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Mammalian oocytes are sensitive to psychological stress at each period of follicular development. Especially, thermal stress interfere with reproductive condition by inducing formation of reactive oxygen species (ROS) and oxidative stress (OS). ROS lead to oocyte apoptosis, weakening oocyte quality and lowering the fertilization rate. As a result, the pregnancy rate is lowered, leading to infertility. Thermal stress also seems to influence zygotes through physiological changes in the maternal environment surrounding them. Loss of developmental competence suggests hyperthermia-induced oxidative stress in embryos. Interest in organic Lonicera caerulea berries has increased in recent years. They are abundant in various health-improving materials. Berries that found from natural products can be as free as possible from the bioactive toxicity of the active ingredient without side effects, and it can be a big advantage because it can work. Mammalian oocytes are arrested at the first meiotic prophase stage and get their meiotic competence to produce offspring during the development of follicle. A series of nuclear and cytoplasmic maturations are involved in this process and these vary in temperature sensitivity. Our study demonstrated that L. caerulea can relieve the negative effects of maternal hyperthermia by reducing ROS level at the developmental stage.

Mitochondria is energy generating organelle. It synthesizes ATP, which is the essential energy source of many cellular processes. During producing energy, some redox centres leak electrons to oxygen and it is contributory to the reactive oxygen species. Besides, mitochondria have significant functions in metabolism, calcium homeostasis, and fatty acid oxidation. Also mitochondria has importance to the breakdown of the ovarian follicles and could be factor determining oocyte of quality adversely. Increasing evidence shows that the number of mitochondria affect oocyte of developmental competence and maturation detrimentally during aging. Oocyte is the mitochondria-rich cell and enable the organelle to have competence for fertilization and early embryonic development. Occurrence of blastomere depends on distribution change of mitochondria which present in the egg. Lonicera caerulea treatment inhibited ovarian mitochondrial oxidative damage by suppressing mitochondrial reactive oxygen species (mROS) generation, decreasing apoptosis, controlling disintegration of mitochondrial membrane potential and conserving respiratory chain complex activities. The purpose of this study is to identify if mouse accepting treatment with L. caerulea could counter age-induced sterility and ovarian mitochondrial oxidative stress in a model organism of ovarian ageing.

In many studies regarding in vitro maturation of mammalian oocytes, flavonoids have been used to improve maturation rate and developmental competence of in vitro-matured oocytes as an one of antioxidants. Myricetin is one of flavonoids that is present in the garlic, tea, and nuts. In human, treatment of myricetin enhanced motility and viability in spermatozoa in vitro. However, study regarding with relation ship betweem myricetin and mammalian oocytes has not been conducted. Therefore, the aim of this study was to investigate the effects of myricetin on the maturation and developmental competence of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated in IVM medium containing 0, 10, 50, and 100 μg/mL myricetin for 44 h. After 44 h of IVM, oocytes were inseminated with spermatozoa and clevage and blastocyst formation rates were evaluated 48 h and 168 h of IVF, respectively. Nuclear maturation stage was assessed by the aceto-orcein method. For measurement of oxidative stress status, intracellular reactive oxygen species (ROS) and glutathione (GSH) levels were measured using carboxy-DCFDA and cell tracker red, respectively. In results, oocytes in metaphase-II stage was no significant difference between control and myricetin treatment groups. However, treatment of myricetin significantly decreased oocyte population in germinal vesicle (GV) stage (p<0.05), whereas oocytes were arrested in germinal vesicle breakdown (GVBD) stage by myricetin. Intracellular GSH level in oocytes was higher in myricetin treatment groups than control group, but there were no significant difference. On the other hand, intracellular ROS level was not changed among the treatment groups. Interestingly, clevage rate was slightly reduced in 50 μg/mL myricetin treatment group compared with control group. Our findings suggested that treatment of myricetin during in vitro maturation of porcine oocytes could disrupt interaction between nucleic and cytoplasmic maturation. However, more studies regarding with mechanism of this phenomenon are needed.

Nicotinamide (NAM) induced growth defects attributing the inhibition of Hst3 and Hst4 and consequent elevation of H3K56ac in yeast. Interestingly, H3K56 acetylation plays an important role in mammalian genomic stability, and the function of this modification in mouse embryos is not known. Hence, we designed to study the effects of NAM on H3K56ac and the possible functions of this modification related to mouse embryo development. We found that 20 mM NAM arrested mouse embryos at 2-cell stage, which possessed constitutive H3K56 acetylation and did not passed into M phase. Treatment using 20 mM NAM did not result in excessive reactive oxygen species( ROS) production and mitochondrial content, and aberrant mitochondrial distribution. Nuclear DNA fragmentation was not detectable in the arrested 2-cell but it was observed in blastocyst. Further we explored the possibility to overcome the NAM mediated arrest of 2-cell stage embryos, by I-CBP112 or resveratrol. Interestingly, I-CBP112 or resveratrol treated mouse embryos shows rescue effects caused by NAM. Finally, we demonstrated that Sirt1, a member of Sirtuins deacetylases family that target histone and non-histone proteins and maintain genome integrity, is involved in mitigating the NAM induced growth defects by promoting deacetylation of H3K56ac.

