Cryopreservation of Pig Semen Using a Quercetin-Supplemented Freezing Extender.

Reactive oxygen species (ROS) produced during freeze−thaw procedures cause oxidative damage to the sperm, reducing fertility. We aimed to improve the post-thaw quality of pig sperm by quercetin (QRN) supplementation to reduce the cryodamage associated with the freeze−thaw procedure. Four equal aliquots of pooled boar semen were diluted with a freezing extender supplemented with different concentrations of QRN (0, 25, 50, and 100 µM) and then were subjected to cryopreservation in liquid nitrogen. Semen analysis was performed following 7 days of cryopreservation. Results demonstrated that the semen samples supplemented with 50 µM QRN significantly improved the post-thaw sperm quality than those subjected to other supplementations (p < 0.05). Semen samples supplemented with 50 µM QRN showed significantly improved plasma membrane functional integrity (47.5 ± 1.4 vs. 43.1 ± 4.1, 45.3 ± 1.7, and 44.1 ± 1.4) and acrosome integrity (73.6 ± 3.4 vs. 66.3 ± 2.4, 66.7 ± 3.6, and 68.3 ± 32.9) as compared to the control, 25 µM, and 100 µM QRN groups, respectively. The mitochondrial activity of the 50 µM QRN group was greater than control and 25 µM QRN groups (43.0 ± 1.0 vs. 39.1 ± 0.9 and 41.9 ± 1.0) but showed no difference with the 100 µM QRN group. Moreover, the 50 µM QRN group showed a higher sperm number displaced to 1 cm and 3 cm points in the artificial mucus than other groups. Therefore, supplementing the freezing extender with QRN can serve as an effective tool to reduce the magnitude of oxidative damage associated with sperm freezing.

Estrus synchronization and artificial insemination in Korean black goat (Capra hircus coreanae) using frozen-thawed semen.

Presently, there is an increased demand for livestock products all over the world which has led to more devotion on improving livestock population. Although goats have been bred for a long time in Korea, but there is not much research conducted on traditional Korean black goat (Capra hircus coreanae) compared to other livestock populations. Mutton consumption has been dramatically changing from medicinal use to edible meat and this trend directs the black goat populations declining and also mutton import quantities are increasing consistently. The present study introduced a new estrus synchronizing technique with subsequent artificial insemination (AI) for Korean black goats to enable crossbreeding with non-native breeds for the small or subsistent farmers. Our data highlighted that, the percentage of motile sperm from the electro-ejaculated samples declined significantly after freezing and melting. In addition, the sperm motility significantly declined with regard to sperm incubation period (0, 5, 60, and 120 min at 37°C) and was negatively correlated (64.2 ± 7.9%, 63.3 ± 5.8%, 49.9 ± 6.3%, and 35.9 ± 7.6%, respectively) in frozen-thawed sperm samples. Moreover, the E2 levels were unchanged even 24 h after controlled internal drug releas (CIDR) withdrawal. But, 48 h and 72 h after CIDR removal, E2 levels increased significantly. These data helps us to consider the two time points for AI; CIDR removal after 24 h, at which E2 decreases, and after 48 h, as the time at which progesterone increases. Additionally, the AI after 48 h of CIDR removal group exhibited significantly higher pregnancy and parturition rates (42.9%) compared to AI after 24 h after CIDR removal 28.6% group. In conclusion, these studies will propose an optimal estrus synchronisation process with subsequent timing of AI and also will promote the Korean black goat breeding industry.

Follistatin Rescues Blastocyst Development of Poor Quality Porcine Cumulus-Oocyte Complexes by Delaying Meiotic Resumption With Decreased cGMP.

Mammalian oocytes resume maturation when removed from follicles and cultured in vitro. During folliculogenesis, oocytes are bathed in follicular fluid (FF), which provides an important and specialized microenvironment for oocyte competence. Follistatin (FST) is one component of FF that may play a role in oocyte maturation and embryo development. This study was conducted to examine possible effects of FST on porcine oocyte competence and embryo development. Exogenous FST in oocyte maturation medium for 22 or 44 hours did not improve nuclear maturation and had no effect on good quality cumulus-oocyte complexes (COCs). However, FST improved blastocyst rates in embryos derived from oocytes with less than 2 layers of cumulus. Follistatin treatment of the poor quality COC group increased transcript levels for genes indicative of oocyte quality. Transcript levels were also altered for cumulus expansion-related genes in response to FST when measured during the germinal vesicle breakdown stage. Interestingly, high-quality oocytes remained at germinal vesicle stage much longer than low-quality oocytes, FST treatment induced temporary blockage of spontaneous meiotic resumption when added during culture of both good and poor quality COCs, and levels of cyclic guanosine monophosphate (cGMP) were higher in FST-treated versus untreated groups for both good and poor quality oocytes. In conclusion, FST treatment of porcine oocytes during in vitro maturation can rescue competency of poor quality oocytes to develop to blastocyst stage following in vitro fertilization. Beneficial effects of addition of FST to culture medium may be mediated by inhibiting degradation of cGMP and temporarily delaying nuclear maturation.

