This study was carried out with the objective to investigate the differentially expressed genes (DEGs) between Jeju native pig (JNP) and Berkshire piglets. The RNA-Seq technique was used to investigate the transcriptomes in the fat, liver and longissimus dorsi muscle from these two breeds. Paired-end reads of the sequences that passed the quality filters were aligned to the Sus scrofa genome using tophat2 (v2.0.2). In this study, 65% of muscle, 20% of fat and 54% of liver genes showed higher expression in the piglets of JNP than in Berkshire. Gene Ontology and signaling pathways showed that immune response and lipid metabolisms were commonly enriched pathways in all three tissues. It was found that the genes pertaining to body growth and immune system are significantly (P < 0.01) more highly expressed in Berkshire piglets. DEGs explored between the piglets of the two breeds might influence the identification of the genetic markers for further breed improvement programs. Our findings provide a new perspective for understanding and identifying candidate genes that are involved in various biological functions. Moreover, transcriptome analysis makes it easier to understand the differences between genetic mechanisms of breeds.
Recent findings have shown that ovaries after birth have germ line stem cells, which were considered as an alternative for the production of an animal model. The present study was therefore aimed to characterise ovarian theca cells and generate oocyte-like cell masses in vitro in porcine. Theca cells isolated from ovarian follicle were cultured in A-DMEM supplemented with 10% FBS at 38.5°C in a humidified atmosphere of 5% CO2 in air. The cells were evaluated the expression of transcriptional factors (Oct3/4, Nanog, and Sox2) by immunocytochemical staining and RT-PCR, and followed by differentiated into osteocytes, adipocytes, and chondrocytes under controlled conditions. Differentiation of multiple mesenchymal lineages was confirmed by RT-PCR and specific marker staining. Differentiated cells into osteocytes, adipocytes, and chondrocytes were characterised by von Kossa and Alizarin Red staining, Oil red O staining, and Alcian Blue staining, respectively. The specific genes of osteocytes (Osteonectin, Osteocalcin and Runx2) and adipocytes (aP2) were analysed by RT-PCR. In vitro oogenesis was induced in DMEM/F12 by the previously described method (Dyce et al. 2006) for 48 days. Expression of transcriptional factors (Oct4, Sox2, and Nanog) and oocyte-specific markers (c-Mos and GDF9b) was analysed by RT-PCR in these differentiated cells. At 48 days of differentiation, the oocyte-like cell masses were further cultured in TCM-199 supplemented with 0.5 μL mL–1 FSH and 0.5 μL mL–1 LH for 15 days. Induced cells were morphologically observed following Hoechst 33342. Expression of Oct3/4 was analysed by immunocytochemical staining in these cells. Among the transcriptional factors, only Sox2 was detected by immunocytochemical staining and RT-PCR in the theca cells. Differentiation to osteocytes, adipocyte, and chondrocytes was confirmed by specific-marker staining and gene expression by RT-PCR, respectively. The morphology of oocyte-like cell masses was distinct by 40 days of differentiation. Granulosa or cumulus-like cells were distributed through the whole surface of oocyte-like cell masses. Transcriptional factors, c-Mos, and GDF9b were detected in the cell masses by RT-PCR. After being transferred oocyte-like cell masses to TCM-199, zona pellucida-like structure was formed around the edge of the cell mass. After 15 days of culture in TCM-199, the morphology of cells was changed into blastocyst-like structure, which surrounded cumulus-like cells. Oct3/4 was expressed by immunocytochemical staining in a blastocyst-like structure. These observations demonstrated that ovarian theca cells have similar characteristics to mesenchymal stem cells in view of multilineage differentiation. Theca cells can be differentiated into oocyte-like cell masses, which expressed oocyte-specific markers. These cell masses were further developed to a blastocyst-like structure, which expressed Oct3/4. Further studies are required to evaluate in vivo differentiation to oocyte-like cells. This work was supported by Grant No. 200908FHT010204005 from Biogreen21 and Grant No. 2007031034040 from Bio-organ.