This study investigated the effect of Charcoal-Dextran Stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in maturation medium on their ability to support in vitro oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos. Oocytes were cultured in TCM-199 supplemented with either 8% BSA, 10% CDS FBS, or 10% HI FBS and 1 μg/mL estradiol-17β, 10 μg/mL FSH, 10 ng/mL EGF, 0.6 mM cysteine, 0.2 mM sodium pyruvate, and followed by in vitro fertilization and the zygotes were cultured in SOF-BE1 medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, immunocytochemistry, and cryo-tolerance. The differences in embryo development between experimental groups were analyzed by one-way ANOVA. We have shown that CDS FBS had a higher significant (p<0.05) effect on the rate of blastocyst formation comparing to HI FBS and BSA (45.2±0.7% vs. 37.4±1.5% and 31.1±3.9%, respectively, six replicates were performed). Culture of oocytes with CDS FBS increased (p<0.05) the expression of gape junction proteins, CX37 and CX43, at both transcriptional and translation levels. We also found that CDS FBS significantly increased total cell number and decreased the apoptotic index in day-8 blastocyst comparing to BSA group. Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocyst. Taken together, these data suggest that supplementation of maturation medium with CDS FBS affect cumulus cell-oocyte gap junctional communication, and subsequently improved in vitro developmental competence of bovine oocytes and embryos.

Bisphenol-A (BPA) as an organic synthetic compound of exhibiting estrogen-mimicking and hormone-like properties, which is commonly used to induce cellular stress or female reproductive toxicity. In addition, BPA induces the increasing of mitochondrial derived reactive oxygen species (ROS) such as superoxide, and production of these ROS affects to the meiotic maturation and cumulus cells expansion on in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COCs). However, anti-oxidative effect of melatonin for reduction of BPA-induced superoxide on porcine oocyte maturation has not been reported. Therefore, in present study, we confirmed that the reduction of BPA-derived superoxide by melatonin related to the reducing of mitochondria mediated apoptosis on meiotic maturation and cumulus cells expansion of porcine COCs. Then, to investigate the effects of superoxide specific scavenger, Mito-TEMPO, during porcine oocyte maturation progression, COCs cultured in maturation medium with Mito-TEMPO (0.1 μM) after pre-treatment of BPA (75 μM) for 22 h. Reduced meiotic maturation rate and cumulus cells expansion of COCs in the BPA (75 μM) treated group were recovered (p<0.01) by Mito-TEMPO treatment. Also, increasing of mitochondria derived apoptotic factors (AIF, Cleaved Caspase 3 and Cleaved PARP 1) protein levels by BPA treatment were reduced by Mito-TEMPO treatment in porcine COCs maturation. Positive effects of Mito-TEMPO for superoxide reduction on oocyte maturation and reducing mitochondrial apoptosis showed the same pattern in melatonin (0.1 μM) treated COCs. In case of supplemented with BPA and melatonin, superoxide production in COCs was not changed compared to control or melatonin treated groups. Based on these results, we concluded that melatonin as a regulator of superoxide such as Mito-TEMPO improves oocyte maturation through reduction of mitochondria derived apoptosis during IVM of porcine COCs.

Mitochondria have vital functions that regulate Ca2+ homeostasis and ATP production in fertilized mammalian embryos. In addition, regulation of mitochondrial Ca2+ is related to the mitochondrial functions and mitochondria mediated apoptosis. However, early embryonic development according to the change of mitochondrial Ca2+ regulation during IVF of porcine oocyte has not been reported. In this study, we investigated the regulation of mitochondrial Ca2+ by ruthenium red (RR) during IVF affects blastocyst development in porcine embryos. Here, we investigated the changes of mitochondrial Ca2+ by using Rhod-2 staining in fertilized oocytes during IVF progression (0, 3 and 6 h). After IVF, Rhod-2 expression significantly increased in most of fertilized oocytes at 3 and 6 h. Therefore, we treated RR into IVF medium for reduction of mitochondrial Ca2+. As expected, expressions of rhod-2 and mito-sox significantly decreased in RR treated zygotes. To confirm the mitochondrial functions, we performed the JC-1 and ATP determination in porcine zygotes. As a result, MMP and ATP increased in fertilized oocytes after RR treatment. Interestingly, blastocyst development rate was significantly increased in 20 μM RR treated fertilizing oocytes compared to other groups (p <0.05; 20 μM RR: 33.2±5.0% vs. 10 μM RR: 22.4±5.0% and Con: 24.7±3.8%). In addition, we confirmed that apoptosis in porcine blastocysts was reduced according to RR treatment during IVF procedure by using a TUNEL assay. These results demonstrated that positive effects of RR for regulation of mitochondrial Ca2+ during IVF progression improve the mitochondrial functions and blastocyst developmental competence in porcine embryos.

