三浦 亮太朗

The objectives of this study were (1) to investigate the distribution of large (≥10 mm) follicle numbers during the estrous cycle and (2) to compare the timing of the estrus expression period after the ovarian examination between cows with one large follicle (1F) and two or more large follicles (2F) with functional corpus luteum (CL) at the ovarian examination in lactating Holstein dairy cows. In experiment 1, we performed 393 ovarian examinations by ultrasonography, addressed the existence of CL (≥20 mm) and large follicle numbers, and classified cows into 1F (n = 229) and 2F (n = 164) groups. The 1F appearance rates were beyond 75% each day during 3 to 12 d after estrus. However, 2F appearance rates were beyond 75% each day during 15 to 24 d after estrus. In experiment 2, we performed 302 ovarian examinations by ultrasonography and classified cows into the 1F (n = 168) and 2F (n = 134) groups. Estrus detection was performed for 24 d after the ovarian examination in each cow. In the 2F group, 75% of estrus occurred within 9 d of the ovarian examination. However, 75% of estrus occurred 10 d after the ovarian examination in 1F. Days from the ovarian examination to estrus were significantly shorter in the 2F (6.0 d; median, 7.2 ± 4.0 d; mean ± SD) than in the 1F (13 d, 12.4 ± 4.3 d) group. In conclusion, focusing on ≥10 mm follicle numbers with CL could be useful for predicting the estrus expression period.

The objectives of this study were to assess the sequential dynamics of the endometrial polymorphonuclear cells (PMN) after calving by endometrial cytology, and clarify the factors that cause prolonged endometrial inflammation in lactating dairy cows. A total of 33 lactating Holstein dairy cows were used from -4 to 8 wk relative to calving (0 wk: the calving week). Endometrial samples were obtained sequentially from 2 to 8 wk. Body condition score and backfat thickness were obtained weekly from -4 to 8 wk. Blood samples collected from -4 to 8 wk were analyzed for indicators of energy status, hepatic function, systemic inflammation, and calcium. Blood amino acids were measured at 2 wk. Daily milk production was determined between 5 and 65 d postpartum. Based on the sequential cytological analysis, the endometrial inflammation threshold was set at ≥5.0% PMN, and the median wk of PMN% lower than 5.0% was 4.5 wk in this study; therefore, we classified the cows into the early group (cows with endometrial inflammation converged within 4 wk: n = 17) and the late group (cows with endometrial inflammation converged at or after 5 wk: n = 16). There were no differences in daily milk production, energy status, hepatic function, blood calcium concentration, and systemic inflammatory response. The late group had lower body condition scores and backfat thickness during the experimental period, and a higher blood concentration of 3-methyl histidine, indicating muscle breakdown, was observed in the late group at 2 wk. Our findings indicated that the lack of body fat reservation during the peripartum period and the increased muscle breakdown after calving were risk factors for prolonged endometrial inflammation.

Trypanosoma theileri is considered a non- or low-pathogenic trypanosome that generally causes latent infection in apparently healthy cattle; however, T. theileri propagates in the bloodstream and may cause clinical disease in pregnant animals or co-infection with bovine leukemia virus or Theileria orientalis. In the current study, a monthly survey of T. theileri infection over one year was carried out in a research dairy farm in Hokkaido, Japan to determine the 1) seasonal variations in the prevalence, 2) genetic characterization of T. theileri, and 3) associations of milk and blood parameters in dairy cattle with T. theileri infection, including data of metabolic profile tests and dairy herd performance tests, using linear mixed models. We found that 1) the prevalence of T. theileri infection was significantly higher in summer and winter than in other seasons; 2) T. theileri possibly showed genetic diversity in Eastern Hokkaido; and 3) T. theileri infection was associated with significantly lower levels of blood urea nitrogen, milk protein, and solids-not-fat, which are caused by a low rumen fermentation level. This is the first study to report the negative impact of T. theileri infection in dairy cattle, and our study indicates that control of T. theileri infection can improve the productivity of dairy cattle.

