Identification of an osteopontin structural element for the restoration of a normal endometrial epidermal growth factor (EGF) profile determined by the EGF concentration on day 3 of estrous cycle and pregnancy outcome in repeat breeder dairy cows.
Takashi Tanida, Takayoshi Tagami, Yojiro Yanagawa, Seiji Katagiri
Theriogenology, 231, 171, 181, 2025年01月01日, ［国際誌］
英語, 研究論文（学術雑誌）, The loss of a cyclic change with two peaks of increased endometrial epidermal growth factor (EGF) concentration on days 2-4 and 13-14 during the estrous cycle has been linked to low fertility in repeat breeder (RB) cows. We have shown that an intravaginal infusion of osteopontin (OPN) restored the EGF profile in RB cows. The present study aimed to determine a structural element of OPN to restore the normal EGF profile and fertility. Holstein RB cows were diagnosed the EGF profile by a single examination of the endometrial EGF concentration on day 3 of the estrous cycle. Those with an altered EGF profile were intravaginally infused with OPN and its fragments on the day of insemination (day 0); the concentration of endometrial EGF was measured on day 3, and pregnancy was diagnosed on days 30-35. In Study 1, recombinant OPN (rOPN) (16 nmol), thrombin-cleaved N- and C-terminal fragments of rOPN (N-rOPN and C-rOPN, respectively), and a combination of these fragments (Th-rOPN) were infused (n = 13-20). The restoration rate of the normal EGF profile of the N-rOPN group (25.0 %) was a level in between the C-rOPN group (7.7 %) and both the rOPN (55.6 %) and Th-rOPN (64.3 %) groups. In Study 2, PBS (n = 47), rOPN (9.5 nmol, n = 83), and peptides of integrin binding motifs, GRGDSVAYGLK (peptide 1; 32, 320, and 1600 nmol), GRGDS (peptide 2; 320 and 1600 nmol), and SVAYGLK (peptide 3; 320 and 1600 nmol), were infused (n = 20-25). Restoration rates of the normal EGF profile of peptide 1 (320 and 1600 nmol) and peptide 3 (1600 nmol) groups (44.0-56.3 %) were comparable with those of the rOPN group (63.9 %) and higher than those of the PBS group (15.6 %). Restoration rates of the other groups were similar to those of the PBS group. Additional cows received infusions to determine the effect on fertility. Conception rates of the peptide 1 (320 and 1600 nmol; n = 50 each), peptide 3 (1600 nmol; n = 55), and rOPN (n = 111) groups (41.8-50.0 %) were comparable and higher than that of the PBS group (21.6 %, n = 75). In Study 3, PBS (n = 24), peptide 1 (320 nmol; n = 78), and GRGESVAYGLK peptide (peptide 4; 320 and 1600 nmol; n = 50 and 26, respectively) were infused. Restoration rates of the normal EGF profile of peptide 4 and PBS groups (16.0-19.2 %) were comparable and lower than those of the peptide 1 group (44.9 %). Thus, the SVAYGLK motif may be an OPN structural element to restore the normal EGF profile and fertility in RB cows, and the RGD motif may enhance its effect.

Physiological high temperatures alter amino acid metabolism of bovine early antral follicles
Kohei Kawano, Kenichiro Sakaguchi, Nattapong Ninpetch, Yojiro Yanagawa, Seiji Katagiri
Journal of Reproduction and Development, 2024年03月, ［査読有り］
英語, 研究論文（学術雑誌）

The value of evaluating luteal blood flow on the day of embryo transfer for recipient selection in Holstein lactating dairy cows
Takashi Tanida, Itsuro Mukono, Mikiko Nakahashi, Daiki Miyano, Kurumi Miyazawa, Yojiro Yanagawa, Seiji Katagiri
Japanese Journal of Veterinary Research, 71, 3, 72, 81, 2024年02月, ［査読有り］

Changes in interleukin-2, -4, -6 and -8 expression in the postovulatory sow endometrium after artificial insemination based on conceived or failed to conceive
Minami W. OKUYAMA, Masaharu MORIYOSHI, Seiji KATAGIRI
Journal of Reproduction and Development, 70, 3, 192, 196, Japanese Society of Animal Reproduction, 2024年
研究論文（学術雑誌）

Effects of recombinant osteopontin expressed in Escherichia coli on the recovery of the endometrial epidermal growth factor profile and fertility in repeat breeder dairy cows
Takashi Tanida, Takayoshi Tagami, Hiroko Sato, Hay Mar Kyaw, Takeshi Fujikawa, Masashi Nagano, Kenji Momozawa, Yojiro Yanagawa, Seiji Katagiri
Theriogenology, Elsevier BV, 2024年01月
研究論文（学術雑誌）

Effects of prostaglandin F2α treatment at follicular wave emergence on endometrial epidermal growth factor concentration on day of the next estrous cycle on fertility in dairy cows
Vuong Tuan Phong, Takeshi Fujikawa, Kohei Kawano, Yojiro Yanagawa, Seiji Katagiri
Theriogenology, 212, 189, 196, Elsevier, 2023年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

The Difference in Subpopulation Structures in Sex-Sorted and Non-Sorted Semen by Discriminant Analysis of Bull Sperm Motility Data Collected by a Computer-Assisted Sperm Analysis System
Chihiro Kanno, Kentaro Q. Sakamoto, Sung-Sik Kang, Yojiro Yanagawa, Seiji Katagiri, Masashi Nagano
Andrologia, 2023, 1, 7, Hindawi Limited, 2023年05月24日
研究論文（学術雑誌）, We examined the motility characteristics of sperm from sex-sorted bull semen as a model for low-quality (expected low conception rate) sperm compared to that of sperm from non-sorted semen (expected average conception rate) using cluster analysis followed by discriminant analysis. The results indicated that sex-sorted semen contained sperm with hyperactivation-like motility as the main subpopulation immediately after thawing, and this subpopulation decreased after 2 h of incubation. The main subpopulation in the non-sorted semen had progressive motility that was maintained during incubation. A conventional comparison of the mean values of kinematic parameters could not distinguish the samples. In conclusion, discriminant analysis using data from a cluster analysis of motile sperm may accurately describe differences in the structures of sperm motility subpopulations between low and normal fertility semen.

Pre-maturational culture promotes the developmental competence of bovine oocytes derived from an 8-day in vitro growth culture system.
Madalitso Chelenga, Yojiro Yanagawa, Seiji Katagiri, Masashi Nagano
The Journal of reproduction and development, 69, 4, 214, 217, 2023年05月18日, ［国内誌］
英語, 研究論文（学術雑誌）, In this study, we evaluated the effects of pre-maturational culture (pre-IVM) on the developmental competence of bovine oocytes derived from an 8-day in vitro growth (IVG) culture system. IVG oocytes were subjected to 5 h pre-IVM before in vitro maturation, followed by in vitro fertilization (IVF). The proportion of oocytes that progressed to the germinal vesicle breakdown stage was similar between groups with and without pre-IVM. Although metaphase II oocytes and cleavage rates after IVF were similar regardless of pre-IVM culture, the blastocyst rate was significantly higher in the group with pre-IVM (22.5%) than without pre-IVM (11.0%, P < 0.05). In conclusion, pre-IVM culture improved the developmental competence of bovine oocytes derived from an 8-day IVG system.

Metataxonomic Analysis of the Uterine Microbiota Associated with Low Fertility in Dairy Cows Using Endometrial Tissues Prior to First Artificial Insemination
Takuya Yagisawa, Jumpei Uchiyama, Iyo Takemura-Uchiyama, Shun Ando, Osamu Ichii, Hironobu Murakami, Osamu Matsushita, Seiji Katagiri
Microbiology Spectrum, 11, 3, 2023年05月
研究論文（学術雑誌）, The deterioration in reproductive performance in association with low fertility leads to significant economic losses on dairy farms. The uterine microbiota has begun to attract attention as a possible cause of unexplained low fertility. We analyzed the uterine microbiota associated with fertility by 16S rRNA gene amplicon sequencing in dairy cows. First, the alpha (Chao1 and Shannon) and beta (unweighted and weighted UniFrac) diversities of 69 cows at four dairy farms that had passed the voluntary waiting period before the first artificial insemination (AI) were analyzed with respect to factors including farm, housing style, feeding management, parity, and AI frequency to conception. Significant differences were observed in the farm, housing style, and feeding management, except parity and AI frequency to conception. The other diversity metrics did not show significant differences in the tested factors. Similar results were obtained for the predicted functional profile. Next, the microbial diversity analysis of 31 cows at a single farm using weighted UniFrac distance matrices revealed a correlation with AI frequency to conception but not with parity. In correlation with AI frequency to conception, the predicted function profile appeared to be slightly modified and a single bacterial taxon, Arcobacter, was detected. The bacterial associations related to fertility were estimated. Considering these, the uterine microbiota in dairy cows can be varied depending on the farm management practices and may become one of the measures for low fertility. IMPORTANCE We examined the uterine microbiota associated with low fertility in dairy cows derived from four commercial farms via a metataxonomic approach using endometrial tissues prior to the first artificial insemination. The present study provided two new insights into the relevance of uterine microbiota with respect to fertility. First, the uterine microbiota varied depending on housing style and feeding management. Next, a subtle change was observed in functional profile analysis: a formation of uterine microbiota was detected to be different in correlation with fertility in one farm studied. Considering these insights, an examination system on bovine uterine microbiota is hopefully established based on continuous research on this topic.

Relationship between Amino Acid Metabolism and Bovine In Vitro Follicle Activation and Growth
Kenichiro Sakaguchi, Kohei Kawano, Yuki Otani, Yojiro Yanagawa, Seiji Katagiri, Evelyn E. Telfer
Animals, 13, 7, 1141, 1141, MDPI AG, 2023年03月23日
研究論文（学術雑誌）, The amino acid metabolism of bovine follicles during in vitro growth (IVG) was evaluated to identify potential indicators of health during culture. The bovine ovarian cortex was sliced, prepared as strips, and cultured for 6 days. Tissue samples were examined histologically before and after 6 days of culture, and the degree of follicle activation was classified as either high or low based on the number of growing secondary follicles present (high: 7~11; low: 0~1). In a separate experiment, secondary follicles (diameter range: 100~200 μm) were manually isolated and cultured, and their growth was monitored for 6 days. Cultured follicles were classified as growth or degenerate based on diameter change during culture (growth: +60.5~74.1 μm; degenerate: −28~15.2 μm). Free amino acids and their metabolites were measured in the spent culture medium from each group. In cultured ovarian cortical strips, the concentration of α-aminoadipic acid was significantly higher in the low activation group than in the high group (p < 0.05), while those of methionine, lysine, and arginine were higher in the high activation group. In cultured isolated secondary follicles, concentrations of methionine, tyrosine, histidine, and hydroxyproline were higher in the degenerate group (p ≤ 0.05). In conclusion, amino acid metabolism has the potential to serve as an indicator of primordial follicle activation and subsequent growth rate during bovine IVG.

Analysis of sequential ruminal temperature sensor data from dairy cows to identify cow subgroups by clustering and predict calving through supervised machine learning.
Eri Furukawa, Yojiro Yanagawa, Akira Matsuzaki, Heejin Kim, Hanako Bai, Masashi Takahashi, Seiji Katagiri, Shogo Higaki
The Journal of reproduction and development, 69, 2, 103, 108, 2023年02月17日, ［国内誌］
英語, 研究論文（学術雑誌）, The present study investigated the applicability of a calving prediction model based on supervised machine learning of ruminal temperature (RT) data in dairy cows. The existence of cow subgroups for prepartum RT changes was also examined, and the predictive performance of the model was compared among these subgroups. RT data were collected from 24 Holstein cows at 10 min intervals using an RT sensor system. The average hourly RT was calculated and data were expressed as residual RTs (rRT = actual RT - mean RT for the same time on the previous three days). The mean rRT decreased beginning at approximately 48 h before calving to a low of -0.5°C at 5 h before calving. However, two cow subgroups were identified: cows with a late and small rRT decrease (Cluster 1, n = 9) and those with an early and large rRT decrease (Cluster 2, n = 15). A calving prediction model was developed using five features extracted from the sensor data (indicative of prepartum rRT changes) through a support vector machine. Cross-validation showed that calving within 24 h was predicted with a sensitivity of 87.5% (21/24) and precision of 77.8% (21/27). A significant difference in sensitivity was observed between Clusters 1 and 2 (66.7 vs. 100%, respectively), while none was observed for precision. Therefore, the model based on RT data with supervised machine learning has the potential to efficiently predict calving, although improvements for specific cow subgroups are required.

Relationship between frame rates and subpopulation structure of bovine sperm divided by their motility analyzed by a computer‐assisted sperm analysis system
Chihiro Kanno, Kang Sun‐Sik, Kentaro Q. Sakamoto, Yojiro Yanagawa, Seiji Katagiri, Masashi Nagano
Animal Science Journal, 93, 1, Wiley, 2022年12月, ［査読有り］
英語, 研究論文（学術雑誌）

Simultaneous free fatty acid elevations and accelerated desaturation in plasma and oocytes in early postpartum dairy cows under intensive feeding management
Eri Furukawa, Zhen Chen, Tomoaki Kubo, Yue Wu, Koichiro Ueda, Madalitso Chelenga, Hitoshi Chiba, Yojiro Yanagawa, Seiji Katagiri, Masashi Nagano, Shu-Ping Hui
Theriogenology, 193, 20, 29, Elsevier BV, 2022年09月
研究論文（学術雑誌）

Effects of the presence of a large follicle and a corpus luteum in bovine ovaries on nuclear maturation of oocytes and steroidogenesis of granulosa cells cultured as oocyte-cumulus-granulosa complexes derived from early antral follicles
Kenichiro Sakaguchi, Kohei Kawano, Yojiro Yanagawa, Seiji Katagiri, Masashi Nagano
Japanese Journal of Veterinary Research, 70, 2, 45, 56, 2022年08月, ［査読有り］
英語, 研究論文（学術雑誌）

乳牛の血漿遊離脂肪酸濃度上昇に伴う卵子エネルギー代謝関連脂質プロファイルの変化　　　　　　　　　　　　　　　
古川 瑛理, 陳 震, 窪 友瑛, Madalitso Chelenga, Yue Wu, 千葉 仁志, 柳川 洋二郎, 片桐 成二, 惠 淑萍, 永野 昌志
日本生殖医学会雑誌, 67, 3, 139, 139, (一社)日本生殖医学会, 2022年07月
日本語

乳牛の血漿遊離脂肪酸濃度上昇に伴う卵子エネルギー代謝関連脂質プロファイルの変化　　　　　　　　　　　　　　　
古川 瑛理, 陳 震, 窪 友瑛, Madalitso Chelenga, Yue Wu, 千葉 仁志, 柳川 洋二郎, 片桐 成二, 惠 淑萍, 永野 昌志
日本生殖医学会雑誌, 67, 3, 139, 139, (一社)日本生殖医学会, 2022年07月
日本語

Relationship between the timing of insemination based on estrus detected by the automatic activity monitoring system and conception rates using sex-sorted semen in Holstein dairy cattle.
Eri Furukawa, Chihiro Kanno, Yojiro Yanagawa, Seiji Katagiri, Masashi Nagano
The Journal of reproduction and development, 68, 4, 295, 298, 2022年05月30日, ［国内誌］
英語, 研究論文（学術雑誌）, We investigated the optimal timing of artificial insemination (AI) for achieving pregnancy according to the onset/end of estrus detected by an accelerometer system in Holstein cattle. The conception rates of conventional semen were used as a reference. The conception rate from AI of sex-sorted semen was higher at -4 to 4 h (57.1%) from the end of estrus than those at -12 to -4 h (37.7%) and 12-20 h (30.3%), whereas AI at 4-12 h showed an intermediate conception rate (47.4%). Conversely, conception rates were similar in AI performed between 0 and 32 h from the onset of estrus. Regarding conventional semen, the interval from the onset and end of estrus did not affect conception rates. The present results suggest that the time of the end of estrus is the better indicator of optimal AI timing for sex-sorted semen than the onset of estrus.

Effect of heat exposure on the growth and developmental competence of bovine oocytes derived from early antral follicles.
Kohei Kawano, Kenichiro Sakaguchi, Chelenga Madalitso, Nattapong Ninpetch, Shintaro Kobayashi, Eri Furukawa, Yojiro Yanagawa, Seiji Katagiri
Scientific reports, 12, 1, 8857, 8857, 2022年05月25日, ［国際誌］
英語, 研究論文（学術雑誌）, In dairy cows, low fertility caused by summer heat stress continues into the cooler autumn season. This can be caused by impaired oocyte quality in small growing follicles during summer. Here, we subjected oocyte-cumulus-granulosa complexes (OCGCs) derived from early antral follicles (0.5-1 mm) to in vitro growth (IVG) culture under two different temperature settings (the control and heat shock groups), and evaluated effects of heat exposure on growth and developmental competence of oocytes, factors affecting the developmental competence of oocytes (steroidogenesis of granulosa cells, oxidative stress in oocytes, and cell-to-cell communication between oocytes and somatic cells). Oocyte diameters after culture were smaller in the heat shock group. Although nuclear maturation and cleavage rates were similar between the groups, blastocyst rates were lower in the heat shock group (0.0%) than in the control group (27.7%), and reduced glutathione (GSH) levels in oocytes were lower in the heat shock group. Supplementation of cysteine, which stimulates GSH synthesis, increased GSH level and improved blastocyst rate of heat shocked oocytes (27.9%). These results suggest that heat exposure impairs the growth and developmental competence of oocytes in early antral follicles through GSH depletion, which can induce low fertility during summer and the following autumn.

Effects of milk osteopontin on the endometrial epidermal growth factor profile and restoration of fertility in repeat breeder dairy cows
Hay Mar Kyaw, Hiroko Sato, Takayoshi Tagami, Yojiro Yanagawa, Masashi Nagano, Seiji Katagiri
Theriogenology, 184, 26, 33, Elsevier BV, 2022年05月, ［国際誌］
英語, 研究論文（学術雑誌）, Endometrial epidermal growth factor (EGF) shows a cyclic change with two peaks on Days 2-4 and 13-14 during the estrous cycle. An altered (i.e., loss of the two peaks) profile has been linked to reduced fertility in repeat breeder cows. We previously demonstrated that a form of osteopontin (OPN), with a molecular weight of 29 kDa and found in bull seminal plasma (SP), normalized the EGF profile and restored fertility in repeat breeder cows. OPN has many molecular forms due to post-translational modifications and is abundant in bovine milk. The purpose of the present study was to investigate whether mOPN normalizes the endometrial EGF profile and restores fertility in repeat breeder dairy cows with an altered EGF profile. OPN was separated by one-step anion-exchange column chromatography from the whey of bovine milk. Purified mOPN was verified by Western blotting and peptide mass fingerprinting analyses. The OPN fraction showed three major protein bands of 61, 37 and 31 kDa (peptides I, II, and III, respectively) on SDS-PAGE. All three major bands were identified as OPNs by Western blotting and their tryptic peptide masses were matched at approximately 50, 40, and 10%, respectively, to the bovine OPN amino acid sequence by a peptide mass finger printing analysis. The three bands accounted for approximately 85% of the total protein content and 6-23 mg of OPN was obtained from 1 L of bovine milk. A lyophilized eluate containing 1.3 mg of mOPN (171 cows), 0.5 mL of frozen SP (62 cows), and PBS (84 cows) was infused at estrus into the vagina of repeat breeder cows with an altered EGF profile. Some of the cows treated with mOPN, SP, and PBS (46, 50, and 45 cows, respectively) were inseminated immediately before the infusion and then examined for pregnancy between Days 60 and 65. The rate at which mOPN to normalize the EGF profile (56.1%) was similar to that of SP (58.1%) and higher than that of PBS (23.8%) (P < 0.05). The conception rate after the infusion of mOPN (43.5%) was similar to that of SP (40.0%) and higher than that of PBS (22.2%) (P < 0.05). The present results indicate that the infusion of mOPN into the vagina is a treatment option for repeat breeder cows with an altered EGF profile. Further studies are needed to compare the capacity of the three OPN molecules in milk to normalize the EGF profile, together with their molecular characteristics due to post-translational modifications.

Semen collection by urethral catheterization and electro-ejaculation with different voltages, and the effect of holding temperature and cooling rate before cryopreservation on semen quality in the Japanese macaque (Macaca fuscata).
Noboru Takaesu, Chihiro Kanno, Kosuke Sugimoto, Masashi Nagano, Akihisa Kaneko, Yoriko Indo, Hiroo Imai, Hirohisa Hirai, Munehiro Okamoto, Mariko Sashika, Michito Shimozuru, Seiji Katagiri, Toshio Tsubota, Yojiro Yanagawa
The Journal of veterinary medical science, 84, 3, 429, 438, 2022年03月15日, ［国内誌］
英語, 研究論文（学術雑誌）, In the Japanese macaque, semen has been collected by electro-ejaculation (EE), using the higher voltage stimuli compared to other species including genus Macaca. Semen coagulates immediately after ejaculation, which makes difficult to produce high-quality semen for artificial insemination. Recently, semen collection using urethral catheterization (UC) has been reported in carnivore and this technique may allow semen collection without coagulation in a less invasive manner. Further, the temporal preservation temperature and cooling rate of semen during cryopreservation affect post thawing sperm quality. In this study, to improve semen quality and quantity, as well as the animal welfare, semen collection was performed by EE with high (5-15 V) or low (3-6 V) voltage, UC and a combination of the two (EE-UC). It has been suggested that a high voltage is necessary for semen collection, but 10 V stimulation was effective enough and 15 V is for additional sperm collection. Also, liquid semen was collected by EE-UC and this could increase the total number of sperm. Further, to improve the post thawing sperm motility, semen was kept at four temperatures (4, 15, 25 and 37°C) for 60 min, and processed with two cooling procedures (slow cooling before second dilution and fast cooling after second dilution). Holding semen at 25°C and fast cooling after the second dilution maintained progressive motile sperm rate. The present results will contribute to the improvement of semen collection and animal welfare of Japanese macaques.

Leptin receptor expression and its change in association with the normalization of EGF profile after seminal plasma treatment in repeat breeder dairy cows.
Nattapong Ninpetch, Dagvajamts Badrakh, Kohei Kawano, Yojiro Yanagawa, Masashi Nagano, Seiji Katagiri
The Journal of reproduction and development, 68, 3, 209, 215, 2022年02月28日, ［国内誌］
英語, 研究論文（学術雑誌）, Factors associated with high milk production levels have been linked to alterations in the endometrial epidermal growth factor (EGF) profile, a cause of reduced fertility in dairy cows. Therefore, we examined the leptin system that connects nutritional status and reproduction in dairy cattle related to reduced fertility in repeat breeder cows. Plasma leptin concentrations were measured in 18 heifers, 20 high-yielding control cows, and 26 repeat breeder cows, showing an altered EGF profile. Then, all repeat breeder cows were infused with seminal plasma (SP) into the vagina at the next estrus to normalize the EGF profile, while heifers and control cows were infused with vehicle alone. All animals were examined for EGF profiles. Eighteen repeat breeder cows, nine heifers, and nine control cows were also determined for leptin receptor (Ob-R) expression levels in the estrous cycle before and after the infusion. SP normalized the EGF profile in 53.8% of the repeat breeder cows. Leptin concentrations were similar in all groups, regardless of the treatment results for the EGF profile. In contrast, Ob-R levels in repeat breeder and control cows were similar and higher than those in heifers before SP treatment. Ob-R in repeat breeders showing a normal EGF profile after treatment decreased to an intermediate level between heifers and control cows and may provide a clue to take measures against repeat breeding in dairy cows.

Ovulatory follicle size investigated by ultrasonography and single artificial insemination with ovulation induction in the Japanese macaque (Macaca fuscata)
Noboru Takaesu, Masako Minami, Masashi Nagano, Akihisa Kaneko, Yoriko Indo, Hiroo Imai, Hirohisa Hirai, Mariko Sashika, Michito Shimozuru, Munehiro Okamoto, Seiji Katagiri, Toshio Tsubota, Yojiro Yanagawa
Japanese Journal of Veterinary Research, 70, 1, 5, 17, 2022年02月, ［査読有り］
英語, 研究論文（学術雑誌）

Effects of heat stress on the endometrial epidermal growth factor profile and fertility in dairy cows.
Kohei Kawano, Yojiro Yanagawa, Masashi Nagano, Seiji Katagiri
The Journal of reproduction and development, 68, 2, 144, 151, 2022年01月28日, ［国内誌］
英語, 研究論文（学術雑誌）, The endometrial epidermal growth factor profile is an indicator of uterine function and fertility in cattle. The present study aimed to investigate the effects of heat stress on the endometrial EGF profile and fertility in lactating Holstein cows. The endometrial EGF profiles of 365 cows in the Hokkaido and Kyushu regions were examined between June and September (heat stress period, n = 211) and between October and January (control period, n = 154). EGF profiles were investigated using uterine endometrial tissues obtained by biopsy 3 days after estrus (Day 3). The proportion of cows with an altered EGF profile was higher between June and September than between October and January (41.2 vs. 16.2%, P < 0.05). The effects of rectal temperature on Days 0 and 3 on the endometrial EGF profile were also assessed in cows (n = 79) between June and September in the Kyushu region. A single embryo was transferred to cow on Day 7 to evaluate fertility (n = 67). Regardless of the rectal temperature on Day 3, the proportion of cows with an altered EGF profile was higher (64.1 vs. 30.0%, P < 0.05) and the pregnancy rate after embryo transfer (ET) was lower (26.7 vs. 51.4%, P < 0.05) in cows with a rectal temperature ≥ 39.5°C on Day 0 than in cows with a rectal temperature < 39.5°C on Day 0. The present results indicate that alterations in the endometrial EGF profile induced by an elevated body temperature on Day 0 contributed to reductions in fertility in lactating dairy cows during the heat stress period.

Plasma profile of follicle-stimulating hormone and sex steroid hormones after a single epidural administration of follicle-stimulating hormone via caudal vertebrae in Holstein dry cows.
Kenichiro Sakaguchi, Tomoko Suda, Nattapong Ninpetch, Kohei Kawano, Yojiro Yanagawa, Seiji Katagiri, Koji Yoshioka, Masashi Nagano
Animal science journal = Nihon chikusan Gakkaiho, 93, 1, e13696, 2022年01月, ［国際誌］
英語, 研究論文（学術雑誌）, The conventional follicle-stimulating hormone (FSH) treatment for bovine superstimulation involves multiple intramuscular injections, which is stressful for animals and onerous. We herein investigated whether a single epidural injection of porcine FSH (pFSH) can induce superovulation and peripheral concentrations of pFSH and steroid hormones after the treatment in Holstein dry cows. We intramuscularly administered pFSH twice daily to three cows for 3 days (control) or a single epidural pFSH administration (epidural). Numbers of follicles (≥10 mm in diameter) at estrus and corpora lutea at luteal phase were counted by ultrasonography. Blood was sampled from 0 to 104 h after the first pFSH administration and plasma pFSH, progesterone, androstenedione, testosterone, and estradiol-17β concentrations were measured. Numbers of follicles (control: 18.3 ± 7.5, epidural: 15.7 ± 4.0; mean ± SD) and corpora lutea (control: 7.3 ± 4.2, epidural: 8.0 ± 2.6) were similar between both treatments. Plasma pFSH concentrations were higher in epidural than in control (p < 0.01). Although no significant differences were observed in progesterone, androstenedione, or estradiol-17β concentrations between the groups, testosterone concentrations were slightly lower with the epidural treatment than with the control treatment (p = 0.08). In conclusion, superovulation was induced by a single epidural injection of pFSH, which achieved higher pFSH level than the multiple injections in Holstein dry cows.

