We demonstrated that in vivo-matured oocytes (mOC) collected by ovum-pick up (OPU) from cows after stimulation of follicular growth (FG) are suitable for producing good quality blastocysts (BL). However, it is not known whether duration of FG affects developmental competence of mOC. The purpose of this study was to examine development of mOC after stimulation with different duration of FG. Japanese black donor cows (n = 4 per each group), were treated with a CIDR at Day 0. Follicle of diameter >8 mm were removed on Day 5. A total 20 AU of FSH was administrated to cows twice daily with decreasing doses from the evening of Day 6 to the morning of Day 10. In the conventional group (48PG), a administration of PGF2α (0.75 mg of cloprostenol), CIDR withdrawal, and administration of GnRH (0.2 mg of fertirelin acetate) were performed on the evening of Day 8, morning of Day 9, and morning of Day 10, respectively. In the experimental group (72PG), administration of PGF2a, CIDR withdrawal, and administration of GnRH were performed on the evening of Day 9, the morning of Day 10, and the morning of Day 11, respectively. The mOC were collected from follicles >5 mm by OPU at 25 to 26 h following GnRH administration. Collected mOC were inseminated with 3 × 106 sperm mL–1 in BO solution on 30 h after GnRH. After 6 h of IVF, presumptive zygotes were cultured for 168 h in 5% CS + CR1aa, using a micro-well culture dish (Dai-Nippon-Print) and time-lapse cinematography (CCM-1.4MZS; Astec) for individual embryo observation. The kinetics of early embryo was analysis by CCM-1.4 software. To assess the quality of BL, prognostic factors were used as follows: (1) less than 27 hpi (hours post-insemination) at the first cleavage (1st CD), (2) 2 blastomeres at the end of 1st CD, and (3) absence of multiple fragments at the end of the 1st CD (Sugimura et al. 2012 PLoS ONE 7, e36627; Imai et al. 2014 Reprod. Fertil. Dev. 26, 182). Data were analysed by Student's t-test or chi-square test. The number of mOC were 12.5 ± 4.7 and 10.3 ± 2.7 (means ± s.e.) oocytes per session in 48PG and 72PG. There was no significant difference in cleavage rate or BL formation rate (97.5 ± 1.5 v. 98.2 ± 1.8%, 66.3 ± 8.2 v. 66.8 ± 3.5%, respectively). The time for 1st CD was shorter in 48PG (26.1 ± 0.3 v. 27.8 ± 0.4; P < 0.01), and the rate of 1st CD less than 27 hpi was superior in 48PG compared with 72PG (74.3 v. 42.9%; P < 0.05). However, the rate of 2 blastomeres and absence of multiple fragments were not different between 48PG and 72PG. The number of BL tended to decrease in 72PG compared with 48PG (28.6 v. 48.6%; P = 0.087). These results indicate that duration of FG did not affect the rate of cleavage and BL formation. However, extension of duration of FG might reduce the quality of BL.
Bovine oocytes obtained by ovum-pick-up (OPU) following follicle growth treatment (FGT) have improved quality and competence (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). However, the effect of the presence of FSH or epidermal growth factor (EGF) like peptide during in vitro maturation (IVM) on the developmental competence of FGT oocytes has not been well known. This study was undertaken to examine the developmental competence of FGT oocytes following IVM in the presence of FSH (recombinant human FSH) or EGF-like peptide (amphiregulin; Areg) and IVF. Japanese Black cows (n = 17) were used as donors. Five days after arbitrary OPU (opu group), follicles ≥8 mm in diameter were aspirated again, a controlled internal drug release (CIDR) was inserted into the vagina, and then pFSH was injected twice a day from the evening of Day 6 to the morning of Day 10 with decreasing doses (total of 20 AU; 4, 4, 3, 3, 2, 2, 1, 1 AU/day). On the evening of Day 8, PGF2α (0.5 mg of cloprostenol) was administered. On Day 11, oocytes were aspirated from follicles with ≥5 mm in diameter of the treated donors by OPU (fgt group). The cumulus-oocyte complexes (COC) were cultured in the absence (opu-cont and fgt-cont groups) or presence of 0.1 IU mL−1 FSH (opu-fsh and fgt-fsh groups) or 100 ng mL−1 Areg (opu-areg and fgt-areg groups) in IVM medium (mTCM199 containing 5 mg mL−1 BSA) for 20 to 22 h (1 COC/5 µL, total of 162–171 COC per group), and then co-cultured with 3 × 106 sperm/mL for 6 h. The presumptive zygotes were continued to culture in mCR1aa supplemented with 5% newborn calf serum for 216 h (1 zygote/5 µL) using micro-well culture dishes (Dai-Nippon-Print). When repeating this opu-fgt session in the same cow, an interval at least for 50 days was kept, and the session was performed 28 times. Statistical analysis was carried out by Mann-Whitney’s U-test (between opu and fgt groups) or Steel-Dwass test after Kruskal-Wallis test (among all groups). The number of follicles ≥5 mm increased in the fgt than opu group (17.8 v. 2.9; P < 0.01). The number of COC collected was not different between the opu and fgt groups (23.1 v. 19.6; P > 0.05). The blastocyst formation rate was higher in the fgt than opu group (36.9 v. 23.1%; P < 0.01). Within 6 groups, the blastocyst formation rate was higher in the fgt-fsh (43.3%; P < 0.01) and fgt-areg (39.5%; P < 0.05) groups than the opu-cont (16.3%) group. The rate in the fgt-fsh group was also higher than that in the opu-fsh group (43.3 v. 18.7%; P < 0.01). These results suggested that FGT improved the developmental competence of bovine oocytes, probably through improving the ability of the COC to react against FSH/Areg.
