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.     

Viability of nuclei of two-cell mouse embryos stored at 4 degrees C and fused with blastomeres of fresh two-cell embryos.

This study compares the resistance of the nuclei and the cytoplasm of two-cell mouse embryos to short-term storage at low temperature above 0 degrees C. Two-cell embryos were stored at 4 degrees C for 24-96 h in PB1 containing 0.25, 0.5, 0.75, and 1.0 M sucrose. The development to blastocysts in culture was highest in the presence of 0.5 M sucrose. However, only 3% of the embryos developed into blastocysts after 96 h of storage. On the other hand, the viability of the nuclei of two-cell embryos stored at 4 degrees C was significantly prolonged when they were transplanted into a blastomere of enucleated fresh F1 (C57BL/6JXCBA) two-cell embryos. The proportions of chimeric embryos that developed to blastocysts were 88, 67, 76, 71, 64, 45, 32, and 20% following storage for 0, 48, 72, 96, 120, 144, 168, and 192 h, respectively. In addition, there was no difference in the coat color of the young derived from nuclei stored at 4 degrees C or fresh nuclei, although the proportions of chimeric embryos that developed into live young after transfer tended to decrease with increased storage time. Moreover, the viability of nuclei stored at 4 degrees C for 192 h was confirmed in the germ cell population of chimeric mice mated with albino mice. These results demonstrated that the nuclei in the two-cell mouse embryos were more resistant to storage at low temperature than the cytoplasm.     

Effect of various extenders and taurine on survival of stallion sperm cooled to 5 degrees C

Stallion semen was diluted in five different extenders (dimitro-poulus onze (Dimitro's), Kenney's modified tryode (Kenney's), modified INRA82 (INRA82), egg yolk-citrate-taurine (Citrate) and EZ-Mixin) and evaluated for motility after cooling and storage at 5 degrees C for 0, 24, 48, 72 and 96 h. EZ-Mixin extender was used as control while 70 and 100 mM of taurine were added to Dimitro's, Kenney's and INRA82 to study its effect under conditions of storage at 5 degrees C and varying processing modifications. Motility in INRA82 was 57.0, 58.4, 61.1, and 56.1% after 24, 48, 72 and 96 h, respectively and was higher (P < 0.05) than in other extenders after 48, 72, and 96 h. Motility decreased over time in Dimitro's (P < 0.05) and Kenney's (P < 0.01). Motility in INRA82 and EZ-Mixin decreased (P < 0.05) after 24 h and then remained unchanged until 96 h. In taurine (70 mM) containing extender Citrate, motility decreased throughout storage. Motility in INRA82 and Kenney's with taurine decreased (P < 0.05 and P < 0.01, respectively) after 48 h, and then remained stable until 96 h only in INRA82 with taurine. Motility in INRA82 with taurine was higher (P < 0.05) than in Citrate throughout incubation, whereas motility in INRA82 with taurine was higher than in EZ-Mixin and Kenney's with taurine after 24 and 48 h, respectively. Motility in INRA82 and Kenney's with taurine improved (P < 0.05) when osmotic pressure was increased with taurine (100 mM) but not when osmotic pressure was increased with Na (+) and K (+) salts. Motility was always higher (P < 0.01) in taurine (70 mM or 100 mM) containing extenders than in non-taurine extenders, Dimitro's, INRA82, and Kenney's, when sperm were incubated for 24 h at 5 degrees C in these extenders, then washed and incubated at 39 degrees C in Sp-TALP for 12 or 24 h. In conclusion 1) INRA82 was a better extender than the other extenders tested. 2) inclusion of taurine (100 mM) in INRA82 and Kenney's improved sperm survival until 96 and 48 h, respectively, and 3) sperm preincubation for 24 h in taurine containing extenders resulted in better sperm survival when washed and stored in Sp-TALP for further 12 or 24 h.     

