Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro

The effects of media (TCM199 vs. synthetic oviduct fluid, SOF), sera (foetal calf serum, FCS vs. human serum, HS), gas atmosphere (5% CO2 in air vs. 5% CO2, 5% O2 and 90% N2) and coculture with bovine oviduct epithelial cells (cells vs. no cells) on the in-vitro development of in-vitro matured and fertilized bovine oocytes were examined. Immature oocytes surrounded with compacted cumulus cells were cultured for 24 h in TCM199 supplemented with 10% FCS, 10 micrograms follicle-stimulating hormone (FSH)/ml and 10 micrograms luteinizing hormone (LH)/ml, 1 microgram oestradiol/ml, and 1 x 10(6) granulosa cells at 39 degrees C under 5% CO2 in air. In-vitro fertilization was performed with frozen-thawed, heparin-treated (100 micrograms/ml, 15 min) spermatozoa from 2 bulls. Oocytes were incubated with 2.5 x 10(6) spermatozoa/ml for 24 h and then cultured in one of 16 treatments for 7 days. Cleavage (2-8-cell) and development to blastocysts were recorded on Days 2 and 7, respectively, after the start of culture. SOF was superior to TCM199 for cleavage (P less than 0.01), development to blastocysts (P less than 0.001) and for proportion of cultured ova resulting in blastocysts with at least 60 or at least 100 nuclei (P less than 0.001). FCS was superior to HS for development to blastocysts (P less than 0.001) and 5% oxygen was superior to air for the proportion of ova reaching at least 60 cells (P less than 0.01). For cleavage and development to blastocysts, there was an interaction between serum and cells (P less than 0.01). In the presence of cells, ova preferred FCS, in their absence, serum had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of gas conditions,time of medium change,and ratio of medium to embryo on in vitro development of horse oocytes fertilized by intracytoplasmic sperm injection

This study was conducted to evaluate the effects of two different gas conditions (5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2), mixed gas), time of medium change (Day 3 or 4) and ratio of medium to embryo (2, 5 or 10 microl per presumptive zygote) on the development of horse oocytes fertilized by intracytoplasmic sperm injection and cultured in G1.2/2.2 medium. Oocytes from slaughterhouse-derived ovaries were matured in vitro for 24 h and fertilized by injection of frozen-thawed sperm using micromanipulation with a Piezo drill. Presumptive zygotes were randomly assigned to 5% CO(2) in air or mixed gas and fixed after 96 h of culture. Cleavage rates between two gas conditions were similar (67 and 63%), but the mean nucleus number of embryos in the mixed gas treatment was significantly (P<0.05) higher than that of embryos cultured in 5% CO(2) in air (15.2 versus 7.0, respectively). Further experiments were done with mixed gas incubation. Development of embryos was compared after change from G1.2 to G2.2 medium at Day 3 or 4. There was no significant difference in cleavage rate (56 and 65%, respectively) or development to the blastocyst stage after 7 days of culture (5% and 46%, respectively) between embryos changed on different days. To evaluate the effect of the ratio of medium to embryo, zygotes were cultured at a ratio of 2, 5 or 10 microl medium per zygote. There were no significant differences among ratio treatments in rates of cleavage or development to blastocyst.     

Improvement of in vitro co-culture systems for bovine embryos using a low concentration of carbon dioxide and medium supplemented with beta-mercaptoethanol

Two experiments were conducted to investigate the effect of carbon dioxide (CO2) gas atmosphere and beta-mercaptoethanol on the development of bovine embryos in an in vitro co-culture system. In Experiment 1, in vitro-matured bovine oocytes were inseminated and then co-cultured with cumulus cells in culture medium (CM; 25 mM HEPES buffered TCM-199 supplemented with 5% superovulated cow serum and 0.5 mM sodium pyruvate). Oocytes matured and fertilized in 2 or 5% CO2 in air exhibited similar cleavage rates, but the proportion of embryos that developed to the blastocyst stage was higher for embryos co-cultured in 2 versus 5% CO2 in air. In Experiment two, 4- to 8-cell embryos produced under the condition of 2% CO2 in air were co-cultured with cumulus cells in CM supplemented with various levels of beta-mercaptoethanol (0, 5, 10, 50 microM). The percentage of embryos that developed to the blastocyst stage in CM with 10 microM beta-mercaptoethanol was higher (P<0.05) than that of embryos co-cultured with 0 or 50 microM beta-mercaptoethanol. These results indicate that cumulus cell co-culture in an atmosphere of 2% CO2 in air has a marked stimulatory effect on in vitro development of bovine embryos and that addition of beta-mercaptoethanol to the co-culture medium 2 d after insemination improved the in vitro development of bovine 4- to 8-cell embryos to the blastocyst stage.     

