Multiple ovulation and embryo transfer with fresh, frozen and vitrified red deer (Cervus elaphus) embryos in Argentina.

Two multiple ovulation and embryo transfer (MOET) programs with fresh, frozen and vitrified red deer embryos were carried out during the reproductive season of 2005 and 2006 in a local breeding farm in Argentina. Multiparous (n=10 and 9, respectively) weaned hinds were used as donors for each year. The estrous synchronization treatment of donors and recipients consisted of inserting an ovine intravaginal sponge containing medroxiprogesterone acetate (MAP) for 12 days. Superovulation was conducted with a total dose of 180 mg of NIH-FSH-P1 (Folltropin-V, Bioniche, Belleville, Ontario, Canada), given i.m. in eight decreasing doses every 12h (40, 40; 27, 27; 15, 15; 8, 8 mg), from days 10 to 13. Donor females were mated with one stag of proven fertility. The recovery rate was 84.1% (122/145), obtaining 45.1% (55/122) of transferable embryos, 24.6% (30/122) of degenerated embryos and 30.3% (37/122) of unfertilized oocytes. Pregnancy rates after transfer of fresh, OPS vitrified/warmed and ethylene glycol (EG) frozen/thawed embryos were 64.3% (18/28), 53.3% (8/15) and 70.0% (7/10), respectively. Vitrification and freezing with ethylene glycol procedures constitute an interesting alternative for red deer embryo cryopreservation.

Interspecific embryo transfer from mouflon (Ovis gmelini musimon) to domestic Corriedale sheep in Argentina.

An interspecific embryo transfer program was conducted for genetic improvement and increasing the number of offspring from a flock of mouflon sheep in Argentina. The female donor mouflons were divided into three groups, G1 (n=5), G2 (n=4) and G3 (n=5). The total NIH-FSH-P1 dose given to each donor on the superovulatory treatment was 260, 200 and 160 mg for G1, G2 and G3, respectively. The mouflons in G3 were maidens, while the others were multiparous. Domestic Corriedale ewes (n=60) were synchronized and used as recipients. The embryo recovery and transfer was performed by a surgical method. Mouflons (n=13) responded to the superovulatory treatment with an average of 9.1+/-2.8 ovulations. A low incidence of early luteal regression was found (1 out of 14 donors). Embryo recovery rates were 60, 31 and 76% in groups G1, G2 and G3, respectively. The percentage of transferable embryos obtained in G1 and in G2 exceeded 80%. None of the embryos obtained from G3 were of transferable quality. In G1, 25 transferable embryos were recovered and transferred to 13 recipients, resulting in a pregnancy rate of 76.9% (10/13). In G2, 10 embryos were transferred to 5 recipients, resulting in a 60% pregnancy rate (3/5). Lambing rate was 60% (15/25) and 30% (3/10) for G1 and G2, respectively. Thirteen lambs were born to the 14 donors following natural service after the embryo recoveries. This study demonstrates that the application of IET technology would have great reproductive impact, especially when the donor mouflon hinds are selected according to age and reproductive history.

Electrogenic bicarbonate secretion in the turtle bladder: apical membrane conductance characteristics.

We have recently shown that stimulation of electrogenic HCO3- secretion is accompanied by a simultaneous increase in short-circuit current (Isc, equivalent to HCO3- secretion rate under these conditions), apical membrane capacitance (Ca, proportional to membrane area), and apical membrane conductance (Ga, proportional to membrane ionic permeability). The current experiments were undertaken to explore the ionic basis for the increase in Ga and the possibility that the rate of electrogenic HCO3- secretion is regulated by changes in Ga. Membrane electrical parameters were measured using impedance-analysis techniques before and after stimulation of electrogenic HCO3- secretion with cAMP in three solutions which contained different chloride concentrations. In another series of experiments, the effects of an anion channel blocker, anthracene-9-carboxylic acid (9-AA), were measured after stimulation of electrogenic HCO3- secretion with cAMP. The major conclusions are: (i) a measurable apical Cl- conductance exists in control hemibladders; (ii) the transport-associated increase in Ga includes a Cl(-)-conductive component; (iii) Ga also appears to reflect a HCO3- conductance; (iv) the relative magnitudes of the apical membrane conductances to Cl- and HCO3- are similar; (v) 9-AA reduces Ga and Isc in cAMP-stimulated hemibladders; and (vi) alterations in Isc appear to be mediated by changes in Ga.

