Makoto Hirako

Evidence of melatonin synthesis in the cumulus oocyte complexes and its role in enhancing oocyte maturation in vitro in cattle

Melatonin is a multifunctional molecule that mediates several circadian and seasonal reproductive processes. The exact role of melatonin in modulating reproduction, however, is not fully understood—especially its effects on the ovarian follicles and oocytes. This study was conducted to investigate the expressions of the ASMT and melatonin‐receptor MTNR1A and MTNR1B genes in bovine oocytes and their cumulus cells, as well as the effects of melatonin on oocyte nuclear and cytoplasmic maturation in vitro. Cumulus‐oocyte complexes (COCs) from abattoir ovaries were cultured in TCM‐199 supplemented with melatonin at concentrations of 0, 10, 50, and 100 ng/ml. The expression of ASMT, MTNR1A, and MTNR1B genes was evaluated by RT‐PCR. Moreover, the effects of melatonin on cumulus cell expansion, nuclear maturation, mitochondrial characteristics and COCs steroidogenesis were investigated. Furthermore, the level of reactive oxygen species (ROS) was evaluated in denuded oocytes. Our study revealed that ASMT and MTNR1A genes were expressed in COCs, while the MTNR1B gene was expressed only in oocytes. Additionally, melatonin supplementation at 10 and 50 ng/ml to in vitro maturation medium significantly enhanced oocyte nuclear maturation, cumulus cell expansion and altered the mitochondrial distribution patterns, but had no effects on oocyte mitochondrial activity and COCs steroidogenesis. Melatonin‐treated oocytes had a significantly lower level of ROS than controls. The presence of melatonin receptors in COCs and its promoting effects on oocyte nuclear and cytoplasmic events, indicate the potentially important roles of this hormone in regulating bovine oocyte maturation. Moreover, the presence of ASMT transcript in COCs suggests the possible involvement of these cells in melatonin biosynthesis. Mol. Reprod. Dev. 78:250–262, 2011.

Effect of fetal mass, number and stage of gestation on pregnancy-specific protein B concentrations in the bovine

In this study we characterized the peripheral plasma pregnancy-specific protein-B (PSPB) profile throughout gestation and examined the effect of stage of gestation, fetal mass and number on this profile in Holstein cows after non surgical embryo transfer. Cows (n = 12) were divided into 2 groups: Group 1 = single embryo recipient cows (n = 5), Group 2 = twin-embryo recipient cows (n = 7). Blood was collected approximately every third day from Day 0 (Day 0 = first day of standing estrus), then daily for the last 10 d of gestation, and sampling was stopped 1 d post partum. Two twin-embryo recipient cows had abnormal pregnancies; therefore, their data were excluded from the group. The time trend concentrations of plasma PSPB were significantly affected by the stage of gestation (P < 0.001) and fetal number (P < 0.001). In both groups PSPB increased gradually, with the mean levels being significantly higher (P < 0.01) in the twin-bearing group from Day 50 onwards (0.7 +/- 0.2 vs 9.2 +/- 4.5 ng/ml, singleton and twin-bearing cows, respectively) except for Day 10 pre-partum. By mid-gestation (Day 140), mean PSPB levels increased in the singleton (P < 0.001) cows by thirty-fold (21.2 +/- 3.2 ng/ml) as opposed to a ten-fold (98.4 +/- 13.2 ng/ml) increase in the twin-bearing (P < 0.001) group. The mean PSPB concentrations between Days 30 to 20 prepartum dramatically increased by about 700 to 200% in singleton (128.8 +/- 46.3 to 745.6 +/- 66.7 ng/ml) and twin-bearing cows (375.6 +/- 130.4 to 861.5 +/- 127.9 ng/ml), respectively. The PSPB levels between Day 10 prepartum to parturition were significantly higher (P < 0.001) in the twin-bearing group than in the singleton group (745.6 +/- 66.7 to 1627.4 +/- 238.9 ng/ml vs 861.5 +/- 127.9 to 3103.0 +/- 643.0 ng/ml in singleton and twin-bearing groups, respectively). Calf birthweight was correlated (P < 0.01) to peripheral PSPB concentration in singleton cows; however, this relationship decreased with the subsequent increase in fetal number. Cows giving birth prematurely to stillborn calves or to a schistosomus reflexus calf exhibited abnormal PSPB profiles. These results indicate that peripheral PSPB levels are correlated to the stage of gestation and fetal number. In addition, the peripheral pattern of PSPB is a valuable guage for predicting fetoplacental viability.