In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres

This study investigated the effect of culture media and gas atmospheres on the development of porcine nuclear transfer embryos. Oocytes derived from a local abattoir were matured for 42-44 h and enucleated. Fetal fibroblasts were prepared from a Day 35 porcine fetus. Confluent stage fetal fibroblasts were introduced into the perivitelline space of enucleated oocytes. Fusion and activation were induced simultaneously with two direct current (1.2 kV/cm for 30 micros) in 0.3 M mannitol medium. For parthenogenetic activation, the same pulses were used. In Experiment 1, parthenogenetically activated oocytes were cultured in North Carolina State University-23 (NCSU-23), Porcine Zygote Medium-3 (PZM-3), or Beltsville Embryo Culture Medium-3 (BECM-3). Parthenogenetically activated oocytes cultured in PZM-3 had a higher (P < 0.05) developmental rate to the blastocyst stage (15.2% versus 3.7-9.6%) as compared to BECM-3 or NCSU-23. The number of nuclei in Day 6 blastocysts was higher (P < 0.05) in PZM-3 (23.6) and NCSU-23 (21.4) than BECM-3 (14.2). In Experiment 2, parthenogenetically activated oocytes were cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air for 6 days (T1), 5% CO(2), 5% O(2), 90% N(2) for 6 days (T2), 5% CO(2) in air for 3 days, then 5% CO(2), 5% O(2), 90% N(2) for 3 days (T3), or 5% CO(2), 5% O(2), 90% N(2) for 3 days, then 5% CO(2) in air for 3 days (T4). Blastocyst formation rates were not different among treatments (12.9 =/-3.6 %, 13.5 +/- 4.2%, 10.8+/-2.4%, and 12.6+/-2.7%, respectively). However, T2 (36.7+/-2.9) and T3 (33.8+/-3.0) resulted in more nuclei per blastocyst than T1 (23.2+/-2.1) or T4 (26.0+/-2.1 ). In Experiment 3, reconstructed porcine nuclear transfer (NT) embryos were cultured in NCSU-23 or PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2). Developmental rates to blastocyst stage for porcine NT embryos cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) were 7.2+/-1.4% and 12.3+/-1.4%, and the number of nuclei was 12.2=/-0.8% and 19.4+/-1.0, respectively. NT embryos cultured in PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) had developmental rates to blastocyst stage of 18.8+/-1.9 %, and 17.8+/-3.8% the nuclei number was 20.9 +/- 1.9 and 21.9+/-3.3, respectively. NT embryos cultured in NCSU-23 had a higher developmental rate to the blastocyst stage in 5% CO(2), 5% O(2), 90% N(2) than in 5% CO(2) in air (P < 0.05). Regardless of gas atmospheres, NT embryos cultured in PZM-3 had a higher developmental rate (18.3 =/- 1.7% versus 16.9 +/- 1.2%) and nuclei number (21.4 +/-1.8 versus 16.9 +/- 1.2) than in NCSU-23 (P < 0.05). In conclusion, a gas atmosphere of 5% CO(2), 5% O(2), 90% N(2) supported a higher development rate of porcine NT embryos than 5% CO(2) in air when the porcine NT embryos were cultured in NCSU-23. Furthermore, regardless of atmosphere, PZM-3 supported a higher development rate of porcine nuclear transfer embryos than NCSU-23.     

Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes

Sera (fetal calf serum: FCS; and oestrous cow serum: ECS), hormones (2.5 FSH micrograms/ml + 5 micrograms LH/ml + 1 microgram oestradiol/ml) and granulosa cells (5 x 10(6)/ml) were added to culture medium to determine the frequencies of in-vitro maturation, fertilization, cleavage (2- to 8-cell) and development into blastocysts of bovine follicular oocytes. The maturation rates after 24 h in culture were not significantly different among the three factors tested (56-72%). The fertilization rates were significantly affected by serum type and the addition of granulosa cells. FCS gave significantly higher rates of fertilization (57-71%) than did ECS (34-52%), but the proportions of polyspermic fertilization were significantly higher in the former (8-19%) than in the latter (2-3%). The addition of hormones did not affect fertilization, cleavage and development. Neither type of serum affected cleavage and development. The highest rates of blastocyst formation were obtained when granulosa cells alone were added (FCS, 17%; ECS, 16%). The cell numbers of the blastocysts obtained were 100-150, similar to those of blastocysts developed in vivo. Transfer of 6 blastocysts to 3 cows resulted in 1 pregnancy. The present results indicate that the co-culture with granulosa cells is the most important factor for in-vitro fertilization to development into blastocysts of bovine oocytes matured in vitro.     

Development of one-cell ovine embryos in two culture media under two gas atmospheres

The objective of this study was to develop a successful system for culturing one-cell ovine embryos through several cleavage divisions. One hundred and four one-cell embryos were collected from synchronized, FSH-treated ewes 48 hr after the onset of estrus and randomly placed in one of four culture treatments. The effect of glucose supplementation and reduced oxygen tension (20% vs. 5%) on embryo development was studied. Embryo development was quantitated by a cleavage index based on the number of completed cell divisions. The number of embryos completing at least two cell divisions when cultured in Brinster's Pyruvate Medium (BPM) was 7 26 and 9 26 , under 5% CO(2) in air and 90% N(2), 5% CO(2), 5% O(2), respectively, while 22 26 and 20 26 embryos divided when cultured in BPM supplemented with 0.1% glucose (BPM-G) under similar atmospheres. Mean cleavage indices for embryos cultured in BPM were 1.2 and 1.6 under 5% CO(2) in air and 90% N(2), 5% CO(2), 5% O(2), respectively, while embryos cultured in BPM-G had mean cleavage indices of 4.6 and 4.0, respectively. Results of this study indicate that one-cell ovine embryos can be successfully cultured through several cleavage divisions. Glucose supplementation was beneficial for one-cell ovine embryo development. Reducing the oxygen tension from 20 to 5% had no effect on embryo development, and there was no media x gaseous atmosphere interaction.     

In-vitro development of zygotes from superovulated prepubertal and mature gilts

Ten prepubertal and 8 mature gilts were superovulated with PMSG and hCG, and inseminated with fresh boar semen. Zygotes were surgically recovered from oviducts 54-60 h after hCG. One and 2-cell zygotes were randomly allotted to Medium PL (modified BMOC-3 supplemented with 0.1 mM-EDTA and 1.5% BSA) or Medium G (Medium PL without pyruvate or lactate). Eggs were washed twice in medium, and placed in microdrops of medium overlaid with silicon oil for culture in an humidified 5% CO2, 5% O2, 90% N2 environment, then observed daily for 6 days. Development of eggs was dependent (P less than 0.001) on the interactive effects of age of gilt (prepubertal versus mature) and medium type (PL versus G) used in culture. A greater proportion of eggs cultured in Medium G developed further than did eggs in Medium PL (P less than 0.001). Additionally, a greater proportion of eggs from mature gilts developed further than did eggs from prepubertal gilts (P less than 0.02). We suggest that these results provide evidence that zygotes resulting from superovulation regimens of prepubertal gilts do not possess the same capacity for in-vitro development as do zygotes from pubertal gilts.     

