Influence of water activity and temperature on survival of and colony formation by heat-stressed Chrysosporium farinicola aleuriospores.

The ability of sublethally heat-stressed aleuriospores of Chrysosporium farinicola to form colonies on yeast extract-glucose agar (YGA) supplemented with sufficient glucose, sorbitol, glycerol, and NaCl to achieve reduced water activity (aw) in the range of 0.88 to 0.95 was determined. The effects of the aw of diluent and incubation temperature during recovery and colony formation were also investigated. Aleuriospores harvested from 14-day-old cultures grown at 25 degrees C were less resistant to heat inactivation compared with aleuriospores from 20-day-cultures. Increased populations of heat-stressed aleuriospores were recovered as the aw of YGA was decreased from 0.95 (glucose and glycerol) and 0.94 (sorbitol) to 0.89 and 0.88, respectively. In NaCl-supplemented YGA, populations recovered at an aw of 0.94 were greatly reduced compared with populations detected at an aw of 0.92; no colonies were formed on NaCl-supplemented YGA at an aw of 0.88. Tolerance to aw values above 0.88 to 0.89 as influenced by solute type was in the order of glucose greater than sorbitol greater than glycerol greater than NaCl. Incubation at 20 degrees generally resulted in an increase in recoverable aleuriospores compared with incubation at 25 degrees C or at 30 degrees C for 14 days followed by 20 degrees C for 10 days. The lethal effect of NaCl on heat-stressed aleuriospores was enhanced at 30 degrees C. The retention of viability of aleuriospores held in sucrose-peptone water diluent (aw, 0.936) for 20 min was essentially the same as that observed when aleuriospores were held in peptone water (aw, 0.997).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of solute, pH, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia

The influences of glucose, sorbitol, and NaCl in a basal enumeration medium at water activities (aw) from 0.82 to 0.97 on colony formation by sublethally heat-stressed Wallemia sebi conidia were determined. Over this aw range, glucose and sorbitol had similar effects on recovery, whereas at an aw of 0.82 to 0.92, NaCl had a detrimental effect. Colony diameters were generally largest on media containing sorbitol and smallest on media containing NaCl. Maximum colony size and viable population of heat-stressed conidia were observed on media at an aw of ca. 0.92. When the recovery incubation temperature was 20 degrees C, the number of uninjured conidia detected at an aw of 0.82 was reduced compared with the number detected at 25 degrees C, while at 30 degrees C, the number recovered at an aw of 0.97 was reduced. The effect on heat-stressed conidia was magnified. This suggests that W. sebi conidia may be more tolerant of aw values higher than the optimum 0.92 when the incubation temperature is decreased from the near optimum of 25 degrees C and less tolerant of aw values greater than 0.92 when the incubation temperature is higher than 25 degrees C. The sensitivity of heat-stressed conidia increased as the pH of the recovery medium was decreased from 6.55 to 3.71. W. sebi conidia dispersed in wheat flour at aw values of 0.43 and 0.71 and stored for up to 65 days at both 1 and 25 degrees C neither lost viability nor underwent sublethal desiccation or temperature injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of solute, pH, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia.

The influences of glucose, sorbitol, and NaCl in a basal enumeration medium at water activities (aw) from 0.82 to 0.97 on colony formation by sublethally heat-stressed Wallemia sebi conidia were determined. Over this aw range, glucose and sorbitol had similar effects on recovery, whereas at an aw of 0.82 to 0.92, NaCl had a detrimental effect. Colony diameters were generally largest on media containing sorbitol and smallest on media containing NaCl. Maximum colony size and viable population of heat-stressed conidia were observed on media at an aw of ca. 0.92. When the recovery incubation temperature was 20 degrees C, the number of uninjured conidia detected at an aw of 0.82 was reduced compared with the number detected at 25 degrees C, while at 30 degrees C, the number recovered at an aw of 0.97 was reduced. The effect on heat-stressed conidia was magnified. This suggests that W. sebi conidia may be more tolerant of aw values higher than the optimum 0.92 when the incubation temperature is decreased from the near optimum of 25 degrees C and less tolerant of aw values greater than 0.92 when the incubation temperature is higher than 25 degrees C. The sensitivity of heat-stressed conidia increased as the pH of the recovery medium was decreased from 6.55 to 3.71. W. sebi conidia dispersed in wheat flour at aw values of 0.43 and 0.71 and stored for up to 65 days at both 1 and 25 degrees C neither lost viability nor underwent sublethal desiccation or temperature injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colony formation by sublethally heat-injured Zygosaccharomyces rouxii as affected by solutes in the recovery medium and procedure for sterilizing medium

