Transcervical artificial insemination of Australian Merino ewes with frozen-thawed semen

We compared conventional methods for laparoscopic and cervical artificial insemination (AI) to a transcervical AI procedure (Guelph System for Transcervical AI; GST-AI) for use with frozen semen in Merino ewes. The GST-AI procedure was performed by an experienced operator in Experiment 1 (771 ewes) and by 2 inexperienced operators in Experiment 2 (555 ewes). In Experiment 1, intrauterine insemination by GST-AI was achieved in 76% of the ewes. The pregnancy rate at Day 70 for ewes inseminated by laparoscopy (48%, 120 251 ) was higher (P<0.01) than for ewes inseminated by either intrauterine GST-AI (32%, 64 201 ) or cervical AI (9%, 24 256 ). The overall (intrauterine and intracervical) pregnancy rate for GST-AI was 26% (68 264 ) and was unaffected by depth of insemination within the cervix. Pregnancy rates were unaffected by ram or day of insemination. In Experiment 2, the operators achieved intrauterine inseminations by GST-AI in 43% (78 182 ) of the ewes, with a significant operator effect (P<0.01) on depth of cervical penetration. The pregnancy rate to intrauterine GST-AI (40%, 31 78 ) did not differ from that to laparoscopic insemination. The total pregnancy rate for GST-AI in Experiment 2 (19%, 34 182 ) was lower (P<0.05) than that for laparoscopic AI (39%, 72 187 ) but superior (P<0.05) to that for cervical AI (1%, 1 186 ). The GST-AI pregnancy rates were affected by depth of AI (P<0.01) and by operator (P<0.05). It is concluded that GST-AI is superior to cervical AI, and may have application in Merinos if cervical penetration rates can be improved.     

Intrauterine insemination enhances fertility of frozen semen in superovulated ewes

Superovulated ewes were inseminated with fresh or frozen semen in a factorial experiment which compared two techniques of artificial insemination; i.e. conventional cervical deposition and intrauterine deposition at laparoscopy. Similar fertilization rates resulted from insemination with fresh semen at cervical (81% of ova from 11/11 ewes) and intrauterine (83% of ova from 10/12 ewes) sites. These results approached those observed in a naturally-mated group (95% of ova from 5/5 ewes). In ewes inseminated with frozen semen, fertilization rate was markedly reduced (P less than 0.05) after cervical insemination (11% of ova from 3/11 ewes) and partly restored (P less than 0.05) after intrauterine insemination (50% of ova from 8/11 ewes).     

CO2 sensitive receptors on labial palps ofRhodogastria moths (Lepidoptera: Arctiidae): physiology,fine structure and central projection

Summary The tips of the labial palps ofRhodogastria possess a pit housing uniform sensilla (Fig. 1), histologically characterized by wall-pores and receptor cells with lamellated outer dendrites (Fig. 2). The receptor cell axons project to glomeruli in the deutocerebrum (cf. Fig. 3) which are not innervated by antennal receptors. From their histology as well as from their central projection these sense organs are identical with palpal pit organs of other Lepidoptera (Lee et al. 1985; Kent et al. 1986; Lee and Altner 1986).Physiologically, the palp-pit receptors respond uniformly; they are most excitable by stimulation with carbon dioxide (Fig. 6) while they exhibit relatively moderate responses to various odorants (Fig. 4). The responses to CO₂ (Fig. 7) show a steep dose-response characteristic. In ambient atmosphere (i.e., ca. 0.03% CO₂) the cells are in an excited condition already; the seeming spontaneous activity exhibited in air is decreased if the preparation is kept under N₂ or O₂ or CO₂-free air (Figs. 7, 10). There is hardly any adaptation of the responses to continuous or repeated stimulation (Fig. 8). Perhaps CO₂ sensitivity is correlated with sensilla characterized by both wall-pores and lamellated dendrites. Pilot tests indicate that CO₂ perception might be widespread in the Lepidoptera (cf. Fig. 12), but the biological significance remains obscure.     