Pregnancy diagnosis during early gestation is important for cattle reproduction. The expression of interferon-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) was studied in embryo-transferred (ET) Japanese Black cattle. ISGs in PBLs—ISG15, MX1, MX2, and OAS1—were detected in multiple ovulation ET cattle using a real-time quantitative polymerase chain reaction, and receiver operating characteristic (ROC) curve analysis was performed. Gestational status was predicted using the average ISG levels during the normal estrous cycle (AVE) and the Youden index from the ROC curve analysis as cutoff values. The ISG15, MX1, and MX2 levels were significantly higher in pregnant cattle (n = 10) than in non-pregnant cattle (n = 23) on gestation day 21, whereas the levels of all ISGs were similar between non-pregnant and non-pregnant cattle with late embryonic death (n = 7). ISG15, MX1, and MX2 appropriately predicted the gestational status of ET cows. The statistical evaluation of the diagnostic accuracy in ET cows on day 21 of gestation presented higher values of sensitivity, specificity, accuracy, and positive predictive values of ISG15, MX1, and MX2 using the Youden index than using the AVE. Therefore, ISG15, MX1, and MX2 are excellent biomarkers of gestational status during the peri-implantation period in ET cattle.
The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.
The aim of this study was to evaluate the effect of administration of eCG and intrauterine infusion of povidone-iodine to promote the early recovery of ovary and uterus functions in early stage after delivery on postpartum beef cows. Thirty-three postpartum Japanese Black cows without retention of placenta were used for this investigation. After delivery, cows nursed colostrums to calves for several hours in a pen, and then were separated from calves and returned into a barn managed in a group. In experimental group (n = 14), on Day 10 (Day 0 = day of parturition), an intramuscular administration of 500 IU of eCG and infusion of 30 mL of 2% povidone-iodine into the uterus body were conducted. The untreated cows delivered in the same period were compared as a control group (n = 19). Cows were inseminated artificially using commercial Japanese Black frozen–thawed semen when standing oestrus was detected until Day 90. Pregnancy diagnosis was performed 30 days after insemination by ultrasonography. For all of the experimental group and 8 cows in the control group, the diameter of follicles >8 mm and the diameter of cross-section between endometrium in uterus of the implanted side at the point of ~2 cm from bifurcation of the uterus were measured using an ultrasound scanning machine connected to a 7.5-MHz convex probe. This monitoring was carried out every 10 days from Day 10 until the day of first oestrus (= insemination) or until Day 80 for non-returned oestrus cows. Data were analysed using Fisher’s exact test and ANOVA. The ratio of returned oestrus by Day 90 was 100% (14/14) in experimental group and 89.5% (17/19) in control group. The first oestrus day postpartum in the experimental group and the control group was the almost same: 52.9 ± 12.1 (24–88) days and 57.2 ± 19.9 (29–78) days, respectively. The conception rate until Day 90 tended to be higher (P = 0.07) in the experimental group (78.6%, 11/14) than in the control group (47.4%, 9/19). The days inseminated to cows that were conceived, was almost same between the experimental group (57.5 ± 18.6, 24–87 d) and the control group (55.4 ± 12.9, 36–78 d). The ratio of cows observed follicles more than 8 mm, was higher in the experimental group than control group on Day 50 (90.9% v. 50.0%; P = 0.09) and Day 60 (66.7% v. 0%; P < 0.05), respectively. The diameter of uterus tended to be lesser in the experimental group than in the control group on Day 30 (16.9 ± 2.8 mm v. 19.2 ± 2.8 mm; P = 0.09), and that regressed linearly to Day 30 in the experimental group however that was prolonged until Day 40 (16.4 ± 1.7 mm) in the control group. These results indicate that administration of eCG and uterus infusion of povidone-iodine at an early stage postpartum may promote the early recovery of ovary and uterus functions in beef cows. This research was supported by a grant from the Research Program of MAFF, (REP1004) Development of Innovative Technology for Animal Reproduction.
