Activation and early parthenogenesis of bovine oocytes treated with ethanol and strontium

Efficient artificial activation is indispensable for the success of cloning programs. Strontium has been shown to effectively activate mouse oocytes for nuclear transfer procedures, however, there is limited information on its use for bovine oocytes. The present study had as objectives: (1) to assess the ability of strontium to induce activation and parthenogenetic development in bovine oocytes of different maturational ages in comparison with ethanol; and (2) to verify whether the combination of both treatments improves activation and parthenogenetic development rates. Bovine oocytes were in vitro matured for 24, 26, 28, and 30 h, and treated with ethanol (E, 7% for 5 min) or strontium chloride (S, 10 mM SrCl2 for 5 h) alone or in combination: ethanol + strontium (ES) and strontium + ethanol (SE). Activated oocytes were cultured in vitro in synthetic oviductal fluid (SOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage (M). Treatment with ethanol and strontium promoted similar results regarding pronuclear formation (E, 20-66.7%; S, 26.7-53.3%; P > 0.05) and cleavage (E, 12.8-40.6%; S, 16.1-41.9%; P > 0.05), regardless of oocyte age. The actions of both strontium and ethanol were influenced by oocyte age: ethanol induced greater activation rates after 28 and 30 h of maturation (48.4 and 66.7% versus 20.0 and 23.3% for 24 and 26 It, respectively; P < 0.05) and strontium after 30 It (53.3%) was superior to 24 and 26h (26.7% for both). Blastocyst development rates were minimal in all treatments (0.0-6.3%; P > 0.05), however, when the mean (+/-S.D.) cell number in blastocysts at the same maturational period was compared, strontium treatment was superior to ethanol for activation rates (82 +/- 5.7 and 89.5 +/- 7.8 versus 54 and 61, at 28 and 30 h, respectively). Improved results were obtained by combined treatments. The combination of ethanol and strontium resulted in similar pronuclear formation (ES, 36.7-83.9%; SE, 53.1-90.3%) and cleavage rates (ES, 31.3-81.3%; SE, 65.6-80.7%). Regarding embryo development, there was no difference (P > 0.05) between treatments, and blastocysts were only obtained in treatment SE at 24 and 26 h (6.5% for both). It is concluded that, SrCl2 induces activation and parthenogenetic development in bovine oocytes. (C) 2003 Elsevier B.V. All rights reserved.

Parthenogenetic activation of bovine oocytes using single and combined strontium, ionomycin and 6-dimethylaminopurine treatments

In vitro-matured (IVM) bovine oocytes were activated with single and combined treatments of strontium (S), ionomycin (1) and 6-DMAP (D). Using oocytes IVM for 26 h, we observed that activation altered cell cycle kinetics (faster progression, MIII arrest, or direct transition from MII to pronuclear stage) when compared to in vitro fertilization. The effect of oocyte age on early parthenogenesis was assessed in oocytes IVM for 22, 26 and 30 h. Better results in pronuclear development were obtained in treatments ISD (81.7%) at 22 h; D (66.7%), IS (63.3%), ID (73.3%) and ISD (76.7%) at 26 h; and D (86.7%), IS (85.0%) and ID (78.3%) at 30 h. Higher cleavage occurred on ISD (80.0%) at 22 h; ID (83.3%) and ISD (91.7%) at 26 h; and 1 (86.7%), IS (90.0%), ID (85.0%) and ISD (95.0%) at 30 h. More blastocysts were achieved in ID (25.0%) and ISD (18.3%) at 22h; and in ID at 26h (45.0%) and 30h (50.0%). We also observed that IS allowed higher haploid (77.4%) embryonic development, whilst ID was better for diploid (89.1%) development. It was concluded that association of S and D without I was not effective for blastocyst development; treatments using S were less influenced by oocyte age, but when S was associated with D there was a detrimental effect on aged oocytes; treatment ISD promoted higher activation and cleavage rates in young oocytes and ID protocol was the best for producing blastocysts.