Changes in membrane conductances and areas associated with bicarbonate secretion in turtle bladder.

Transepithelial impedance-analysis studies were performed in turtle bladder epithelium in order to measure changes in the different epithelial membranes resulting from stimulation of electrogenic bicarbonate secretion. Changes in membrane conductance relate to changes in ionic permeability, whereas changes in membrane capacitance relate to changes in membrane area, since most biological membranes exhibit a specific capacitance of approximately 1 muF/cm2. The results of this investigation are summarized as follows: (i) cAMP and carbachol, agents which have been shown previously to stimulate electrogenic bicarbonate secretion, result in increases in apical-membrane conductance and capacitance; (ii) these changes occur concomitantly with the observed change in transport (measured using the short-circuit-current technique), thereby suggesting that bicarbonate secretion may be regulated in part by changes in the chloride conductance of the apical membrane; (iii) the increase in conductance does not reflect an increase in the membrane's specific conductance, thereby indicating that it results from the addition of membrane possessing similar ionic permeability as the existing apical membrane; (iv) the magnitude of the changes in capacitance indicate that a minor cell population (beta-type carbonic-anhydrase-rich cells) increase their apical-membrane area by several-fold; (v) a lack of transport-associated changes in the basolateral-membrane parameters suggest that transport is not regulated by alterations in basolateral-membrane ionic conductance or area; (vi) a lack of colchicine sensitivity, coupled with the magnitude of the changes in apical-membrane capacitance, indicate that the membrane remodeling processes are different from those involved in the regulation of proton secretion in a different cell population (alpha-type carbonic-anhydrase-rich cells).

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium.

Proton secretion in the urinary bladder of the freshwater turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo- and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15 degrees C. In addition, serosal application of 0.5 mM acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15 degrees C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1 mM serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.

Proton transport and membrane shuttling in turtle bladder epithelium.

Proton secretion in the urinary bladder of the fresh-water turtle is mediated by proton pumps located in the apical membrane of carbonic-anhydrase (CA)-rich cells. It has been proposed that the rate of proton transport is regulated by endocytotic and exocytotic fusion processes which alter the apical membrane area, and hence number of exposed pumps. Three techniques were used to study this process. Analyses of transepithelial impedance provided estimates of transport-associated changes in net membrane area, as well as other electrical parameters. Electron microscopy allowed visualization of the endocytotic vesicles thought to be involved in the process. Finally, uptake of a fluorescent fluid-phase marker provided measurements of the rates of endocytosis. We report the following: endocytotic and exocytotic processes occur primarily in the CA-rich cells; inhibition of proton transport resulting from 0.5 mM acetazolamide (AZ) results in a decrease in the apical membrane area of approximately 0.47 cm2/cm2 tissue; the apical membrane specific conductance of the CA-rich cells is approximately 220 microS/microF, and possibly represents a Cl- conductance that may function in counter-ion flow; the decline in transport following AZ is not directly proportional to the decline in apical membrane area, suggesting that changes in pump kinetics are also involved in the regulation of transport; the CA-rich cells exhibit a high rate of constitutive pinocytosis, and hence membrane shuttling, which appears to be independent of the rate of transport; AZ induces a transient increase in the rates of endocytosis and shuttling; and the transport-associated changes in apical membrane area may reflect an effect of AZ on a regulated endocytotic pathway which is distinct from the pinocytotic process.