Peripheral changes in estrone sulfate concentration during the first trimester of gestation in cattle: comparison with unconjugated estrogens and relationship to fetal number

Estrone sulfate originates mainly in the conceptus during gestation in cattle. Its concentration in maternal body fluids is a useful indicator of placental function. The objective of this study was to determine the profiles of estrone sulfate during early gestation in singleton and twin bearing cows using a newly developed extraction method. One or two blastocysts produced in vitro were nonsurgically transferred to regularly cycling Holstein cows on Day 7 (Day 0 was defined as the first day of standing estrus). Pregnancy was diagnosed on Days 30, 45 and 60 by transrectal ultrasonography and finally confirmed at parturition. Six cows with singleton and six with twin pregnancies were used in the experiment. Blood was collected every other morning by jugular venipuncture from the day after transfer to Day 100. Harvested plasma was applied to reversed-phase C18 cartridges. Estrone sulfate and unconjugated estrogens (estrone and estradiol-17beta) retained in the cartridge were eluted separately by methanol stepwise gradient and each measured by validated radioimmunoassay. On average, estrone sulfate concentrations fluctuated between 2 and 6 pg/ml until Day 50 in both groups and then gradually increased. However, the levels of estrone and estradiol- 17beta remained low (1-5 pg/ml) until Day 80. The concentration of estrone sulfate after Day 50 was significantly affected by the day of gestation (P < 0.0001) and the number of fetuses (P < 0.01). After Day 80. estrone sulfate increased drastically, followed by increases in estrone and estradiol-17beta concentrations. The rate of increase in estrone sulfate during Days 80-100 was the greatest among all estrogens (P < 0.05). The rates of increase in estrone sulfate during Days 50-80 and 80-100 were 1.7 times greater in twin pregnancies than in cows having one fetus. These results suggest that the concentration of estrone sulfate in bovine peripheral blood plasma during early gestation has potential application in monitoring embryonic growth as well as fetoplacental development.

Effect of Ovary Storage on Development of Bovine Oocytes after Intracytoplasmic Sperm Injection, Parthenogenetic Activation, or Somatic Cell Nuclear Transfer

ABSTRACT The purpose of this study was to examine the effect of ovary storage on the development of bovine oocytes after intracytoplasmic sperm injection (ICSI), parthenogenetic activation, or somatic cell nuclear transfer (SCNT). Oocytes were obtained from ovaries stored in PBS for 2 to 6 h (control group) or 26 to 30 h (stored group) at 15°C. The maturation rate of the oocytes was significantly lower in the stored group (67%) than in the control group (78%). The degeneration rate of the oocytes was significantly higher in the stored group (24%) than in the control group (2%). ICSI and parthenogenetic oocytes from stored ovaries had a significantly decreased development to the blastocyst stage compared with the control (ICSI 8% vs. 24%, parthenogenetic activation 15% vs. 31%). However, the development rate to blastocysts of SCNT embryos derived from cumulus cells was not different between the two groups (38% vs. 38%). Also, the storage period of ovaries did not decrease the pregnancy rate of SCNT embryos, and cloned calves were produced in both groups with the same efficiency (21% vs. 21%). In summary, ovary storage at 15°C for 26 to 30 h reduced the maturation rate and in vitro development rate of bovine oocytes after ICSI or parthenogenetic activation, but did not decrease the blastocyst formation rate or survival rate after embryo transfer in SCNT.

Oestrone sulfate commences an increase around 50 days of gestation in bovine peripheral blood

The objective of this study was to determine the peripheral plasma concentration of oestrone sulfate during early gestation in the cow with a sensitive assay system. Five Holstein heifers were inseminated on oestrus and bled from the jugular vein at regular intervals until 100 days of gestation. Oestrone sulfate, oestrone and oestradiol-17beta in blood plasma were extracted with a reverse-phase cartridge and each measured by specific radioimmunoassay. Oestrone sulfate in bovine circulation started to increase around 50 days of gestation, whereas oestrone and oestradiol-17beta remained at basal concentrations for 80 days. The plasma concentration of oestrone sulfate increased gradually and linearly from 50 days of gestation, and was drastically elevated after 80 days of gestation before the increase in unconjugated oestrogens. The rate of increase from 80 to 100 days was the greatest for oestrone sulfate concentration among all oestrogens. These results suggest that plasma oestrone sulfate concentration could be a useful indicator of pregnancy within the first trimester of gestation in the cow.