Effects of cooling rate and storage temperature on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storage at 4 degrees C, and 5) fast cooling rate to and storage at 4 degrees C. Total spermatozoal motility (TSM), progressive spermatozoal motility (PSM), and spermatozoal velocity (SV) were estimated at 6, 12, 24, 48, 72, 96 and 120 h postejaculation. The longevity of spermatozoal motility was greatly reduced when spermatozoa were stored at 37 degrees C as compared to lower spermatozoal storage temperatures. At 6 h postejaculation, TSM values (mean % +/- SEM) of semen stored at 37 degrees C, slowly cooled to and stored at 25 degrees C or slowly cooled to and stored at 4 degrees C were 5.4 +/- 1.1, 79.8 +/- 1.6, and 82.1 +/- 1.6, respectively. Mean TSM for semen that was cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a moderate rate for four of seven time periods (6, 24, 72 and 120 h), and it was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a fast rate for five of seven time periods (6, 12, 24, 72 and 120 h). Mean TSM of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 25 degrees C for five of seven time periods (24 to 120 h). A similar pattern was found for PSM. Mean SV of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean SV of semen cooled to 25 degrees C for all time periods. A slow cooling rate (initial cooling rate of -0.3 degrees /min) and a storage temperature of 4 degrees C appear to optimize liquid preservation of equine spermatozoal motility in vitro.     

Effect of storage time and temperature and the variation among replicate tests (on different days) on the performance of spore disks and strips.

Laboratory-prepared spore disks were stored for 96 weeks at 22 degrees C with 50% relative humidity (RH) and at 4 degrees C with less than 1% RH. At the same time commercial spore strips were stored for 64 weeks at 22 degrees C with 50% RH. The spore count per unit and the heat resistance were measured at the beginning of the experiment and after 16, 32, 48, 64, 80, and 96 weeks of storage. The laboratory-prepared spore disks stored at 4 degrees C with less than 1% RH showed less change in numbers of spores per disks and decrease in the survival time than did the disks stored at 22 degrees C with 50% RH. Both the laboratory-prepared spore disks and the commercial spore strips stored at 22 degrees C with 50% RH decreased in survival times with increased storage time. The relative change in the survival times with storage was less for the commercial spore strips than for the laboratory-prepared spore disks.     

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)     

Effects of long-term chilled storage of red deer epididymides on DNA integrity and motility of thawed spermatozoa

We have carried out a study on the influence of prolonged cold storage (5 degrees C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5 degrees C and stored for 24, 96 and 192 h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37 degrees C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96 h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192 h at 5 degrees C (P<0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96 h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192 h.     

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 x 10(6) progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20 degrees C or from semen stored for 24 h at 5 degrees C were the same (11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 +/- 2.9, 19.6 +/- 2.6 and 20.5 +/- 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 +/- 2, semen stored for 24 h at 20 degrees C was 57 +/- 11 and semen stored for 24 h at 5 degrees C was 80 +/- 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5 degrees C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20 degrees C than that stored at 5 degrees C.     

Viability and fertility of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15 degrees C

Artificial insemination (AI) in rabbits is not extensive in the breeding programs of the rabbit meat industry. A limiting factor is related to the semen preservation. In order to improve the use of AI, two experiments have been conducted to evaluate sperm viability and fertility of rabbit semen chilled and stored at 15 degrees C after dilution in Tris-based extenders. In Experiment 1, pooled semen samples were diluted 1:10 (semen/extender) in four different Tris-based extenders (Tris-citric-glucose (TCG), TES-Tris-glucose (TTG), Tris-citric-fructose (TCF) and TES-Tris-fructose (TTF)) and stored at 15 degrees C. Sperm viability was evaluated at 0, 24, 48, 72 and 96 h following dilution for total sperm motility (TSM), forward progressive motility (FPM), plasma membrane integrity (PMI) and acrosome integrity (NAR). Viability of spermatozoa declined with time of storage (P<0.05), irrespective of the extender used. There were interactions between extender and time of storage (P<0.05) in all viability parameters evaluated. After 96 h of storage, TCG provided the highest sperm viability (P<0.05) and TTG the lowest (P>0.05). In Experiment 2, a field trial was conducted at a commercial farm to evaluate the conception and farrowing rates of rabbit spermatozoa extended in TCG. After synchronization of oestrous and induction of ovulation, 3713 does with different physiological conditions (nulliparous, primiparous, lactating and re-breeding) were inseminated one time (15x10(6) sperm per doses) with semen stored at 0 (n: 1275), 24 (n: 1503) and 48 h (n: 935) at 15 degrees C. Overall conception and farrowing rates were 77.1+/-0.7 and 70.4+/-0.7, respectively, and the mean litter size was 7.6+/-0.1. Fertility results were unaffected by the time of semen storage (P>0.05). Regardless of time of semen storage, fertility results were affected by the physiological conditions of does (P<0.05). Nulliparous and lactating does showed the highest fertility and primiparous the lowest. In summary, these results indicate that Tris-buffer extenders are effective for preserving viability and fertilizing capability of rabbit spermatozoa stored at 15 degrees C.     