Recovery and colony formation by healthy and sublethally heat-injured cells of Zygosaccharomyces rouxii as influenced by the procedure for sterilizing recovery media (YM agar [YMA], wort agar, cornmeal agar, and oatmeal agar) were investigated. Media were supplemented with various concentrations of glucose, sucrose, glycerol, or sorbitol and sterilized by autoclaving (110 degrees C, 15 min) and by repeated treatment with steam (100 degrees C). An increase in sensitivity was observed when heat-injured cells were plated on glucose-supplemented YMA at an aw of 0.880 compared with aws of 0.933 and 0.998. Colonies which developed from unheated and heated cells on YMA at aws of 0.998 and 0.933 generally exceeded 0.5 mm in diameter within 3.5 to 4 days of incubation at 25 degrees C, whereas colonies formed on YMA at an aw of 0.880 typically did not exceed 0.5 mm in diameter until after 5.5 to 6.5 days of incubation. The number of colonies exceeding 0.5 mm in diameter which were formed by heat-injured cells on YMA at an aw of 0.880 was 2 to 3 logs less than the total number of colonies detected, i.e., on YMA at an aw of 0.933 and using no limits of exclusion based on colony diameter. A substantial portion of cells which survived heat treatment were sublethally injured as evidenced by increased sensitivity to a suboptimum aw (0.880). In no instance was recovery of Z. rouxii significantly affected by medium sterilization procedure when glucose or sorbitol was used as the aw-suppressing solute.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colony formation by sublethally heat-injured Zygosaccharomyces rouxii as affected by solutes in the recovery medium and procedure for sterilizing medium.

Recovery and colony formation by healthy and sublethally heat-injured cells of Zygosaccharomyces rouxii as influenced by the procedure for sterilizing recovery media (YM agar [YMA], wort agar, cornmeal agar, and oatmeal agar) were investigated. Media were supplemented with various concentrations of glucose, sucrose, glycerol, or sorbitol and sterilized by autoclaving (110 degrees C, 15 min) and by repeated treatment with steam (100 degrees C). An increase in sensitivity was observed when heat-injured cells were plated on glucose-supplemented YMA at an aw of 0.880 compared with aws of 0.933 and 0.998. Colonies which developed from unheated and heated cells on YMA at aws of 0.998 and 0.933 generally exceeded 0.5 mm in diameter within 3.5 to 4 days of incubation at 25 degrees C, whereas colonies formed on YMA at an aw of 0.880 typically did not exceed 0.5 mm in diameter until after 5.5 to 6.5 days of incubation. The number of colonies exceeding 0.5 mm in diameter which were formed by heat-injured cells on YMA at an aw of 0.880 was 2 to 3 logs less than the total number of colonies detected, i.e., on YMA at an aw of 0.933 and using no limits of exclusion based on colony diameter. A substantial portion of cells which survived heat treatment were sublethally injured as evidenced by increased sensitivity to a suboptimum aw (0.880). In no instance was recovery of Z. rouxii significantly affected by medium sterilization procedure when glucose or sorbitol was used as the aw-suppressing solute.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solute stresses affect growth patterns, endogenous water potentials and accumulation of sugars and sugar alcohols in cells of the biocontrol yeast Candida sake

AIM: To evaluate the effect of modifications of water activity (aw 0. 996-0.92) of a molasses medium with different solutes (glycerol, glucose, NaCl, proline or sorbitol) on growth, intracellular water potentials (psi(c)) and endogenous accumulation of polyols/sugars in the biocontrol yeast Candida sake. METHODS AND RESULTS: Modification of solute stress significantly influenced growth, psi(c) and accumulation of sugars (glucose/trehalose) and polyols (glycerol, erythritol, arabitol and mannitol) in the yeast cells. Regardless of the solute used to modify aw, growth was always decreased as water stress increased. Candida sake cells grew better in glycerol- and proline-amended media, but were sensitive to NaCl. The psi(c) measured using psychrometry showed a significant effect of solutes, aw and time. Cells from the 0.96 aw NaCl treatment presented the lowest psic value (- 5.20 MPa) while cells from unmodified media (aw = 0. 996) had the highest value (- 0.30 MPa). In unmodified medium, glycerol was the predominant reserve accumulated. Glycerol and arabitol were the major compounds accumulated in media modified with glucose or NaCl. In proline media, the concentration of arabitol increased. In glycerol- and sorbitol-amended media, the concentration of glycerol rose. Some correlations were obtained between compatible solutes and psi(c). CONCLUSIONS AND SIGNIFICANCE: This study demonstrates that subtle changes in physiological parameters significantly affect the endogenous contents of C. sake cells. It may be possible to utilize such physiological information to develop biocontrol inocula with improved quality.     