The purpose of this study was to evaluate the viability, acrosome integrity, mitochondrial activity, and fertility of frozen-thawed fowl semen using different cryoprotective agents. The experiments were carried out on 10 sexually adult roosters of the Ogye breed (Korean native black chicken). The semen was collected twice per week and diluted in a 1 : 1 ratio with EK extender without cryoprotectant at 5°C. After equilibration for 30 min, diluted semen was added with an equal volume of diluents containing 14% dimethylacetamide (DMA), 14% dimethylformamide (DMF), or 15% methylacetamide (MA). The semen were packed into 0.5-mL plastic straws, frozen for 30 min by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, and then plunged into liquid nitrogen at –196°C. Frozen semen was thawed in a cold water bath at 5°C for 2 min. For fluorescence-assisted cell sorting (FACS) analysis, the frozen-thawed semen was adjusted to a final concentration of 90 million spermatozoa per milliliter with EK extender. Sperm membrane integrity was evaluated by the live-dead staining method with SYBR-14 dye and propidium iodide (PI). Acrosome integrity was measured with fluorescein isothiocyanate-labelled Pisum sativum agglutinin (PSA) and PI. The percentage of functional mitochondria was estimated using Rhodamine 123 (R123) dye and PI. After intravaginal AI was performed twice per week by injecting 0.2 mL of thawed semen directly into the vagina within 2 min after thawing, the resulting eggs were incubated for 7 days to confirm fertilization. The survival rates of cryopreserved sperm with DMF, DMA, and MA were 52.1 ± 5.5, 46.9 ± 5.1, and 36.6 ± 4.7%, respectively. The survival rate of DMF was significantly higher than those of DMA and MA (P < 0.05). The percentages of sperm that had damaged acrosomal membranes using DMF, DMA, and MA were 36.6 ± 1.4, 47.5 ± 1.9, and 61.2 ± 1.9% (P < 0.05), respectively. The percentages of live sperm with intact mitochondrial membranes cryopreserved with DMF, DMA, and MA were 52.7 ± 1.1, 44.5 ± 1.0, and 38.4 ± 1.9%, respectively, with significant differences (P < 0.05). The fertilization rates of resulting eggs after AI were 68.4% in DMF, 67.9% in DMA, and 61.9% in MA, without significant differences. These results indicate that cryopreserved rooster semen with 7% DMF showed a significantly higher survival rate and mitochondrial functionality but a lower rate of damaged acrosomes. As a cryoprotective agent, DMF has the lowest influences on Ogye rooster sperm membranes and acrosome integrity and thus could be used as a conservation method for poultry genetic resources.
The dilution of chicken semen is one of the important processes for low-temperature storage and cryopreservation and is considered to be a highly applicable method for semen exchanges between small farms in developing countries. However, studies on chicken semen preservation have been limited for several reasons. First, dilution shocks chicken semen, with more than 4 to 6 times deterioration of sperm qualities. For example, approximately 8 to 10 times dilution reduces the activity and vitality of chicken semen. The dilution factor of chicken semen could not compete with that of mammals such as cow, ram, and goat. Second, the use of glycerol as a cryoprotectant causes serious problems such as infertility of spermatozoa; therefore, farmers are reluctant to use cryopreserved semen. To increase dilution factors of chicken semen without loss of fertility, the use of fatty acid-free BSA (FAF-BSA) in Beltsville poultry semen extender (BPSE) diluent was studied with Leghorn semen. In this study, fresh semen of 9 leghorn cocks was obtained using the side collection method. The semen was diluted 10 times using BPSE medium supplemented with 0.1% FAF-BSA as a final concentration and preserved at 17°C to test fertility and hatchability of diluted chicken semen. The semen was stored at 17°C for 6h to test transportation time and used for AI to 18 layers with 3 repeats. After AI with 100µL of preserved semen (total: 60~120×106 spermatozoa), the fertilized eggs were harvested for 10 days and incubated at 37.8°C for 21 days. The fertility of the eggs was confirmed at 5 days and the hatchability was confirmed at 21 days of incubation. The supplementation of FAF-BSA in dilution medium induced higher fertility rates (53.2% v. 38.3%; P<0.05), and hatching rates also increased (90.9% v. 78.33%; P<0.05) compared with the control group. All data were analysed by Student’s t-test. These results showed that FAF-BSA supplementation could improve semen utilisation by low-temperature preservation. Small farmers who want to increase the ability of their herd could utilise FAF-BAS containing diluents to exchange the semen.
The successful cryopreservation of spermatozoa of the beagle dog for AI is essential for the establishment of the genetic banks of drug detection dogs. The beagle dog is widely used for drug testing and chosen for breeding by breeders. However, the use of cryopreserved beagle semen is limited by the lower number of offspring of dog species. In this study, 3 highly trained beagle dogs were chosen and their semen was cryopreserved for the next generation. The effects of dilution methods of beagle semen were tested using a direct dilution method at RT and a 2-step dilution method at 5°C. As a control group, the effects of a direct dilution method of semen on the percentage of motile sperm and progressive motility were analysed by computer-assisted semen analysis system (SAIS, Korea), and abnormality of spermatozoa was examined by Diff Quik staining. A total of 9 samples from 3 dogs were extended in 4% glycerol containing Tris-egg yolk diluents at approximately 22 to 25°C. The diluted semen was cooled to 5°C within 2h. The packed 0.5-mL straws were placed 5cm above the surface of LN for 10min and then plunged in. A 2-step dilution method was conducted using the same procedures of freezing, but the first dilution was done with glycerol-free diluent. After cooling to 5°C within 2h, the second diluent with 8% glycerol was added to the same volume of diluted semen at 5°C and stabilised for 1h. After thawing for 45s at 37°C, the semen from the 2-step dilution method showed the higher percentage of motile sperm (65.4±6% v. 45.3±8%; P<0.05) and progressive motility (41.6±5.3% v. 32.3±3.7%; P<0.05). However, the abnormalities between groups showed no differences. The results suggest that the optimal method for freezing beagle dog spermatozoa is a 2-step dilution process that consists of the first dilution at RT and the second dilution with glycerol at 5°C into diluted semen.
