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Biomedical subjects

E F Graham

Publications and source records attributed to E F Graham.

At least 19 recordsLinked to original sources
PubMed1990

Thermographic studies of phantom and canine kidneys thawed by microwave radiation.

Whole organs, such as kidneys, must be thawed quickly and uniformly to prevent damage during thawing due to excessive heating. Electromagnetic heating with microwaves thaws the kidneys quickly but frequently produces "hot spots" with heat damage. To study heat damage, phantom gelatin kidneys with different dielectric constants and canine kidneys perfused with 12.5% glycerol, ethylene glycol, or dimethyl sulfoxide before freezing were microwave thawed, and the interior temperature was measured by thermography. Phantom kidneys were thawed free standing and canine kidneys were either free standing or packed in a gel mixture. Both phantom and canine kidneys were split symmetrically and separated with a sheet of Styrofoam to facilitate immediate separation and evaluation of the halves after thawing (approximately 3 sec). All the phantoms, regardless of dielectric properties, had areas less than 0 degrees C or greater than 37 degrees C after thawing. The free-standing canine kidneys and the gel-packed ethylene glycol-perfused kidneys had frozen areas (less than 0 degrees C) and hot spots (greater than 37 degrees C). However, glycerol- and dimethyl sulfoxide-perfused kidneys packed in gel before thawing had no areas less than 0 degrees C or greater than 37 degrees C. Altering the geometry from a "kidney shape" to a cylindrical shape with increased volume improved the uniformity of thawing and was more effective than altering the dielectric constant over the range evaluated.

Animals↗
PubMed1990

Effect of season on seminal traits and serum hormone concentrations in captive male Siberian tigers (Panthera tigris).

Electroejaculates and serum samples were collected throughout the year from 5 male Siberian tigers. Semen quality, seminal plasma chemistry and serum hormone concentrations were evaluated and analysed as a function of season. Semen volume, concentration, motility, viability and morphology were not influenced by season. The ability of spermatozoa to undergo capacitation and penetrate zona-free hamster eggs did not differ due to season. Season had no influence on seminal plasma concentrations of acid phosphatase, calcium, glucose, potassium and phosphorus. Serum values of thyroxine and triiodothyronine were lowest (P = 0.002 and P = 0.03, respectively) during the summer months while cortisol showed no seasonal variation. Serum testosterone concentrations were higher in fall and early winter (P less than 0.001) than at other times of the year but were not correlated with semen quality. These results demonstrate that season has no effect on the semen production and quality of captive male Siberian tigers.

Animals↗
PubMed1990

The effect of whole ejaculate filtration on the morphology and the fertility of bovine semen.

A Sephadex G-15 filtration method was developed to remove abnormal and nonmotile bull sperm from an entire ejaculate. The efficiency of filtration was determined by adding freeze-killed sperm to the ejaculate or using ejaculates with elevated numbers of abnormal cells induced by scrotal insulation. A fertility trial, using split ejaculates, compared fertility of filtered and unfiltered semen. After filtration, samples with 0, 25, and 50% killed cells added contained 77 to 81% motile cells. Addition of 75% killed sperm resulted in significant decrease (52%) of motile cells following filtration. Morphologic examination of semen with elevated numbers of abnormal cells revealed higher percentages of sperm with normal shaped heads and normal or swollen acrosomes after filtration than in unfiltered samples. Percentage of pear-shaped heads, lifted acrosomes, and bent tails decreased after filtration. Six high fertility and six low fertility bulls were used to evaluate fertility of filtered semen. Filtering increased motile sperm from 51 to 57% and from 36 to 50% for the high and low fertility bulls, respectively. The 60- to 90-d nonreturn rates for high fertility bulls were not increased by filtering (73 vs. 72%). However, filtering significantly improved the nonreturn rates for the low fertility bulls (61 vs. 67%).

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PubMed1990

Cryopreservation of semen from unique lines of chicken germ plasm.

