Survival of bovine embryos stored at 4 degrees C

These experiments were designed to test the efficacy of storing bovine embryos at 4 degrees C. Of particular interest were the age of embryo at which maximum post-storage survival could be achieved and longevity at 4 degrees C. A greater proportion of day 8 blastocysts developed in vitro at 37 degrees C following refrigeration for 48 hr than did embryos collected 2, 4 or 6 days after estrus (P<0.01). Survival of blastocysts stored at 4 degrees C for 48 hr was similar to that of nonstored blastocysts. In a subsequent experiment, day 8 blastocysts were recovered nonsurgically and assigned to one of the following treatments: (a) immediate transfer; (b) culture at 37 degrees C; or (c) storage at 4 degrees C for 1, 2, 3 or 5 days. Post-storage viability was assessed by either development in culture at 37 degrees C or embryo survival following nonsurgical transfer to synchronized recipients. In vitro survival of nonstored embryos and embryos stored 1 day did not differ. Survival decreased after storage for 2 days (P<0.10) or longer (P<0.05). Similar results were observed for survival after transfer, but embryo viability decreased even more rapidly with increasing duration of storage. In vitro survival was approximately 50% for blastocysts stored for 3 and 5 days, but few pregnancies resulted from transfer of embryos stored for these periods. In another experiment survival after transfer of blastocysts stored at 4 degrees C for up to 2 days was similar to that of nonstored blastocysts.     

Preliminary studies on bovine embryo survival following short-term storage at 4 degrees C

A total of 113 non-surgically collected bovine embryos, 5-8 days of age, were stored for 48 hours at 4 degrees C in a modified phosphate-buffered saline solution (PBS). Following storage, embryos were cultured for 8-12 hours at 37 degrees C, and those which were morphologically normal were transferred to synchronized recipients by several methods designed to achieve twin pregnancies. Embryos which were collected and transferred on the same day served as controls. Of 113 embryos stored, 47 (42%) appeared to be transferable after the brief culture period. There was a marked breed effect on viability after refrigeration, with Hereford embryos surviving significantly better than Angus embryos (71% vs. 12%, respectively, p < .001). Post-transfer embryo survival of stored and control embryos, based on actual calvings, was 34 and 48 percent, respectively, a difference which was not significant (p=0.3). A marked difference in pregnancy rate following non-surgical transfer by 2 different technicians was noted (50% vs. 21.7%, respectively).     

Development, DNA fragmentation and cell death in porcine embryos after 24 h storage under different conditions

For practical applications of porcine embryo transfer (ET) it is important to develop feasible embryo storage conditions. The aim of the present study was to evaluate the effect of short-term storage (24 h) on the quality of in vivo produced porcine embryos. Three temperatures 18, 25 and 38 degrees C and three different media: Dulbecco's phosphate buffered saline (DPBS), TCM199 and Emcare, were tested for two different embryo ages: D4 embryos (collected 144 h after hCG treatment) and D5 embryos (collected 168 h after hCG). After slaughter of the donor gilts, embryos were collected and transported at 25 degrees C to the lab where morulas and blastocyst were selected (D4 n = 222; D5 n = 167) and randomly used as controls or distributed over the treatment groups. Developmental stage and embryo diameter were assessed by normal light microscopy, while total number of cells and incidence of apoptosis were assessed using a fluorescent embryo quality staining technique that combines three different dyes: Ethidium Homodimer (EthD-1), TUNEL and Hoechst 33342. Following 24 h storage, D5 embryos had higher rates of hatching (24%) and degeneration (9%) compared to D4 embryos (10 and 4%, respectively; P < 0.05). Embryos stored at 38 degrees C had higher rates of hatching (37%) compared to those ones stored at 25 degrees C (13%) or 18 degrees C (0%; P < 0.01). More embryos hatched when stored in medium Dulbecco's phosphate buffered saline (DPBS) or in TCM199 compared to those stored in Emcare (P < 0.05). A higher percentage of embryos stored at 18 degrees C degenerated compared to those stored at 25 or 38 degrees C (P < 0.01). No significant increase in apoptosis was observed after storage compared to the rates of apoptosis at 0 h (controls) or between the different storage groups. Based on the results we conclude that D4 porcine embryos produced in vivo, selected under normal light microscopy and stored at 25 degrees C in a serum free medium for 24 h will have a suitable developmental stage for ET and a high embryo quality.     

