When sperm penetrates into the ovum, hyaluronidase plays a role of hydrolyzing the hyaluronic acid present in the membrane surrounding the oocytes. The zona pelucida of the ovum is hydrolysed to facilitate sperm entry. Therefore, the aim of this study was to investigate the effects of hyaluronidase during the in vitro maturation in porcine oocytes. The cumulus-oocyte complexes (COCs) were cultured during in vitro maturation (IVM) medium containing 0 and 0.1mg/ml hyaluronidase for 44 h. Representative images of oocytes were captured after cultured for 0 h and 22 h by using a microscope. The area was quantified using a image J software. After 44 h of IVM, nuclear maturation stage was assessed by the aceto-orcein method. In results, cumulus cells expansion was no significant difference between control and hyaluronidase treatment groups in 0 h. However, after 22 h of IVM, in 0.1mg/ml hyaluronidase group, cumulus cells diffusion was significantly reduced than control group (p<0.05). After 22 h matured COCs, the cumulus cells were normally expanded in the control group, but there was a significantly lower 0.1mg/ml hyaluronidase group than control group (p<0.05). The nuclear maturation rate was treated with 0.1mg/ml hyaluronidase, it was significantly decrease than control group (p<0.05). In conclusion, our study indicated that hyaluronidase exposure could reduce nuclear maturation in vitro by reducing the expansion of cumulus cells. According to the results, we conjectured that hyaluronidase treatment disrupted the oocyte maturation by hydrolyzing the hyaluronic acid around the oocytes and it reduces the activity of the intercellular gap junction because it weakens cumulus cell bonds and interferes with communication. However, additional studies on hyaluronidase are needed. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

Preimplantation embryonic production in vitro is important in human assisted reproductive technology and animal embryo engineering. Icariin (ICA) is one type of flavonoids and a main component isolated from the stem leaf of Epimedium brevicornum. Flavonoids, which are among the best well-studied natural antioxidants, have been demonstrated to be active in clearing reactive oxygen species (ROS). The purpose of this study was to investigate the effects of ICA treatment during porcine oocyte in vitro aging and their in vitro developmental competency after parthenogenetic activation (PA). Porcine oocytes were matured in vitro for 44 h (control) and for an additional 24 h in the presence of 0, 5 μM ICA (aging, ICA-5), respectively. This study investigated the effects of ICA on nuclear maturation, ROS level, apoptosis index, and the developmental capacity of aged porcine oocytes. Oocyte survival was not different in aging group compared to control or ICA-5 group. The increased ROS level during in vitro aging was prevented in ICA-5 group, while GSH level was not decreased. The decrease of normal spindle formation during in vitro aging was prevented in ICA-5 group. After PA, although the cleavage rate was not different among treatment groups, the blastocyst formation was significantly higher in control and ICA-5 group than in aging group. However, there was significantly difference in the apoptotic index of the ICA-5 group, while it was no difference in the total cell number of the ICA-5 group. (p<0.05). Therefore, this result demonstrated that the ICA is an effective agent to prevent the deterioration during in vitro aging of porcine oocytes.