Fertilization and ovum recovery rates in superovulated ewes following cervical insemination or laparoscopic intrauterine insemination at different times after progestagen withdrawal and in one or both uterine horns

In Exp. 1, 40 ewes were used in a 2 x 2 factorial design to investigate the effects of intrauterine versus cervical insemination and superovulation using pig FSH or PMSG and GnRH on egg recovery and fertilization rate. Cervical inseminations were carried out at 48 and 60 h (N = 20 ewes) and intrauterine insemination at 52 h (N = 20 ewes) after progestagen pessary withdrawal. Eggs were recovered on Day 3 of the oestrous cycle. Ovulation, egg recovery and fertilization rates were independent of the type of superovulatory hormone used. Fertilization rate was high irrespective of insemination site but intrauterine insemination at 52 h was associated with a significant (P less than 0.01) decrease in egg recovery of over 40% compared with cervically inseminated ewes. In Exp. 2 ewes were inseminated at 36 (N = 5), 48 (N = 6) or 60 (N = 6) h after pessary withdrawal to determine the optimum intrauterine insemination time to maximize both fertilization rate and egg recovery. Egg recovery per ewe flushed was 23, 59 and 67% after intrauterine insemination at 36, 48 and 60 h respectively. Correspondingly, 0, 85 and 100% of the eggs recovered were fertilized. The results of Exps 1 and 2 suggest that when intrauterine insemination occurs before or during ovulation it interferes with oocyte collection by the fimbria. In Exp. 3 egg recovery and fertilization rates were determined after cervical insemination at 48 and 60 h (N = 8) or intrauterine insemination at 48 (N = 9) or 60 (N = 8) h after progestagen withdrawal. Ewes in the last two groups were subdivided and inseminated unilaterally or bilaterally. Egg recovery was high after cervical insemination (95%) but only 36% of these eggs were fertilized. Unilateral intrauterine insemination was as effective as bilateral in ensuring high fertilization rates (100 versus 97%). Intrauterine insemination at 48 h compared with 60 h resulted in a significantly lower (P less than 0.05) percentage of eggs recovered (42 versus 90% respectively). However, reducing the degree of interference by adopting unilateral rather than bilateral insemination did not alleviate the detrimental effects of the 48-h insemination time on egg recovery. From these results we advocate the adoption of intrauterine insemination at 60 h after progestagen withdrawal to maximize fertilization rate and egg recovery in superovulated ewes.     

Effects of intracervical PGE2-gel on myometrial activity and cervical state in first trimester pregnancy

In 7 primigravidae admitted for first trimester abortions by dilatation and evacuation, 0.5 mg PGE₂ in viscous gel (5 patients) or placebo gel (2 patients) was applied intracervically 6 hours prior to the operation. Throughout the treatment period intrauterine pressure was recorded. Application of placebo gel induced no cervical ripening or myometrical activation. In all patients receiving active gel, a marked improvement of the cervical state was induced by the treatment. In three cases, this priming occurred in parallel to minimal changes in myometrial activity, without regular uterine contractions. In two patients, marked uterine activation was registered due to partly extraamniotic application. It is suggested, that the PGE₂-gel has a direct effect on the cervical tissues. Further, the risk of partially applying the gel in the extraamniotuc space, thus stimulating the myometrium, depends on the gel volume relative to the dimensions of the cervical canal and the application technique.     