Hypothermic storage of sheep embryos with antifreeze proteins: development in vitro and in vivo

Antifreeze proteins (AFPs) non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. In Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4 degrees C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viability rates were not different between controls (18 21 ); BSA (9 15 ); WF at 1 mg/ml (14 15 ); WF at 10 mg/ml (13 15 ) or OP at I mg/n-d (15 21 ), but were decreased (P < 0.05) in embryos stored in OP at 1 0 mg/ml (I 1 20 ). Pooled data showed higher (P < 0.05) viability rates for WF (27 30 ) than for OP (26 41 ) or BSA (9 15 ). There was no effect of protein source on hatching rates, but mean hatched diameters of embryos were lower (P < 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0 degrees or 4 degrees C in 4 mg/n-d BSA or 1 mg/ml WF. Embryos stored in WF at 4 degrees C (WF/4 degrees C) had comparable hatching rates (8 12 ) to that of controls (10 10 ), but embryos in the other treatments (WF 0 degrees C, 5 11 , BSA 4 degrees C, 6 11 and BSA 0 degrees C, 3 10 ) had significantly lower hatching rates (P < 0.01) compared with controls. Hatched diameters were comparable between controls and embryos stored in WF 4 degrees C, but embryos stored in WF 0 degrees C and BSA at both temperatures had smaller diameters (P < 0.05). In Experiment 3, Grade 1 to 3 embryos were either transferred fresh or were stored for 4 d at 4 degrees C in 4 mg/ml BSA or 1 mg/ml WF at different cooling rates (T1, BSA > 2 degrees C/min; T2, WF > 2 degrees C/min and T3, WF < 1 degrees C/min) prior to transfer. There were no differences in the number of ewes pregnant (T1, 10 1 1; T2, 6 10 and T3, 8 10 ) or in the number of viable fetuses recovered per treatment (T1, 14 25 ; T2, 10 1 4 and T3, 15 2 1) to indicate a negative effect of cooling rate or protein on embryo survival. In conclusion, ovine embryos can be stored in WF or BSA at 4 degrees C for 4 d, yielding similar pregnancy and embryo survival rates as fresh embryos following transfer to recipient ewes.     

Deep freezing of sheep embryos.

Sheep embryos, collected 1-8 days after oestrus, were placed in Dulbecco's phosphate-buffered saline medium (PBS). After treatment, the viability of the embryos was tested by temporary transfer to ligated rabbit oviducts. In Exp. 1, Days 5-8 embryos survived for at least 15 min at 0 degrees C in the presence of 1-5 M-DMSO. In Exp. 2, 12/14 Days 5-8 embryos survived after being frozen in 1-5 M-DMSO at 0-3 degrees C/min to temperatures ranging between-15 degrees and -60 degrees C and then thawed at 12 degrees C/min. In Exp. 3, Days 5-8 embryos were frozen in 1-5 M-DMSO at 0-3 degrees C/min to below-65 degrees C before being transferred to liquid nitrogen (-196 degrees C), and stored for 12 hr to 1 month. The embryos were thawed at 3 degrees C/min, 12 degrees C/MIN or 360 degrees C/min and, after transfer to rabbit oviducts, 0/4, 10/36 and 1/4, respectively, developed normally. The 11 embryos which were considered normal when recovered from the rabbit oviducts plus 1 slightly retarded embryo were transferred to 7 recipient ewes. Four ewes subsequently lambed, producing 5 lambs. In addition, 8 embryos were transferred to 4 ewes directly after thawing. Three of these ewes subsequently lambed, producing 3 lambs.     

Cold storage of mouse embryos of different stages of development

Experiments were designed to evaluate the survival rates of preimplantation mouse embryos of different stages of development in cold culture at 4 degrees C. Several developmental stages, from one-cell to the blastocyst, were stored at 4 degrees C from 1 to 8 d. Viability following cold culture was determined by blastocyst expansion during culture in Whitten's medium at 37 degrees C. Blastocyst formation of nonstored controls ranged from 93 to 100% for all developmental stages tested. Only 3% of one-cell embryos survived 1 d and none survived 2 days at 4 degrees C. Survival improved using two-cell embryos, with 84, 69 and 15% forming expanded blastocysts following storage for 1, 2 and 3 d, respectively. Eighty five and 38% of eight-cell embryos formed expanded blastocysts following cold storage for 3 and 4 d, respectively. Survival rates for cold stored morulae and blastocysts remained above 75% for 6 d but decreased significantly to 30 and 36%, respectively, when stored for 8 d. A large percentage of blastocysts were observed to collapse when placed in cold storage from 1 to 8 d but almost all expanded when placed in culture at 37 degrees C. This study showed that one-cell embryos were particularly sensitive to cold storage compared to later-stage mouse embryos. Cold storage survival increased with increasing age of the embryo; morula and blastocyst survival rate was similar.     