Generally, in vivo, primary oocytes are grown and matured into secondary oocytes in the ovarian follicles. Quality of the oocytes matured in vivo is higher than that of oocytes matured in vitro, indicating the importance of materializing the microenvironment of ovarian follicles for production of high quality oocyte. Therefore, we tried to mimic the stiffness of ovarian follicles using an agarose as a biocompatible natural polymer. Unfortunately, to date, there are no many reports on whether the quality of porcine oocytes can be increased effectively under the soft matrix. Accordingly, we tried to evaluate the effects of IVM using different mechanical properties of agarose substrate on developmental competence of porcine oocytes. Agarose substrate was constructed and cumulus-oocyte-complexes (COCs) retrieved from porcine medium antral follicles were matured on non-coated (control) culture dish or dishes coated with 1% and 2% (w/v) agarose substrate. Then, cumulus expansion, embryonic development after parthenogenetic activation, and gene expression level were analyzed and compared. As the results, significant increase in blastocyst formation and cumulus expansion were detected in COCs matured on 1% (w/v) agarose substrate compared with control. Moreover, oocytes of COCs matured on 1% (w/v) agarose substrate showed significantly higher BMP15 expression level compared with control. Pro-apoptotic gene BAX expression was significantly increased in oocytes of COCs matured on 2% (w/v) agarose substrate compared with control. In the glycolytic enzyme phosphofructokinase (PFKP) gene expression, cumulus cells of COCs matured on agarose substrate showed significantly higher PFKP expression than control while they showed significantly lower BAX expression than control. These results demonstrated that quality of porcine oocytes could be increased efficiently by the IVM of immature oocytes on the soft culture matrix using agarose.

This study was conducted to evaluate the effects of insulin and epidermal growth factor (EGF) in a in vitro growth (IVG) medium on oocyte growth, in vitro maturation (IVM) and embryonic development of pig oocytes derived from small antral follicles (SAF) less than 3 mm in diameter. SAF oocytes were cultured for 2 days to induce IVG in alpha-minimal essential medium supplemented with 1 mM dbcAMP and 15% (v/v) fetal bovine serum. After IVG culture, oocyte maturation was induced by culturing IVG oocytes in IVM medium for 44 h. IVM oocytes that extruded the first polar body were selected and induced for parthenogenesis (PA) by applying electric stimulus. SAF oocytes cultured under the insulin treatment showed a significantly increased (P < 0.05) nuclear maturation (73.8%) compared to those cultured with insulin and EGF (59.8%). After PA, the proportions of blastocysts based on the number of metaphase II oocytes were significantly higher (P < 0.05) in oocytes that were cultured for IVG with insulin, EGF, and insulin + EGF (32.4%, 35.2%, and 34.8%, respectively) than in control (22.9%). IVG oocytes treated with insulin showed an increased oocyte diameter (116.3 μm) compared to those treated with insulin and EGF (114.0 μm) (P < 0.05). Intra-oocyte GSH content significantly increased (1.07 pixels/oocyte) by insulin treatment during IVG compared to that of oocytes treated with insulin + EGF (0.78 pixels/oocyte). These results demonstrate that IVG culture of SAF oocytes under insulin or/and EGF treatment supports oocyte maturation and improves embryonic development to the blastocyst stage after PA in pigs.

U0126 is a highly selective inhibitor of both MEK1 and MEK2, a type of MAPK/ERK kinase. This study was conducted to evaluate the effect of U0126 treatment during in vitro maturation (IVM) on nuclear maturation, intra-oocyte glutathione content, and embryonic development after parthenogenesis (PA). U0126 (5 μM) was supplemented to IVM medium during the first 0 (control), 2, and 4 h. The basic medium used for IVM was medium-199 supplemented with 10% (v/v) porcine follicular fluid (standard), 0.6 mM cysteine, 0.91 mM pyruvate, 75 μg/ml kanamycin, and 1 μg/ml insulin. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II stage were electrically activated to induce PA. The in vitro culture medium for embryonic development was porcine zygote medium-3 containing 0.3% (w/v) fatty acid-free BSA. When immature oocytes were treated with U0126 during the first 0, 2, 4 h of IVM culture, nuclear maturation was significantly (P < 0.05) increased by the U0126 treatment for 4 h (96.2 ± 1.3%) compared to standard IVM (90.6 ± 2.1%). Cleavage of PA embryos was significantly increased by 4 h- treatment (90.6 ± 2.2%) compared to standard medium (83.9 ± 1.8%). In addition, blastocyst formation of PA embryos was significantly (P < 0.05) increased by the treatment for 4 h (55.8 ± 5.7%) compared to 2 h (38.1 ± 6.1%). The glutathione contents in IVM oocytes were not altered by the U0126 treatments for 0, 2, and 4 h (1.28 ± 0.10, 1.16 ± 0.09, and 1.10 ± 0.09, respectively). Our results demonstrated that 5 μM U0126 treatment during the first 4 h of IVM showed positive effects on nuclear maturation, cleavage, and embryonic development in pigs.

