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PubMed2006

Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes.

Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.

Animals↗
PubMed1979

Influence of dietary protein restriction on ovulation, fertilization rates and pre-implantation embryonic development in mice.

Eggs were recovered from 153 adult female mice fed either a normal (27% protein) or a protein-deficient (8% protein) diet from day 15 (Group I) or 30 (Group II) before mating until sacrifice. The females were mated with fertile males and sacrificed 48, 72, 96 or 120 hours after observation of vaginal plugs. The study was performed with females that ovulated spontaneously and with females induced to ovulate by injection of pregnant mare's serum gonadotrophin and human chorionic gonadotrophin. Without gonadotrophin treatment, significantly fewer eggs were recovered from malnourished than from normal females. However, after hormonal treatment, the number of eggs ovulated by malnourished and normal females increased similarly. About 50% of the eggs recovered from malnourished females in both groups I and II were cleaved whereas 90% of the eggs recovered from well-nourished females were cleaved. The development of the cleaved eggs from protein deficient females was delayed and asynchronous in comparison with that of eggs from normal females. Embryos recovered from malnourished females also were retarded in differentiation of morulae to blastocysts. It is concluded that a protein-restricted diet adversely affects the fertilization of eggs as well as their early development and differentiation.

Animals↗
PubMed2003

Effect of phosphate fertilizer application on phosphorus (P) losses from paddy soils in Taihu Lake Region. I. Effect of phosphate fertilizer rate on P losses from paddy soil.

A field plot study was conducted on two types of paddy soils in the Taihu Lake Region, during the rice season of year 2000 in order to assess phosphorus (P) losses by runoff and vertical leaching, which are considered the two main pathways of P movement from paddy soil into its surrounding water course. Commercial NPK compound fertilizer and single superphosphate fertilizer were applied to furnish 0, 30, 150, and 300 kg applied P ha m(-2). The experiments consisted of three replicates of each treatment in Changshu site and four replicates in Anzhen site, with a plot size of 5 x 6 m2 in a randomized block. Results revealed that the average concentration range for total P (TP) in runoff was 1.857-7.883, 1.038-5.209, 0.783-1.255 and 0.572-0.691 mg P l(-1) respectively for P300, P150, P30 and P0 in Anzhen, while it was 2.431-2.449, 1.578-1.890, 1.050-1.315 and 0.749-0.941 mg P l(-1) respectively in Changshu. In all treatments, particulate P (PP) represented a major portion of the TP lost in runoff, it was 80% in Anzhen, and it was even more (>90%) in Changshu. Phosphate fertilizer treatments significantly affected P concentrations and P loads in the runoff. The mean concentration and average seasonal TP load from the P150 plots were 1.809 mg P l(-1) and 395 g P ha m(-2) season(-1) respectively, and lower than that from the P300 plots (2.957 mg P l(-1) and 652 g P ha m(-2) season(-1)). These were obviously higher than from the P30 (0.761 mg P l(-1) and 221 g P ha m(-2) season(-1)) and P0 (0.484 mg P l(-1) and 146 g P ha m(-2) season(-1)) respectively. There was no significant difference found between the P30 and the P0 in both sites. Under usual P application rate, there were total 31.7 and 20.6 tones P removed by runoff from permeable (Anzhen site) and waterlogged (Changshu site) paddy soils in the southern Jiangsu region (major part of the TLR) in the rice season of the year 2000. But if the P application rate is unusual high, or the Olsen P in soil accumulates to above a certain level, then this could sharply increase in the future. The average concentration of molybdate reactive phosphorus (MRP) in the vertical leachate from the four different P treatments ranged from 0.058 to 0.304 mg P l(-1) in Anzhen and from 0.048 to 0.394 mg P l(-1) in Changshu. P application rate significantly affected the MRP concentration at each depth in both sites, except for the 90 cm in Anzhen. The average MRP loads during the rice season moved by vertical leaching from the four treatments ranged from 163 to 855 g P ha m(-2) season(-1) in Anzhen and 208-1,825 g P ha m(-2) season(-1) in Changshu. Vertical leachate movement does not necessarily mean that it moves towards surface water and contaminate the watercourses in this flat plain paddy soil region, it does, however, imply that P can move down from surface layers of soil to deeper levels.

Crops, Agricultural↗
PubMed2006

Chemical activation of oocytes in different types of sperm abnormalities in cases of low or failed fertilization after ICSI: a prospective pilot study.

This study evaluated the influence of chemical activation of oocytes in cases with abnormal sperm morphology and previous low intracytoplasmic sperm injection (ICSI) fertilization rate. Fertilization, early cleavage and embryo quality were assessed according to individual abnormal sperm morphology with or without chemical activation of oocytes after ICSI in 56 consecutive cycles with different types of abnormal sperm morphology (A = amorphous head, B = tapered head, C = bent neck) having a history of previous failed or low fertilization rate. Chemical activation significantly increased fertilization rate in subgroup A (82.7 versus 36.7%, P = 0.0008) and subgroup B (81.7 versus 39.3%, P = 0.005), but not in subgroup C (48.2 versus 49.4%). There was no effect on early cleavage (25 h after injection) or on the number of good embryos on day 2 (48 h after injection) in the different subgroups. Chemical activation of oocytes after ICSI in selected cases with previous history of failed or low fertilization rate and 100% sperm abnormality may increase fertilization rate in some sperm morphological abnormalities (amorphous head and tapered head) but not in others (bent neck). However, there was no significant effect on early cleavage or number of good embryos in all subgroups.

Adult↗
PubMed1994

In vitro fertilization rate of horse oocytes with partially removed zonae.

Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 micro M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under x 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (2/57), 12 (7/58), 52 (31/60), and 86% (44/51) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (2/2), 57 (4/7), 58 (18/31), and 34% (15/44), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 11/49), and increased to 38% (21/55) at 5 h, to 46% (23/50) at 10 h, and to 56% (27/48) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.

Journal Article↗