Developmental competence of bovine oocytes frozen at different cooling rates.

Experiments were conducted to determine the optimal cooling conditions for improving the developmental competence of in vitro-matured bovine oocytes when frozen in 1.6 M 1,2-propanediol. Bovine oocytes were cooled from 0 degree to -5.5 degrees C at 1 degree C/min, seeded, and then cooled to -30 degrees C at different cooling rates (0.3 degree, 0.6 degree or 0.9 degree C/min) and then plunged into LN2. When frozen-thawed oocytes were fertilized in vitro, the proportions of fertilization and developmental competence did not differ among the three groups frozen at different cooling rates. The findings indicated that a cooling rate of less than 1.0 degree C/min in the freezing procedure did not affect the developmental competence of postthaw in vitro-matured bovine oocytes.


Postthaw viability of the inner cell mass of in vitro-matured/in vitro-fertilized bovine embryos frozen in various cryoprotectants.

A study was conducted to examine the viability of inner cell mass (ICM) cells of frozen-thawed in vitro-matured (IVM)/in vitro-fertilized (IVF)-derived embryos using various cryoprotectants. Expanded blastocysts were frozen and thawed in 1.4 M glycerol with 0.25 M sucrose (GL), 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), or 1.3 M ethylene glycol monomethyl ether (EME) as cryoprotectants using a one-step method. After thawing, the embryos were cocultured for 24 h with cumulus cells in TCM199. Embryos which were viable after thawing and developed beyond the blastocyst stage were treated by immunosurgery and differential fluorochrome staining for ICM cell counts. Overall, there were no significant differences in the development to blastocyst stage after 24 h culture in each cryoprotectant (P < 0.05, chi 2 analysis). The viability of ICM cells of frozen-thawed embryos with each cryoprotectant was lower (GL, 72.7%; PG, 67.8%; EG, 77.5%; EME, 74.7%) than that of unfrozen embryos (84.4%). In the case of PG as a cryoprotectant, viability of ICM cells was significantly lower than that of unfrozen embryos (P < 0.05, ANOVA analysis). Our results suggest that the viability of ICM cells of frozen-thawed bovine embryos tend to be lower than that of unfrozen embryos irrespective of the cryoprotectants used. PG was significantly more toxic to the ICM cells compared with the other cryoprotectants.


Viability of frozen-thawed bovine IVM/IVF embryos in relation to aging using various cryoprotectants.

Bovine IVF embryos developed on Days 7, 8 and 9 were equilibrated with 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), 1.1 M diethylene glycol (DEG) or 1.3 M ethylene glycol monomethyl ether (EME) for 10 to 20 min in modified phosphate buffered saline. (mPBS) supplemented with 10% superovulated cow serum. The embryos were loaded into 0.25-ml plastic straws and were placed directly into a 0 degrees C alcohol bath chamber and held for 2 min. They were cooled from 0 degrees C to -5.5 degrees C at 1 degrees C/min and then seeded, followed by a 10-min holding period at -5.5 degrees C. The straws were then cooled to -30 degrees C at 0.3 degrees C/min before plunging into liquid nitrogen. Embryos were thawed and placed directly into the culture medium and washed 3 times. The survival rates of the Day-9 embryos based on reappearance of blastocoele, expansion, and hatching after 48 h of post-thaw culture were significantly lower (P<0.01) than those of the Day-7 and 8 embryos, in all of the cryoprotectants tested. On the other hand, while the reappearance of blastocoele and expansion of blastocysts after 48 h of post-thaw culture were not significantly different among each cryoprotectant, the percentage of hatching blastocysts were significantly different between DEG and EME (P<0.05), between DEG and EG (P<0.01) and between PG and EG (P<0.05). These findings demonstrate that the age of the embryo (Day 7 and 8) is very important for the successful freezing of IVF bovine embryos. Also, as to the hatching rates, EME and EG are superior as cryoprotectants than the other 2 cryoprotectants tested.


Effect of washing, antibiotics and trypsin treatment of bovine embryos on the removal of adhering K99+ Escherichia coli.