Growth and survival of uninjured and sublethally heat-injured Escherichia coli O157:H7 on beef extract medium as influenced by package atmosphere and storage temperature.

The effect of atmospheric composition and storage temperature on growth and survival of uninjured and sublethally heat-injured Escherichia coli O157:H7, inoculated onto brain heart infusion agar containing 0.3% beef extract (BEM), was determined. BEM plates were packaged in barrier bags in air, 100% CO2, 100% N2, 20% CO2: 80% N2, and vacuum and were stored at 4, 10, and 37 degrees C for up to 20 days. Package atmosphere and inoculum status (i.e., uninjured or heat-injured) influenced (P < 0.01) growth and survival of E. coli O157:H7 stored at all test temperatures. Growth of heat-injured E. coli O157:H7 was slower (P < 0.01) than uninjured E. coli O157:H7 stored at 37 degrees C. At 37 degrees C, uninjured E. coli O157:H7 reached stationary phase growth earlier than heat-injured populations. Uninjured E. coli O157:H7 grew during 10 days of storage at 10 degrees C, while heat-injured populations declined during 20 days of storage at 10 degrees C. Uninjured E. coli O157:H7 stored at 10 degrees C reached stationary phase growth within approximately 10 days in all packaging atmospheres except CO2. Populations of uninjured and heat-injured E. coli O157:H7 declined throughout storage for 20 days at 4 degrees C. Survival of uninjured populations stored at 4 degrees C, as well as heat-injured populations stored at 4 and 10 degrees C, was enhanced in CO2 atmosphere. Survival of heat-injured E. coli O157:H7 at 4 and 10 degrees C was not different (P > 0.05). Uninjured and heat-injured E. coli O157:H7 are able to survive at low temperatures in the modified atmospheres used in this study.     

Relationship between dead cells and DNA fragmentation in bovine embryos produced in vitro and stored at 4 degrees C.

DNA fragmentation and its relationship with dead cells were examined in bovine blastocysts produced in vitro and stored at 4 degrees C for 1-5 days. Survival and development to the hatching and hatched blastocyst stage decreased with increasing storage time. Both were significantly lower at 72 hr than at 48 hr. None of the embryos stored for 120 hr developed to the hatching or hatched blastocyst stage. The proportion of dead cells per embryo increased progressively as the time of storage increased, until 69% of embryonic cells were dead after 120 hr of storage. There was no significant difference between the proportions of DNA fragmentation per embryo stored for 0 and 24 hr (12% vs 16%). However, the proportion of DNA fragmentation in embryos stored for longer than 48 hr was significantly greater than that in embryos stored for less than 24 hr. There were no significant differences among those stored for longer than 48 hr (28-33%). These results suggest that the reduced developmental competence of bovine embryos stored at 4 degrees C is characterized by necrotic change rather than apoptotic change.     

Freezing of epididymal spermatozoa from dogs after cool storage for 2 or 4 days

An experiment was conducted to investigate the freezing ability of canine epididymal spermatozoa after cool storage at 5 degrees C for 2 or 4 days. Spermatozoa were collected from the caudae epididymidis from 16 dogs. Total motility, plasma membrane integrity and acrosome integrity were evaluated immediately on harvesting, and after 2 and 4 days of storage at 5 degrees C, and at 0 and 2 h post-thaw at 37 degrees C. Sperm motility decreased significantly during cold storage, compared to freshly harvested spermatozoa (P < 0.001). Although there was no significant effect of pre-freeze storage time on post-thaw motility, there was a tendency towards decreased motility in spermatozoa that had been stored for 4 days, compared to spermatozoa that were frozen immediately after collection (P = 0.09). The number of post-thaw spermatozoa with an intact plasma membrane was decreased in spermatozoa cold-stored for 4 days (P < 0.001). There was no significant effect of pre-freeze storage time on the acrosomal status of post-thaw spermatozoa. In conclusion, canine epididymal spermatozoa were stored at 5 degrees C for up to 4 days without a clear detrimental effect on post-thaw motility and acrosome integrity, but storage may have decreased post-thaw motility. Results were, however, generally low.     

