastocysts. In frozen-thawed blastocysts increased extent of DNA fragmentation was associated with reduced numbers and proportion of TE cells compared with vitrified and control embryos. The number and ratio of ICM cells and the extent of DNA fragmentation in ICM did not differ among control, frozen and vitrified groups. In conclusion, compared with vitrified embryos, blastocysts preserved by slow-freezing showed a delayed timing of re-expansion which was associated with an increased frequency of DNA fragmentation in TE cells.

The antioxidant effect of N, N-dimethylglycine (DMG) on in vitro-produced (IVP) bovine embryos was examined. After in vitro fertilization, presumptive zygotes were cultured with or without 0.1 μM DMG under different oxygen tensions. The percentage of embryos developing to the blastocyst stage was lowest under a 20% oxygen concentration without DMG, and it was significantly increased (P < 0.05) by applying a 5% oxygen concentration. Under the 20% oxygen concentration, supplementation of the medium with DMG significantly improved blastocyst development, which was nearly equal to that achieved under 5% oxygen without DMG. Furthermore, a tendentious increase (P = 0.06) in blastocyst cell numbers was observed when DMG was applied. In the second experiment, addition of H2O2 (0.5 mM) to the culture medium significantly (P < 0.01) reduced the percentage of embryos developing to the blastocyst stage. However, DMG supplementation prevented this reduction. In conclusion, DMG enhanced the in vitro development of IVP bovine embryos by acting as an antioxidant.

The aim of the present study was to clarify if flow-cytometric sex-sorting of bovine sperm affected in vitro blastocyst production in different bulls, either in terms of its ability to fertilize the oocyte or by interfering with post-fertilization embryo development. We performed in vitro fertilization (IVF) using both commercially available frozen-thawed X-sorted and non-sorted sperm of 4 Holstein bulls at 3 concentrations (1 × 106, 2 × 106, and 5 × 106 sperm/ml). When fertilization rates were compared, a variation in fertilization rates among different sperm concentrations was detected in 2 bulls, with similar results for X-sorted and non-sorted sperm. However, we found no evidence that the fertilization rates were affected by the sorting process. To investigate effects on embryo development, we determined the optimum sperm concentration for IVF in each bull, which resulted in similar fertilization rates among bulls. We next performed IVF using both X-sorted and non-sorted sperm of the 4 bulls at their optimum sperm concentration and compared in vitro embryo development. Cleavage rates with X-sorted sperm were similar to their non-sorted counterparts. However, significantly reduced blastocyst development was associated with the use of X-sorted sperm in one bull, whereas in the other three bulls, blastocyst development after IVF with X-sorted and non-sorted sperm was similar. In conclusion, in our system, X-sorting affects in vitro blastocyst production by reducing the developmental competence of fertilized oocytes rather than affecting the fertilization ability of the sperm. However, the occurrence of this phenomenon varies among bulls.

The antioxidant effect of N, N-dimethylglycine (DMG) on in vitro-produced (IVP) bovine embryos was examined. After in vitro fertilization, presumptive zygotes were cultured with or without 0.1 μM DMG under different oxygen tensions. The percentage of embryos developing to the blastocyst stage was lowest under a 20% oxygen concentration without DMG, and it was significantly increased (P < 0.05) by applying a 5% oxygen concentration. Under the 20% oxygen concentration, supplementation of the medium with DMG significantly improved blastocyst development, which was nearly equal to that achieved under 5% oxygen without DMG. Furthermore, a tendentious increase (P = 0.06) in blastocyst cell numbers was observed when DMG was applied. In the second experiment, addition of H2O2 (0.5 mM) to the culture medium significantly (P < 0.01) reduced the percentage of embryos developing to the blastocyst stage. However, DMG supplementation prevented this reduction. In conclusion, DMG enhanced the invitro development of IVP bovine embryos by acting as an antioxidant.