Bovine embryos with the intact zona pellucida were exposed in vitro to K99+ Escherichia coli (K99 E. coli). The recommended procedures for washing and treating embryos were then evaluated for their effectiveness in removing or killing the adherent bacteria. After 10-step washing, bacteria were recovered not only from the embryos exposed to E. coli suspensions but also from those treated with trypsin during washing. On the other hand, no bacteria were recovered from any embryos treated with antibiotics (gentamicin; 50 micrograms/ml) in culture medium before washing, indicating that the recommended washing procedures with appropriate antibiotics assure that zona pellucida-intact bovine embryos are free from E. coli.

The Journal of Veterinary Medical Science


Survival rate of frozen-thawed bovine IVM/IVF embryos in relation to post-thaw exposure time in two cryoprotectants.

The relationship between post-thaw exposure time in cryoprotectants and the viability of in vitro fertilized (IVF)-derived bovine embryos after culture was examined. Good- and excellent-quality Day 7 to Day 8 blastocysts and expanded blastocysts were frozen by the nonstepwise method using 1.6 M propylene glycol (PG) or ethylene glycol (EG) as the cryoprotectant. After the straws were thawed, the embryos were kept in the cryoprotectants for 1, 10, or 30 min. Then the embryos were placed directly into culture medium and washed three times, followed by coculture with cumulus cells. After 48 h of culture, there were no significant differences in survival rate among the various exposure times in PG (79-81%) and EG (71-75%). After 72 h of culture, there were no significant differences among the various exposure times in the fully expanded (PG 76-81%; EG 71-72%), hatching (PG 69-74%; EG 58-71%), and hatched blastocyst rates (PG 60-71%; EG 47-65%). Our findings suggest that different post-thaw exposure times up to 30 min using PG or EG as the cryoprotectant are not harmful to IVF bovine embryos.


Developmental capacity of bovine oocytes frozen in different cryoprotectants.

Iso-osmolar concentrations of 1.6 M of 1,2-propanediol, glycerol and dimethylsulfoxide were compared for their effectiveness as cryoprotectants for in vitro-matured bovine oocytes. Survival, defined as the number of morphologically normal oocytes, after freeze-thawing in 1,2-propanediol (49.8%) was significantly higher (P<0.01) than in glycerol (28.6%) or dimethylsulfoxide (32.6%). After insemination, the fertilization rate of morphologically normal oocytes frozen-thawed in 1,2-propanediol (57.9%) was higher (P<0.05) than that of those frozen in dimethylsulfoxide (38.3%). However, the proportion of surviving oocytes developing to blastocysts was not different among the 3 groups.


Effect of trypsin treatment of in vitro fertilized bovine embryos on their subsequent survival and development.

The object of this study was to determine whether several washings with trypsin affected the survival and development of in vitro fertilized (IVF) bovine embryos. The embryos developed to blastocysts 7-8 days after insemination, were washed 12 times in washing medium (modified Dulbecco's phosphate-buffered saline (mPBS) containing 20% fetal bovine serum (FBS): 25 mM Hepes TCM199 with Earle's salts = 1:1), or in a series of three washes in the washing medium, two in mPBS containing 0.3% bovine serum albumin (BSA), two in 0.25% trypsin in Hank's solution (without Ca++ and Mg++) for a total time in the trypsin of 60 to 90 sec and five in the washing medium. After washing, the embryos were either cultured in vitro or cryopreserved. The fresh and frozen-thawed embryos were either cultured for 72 hr in vitro for evaluating development or transferred nonsurgically to recipient cows. Development of fresh and frozen-thawed embryos in vitro in control-washed and trypsin-washed embryos did not differ. Pregnancy rates did not differ (P > 0.05) among recipient cows receiving control-washed or trypsin-washed embryos, and transferring fresh or frozen-thawed embryos. These results indicate that the treatment of bovine IVF embryos with trypsin during washing did not have a positive effect on embryonic development.


Successful vitrification of bovine blastocysts, derived by in vitro maturation and fertilization.