We have reported monozygotic twin calves that can be produced efficiently by blastomere separation of 2-cell stage embryos and by the use of a commercially provided well-of-the-well culture dish (Hashiyada 2017 J. Reprod. Dev.). The present study was conducted to evaluate the effect of a culture supplement, l-ascorbic acid 2-phosphate (AA-2P), a sustained antioxidant substance that reduces reactive oxygen species. Embryos were produced using oocytes from ovaries collected at an abattoir by in vitro maturation, IVF, and in vitro culture (IVC). TCM199 supplemented with 5% calf serum, Brackett-Oliphant solution supplemented with 10mg mL−1 BSA, and CR1aa containing 5% calf serum were used for each culture step. Two-cell stage embryos were obtained 24 to 27h post-insemination (hpi). Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into each blastomere by gentle pipetting in IVC medium. Each blastomere was introduced into a single conical micro-well of 25 wells, each having a diameter and depth of ~287 and 168µm (Dai Nippon Printing, Tokyo, Japan). Culture of blastomeres in wells was performed covered with a droplet of 2.5 µL/well IVC medium supplemented with 0 (n=212), 250 (n=214), 500 (n=206), and 750 µM (n=204) of AA-2P. The blastocyst formation rate at Day 8 after IVF, the quality of blastocysts assessed by morphological observation, and the cell numbers were compared among each concentration of AA-2P. In addition, the developmental speed to the blastocyst stage was analysed using time-lapse cinematography for 0 and 500 µM of AA-2P (n=40, respectively). Statistical analysis was performed using Fisher’s exact test and ANOVA. The blastocyst formation rate (32-40%), the total cell number (108-114), and inner cell mass cell number (26-28) did not differ among groups. The time to reach the 4-cell stage was significantly shorter in media supplemented with 0 µM (43 hpi) than with 500 µM (52 hpi); however, the time to reach the blastocyst stage did not differ (150 and 155 hpi, respectively). Regarding the proportion of quality grade 1 to 3 blastocysts and the developmental speed to the blastocyst stage, high-quality grade 1 embryos were significantly faster than those of middle and low-quality grade 2 and 3 ones in 0 (145 v. 154 hpi; P<0.05) and 500 µM (150v. 158 hpi; P<0.05) supplemented medium. In this experiment, no effect of AA-2P was observed for the culture of isolated blastomeres from 2-cell stage embryos, although it was suggested that blastomeres with high developmental competence reach the blastocyst stage faster, which might reflect the quality of the embryos.
Monozygotic twin bovine embryos can be produced by blastomere separation of 2-cell embryos and commercial well-of-the-well (WOW) culture dish (Hashiyada et al. 2016 Reprod. Fertil. Dev. 28, 178) obtaining 60% and 48% of blastocyst formation and monozygotic blastocyst pairs, respectively. The present study was conducted to evaluate the fertility of blastocysts derived from this production system in Japanese Black beef cattle. Embryos were produced using oocytes collected by ovum pick-up technique. TCM-199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10 mg mL−1 BSA, and CR1aa containing 5% CS, were used for each culture step: in vitro maturation, fertilization, and culture (IVM,IVF, and IVC). Two-cell stage embryos were obtained 24 to 27 h post-insemination. Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into blastomeres by gentle pipetting in IVC medium. Each blastomere was introduced into a single conical microwell of 25 wells, each having a diameter and depth of ~287 μm and 168 μm (Dai Nippon Printing, Tokyo, Japan). Blastomeres in wells were cultured covered with a droplet of 2.5 μL of IVC medium/well. The developed blastocysts in pairs on 7 days post-insemination were used for transfer. Single embryos of monozygotic twin embryos were transferred to Japanese Black cattle with a generally small body frame to produce twin calves from a set of recipients. Twin embryos were transferred in pairs to unilateral of uterus of non-lactating Holstein cows. Pregnancy and twin pregnancy were determined at 30 days of gestation by ultrasonography and were reconfirmed at 60 days with detection of fetal loss. Statistical significance was analysed by Fisher’s exact test. There was no significant difference in pregnancy rate or twin pregnancy rate between single embryo transfer (7/14, 50% and 2/7, 28.6%) and twin embryo transfer (9/21, 42.9% and 4/21, 19%). In either transfer method, fetal loss was not observed in diagnosis carried out at 60 days by ultrasonography. To date, 2 pairs of twin calves have been obtained from twin pregnant cows by twin embryo transfer within the normal range of gestation length (286 and 288 days) and birth weight (31-40 kg). These results indicate that blastocysts developed from blastomeres separated from 2-cell embryos by culturing with commercial WOW culture dish had fertility similar to that of intact embryos derived from standard in vitro culture and further demonstrate the possibility of production of normal twin calves.