Activation of bovine oocytes by strontium combined or not with an electric pulse

O objetivo deste trabalho foi avaliar as taxas de ativação e de clivagem de oócitos bovinos tratados com estrôncio (10mm de SrCl2), após maturação in vitro por 27-28 horas. No experimento 1, os tratamentos foram: S4 - ativação pelo estrôncio por 4 horas; S12 - ativação pelo estrôncio por 12 horas; S30 - ativação pelo estrôncio por 30 horas; e P - ativação por pulso elétrico (3 pulsos de 1,0kv/cm). No experimento 2 os tratamentos foram: PS4 - ativação combinada pelo pulso elétrico e pelo estrôncio por 4 horas; S4P - ativação pelo estrôncio por 4 horas e pelo pulso elétrico; e PS30 - ativação pelo pulso elétrico e pelo estrôncio por 30 horas. No experimento 1, todos os tratamentos apresentaram taxas similares de ativação (83-90%; P>0,05). Para clivagem, P foi melhor (53%; P<0,05) do que todos os tratamentos com estrôncio (6 a 28%). No experimento 2, o tratamento S4P apresentou melhor taxa de ativação (88%; P<0,05) do que PS4 e PS30 (60 e 68%, respectivamente). Para clivagem, observou-se o mesmo padrão, S4P (65%; P<0,05) e PS4 e PS30 (37% e 44%, respectivamente). Conclui-se que o estrôncio é capaz de ativar oócitos bovinos e sua combinação com pulso elétrico não melhora a ativação. Este é o primeiro relato demonstrando que o estrôncio ativa oócitos bovinos.

Strontium isotope ratios of pore waters and planktonic foraminifera from DSDP holes

A detailed record of the strontium-87 to strontium-86 ratio in seawater during the last 100 million years was determined by measuring this ratio in 137 well-preserved and well-dated fossil foraminifera samples. Sample preservation was evaluated from scanning electron microscopy studies, measured strontium-calcium ratios, and pore water strontium isotope ratios. The evolution of the strontium isotopic ratio in seawater offers a means to evaluate long-term changes in the global strontium isotope mass balance. Results show that the marine strontium isotope composition can be used for correlating and dating well-preserved authigenic marine sediments throughout much of the Cenozoic to a precision of +/- 1 million years. The strontium-87 to strontium-86 ratio in seawater increased sharply across the Cretaceous/Tertiary boundary, but this feature is not readily explained as strontium input from a bolide impact on land.

Strontium isotopic data for calcareous ooze, chalk, and pore waters from DSDP Hole 590B

A detailed study of strontium isotope variations in Neogene marine carbonate sediments from Deep Sea Drilling Project Site 590B, using techniques that allow the 87Sr/86Sr ratio to be determined to better than +/-0.00001, gives a high-resolution record of the Sr isotopic evolution of seawater. The data show that the rate of change of the marine 87Sr/86Sr ratio has varied significantly even on time scales as short as 1 m.y. Periods of particularly rapid growth appear to follow major marine regressions and probably reflect an increase in the delivery of radiogenic Sr from the continents coupled with a decreased submarine carbonate dissolution rate (greater carbonate compensation depth). Periods of relatively slowly changing 87Sr/86Sr follow major marine transgressions. On the basis of correlations with the marine oxygen isotope record and the times of major continental glacier growth, it is inferred that the effects of sea-level variations are modified by climatic factors that affect the intensity of continental weathering and runoff. The effects of sea-floor generation rate variations are not discernible for the Neogene. The maximum attainable stratigraphic resolution using Sr isotopes is between 0.1 and 2 m.y. for this time period.

Strontium isotope ratios of DSDP samples

The isotopic ratio of strontium-87 to strontium-86 shows no detectable variation in present-day ocean water but changes slowly over millions of years. The strontium contained in carbonate shells of marine organisms records the ratio of strontium-87 to strontium-86 of the oceans at the time that the shells form. Sedimentary rocks composed of accumulated fossil carbonate shells can be dated and correlated with the use of high precision measurements of the ratio of strontium-87 to strontium-86 with a resolution that is similar to that of other techniques used in age correlation. This method may prove valuable for many geological, paleontological, paleooceanographic, and geochemical problems.