Semen from Korean Native Black roosters (Ogye) was cryopreserved with 8% N-Methylacetamide (MA) in HS-1 diluent, of the following composition (per 100 mL): glucose 0.2 g, trehalose dihydrate 3.8 g, l-glutamic acid monosodium salt 1.2 g, potassium acetate 0.3 g, magnesium acetate tetrahydrate 0.08 g, potassium citrate monohydrate 0.05 g, BES 0.4 g, Bis-Tris 0.4 g, and gentamicin sulfate 0.001 g. Ogye semen was collected 2 times a week by dorsal-abdominal massage and cooled into ice slurry. During dilution, semen was diluted by 2 steps. First, HS-1 diluent that was supplemented with respective concentration of MitoTEMPOL, mitochondria-specific antioxidant was added into fresh semen with same volume. Second, after waiting for 10 min, 16% MA containing diluent was added into first diluted semen so the final concentrations of MA were adjusted to 8%. The concentration of the MitoTEMPOL of first diluent was adjusted to 0.1, 1, 5, and 10 μM. After freezing for 30 min by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, the semen was thawed 5°C for 2 min 1 to 3 weeks later. The viability and longevity of thawed Ogye semen were analysed by sperm movement methods. Briefly, 5 μL of thawed semen was placed onto a Makler chamber and the movement of spermatozoa was recorded for 10 s by digital camera and saved as movie files on the computer. With digital rewinding for 1 s of saved movies, the non-motile and motile sperm was counted using a manual counter. The resulting numbers of respective spermatozoa were analysed by Student t-test. The 0.1 and 1 μM treated semen showed significant increases in viability compared with control, 5 μM and 10 μM MitoTempol (77.3 and 84.2% v. 65.4, 53.2, and 21.5%; P < 0.05). The longevity of frozen/thawed Ogye semen for 3 h was also higher in 0.1 and 1 μM treated groups than control, 5 μM, and 10 μM (67.5 and 54.2% v. 36, 5.2, and 1.2%; P < 0.05). With these results, utilisation of mitochondria-specific antioxidant for freezing of Ogye spermatozoa could increase the viability and longevity of frozen-thawed semen. However, treatment with concentrations >1 μM showed negative effects on freezing of Ogye chicken semen. These findings could be helpful for cryobanking of rooster semen for preservation of selected breeders from malignant viral avian disease.
The abnormality of Ogye rooster sperm chromatin could be detected by simple sperm staining. In this abstract, a Diff-Quick staining kit was tested for assessment of chicken sperm quality. Using a standard bright-field microscope, Diff-Quik stains can be reproducibly, easily, and routinely monitored with simple staining. The presence of abnormal chromatin staining of rooster sperm was determined by darker stain in head. In the fresh semen, the viabilities of 3 tested Ogye spermatozoa were 93.53, 82.42, and 90.63%, and normal chromatin rates were 87.96, 74.25, and 85.10%, respectively. However, after cryopreservation, the rates of viability of thawed semen were reduced to 69.58, 61.98, and 72.20%, and normal chromatin rate also reduced to 58.91, 48.49, and 63.34%. A significant correlation between live sperm and normal sperm nuclei was 0.875 in fresh semen and 0.513 in frozen semen. After incubation of sperm at 37°C for 5 min, the rates of viability, chromatin normality, and sperm head activity were shown as 90.63 ± 1.28%, 82.44 ± 8.09%, and 66.68 ± 10.29% in fresh semen. However, the rates of thawed semen were reduced to 67.92 ± 7.55%, 56.92 ± 12.15%, and 47.32 ± 5.02%, respectively. The relationship between chromatin normality and sperm head movements in fresh and thawed semen were 0.564 and 0.540, respectively. With these results, the chicken sperm normality could be assessed by the Diff-Quik staining, which could be used for chromatin status of sperm head and activated morphology of live spermatozoa, as a simple and rapid staining method.
Germplasm cryopreservation from a desired species with agricultural and genetic importance would protect them from the risk for extinction. Semen freezing from Korean native cattle would be a good approach for protecting genetic resources due to their limited numbers. It has been known that sperm could resist cryo-damages by freeze-thaw cycles. Thus, we performed 2 refreezing experiments with different initial thawing temperatures using frozen Korean native cattle semen. A total of 5 Hanwoo, Korean Albino, and brindle cattle were used as semen donors. After thawing by using 5°C/2 min or 37°C/40 s with cooling rates, the semen was diluted with the same volume of cryo-media in the first thawing temperature and refrozen. Sperm motilities were determined and compared between animals and groups after rethawing. The mean sperm concentration and motility was 45 × 106 mL–1 (range 2.3 to 89 × 106 mL–1) and 40% (range 13 to 55%). Mean values of motility and viability of sperm that underwent second preservation were significantly higher in 5°C than in 37°C (P < 0.01). However, the activity of viable sperm thawed at 5°C was significantly decreased before refreezing. It is estimated that refreezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa. The higher motility and viability of refrozen semen could be obtained with 5°C thawing procedure for reuse of frozen semen.
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