Effect of dimethyl sulfoxide and protein concentration on the viability of two-cell mouse embryos frozen with a rapid freezing technique.

Two-cell mouse embryos were frozen by direct plunging into liquid nitrogen after a 3-min exposure to solutions containing 0.25 M sucrose with 1.5, 3, or 4.

Development of preimplantation goat (Capra hircus) embryos in vivo and in vitro.

Preimplantation goat embryos were cultured with or without goat oviduct epithelial cells in Earle's 199 medium + 10% goat serum (E199 + 10%GS), and in three different simple chemically defined media. In-vivo development was characterized by an extended 8- to 16-cell stage followed by a rapid cleavage rate in the next 3 cell cycles. Culture of 1-8-cell embryos in Medium E199 + 10%GS led to cleavage arrest at the 8-16-cell stage, while in the chemically defined media embryos developed poorly and a high percentage failed to pass the 8-16-cell stage.

Human micro-insemination by injection of single or multiple sperm: ultrastructure.

The process of micro-insemination by single or multiple sperm transfer into the perivitelline space (PVS) or by direct sperm injection into oocytes was examined by transmission electron microscopy. Spermatozoa from normal and oligozoospermic men were injected into oocytes, obtained from consenting IVF patients, mostly by zona-puncture using micromanipulators. Spermatozoa were washed by the Percoll or Ficoll methods and capacitated using Whittingham's T6 or modified Tyrode's medium or incubated in strontium medium before injection.

Left and right ventricular myocardial morphometry in fetal, neonatal, and adult sheep.

This study has examined left (LV) and right ventricular (RV) myocardial morphometry in perfusion-fixed hearts of late-gestation sheep fetuses, neonatal lambs, and adult sheep. During development, myocyte size, intercapillary distance, and myocyte myofibrillar and mitochondrial volume densities increased, whereas capillary density, the myocyte-to-capillary ratio, and the myocyte matrix volume density decreased. RV myocytes were larger than LV myocytes in cross section in fetuses and 4-day-old lambs.

Co-administration of ascorbic acid with cyclophosphamide (CPA) to pregnant mice inhibits the clastogenic activity of CPA in preimplantation murine blastocysts.

Cyclophosphamide (CPA) administered at a dose of 15 mg/kg body weight to pregnant inbred CBA/CaH mice 60 h after copulation significantly elevated the incidence of structural chromosomal aberrations and sister-chromatid exchanges in 96 h blastocysts. When ascorbic acid (800 mg/kg body weight) was co-administered with CPA (15 mg/kg) using different injection sites, the incidence of structural chromosomal aberrations and the number of aberrant metaphases were significantly lower as compared to the CPA-exposed embryos, but still significantly higher than untreated controls. Sister-chromatid exchanges were not significantly different in embryos exposed to CPA only when compared to those exposed to CPA and ascorbic acid.

Fertilization and development of mouse eggs injected under the zona pellucida with single spermatozoa treated to induce the acrosome reaction.

Mouse spermatozoa were capacitated and acrosome reacted by incubation for 30 min to 6 h in modified Tyrode's medium (T6) and in cGMP. The fertilization rates of eggs microinjected with a spermatozoon from samples incubated for 30 min, 2 h, and 6 h in T6 and in cGMP were 36%, 34%, 29%, and 43%, respectively. These rates were not correlated to the percentage of acrosome-reacted spermatozoa identified after these treatments.

Cardio-respiratory responses to cool ambient temperature differ with sleep state in neonatal lambs.

1. Responses to cool ambient temperature were tested with reference to the sleep-wakefulness cycle in six chronically instrumented newborn lambs which were exposed to warm (20-25 degrees C) and cool (10-15 degrees C) ambient temperatures (Ta) in fifteen studies. 2.

Blood flow to the respiratory muscles during hypercapnic hyperpnoea in the newborn lamb.

The blood flow to the diaphragm, external and internal intercostal muscles, abdominal oblique muscles, and other rib-cage and abdominal muscles was measured, using radio-labelled microspheres, in 6 newborn lambs quietly breathing in air and during 3 different levels of CO2 induced hypercapnic hyperpnoea (inspired gas containing 4%, 5.5%, or 7% CO2). We also calculated the oxygen uptake of the diaphragm (VO2di).

Diaphragm VO2, diaphragm EMG, pressure-time product and calculated ventilation in newborn lambs during hypercapnic hyperpnoea.

We examined the relationship between diaphragmatic EMG (EMGdi) and pressure-time index (Pt'), and diaphragmatic oxygen consumption (VO2di) in 6 newborn lambs quietly breathing air and during 3 levels of hypercapnic hyperpnoea (PaCO2 was equal to 48, 53, or 60 Torr). We also determined minute ventilation (VI), whole body oxygen consumption (VO2), diaphragmatic oxygen consumption (VO2di), and respiratory muscle oxygen consumption (VO2resp). VO2 did not change significantly throughout the experiment.

Successful single-cell biopsy and cryopreservation of preimplantation mouse embryos.

We have previously observed that preimplantation embryo biopsy in the mouse causes a reduction in implantation rate in utero. After minor modifications to the technique, we now find that sampling a single blastomere from the 4-cell mouse embryo does not compromise continued development in vitro or in vivo. When transferred to pseudopregnant foster mice, 60.

Effect of sinoaortic denervation on arousal responses to hypotension in newborn lambs.

