Evaluating methods of mouse euthanasia on the oocyte quality: cervical dislocation versus isoflurane inhalation

Cervical dislocation is a commonly used method of mouse euthanasia. Euthanasia by isoflurane inhalation is an alternative method which allows the sacrifice of several mice at the same time with an anaesthesia, in the aim to decrease pain and animal distress. The objective of our study was to assess the impact of these two methods of euthanasia on the quality of mouse oocytes. By administering gonadotropins, we induced a superovulation in CD1 female mice. Mice were randomly assigned to euthanasia with cervical dislocation and isoflurane inhalation. Oviducts were collected and excised to retrieve metaphase II oocytes. After microscopic examination, oocytes were classified into three groups: intact, fragmented/cleaved and atretic. Intact metaphase II oocytes were employed for biomedical research. A total of 1442 oocytes in the cervical dislocation group were compared with 1230 oocytes in the isoflurane group. In the cervical dislocation group, 93.1% of the oocytes were intact, versus 65.8% in the isoflurane group (P ≤ 0.001). In light of these results, we conclude that cervical dislocation is the best method of mouse euthanasia for obtaining intact oocytes for biomedical research.     

Loss of cortical function in mice after decapitation, cervical dislocation, potassium chloride injection, and CO2 inhalation

Electroencephalograms (EEG) and visual evoked potentials (VEP) in mice were recorded to evaluate loss of cortical function during the first 30 s after euthanasia by various methods. Tracheal cannulae (for positive-pressure ventilation, PPV) and cortical surface electrodes were placed in mice anesthetized with inhaled halothane. Succinylcholine was used to block spontaneous breathing in the mice, which then underwent continuous EEG recording. Photic stimuli (1 Hz) were presented to produce VEPs superimposed on the EEG. Anesthesia was discontinued immediately before euthanasia. Compared with that obtained before euthanasia, EEG activity during the 30-s study period immediately after euthanasia was significantly decreased after cervical dislocation (at 5 to 10 s), 100% PPV-CO2 (at 10 to 15 s), decapitation (at 15 to 20 s), and cardiac arrest due to KCl injection (at 20 to 25 s) but not after administration of 70% PPV-CO2. Similarly, these euthanasia methods also reduced VEP amplitude, although 100% PPV-CO2 treatment affected VEP amplitude more than it did EEG activity. Thus, 100% PPV-CO2 treatment significantly decreased VEP beginning 5 to 10 s after administration, with near abolition of VEP by 30 s. VEP amplitude was significantly reduced at 5 to 10 s after cervical dislocation and at 10 to 15 s after decapitation but not after either KCl or 70% PPV-CO2 administration. The data demonstrate that 100% PPV-CO2, decapitation, and cervical dislocation lead to rapid disruption of cortical function as measured by 2 different methods. In comparison, 70% PPV-CO2 and cardiac arrest due to intracardiac KCl injection had less rapid effects on cortical function.     

Lung mechanics and neuromuscular output during CO2 inhalation after airway anesthesia

Airway anesthesia with aerosolized lidocaine has been associated with an increase in minute ventilation (VE) during CO2 inhalation. The increase in VE may be due to increased neuromuscular output or decreased mechanical load on breathing. To evaluate this we measured VE, breathing pattern, mouth occlusion pressure, and lung mechanics in 20 normal subjects during room-air breathing and then inhalation of 6% CO2-94% O2, before and after airway anesthesia. Measurements of lung mechanics included whole-lung resistance, dynamic and static compliance, and functional residual capacity. Airway anesthesia had no detectable effect on any measurements during room-air breathing. During CO2 inhalation, airway anesthesia produced increases in VE and mean inspiratory flow rate (VT/TI) and more negative inspiratory pleural pressure but had no detectable effect on lung mechanics or mouth occlusion pressure. Pleural pressure was more negative during the latter 25% of inspiration. We concluded that airway receptors accessible to airway anesthesia play a role in determining neuromuscular output during CO2 inhalation.     

