Profiling of seminal plasma proteins to identify the fertility of Simmental bull with low semen quality.

Objective: The present study analyzed the seminal plasma proteome and possible relationships between proteins and semen quality in azoospermic and normal Simmental bulls. Materials and Methods: Fresh semen plasma samples from the Lembang Artificial Insemination Center were used for this study, including one bull (76´ ejaculate) with very poor semen quality/azoospermia (poor fresh semen/infertile; PFS) and three bulls with normal semen quality (normal fresh semen; NFS) for proteomic analysis using a pooled system (NFS-Stud) (60´ ejaculate). The only males obtained with very low quality or azoospermia (PFS) had sperm motility of <10% (one head). Bulls with azoospermic conditions produce fresh semen without sperm or with very little sperm concentration. A total of 109 proteins were identified in the seminal plasma of Simmental bulls analyzed using liquid chromatography-mass spectrometry. Bioinformatics analysis was used to explore total protein, expression, function, and protein mechanism in the seminal plasma of Simmental bulls. Results: The results showed that the seminal plasma proteins expressed in NFS bulls include ELSPBP1, SIL1, HSPA13, angiotensin-1 covering enzyme, and CRISP1. On the other hand, B2M, C3, CFB, venin-2, and cathepsin S contribute significantly to PFS. The NFS bull proteins play important roles in sperm capacitation, protein transport, sperm motility, spermatogenesis, immune tolerance, and fertilization, while the PFS proteins perform apoptotic and antigen pathway functions. Conclusion: There is an interaction between proteins in the seminal plasma of males with poor semen quality (PFS) and cases of infertility (azoospermia) that cause a decrease in sperm quality in PFS bulls.

Conditioned medium of E17 rat brain cells induced differentiation of primary colony of mice blastocyst into neuron-like cells.

BACKGROUND: Conditioned medium is the medium obtained from certain cultured cells and contained secretome from the cells. The secretome, which can be in the form of growth factors, cytokines, exosomes, or other proteins secreted by the cells, can induce the differentiation of cells that still have pluripotent or multipotent properties. OBJECTIVES: This study examined the effects of conditioned medium derived from E17 rat brain cells on cells with pluripotent properties. METHODS: The conditioned medium used in this study originated from E17 rat brain cells. The CM was used to induce the differentiation of primary colonies of mice blastocysts. Primary colonies were stained with alkaline phosphatase to analyze the pluripotency. The morphological changes in the colonies were examined, and the colonies were stained with GFAP and Neu-N markers on days two and seven after adding the conditioned medium. RESULTS: The conditioned medium could differentiate the primary colony, beginning with the formation of embryoid-body-like structure; round GFAP positive cells were identified. Finally, neuron-like cells testing positive for Neu-N were observed on the seventh day after adding the conditioned medium. CONCLUSIONS: Conditioned medium from different species, in this case, E17 rat brain cells, induced and promoted the differentiation of the primary colony from mice blastocysts into neuron-like cells. The addition of CM mediated neurite growth in the differentiation process.

Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes.

CD163 is a putative fusion receptor for virus of porcine reproductive and respiratory syndrome (PRRS). In this study, we introduced a CRISPR/Cas9 system [guide RNAs (gRNAs) with Cas9 protein] targeting the CD163 gene into in vitro-fertilized porcine zygotes by electroporation to generate CD163-modified pigs. First, we designed four types of gRNAs that targeted distinct sites in exon 7 of the CD163 gene. Cas9 protein with different gRNAs was introduced into in vitro-fertilized zygotes by electroporation. When the electroporated zygotes were allowed to develop to blastocysts in vitro and the genome editing efficiency was evaluated using these blastocysts, three (gRNA1, 2, and 4) of the four gRNAs tested successfully edited the CD163 gene. To generate CD163-knockout pigs, a total of 200 electroporated zygotes using these three gRNAs were transferred into the oviducts of oestrous-synchronized surrogate and the surrogate gave birth to eight piglets. Subsequent sequence analysis revealed that one of the piglets carried no wild-type sequence in CD163 gene. The other seven piglets carried only wild-type sequence. Thus, we successfully generated a CD163-edited pig by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes, although further improvement is required to generate genetically modified pigs with high efficiency.

Characteristics of testicular cell development of 5-day-old mice in culture in vitro.