The citrus flavonoid hesperetin has various pharmacological actions, including antioxidant, anti-inflammatory, and anticancer activities. The purpose of this study is to confirm whether the treatment of hesperetin can protect the oocyte from in vitro aging. Porcine oocytes were matured in vitro for 44 h (control) and for an additional 24 h in the presence of 0, 1, 10, 100, and 250 μM hesperetin (aging, H-1, H-10, H-100 and H-250, respectively). This study investigated the effect of different concentration of hesperetin on maturation, and reactive oxygen species (ROS) level, apoptosis index, and the developmental capacity of aging porcine oocytes. In the results, the percentage of cleaved oocytes that reached to the blastocyst stage of H-100 group (37.9 ± 1.1%) was similar to control (38.1 ± 0.8%), and also significantly higher than other aging groups (23.2 ± 0.8%; H-1, 19.7 ± 1.3%; H-10, 26.7 ± 0.6%; and H-250, 18.4 ± 1.6%.)(p<0.05). The H-100 group was significantly decreased ROS activity, and increased the level of glutathione (GSH) and expression of the antioxidant genes (PRDX5, NFE2L, SOD1 and SOD2) compared to the aging group. The H-100 groups prevented aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK), and increased the mRNA expression of cytoplasmic maturation factor genes (GDF9, CCNB1, BMP15 and MOS). Also, it was confirmed that the H-100 group expressed higher level of estrogen receptor than the aging group. Therefore, this result indicated that hesperetin is an effective agent to protect from the oxidative stress during in vitro aging of porcine oocytes.

Mammalian fetal ovaries contains numerous primordial germ cells, however fewer ones can yield mature oocytes due to apoptosis and follicle atresia. Successful in vitro reconstitution of primordial germ cells has recently had a significant effect in the field of assisted reproductive technologies. However, the regulatory mechanisms underlying oogenesis remain unknown and recapitulation of oogenesis in vitro remains unachieved. Therefore, development of methods for obtaining mature oocytes by culturing the fetal ovaries in vitro could contribute to clarify these mechanisms. We adapt an in vitro system for culturing mouse fetal ovaries that support successful follicle assembly and improve oocyte growth and maturation. Ovarian tissues from 12.5 days postcoitum (dpc) fetal mice were cultured in vitro and the matured oocytes were differentiated from primordial germ cells after a 31 days culture period. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with artificial ovarian cells, and that oocytes within the growing follicles are able to mature normally in vitro. Taken together, this in vitro culture system is expected to aid in the development of new strategies to identify the reasons behind failure of follicle assembly and offer a platform for innovative research into preservation of female germ cells and conservation of endangered species.

