Akira Hanada

In vitro fertilization and cleavage capability of bovine follicular oocytes classified by cumulus cells and matured in vitro

Bovine oocytes were collected from ovaries obtained from an abattoir. They were classified according to the character of the cumulus cells using a stereomicroscope, and cultured in 25 mM Hepes buffered Tissue Culture Medium 199 supplemented with 10% fetal calf serum at 39 degrees C and inseminated by capacitated sperm. Maturation rates of Class A oocytes, with compact, dense cumulus cells; Class B, partially naked oocytes with thin cumulus layers or small remnants of cumulus cells and Class C, naked oocytes were 97.4% (38/39), 89.8% (106/118) and 52.9% (45/85), respectively. The fertilization rates for the three classes were 86.8%, 85.8% and 53.3%, respectively. The naked oocytes had a significantly lower fertilization rate than oocytes of the other two classes. Significantly more Class A oocytes cleaved (63.7%, 232/364) than those of Class B (29.5%, 36/122) and Class C (17.7%, 28/158).

EFFECT OF NUCLEAR STAGES DURING IVM ON THE SURVIVAL OF VITRIFIED-WARMED BOVINE OOCYTES

The effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h in TCM199 supplemented with 5% fetal bovine serum (FBS) in 3% CO2 in air. The oocytes were first exposed to 20% ethylene glycol solution and were subjected to vitrification in a solution containing 40% ethylene glycol, 18% Ficoll-70 and 0.3 M sucrose. After warming in 20 degrees C water, oocytes which had been vitrified at less than 24-h of IVM were again cultured to complete the 24-h of IVM period. Oocytes were then incubated with frozen-thawed spermatozoa in Brackett and Oliphant (BO) medium containing 60 micrograms/ml heparin and 0.25% BSA for 20 h. In vitro fertilization rates of oocytes vitrified-warmed at 0, 6, 12 and 24-h IVM were 75.2, 68.0, 82.0 and 72.4%, respectively, comparable to the rates for unvitrified control oocytes (80.6%). A higher incidence of polyspermic fertilization was observed in oocytes vitrified at 24-h IVM (44.9 vs 22.6% in the control group, P < 0.05). Vitrification of oocytes at 12-h IVM seemed to be better than that of other IVM groups, since the normal fertilization rate of all treated oocytes was the highest (36.0%) among the vitrification groups. Developmental competence of the oocytes following vitrification and in vitro fertilization (12-h IVM group) was examined by cell-free culture of presumptive zygotes up to 9 d in modified synthetic oviduct fluid (mSOF) in 5% CO2, 5% O2 and 90% N2. The cleavage rate of zygotes from vitrified oocytes 48 h after insemination was 29.8%, which was lower than that of the control group (57.0%, P < 0.05). Development to blastocysts from the vitrified oocytes (4.8%) was much lower than that of the control group (27.0%, P < 0.05). These results indicate that cryopreservation of bovine oocytes by vitrification may be affected by their maturation stage in vitro, and that developmental competence to blastocysts of cleaved oocytes following vitrification may be impaired compared with unvitrified control oocytes.

Effect of linoleic acid-albumin in the culture medium on freezing sensitivity of in vitro-produced bovine morulae

The objective of this study was to improve the survival of in vitro-produced bovine morulae after cry opreservation. In Experiment 1, presumptive zygotes at 20 h post-insemination (hpi) were cultured in a mixture of modified synthetic oviduct fluid (m-SOF)/0.3% BSA and m-SOF/0.3% linoleic acid-albumin from bovine serum (LAA) at 39.0 degrees C in 5% O2, 5% CO2 and 90% N2 (final LAA concentration: 0, 0.01, 0.03, 0.1 or 0.3%). Morulae harvested at 138 hpi were frozen and thawed in m-PBS/0.3% BSA containing 1.5 M ethylene glycol and were cultured for 96 h in m-SOF/10% FBS to assess further development. The post-thaw survival of morulae derived from culture in 0.1% LAA (60%, P < 0.01) and in 0.03% LAA (55%, P < 0.05) was higher than that in 0% LAA (32%). Lowering the LAA concentration below 0.1% resulted in similar rates of morula development as in m-SOF/0.3% BSA. In Experiment 2, zygotes were cultured in m-SOF/0.1% LAA from 20 to 90 hpi and/or from 90 to 138 hpi. Post-thaw survival of morulae that had been exposed to LAA from 20 to 90 hpi (39%) or from 90 to 138 hpi (56%) was higher than that of morulae cultured without LAA from 20 to 138 hpi (12%, P < 0.02). These survival rates were lower than that of morulae cultured with LAA over a period of 20 to 138 hpi (76%, P < 0.001). The results indicate that cell-free culture of IVM/IVF bovine zygotes in m-SOF supplemented with LAA produces morula-stage embryos relatively tolerant to the process of freezing and thawing.