The dynamics of indole-3-acetic acid in Acer platanoides seeds during stratification and germination

During stratification at 5°C indole-3-acetic acid (IAA) levels in embryos of Acer platanoides decreased during the early stages but subsequently increased again throughout the remainder of a 144 day period. The reduction in IAA levels in embryos of fruits stored at 17°C was even more pronounced, and in addition, no increase was observed after longer storage periods at this temperature, the levels of IAA remaining very low. Germination in seeds maintained at 5°C was not observed until after 120 days or longer, but germination potential increased at an earlier stage, as shown by the fact that seeds transferred to 20°C gave appreciable increases in germination after much shorter chilling periods. Endogenous IAA levels in embryos from seeds transferred to 20°C after a chilling period, long enough to break dormancy, increased within 24 h, i.e. before visible germination, to levels similar to those observed in embryos from seeds chilled continuously for 144 days. Embryos from seeds chilled for 120 days, i.e. when the samples already showed visible germination and when the endogenous IAA content was already high, showed no further increase in endogenous IAA during a three day incubation at 20°C. None of the treatments employed was effective in inducing germination of seeds or embryos from fruits stored at 17°C.     

Cold culture of different stage mouse embryos in bicarbonated and bicarbonate-free media

Mouse embryos of different stages of development were cultured to expanded blastocysts following storage (1 to 8 d) at 4 degrees C in the presence or absence of HCO(3)(-). The effect of oxygen tension on the cold storage of one- and two-cell mouse embryos at 4 degrees C was evaluated by 37 degrees C culture and transfer to pseudopregnant recipients. Survival at 4 degrees C of early, one- to four-cell mouse embryos was improved with HCO(3)(-) in the medium. The presence of HCO(3)(-) was not of benefit for morulae or blastocyst survival following cold storage. Reducing the oxygen atmosphere from 20 to 5% O(2) improved survival of one-cell mouse embryos stored at 4 degrees C. The survival of two- and four-cell embryos, morulae and blastocysts at 4 degrees C was similar in 90% N(2), 5% CO(2) and 5% CO(2) in air, but it was significantly poorer in air alone. The collapse of morulae and blastocysts during cold storage up to 5 d was reduced with HCO(3)(-) in the storage medium. Blastocysts stored for 6 d at 4 degrees C failed to survive following immediate transfer to pseudopregnant recipients. Blastocyst survival was improved compared to controls (direct transfer of unstored blastocysts to recipients) when cultured for 36 h at 37 degrees C following 6 d of cold storage. This result suggests that cold-stored mouse blastocysts may require a metabolic period of readjustment to survive following transfer to synchronized recipients.     

Use of sucrose during removal of cryoprotectants after thawing eight-cell mouse embryos

Eight-cell mouse embryos were frozen in 0.5-ml plastic straws in modified Dulbecco's phosphate buffered saline (PBS) plus 5% steer serum plus either 1.32 M dimethyl sulfoxide (DMSO) or 1.32 M glycerol. Upon thawing, embryos were diluted 1:4 with 0.0, 0.2, 0.6, or 1.0 M sucrose solutions within the straws. Thawing was either in air at ambient temperature or in 8 degrees C or 38 degrees C water. After 48 h of culture, more embryos frozen in DMSO and thawed in 8 degrees C and 37 degrees C water developed to blastocysts (87 and 93%, respectively) than embryos thawed in air (75%; P < 0.05). No significant differences in development were noted among the three thawing regimens when embryos were frozen with glycerol. There was no significant effect of concentration of sucrose during dilution on development of embryos postthaw. With glycerol as the cryoprotectant, damage to zonae pellucidae increased as thawing rates increased, whereas the opposite was observed with DMSO as the cryoprotectant (P < 0.05).     

Fate of Salmonella montevideo on and in raw tomatoes as affected by temperature and treatment with chlorine.