Alanine known as non-essential amino acid was detected at high concentration in reproductive tracts and follicular fluid in developing porcine antral follicle. The purpose of this study was to determine the effect of alanine supplementation during in vitro maturation (IVM) of porcine oocyte. We investigated nuclear maturation, intraoocyte glutathione (GSH) contents in metaphase II (MII) oocytes, and subsequent embryonic development. And also, We detected the gene expression pattern in MII oocytes, early embryo and blastocyst derived parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). The base medium for IVM was North Carolina State University-23 (NCSU-23) medium, modified by supplementing 10 ng/mL EGF, 0.5 ug/mL FSH, and 0.5 ug/ LH, replacing BSA with 0.1% (w/v) PVA, and deleting glutamine. Alanine of various concentrations (0, 0.363, 1, 5, and 10 mM) were added to base IVM medium. The proportion of mature oocyte after IVM did not increase by alanine treatment at various concentrations. However, The intraoocyte GSH content was higher (p<0.05) in oocytes treated with 0.363 mM alanine (1.17±0.01 pixels per oocyte) than non-treated oocytes (1.00±0.02 pixels per oocyte). Blastocyst formation of PA (31.2±2.5% vs. 19.8±2.5%) and SCNT (20.5±1.9% vs. 10.1±2.0%) embryos was significantly (p<0.05) improved by treatment with 0.363 mM alanine during IVM compared with embryos derived from the non-treated oocytes. Supplementation of oocytes IVM medium with 0.363 mM alanine significantly (p<0.05) increased the gene expression of POU5F1 and FGFR2 levels in early embryo and blastocyst derived PA and SCNT embryos. In MII oocytes, transcript levels of POU5F1 and FGFR2 as well as CDK1 gene were significantly (p<0.05) increased in 0.363 mM alanine-treated oocytes. Our results demonstrate that treatment with 0.363 mM alanine during pig oocyte maturation improves developmental competence after PA and SCNT by influencing cytoplasmic maturation, such as improved GSH content in IVM oocyte and increasing gene expression associated with embryonic development in oocyte and embryo.

Hesperetin (H), one of the citrus flavonoids, shows a various of pharmacological properties including antioxidant, anti‐inflammatory, and anticancer. The purpose of this study is to prevent in vitro aging by treating hesperetin to protect it from oxidative stress. Porcine oocytes were matured in vitro for 44 h (control) and for an additional 24 h in the presence of 0, 0.1, 1, 10, and 100 μM hesperetin (aging, H-1, H-10, H-100 and H-250, respectively). The study investigated the effect of proper concentration of hesperetin on nuclear maturation rate, and ROS level, apoptosis proportion, and the developmental capacity of aging porcine oocytes. The H-100 group was the most effective of protect for oxidate stress. The all results of the investigated showed that the aging group was significantly worse than the control group. During in vitro aging, normal spindle formation, apoptotic proportion, and expression of maturation marker genes were protected by the H-100 group, that result then the H-100 group was similar to the control group. The ROS level was the H-100 group better than the aging group. The estrogen receptor gene was examined to determine how hesperetin enters the porcine oocytes. As a result, it was confirmed that estrogen receptor was used because the H-100 group was expressed more than the aging group. Thus, the hesperetin is effective agent to protect from the oxidative stress during in vitro aging of porcine oocytes. Therefore, hesperetin will improve the quality of mature oocytes by allowing in vitro experiments to be protected from oxidative stress.

Allicin, one of the chemical substances from garlic, has strong antioxidant activity and considered to represent anti-aging effect in vitro. The objective of this study was to investigate the effects of allicin treatment during porcine oocyte in vitro aging and their in vitro developmental competency after parthenogenetic activation (PA). Porcine oocytes were maturated in vitro for 44 h (control) and 44+24 h with 0, 0.1, 1, 10 and 100 μM allicin (0 AL, 0.1 AL, 1 AL, 10 AL and 100 AL, respectively). We investigated the effects of appropriate concentration of allicin on nuclear, cytoplasmic maturation and the developmental capacity of aged porcine oocytes. Oocyte survival and polar body extrusion were significantly decreased in 0 AL compared with control or 1 AL. The decrease of normal spindle formation, chromosome alignment and expression of maturation marker genes during in vitro aging was prevented in 1 AL. The allicin acted as not anti-oxidant but an autophagy regulator during in vitro aging. After PA, although the cleavage rate were similar among these groups, control, 0 AL and 1 AL, the blastocyst formation was higher in control and 1 AL than 0 AL. Thus, the allicin is effective agent to prevent the deterioration during in vitro aging of porcine oocytes. Therefore, allicin may be helpful for improve the aged oocytes quality to use on in vitro experiments.

The optimization of culture condition is both basic and important for improving oocyte maturation, subsequent development of preimplantation embryo, and further implantation. This study performed to confirm effect of pioglitazone (PIO) treatment directly on porcine oocyte during in vitro maturation, further development of embryo. Immature oocytes were cultured in IVM medium, containing 0, 0.01, 0.1, 1, and 10 μM pioglitazone (0, 0.01, 0.1, 1, and 10 PIO, respectively) for 44 h. Although both oocyte survival and polar body emission did not significantly differ between the control and PIO-treated groups, percentage of normal spindle formation and chromosome alignment was increased in the 1 PIO-treated group than in the control group (control, 60.9±8.2%; and 1 PIO, 81.5±5.9%, p<0.1). Level of GSH was significantly increased in 1 PIO-treated group than in the control group (control, 100.0±5.0%; and 1 PIO, 119.8±5.2%, p<0.05), while the level of ROS was not differ in all groups. The rate of blastocyst was significantly higher in the 1 PIO-treated group than in the control at day 6 (control, 29.6±2.5%; and 1 PIO, 37.9±2.3%, p<0.05) and 7 (control, 35.1±1.6%; and 1 PIO, 41.8±2.7%, p<0.05), respectively. These results indicated that PIO treatment during IVM showing beneficial effects on oocyte and embryonic development. Pioglitazone will contribute to enhancing efficiency of in vitro production.