In this study, we examined sperm penetration and blastocyst developmental rate of oocytes to determine fertilizability of cauda epididymal spermatozoa in Hanwoo bull. One testicle with epididymides were castrated from one Hanwoo bull (14 months of age) and transported to laboratory. Spermatozoa recovered from cauda epididymis by mincing with semen extender (Optixcell, IMV, France) and cryporeserved in liquid nitrogen tank until use. As control, frozen Hanwoo semen was used. Cumulus oocyte complexes (COCs) were collected from follicles (2-8 mm) of slaughtered ovaries and 10 to15 COCs were matured in 50μl droplet with M-199 media supplemented with 10% fetal bovine serum, 10μg/ml FSH, 10μg/ml LH, 10μg/ml EGF for 22 to 24 hours in a humidified atmosphere of 5% CO2 in air. After maturation of COCs, matured COCs were co-incubated with cauda epididymal spermatozoa in 100μl droplet in modified Brackett and Oliphant media supplemented with 2.5 mM theophylline for 12 or 18 hours under 5% CO2 in air. Sperm concentration was adjusted to 5 × 106cells/ml. After IVF for 18 hours, presumptive zygotes were cultured in modified synthetic oviductal fluid with 1mM glutamine, 12 essential amino acids, 10 μg/ml insulin under 5% CO2, 5% O2 in air. In experiment 1, we examined sperm penetration rate at 12 hours of IVF of frozen-thawed epididymal sperm. Total penetration rate among cauda epididymis and control were not significantly different (mean±standard error, cauda epididymis and control vs. 49.7±11.3 and 54.4±12.8%) In experiment 2, cleavage and blastocyst development rate were evaluated at day 2 and day 8 after IVF for 18 hours. Cleavage rate among cauda epididymis and control was similar different (cauda epididymis and control vs. 81.2±3.4 and 82.7±2.5%). However, blastocyst developmental rate of cauda epididymis group was significantly higher than that of control group (cauda epididymis and control vs. 24.4±1.6 and 12.2±2.8%, p<0.05). In conclusion, cauda epididymal spermatozoa in Hanwoo bull has high fertilizability and embryo development. Cauda epididymal sperm can be used as an alternative to ejaculated frozen sperm in vitro.

Alpha-linolenic acid (ALA; n-3 18:3), a one of omega-3 fatty acid, is mainly contained in chloroplast of plant and ALA is an essential fatty acid, not synthesized in mammalian body, it must be supplied from foods. Polyspermy is especially high on in vitro fertilization (IVF) in pigs, which is a major obstacle to in vitro embryo production systems. In our previous study, when ALA was supplemented during in vitro maturation (IVM), the methaphase-II rate and gluthathione level was increased. The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) supplementation during IVM and subsequent of IVF in pigs. The cumulus-oocyte complexes (COCs) were submitted to IVM medium containing 0, 25, 50, and 100 μM ALA for 44 h. After 44 h of IVM, denuded oocytes were co-cultured with spermatozoa during 18 h. After 18 h of in vitro fertilization, oocyte were using aceto-orcein method, to evaluated penetration rate, monospermy (number of monospermy oocytes/total oocytes), and the IVF efficiency (number of monospermy/total penetrated oocytes). In results, 25 and 50 μM ALA groups were significantly increased on penetration rate compared with 100 μM ALA group (p<0.05). Similarly, monospermy rate were significantly increased 25 and 50 μM ALA groups than control group (p<0.05). IVF efficiency was no significant difference between control and ALA treatment groups. Our findings suggested that treatment of ALA supplementation during in vitro maturation (IVM) and subsequent of in vitro fertilization in pigs, ALA can increase IVF efficiency by effectively blocking polyspermy and increasing monospermy some mechanism in porcine oocytes. However, the study of mechanism by which ALA blocks polyspermy are needed, and this study suggests that ALA has a positive effect on in vitro production of porcine oocytes by decreasing polyspermy. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

The national natural monument of Korea, Jeju Black Cattle (JBC), it is a native species with unique blood line. This cattle breed needs mass production and industrialization to further improve and preserve their characteristics. This study was to examine whether there were differences in in vitro developmental rates according to body weight (<300, 300 ~ 350, 350 ~ 400 and >400 kg) and grade (1++, 1+, 1, 2 and 3), and oocyte donors or non-donors. As a method of IVM, groups of ten cumulus oocyte complexes (COCs) were cultured in 50 μl droplets of maturation medium (TCM199 supplemented with 10% FBS, 0.2 mM sodium pyruvate, 1 μg/ml follicle-stimulating hormone, 1 μg/ml estradiol-17β) under mineral oil at 38.8℃ in an incubator with a 5% CO2 atmosphere for 22 to 24 h. For IVF, 44 ul IVF drop contained 10 oocytes with sperm concentration of 1 × 106 cells/ml, and then 2 μl heparin and 2 μl PHE (20 μM peicillamine, 10 μM hypotaurine, 2 μM epinephrine) were added. For IVC, after 44±2 h of incubation, cleaved embryos were incubated in CR1aa medium containing 3 mg/ml FAF-BSA until day 4 at 38.8℃ in a 5% CO2 incubator. Embryos were then cultured in CR1aa medium containing 10% FBS until day 8. As a result, in vitro development rates were the highest in 350 ~ 400 kg body weight group and in 1++ grade group than other groups (p<0.05). However, there was no difference in in vitro developmental capacity of classified donor and non-donor oocyte groups. This result demonstrated that the better in vitro developmental capacity was obtained in high level originated oocyte groups (350 ~ 400kg, 1++ grade) than in others, while there was no different in donor types.