A study was undertaken to determine the survival patterns of Salmonella montevideo G4639 on and in tomatoes during storage and the efficacy of chlorine treatment on inactivation of the pathogen. The population of S. montevideo on the surfaces of inoculated tomatoes stored at 10 degrees C did not change significantly (P < 0.05) throughout an 18-day storage period. Significant increases in population occurred within 7 days and within 1 day when tomatoes were stored at 20 and 30 degrees C, respectively. A significantly higher number of cells was taken up by the core tissue of tomatoes tempered at 25 degrees C when the tomatoes were dipped in a suspension at 10 degrees C compared with the number taken up when the tomatoes were dipped in cell suspensions tempered at 25 or 37 degrees C. Populations remained constant throughout subsequent storage for 8 days at 10 degrees C, regardless of the temperature differential between tomatoes and the dip suspension. Storage of tomatoes at 20 degrees C, however, resulted in significant increases in populations of S. montevideo. Populations of the pathogen on the surfaces and in the core tissues of tomatoes were significantly reduced by dipping for 2 min in a solution containing 60 or 110 ppm (60 or 110 micrograms/ml) chlorine, respectively; however, treatment in solution containing 320 ppm chlorine did not result in complete inactivation. Populations of S. montevideo remained unchanged in chopped tomatoes stored at 5 degrees C for 216 h (9 days) but increased significantly after storage for 96 or 22 h at 20 or 30 degrees C, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Advances in cooled semen technology.

In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks' salts, supplemented with NPPC was then developed for use with cooled/stored semen.Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 degrees C, and anaerobic conditions when stored at 4 degrees C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 degrees C or diluted in Kenney or INRA82 extenders and then stored at 4 degrees C for 24h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 degrees C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 degrees C for 24h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E-Z Mixin extenders then stored at 4 degrees C for 24h until insemination. Experiment 1 demonstrated that at 15 degrees C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 degrees C after 24h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 degrees C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% (n=52) compared to 68% (n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 degrees C (54%, n=37) as at 4 degrees C (54%, n=35) after 24h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 degrees C (59%, n=39) seems to improve fertility per cycle compared to E-Z Mixin at 4 degrees C (49%, n=39, P=0.25), but this result has to be confirmed.These results demonstrate that semen diluted in INRA96 extender and stored at 15 degrees C can be an alternative to semen diluted in milk-based extenders and stored at 4 degrees C for "poor cooler" stallions. Furthermore, INRA96 extender can be as efficient at 15 degrees C as at 4 degrees C, for preserving sperm motility and fertility.     

Development and viability of frozen-thawed cattle embryos

Embryos recovered 7 to 8 days after estrus were frozen from -7 to -30 degrees C at 0.3 degrees C/min, from -30 to -33 degrees C at 0.1 degrees C/min, and then plunged into liquid nitrogen. They were thawed in a 25 degrees C waterbath. In a preliminary study, 15 of 18 embryos continued to develop during the 24-hour culture post-thaw in either Ham's F-10 or modified Dulbecco's phosphate buffered saline (PBS). In the main study, 5 of 20 embryos developed to 60-day pregnancies when embryos were transferred within 5 hours after thawing. The incidence of extended estrous cycles (pregnancy or presumed embryonic mortality) was 10 of 14, when the zona pellucida was intact after thawing, and 0 of 6, when it was ruptured or absent (P<.05). Embryos cultured in PBS tended to develop more readily than those in Ham's F-10 (15 of 20 vs 9 of 20, respectively, P reverse similar.1). Quality of the embryos, at recovery from the donor and after thawing, affected development in culture (19 of 27 embryos excellent at recovery developed vs 5 of 13 poor to very good, P reverse similar.1; 23 of 33 embryos good to excellent after thawing developed vs 1 of 7 poor, P<.05). The proportion of pyknotic nuclei in embryos which were cultured ranged from 18 to 100%. The pregnancy rate from embryos which were cultured was low (2 of 20). Thirty percent of frozen and thawed embryos had damaged zonae pellucidae. The study showed that: the pregnancy rate from frozen embryos was approximately half that achieved with unfrozen embryos; culturing embryos for 24 hours before transfer was not beneficial; the PBS culture system appears to be the system of choice for assessing embryo viability in vitro .     

Impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 populations to undercooking and simulated exposure to gastric fluid

