O. Dochi

Factors affecting nuclear maturation, cleavage and embryo development of vitrified bovine cumulus-oocyte complexes

The objective was to investigate the effects of cryodevice, vitrification solutions, and equilibration time on in vitro maturation, cleavage, and embryo development of vitrified bovine oocytes. In Experiment 1, the nuclear maturation (MII) rate of immature bovine COCs vitrified was compared between two equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P < 0.0001). Equilibration time did not affect MII rate (P = 0.964); however, the MII rate was higher for COCs vitrified on cryotops than in straws (23 vs 9%, P = 0.007). In Experiment 2, bovine COCs were vitrified on cryotops using two equilibration times (0 vs 5 min) in VS1 and two kinds of vitrification solutions (freshly prepared vs frozen). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocysts rate 31 vs 0.4%). Cleavage rate of COCs vitrified using frozen solutions with 5 min equilibration was higher (P = 0.05) than other treatment groups. However, blastocyst rate did not differ (P = 0.993) among treatment groups. In conclusion, cryotop was a better cryodevice than 0.25 mL straw for vitrification of bovine COCs. Furthermore, 5 min equilibration in VS1 improved cleavage. Compared with control, the vitrification procedure per se damaged bovine COCs, resulting in poor nuclear maturation and embryo development. However, vitrification did not immediately kill oocytes, as the cleavage rate was acceptable.

Gene expression of leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF) in bovine endometrium during early pregnancy

Abstract Leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF), members of the group of hemopoietic cytokines, play a primary role in the control of embryo development and implantation and in the growth of the placenta in humans and mice. Gene expressions of LIF and M-CSF were investigated using quantitative RT-PCR in bovine endometrial tissues during early and mid-pregnancy (Days 16–17, 20–21, 30–36, 48–49 and 74–140) and during the estrous cycle (Days 13–14). Leukemia inhibitory factor and M-CSF genes were expressed in all samples examined. Significant differences were found between the gene expression patterns of LIF and M-CSF. Leukemia inhibitory factor expression level at Days 48–49 was the highest in caruncular endometrium, however, the large variability negated any significant differences. Leukemia inhibitory factor expression levels in intercaruncular endometrium at Days 48–49 and 74–140 of pregnancy were greater than at Days 13–14 of the estrous cycle and at other days of pregnancy. No significant change was recognized in M-CSF expression levels in caruncular endometrium. Macrophage colony stimulating factor expression level in intercaruncular endometrium at Days 74–140 was greater than those of the other samples. These results suggest that LIF and M-CSF are produced in the endometrium and may play different roles in early and mid-pregnancy.

Effect of length of progesterone exposure during ovulatory wave development on pregnancy rate.

The objective was to determine the effects of the duration of progesterone exposure during the ovulatory wave on fertility (pregnancy rate) in beef cattle. We tested the hypothesis that short-progesterone exposure during the growing and early-static phase of the ovulatory follicle (analogous to the ovulatory wave of 3-wave cycles) is associated with higher fertility than a longer duration of exposure (analogous to the ovulatory wave of 2-wave cycles). Three to 5 days after ovulation, beef heifers (n = 172) and suckled beef cows (n = 193) were given an intravaginal progesterone-releasing device (CIDR) and 2.5 mg estradiol - 17β +50 mg progesterone im to induce a new follicular wave. Cattle were allocated to short- or long-progesterone exposure groups (for 3 and 6 d after wave emergence, respectively) after which prostaglandin F(2α) was administered and CIDR were removed. Forty-eight hours later, all cattle were given 12.5 mg pLH and artificially inseminated (AI) with frozen-thawed semen. The diameter of the two largest follicles and the corpus luteum were measured by transrectal ultrasonography at CIDR removal, insemination, and 36 h after insemination. Pregnancy diagnosis was done ultrasonically 38 and 65 d post-AI. There was no difference in pregnancy rates in short- vs long-progesterone exposure in heifers (53 vs 47%, P = 0.44) or cows (63 vs 58%, P = 0.51). However, the diameter of the ovulatory follicle at CIDR removal and AI was smaller in short- than in long-progesterone groups (P < 0.02), and larger in cows than in heifers (P < 0.006). In conclusion, short-progesterone exposure during the growing and early-static phase of the ovulatory follicle (similar to 3-wave cycles) was not associated with higher fertility than a longer progesterone exposure (similar to 2-wave cycles).