Postpartum cows showed high oocyte triacylglycerols concurrently with high plasma free fatty acids
Eri Furukawa, Zhen Chen, Hiroki Ueshiba, Yue Wu, Hitoshi Chiba, Yojiro Yanagawa, Seiji Katagiri, Masashi Nagano, Shu-Ping Hui
Theriogenology, 176, 174, 182, Elsevier BV, 2021年12月, ［査読有り］
研究論文（学術雑誌）

Low oxygen environment and astaxanthin supplementation promote the developmental competence of bovine oocytes derived from early antral follicles during 8 days of in vitro growth in a gas-permeable culture device
Madalitso Chelenga, Kenichiro Sakaguchi, Kohei Kawano, Eri Furukawa, Yojiro Yanagawa, Seiji Katagiri, Masashi Nagano
Theriogenology, Elsevier BV, 2021年10月
研究論文（学術雑誌）

ホルスタイン種泌乳牛の分娩後日数及びボディコンディションスコアと卵子中トリアシルグリセロール量の関係　　　　　　　　　　　　　　　
古川 瑛理, 陳 震, 窪 友瑛, Madalitso Chelenga, Yue Wu, 千葉 仁志, 柳川 洋二郎, 片桐 成二, 惠 淑萍, 永野 昌志
日本獣医学会学術集会講演要旨集, 164回, [GO, 5], (公社)日本獣医学会, 2021年09月
日本語

第二胃内留置型自動体温測定器で測定したホルスタイン種経産牛の分娩前1週間の体温変化と分娩時間帯の関係　　　　　　　　　　　　　　　
古川 瑛理, 高橋 昌志, 松崎 明, 唄 花子, 永野 昌志, 片桐 成二, 柳川 洋二郎
日本畜産学会大会講演要旨集, 129回, 117, 117, (公社)日本畜産学会, 2021年09月
日本語

Effect of increased oxygen availability and astaxanthin supplementation on the growth, maturation and developmental competence of bovine oocytes derived from early antral follicles
Madalitso Chelenga, Kenichiro Sakaguchi, Mohammed A. Abdel-Ghani, Yojiro Yanagawa, Seiji Katagiri, Masashi Nagano
Theriogenology, 157, 341, 349, Elsevier BV, 2020年08月, ［査読有り］
研究論文（学術雑誌）

Identification of bovine seminal plasma proteins with an activity to normalize endometrial epidermal growth factor concentrations in repeat breeder cows
Dagvajamts Badrakh, Atsushi Shirasawa, Yojiro Yanagawa, Masashi Nagano, Seiji Katagiri
JAPANESE JOURNAL OF VETERINARY RESEARCH, 68, 2, 91, 103, HOKKAIDO UNIV, 2020年05月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, Epidermal growth factor (EGF) concentrations in the bovine uterus show two peaks on Days 2-4 and 13-14 during the estrous cycle in normal cows; however, these peaks were not found in about 70% of Holstein repeat breeder cows. We have demonstrated the effect of seminal plasma (SP) to normalize the endometrial EGF concentrations on Day 3 and restore fertility in repeat breeder cows. The objective of this study was to identify SP protein(s) with the activity of normalizing the endometrial EGF concentrations. Semen was collected from 5 Holstein bulls and pooled SP obtained from 30 ejaculates were used for this study. The SP protein were separated by gel filtration and 2-dimensional electrophoresis. SP fractions with molecular weight of 16-29 kDa and pI5.8-7.0 showed an activity to normalize the endometrial EGF concentrations on Day 3. Then, protein spots in these area on electrophoresis gels were extracted and subjected to liquid chromatography with tandem mass spectrometry analysis. Twelve protein spots that include four spots of osteopontin (OPN) with different molecular weights and isoelectric points were identified. Protein extracts of one of these OPN spots normalized the endometrial EGF concentrations on Day 3 in 41.9% of repeat breeder cows. The present results indicated that OPN may be the molecule responsible for the activity normalizing the EGF concentrations on Day 3 in the endometrium of repeat breeder cows.

Effect of seminal plasma infusion into the vagina on the normalization of endometrial epidermal growth factor concentrations and fertility in repeat breeder dairy cows.
Dagvajamts Badrakh, Yojiro Yanagawa, Masashi Nagano, Seiji Katagiri
The Journal of reproduction and development, 66, 2, 149, 154, 2020年01月28日, ［査読有り］, ［最終著者, 責任著者］, ［国内誌］
英語, 研究論文（学術雑誌）, Epidermal growth factor (EGF) concentrations in the uterus show two peaks on days 2-4 and 13-14 during the estrous cycle in fertile cows. Loss of the two peaks has been linked to reduced fertility in repeat breeder cows. This study aimed to examine the effect of seminal plasma (SP) on normalizing endometrial EGF concentrations and restoring fertility in repeat breeder cows with low EGF concentrations on day 3. In study 1, we examined the effect of the deposition sites (the vagina and uterus) of SP on the endometrial EGF concentrations in repeat breeder cows. SP infusion into the vagina, but not uterus, on the first day of the estrus cycle (day 0) normalized the endometrial EGF concentrations (≥ 4.7 ng/g tissue weight) on day 3. In study 2, the effect of SP volume (0.5 and 10 ml of SP and 0.5 ml of SP diluted to 10 ml) on EGF concentration was examined. All groups with SP infusion had increased EGF concentrations on day 3, and cows with 10 ml of SP and 0.5 ml of SP diluted to 10 ml showed the highest levels of EGF concentrations. In study 3, we examined the effect of SP infusion on fertility. SP infusion normalized two peaks of endometrial EGF concentrations in about 60% of repeat breeder cows and produced more pregnancies than the controls (44.4 vs. 19.4%). Therefore, we concluded that SP may contain an activity to normalize the EGF profile and restore fertility in repeat breeder cows with altered EGF profiles.

Monitoring follicular dynamics to determine estrus type and timing of ovulation induction in captive brown bears (Ursus arctos)
Yoshiko TORII, Naoya MATSUMOTO, Hideyuki SAKAMOTO, Masashi NAGANO, Seiji KATAGIRI, Yojiro YANAGAWA
Journal of Reproduction and Development, 66, 6, 563, 570, Japanese Society of Animal Reproduction, 2020年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, It is important to understand ovarian physiology when developing an artificial insemination (AI) protocol. Brown bears (Ursus arctos) have a breeding season from May to July, although the type of estrus (polyestrus or monoestrus) is still contested. The present study aimed to define the ovarian dynamics, including follicular waves and ovulatory follicle size, and estrus type in brown bears. Six brown bears were used for ovarian ultrasonography; four were observed between April and October (before the start and after the end of the breeding season) and two in June (breeding season). In addition, we attempted to induce ovulation by administering a gonadotropin releasing hormone (GnRH) agonist. We observed follicular development in April in four bears, but follicles did not develop to greater than 6.0 mm in diameter until May. Thereafter, a group of follicles developed to more than 6.0 mm and grew as dominant follicles, except in one bear. After ovulation and subsequent corpus luteum (CL) formation, the follicular waves disappeared. Furthermore, in three bears treated with GnRH, follicles between 8.2 to 11.2 mm in diameter at the time of treatment ovulated and formed CLs. In two bears, follicles between 5.8 to 8.8 mm ovulated spontaneously within the observation interval. Our results suggest that brown bears may be monoestrous animals. Therefore, AI can only be performed once during the breeding season. Our results also suggest that dominant follicles larger than 8.0 mm are a suitable size for inducing ovulation.

Theca cells can support bovine oocyte growth in vitro without the addition of steroid hormones
Yang Y, Kanno C, Sakaguchi K, Katagiri S, Yanagawa Y, Nagano M
Theriogenology, 142, 41, 47, 2020年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Theca cells (TCs) are essential to folliculogenesis by contributing to steroidogenesis. However, the in vitro growth (IVG) of oocytes co-cultured with TCs has not yet been examined. In the present study, we investigated the feasibility of the IVG of bovine oocyte-cumulus-granulosa cell complexes (OCGCs) co-cultured with TCs and the developmental competence of co-cultured oocytes. OCGCs and TCs were co-cultured without steroid hormone addition for 12 days. Steroidogenesis, the viability of OCGCs, and TC numbers during co-culture were assessed every 4 days. After IVG, oocytes were matured and the nuclear status was evaluated. Some oocytes were inseminated and cultured to examine blastocyst development. During the co-culture, androstenedione production by TCs was only observed during the first 4 days (1.1 ng/well) while estradiol-17β was continuously produced, peaking during the second 4 days (0.5 ng/well). The number of TCs decreased to ∼60% of the seeding number (4.0 × 104 cells/well) during the first 4 days, and was maintained thereafter. The majority of co-cultured OCGCs (82.7%) survived after 12-day IVG. Only a few OCGCs (6.2%) survived in the OCGC culture without TCs (p < 0.01); however, the addition of androstenedione to the culture medium markedly improved survivability to 80.1%, which was similar to that in the co-culture with TCs. In the subsequent development of oocytes derived from the co-culture, 58.3% reached metaphase II stage, 58.7% cleaved, and 17.3% developed to blastocysts, which were similar values to those of oocytes cultured with the addition of androstenedione. In conclusion, TC-produced androgen contributes to OCGC growth and the acquisition of subsequent embryonic developmental competence.

Relationships between the antral follicle count, steroidogenesis, and secretion of follicle-stimulating hormone and anti-Müllerian hormone during follicular growth in cattle.
Sakaguchi K, Yanagawa Y, Yoshioka K, Suda T, Katagiri S, Nagano M
Reproductive biology and endocrinology : RB&E, 17, 1, 88, 88, 2019年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: The antral follicle count (AFC) in mammalian ovaries positively correlates with female fertility. To clarify the causes of differences in fertility between low and high AFC cows, we investigated follicular growth dynamics and hormone concentrations in plasma, follicular fluid, and in vitro growth (IVG) media at different stages of follicular growth. METHODS: Seven cows were divided into high AFC (n = 4, > 30 follicles) and low AFC (n = 3, < 30 follicles) groups based on the peak AFC detected by ultrasonography. These cows were subjected to estrous synchronization, daily ovarian ultrasonography, and blood collection. Their follicular fluid was collected from dominant follicles at different stages (selection, luteal, and ovulatory phases). In another experiment, we cultured oocyte-cumulus-granulosa cell complexes collected from early antral follicles (< 1 mm) for 12 days. Estradiol-17β (E2), testosterone (T), progesterone (P4), and anti-Müllerian hormone (AMH) concentrations in follicular fluids and plasma were measured. Plasma follicle-stimulating hormone (FSH) concentrations were examined. E2, P4, and AMH concentrations were also measured in IVG media. RESULTS: The numbers of small (< 4 mm) and intermediate (4-8 mm) follicles were larger in the high AFC group than in the low AFC group (P < 0.05). The number of intermediate follicles was stable in the low AFC group, indicating consistent development. However, the number of these follicles fluctuated in the high AFC group. Plasma FSH concentrations were higher, whereas E2 and T concentrations were lower in the low AFC group (P < 0.05). E2 concentrations and the E2/P4 ratio in ovulatory follicles and IVG media on day 8 were higher in the high AFC group (P < 0.05). AMH concentrations in plasma and IVG media (P < 0.01) were higher in the high AFC group. CONCLUSIONS: The weaker response to FSH of granulosa cells caused low E2 production in the low AFC group, resulting in high FSH concentrations and the consistent development of intermediate follicles. Conversely, higher E2 concentrations suppressed FSH secretion in the high AFC group. Granulosa cells in the high AFC group had the ability to produce more AMH than those in the low AFC group throughout IVG culture.

若手研究者の描く獣医繁殖学の未来 牛子宮内膜厚の変化とその調節機序　　　　　　　　　　　　　　　
杉浦 智親, 奥山 みなみ, 中田 健, 片桐 成二
日本獣医学会学術集会講演要旨集, 162回, 264, 264, (公社)日本獣医学会, 2019年08月
日本語

Monitoring follicular dynamics using ultrasonography in captive brown bears (Ursus arctos) during the breeding season
Torii Y, Matsumoto N, Sakamoto H, Nagano M, Katagiri S, Yanagawa Y
Theriogenology, 140, 164, 170, 2019年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Artificial insemination (AI) may be a useful tool in the reproductive management of endangered animals, including bears. To establish an AI program for bears, we investigated follicular dynamics using weekly transrectal ultrasonography in six captive brown bears. Along with ultrasonography, we monitored plasma progesterone (P4) and estradiol-17β (E2) concentrations. Furthermore, two bears were administered a gonadotropin releasing hormone (GnRH) agonist to induce ovulation on the first day on which the largest follicle reached more than 10.0 mm in diameter. Brown bears showed two patterns of follicular development in the early and late periods of the breeding season. In the early period (May to mid-June), multiple follicular waves were observed; namely, many follicles developed, and the largest follicles grew to less than 6.0 mm in diameter then regressed. In the late period (mid-June to July), one or two follicles grew to greater than 6.0 mm in diameter and developed as dominant follicles. Moreover, the growth rate of the largest follicle in the late period was faster than that in the early period of the breeding season. One bear with a follicle of 13.1 mm ovulated spontaneously, and one bear ovulated when the follicle was 10.2 mm in diameter after GnRH agonist treatment. Plasma E2 concentrations increased and showed peaks five to seven days before the largest follicles reached their maximum size. Plasma P4 concentrations increased on the day the corpus luteum could be detected using ultrasonography. This is the first study that showed there are two patterns of follicular development in brown bears. Furthermore, the largest follicle reaching greater than 10.0 mm in diameter could be an indicator of the appearance of ovulatory follicles.

Effects of follicle-stimulating hormone followed by gonadotropin-releasing hormone on embryo production by ovum pick-up and in vitro fertilization in the river buffalo (Bubalus bubalis).
Sakaguchi K, Maylem ERS, Tilwani RC, Yanagawa Y, Katagiri S, Atabay EC, Atabay EP, Nagano M
Animal science journal = Nihon chikusan Gakkaiho, 90, 5, 690, 695, 2019年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In this study, we examined the effects of superstimulation using follicle-stimulating hormone (FSH) followed by gonadotropin-releasing hormone (GnRH) on buffalo embryo production by ultrasound-guided ovum pick-up (OPU) and in vitro fertilization (IVF). Nine Murrah buffaloes were subjected to OPU-IVF without superstimulation (control). The morphologies of the oocytes collected were evaluated, and oocytes were then submitted to in vitro maturation (IVM). Two days after OPU, same nine buffaloes were treated with twice-daily injections of FSH for 3 days for superstimulation followed by a GnRH injection. Oocytes were collected by OPU 23-24 hr after the GnRH injection and submitted to IVM (the superstimulated group). The total number of follicles, number of follicles with a diameter > 8 mm, and number of oocytes surrounded by multi-layered cumulus cells were higher in the superstimulated group than in the control group (p ≤ 0.05). After IVF, the percentages of cleavage and development to blastocysts were higher in the superstimulated group than in the control group (p < 0.05). In conclusion, superstimulation improved the quality of oocytes and the embryo productivity of OPU-IVF in river buffaloes.

Astaxanthin improves the developmental competence of in vitro grown oocytes and modifies the steroidogenesis of granulosa cells derived from bovine early antral follicles
Abdel-Ghani MA, Yanagawa Y, Balboula AZ, Sakaguchi K, Kanno C, Katagiri S, Takahashi M, Nagano M
Reproduction, fertility and development, 31, 2, 272, 281, 2019年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In this study we investigated the effect of astaxanthin (Ax), which exhibits strong antioxidant activity, during invitro growth (IVG) on the developmental competence of oocytes and steroidogenesis of granulosa cells derived from early antral follicles. Bovine oocyte-cumulus-granulosa complexes collected from early antral follicles were cultured for 12 days in the presence or absence (control) of 500µM Ax. The viability of oocytes and antrum formation in the granulosa cell layer during IVG culture were greater in the presence than absence of Ax (P<0.05). Regardless of Ax treatment, 17β-oestradiol production increased during IVG culture; however, progesterone production was significantly lower in the presence than absence of Ax (P<0.05). Reactive oxygen species levels were lower in Ax-treated oocytes than in controls after IVG (P<0.05). Although nuclear maturation and cleavage rates did not differ between the Ax-treated and control groups, Ax treatment led to weaker cathepsin B activity in oocytes and better blastocyst rates than in controls (P<0.05). Accordingly, Ax treatment during IVG increased the total number of cells in blastocysts (P<0.05). These results indicate that Ax supplementation of IVG medium improves the quality of bovine oocytes due to its antioxidative effects on growing oocytes and its suppression of the luteinisation of granulosa cells.

Relationship between the antral follicle count in bovine ovaries from a local abattoir and steroidogenesis of granulosa cells cultured as oocyte-cumulus-granulosa complexes.
Sakaguchi K, Tanida T, Abdel-Ghani MA, Kanno C, Yanagawa Y, Katagiri S, Nagano M
The Journal of reproduction and development, 64, 6, 503, 510, 2018年12月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The antral follicle count (AFC) is used as an indicator of cow fertility. We herein investigated the relationship between AFC and the steroidogenesis of granulosa cells and confirmed the developmental competence of oocytes derived from early antral follicles (0.5-1.0 mm) using in vitro growth culture. Slaughterhouse-derived ovaries were divided into high (≥ 25) and low (< 25) AFC groups based on AFC (≥ 2.0 mm). Oocyte-cumulus-granulosa complexes (OCGCs) collected from early antral follicles were cultured for 12 days. The total number, viability, and diameter of granulosa cells and estradiol-17β and progesterone production during the culture were evaluated. Surviving oocytes on day 12 were subjected to in vitro maturation, and their volume and nuclear status were evaluated. Some oocytes were subjected to the evaluation of developmental competence to blastocysts. Although the total number and viability of granulosa cells did not differ between the groups, granulosa cell diameters were smaller in the high AFC group than in the low AFC group. The estradiol-17β and progesterone ratio on day 8 was higher in the high AFC group than in the low AFC group. Oocyte volumes and nuclear maturation rates were greater in the high AFC group than in the low AFC group. The development rate to blastocysts was 9.1% in the high AFC group, while no oocytes developed to blastocysts in the low AFC group. Therefore, estradiol-17β production by granulosa cells appears to be greater in high AFC cattle than in low AFC cattle, thereby promoting the acquisition of oocyte competence.

Effect of astaxanthin addition to an individual culture system for in vitro maturation of bovine oocytes on accumulation of reactive oxygen species and mitochondrial activity
Erdenetogtokh P, Kanno C, Sakaguchi K, Yanagawa Y, Katagiri S, Nagano M
Japanese Journal of Veterinary Research, 66, 4, 325, 329, 2018年11月, ［査読有り］
英語, 研究論文（学術雑誌）

乳牛の人工授精時の子宮内膜厚と受胎性との関係　　　　　　　　　　　　　　　
杉浦 智親, 富永 翔太, 森好 政晴, 片桐 成二, 中田 健
日本獣医学会学術集会講演要旨集, 161回, 406, 406, (公社)日本獣医学会, 2018年08月
日本語

Effect of a single epidural administration of follicle-stimulating hormone via caudal vertebrae on superstimulation for in vivo and in vitro embryo production in Japanese black cows.
Sakaguchi K, Ideta A, Yanagawa Y, Nagano M, Katagiri S, Konishi M
The Journal of reproduction and development, 64, 5, 451, 455, 2018年06月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Here, we describe a simplified procedure for embryo production in the Japanese black cow that uses a single caudal epidural injection of follicle-stimulating hormone (FSH). First, we compared the efficiency of superovulation for in vivo embryo production between conventional multiple FSH treatment (control, n = 10) and single epidural administration (epidural, n = 5). The number of transferable blastocysts was similar between control and epidural groups (4.7 ± 3.5 and 9.0 ± 6.0, respectively). Next, we compared in vitro embryo production by ovum pick-up and in vitro fertilization (OPU-IVF) between control (n = 12) and epidural groups (n = 12). The rate of development to transferable blastocysts was higher in the epidural group than in the control (23.3 vs. 10.5%, P < 0.001). In conclusion, a single epidural administration of FSH can induce follicular development comparable to that of the conventional superovulation protocol and may improve the productivity of OPU-IVF.

Effects of pre-maturational culture duration on developmental competence of bovine small-sized oocytes.
Abdel-Ghani MA, Sakaguchi K, Kanno C, Yanagawa Y, Katagiri S, Nagano M
The Journal of reproduction and development, 64, 4, 365, 369, 2018年05月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, We investigated the effects of pre-maturational (pre-IVM) culture on the developmental competence of small-sized bovine oocytes (110 and < 115 µm). Oocytes were cultured with 3-isobutyl-1-methylxanthine (IBMX) for 0, 5, or 10 h and subjected to in vitro maturation, fertilization, and culture. The cleavage rate (73%) of small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (61 and 62%, respectively). The blastocyst rate (16%) of embryos derived from small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (9 and 8%, respectively). In addition, small-sized oocytes with 5 h pre-IVM had a higher mean cell number in blastocysts (134.1 ± 34.8) than those with 0 and 10 h pre-IVM (100.2 ± 17.2 and 107.8 ± 23.7, respectively). In conclusion, the pre-IVM of small-sized oocytes with IBMX for 5 h improved the developmental competence of bovine oocytes, as well as the quality of blastocysts.

Relationship between bovine endometrial thickness and plasma progesterone and estradiol concentrations in natural and induced estrus.
Tomochika Sugiura, Shun Akiyoshi, Fumihiro Inoue, Yojiro Yanagawa, Masaharu Moriyoshi, Motoshi Tajima, Seiji Katagiri
The Journal of reproduction and development, 64, 2, 135, 143, 2018年04月13日, ［国内誌］
英語, 研究論文（学術雑誌）, The objective of this study was to investigate cyclical changes in endometrial thickness in relation to progesterone (P4) and estradiol-17β (E2) concentrations during natural and induced estrus in 15 cows. In the prostaglandin (PG) F2α-induced estrus group, ultrasonography (USG) at 6-h intervals was used to determine endometrial thickness 48-24 h before the PGF2α treatment until 24 h after ovulation (ovulation = Day 0). In the natural estrus group, USG was performed every 48 h from Day 3 to Days 15-18 after the first ovulation, and then every 6 h until 24 h after ovulation. Endometrial thickness was standardized using Day 13 as a reference day. Blood was collected during every USG examination and plasma P4 and E2 concentrations were determined. Endometrial thickness of the induced estrus group (n = 11) was greater than that of the natural estrus group (n = 9) between 60 and 12 h before ovulation (P < 0.05). In the natural estrus group, prior to an increase in endometrial thickness, a decrease in P4 and an increase in E2 were detected. In the induced estrus group, based on the time of ovulation, an increase in endometrial thickness was detected at the same time of a decrease in P4 before an increase in E2. These results suggest that decreases in P4 concentrations may be a cue to changes in endometrial thickness, while increases in E2 concentrations appear to sustain and/or enhance these changes.

Evaluation of the chemiluminescent enzyme immunoassay system for the measurement of testosterone in the serum and whole blood of stallions
Toishi Y, Tsunoda N, Nagata SI, Kirisawa R, Nagaoka K, Watanabe G, Yanagawa Y, Katagiri S, Taya K
J Reprod Dev., 64, 1, 41, 47, 2018年02月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Testosterone (T) concentration is a useful indicator of reproductive function in male animals. However, T concentration is not usually measured in veterinary clinics, partly due to the unavailability of reliable and rapid assays for animal samples. In this study, a rapid chemiluminescent enzyme immunoassay system (CLEIA system) that was developed for the measurement of T concentration in humans use was validated for stallion blood samples. First, serum T concentrations were measured using the CLEIA system and compared with those measured by a fluoroimmunoassay that has been validated for use in stallions. The serum T concentrations measured by the two methods were highly correlated (r = 0.9865, n = 56). Second, to validate the use of whole blood as assay samples, T concentrations in whole blood and in the serum were measured by the CLEIA system. T concentrations in both samples were highly correlated (r = 0.9665, n = 64). Finally, to evaluate the practical value of the CLEIA system in clinical settings, T concentrations were measured in three stallions with reproductive abnormalities after the administration of human chorionic gonadotropin (hCG). Two stallions with small or absent testes in the scrotum showed an increase in T production in response to hCG administration and one stallion with seminoma did not. In conclusion, the CLEIA system was found to be a rapid and reliable tool for measuring T concentrations in stallions and may improve reproductive management in clinical settings and in breeding studs.

A transvaginal endoscopy-based technique for performing ovarian examinations in sows
Minami W. Okuyama, Tomochika Sugiura, Masaharu Moriyoshi, Kazuto Yamashita, Jun Tamura, Seiji Katagiri
JOURNAL OF REPRODUCTION AND DEVELOPMENT, 63, 6, 617, 622, SOCIETY REPRODUCTION & DEVELOPMENT-SRD, 2017年12月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, For examining pig ovaries, which have complex structures, laparoscopy is a useful technique, but requires general anesthesia; therefore, it cannot be performed repeatedly within a short period of time. We report a transvaginal endoscopy-based technique for conducting ovarian examinations without general anesthesia. Sows were sedated in pig stalls. Using a colonoscope, the vaginal wall was punctured with a trocar. To avoid the trocar being caught in the broad ligament of the uterus or the connective tissue around the vagina, the trocar was inserted close to the external uterine os and between the 2:00 and 3:00 or the 9:00 and 10:00 positions (in a clockwise direction). Then, a urethroscope was inserted into the abdomen, and an examination was carried out after the ovaries had been moved towards the urethroscope camera via rectal palpation. This less invasive procedure may allow repeated examinations and will increase our understanding of ovarian dynamics in pigs.

Extension of the culture period for the in vitro growth of bovine oocytes in the presence of bone morphogenetic protein-4 increases oocyte diameter, but impairs subsequent developmental competence
Yang Y, Kanno C, Sakaguchi K, Yanagawa Y, Katagiri S, Nagano M
Animal Science Journal, 88, 11, 1686, 1691, 2017年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Bone morphogenetic protein-4 (BMP-4) inhibits luteinization of granulosa cells during in vitro growth (IVG) culture of bovine oocytes; however, oocytes derived from a 12 day IVG were less competent for development than in vivo-grown oocytes. We herein investigated whether an extended IVG culture with BMP-4 improves oocyte growth and development to blastocysts after in vitro fertilization. Oocyte-granulosa cell complexes (OGCs) were cultured for 14 or 16 days with BMP-4 (10 ng/mL), while a 12 day culture with BMP-4 served as the in vitro control. OGC viability was maintained for the 16 day culture with BMP-4 (83.2%), but was significantly lower without BMP-4 (58.9%) than the control (83.0%). Prolong-cultured oocytes at 16 days had statistically greater diameter (114.6 μm) than the control (111.7 μm). IVG oocytes with BMP-4 for the 16 day culture had a similar nuclear maturation rate to the control (approximately 67%); however, blastocyst rates in BMP-4 treated oocytes of 14 (1.8%) and 16 day (0%) IVG were statistically lower than that of 12 day IVG (9.0%). In conclusion, BMP-4 maintained OGC viability and promoted oocyte growth in a prolonged culture, but impaired the developmental competence of oocytes. Prolonged culture may not be an appropriate strategy for enhancing the developmental competence of IVG oocytes.

