Background: Largemouth bass (Micropterus salmoides) is introduced species that has caused aquatic ecology activity both in vitro and in vivo were investigated for the possibility of application of the bass extract as an alternative feed ingredient. Methods: The bass oil was extracted using a 1-L supercritical extractor, while the protein was extracted from 250 g of bass dry matter, which was dissolved in 1 mL of H2O at 50℃. Both oil and protein extracts were evaluated antioxidant activities and the level of DPPH radical scavenging assay and nitric oxide (NO) production assay with lipopolysaccharide response. Oral administration of 6.6 μL/g bass protein and 5.38 μL/g bass oil conducted for investigating serological and physiological effect. Results: DPPH radical scavenging showed similar radical scavenging ability of 50 μM of ascorbic acid at 200 μg of protein and 10% of oil treatment. NO concentration was diminished by the treatment of bass oil. Oral administration of both bass oil and proteins to mice showed that the body weight increase rate of the bass oil treated group was significantly reduced by 1.55 g compared to the other groups. The number of white blood cells (WBC) was increased by 4.52 k/μL in the bass protein-treated group and 4.44 k/μL in the bass oil-treated group compared to the control group. However, the serum IgG level did not show a significant difference between the bass extract-treated groups and the control group. Conclusions: These studies demonstrate that both bass oil and proteins extracted from the bass not only provide excellent effects of antioxidant and immune activity but can also be used as functional food supplements.

The identification of biomarkers of a living tissues is essentially required to understand specific functions of the cells. In previous study, we reported IGFBP 3 as one of the putative biomarkers, by showing specific expression at porcine spermatogonial stem cells (SSCs) of early stage of porcine testis. In this study, we analyzed the expression of seven members of IGFBP family (IGFBPs) in SSCs and histological expression pattern of pregnancy-associated plasma protein-A (PAPP-A), which plays a role on the growth promoting enzyme by cleavage of IGFBPs in testis of 5 days old pig. RT-PCR analysis showed that IGFBP 1, 2, 3, 4, and 6 were expressed at high level specifically in porcine SSCs compared with whole testis. We performed immunohisotochemical staining of testis sections with PAPP-A and protein gene product 9.5 (PGP9.5) which are the known biomarkers for SSCs. We were not able to find co-expression of PAPP-A and PGP9.5; PAPP-A was expressed only in Sertoli cells and PGP9.5 expression was confirmed in spermatogonium. Additionally, we were able to confirm the GATA4 expression in Sertoli and Leydig cells as a regulator of Sertoli cell function was not detected PGP9.5 expressing cells, indicating indirect evidence of that cytolocalization of PAPP-A expression is limited in Sertoli cells. These results suggested that the PAPP-A expressed in Sertoli cells may play role on regulation of development and differentiation of testicular cells through the IGF axis in neonatal porcine testis.

The mammary gland is a complex organ, made up of various cell types that work together for mil synthesis. A previous study had established a clonal cell line from primary bovine mammary alveolar cells (MAC-T) for the study of bovine milk production and synthesis. In this study, we transplanted MAC-T bovine alveolar cell line to BALB/C nude mice for regeneration of bovine mammary gland alveolar ducts. The MAC-T cells were suspended with matrigel and injected into the dorsal tissue of 8 weeks old male BALB/C nude mice. After 6 weeks, the injected cells were showed typical morphology of the tubuloalveolar female glands by histological analysis. It was made the branching ducts that were surrounded by smooth muscle with small alveoli budding off the ducts. In addition, the epithelial markers CK14 and CK18 were expressed within the duct-like structure. Prolactin was detected in the duct interior in these CK14 and CK18 positive cells but not in the non-transplanted MAC-T cells. These results showed that duct-like tissue had been successfully formed after 6 weeks of transplantation of the CK14 and CK18 positive MAC-T cells into mice dorsal tissue. This mouse model will be a useful tool for further research on the bovine mammary gland.