Effect of duration of the growing phase of ovulatory follicles on oocyte competence in superstimulated cattle

In the present study, we tested the hypotheses that oocyte competence is compromised by a longer duration of follicular growth and that it is not affected by FSH starvation. Cows were allocated to short FSH (n=14), FSH starvation (n=13) and long FSH (n=13) groups. The first two groups were given eight doses of FSH, whereas the third group was given 14 doses of FSH, starting from the day of wave emergence (Day 0). A progesterone-releasing device (controlled internal drug release; CIDR) was placed intravaginally at the start of the experiment in all groups. The short FSH group was given prostaglandin (PG) F2α on Day 3, whereas the two other groups received PGF2α on Day 6. In all cows, the CIDR was removed at the time of PGF treatment; porcine (p) LH was given 24h after CIDR removal and cows were inseminated 24 and 36 h later. Reproductive tracts were collected 4 days after insemination and ova and/or embryos were cultured for ≥6 days. The FSH starvation group had fewer ovulations (P=0.001), and ova and/or embryos (P<0.05). No difference in embryo quality was detected between long and short FSH groups at 7, 9 or 10 days after artificial insemination. In conclusion, oocyte competence was not altered by the duration of the follicular growth phase in superstimulated cows, whereas FSH starvation substantially reduced the ability of superstimulated follicles to ovulate.

Developmental Competence of Bovine Oocytes Selected by Brilliant Cresyl Blue Staining: Effect of the Presence of Corpus Luteum on Embryo Development

ABSTRACT The local circumstances of the ovary may influenced by the presence of corpus luteum (CL). We investigated the cleavage rate and blastocyst development of bovine oocytes collected from ovaries with or without CL. Oocytes were incubated with brilliant cresyl blue (BCB) for 90 min and separated on the basis of low (BCB+) or high (BCB–) activity of glucose-6-phosphate dehydrogenase. For both types of ovaries, the embryo cleavage rates in the case of BCB+ oocytes were significantly higher than those in the case of BCB– oocytes, but were not higher than the rates in the control group. The percentage of blastocysts developing from BCB+ and BCB– oocytes from the ovaries with CL did not significantly differ. However, in the case of ovaries without CL, a significantly higher (P<0.05) percentage of blastocysts developed from BCB+ oocytes than from BCB– and control group oocytes. The presence or absence of CL did not significantly influence the cleavage rate and blastocyst development. Based on our results, we conclude that BCB staining facilitates the selection of competent oocytes that will develop into blastocysts for IVF better than conventional morphological selection methods. We also conclude that the CL does not significantly influence blastocyst development.

Effect of Meiotic Arrest by Cycloheximide on the In Vitro Maturation of Bovine Oocytes and Their Subsequent Development Following In Vitro Fertilization

ABSTRACT Cycloheximide (CHX) is a reversible inhibitor of bovine meiotic resumption. This study examined the timing of nuclear maturation in bovine oocytes treated with CHX and determined the optimum maturation interval in cultures for their subsequent development. CHX prevented the nuclear maturation of nearly all oocytes for 24 h. In an inhibitor-free medium, the majority of the CHX-treated oocytes underwent germinal vesicle breakdown (GVBD) after 6 h of culture, while the control oocytes did so at 10 h. In the maturation culture, the majority of the CHX-treated oocytes had reached metaphase II by 16 h whereas the control oocytes took 20 h. However, the CHX-treated oocytes that matured for 16 h developed into blastocysts at low rates while those that were matured for 20 h had a development rate similar to that of the control oocytes. These results indicate that the nuclear maturation of CHX-treated oocytes did accelerate, but these oocytes needed the same maturation time as required by the control oocytes for their subsequent development to blastocysts.

Establishment of protocol for preparation of gene-edited bovine ear-derived fibroblasts for somatic cell nuclear transplantation

Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbeccos modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique.