There was examination of effects of accessory corpus lutea (CLs) formation and regression during pregnancy on circulating progesterone (P4) concentrations and pregnancy maintenance in beef heifers. Heifers (Experiment 1, n = 75; Experiment 2, n = 496) were randomly assigned to either a human chorionic gonadotropin (hCG) treatment or untreated group 5 days post-estrus, followed by embryo transfer (ET) on Days 6-8 (Day 0 = Estrus). In Experiment 1, blood samples were collected from pregnant heifers on Days 33, 40, and 47 for conducting P4 assays. Plasma P4 concentrations were greater in hCG-treated heifers than in untreated heifers on Day 33. In hCG-treated heifers with accessory CL regression between Days 33 and 47, plasma P4 decreased to concentrations similar to those of untreated heifers after Day 40. In hCG-treated pregnant heifers in Experiment 2, CL regression by Day 50 of gestation was more frequent when CLs were contralateral (49.3 %) rather than ipsilateral (4.4 %, P < 0.001) to the original CL. The hCG treatment resulted in a greater pregnancy percentages on Days 30 (80.5 % and 68.6 %, P = 0.002) and 50 (76.2 % and 65.3 %, P = 0.007) compared with untreated heifers. There, however, were no differences in either pregnancy percentages on Days 30 and 50 or pregnancy losses between hCG-treated heifers with ipsilateral and contralateral accessory CLs. These results indicate accessory CL formation improves pregnancy percentages resulting from ET. Furthermore, plasma P4 decreases associated with accessory CL regression does not affect pregnancy loss in beef heifers.

<title>Abstract</title>Bovine-derived cultured cells, including Madin-Darby bovine kidney cells, are used worldwide; however, lipofection tend to result in low transfection efficiency, which has impeded the progress of veterinary research. We performed experiments to confirm the lipofection efficiency of bovine-derived cultured cells, to identify cells that suitable for lipofection. Several bovine tissues (endometrium, testis, ear tissue and foetal muscle) were collected, and primary cultured cells were prepared. Lipofection assay showed that only bovine endometrium (BE)-derived cells could be transfected efficiently (50‒70%). BE cells can be divided into at least two types of cell populations (BE-1 and BE-2). The BE-1 cells, which were suitable for lipofection, were obtained by passages at short intervals and were negative for cytokeratin- and positive for vimentin-expression; the BE-2 cells did not have these characteristics and were not suitable for lipofection. Furthermore, the BE-1 cells and artificially immortalised cells of BE-1, iBE-1 cells, were utilised in a reporter assay requiring the introduction of multiple DNAs. Endometrial tissues can be collected from living cows, and BE-1 cells can be obtained easily by controlling passaging timing. The production of BE-1 cells and sharing the methods required to prepare them will contribute to the development of veterinary research.

<title>Abstract</title><sec> <title>Background</title> Repeat breeding is a critical reproductive disorder in cattle. The problem of repeat breeder cattle remains largely unmanageable due to a lack of informative biomarkers. Here, we utilized metabolomic profiling in an attempt to identify metabolites in the blood plasma and uterine luminal fluids. We collected blood and uterine fluid from repeat breeder and healthy cows on day 7 of the estrous cycle. </sec><sec> <title>Results</title> Metabolomic analysis identified 17 plasma metabolites detected at concentrations that distinguished between the two groups, including decreased various bile acids among the repeat breeders. However, no metabolites that varied significantly were detected in the uterine luminal fluids between two groups. Among the plasma samples, kynurenine was identified as undergoing the most significant variation. Kynurenine is a metabolite produced from tryptophan via the actions of indoleamine 2,3-dioxygenase (IDO). As IDO is key for maternal immune tolerance and induced in response to interferon tau (IFNT, ruminant maternal recognition of pregnancy factor), we examined the responsiveness to IFNT on peripheral blood mononuclear cells (PBMC) isolated from healthy and repeat breeder cows. The mRNA expression of IFNT-response makers (<italic>ISG15</italic> and <italic>MX2)</italic> were significantly increased by IFNT treatment in a dose-dependent manner in both groups. Although treatment with IFNT promoted the expression of <italic>IDO</italic> in PBMCs from both groups, it did so at a substantially reduced rate among the repeat breeder cows, suggesting that decreased levels of kynurenine may relate to the reduced IDO expression in repeat breeder cows. </sec><sec> <title>Conclusions</title> These findings provide valuable information towards the identification of critical biomarkers for repeat breeding syndrome in cattle. </sec>