Monozygotic twin embryos can be produced using the technique of blastomere separation and well of the well (WOW) dish having handmade micro-wells by the needle depression (Tagawa et al. 2008). We have recently reported that developed commercial WOW dish enhances embryo competence in individual culture (Sugimura et al. 2010). The present study was conducted to evaluate the availability of commercial WOW dish for production of monozygotic twin embryos in bovine. Embryos were produced using oocytes from ovaries collected at an abattoir by IVM, IVF, and IVC. For each culture, TCM-199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10 mg mL–1 BSA, and CR1aa containing 5% CS were used. To evaluate the adaptability of dishes on culture of isolated blastomeres from different cell stage, 2- (n = 63), 4- (n = 94), 8- (n = 137), and 10- to 14- (n = 116) cell stages were obtained on 24–27 h, 30–36 h, 48–54 h, and 48–54 h from the beginning of fertilization, respectively. The zona pellucida was removed by exposure of 0.25% pronase, followed by gentle pipetting by inspiration and expiration in the IVC medium. Then, two halves separated from the original number of blastomeres were randomly allocated to the conical micro-wells of commercial dish (Dai Nippon Printing, Tokyo, Japan) or created micro-wells by pressing the bottom of the dish with an eyeleteer (control). The approximate diameter and depth of each 25 wells in a commercial dish was 287 and 168 μm, and each 20 wells in the control were 800 and 600 μm. The blastomeres were cultured in wells covered with a droplet of 2.5 μL well–1 IVC medium until Day 8 (IVF = Day 0). Expanded blastocysts (n = 28) derived from tetra-blastomeres of 8-cell stage were stained to determine the number of the inner cell mass (ICM) and trophectoderm (TE) in each group. Statistical significance of the blastocyst formation rates and the number of cells were analysed by the chi-square test and the Student’s t-test, respectively. In the 2-cell stage, blastocyst formation rate in commercial dish tended to be higher than that in the control (60.0% v. 46.1%). The rate of monozygotic blastocyst pairs in commercial dish was higher compared with the control (48.0% v. 26.3%, P < 0.05). In the 4-cell stage, rates of blastocyst formation (50.0% v. 33.0%, P < 0.05) and the pairs (39.5% v. 12.5%, P < 0.01) in the commercial dish, both were higher compared with the control. In the 8-cell stage, there were no differences between two groups in rates of blastocyst formation (53.1% v. 59.0%) and the pairs (41.8% v. 48.7%), similarly in the 10- to 14-cell stage (47.9% v. 56.8% and 36.2% v. 40.9%, respectively). The ICM, TE, and total cell numbers were not different between the commercial and the control dish (28.0 ± 3.2 v. 26.0 ± 3.8, 64.6 ± 4.3 v. 76.0 ± 7.9, and 92.6 ± 6.2 v. 102.0 ± 11.0, respectively). These results indicate that separated blastomeres could be developed to blastocysts efficiently and stably regardless of embryo cell stage with a commercial WOW culture dish.
During antral folliculogenesis, developmental competence of prospective oocytes is regulated in large part by the follicular somatic component to prepare the oocyte for the final stage of maturation and subsequent embryo development. The underlying molecular mechanisms are poorly understood. Oocytes reaching the advanced stage of follicular growth by administration of exogenous follicle-stimulating hormone (FSH) possess higher developmental competence than oocytes in FSH-untreated smaller follicles. In this study, the transcriptomic profile of the cumulus cells from cows receiving FSH administration (FSH-priming) was compared, as a model of high oocyte competence, with that from untreated donor cows (control). Ingenuity Pathway Analysis showed that cumulus cells receiving FSH-priming were rich in down-regulated transcripts associated with cell movement and migration, including the extracellular matrix-related transcripts, probably preventing the disruption of cell-to-cell contacts. Interestingly, the transcriptomic profile of up-regulated genes in the control group was similar to that of granulosa cells from atretic follicles. Interferon regulatory factor 7 was activated as the key upstream regulator of FSH-priming. Thus, acquisition of developmental competence by oocytes can be ensured by the integrity of cumulus cells involved in cell-to-cell communication and cell survival, which may help achieve enhanced oocyte-somatic cell coupling.