Comparison of sperm subpopulation structures in first and second ejaculated semen from Japanese black bulls by a cluster analysis of sperm motility evaluated by a CASA system
Kanno C, Sakamoto KQ, Yanagawa Y, Takahashi Y, Katagiri S, Nagano M
The Journal of Veterinary Medical Science, 79, 8, 1359, 1365, 公益社団法人 日本獣医学会, 2017年06月, ［査読有り］
英語, 研究論文（学術雑誌）,

In the present study, bull sperm in the first and second ejaculates were divided into subpopulations based on their motility characteristics using a cluster analysis of data from computer-assisted sperm motility analysis (CASA). Semen samples were collected from 4 Japanese black bulls. Data from 9,228 motile sperm were classified into 4 clusters; 1) very rapid and progressively motile sperm, 2) rapid and circularly motile sperm with widely moving heads, 3) moderately motile sperm with heads moving frequently in a short length, and 4) poorly motile sperm. The percentage of cluster 1 varied between bulls. The first ejaculates had a higher proportion of cluster 2 and lower proportion of cluster 3 than the second ejaculates.

Effects of semen preservation procedure in egg yolk-tris based extender on bull spermatozoa characteristics
Kanno C, Yanagawa Y, Takahashi Y, Katagiri S, Nagano M
Japanese Journal of Veterinary Research, 65, 3, 151, 157, Faculty of Veterinary Medicine, Hokkaido University, 2017年05月, ［査読有り］
英語, 研究論文（学術雑誌）, To verify the dynamics of damages to spermatozoa during semen freezing, characteristics of spermatozoa collected from 3 Japanese black bulls were evaluated by using fluorescent staining. Pre-diluted sample showed the highest proportion of spermatozoa with intact plasma membrane, intact acrosome and high mitochondrial potential. The proportion of spermatozoa with intact plasma membrane, intact acrosome, and low mitochondrial membrane potential were higher after cooling to 4°C than the other processes. During cooling preservation examined in this study, the proportion of spermatozoa with damaged acrosome increased. These results lead us to speculate that, during cooling process, spermatozoa may be firstly injured to mitochondrial membrane, and low mitochondrial function may cause the impairment of plasma membrane and subsequent cell death with acrosomal damage.

Early germinal vesicle breakdown is a predictor of high preimplantation developmental competent oocytes in mice.
Higaki S, Kishi M, Koyama K, Nagano M, Katagiri S, Takada T, Takahashi Y
Zygote (Cambridge, England), 25, 1, 41, 48, 2017年02月, ［査読有り］

Endometrial epidermal growth factor profile and its abnormalities in dairy cows
Katagiri Seiji, Moriyoshi Masaharu, Yanagawa Yojiro
JOURNAL OF REPRODUCTION AND DEVELOPMENT, 62, 5, 465, 470, 2016年10月, ［査読有り］, ［筆頭著者, 責任著者］, ［国内誌］
英語, 研究論文（学術雑誌）, Normal cows have 2 peaks in endometrial epidermal growth factor (EGF) concentrations on Days 2-4 and 13-14, and the absence of peaks has been linked to reduced fertility in repeat breeder (RB) cows. However, the timing of the peaks (i.e., day of examinations) was estimated for a few cows per cycle day. Therefore, the present study characterized EGF peaks and examined if the absence of peaks in RB cows indicate either peak loss or changed timing. In Study 1, 20 Holstein cows were examined for EGF concentrations between Days 1 and 6 using repeated biopsy of the uterine endometrial tissues. Sixteen cows exhibited increased EGF concentrations for 2-3 days between Days 2 and 5. All 16 cows exhibited increased EGF concentrations on Day 3. In Study 2, 10 cows were examined for EGF concentrations between Days 11 and 16. Increased EGF concentrations for 2-3 days were found in 7 cows between Days 12 and 15. All 7 cows exhibited increased EGF concentrations on Days 13 and 14. In Study 3, 12 RB cows were examined for endometrial EGF concentrations between Days 1 and 6. Four cows exhibited an increase of EGF concentrations on Days 3 and 4, whereas 8 cows (66.7%) exhibited low EGF concentrations throughout the study period. In conclusion, Days 3 and 13-14 are suitable days to examine a cyclic change of endometrial EGF concentrations. Further, low EGF concentrations on Day 3 in RB cows indicated an absence, but not altered timing, of the EGF peak.

北海道内における乳牛への性選別精液使用が後継牛生産と母牛の生存率に与える影響
菅野 智裕, 萩原 精一, 伊藤 純一, 廣田 和久, 片桐 成二, 永野 昌志
北海道獣医師会雑誌, 60, 8, 378, 378, (公社)北海道獣医師会, 2016年08月
日本語

北海道における乳牛への性選別乳牛精液の使用が産子の性別及び母牛の生存率に及ぼす影響
萩原 精一, 菅野 智裕, 伊藤 純一, 廣田 和久, 永野 昌志, 片桐 成二
日本獣医師会雑誌, 69, 7, 383, 388, (公社)日本獣医師会, 2016年07月
日本語, 性選別乳牛精液が産子の性別、母牛の分娩後生存率に与える影響を明らかにするために道内16農業共済組合により人工授精されたホルスタイン種母牛379,468頭と、その産子の診療履歴を調査した。性選別乳牛精液による雌産子の出生割合(初産牛92.3%、経産牛89.4%)は、通常乳牛精液(初産牛49.1%、経産牛45.4%)より有意に高かった(P<0.01)。性選別乳牛精液により受胎した経産牛の死産発生率(6.1%)は通常乳牛精液で受胎した場合(7.7%)より有意に低かった(P<0.01)。性選別乳牛精液により受胎・分娩した初産・経産牛の分娩後1年生存率は、通常乳牛精液より有意に高かった(P<0.01)。本研究の結果から性選別乳牛精液の利用により、雌産子の出生は増加し、かつ母牛の供用年数は高まることが示唆された。(著者抄録)

The relationship between antral follicle count in a bovine ovary and developmental competence of in vitro-grown oocytes derived from early antral follicles.
Nagai K, Yanagawa Y, Katagiri S, Nagano M
Biomedical research (Tokyo, Japan), 37, 1, 63, 71, 2016年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, To clarify the relationship between ovarian reserve and the developmental competence of bovine oocytes, oocyte-granulosa complexes (OGCs) collected from early antral follicles (≤ 1 mm) in ovaries with high (≥ 25) and low (<25) antral follicle counts (AFCs) were used. OGCs derived from different AFC groups were cultured for growth followed by maturation, fertilization and blastocyst formation. Viability of OGCs during growth culture was similar between groups; however, OGCs in the high-AFC group had a larger number of granulosa cells than the low-AFC group at 12 days of growth. The proportion of matured oocytes in the high-AFC group was higher than that in the low-AFC group. Mitochondrial activity of oocytes before maturation in the high-AFC group was higher than that in the low-AFC group; however, accumulation of reactive oxygen species was similar between groups. Cleavage rate in the high-AFC group tended to be higher than that in the low-AFC group, although blastocyst development was similar between groups. In conclusion, oocytes derived from ovaries with high AFC have higher maturational ability and fertilizability than those from low AFC. The difference may be caused by high proliferation of granulosa cells from ovaries with high AFC.

個別管理培養ディッシュを用いた牛体外受精胚の培養個数が胚の発生率に与える影響
高山茉莉, 片桐成二, 片桐成二, 森好政晴, 堂地修, 今井敬
繁殖技術, 35, 3, 59, 61, 北海道家畜人工授精師協会, 2015年12月
日本語

Fertilizability of oocytes derived from Holstein cows having different antral follicle counts in ovaries.
Nagai K, Yanagawa Y, Katagiri S, Nagano M
Animal reproduction science, 163, 172, 178, 2015年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In this study, to clarify the relationship between ovarian reserve and oocyte quality, cumulus-oocyte complexes (COCs) were collected repeatedly by ovum pick-up (OPU) from cows with high and low antral follicle counts (AFCs) at short (3-4 days) and long (7 days) intervals, and COC morphologies and oocyte fertilizability were examined. The relationship between AFC and follicular growth after OPU was also investigated. Cows showing AFC of ≥30 in at least one OPU session were grouped into the high-AFC group. At a short interval, follicular sizes and COC morphologies were similar between the different AFC groups. However, the normal fertilization rate was higher in the high-AFC group than in the low one, although total penetration rates were similar. At a long interval, the percentage of COCs with poor morphology in the high-AFC group was higher and the normal fertilization rate was lower than in the low one. In the low-AFC group, normal fertilization rates at short and long intervals were similar, and mean follicular size became larger at a long than at a short interval. However, mean follicular sizes at short- and long-interval OPU were similar in the high-AFC group. In conclusion, it is suggested that oocytes derived from cows with high AFC had higher fertilizability than those from cows with low AFC when OPUs were performed at a short (3-4 days) interval. However, oocyte quality in high-AFC cows was impaired by long-interval (7 days) OPU, possibly due to the degradation of follicles.

乳牛の子宮における上皮成長因子発現の異常による受胎性低下とその対策
片桐 成二
繁殖技術 = Technic for animal reproduction, 34, 4, 50, 53, 北海道家畜人工授精師協会, 2015年03月
日本語

分娩後のホルスタイン種への黒毛和種精漿の投与効果
板橋知子, 片桐成二, 柳川洋二郎, 佐藤秀俊, 及川俊徳
宮城県獣医師会会報, 68, 1, 13‐15, 2015年01月
日本語, 研究論文（大学，研究機関等紀要）

Accessory corpora lutea formation in pregnant Hokkaido sika deer (Cervus nippon yesoensis) investigated by examination of ovarian dynamics and steroid hormone concentrations.
Yanagawa Y, Matsuura Y, Suzuki M, Saga S, Okuyama H, Fukui D, Bando G, Nagano M, Katagiri S, Takahashi Y, Tsubota T
The Journal of reproduction and development, 61, 1, 61, 66, SOCIETY REPRODUCTION & DEVELOPMENT-SRD, 2015年, ［査読有り］
英語, 研究論文（学術雑誌）, Generally, sika deer conceive a single fetus, but approximately 80% of pregnant females have two corpora lutea (CLs). The function of the accessory CL (ACL) is unknown; moreover, the process of ACL formation is unclear, and understanding this is necessary to know its role. To elucidate the process of ACL formation, the ovarian dynamics of six adult Hokkaido sika deer females were examined ultrasonographically together with peripheral estradiol-17 beta and progesterone concentrations. ACLs formed in three females that conceived at the first estrus of the breeding season, but not in those females that conceived at the second estrus. After copulation, postconception ovulation of the dominant follicle of the first wave is induced by an increase in estradiol-17 beta, which leads to formation of an ACL. A relatively low concentration of progesterone after the first estrus of the breeding season is considered to be responsible for the increase in estradiol-17 beta after copulation.

ホルスタイン種リピートブリーダー牛に対する黒毛和種精漿注入の効果
板橋 知子, 片桐 成二, 柳川 洋二郎, 及川 俊徳
東北農業研究, 67, 75, 76, [東北農業試験研究協議会], 2014年12月
日本語, 乳牛では受胎率の低下が問題となっており、その原因の一つとして子宮内膜における上皮成長因子(EGF)の発現異常が高産乳牛やリピートブリーダーで確認されている。正常牛の子宮内膜EGF濃度は、発情2～4日目および13～14日目に上昇し二峰性のピークを示すが、リピートブリーダー牛ではこのピークが低下し、周期的変動が消失することが報告されている。最近、牛精漿をリピートブリーダー牛の膣内に注入することによって子宮内膜のEGF発現が正常化し、受胎性が向上したとの報告がある。そこで今回は、場内で繋養している黒毛和種種雄牛の精漿をホルスタイン種リピートブリーダー牛に投与し、その効果について検討した。

Effects of ovarian storage condition on in vitro maturation of Hokkaido sika deer (Cervus nippon yesoensis) oocytes
Tulake Kuerban, Yanagawa Yojiro, Takahashi Yoshiyuki, Katagiri Seiji, Higaki Shogo, Koyama Keisuke, Wang Xuguang, Li Heping
JAPANESE JOURNAL OF VETERINARY RESEARCH, 62, 4, 187, 192, 2014年11月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The effects of different preservation conditions (temperature 20-25 or 10-15 degrees C; duration 12 or 24 h) for ovaries of Cervus nippon yesoensis on in vitro maturation of oocytes were examined. When ovaries were kept for 12 h at 20-25 degrees C, maturation rate of oocytes was highest (71%); however, it declined when the preservation time was extended to 24 h (31%). When the preservation temperature decreased to 10-15 degrees C, the maturation rate after 12 h preservation decreased (51%) but it remained in same level even though preservation time is prolonged to 24 h (55%).

Cryopreservation of zebrafish (Danio rerio) primordial germ cells by vitrification of yolk-intact and yolk-depleted embryos using various cryoprotectant solutions.
Higaki S, Kawakami Y, Eto Y, Yamaha E, Nagano M, Katagiri S, Takada T, Takahashi Y
Cryobiology, 67, 374, 382, 3, 2013年12月, ［査読有り］

Effects of parity and litter size on the energy contents and immunoglobulin G concentrations of Awassi ewe colostrum
HIGAKI SHOGO, NAGANO MASASHI, KATAGIRI SEIJI, TAKAHASHI YOSHIYUKI
Turkish Journal of Veterinary and Animal Sciences, 37, 1, 109, 112, TÜBİTAK, 2013年02月
英語, This study determined the energy content and immunoglobulin G (IgG) concentration of Awassi ewe colostrum and examined the effects of parity and litter size on them. Colostral samples were collected from 57 ewes from the 1st to 7th parities with a litter size of 1 or 2, within 12 h after lambing. Concentrations of colostral fat, lactose, and protein were measured as energy sources, and IgG was measured as representative of the immunoglobulin content of the colostrum. Mean values obtained from analyses of the ewe colostrum samples were 7.4±2.1 kJ/g for the energy value and 60.9±21.4 mg/mL for IgG. Ewe parity did not influence colostral energy content, whereas the IgG concentration of the colostrum obtained from the primiparous ewes was higher than that from the multiparous ewes (P < 0.05). Among the multiparous ewes, the colostrum obtained from ewes carrying twins showed higher energy content and IgG concentrations than that from those carrying a single lamb (P < 0.05).

精漿成分による牛の子宮機能調節
片桐 成二
日本胚移植学雑誌 = Japanese journal of embryo transfer, 35, 1, 21, 24, 2013年01月21日
日本語

Alteration of the Endometrial EGF Profile as a Potential Mechanism Connecting the Alterations in the Ovarian Steroid Hormone Profile to Embryonic Loss in Repeat Breeders and High-producing Cows
KATAGIRI Seiji, MORIYOSHI Masaharu
Journal of Reproduction and Development, 59, 5, 415, 420, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT, 2013年, ［国内誌］
英語, 研究論文（学術雑誌）, Poor reproductive efficiency is a worldwide problem that has affected the dairy industry during the last several decades. In an attempt to explain the changes in reproductive physiology caused by high milk production, a model of elevated steroid metabolism in lactating dairy cows has been proposed. A slow increase in levels and low peak levels of estradiol (E₂) and progesterone (P₄) characterize endocrine changes in high producing cows. Similar changes have been reported in the repeat breeder cows. The abnormal changes in E₂ and P₄ concentrations of these cows may cause an improper uterine environment due to disturbed expression of growth factors and cytokines in the endometrium. This review focuses on the alteration in epidermal growth factor (EGF) profile in the endometrium during the estrous cycle. The normal cow has two peaks of EGF concentrations on days 2–4 and 13–14. Low concentrations of EGF on these days distinguished both high-producing and repeat breeder cows from normal cows. Alteration of the EGF profile could be found in 70 and 40% of the repeat breeder and high-producing cows, respectively. Treatment with a high dose of estradiol benzoate and an intravaginal progesterone-releasing device restored the normal EGF profile in about 70% of the affected cows. The cows having a normal EGF profile after treatment showed a higher pregnancy rate than the cows with the altered profile. Further studies to understand the etiology of the alteration in the EGF profile are needed to develop another treatment option and preventive management for this problem.

Low Incidence of an Altered Endometrial Epidermal Growth Factor (EGF) Profile in Repeat Breeder Holstein Heifers and Differential Effect of Parity on the EGF Profile Between Fertile Holstein (Dairy) and Japanese Black (Beef) Cattle
KATAGIRI Seiji, MORIYOSHI Masaharu, TAKAHASHI Yoshiyuki
Journal of Reproduction and Development, 59, 6, 575, 579, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT, 2013年, ［国内誌］
英語, 研究論文（学術雑誌）, A high incidence (about 70%) of alteration in endometrial epidermal growth factor (EGF) profile, i.e., loss of 2 peaks on days 2–4 and 13–14, has been linked to a reduced fertility in multiparous repeat breeder Holstein cows. However, the EGF profile in Holstein heifers and other breeds (types) of cattle has not been investigated. In study 1, EGF concentrations were determined using endometrial tissues obtained by biopsy on days 3, 7 and 14 from 84 fertile Holstein heifers to obtain a normal range and 53 repeat breeder Holstein heifers to estimate incidence of alterations in the EGF profile. In repeat breeder heifers, EGF concentrations were similar to fertile controls on 3 days and five animals (9.4%) had an altered EGF profile with EGF concentrations below the normal range on days 3 and 14. In study 2, EGF concentrations on day 3 were repeatedly examined from the nulliparous period to the third postpartum period in 28 Holstein (dairy) and 47 Japanese Black (beef) cattle. The effect of parity on EGF concentrations on day 3 was different between Holstein and Japanese Black cattle. In Japanese Black cows, the EGF concentrations were consistently high throughout the study period, while in Holstein cows, the EGF concentrations decreased after the second calving. In conclusion, unlike multiparous repeat breeder Holstein cows, an altered EGF profile may not be a major cause of repeat breeding in Holstein heifers, and the peak EGF concentrations around day 3 may decrease even in fertile populations of multiparous dairy cows, but not in beef cows.

Cryopreservation of Primordial Germ Cells by Rapid Cooling of Whole Zebrafish (Danio rerio) Embryos
Shogo Higaki, Kentaro Mochizuki, Yuichiro Akashi, Etsuro Yamaha, Seiji Katagiri, Yoshiyuki Takahashi
JOURNAL OF REPRODUCTION AND DEVELOPMENT, 56, 2, 212, 218, SOCIETY REPRODUCTION & DEVELOPMENT-SRD, 2010年04月, ［査読有り］
英語, 研究論文（学術雑誌）, The feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells (PGCs) by rapid cooling (i.e., vitrification) of dechorionated whole embryos at the 14- to 20-somite stage was investigated. Initially, we examined the glass-forming properties and embryo toxicities of six cryoprotectants: methanol (MeOH), ethylene glycol (EC), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG) and 1,3-butylene glycol (1,3-BG). According to the results of glass-forming and embryo toxicity tests, pretreatment solution (PS) containing 2 or 3 M cryoprotectant and vitrification solution (VS) containing 5 M cryoprotectant and 0.5 M sucrose were prepared using each cryoprotectant. Dechorionated embryos, the PGCs of which were visualized by injection of green fluorescence protein-nos1 3'UTR mRNA, were cooled rapidly by plunging into liquid nitrogen after serial exposure to PS and VS. All embryos cooled with MeOH, PG and 1,3-BG showed ice formation during cooling, and few embryos had live PGCs after warming. Most embryos cooled with GC did not show ice formation; however, few embryos had live PGCs. All embryos cooled with EC and most embryos cooled with DMSO had live PGCs when the embryos did not show ice formation during cooling. Based on the number of live PGCs in fresh embryos, the maximum survival rates of PGCs recovered from embryos cooled with EC, and DMSO were estimated to be about 40 and 20%, respectively. The present study indicates that rapid cooling of dechorionated whole embryos, especially using EG-based solutions, could be utilized as a simple and promising tool for cryopreservation of PGCs.

Production of fertile zebrafish (Danio rerio) possessing germ cells (gametes) originated from primordial germ cells recovered from vitrified embryos.
Higaki S, Eto Y, Kawakami Y, Yamaha E, Kagawa N, Kuwayama M, Nagano M, Katagiri S, Takahashi Y
Reproduction (Cambridge, England), 139, 733, 740, 4, 2010年04月, ［査読有り］

Measurement of pO2 in cultured mouse oocytes using electron paramagnetic resonance oximetry.
Higaki S, Fujii H, Nagano M, Katagiri S, Takahashi Y
Biomedical research (Tokyo, Japan), 31, 2, 165, 168, 2, 2010年04月, ［査読有り］

Feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells by whole embryo freezing
Shogo Higaki, Kentaro Mochizuki, Hiroko Baba, Yuichiro Akashi, Etsuro Yamaha, Seiji Katagiri, Yoshiyuki Takahashi
JAPANESE JOURNAL OF VETERINARY RESEARCH, 57, 2, 119, 128, HOKKAIDO UNIV, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, We investigated the feasibility of cryopreservation of zebrafish (Danio rerio) blastomeres and primordial germ cells (PGCs) by rapid freezing of dechorionated whole embryos at the blastula, gastrula and segmentation stages. Initially we examined the glass-forming properties and embryo toxicities of 5 cryoprotectants: methanol (MeOH), ethylene glycol (EG), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (1,3-BG). Embryos at the blastula and gastrula stages had high sensitivities to cryoprotectant toxicities and were fragile against mechanical damage. Thus the segmentation stage embryos, the PGCs of which were visualized by injecting green fluorescence protein-nos1 3'UTR mRNA, were frozen using solutions containing each cryoprotectant at 6 M (first trial) and 2 types of cryoprotectants at 3 M each (second trial). In the first trial, live PGCs were recovered from most of the embryos frozen with EG (about 2 cells/embryo); however, a few embryos had live PGCs when embryos were frozen with other cryoprotectants. In the second trial, a mixture of EG + PG better preserved the viability of PGCs in frozen embryos. Live PGCs were recovered from all embryos frozen with EG + PG (about 3 cells/embryo), and the survival rate of PGCs was estimated to be about 25% based on the number of live PGCs in fresh embryos (about 12 cells/embryo). The present study indicates that we can utilize rapid freezing of dechorionated whole embryos at the segmentation stage for the cryopreservation of PGCs.

Fetal Age Estimation of Hokkaido Sika Deer (Cervus nippon yesoensis) Using Ultrasonography During Early Pregnancy
Yanagawa Yojiro, Matsuura Yukiko, Suzuki Masatsugu, Saga Shin-ichi, Okuyama Hideto, Fukui Daisuke, Bandou Gen, Katagiri Seiji, Takahashi Yoshiyuki, Tsubota Toshio
JOURNAL OF REPRODUCTION AND DEVELOPMENT, 55, 2, 143, 148, SOCIETY REPRODUCTION & DEVELOPMENT-SRD, 2009年04月, ［査読有り］
英語, 研究論文（学術雑誌）, In sika deer, the normal method of estimating fetal age, based on fetal weight, is not applicable during the early pregnancy period. The objective of the present study was to describe the growth and development of sika deer fetuses and to establish a method for fetal age estimation during early pregnancy using ultrasonography. Five captive female Hokkaido sika deer (Cervus nippon yesoensis) were observed for estrus and mated (day 0) with an intact male. At two- or three-day intervals, fetuses were observed by rectal ultrasonographic scans until 59-61 days of gestation. The straight crown-rump length (SCRL), curved crown-rump length (CCRL), head length (HL), trunk depth (TD) and heart rate (HR) of the fetuses were measured. Linear regression equations were computed for each measurement together with fetal age. Analyses were conducted after transformation to a natural logarithm for SCRL and CCRL. All equations were significant (P<0.001), with SCRL becoming measurable earlier (day 20) than the others and yielding the best correlation (Days = -2.08+14.15 LnX: X=SCRL, Ln=natural logarithm). Therefore, we concluded that a precise estimation of fetal age in early gestation is best performed using SCRL measurements.

A Progestin-Based Treatment with a High Dose of Estradiol Benzoate Normalizes Cyclic Changes in Endometrial EGF Concentrations and Restores Fertility in Repeat Breeder Cows
Katagiri Seiji, Takahashi Yoshiyuki
The Journal of reproduction and development, 54, 6, 473, 479, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT, 2008年12月01日, ［国内誌］
英語, 研究論文（学術雑誌）, The objective of the present study was to examine the efficacy of a progestin-based treatment with a high dose of estradiol benzoate (EB) to normalize the epidermal growth factor (EGF) profile in the uterine endometrium and restore fertility in repeat breeder cows. Repeat breeder cows without peaks in their endometrial EGF concentrations on Days 3 and 14 of the estrous cycle were used throughout the study. The effect of 1 (standard dose), 2.5 and 5 mg of EB in a progestin-based treatment protocol (EB1, EB2.5 and EB5 treatments, respectively; n=5 for each group) on endometrial EGF concentrations was first examined. The EB1 and EB2.5 treatments in the repeat breeder cows produced a suppressed response in endometrial EGF compared with EB1 treatment in the fertile controls (n=5) and failed to restore the normal EGF profile during the next estrous cycle. However, EB5 treatment produced an increase in EGF concentrations similar to the fertile controls and normalized the endometrial EGF profile. The effects of the EB1 and EB5 treatments (n=30 for each group) on the endometrial EGF profile and fertility were then examined in the repeat breeder cows. The proportion of cows, with an EGF profile normalized by the treatments was higher in the EB5 group (66.7%) than in the EB1 (30.0%) and untreated control (13.3%; n=30) groups (P<0.01). The pregnancy rates of the cows having a normal EGF profile after treatment in the EB1 and EB5 groups were similar (88.9 and 85.0%, respectively) and higher than those of the cows having an abnormal profile within the same groups (19.0 and 30.0%, respectively, P<0.01). In summary, the endometrial response to EB in terms of the EGF concentration was suppressed in repeat breeder cows. A high dose (5 mg) of EB in a progestin-based treatment was found to be effective for restoration of a normal EGF profile and fertility in repeat breeder cows having lesser endometrial EGF concentrations on Days 3 and 14.

Immunohistochemical localization of the estrogen receptor alpha (ERα) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) during pregnancy
Yanagawa Yojiro, Matsuura Yukiko, Suzuki Masatsugu, Katagiri Seiji, Tsubota Toshio
Japanese Journal of Veterinary Research, 56, 3, 139, 149, The Graduate School of Veterinary Medicine, Hokkaido University, 2008年11月, ［国内誌］
英語, 研究論文（学術雑誌）, Information on steroid hormone receptor distribution in the uterus is essential to understand the roles of their ligands in pregnancy. This study examined the spatio-temporal localization of estrogen receptor alpha (ERα) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) to determine the estrogen and progesterone action site during pregnancy. Ovaries and uteri were collected from 21 pregnant sika deer with single fetus and two corpora lutea, ranging from Day 20 to Day 207 of pregnancy. In addition, genital organs were also collected from three sika deer whose gestatio...