Ventilatory response during CO2 inhalation after airway anesthesia with lidocaine

Airway anesthesia with inhaled aerosolized lidocaine has been associated with increases in minute ventilation (VE) and mean inspiratory flow rate (VT/TI) during CO2 inhalation. However, it is unclear whether these increases are local effects of the anesthesia or systemic effects of absorbed and circulating lidocaine. To evaluate this 20 normal subjects were treated on separate days with aerosolized lidocaine, intravenous lidocaine, aerosolized control solution, or intravenous control solution, and the effects of each treatment on VE and VT/TI were determined and compared during room-air breathing and inhalation of 5% CO2-95% O2. None of the treatments altered VE or VT/TI during room-air breathing. Aerosolized lidocaine produced small (5.9-6.0%) increases in VE and VT/TI during CO2 inhalation, but these effects were not present after intravenous lidocaine despite equivalent lidocaine blood levels. We concluded that the increases in VE and VT/TI after aerosolized lidocaine were local effects of airway anesthesia rather than systemic effects of absorbed and circulating lidocaine.     

Euthanasia via CO2 inhalation causes premature cortical granule exocytosis in mouse oocytes and influences in vitro fertilization and embryo development

Generation of high quality mouse metaphase II oocytes is an integral part for efficient in vitro fertilization (IVF), and subsequent embryo production for reproductive studies and genome banking. The main objectives of this study were to investigate the impact of various euthanasia methods on IVF, embryo development, and subcellular structures of MII mouse oocytes. Following superovulation regimen, female mice were euthanized by high flow CO₂ (H CO₂), low flow CO₂ (L CO₂), or cervical dislocation (CD). The MII oocytes obtained from these mice were evaluated for subcellular integrity by assessing their cortical granules and F‐actin. Furthermore, fertilization and subsequent embryonic development competence up to blastocyst stage were also evaluated in vitro. The oocytes collected from females euthanized by CD resulted in significantly higher two‐cell development rates (p = 0.028) and subsequently lead to in higher embryo development rates (p = 0.027) compared with oocytes from females euthanized by L CO₂. The cortical granule integrity analysis revealed significantly higher rate of premature cortical granules exocytosis (PCGE) for L CO₂ group compared with CD and H CO₂ groups (p < 0.001). These data collectively suggest that CO₂ associated PCGE during euthanasia procedure is the main cause of decreased IVF rates and CD is the optimal euthanasia method for the purpose of obtaining good quality MII oocytes for mouse IVF and other reproductive studies.     

Mouse meningiocytes express Sox2 and yield high efficiency of chimeras after nuclear reprogramming with exogenous factors

Induced pluripotent stem cell technology, also termed iPS, is an emerging approach to reprogram cells into an embryonic stem cell-like state by viral transduction with defined combinations of factors. iPS cells share most characteristics of embryonic stem cells, counting pluripotency and self-renewal, and have so far been obtained from mouse and humans, including patients with genetic diseases. Remarkably, autologous transplantation of cell lineages derived from iPS cells will eliminate the possibility of immunological rejection, as well as current ethical issues surrounding human embryonic stem cell research. However, before iPS can be used for clinical purposes, technical problems must be overcome. Among other considerations, full and homogeneous iPS reprogramming is an important prerequisite. However, despite the fact that cells from several mouse tissues can be successfully induced to iPS, the overall efficiency of chimera formation of these clones remains low even if selection for Oct4 or Nanog expression is applied. In this report, we demonstrate that cells from the mouse meningeal membranes express elevated levels of the embryonic master regulator Sox2 and are highly amenable to iPS. Meningeal iPS clones, generated without selection, are fully and homogeneously reprogrammed based on DNA methylation analysis and 100% chimera competent. Our results define a population of somatic cells that are ready to undergo iPS, thus highlighting a very attractive cell type for iPS research and application.     

Breathing pattern in anesthetized chickens: CO2 inhalation and vagotomy

The breathing patterns of 20 anesthetized chickens were studied during progressive suppression of intrapulmonary chemoreceptors (IPC) by various concentrations of CO2 and following bilateral vagotomy. The vagotomy breathing pattern, characterized by a marked accentuation of expiratory times with prolonged expiratory pauses, was markedly different from that induced by CO2 inhalation. Removal of neural input to the central respiratory centers from IPC does not appear to be solely responsible for the altered breathing pattern following vagotomy in birds.     