Metabolism of glucose by human embryos

Glucose turnover, as measured by CO2 production, lactate accumulation and carbon incorporation from [U-14C]glucose as sole energy substrate, was low on the 2nd day of culture of human embryos resulting from in-vitro fertilization but above that of unfertilized oocytes. In general, all parameters of metabolism increased substantially during the following 2 days of development but the rate of increase in lactate production was greater than that of CO2, especially between Days 3 and 4. Within developing embryos, no correlation was evident between the metabolic turnover of glucose and the method of patient stimulation, the morphological quality of embryos or the apparent rate of cleavage in culture. The results indicate that, before Day 3 of development, glucose is not effective as an energy source for the human embryo because of a blockade to glycolysis similar to that in mouse embryos.     

Increasing carbon dioxide from five percent to ten percent improves rabbit blastocyst development from cultured zygotes.

One-cell rabbit zygotes were cultured at 39 degrees C in basal synthetic medium II (BSM-II) with 5%, 10%, or 15% CO2 and humidified air to determine the effect of CO2 concentration on development in vitro. After 4 days in culture, 37% of the embryos grown in 10% or 15% CO2 had reached the hatching blastocyst stage, but only 10% of the embryos were hatching when cultured under 5% CO2 (P = 0.01). Over all blastocysts, cell numbers were 207, 246, and 205 for the 5%, 10%, and 15% CO2 treatments, respectively. In a second experiment to determine if there was a beneficial effect, particularly at the blastocyst stage, of a higher concentration of CO2, embryos were cultured 4 days in either 5% or 10% CO2 or for 2 days in 5% CO2 followed by 2 days in 10% CO2. The numbers of blastomeres per embryo and embryo diameter were greater (P < 0.05) in embryos cultured continuously in 10% CO2 or in 10% CO2 only during days 3 and 4 of culture than in embryos cultured continuously in 5% CO2. In a third experiment, one-cell rabbit zygotes were cultured with 5% or 10% CO2 in a defined, protein-free medium consisting of 1:1 RPMI 1640 and Dulbecco's modified Eagle's medium. The proportion of embryos hatching and cell counts were significantly greater (P < 0.01) when cultured in the presence of 10% CO2. These data indicate that a 10% CO2 atmosphere exerts a beneficial effect on the development of zygotes into expanding and hatching rabbit blastocysts in vitro.     

In vitro culture of Dirofilaria immitis third- and fourth-stage larvae under defined conditions

The objective of the present study was to define culture conditions under which larval Dirofilaria immitis would molt, grow, and survive. Third-stage larvae (L3) survived for over 3 wk with a molt rate of up to 95% in a variety of media supplemented with fetal calf serum. Bovine albumin, added to several media at concentrations of 10-30 mg/ml, also proved to be an effective culture supplement for the induction of molting and for supporting larval survival. Two gas phases were tested, 5% CO2/95% N2 and 5% CO2/air; no differences were noted in larval development based on gas phase. Larvae, maintained in media with FCS or albumin for 48 hr, were capable of completing the molting process and growing in length in unsupplemented media. If the temperature at which cultures were maintained was changed from 37 C to 27 C, L3 did not molt but did survive for several weeks. Two factors required for larval D. immitis molting and growth have been identified, temperature of approximately 37 C and the presence of albumin in the culture medium. The defined culture system developed for D. immitis L3 may provide a source for collection of excretory-secretory antigens, which could prove useful in immunodiagnosis or immunoprophylaxis as well as provide a means of studying the process and requirements of filarial larval molting.     

Optimization of culture conditions for in vitro fertilization and reproduction of Microphallus turgidus (Trematoda: Microphallidae)