Effect of polyethylene glycol on lipid peroxidation in cold-stored rat hepatocytes

A mechanism suggested to cause injury to preserved organs is the generation of oxygen free radicals either during the cold-storage period or after transplantation (reperfusion). Oxygen free radicals can cause peroxidation of lipids and alter the structural and functional properties of the cell membranes. Methods to suppress generation of oxygen free radicals of suppression of lipid peroxidation may lead to improved methods of organ preservation. In this study we determined how cold storage of rat hepatocytes affected lipid peroxidation by measuring thiobarbituric acid reactive products (malondialdehyde, MDA). Hepatocytes were stored in the UW solution +/- glutathione (GSH) or +/- polyethylene glycol (PEG) for up to 96 h and rewarmed (resuspended in a physiologically balanced saline solution and incubated at 37 degrees C under an atmosphere of oxygen) after each day of storage. Hepatocytes rewarmed after storage in the UW solution not containing PEG or GSH showed a nearly linear increase in MDA production with time of storage and contained 1.618 +/- 0.731 nmol MDA/mg protein after 96 h. When the storage solution contained PEG and GSH there was no significant increase in MDA production after up to 72 h of storage and at 96 h MDA was 0.827 +/- 0.564 nmol/mg protein. When freshly isolated hepatocytes were incubated (37 degrees C) in the presence of iron (160 microM) MDA formation was maximally stimulated (3.314 +/- 0.941 nmol/mg protein). When hepatocytes were stored in the presence of PEG there was a decrease in the capability of iron to maximally stimulate lipid peroxidation. The decrease in iron-stimulated MDA production was dependent upon the time of storage in PEG (1.773 nmol/mg protein at 24 h and 0.752 nmol/mg protein at 48 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of blood storage on radiation-induced micronuclei in human lymphocytes.

To evaluate the effect of blood storage on the yield of micronuclei (MN) in both irradiated (in vivo and ex vivo) and unirradiated peripheral blood lymphocytes (PBL), we applied the MN assay in cytokinesis-blocked (CB) PBL obtained from healthy subjects (n=11), and from cancer patients (n=10) who were undergoing fractionated partial-body radiotherapy (xRT). The heparinized blood samples were exposed to 137Cs-irradiation (0 Gy or 2 Gy) immediately after blood collection and were stored upright in test tubes either at room temperature (22 degrees C) or in the refrigerator (5 degrees C). Duplicate whole blood cultures from each sample were set up at 0 h, 96 h, and 120 h after ex vivo irradiation. Giemsa (10%) stained slides were prepared from each culture. MN yield was determined per 1000 binucleated cells. As compared to that obtained from the corresponding fresh blood samples, we found that (1) the 22 degrees C blood storage temperature did not affect MN yields in PBL of either healthy subjects or cancer patients up to 96 h, either with or without ex vivo irradiation; and (2) while blood samples were stored at 5 degrees C, the MN yield increased significantly in PBL of healthy subjects (with or without ex vivo irradiation) at 120 h, and in cancer patients (with ex vivo irradiation) at 96 h and 120 h. Since handling of the blood sample is important for CBMN assay during shipment or in the laboratory, our findings showed that blood storage at 22 degrees C or at 5 degrees C up to 96 h appeared to provide insignificant variations of the MN results as compared to fresh blood samples. However, the 96 h of blood storage at 5 degrees C elevated the MN frequency in ex vivo irradiated PBL of cancer patients who were undergoing xRT.     

DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water.