Effects of prostaglandin E2 (PGE2) on estradiol-17β-induced luteolysis in the nonpregnant ewe

Fifteen ewes were assigned as they came into estrus to the following randomized treatment groups: 1) Vehicle (1 ml corn oil + vehicle Na₂CO₃ buffer), 2) Estradiol-17β + vehicle and 3) Estradiol-17β + PGE₂ (500 μg) in Na₂CO₃ buffer (5 ewes/treatment group). Prostaglandin E₂ was given through an intrauterine cannula every four hours from days 8 through 15 postestrus. PGE₂ prevented a luteolytic dose of estradiol-17β given on days 9 and 10 from causing a precious luteolysis. PGE₂ maintained concentrations of progesterone in peripheral blood (days 8 through 15) and weights and concentrations of progesterone in corpora lutea on day 15 postestrus of ewes receiving estradiol-17β. It is concluded that chronic intrauterine infusions of PGE₂ can prevent an estradiol-17β-induced premature luteolysis.     

Spinal cord transection inhibits HR reduction in anesthetized rats immersed in an artificial CO<Subscript>2</Subscript>-hot spring bath

Like humans, the heart rate (HR) of anesthetized rats immersed in CO₂-water is lower than that when immersed in tap water at the same temperature. To investigate the afferent signal pathway in the mechanism of HR reduction, Wistar rats were anesthetized with urethane and then the spinal cord was transected between T₄ and T₅. The animals were immersed up to the axilla in a bathtub of tap-water (CO₂ contents: 10–20 mg·l⁻¹) or of CO₂-water (965–1,400 mg·l⁻¹) at 35°C while recording HR, arterial blood pressure, and arterial blood gas parameters (PaCO₂, PaO₂, pH). Arterial blood gas parameters did not change during immersion, irrespective of CO₂ concentration of the bath water, whereas the HR was reduced in the CO₂-water bath. The inhalation of CO₂-mixed gas (5 % CO₂, 20 % O₂, 75 % N₂) resulted in increased levels of blood gases and an increased HR during immersion in all types of water tested. The HR reduction observed in sham transected control animals immersed in CO₂-water disappeared after subsequent spinal cord transection. These results show that the dominant afferent signal pathway to the brain, which is involved in inducing the reduced HR during immersion in CO₂-water, is located in the neuronal route and not in the bloodstream.     

An estimation of CO<Subscript>2</Subscript> fixation capacity in mangrove forest using two methods of CO<Subscript>2</Subscript> gas exchange and growth curve analysis

In many coastal areas of South-East Asia, attempts have been made to revive coastal ecosystem by initiating projects that encourage planting of mangrove trees. Compared to the terrestrial trees, mangrove trees possess a higher carbon fixation capacity. It becomes a very significant option for clean development mechanism (CDM) program. However, a reliable method to estimate CO₂ fixation capacity of mangrove trees has not been established. Acknowledging the above fact, we decided to set up an estimation method for the CDM program, using gas exchange analysis to estimate mangrove productivity, we put into consideration the net CO₂ fixation of reforested Kandelia candel (5-, 10-, and 15-year-old stand). This was estimated by gas exchange analysis and growth curve analysis. In growth curve analysis, we drew a growth curve of a single stand using data of above- and below-ground biomass. In the gas exchange analysis, we calculated CO₂ fixation capacity by (1) measuring respiration rate of each organ of stand and calculating respiratory CO₂ emission from above- to below-ground biomass. (2) Measuring the single-leaf photosynthetic rate in response to light intensity and calculating the photosynthetic CO₂ absorption. (3) We also developed a model for the diurnal changes in temperature, and monthly averages based on one-day estimation of CO₂ absorption and emission, which we corrected by this model in order to estimate the net CO₂ fixation capacity in response to temperature. Comparing the biomass accumulation of the two methods constructed for the same forest, the above-ground biomass accumulation of 10-year-old forest (34.3 ton ha⁻¹ yr⁻¹) estimated by gas exchange analysis was closely compared to those of growth curve analysis (26.6 ton ha⁻¹ yr⁻¹), suggesting that the gas exchange analysis was capable of estimating mangrove productivity. The validity of the estimated CO₂ fixation capacity by the gas exchange analysis and the growth curve analysis was also discussed.     