Rapid sex chromosomal chimerism analysis in heterosexual twin female calves by Loop-mediated Isothermal Amplification
Hiroki Hirayama, Seiji Katagiri, Soichi Kageyama, Akira Minamihashi, Satoru Moriyasu, Ken Sawai, Sadao Onoe, Yoshiyuki Takahashi
ANIMAL REPRODUCTION SCIENCE, 101, 1-2, 38, 44, ELSEVIER SCIENCE BV, 2007年09月, ［査読有り］
英語, 研究論文（学術雑誌）, We attempted to apply an embryo sexing kit with Loop-mediated Isothermal Amplification (LAMP) to sex chromosomal chimerism analysis in heterosexual twin female calves. Peripheral blood was used for the amplification of male-specific DNA, derived from XY leukocytes. When blood samples were diluted 1: 1000 in LAMP reaction mixture, hemoglobin or blood coagulation did not influence the turbidity measurement of the reaction mixture for detection of amplified DNA. This procedure detected the existence of XY leukocytes of 0.01% in female blood. Furthenmore, all heterosexual twin female calves, bearing sex chromosomal chimerism based on karyotyping and PCR, showed male-specific DNA from peripheral blood by LAMP. These results indicated that the embryo sexing kit with LAMP was available for sensitive detection of sex chromosomal chimerism. This procedure made it possible to detect easily Y-chromosome specific DNA in a short interval compared with PCR, and was convenient for field application of freemartin diagnosis. (C) 2006 Elsevier B.V. All rights reserved.

The relationship between oocyte morphology and ovarian status in cattle.
Nagano M, Hishinuma M, Katagiri S, Takahashi Y
The Journal of reproduction and development, 53, 4, 953, 958, 4, 2007年08月, ［査読有り］
英語, We investigated the relationships between oocyte morphology, follicular size and follicular waves using bovine ovaries derived from local abattoirs. Ovaries at the recruitment and selection phases contained larger numbers of oocytes with good developmental ability, although ovaries at the recruitment phase contained the largest numbers of follicles compared with ovaries at the selection and dominant phases. Dominant phase ovaries contained a high percentage of oocytes with as good developmental ability as selection phase ovaries; however, they contained the lowest total number of oocytes with good developmental ability. Small follicles under 3.0 mm in diameter contained large numbers of small and degenerating oocytes. In contrast, follicles more than 3.0 mm in diameter contained a higher percentage of oocytes with good developmental ability.

Effects of isolation method and pre-treatment with ethylene glycol or raffinose before vitrification on in vitro viability of mouse preantral follicles.
Nagano M, Atabay EP, Atabay EC, Hishinuma M, Katagiri S, Takahashi Y
Biomedical research (Tokyo, Japan), 28, 3, 153, 160, 3, 2007年06月, ［査読有り］
英語, Effects of isolation and vitrification protocols on follicular survival after warming were examined. Mouse preantral follicles enzymatically or mechanically isolated from ovaries of 12-day-old mice were exposed either to 2 M ethylene glycol (EG) for 2 or 5 min, or to ascending concentrations (0.15 then 0.3 M) of raffinose for 2 or 5 min each (2-2 and 5-5 min). They were then exposed to a vitrification solution (VS) composed of 6 M EG and 0.3 M raffinose for 0.5, 1, or 2 min before vitrification. Mechanically isolated follicles showed higher survival than enzymatically isolated follicles, regardless of periods of exposure to EG or raffinose and subsequent exposure to VS. After 10 days of culture, follicular growth and maturational ability of oocytes derived from vitrified follicles exposed to 2 M EG for 5 min and to VS for 1 min were higher than those from follicles exposed to raffinose solutions for 2-2 min and to VS for 1 min. Histological evaluation revealed that exposure of preantral follicles to raffinose solutions caused cytoplasmic vacuolation in granulosa cells which could be due to cellular shrinkage during dehydration; whereas, exposure to 2 M EG induced morphological alterations in follicles only to a lesser extent.

ATP content and maturational/developmental ability of bovine oocytes with various cytoplasmic morphologies.
Nagano M, Katagiri S, Takahashi Y
Zygote (Cambridge, England), 14, 4, 299, 304, 4, 2006年11月, ［査読有り］
英語, We examined the relationship among morphological appearance (six groups) of bovine oocytes, ATP content and maturational/developmental ability. Oocytes with a brown ooplasm (with or without a dark region) had intermediate levels of ATP at the germinal vesicle (GV) stage and showed higher rates of first polar body (PB) extrusion than the other groups. Oocytes with a low level of ATP (oocytes with a pale ooplasm without dark clusters) and oocytes with a high level of ATP (oocytes with a black ooplasm) showed lower rates of PB extrusion. During in vitro maturation, ATP levels in oocytes decreased at around GV breakdown and increased toward metaphase II (MII). MII oocytes having a brown ooplasm with a dark region, which had good developmental capacity, had a relatively high level of ATP. MII oocytes with a brown or pale ooplasm without dark clusters, which had poor developmental capacity, had low ATP levels. MII oocytes with a black ooplasm, which had poor developmental capacity, had an unusually high level of ATP. These results suggest that the morphological appearance of bovine oocytes is closely related to their ATP levels and that cytoplasmic morphology will give an advantage for the selection of oocytes with a high maturational and developmental ability.

Potential relationship between normalization of endometrial epidermal growth factor profile and restoration of fertility in repeat breeder cows.
Seiji Katagiri, Yoshiyuki Takahashi
Animal reproduction science, 95, 1-2, 54, 66, 2006年09月, ［国際誌］
英語, 研究論文（学術雑誌）, The present study characterized alterations of the endometrial epidermal growth factor (EGF) and examined potential relationships between normalization of the EGF and restoration of fertility in repeat breeder cows. In Experiment 1, endometrial tissues were obtained by biopsy on Days 3, 7 and 14 of the estrous cycle from 99 fertile dairy cows and normal ranges of uterine tissue EGF concentrations were determined. Then, eight fertile cows were examined for endometrial EGF concentrations on Days 3, 7 and 14 during the three consecutive estrous cycles. All eight cows had a normal EGF profile (EGF concentrations were within the normal ranges of all 3 days) in all three estrous cycles and endometrial EGF concentrations were similar among the three estrous cycles. In Experiment 2, 61 repeat breeder cows were examined EGF profile and 43 (70.5%) cows had altered EGF profiles compared with control profiles. A typical alteration was characterized by decreased concentrations on Days 3 and 14 and accounted for 86.0% of all alterations. When repeat breeder cows with altered EGF profiles (n=17) were left untreated, abnormality persisted in 14 (82.4%) cows at the second examination and only 3 animals became pregnant within the next two estrous cycles. Among 15 cows showing a typical alteration at the first examination, 11 (73.3%) cows had the same alteration until the third examination performed after two infertile inseminations following the second examination. When repeat breeder cows with altered EGF profiles (n=26) were given one of four therapeutic treatments, cows in which the EGF profile normalized after treatment had a greater pregnancy rate than those with altered profiles (11/14 animals versus 3/12 animals pregnant; P<0.05). In conclusion, suppressed endometrial EGF concentrations on Days 3 and 14 may be common alterations and persist between estrous cycles in repeat breeder cows. Potential relationship between normalization of the endometrial EGF profile and restoration of fertility was observed. Additional study to confirm results of the treatment is, however, necessary because the present study used a limited number of animals. Nevertheless, current data suggest that normalization of the EGF profile is one of the prerequisite factors to restore fertility.

Relationship between bovine oocyte morphology and in vitro developmental potential.
Nagano M, Katagiri S, Takahashi Y
Zygote (Cambridge, England), 14, 53, 61, 1, 2006年02月, ［査読有り］

Sucrose-exposed chemically enucleated mouse oocytes support blactocyst development of reconstituted embryos.
ELSHEIKH Adil Salim, TAKAHASHI Yoshiyuki, KATAGIRI Seiji, KANAGAWA Hiroshi
Reprod Fert Dev, 18, 6, 697, 701, 2006年

乳用牛における分娩後の卵胞発育ウェーブの発現時期と分娩前後のFSH分泌動態　　　　　　　　　　　　　　　
笹本 良彦, 坂口 実, 片桐 成二, 中田 健, 高橋 芳幸
The Journal of Reproduction and Development, 50, Suppl., j79, j79, (公社)日本繁殖生物学会, 2004年08月
日本語

Changes in EGF concentrations during estrous cycle in bovine endometrium and their alterations in repeat breeder cows.
Seiji Katagiri, Yoshiyuki Takahashi
Theriogenology, 62, 1-2, 103, 12, 2004年07月, ［国際誌］
英語, 研究論文（学術雑誌）, The objective of the present study was to determine if abnormalities in the cyclic changes of endometrial EGF concentrations can be a diagnostic tool for repeat breeder cows. First, the profile of EGF concentrations during the estrous cycle was determined using endometrial tissues obtained from 31 Holstein cows after slaughter. Cyclic cows had two peaks of EGF concentrations. Then, endometrial tissues were obtained from 12 control and 20 repeat breeder cows by biopsy on Days 3, 7, and 14 of the same estrous cycle. Endometrial EGF concentrations in biopsied samples of the controls were similar to those found in slaughterhouse materials; they were high on Days 3 and 14 (9.2 and 10.4 ng/g tissue, respectively) and low on Day 7 (3.8 ng/g tissue). Concentrations of EGF in repeat breeder cows had a different profile; they were similar on Days 3, 7, and 14 (4.4, 3.4, and 4.0 ng/g tissue, respectively). In conclusion, changes in endometrial EGF concentrations were altered in repeat breeders; these alterations may be a potential diagnostic marker for repeat breeder cows.

Serum Leptin Levels during the Periparturient Period in Cows
SOLIMAN Mohamed, ISHIOKA Katsumi, YOSHIDA Ryusuke, KOMABAYASHI Kenichi, HATAI Hitoshi, MATSUI Yoshitaka, HIRAI Tuneo, KATAGIRI Seiji, TAKAHASHI Yoshiyuki, KAWAKITA Yumi, ABE Hiroyuki, KITAMURA Hiroshi, KIMURA Kazuhiro, SAITO Masayuki
The journal of veterinary medical science, 64, 11, 1053, 1056, JAPANESE SOCIETY OF VETERINARY SCIENCE, 2002年11月25日
英語, Serum leptin concentrations were measured in antenatal and postnatal cows housed at two different locations. The mean serum leptin concentration was 9.2 ± 0.6 ng/m l (n=22) in one group, and was slightly lower in the other (7.4 ± 0.4 ng/ml, n=54), probably because of the different nutritional conditions between the two groups. There was no consistent variation in relation to the menstrual cycle and the periparturient period in both groups. Moreover, serum leptin concentrations during the periparturient period were independent of the number of delivery and the incidence of mastitis and milk fever. These results are quite different from those in rodents and human, suggesting the different regulatory mechanism of circulating leptin concentration in cows.

Steroid Hormones Do Not Reactivate Neospora caninum in Ovariectomized Mice.
KOBAYASHI Atsushi, KATAGIRI Seiji, KIMURA Takashi, OCHIAI Kenji, UMEMURA Takashi
Journal of Veterinary Medical Science, 64, 9, 773, 777, JAPANESE SOCIETY OF VETERINARY SCIENCE, 2002年
英語, The direct effects of three steroid hormones (progesterone, estradiol-17β and corticosterone) on the growth of Neospora caninum (N. caninum) tachyzoite were examined in Vero cells. Subsequently, ovariectomized BALB/c mice infected with N. caninum were treated with physiological concentrations of the steroid hormones for 1 or 2 weeks. These hormones had no direct effect on the parasite growth in vitro. In the infected mice, there was no significant difference in the parasite distribution and histopathological changes between the hormone-injected and control groups. No mice showed parasitemia at the time of autopsy. These results suggest that physiological levels of steroid hormones (progesterone, estradiol-17β and corticosterone) do not reactivate N. caninum in mice.

In vitro viability of mouse oocytes vitrified in an ethylene glycol-based solution
BAUTISTA Jose Arceo N., DELA PENA Eufrocina C., KATAGIRI Seiji, TAKAHASHI Yoshiyuki, KANAGAWA Hiroshi
Japanese journal of veterinary research, 46, 1, 13, 18, 北海道大学, 1998年05月30日
英語, Ovulated mouse oocytes denuded of their cumulus cells, were vitrified in a solution containing 7 M ethylene glycol as the sole cryoprotectant using one or two steps of exposure before vitrification and were diluted in 1 M sucrose solution in 5 or 10 min after warming. The results proved that the viability of oocytes are detrimentally affected by exposure to the vitrification solution even without vitrification. At 5 min dilution time, the two-step exposure was superior to the one-step in terms of the post-warming recovery rate of vitrified oocytes with normal morphology and their subsequent development to the blastocyst stage (p<0.01) after fertilization in vitro. At 10 min dilution time, no significant difference between one- or two-step exposure was found. The effect of the addition of 0.5 M sucrose to the vitrification solution was also determined and did not result in a significant improvement in the viability of oocytes vitrified in one-step and diluted for 10 min. In conclusion, the results in this study indicate that oocytes can be vitrified with 7 M ethylene glycol as the sole cryoprotectant in the vitrification solution, and that the recovery of normal oocytes after one-step exposure in the vitrification solution can be improved by 10 min dilution time. However, the improvement in the recovery rate of oocytes with normal morphology and their subsequent developmental in vitro was not improved by the addition of 0.5 M sucrose to the vitrification solution.

Functional enucleation of mouse metaphase II oocytes with etoposide
ELSHEIKH Adil Salim, TAKAHASHI Yoshiyuki, KATAGIRI Seiji, KANAGAWA Hiroshi
Japanese journal of veterinary research, 45, 4, 217, 220, 北海道大学, 1998年02月27日
英語, Mouse metaphase II (M II) oocytes were exposed to 50 μg/ml etoposide (ETO) before and after parthenogenetic activation with 7% ethanol and they were washed with 0.75 M sucrose. The ETO treated parthenogenetically activated oocytes were cultured or fused to single blastomeres of late 2-cell stage mouse embryo to test their ability to support development in vitro. In parallel untreated parthenogenetically activated oocytes were cultured to serve as control. None of ETO treated oocytes developed beyond the 2-cell stage, whereas 4% of the reconstituted embryos and 35% of control developed to blastocysts. It is concluded that mouse M II oocytes can be functionally enucleated by ETO treatment and can be used for nuclear transfer experiments.

めん羊の発情同期化に用いる腟内プロジェステロン徐放剤処置が人工授精時の腟内細菌叢に与える影響
齋藤希実, 佐川里子, 白鳥麗奈, 健山幸乃, 鈴木康規, 久保田寛顕, 谷田孝志, 柳川洋二郎, 片桐成二, 菅野智裕, 桃沢健二, 永野昌志, 東北畜産学会報, 74, 2, 2024年

乳牛の分娩前の体温低下,ホルモン動態および分娩経過の関連について
亀谷牧子, 古川瑛理, 古川瑛理, 池中良徳, 松崎明, 柳川洋二郎, 片桐成二, 日本獣医学会学術集会講演要旨集, 166th, 2023年

乳用牛の発情後期および発情休止期における耐糖能の違い
谷田孝志, 池田侑樹, 田嶋彩野, 田嶋彩野, 菅野智裕, 菅野智裕, 坂口謙一郎, 柳川洋二郎, 永野昌志, 永野昌志, 片桐成二, 日本獣医学会学術集会講演要旨集, 166th, 2023年

生理的な範囲の暑熱負荷が牛初期胞状卵胞由来卵子の発生能および抗酸化能に及ぼす影響
河野光平, 坂口謙一郎, 坂口謙一郎, CHELENGA Madalitso, NATTAPONG Ninpetch, 小林進太郎, 古川瑛理, 柳川洋二郎, 片桐成二, 日本生殖医学会雑誌, 67, 3, 2022年

ルーメンセンサから得られる温度データの機械学習による乳牛の分娩予測
古川瑛理, 檜垣彰吾, 松崎明, 唄花子, 高橋昌志, 片桐成二, 柳川洋二郎, 日本畜産学会大会講演要旨, 130th, 2022年

暑熱負荷が牛の初期胞状卵胞に由来する卵子中の活性酸素種および還元型グルタチオン量に及ぼす影響
河野光平, 坂口謙一郎, 坂口謙一郎, CHELENGA Madalitso, 古川瑛理, NINPETCH Nattapong, 柳川洋二郎, 片桐成二, 日本繁殖生物学会講演要旨集(Web), 114th, 2021年

第二胃内留置型自動体温測定器で測定したホルスタイン種経産牛の分娩前1週間の体温変化と分娩時間帯の関係
古川瑛理, 高橋昌志, 松崎明, 唄花子, 永野昌志, 片桐成二, 柳川洋二郎, 日本畜産学会大会講演要旨, 129th, 2021年

乳汁中プロジェステロン濃度測定による発情回帰牛の摘発
古川瑛理, 安田奈絵, 山田淑子, 石田晃彦, 渡慶次学, 渡邊龍之介, 篠原禎忠, 松田憲司, 野内繁広, 片桐成二, 柳川洋二郎, 日本獣医学会学術集会講演要旨集, 163rd, 2020年

暑熱が乳牛の初期胞状卵胞に由来する卵子の発育,成熟および顆粒層細胞の性ステロイドホルモン産生能に及ぼす影響
河野光平, 坂口謙一郎, 坂口謙一郎, 古川瑛理, CHELENGA Madalitso, 柳川洋二郎, 片桐成二, 日本獣医学会学術集会講演要旨集, 163rd, 2020年

精漿が豚の末梢血単核球中のサイトカイン発現および制御性T細胞に与える影響
阪井紀乃, 奥山みなみ, 奥山みなみ, 對馬隆介, 對馬隆介, 南家夕子, 篠原明, 古川瑛理, 河野光平, 柳川洋二郎, 楢原久司, 片桐成二, 日本獣医学会学術集会講演要旨集, 163rd, 2020年

若手研究者の描く獣医繁殖学の未来 クマ科動物の人工授精技術の開発に向けた研究
鳥居 佳子, 永野 昌志, 片桐 成二, 柳川 洋二郎, 日本獣医学会学術集会講演要旨集, 162回, 269, 269, 2019年08月
(公社)日本獣医学会, 日本語

牛性選別精子の卵管上皮細胞結合能および受精能獲得のフローサイトメトリーによる解析
中島愛理, 菅野智裕, 菅野智裕, 片桐成二, 永野昌志, 柳川洋二郎, 日本獣医学会学術集会講演要旨集, 162nd, 2019年

ニホンザルにおけるプロジェステロン作動薬による月経周期同期化
黒澤拓斗, 黒澤拓斗, 兼子明久, 夏目尊好, 森本真弓, 愛洲星太郎, GRIS Vanessa, TAKESHITA Rafaela Sayuri, 宮部貴子, 岡本宗裕, 永野昌志, 片桐成二, 柳川洋二郎, 日本野生動物医学会大会・講演要旨集, 25th, 2019年

液体クロマトグラフィー高分解能質量分析を用いた牛卵子中遊離脂肪酸およびトリアシルグリセロールの分析　　　　　　　　　　　　　　　
植芝 滉己, 呉 ユエ, 陳 震, 坂口 謙一郎, 柳川 洋二郎, 片桐 成二, 千葉 仁志, 惠 淑萍, 永野 昌志, 日本獣医学会学術集会講演要旨集, 161回, 405, 405, 2018年08月
(公社)日本獣医学会, 日本語

LC/Orbitrap-MSを用いた牛卵子中脂質の網羅的解析(Lipidomic profiling in bovine oocytes by LC/Orbitrap-MS)　　　　　　　　　　　　　　　
呉 げつ, 植芝 滉己, 陳 震, 坂口 謙一郎, 柳川 洋二郎, 片桐 成二, 永野 昌志, 千葉 仁志, 惠 淑萍, JSBMS Letters, 43, Suppl., 105, 105, 2018年08月
(一社)日本医用マススペクトル学会, 英語

乳牛における暑熱ストレスと子宮内膜中上皮成長因子濃度異常との関係および暑熱の子宮内環境への影響評価のための子宮内温度と子宮内膜血流量の測定法の検討
河野光平, 柳川洋二郎, NATTAPONG Ninpetch, 坂口謙一郎, 菅野智裕, 菅野智裕, 鳥居佳子, 植芝滉己, 宮本祥代, 永野昌志, 片桐成二, 日本獣医学会学術集会講演要旨集, 161st, 2018年

クラスター解析の精子受胎性評価への応用:性選別および通常凍結精液中の精子運動性サブポピュレーション構成の比較
菅野智裕, 菅野智裕, 柳川洋二郎, 片桐成二, 永野昌志, 日本獣医学会学術集会講演要旨集, 161st, 2018年

ヒグマ精液の凍結保存液に添加する糖の種類および冷却速度が凍結融解後の精液性状に及ぼす影響
鳥居佳子, 菅野智裕, 蓑原悠太朗, 坂元秀行, 松本直也, 冨安洵平, 松井基純, 永野昌志, 片桐成二, 柳川洋二郎, 日本獣医学会学術集会講演要旨集, 160th, 440, 440, 2017年08月30日
(公社)日本獣医学会, 日本語

Astaxanthin improves the growth parameters, developmental competence, and quality of in vitro grown oocytes derived from bovine early antral follicles(和訳中)　　　　　　　　　　　　　　　
Abdel-Ghani Mohammed.A, 坂口 謙一郎, 菅野 智裕, 柳川 洋二郎, 片桐 成二, 永野 昌志, 日本獣医学会学術集会講演要旨集, 160回, 431, 431, 2017年08月
(公社)日本獣医学会, 英語

Mitochondrial activity and reactive oxygen species(ROS) accumulation in bovine oocytes derived from different culture system for in vitro maturation(和訳中)　　　　　　　　　　　　　　　
Purevdorj Erdenetogtokh, 柳川 洋二郎, 片桐 成二, 永野 昌志, 日本獣医学会学術集会講演要旨集, 160回, 432, 432, 2017年08月
(公社)日本獣医学会, 英語

牛卵巣の大きさが顆粒層細胞の性ステロイドホルモン産生能および卵子核成熟能に与える影響
坂口 謙一郎, 菅野 智裕, 楊 応華, 柳川 洋二郎, 片桐 成二, 永野 昌志, 北海道獣医師会雑誌, 61, 8, 300, 300, 2017年08月
(公社)北海道獣医師会, 日本語

精子運動能自動解析装置(CASA)を用いた受精能獲得精子の運動性評価
菅野智裕, 柳川洋二郎, 片桐成二, 永野昌志, 日本獣医学会学術集会講演要旨集, 160th, 2017年

ニホンザルにおける排卵誘起処置を伴う単回人工授精プログラムの検討
柳川洋二郎, 菅野智裕, 南晶子, 兼子明久, 印藤頼子, 佐藤容, 木下こづえ, 岡本宗裕, 片桐成二, 永野昌志, 日本獣医学会学術集会講演要旨集, 160th, 2017年

内視鏡を用いたヒグマ(Ursus arctos)における子宮内授精の試み
柳川洋二郎, 鳥居佳子, 菅野智裕, 田嶋彩野, 冨安洵平, 松本直也, 坂元秀行, 亀井利活, 間野勉, 片桐成二, 永野昌志, 日本野生動物医学会大会・講演要旨集, 23rd, 2017年

飼育下アフリカゾウ1個体から採取した精液の輸送後および凍結融解後の精子性状
鳥居佳子, 菅野智裕, 曽地千尋, 宗内一平, 牛尾佳名子, 吉住和規, 千田圭, 橋本渉, 永野昌志, 片桐成二, 柳川洋二郎, 日本野生動物医学会大会・講演要旨集, 23rd, 2017年

卵胞刺激ホルモンの尾椎硬膜外腔内単回投与が黒毛和種牛の体内・体外胚生産に及ぼす影響
坂口 謙一郎, 出田 篤司, 土屋 加那美, 真方 文絵, 小牧 春菜, 佐藤 正明, 酒井 伸一, 馬塲 貴大, 柳川 洋二郎, 永野 昌志, 片桐 成二, 小西 正人, 北海道獣医師会雑誌, 60, 8, 377, 377, 2016年08月
(公社)北海道獣医師会, 日本語

卵胞膜細胞と共培養した牛卵母細胞-顆粒膜細胞複合体の増殖、ステロイド産生とその後の発生能(Growth, steroidogenesis and subsequent developmental competence of bovine oocyte-granulosa cell complex co-cultured with theca cells)　　　　　　　　　　　　　　　
楊 応華, 菅野 智裕, 坂口 謙一郎, 柳川 洋二郎, 片桐 成二, 永野 昌志, 日本獣医学会学術集会講演要旨集, 159回, 428, 428, 2016年08月
(公社)日本獣医学会, 英語

牛の小型卵母細胞の発生能に対する成熟前培養時間の影響(Effect of prematuration culture duration on developmental competences of bovine small sized oocytes)　　　　　　　　　　　　　　　
Mohammed.A. Abdle-Gahni, 坂口 謙一郎, 菅野 智裕, 楊 応華, 柳川 洋二郎, 片桐 成二, 永野 昌志, 日本獣医学会学術集会講演要旨集, 159回, 429, 429, 2016年08月
(公社)日本獣医学会, 英語

牛精液の射出順序が精子運動性サブポピュレーション構成に与える影響
菅野 智裕, 柳川 洋二郎, 片桐 成二, 高橋 芳幸, 永野 昌志, 日本獣医学会学術集会講演要旨集, 159回, 430, 430, 2016年08月
(公社)日本獣医学会, 日本語

ペレット法による凍結保存が融解後のニホンザル精液の運動性に与える影響
柳川 洋二郎, 菅野 智裕, 兼子 明久, 印藤 頼子, 岡本 宗裕, 片桐 成二, 永野 昌志, 日本獣医学会学術集会講演要旨集, 159回, 431, 431, 2016年08月
(公社)日本獣医学会, 日本語

牛の卵子品質と卵巣内卵胞数の関係 体内および体外発育卵子を用いた予備的検討　　　　　　　　　　　　　　　
永井 克尚, 杉山 ちさと, 楊 応華, 柳川 洋二郎, 片桐 成二, 永野 昌志, The Journal of Reproduction and Development, 61, Suppl., j91, j91, 2015年09月
日本繁殖生物学会, 日本語

ニホンザルにおける電気刺激時の電圧が精子採取に与える影響および精子凍結保存法の改善について
杉本 幸介, 柳川 洋二郎, 菅野 智裕, 高江洲 昇, 兼子 明久, 印藤 頼子, 岡本 宗裕, 片桐 成二, 永野 昌志, 日本獣医学会学術集会講演要旨集, 158回, 381, 381, 2015年08月
(公社)日本獣医学会, 日本語

乳牛の子宮内膜における上皮成長因子濃度の周期的変化と受胎性の関係 (特集 酪農学園大学の教育・研究から)
片桐 成二, 酪農ジャーナル, 66, 1, 32, 34, 2013年01月
酪農学園大学エクステンションセンター, 日本語, 速報，短報，研究ノート等（大学，研究機関紀要）

マウス胞状卵胞由来卵母細胞の成熟培養時の基礎培地と気相中の酸素濃度が減数分裂の進行と発生能に及ぼす影響
古山 敬祐, 片桐 成二, 高橋 芳幸, The Journal of Reproduction and Development, 54, Suppl., j94, j94, 2008年08月
(一社)日本繁殖生物学会, 日本語

マウス卵子の成熟培養時の酸素濃度と培養時間が核成熟動態と発生能に及ぼす影響
檜垣彰吾, 岸昌生, 永野昌志, 片桐成二, 高橋芳幸, J Reprod Dev, 53, Supplement, J197, 2007年09月25日
日本語