We assessed the effect of pretreating sperm with dithiobutylamine (DTBA) to improve embryo development by intracytoplasmic sperm injection (ICSI) in cows. Acridine Orange staining revealed that when applied at different concentrations (2.5, 5, and 10 mM) and exposure times (5 min, 20 min, 1 h, and 2 h), DTBA reduced disulfide bonds in spermatozoa with the highest efficacy at 5 mM for 5 min. DTBA enhanced the percentage of spermatozoa with free protamine thiol groups compared with untreated spermatozoa (control) (P < 0.05); however, this result did not differ from that of dithiothreitol (DTT) treatment. The percentage of live spermatozoa after DTBA treatment was identical to that in the control, but significantly higher than that after DTT treatment (P < 0.05). After ICSI, DTBA treatment tended to improve male pronuclear formation rate (P = 0.071) compared with non-treated sperm injection. Blastocyst formation rate was significantly improved by DTBA treatment compared with that in DTT, control, and sham injection groups (P < 0.05). Blastocyst quality in terms of cell numbers and ploidy was not different among these groups. In conclusion, DTBA increases the efficacy of blastocyst production by ICSI even if DTT treatment does not work.

Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling.

The aim of the present study was to improve the penetration during in vitro fertilization (IVF) of a frozen lot of epididymal sperm with a notoriously low fertilization ability of a Ban boar which is a native Vietnamese breed by optimizing different parameters of the IVF system. In Experiment 1, we determined that Pig-fertilization medium was superior medium to Tyrode's albumin lactate pyruvate-polyvinyl alcohol medium for IVF and defined the optimum the sperm concentration (1 × 106 sperm/ml). In Experiment 2, we clarified that partial removal of cumulus cells from cumulus-oocyte complexes by hyaluronidase treatment before IVF enhances sperm penetration, whereas complete cumulus removal reduces penetration. Finally, in Experiment 3 the elevation of concentration of caffeine in Pig-fertilization medium from 2 to 5 mmol/L and the prolongation of the co-culture of gametes from 3 to 5 hr significantly increased the total penetration rate from 15.2% to over 50%. In conclusion, the combination of partial oocyte denudation, an elevated caffeine concentration in Pig-fertilization medium and an extended interval of IVF with using an optimized sperm concentration was a potent way to improve the fertilization results for a frozen epididymal Ban sperm lot with low fertility.

This study determined the optimum storage vessel and the effects of resveratrol for the storage of in vitro matured (IVM) bovine oocytes. After IVM, the oocytes were kept in a Hepes-buffered medium at 25 °C for 20 h in different containers including Eppendorf tubes (ET) made of polypropylene (PP) and polystyrene (PS), and tissue culture tubes (TCT) made of PP, PS, and glass. Then oocytes were subjected to IVF and subsequent in vitro embryo development was compared among the groups and to that of a control group without storage. The percentage of blastocyst development in the control group was significantly higher than in the stored groups (P < 0.05). Among oocytes stored in TCT, the percentage of blastocyst development of oocytes stored in glass TCT was significantly higher than that of oocytes stored in PP and PS TCT (P < 0.05); however, it did not differ from that of oocytes stored in ET. The quality of blastocysts did not differ among the control and stored groups. Embryo development was not affected when 0.1, 1 or 10 μM resveratrol was added to the medium during oocyte storage. In conclusion, glass tubes were optimal for oocyte storage and resveratrol did not improve the development of stored oocytes.

Co-culture of cumulus-oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte-secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β-mercaptoethanol. Cumulus-oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co-culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus-enclosed porcine oocytes in a defined system.