The endemic spring snail Pyrgulopsis montezumensis in a high CO2 environment: Importance of extreme chemical habitats as refugia

1. We examined the distribution of the endemic spring snail, Pyrgulopsis montezumensis, along a gradient of dissolved free CO₂ and on selected substrata in the collapsed travertine springmound of Montezuma Well, Arizona, U.S.A. 2. Dissolved CO₂ concentration ranged from 480 (SE ± 56) mg L^??1 near the bottom spring vents in the limnetic zone (12 m deep) of the well to < 20 mg L^??1 in an irrigation canal about 1400 m downstream from the well outlet. Spring snails occurred in habitats with 110–315 mg L^??1 dissolved CO₂. 3. Laboratory experiments showed that P. montezumensis tolerated a dissolved CO₂ concentration up to about 400 mg L^??1 for 11 days, but displayed 40% (SE ± 4) mortality after 3 days at 728 mg L^??1 and 100% mortality after 24 h at 1200 mg L^??1. We also measured 100% mortality after 10 days at ambient concentrations (< 10 mg L^??1) of dissolved CO₂. 4. Although the concentration of dissolved CO₂ was within the tolerance range for spring snails in the irrigation canal, inadequate substrata and increased predation probably restricted their distribution in the lower reaches of the canal. Both field measurements and laboratory selection experiments showed a significant (P < 0.001) preference for limestone substrata over submerged vegetation and fine sediments. 5. We propose that springs with high dissolved CO₂ may provide refugia from invertebrate and/or vertebrate predators not physiologically adjusted to the extreme chemical environment. These extreme chemical habitats probably reduce competition for resources and predation and allow resistant populations like P. montezumensis to attain high density.     

Effects of temperature on respiration and acid-base balance in a monitor lizard

Summary Ventilation, gas exchange, blood gas tensions and arterial pH were measured simultaneously in monitor lizards,Varanus exanthematicus. In contrast to previously studied poikilotherms, the arterial pH is independent of body temperature within the normally encountered temperature range (Fig. 1). This exception to the relative alkalinity concept (Rahn, 1966) is correlated with the finding thatV. exanthematicus maintains a constant ratio of ventilation to oxygen uptake (and CO₂ production) at different temperatures (Fig. 3). The increase in arterial (Fig. 1) is related to an increase in physiological dead space; i.e., alveolar ventilation increases less with temperature than total ventilation (Fig. 4). This may result from the increased frequency of breathing which results in a reduced breath holding time (Fig. 2). Varanid lizards have a higher oxygen requirement than other reptiles. This is reflected in the control of ventilation, the specialized lung morphology, the high arterial saturation due to low intracardiac shunting, pH regulation and other mammal-like features ofVaranus.     

Zur Glykolatoxydation einzelliger Grünalgen

Summary O₂-uptake and CO₂-release by a chlorophyll-free, carotenoid-containing mutant of Chlorella vulgaris increase on addition of Na-glycolate by factors of 4–5 and 5–6, respectively (Fig. 1). In an enzyme preparation of that alga (sonification, centrifugation, precipitation with 0–30% (NH₄)₂SO₄, dialysis) activity of glycolate oxidase can be demonstrated by O₂-uptake (Fig. 2a) as well as by reduction of the artificial electron acceptor DCPIP (Fig. 2b). The same holds true for whole cells as well as equally prepared enzyme preparations of heterotrophically or autotrophically grown wildtype Chlorella vulgaris, provided the cells are cracked by a French press instead of a sonicator (Figs. 3a-c and 4a-c). Glyoxylate is the main reaction product (Table). Oxidation of exogenous glycolate is rapidly performed by whole cells of Scenedesmus quadricauda and of Ankistrodesmus convolutus, too, but hardly or not at all by Chlorella pyrenoidosa and Ankistrodesmus braunii. No definite influence of the level of CO₂ applied during growth is found: Chlorella vulgaris and Ankistrodesmus convolutus show a rapid oxidation of glycolate after growth under 0,03 and 1,5% CO₂ in air, whereas Chlorella pyrenoidosa and Ankistrodesmus braunii do not show an enhanced O₂-uptake on addition of glycolate after either condition (Fig. 5). Various developmental stages of Chlorella pyrenoidosa respond differently to addition of glycolate, the extra O₂-consumption varying between about 25% (mature cells) and 50–60% (young cells) of the endogenous rate (Fig. 6). It thus appears that species of unicellular green algae within the same genus have strong or weak glycolate oxidase activity and that several external factors have only a modifying effect on that enzyme.     