The crude testicular cells (CTCs) contain many cell types, such as Sertoli cells, leydig cells, spermatogonial stem cells (SSCs), spermatocytes, and other somatic testicular cells, that secrete various growth factors needed in spermatogenesis. The objective of this study was to characterize development of 5-day-old mice testicular cells cultured. Crude testicular cells prepared from the testes of 5-day-old male mice were cultured in Dulbecco's Modified Eagle Medium and incubated at 37°C in a 5% CO2 atmosphere for 6 days. The results demonstrated that the testicular cells developed rapidly with a population doubling time (PDT) of 0.63 days and more than 90% of cells were viable after being cultured for 3 days. The number of Sertoli-like cells increased significantly over days 1, 3, and 6 to 22.1%, 34.6%, and 50.1%, respectively. A significant increase was also observed in fibroblast-like cells (15.5% on day 1 to 28.8% on day 3 and to 26.6% on day 6). In contrast, the number of spermatogonia-like cells decreased significantly (54.3%, 30.4%, and 18.7%, on days 1, 3, and 6, respectively). These data indicated that the developmental pattern of the testicular cell in this study might be affected by the niche provided by the cultured testicular cells.

The effect of propolis administration on fetal development.

Propolis is one of the bee products that widely used in health therapy. However, there has no study evaluating the developmental toxicity of propolis. This study was aimed to analyze the effect of propolis administration during pregnancy on fetal development. The pregnant mice were divided into five groups including control group (Tween 80 1%), low-dose (380 mg/kg b.wt.) and high-dose (1400 mg/kg b.wt.) of water extract of propolis from Banten (WEB), and low-dose (380 mg/kg b.wt.) and high-dose (1400 mg/kg b.wt.) of ethanol extract of propolis from South Sulawesi (EES). Propolis was administered for 18 days of gestation and then sacrificed to analyze the fetal development by examining external and skeletal abnormalities. The histopathological examination of placenta was also conducted. The result showed both low-dose groups did not inhibit fetal development. However, the high-dose of EES significantly reduced the weight, crown-rump of fetuses and increased the number of resorption (p < 0.05). Fetal weight was the only significantly reduced parameter of fetal growth in the highdose group of WEB (p < 0.05). The histopathological examination of placenta showed a reduction of labyrinth development in both high-dose groups. Dose of 380 mg/kg dose of Indonesian propolis is relatively safe for consumption during pregnancy.

The Relationship between Embryonic Development and the Efficiency of Target Mutations in Porcine Endogenous Retroviruses (PERVs) Pol Genes in Porcine Embryos.

Porcine endogenous retrovirus (PERV) is a provirus found in the pig genome that may act as an infectious pathogen in humans who receive pig organ xenotransplantation. Inactivation of the PERV pol gene in porcine cells reportedly affects cell growth. Therefore, the mutation of PERV pol gene in porcine embryos using genome editing may affect the embryonic development. The present study was carried out to investigate the relationship between the mutation of the PERV pol gene in porcine embryos and their development. We introduced, either alone or in combination, three different gRNAs (gRNA1, 2, and 3) into porcine zygotes by genome editing using electroporation of the Cas9 protein (GEEP) system. All three gRNAs targeted the PERV pol gene, and we assessed their effects on porcine embryonic development. Our results showed that the blastocyst formation rates of zygotes electroporated with gRNA3-alone and in combination-were significantly lower (p < 0.05) than those of zygotes electroporated with gRNA1. The mutation rates assessed by the PERV pol gene target site sequencing in individual blastocysts and pooled embryos at the 2-to-8-cell stage did not differ among the three gRNAs. However, the frequency of indel mutations in mutant embryos at the 2-to-8-cell stage trended higher in the embryos electroporated with gRNA3 alone and in combination. Embryonic development may be affected by gRNAs that induce high-frequency indel mutations.

Genome mutation after the introduction of the gene editing by electroporation of Cas9 protein (GEEP) system into bovine putative zygotes.

