In Experiment I, a suitable combination of a vitrification solution (VS) and a rehydration solution (RS) was investigated for efficient cryopreservation of in vitro fertilized (IVF) bovine blastocysts on day 7. The variable of the VS is the concentration of polyvinylpyrrolidone (PVP) and that of the RS is the concentration of trehalose. Embryo development of 79% and hatching of 73% were obtained with VS4, 40% ethylene glycol (EG) + 11.3% trehalose + 12% PVP and RS1, 0% trehalose combination. In Experiment II, IVF bovine embryos on days 7, 8 and 9 were vitrified in VS4 and rehydrated in RS1. The highest proportions of embryos reexpanded (84%) and hatched (68%) during culture when the age was day 7. In Experiment III, day 7 embryos which had been vitrified in VS4 and warmed in 30 C waterbath were kept in situ inside the straw at 5 C or 20 C for 5 min before rehydration in RS1. Significantly higher proportions of embryos developed in culture when they were kept at 5 C than at 20 C. Furthermore, 3 out of 5 recipients in which vitrified embryos had been directly transferred gave birth to normal calves. Therefore, day 7 IVF bovine blastocysts can be successfully vitrified in VS4 and can be directly transferred into an isotonic environment.
A cDNA clone encoding preprorelaxin was isolated from the equine placental cDNA library and its nucleotide sequence was determined. Analysis of the sequence revealed that the cDNA contained a polyadenylation site and an open reading frame encoding 182 amino acids, including a signal peptide of 25 amino acids, a B-chain of 28, a C-peptide of 109 and an A-chain of 20 amino acids. The nucleotide sequence of preprorelaxin shows homology with those of the pig (75.5%), monkey (67.4%), human (67.4%), rat (60.7%) and mouse (54.6%). The main differences between equine preprorelaxin and that of these other species were the insertion of a Leu residue at position 87 in the C-peptide and also the deletion of two residues from the C-peptide and A-chain. Northern blot analysis of mRNA obtained from pregnancy day 70 placenta showed that there were two different sizes of mRNA (1.0 and 1.8 kb).
Transferring the nucleus from a fertilized mouse 2-cell embryo causes oocyte activation. Here, the sequential changes in a transferred nucleus in reconstructed oocytes were investigated and the histone H1 kinase activity of these oocytes was studied. Diploid nuclei from fertilized or parthenogenetic 2-cell embryos placed in oocytes underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) and the histone H1 kinase activity was high within 1 h after fusion. Two polar bodies were extruded when the oocytes received fertilized 2-cell nuclei, and two pronuclei were formed 5 to 6 h after fusion. Histone H1 kinase activity then significantly decreased and reached the basal level (35.6% of the initial level). However, oocytes receiving a nucleus from parthenogenetic 2-cell embryos arrested at metaphase, although histone H1 kinase activity was diminished only slightly (71.6% of the initial level). These results showed that nuclei from early fertilized embryos induced oocyte activation and that various events in oocytes activated by these nuclei were similar to fertilization in some respects.
The role of estradiol in regulating the secretion of LH and FSH from diestrus to proestrus of rats was examined by neutralizing estradiol using antiserum against estradiol (estradiol-AS). An IV injection of varying doses of estradiol-AS at 1100 h on diestrus caused a significant rise in plasma LH (maximally 318.2 ± 27.3% vs control) and non-significant rise in plasma FSH (maximally 144.0 ± 22.0% vs control) at 6 hours after the injection. Administration of the antiserum prolonged the estrous cycle and the day of ovulation by 1 day. The prolongation was caused by a 1-day delay of the LH surge. A small but significant increase in the number of tubal oocytes shed was noted in the estradiol-AS treated group (16.3 ± 0.8, n=12) compared with controls (14.5 ± 0.4, n=15). These results indicated that estradiol suppresses tonic secretion of LH and that the steroid may also be concerned with the regulation of FSH. They also confirmed that the rise in plasma estradiol from diestrous morning is necessary for triggering the LH surge on the following afternoon. Further, administration of estradiol-AS on diestrus resulted in a significant increase in the number of oocytes shed, probably due to the increase in tonic secretion of LH and FSH between diestrus to proestrus.