The metabolism of prostaglandin D2 after inhalation or intravenous infusion in normal men

Tritium-labelled prostaglandin D2 (PGD2) was administered to normal volunteers by either intravenous infusion or inhalation in order to establish which metabolites of PGD2 are initially found in human plasma. Inhaled PGD2 was rapidly absorbed from the airways, as indicated by the rapid appearance of tritium in the plasma. Metabolites chromatographically similar to 9 alpha,11 beta-PGF2 and 13,14-dihydro-15-keto-9 alpha,11 beta-PGF2 were found after both routes of administration. At later time points, other unidentified compounds were present. Only after intravenous infusion was there evidence of metabolites with 9 alpha,11 alpha stereochemistry of the ring hydroxyl functions. In human lung, 9 alpha,11 beta-PGF2 was metabolized in the presence of NAD+ to compounds tentatively identified by gas chromatography/mass spectrometry (GC/MS) as 15-keto-9 alpha,11 beta-PGF2 and 13,14-dihydro-15-keto-9 alpha,11 beta-PGF2. Thus, after 11-ketoreductase-dependent metabolism of PGD2 to the biologically active compound 9 alpha,11 beta-PGF2, further metabolism probably proceeds by the combined action of 15-hydroxyprostaglandin dehydrogenase/15-ketoprostaglandin-delta 13-reductase (15-PGDH/delta 13R). Both 9 alpha,11 beta-PGF2 and its 13,14-dihydro-15-keto metabolite may be useful analytes for the measurement of PGD2 turnover, and may therefore prove to be important in understanding the pathophysiological significance of this putative mediator.     

Cyclooxygenase-2-derived endogenous prostacyclin reduces apoptosis and enhances embryo viability in mouse

The role of prostaglandins (PGs) in apoptosis in preimplantation mice embryo development is reported in this study. It is known that apoptosis plays a very important role in normal mice embryo development. Very few reports are available on this subject. Embryos (6-8 cells) were cultured in the presence of a selective cyclooxygenase (COX)1 inhibitor (SC560), a selective COX2 inhibitor (NS398) and a selective prostacyclin synthase (PGIS) inhibitor (U51605) in a 48-h culture. In another experiment, culture media were supplemented with prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2 or prostacyclin) analogues. The apoptosis was evaluated by detection of active caspase-3. It was strongly detected in the presence of selective COX-2 and PGIS inhibitors, which can be decreased by a PGI2 analogue. In our embryo transfer experiment, the implantation rate decreased with exposure to either the COX2 or the PGIS inhibitor which is increased further after PGI2 supplementation. The level of PGI2 is also higher at the 8-16-cell stage, compaction and blastocyst stage than PGE2. All these results indicate that COX2-derived PGI2 plays an important role in preimplantation embryo development and acts as an antiapopetic factor in in vitro culture.     

Perturbations in mouse embryo development and viability caused by ammonium are more severe after exposure at the cleavage stages

The presence of ammonium in culture medium has a detrimental effect on embryo physiology and biochemistry; however, the stage at which the embryo is most sensitive to this effect is unknown. The aim of this study was to determine the exact stage at which the embryo is most vulnerable to ammonium by exposing the preimplantation embryo to 300 muM ammonium either at the precompaction stage (between the zygote and two-cell or the two-cell to eight-cell) or at the postcompaction stage (between the eight-cell and blastocyst). This study determined that exposure of embryos to ammonium at the precompaction stage from either the zygote to two-cell stage or from the two-cell to the eight-cell stage did not affect the rate of development to the blastocyst stage; however, the resultant blastocysts had decreased cell numbers and inner cell mass cells. Furthermore, these blastocysts had increased levels of cellular apoptosis and perturbed levels of Slc2a3 expression and glucose uptake. Transfer of these blastocysts revealed that, while implantation was not affected, the number of fetuses was reduced by culture with ammonium at the precompaction stage and fetal development was delayed, as observed by reduced crown-rump length and maturity. In contrast, the later stage embryo was more resistant to the negative effects of ammonium, with only Slc2a3 expression and fetal maturity affected. This raises the possibility that the later stage embryo is more able to protect itself from in vitro-derived stress and that the majority of in vitro-induced damage to mouse embryos is inflicted at the early stages of development.     

Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SYBR-14

The objective of these experiments was to determine the efficacy of the new membrane permeant nucleic acid stain, SYBR-14, for assessing boar sperm viability and to determine it's effect on fertilization and early embryonic development using the pig as a model. We examined the staining patterns of SYBR-14 and another vital stain, Hoechst 33342, both in combination with the dead cell stain, propidium iodide (PI), to quantify the proportion of living and dead spermatozoa in ejaculated and epididymal semen. Flow cytometry analyses of semen from 4 boars revealed significant differences among boars for the proportion of SYBR-14-stained spermatozoa in both epididymal and ejaculated samples, but not for Hoechst 33342 or PI stained spermatozoa. Gilts were inseminated with unstained spermatozoa or spermatozoa stained with 2 levels of SYBR-14 or 2 levels of the reference stain, Hoechst 33342. Embryos recovered at 42 to 48 h postinsemination were morphologically evaluated, and only 4 to 8-cell embryos were continued in culture. Overall, fluorescent staining of boar spermatozoa with SYBR-14 or Hoechst 33342 neither affected their ability to fertilize oocytes, nor the developmental competence of the resultant embryos.     

Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen-thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen-thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n=28 and 31; year 2: n=16 and 15, respectively) or laparoscopically inseminated (year 2: n=13 and 14). In experiment 2, superovulated Belclare (n=4) and Suffolk (n=13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P<0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P<0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P<0.02) but there was no difference after laparoscopic AI (62% versus 64%, P>0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI (P<0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.     

Effect of hilar nerve denervation on breathing and arterial PCO2 during CO2 inhalation

We determined the effects of denervating the hilar branches (HND) of the vagus nerves on breathing and arterial PCO2 (PaCO2) in awake ponies during eupnea and when inspired PCO2 (PICO2) was increased to 14, 28, and 42 Torr. In five carotid chemoreceptor-intact ponies, breathing frequency (f) was less, whereas tidal volume (VT), inspiratory time (TI), and ratio of TI to total cycle time (TT) were greater 2-4 wk after HND than before HND. HND per se did not significantly affect PaCO2 at any level of PICO2, and the minute ventilation (VE)-PaCO2 response curve was not significantly altered by HND. Finally, the attenuation of a thermal tachypnea by elevated PICO2 was not altered by HND. Accordingly, in carotid chemoreceptor-intact ponies, the only HND effect on breathing was the change in pattern classically observed with attenuated lung volume feedback. There was no evidence suggestive of a PCO2-H+ sensory mechanism influencing VE, f, VT, or PaCO2. In ponies that had the carotid chemoreceptors denervated (CBD) 3 yr earlier, HND also decreased f, increased VT, TI, and TT, but did not alter the slope of the VE-PaCO2 response curve. However, at all levels of elevated PICO2, the arterial hypercapnia that had persistently been attenuated, since CBD was restored to normal by HND. The data suggest that during CO2 inhalation in CBD ponies a hilar-innervated mechanism influences PaCO2 by reducing physiological dead space to increase alveolar ventilation.     

Life with CO or CO2 and H2 as a source of carbon and energy

An account is presented of the recent discovery of a pathway of growth by bacteria in which CO or CO2 and H2 are sources of carbon and energy. The Calvin cycle and subsequently other cycles were discovered in the 1950s, and in each the initial reaction of CO2 involved adding CO2 to an organic compound formed during the cyclic pathway (for example, CO2 and ribulose diphosphate). Studies were initiated in the 1950s with the thermophylic anaerobic organism Clostridium thermoaceticum, which Barker and Kamen had found fixed CO2 in both carbons of acetate during fermentation of glucose. The pathway of acetyl-CoA biosynthesis differs from all others in that two CO2 are combined with coenzyme A (CoASH) forming acetyl CoA, which then serves as the source of carbon for growth. This mechanism is designated the acetyl CoA pathway and some have called it the Wood pathway. A unique feature is the role of the enzyme carbon monoxide dehydrogenase (CODH), which catalyzes the conversion of CoASH, CO, and a methyl group to acetyl CoA, the final step of the pathway. The pathway involves the reduction of CO2 to formate, which then combines with tetrahydrofolate (THF) to form formyl THF. It in turn is reduced to CH3-THF. The methyl is then transferred to the cobalt on a corrinoid-containing enzyme. From there the methyl is transferred to CODH, and CO and CoASH bind with the enzyme at separate sites. Acetyl CoA is then synthesized. CODH would more properly be called carbon monoxide dehydrogenase-acetyl CoA synthase as it catalyzes oxidation of CO to CO2 and the synthesis of acetyl CoA. The solution of the mechanism of this pathway required more than 30 years, in part because the intermediate compounds are bound to enzymes, the enzymes are extremely sensitive to O2 and must be isolated under strictly anerobic conditions, and the role of a corrinoid and CODH was unprecedented. It is now apparent that this pathway occurs (perhaps with some modification) in many bacteria including the methane and sulfur bacteria. In some humans this pathway is catalyzed by the bacteria of the gut and acetate is produced rather than methane; it is calculated that 2.3 x 10(6) metric tons of acetate are formed daily from CO2. A similar synthesis occurs in the hind gut of termites. It is becoming apparent that the acetyl CoA pathway plays a significant role in the carbon cycle.(ABSTRACT TRUNCATED AT 400 WORDS)