Patency of the cervical canal in cross-bred female Zebu x Brown Swiss selected for non-surgical recovery or transfer of embryos

A test to evaluate the degree of patency of the cervical canal was carried out on 260 Zebu x Brown Swiss cows and heifers that were assigned for non-surgical recovery or transfer of embryos. A total of 204 females (78.5%) were found to have a patent cervix (P <0.05), compared with 56 (21.5%) which had a blockage or obstruction when an attempt was made to pass a catheter through the cervical canal or the "cervical knot" of the purebred and cross-bred Zebu cows. A smaller percentage (12.89%) of non-patent cervices (P <0.05) were detected in multiparas, compared with 25 and 27.5% in primiparas and nullipara, respectively. The test for cervical patency is carried out on Bos indicus cattle to screen the animals before non-surgical embryo transfer manipulations and to attain better results in the application of non-surgical recovery and transfer of embryos.     

Calibration of Infra-red CO(2) Gas Analyzers

Precision gas mixing pumps produce CO₂ gas mixtures for the calibration of infra-red CO₂ gas analyzers equivalent in accuracy to the standard CO₂ gas mixtures (± 1%) supplied by the National Bureau of Standards, Washington, D. C.     

Enhancement of CO2 tolerance of Chlorella vulgaris by gradual increase of CO2 concentration

Summary Chlorella vulgaris UTEX259 was cultivated using two different methods of gas supply. In one method the CO₂ concentration in bubbled gas was held constant and in the other method it was increased gradually. Algal growth was almost linear after a short period of lag phase in both methods. With the constant CO₂ concentration, the CO₂ fixation rate in the linear growth phase decreased over 10%(v/v) CO₂, while the rate increased up to 6% CO₂. However, the rate was enhanced by using the latter incremental increase method, especially under a higher concentration of CO₂. The maximum rate of CO₂ fixation was 52 mg CO₂/l·h at 20% CO₂ during the gradual increase of CO₂ concentration.     

Laparoscopy for intrauterine insemination and embryo recovery in superovulated ewes at a commercial embryo transfer unit

Of 111 variable age, pedigree ewes subjected to a range of superovulatory regimens and then submitted to embryo recovery by laparoscopy, nine had adhesions corresponding to a mid-line laparotomy (presumably from a previous attempt to recover embryos) and could not have their embryos recovered by the laparoscopic technique. Of the remainder, 27 ewes (26.5%) had less than three ovulations or had prematurely regressing corpora lutea at the selected time for embryo recovery (Days 5 to 6 following insemination), and no attempt was made to recover embryos from them. For the 75 ewes subjected to laparoscopic ovum recovery following laparoscopic intrauterine insemination, the average number of ovulations (+/- SEM) was 7.9 +/- 0.6; the average ovum recovery (mean of values for each ewe) was 51.7% +/- 3.5; and the percentage of recovered ova that were fertilized was 87.3%. For a further nine 3-yr-old crossbred ewes the mean values for ovulation and ovum recovery were 7.6 +/- 1.2 and 70.1 +/- 7.7, and were not significantly different for the two insemination methods used (laparoscopic intrauterine vs cervical). In general, ovulation rates for ewes given pregnant mare serum gonadotrophin (PMSG) tended to be lower (5.2 +/- 0.7) than for those given porcine follicle stimulating hormone (pFSH, 7.7 +/- 0.8) or human menopausal gonadotrophin (hMG, 7.7 +/- 2.3). Ova recovery rates were similar on Days 5 and 6 (Day 0 = insemination), and were not affected by method of insemination (laparoscopic intrauterine vs cervical).     