We investigated changes in cortisol (COR) concentration, which is well known as an index of stress in the serum of dairy cattle. The COR concentrations in serum obtained from dairy cattle were collected during practical training of first-year students on a farm attached to the Nippon Veterinary and Life Science University. Mean COR concentration in serum determined after practical training was significantly higher (P &lt 0.001) than that in serum collected before training. Discriminant analysis was used to classify the relation between COR concentration of serum collected before and after practical training. In conclusion, the data was bipartite according to the percentage of rise (rise rate) of COR concentration. Although the percentage of the rise was more than 300% in the high-rise-rate group, there was a significant negative correlation (P &lt 0.05) between age and COR concertation. It was thought that the high-rise-rate group has a chance to decrease stress after more experience. In contrast, the low-rise-rate group included 3 cattle indicating high COR concentration before and after practical training. Those 3 cattle were thought to be stressed easily. It is suggested that there was individual difference to stress.

In this study, we examined the locational effect (left or right ovary) of the preovulatory follicle (PF) on fertility in dairy heifers. In total, 1,111 artificial inseminations (AI) were analyzed. At AI, PF locations were examined using rectal palpation, and heifers were divided into two groups on their PF locations: (i) the PF located in the left ovary (L-PF) and (ii) the PF located in the right ovary (R-PF). Pregnancy was diagnosed by rectal palpation 60 days after AI. The conception rate was 50.7% in all heifers. Conception rate was significantly higher in the L-PF (60.1%) than in the R-PF (46.2%). The conception rate was significantly lower by sexed semen (48.6%) than conventional semen (59.1%). Conception rates divided by the semen type (sexed: n = 896, conventional: n = 215) were significantly higher in the L-PF than in the R-PF for both semen types (sexed L-PF vs. R-PF: 57.3% vs. 44.4%, conventional L-PF vs. R-PF: 72.3% vs. 53.3%). In addition, season, age, AI number, and the number of re-inseminations at the same estrus did not affect conception rates. In summary, PF development in the left ovary was associated with increased conception rates in dairy heifers.

In the present study, the ventral tail base surface temperature (ST) was monitored using a wearable wireless sensor for estrus detection in cattle. Relationships among ST, behavioral estrus expression, ovulation, and changes in hormone profiles during the estrous cycle were examined. Holstein Friesian or Japanese Black female cattle were used in summer (August September), autumn (October November) and winter (January February; three animals per season). On Day 11 of the estrous cycle (Day 0 = the day of ovulation), the sensor was attached to the surface of the ventral tail base and ST was measured every 2 min until Day 11 of the next estrous cycle. Hourly maximum ST values were used for analysis. To exclude circadian rhythm and seasonal effects, ST changes were expressed as residual temperatures (RT = actual ST mean ST for the same hour on the previous 3 days). Obvious circadian rhythms of the ST were observed and daily changes in the ST significantly differed among seasons. There was no significant seasonal difference, however, in the RT. The mean RT increased significantly similar to 24 compared with similar to 48 h before ovulation. The mean maximum RT was 1.27 +/- 0.30 degrees C, which was observed 5.6 +/- 2.4 h after the onset of estrus, 2.4 +/- 1.3 h before LH peak, and 26.9 +/- 1.2 h before ovulation. The ST of the ventral tail base could be monitored throughout the estrous cycle and could detect a substantial change around the time of expression of behavioral estrus. Calculation and analysis of the RT could be useful for automatic estrous detection. (C) 2017 Elsevier B.V. All rights reserved.

Breeding is an important part of all livestock farming. Accurately detecting estrus, the period during which insemination should occur, is critical in order to maintain production and profit. However, conventional estrus detection depends on ocular inspection of the animals by skilled labour and this practice is expensive and relatively inefficient. Here, a wireless intravaginal probe for cattle capable of automatizing the process based on measurements of conductivity and temperature as well as movement sensing is presented and tested in-situ. These parameters can all be used independently to detect estrus. A good conformity between the data collected with this probe and established estrus patterns is demonstrated. Furthermore, the magnitude of natural daily variations and their impact on the individual parameters are discussed together with the impact of extraordinary events such as stress. Compared to existing alternatives, a multi-parameter approach like this is shown to be capable of much higher reliability, and also to be much more resistant to disturbances. The demonstrated system is very power efficient and capable of years of continuous isolated operation. Small to intermediate farm environments can be covered by the probe transmitters themselves with a single receiver unit, while bigger areas are handled with battery powered repeater units. (C) 2016 Elsevier B.V. All rights reserved.