This study was conducted to examine the effect of new inoculants on in vitro digestibility and fermentation characteristics of high moisture rye silage. Rye was harvested at heading stage and divided into 5 treatments, following: No additives(CON); L. plantarum R48-27(NI1); L. buchneri R4-26(NI2); mixture of NI1 and NI2 at 1:1 ratio(MIX); and L. buchneri(LB). The rye forage was ensiled into 10 L bucket silo for 100 days. In vitro digestibility of dry matter and neutral detergent fiber were highest(p<0.05) in NI2 silage. The pH in NI2 and LB silages were lower(p<0.05) than CON silage. Lactate concentration was highest(p<0.05) in NI1 silage. While concentrations of acetate and propionate were highest(p<0.05) in MIX silage. Lactates : acetate ratio was highest(p<0.05) in NI1 silage, but lowest in LB silage. Butyrate concentrations of NI2 and LB silages were lower(p<0.05) than that in CON and NI1 silages. Lactic acid bacteria (LAB) count in all inoculated silages was higher(p<0.05) than that in CON silage, while yeast count in LB silage was lower than in CON, NI1, and MIX silages. In conclusion, application of NI2 inoculant could improve potentially fermentation quality and digestibility of high moisture rye silage.

The objectives of this study were to determine in vitro dry matter and energy utilization of hatchery waste products and to confirm whether in vivo energy digestibility of hatchery waste products could be estimated using in vitro data. Two in vitro assays were conducted for infertile eggs, unhatched eggs, culled chicks, and a mixture(20% dried infertile eggs, 20% dried unhatched eggs, and 60% dried culled chicks). In Exp.1, in vitro dry matter disappearance (IVDMD) of hatchery waste products was determined. In Exp.2, in vitro energy disappearance (IVED) was determined using undigested residues from Exp.1. The IVDMD of infertile eggs, unhatched eggs, culled chicks, and the mixture were 81.7, 88.7, 83.9, and 85.4%, respectively. The IVED of the test ingredients were 74.4, 85.1, 77.6, and 79.8%, respectively. Both IVDMD and IVED were greater in unhatched eggs compared with infertile eggs and culled chicks (p<0.05). In vivo energy digestibility was estimated well using prediction equations for hatchery waste products developed in the present study: In vivo energy digestibility(%) = 2.52 × IVDMD (%) – 133.95 with r2 = 0.70 and in vivo energy digestibility(%) = 1.63 × IVED(%) – 50.03 with r2 = 0.67. In conclusion, energy utilization of unhatched eggs was the greatest among test ingredients and energy utilization of hatchery waste products can be estimated using data from in vitro procedures.