A technique for transcervical intrauterine insemination of ewes

In commercial artificial insemination (AI) of sheep, fresh extended semen is deposited into the vagina or cervical os, or fresh extended or frozen semen is placed laparoscopically into the uterus. Transcervical intrauterine insemination of the ewe is not used commercially. In this study, methods of restraint and instrumentation for AI were evaluated and modified to produce a transcervical intrauterine technique suitable for commercial application. Four methods of restraint, four vaginal specula, three forceps and four instruments suitable for transcervical passage were compared. From these comparisons a technique was developed in which the ewes were positioned in dorsal recumbency with their hindquarters elevated. The vagina was dilated using a duck-billed speculum, the cervix was grasped and retracted using forceps, and an inseminating instrument was introduced into the cervical opening and manipulated through the cervical canal. The technique was repeated on 89 mature, multiparous ewes: the difficulty in locating the cervical opening, the force required to retract the cervix and the time required to penetrate into the uterus were recorded. Uterine penetration was achieved in 82% of the ewes. This technique has the potential to be applied in commercial artificial insemination programs of sheep.     

The growth of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> as influenced by CO<Subscript>2</Subscript>, light,and flue gases

In order to achieve recognition as environmentally friendly production, flue gases should be used as a CO₂ source for growing the microalgae Chlorella sorokiniana when used for hydrogen production. Flue gases from a waste incinerator and from a silicomanganese smelter were used. Before testing the flue gases, the algae were grown in a laboratory at 0.04, 1.3, 5.9, and 11.0 % (v/v) pure CO₂ gas mixed with fresh air. After 5 days of growth, the dry biomass per liter algal culture reached its maximum at 6.1 % CO₂. A second experiment was conducted in the laboratory at 6.2 % CO₂ at photon flux densities (PFD) of 100, 230, and 320 μmol photons m^?2 s^?1. After 4 days of growth, increasing the PFD increased the biomass production by 67 and 108 % at the two highest PFD levels, as compared with the lowest PFD. A bioreactor system containing nine daylight-exposed tubes and nine artificial light-exposed tubes was installed on the roof of the waste incinerator. The effect of undiluted flue gas (10.7 % CO₂, 35.8 ppm NO _x , and 38.6 ppm SO₂), flue gas diluted with fresh air to give 4.2 % CO₂ concentration, and 5.0 % pure CO₂ gas was studied in daylight (21.4?±?9.6 mol photons m^?2 day^?1 PAR, day length 12.0 h) and at 135 μmol photons m^?2 s^?1 artificial light given 24 h day^?1 (11.7?±?0.0 mol photons m^?2 day^?1 PAR). After 4 days’ growth, the biomass production was the same in the two flue gas concentrations and the 5 % pure CO₂ gas control. The biomass production was also the same in daylight and artificial light, which meant that, in artificial light, the light use efficiency was about twice that of daylight. The starch concentration of the algae was unaffected by the light level and CO₂ concentration in the laboratory experiments (2.5–4.0 % of the dry weight). The flue gas concentration had no effect on starch concentration, while the starch concentration increased from about 1.5 % to about 6.0 % when the light source changed from artificial light to daylight. The flue gas from the silicomanganese smelter was characterized by a high CO₂ concentration (about 17 % v/v), low oxygen concentration (about 4 %), about 100 ppm NO _x , and 1 ppm SO₂. The biomass production using flue gas significantly increased as compared with about 5 % pure CO₂ gas, which was similar to the biomass produced at a CO₂ concentration of 10–20 % mixed with N₂. Thus, the enhanced biomass production seemed to be related to the low oxygen concentration rather than to the very high CO₂ concentration.     