The objective of our study was to compare the characteristics of the corpus luteum (CL) formed after ovulation of the dominant follicle (DF) of the first follicular wave (W1) and those of the CL formed after ovulation of the DF of the second (induced) follicular wave (W2). Non-lactating Holstein cows were used for this study. In Experiment 1, cows were treated with PGF2α and GnRH on days 6 and 8 (day 0 = day of follicular wave emergence) for W1 (n = 6) and W2 (n = 6), respectively. Dominant follicles were aspirated on day 9 to quantify the amounts of mRNA (VEGF120, VEGF164, FGF-2, StAR, P450-scc and 3β-HSD) in granulosa cells (GC). In Experiment 2, the size and blood flow area of the CL formed after ovulation of the DF in W1 (W1CL n = 6) and W2 (W2CL n = 6) (the day of DF ovulation in W1 and W2 was day 10) were evaluated on days 12, 15, 18 and 21. The plasma P4 concentration was measured on days 10 to 21. The amounts of VEGF164, P450-scc and 3β-HSD mRNA were higher (P &lt 0.05) in the DF in W1, and those of VEGF120,FGF-2 and StAR mRNA tended to be higher (P &lt 0.1) in the DF in W1. The size of the CL was greater in the W1CL on days 15, 18 and 21. The blood flow area of the CL was greater in the W1CL on days 12 and 15. The plasma P4 concentrations were higher in the W1CL. These results indicate that the CL formed after ovulation of the DF in W1 was greater in terms of size, blood flow and plasma P4 concentration.

【目的】近年，ウシの繁殖成績低下が問題となっており，低い発情発見率および適期授精が行われていないことが要因の一つと考えられる。過去の報告から，ウシにおいて発情時に一過性の体温上昇が確認されている。今回，小型の無線式体表温測定センサをウシの尾根部腹側に装着，体表温を連続測定し，発情および排卵時期との関連性を調査した。【方法】実験には，黒毛和種雌牛4頭およびホルスタイン種未経産牛3頭を用いた。発情周期の10日目に体表温センサを尾根部腹側に装着し，体表温を2分間隔で測定した。卵巣の超音波検査は1日1回，採血は1日2回行った。黄体退行が開始した後，3 h間隔で試情により発情を確認し，発情終了時点から1 h間隔で超音波検査により卵巣を観察して排卵時期を特定するとともに，この間3 h間隔で採血を行った。得られた血漿はTR-FIA法により血中LH濃度を測定した。体表温計測値は30分ごとの最高温を抽出し，解析に用いた。体表温に日内変動が観察されたため，それぞれの時間の前3日間の同時刻の平均に対する差（平均日内変動値との差）を算出し，発情開始時期，LHサージおよび排卵時期との関連性を調べた。【結果】体表温センサのデータ取得率は90.7±5.0％（平均±標準誤差）であった。発情開始およびLHサージのピークから排卵までの時間は，それぞれ28.1±0.8 hおよび23.1±0.9 hであった。平均日内変動値との差が最大値（1.7±0.4℃）に達した5.5±7.9 h後に発情が開始し，9.3±7.8 h 後にLHサージのピークが認められた。平均日内変動値との差が最大値を示してから排卵までの時間は33.7±7.8 hであった。【考察】尾根部腹側の体表温を連続計測する侵襲性の極めて低いセンシング技術を用いて，体温変動をモニタリングできることが判明した。また，平均日内変動値との差が最大となる時点を算出することで，客観的な数値変動に基づいて排卵時期を予測できる可能性が示された。 本研究は，戦略的イノベーション創造プログラム（次世代農林水産業創造技術）により実施された。