The present study was designed to investigate the effects of voltage strength on embryonic developmental rate and mutation efficiency in bovine putative zygotes during electroporation with the CRISPR/Cas9 system to target the MSTN gene at different time points after insemination. Results showed that there was no significant interaction between electroporation time and voltage strength on the embryonic cleavage and blastocyst formation rates. However, increasing the voltage strength to 20 V/mm to electroporate the zygotes at 10 h after the start of insemination yielded significantly lower blastocyst formation rates (P < 0.05) than those of the 10-V/mm electroporated zygotes. Mutation efficiency was then assessed in individual blastocysts by DNA sequence analysis of the target sites in the MSTN gene. A positive correlation between mutation rate and voltage strength was observed. The mutation efficiency in mutant blastocysts was significantly higher in the zygotes electroporated with 20 V/mm at 10 h after the start of insemination (P < 0.05) than in the zygotes electroporated at 15 h, irrespective of the voltage strength. We also noted that a certain number of blastocysts from zygotes that were electroporated with more than 15 V/mm at 10 h (4.8-16.7%) and 20 V/mm at 15 h (4.8%) were biallelic mutants. Our results suggest that the voltage strength during electroporation as well as electroporation time certainly have effects on the embryonic developmental rate and mutation efficiency in bovine putative zygotes.

Heterogeneity of Cells Population and Secretome Profile of Differentiated Cells from E17 Rat Neural Progenitor Cells.

Conditioned medium has now gained increasing interest since the development of secretome-based therapy. Various types of cells have been studied as a source of the secretome. One of them is neural progenitor cells (NPCs). These are cells that capable of differentiating into neurons as well as glial cells. Indeed, the study on NPCs has risen in the last few decades, but the study on the differentiated cells has not clearly described. The most common procedures that widely used to get the conditioned medium is starvation. However, cell starvation may cause environmental stress and become an apoptotic trigger for the cells. In this study, we analyzed the effect of starvation on differentiated cells from E17 rat neural progenitor cells (NPCs) based on cells characteristics and secretome profile. We found that starvation decreased cells viability and affected the heterogeneity of the cell population. Astrocytes survived more under nutrient deprivation conditions, and the progenitor cells showed a higher tendency to differentiate to glial cells than neurons. Duration of starvation also influenced the secretome profile, alterations found in protein types and also their function in the biological process. During 24 hours of starvation, cells secreted proteins that were used to maintain cell growth, stimulate differentiation, and produce energy, but there were also proteins that identified and involved in autophagy activation. After 48 hours of starvation, astrocytes that became the dominant cells secreted proteins that try to keep protecting the remaining neurons.

CHARACTERISTICS AND FERTILITY OF SUMATRAN TIGER SPERMATOZOA CRYOPRESERVED WITH DIFFERENT SUGARS.

BACKGROUND: Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. OBJECTIVE: This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). MATERIALS AND METHODS: The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. RESULTS: All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. CONCLUSION: Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.

Comparison between effects of 3-isobutyl-1-methylxanthine and FSH on gap junctional communication, LH-receptor expression, and meiotic maturation of cumulus-oocyte complexes in pigs.

We investigated cAMP content, gap junctional communications (GJCs) status, and LH-receptor (LH-R) expression in porcine cumulus-oocyte complexes (COCs) during in vitro maturation treated with the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) or with FSH. COCs were cultured for 20 hr (1st culture) in M199 containing 10% FBS (basic medium, BM group) or BM supplemented with FSH (FSH group) or IBMX (IBMX group). Each COC was then transferred into BM containing both FSH and LH and cultured for an additional 24 hr (2nd culture). The proportions of metaphase-II (M-II) oocytes at the end of the 2nd culture did not differ between the FSH (75.7%) and IBMX (68.2%) groups, whereas only 10.1% of oocytes in the BM group reached the M-II stage. During the 1st culture, the cAMP content of COCs and oocytes became significantly higher in the FSH and IBMX groups than in the BM group; the FSH group had a far greater increment than did the IBMX group. GJCs in the FSH and BM groups gradually closed with increasing duration of the 1st culture, whereas a significantly higher proportion of COCs in the IBMX group still had open GJCs than in the other two groups. Furthermore, LH-R mRNA expression significantly increased in both the FSH and IBMX groups compared with the BM group. These results suggest that inhibition of PDEs in porcine COCs make the oocyte ready for release from meiotic arrest, and that maintenance of a moderate cAMP content may prolong GJCs and stimulate LH-R expression.

In vitro development of polyspermic porcine oocytes: Relationship between early fragmentation and excessive number of penetrating spermatozoa.