Bovine blastocysts were produced through maturation, fertilization, and development in vitro. For vitrification, solutions designated EFS, GFS, and PFS were prepared; these were 40% ethylene glycol, 40% glycerol, and 40% propylene glycol, respectively, diluted in modified phosphate-buffered saline (PBS) containing 30% Ficoll + 0.5 M sucrose. The embryos were exposed to the solutions in one step at room temperature, kept in the solutions for various times, vitrified in liquid nitrogen, and warmed rapidly. When the embryos were vitrified in EFS solution after 1 or 2 min exposure, the postwarming survival rate, assessed by the reexpansion of the blastocoel, was 74-77%. However, when the exposure time was extended to 3 min or longer, this rate dropped to 7-0%. This reduction was attributed to the toxicity of ethylene glycol. Of the embryos vitrified in GFS solution, 53% survived when they were cooled after 1 min exposure; as the duration of the exposure increased, the survival rate increased, reaching a peak (72%) at 4 min. The rate then decreased gradually with exposure time. In PFS solution, embryos surviving after vitrification were recovered only with 1 min exposure (33%), reflecting the high toxicity of propylene glycol. After vitrification in EFS or GFS solution, two embryos were nonsurgically transferred into each of 14 recipient animals. Of the 14 recipients, ten (71%) became pregnant; two resulted in early stillbirths, four recipients delivered twins (four alive and four stillborn), and two delivered single live calves, demonstrating the effectiveness of this simple vitrification method for the cryopreservation of in-vitro-produced bovine blastocysts.


Effects of different lots of semen from the same bull on in vitro development of bovine oocytes fertilized in vitro.

The effectiveness of 8 different lots of semen from the same bull on the in vitro development of bovine oocytes fertilized in vitro was evaluated. Cleavage and development rates to the blastocyst stage were not significantly different among the 8 lots of semen. However, the cleavage and development rates varied no more than +/-11 and +/-6%, respectively, in overall rates. A maximum of 35.2 and 27.9% difference in cleavage and development rates to the blastocyst stage, respectively, was observed using a single straw from the same semen lot for insemination in 4 trials. This variation tended to be higher than that observed for the cleavage and development rates of oocytes after insemination with double straws from a single lot. These results indicate that the developmental capacity of embryos after insemination are affected by factors associated with a different semen lot from the same bull and with different straws from a single lot.


Developmental capacity of bovine oocytes cryopreserved after maturation in vitro and of frozen-thawed bovine embryos derived from frozen mature oocytes.

The present study was conducted 1) to investigate the post-thaw developmental capacity of in vitro mature bovine oocytes (Metaphase II) frozen by 1.6 M of 1,2-propanediol and 2) to confirm the viability of frozen bovine embryos derived from frozen mature oocytes. The cleavage and developmental rates to the blastocyst stage of frozen-thawed mature oocytes were significantly lower (P<0.01) than that of nonfrozen oocytes. When mature oocytes were treated with hyaluronidase, trypsin, or base solution (solution control) before processing to remove the cumulus cells, the developmental rates to the blastocyst stage of frozen-thawed oocytes were 2.8% (5/180), 3.1% (9/295) and 1.1% (1/89), respectively. The viability and developmental capacity of frozen-thawed bovine embryos derived from frozen mature oocytes were not different from those of frozen-thawed bovine embryos derived from nonfrozen mature oocytes (control). Furthermore, nonfrozen and frozen-thawed embryos derived from frozen-thawed mature oocytes were nonsurgically transferred to recipient cows. One of the four and one of the two recipient cows became pregnant, respectively. The results of this study demonstrated the viability of embryos obtained from frozen-thawed bovine oocytes at Metaphase II followed by in vitro fertilization and culture to the blastocyst stage in vitro.


Effect of antibiotics treatment of in vitro fertilized bovine embryos to remove adhering bacteria.


The time of insemination affects the sex ratio of Japanese black cow offspring.


Influence of large follicles on oestrus induction and ovulation after embryo collection in superovulated Japanese Black Cows.