A probe for wireless monitoring of the vaginal conductivity and temperature in cows has been developed, manufactured, and tested in-situ for approximately 48 hours. The cow was filmed during testing, and events and activities correlated with the obtained data. This allowed systematic variations due to stress, position and activity, as well as circadian patterns, to be identified. The probe has three electrode pairs for conductivity measurements that are probed consecutively, which allow electrode position related variations to be investigated. One of the main applications of this type of sensor is automated estrus detection, which is of high economic importance to farmers. Prior work has mainly relied on manual and/or implanted probes, both of which are inconvenient for commercial applications, and the collected data has usually consisted of a relatively limited number of points. This probe collects data from multiple positions at user defined intervals for extended periods of time, thus giving a much more complete picture of the behavior of the investigated quantities.

In this study, we examined the effect of the locations of the first-wave dominant follicle (DF) and corpus luteum (CL) on fertility. In total, 350 artificial insemination (AI) procedures were conducted (lactating dairy cows: n = 238, dairy heifers: n = 112). Ovulation was confirmed 24 h after AI. The locations of the first-wave DF and CL were examined 5 to 9 d after AT using rectal palpation or transrectal ultrasonography. Lactating dairy cows and dairy heifers were divided into 2 groups: (1) the ipsilateral group (IG), in which the DF was ipsilateral to the CL; and (2) the contralateral group (CG), in which the DF was contralateral to the CL. Pregnancy was diagnosed using transrectal ultrasonography 40 d after AI. Conception rates were 54.0% in all cattle: 48.9% in lactating dairy cows, and 58.9% in dairy heifers. The incidence of the first-wave DF location did not differ between IG and CC (all cattle: 184 vs. 166; lactating cows: 129 vs. 109; heifers: 55 vs. 57 for IC vs. CG). Conception rates were lower in IG than in CG (all cattle: 40.2 vs. 69.3%; lactating dairy cows: 38.0 vs. 67.0%; dairy heifers: 45.5 vs. 73.7%, for IC vs. CG). Conception rate was not affected by season or live weight in heifers and lactating cows. In addition, days in milk at AI, milk production, body condition score, and parity did not affect conception in lactating cows. In summary, development of the first-wave DF in the ovary ipsilateral to the CL was associated with reduced conception rates in both lactating cows and heifers.

To clarify the wave (DF-1) after spontaneous ovulation and DF in second follicular wave (DF-2) and after induced ovulation of the first-wave DF by GnRH were examined in non-lactating Holstein cows. Follicular maturation of DF-1 and DF-2 were induced by PGF2 alpha and GnRH treatment on Day 6 and 8 (Day 0 = Day of follicular wave emergence), respectively. Follicular growth and blood flow (BF) in the follicular wall of DF-1 and DF-2 were examined. To analyze sex steroids in follicular fluid (FF) and amount of mRNA in granulosa cells, DF-1 and DF-2 were aspirated on Day 8 or 9 in different estrous cycle. Diameter in DF-1 was larger than DF-2 on Day 8 and 9. From Day 8 to 9, BF area (BFA) and percentage of the follicular wall with BF, which represents the degree of distribution of BF, increased in DF-1 but not in DF-2. BFA per length of follicle circumference with BF, which represents the thickness of BF, was not different between DF-1 and DF-2. Concentrations of 17 beta-estradiol (E2) in plasma, E2 and androstendione in FF and amounts of LH receptor mRNA were greater in the DF-1 on Day 8. Gene expression for steroidogenesis, prostaglandin synthesis and angiogenesis did not differ between DF-1 and DF-2. These results indicated that DF-1 were more active than DF-2 in growth, BF supply and steroidogenesis. The greater BFA observed in the DF-1 may be derived from as a result of the greater vascularity in the follicular wall. (C) 2014 Elsevier B.V. All rights reserved.