Most attempts to culture adult digeneans in vitro are unsuccessful. Even progenetic digeneans typically fail to produce infective eggs in axenic culture. However, metacercariae of Microphallus turgidus grown in vitro mature into adults and release eggs infective to the hydrobiid snail Spurwinkia salsa. The objectives of the present study were to verify the reproducibility of the M. turgidus culture protocol, to define optimal culture conditions for M. turgidus further, and to investigate why the parasite can be grown successfully in the absence of the definitive host. In the original cultivation protocol, excysted M. turgidus metacercariae from grass shrimp (Palaemonetes pugio) were incubated overnight in a conical-bottom centrifuge tube containing Hank's balanced salt solution and then cultivated in flat-bottom culture plate wells containing RPMI-1640 plus 20% horse serum. The gas phase was air. Worms cultured under this protocol consistently deposited eggs infective to snails. Worms grown in anaerobic conditions deposited few eggs, and those cultured in a gas phase of 5% CO(2) survived longer and produced more eggs than those cultured in air. However, snails were less likely to become infected when fed eggs deposited by worms cultured in 5% CO(2). Additionally, worms incubated with conspecifics in conical-bottom tubes prior to cultivation were more likely to be inseminated than worms incubated in flat-bottom culture wells; the highest percentages of inseminated worms occurred when metacercariae were incubated 24 hr in conical-bottom tubes at a density of 50 worms/tube and at a temperature of 37 C. Worms incubated in the absence of conspecifics were not fertilized and failed to produce infective eggs.     

Bovine blastocysts obtained from reconstructed cytoplast and karyoplasts using a simple portable CO2 incubator

To enable both the multiplication of elite livestock and the engineering of transgenic animals for various agricultural and biochemical purposes, scientists around the world are intensively studying efficient ways of improving developmental competency of bovine embryos reconstructed by somatic cell nuclear transfer. Because it is widely accepted that culture conditions along with many other factors contribute to the developmental competency of reconstructed embryos, this preliminary study was designed to test whether or not bovine reconstructed embryos could develop in vitro using a simple portable CO(2) incubator. CO(2) and O(2) gas tensions and air pressure can be varied using this system. The parameters used in the five conducted trials were low CO(2) (2-5%) and O(2) (8-10%) gas tensions, and negative air pressure (of 300 mm Hg). Chamber temperature was maintained at 38.5 degrees C. Bovine fetal fibroblasts were used as donor karyoplasts and were fused into microsurgically enucleated M II oocytes followed by activation and culture. From the 250 enucleated oocytes, 217 (86.8%) fused, 183 (73.2%) cleaved, and 43 (17.2%) developed to the blastocyst stage. While relatively low developmental rates were achieved, technical proficiency may have been a contributing factor. Further studies using this system are needed to determine optimal levels of O(2), CO(2), and air pressure.     

Growth of Legionella pneumophila in continuous culture

A method was developed to grow Legionella pneumophila in continuous culture. A chemostat was used to simulate nutrient-limited, submaximal growth in the natural environmental and to provide a precisely controlled growth regimen. Cultures grew under forced aeration under conditions yielding up to 38% saturation of dissolved oxygen; supplemental CO2 (5%) at the same gas flow rates as ambient air had no effect on culture growth. Pleomorphism was observed during growth under all conditions. Pigment was produced only at D less than 0.03 h-1. Catalase was produced at higher growth rates but not at higher temperatures. The pathogenicity was unaffected by altering either the growth rate or the growth temperature.     

Growth of Legionella pneumophila in continuous culture.

A method was developed to grow Legionella pneumophila in continuous culture. A chemostat was used to simulate nutrient-limited, submaximal growth in the natural environmental and to provide a precisely controlled growth regimen. Cultures grew under forced aeration under conditions yielding up to 38% saturation of dissolved oxygen; supplemental CO2 (5%) at the same gas flow rates as ambient air had no effect on culture growth. Pleomorphism was observed during growth under all conditions. Pigment was produced only at D less than 0.03 h-1. Catalase was produced at higher growth rates but not at higher temperatures. The pathogenicity was unaffected by altering either the growth rate or the growth temperature.     