A simple assay is described using bisbenzimidazole (Hoechst 33258) to determine cellular DNA content in 96-well tissue cultures plates. At time points of interest, the plates are emptied of media and stored frozen. When the assay is to be performed, cultures are briefly incubated in distilled water and frozen again. This process lyses the cells and allows rapid and thorough mixing of the fluorochrome and cellular DNA. Freezing permits convenient storage of cultures until the time of assay. Experiments can be batched, further reducing processing time and giving better intra- and interexperimental standardization. The assay generates a linear standard curve for DNA fluorescence versus cell number, the range of which is appropriate for microculture wells. This enables the rapid and accurate measurement of cell number involving minimal processing time, making this assay well suited for cell proliferation studies.     

Immobilization as a technical possibility for long-term storage of bacterial biosensors

For applications in field experiments, the recombinant strain Salmonella typhimurium TA1535 was immobilized to permit its immediate utilization after long storage periods. Salmonella typhimurium TA1535 cells contain the plasmid that has an inducible SOS promoter fused to a promoterless luxCDABFE operon from Photobacterium leiognathi. The induction of bioluminescence occurs in the presence of the DNA-damaging agent mitomycin C which stimulates the bacterial SOS response. Early stationary phase cells were immobilized at a cell concentration of 10(10) CFU/ml in microtiter plates and stored up to 6 weeks at 4 degrees C in a sealed container. Even after 4 weeks of storage, the bioluminescence kinetics and yield in response to different concentrations of mitomycin C were not significantly different from those of freshly prepared samples.     

Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoal motility

Ejaculates were collected form three mixed-breed male dogs daily for 3 d. The semen was diluted in either a nonfat dried milk solid-glucose (NFDMS-G) or egg yolk citrate (EYC) extender at a concentration of 25 x 10(6) sperm/ml. The diluted samples were exposed to three different storage temperatures (35, 22 and 4 degrees C). Three cooling rates (-1.0, -0.3 and -0.1 degrees C/min) were also investigated at the lowest storage temperature (4 degrees C). The semen was evaluated for total motility, progressive motility and velocity at 0, 6, 12, 24, 48, 72, 96 and 120 h after collection by two independent observers. Interactions between extenders, temperatures and time after collection were found for each of the variables. Nonfat dried milk solid-glucose diluent was superior to EYC (P<0.05) in preservating sperm motility parameters that were evaluated for most of the observations. The evaluated sperm motility parameters were also significantly superior (P<0.05) in semen stored at 4 degrees C than at 35 or 22 degrees C for most of the observations. The progressive motility and velocity of sperm in semen cooled at 4 degrees C in NFDMS-G were higher (P<0.05) at the fast and medium cooling rates (-1.0 and -0.3 degrees C) than at the slow cooling rate (-0.1 degrees C/min) at 24 and 72 h, and at 48 h, respectively. In conclusion, the present study suggests that canine spermatozoal motility is well preserved when a NFDMS-glucose extender is added to the semen and the semen is cooled at a medium or fast rate to a storage temperature of 4 degrees C. Additional studies are needed to evaluate the fertility of semen stored in this manner.     

Effect of pre-analytical handling on haematological variables in minipigs

Pre-analytical handling may be an important determinant of haematological variables, if analysis is delayed. We investigated the effect of anticoagulants, i.e. tripotassium ethylenediamine-tetraacetic acid (EDTA) and citric acid, theophylline, adenosine, dipyridamole (CTAD), storage time (0.5, 1.5, 3.5, 5.5, 7.5, 25.5 and 27.5 h after blood sampling), and storage temperature (5 degrees C and 20 degrees C) on the variation in haemoglobin (HGB), red blood cell count (RBC), haematocrit (HCT), white blood cell count (WBC), and platelet count (PLT) in minipigs. Medians of HGB, RBC, HCT, WBC and PLT were significantly higher in EDTA tubes than in CTAD tubes due to the dilution effect of the anticoagulant. We found a minor significant increase in HCT after 25.5 h in blood stored at 20 degrees C, and at the same time a minor significant increase in WBC in EDTA tubes stored at 20 degrees C. We found a significant decrease in PLT in blood stored at 5 degrees C, especially in EDTA tubes. Minor variations were also observed in HGB and RBC. Our results indicate that PLT should only be measured in tubes placed at room temperature. If HCT or WBC analyses are to be performed on the day after blood sampling, the samples must be stored in a refrigerator until analysis. Our studies underline that time delay before analysis of haematological variables can cause increased variation, and should therefore be limited as far as possible in order to reduce the number of animals needed to make reliable conclusions.     

