S. Matoba

274 SIMILARITY OF EMBRYOS PRODUCED BY OVUM PICK-UP AND INVITRO FERTILIZATION IN IDENTICAL TWIN CATTLE

The present study was designed to assess the similarity of follicular development, oocyte quality, and their subsequent development on ovum pick-up (OPU)-IVF in identical twin cattle. Four pairs of identical twin Japanese black cows (A, B pairs at 5 years old and C, D pairs at 3 years old) were kept under the same feeding and environmental conditions. OPU was performed for these cows once a week for seven continuous weeks. OPU was done by using a 7.5-MHz linear transducer with needle (17 G, 530-mm length) connected to an ultrasound scanner (SSD-1200, ALOKA, Tokyo, Japan). Oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. To assess the development, collected COCs were cultured for 20 h in TCM-199 supplemented with 5% calf serum (CS) in a microdroplet (volume was adjusted to 5 μL/oocyte) at 38.5°C under atmosphere of 5% CO2 in air. After maturation, the COCs were inseminated with frozen-thawed semen collected from the same ejaculation of a single bull. The fertilization was performed with BO solution as described by Imai et al (J. Vet. Med. Sci., 2002, 64(10), 887–891). The zygotes were then cultured in CR1aa supplemented with 5% CS under the same condition of maturation for nine days. Embryo development was assessed by the cleavage rate on Day 2 and the blastocyst production rate on Days 7 to 9 (insemination day = Day 0). Blastocysts were classified according to the IETS creteria. Data were analyzed by ANOVA. A total 56 sessions of OPU were performed in this study. The overall mean number of developing follicles (larger than 2 mm in diameter), collected oocytes, and produced blastocysts were 30.3 ± 9.2, 20.1 ± 9.2 and 6.3 ± 3.8 (mean ± SD) per session, respectively. The mean number of developing follicles on the day of OPU were significantly different between B and D pairs (38.6 ± 7.5 and 21.9 ± 6.5, P < 0.01); however, no significant difference was found within each twin. In oocyte quality, C and D pairs were significantly higher grade than the A pair. The percentages of cleaved oocytes and embryos developed to the blastocyst stage (34 ± 16, 27 ± 10, 41 ± 17 and 39 ± 24) showed no differences among 4 pairs and within each twin. However, the percentage of Grade 1 blastocyst of B pair was significantly lower (P < 0.01) than that of other pairs, and C pair was significantly higher (P < 0.05) than that of A and D pairs (67 ± 25, 41 ± 22, 93 ± 10 and 71 ± 25; A, B, C and D pairs, respectively). There was no significant difference within twins. These results show little statistical variation between cows of the same genetic background in the production of embryos in vitro.

252 EFFECT OF SUPERSTIMULATORY TREATMENT TO DONOR COWS IN OXYGEN CONSUMPTION OF MATURED OOCYTES

We previously reported that follicular wave synchronization and follicular growth treatment (FGT) before ovum pick-up (OPU) were effective in improving oocyte competence, which was associated with an increase in related embryos obtained by somatic cell nuclear transfer (Sugimura et al. 2012 Cell. Reprogram. 14, 29–37). However, oxygen consumption in oocytes remained unknown. The present study was designed to examine the differences in oxygen consumption between bovine oocytes obtained by OPU with or without FGT after in vitro maturation. Holstein dry cows (n = 8) were reared under the same feeding and environmental conditions. Two OPU sessions were conducted in each cow to collect immature oocytes, as described by Sugimura et al. (2012). The first OPU session (OPU group) was performed in cows on arbitrary days of the oestrous cycle, using a 7.5-MHz linear transducer with the needle connected to an ultrasound scanner. Follicles larger than 8 mm in diameter were then aspirated and a controlled internal drug release device (CIDR) was inserted on Day 5 (the day of the first OPU session = Day 0). Then 30 Armour units (AU) of FSH (Antrin, Kyoritsu Seiyaku, Tokyo, Japan) was administrated to cows twice a day from Day 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 AU day–1). Cloprostenol (prostaglandin F2α; 0.75 mg) was administered in the morning of Day 9. The second OPU session (FGT-OPU group) was performed 48 h after prostaglandin F2α administration (Day 11), and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Collected cumulus–oocyte complexes in the OPU and FGT-OPU groups were matured in vitro as described by Imai et al. [2006 J. Reprod. Dev. 52(Suppl.), S19–S29]. To collect in vivo-matured oocytes (control group), the CIDR was inserted into the cows on arbitrary days of the oestrous cycle (= Day 0), and oestradiol benzoate (0.8 mg) was administered on Day 1. The cows received the FGT treatment (as described above) from Day 6 to 10; however, the CIDR was removed in the evening of Day 8. Buserelin (gonadotropin-releasing hormone; 200 µg) was then administrated in the morning of Day 10, and OPU was performed at 24 h after gonadotropin-releasing hormone administration (Day 11). Oxygen consumption of matured oocytes was measured noninvasively with a scanning electron microscopy system (HV-405SP; Hokuto Denko Co., Tokyo, Japan). Data were analysed by ANOVA followed by a Tukey-Kramer test. There was no difference in the mean oxygen consumption between the FGT-OPU group (0.34 ± 0.02 × 10–14 mol–1, mean ± SEM) and control group (0.40 ± 0.01 × 10–14 mol–1). However, oxygen consumption in the FGT-OPU and control groups was significantly lower (P < 0.01) than that in the OPU group (0.50 ± 0.02 × 10–14 mol–1). These results revealed significantly lower oxygen consumption in OPU-derived in vitro-matured bovine oocytes after FGT treatment compared with those obtained without FGT treatment. Oxygen consumption of oocytes obtained from FGT-OPU was similar to that of in vivo-matured oocytes, which may reflect their cytoplasmic maturation status with high developmental competence.