본 연구는 국화과 추출물 첨가가 in vitro 반추위 발효성상 및 메탄 발생에 미치는 영향을 구명하고자 수행하였다. 반추위액은 cannula 장착된 한우 암소 1두에서 채취하였으며, 공시축의 사양관리는 timothy와 농후사료를 6:4 비율로 하였다. 배양액은 Mcdougall buffer 10mL, 위액 5mL 섞어 50mL serum bottle에 혐기상태로 분주하였다. 티모시 0.3g를 각각 넣고 국화과 추출물인 국화, 민들레, 씀바귀, 제비쑥, 해바라기를 기질의 5%를 첨가한 뒤 발효시간대별(3, 6, 9, 12, 24, 48 및 72시간)로 7처리 3반복 수행하였다. 배양액 pH값은 6.29~7.44으로 반추위 적정 pH범위에 속하였다. 건물 소화율은 발효시간대가 늘어날수록 처리구에서 대조구에 비해 유의적(p<0.05)으로 증가하였다. 총가스 발생량은 발효 48시간대에서 민들레, 씀바귀, 제비쑥, 해바라기에서 대조구에 비해 유의적(p<0.05)으로 높게 측정 되었다. 이산화탄소 발생량은 발효 48시간대에서 민들레, 씀바귀, 제비쑥, 해바라기에서 대조구에 비해 유의적(p<0.05)으로 높게 측정 되었으며, 메탄 발생량도 발효 48시간대에서 민들레, 씀바귀, 제비쑥, 해바라기에서 대조구에 비해 유의적(p<0.05)으로 높게 측정 되었다. 미생물 성장량은 발효 48시간대에서 제비쑥과 해바라기 처리구에서 대조구에 비해 유의적(p<0.05)으로 높게 측정되었다. 발효 48시간대 에서 Acetic acid 생성량이 민들레, 씀바귀, 제비쑥, 해바라기 처리구에서 대조구에 비해 유의적(p<0.05)으로 높았다. 발효 12시간대에서 Propionic acid 생성량은 씀바귀, 제비쑥, 해바라기 처리구에서 대조구에 비해 유의적(p<0.05)으로 높았다. 발효 24시간대에서 A/P ratio는 민들레, 제비쑥, 해바라기 처리구에서 대조구에 비해 유의적(p<0.05)으로 감소하였다. 본 연구의 결과에 의해 국화과 추출물 첨가가 반추위 발효에 부정적인 영향을 미치지 않는 것으로 생각되나 메탄 발생량을 감소시키는데 영향이 없는 것으로 생각된다.

It has been claimed that artificial insemination (AI) of cows with frozen-thawed semen treated with commercially produced kits, Wholemom (in favour of female gender) increases the birth chance of calves with desired sex ratio by approximately 85% without decrease of pregnancy rates. Hence, this study was conducted to investigate the efficacy of wholemom kits as combined with frozen-thawed bovine semen during in vitro fertilization on the in vitro fertilization and developmental efficiency and sex ratios such as some reproductive parameters in bovine. For this, 1,737 oocytes were in vitro fertilized and developed. Agglutination effects on bovine after treatment of Wholemom kit were observed by time passage and dose respectively. To determine sex of embryos, Bovine embryo Y-specific gene primers(ConEY) and Bovine specific universal primer(ConBV) were used as multiple PCR method. Fertilization rate of wholemom-treated group was significantly lower than its of control group[66.9% (1,156/1,737) in Wholemom-treated group; 75.0% (610/813) in control group]. However, developmental rate after fertilization of both wholemom-treated and control groups were not significantly different [26.1% (404/1,156) in Wholemom-treated group; 27.4% (224/610) in control group]. Sex ratio of in vitro fertilized embryo with frozen-thawed semen treated with wholemom kit was determined by multi PCR. Female ratio in wholemom-treated group [85.4% (173/201)] was significantly higher than its of control group [47.2% (66/141)]. In conclusion, wholemom treatments of semen used in the in vitro fertilization and development of bovine oocytes provided increase in female ratio with decrease of fertilization rate.

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for animal cloning. Until now, SCNT has been inefficient, especially in dog. It is believed that an embryo developmental block in SCNT embryos is cause of low production efficiency. However, no studies have been performed on canines for embryo developmental block. In this study, we attempted to evaluate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into 0.01 mM EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, Reactive Oxygen Species (ROS) activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Furthermore, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Moreover, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, we elucidated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.