Effects of supplementing an active dry yeast product on rumen microbial community composition and on subsequent rumen fermentation of lactating cows in the mid‐to‐late lactation period

The effects of supplementing feed of cows in mid-to-late lactation with an active yeast product (Actisaf Sc 47) were evaluated using 15 Holstein cows in a replicated 3 × 3 Latin square design. The animals were fed a mixed ration with 33% neutral detergent fiber, consisting of timothy hay (29.8%), a commercial concentrate (70.0%) and commercial calcium triphosphate (0.2%), twice daily to meet 105% of their energy requirement. Yeast supplement was set at 0, 5 and 10 g per day over 21-day periods, each of which consisted of 14 days for adaptation followed by 7 days of data collection. Milking performance, plasma metabolite parameters, rumen volatile fatty acids, lipopolysaccharide and microbial properties were measured. Although there were no significant differences in feeding and milking performance or blood parameters associated with supplementation, the acetate to propionate ratio in the rumen fluid tended to decrease (P = 0.08). The population of Bacteroidetes tended to be less prominent (P = 0.07) and the fibrolytic bacterium Fibrobacter significantly increased (P < 0.05) in the rumen fluid of the yeast 10 g group compared with that of the control. These data suggest that effects of supplementing live yeast to cows in mid-to-late lactation may be limited to microbial composition and fermentation characteristics in the rumen.

Biological Activity of Recombinant Bovine Interferon τ Produced by a Silkworm-Baculovirus Gene Expression System

ABSTRACT Bovine interferon (bIFN) τ plays a crucial role in maternal-fetal recognition and was expressed using a Bombyx mori (Bm) nuclear polyhedrosis virus (silkworm baculovirus) gene expression system. The biological effects of Bm-recombinant bIFNτ (rbIFNτ) on prostaglandin (PG) F2α synthesis were investigated in cultured bovine endometrial epithelial cells with oxytocin (OT, 100 nM) and on the in vitro development of bovine embryos. Bm-rbIFNτ and OT were shown to suppress PGF2α production in a dose-dependent manner. When in vitro produced morula stage embryos were cultured for 72 hr in modified CR1aa medium supplemented with or without rbIFNτ, Bm-rbIFNτ (10 ng/ml) significantly promoted development to the expanded blastocyst stage. In conclusion, Bm-rbIFNτ was suggested to have the same bioactivity as native IFNτ.