236 KINETICS AND PATTERN OF THE FIRST CLEAVAGE OF IN VITRO-FERTILIZED EMBRYOS BY IN VIVO-MATURED OOCYTES AND X-SORTED SPERMATOZOA IN DAIRY CATTLE

Recently, we reported that high rates of good-quality blastocysts can be produced by IVF of in vivo-matured oocytes, obtained by ovum pick-up (OPU) after superstimulation in Holstein cows, with X-sorted sperm [Matoba et al. 2012 Reprod. Domest. Anim. 47(Suppl. 4), 515]. However, we have limited knowledge concerning the normality of embryonic cleavages in such embryos. The present study examined their kinetics and pattern of the first cell cycle. In vivo-matured oocytes were collected by OPU from non-lactating Holstein cows just before ovulation after superstimulation and ovulation induction by gonadotropin-releasing hormone. The oocytes were inseminated with 5 × 106 sperm mL–1 of X-sorted sperm and cultured in CR1aa supplemented with 5% newborn calf serum and 0.25 mg mL–1 of linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually using a microwell culture dish and time-lapse cinematography (CCM-1.4MZS, Astec, Fukuoka, Japan) (Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken every 15 min during the in vitro culture period, and images were analysed by CCM-1.4 software (Astec). The cleavage pattern was categorised into normal cleavage (2 even blastomeres without fragment or protrusion) or abnormal cleavage (those with 2 uneven blastomeres, with fragments or protrusions and those dividing into 3 to 5 blastomeres at the first cleavage). Data were analysed by ANOVA, chi-square, and discriminant function. A total of 117 embryos were examined; of this number, 63.2% developed to the blastocyst stage and the rest were degenerated. A high rate of normal cleavage and a low rate of abnormal cleavage, including those with 2 uneven blastomeres and those with fragments or protrusions in the first cleavage pattern, were recorded in embryos that could develop to blastocysts compared with degenerated ones (P < 0.01 or P < 0.05, respectively; Table 1). No significant difference was found in those dividing into 3 to 5 blastomeres between the blastocysts and degenerated embryos (Table 1). Embryos developing to the blastocyst stage had a shorter duration of the first cell cycle [27.2 ± 2.3 h post-insemination (hpi)] compared with those undergoing degeneration (30.6 ± 5.7 hpi; P < 0.001). The threshold of duration of the first cell cycle was calculated by (X – 27.2)/2.3 = (30.6 – X)/5.7, resulting in X = 28.2. Blastocysts with a short duration of the first cell cleavage (≤28.2 hpi) showed a higher frequency of the normal cleavage pattern than those with a duration of the first cell cleavage longer than 28.2 hpi (71.7 and 53.6%, respectively; P < 0.05). Our results revealed that those IVF embryos that finished their first cleavage before 28.2 h of IVF and showed a normal cleavage pattern had superior developmental competence. Table 1.The first cleavege pattern reflects the developmental competence: blastocysts versus degenerated embryos This work was supported by the Research and Development Projects for Application in Promoting New Policy of Agriculture, Forestry and Fisheries (22016).