Identification of specific marker proteins in cells is useful for isolating cells and determining their cellular characteristics and functions. Based on our previous study showing that matrix metalloproteinase 9 (MMP-9) can be used as a marker for porcine spermatogonia, the expression pattern of MMP-9 was determined in both pre- (5-month old) and post-pubertal (11–month old) bovine testes. Histological analysis revealed that spermatogonia were located near the basement membrane in both testes, while spermatozoa were not detected in the 5-month old pre-pubertal bovine testes and epididymides. Mature spermatozoa were observed in the 11-month bovine testes and epididymides, and MMP-9 expression in 11-month old bovine testes was lower than 5-month old testes, according to reverse transcription-PCR and real-time-PCR data. To determine the specific expression sites of MMP-9 in the bovine testes, immunohistochemistry was performed. Expression of MMP-9 was observed in cells near the basement membrane of seminiferous tubules in both 5- and 11-month old testes. Furthermore, MMP-9 positive cells expressed protein gene product 9.5 (PGP9.5) and deleted in azoospermia (DAZL) that are already known as bovine spermatogonial stem cells markers. In the present study, MMP-9 expression was identified in both pre- and post-pubertal bovine spermatogonia expressing PGP9.5 and DAZL, and located near the basement membrane of seminiferous tubules. Thus, MMP-9 can be used as a marker for bovine spermatogonia, and may provide useful platforms for understanding the interaction between germ cells and extracellular matrix during spermatogenesis in the seminiferous tubules.

Glucose is universal and essential fuel of energy metabolism and in the synthesis pathways of all mammalian cells. Glucose is the one of the major precursors of lactose synthesis using glycolysis result in producing milk fat and protein. During the milk fat synthesis, lipoprotein lipase (LPL) and CD36 are required for glucose uptake. Various morecules such as acyl-CoA synthetase 1 (ACSL1) activity of acetyl-CoA synthetase 2 (ACSS2), ACACA, FASN AGPAT6, GPAM, LPIN1 are closely related with milk fat synthesis. Additionally, glucose plays a major role for synthesizing lactose. Activations of lactose synthesize enzymes such as membranebound enzyme, beta-1,4-galactosyl transferase (B4GALT), glucose-6-phosphate dehydrogenase (G6PD) are changed by concentration of glucose in blood resulting change of amount of lactose production. Glucose transporters are a wide group of membrane proteins that facilitate the transport of glucose over a plasma membrane. There are 2 types of glucose transporters which consisted facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Among them, GLUT1, GLUT8, GLUT12, SGLT1, SGLT2 are main glucose transporters which involved in mammary gland development and milk synthesis. However, more studies are required for revealing clear mechanism and function of other unknown genes and transporters. Therefore, understanding of the mechanisms of glucose usage and its regulation in mammary gland is very essential for enhancing the glucose utilization in the mammary gland and improving dairy productivity and efficiency.

The purpose of this study is to develop transgenic cell line expressing targeted human granulocyte colony stimulating factor (hGCSF) and green fluorescence protein (GFP) genes as well as production of Somatic Cell Nuclear Transfer (SCNT) embryos derived from co-expressed transgenic donor cells. Constructed pPiggy-mWAP-hGCSF-EF1-GFP vector was chemically transfected into bovine fetus cells and then, only GFP expressed cells were selected as donor cells for SCNT. Cleavage and blastocyst rates of parthenogenetic, SCNT embryos using non-TG cell and hGCSF-GFP dual expressed SCNT embryos were examined (cleavage rate: 78.0±2.8 vs. 73.1±3.2 vs. 70.4±4.3%, developmental rate: 27.2 ±3.2 vs. 21.9±3.1 vs. 17.0±2.9%). Result indicated that cleavage and blastocyst rates of TG embryos were significantly lower (P<0.05) than those of parthenogenetic and non-TG embryos, respectively. In this study, we successfully produced hGCSF-GFP dual expressed SCNT embryos and cryopreserved to produce transgenic cattle for bioreactor system purpose. Further process of our research will transfer of transgenic embryos to recipients and production of hGCSF secreting cattle.