Membrane electrical parameters in turtle bladder measured using impedance-analysis techniques.

Equivalent-circuit impedance analysis experiments were performed on the urinary bladders of freshwater turtles in order to quantify membrane ionic conductances and areas, and to investigate how changes in these parameters are associated with changes in the rate of proton secretion in this tissue. In all experiments, sodium reabsorption was inhibited thereby unmasking the electrogenic proton secretion process. We report the following: transepithelial impedance is represented exceptionally well by a simple equivalent-circuit model, which results in estimates of the apical and basolateral membrane ionic conductances and capacitances; when sodium transport is inhibited with mucosal amiloride and serosal ouabain, the apical and basolateral membrane conductances and capacitances exhibit a continual decline with time; this decline in the membrane parameters is most likely caused by subtle time-dependent changes in cell volume, resulting in changes in the areas of the apical and basolateral membranes; stable membrane parameters are obtained if the tissue is not treated with ouabain, and if the oncotic pressure of the serosal solution is increased by the addition of 2% albumin; inhibition of proton secretion using acetazolamide in CO2 and HCO3- -free bathing solutions results in a decrease in the area of the apical membrane, with no significant change in its specific conductance; stimulation of proton transport with CO2 and HCO3- -containing serosal solution results in an increase in the apical membrane area and specific conductance. These results show that our methods can be used to measure changes in the membrane electrophysiological parameters that are related to changes in the rate of proton transport. Notably, they can be used to quantify in the live tissue, changes in membrane area resulting from changes in the net rates of endocytosis and exocytosis which are postulated to be intimately involved in the regulation of proton transport.

Environmental exposure to hexachlorobenzene in the USA.

Hexachlorobenzene (HCB) is a chlorinated hydrocarbon chemical which has various industrial and agricultural uses in the USA. The agricultural use of HCB as a fungicide is relatively minor and is being phased out, but HCB is still used directly in a few industrial processes and is a waste product or impurity associated with the manufacture of various chlorinated solvents and other registered pesticides. A review of available HCB data from ambient environmental monitoring networks (agricultural and urban soils, ambient air, fresh-water fish, starlings, ducks and ready-to-eat foods) of the National Pesticide Monitoring Program resulted in the following general conclusions: Actual mean residue levels of HCB were erratic or steady during the 1970s and showed no major upward or downward trends; The percentages of detectable HCB did show a trend in most of the environmental monitoring networks. The percentage of HCB in the environmental components examined generally increased from the early 1970s to the late 1970s, when it peaked, and then declined into the early 1980s, the most recently available data sets. The reason for the increase of HCB in several environmental components is not clear.

Fluorescence identifies an alkaline cell in turtle urinary bladder.

Intracellular pH (pHi) of turtle bladder mucosal cells was studied by the trapped fluorescent indicator technique. Bladders efficiently accumulated and converted 4-methylumbelliferyl acetate to its pH-sensitive derivative 4-methylumbelliferone (4MU). Excited at the pH-indifferent wavelength 334 nm, bladders fluoresced a uniform blue. Using pH-sensitive 365-nm excitation, 10-20% of the mucosal cells fluoresced distinctly brighter, suggesting a more alkaline pHi. Using the 365/334 ratio to quantitate pHi, this difference averaged 0.1 pH units. Bright cells were more distinct after SITS or acetazolamide but disappeared after digitonin permeabilization, dinitrophenol, or treatment with propionate, DMO, and NH4Cl. Essentially the same population of bright cells was identified by carboxyfluorescein diacetate. The brighter cell corresponded exactly to a population of cells with distinctive acridine orange staining and bright costaining with the potential-sensing probes Di-O-C5, Di-S-C3, and 4-Di-5-Asp. Two extremes of bright cell shape were seen: an elongate cell, prevalent under conditions stimulating H+ secretion, and a more compact cell, when acidification was inhibited. These observations support the hypothesis that acidification represents H+ secretion via the luminal membrane and that a primary role of carbonic anhydrase in this process is to support the exit of base from the cell. The more alkaline cells appear to be the carbonic anhydrase-rich cells. These cells are chemically isolated from the surrounding granular cells and change their morphology in response to changes in acidification. These special properties indicate a unique role for the carbonic anhydrase cell in H+ secretion.