Metabolism of Radiolabeled beta-Guaiacyl Ether-Linked Lignin Dimeric Compounds by Phanerochaete chrysosporium

Phanerochaete chrysosporium metabolized the radiolabeled lignin model compounds [gamma-C]guaiacylglycerol-beta-guaiacyl ether and [4-methoxy-C]veratrylglycerol-beta-guaiacyl ether (VI) to CO(2) in stationary and in shaking cultures. CO(2) evolution was greater in stationary culture. CO(2) evolution from [gamma-C]guaiacyl-glycerol-beta-guaiacyl ether and [4-methoxy-C]veratrylglycerol-beta-guaiacyl ether in stationary cultures was two- to threefold greater when 100% O(2) rather than air (21% O(2)) was the gas phase above the cultures. CO(2) evolution from the metabolism of the substrates occurred only as the culture entered the stationary phase of growth. The presence of substrate levels of nitrogen in the medium suppressed CO(2) evolution from both substrates in stationary cultures. [C]veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol were formed as products of the metabolism of VI and 4-ethoxy-3-methoxyphenylglycerol-beta-guaiacyl ether, respectively.     

Anisakis simplex: CO(2)-fixing enzymes and development throughout the in vitro cultivation from third larval stage to adult

We studied the effect of CO(2) on the in vitro cultivation of Anisakis simplex, an aquatic parasitic nematode of cetaceans (final hosts) and fish, squid, crustaceans and other invertebrates (intermediate/paratenic hosts), and, occasionally, of man (accidental host). The results showed that a high pCO(2), at a suitable temperature, is vital for the optimum development of these nematodes, at least from the third larval stage (L3) to adult. After 30 days cultivation in air, molting to L4 (fourth larval stage) was reduced to 1/3, while survival was about 1/3 of that when cultivated in air + 5% CO(2). The activity of the CO(2)-fixing enzymes, PEPCK and PEPC, was also studied. Throughout the development of the worms studied, PEPCK activity was much higher than that of PEPC (e.g., 305 vs. 6.8 nmol/min.mg protein, respectively, in L3 collected from the host fish). The activity of these enzymes in the worms cultivated in air + 5% CO(2) was highest during M3, and was also generally higher than that of those cultivated in air only, especially during molting from L3 to L4 (e.g., in recently molted L4, PEPCK activity was 3.7 times greater than that of PEPC 2.9 times greater than when cultivated in air).     

Inhibitory effect of carbon dioxide on the fed-batch culture of Ralstonia eutropha: evaluation by CO2 pulse injection and autogenous CO2 methods

In order to see the effect of CO(2) inhibition resulting from the use of pure oxygen, we carried out a comparative fed-batch culture study of polyhydroxybutyric acid (PHB) production by Ralstonia eutropha using air and pure oxygen in 5-L, 30-L, and 300-L fermentors. The final PHB concentrations obtained with pure O(2) were 138.7 g/L in the 5-L fermentor and 131.3 g/L in the 30-L fermentor, which increased 2.9 and 6.2 times, respectively, as compared to those obtained with air. In the 300-L fermentor, the fed-batch culture with air yielded only 8.4 g/L PHB. However, the maximal CO(2) concentrations in the 5-L fermentor increased significantly from 4.1% (air) to 15.0% (pure O(2)), while it was only 1.6% in the 30-L fermentor with air, but reached 14.2% in the case of pure O(2). We used two different experimental methods for evaluating CO(2) inhibition: CO(2) pulse injection and autogenous CO(2) methods. A 10 or 22% (v/v) CO(2) pulse with a duration of 3 or 6 h was introduced in a pure-oxygen culture of R. eutropha to investigate how CO(2) affects the synthesis of biomass and PHB. CO(2) inhibited the cell growth and PHB synthesis significantly. The inhibitory effect became stronger with the increase of the CO(2) concentration and pulse duration. The new proposed autogenous CO(2) method makes it possible to place microbial cells under different CO(2) level environments by varying the gas flow rate. Introduction of O(2) gas at a low flow rate of 0.42 vvm resulted in an increase of CO(2) concentration to 30.2% in the exit gas. The final PHB of 97.2 g/L was obtained, which corresponded to 70% of the PHB production at 1.0 vvm O(2) flow rate. This new method measures the inhibitory effect of CO(2) produced autogenously by cells through the entire fermentation process and can avoid the overestimation of CO(2) inhibition without introducing artificial CO(2) into the fermentor.     