Oral exposure of humans by excess amounts of arsenic may cause disturbances of the reproductive system. In the present study, such exposure was modelled in rats, with the support of sperm principal parameters and histopa-thological observations. Male Sprague–Dawley rats were randomly divided into three groups where the group I was served as a normal control, group II was received sodium meta-arsenite as arsenic (10 mg/kg b.w/day) and a combi-nation of sodium meta- arsenite and sodium selenite (3 mg/kg b.w/day) in group III. After 6 weeks, there was no significant change in testis weight and in total motility of all the three experimental groups, whereas, rapid moving spermatozoa, moderately moving spermatozoa and slow moving spermatozoa were significantly decreased in arsenic treated rats as compared to control rats. The other sperm principal parameters like progressiveness, average path velocity, straightness linear velocity (VSL), curvilinear velocity (VCL), straightness, linearity sperm head elongation ratio, area, linearity amplitude of lateral head department (ALH) and beat cross frequency (BCF) were found to be reduced in arsenic intoxicated rats. These results are not correlated with the histological studies. On oral admini-stration of selenium ameliorated the adverse effects of arsenic as compared to arsenic alone treated rats. Our findings clearly demonstrate that administration of selenium could prevent some of the deleterious effects of arsenic in the testis.

Spermatogenesis is initiated from spermatogonial stem cells (SSCs) that has an ability of self-renewal and unipotency to generate differentiating germ cells. The objective of this study is to develop the simple method for derivation of SSCs using non-sorting of both spermatogonia and feeder cells. Simply uncapsulated mouse testes were treated with enzymes followed by surgical mincing, and single cells were cultured in stempro-34TM cell culture media at 37℃. After 5 days of culture, aciniform of SSC colony was observed, and showed a strong alkaline phosphatase activity. Molecular characterization of mouse SSCs showed that most of the mouse SSC markers such as integrin α6 and β1, CD9 and Stra8. In addition, pluripotency embryonic stem cell (ESC) marker Oct4 were expressed, however Sox2 expression was lowered. Interestingly, expression of SSC markers such as Vasa, Dazl and PLZF were stronger than mouse ESC (mESC). This data suggest that generated mouse SSCs (mSSCs) in this study has at least similar biomarkers expression to mESC and mSSCs derived from other study. Immunocytochemistry using whole mSSC colony also confirmed that mSSCs generated from this study expressed SSC specific biomarkers such as c-kit, Thy1, Vasa and Dazl. In conclusion, mSSCs from 5 days old mouse testes were successfully established without sorting of spermatogonia, and this cells expressed both mESC and SSC specific biomarkers. This simple derivation method for mSSCs may facilitate the study of spermatogenesis.

Artificial insemination (AI) has been performed widely in swine industry using fresh liquid sperm instead of frozen type of sperm. However fresh sperm are not able to preserve more than three days with optimal motility and other sperm parameters for the successful fertilization, since in vitro stored sperm has an oxidative stress that resulted increase of abnormality and acrosome reation. To overcome these major problems, novel preservative formulation is needed to neutralize the oxidative stress and to provide suitable physiological environment for sperm in in vitro. In this study, naturally derived substances such as Poncirus trifoliate (Trifoliate orange), Garcinia mangostana (Mangosteen), pig placenta and testis extracts were tested as sperm preservative agents. Placenta extracts (PE), trifoliate orange extracts (TOE), testes extracts (TE) and mangosteen extracts (ME) were applied to analyze specific parameters for sperm motion characteristics individually and combinatorial. Each individual extract treatment can accelerate the sperm motility but noticeably TOE, TE and ME treatments exhibited the considerable and significant preservation of sperm motility. PE, TE and ME showed a significant (p<0.05) increase in ALH after one week. Further we evaluated the five different combinations of these extracts on sperm motility and its motion characteristics. Surprisingly even after one week ME, TOE and TE combination significantly preserved the sperm motility about 75%. It is noteworthy that unlike individual extract treatment, combination of ME, TOE and TE simultaneously protect the sperm motility and its motion characteristics. Taken together these data conclude that addition of ME, TOE and TE can be effective for preservation of pig sperm.