Effect of Acteoside as a Cell Protector to Produce a Cloned Dog.

Somatic cell nuclear transfer (SCNT) is a well-known laboratory technique. The principle of the SCNT involves the reprogramming a somatic nucleus by injecting a somatic cell into a recipient oocyte whose nucleus has been removed. Therefore, the nucleus donor cells are considered as a crucial factor in SCNT. Cell cycle synchronization of nucleus donor cells at G0/G1 stage can be induced by contact inhibition or serum starvation. In this study, acteoside, a phenylpropanoid glycoside compound, was investigated to determine whether it is applicable for inducing cell cycle synchronization, cytoprotection, and improving SCNT efficiency in canine fetal fibroblasts. Primary canine fetal fibroblasts were treated with acteoside (10, 30, 50 µM) for various time periods (24, 48 and 72 hours). Cell cycle synchronization at G0/G1 stage did not differ significantly with the method of induction: acteoside treatment, contact inhibition or serum starvation. However, of these three treatments, serum starvation resulted in significantly increased level of reactive oxygen species (ROS) (99.5 ± 0.3%) and apoptosis. The results also revealed that acteoside reduced ROS and apoptosis processes including necrosis in canine fetal fibroblasts, and improved the cell survival. Canine fetal fibroblasts treated with acteoside were successfully arrested at the G0/G1 stage. Moreover, the reconstructed embryos using nucleus donor cells treated with acteoside produced a healthy cloned dog, but not the embryos produced using nucleus donor cells subjected to contact inhibition. In conclusion, acteoside induced cell cycle synchronization of nucleus donor cells would be an alternative method to improve the efficiency of canine SCNT because of its cytoprotective effects.

Effect of acteoside on the re-localization and abnormal morphology of mitochondria in porcine oocytes during in vitro maturation.

PURPOSE: The aim of this study is to investigate the effect of acteoside, an antioxidant, on in vitro maturation (IVM) of oocytes to improve early parthenogenetic embryonic developmental competence. METHODS: Porcine immature oocytes (total 770) were cultured in IVM medium with acteoside at various concentrations, 0 (control), 10, 30, and 50 µM. Each group was assessed for maturation and subsequent development rates, reactive oxygen species (ROS) level (15 oocytes per group and four independent experiments performed), ultrastructure observation (15 oocytes per group), mitochondrial activity (30 oocytes per groups and three independent experiments performed), and expression patterns of apoptosis-related genes (100 expended parthenogenetic embryos per group and three independent experiment performed). Main outcome measures were the rates of IVM, blastocyst formation, ROS, mitochondria, and expression of apoptosis-related genes in oocytes treated with acteoside. RESULT(S): Addition of acteoside during IVM did not change the maturation efficiency of oocytes but improved the rate of blastocyst formation with significantly decreased ROS level. Moreover, in acteoside-treated oocytes, cytoplasmic maturation was improved with morphologically uniform distribution of mitochondria and lipid droplets in cytoplasm. Acteoside supplementation also increased the mRNA expression levels of antiapoptotic genes and reduced those of pro-apoptotic genes. CONCLUSION(S): Acteoside supplementation in IVM medium improves the oocyte quality and subsequent development of pre-implantation embryos that would eventually contribute to produce embryos with high embryonic development competence.

Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into ß-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein.

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine ß-casein gene locus using a knock-in vector for the ß-casein gene locus. We developed the knock-in vector on the porcine ß-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine ß-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using ß-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous ß-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.

Neuronal cell differentiation of mesenchymal stem cells originating from canine amniotic fluid.