Effect of solid storage at 15 degrees C on the subsequent motility and fertility of rabbit semen

We conducted two studies to improve preservation of rabbit semen. The objective of the first study was determine whether a glucose- and fructose-based extender with two different amounts of gelatin would solidify at 15 degrees C, and to evaluate the influence of gelatin supplementation on sperm motility parameters after storing semen up to 10 days at 15 degrees C. The fertility of rabbit semen diluted in the best gelatin-supplemented extender established in Study 1 and stored for up to 5 days was evaluated in the second study. In Study 1, semen was collected with an artificial vagina from 40 bucks. Each ejaculate was diluted to (80-100) x 10(6) spermatozoa/mL (1:3, semen/extender) at 37 degrees C in one of the three following glucose- and fructose-based extenders: control (standard liquid extender), semi-gel or gel (0.7 or 1.4 g gelatin in 100 mL extender, respectively). Pools of semen were allocated among 0.6 mL plastic artificial insemination (AI) guns. Thirty (10 per extender group) AI doses were immediately analyzed (0 h) and the remainder stored in a refrigerator (15 degrees C) for 12, 24, 36, 48, 72, 96, or 240 h. All doses with gelatin extenders solidified at 15 degrees C. Semen samples, prewarmed to 37 degrees C, were evaluated with a computer-assisted sperm analysis (CASA) system. The percentage of motile cells was significantly lower using the liquid compared to the gel extenders during semen storage from 0 to 96 h. Although significance was lost, these differences persisted after 240 h of storage. Motility of spermatozoa in the semi-gel extender was intermediate between that of liquid and gel extender throughout the study. Study 2 was performed on 1250 multiparous lactating does. Five homogeneous groups of 250 does previously synchronized were inseminated using semen previously stored for 120, 96, 72, 48 or 24 h, respectively. Rabbit does receiving 24 h-stored semen (diluted with the control extender used in Study 1) served as controls. The remaining females received seminal doses supplemented with 1.4 g/100mL gelatin (gel extender used in Study 1). Kindling rates for rabbit does inseminated with gelatin-supplemented (solid) semen doses stored for 48 h (88%) or 72 h (83%) were similar to those recorded for liquid controls stored for 24 h (81%), whereas rates significantly decreased when the semen was solid and stored for 96 h (64%) or 120 h (60%) before AI. In conclusion, rabbit spermatozoa were effectively stored in the solid state at 15 degrees C, with fertility preserved for up to 5 days. Solid storage of rabbit semen would facilitate commercial distribution.     

Characteristics of Iberian red deer (Cervus elaphus hispanicus) spermatozoa cryopreserved after storage at 5 degrees C in the epididymis for several days

The aim of this study was to assess the influence of prolonged cold storage of Iberian red deer epididymides on post-thaw sperm characteristics. Thirty-seven pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control group). The remaining epididymides were cooled to 5 degrees C and stored for 12, 24, 48, 72 and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. After thawing, sperm motility, membrane and acrosome integrities, mitochondrial function and DNA damage were evaluated. The motility of spermatozoa stored in the epididymis for up to 96 h did not decrease significantly (P>0.05) but, after cryopreservation, a decline in sperm motility was seen in spermatozoa stored for 48 h, or later. A slower decrease in sperm membrane and acrosome integrities after cryopreservation were seen as storage time progressed. Some differences were seen when different methods were used to assess the same sperm parameter although changes followed similar patterns. This was the case for acrosome integrity (phase contrast microscopy versus fluorescent lectin) or membrane integrity (hypo-osmotic swelling test or nigrosin-eosin stain versus propidium iodide). We conclude that frozen-thawed spermatozoa of Iberian red deer recovered from epididymides stored at 5 degrees C have a good sperm quality (including motility) during less than 48 h of storage for most of the sperm parameters assessed.     