Water relations of solute accumulation in Pseudomonas fluorescens

When Pseudomonas fluorescens was grown in a glucose salts medium adjusted with NaCl to a water activity (aw) value of 0.980, the intracellular glutamic acid concentration increased 23-fold and comprised 90% of the total amino acid pool. This increase was not observed when the aw of the medium was reduced to 0.980 with sorbitol. Sorbitol was taken up rapidly over a 30 min period and accumulated intracellularly to a level approximately two-fold greater than the concentration in the growth medium. In continuous culture, the specific rate of glutamic acid production and glucose uptake was greater at 0.980 (NaCl) than at 0.997 aw. The maintenance coefficients for glucose uptake were similar at both aw values but were 2.4-fold greater for glutamic acid production at 0.980 (NaCl) than at 0.997 aw.     

Survival of Escherichia coli O157:H7 in broth and processed salami as influenced by pH, water activity, and temperature and suitability of media for its recovery.

The survival of unheated and heat-stressed (52 degrees C, 30 min) cells of Escherichia coli O157:H7 inoculated into tryptic soy broth (TSB) adjusted to various pHs (6.0, 5.4, and 4.8) with lactic acid and various water activities (a(w)s) (0.99, 0.95, and 0.90) with NaCl and incubated at 5, 20, 30, and 37 degrees C was studied. The performance of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), and modified eosin methylene blue agar in supporting colony development of incubated cells was determined. Unheated cells of E. coli O157:H7 grew to population densities of 10(8) to 10(9) CFU ml-1 in TSB (pHs 6.0 and 5.4) at an a(w) of 0.99. Regardless of the pH and a(w) of TSB, survival of E. coli O157:H7 was better at 5 degrees C than at 20 or 30 degrees C. At 30 degrees C, inactivation or inhibition of growth was enhanced by reduction of the a(w) and pH. A decrease in the a(w) (0.99 to 0.90) of TSB in which the cells were heated at 52 degrees C for 30 min resulted in a 1.5-log10 reduction in the number of E. coli O157:H7 cells recovered on TSA; pH did not significantly affect the viability of cells. Recovery was significantly reduced on MSMA when cells were heated in TSB with reduced pH or a(w) for an increased length of time. With the exception of TSB (a(w), 0.90) incubated at 37 degrees C, heat-stressed cells survived for 24 h in recovery broth. TSB (a(w), 0.99) at pH 6.0 or 5.4 supported growth of E. coli O157:H7 cells at 20 or 37 degrees C, but higher numbers of heated cells survived at 5 or 20 degrees C than at 37 degrees C. The ability of unheated and heat-stressed E. coli O157:H7 cells to survive or grow as affected by the a(w) of processed salami was investigated. Decreases of about 1 to 2 log10 CFU g-1 occurred soon after inoculation of salami (pHs 4.86 and 4.63 at a(w)s of 0.95 and 0.90, respectively). Regardless of the physiological condition of the cells before inoculation into processed salami at an a(w) of either 0.95 or 0.90, decreases in populations occurred during storage at 5 or 20 degrees C for 32 days. If present at < or = 100 CFU g-1, E. coli O157:H7 would unlikely survive storage at 5 degrees C for 32 days. However, contamination of salami with E. coli O157:H7 at 10(4) to 10(5) CFU g-1 after processing would pose a health risk to consumers for more than 32 days if storage were at 5 degrees C. Regardless of the treatment conditions, performance of the media tested for the recovery of E. coli O157:H7 cells followed the order TSA > modified eosin methylene blue agar > MSMA.     

Interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii.

The interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii were investigated. Growth rates in media supplemented with glucose, sucrose or NaCl to aw 0.93 were more rapid than in unsupplemented media (aw 0.99). Although growth in unsupplemented medium was lower at 35 degrees C, incubation at 21 degrees C or 35 degrees C had little effect on growth in media supplemented with glucose and sucrose. The addition of 300 micrograms potassium sorbate/ml to media resulted in reduced growth rates, particularly at 35 degrees C. Heat resistance of Z. rouxii was substantially greater in cultures previously incubated at 35 degrees C than in cultures incubated at 21 degrees C in media both with and without 300 micrograms potassium sorbate/ml. Zygosaccharomyces rouxii was tolerant to freezing at -18 degrees C for up to 120 d in all test media supplemented with glucose, sucrose or NaCl. The addition of 300 micrograms potassium sorbate/ml to sucrose-supplemented media resulted in increased resistance to freezing in cultures previously incubated at 21 degrees C. Sensitivity to freezing increased when cultures were incubated at 21 degrees C in media not supplemented with solutes. Glucose and sucrose provided the best protection against inactivation by heating and freezing, regardless of the presence of potassium sorbate in growth media.     

Liquid formulation of the biocontrol agent Candida sake by modifying water activity or adding protectants

AIMS: To evaluate the effect of modification of water activity (aw) and the addition of protective substances in the preservation medium of liquid formulations of the biocontrol agent Candida sake stored at 4 and 20 degrees C. METHODS AND RESULTS: The aw of the preservation medium of C. sake was modified from 0.72 to 0.95 by adding glycerol or polyethylene glycol (PEG). Moreover, several protectant substances at different concentrations were evaluated. Modification of lower aw-levels (0.721-0.901) with glycerol did not maintain the viability of the yeast cells. Higher aw-levels (0.93-0.95) with either glycerol or PEG improved the viability but not at acceptable viability levels. C. sake cells maintained viabilities >60% when sugars, such as trehalose, and polyols, such as glycerol and PEG were used as protectants in liquid formulations. Moreover, liquid formulations of C. sake stored at 4 degrees C showed higher number of viable counts than at 20 degrees C. When different sugars were tested, all of them, except 10% fructose, resulted in a viability higher than 50% of the C. sake formulations. Biocontrol of liquid formulation treatments was similar to fresh cells in controlling Penicillium expansum on wounded apples. CONCLUSIONS: Sugars such as lactose and trehalose could be considered as good protectants in order to obtain liquid formulations of C. sake cells as they maintain the viability >70% for 4 months at 4 degrees C. SIGNIFICANCE AND IMPACT OF STUDY: This study shows that a suitable liquid formulation for commercial application can be produced with high viability and conservation of biocontrol efficacy. Moreover, if 10% lactose is the protectant used in the formulation, the economic costs would not be limiting for industrial production.     

The Effect of Water Activity and aw-Controlling Solute on Sporulation of Bacillus cereus

The influence of water activity (aw) on the formation of phase bright, heat stable, and dipicoiir.ic acid-containing spores of Bacillus cereus T from stage III to stage IV forespores has beer. investigated. Decreasing a_w levels reduced the rate of sporulation and the number of forespores which lysed was determined by the a_w-controlling solute used. The limiting a_w value for ir.e formation of mature spores was about 0·95 for glucose, sorbitol and NaCl whereas it was about 0·91 for glycerol. The development of refractility. the synthesis of dipicolinic acid, and acquisition of heat stability were affected equally by decrease in a_w during sporulation. With the range of a_w value where spores could be formed NaCl and glycerol had no signifcant: influence on the D value of the resulting spores whereas at all a_w levels, when sorbitol was use: as the a_w-controlling solute, the heat resistance was greater than in the basal medium. It Is suggested that the a_w of the sporulation medium determines the quantity of spores rather than. the spore properties.     

Factors affecting inactivation of Moraxella-Acinetobacter cells in an irradiation process.

The effect of various stages of the irradiation processing of beef on the injury and inactivation of radiation-resistant Moraxella-Acinetobactor cells was studied. Moraxella-Acinetobacter cells were more resistant to heat inactivation and injury when heated in meat with salts (0.75% NaCl and 0.375% sodium tripolyphosphate) then in meat without salts. These salts had no effect on radiation resistance. Both radiation- and heat-injured cells were unable to form colonies at 30 degrees C in plate count agar containing 0.8% NaCl. Neither unstressed nor heat-stressed cells were able to multiply in minced beef incubated at 30 degrees C for 12 h. Only after the beef was diluted 1:10 with peptone water were the heat-injured cells able to repair and eventually multiply. Heated cells were more sensitive to radiation inactivation and injury than unheated cells. After repair, the cells regained their resistance to both NaCl and irradiation. Freezing and storage at -40 degrees C for 14 days had only a slight effect on either unstressed or heat-stressed cells.     