The amniotic fluid contains mesenchymal stem cells (MSCs) and can be readily available for tissue engineering. Regenerative treatments such as tissue engineering, cell therapy, and transplantation show potential in clinical trials of degenerative diseases. Disease presentation and clinical responses in the Canis familiaris not only are physiologically similar to human compared with other traditional mammalian models but is also a suitable model for human diseases. The aim of this study was to investigate whether canine amniotic-fluid-derived mesenchymal stem cells (cAF-MSCs) can differentiate into neural precursor cells in vitro when exposed to neural induction reagent. During neural differentiation, cAF-MSCs progressively acquire neuron-like morphology. Messenger RNA (mRNA) expression levels of neural-specific genes, such as NEFL, NSE, and TUBB3 (ßIII-tubulin) dramatically increased in the differentiated cAF-MSCs after induction. In addition, protein expression levels of nestin, ßIII-tubulin, and tyrosine hydroxylase remarkably increased in differentiated cAF-MSCs. This study demonstrates that cAF-MSCs have great potential for neural precursor differentiation in vitro. Therefore, amniotic fluid may be a suitable alternative source of stem cells and can be applied to cell therapy in neurodegenerative diseases.

Glutathione and cysteine enhance porcine preimplantation embryo development in vitro after intracytoplasmic sperm injection.

Because intracytoplasmic sperm injection (ICSI) had been introduced to animal science, not only reproductive biology of domestic animals, but also medicine to treat infertility has been developed. This assisted reproductive technology is beneficial for generating transgenic animals, especially pigs, because polyspermy is the greatest hurdle in porcine IVF when researchers make highly qualified preimplantation embryos. However, ICSI-derived embryos expressed high level of reactive oxygen species (ROS), which are known to cause serious dysfunction during preimplantation development. The objective of this study was to investigate the developmental competence, ROS level, and apoptosis index when glutathione (GSH) or cysteine was supplemented into the in vitro culture medium for ICSI-derived porcine embryos. First, we evaluated the effect of different concentrations of GSH or cysteine on developmental ability of porcine ICSI-derived embryos. The cleavage rate (79.6%) and the blastocyst formation rate (20.9%) were significantly improved in culture medium supplemented with 1 mmol/L GSH compared with other concentrations or no supplementation. Also, 1.71 mmol/L cysteine showed a significantly higher proportion of cleavage (80.7%) and blastocyst formation (22.5%) than other cysteine-supplemented groups. Next, we confirmed that intracellular ROS level was significantly reduced in the group of blastocysts cultured with GSH or cysteine after ICSI compared with the no supplementation group. Finally, we found that terminal uridine nick-end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased in blastocysts when ICSI-derived embryos were cultured with supplementation of 1.71 mmol/L cysteine or 1 mmol/L GSH. Taken together, these results strongly indicate that GSH or cysteine can improve the developmental competence of porcine ICSI-derived embryos by reducing intracellular ROS level and the apoptosis index.

Develop to term rat oocytes injected with heat-dried sperm heads.

This study investigated the development of rat oocytes in vitro and in vivo following intracytoplasmic injection of heads from spermatozoa heat-dried at 50°C for 8 h and stored at 4°C in different gas phases. Sperm membrane and chromosome are damaged by the process of heat-drying. Oocyte activation and cleavage of oocytes were worse in oocytes injected with spermatozoa heat-dried and stored for 1 week than unheated, fresh spermatozoa, but in heat-dried spermatozoa, there were no differences in these abilities of oocytes between the samples stored in nitrogen gas and in air. The oocytes injected with heat-dried spermatozoa stored for 1 week could develop to the morula and blastocyst stages without difference between the samples stored in nitrogen gas and in air after artificial stimulation. Cleavage of oocytes and development of cleaved embryos were higher when heat-dried spermatozoa were stored for 3 and 6 months in nitrogen gas than in air. However, the ability of injected oocytes to develop to the morula and blastocyst stages was not inhibited even when heat-dried spermatozoa stored in both atmosphere conditions for as long as 6 months were used. When 2-cell embryos derived from oocytes injected with heads from spermatozoa heat-dried and stored for 1 week and 1 month were transferred, each 1 of 4 recipients was conceived, and the conceived recipients delivered 1 live young each. These results demonstrate that rat oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop to term.