This study evaluated the impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 exposed to consumer-induced stresses simulating undercooking and digestion. Lean beef tissue samples were inoculated with E. coli O157:H7 cultures prepared in tryptic soy broth or meat decontamination runoff fluids (WASH) or detached from moist biofilms or dried biofilms formed on stainless steel coupons immersed in inoculated WASH. After inoculation, the samples were left untreated or dipped for 30 s each in hot (75 degrees C) water followed by lactic acid (2%, 55 degrees C), vacuum packaged, stored at 4 (28 days) or 12 degrees C (16 days), and periodically transferred to aerobic storage (7 degrees C for 5 days). During storage, samples were exposed to sequential heat (55 degrees C; 20 min) and simulated gastric fluid (adjusted to pH 1.0 with HCl; 90 min) stresses simulating consumption of undercooked beef. Under the conditions of this study, cells originating from inocula of planktonic cells were, in general, more resistant to heat and acid than cells from cultures grown as biofilms and detached prior to meat inoculation. Heat and acid tolerance of cells on meat stored at 4 degrees C was lower than that of cells on nondecontaminated meat stored at 12 degrees C, where growth occurred during storage. Decontamination of fresh beef resulted in injury that inhibited subsequent growth of surviving cells at 12 degrees C, as well as in decreases in resistance to subsequent heat and acid stresses. The shift of pathogen cells on beef stored under vacuum at 4 degrees C to aerobic storage did not affect cell populations or subsequent survival after sequential exposure to heat and simulated gastric fluid. However, the transfer of meat stored under vacuum at 12 degrees C to aerobic storage resulted in reduction in pathogen counts during aerobic storage and sensitization of survivors to the effects of sequential heat and acid exposure.     

Relationship between variations in pathogenicity and lag phase at 37°C of Listeria monocytogenes previously stored at 4°C

s. BUNCIC AND S.M. AVERY. 1996. Three haemolytic, pathogenic strains of Listeria monocytogenes (a reference strain, a food-derived strain and a human strain) were held at 4°C for 4 weeks in phosphate-buffered saline pH 5.5 or 7.0, with and without 0.2% potassium sorbate or 0.3% sodium acetate. The number of viable cells did not change significantly during this storage. Pathogenicity of non-growing L. monocytogenes cells for 14-d-old chick embryos was determined before and after storage. Storage at 4°C resulted in decreased pathogenicity, but effects were strain-, pH- and substrate-dependent. After 4 weeks storage at 4°C non-growing bacterial cells were transferred to Brain Heart Infusion broth and growth characteristics were determined during incubation at 37°C. Strains that showed decreased pathogenicity had significantly longer lag phases at 37°C than strains that maintained pathogenicity. It is concluded that decreased pathogenicity of L. monocytogenes stored without growth at 4°C for 4 weeks and subsequent long lag phase at 37°C are correlated.     

Toxoplasma gondii in vitro culture for experimentation

The aim of this study was to develop a culture method for Toxoplasma gondii that could provide fresh viable tachyzoites as and when required. When T. gondii was continuously maintained in HeLa cell cultures at 37 degrees C, the time to harvest varied from 48 to 144 h. Tachyzoite yields of > or = 1 x 10(6)/ml and > or = 90% viable were obtained from 519/882 (58.8%) cultures and 120/155 (77.4%) harvests were successfully used in the dye test. When cultures were transferred from 37 to 25 degrees C when maximally infected (48-54-h post-infection), they could be stabilised and tachyzoites could be harvested as required, up to 168 h later. When harvested from 25 degrees C, significantly more cultures 783/811 (96.5%) produced tachyzoite yields > or = 1 x 10(6)/ml > or = 90% viable (p < 0.001). Tachyzoite quality also significantly improved and 206/224 harvests (91.9%) (p < 0.001) were successfully used in the dye test. We have demonstrated that tachyzoites can be maintained at dye test quality for at least 7 days in HeLa cultures at 25 degrees C. The system is flexible and robust and provides a means whereby tachyzoites of standard quality can be stored for use in experimental models as and when required.     

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.     

A successful technique for the preservation of rabbit embryos

A technique for successfully freezing, thawing and transferring rabbit embryos has been developed. Morula stage embryos were collected from super-ovulated female rabbits by flushing both oviducts and uterine horns with a tissue culture medium. Well developed, viable embryos were then transferred to freezing vials and a cryoprotectant, dimethyl sulfoxide (DMSO) was added in several steps to bring its final concentration to 1.6 molar. To freeze the embryos the temperature was lowered slowly (either 0.5 degrees C/min or 1.0 degrees C/min) to -80 degrees C at which point the vials were transferred directly to liquid nitrogen (-196 degrees C). Thawing was done at 8 degrees C/min. After thawing, phosphate buffered saline was added in a stepwise manner to dilute the DMSO. The thawed embryos were then cultured at 37 degrees C. Transfer of the embryos was accomplished by laparotomizing a pseudopregnant doe and introducing the embryos into the fimbriated ends of the oviducts. The 101 positively transferred embryos resulted in 45 implantations and 34 live born young.