Follicle stimulation by follicular stimulating hormone (FSH) is known to improve developmental competence of bovine oocytes obtained by Ovum Pick-Up (OPU); however, the exact factors in oocytes affected by this treatment have remained unclear. We compared in vitro matured (IVM) oocytes obtained at the immature stage from cows by OPU either without or with stimulation with FSH (non-stimulated and stimulated OPU, respectively) to those obtained by superstimulation and in vivo maturation in terms of cytoskeleton morphology, mitochondrial distribution, intracellular adenosine triphosphate (ATP) content and H2 O2 levels at the metaphase-II stage and intracellular Ca(2+) levels after in vitro fertilization (IVF). Confocal microscopy after immunostaining revealed reduced size of the meiotic spindle, associated with increased tendencies of microfilament degradation and insufficient mitochondrial re-distribution in non-stimulated OPU-derived IVM oocytes compared with those collected by stimulated OPU, which in turn resembled in vivo matured oocytes. However, there was no difference in mitochondrial functions between oocytes obtained by stimulated or non-stimulated OPU in terms of ATP content, cytoplasmic H2 O2 levels, base Ca(2+) levels and the frequencies and amplitudes of Ca(2+) oscillations after IVF. Larger size of metaphase spindles in oocytes obtained by stimulated OPU may reflect and potentially contribute to their high developmental competence.

The aim of the present study was to clarify interactions between oocytes and cumulus cells (CCs) on the level of cumulus expansion and oocyte maturation during IVM of cumulus-oocyte complexes (COCs) in a chemically defined medium using a system that allows individual tracking of oocytes. Especially, the influence of oocyte-secreted factors was investigated by the aid of addition of denuded oocytes (DOs) as a possible approach to improve the IVM system. The basic maturation medium was porcine oocyte medium with addition of gonadotropins only during the first 20 hours of IVM. During IVM, COCs were kept fixed to the bottom of culture dish by adhesive Cell-Tak coating, which enabled individual tracking of COCs during IVM. Size changes in COCs during IVM were measured by digital image analysis. Cumulus expansion in a porcine oocyte medium of intact COCs increased in a typical manner until 20 hours and decreased in size subsequently until 48 hours of IVM (P 0.05). Removal of oocytes from COCs by oocytectomy allowed the expansion of CCs to some extent, although their expansion ability was lower than that of COCs (P 0.05). Addition of DOs (COCs to DOs ratio of 9:16) did not improve cumulus expansion and oocyte maturation rates of intact COCs (P 0.05) but did enhance cumulus expansion of oocytectomized complexes (P 0.05). Furthermore, removal of CCs before IVM increased oocyte maturation rates compared with COCs (52.3% and 32.9%, respectively) (P 0.05) and a similar effect was observed in COCs when the gap junction inhibitor carbenoxolone was added to the IVM medium: carbenoxolone repressed the expansion of COCs at 20 hours of IVM. In conclusion, the porcine oocyte enhances cumulus expansion both by gap junctional communications and presumably by oocyte-secreted factor production. Nevertheless, the presence of oocytes is not a prerequisite for this process. In return, CCs maintain meiotic arrest in cumulus-enclosed oocytes during the initial culture through gap junctions. On the basis of these findings, future research could investigate if coculture with DOs during IVM is beneficial for fertilization and embryo development.

Our aim was to optimize the cryoprotectant treatment for the preservation of immature porcine cumulus-oocyte complexes (COCs) by solid surface vitrification. In each experiment, the vitrification solution consisted of 50 mg/ml polyvinyl pyrrolidone, 0.3 M of the actual sugar and in total 35% (v/v) of the actual permeating cryoprotectant (pCPA) combination. After warming, the COCs were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, trehalose and sucrose were equally effective during vitrification and warming in terms of facilitating oocyte survival and subsequent embryo development. In Experiment 2, when equilibration was performed at 38.5 C in a total of 4% (v/v) pCPA for 15 min, the combination of ethylene glycol and propylene glycol (EG + PG = 1:1) was superior to EG and dimethyl sulfoxide (EG + DMSO = 1:1) in terms of oocyte survival after vitrification and the quality of resultant blastocysts. In Experiment 3, equilibration in 4% (v/v) pCPA for 15 min before vitrification was superior to that in 15% (v/v) CPA for 5 min for achievement of high survival rates irrespective of the pCPA combination used. In Experiment 4, when equilibration was performed in 4% EG + PG for 5 min, 15 min or 25 min, there was no difference in oocyte survival and subsequent embryo development after vitrification and warming; however, the developmental competence of cleaved embryos was tendentiously reduced when equilibration was performed for 25 min. In conclusion, trehalose and sucrose were equally effective in facilitating vitrification, and the optimum pCPA treatment was 5-15 min equilibration in 4% (v/v) of EG + PG followed by vitrification in 35% (v/v) EG + PG.