Leptin and nonessential amino acids enhance porcine preimplantation embryo development in vitro by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) has been considered one of the strong assisted reproductive technologies for producing transgenic animals as well as treating infertility in animals and humans. However, in porcine ICSI, embryos produced by in vitro methods show low pregnancy rates with high abnormal offspring and blastocyst formation rate as well as quality are poor compared with those in other species. For these reasons, developing a protocol for porcine ICSI is essential to efficiently generate transgenic pigs. Since amino acids were introduced to embryo development because of their beneficial effects, many embryologists have been using nonessential amino acid (NEAA) in culture medium for embryonic development in pig and other species. Leptin also has been shown to be beneficial in embryonic development for increasing rate of cleavage and blastocyst development. However, the effects of NEAA and leptin were not fully understood in the development of porcine ICSI-derived embryos. Here we investigated the optimization of NEAA and leptin supplementation in culture medium to improve developmental competence and quality of preimplantation embryos after ICSI in pig. The proportion of embryos that developed to the blastocyst stage was significantly greater when 1% vol/vol NEAA (24.6%) or 100 ng/mL leptin (27.1%) was supplemented in the culture medium compared with other concentrations or no supplement. When NEAA and leptin (24.8%) were supplemented together, blastocyst formation was significantly higher than other single supplementation groups. We also evaluated the effects of different supplementation periods of NEAA or leptin on the preimplantation embryonic development after ICSI. Both NEAA and leptin showed that supplementation for the entire 7 days significantly increased the blastocyst formation rate compared with the other groups of supplementation for the first 4 days and for the subsequent 3 days. A second goal of our research was to evaluate the quality of developed blastocysts after ICSI. The supplementation of 100 ng/mL leptin in culture medium made blastocysts express less of the proapoptosis genes BAX and BAK and more of the antiapoptosis genes BCL-XL and BCL-2 after the ICSI procedure. Furthermore, terminal deoxynucleotidyl transferase dUTP nick end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased when the ICSI-derived embryos were cultured to blastocyst stage in the presence of the combination of NEAA and leptin. These results suggest that NEAA and leptin could improve not only the quantity but also quality of ICSI-derived porcine embryos during in vitro culture with the optimal concentration of each reagent.

Isolation of canine mesenchymal stem cells from amniotic fluid and differentiation into hepatocyte-like cells.

Recent findings have demonstrated that amniotic fluid cells are an interesting and potential source of mesenchymal stem cells (MSCs). In this study, we isolated MSCs from canine amniotic fluid and then characterized their multilineage differentiation ability. Canine amniotic fluid stem (cAFS) cells at passage 5 had a fibroblast-like morphology instead of forming colonies and were positive for pluripotent stem cell markers such as OCT4, NANOG, and SOX2. Flow cytometry analysis showed the expression of MSC surface markers CD44, CD29, and CD90 on the cAFS cells. In addition, these cells were cultured under conditions favorable for adipogenic, chondrogenic, and osteogenic induction. The results of this experiment confirmed the mesenchymal nature of cAFS cells and their multipotent potential. Interestingly, although the cells exhibited a fibroblast-like morphology after hepatogenic induction, reverse transcription-polymerase chain reaction revealed that the expression of several hepatic genes, such as albumin, tyrosine aminotransferase, and alpha-1 antiproteinase, increased following maturation and differentiation. These findings indicated that cAFS cells have functional properties similar to those of hepatocytes. Taken together, the results of our study demonstrated that cAFS cells with mesenchymal characteristics can be successfully isolated from canine amniotic fluid and possess functional properties characteristic of hepatocytes. The findings of our work suggest that cAFS cells have the potential to be a resource for cell-based therapies in a canine model of hepatic disease.

Histological and genetic characterization of true hermaphroditism in Korean pigs.

True hermaphrodites are animals of equivocal sex in which both male and female gonads develop simultaneously in the same individual. The frequency of true hermaphroditism is relatively higher in pigs than in other domestic animals. Two Korean pigs were diagnosed with true hermaphroditism showing ovotestes, epididymides, a penis and uteri. The testicular tissues consisted of Sertoli cells that were devoid of spermatogenic cells and showed proliferation of interstitial cells. However, uteri looked normal and had well-developed endometrial glands. Samples showed the interpretation of 38, XX female karyotype and sex-determining region Y (SRY) gene expression was negative. These findings could be helpful to understand true porcine hermaphroditism for animal research as well as for the industry of Korean domestic animals.

DNA sexing of Nipponia nippon by duplex polymerase chain reaction.

The aim of this study was to develop a rapid, simple, sensitive, and accurate duplex polymerase chain reaction (PCR) to sex Nipponia nippon, a monomorphic bird. Amplification by duplex PCR of a sex-related gene on the female chromosome and the 12S rRNA gene yielded good results using genomic DNA extracted from a feather follicle or the membranes of eggshell samples. To simplify the DNA extraction procedure, a simple boiling method was used. Our simple boiling DNA extraction method produced similar PCR amplification results as when using DNA extracted using TRIzol. Sex determination in the endangered Nipponia nippon is of crucial value to breeding programs. The duplex PCR protocol that we developed provides a simple sex identification method that is based on amplification of a sex-related gene, and we anticipate that it will facilitate effective conservation and management of Nipponia nippon.