In postpartum dairy cows, various inflammatory diseases depress reproductive performance. Lipopolysaccharide (LPS) derived from infections of the uterus or mammary gland with Gram-negative bacteria was shown to suppress steroid production in the granulosa cells of follicles in vitro. The aim of the study was to investigate the relationship between LPS in ovarian follicular fluid and steroidogenesis by the theca and granulosa cells of the large follicles in vivo. Bovine ovaries were collected from a slaughterhouse, and the largest (F1) and the second largest (F2) follicles were used (&gt;8 mm in diameter, n=38). LPS concentration in the follicular fluid was measured using quantitative kinetic assay. Follicular steroidogenesis was evaluated by measuring the estradiol (E2) and progesterone (P4) concentration in follicular fluid and by analysing transcription levels of steroidogenesis-related genes in theca and granulosa cells. LPS concentration detected in follicular fluid ranged from 0.2 to 2.0 EU/mL. In follicles with a high level of LPS (&gt;0.5 EU/mL, n = 15), the concentration of E2 was lower and that of P4 was higher when compared to those in follicles with a low level of LPS (&lt;0.5 EU/mL, n = 23), which was observed both in F1 and F2 follicles. Furthermore, in follicles with a high level of LPS, transcripts of steroidogenic enzymes such as CYP17 and P450arom were lower. In those follicles, the expression of caspase-3 was high, suggesting an association with follicular atresia. These findings indicate that LPS present in follicular fluid may cause ovarian dysfunction by inhibiting follicular activity. (C) 2013 Elsevier B.V. All rights reserved.

The objective of this study was to examine whether an oxytocin challenge test (OCT), evaluated by measuring the changes in uterine blood flow using transrectal colour Doppler ultrasonography, is a suitable non-invasive method to determine uterine contractility in puerperal dairy cows. The changes in uterine blood flow during uterine contractions induced by oxytocin were evaluated on days 2 and 5 postpartum (pp). Twelve clinically healthy Holstein cows were randomly assigned into two groups: (1) oxytocin group (n= 7), 50. IU oxytocin injected IM and (2) control group (n= 5), 5. mL saline injected IM. Blood flow volume (BFV) and pulsatility index (PI) in the uterine arteries were determined before and after injection for 120. min on days 2 and 5 pp.BFV declined and PI increased rapidly after oxytocin injection on day 2 (P&lt . 0.05), whereas oxytocin on day 5 pp did not cause changes in blood flow parameters. The result confirmed that uterine responsiveness to oxytocin decreases with time postpartum in healthy cows. The same OCT was applied in cows with retained fetal membranes (n= 6) on day 2 pp, however uterine blood flow showed no change after oxytocin injection. The results showed that an OCT on day 2 pp may be a useful method for investigating the uterine contractile response to oxytocin (reflected as the decrease of uterine blood flow) and the potential pathophysiology of uterine involution in cows. © 2012 Elsevier Ltd.

【目的】ウシでは排卵直後に新たな卵胞波が形成され，これは第1卵胞波と呼ばれるが，この第1卵胞波が繁殖生理や受胎性に与える影響はよく分かっていない。そこで，今回，第1卵胞波主席卵胞が黄体と同一卵巣内に共存するまたは共存しないということが，受胎率にどのように影響を与えるかを検討した。【方法】試験1では，帯広畜産大学畜産フィールド科学センターにおいて，2008年3月～2009年6月に，人工授精後1日以内に排卵が確認され，排卵後5から9日の間に黄体と第1卵胞波主席卵胞が確認された，経産泌乳牛（71頭）および未経産牛（43頭）のデータを用いた。全授精頭数に対する受胎頭数（総受胎率），経産泌乳牛授精頭数に対する受胎頭数（経産受胎率），未経産牛授精頭数に対する受胎頭数（未経産受胎率）を調べた。また，黄体が存在する卵巣に第1卵胞波主席卵胞が共存する牛（共存群：54頭）の受胎率および共存しない牛（非共存群：60頭）の受胎率を調べた。試験2では，2010年3～7月に，経産泌乳牛（14頭）を用い，排卵確認日から3，6および12日目に採血と超音波画像診断装置を用いて卵巣の観察を行い，血中プロジェステロン（P4）濃度および黄体と第1卵胞波主席卵胞との共存および非共存を確認した。【結果】総受胎率57.0％，経産受胎率47.9％，未経産受胎率72.1％であった。非共存群（72.2％）が共存群（40.4％）に比べ有意に高い受胎率を示した（P＜0.01）。また経産牛と未経産牛別で検討したとき，双方とも非共存群で有意に高い（P＜0.05）受胎率を示した（経産牛；非共存群：62.1％ vs 共存群：27.6％，未経産；非共存群：84.0％ vs 共存群：55.6％）。血中Ｐ4濃度は排卵確認日から3日目において非共存群で有意に高く（P＜0.05），6および12日目では差はなかった。【考察】人工授精後における，同一卵巣内での第1卵胞波主席卵胞と黄体共存の有無は，その後の受胎性に大きく関与することが示され，その要因の1つとして主席卵胞が黄体と共存することで黄体のＰ4産生能に影響を与えることと示唆された。