Embryo development during in vitro culture of polyspermic porcine oocytes was investigated in the present study. After in vitro fertilization (IVF) of in vitro matured oocytes, putative zygotes were centrifuged to visualize pronuclei. Two pronuclear (2PN) and poly-pronuclear (PPN) zygotes were selected and cultured in vitro. Their development to the blastocyst stage and total cell numbers, dead cell rates and ploidy at the blastocyst stage and morphology of resultant embryos after first cleavage were compared. A cleavage rate of PPN embryos was lower than that of 2PN (61.3% and 82.2%, respectively), however, the ability of cleaved embryos to develop to the blastocyst stage did not differ between the PPN and the 2PN groups (22.4% and 32.9%, respectively). Also there was no difference in total cell numbers and rates of dead cells between PPN and 2PN blastocysts. The majority of blastocysts in 2PN group were found to be diploid. In contrast, blastocysts in PPN group showed heterogeneous status in their ploidy including polyploidy and mixoploidy, whereas a remarkable proportion (31.3%) of them was found to be diploid. After the first cleavage (at 36 h after IVF), there was no difference in the number of nuclei/embryo between the two groups, nevertheless embryos in PPN group had significantly higher numbers of blastomeres than that of embryos in 2PN group, mainly due to an increased frequency of anuclear blastomeres. The present results indicate that correction of embryo ploidy in polyspermic embryos can occur during IVC. Nevertheless the frequency of partial fragmentation in polyspermic embryos is increased.

Development to the blastocyst stage of porcine somatic cell nuclear transfer embryos reconstructed by the fusion of cumulus cells and cytoplasts prepared by gradient centrifugation.

The present study was designated to examine the possibility of producing somatic cell nuclear transfer (SCNT) embryos in pigs using oocyte cytoplasm fragments (OCFs), prepared by centrifugations, as recipient cytoplasts. In Experiment 1, in vitro matured oocytes were centrifuged at 13,000 x g for 3, 6, and 9 min to stratify the cytoplasm, and then the oocytes were freed from zona pellucida and recentrifuged at 5,000 x g for 4 sec in Percoll gradient solution to produce OCFs as the source of recipient cytoplasts. It was found that a long duration of the first centrifugation tends to produce large-sized OCFs after the second centrifugation. In Experiment 2, two or three cytoplasts without chromosomes were aggregated, and then they were fused with a cumulus cell to produce SCNT embryos. The results showed that 66.4 +/- 9.4% of the reconstructed embryos underwent premature chromosome condensation at 1 h after activation, and 85.2 +/- 7.1% and 61.6 +/- 7.0% of them had pseudopronuclei at 10 and 24 h after activation, respectively. In Experiment 3, when SCNT embryos reconstructed by the fusion of three cytoplasts and one cumulus cell, a significantly higher (p < 0.05) rate of reconstructed embryos developed to the blastocyst stage (10.6 +/- 1.8%) than that of reconstructed with two cytoplasts and one cumulus cell (5.2 +/- 1.5%). These results indicate that cytoplasts obtained by two centrifugations can support the remodeling of a transferred somatic nucleus, resulting in the development of the reconstructed porcine embryos to the blastocyst stage.

Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions.

BACKGROUND: Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined. METHODS: In vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU-37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6. RESULTS: Under 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA fragmentation rate than those of Pyr-Lac blastocysts. CONCLUSION: These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos. Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

Diploid porcine parthenotes produced by inhibition of first polar body extrusion during in vitro maturation of follicular oocytes.

We investigated nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to cytochalasin B (CB) during in vitro maturation (IVM). Nuclear progression was similar in control oocytes and oocytes matured in the presence of 1 microg/ml CB (IVM-CB group) by 37 h IVM; at this time the proportion of oocytes that had reached or passed through the anaphase-I stage did not differ significantly between the IVM-CB and the control groups (61.3 and 69.9% respectively; P < 0.05). After IVM for 37 h, no polar body extrusion was observed in the IVM-CB group. In these oocytes, the two lumps of homologous chromosomes remained in the ooplasm after their segregation and turned into two irregular sets of condensed chromosomes. By 41 h IVM, the double sets of chromosomes had reunited in 89.5% IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached the metaphase-II stage by this time. When IVM-CB oocytes cultured for 46 h were stimulated with an electrical pulse and subsequently cultured for 8 h without CB, 39.0% of them extruded a polar body and 82.9% of them had a female pronucleus. Chromosome analysis revealed that the majority of oocytes that extruded a polar body were diploid in both the control and the IVM-CB groups. However, the incidence of polyploidy in the IVM-CB group was higher than that in the control group (P < 0.05). In vitro development of diploid parthenotes in the control and the IVM-CB groups was similar in terms of blastocyst formation rates (45.8 and 42.8% respectively), number of blastomeres (39.9 and 44.4 respectively), the percentage of dead cells (4.3 and 2.9% respectively), and the frequency of apoptotic cells (7.3 and 6.3% respectively). Tetraploid embryos had a lower blastocyst formation rate (25.5%) and number of cells (26.2); however, the proportion of apoptotic nuclei (7.0%) was similar to that in diploid parthenotes. These results suggest that the proportion of homozygous and heterozygous genes does not affect in vitro embryo development to the blastocyst stage.