Contents We tested the effects of resveratrol both as a pre-treatment and as a recovery treatment after warming during in vitro maturation (IVM) on the viability and developmental competence of porcine oocytes vitrified at the germinal vesicle stage. Pre-treatment before vitrification of oocytes for 3 hr with 2 μM resveratrol did not affect survival, oocyte maturation and embryo developmental competence to the blastocyst stage after parthenogenetic activation. However, supplementation of the medium with resveratrol during subsequent IVM after vitrification and warming significantly improved the ability of surviving oocytes to develop to the blastocyst stage, and this effect was observed only on vitrified, but not on non-vitrified oocytes. The intracellular levels of glutathione and hydrogen peroxide in oocytes were not affected by vitrification and resveratrol treatment. Also, there was no significant difference in the occurrence of apoptosis measured by annexin V binding between vitrified and non-vitrified oocytes, regardless of the resveratrol treatment. In conclusion, resveratrol did not prevent the cellular damages in immature porcine oocytes during vitrification; however, when added to the IVM medium, it specifically improved the developmental competence of vitrified oocytes. Further research will be necessary to clarify the mechanisms of action of resveratrol on the recovery of vitrified oocytes from vitrification-related damages.

In the present study, we examined the development to blastocysts of large and small blastomeres from unevenly cleaved 2-cell embryos (uneven 2-cell embryos) in pigs. Proportion of blastocysts derived from large blastomeres (52.8 ± 6.4%) was significantly higher (P

Cryopreservation of growing oocytes enriches the choice of timing and location of artificial embryo production. However, completion of oocyte growth after warming is crucial when using such cryopreserved oocytes. Our research objective was to develop a sequential system that incorporates cryopreservation of growing bovine oocytes and their subsequent in vitro growth. Oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 µm were vitrified-warmed and then cultured for 14 days. The percentage of surviving oocytes following cryopreservation and 14-day culture was approximately 80%. More than half of the surviving oocytes were capable of maturing to metaphase II after in vitro maturation; the rate was comparable to that of control oocytes grown in vitro without cryopreservation. Taken together, the combined protocols for vitrification-warming of growing oocytes and subsequent in vitro growth can produce oocytes capable of undergoing meiotic maturation.

Previous studies reported the activation of the apoptotic cascade by vitrification in mature porcine oocytes (Vallorani et al. 2012 Anim. Reprod. Sci. 135, 68-74) and that the cathepsin B inhibitor E-64 improved developmental competence of bovine oocytes via an antiapoptotic effect (Balboula et al. 2013 Reproduction 146, 407-417). The present study was carried out to test whether E-64 affected the developmental competency of porcine oocytes vitrified at the germinal vesicle stage. Cumulus-enclosed porcine oocytes were vitrified in microdrops and warmed by our method (Somfai et al. 2015 J. Reprod. Dev. 61, 571-579). Then, the oocytes were subjected to in vitro maturation (IVM) for 46h in a chemically defined porcine oocyte medium supplemented with 10ng mL−1 of epidermal growth factor, 10IU mL−1 of eCG, and 10IU mL−1 of hCG and during the first 22h of IVM with 1mM dibutyryl cyclic adenosine monophosphate. Then, cumulus-oocyte complexes were fertilized in vitro and presumptive zygotes were cultured in 50-µL drops of porcine zygote medium-3 for 7 days in 6-well dishes covered by paraffin oil in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. On Day 5 (Day 0=IVF), the porcine zygote medium-3 was supplemented with 10% (vol/vol) FCS. The effects of 1.0μM of E-64 supplementation during IVM of non-vitrified and vitrified cumulus-oocyte complexes were investigated in a 2×2 factorial design. Survival rates after IVM, cleavage rates on Day 2, blastocyst rates, and total cell numbers in blastocysts on Day 7 were compared among groups. The experiment was replicated 5 times. Results were analysed by ANOVA and Tukey’s multiple comparison test. The percentages of live oocytes were statistically similar when oocytes were matured in the absence or presence of E-64 both in non-vitrified (99.2% v. 99.6%, respectively) and vitrified (94.3% v. 90.8%, respectively) groups. Similarly, IVM without or with E-64 supplementation had no effect on subsequent cleavage and blastocyst development rates in non-vitrified (67.4% v. 71.2% and 38.7% v. 43.2%, respectively) and vitrified (46.8% v. 48.8% and 14.6% v. 22.8%, respectively) oocytes. Irrespective of E-64 treatment, all survival and developmental rates in the vitrified groups were significantly lower (P