Effects of Chemical Zona Pellucida Thinning with Pronase on the Development of Bovine Embryos

プロナーゼを用いた透明帯菲薄化処理が牛胚の発育及び受胎率に及ぼす影響

Journal of the Japan Veterinary Medical Association


ホルスタイン種牛の過剰排卵処置における雌雄選別精液の有用性の検討

ホルスタイン種牛の過剰排卵処置における雌雄選別精液の有用性の検討


異なる光学異性体から構成されるPGF2α製剤の過剰排卵処置後の採胚成績および発情回帰に及ぼす影響


黒毛和種牛における分娩後の発情誘起が子宮修復と胚回収およびその後の受胎成績に及ぼす影響


Influence of Recipient Dams of Different Breeds and Artificial Colostrum Feedings on Performance of Japanese Black Male Calves Produced by Embryo Transfer

受胚牛の品種および人工初乳の給与が胚移植により生産された黒毛和種雄子牛の発育および血液成分に及ぼす影響


犬の血圧に及ぼす暑熱ストレスと反復運動の影響

犬の血圧に及ぼす暑熱ストレスと反復運動の影響


Development of vitrified cat oocytes and effects of glucose supplementation on in vitro development of embryos

ネコ卵母細胞のガラス化保存と体外発育に及ぼすグルコースの影響


Morphological and Functional Standard Parameters of Echocardiogram in Beagles

ビーグル犬の心エコー法における形態・機能学的な標準値


牛卵母細胞の体外成熟，受精と発生に係るアラニン，グリシンと成長ホルモンとの関係

牛卵母細胞の体外成熟，受精と発生に係るアラニン，グリシンと成長ホルモンとの関係


簡易炭酸ガス培養器を用いた牛体外受精胚の作出 -陰圧気相条件が胚の発生におよぼす影響


黄体ホルモン製剤を膣内留置した黒毛和種牛への反復過剰排卵処置におけるGnRH投与の効果


Effect of growth hormone and type of incubators on cleavage rate and development of in vitro fertilized bovine embryo.

Journal of the Egyptian Veterinary Medical Association


Effect of Glucose and Lactate on Development of In Vitro Produced Bovine Embryos in a Modified Synthetic Oviduct Fluid Medium.

修正合成卵管液(m-SOF)に添加した乳酸とグルコ-スが牛体外受精胚の発生に及ぼす効果


Isolation of Bacteria from In Vitro Bovine Embryo Production System and Effect of Bacterial Contamination on the Developmental Capacity of In Vitro Fertilized Embryo

食肉処理場で採取した卵巣を用いた牛体外受精胚作出過程での細菌分離と細菌汚染が体外受精胚の発生能に及ぼす影響

The Journal of Reproduction and Development


Effect of Thawing Rate on The Viability of Frozen Bovine Embryos Derived from In Vitro Fertilization

凍結牛体外受精由来胚の生存性に及ぼす融解速度の影響


Direct transfer of vitrified bovine IVF embryos using ethylene glycol supplemented with polyvinylpyrrolydone


In vitro development of bovine oocytes collected from ovaries in every month through the year after in vitro fertilization.


牛における未成熟卵子の細菌曝露が体外発生能に及ぼす影響

牛における未成熟卵子の細菌曝露が体外発生能に及ぼす影響


Inhibitory Effect of Prifinium Bromide after Intrarectal Adminis-tration on the Rectal Peristalic Movement in Bovine

牛直腸内への臭化プリフィニウム投与による直腸の蠕動運動抑制効果


In vitro exposure of bovine immature oocytes to K99+Escherichia coli and elimination of exposed bacteria by washing

牛未成熟卵子のK99保有大腸菌への曝露と洗浄による細菌除去 -特に卵丘細胞の有無による洗浄効果の比較-


プロピレングリコ-ルを用いた牛体外受精胚の1段階希釈凍結保存法の開発

プロピレングリコ-ルを用いた牛体外受精胚の1段階希釈凍結保存法の開発


Outbreak of K99+ Escherichia coli Infection and Serological Survey in Calves

子牛におけるK99保有大腸菌症の発生と抗体調査

Journal of the Japan Veterinary Medical Association