Effects of electric field strengths on fusion and in vitro development of domestic cat embryos derived by somatic cell nuclear transfer.

The present study was conducted to determine the effect of electric field strength on the rate of membrane fusion between the somatic cell and cytoplast and on subsequent in vitro development of reconstructed embryos. Additionally, the in vitro developmental competence of cat oocytes artificially activated after 44 h of maturation culture was examined. An efficient fusion rate (64.2%) was obtained by applying a single pulse of 1.5 kV/cm for 50 micros, and the fusion rate remained almost constant at the higher field intensity (59.8 and 54.9% at 1.7 and 2.0 kV/cm, respectively). Although the cleavage rate of fused embryos increased with an increase of the electric field strength, there were no differences among the groups with respect to the proportion of development to the morula and blastocyst stages. In the additional experiment, oocytes at the metaphase II stage after culture for 44 h were activated by the combination of calcium ionophore (CaI) with cycloheximide (CHX). Some (11.8%) of activated oocytes developed to the blastocyst stage. Results from this study indicated that electric field strength affects the rates of fusion and cleavage but has no significant effects on the development to the blastocyst stage of reconstructed embryos. Prolonged maturation culture of cat oocytes (up to 44 h) decreased their ability to develop to the blastocyst stage.

Addition of glutathione or thioredoxin to culture medium reduces intracellular redox status of porcine IVM/IVF embryos, resulting in improved development to the blastocyst stage.

The present series of experiments investigated the effect of a reducing environment created by addition of reduced glutathione (GSH) or thioredoxin (TRX) to in vitro culture medium on the developmental competence of in vitro produced porcine embryos, and their intracellular redox status. Porcine cumulus-oocyte complexes were collected from ovaries matured and fertilized in vitro. The putative zygotes were then cultured for 6 days in modified NCSU-37 medium with or without (control) GSH or TRX, and their developmental competence was evaluated. In addition, the intracellular redox status of the cultured embryos was compared quantitatively using an index based on the ratio of the intracellular GSH content relative to the intracellular H(2)O(2) level. The proportion of embryos that developed to the blastocyst stage was significantly increased when 0.5 or 1.0 microM GSH (29.6% or 30.4%, P < 0.05 or 0.01, respectively) or 1.0 mg/ml TRX (30.6%, P < 0.01) was added to the medium compared to that without any supplementation (control; 20.1%). The intracellular redox status of embryos at the 8- to 12-cell stage or the blastocyst stage in the group cultured in the presence of GSH or TRX was significantly reduced in comparison with the control (P < 0.05 to 0.001). Furthermore, administration of GSH or TRX enhanced the total cell number (from 48.3 to 49.2) and lowered the proportion of apoptotic cells (from 6.2% to 7.0%) in blastocysts compared with the control (cell number 39.3; apoptosis rate 11.1%, P < 0.05). These results suggest that GSH or TRX can improve the in vitro development of porcine embryos, while maintaining an intracellular reductive status.

Successful long term culture of immature porcine sertoli cells in the reconstructed testicular cell cord.

The ultimate goal of this study was to establish an in vitro system to produce sperms. To pursue this goal, immature porcine testicular cells were cultured in stereostructural form and cultured testicular cord was investigated morphologically. At 4 weeks of age, the seminiferous tubules of the porcine testes consisted of undifferentiated germ cells (gonocytes and undifferentiated spermatogonia) and immature Sertoli cells. The interstitial tissue was largely occupied by Leydig cells. The testes were enzymatically digested, and the dispersed cells were encapsulated with alginate either immediately or after freeze-thawing. The resulting testicular cell cords were cultured for up to 10 weeks. After 2 weeks of culture, Sertoli cells, which were identified by their inhibin-positive reaction in immunohistochemistry, and Leydig cells, which were identified by their morphological characteristics, were observed in the cords. Neither undifferentiated nor differentiated types of germ cells were detected. The number of cells in the cords progressively decreased during the culture period. In order to discover the fate of the Sertoli cells, the level of inhibin in the spent media was determined. Inhibin in the media was at a detectable level after 2 days of culture. The levels increased and peaked at 2 weeks. When frozen-thawed testicular cells were applied to the culture, the peak level was maintained for over 8 weeks, in contrast to the gradual decrease of inhibin level when fresh cells were cultured. These results indicate that the culture conditions can sustain the survival of Sertoli cells. Further improvement is required for proliferation and differentiation of germ cells.