The present study was conducted to compare glycerol and ethylene glycol as cryoprotectants for porcine in vivo derived and in vitro pre-cultured peri-hatching blastocysts. Embryos were surgically collected from superovulated gilts on Day 6. In vivo derived hatched blastocysts (in vivo HB) were frozen with 1.5 M glycerol (1.5 G) or 1.5 M ethylene glycol (1.5 EG). In vitro pre-cultured peri-hatching blastocysts (in vitro pHB), obtained by culturing Day 6 embryos for 18 h in Whitten's medium supplemented with 1.5% bovine serum albumin and with or without 5% fetal calf serum (FCS), were frozen with 1.5 G or 1.8 EG. All embryos were frozen by a slow cooling method using a programmable freezer. The survival of in vivo HB frozen with 1.5 G (82%) was higher (P<0.01) compared with embryos frozen with 1.5 EG (36%). More blastocysts cultured with FCS developed to the hatched blastocyst stage compared with those cultured without FCS (73% vs 33%, P<0.01) within 18 h. The survival of embryos pre-cultured with or without FCS however was not different following freezing with 1.5 G (23% vs 34%) or 1.8 EG (74% vs 81%). The survival rates for embryos frozen with 1.8 EG were significantly higher (P<0.01) than those for the 1.5 G groups. These results demonstrate that more than 80% of in vivo HB can be successfully frozen with 1.5 G and that similar survival rates for in vitro pHB can be achieved by using 1.8 EG. The study also indicates inherent differences in the post-thaw survival of in vivo derived and in vitro pre-cultured peri-hatching blastocysts frozen with 1.5 G.
The aim of the present study was to investigate the effect of oxytocin (OT) on progesterone secretion by bovine corpus luteum (CL) in different culture systems. When luteal cells were cultured for 1, 2, 4 and 15 h at a density of 6 × 104 cells/ml, OT (10-7 M) did not affect progesterone secretion. On the other hand, bovine LH strongly stimulated progesterone secretion by the cells 2-15 h after incubation (P<0.01). In another experiment, a novel luteal slice culture system was used, in which luteal cells maintained cell-to-cell contact. Three slices (15-20 mg/a slice) from a CL were pre-incubated for 1 h, and then each slice was incubated for 1 h in a culture tube with a medium (3 ml) supplemented with and without bovine LH (100 ng/ml) or OT (10-7 M). Slices were replaced every 1 h in a fresh medium during the 5 h incubation experiment. A 1-h stimulation with bovine LH and OT induced significant but different effects on the release of progesterone. OT acutely stimulated progesterone release at doses 10-7 M (155%; P<0.05), and bovine LH significantly increased progesterone release during and after stimulation (223%; P<0.01). These data support the hypothesis that OT plays a role in modulating the mechanisms of progesterone secretion as an intraluteal regulator. Differences observed between the two systems might be due to the presence or absence of cell-to-cell contact.
The objective of this study was to determine if bovine fetal cell DNA is present in pregnant maternal blood as human fetal cells are present in the blood of pregnant women after they cross the placenta and penetrate into the v. uterina. The btDYZ PCR amplification was employed to detect male-specific DNA of bovine male fetal cells. Blood samples were collected from the v. uterina and v. jugularis externa of 3 pregnant cows (2 with male fetuses and 1 with a female fetus 12 to 20 wks of age) and from only the v. jugularis externa of 33 pregnant cows (with 18 male and 15 female fetuses 10 to 40 wks of age). All blood samples were male-specific DNA negative, which indicated that fetal cells were absent in bovine maternal blood at a level of 1 in 10, 000 and, therefore, cannot be used for prenatal sexing with PCR.
Fifty-eight anestrous Suffolk ewes, raised in Hokkaido, were randomly divided into 2 groups, with or without melatonin feeding as follows: Group 1 ewes (n=33) were fed melatonin daily for 45-90 days from the early or mid-anestrous season (late March to June) and Group 2 ewes (n=25) were not. All the ewes were introduced to fertile rams for 6-12 weeks starting between April and July. The incidence of estrus in Group 1 ewes during the joining of the rams was higher than that in Group 2 ewes (100% vs. 32%, p<0.01). The first estrus in Group 1 was observed 7-10 weeks after the beginning of melatonin feeding. The intervals from the ram introduction to the onset of estrus in 8 ewes that showed estrous response in Group 2 were not distinguishable from those in the ewes fed melatonin for over 5 weeks before the ram introduction in Group 1. The conception rate in Group 1 was higher than that in Group 2 (93.9% vs. 8%, p<0.01). The prolificacy in the Group 1 and 2 ewes was 148% and 8%, respectively. These results indicates that Suffolk ewes were responsive to the 'male effect' 5 weeks onward after the start of the melatonin feeding during early and mid- anestrous season.
To analyze mitotic activity of fetal tissues, pregnant hamsters were continuously injected with [2-14C]thymidine through a cannula installed in the abdominal aorta. Controls received periodic injections of the same amount of the drug into the sublingual vein under anesthesia. Both the relative concentration and intensity of radioactivity in various tissues of the 12th day fetus, 3.5 h after administration, were significantly higher in the experimental group than in controls. Positive images of autoradioluminographs and of light microscopic autoradiographs in the eye, lung and liver of the experimental group were also remarkably increased when compared with controls. These results suggest that this procedure will be useful for studies on proliferating cells of fetuses