298 EFFECT OF TWO DIFFERENT DOSES OF FOLLICLE-STIMULATING HORMONE IN ALUMINUM HYDROXIDE GEL BY A SINGLE INJECTION ON ESTRUS AND SUPEROVULATORY RESPONSE IN HOLSTEIN CATTLE

We investigated the effect of administration of different dosage of FSH in aluminum hydroxide gel (Gel; an adjuvant and absorbent of large molecules) by a single injection on induction of estrus, superovulatory response, and embryo recovery in Holstein cattle. In this study, six cloprostenol-synchronized [PGF (0.75 mg, IM); Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan], nonlactating Holstein cows were divided into three groups (Gel-40, Gel-30, Controls) between days 8 and 11 (day 0 = the day of estrus) in a Latin square design. Forty mg (Gel-40 group) FSH (Antrin-R10; Kawasaki Mitaka Pharmaceutical Co., Tokyo, Japan) or 30 mg (Gel-30 group) FSH were mixed with 5 mL Gel and administered by a single injection into fat in the ischiorectal fossa. The control group received 40 mg pFSH twice daily for 4 d in decreasing doses (8, 8, 6, 6, 4, 4, 2, and 2 mg) into the ischiorectal fossa. Then, all cows received PGF (0.75 mg, IM) twice on the third day of the treatment, and cows were inseminated at 48 and 60 h after second PGF treatment; embryo recovery was performed 7 d later. Follicular development was monitored ultrasonically every 24 h from the first FSH treatment to ovulation, and the numbers of medium (MF, 5–8 mm) and large (LF, >8 mm) sized follicles, and corpora lutea (CL) were counted on the day of embryo recovery. Estrous status was determined by walking activity of cows as an estrous index. Each cow was fitted with a pedometer (Gyuho; Comtec, Miyazaki, Japan) that measures total amount of walking activity per hour, and data of individual cows were sent to a personal computer in real time. Estrus was defined as an increase in number of steps to more than the value of mean steps plus standard deviation recorded for the preceding 240 h. Data were analyzed by one-way ANOVA. There was no significant difference among the Gel-40, Gel-30, and control groups for the mean ( ± SD) duration of estrus (22.3 ± 5.2 h, 18.0 ± 2.8 h and 18.5 ± 5.3 h, respectively), increments of estrous activity (398.3 ± 135.3%, 388.9 ± 44.4% and 393.9 ± 82.1%, respectively), and the mean interval from the first PGF to onset of estrus (35.5 ± 2.2 h, 37.8 ± 5.7 h and 39.3 ± 5.6 h, respectively). There was no significant difference among the Gel-40, Gel-30, and control groups for the mean number of large follicles at the time of estrus (21.3 ± 10.2, 21.5 ± 7.6 and 21.0 ± 9.4, respectively), number of CL at the time of ova/embryo collection (21.5 ± 10.2, 19.2 ± 4.9, and 20.5 ± 9.5, respectively), the total number of ova/embyos recovered (12.3 ± 8.3, 12.5 ± 7.8, and 13.8 ± 9.2, respectively), or transferable embyos (3.3 ± 4.8, 4.5 ± 3.5, and 5.5 ± 4.5, respectively). Results suggested that administration of 30 mg of FSH in 5 mL Gel by a single injection in the ischiorectal fossa was effective in inducing superstimulation in Holstein cattle. This work was supported by a grant of the Research Project for Utilizing Advanced Technology (04–1676).