Survival of Salmonella enteritidis and Salmonella typhimurium in chicken manure at different levels of water activity

Survival of Salmonella enteritidis and Salmonella typhimurium in chicken manure at different levels of water activity (aw) was determined. The aw was adjusted by means of saturated salts with defined equilibrium relative humidity and the manure samples were stored aerobically at 20 degrees C. At aw levels higher than 0.93, a moderate increase in colony-forming units over 8-9 h was found for both strains; at aw levels of 0.89-0.75, there was a thousand-fold reduction. Extended storage resulted in a million-fold reduction of Salmonella enteritidis in 8 days at an aw of 0.89. At higher and lower levels of aw, the reduction was less extensive.     

Improving water stress tolerance of the biocontrol yeast Candida sake grown in molasses-based media by physiological manipulation

The biocontrol agent Candida sake was cultured on either an unmodified molasses-based medium (water activity, a(w) 0.996) or on water stressed media produced by the addition of glycerol, glucose, NaCl, sorbitol, or proline to 0.98, and 0.96 a(w) for 24, 48, and 72 h, to study their impact on subsequent cell viability, and on concentrations of endogenous sugars (trehalose and glucose) and polyols (glycerol, erythritol, arabitol, and mannitol). The viability of cells of different ages cultured on these media was evaluated on NYDA medium with freely available water (a(w) 0.995), and on medium modified with polyethylene glycol to a(w) 0.95. Regardless of solute used, viable counts of cells grown on molasses-based medium (a(w) 0.98) were equal to or higher than those obtained from the medium with water freely available. The amino acid proline stimulated growth at 10% concentration. In contrast, water stress induced by addition of NaCl, glucose, or sorbitol at a(w) 0.96 caused a significant reduction in viable counts. Older cultures were more resistant to water stress. Glycerol and arabitol were the main solutes accumulated by C. sake cells in response to lowered a(w). Intracellular concentration of these polyols depended more on the solute used to adjust the a(w) than on the a(w) itself. Candida sake was more resistant to water stress with higher intracellular concentration of glycerol and erythritol.     

Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour

The toxic effects of sucrose and the conditions of in-straw glycerol removal after freezing and thawing were studied using Day-3 mouse embryos. At 20 degrees C, exposure to less than or equal to 1.0 M-sucrose for periods up to 30 min had no adverse effects on freshly collected embryos. At 25 and 36 degrees C, however, greater than or equal to 1.0 M-sucrose significantly reduced the developmental potential (P less than 0.001). In the freezing experiments the embryos were placed in 0.5 ml straws containing 40 microliters freezing medium separated by an air bubble from 440 microliters sucrose solution. The straws were frozen rapidly in the vapour about 1 cm above the surface of liquid nitrogen. The post-thaw viability was substantially better after sucrose dilution at 20 degrees C than at 36 degrees C. Mixing the freezing medium with the sucrose diluent immediately after thawing further improved the rate of survival relative to mixing just before freezing (P less than 0.001). The best survival was obtained when the freezing medium contained 3.0 M-glycerol + 0.25 M-sucrose; it was mixed with the diluent after thawing and the glycerol was removed at 20 degrees C. Under such conditions the sucrose concentration in the diluent had no significant effect on the rate of development (0.5 M, 69%; 1.0 M, 73%; 1.5 M, 64%). The results show that during sucrose dilution the temperature should be strictly controlled and suggest that intracellular and extracellular concentrations of glycerol are important in the cryoprotection of embryos.     

Radiation resistance and injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25 degrees C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and -30 degrees C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at -20 degrees C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at -20 degrees C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at -20 degrees C, nor did storage at -20 degrees C alter the cell's resistance to irradiation at 25 degrees C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36 degrees C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36 degrees C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5 degrees C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36 degrees C for 1 day than at 5 degrees C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Improving low water activity and desiccation tolerance of the biocontrol agent Pantoea agglomerans CPA-2 by osmotic treatments