Embryo development is the most important time in the life of a multicellular organism because the limited cells of the embryo produce all the cells of the adult body. And transcription factors are transcriptional regulatory proteins that specifically bind to the transcriptional regulatory region DNA and activate or inhibit the transcription of that gene. It is change in the stage of embryo development that positions and levels of expression of transcription factors. Therefore, we know that transcription factors conduct vary crucial role in the stage of embryo development. So, if we could identify clearly changes of transcription factors, it can be very helpful in understanding the mechanism of embryo development. We experimented in transcription factors from GV oocyte to blastocyst how are expressed in porcine. We focused on especially pluripotency genes, which play an important role in early embryo development. So we conducted to immuno- staining using antibodies of Cdx2, Oct4, Sox2, Nanog, and E-Cadherin. Unfortunately we used mouse’s antibody because pig`s antibody against transcription factors did not exist. We checked five transcription factors (Cdx2, Oct4, Sox2, Nanog and E-Cadherin) in early embryo development in porcine. We found different expression patterns between mouse and pig in some stages. Therefore, we need to experiment that different patterns of transcription factors play a role in species, and why occur.

Poor-quality oocytes (those with 1 to 2 layers of cumulus cells) typically possess low meiotic competence and development. Prolonging the duration of in vitro maturation (IVM; 52 h) can enhance the maturation rate of poor-quality oocytes, but it does not improve subsequent embryonic development. This likely reflects the increased reactive oxygen species (ROS) production and apoptosis seen in these oocytes compared with the non-prolonged IVM (44 h) group. Melatonin is a free radical scavenger, anti-oxidant and anti-apoptotic agent that has been reported to enhance the quality of embryos by inhibiting ROS generation and apoptosis. Therefore, we herein investigated whether melatonin combined with prolonged IVM (52 h) could improve the quality and development of poor-quality oocytes. We supplemented IVM and/or in vitro culture (IVC) media with various concentrations (0, 10-7, 10-6 and 10-5 M) of melatonin, and estimated parameters related to oocyte quality and development. The addition of melatonin (10-6 M) to a prolonged IVM system improved the oocyte quality and development compared to those of the melatonin-free oocytes group, and that this was due to decreases in ROS generation, apoptosis, and DNA damage. When melatonin was added during both IVM (10-6 M) and IVC (10-6 M), we observed a cumulative positive influence on embryonic development and quality; this treatment enhanced the expression level of Oct4, and decreased the levels of ROS, DNA damage and apoptosis. Together, these findings suggest that the combination of melatonin plus prolonged IVM can improve the quality and development of poor-quality porcine oocytes via anti-oxidative and anti-apoptotic effects.

Porcine litter size is a quantitative trait and its heritability is especially low. So it is necessary to identify porcine reproductive gene and protein. The establishment of pregnancy requires performance of a receptive endometrium and ovary. The endometrium secretes a wide array of growth factors, cytokines and proteins. Also, the ovaries secrete hormones that play a role in the estrous cycle and fertility. Based on these background, we analyzed the endometrial tissue and ovary protein of porcine and would find out biomarker proteins related to porcine litter size. We sorted the two groups according to litter size of porcine: a small litter size group (SLSG) (n=2) and a large litter size group (LLSG) (n=2). The porcine endometrial tissue samples were preprocessed for proteomic analysis. In order to comparison, samples of each 2 mg endometrium protein were separated form pI and molecular weight in the same conditions by applying a pH 3.0~10.0 IPG gels for the first dimension and then 8~16% SDS-PAGE gel for the second dimension. After proteins were visualized by staining with Commassie brilliant blue (CBB), imange analysis was performed with Image Master detect variations in protein spots between large litter size group and small litter size group endometrium. And then differential proteins were identified using MALDI-TOF analysis. The master images of 2-DE gel images obtained from 2 mg samples of large litter size group and small litter size group endometrial proteins at pH 3.0~10.0 revealed more than 400 protein spots in pH 3.0~10.0 range. When we analyzed the levels of expression of proteins that protein spots appeared more than 1.5-fold difference in endometrial tissue from porcine. In comparison of SLSG (small litter size group) with LLSG (large litter size group), a total of 9 protein spots differentially expressed on porcine endometrial tissue 2-DE gels, among which 5 spots were up-regulated proteins as retinol dehydrogenase 16-like isoform 1, Acrosin-binding protein, alpha-N-acetylgalactosaminidase. phosphoglycerate kinase 2, Acrosin-binding protein in LLSG. And 4 spots were up-regulated proteins as phosphoglycerate kinase 2, prenylcysteine oxidase in SLSG.