【目的】乳牛の子宮内膜炎は分娩後の細菌感染によって引き起こされ，繁殖障害の主要な要因となっている。起因菌であるグラム陰性細菌の細胞壁構成成分であるLPSは，卵胞発育を制御するゴナドトロピンの分泌を阻害し，卵巣機能を障害することが報告されている。さらに，子宮内膜炎罹患牛ではその卵胞液中から高濃度のLPSが検出されており，LPSが卵巣機能に直接影響を及ぼす可能性が示唆されている。本研究では，卵胞液中のLPSが卵胞のE2産生に与える影響を検証した。【方法】食肉処理場よりウシ卵巣を採取し，直径8 mm以上の大卵胞を解析に用いた。［実験1］卵胞液中のLPS濃度を測定し，高LPS卵胞（0.5 EU/ml以上）と低LPS卵胞（0.5 EU/ml未満）に分類した。それぞれの卵胞において卵胞液中のE2濃度を測定，さらに卵胞膜細胞および顆粒層細胞でのステロイドホルモン産生関連遺伝子の発現を解析した。［実験2］LPSが卵胞のE2産生に与える影響を細胞レベルで調べるために，E2産生の基質となるアンドロステンジオン（A4）を産生する卵胞膜細胞に着目した。卵胞膜細胞をLPSで処理し，A4産生量およびA4産生関連遺伝子の発現解析を行った。【結果】［実験1］高LPS卵胞の卵胞液中E2濃度は，低LPS卵胞に比べ有意に低かった。また，顆粒層細胞のP450arom遺伝子および卵胞膜細胞のCYP17遺伝子の発現は，高LPS卵胞で有意に低かった。さらに，LH受容体（LHr）遺伝子の発現低下も認められた。［実験2］培養96時において，LPSは卵胞膜細胞でのA4産生を抑制した。また，LPS処理によりCYP17遺伝子およびLHｒ遺伝子の発現が有意に減少した。【考察】高LPS卵胞のE2産生抑制は，顆粒層細胞のE2産生阻害に加え，LPSによる卵胞膜細胞のA4産生の阻害によって引き起こされることが明らかとなった。本研究より，卵胞液中のLPSによる局所的な卵胞機能の抑制が，乳牛の子宮内膜炎における卵巣機能障害の一因となっている可能性が示された。

We conducted this study to analyze apoptotic changes in the bovine placentome at spontaneous and induced parturition. Cows delivered i) after the administration of dexamethasone followed by prostaglandin F-2 alpha and estriol, ii) after the administration of prostaglandin F-2 alpha and estriol or iii) spontaneously. Prepartum changes in plasma progesterone and estradiol-17 beta concentrations were similar between spontaneous and induced parturition. Messenger RNA of BCL2-related protein A1 (BCL2A1), an antiapoptotic gene, was expressed by trophoblast binucleate cells and caruncular epithelial cells. Quantitative RT-PCR showed that the expression of BCL2A1 mRNA in cotyledonary and caruncular portions was significantly lower in spontaneous parturition than induced parturition. The expression of BCL2-associated X protein (BAX) mRNA, a proapoptotic gene, was significantly higher in cotyledons at spontaneous parturition than parturition induced without dexamethasone. Caspase-3 (CASP3) mRNA and pre-activated CASP3 protein were predominantly detected in caruncular epithelial cells regardless of how parturition proceeded. Activated CASP3 protein was found in trophoblast uninucleate cells and binucleate cells rather than caruncular epithelial cells. In spontaneous parturition, intense staining of activated CASP3 was detected in caruncular epithelial cells. Spontaneous and dexamethasone-induced parturition increased apoptotic cells in the placentome compared with parturition induced without dexamethasone. The number of binucleate cells was significantly decreased in spontaneous parturition. The present results suggest that although the clinical dose of dexamethasone induces apoptosis in the placentome at term, neither dexamethasone nor prostaglandin F-2 alpha evoke normal physiological changes in the placentome during delivery such as a change in the balance of apoptosis-related genes and disappearance of binucleate cells.