Metabolic pathways coupled to H+ transport in turtle urinary bladder.

Active H+ transport in the turtle urinary bladder is mediated by an ATPase. Although the source of ATP is usually mitochondrial oxidative phosphorylation, it is possible because of intracellular compartmentalization or cellular heterogeneity that one metabolic pathway exclusively provides ATP to the pump. To examine this we performed several types of experiments. In one, the coupling between the rate of transport and the rate of oxidation of 14C-labeled substrates was studied. We found that there was coupling between H+ transport and glucose, butyrate oleate, and beta-OH-butyrate oxidation. In another set of experiments we depleted turtle bladders of their endogenous substrates and tested the effect of a number of substrates on the rate of transport. We found that glucose, pyruvate, lactate, actetate, butyrate and beta-OH butyrate all stimulated H+ transport. In a third set of experiments we found no coupling between H+ transport and lactate production. Finally, we found that reduction of H+ transport by mucosal acidification resulted in an increase in epithelial cell ATP concentrations and a decrease in ADP levels. These results suggest that the H+ pump receives its ATP from carbohydrate and fatty acid oxidation. The changes in ATP and ADP levels provide an initial explanation for the coupling of H+ transport to the rate of cellular oxidative metabolism.

H+/ATP stoichiometry of proton pump of turtle urinary bladder.

Urinary acidification in the turtle urinary bladder is due to a reversible proton-translocating ATPase. To estimate the H+/ATP stoichiometry of this pump, we measured the delta G'ATP in the epithelial cells and the maximum e.m.f. generated by the pump. The latter is the maximal transepithelial electrochemical gradient for protons placed across the epithelium that is needed to nullify the rate of transport and averaged 179 +/- 7 mV. The delta G'ATP averaged 50.1 kJ/mol. The H+/ATP stoichiometry of these bladders was 2.92 +/- 0.1. In other experiments, the bladders were poisoned by iodoacetate and cyanide and a variable transepithelial electrochemical gradient for protons was placed across them. It was noted that ATP synthesis occurred at a transepithelial electrochemical gradient for protons greater than 120 mV. The delta G'ATP in other bladders treated identically averaged 40.0 kJ/mol, giving a H+/ATP stoichiometry of 3.4 +/- 0.1. We conclude that the H+/ATP stoichiometry of the proton pump of turtle urinary bladder is approximately 3.

Urinary acidification in turtle bladder is due to a reversible proton-translocating ATPase.

Adverse proton electrochemical gradients (delta muH) applied across the turtle urinary bladder decrease active H+ transport in this epithelium. A delta muH of 180 mV abolishes both transport and its tightly coupled metabolic reaction. Larger gradients should, in theory, reverse the direction of H+ transport and the metabolic reaction leading to synthesis of ATP if the pump is an ATPase, or cause an increase in the oxidized state of a redox pair if it is a redox pump. To distinguish between these two possibilities, we measured ATP levels in epithelial cells that were poisoned to inhibit cellular mechanisms of ATP synthesis. At delta muH of 120 mV or less no ATP synthesis was found. At delta muH of greater than 120 mV there was a linear increase in ATP synthesis. Dinitrophenol, a H+ carrier, prevented synthesis at delta muH of 310 mV. Dicyclohexylcarbodiimide, an inhibitor of H+ transport that works at the cell surface, prevented ATP synthesis at delta muH of 310 mV. These results demonstrate that a reversible proton-translocating ATPase in the mucosal border of the bladder is the H+ pump responsible for urinary acidification.