Proliferative and secretory activity of human umbilical endothelial cells cultivated under various hypoxia conditions

In this study, we examined the impact of 3-day hypoxia of different degrees on the viability, proliferation, and secretory activity of endothelial cells from human umbilical vein (HUVEC). A gas mixture of three components was used (%): 1) 10 O₂, 5 CO₂, and 85 Ar; 2) 5 O₂, 5 CO₂, and 80 Ar; and 3) 1 O₂, 5 CO₂, and 94 Ar. Cells cultivated in a CO₂ incubator in atmospheric oxygen (21% O₂) served as control. It was found that 3-day HUVEC cultivation at 1% O₂ increased NO synthesis; enhanced secretion of endothelin-1, IL-6, IL-8, TNF-alpha, sVCAM-1, sE-cadherin, sE-selectin, VEGF-A, and bFGF; and inhibited proliferation. HUVEC cultivated under 10% O₂ and 5% O₂ exhibited the lowest level of basal secretion of these substances and increased proliferative activity. These cells cultivated under conditions of atmospheric oxygen for 3 days displayed activated secretion of NO, IL-6, IL-8, and von Willebrand factor; the activation was higher than at 10% O₂ and 5% CO₂. Thus, the gaseous medium with reduced oxygen content (5%) is a more physiological condition for HUVEC cultivation. An increase in the amount of oxygen up to the atmospheric level causes endotheliocyte activation; the cells exhibit the features of endothelial dysfunction. Oxygen content reduced to 1% induces endothelial dysfunction and reduced proliferative potential.     

Effects of carbon dioxide acidification of mouse oocytes before in vitro fertilization,culture, and transfer

The effects of varying pH levels on superovulated mouse oocytes before in vitro fertilization, culture, and transfer were investigated. Mouse oocytes acidified for 1 hour with 20% CO₂ (pH 6.9) exhibited a wide range of alterations. Five hours after insemination, 25% of these eggs showed different degrees of cytolysis and their perivitelline space contained numerous spermatozoa. At 12 hours normal-looking eggs from this group showed a higher proportion of polypronuclear zygotes than the control group (exposed, 51%; controls, 18%). Acidification thus markedly increased the permeability of the zona pellucida and/or interfered with the normal block to polyspermy. Fewer eggs achieved the two-cell stage among groups submitted to acidotic pH levels (6.9 to 6.6). In contrast, alkaline conditions (pH 7.8) did not reduce the proportion of two-cell embryos. However, only eggs maintained at pH 7.5 were capable of producing 80% of blastocysts. In other groups developmental blockage occurred mainly between stages 2 and 4. A significant reduction was found in the proportion of recipient females becoming pregnant after intrauterine transfer of blastocysts originating from CO₂-treated oocytes. This difference might be related to the existence of a large polyploid population among experimental eggs.     

Influence of buffer system and pH on the amount of oxygen exchanged between solvent H2O and the CO2 produced in the aerobic oxidation of Cypridina luciferin catalyzed by Cypridina luciferase.

Incorporation of ¹⁸O into CO₂ was measured under various buffer conditions when the bioluminescent oxidation of Cypridina luciferin, catalyzed by luciferase, was carried out either in H₂¹⁶O medium with ¹⁸O₂ gas, or in H₂¹⁸O medium with ¹⁶O₂ gas. The results indicate that (1) the exchange of oxygen between CO₂ and solvent H₂O is significantly influenced by the kind of buffer as well as by pH, (2) the exchange of oxygen between solvent H₂O and CO₂ produced from luciferin in a neutral buffer can be reasonably well estimated from the exchange that takes place when the same amount of CO₂ gas is introduced into the same buffer by the presently employed method, and (3) in the Cypridina bioluminescent reaction, one of two oxygens of O₂ is quantitatively incorporated into the product CO₂ prior to the exchange of oxygen between CO₂ and solvent H₂O.