10 PREDICTION OF CALVING TIME USING BODY-SURFACE TEMPERATURE SENSORS AND PEDOMETERS IN BEEF CATTLE

Predicting the onset of calving is important for the prevention of stillbirths. This study aimed to investigate whether using body-surface temperature and number of steps at the time of calving could be used to predict calving time. Three heifers and 4 cows of the beef cattle variety were used in this study. All animals were housed in calving pens measuring 4.2×3.6m each. Body-surface temperature was recorded using a sensor that was fixed ventrally to the body of the tail head (NIAH, NARO, Tsukuba, Japan). The number of steps the beef cattle took was measured using pedometers (Gyuho, Comtec, Miyazaki, Japan), where each 10 steps resulted in 1 count. The temperature sensors and pedometers were placed on the animals for at least 10 days before the estimated date of delivery. Body-surface temperature and pedometer readings were automatically collected every 2min and every hour. The data used to predict calving were the means of the body-surface temperature and steps taken, which were recorded from the day the sensors were mounted until 1 day after calving. All data were statistically analysed using t-tests. The mean body-surface temperature before calving was 35.4°C (10h), which was significantly lower than that recorded 3 days before calving (36.2°C; P<0.05). The mean body-surface temperature of beef cattle 10 to 6h before calving (36.0°C) was not significantly different from the mean temperature 3 days before calving. However, from 5 to 1h before calving, the mean body-surface temperature (34.8°C) was significantly lower than that 3 days before calving (P<0.05). The mean step number of 101.4 counts, taken 10h before calving, was significantly increased compared with the mean step number of 44.7 counts 3 days before calving (P<0.05). The mean step number of 59.7 counts from 10 to 6h before calving was not significantly different from readings taken 3 days before calving. The mean step number count from 5 to 1h before calving was 143.1 counts, which increased significantly from readings taken 3 days before calving (P<0.05). In conclusion, ventral tailhead body-temperature sensors and pedometers can be used to predict the onset of calving in beef cattle. Their body-surface temperature, along with the number of steps taken before calving, decreased and increased, respectively, in the 5h leading up to calving.

Gene expression of leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF) in bovine endometrium

Leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF), members of the group of hemopoietic cytokines, play a primary role in the control of embryo development and implantation and in the growth of the placenta in humans and mice. Gene expressions of LIF and M-CSF were investigated using quantitative RT-PCR in bovine endometrial tissues during early and mid-pregnancy (Days 16-17, 20-21, 30-36, 48-49 and 74-140) and during the estrous cycle (Days 13-14). Leukemia inhibitory factor and M-CSF genes were expressed in all samples examined. Significant differences were found between the gene expression patterns of LIF and M-CSF. Leukemia inhibitory factor expression level at Days 48-49 was the highest in caruncular endometrium, however, the large variability negated any significant differences. Leukemia inhibitory factor expression levels in intercaruncular endometrium at Days 48-49 and 74-140 of pregnancy were greater than at Days 13-14 of the estrous cycle and at other days of pregnancy. No significant change was recognized in M-CSF expression levels in caruncular endometrium. Macrophage colony stimulating factor expression level in intercaruncular endometrium at Days 74-140 was greater than those of the other samples. These results suggest that LIF and M-CSF are produced in the endometrium and may play different roles in early and mid-pregnancy.

201 EFFECT OF A SINGLE SUBCUTANEOUS INJECTION OF FSH AND TIMING OF PROSTAGLANDIN F2α ADMINISTRATION ON SUPEROVULATORY RESPONSE IN JAPANESE BLACK COWS

Superovulation treatment using FSH requires injection twice a day, for 3 to 4 days. This conventional method requires frequent handling of donors and higher labour costs. Therefore, simplification of the superovulation treatment protocol is needed to reduce animal handling and labour costs. The objective of this study was to investigate the effect of a single subcutaneous FSH injection and the timing of prostaglandin F2α (PGF; cloprostenol) administration on the superovulatory response in Japanese Black cows (Hiraizumi et al. 2015 Theriogenology 83, 466-473) and to determine whether the superovulation treatment protocol can be used in on-farm conditions. A total of 270 Japanese Black cows were used in this study. Twenty Armour units of pFSH dissolved in 30mL of saline was injected subcutaneously in the neck region. In Experiment 1, 32 cows received an intravaginal progesterone device (CIDR) at random stages of the oestrous cycle (Day 0), and 2mg of oestradiol benzoate on Day 1 (24h after CIDR insertion). On Day 6, FSH was injected subcutaneously, and 16 cows were simultaneously injected with 0.5mg of PGF (0-h PGF, Group A); the other 16 cows were injected with 0.5mg of PGF at 48h (on Day 8) after FSH injection (48-h PGF, Group B). The CIDR was removed at 60h after FSH injection and AI was done 42 to 48h after CIDR removal. Embryo collections were performed 7 days after AI. In Experiment 2, 238 cows were used in farm conditions. The cows were superstimulated using the same protocol as that used for Group A. Data were analysed by ANOVA for the mean numbers of collected ova/embryos and transferrable embryos and chi-square test for the proportion of transferrable embryos. In Experiment 1, there were no differences in the mean numbers of ova/embryos collected (16.9±12.3v. 16.1±17.1) or transferrable embryos (11.1±9.5v. 7.2±6.2). However, the proportion of transferrable embryos for Group A was significantly higher than that of Group B (65.9v. 44.7%; P<0.01). In Experiment 2, the mean numbers of ova/embryos collected and transferrable embryos were 15.7±13.3 and 6.8±7.8, respectively. These results showed that a superovulation treatment protocol involving a single subcutaneous injection of FSH with simultaneous PGF injection can be effectively used for Japanese Black cows under on-farm conditions.