Interferon induced transmembrane protein-1 (Ifitm-1) has been reported to have an important role in primordial germ cell formation, and it has expressed in female reproductive organ. In the present study, Ifitm-1 gene expression was identified in testes and all part of epididymis using western immunoblot and immunohistochemistry. Interestingly, Ifitm-1 expression was observed on the head of spermatozoa. To investigate the role of Ifitm-1 gene expression in behavior of spermatozoa after acrosome reaction, fresh sperm was incubated with calcium ionophore to induce acrosome reaction, whereas the expression of Ifitm-1 was not altered after the acrosome reaction. Then to identify the effect of Ifitm-1 in sperm motility and other seminal parameters, different concentration of Ifitm-1 antibody was incubated with spermatozoa, and seminal parameters were assessed using computer-assisted semen analysis (CASA). Interestingly, motility, progressive, and VAP were increased in the sperm with Ifitm-1 antibody treated compared to rabbit serum, however other parameters such as straightness were not changed. In order to identify the functional significance of Ifitm-1 in fertilization, capacitated spermatozoa were pre-incubated with anti- Ifitm-1 antibody and subsequently examined the ability to adhere to mouse oocytes. However, any defection or alteration in sperm-egg fusion was not found, Ifitm-1 antibody treated or non-treated spermatozoa showed a normal penetration. Although the precise role of Ifitm-1 in sperm motility and following fertilization need to be elucidated, this study suggests that the activation of Ifitm-1 on the sperm may enhance the motility of spermatozoa in mice.

Althogh Spermatogonial stem cells (SSCs) are widely studied in mice, study of pig SSCs is not sufficient for the isolation, long-term culture, and characterization. To identify the effect of growth factor in cultured pig SSC, newly generated pSSCs like cell from neonatal 5days porcine testis were cultured and investigated for the pSSCs like cell formation. Glial derived neurotrophic gactor (GDNF), fibroblast growth factor (FGF), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF) were applied to culture media to identify the pSSC like cell growth and stem cell formation. The criteria for the determining of stem cell characters, morphology, number of colonies, putative stem cell marker were analysed by microspic, polymerase chain reaction (PCR) and immunocytochemistry (ICC) methods. Most of the pSSCs like cells were formed approximately 100 μm size with sphere shape. Most of the feeder cells were highly dependent on FGF that feeder cells were not stably attached on plate without FGF and colony formation of pSSC was not observed consequently. Immunocyto chemistry data revealed that this cells expressed the ubiquitin-C-terminal hydrolase 1 (UCHL-1, PGP9.5) and Dolichos Biflorus Agglutinin (DBA) in addition of 20 ng/ml EGF, 10 ng/ml FGF, 10 ng/ml GDNF, 10 U3/ml LIF. In addition, Alkaline Phosphatase ()was positive in all period of culture. This study suggest that various growth factorsinp SSC culture system is important to regulate and maintain the pSSC. In conculsion, although the precise role of growth factor in pSSC proliferation need to be clarified, combination of growth factor might be critical in order to derivation and proliferation of neonatal pSSCs and spermatogenesis.