In vitro development and post-thaw survival of blastocysts derived from delipidated zygotes from domestic cats.

The ability to cryopreserve in vitro-produced feline embryos was investigated. To improve the survival rate of cryopreserved embryos, first the developmental ability of in vitro fertilized feline zygotes (after removal of intracellular lipids) was determined, followed by the post-thaw survival of cryopreserved blastocysts derived from delipidated zygotes. More than 67% of the delipidated zygotes cleaved and 36% of them developed to the morula stage. The developmental ability of delipidated zygotes to the blastocyst stage (26%) was similar to that of sham-operated (30.5%) or control embryos (31.3%). Although the survival rate of delipidated blastocysts (81.8%) after freezing and thawing tended to be higher than that of control embryos without delipidation (60.6%), rates were not significantly different between the both groups. In conclusion, in vitro-produced feline blastocysts were successfully frozen, removal of the cytoplasmic lipid content in feline zygotes did not impair their in vitro developmental competence (up to the blastocyst stage), and reduction of cytoplasmic lipids by aspiration had no apparent effects on the survival of in vitro-derived blastocysts after cryopreservation.

Effect of cycloheximide on in vitro development of electrically activated feline oocytes.

This study was conducted to improve parthenogenetic development in vitro of feline oocytes following a combined activation treatment of electrical stimulation and cycloheximide. In vitro matured (IVM) oocytes were stimulated electrically by a DC electrical pulse of 2 kV/cm for 50 micros. The stimulated oocytes were then incubated in MK-1 medium with or without cycloheximide and subsequently cultured in vitro for 6 days. No significant differences were observed between the two groups with respect to the proportions of cleavage, development to the morula stage, and the cell number of blastocysts. However, exposure of electrically stimulated oocytes to cycloheximide significantly increased the rate of development of the stimulated oocytes into the blastocyst stage compared with oocytes stimulated by electrical stimulation alone (31.0% vs 6.7%). The results from the present study suggested that a single electrical stimulation was insufficient to activate the IVM cat oocytes at 24 h of maturation and that exposure to cycloheximide following electrical stimulation improved the efficacy of the parthenogenetic development of domestic cat oocytes.

Effects of oxygen tension on the development and quality of porcine in vitro fertilized embryos.

The present study was conducted to examine the effect of oxygen tension during in vitro culture (IVC) of porcine oocytes/embryos on their development and quality using two different culture systems. Porcine cumulus oocyte complexes (COCs) were matured (IVM) and fertilized (IVF) in vitro, and subsequently cultured for 6 days in a simple and economical portable incubator or a standard CO(2) incubator. While the same temperature (38.5 degrees C) and CO(2) concentration (5%) were used in the both systems, the portable incubator was operated in a negative air pressure (- 300 mmHg) to create an O(2) level at 8-10% (low O(2) concentration), or in a positive air pressure (high O(2) concentration). To compare the two culture systems, IVM and IVF of COCs and subsequent IVC of in vitro produced (IVP) embryos were carried out in the portable incubator with a low O(2) concentration (Group I) or in the standard incubator with a high O(2) concentration (Group II). To assess the effect of O(2) concentration on IVC of IVP embryos, some oocytes that had been cultured in the standard incubator for IVM and IVF were subsequently cultured in the portable incubator with a low O(2) concentration (Group III) or a high O(2) concentration (Group IV). The occurrence of DNA fragmentation in the blastocysts produced under different culture conditions was examined by TUNEL staining to assess embryo quality. The rates of oocytes that reached MII and were penetrated by spermatozoa following IVF did not differ between the two incubation systems. In contrast, the proportions of development to blastocysts and the mean cell number of blastocysts in Group I were higher than those in Group II and Group IV. The index of DNA-fragmented nucleus in the blastocysts of Group I was significantly lower than that in the blastocysts of Group II. Therefore, low oxygen tension during IVM, IVF and IVC enhanced the subsequent development of IVP embryos to the blastocyst stage and improved their quality.