We produced recombinant porcine leukemia inhibitory factor (pLIF) and examined its effect on in vitro maturation (IVM) of porcine oocytes and their developmental competence after in vitro fertilization. Porcine cumulus-oocyte complexes (COCs) were matured in a medium supplemented with pLIF during the first 22 hr, last 22 hr, or entire 44 hr duration of IVM. Oocytes in all groups tended to show enhanced nuclear maturation rates by the metaphase II (MII) stage (76.1%, 82.1%, and 86.6%, respectively) compared to the without-pLIF treatment group (69.6%, control). A significant increase in MII rate (P < 0.05) and obvious induction of cumulus expansion were observed over the whole time span (44 hr) in the IVM group. When cumulus cells were removed at 22 hr and denuded oocytes were further cultured, pLIF showed no effect on maturation rate. Oocytes matured in pLIF-supplemented medium showed a tendency for more rapid blastocyst development (21.1% vs. 16.2%, P = 0.0715). Examination of transcripts and proteins of the LIF signaling pathway in COCs revealed that LIF, LIF receptors, and signal transducer and activator of transcription 3 (STAT3) are present in both cumulus cells and oocytes. The amount of phosphorylated STAT3 (p-STAT3) markedly increased in both cumulus cells and oocytes cultured in pLIF-supplemented media, although oocyte p-STAT3 disappeared after 44 hr of IVM. These results suggest that the LIF/STAT3 pathway is functional during IVM of porcine oocytes, and supplementing pLIF in the IVM medium can improve oocyte maturation by activating this pathway.

The purposes of the present study were to examine the effect of naloxone, a mu-opioid receptor (MOR) antagonist, on porcine oocyte maturation and embryo development. MOR gene was expressed in germinal vesicle (GV) and metaphase II (M-II) porcine oocytes, one-, four-cell stage embryos and blastocysts. In blastocysts, MOR gene was mainly expressed in inner cell mass (ICM) cells. Supplementation of 10(-8) mol/L naloxone in in vitro maturation (IVM) medium increased the maturation rate (P < 0.05). However, 10(-4) mol/L naloxone reduced the maturation rate (P < 0.05) compared with the control. The presence of naloxone during IVM had no effects on fertilization status and subsequent embryonic development after in vitro culture (IVC). The addition of 10(-3) mol/L dibutyryl cyclic adenosine monophosphate (dbcAMP), and 10(-8 ) mol/L naloxone together into IVM medium increased nuclear maturation (P < 0.05) compared with the addition of either dbcAMP or naloxone alone. Supplementation with naloxone in IVC medium did not improve embryonic development. However, at the concentrations of 10(-6) mol/L and 10(-8) mol/L, naloxone increased the ratio of ICM to total cells in blastocysts (P < 0.05). In conclusion, at low concentration, naloxone increases maturation rate and the ratio of ICM to total cells in blastocysts. Naloxone and cAMP have a synergistic effect on oocyte maturation.