乳用牛における分娩後の卵巣活動および子宮修復と子宮内膜上皮成長因子(EGF)発現の関係
片桐成二, 奥村嘉子, 永野昌志, 高橋芳幸, 日本獣医学会学術集会講演要旨集, 140th, 152, 2005年08月31日
日本語

乳用牛における卵胞発育ウェーブと子宮内膜上皮成長因子(EGF)発現の関係
片桐成二, 奥村嘉子, 永野昌志, 高橋芳幸, J Reprod Dev, 51, Supplement, J93, 2005年08月25日
日本語

In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes
AAG Adam, Y Takahashi, S Katagiri, M Nagano, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 50, 5, 579, 586, 2004年10月
To develop a reliable follicle culture system, mouse preantral follicles 150-200 am in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P < 0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro., SOCIETY REPRODUCTION & DEVELOPMENT-SRD, 英語

メンブレンインサートを用いたマウス二次卵胞の体外培養と培養卵胞から排卵された卵子の発生能
高橋 芳幸, 片桐 成二, Adam A.A.G., ほか1名, The Journal of reproduction and development, 50, 5, 579, 586, 2004年10月
マウスの二次卵胞（直径150-200μm）を卵巣から機械的に単離してメンブレンインサートあるいは微小滴中で個別に5-6日間培養したのち、培地（αMEM）にhCGを添加して排卵を誘起した。その結果、メンブレンインサートおよび微小滴中で培養した卵胞の生存率（83 vs. 77％）に有意差は認められなかった。しかし、メンブレンインサート中で培養した卵胞の方が排卵率は高かった（63 vs. 39％、P, Japanese Society of Animal Reproduction, 日本語

Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification
H Hirayama, S Kageyama, S Moriyasu, K Sawai, S Onoe, Y Takahashi, S Katagiri, K Toen, K Watanabe, T Notomi, H Yamashina, S Matsuzaki, A Minamihashi, THERIOGENOLOGY, 62, 5, 887, 896, 2004年09月
Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was < 1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application. (c) 2004 Elsevier Inc. All rights reserved., ELSEVIER SCIENCE INC, 英語

CIDR処理に併用する高用量エストロジェン製剤が乳用牛の卵胞発育動態に及ぼす影響
奥村嘉子, 片桐成二, 永野昌志, 高橋芳幸, J Reprod Dev, 50, Supplement, J81, 2004年08月25日
日本語

Effects of oxygen tension in the gas atmosphere during in vitro maturation, in vitro fertilization and in vitro culture on the efficiency of in vitro production of mouse embryos
AAG Adam, Y Takahashi, S Katagiri, M Nagano, JAPANESE JOURNAL OF VETERINARY RESEARCH, 52, 2, 77, 84, 2004年08月
Effects of oxygen (O-2) tension in the gas atmosphere during in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) on the efficiency of in vitro production of mouse embryos were examined. Mouse oocytes recovered from large antral follicles were subjected to IVM in Waymouth medium for 15, 16 and 17 hr under 5 or 20% O-2 and then subjected to IVF and IVC under 5 or 20% O-2 tension. Lowering the O-2 tension in the gas atmosphere for IVM from 20 to 5% improved the cleavage rate after IVF when the oocytes were subjected to IVM for 15 hr; however, no improvement in the cleavage rate was observed when the culture period for IVM was extended to 16 and 17 hr. Lowering the O-2 tension to 5% for IVM and IVC improved the development of the cleaved oocytes to the blastocyst stage, regardless of the culture period for IVM. However, the O-2 tension for IVF had no remarkable effect on the subsequent embryonic development. These results demonstrate that 5% O-2 is superior to 20% O-2 for IVM and IVC, and suggest that 20% O-2 for IVM may delay oocyte maturation and/or the acquisition of fertilizability and impair the developmental competence of oocytes., HOKKAIDO UNIV, 英語

血尿を主訴とした雌ウサギの子宮疾患例
永野昌志, 片桐成二, 滝口満喜, 小松智彦, 永島太郎, 井川真一, 高橋芳幸, 北海道獣医師会雑誌, 48, 8, 318, 318, 2004年08月01日
(公社)北海道獣医師会, 日本語

犬の真性半陰陽の1例
藤木亮介, 永野昌志, 小西郁男, 穴川明子, 奥村嘉子, 斉藤千寛, 片桐成二, 高橋芳幸, 北海道獣医師会雑誌, 48, 8, 318, 2004年08月01日
日本語

Changes in EGF concentrations during estrous cycle in bovine endometrium and their alterations in repeat breeder cows
S Katagiri, Y Takahashi, THERIOGENOLOGY, 62, 1-2, 103, 112, 2004年07月, ［国際誌］
The objective of the present study was to determine if abnormalities in the cyclic changes of endometrial EGF concentrations can be a diagnostic tool for repeat breeder cows. First, the profile of EGF concentrations during the estrous cycle was determined using endometrial tissues obtained from 31 Holstein cows after slaughter. Cyclic cows had two peaks of EGF concentrations. Then, endometrial tissues were obtained from 12 control and 20 repeat breeder cows by biopsy on Days 3, 7, and 14 of the same estrous cycle. Endometrial EGF concentrations in biopsied samples of the controls were similar to those found in slaughterhouse materials; they were high on Days 3 and 14 (9.2 and 10.4 ng/g tissue, respectively) and low on Day 7 (3.8 ng/g tissue). Concentrations of EGF in repeat breeder cows had a different profile; they were similar on Days 3, 7, and 14 (4.4, 3.4, and 4.0 ng/g tissue, respectively). In conclusion, changes in endometrial EGF concentrations were altered in repeat breeders; these alterations may be a potential diagnostic marker for repeat breeder cows. (C) 2003 Elsevier Inc. All rights reserved., ELSEVIER SCIENCE INC, 英語

Follicular development after ovum pick-up and fertilizability of retrieved oocytes in postpartum dairy cattle
Y Sasamoto, M Sakaguchi, M Nagano, S Katagiri, Y Takahashi, JAPANESE JOURNAL OF VETERINARY RESEARCH, 51, 3-4, 151, 159, 2004年02月
This study aimed to evaluate gonadotropin secretion and the developmental competence of follicular oocytes in dairy cattle during the early postpartum (PP) period. The number of follicles developed after transvaginal ultrasound -guided ovum pick-up (OPU) and fertilizability of retrieved oocytes were compared between cows in which the first dominant follicle (DF) ovulated (ovulated group, n = 4) and did not ovulate (non-ovulated group, n = 3), and between early PP (early PP group, n = 2) and after the resumption of the estrous cycle (cyclic group, n = 2). Follicular ablation was performed 2 - 4 days after the detection of DF in the second follicular wave PP. OPU was repeated 3 - 5 times at 3 or 4 -day intervals from 3 - 4 days after the follicular ablation. At OPU, the follicles were enumerated and all those greater than or equal to5 mm in diameter were aspirated. Recovered oocytes were subjected to in vitro maturation and fertilization. Both criteria were similar between ovulated and non-ovulated groups, and between early PP and cyclic groups. These results suggest that FSH/LH secretions required for follicle recruitment and subsequent follicular growth during the early PP period are similar to those after resumption of the estrous cycle. They also indicate that follicular oocytes during the early PP period have developmental competence., HOKKAIDO UNIV, 英語

母体の妊娠認識メカニズムと影響する要因　　　　　　　　　　　　　　　
酪農ジャーナル 臨時増刊号, 2004年

Vitrification of bovine oocytes and its application to intergeneric somatic cell nucleus transfer
EC Atabay, Y Takahashi, S Katagiri, M Nagano, A Koga, Y Kanai, THERIOGENOLOGY, 61, 1, 15, 23, 2004年01月
We determined the efficacy of a microdrop vitrification procedure for cryopreservation of bovine oocytes, using vitrified oocytes as cytoplasts for intraspecies and intergeneric somatic cell nucleus transfer (NT). In vitro matured bovine MII oocytes were vitrified in microdrops with a vitrification solution containing 35% ethylene glycol, 5% polyvinyl pyrrolidone, and 0.4 M trehalose. After warming, approximately 80% of the vitrified oocytes were morphologically normal, and their enucleation rate was similar to that of fresh oocytes. The NT embryos constructed with bovine cumulus cells and the vitrified oocytes developed similar to blastocysts constructed with fresh oocytes, although the cell number of NT blastocysts originating from vitrified oocytes was lower than that of the fresh control. In a second experiment, we examined the development of NT embryos constructed with vitrified bovine oocytes and bovine fibroblasts (intraspecies NT embryos) or swamp buffalo fibroblasts (intergeneric NT embryos). There were no differences between the intraspecies and intergeneric NT embryos in fusion, cleavage and development to blastocysts, except for lower cell numbers in the intergeneric NT blasocysts. In conclusion, the efficacy of this microdrop vitrification procedure and the production of swamp buffalo NT blastocysts using vitrified bovine oocytes was demonstrated. (C) 2003 Elsevier Inc. All rights reserved., ELSEVIER SCIENCE INC, 英語

Relationship between endometrial concentrations of epidermal growth factor (EGF) and preimplantation embryo development in dairy cattle.
S Katagiri, Y Takahashi, BIOLOGY OF REPRODUCTION, 211, 211, 2004年
SOC STUDY REPRODUCTION, 英語, 研究発表ペーパー・要旨（国際会議）

The effects of twisting and type of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up in cattle
Y Sasamoto, M Sakaguchi, S Katagiri, Y Yamada, Y Takahashi, JOURNAL OF VETERINARY MEDICAL SCIENCE, 65, 10, 1083, 1086, 2003年10月
The effects of twisting and type (single- or double-lumen) of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up (US-guided OPU) were investigated in cattle. The first study using slaughterhouse ovaries revealed that twisting of the needle during follicle aspiration improved the oocyte recovery rate without deleterious effects on the attachment of cumulus layers. Vacuum pressure affected the oocyte recovery and cumulus attachment, regardless of the needle type. The needle type did not affect the Oocyte recovery or cumulus attachment with an optimized vacuum pressure. In the second study, US-guided OPU was performed in live cows using two types of needles with a vacuum pressure of 75 mmHg. The needle type did not affect the oocyte recovery or cumulus attachment of the recovered oocytes. The results revealed that twisting of the needle is effective in follicle aspiration, and suggested that a single-lumen needle is as useful as a double-lumen needle for US-guided OPU in cattle., JAPAN SOC VET SCI, 英語

Effect of fusion･activation protocol on in vitro development of porcine nuclear transfer embryos constructed with foreign gene-transfected fetal fibroblast
MAM Diaz, T Mori, M Nagano, S Katagiri, Y Takahashi, J Vet Med Sci, 65, 9, 989, 994, 2003年09月
The effect of fusion/activation protocol on in vitro development of porcine nuclear transfer (NT) embryos constructed with foreign gene-transfected somatic cells were investigated. NT embryos were produced by using enucleated M 11 oocytes and enhanced green fluorescence protein (EGFP) gene-transfected or non-transfected porcine fetal fibroblasts. One group of NT embryos received a single electrical pulse to induce fusion and activation simultaneously (FAS). The other group was fused 2 hr before activation (FBA) using two kinds of electrical pulses. Electrically activated NT embryos in both groups were treated with cycloheximide (CHX) before culture to assess the development to the blastocyst stage. After 6 days of culture, all morulae and blastocysts derived from EGFP-transfected fibroblasts emitted green fluorescence without mosaicism, and EGFP-gene product was also detected in all morulae and blastocysts examined. NT embryos undergoing FAS showed higher developmental capacity to blastocysts than those undergoing FBA, regardless of the EGFP transfection into the nuclear donor cells. The results also indicated that EGFP-gene transfection into nuclear donor cells has no obvious deleterious effect on the development of NT embryos to blastocysts., JAPAN SOC VET SCI, 英語

分娩後のウシ子宮内膜上皮におけるPAGファミリーの遺伝子発現について　　　　　　　　　　　　　　　
橋爪 一善, 石渡 広子, 坂口 実, 片桐 成二, 木崎 景一郎, 今井 敬, 高橋 透, 平澤 明, 塩島 聡, 勝間 進, 辻本 豪三, 日本畜産学会大会講演要旨集, 101回, 86, 86, 2003年03月
(公社)日本畜産学会, 日本語

Effect of activation treatments of recipient oocytes on subsequent development of bovine nuclear transfer embryos
EC Atabay, S Katagiri, M Nagano, Y Takahashi, JAPANESE JOURNAL OF VETERINARY RESEARCH, 50, 4, 185, 194, 2003年02月
Effects of recipient oocyte activation methods on the development of nuclear transfer (NT) embryos were investigated. In Exp. 1, cell-cycle phase of serum-starved bovine cumulus cells was examined by flow cytometry. Majority (95.5%) of medium-sized (16-20 mum) cells that made up 56% of total cells was at the G(0)/G(1) phase. NT embryos were constructed by electric fusion with the medium-sized serum-starved cumulus cells and bovine oocytes of 3 different preparations: enucleated oocytes treated with calcium ionophore A 23187 for 5 min and cycloheximide for 5 hr (A 23187/CHX), those treated with ethanol for 7 min and cycloheximide for 2 hr (ethanol/CHX) and those without treatment. In Exp. 2 and 3, developmental competence of NT embryos constructed with A 23187/CHX- and ethanol/CHX-treated oocytes was compared to that of NT embryos constructed with non-treated oocytes, respectively. Further, nuclear behavior in 3 different NT embryos was examined in Exp. 4. Within 1 hr after fusion, majority of the NT embryos constructed with non-treated oocytes showed condensed chromosome. Three hours after fusion, about 50% of NT embryos constructed with non-treated or ethanol/CHX-treated oocytes showed a single pronucleus-like structure. NT embryos constructed with ethanol/CHX-treated oocytes showed similar rates of fusion, cleavage and blastocyst formation to those of the non-treated oocytes. In contrast, NT embryos constructed with A 23187/CHX-treated oocytes did not show any pronucleus-like structure and showed lower cleavage rate and no development to blastocysts. The results indicate that ethanol/CHX-treated oocytes could support development of somatic cell NT embryos to the blastocyst stage at a similar rate to that of non-treated oocytes., HOKKAIDO UNIV, 英語

Changes in sexual behavior and fecal steroid hormone concentrations during the breeding season in female Hokkaido brown bears (Ursus arctos yesoensis) under captive condition
A Ishikawa, H Sakamoto, S Katagiri, Y Takahashi, JOURNAL OF VETERINARY MEDICAL SCIENCE, 65, 1, 99, 102, 2003年01月
The longitudinal changes in fecal steroid hormone concentrations and sexual behavior in 2 mated/pregnant and 3 non-mated female Hokkaido brown bears were investigated during the breeding season. Behavioral estrus (standing) lasted for 14 and 32 days in the mated females and for 25 to 36 days in the non-mated females. In non-mated females, sexual behavior, such as female-female mounting and masturbation, was observed for several days before and after the estrous period. In mated females, mean fecal estradiol-17beta concentrations were higher in the estrous period than in the post-estrous period, while fecal progesterone concentrations were higher in the post-estrous period than in the estrous period. The similar trends of steroid hormone changes were observed in the non-mated females., JAPAN SOC VET SCI, 英語

連続ちつ温測定による乳牛の分娩時刻予知
関塚次郎, 高橋芳幸, 片桐成二, 永野昌志, 坂口実, 山田豊, 中辻浩喜, 北海道獣医師会雑誌, 46, 8, 239, 2002年08月01日
日本語

Efficiency of fecal steroid hormone measurement for assessing reproductive function in the Hokkaido brown bear (Ursus arctos yesoensis)
A Ishikawa, S Kikuchi, S Katagiri, H Sakamoto, Y Takahashi, JAPANESE JOURNAL OF VETERINARY RESEARCH, 50, 1, 17, 27, 2002年05月
The present study aimed to establish simple systems for measuring fecal steroid hormones in order to monitor the reproductive profiles of captive Hokkaido brown bears. The efficiency of fecal sample processing at the steps of dehydration and extraction and the correlation between steroid concentrations in matched fecal and blood samples were studied. Then, monthly changes in fecal estradiol-17beta and progesterone in female bears, and testosterone in male bears were examined. The procedure was finalized as follows. Fecal samples were dried at 100degreesC for 3 hr and extracted with diethyl ether. The diethyl ether in the extracts was evaporated and residues were reconstituted in ethanol for the assays. Hormone concentrations were quantified using enzyme immunoassays. Concentrations of progesterone and testosterone in fecal and plasma samples were correlated in the systems. The changes in fecal progesterone and testosterone concentrations were similar to those in serum concentrations of bears as reported previously. In contrast, fecal estradiol concentrations did not correlate with plasma levels probably because of the time lag in excretion. However, the changes in estradiol-17beta concentrations in feces in the present study were similar to those reported in serum. In conclusion, fecal progesterone and testosterone assay systems appear practical for monitoring ovarian and testicular activities without immobilization, though methodological improvements and further validation may be required. For the fecal estradiol-17beta assay, there is a need to solve the problem of excretion time lag before the system can be used in the study of reproductive physiology., HOKKAIDO UNIV, 英語

Efficiency of fecal steroid hormone measurement for assessing reproductive function in the Hokkaido brown bear (Ursus arctos yesoensis)
A Ishikawa, S Kikuchi, S Katagiri, H Sakamoto, Y Takahashi, JAPANESE JOURNAL OF VETERINARY RESEARCH, 50, 1, 17, 27, 2002年05月
The present study aimed to establish simple systems for measuring fecal steroid hormones in order to monitor the reproductive profiles of captive Hokkaido brown bears. The efficiency of fecal sample processing at the steps of dehydration and extraction and the correlation between steroid concentrations in matched fecal and blood samples were studied. Then, monthly changes in fecal estradiol-17beta and progesterone in female bears, and testosterone in male bears were examined. The procedure was finalized as follows. Fecal samples were dried at 100degreesC for 3 hr and extracted with diethyl ether. The diethyl ether in the extracts was evaporated and residues were reconstituted in ethanol for the assays. Hormone concentrations were quantified using enzyme immunoassays. Concentrations of progesterone and testosterone in fecal and plasma samples were correlated in the systems. The changes in fecal progesterone and testosterone concentrations were similar to those in serum concentrations of bears as reported previously. In contrast, fecal estradiol concentrations did not correlate with plasma levels probably because of the time lag in excretion. However, the changes in estradiol-17beta concentrations in feces in the present study were similar to those reported in serum. In conclusion, fecal progesterone and testosterone assay systems appear practical for monitoring ovarian and testicular activities without immobilization, though methodological improvements and further validation may be required. For the fecal estradiol-17beta assay, there is a need to solve the problem of excretion time lag before the system can be used in the study of reproductive physiology., HOKKAIDO UNIV, 英語

Cryopreservation of the semen collected by electroejaculation from the Hokkaido brown bear (Ursus arctos yesoensis)
A Ishikawa, M Matsui, H Sakamoto, S Katagiri, Y Takahashi, JOURNAL OF VETERINARY MEDICAL SCIENCE, 64, 4, 373, 376, 2002年04月
Four adult Hokkaido brown bears were used as semen donors, and semen characteristics were examined before freezing and after thawing. A total of 10 electroejaculates were diluted with Tris-egg yolk extender and cooled to 4degreesC over 90 min. Spermatozoa were equilibrated with 4.7% glycerol for 80 min. Semen packed in 0.2-5 ml plastic straws were frozen with liquid nitrogen vapor, Percentages (mean +/- SD) of motile and live sperm were 96 +/- 2 and 86.5 +/- 7.2% before freezing, and 43 +/- 5 and 67.4 +/- 3.9% after thawing, respectively. Although the number of progressively motile sperm after thawing varied among samples (1.8 +/- 1.2 x 10(8) cells/ejaculate), frozen semen in the present study might serve for artificial insemination., JAPAN SOC VET SCI, 英語

Birth of pups after transfer of mouse embryos derived from vitrified preantral follicles
EC dela Pena, Y Takahashi, S Katagiri, EC Atabay, M Nagano, REPRODUCTION, 123, 4, 593, 600, 2002年04月
Preantral follicles mechanically isolated from the ovaries of 12-day-old mice were exposed to 2 mol ethylene glycol l(-1) for 2 or 5 min and then to a vitrification solution containing 6 mol ethylene glycol l(-1) and 0.3 mol raffinose l(-1) for 0.5, 1.0 or 2.0 min before vitrification. The vitrified and fresh preantral follicles were treated with collagenase, and the oocyte-granulosa cell complexes (OGCs) obtained were cultured in vitro for 10 days in membrane inserts. Preantral follicles exposed to 2 mol ethylene glycol l(-1) for 5 min and then to the vitrification solution for 0.5 or 1.0 min showed the highest survival rates after warming. The follicular loss after warming was approximately 20%. After in vitro culture, the proportion of viable OGCs from the vitrified follicles was 10% lower than that of the fresh preantral follicles. There were no differences in the rates of maturation, fertilization and subsequent development to blastocysts between the oocytes derived from vitrified follicles and those derived from fresh preantral follicles; however, the developmental competence of the oocytes derived from both vitrified and fresh preantral follicles grown in vitro was lower than that of oocytes grown in vivo. One of the five recipient mice that received 20 blastocysts derived from vitrified preantral follicles gave birth to six live pups. The results of the present study demonstrate for the first time that mouse preantral follicles can be vitrified and that some of the embryos derived from vitrified preantral follicles can develop to live pups., SOC REPRODUCTION FERTILITY, 英語

Cryopreservation of the semen collected by electroejaculation from the Hokkaido brown bear (Ursus arctos yesoensis)
A Ishikawa, M Matsui, H Sakamoto, S Katagiri, Y Takahashi, JOURNAL OF VETERINARY MEDICAL SCIENCE, 64, 4, 373, 376, 2002年04月
Four adult Hokkaido brown bears were used as semen donors, and semen characteristics were examined before freezing and after thawing. A total of 10 electroejaculates were diluted with Tris-egg yolk extender and cooled to 4degreesC over 90 min. Spermatozoa were equilibrated with 4.7% glycerol for 80 min. Semen packed in 0.2-5 ml plastic straws were frozen with liquid nitrogen vapor, Percentages (mean +/- SD) of motile and live sperm were 96 +/- 2 and 86.5 +/- 7.2% before freezing, and 43 +/- 5 and 67.4 +/- 3.9% after thawing, respectively. Although the number of progressively motile sperm after thawing varied among samples (1.8 +/- 1.2 x 10(8) cells/ejaculate), frozen semen in the present study might serve for artificial insemination., JAPAN SOC VET SCI, 英語

Birth of pups after transfer of mouse embryos derived from vitrified preantral follicles
EC dela Pena, Y Takahashi, S Katagiri, EC Atabay, M Nagano, REPRODUCTION, 123, 4, 593, 600, 2002年04月
Preantral follicles mechanically isolated from the ovaries of 12-day-old mice were exposed to 2 mol ethylene glycol l(-1) for 2 or 5 min and then to a vitrification solution containing 6 mol ethylene glycol l(-1) and 0.3 mol raffinose l(-1) for 0.5, 1.0 or 2.0 min before vitrification. The vitrified and fresh preantral follicles were treated with collagenase, and the oocyte-granulosa cell complexes (OGCs) obtained were cultured in vitro for 10 days in membrane inserts. Preantral follicles exposed to 2 mol ethylene glycol l(-1) for 5 min and then to the vitrification solution for 0.5 or 1.0 min showed the highest survival rates after warming. The follicular loss after warming was approximately 20%. After in vitro culture, the proportion of viable OGCs from the vitrified follicles was 10% lower than that of the fresh preantral follicles. There were no differences in the rates of maturation, fertilization and subsequent development to blastocysts between the oocytes derived from vitrified follicles and those derived from fresh preantral follicles; however, the developmental competence of the oocytes derived from both vitrified and fresh preantral follicles grown in vitro was lower than that of oocytes grown in vivo. One of the five recipient mice that received 20 blastocysts derived from vitrified preantral follicles gave birth to six live pups. The results of the present study demonstrate for the first time that mouse preantral follicles can be vitrified and that some of the embryos derived from vitrified preantral follicles can develop to live pups., SOC REPRODUCTION FERTILITY, 英語

獣医繁殖学マニュアル　　　　　　　　　　　　　　　
文永堂出版, 59-61, 2002年

Thatcher WW, Moreira F, Santos J, Mattos R. Novel new concepts for reproductive management and strategies to improve reproductive efficiency in dary cattle（乳牛の新しい繁殖管理法と繁殖効率改善の戦略、訳 片桐成二）　　　　　　　　　　　　　　　
日本獣医師会雑誌, 55:203-207, 2002年

Soliman M, Ishioka K, Yoshida R, Komabayashi K, Hatai H, Matsui Y, Hirai T, Katagiri S, Takahashi Y, Kawakita Y, Abe H, Kitamura H, Kimura K, Saito M. Serum leptin levels during the periparturient period in cows.　　　　　　　　　　　　　　　
J Vet Med Sci, 64:1053-1056, 2002年

Kobayashi A, Katagiri S, Kimura T, Ochiai K, Umemura T. Steroid hormones do not reactivate Neospora caninum in ovariectomized mice.　　　　　　　　　　　　　　　
J Vet Med Sci, 64:773-777, 2002年

Soliman M, Ishioka K, Yoshida R, Komabayashi K, Hatai H, Matsui Y, Hirai T, Katagiri S, Takahashi Y, Kawakita Y, Abe H, Kitamura H, Kimura K, Saito M. Serum leptin levels during the periparturient period in cows.　　　　　　　　　　　　　　　
J Vet Med Sci, 64:1053-1056, 2002年

Kobayashi A, Katagiri S, Kimura T, Ochiai K, Umemura T. Steroid hormones do not reactivate Neospora caninum in ovariectomized mice.　　　　　　　　　　　　　　　
J Vet Med Sci, 64:773-777, 2002年

乳牛における分娩後の卵胞発育と卵胞内卵子の受精能
笹本良彦, 坂口実, 山田豊, 永野昌志, 片桐成二, 高橋芳幸, 日本獣医学会学術集会講演要旨集, 132nd, 228, 2001年09月07日
日本語

マウスの卵子,はいおよび胎子臓器における脱共役タンパクの発現
古畑ちひろ, 片桐成二, 高橋芳幸, 永野昌志, 木村和弘, 斉藤昌之, 日本不妊学会雑誌, 46, 3, 183, 2001年07月01日
日本語

ウシ子宮膜におけるプロスタグランジン(PG)産生における上皮成長因子(EGF)の役割 単離培養細胞および組織片を用いた検討
福井秀樹, 片桐成二, 高橋芳幸, 永野昌志, 日本不妊学会雑誌, 46, 3, 182-183, 2001年07月01日
日本語