Control of Penicillium martensii Development and Penicillic Acid Production by Atmospheric Gases and Temperatures

The effects of various gaseous environments and temperatures on development of Penicillium martensii NRRL 3612 and production of penicillic acid (PA) were determined. Accumulation of PA in mold-inoculated corn was measured following incubation under air; 20% CO(2), 20% O(2), 60% N(2); 40% CO(2), 20% O(2), 40% N(2); and 60% CO(2), 20% O(2), 20% N(2). Although reduced temperature initially inhibited PA production, at the end of the trial the largest quantity of PA (120 mug/g of corn) was found in air-incubated corn at the lowest test temperature (5 C). Atmospheres enriched with 60% CO(2) reduced PA accumulation below a detectable level at 5 and 10 C after a 4-week incubation period. Spore germination tests were carried out in a liquid growth medium incubated for 16 hr under several test conditions. Germ tube outgrowth at 30 C ranged from 36% in air to 2% in 60% CO(2), whereas no germination was observed in CO(2)-enriched gases at 10 C. When spore respiration rates were measured in air and O(2) in a liquid growth medium, complete removal of CO(2) from the reaction atmosphere did not reduce O(2) uptake.     

In vitro embryo production in camel (Camelus dromedarius) from in vitro matured oocytes fertilized with epididymal spermatozoa stored at 4 degrees C

Experiments were conducted to study the effect of storing epididymal spermatozoa, in tris-tes- and tris-lactose egg yolk extenders, on their fertilizing ability and subsequent in vitro embryo development. Ovaries and testes were collected from a local slaughterhouse in normal saline solution (NSS) at 37 degrees C and on ice (0-1 degrees C), respectively. Cumulus oocyte complexes (COCs) aspirated from the follicles were randomly distributed to 4-well culture plates (20-25COCs/well) containing 500 microL of maturation medium and cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in air for 36 h. Spermatozoa were collected from the cauda epididymides in syringes containing 2-3 mL of either tris-tes- or tris-lactose egg yolk extender. They were cooled down slowly and stored at refrigeration (4 degrees C) temperature. The spermatozoa were evaluated for motility and used for IVF of IVM oocytes on the day of collection and after 2, 4, 6 and 8 days of storage. On the day of IVF, spermatozoa were prepared by the swim up technique and both spermatozoa and oocytes were co-incubated at 38.5 degrees C in a humidified atmosphere of 5% CO(2) in air for 15-16 h. Presumptive zygotes were either fixed and stained with Hoechst 33342 for evaluation of fertilization or were cultured in 500 microL of the culture medium at 38.5 degrees C in an atmosphere of 5% CO(2), 5% O(2) and 90% N(2) in air. There was no significant difference (P>0.05) in the proportion of oocytes fertilized with spermatozoa stored in either of the two extenders for up to 8 days. The proportion of oocytes that cleaved (43-60%) and those that developed to blastocysts (14-21%) did not show any difference (P>0.05) either, when spermatozoa from different days of storage were used. First cleavage was observed as early as 16 h after IVF, early blastocysts had developed by day 4, expanded blastocysts after day 5 and hatching of blastocysts started after day 6 of culture. It may be concluded that dromedary epididymal spermatozoa survive in storage for at least 8 days in tris-lactose- and tris-tes egg yolk diluents at 4 degrees C. These spermatozoa maintain fertilizing ability and may be suitable for use in IVF and other assisted reproductive procedures.     

Effect of hypercapnia on thermoregulation at high ambient temperature

The reported investigations were carried out on rabbits exposed for three hours to ambient temperature of 25 degrees C or 35 degrees breathing athmospheric air (controls) or gas mixtures containing 4% or 7% of CO2. During the exposure to 35 degrees C in rabbits breathing the gas mixture with 7% of CO2 the rise of rectal temperature was significantly greater, heat elimination from the auricular surface was increased, whereas the oxygen uptake was increased insignificantly. In tracheostomized rabbits breathing the gas mixture with 7% of CO2 at 32 degrees C the respiratory rate decreased but the respiration volume increased as compared with the animals breathing atmospheric air. It seems that the hyperthermic effect of hypercapnia demonstrated in this work can be attributed to the impairment of heat elimination through the upper airways due to an inhibition of thermal panting.