Equine follicle-stimulating hormone (eFSH) is a member of the glycoprotein family with luteinizing hormone (LH), thyroid stimulating hormone (TSH), and equine chorionic gonaodotropin (eCG). These receptors called G protein-coupled (GPCR) receptor were synthesized in granulosa/theca and Sertoli/Leydig cells in ovary/testis. GPCRs are key signaling proteins that regulate nearly every aspect of cell function. GPCRs relay information from extracellular stimuli to intracellular responses in a wide range of physiological and pathological processes. All of the receptors in this superfamily transverse the plasma membrane with seven highly conserved a-helices oriented with an extracellular amino terminus and an intracellular carboxy terminus. To access the functional effect of the activation mutant (D566G) and inactivations (A189V, N191I, R572C, A574V, and R633H) in the eFHSR, wild type and the mutated eFSHR were transiently expressed in CHO-K1 cells and cAMP accumulation was measured. The activity of activation mutant at residue 556 exhibits a 9.4-fold increase in basal cAMP accumulation. In the inactivation mutants, EC50 values of the 189 site was 73.2% compared to that of wild type receptor. The other site (191) does not affect in the cAMP responsiveness. Other 3 sites (572, 574, and 633), the EC50 values were detected 109.7, 118.9, and 115.3%, respectively. The mutation is localized in a crucial region of the transmembrane domain, highly conserved in all glycoprotein hormone receptors, and within the FSH receptor of different species. The same site (Asp-Gly) has been previously reported to lead to constitutive activation of these receptors (LHR, and TSHR). Those receptors were found in patients with pseudoprecocious puberty and thyroid adenoma, respectively. In summary, we have identified constitutively activating point mutations and inactivating mutations of eFSHR in CHO-K1 cells. Knowledge of those mutations will facilitate genetic counseling and diagnosis, as well as provide the basis to study the three-dimensional conformation of the receptor domains involved in ligand-binding and G protein activation. To our knowledge, this remains the sole example of a naturally occurring activating mutation of the FSH receptor described so far. Thus, the significance of naturally occurring polymorphisms in the FSH receptor is still unknown. We insist that some receptor variant or combination of variants is related to a higher incidence of reproductive disorders.

Lonicera Caerulea (Honey berry) has been used in medical treatment in Russia, Japan, China and Korea. It has high level of vitamin C and polyphenolics. Polyphenolics can improve anti-inflammatory effect and prevent cancer, diabetes mellitus type 2. Also, Vitamin C is a representative anti-oxidant. however, It is still unknown what effect it will have on the oxidation stress of the reproductive system. In previous studies, ROS can be produced when it is exposed to heat stress and has negative effect on sperm’s maturation, capacitation, hyperactivation, acrosome reaction and fusion of egg and sperm. Therefore, The purpose of this study is to investigate the antioxidant effects of L.caerulea on the sperm and egg cells of mice. At first, it conducted using ICR mouse(n=20) during 4 weeks. There are four groups of mouse(n=5 per group). Also, L.caerulea was taken by oral gavage. Group Ⅰ(control) kept at 23~27℃ and administer D.W(0.5 mL/day), Likewise, Group Ⅱ(HB) kept at room temperature but gave HB(0.5 mL/day), Group Ⅲ(HB+HS) received heat stress (42℃) using hyperthermia induction chamberand gave HB at same dose. and Group Ⅳ(HS) exposed heat stress only. Mainly, We showed degree of gene expression using Western blot in SOD, HSP 70, 17β-HSD and Real time PCR. It can find correlation between intracellular activity like steroid hormone, apoptosis under ROS and antioxidant activity of L. caerulea.

As an one of detrimental effect during freezing and thawing process, fatty acids in plasma membrane of spermatozoa were released and sperm membrane was damaged. And several studies had reported that spermatozoa could absorb fatty acid in freezing extender. We expected that supplement of alpha-linolenic acid (ALA) during freezing process could improved efficiency of cryopreservation for boar sperm. Therefore, the aim of this study was to evaluate effect of ALA combined with Bovine Serum Albumin (BSA) and Methyl-β-Cyclodextrin (MβCD) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. For preparation of ALA-carrier protein complex, 3 ng/mL ALA was mixed with 0.7 μg/mL Bovine Serum Albumin (BSA) or 14 ng/mL Methyl-β-Cyclodextrin (MβCD) in distilled water. The boar semen was purchased from GUMBO Company. Boar semen was cryo-preserved in lactose-egg yolk (LEY) containing ALA, BSA, MβCD, ALA+BSA, ALA+MβCD and frozen sperm was thawed at 37.5℃ for 45 sec in water-bath. Sperm viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed sperm was increased in all treatment groups. However, there was no significant difference. Acrosome reaction and mitochondrial damage in frozen-thawed sperm was decreased in all treatment groups compared with control group. However, there was no significant difference. In all sperm characteristics, ALA+BSA treatment group was higher than all treatment groups. In conclusion, addition of ALA with carrier proteins during cryopreservation did not improves viability and acrosomal membrane and mitochondrial integrity in boar sperm. Therefore, it is necessary to continue the study on the effects of ALA with carrier protein on boar spermatozoa during cryopreservation.