In vitro growth of mouse ovarian preantral follicles and the capacity of their oocytes to develop to the blastocyst stage
C Bishonga, Y Takahashi, S Katagiri, M Nagano, A Ishikawa, JOURNAL OF VETERINARY MEDICAL SCIENCE, 63, 6, 619, 624, 2001年06月
TWO groups Of mouse preantral follicles with diameters of 125-150 and 151-175 mum were cultured individually for 6 days in a medium supplemented with FSH and fetal calf serum to determine their in vitro growth characteristics. Their oocyte capacity for maturation and development to the blastocyst stage following in vitro fertilization was also assessed. Antral formation rare at the end of culture was higher in the Follicles of 151-175 mum (89%) than 125-150 mum (76%). The timing of antrum formation was different between the two follicle categories: most 151-175 m follicles formed antra earlier than 125-150 mum follicles (days 1 and 5 vs. 5 and 6). However. follicle diameters at the time of antrum formation were the same regardless of the initial size and the culture period. Maturation rates of the oocytes derived from both categories of in vitro grown follicles (70 and 62%) were not different from those of oocytes from in vivo grown follicles (74%). The in vitro derived oocytes, however. showed less cleavage (30 and 35%) than the in vivo derived oocytes(89%). Although the oocytes from both follicle categories developed to the morula stage after in vitro fertilization. blastocysts were only obtained from oocytes derived from the 151-175 mum category. These results demonstrate that an individual follicle culture system using a medium with FSH and fetal calf serum supports in vitro growth of mouse preantral follicles with diameters of 151-175 mum to the preovulatory stage, and that their oocytes have the capability to develop to the blastocyst stage., JAPAN SOC VET SCI, 英語

In vitro growth of mouse ovarian preantral follicles and the capacity of their oocytes to develop to the blastocyst stage
C Bishonga, Y Takahashi, S Katagiri, M Nagano, A Ishikawa, JOURNAL OF VETERINARY MEDICAL SCIENCE, 63, 6, 619, 624, 2001年06月
TWO groups Of mouse preantral follicles with diameters of 125-150 and 151-175 mum were cultured individually for 6 days in a medium supplemented with FSH and fetal calf serum to determine their in vitro growth characteristics. Their oocyte capacity for maturation and development to the blastocyst stage following in vitro fertilization was also assessed. Antral formation rare at the end of culture was higher in the Follicles of 151-175 mum (89%) than 125-150 mum (76%). The timing of antrum formation was different between the two follicle categories: most 151-175 m follicles formed antra earlier than 125-150 mum follicles (days 1 and 5 vs. 5 and 6). However. follicle diameters at the time of antrum formation were the same regardless of the initial size and the culture period. Maturation rates of the oocytes derived from both categories of in vitro grown follicles (70 and 62%) were not different from those of oocytes from in vivo grown follicles (74%). The in vitro derived oocytes, however. showed less cleavage (30 and 35%) than the in vivo derived oocytes(89%). Although the oocytes from both follicle categories developed to the morula stage after in vitro fertilization. blastocysts were only obtained from oocytes derived from the 151-175 mum category. These results demonstrate that an individual follicle culture system using a medium with FSH and fetal calf serum supports in vitro growth of mouse preantral follicles with diameters of 151-175 mum to the preovulatory stage, and that their oocytes have the capability to develop to the blastocyst stage., JAPAN SOC VET SCI, 英語

Vitrification of mouse oocytes in ethylene glycol-raffinose solution: Effects of preexposure to ethylene glycol or raffinose on oocyte viability
EC dela Pena, Y Takahashi, EC Atabay, S Katagiri, M Nagano, CRYOBIOLOGY, 42, 2, 103, 111, 2001年03月
We investigated the effects of preexposure to ethylene glycol (EG) or raffinose on the viability of vitrified mouse oocytes. Ovulated oocytes at the metaphase II stage were preexposed either to 2 M EG for 0, 2, or 5 min or to ascending concentrations (0.15 followed by 0.3 M) of raffinose solution for 2, 5, or 10 min each (here referred to as 2-2,5-5, and 10-10 min, respectively). The oocytes were then exposed to a vitrification solution (VS), 6 M EG + 0.3 M raffinose, for 0.5, 1, 2, or 5 min and then vitrified or immediately diluted. After warming, the developmental capacity of oocytes was determined after irt vitro fertilization. Volume changes in oocytes during preexposures and exposure to the VS were also investigated. The results demonstrated that preexposure to 2 M EG allowed shorter exposure times of oocytes to the VS and that predehydration in raffinose solutions for 5-5, but not 2-2 or 10-10 min, allowed a wider range of exposure times to the VS. Experiments on volume change suggested that the optimum time of exposure to the VS depends on the amount of EG permeation after preexposure to 2 M EG or to raffinose solutions. Preexposures to 2 M EG or raffinose under optimized conditions increased thc viability of vitrified-warmed oocytes compared to direct exposure to VS without preexposures. (C) 2001 Academic Press., ACADEMIC PRESS INC, 英語

Vitrification of mouse oocytes in ethylene glycol-raffinose solution: Effects of preexposure to ethylene glycol or raffinose on oocyte viability
EC dela Pena, Y Takahashi, EC Atabay, S Katagiri, M Nagano, CRYOBIOLOGY, 42, 2, 103, 111, 2001年03月
We investigated the effects of preexposure to ethylene glycol (EG) or raffinose on the viability of vitrified mouse oocytes. Ovulated oocytes at the metaphase II stage were preexposed either to 2 M EG for 0, 2, or 5 min or to ascending concentrations (0.15 followed by 0.3 M) of raffinose solution for 2, 5, or 10 min each (here referred to as 2-2,5-5, and 10-10 min, respectively). The oocytes were then exposed to a vitrification solution (VS), 6 M EG + 0.3 M raffinose, for 0.5, 1, 2, or 5 min and then vitrified or immediately diluted. After warming, the developmental capacity of oocytes was determined after irt vitro fertilization. Volume changes in oocytes during preexposures and exposure to the VS were also investigated. The results demonstrated that preexposure to 2 M EG allowed shorter exposure times of oocytes to the VS and that predehydration in raffinose solutions for 5-5, but not 2-2 or 10-10 min, allowed a wider range of exposure times to the VS. Experiments on volume change suggested that the optimum time of exposure to the VS depends on the amount of EG permeation after preexposure to 2 M EG or to raffinose solutions. Preexposures to 2 M EG or raffinose under optimized conditions increased thc viability of vitrified-warmed oocytes compared to direct exposure to VS without preexposures. (C) 2001 Academic Press., ACADEMIC PRESS INC, 英語

Relationship among growth, steroid production and immunolocalization of transforming growth factor-beta 1 in the normally developing mouse follicles cultured in vitro
C Bishonga, Y Takahashi, S Katagiri, M Nagano, A Ishikawa, JAPANESE JOURNAL OF VETERINARY RESEARCH, 48, 4, 169, 176, 2001年02月
This study examined the relationship among growth, steroid production and transforming growth factor-beta1 (TGF-beta1) immunolocalization in the mouse follicles cultured in vitro to evaluate the hypothesis that normally developing follicles should express TGF-beta1 in the granulosa cells around the time of antrum formation. Preantral follicles with 151-175 mum (large category) and 125 -150 mum (small category) of initial diameters were used as models for normal and retarded follicles, respectively. Growth rate and timing of antrum formation in both categories were comparable to those of in-vivo grown follicles. At the time of antrum formation, follicular diameters were similar between the two follicle categories; however, antral follicles from the large category showed larger number of granulosa cells, higher estradiol production and proportion of follicles with TGF-beta1 positive granulosa cells. Two days after antrum formation, there were no differences in the number of granulosa cells and the proportions of follicles with TGF-beta1 positive granulosa or theca cells between the two categories. Temporal association in large follicles between the increase in estradiol production and proportion of follicles with TGF-beta1 positive granulosa cells at the time of antrum formation supports our hypothesis. Furthermore, this study demonstrated the usefulness of the follicle culture system in the investigations of follicular physiology., HOKKAIDO UNIV, 英語

Heath SE. 災害発生に伴う動物の救護について (訳 片桐成二)　　　　　　　　　　　　　　　
北海道獣医師会雑誌, 45:15-18, 2001年

Wolfgang Jochle. End user attitude toward animal welfare and the environmental issues involved（動物福祉とそれに関連する環境問題に対する消費者の姿勢、訳 片桐成二)　　　　　　　　　　　　　　　
臨床獣医, 19:43-47, 2001年

片桐成二. 卵子形成と成熟、獣医繁殖学 第2版（森純一、金川弘司、浜名克己 編）　　　　　　　　　　　　　　　
永堂出版, 45-56, 2001年

片桐成二. 暑熱ストレス環境下における繁殖成績向上のためのマネージメント　　　　　　　　　　　　　　　
臨床獣医, 19: 30-34, 2001年

Bishonga C, Takahashi Y, Katagiri S, Nagano M. Immunolocalization of transforming growth factor-β1 in the ovarian follicular compartments of the adult mouse at diestrus, proestrus and after treatment with human chorionic gonadotropin.
Christopher Bishonga, Yoshiyuki Takahashi, Seiji Katagiri, Masashi Nagano, J Reprod Dev, 47, 2, 91, 96, 2001年
This study determined the immunolocalization of transforming growth factor-β1 (TGF-β1) in the ovarian follicles of the adult mouse at diestrus, proestrus and estrus (10 and 12.5 h after treatment with hCG). The majority (≥72%) of the oocytes were stained positive in type 3b through to type 8 follicles. TGF-β1 staining in the granulosa and theca cells was first observed in type 5b follicles. The percentage of follicles with positively stained granulosa cells increased with follicular development from type 6 to 8 (13 to 82%). The percentage of follicles with positively stained theca cells did not show a clear trend (∼30%) up to 10 h after hCG treatment, but increased in type 7 (79%) and unovulated type 8 (55%) follicles during the ovulation period (12.5 h after hCG). These results indicate that TGF-β1 is involved in follicular development and differentiation., The Japanese Society of Animal Reproduction (JSAR), 英語

Bishonga C, Takahashi Y, Katagiri S, Nagano M, Ishikawa A. Relationship among growth, steroid production and immunolocalization of transforming growth factor-β1 in the normally developing mouse follicles cultured in vitro.　　　　　　　　　　　　　　　
Jpn J Vet Res, 48: 169-176, 2001年

Immunolocalization of transforming growth factor-β1 in the ovarian follicular compartments of the adult mouse at diestrus, proestrus and after treatment with human chorionic gonadotropin
Christopher Bishonga, Yoshiyuki Takahashi, Seiji Katagiri, Masashi Nagano, Journal of Reproduction and Development, 47, 2, 91, 96, 2001年
This study determined the immunolocalization of transforming growth factor-β1 (TGF-β1) in the ovarian follicles of the adult mouse at diestrus, proestrus and estrus (10 and 12.5 h after treatment with hCG). The majority (≥72%) of the oocytes were stained positive in type 3b through to type 8 follicles. TGF-β1 staining in the granulosa and theca cells was first observed in type 5b follicles. The percentage of follicles with positively stained granulosa cells increased with follicular development from type 6 to 8 (13 to 82%). The percentage of follicles with positively stained theca cells did not show a clear trend (∼30%) up to 10 h after hCG treatment, but increased in type 7 (79%) and unovulated type 8 (55%) follicles during the ovulation period (12.5 h after hCG). These results indicate that TGF-β1 is involved in follicular development and differentiation., The Japanese Society of Animal Reproduction (JSAR), 英語

ウシの体細胞核移植におけるドナー細胞とレシピエント卵子の細胞周期が再構築はいの発生および初期化に及ぼす影響
高橋芳幸, 片桐成二, 永野昌志, NOUR M S M, 食肉に関する助成研究調査成果報告書, 18, 11-15, 2000年12月
日本語

ウシの体外受精 廃用決定された優良雌牛からのはい生産例
永野昌志, 高橋芳幸, 片桐成二, 竹下章, 森好政晴, 横田修, 北海道獣医師会雑誌, 44, 7, 196, 2000年07月01日
日本語

ウシ子宮内膜間質細胞にとけるプロスタグランジンの合成制御 プロジェストロン,エストロジェンおよび上皮成長因子の役割
山下傑夫, 片桐成二, 永野昌志, 高橋芳幸, 日本不妊学会雑誌, 45, 2, 165, 2000年04月01日
日本語

ウシ一次卵胞の発育能の検討
山本直人, 永野昌志, 片桐成二, 高橋芳幸, 日本不妊学会雑誌, 45, 2, 165, 2000年04月01日
日本語

初期胞状卵胞由来ウシ卵母細胞の体外発育および成熟
猪川真紀, 永野昌志, 片桐成二, 高橋芳幸, 日本不妊学会雑誌, 45, 2, 165, 2000年04月01日
日本語

Cheong HT, Ikeda K, Martinez Diaz MA., Katagiri S and Takahashi Y. Development of reconstituted pig embryos by nuclear transfer of cultured cumulus cells.　　　　　　　　　　　　　　　
Reprod Fertil Dev, 12: 15-20, 2000年

dela Pena EC、Atabay EC、永野昌志、片桐成二、高橋芳幸. エチレングリコールとラフィノースを用いたマウス卵子のガラス化保存における前処理の効果　　　　　　　　　　　　　　　
北海道牛受精卵移植研究会会報, 19: 31-34, 2000年

永野昌志、猪川真紀、山本直人、片桐成二、高橋芳幸. ウシの初期胞状卵胞および一次卵胞の体外培養　　　　　　　　　　　　　　　
北海道牛受精卵移植研究会会報, 19: 8-13, 2000年

Effect of medium change on the development of in vitro matured and fertilized bovine oocytes cultured in medium containing amino acids
K Ikeda, Y Takahashi, S Katagiri, JOURNAL OF VETERINARY MEDICAL SCIENCE, 62, 1, 121, 123, 2000年01月
Bovine in vitro matured and fertilized oocytes were cultured for 153 hr in groups of 3 or 30 in 30 mu l of modified synthetic oviduct fluid medium supplemented with amino acids. The concentration of ammonium in culture medium at 153 hr of culture was significantly decreased by medium change at 72 hr of culture. However, regardless of embryo density, medium change had no beneficial or detrimental effect on the development of bovine embryos. Increase in the development to blastocysts and production of ammonium were observed when embryos were cultured in groups of 30, These results indicated that the ammonium concentration detected in this culture system has a negligible effect on the development of bovine embryos to blastocysts., JAPAN SOC VET SCI, 英語

Cheong HT, Ikeda K, Martinez Diaz MA., Katagiri S and Takahashi Y. Development of reconstituted pig embryos by nuclear transfer of cultured cumulus cells.　　　　　　　　　　　　　　　
Reprod Fertil Dev, 12: 15-20, 2000年

Effect of medium change on the development of in vitro matured and fertilized bovine oocytes cultured in medium containing amino acids
K Ikeda, Y Takahashi, S Katagiri, JOURNAL OF VETERINARY MEDICAL SCIENCE, 62, 1, 121, 123, 2000年01月
Bovine in vitro matured and fertilized oocytes were cultured for 153 hr in groups of 3 or 30 in 30 mu l of modified synthetic oviduct fluid medium supplemented with amino acids. The concentration of ammonium in culture medium at 153 hr of culture was significantly decreased by medium change at 72 hr of culture. However, regardless of embryo density, medium change had no beneficial or detrimental effect on the development of bovine embryos. Increase in the development to blastocysts and production of ammonium were observed when embryos were cultured in groups of 30, These results indicated that the ammonium concentration detected in this culture system has a negligible effect on the development of bovine embryos to blastocysts., JAPAN SOC VET SCI, 英語

In vitro fertilization and cortical granule distribution of bovine oocytes having heterogeneous ooplasm with dark clusters
M Nagano, Y Takahashi, S Katagiri, JOURNAL OF VETERINARY MEDICAL SCIENCE, 61, 5, 531, 535, 1999年05月
In vitro maturation, fertilization and subsequent development of oocytes with homogeneous (category 1), or heterogeneous ooplasm (category 2) were investigated. No significant differences were observed in the nuclear maturation and total fertilization rates between the two categories. However, category 2 oocytes showed a higher normal fertilization rate due to their lower incidence of polyspermy as compared to category 1 oocytes. Electron microscopic study revealed that all category 2 oocytes had cortical granules lined up next to the plasma membrane, and that some category 1 oocytes still had small clusters of cortical granules after maturation. Although the proportion of cleaved zygotes was higher in category 2, the percentages of cleaved zygotes that developed to the blastocyst stage did not differ between the two categories. These results demonstrate that oocytes with heterogeneous ooplasm have a higher capacity for normal fertilization due to the reduction in polyspermy. This can be attributed to the normal distribution of cortical granules in category 2 oocytes after maturation., JAPAN SOC VET SCI, 英語

In vitro fertilization and cortical granule distribution of bovine oocytes having heterogeneous ooplasm with dark clusters
M Nagano, Y Takahashi, S Katagiri, JOURNAL OF VETERINARY MEDICAL SCIENCE, 61, 5, 531, 535, 1999年05月
In vitro maturation, fertilization and subsequent development of oocytes with homogeneous (category 1), or heterogeneous ooplasm (category 2) were investigated. No significant differences were observed in the nuclear maturation and total fertilization rates between the two categories. However, category 2 oocytes showed a higher normal fertilization rate due to their lower incidence of polyspermy as compared to category 1 oocytes. Electron microscopic study revealed that all category 2 oocytes had cortical granules lined up next to the plasma membrane, and that some category 1 oocytes still had small clusters of cortical granules after maturation. Although the proportion of cleaved zygotes was higher in category 2, the percentages of cleaved zygotes that developed to the blastocyst stage did not differ between the two categories. These results demonstrate that oocytes with heterogeneous ooplasm have a higher capacity for normal fertilization due to the reduction in polyspermy. This can be attributed to the normal distribution of cortical granules in category 2 oocytes after maturation., JAPAN SOC VET SCI, 英語

笹本良彦、片桐成二、高橋芳幸. ウシの超音波誘導経腟採卵において卵胞吸引時の吸引針の回転と吸引針の違いが卵子回収成績に及ぼす影響　　　　　　　　　　　　　　　
北海道牛受精卵移植研究会会報, 18: 19-22, 1999年

The efficacy of the water purification system with an ultra violet lamp and ultrafilter for the preparation of bovine embryo culture media
Masashi Nagano, Yoshiyuki Takahashi, Seiji Katagiri, Journal of Reproduction and Development, 45, 3, 239, 242, 1999年
The efficacy of a water purification system including a high-intensity 185 nm ultra violet lamp with ultrafilter (UV-UF) for eliminating endotoxins was examined. The activity of endotoxins was below the detectable level in UV-UF water. In contrast, ultra-purified water from a system without a UV lamp (UP) showed a high level of endotoxins, despite water resistivity of more than 18 MQ-cm. Bovine IVM/IVF zygotes were cultured for 174 h in protein-free media prepared with UV-UF and UP water. The developmental rate to blastocysts at 150 h was significantly higher in the medium prepared with UV-UF water. The results demonstrate that the UV-UF system is beneficial for eliminating endotoxins from ultra-purified water, and for preparing protein-free media for culture of bovine embryos., The Japanese Society of Animal Reproduction (JSAR), 英語

The efficacy of the water purification system with an ultra violet lamp and ultrafilter for the preparation of bovine embryo culture media
Masashi Nagano, Yoshiyuki Takahashi, Seiji Katagiri, Journal of Reproduction and Development, 45, 3, 239, 242, 1999年
The efficacy of a water purification system including a high-intensity 185 nm ultra violet lamp with ultrafilter (UV-UF) for eliminating endotoxins was examined. The activity of endotoxins was below the detectable level in UV-UF water. In contrast, ultra-purified water from a system without a UV lamp (UP) showed a high level of endotoxins, despite water resistivity of more than 18 MQ-cm. Bovine IVM/IVF zygotes were cultured for 174 h in protein-free media prepared with UV-UF and UP water. The developmental rate to blastocysts at 150 h was significantly higher in the medium prepared with UV-UF water. The results demonstrate that the UV-UF system is beneficial for eliminating endotoxins from ultra-purified water, and for preparing protein-free media for culture of bovine embryos., The Japanese Society of Animal Reproduction (JSAR), 英語

A delayed-implantation-associated protein prevents resumption of DNA synthesis by the trophectoderm of delayed-implanting mouse blastocysts in vitro
S Katagiri, Y Takahashi, H Kanagawa, BH Yuen, YS Moon, JOURNAL OF VETERINARY MEDICAL SCIENCE, 60, 7, 791, 794, 1998年07月
Objective of this study was to determine the ability of a delayed-implantation-associated protein (MW 170,000, DIAP170K) to inhibit DNA synthesis by mouse blastocysts. Mice were ovariectomized on day 3 of pregnancy and treated with daily injections with I mg progesterone till day 7 to induce delayed implantation. Blastocysts were collected on day 8 with or without a single injection of 25 ng estradiol-17 beta on day 7 that activates blastocyst metabolisms (activated blastocysts and delayed-implanting blastocysts respectively). DNA synthesis was determined by measuring [H-3]thymidine incorportion by blastocysts. DIAP170K at 10 mu g/ml suppressed resumption of DNA synthesis by delayed-implanting blastocysts and suppression was maximal at 50 mu g/ml. However, DIAP170K did not affect DNA synthesis by blastocysts obtained on day 5 of pregnancy (normal blastocysts) and activated blastocysts. Resumption of DNA synthesis in the inner cell mass (ICM) and trophectoderm from delayed-implanting blastocysts was then separately assessed. DNA synthesis resumed in the trophectoderm of intact blastocysts during 24-hr culture but not in the trophectoderm cultured apart from the ICM. DIAP170K inhibited the resumption of DNA synthesis by the trophectoderm of intact delayed-implanting blastocysts but did not affect DNA synthesis by the ICM. In conclusion, DIAP170K inhibits resumption of DNA synthesis by trophectoderm of delayed-implanting blastocysts. This action of DIAP170K may play a central role in maintaining, but not achieving, dormancy of DNA synthesis by delayed-implanting blastocysts in mice., JAPAN SOC VET SCI, 英語

A delayed-implantation-associated protein prevents resumption of DNA synthesis by the trophectoderm of delayed-implanting mouse blastocysts in vitro
S Katagiri, Y Takahashi, H Kanagawa, BH Yuen, YS Moon, JOURNAL OF VETERINARY MEDICAL SCIENCE, 60, 7, 791, 794, 1998年07月
Objective of this study was to determine the ability of a delayed-implantation-associated protein (MW 170,000, DIAP170K) to inhibit DNA synthesis by mouse blastocysts. Mice were ovariectomized on day 3 of pregnancy and treated with daily injections with I mg progesterone till day 7 to induce delayed implantation. Blastocysts were collected on day 8 with or without a single injection of 25 ng estradiol-17 beta on day 7 that activates blastocyst metabolisms (activated blastocysts and delayed-implanting blastocysts respectively). DNA synthesis was determined by measuring [H-3]thymidine incorportion by blastocysts. DIAP170K at 10 mu g/ml suppressed resumption of DNA synthesis by delayed-implanting blastocysts and suppression was maximal at 50 mu g/ml. However, DIAP170K did not affect DNA synthesis by blastocysts obtained on day 5 of pregnancy (normal blastocysts) and activated blastocysts. Resumption of DNA synthesis in the inner cell mass (ICM) and trophectoderm from delayed-implanting blastocysts was then separately assessed. DNA synthesis resumed in the trophectoderm of intact blastocysts during 24-hr culture but not in the trophectoderm cultured apart from the ICM. DIAP170K inhibited the resumption of DNA synthesis by the trophectoderm of intact delayed-implanting blastocysts but did not affect DNA synthesis by the ICM. In conclusion, DIAP170K inhibits resumption of DNA synthesis by trophectoderm of delayed-implanting blastocysts. This action of DIAP170K may play a central role in maintaining, but not achieving, dormancy of DNA synthesis by delayed-implanting blastocysts in mice., JAPAN SOC VET SCI, 英語

永野昌志、高橋芳幸、片桐成二、金川弘司. ウシの胞状卵胞から回収した未成熟卵子の超微形態　　　　　　　　　　　　　　　
北海道牛受精卵移植研究会会報, 17: 1-6, 1998年

Bautista JAN, Dela Pena EC, Katagiri S, Takahashi Y, Kanagawa H. In vitro viability of mouse oocytes vitrified in an ethylene glycol-based solution.　　　　　　　　　　　　　　　
Jpn J Vet Res, 46: 13-18, 1998年

Elsheikh AS, Takahashi Y, Katagiri S, Kanagawa H. Functional enucleation of mouse metaphase II oocytes with etoposide.　　　　　　　　　　　　　　　
Jpn J Vet Res, 45: 217-220, 1998年

Insulin-like growth factor-1 improves development of 8-cell rat embryos in vitro and subsequent development in vivo
Seiji Katagiri, Basil H.O. Yuen, Young S. Moon, Journal of Reproduction and Development, 44, 1, 15, 20, 1998年
The present study examined the role for insulin-like growth factor-I (IGF-I) in preimplantation development in rats. The 8-cell stage rat embryos were cultured with 0 (control), 0.02, 0.2, or 2.0 nM human recombinant (hr) IGF-I for 36 h. Morphological development of embryos to the blastocyst stage was stimulated by hrIGF-I at all examined concentrations. Human recombinant IGF-I at ≥0.2 nM increased the number of live cells in the inner cell mass (ICM), the embryonic lineage, of resulting blastocysts by increasing total cell number and decreasing the rate of dead cells in the ICM. Levels of protein synthesis by blastocysts cultured with ≥0.2 nM hrIGF-I increased to the same levels as that of in vivo grown counterparts. When blastocysts were transferred to a receptive uterus, the rates of implantation and fetal development to day 18 of pregnancy in the 0.2 and 2.0 nM hrIGF-I groups were greater than those of the control group. In conclusion, hrIGF-I at ≥ 0.2 nM may promote development of 8-cell rat embryos to blastocysts that are fully-potent to postimplantational development., The Japanese Society of Animal Reproduction (JSAR), 英語

Bautista JAN, Dela Pena EC, Katagiri S, Takahashi Y, Kanagawa H. In vitro viability of mouse oocytes vitrified in an ethylene glycol-based solution.　　　　　　　　　　　　　　　
Jpn J Vet Res, 46: 13-18, 1998年

Elsheikh AS, Takahashi Y, Katagiri S, Kanagawa H. Functional enucleation of mouse metaphase II oocytes with etoposide.　　　　　　　　　　　　　　　
Jpn J Vet Res, 45: 217-220, 1998年

Insulin-like growth factor-1 improves development of 8-cell rat embryos in vitro and subsequent development in vivo
Seiji Katagiri, Basil H.O. Yuen, Young S. Moon, Journal of Reproduction and Development, 44, 1, 15, 20, 1998年
The present study examined the role for insulin-like growth factor-I (IGF-I) in preimplantation development in rats. The 8-cell stage rat embryos were cultured with 0 (control), 0.02, 0.2, or 2.0 nM human recombinant (hr) IGF-I for 36 h. Morphological development of embryos to the blastocyst stage was stimulated by hrIGF-I at all examined concentrations. Human recombinant IGF-I at ≥0.2 nM increased the number of live cells in the inner cell mass (ICM), the embryonic lineage, of resulting blastocysts by increasing total cell number and decreasing the rate of dead cells in the ICM. Levels of protein synthesis by blastocysts cultured with ≥0.2 nM hrIGF-I increased to the same levels as that of in vivo grown counterparts. When blastocysts were transferred to a receptive uterus, the rates of implantation and fetal development to day 18 of pregnancy in the 0.2 and 2.0 nM hrIGF-I groups were greater than those of the control group. In conclusion, hrIGF-I at ≥ 0.2 nM may promote development of 8-cell rat embryos to blastocysts that are fully-potent to postimplantational development., The Japanese Society of Animal Reproduction (JSAR), 英語