Liquid storage of flow cytometrically sorted ram spermatozoa

This study investigated the optimum short-term storage conditions for ram spermatozoa before and after flow cytometric sorting. Prior to sorting, semen from four rams (n = 3 ejaculates per ram) was diluted in either a Tris-based diluent (TRIS) or AndroHep (AH) and stored at 5, 15 or 21 degrees C for 0, 6 or 24h. Sperm characteristics were assessed during storage and after sorting, freeze-thawing and incubation (6h, 37 degrees C). Functional capacity and migration ability in artificial cervical mucus (sperm migration test (SMT)) of stored, sorted and non-sorted (control) spermatozoa were assessed after freeze-thawing. After sorting, semen from three rams (n = 3 ejaculates per ram) was diluted in four different extenders: ultra-heat-treated (UHT) long life milk, TRIS containing 10% (v/v) egg yolk (TRIS-EY), AH (pH 7.4), or TEST buffer containing 10% (v/v) egg yolk (TYB). Sorted and non-sorted (control) spermatozoa were stored at 15 degrees C for 24h or 5 degrees C for 6 days. Sperm characteristics were evaluated at 0, 6 and 24h for samples stored at 15 degrees C and daily for samples stored at 5 degrees C. The SMT was performed on sorted and non-sorted (control) spermatozoa after 6h and 3 days storage at 15 and 5 degrees C, respectively. Spermatozoa stored in TRIS were sorted more efficiently, had higher motility after sorting, freezing, thawing and incubation and had greater numbers of spermatozoa penetrating into the SMT than spermatozoa stored in AH prior to sorting. Spermatozoa stored in UHT at both temperatures had higher motility, acrosome integrity and traveled greater distances in the SMT than spermatozoa stored in all other diluents. In summary, storage in TRIS at 21 degrees C was optimal for transport of ram spermatozoa to the sorting site, and storage of spermatozoa in UHT diluent (after sorting) preserved sperm viability and migration ability best at both 15 and 5 degrees C.     

Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products

The growth and survival of pathogenic and non-pathogenic strains of Escherichia coli was determined in traditionally fermented pasteurized and unpasteurized milk and in Lacto, an industrially fermented milk. Each milk treatment was incubated at 20 degrees C for 24 h and then stored at either 20 degrees C or 5 degrees C for 96 h. Lacto inhibited all the three E. coli strains. Two strains could not be recovered and the third survived only in very low numbers after 24 h storage of Lacto at both 20 degrees C and 5 degrees C. All three E. coli strains survived and multiplied to maximum cell numbers in the range 10(7)-10(9)/ml during traditional fermentation of unpasteurized milk. Cell numbers decreased to 10(3)-10(6) and 10(2)-10(5) during storage of the fermented product at 20 degrees C and 5 degrees C respectively. Higher maximum numbers, 10(9)-10(10), of the three strains of E. coli were attained during traditional fermentation of pasteurized milk. The numbers decreased to 10(5)-10(8) and 10(4)-10(7) during storage of the fermented product at 20 degrees C and 5 degrees C respectively. Generally, fewer E. coli survived when the fermented milk products were stored at refrigeration temperature.     

Survival of honey bee (Hymenoptera: Apidae) spermatozoa stored at above-freezing temperatures

The development of practical techniques for the storage of honey bee, Apis mellifera L., semen would significantly improve our ability to breed for desirable genotypes and maintain genetic diversity in populations. Artificial insemination of queens has been possible for some time, but the semen used is usually freshly collected, or held for < 1 wk at room temperature. I examined the limitations of spermatozoal survival at nonfrozen temperatures. Pooled, diluted semen was stored in sealed capillary tubes at room temperature (25 degrees C) or in a refrigerator set to 12 degrees C, for periods up to 1 yr. Survival of spermatozoa was assayed by a dual fluorescent staining technique using SYBR-14 and propidium iodide stains, which readily distinguishes live and dead cells. No significant loss of viable spermatozoa occurred within the first 6 wk. Between weeks 6 and 9, the percent live spermatozoa fell from 80 to 58%, and remained at that level until after 39 wk. By week 52, samples at room temperature, but not at 12 degrees C, fell to 18.9% live spermatozoa. Nonfrozen storage of honey bee semen has potential for short-term preservation of germplasm, however several factors need to be studied further to optimize survival rates.