129 Embryo Production from Fully Grown and Growing Stage Oocytes in Japanese Black Calves

Before fattening of Japanese Black female calves, the ovaries are sometimes removed and discarded. Production of embryos from the oocytes residing in such ovaries is beneficial for the rescue of genetic resources. The aim of this study was to establish an embryo production system using oocytes collected from the ovaries of calves just before fattening and to investigate the correlation between the developmental competence of oocytes and the onset of puberty. Ovaries were collected from Japanese Black calves (9.5 ± 0.1 months old, n = 30, 3 replicates) in a fattening farm and separated according to the presence or absence of corpus luteum as the indicator of puberty (CL+ and CL– groups, respectively). Immature fully grown oocytes (IM oocytes), ~120 μm in diameter, were aspirated from follicles of 2 to 6 mm in diameter (CL+; n = 132, CL–; n = 41) and cultured for 22 to 23 h for maturation (IVM). After in vitro fertilization (IVF) for 6 h (designated Day 0), the oocytes were cultured for 9 days (in vitro culture, IVC) (Matoba et al. 2014 J. Dairy Sci. 97, 743-753). Growing oocytes, ~100 μm in diameter, were also collected by dissecting the follicles smaller than 1 mm in diameter. The growing oocytes were cultured for 14 days on membrane inserts for in vitro growth (IVG) (Hirao et al. 2013 Biol. Reprod. 89, 1-11). Then, IVG oocytes (CL+; n = 29, CL–; n = 32) were subjected to IVM, IVF, and IVC. Presumptive zygotes were cultured individually in microwells in culture dishes. Mitochondrial DNA (mtDNA) copy numbers (COX1 gene) of oocytes were examined (Takeda et al. 2010 Mitochondrion 10, 137-142). A comparison was made between the oocytes derived from calf ovaries and those of oocytes collected from cow ovaries by transvaginal ovum pick-up or by aspiration of the ovaries obtained at a local slaughterhouse. In IM oocytes, the rate of embryos developed to the blastocyst stage on Day 7 to 9 was higher in the CL+ group than in the CL– group (38.9 ± 2.6 v. 10.0 ± 7.1%, respectively; P < 0.05, t-test). However, IVG oocytes were compared, there was no significant difference in the blastocyst formation rate between the CL+ or CL– groups (34.7 ± 5.2 v. 19.8 ± 10.1%, respectively). The mtDNA copy numbers of matured oocytes were similar between IM and IVG oocytes irrespective of the maturity of the donor animals. In conclusion, we demonstrated the possibility of embryo production by IVM/IVF/IVC using fully grown and growing oocytes that are present in the ovaries of calves before fattening. Puberty positively affected the developmental competence of IM oocytes but the effect was not significant in IVG oocytes. Utilisation of both fully grown oocytes and growing oocytes may double the chance of rescuing genetic resources of high-breeding-value calves. This study was partly supported by grants from the Ito Foundation. We thank staff at Mie-Katoubokujou for allowing access to calves’ ovaries.

57 RELATIONSHIP BETWEEN FETAL ABNORMALITIES AND PERIPHERAL STEROID CONCENTRATIONS DURING GESTATION IN COWS TRANSFERRED WITH EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

Cloning of mammals by nuclear transfer frequently results in gestational or neonatal failure with a variety of abnormalities that are likely caused by inappropriate epigenetic reprogramming. Early diagnosis of fetal abnormality is important for efficient production of cloned animals. Sex steroids are produced in the bovine placenta and their levels in the blood might be useful as a measure of fetal well-being, as in humans. The objective of this study was to investigate whether changes in peripheral levels of progesterone and estrogens reflect fetal abnormalities. Donor cells for nuclear transfer were obtained from subculture of cumulus cells retrieved from ovarian follicles of a Japanese Black cow. Recipient oocytes were derived from ovaries obtained at an abattoir and matured in vitro. Metaphase II oocytes were enucleated and each fused with a donor cell by DC pulses. Nuclear transferred oocytes were activated and cultured for 7 days. Embryos developed to the blastocyst stage were transferred into the uterine horn ipsilateral to the ovary bearing the CL of 32 multiparous Japanese Black and Holstein crossbred cows at 7 to 8 days after the day of standing estrus (Day 0). Blood was collected from Day 40 until parturition. Progesterone and estrogens in the blood plasma of 6 recipient cows with full-term delivery were measured by RIA. These profiles were compared with each other and with the changes in a cow made pregnant by MOET. Statistical differences were analyzed with repeated measures ANOVA. Parturition was induced on Day 290. Stillborn, dead, or euthanized calves were subjected to necropsy and histopathological analysis. Pregnant cows were 14, 13, and 9 on Days 30, 60, and 90, respectively. Thereafter, 3 aborted around Days 110, 120, and 190. Six cows delivered calves weighing 45.8 ± 2.1 kg (mean ± SEM) on Days 291 to 293. Their birth weights were greater than those of female calves (31.2 ± 0.4 kg, n = 6) produced by MOET in the same breed. Three calves grew normally until weaning. One was stillborn due to dystocia, but no abnormalities were observed except for large offspring syndrome. One was euthanized 2 days after natural delivery due to ananastasia. Thymic and thyroid hypoplasia, left ventricular dysplasia, and pulmonary fibrosis were found by necropsy. Another calf delivered by Caesarean section died of infirmity 2 days later and had thymic and thyroid hypoplasia. Changes in plasma steroid concentrations were consistent with each other and with those in a MOET cow except the last one, in which progesterone levels tended to be higher during the first trimester and estrogens were lower during the last half of gestation. Progesterone levels tended to be lower in cows bearing a healthy calf than in cows bearing a weak calf before parturition. These results imply that peripheral steroid levels may reflect fetal normality, although large offspring syndrome does not affect their concentrations.