A somatostatin analogue decreases embryonic loss following superovulation in rats by normalizing insulin-like growth factor-I action in the uterus
S Katagiri, YS Moon, BH Yuen, HUMAN REPRODUCTION, 12, 4, 671, 676, 1997年04月
This study was designed to determine whether the somatostatin analogue, octreotide, could prevent embryonic loss by normalizing increased uterine insulin-like growth factor-I (IGF-I) action related to hyperoestrogenaemia following superovulation, Superovulated immature and oestradiol-17 beta-treated adult rats were infused with 100 or 300 mu g/ml of octreotide respectively, or injected daily with 1 or 10 mu g of octreotide from day 1 to day 3 of pregnancy. On day 3, embryos were collected from the oviducts and uteri, Uterine luminal fluid was subjected to embryo culture, The amounts of uterine IGF-I and IGF binding proteins (IGFBP) were determined by radioimmunoassay and ligand binding assay respectively, Octreotide infusion normalized uterine IGF-I action following superovulatory and oestradiol-17 beta treatment, by reducing IGF-I concentrations and increasing IGFBP concentrations, Octreotide infusion increased the number of normal embryos by 2.7-fold and 1.7-fold in superovulated and oestradiol-17 beta-treated rats respectively, and reversed the detrimental effects of uterine luminal fluid on embryonic development caused by superovulatory and oestradiol-17 beta treatment, Daily injections with octreotide had similar but reduced effects in all parameters examined in both treatment groups, In conclusion, octreotide may reduce embryonic loss, at least in part, by normalizing IGF-I action following superovulation., OXFORD UNIV PRESS, 英語

A somatostatin analogue decreases embryonic loss following superovulation in rats by normalizing insulin-like growth factor-I action in the uterus
S Katagiri, YS Moon, BH Yuen, HUMAN REPRODUCTION, 12, 4, 671, 676, 1997年04月
This study was designed to determine whether the somatostatin analogue, octreotide, could prevent embryonic loss by normalizing increased uterine insulin-like growth factor-I (IGF-I) action related to hyperoestrogenaemia following superovulation, Superovulated immature and oestradiol-17 beta-treated adult rats were infused with 100 or 300 mu g/ml of octreotide respectively, or injected daily with 1 or 10 mu g of octreotide from day 1 to day 3 of pregnancy. On day 3, embryos were collected from the oviducts and uteri, Uterine luminal fluid was subjected to embryo culture, The amounts of uterine IGF-I and IGF binding proteins (IGFBP) were determined by radioimmunoassay and ligand binding assay respectively, Octreotide infusion normalized uterine IGF-I action following superovulatory and oestradiol-17 beta treatment, by reducing IGF-I concentrations and increasing IGFBP concentrations, Octreotide infusion increased the number of normal embryos by 2.7-fold and 1.7-fold in superovulated and oestradiol-17 beta-treated rats respectively, and reversed the detrimental effects of uterine luminal fluid on embryonic development caused by superovulatory and oestradiol-17 beta treatment, Daily injections with octreotide had similar but reduced effects in all parameters examined in both treatment groups, In conclusion, octreotide may reduce embryonic loss, at least in part, by normalizing IGF-I action following superovulation., OXFORD UNIV PRESS, 英語

Mohamed Mohamed Nour、高橋芳幸、片桐成二、金川弘司. ウシ核移植胚の発育に及ぼすレシピエント卵子の品質の影響　　　　　　　　　　　　　　　
北海道牛受精卵移植研究会会報, 16: 40-43, 1997年

Role for insulin-like growth factor I in the regulation of electrolyte composition of uterine luminal fluid
S Katagiri, S Ma, BH Yuen, YS Moon, JOURNAL OF REPRODUCTION AND FERTILITY, 109, 1, 115, 120, 1997年01月
A potential role for insulin-like growth factor I (IGF-I) in the regulation of the uterine electrolyte environment was studied in conjunction with hyperoestrogenaemia caused by superovulation. Uterine luminal fluid from immature rats treated with 4 (control), 10, 20 and 40 iu (superovulation) pregnant mares' serum gonadotrophin (PMSG, day - 2) and the electrolyte composition was determined on day 3 of pregnancy. Superovulation increased total cation content in uterine flushes by more han twofold, suggesting a comparable increase in the uterine luminal fluid volume. Percentages of K+ and HCO3- content to total cations or anions increased by 27% and 16%, respectively, and those of Na+ and Cl- decreased by 26% and 15%, respectively, after superovulation. Daily injections with 1.0 mu g or more oestradiol, from day 0 to day 2, in the 4 iu PMSG-primed immature rats caused similar changes in total cation content and electrolyte composition of uterine luminal fluid. Anti-IGF-I antibody infusion in the superovulated or oestradiol-treated immature rats restored the alterations in cation composition but had no effect on anion composition and total cation content. IGF-I was infused into adult rats to achieve increased IGF-I action observed after superovulation. IGF-I infusion altered electrolyte composition, as is observed after superovulation or oestradiol treatment, but had no effect on total cation content. In conclusion, hyperoestrogenaemia caused by superovulation may alter the uterine electrolyte environment for preimplantation embryonic development. IGF-I appears to play a central role in mediating this action of oestrogen., J REPROD FERTIL INC, 英語

Role for insulin-like growth factor I in the regulation of electrolyte composition of uterine luminal fluid
S Katagiri, S Ma, BH Yuen, YS Moon, JOURNAL OF REPRODUCTION AND FERTILITY, 109, 1, 115, 120, 1997年01月
A potential role for insulin-like growth factor I (IGF-I) in the regulation of the uterine electrolyte environment was studied in conjunction with hyperoestrogenaemia caused by superovulation. Uterine luminal fluid from immature rats treated with 4 (control), 10, 20 and 40 iu (superovulation) pregnant mares' serum gonadotrophin (PMSG, day - 2) and the electrolyte composition was determined on day 3 of pregnancy. Superovulation increased total cation content in uterine flushes by more han twofold, suggesting a comparable increase in the uterine luminal fluid volume. Percentages of K+ and HCO3- content to total cations or anions increased by 27% and 16%, respectively, and those of Na+ and Cl- decreased by 26% and 15%, respectively, after superovulation. Daily injections with 1.0 mu g or more oestradiol, from day 0 to day 2, in the 4 iu PMSG-primed immature rats caused similar changes in total cation content and electrolyte composition of uterine luminal fluid. Anti-IGF-I antibody infusion in the superovulated or oestradiol-treated immature rats restored the alterations in cation composition but had no effect on anion composition and total cation content. IGF-I was infused into adult rats to achieve increased IGF-I action observed after superovulation. IGF-I infusion altered electrolyte composition, as is observed after superovulation or oestradiol treatment, but had no effect on total cation content. In conclusion, hyperoestrogenaemia caused by superovulation may alter the uterine electrolyte environment for preimplantation embryonic development. IGF-I appears to play a central role in mediating this action of oestrogen., J REPROD FERTIL INC, 英語

An improved technique for molecular cytogenetic analysis of human preimplantation embryos with fluorescence in situ hybridization
S Ma, DK Kalousek, BH Yuen, S Katagiri, YS Moon, JOURNAL OF REPRODUCTIVE MEDICINE, 41, 6, 379, 383, 1996年06月
OBJECTIVE: To develop a method for the cytogenetic evaluation of preimplantation embryos using nonradioactive centromeric probes for chromosomes 1, 16 and X.
STUDY DESIGN: The embryos used for this study were either fragmented or polyploid embryos rejected from art in vitro fertilization program. Prior to in situ hybridization, the embryos were treated with 0.5% protease. After application of gradual fixation, conventional hybridization protocol was followed.
RESULTS: Ten of 11 embryos showed hybridization signals suggesting that the success rate of in situ hybridization of human embryos is improved when a modified method of digesting the zona pellucida and gradual fixation with removal of the cytoplasm are used.
CONCLUSION: The method described in this study demonstrates that the iona pellucida is the key to successful in situ hybridization of whole human embryos. When the zona pellucida is removed, penetration by a probe becomes possible., SCI PRINTERS & PUBL INC, 英語

An improved technique for molecular cytogenetic analysis of human preimplantation embryos with fluorescence in situ hybridization
S Ma, DK Kalousek, BH Yuen, S Katagiri, YS Moon, JOURNAL OF REPRODUCTIVE MEDICINE, 41, 6, 379, 383, 1996年06月
OBJECTIVE: To develop a method for the cytogenetic evaluation of preimplantation embryos using nonradioactive centromeric probes for chromosomes 1, 16 and X.
STUDY DESIGN: The embryos used for this study were either fragmented or polyploid embryos rejected from art in vitro fertilization program. Prior to in situ hybridization, the embryos were treated with 0.5% protease. After application of gradual fixation, conventional hybridization protocol was followed.
RESULTS: Ten of 11 embryos showed hybridization signals suggesting that the success rate of in situ hybridization of human embryos is improved when a modified method of digesting the zona pellucida and gradual fixation with removal of the cytoplasm are used.
CONCLUSION: The method described in this study demonstrates that the iona pellucida is the key to successful in situ hybridization of whole human embryos. When the zona pellucida is removed, penetration by a probe becomes possible., SCI PRINTERS & PUBL INC, 英語

The role for the uterine insulin-like growth factor I in early embryonic loss after superovulation in the rat
S Katagiri, YS Moon, BH Yuen, FERTILITY AND STERILITY, 65, 2, 426, 436, 1996年02月
Objectives: To examine possible roles of the insulin-like growth factor (IGF) system in increased early embryonic loss after superovulation.
Design: Changes in the uterine IGF system were examined in superovulated rats. Insulinlike growth factor I (IGF-I) was infused to the right uterine horns to mimic enhanced IGF-I actions after superovulation. Uterine luminal fluids were collected after IGF-I infusions and embryos were cultured with uterine luminal fluids.
Main Outcome Measures: Steroid hormones, IGF-I, IGF binding protein (IGFBP), and IGF-I receptor levels, developmental rate, and cell numbers of embryos.
Results: Elevated IGF-I levels and suppressed IGFBP levels were found from days 1 to 3 of pregnancy after superovulation. Uterine luminal fluids of the ICE-I infusion and superovulation groups impaired embryo development in vitro. Anti-IGF-I antibody infusions after superovulation reversed detrimental effects of superovulation. Dialysis of uterine luminal fluids of the IGF-I infusion and superovulation groups before culture improved embryo development.
Conclusions: Enhanced IGF-I actions in the uterus after superovulation may be responsible for the increase of early embryonic loss. The detrimental factor for embryo development seems a small molecule and is likely a local product of the uterus in which IGF-I actions are enhanced., AMER SOC REPRODUCTIVE MEDICINE, 英語

The role for the uterine insulin-like growth factor I in early embryonic loss after superovulation in the rat
S Katagiri, YS Moon, BH Yuen, FERTILITY AND STERILITY, 65, 2, 426, 436, 1996年02月
Objectives: To examine possible roles of the insulin-like growth factor (IGF) system in increased early embryonic loss after superovulation.
Design: Changes in the uterine IGF system were examined in superovulated rats. Insulinlike growth factor I (IGF-I) was infused to the right uterine horns to mimic enhanced IGF-I actions after superovulation. Uterine luminal fluids were collected after IGF-I infusions and embryos were cultured with uterine luminal fluids.
Main Outcome Measures: Steroid hormones, IGF-I, IGF binding protein (IGFBP), and IGF-I receptor levels, developmental rate, and cell numbers of embryos.
Results: Elevated IGF-I levels and suppressed IGFBP levels were found from days 1 to 3 of pregnancy after superovulation. Uterine luminal fluids of the ICE-I infusion and superovulation groups impaired embryo development in vitro. Anti-IGF-I antibody infusions after superovulation reversed detrimental effects of superovulation. Dialysis of uterine luminal fluids of the IGF-I infusion and superovulation groups before culture improved embryo development.
Conclusions: Enhanced IGF-I actions in the uterus after superovulation may be responsible for the increase of early embryonic loss. The detrimental factor for embryo development seems a small molecule and is likely a local product of the uterus in which IGF-I actions are enhanced., AMER SOC REPRODUCTIVE MEDICINE, 英語

SEPARATION OF A 170-K DELAYED-IMPLANTATION-ASSOCIATED PROTEIN WITH INHIBITORY ACTIVITY ON TROPHOBLAST OUTGROWTH AND RNA-SYNTHESIS BY MOUSE EMBRYO
S KATAGIRI, BH YUEN, YS MOON, Y TAKAHASHI, H KANAGAWA, AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, 31, 2-3, 141, 150, 1994年03月
PROBLEM: To screen the uterine protein responsible for embryonic dormancy associated with delayed implantation.
METHOD: Uterine protein extracts and sera from mice in which delayed implantation had been induced and those from pregnant mice were separated by three steps of chromatogra hy and SDS-PAGE by monitoring an inhibitory activity on trophoblast outgrowth. The presence of the separated protein in the uterine luminal fluid was assessed. Effect of the protein on cell proliferation and RNA synthesis by blastocysts were assessed.
RESULTS: A 170-K protein was found in the uterine tissue as well as uterine luminal fluid associated with delayed implantation. The 170-K protein suppressed RNA synthesis by approximately 50% and cell proliferation in blastocysts.
CONCLUSION: A 170-K protein is secreted into the uterine lumen during delayed implantation period. The ability of 170-K protein to suppress RNA synthesis and cell proliferation may play a role in regulation of embryonic dormancy associated with delayed implantation., MUNKSGAARD INT PUBL LTD, 英語

SEPARATION OF A 170-K DELAYED-IMPLANTATION-ASSOCIATED PROTEIN WITH INHIBITORY ACTIVITY ON TROPHOBLAST OUTGROWTH AND RNA-SYNTHESIS BY MOUSE EMBRYO
S KATAGIRI, BH YUEN, YS MOON, Y TAKAHASHI, H KANAGAWA, AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, 31, 2-3, 141, 150, 1994年03月
PROBLEM: To screen the uterine protein responsible for embryonic dormancy associated with delayed implantation.
METHOD: Uterine protein extracts and sera from mice in which delayed implantation had been induced and those from pregnant mice were separated by three steps of chromatogra hy and SDS-PAGE by monitoring an inhibitory activity on trophoblast outgrowth. The presence of the separated protein in the uterine luminal fluid was assessed. Effect of the protein on cell proliferation and RNA synthesis by blastocysts were assessed.
RESULTS: A 170-K protein was found in the uterine tissue as well as uterine luminal fluid associated with delayed implantation. The 170-K protein suppressed RNA synthesis by approximately 50% and cell proliferation in blastocysts.
CONCLUSION: A 170-K protein is secreted into the uterine lumen during delayed implantation period. The ability of 170-K protein to suppress RNA synthesis and cell proliferation may play a role in regulation of embryonic dormancy associated with delayed implantation., MUNKSGAARD INT PUBL LTD, 英語

Ma S, Kalousek DK, Ho Yuen B, Gomel V, Katagiri S, Moon YS. Chromosome investigation in in vitro fertilization failure.　　　　　　　　　　　　　　　
J Assis Reprod Genetics, 11: 445-451, 1994年

Ma S, Kalousek DK, Ho Yuen B, Gomel V, Katagiri S, Moon YS. Chromosome investigation in in vitro fertilization failure.　　　　　　　　　　　　　　　
J Assis Reprod Genetics, 11: 445-451, 1994年

PMSG PROFILES IN SUPEROVULATED AND ANTI-PMSG ANTISERUM TREATED MICE AND HEIFERS WITH ENZYME-IMMUNOASSAY
S KATAGIRI, Y TAKAHASHI, M HISHINUMA, H KANAGAWA, O DOCHI, H TAKAKURA, JAPANESE JOURNAL OF VETERINARY RESEARCH, 39, 1, 11, 21, 1991年05月
A sandwich enzymeimmunoassay (EIA) for pregnant mare serum gonadotropin (PMSG) using a microtiter plate was developed. Sensitivity of the assay to PMSG was 15.6 mIU/ml (0.2 ng/well). The PMSG levels in serum were measured with the EIA in superovulated and anti-PMSG rabbit antiserum treated mice and heifers. In mice, the PMSG blood level was measurable in the serum 4-6 days after intraperitoneal injection of 5-30 IU of PMSG. The administration of anti-PMSG antiserum at the same dose level as PMSG caused a rapid decrease in the PMSG blood level, declining to undetectable levels within 17 hours. In heifers, the PMSG level was measurable at 10-11 days after the injection of 2500 or 3000 IU of PMSG. When antiserum was injected 48 hours after the PMSG injection, the clearance rate of PMSG was affected by the route of the administration. The administration of 3000 units of anti-PMSG antiserum intravenously caused a rapid decline and the disappearance of circulating PMSG within 17 hours. When 3000 units of anti-PMSG antiserum was injected intramuscularly, the PMSG blood level also decreased and became unmeasurable 24 hours after administration; however, it was still detectable for up to 17 hours.
These results indicate that the administration of anti-PMSG antiserum at the proper timing and dosage could lead to successful superovulation through the improvement of hormonal conditions., HOKKAIDO UNIV, 英語

PMSG PROFILES IN SUPEROVULATED AND ANTI-PMSG ANTISERUM TREATED MICE AND HEIFERS WITH ENZYME-IMMUNOASSAY
S KATAGIRI, Y TAKAHASHI, M HISHINUMA, H KANAGAWA, O DOCHI, H TAKAKURA, JAPANESE JOURNAL OF VETERINARY RESEARCH, 39, 1, 11, 21, 1991年05月
A sandwich enzymeimmunoassay (EIA) for pregnant mare serum gonadotropin (PMSG) using a microtiter plate was developed. Sensitivity of the assay to PMSG was 15.6 mIU/ml (0.2 ng/well). The PMSG levels in serum were measured with the EIA in superovulated and anti-PMSG rabbit antiserum treated mice and heifers. In mice, the PMSG blood level was measurable in the serum 4-6 days after intraperitoneal injection of 5-30 IU of PMSG. The administration of anti-PMSG antiserum at the same dose level as PMSG caused a rapid decrease in the PMSG blood level, declining to undetectable levels within 17 hours. In heifers, the PMSG level was measurable at 10-11 days after the injection of 2500 or 3000 IU of PMSG. When antiserum was injected 48 hours after the PMSG injection, the clearance rate of PMSG was affected by the route of the administration. The administration of 3000 units of anti-PMSG antiserum intravenously caused a rapid decline and the disappearance of circulating PMSG within 17 hours. When 3000 units of anti-PMSG antiserum was injected intramuscularly, the PMSG blood level also decreased and became unmeasurable 24 hours after administration; however, it was still detectable for up to 17 hours.
These results indicate that the administration of anti-PMSG antiserum at the proper timing and dosage could lead to successful superovulation through the improvement of hormonal conditions., HOKKAIDO UNIV, 英語

片桐成二、首藤文栄、高橋芳幸、金川弘司. ボツリヌス菌産生C1型（C-ST）毒素および酵素（C3）が胚の発育に及ぼす影響　　　　　　　　　　　　　　　
北海道牛受精卵移植研究会会報, 10: 81-84, 1991年

浦野浩司、片桐成二、菱沼 貢、高橋芳幸、金川弘司. ウシ胚回収用フィルターの検討
浦野 浩司, 片桐 成二, 菱沼 貢, 高橋 芳幸, 金川 弘司, Jpn J Anim Reprod, 33: 48-50, 1, 48, 50, 1987年
2種類の牛胚回収用フィルターの有用性について検討を加えた。供試卵を一定量の灌流液とともにフィルターを用いてろ過した結果,いずれのフィルターでも98.3%の供試卵を回収することができた。しかし,少数(7.6%)ではあるがフィルターを洗浄した液からも供試卵が回収され,フィルター内を洗浄する必要のあることが示唆された。, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT, 日本語

菱沼 貢、片桐成二、橘田達慶、西山栄治、高橋芳幸、金川弘司. 犬および猫子宮蓄膿症の発症傾向、臨床症状と血液検査所見について　　　　　　　　　　　　　　　
北海道獣医師会雑誌, 29: 272-276, 1985年

乳牛の血漿遊離脂肪酸濃度上昇に伴う卵子エネルギー代謝関連脂質プロファイルの変化　　　　　　　　　　　　　　　
古川瑛理, 陳震, 窪友瑛, CHELENGA Madalitso, WU Yue, 千葉仁志, 柳川洋二郎, 片桐成二, 惠淑萍, 永野昌志
日本生殖医学会雑誌, 2022年
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第二胃内留置型自動体温測定器で測定したホルスタイン種経産牛の分娩前1週間の体温変化と分娩時間帯の関係　　　　　　　　　　　　　　　
古川 瑛理, 高橋 昌志, 松崎 明, 唄 花子, 永野 昌志, 片桐 成二, 柳川 洋二郎
日本畜産学会大会講演要旨集, 2021年09月, (公社)日本畜産学会, 日本語
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乳牛における子宮内膜上皮増殖因子(EGF)回復およびその後の受胎率の変化に対する危険因子【JST・京大機械翻訳】　　　　　　　　　　　　　　　
NATTAPONG Ninpetch, 河野光平, 柳川洋二郎, 片桐成二
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ホルスタイン種泌乳牛の分娩後日数及びボディコンディションスコアと卵子中トリアシルグリセロール量の関係　　　　　　　　　　　　　　　
古川瑛理, 陳震, 窪友瑛, MADALITSO Chelenga, YUE Wu, 千葉仁志, 柳川洋二郎, 片桐成二, 惠淑萍, 永野昌志
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2021年 - 2021年

液体クロマトグラフィー質量分析法によるアフリカゾウの血中ステロイドホルモン濃度測定法の確立および無排卵雌における排卵誘起の試み　　　　　　　　　　　　　　　
篠原明, 篠原明, 鳥居佳子, 入江陽一, 牛尾佳名子, 曽地千尋, 宗内一平, 楠田哲士, 東達也, 一瀬貴大, 中山翔太, 石塚真由美, 池中良徳, 片桐成二, 柳川洋二郎
日本獣医学会学術集会講演要旨集, 2021年
2021年 - 2021年

リピートブリーダー牛にて、子宮内膜の上皮成長因子profileinを正常化する乳由来オステオポンチンの効果(Capacity of milk osteopontin to normalize endometrial epidermal growth factor profilein repeat breeder cows)　　　　　　　　　　　　　　　
Kyaw Hay Mar, 田上 貴祥, 柳川 洋二郎, 永野 昌志, 片桐 成二
日本獣医学会学術集会講演要旨集, 2020年10月, (公社)日本獣医学会, 英語
2020年10月 - 2020年10月

卵巣内の主席卵胞と黄体の存在が初期胞状卵胞由来卵子の核成熟能と顆粒層細胞の性ステロイドホルモン産生能に及ぼす影響　　　　　　　　　　　　　　　
坂口 謙一郎, 河野 光平, 柳川 洋二郎, 片桐 成二, 永野 昌志
日本獣医学会学術集会講演要旨集, 2020年10月, (公社)日本獣医学会, 日本語
2020年10月 - 2020年10月

非泌乳期ホルスタイン種経産雌牛における卵胞刺激ホルモンの尾椎硬膜外腔投与後の血漿中性ステロイドホルモン動態　　　　　　　　　　　　　　　
坂口 謙一郎, 柳川 洋二郎, Ninpetch Nattapong, 河野 光平, 須田 智子, 吉岡 耕治, 片桐 成二, 永野 昌志
The Journal of Reproduction and Development, 2020年09月, (公社)日本繁殖生物学会, 日本語
2020年09月 - 2020年09月

組換えオステオポンチンによるリピートブリーダー牛の子宮内膜上皮成長因子(EGF)濃度正常化と受胎性回復効果の検証
佐藤 弘子, Hay Mar Kyaw, 柳川 洋二郎, 永野 昌志, 田上 貴祥, 片桐 成二
Journal of Mammalian Ova Research, 2020年07月, (一社)日本卵子学会, 日本語
2020年07月 - 2020年07月

組み換えオステオポンチンによる牛子宮内膜上皮成長因子濃度の正常化効果の検証
佐藤弘子, KYAW Hay Mar, 柳川洋二郎, 永野昌志, 田上貴祥, 片桐成二
Journal of Reproduction and Development, 2019年09月05日, 日本語
2019年09月05日 - 2019年09月05日

牛性選別精子の卵管上皮細胞結合能および受精能獲得のフローサイトメトリーによる解析
中島愛理, 菅野智裕, 菅野智裕, 片桐成二, 永野昌志, 柳川洋二郎
日本獣医学会学術集会講演要旨集, 2019年08月20日, 日本語
2019年08月20日 - 2019年08月20日

クマ科動物の人工授精技術の開発に向けた研究
鳥居佳子, 永野昌志, 片桐成二, 柳川洋二郎
日本獣医学会学術集会講演要旨集, 2019年08月20日, 日本語
2019年08月20日 - 2019年08月20日

乳牛の子宮内膜組織血流量に影響を及ぼす因子の解析
河野光平, 柳川洋二郎, NATTAPONG Ninpetch, 永野昌志, 片桐成二
日本獣医学会学術集会講演要旨集, 2019年08月20日, 日本語
2019年08月20日 - 2019年08月20日

Identification of seminal plasma proteins to normalize endometrial epidermal growth factor concentrations in repeat breeder cows
BADRAKH Dagvajamts, 柳川洋二郎, 永野昌志, 片桐成二
日本獣医学会学術集会講演要旨集, 2019年08月20日, 英語
2019年08月20日 - 2019年08月20日

精漿蛋白による子宮機能調節とその低受胎対策への応用　　　　　　　　　　　　　　　
片桐成二
Journal of Reproduction and Development, 2019年
2019年 - 2019年

モンゴル国における羊体外受精卵由来産子作出の試み：体外成熟培地への抗酸化剤アスタキサンチンの添加効果　　　　　　　　　　　　　　　
永野昌志, Purevdorj Erdenetogtokh, 栁川洋二郎, 片桐成二
北海道牛受精卵移植研究会会報, 2018年09月, 日本語
2018年09月 - 2018年09月

Lipidomic profiling in bovine oocytes by LC/Orbitrap-MS
WU Yue, UESHIBA Hiroki, CHEN Zhen, SAKAGUCHI Kenichiro, YANAGAWA Yojiro, KATAGIRI Seiji, NAGANO Masashi, CHIBA Hitoshi, CHIBA Hitoshi, HUI Shu‐Ping
JSBMS Letters, 2018年08月25日, 英語
2018年08月25日 - 2018年08月25日

液体クロマトグラフィー高分解能質量分析を用いた牛卵子中遊離脂肪酸およびトリアシルグリセロールの分析
植芝滉己, 呉ユエ, 陳震, 坂口謙一郎, 柳川洋二郎, 片桐成二, 千葉仁志, 千葉仁志, 惠淑萍, 永野昌志
日本獣医学会学術集会講演要旨集, 2018年08月21日, 日本語
2018年08月21日 - 2018年08月21日

乳牛の卵巣内胞状卵胞数が血中卵胞刺激ホルモン動態と卵胞における性ステロイドホルモン産生に与える影響
坂口謙一郎, 柳川洋二郎, 吉岡耕治, 須田智子, 植芝滉己, 河野光平, 宮本祥代, 片桐成二, 永野昌志
日本獣医学会学術集会講演要旨集, 2018年08月21日, 日本語
2018年08月21日 - 2018年08月21日