To examine whether hypotension reflexly initiates arousal from sleep and the mechanisms involved, we subjected sleeping lambs to hypotensive stimuli of 1-min duration, before and after sinoaortic denervation (SAD). In intact lambs, hypotension increased the probability of arousal from both quiet sleep (QS) and rapid-eye-movement (REM) sleep. Hypotension resulted in nonarousal in 42% (QS) and 47% (REM) of tests.

Biopsy of preimplantation mouse embryos: development of micromanipulated embryos and proliferation of single blastomeres in vitro.

We have developed a technique to sample the preimplantation embryo, which may, in the future, be applied to prenatal diagnosis of genetic disease. Using micromanipulation, we aspirated a single blastomere from 4-cell mouse embryos. This procedure had no effect on in vitro development; 98% of control and 94% of biopsied embryos reached the blastocyst stage after 48 h in culture.

In vivo cleavage rates and viability obtained for early cleavage mouse embryos in co-culture with oviduct cells.

The cleavage rate and development of two-cell mouse embryos to the morulae stage in co-culture with mouse oviduct cells was studied in vitro and compared with those achieved in vivo. Embryos were cultured in Whittingham's T6 (T6), T6 supplemented with fetal calf serum (FCS) and in co-culture with either Dulbecco's Modified Eagles Medium supplemented with sodium lactate (DMEM + 1a) or a modification of T6 medium containing vitamins and amino acids (T6 + v + aa). Co-culture of oviductal cells with DMEM + la medium supported two-cell mouse embryo development to eight cells at a rate significantly better (P less than 0.

Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses.

Vitrification of mouse oocytes adversely affected the subsequent developmental potential of embryos and fetuses derived from the fertilization of such oocytes after thawing. Only 5% of oocytes vitrified formed viable fetuses on the 15th day of gestation as compared to 47% in the controls. The incidence of chromosomally aneuploid zygotes, derived from cryopreserved oocytes, was approximately threefold higher than the controls irrespective of whether the oocytes were cryopreserved by vitrification or DMSO slow-freezing.

The effects of cooling human oocytes.

Preovulatory human oocytes were cooled to 0 degrees C at 1 degree C/min, with or without the cryoprotectant dimethyl sulphoxide (DMSO), to assess the effects of cooling on the meiotic spindles and on oocyte structure. Batches of oocytes, cultured for 3-9 h, were held at 0 degrees C for 20 or 60 min and then fixed for transmission electron microscopy (TEM) either at 0 or 8 degrees C. Control oocytes were not cooled and were fixed at 22 or 37 degrees C for comparison.

Evidence for prostaglandin involvement in early luteal regression of the superovulated nanny goat (Capra hircus).

Feral does of various ages were treated with intravaginal progestagen sponges for 16 days to synchronize oestrus. On Day 2 before sponge removal the goats were given 1200 i.u.

Ultrarapid embryo freezing: effect of dissolved gas and pH of the freezing solutions and straw irradiation.

While optimizing the ultrarapid embryo freezing procedure we noted that embryo survival was lowest when gas bubbles formed in the straws. Here we report the influences of gassing the freezing solutions with 5% CO2 in air, degassing the solution, the pH of the medium and straw irradiation on the survival and development in vitro of 2-cell mouse embryos. Embryos were ultrarapidly frozen in medium M2 containing 3 M dimethyl sulphoxide and 0.

Time of ovulation in goats (Capra hircus) induced to superovulate with PMSG.

The timing of ovulation in feral goats treated with 1200 i.u. PMSG +/- 50 micrograms GnRH was studied by repeated laparoscopy.

Full term development of mouse eggs fertilized by a spermatozoon microinjected under the zona pellucida.

A mature motile mouse spermatozoon was microinjected under the zona pellucida of mouse eggs. Twenty-five percent of eggs were fertilized, and 54% of these developed to normal fetuses or to term after transfer to pseudopregnant recipients. These results provide a quantitative estimate of the minimum proportion of spermatozoa in a population that are able to contribute to normal development--at least 54% of mature individuals that were able to fertilize the egg after microinjection, or at least 13 1/2% (25% of 54%) of the total population of mature sperm.

Ultrarapid freezing of early cleavage stage human embryos and eight-cell mouse embryos.

Early cleavage stage human embryos and 8-cell mouse embryos were snap-frozen after a brief exposure to high concentrations of dimethyl sulfoxide (DMSO; 2 or 3.5 M) and 0.25 M sucrose and thawed in a warm water bath.

Effect of changing lung liquid volume on the pulmonary circulation of fetal lambs.

During fetal life the lung develops as a liquid-filled structure with low blood flow compared with postnatal life. We studied the effects of liquid expansion of the fetal lung by measuring vascular conductance in perfused lungs in situ and arterial diameters in excised lungs of fetal lambs. Pulmonary vascular conductance invariably rose as the lung was deflated from its initial volume; maximal deflation to residual volume increased conductance 122%.

Ultrarapid freezing: a new low-cost and effective method of embryo cryopreservation.

The authors describe a rapid freezing method (ultrarapid freezing) that has been developed for cryopreservation of early cleavage stage embryos. In the present experiments, 2-cell mouse embryos were frozen under a wide range of conditions in an attempt to optimize their survival and viability in vitro and in vivo. The experiments show that embryos exposed briefly (2 to 2.