4-Octylphenol is final product derived from alkylphenol polyethoxylates that are used as a surfactant in rubber synthesis, or as a surface adhesive. In addition, Octylphenol mainly reaches the marine environment in waste waters form factories. Here, we were evaluated the effect of 4-Octylphenol for reproductive ability. We performed an in vitro study to comprehensively evaluate the toxicity of 4-Octylphenol on mouse testicular cells (TM4 Sertoli cells and TM3 Leydig cells) by examining the influence of 4-Octylphenol on the cell viability, cell death and apoptosis-related gene expression. The results obtained from the MTT assay showed that 4-Octylphenol suppressed both TM3 and TM4 cell viability in dose-and time-dependent manner. Moreover, the number of apoptotic and dead TM3 and TM4 cells was significantly increased after 4-Octylphenol. Based on this our result, we analyzed the expression of the apoptosis related gene in 4-Octylphenol exposed testicular cells (both TM3 and TM4 cells). The 4-Octylphenol significantly upregulated the mRNA levels of Bax, Bad, Bak gene and downregulated the Bcl-2 gene expression in dose-dependent manner. In conclusion, 4-Octylphenol could induce both TM3 leydig cells and TM4 sertoli cell apoptosis, and the cytotoxic effect of 4-Octylphenol on testicular cells may be associated upregulated apoptosis gene such as Bax, Bad and Bak, which is required for mitochondrial permeabilisation during apoptosis.

Temephos is recommended a non-systemic organophosphorus insecticide by WHO because it is safe for humans and has low toxicity to mammals compared with other chemical compounds such as chlorine, potassium permanganate, zinc carbamate, and dichlorodiphenyltrichloroethane (DDT). However, Temephos is involved in the hazardous pesticide category PAN Bad Actor (Pesticide Action Network, USA). Moreover, no study was performed to evaluate toxicity of Temephos in mammalian germ cell. The objectives of this study was to assess the effect of Temephos on sperm functions. First, caudal epididymis spermatozoa from ICR mice were incubated with Temephos (0, 0.1, 1, 10, and 100 μM). Then sperm motility and motion kinematics were assessed using computer-assisted sperm analysis. In addition, capacitation status and adenosine triphosphate (ATP) level were assessed using combined Hoechst 33258/chlortetracycline fluorescence and ATP detection kit, respectively. The acrosome reaction and capacitation status were significantly increased and decreased in the highest concentration of Temephos (100 μM), respectively. In addition, ATP levels in spermatozoa were significantly decreased in higher concentration of Temephos (1, 10, and 100 μM). However, Sperm motility and motion kinematics (motility, medium, slow, progressive, hyperactivated, curvilinear velocity, straight-line velocity, average path velocity, beatcross frequency, dance mean, amplitude of lateral head displacement) were not significantly altered with Temephos. The results were shown that Temephos affect capacitation status, the acrosome reaction and ATP level. Taken together, we suggest that the present study may be applied to evaluate risk of Temephos on male fertility.

Fipronil is widely used pesticide for insects, such as granular turf products, pet care flea and tick solutions. Although the Fipronil presents moderately risky, only for several researches have been reported that the Fipronil affects fertility of insects, fish, and so on. However, effects of Fipronil on mammalian fertility are not fully understood yet. Therefore, the aims of this study are investigating the effects of Fipronil on sperm functions. Spermatozoa were incubated with various concentrations of Fipronil (0, 0.1, 1, 10, 100, and 300 μM). Then sperm motility and motion kinematics were evaluated using computer-assisted sperm analysis. In addition, capacitation status and intracellular ATP level were evaluated using combined Hoechst 33258/chlortetracycline fluorescence and ATP Assay kit, respectively. Sperm motility, rapid speed, progressive, curvilinear velocity, average path velocity, and mean amplitude of head lateral displacement were significantly decreased in higher concentration of Fipronil (1, 10, 100, and 300 μM) treatments. Beat-cross frequency was significantly decreased in presence of 10, 100, and 300 μM Fipronil. Straight-line velocity and the acrosome reaction were significantly decreased and increased in 100 and 300 μM Fipronil treatments, respectively. In addition, the highest concentration of Fipronil (300 μM) was significantly decreased capacitation status and intracellular ATP level. Our results showed that Fipronil suppress sperm motility, motion kinematics, the acrosome reaction, capacitation status and intracellular ATP level. Therefore, Fipronil can negatively affect male fertility and the concentration of Fipronil need to be controlled when Fipronil be used for pesticide.