クラスター解析の精子受胎性評価への応用:性選別および通常凍結精液中の精子運動性サブポピュレーション構成の比較
菅野智裕, 菅野智裕, 柳川洋二郎, 片桐成二, 永野昌志
日本獣医学会学術集会講演要旨集, 2018年08月21日, 日本語
2018年08月21日 - 2018年08月21日

乳牛における暑熱ストレスと子宮内膜中上皮成長因子濃度異常との関係および暑熱の子宮内環境への影響評価のための子宮内温度と子宮内膜血流量の測定法の検討
河野光平, 柳川洋二郎, NATTAPONG Ninpetch, 坂口謙一郎, 菅野智裕, 菅野智裕, 鳥居佳子, 植芝滉己, 宮本祥代, 永野昌志, 片桐成二
日本獣医学会学術集会講演要旨集, 2018年08月21日, 日本語
2018年08月21日 - 2018年08月21日

卵胞刺激ホルモンおよび性腺刺激ホルモン放出ホルモン投与による卵巣刺激が河川型水牛 (Bubalus bubalis) における経腟採卵-体外受精成績に及ぼす影響　　　　　　　　　　　　　　　
坂口謙一郎, Excel Rio S. Maylem, Ramesh C. Tilwani, 栁川洋二郎, 片桐成二, Edwin C. Atabay, Eufrocina P. Atabay, 永野昌志
北海道牛受精卵移植研究会会報, 2018年08月, 日本語
2018年08月 - 2018年08月

ペレット法により凍結したニホンザル精液に対する融解法の違いが精子性状に与える影響　　　　　　　　　　　　　　　
栁川洋二郎, 菅野智裕, 兼子明久, 印藤頼子, 佐藤容, 木下こづえ, 今井啓雄, 平井啓久, 片桐成二, 永野昌志, 岡本宗裕
Cryopreservation conference 2017, 2017年11月, 日本語
2017年11月 - 2017年11月

ニホンザルにおける排卵誘起処置を伴う単回人工授精プログラムの検討
柳川洋二郎, 菅野智裕, 南晶子, 兼子明久, 印藤頼子, 佐藤容, 木下こづえ, 岡本宗裕, 片桐成二, 永野昌志
日本獣医学会学術集会講演要旨集, 2017年08月30日, 日本語
2017年08月30日 - 2017年08月30日

牛卵巣内胞状卵胞数と初期胞状卵胞由来卵子‐顆粒層細胞複合体の顆粒層細胞増殖能および卵子発生能の関連性
坂口謙一郎, 谷田孝志, 谷田孝志, 楊応華, 菅野智裕, 永井克尚, 永井克尚, MOHAMED. A. Abdel‐Ghani, 柳川洋二郎, 片桐成二, 永野昌志
日本獣医学会学術集会講演要旨集, 2017年08月30日, 日本語
2017年08月30日 - 2017年08月30日

ヒグマ精液の凍結保存液に添加する糖の種類および冷却速度が凍結融解後の精液性状に及ぼす影響
鳥居佳子, 菅野智裕, 蓑原悠太朗, 坂元秀行, 松本直也, 冨安洵平, 松井基純, 永野昌志, 片桐成二, 柳川洋二郎
日本獣医学会学術集会講演要旨集, 2017年08月30日, 日本語
2017年08月30日 - 2017年08月30日

精子運動能自動解析装置(CASA)を用いた受精能獲得精子の運動性評価
菅野智裕, 柳川洋二郎, 片桐成二, 永野昌志
日本獣医学会学術集会講演要旨集, 2017年08月30日, 日本語
2017年08月30日 - 2017年08月30日

黒毛和種リピートブリーダー牛の子宮内膜上皮成長因子発現異常に対する治療への抵抗性と肥満との関係
谷田孝志, 谷田孝志, 坂口謙一郎, 菅野智裕, 池田侑樹, 田嶋彩野, 柳川洋二郎, 永野昌志, 片桐成二
日本獣医学会学術集会講演要旨集, 2017年08月30日, 日本語
2017年08月30日 - 2017年08月30日

牛卵巣の大きさが顆粒層細胞の性ステロイドホルモン産生能および卵子核成熟能に与える影響　　　　　　　　　　　　　　　
坂口 謙一郎, 菅野 智裕, 楊 応華, 柳川 洋二郎, 片桐 成二, 永野 昌志
北海道獣医師会雑誌, 2017年08月, (公社)北海道獣医師会, 日本語
2017年08月 - 2017年08月

Mitochondrial activity and reactive oxygen species(ROS) accumulation in bovine oocytes derived from different culture system for in vitro maturation(和訳中)　　　　　　　　　　　　　　　
Purevdorj Erdenetogtokh, 柳川 洋二郎, 片桐 成二, 永野 昌志
日本獣医学会学術集会講演要旨集, 2017年08月, (公社)日本獣医学会, 英語
2017年08月 - 2017年08月

Astaxanthin improves the growth parameters, developmental competence, and quality of in vitro grown oocytes derived from bovine early antral follicles(和訳中)　　　　　　　　　　　　　　　
Abdel-Ghani Mohammed.A, 坂口 謙一郎, 菅野 智裕, 柳川 洋二郎, 片桐 成二, 永野 昌志
日本獣医学会学術集会講演要旨集, 2017年08月, (公社)日本獣医学会, 英語
2017年08月 - 2017年08月

ペレット法による凍結保存が融解後のニホンザル精液の運動性に与える影響　　　　　　　　　　　　　　　
柳川 洋二郎, 菅野 智裕, 兼子 明久, 印藤 頼子, 岡本 宗裕, 片桐 成二, 永野 昌志
日本獣医学会学術集会講演要旨集, 2016年08月, (公社)日本獣医学会, 日本語
2016年08月 - 2016年08月

牛精液の射出順序が精子運動性サブポピュレーション構成に与える影響　　　　　　　　　　　　　　　
菅野 智裕, 柳川 洋二郎, 片桐 成二, 高橋 芳幸, 永野 昌志
日本獣医学会学術集会講演要旨集, 2016年08月, (公社)日本獣医学会, 日本語
2016年08月 - 2016年08月

牛の小型卵母細胞の発生能に対する成熟前培養時間の影響(Effect of prematuration culture duration on developmental competences of bovine small sized oocytes)　　　　　　　　　　　　　　　
Mohammed.A. Abdle-Gahni, 坂口 謙一郎, 菅野 智裕, 楊 応華, 柳川 洋二郎, 片桐 成二, 永野 昌志
日本獣医学会学術集会講演要旨集, 2016年08月, (公社)日本獣医学会, 英語
2016年08月 - 2016年08月

牛卵巣内胞状卵胞数と初期胞状卵胞由来卵子顆粒層細胞複合体の性ステロイドホルモン産生能の関係　　　　　　　　　　　　　　　
坂口 謙一郎, 谷田 孝志, 永井 克尚, 楊 応華, 柳川 洋二郎, 片桐 成二, 永野 昌志
日本獣医学会学術集会講演要旨集, 2016年08月, (公社)日本獣医学会, 日本語
2016年08月 - 2016年08月

卵胞膜細胞と共培養した牛卵母細胞-顆粒膜細胞複合体の増殖、ステロイド産生とその後の発生能(Growth, steroidogenesis and subsequent developmental competence of bovine oocyte-granulosa cell complex co-cultured with theca cells)　　　　　　　　　　　　　　　
楊 応華, 菅野 智裕, 坂口 謙一郎, 柳川 洋二郎, 片桐 成二, 永野 昌志
日本獣医学会学術集会講演要旨集, 2016年08月, (公社)日本獣医学会, 英語
2016年08月 - 2016年08月

北海道内における乳牛への性選別精液使用が後継牛生産と母牛の生存率に与える影響　　　　　　　　　　　　　　　
菅野 智裕, 萩原 精一, 伊藤 純一, 廣田 和久, 片桐 成二, 永野 昌志
北海道獣医師会雑誌, 2016年08月, (公社)北海道獣医師会, 日本語
2016年08月 - 2016年08月

卵胞刺激ホルモンの尾椎硬膜外腔内単回投与が黒毛和種牛の体内・体外胚生産に及ぼす影響　　　　　　　　　　　　　　　
坂口 謙一郎, 出田 篤司, 土屋 加那美, 真方 文絵, 小牧 春菜, 佐藤 正明, 酒井 伸一, 馬塲 貴大, 柳川 洋二郎, 永野 昌志, 片桐 成二, 小西 正人
北海道獣医師会雑誌, 2016年08月, (公社)北海道獣医師会, 日本語
2016年08月 - 2016年08月

凍結前精液の一時保存方法および冷却方法が融解後のマカク属精子の性状に与える影響　　　　　　　　　　　　　　　
栁川洋二郎, 杉本幸介, 菅野智裕, 高江州昇, 印藤頼子, 兼子明久, 木下こづえ, 今井啓雄, 岡本宗裕, 片桐成二, 永野昌志
Cryopreservation Conference 2014, 2015年10月, 日本語
2015年10月 - 2015年10月

牛の卵子品質と卵巣内卵胞数の関係 体内および体外発育卵子を用いた予備的検討　　　　　　　　　　　　　　　
永井 克尚, 杉山 ちさと, 楊 応華, 柳川 洋二郎, 片桐 成二, 永野 昌志
The Journal of Reproduction and Development, 2015年09月, 日本繁殖生物学会, 日本語
2015年09月 - 2015年09月

ニホンザルにおける電気刺激時の電圧が精子採取に与える影響および精子凍結保存法の改善について　　　　　　　　　　　　　　　
杉本 幸介, 柳川 洋二郎, 菅野 智裕, 高江洲 昇, 兼子 明久, 印藤 頼子, 岡本 宗裕, 片桐 成二, 永野 昌志
日本獣医学会学術集会講演要旨集, 2015年08月, (公社)日本獣医学会, 日本語
2015年08月 - 2015年08月

エゾシカにおけるPAGを用いた早期妊娠診断方法の検討　　　　　　　　　　　　　　　
柳川 洋二郎, 宮澤 千鶴, 松浦 友紀子, 下鶴 倫人, Noelita Melo de Sousa, Beckers Jean-Francis, 片桐 成二, 坪田 敏男
北海道獣医師会雑誌, 2009年08月, (公社)北海道獣医師会, 日本語
2009年08月 - 2009年08月

マウス胞状卵胞由来卵母細胞の成熟培養時の基礎培地と気相中の酸素濃度が減数分裂の進行と発生能に及ぼす影響
古山 敬祐, 片桐 成二, 高橋 芳幸
The Journal of Reproduction and Development, 2008年08月, (一社)日本繁殖生物学会, 日本語
2008年08月 - 2008年08月

マウス卵子の成熟培養時の酸素濃度と培養時間が核成熟動態と発生能に及ぼす影響
檜垣彰吾, 岸昌生, 永野昌志, 片桐成二, 高橋芳幸
J Reprod Dev, 2007年09月25日, 日本語
2007年09月25日 - 2007年09月25日

マウス前胞状卵胞の体外培養とガラス化保存
岸昌生, 永野昌志, 檜垣彰吾, 片桐成二, 高橋芳幸
日本生殖医学会雑誌, 2007年04月20日, 日本語
2007年04月20日 - 2007年04月20日

1996年01月 - 現在
日本繁殖生物学会　　　　　　　　　　　　　　　

1996年01月 - 現在
日本獣医学会　　　　　　　　　　　　　　　

牛子宮での上皮成長因子異常におけるToll様受容体4活性化の意義と治療への応用
科学研究費助成事業
2023年04月01日 - 2028年03月31日
片桐 成二, 柳川 洋二郎, 杉浦 智親, 奥山 みなみ, 平山 博樹, 永野 昌志, 田上 貴祥
日本学術振興会, 基盤研究(A), 北海道大学, 23H00353

受胎に必要な免疫制御に精漿が果たす役割とリンパ球の働きに関する研究
科学研究費助成事業
2024年04月01日 - 2027年03月31日
奥山 みなみ, 片桐 成二, 杉浦 智親, 河野 康志
日本学術振興会, 基盤研究(C), 大分大学, 24K09212

牛の精漿蛋白質による子宮機能調節機序の解明と受胎性改善技術への応用
科学研究費助成事業 基盤研究(A)
2019年04月01日 - 2024年03月31日
片桐 成二, 柳川 洋二郎, 田上 貴祥, 平山 博樹, 杉浦 智親, 永野 昌志, 奥山 みなみ
牛の低受胎（リピートブリーディング）の原因の1つとされる子宮での上皮成長因子（Epidermal growth factor, EGF）の濃度異常を解消し、受胎性を回復させる効果を持つと考えられる精漿タンパク質の1つがオステオポンチンである。今年度は、このオステオポンチンを遺伝子組換えタンパク質として調製してEGF濃度の低下がみられるリピートブリーダー牛への投与を行い、その効果を検証した。また、オステオポンチンを豊富に含む生乳からもオステオポンチンを調製し、同様に効果を検証した。
組換えタンパク質は、大腸菌をベクターとして調製し、牛のオステオポンチンcDNAの塩基配列をもとに大腸菌ベクターに合わせたコドンの最適化を行った。予備的検討の結果からオステオポンチンの投与量は1 mgとし、発情日の人工授精実施後に腟深部に投与した。乳中オステオポンチンはイオン交換クロマトグラフィーにより調製し、組換えタンパク質と同様に1 mgを腟内に投与した。
その結果、いずれのオステオポンチンも50%から60％のリピートブリーダー牛で子宮でのEGF濃度異常を解消した。治療後のEGF濃度正常化率には両群間で差異はみられなかったが、治療処置3日後の子宮内膜EGF濃度は組換えタンパク質を投与した群で高かった。遺伝子組換えオステオポンチンを投与した牛では、多くがEGF濃度の正常範囲の高い領域を示したのに対し、乳中オステオポンチンを投与した群では、EGF濃度は異常値（低値）から連続して正常範囲内の全域に広く分布する傾向がみられた。
両オステオポンチンによる治療効果には、ホルモン製剤によるEGF濃度異常牛の治療でみられる農家あるいは牛群間での差異はみられず、全ての牛群でほぼ同等の効果がみられた。
日本学術振興会, 基盤研究(A), 北海道大学, 19H00964

乳牛の低受胎対策技術の実行可能性検証事業　　　　　　　　　　　　　　　
令和3年度日本中央競馬会畜産振興事業
2021年04月 - 2024年03月
栁川洋二郎;田上貴祥;
日本中央競馬会, 研究開発事業, 北海道大学大学院獣医学研究院, 研究代表者

精漿による子宮機能調節作用を利用した受胎促進技術の開発
科学研究費助成事業 基盤研究(B)
2016年04月01日 - 2019年03月31日
片桐 成二, 柳川 洋二郎, 藤井 貴志
本研究では不妊牛に見られる子宮での上皮成長因子（EGF）異常を解消する精漿中の有効成分を同定し、同定した蛋白を用いて投与を行うプロトコールを開発した。また、精漿によるEGF発現正常化の機序にリンパ節でのサイトカイン発現の変化が関与することを示した。さらに、肥満牛におけるEGF発現異常と牛の代謝状態の関係を検討し、EGF発現異常と糖負荷への反応性低下および血中テストステロン濃度増加とがこの病態に関係することも明らかにした。併せて子宮でのEGF発現異常による不妊症の経済的損失とその治療による経済効果を試算してモデル農場による本異常の治療効果がもたらす経済効果を試算し公表した。
日本学術振興会, 基盤研究(B), 北海道大学, 16H05032

乳牛の低受胎対策新規技術開発事業　　　　　　　　　　　　　　　
平成29年度日本中央競馬会畜産振興事業
2017年04月 - 2019年03月
永野昌志, 栁川洋二郎, 田上貴祥, Su Thanh Ling
日本中央競馬会, 研究開発事業, 北海道大学大学院獣医学研究院, 研究代表者

牛精漿中に含まれる子宮機能調節蛋白の同定とその作用経路の解析
科学研究費助成事業 基盤研究(C)
2011年 - 2013年
片桐 成二
腟内に投与した牛の精漿が子宮機能を調節し、受胎性を改善する機序を明らかにするため、精漿のシグナルが子宮に伝達される経路を調べた。その結果、精漿のシグナルはリンパ系路を介して内腸骨および深鼡径リンパ節に運ばれ、リンパ節でのサイトカイン環境を変化させることにより、子宮に伝達されることを示した。また、精漿中の蛋白質を分離・精製することにより、オステオポンチン類似の蛋白質に精漿による子宮機能調節効果のあることを示した。
日本学術振興会, 基盤研究(C), 酪農学園大学, 研究代表者, 23580446

高泌乳牛・低受胎における卵子成熟精動態の解明
科学研究費助成事業 基盤研究(C)
2009年 - 2011年
高橋 芳幸, 片桐 成二, 永野 昌志
現代の高泌乳牛にみられる繁殖性低下の原因となる卵子の成熟・受精の動態の特性・異常について検討、排卵直前の成熟卵子微細構造の特徴と排卵時期が確認され、体内における精子移送・貯蔵時間、排卵卵子の老化時期を推定できた。また、体外成熟卵子を用いて発生能獲期・喪失期の特徴を捉えるとともに、初期胞状卵胞卵子の個別発育培養法を開発した。さらに、腟温変動を指標にした排卵時期と異常排卵の予知法、精漿成分投与による低受胎牛の治療法を開発した
日本学術振興会, 基盤研究(C), 北海道大学, 21580339

国際標準プロトコールによる高産乳牛における妊娠喪失発生状況の解析
科学研究費助成事業 基盤研究(C)
2008年 - 2010年
片桐 成二
乳牛の不妊原因を特定するための診断および研究手法の標準化を実現するため、EUおよび北米の研究者との国際共同研究により、妊娠が中断する時期を特定するための国際標準プロトコールを開発した。また、この標準プロトコールを用いて実施した予備調査の結果から、わが国と海外の乳牛における胚死滅発生時期および頻度の違いを明らかにし、わが国が独自で開発し、重点的に実施しなければならない不妊症対策の領域を明らかにした。
日本学術振興会, 基盤研究(C), 酪農学園大学, 20580350

牛ウイルス性下痢ウイルス感染症の清浄化対策:コホート研究によるリスク分析
科学研究費助成事業 基盤研究(B)
2006年 - 2009年
田島 誉士, 門平 睦代, 片桐 成二, 小沼 操, 片桐 成二, 小沼 操
牛ウイルス性下痢ウイルス(BVDV)感染経路の分析ための疫学調査を、バルク乳検査によって陽性となった農家と陰性であった農家を無作為に抽出して実施した。分離ウイルスの分子系統樹解析結果からは、局地的に同一のBVDVの感染によって流行が生じている危険性が高いことが示された。また、各種疫学情報を各方面から入手し、リストアップされた因子についての危険度を統計学的にリスク分析した。その結果、牛の導入、牛舎内へのカラスや猫の侵入、などいくつかの危険因子が抽出された。(221 文字)
日本学術振興会, 基盤研究(B), 北海道大学, 18380184

子宮での細胞増殖因子発現異常検出によるリピートブリーダー牛の診断および治療法開発
科学研究費助成事業 基盤研究(C)
2003年 - 2005年
片桐 成二
酪農業に重大な経済被害をもたらすリピートブリーディングの対策を講じるため子宮機能異常による不妊牛の摘発および治療法を検討した。まず、子宮における主要な増殖因子とサイトカインの周期的変動を調べた。そのうち上皮成長因子(epidermal growth factor, EGF)は発情後2-4および13-14日目に明瞭なピークを持つ周期的変動を示し、リピートブリーダー牛の約70%ではこれらのピークが消失していることを明らかにした。また、この異常は治療処置を行わなければ持続し、治療処置により受胎したリピートブリーダー牛の80〜90%では、妊娠に先立ちEGF発現が正常化していることも明らかにして、EGF発現異常がリピートブリーダー牛の不妊原因の一つであることを示した。次いで、EGF濃度異常の治療法を検討し、リピートブリーダー牛に対しプロジェステロン徐放剤と高用量(5mg)の安息香酸エストラジオールを組み合わせた治療を施すと約70%の牛でEGF発現が正常化し、その内70-80%の牛は治療後2回までの授精で受胎することを示し、効果的な治療法開発に成功した。一方、授精待機牛においてはEGF発現が受胎性の指標となることを示し、子宮内膜EGF濃度測定を受精卵移植のレシピエント選抜に応用することで受精卵移植後の受胎率が向上することを示した。また、分娩後の子宮におけるEGFの周期的変動が回復する時期とその異常を来す機序を明らかにするため、分娩後の子宮内膜でのEGF発現に影響を及ぼす因子を調べた。その結果、EGFの周期的変動回復には少なくとも8日以上の黄体期の出現が必要であることが明らかとなり、産褥期に短い間隔で排卵を繰り返す牛および子宮回復前に長い黄体期の出現した牛ではEGF発現異常の頻度が高まることが示唆された。
日本学術振興会, 基盤研究(C), 北海道大学, 15580284

ウシ卵胞発育および排卵障害機構解析モデル(卵胞体外培養法)の開発
科学研究費助成事業 基盤研究(B)
2001年 - 2003年
高橋 芳幸, 永野 昌志, 片桐 成二
ウシの卵胞発育および排卵障害の実態を把握するとともに、その機構を体外で解析できる卵胞培養法の開発を目的として研究を行った。
まず、分娩後の乳牛の卵胞発育動態を調べた結果、卵胞発育ウェーブは分娩後4〜6日目頃から再開され、約半数のウシでは初回優勢卵胞が排卵せずに卵胞発育ウェーブを繰り返し、初回排卵の後の卵巣周期が短くなることが分かった。また、初回卵胞発育ウェーブの発現と分娩前後の血中卵胞刺激ホルモン(FSH)濃度を調べた結果、初回排卵の遅いウシでは分娩後の最初のFSH分泌が遅いために卵胞発育ウェーブの発現が遅延することが明らかになった。さらに、超音波誘導経膣採卵技術に改良を加えて分娩後早期の卵胞内卵子の受精能を調べた結果、妊娠末期に発育を停止していた卵胞に由来する卵子でも正常な発生能を保有していることも明らかになった。
卵胞培養法については、まず、マウス卵胞をモデルにして検討した結果、メンブラン・インサートを用いた培養法が優れていることを突きとめ、その培養法を用いてガラス化保存した前胞状卵胞に由来するマウス産子を世界で初めて得ることができた。また、インサート内で培養した前胞状卵胞の発育と機能を調べた結果、80%以上が成熟卵胞へ発育し、その約70%は人絨毛性性腺刺激ホルモン処理によって排卵し、かつ排卵卵子の約50%が胚盤胞へ発育することが確認された。さらに、培養卵胞を用いて顆粒層細胞におけるトランスフォーミング増殖因子の発現が卵胞の発育にとって重要な役割を果たしていることも解析することができ、この培養法が卵胞発育や排卵の障害機構の解析モデルとして利用可能なことも実証された。また、ウシの卵胞はコラーゲン溶液に包埋して培養した結果、一次卵胞の発育と初期胞状卵胞由来卵子の成熟が確認された。
日本学術振興会, 基盤研究(B), 北海道大学, 13460136

ウシの子宮内細胞増殖因子およびレセプターの発現異常による着床不全に関する研究
科学研究費助成事業 奨励研究(A)
1999年 - 2000年
片桐 成二
牛の卵巣および子宮に生理活性物質を投与するシステム(手術、カニュレーション法など)を検討した。その結果、卵巣動脈および子宮動・静脈を介して2〜3発情周期にわたり持続的または断続的に生理活性物質を投与でき、子宮静脈および卵巣動・静脈からの連続採血も可能なシステムを開発できた。また、この状態で牛を妊娠させ、授精後30〜45日目まで妊娠の維持が可能であることを確認した。
上記システムを用い、子宮静脈および卵巣動脈内にプロスタグランジン(PG)F_<2α>とPGE_2を種々の濃度で組み合わせて投与した結果、PGF_<2α>は一過性大量投与よりも、少量反復投与により、より確実に黄体退行を誘起することを確認した。さらに、PGF_<2α>単独の投与に比べ低濃度のPGE_2を同時に投与することにより、確実で急速な黄体退行を誘起できることを明らかにした。
上記試験と平行して、臨床の現場において原因が特定できなかった繁殖障害牛を中心に、発情周期内での子宮内膜における上皮成長因子(EGF)およびそのレセプターの発現と、子宮内膜でのEGF機能について検討した。不妊原因の精査後、リピートブリーダー牛と診断された牛では、EGFレセプター発現量の発情周期内変化に異常はみられないが、多くの例(約80%)で子宮内膜EGF濃度(遺伝子およびタンパク量)の周期的な変化が失われていることを明らかにした。発情後14〜17日目の対照牛からバイオプシーにより採取した子宮内膜組織では、EGFがPGF_<2α>/PGE_2産生量比を抑制するのに対し、リピートブリーダー牛から得られた材料ではEGFによるPGF_<2α>/PGE_2産生量比抑制効果の失われていることを明らかにした。
以上の結果から、リピートブリーダー牛ではEGFの発現および機能の異常により子宮でのPG産生調節機構に異常がみられ、黄体機能を維持できないため胚の死滅や着床不全により不妊となることが示唆された。
日本学術振興会, 奨励研究(A), 北海道大学, 11760188

牛の核移植(クローン)技術の実用化に関する総合的研究
科学研究費助成事業 基盤研究(B)
1998年 - 2000年
高橋 芳幸, 森安 悟, 永野 昌志, 片桐 成二, 岸 昌生
本研究では、割球および体細胞を用いた牛の核移植について検討を加えるとともに、卵子の凍結や卵胞培養も含めたクローン技術に関する総合的な研究を実施した。まず、割球を用いた核移植技術について種々の検討を加えた結果、桑実胚の割球を用いた核移植では、予め活性化処理を施した卵子を除核する効率的な除核方法を開発した。また、体外受精由来胚の割球から作出されなクローン胚の受胎率が体内受精由来胚を用いて作出した核移植胚に比べて低いことや、双子および四つ子のクローン牛の斉一な発育と雌牛の妊孕性が確認された。
体細胞核移植では、乳腺上皮細胞や卵管上皮細胞を用いて作出した核移植クローン胚の産子への発生が確認された。また、体細胞の培養処理やレシピエント卵子との細胞周期の組合せなどを検討し、飢餓培養あるいはコンフルエント培養処理した卵丘や繊維芽細胞の約95%がG0/G1期に同調しており、M期の除核卵子と融合させて作出した核移植胚の発生率が高いことが分かった。しかし、S期の除核卵子はS期に同調させた体細胞と融合させてもほとんど発生しないことも分かった。さらに、牛の核移植の実験で得られた知見をもとに豚の体細胞核移植胚を作出し、胚盤胞への発生を確認できた。
前胞状期卵胞をレシピエント卵子の供給源として活用する技術の開発では、牛の二次卵胞の採取・培養法と初期三次の発育培養法について検討し、初期三次卵胞の培養によりMII期の卵子の得られることや、二次卵胞の採取・培養法ならびに生存卵胞の選抜法に関する基礎的知見も得た。また、前胞状卵胞の低温保存法についてはマウスの卵胞を用いて検討を加え、ガラス化保存した前胞状卵胞由来の卵子が体外受精後に胚盤胞へ発生することが判明した。
日本学術振興会, 基盤研究(B), 北海道大学, 10556058