Effects of timing of oocyte cryopreservation on in vitro development of nuclear-transferred bovine zygotes

The utility of cryopreserved bovine oocytes as recipient cytoplasts for nuclear transfer (NT) was examined. In vitro‐matured (IVM), metaphase‐II oocytes were enucleated by mechanical suction and activated parthenogenetically. The cytoplasts were fused with blastomeres of in vitro‐produced day‐5 morulae by a DC electropulse, and then cultured up to 8 days (non‐frozen controls; group I). Oocytes were frozen‐thawed in 1.5‐M ethylene glycol and 0.1‐M sucrose before enucleation (group II), after enucleation (group III), after enucleation and aging culture (group IV), or after activation (group V). In group I, 91% of IVM oocytes could be used for NT and 89% of them fused successfully. Finally, 36% of the fused zygotes developed into blastocysts. The proportions of morphologically normal oocytes after thawing in groups IV and V (70 and 69%, respectively) were higher than in group III (56%), and the proportion of IVM oocytes used for NT in group IV (56%) was higher than those in groups II, III, and V (33%, 35%, and 38%, respectively). Fusion rates of the NT zygotes in groups III, IV, and V (90%, 88%, and 88%, respectively) were higher than the rate in group II (75%). Rates of development into blastocysts of the fused zygotes in groups II, III, IV, and V were 0%, 3%, 2%, and 6%, respectively (P < 0.05, group II vs. groups III, IV, and V). Developmental kinetics and cell numbers of the blastocysts were similar among the groups. It was suggested that timing of oocyte cryopreservation is among the factors influencing efficiency of production of cloned embryos in cattle. Mol. Reprod. Dev. 54:81–85, 1999.

Repeated superovulation of Holstein heifers with FSH and PGF2.ALPHA. analogue for non-surgical embryo collection.

ホルスタイン種未経産牛18頭にFSHとPGF2αアナログによって連続して2回過排卵処理を行い,人工授精後7日目に非手術的に胚採取を行なった。1. 胚採取率は第1回目の過排卵時には88.9%(16/18頭),第2回目には55.6%(10/18頭)であった。2. 第1回目と第2回目の過排卵を比較すると推定黄体数はそれぞれ9.3,7.9,採取胚胚数はそれぞれ5.6,3.1,で両者の間には差がみられなかったが,発育胚数はそれぞれ3.6,1.3で第2回目の過排卵時の成績が悪かった。第1回目と第2回目の過排卵時の推定黄体数の間には有意な関係が認められず,第1回目の過排卵反応は第2回目の反応の指標にはなりえなかった。3. 胚回収後10~11日にPGF2αアナログを投与することによって妊娠を防止でき,次の発情の発現をはやめることができた。

Variation of diphenylamine-positive materials in seminal plasma and spermatozoal acidsoluble frac-tion during in vitro storage of semen

精子DNAの安定性を調べるため,5種の動物精液の体外保存中に精しょう分画ならびに精子の酸可溶性分画中に検出されるジフェニルアミン反応陽性物質(DPM,主としてDNA由来のデオキシリボースと推察される)を調べ,次の成績を得た。1. 採取直後の精液材料では,調べた全ての動物で,両分画中にいろいろな量のDPMが検出された。とくに馬精しょう中のDPM量は精子1億あたりに換算すると最高であった。2. 原精液の4°Cまたは15°C(豚精液のみ)保存中,精しょうDPM量の増加が鶏(4時間後),馬とうさぎ(各48時間後)で有意に認められた。しかし,牛と豚では保存48時間までの間に採取直後例との有意差は認められなかった。酸可溶性分画DPM量はどの動物の精液を保存した場合でも有意の変動はみられなかった。3. 原精液を液体窒素で5分間凍結後室温で融解し,4°Cに保存した場合,精しょうDPMの増加は鶏と馬で促進されたが,うさぎ,牛ならびに豚では凍融処理の影響はみられなかった。4. 両分画のDPMの総量についてみると,その変動傾向は精しょう量の変動にほとんど一致したが,馬では総量の変動は精しょうにおけるよりも早く現われ,うさぎは総量の変動は保存中認められなかった。