388 SUPEROVULATION TREATMENT BY A SINGLE INJECTION OF FSH WITH ALUMINUM HYDROXIDE GEL IN HOLSTEIN CATTLE

The objective of this study was to examine the effect of administration of FSH in aluminum hydroxide gel (Gel; an adjuvant and adsorbent of large molecules) by a single injection on superovulatory response and embryo recovery in cattle. In this study, cloprostenol-synchronized (PGF; Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan) nonlactating Holstein cows were divided into 2 groups (Gel-treated and Controls) between Days 8 and 11 after estrus in 2 experiments in a crossover design. In Experiment 1, 40 mg FSH (Antrin-R10; Kawasaki Mitaka Pharmaceutical Co., Tokyo, Japan) was mixed with 10 mL aluminum hydroxide suspended in saline at a concentration of 3 mg Al mL-1 and administered by a single IM injection (n = 12; 10 mL-Gel group). Blood samples were collected at 0, 1, 2, 4, 8, and 12 h, and thereafter every 24 h until embryo recovery for measurement of FSH levels in a time-resolved fluoroimmunoassay. In Experiment 2, 40 mg FSH was mixed with 5 mL Gel and injected into fat in the inguinal region (n = 8, 5 mL-Gel group). The Control group in both experiments (n = 2 and n = 8, respectively) received 40 mg FSH twice daily for 4 days in decreasing doses (8, 8, 6, 6, 4, 4, 2, and 2 mg) by IM injection (Experiment 1), or in the inguinal region (Experiment 2). PGF (0.75 mg) was administered twice on the third day of treatment, and cows were inseminated 48 and 60 h after second PGF dose; embryo recovery was performed 7 days later. Follicular development was monitored ultrasonically every 24 h from the first FSH treatment to ovulation, and the numbers of medium (MF, 5–8 mm) and large (LF, >8 mm) follicles, and corpora lutea (CL) were counted on the day of embryo recovery. Data were analyzed by Students t-test. In Experiment 1, there were no differences in the mean (± SD) number of CL or transferable embryos between the 10 mL-Gel (10.5 ± 9.6 and 6.0 ± 8.0, respectively) and Control (14.8 ± 9.6 and 9.0 ± 6.6, respectively) groups (P > 0.1). The growth of LF and MF in the 10 mL-Gel group occurred 24 h earlier than in the Control group (24 and 48 h after initiation of FSH treatment, respectively; P 0.4). Results suggest that administration of FSH in 5 mL Gel by a single injection in the inguinal region is effective in inducing superstimulation in Holstein cattle. This work was supported by a grant of the Research Project for Utilizing Advanced Technology (04-1676).

246 Follicular development and oocyte quality after ovum pickup in donor cows.

The present study was designed to assess the renewal of follicular development and oocyte quality after ovum pickup (OPU) in Holstein dry cows. Cows were kept under the same feeding and environmental conditions. In Experiment 1, follicle aspiration (more than 2 mm) by OPU using a 7.5 MHz linear transducer with needle (cova needle, Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200, ALOKA, Tokyo, Japan) was performed in four cows. After OPU ovaries were observed from Day 4 (Day 0 = the day of OPU) to Day 11 to assess the number of follicles that developed. In Experiment 2, two sessions of OPU (n = 11) were performed with a 7-day interval between to assess the quality of developing follicles and oocytes. Oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. To assess the developmental competence of oocytes, collected cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM-199 supplemented with 5% calf serum (CS) in a microdroplet (volume was adjusted to 5 μL/oocyte) at 38.5°C under atmosphere of 5% CO2 in air. After maturation, the COCs were inseminated with frozen-thawed semen collected from the same ejaculation of a single bull. The fertilization was performed with BO solution as described by Imai et al. (2002 J. Vet. Med. Sci. 64(10), 887–891). The putative zygotes were then cultured in CR1aa supplemented with 5% CS under the same conditions as maturation culture for nine days. Embryo development was assessed by the cleavage rate on Day 2 and the blastocyst formation rate on Days 7 to 9 (the day of insemination = Day 0). Data were analyzed by ANOVA or Students t-test. In Experiment 1, the mean number of developing follicles (larger than 2 mm in diameter) were increased from Day 4 to Day 11 (Day 4: 19.8 ± 10.0, Day 7: 32.5 ± 9.5; Day 11: 39.5 ± 10.7 (mean ± SD), respectively, P < 0.05). In Experiment 2, the mean number of developing follicles and collected oocytes on the day of OPU were significantly (P < 0.05) different between the first and second sessions (54.2 ± 12.4 and 40.8 ± 12.7, 45.7 ± 20.2 and 27.7 ± 8.7, respectively). The percentage of Grade 1 and 2 oocytes for the first session was significantly lower (P < 0.05) than those for the second session (59.1 ± 8.4 and 69.0 ± 11.8), and no significant differences were found within cleavage and blastocyst rates. The mean numbers of blastocysts obtained per session were 14.2 ± 8.9 and 9.7 ± 6.3 in the first and second sessions, respectively. These results indicate that populations of follicles were increased till Day 11 after OPU, and proportion of normal oocytes were increased in the renewal follicles.