Accumulating evidence suggests that chemotherapy can cause long‐term detrimental effects and alter the biology of the recipient environment. Therefore, a subsequent report claimed that the transplantation of female germline stem cells (FGSCs) into the ovaries of recipient mice that were pretreated with a high dose of busulfan and cyclophosphamide (B/C) resulted in the successful production of offspring. Therefore, this study was conducted to further clarify the impact of female germ cell transplantation on female ovaries after B/C treatment. RT‐PCR analysis showed that the period of germ cell depletion coincide with decreased Figla, Lhx8, Nobox, Kit, and Sox3 gene expression in the B/Ctreated ovary. However, depletion of female germ cells is mediated by a Fas/FasL‐, Kit/ Kitl‐, TNF‐, p53‐ and autophagy‐ independent pathway. Also, histological analysis is similar to that of Nobox null‐derived ovaries, indicating that follicle death after B/C treatment might be caused by down‐regulating of Nobox pathway. When female mice during 15 weeks after B/C treatment were checked for reproductive activity, B/C treated mice did not produce their pups. In addition, when 3×106 GFP positive primordial follicles were injected into B/C treated female mouse ovaries, donor follicle were not able to colonize into the ovaries of recipients. In conclusion, these data from a preclinical mouse model strongly suggested that female ovary until 15 weeks after B/C treatment could not support environment for maturing of exogenous FGSCs.

Spermatogonial stem cells (SSC) undergo self-renewal division and generate spermatogenesis to produce mature spermatozoa. Very recently in some species, include rodent and farm animals, SSC has been isolated and cultured in vitro. In this study, we analysed SSC marker of both 5 days old and pubertal pig testis by histological method. In 5day pig testis, prior to set of spermatogenic differentiation, the seminiferous tubules contain a relatively large number of SSCs than in pubertal pig testis. Then putative pig SSCs were successfully isolated from 5 day pig testis, and cultured long term using CD34 positive cell culture media contained GDNF, bFGF, LIF and EGF. The SSC colonies were appeared at 3 days after cells were seed. The SSC colonies were alkaline phosphatase positive and strongly expressed PGP 9.5, PLZF and DBA, but not expressed GATA4 and LH receptors. The SSC colonies were stably proliferated in GDNF, bFGF, LIF and EGF contained CD34 positive cell culture media up to 60 days. This study will be facilitated to study of in vitro and ex vivo spermatogenesis and of production of transgenic pigs using sperm vector.

INTRODUCTION In rodent, molecular markers of spermatogonia, spermatocyte, spermatid and sperm have been identified. It has been reported that DBA, PGP 9.5 and NanpG can be the markers for spermatogonia in pig. For further understanding the spermatogenesis of the pig on morphological and molecular level, we report identification of testicular cells in neonatal and pubertal pig testis, and a putative marker for spermatogonia and spermatid in pig testis. MATERIALS AND METHODS Neonatal (3 day) and pubertal testis (150 day) was cut and fixed in Bouin’s solution for immunohistochemistry (IHC). Gonocytes were isolated from neonatal testis for immunocytochemistry (ICC). Based on references (Phillips et al., 2010), thirteen antibodies (VASA, Oct4, NanoG, PGP 9.5, DAZL, SCF, GFR-alpha 1, PLZF, c-kit, integrin-beta1, Thy1, Sohlh1 and CD9) were used for IHC and ICC. Paraffin section was performed for IHC. Gonocytes were attached to the APS-coated slides for ICC. HRP-conjugated and florescent-labeled secondary antibody was used for IHC and ICC, respectively. RESULTS In histological analysis, spermatogonia and Sertoli cells, which are enclosed by seminiferous tubule and connective tissue, were observed in neonatal testis. However, complete spermiogenesis, including spermatocyte, spermatid and spermatozoa, was not observed in neonatal testis. Spermatocyte, spermatid and elongated spermatid were observed in pubertal testis but matured spermatozoa were not observed. As a result, two antibodies (PGP 9.5 and GFR-alpha1) of thirteen antibodies were available for IHC and ICC. As reported in other studies, PGP 9.5 protein was detected in spermatogonia of ne-onatal in IHC. In addition, it was observed in spermatogonia of pubertal testis. GFR- alpha1 protein was detected in spermatogonia of neonatal testis and spermatid of pubertal testis. In ICC, PGP 9.5 protein was detected in gonocytes as reported in other studies. GFR-alpha1 was also observed in gonocyte isolated from pig testis. In this study, we found that 1) only spermatogonia exist in neonatal pig testis (3 day), 2) GFR-alpha1 is a new marker for spermatogenesis in pig testis.