In peripartum dairy cows, insulin resistance (IR) increases to adjust the direction of energy to lactation after calving. To investigate the effect of prepartum IR on postpartum reproductive performance, the insulin tolerance test (ITT) was applied to 15 cows at 3 weeks (Pre21) and 10 clays (Pre10) before the predicted calving date. Blood glucose area under the curve (AUC(glu)) within 120 min after administration of 0.05 IU/kg-BW insulin was calculated. The occurrence of first ovulation, days to first artificial insemination (Al) and first Al conception rate were recorded. Nutritional status postpartum was evaluated by blood chemical analysis. Based on AUC(glu) changes from Pre21 to Pre10, cows were classified into either the AUC-up group (AUC(glu) increase, n=5) or the AUC-down group (AUC(glu) decrease, n=10). There was no difference in the decrease in blood glucose at 30 min after insulin injection between groups, although glucose recovery from 30 to 60 min during the ITT was slow at Pre10 in the AUC-up group. The AUC-up group had a higher number of days to first Al and high glucose, total protein, globulin, gamma-glutamyltransferase, triacylglycerol levels and a low albumin-globulin ratio at the 14th day postpartum. The present study infers that prepartum slow glucose recovery rather than insulin sensitivity might increase the potential for subclinical health problems postpartum and thus suppress reproductive performance. During the prepartum transition period, glucose dynamics in the ITT can be considered as a new indicator for the postpartum metabolic status and reproductive performance of dairy cows.

【目的】我々は以前の報告で，黄体形成期または開花期に発育させた卵胞に対して排卵誘起を施した場合，形成期に発育した卵胞においてLHサージ後の卵胞壁における血流分布の上昇が大きいことを示した。今回，排卵直前の卵胞壁での血流分布の違いを産み出す要因および黄体形成期あるいは開花期に発育させた排卵前卵胞の機能の差異について調べるために，顆粒層細胞における各種因子のmRNA発現量を測定した。【方法】非泌乳牛（黄体形成期群：n=7，黄体開花期群：n=7）を実験に供した。黄体形成期群では第1卵胞波に着目し，D7（排卵確認日：D1）にPGF_2&alpha;，D9にGnRHを投与し，D10に主席卵胞を吸引し顆粒層細胞を回収した。黄体開花期群ではD6に新たな卵胞波誘起を目的にGnRHを投与し，36時間後のD8に排卵確認した。D14（排卵確認日：D8）にPGF_2&alpha;，D16にGnRHを投与し，D17に卵胞を吸引し顆粒層細胞を回収した。顆粒層細胞からtotal RNAを抽出し，real-time PCR法を用いてmRNA発現量を測定した。血流分布に関わる因子として血管拡張性因子である一酸化窒素合成酵素（iNOS，eNOS）の発現を調べた。卵胞の機能に関わる因子としてLH受容体（LHr），COX-2，PGES，VEGF120，VEGF164，StAR，P450-sccおよび3&beta;-HSDの発現を調べた。【結果】LHｒ，PGESおよびiNOSのmRNA発現量は両群で差はなかった。COX-2，eNOS，VEGF120およびP450-sccのmRNA発現量は黄体形成期群で高い傾向がみられた。VEGF164，StARおよび3&beta;-HSDにおいて，mRNA発現量が黄体形成期群で開花期群に比較して有意に高かった。【考察】卵胞の血流分布に顆粒層細胞のiNOSおよびeNOSは関与しないことが示唆された。黄体形成期群で血管新生因子のVEGF164およびステロイド合成酵素のStAR，3&beta;-HSDの発現量が多いことから，排卵後の黄体形成やプロジェステロン合成能が高まることが示唆された。以上のことから，黄体形成期と開花期で発育する卵胞を比較したところ，LHサージ後の卵胞の機能に差異があることが明らかになった。