Depletion of endogenous germ cells in the recipient model is the key process for successful SSC transplantation. The main objectives of this study are 1) to evaluate the effect of local irradiation or testicular infusion of 70% glycerin on depletion of germ cells, 2) to evaluate germ cell transplantation in the goat testes using ultrasound guided rete-testis transplantation technique. The testes of two recipient goats were treated with irradiation with does of 10 or 15 Gy, respectively. Testicular tissue biopsy was performed to analyze patterns of spermatogenesis at 6 weeks after treatment. Fifteen milliters of 70% glycerin was infused into the core of each testes at 1 cc/min. After 4 weeks, germ cells (500×106) stained with PKH-26 dye were transplanted using ultrasound guided rete testis injection technique. Testes were castrated at 2 weeks after transplantation and seminiferous tubules were isolated to monitor the presence of PKH-26 positive germ cells. Spermatogenesis patterns in cross section of seminiferous tubules at each dose of 10 and 15 Gy were categorized and quantified as following; normal (5.1 and 25.3%), Sertoli cell only (35.3 and 11.3%), and destroyed (59.4 and 63.3%). However, abnomal testicular tissue condition was noticed; tissue was not as firm as normal testes as well as effusion was evidenced. The testes treated with 70% glycerin also showed 3 different patterns of normal (19.6%), destroyed (72.8%), and Sertoli cell only (7.6%). In this testis, the severe inflammation area was evidenced. Germ cells stained with PKH-26 were successfully identified within seminiferous tubules. In conclusion, the local radiation and testicular infusion of 70% glycerin are not ideal method for the depletion of endogenous germ cells in the goat testes. The ultrasound guided rete-testis transplantation technique can be utilized.

이 조사는 국내 양돈전산프로그램인 피그플랜을 2017년에 사용하는 양돈농장의 번식관 련 생산성 자료를 수집하여 생산지표를 비교 분석하였다. 자료가 입력된 554개 양돈농장 중에서 모돈 1두당 연간 이유자돈두수(PSY)를 기준으로 상위 30%은 A그룹 162개 양돈 농장, 하위 30%는 B그룹 162개 양돈농장의 모돈 관련 전산성적 자료를 수집하여 비교 분석하였다. A그룹의 평균 총산자수는 13두로 B그룹의 11.5두보다 1.5두 높았다. 생시자 돈 사고율은 A그룹은 7.7%로 B그룹의 8.7%보다 1.0% 낮았으며, 평균 실산자수는 A그룹 은 12.0두로 B그룹의 10.5두보다 1.5두 높았다. 이유 전 폐사율은 A그룹은 10.0%로 B그 룹의 12.4%보다 2.4% 낮았다. 평균 이유두수는 A그룹이 10.8두로 B그룹의 9.2두보다 1.6두 높았다. 포유기간은 A그룹이 24.1일로 B그룹의 25.3일보다 1.2일 짧았고, 임신기간 도 A그룹이 114.8일로 B그룹의 115.4일보다 0.6일이 짧았다. 모돈 1두당 연간 비생산일 수는 A그룹이 32.5일로 B그룹의 68.8일보다 36.3일이 짧았다. 비생산일수는 이유에서 교 배일, 교배 후 기간, 도태기간으로 3단계로 구분하여 비교하였다. 1단계 이유 후 교배일 까지는 A그룹이 13.6일로 B그룹의 15.5일보다 1.9일이 짧았고, 2단계 교배 후 기간에서 는 A그룹이 16.0일로 B그룹의 43.1일보다 27.1일의 큰 차이를 보였다. 3단계 도태기간에 서는 A그룹은 3.0일로 B그룹의 10.2일보다 7.2일이 짧았다. 모돈회전율은 A그룹은 2.40 회전으로 B그룹의 2.12회전 보다 0.28회전이 길었다. 모돈 1두당 연간 이유자돈두수 (PSY)는 A그룹은 26.0두로 B그룹의 19.5두보다 6.5두 높았다. 양돈농장의 상 ‧ 하위 30% 그룹간 PSY의 차이는 평균 이유두수와 함께 비생산일수가 주요 원인으로 분석되었다. 비생산일수에서는 교배 후 사고까지의 기간이 주요 원인이므로 양돈농장의 생산성을 높 이기 위해서는 철저한 재발체크와 신속한 임신진단이 중요할 것으로 사료된다.

이 조사는 국내 양돈전산프로그램인 피그플랜을 2016년과 2017년에 사용하는 양돈농 장의 번식관련 모돈의 생산성 자료를 수집하여 분석하였다. 전체 사용자 중에서 모돈 관 련 데이터를 모두 입력한 2016년 524농가, 2017년 554농가를 선발하였다. 후보 모돈을 제외한 2016년 258,041두, 2017년 276,104두의 자료를 비교 분석하였다. 2017년의 평균 총산자수는 12.2두로 2016년의 12.1두보다 0.1두가 증가하였다. 생시자돈의 사고율은 2017년 8.4%로 2016년 8.0%보다 0.4% 상승하였으나, 평균 실산자수는 2017년 11.2두로 2016년 11.1두보다 0.1두 상승하였다. 2017년의 이유 전 폐사율은 10.0%로 2016년의 10.5%보다 0.5% 개선되었다. 포유기간은 2017년이 24.6일로 2016년의 24.7일보다 0.1일 짧아졌으나, 임신기간은 115.2일로 2016년의 115.1일보다 0.1일이 증가하였다. 모돈 1두 당 연간 비생산일수는 2017년이 48.6일로 2016년의 46.8일보다 0.2일이 짧아졌다. 2017 년 비생산일수는 48.6일로 2016년의 46.8일보다 1.8일 증가하였다. 임신기간은 2017년과 2016년이 115.2일과 115.1일로 거의 유사하였다. 2017년의 모돈회전율은 2.3회전으로 2016년과 동일하였다. 2017년의 모돈 1두당 연간 이유자돈두수(PSY)는 22.7두로 2016년 의 22.8두보다 0.1두 하락되었다. 양돈농가에서 생산성을 높이기 위해서는 비생산일수를 줄여서 모돈회전율을 높이는 방법과 이유 전 ‧ 후의 폐사율을 낮추는 것이 중요한 것으로 분석되었다. 향후 국내 양돈농가의 농장규모와 함께 경영분석에 대한 비교분석이 필요할 것으로 사료된다.