Strontium isotopes in pore waters of east equatorial Pacific sediments

Pore-water samples from the equatorial sedimentary bulge area show reversals in depth profiles of 87Sr/86Sr ratios at the sediment/basement interface. Results of this work support inferences made from previous pore-water data (from DSDP drilling in the area) that large-scale horizontal advection of seawater has occurred through the basement underlying the thick sedimentary sequence in this region. The area of apparent advection includes the eastern part of the equatorial high-productivity zone and part of the Guatemala Basin. We attempted to find links between the observed near-basement reversals in pore-water chemistry and sedimentary thickness, age, and topography of the area. Most of the sites that show horizontal advection have disturbed basement topography or outcrops within 10 to 20 km, suggesting that the cooling effects of outcrops may extend for at least 20 km horizontally. Heat-flow data from the area were compared to determine whether sites showing near-bottom chemistry reversals were consistent with areas of low conductive heat flow. This was generally true for the area of the sedimentary bulge and Guatemala Basin. Not enough pore-water data from the Nazca Plate were available to establish any reliable systematics. Because the high-productivity area is well-sealed from hydrothermal circulation, the missing heat must be lost by horizontal advective heat transport. From profiles of strontium isotopes and other elements that show departure from seawater values with increasing depth in the sediments, but return to seawater values near the basement, it appears that water flows relatively freely through much of the oceanic crust, even when sealed by considerable sedimentary cover.

87Sr/86Sr ratios of planktonic foraminifers and interstitial waters at DSDP Hole 90-593

Detailed profiles of the Sr isotopic compositions of fossil planktonic foraminifers and interstitial waters have been measured from DSDP Site 593 to determine the Sr isotopic composition of seawater during the last 40 m.y. Foraminiferal recrystallization was assessed through scanning electron microscopy (SEM) and Sr/Ca ratios. Foraminifers were shown to be well preserved. Results document that the seawater 87Sr/86Sr has increased continuously but not uniformly, since the latest Eocene.

Strontium isotope ratios of planktonic foraminifera from late Neogene sediment samples

A curve describing the variation of the strontium isotopic composition of seawater for the late Neogene (9 to 2 Ma) was constructed from 87Sr/86Sr analyses of marine carbonate in five Deep Sea Drilling Project (DSDP) sites: 502, 519, 588, 590, and 593. The strontium isotopic composition of the oceans increased between 9 and 2 Ma with several changes in slope. From 9 to 5.5 Ma, 87Sr/86Sr values were nearly constant at ~0.708925. Between 5.5 and 4.5 Ma, 87Sr/86Sr ratios increased monotonically at a rate of not, vert, similar 1 * 10**-4 per million years. The steep slope during this interval provides the potential for high resolution strontium isotope stratigraphy across the Miocene/Pliocene boundary. The rate of change of 87Sr/86Sr decreases to near zero again during the interval 4.5-2.5 Ma, and ratios average 0.709025. The relatively rapid increase of 87Sr/86Sr between 5.5 and 4.5 Ma must be related to changes in the flux or average 87Sr/86Sr ratios of the major inputs of Sr to the oceans. Quantitative modelling of these inputs suggests that the increase was most probably caused by an increase in the dissolved riverine flux of strontium to the oceans, an increase in the average 87Sr/86Sr composition of river water, or some combination of these parameters. Modelling of this period as a transient-state requires a pulse-like increase in the input of 87Sr to the oceans between 5.5 and 4.5 Ma. Alternatively, the 5.5-4.5 Ma period can be modelled as a simple transition from one steady-state to another if the oceanic residence time of strontium was eight times less than the currently accepted value of 4 Ma. During the time interval of steep 87Sr/86Sr increase, other geochemical and sedimentologic changes also occur including an increase in sediment accumulation rates, a drop in the calcite compensation depth (CCD), and a decrease in the delta13C of dissolved bicarbonate (i.e., "carbon shift"). The simplest mechanism to explain 87Sr/86Sr variation and these related geochemical changes is to invoke an increase in the dissolved chemical fluxes carried by rivers to the oceans. This, in turn, implies increased chemical denudation rates of the continents and shelves during the late Neogene. The increase in chemical weathering rates is attributed to increased exposure of the continents by eustatic regression, intensified glacial/interglacial cycles, and accelerated rates of global tectonism beginning at 5.5 Ma during the latest Miocene.