Kazufumi Goto

Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos.

We have evaluated the effects of embryo density and the co-culture of unfertilized (degenerating) oocytes on the development of in-vitro fertilized (IVF) mouse embryos. In experiment 1, groups of one, five, 10 or 20 zygotes were cultured in 20 μl drops of modified human tubal fluid (HTF) medium for 168 h at 38.7°C in 5% CO 2 and 95% air. As the embryo density increased, significantly (P < 0.05) higher rates of embryos reached hatched blastocyst stage. In addition, the time required for hatching after IVF was significantly (P < 0.05) shortened by the increase in embryo density. In experiment 2, 10 IVF zygotes were cultured with or without 10 unfertilized (degenerating) oocytes in 20 μl drops of HTF medium. The rates of IVF embryos that developed to morula, blastocyst, expanded blastocyst and hatched blastocyst stages were decreased significantly (P < 0.01) by culturing embryos with unfertilized oocytes compared with culturing embryos alone. In experiment 3, groups of one or 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocytes were cultured in 20 μl drops of HTF medium and the number of cells per blastocyst was examined at 120 h after IVF. Increasing embryo density resulted in a significant (P < 0.05) increase in the number of cells per blastocyst. In contrast, the cell number of IVF embryos that developed to blastocyst decreased significantly (P < 0.05) when they were cultured with unfertilized oocytes. The results suggest that in-vitro development of IVF mouse embryos is enhanced by increasing embryo density and is impaired by co-culture with unfertilized (degenerating) oocytes.

Examination of the safety of intracytoplasmic injection procedures by using bovine zygotes

We have evaluated the safety of intracytoplasmic sperm injection(ICSI) procedures by using bovine zygotes. Bovine zygotes were injected with a small amount (2-3 pl) of either medium alone or medium containing polyvinylpyrrolidone (PVP) (sham-ICSI, without spermatozoon) using the same procedure as ICSI, and the subsequent in-vitro embryonic development and embryo quality (number of cells/blastocyst) were examined. Control zygotes which had not been injected were similarly evaluated after in-vitro development. The sham-ICSI of either medium alone or medium containing PVP into bovine zygotes had no harmful effects on the rate of normal fertilization and on the rate of development to hatched blastocyst stage compared with those of controls (P > 0.05). In addition, no harmful effects were observed in the number of cells per blastocyst (embryo quality). The results suggest, for the first time, that the ICSI procedures currently used for animal and human ICSI are neither detrimental to embryonic development nor detrimental to embryo quality.

Endocrine profiles and embryo quality in superovulated Japanese Black cattle

The relationships between plasma concentrations of progesterone (P4), estradiol-17beta (E2) and lutenizing hormone (LH) and embryo yield and quality were examined in 40 superovulated Japanese Black cattle. The results indicated that the ovarian function, especially the function of corpus luteum on the first treatment day, is an important factor for reliable superovulation in cattle. The levels of plasma E2 and LH at estrus were related to embryo yield and quality.

DETERMINATION OF BOVINE FETAL SEX BY PCR USING FETAL FLUID ASPIRATED BY TRANSVAGINAL ULTRASOUND-GUIDED AMNIOCENTESIS

Fetal sex can be determined by the polymerase chain reaction (PCR) using cells from fetal fluid collected by transvaginal ultrasound-guided amniocentesis. A total of 35 aspirates from 30 cows, 15 Holsteins and 15 Japanese Blacks at 59 to 250 d of pregnancy were used. Five cows were aspirated twice at a 10-d interval. A 5.0 MHz convex array transducer connected to a scanner was inserted into the vagina under caudal epidural anesthesia. The transducer was equipped with a 65-cm long, 21-g needle within the probe carrier. A bovine male-specific primer and a bovine gender-neutral primer were used. Fetal fluid was obtained from all except 2 cows in early pregnancy. Five animals aborted within 1 wk following aspiration. A total of 33 samples, 29 of amniotic fluid and 4 of allantoic fluid, was subjected to PCR analysis. Fetal gender was verified in 31 33 samples (18 females and 13 males). Gender was also determined by gross examination of external genitalia of offspring after calving or abortion. Fetal gender was correctly identified by PCR analysis of aspirated fetal fluid in 16 16 females and in 13 15 males. Transvaginal ultrasound-guided amniocentesis followed by PCR analysis of aspirated cell DNA can be used accurately to determine fetal sex in cows at 70 to 100 d of gestation. The procedure requires considerable skill and is not without some risk to fetal viability.

Plasma progesterone profiles and embryo quality in superovulated Japanese Black cattle

The relationship between plasma progesterone (P(4)) levels before and after superovulation treatment and embryo yield and quality was examined in Japanese Black cattle. It is concluded that a plasma P(4) level at the start of follicle stimulating hormone (FSH) injections is related to embryo quality. The P(4) level of over 3.0 ng/ml appears to be useful in preselection of donors when we want to get more than 8 normal (transferable) embryos on the average.

A Globosus amorphus from an in vitro fertilized embryo transferred to a Japanese black cow

A Globosus amorphus along with a living calf was encountered following the transfer of blastocysts obtained by in vitro fertilization of in vitro-matured follicular oocytes in Japanese black cattle. Two embryos obtained 9 days after in vitro fertilization developed into either a hatched blastocyst with distinct inner cell mass or an expanded blastocyst with indistinct inner cell mass. The embryos were loaded into a 0.25-ml plastic straw and were nonsurgically transferred to the uterus of a heifer on Day 8 (Day 0 = estrus). On Day 75, a twin pregnancy was ultrasonically diagnosed in the right uterine horn, in which a live fetus with distinct limbs and a concomitant ovoid mass were detected. On Day 287, the dam developed parturient paralysis with dropsy of the fetal membranes. By palpation per rectum an ovoid mass was detected in the body of the uterus [corpus uteri] and a larger live fetus was in the uterine horn. A cesarean section was performed to extract a live fetus and a Globosus amorphus. The live fetus was female with the 60, XX female complements.

Methodological Investigation on Chromosomal Preparation of Bovine Embryos

過排卵処理牛から得られた子宮内胞胚と,体外受精後,培養によって得られた牛の培養胞胚を用い,染色体標本作製のためのコルセミド処理時間および低張処理法の検討を行なった.また,染色体検査により性判別を行なうとともに,染色体数の異常の有無についても観察した.子宮内胞胚では,コルセミド処理時間を4,6および8時間に延長しても中期核板を有する胚の出現率と性判別率は2時間処理と変わらなかった.しかし培養胞胚では,コルセミド処理を2時間以上行なうと退行胚が多くなり,処理時間を6時間に延長すると中期核板を有する胚の出現率は減少した.培養胞胚における性判別率は,2時間処理区で高く,処理時間の延長に伴い減少する傾向がみられた.0.5%クエン酸ナトリウムを用いた短時間低張処理法による性判別率は,中期核板を持った胚のうち,子宮内胞胚で80.0%,培養胞胚で74.5%であり,常法より高い判別率であった.このことから,子宮内胞胚および培養胞胚において性判別可能な中期核板像を得るためには,コルセミド(0.04μg/ml)で1～4時間処理し,0.5%クエン酸ナトリウムで洗浄を含め5分間低張処理する方法が適していると考えられた.また染色体検査の結果,発育段階および形態的検査による卵質の各ランクにおいて性判別率に差はみられなかった.染色体数異常胚の出現率は,子宮内胞胚で4.8%,培養胞胚で20.0%で,培養によって染色体数の異常が増加することが観察された.