In the present study, we compared cauda epididymal sperm recovery methods in Hanwoo bulls by flushing and floating techniques. Fourteen testicles with epididymides were collected from 7 Hanwoo bulls (months of age=15.1±0.2, scrotal circumference (cm)=31.6±1.1, weight (kg)=391.1±16.6. Epididymis with vas deferens in each bull was isolated from testis and blood vessels on surface of cauda epididymis. To recover cauda epididymal sperm by flushing and floating, each testis was randomly selected from a pair of testicles. The mean weight, length, width, circumference of testicles were similar. For flushing method, a 26 gauge needle connected with 10 mL syringe was inserted into the duct of vas deference. About 12 to 15 incisions were made in cauda epididymis. Pre-warmed semen freezing medium (OptixCell, IMV, France) flushed and epididymal sperm were recovered. For floating method, cauda epididymis was cut and minced using blades in a 100 mm dish. Minced cauda epididymis tissues were incubated with 10 mL of semen freezing medium for 15 min at room temperature. Sperm suspension recovered by filteration with a cell strainer (100 μM nylon mesh). Sperm concentration adjusted to 40 to 50×106 cells/mL with semen freezing medium, semen dilution loaded to 0.5 mL straw and cryopreserved in liquid nitrogen tank. Frozen-semen was thawed at 37°C for 40 sec and measured sperm motility and motility parameters using a CASA system (sperm class analyzer, Spain). Five replicates were conducted in each experimental group of bull. Recovered semen volume (63.3±28.0 vs. 61.7±23.0 mL), motility (89.5±12.8 vs. 91.4±7.9%) among flushing and floating methods were not significantly different. In conclusion, the both of flushing and floating techniques can be used for cauda epididymal sperm recovery without difference of sperm quality.

Artificial insemination with frozen-thawed epdidymal sperm showed low motility and conception rate compared to those with ejaculated frozen-thawed sperm. Cauda epididymal sperm has not contained seminal plasma. We hypothesized that addition of semenial plasma will affects epididymal sperm motility. Therefore, we examined the effect of different seminal plasma concentrations on frozen-thawed epididymal sperm motility. Two testicles were recovered by castration from one Hanwoo bull (age=441 days, scrotal circumference (cm)=31, weight (kg)=420). Epididymis with vas deferens in each bull was isolated from testis and blood vessels on surface of cauda epididymis. Cauda epididymal sperm were recovered by flushing and floating methods with semen freezing medium (OptixCell, IMV, France). Sperm concentration adjusted to 40×106 cells/mL, semen dilution loaded to 0.5 mL straw and crypreserved in LN2 tank until use. Seminal plasma was collected from a Hanwoo bull. In brief, semen collected by artificial vagina and centrifuged at 5,000 ×g for 10 min. The supernatant recovered and centrifuged 5,000 ×g for 10 min for 30 min. The supernatant was cryopreserved at − 80°C until use. Six straws of frozen-semen were thawed at 37°C for 40 sec, mixed and divided into 5 groups (500 uL/group). Frozen-thawed seminal plasma added to prepared 5 frozen-thawed epididymal sperm groups with 0, 1, 2, 5 and 10% of seminal plasma. Sperm motility was evaluated at 0, 2, 4, 6 and 8 h of incubation periods using a computer assisted sperm analysis system (MicroOptic, Spain). Five replicates in each groups were conducted. Addition of 2, 5 and 10% of seminal plasma to frozen- thawed epididymal sperm improved VSL (μM/sec), VAP (μM/sec), LIN (%) and decreased BCF (Hz) compared to those of 0% of seminal plasma group at 4 to 8 h of incubation periods. In conclusion, addition of seminal plasma to frozen-thawed epididymal sperm will improve longevity of sperm in vitro. It suggested that artificial insemination with epididymal sperm and seminal plasma will improve conception rate in vivo.

The aim of the present study was investigated to determine optimal culture condition for IVP embryos using oocytes derived from follicles of ovum pick-up (OPU). Follicular oocytes were aspirated from Hanwoo native cows by the OPU method. After follicular aspiration, the number and morphology of cumulus-oocytes complexes (COCs) and the developmental competence of oocytes after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture were evaluated. The quality of aspirated COCs did not differ among the individual donor. The rates of oocytes recovery from the follicles by ultasound-guided aspiration were not significantly different among individual, but the rate of oocytes useful for IVF was significantly higher (p<0.05) in cows (67%) than heifers (45%) The cleavage and the developmental rate to blastocysts was not significantly different between CR1aa and IVMD medium. In conclusion, the culture medium did not have any apparent effects on the quality of aspirated COCs and developmental competence of oocytes after IVM-IVF, but it may affect follicular recruitment in non-pregnant cows. Moreover, the reproductive phase may influence the developmental competence of the recovered oocytes.