AIMS: To study the improvement of tolerance to low water activity (aw) and desiccation during spray drying in Pantoea agglomerans cells subjected to mild osmotic stress during growth. METHODS AND RESULTS: The micro-organism was cultured in an unmodified liquid (control) or in aw-modified media, and viability of these cells was evaluated on unstressed (0.995) and 0.96 aw stressed solid media, in order to check total viability and aw stress tolerance respectively. Significant improvements in viability on unmodified medium were observed with cells grown for 24 h in NaCl 0.98 aw, glycerol 0.98 aw and 0.97 aw and for 48 h in NaCl 0.98 aw and 0.97 aw modified media. Both yield improvements and water stress tolerance were achieved with low aw media. Cells grown for 24 h in NaCl 0.98 aw or for 48 h in NaCl 0.98 aw, 0.97 aw and 0.96 aw, glucose 0.97 aw and glycerol 0.97 aw showed improved aw stress tolerance in comparison with control cells. The best results were obtained with NaCl treatments (0.98 aw and 0.97 aw) which also exhibited better survival rates than control cells during spray-drying process and maintained their efficacy against postharvest fungal pathogens in apples and oranges. CONCLUSIONS: NaCl treatments are very appropriate for improving P. agglomerans low aw tolerance obtaining high production levels and maintaining biocontrol efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: Improving stress tolerance of biocontrol agents could be an efficient way to obtain consistency and maintain efficacy of biological control under practical conditions.     

Sperm survival during short-term storage and after cryopreservation of semen from striped trumpeter (Latris lineata)

Methods of short-term storage and cryopreservation were examined for semen from striped trumpeter (Latris lineata). For fresh semen at 18 degrees C, the percentage of motile sperm declined rapidly from over 80% immediately after activation with sea water to less than 2% within 9 min after activation. The motility after activation of undiluted fresh sperm stored at 5 degrees C was maintained for two days and then declined markedly so that by the eighth day, sperm were mostly immotile after activation. The post-thawing motility was higher for sperm frozen with a non-activating diluent containing 2.84 M DMSO in saline (117 mM NaCl) than in an activating glycerol (2 M) medium in dilute sea water (300 mOsm). Post-thawing motility was higher for a dilution rate of 1:5 (semen:diluent) than 1:2 or 1:11 but was similar when frozen semen was thawed at 10 degrees, 20 degrees or 30 degrees C. For semen stored at a range of volumes as pellets frozen on dry ice (0.2 to 2.0 mL) or straws frozen in liquid nitrogen vapor (0.25 to 0.5 mL) and thawed in a waterbath at 20 degrees C, the post-thawing motilities were similar even though the patterns of cooling and thawing differed markedly between methods of freezing and sizes of pellets and straws.     

Comparative semen cryopreservation in ferrets (Mustela putorius furo) and pregnancies after laparoscopic intrauterine insemination with frozen-thawed spermatozoa

A study was conducted to determine an optimum technique for semen cryopreservation and the biological competence of frozen-thawed ferret spermatozoa. Fifty-two fresh electroejaculates from 4 males were evaluated for sperm percentage motility, forward progressive motility, motility index (SMI) and acrosomal integrity. To determine the optimum temperature for maintaining sperm motility in vitro and the influence of glycerol on sperm motility, seminal aliquants were diluted in TEST diluent (containing either 0 or 4% glycerol) and maintained at 25 degrees or 37 degrees C. For cryopreservation, semen was diluted in each of 3 cryodiluents (TEST, PDV, BF5F), cooled for 30 min at 5 degrees C and pelleted on solid CO2 or frozen in 0.25 ml straws (20 degrees C/min to -100 degrees C). Following thawing, SMI and acrosomal integrity were determined. Ten females with maximum vulval swelling were given 90 i.u. human chorionic gonadotrophin and laparoscopically inseminated in utero with spermatozoa previously frozen using the optimum diluent and freeze-thaw method. The maintenance temperature of 25 degrees C was superior (P less than 0.05) to 37 degrees C for sustaining sperm motility, and glycerol did not influence (P greater than 0.05) motility for up to 11 h of culture. After thawing, motile spermatozoa were recovered in all treatment groups, but sperm motility and normal acrosomal ratings were highest using the PDV diluent, the pelleting method and thawing at 37 degrees C (P less than 0.05). Seven of the 10 ferrets (70%) inseminated with spermatozoa frozen by this approach became pregnant and produced 31 kits (mean litter size 4.4; range 1-9 kits).(ABSTRACT TRUNCATED AT 250 WORDS)     

Freezing rabbit semen by the use of BF5 diluent

Three experiments were carried out to find the optimal concentration of DMSO and glycerol in BF5 diluent for freezing rabbit spermatozoa. Semen was diluted 1:1 with diluent A (BF5 + DMSO) at 25 degrees C and diluted further 1:1 with diluent B (diluent A + glycerol) after cooling down to 5 degrees C. Diluted semen was frozen immediately and stored in liquid nitrogen. Maximum percentages for motility and normal acrosomes were obtained in the presence of 12% DMSO (as expressed in diluent A) and 3% glycerol (final concentration) after thawing.