Interferon induced transmembrane protein-1 (IFITM1) is one of transmembrane protein which is differentially expressed in uterus during estrus cycle and pregnancy, that IFITM1 gene is highly expressed in estrus stage by the effect of estrogen, and in parturition by the effect of PGF2 alpha. This genes are also up-regulated in cells with hyperactivation of the WNT/β-catenin signaling pathway. In this study, to identify a function of IFITM1, the binding partner of IFITM1 were determined using immunoprecipitation and LC- MASSMASS methods. 1, 3 and 5 ug of polyclonal anti-IFITM1 antisera were used for immunoprecipitation, and the 75 kDa of specific band was detected in silver stained polyacylamide gel. This band were chracterized using LC-MASS-MASS, and revealed this band is glucose regulated protein 75 (GRP75) which binds to p53 and inhibits the p53 action in nucleus. To identify the localization of GRP75 in cells, immunocytochemical approach has been applied, and GRP75 is expressed in mitochondria of L929 murine connective tissue cells. Co-localization study between IFITM1 and GRP75 in L929 cell identified that these two proteins were closely expressed in mitochondria. Although the role of the interaction of these two protein need to be clarified in various biological phenomena, this data suggest that close interaction of IFITM1 and GRP75 may regulate cellular functions in uterus on sets of estrus cycle and pregnancy.

A member of mice interferon inducible transmembrane protein like family, IFITM1, is reported to play an important role in primordial germ cell (PGC) formation and this protein reported for anti‐proliferation. This study conducted to investigate the mIFITM1 expression in reproductive organs of male mice. mIFITM1 expression in testis and epididymis were revealed by immunostaining and immuno blot. Moreover, we showed that mIFITM1 is related to development of mouse testis. mIFITM1 protein expression as markedly upregulated around 5‐ 15day after birth in testis, but postnatal 21 56 day detected pattern was decreasing by immunoblot. In the other reproductive organ, epididymis, we observed that mIFITM1 protein was strongly expressed in caput, corpus and cauda. We analyzed IFITM1 gene expression under conditions of androgen manipulation. Total RNAs were obtained from the epididymis of adult mice that castrated and injected. Testosterone replacement for animals 7day after surgery. In castrated animals, A clear decrease was found in the IFITM1 protein level on Interestingly, castrated mice was revealed that mIFITM1 expression is strong. At that time, the injected catration mice decrease in IFITM1 gene expression. We also found same result from the immunoblot analysis. Our data suggest that the function of the IFITM1 expression in sperm is remain unclear but mIFITM1 expression will be important to postnatal development of mice testis and spermatogenesis. Also, mIFITM1 regulated not only sexual maturation by gene expression in the reproductive organs but also by hormone.

The purpose of this study is to evaluate the effects of alcohol or cigarette smoking on seminal parameters in a large group of mice model. Nine groups (n=20/group) of mice were treated intensive noxious materials that abdominal injection of 21% (v/v) of ethanol, cigarette smoke (10, 20, 30 minutes/day), and combination of ethanol and 30 minutes of smoking. In addition, vitamin C and selenium were also treated to mice exposed to combination of alcohol and smoking to identify the recovering effect. Sperm viability and motility were significantly decreased in either alcohol consumption or smoking exposed group, and combination of both materials have additive detrimental effects on seminal parameters. Mice groups that exposed to alcohol and smoking showed statistically significant decrease in motility and increase of static spermatozoa. Moreover, combination of both treatments showed cumulative effect in increase of static spermatozoa. Treatments of either vitamin C or selenium dramatically recovered detrimental effects of alcohol and smoking on seminal quality, although combination of both antioxidant molecules did not show any additive effect. In conclusion, detrimental effects of alcohol and cigarette consumption on sperm quality and motility were identified in mice model, and these detrimental effects can be compensated to uptake of anti‐oxidant molecules.