Frozen semen is a practical means of preserving valuable germ plasm. Monitored samples of semen cryopreserved with glycerol for heterozygous, dominant marker stocks and for nine chromosomal rearrangement lines had sufficiently high fertility for germ-line retrieval. The results also indicated a potential for the genetic selection of certain lines for the freezability of spermatozoa, since stock and line differences in fertility occurred when previously frozen semen was used for insemination. Freezing the semen of stocks routinely reproduced provides insurance against possible disasters.

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PubMed1989

Estimation of the relative fertilizing ability of frozen chicken spermatozoa using a heterospermic competition method.

Semen was collected from Rhode Island Red and White Leghorn roosters. After adjusting the sperm number from both breeds, half of the semen was frozen-thawed in the presence of glycerol. Frozen and unfrozen semen from both breeds was mixed 1:1 with all four possible combinations and inseminated into Rhode Island Red hens. Feather colour of chicks was used to determine which breed fertilized the eggs. Results showed that sperm cells retained 19.7% [95% confidence interval = (12.8, 30.4)] of the relative fertilizing ability after freezing. Furthermore, Rhode Island Red spermatozoa had 1.5 times [95% confidence interval = (1.1, 2.0)] higher relative fertilizing ability than did White Leghorn spermatozoa. The heterospermic competition assay method is a powerful tool for estimating the relative fertilizing ability of the sperm cells.

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PubMed1989

In-vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris).

Electroejaculates from 5 tigers were split and half of each was assayed fresh while the remainder was frozen and thawed before being assayed. Preincubation time, temperature and removal of seminal plasma were evaluated for their effect on in-vitro capacitation. Ability of spermatozoa to penetrate oocytes, as measured by the zona-free hamster egg-sperm penetration assay (SPA), was used as verification of capacitation. Results of the experiments with fresh semen indicate that: (1) preincubation time affects the fertilizability of tiger spermatozoa with 2 h appearing optimal, (2) a preincubation temperature of 37 degrees C results in significantly higher penetration rates than does a 22 degrees C treatment, and (3) tiger seminal plasma does not appear to contain decapacitation factors, as has been reported for several other species. Frozen semen experiments indicate that (1) frozen-thawed tiger spermatozoa must be removed from the environment of the semen extender before capacitation can take place, and (2) the freeze-thaw procedure results in a shortening of the required capacitation time.

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PubMed1989

Identification of the capacitating agent for bovine sperm in egg yolk-TEST semen extender.

Bovine sperm can be capacitated in egg yolk-TEST buffer. To determine what constituent of the buffer was responsible, ejaculated semen was diluted 1:10 at 37 degrees C with the following 20% egg yolk (vol/vol)-containing buffers: TES-Tris, TES-tetramethylammonium hydroxide, taurine-Tris, citric acid-Tris, citrate, egg yolk salts, egg yolk proteins Tris, and citrate-taurine. Buffers were pH 7.6 and 321 to 325 mOsmol/kg. Extended semen was cooled slowly to 4 degrees C and stored 8 h. Sperm taken at 0 and 8 h were washed in pH 7.6 bovine serum albumin-saline and assessed for motility and capacitation using zona-free hamster eggs. Sperm motilities at 0 and 8 h were similar (60 to 73%) in all extenders except citric acid-Tris (54%) and egg yolk proteins Tris (15%). Bull sperm, stored 8 h in egg yolk-TEST, became capacitated. Because sperm storage in egg yolk-citrate did not result in penetration, both egg yolk and citrate were ruled out as capacitating agents. Capacitating activity resided in the TES and Tris molecules. The TES molecule contains a Tris component and this capacitated bull sperm. The TES molecule also contains a taurine component. However, taurine was not a capacitating agent for bull sperm. In conclusion, both TES- and Tris-containing buffers, alone or together (TEST), were equally effective in capacitating bull sperm.

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PubMed1987

Comparative ultrastructure of the zona radiata from eggs of six species of salmonids.

The zona radiata from unactivated and activated eggs from chinook salmon (Oncorhynchus tshawytscha), chum salmon (O. kisutch), pink salmon (O. gorbuscha), brown trout (Salmo trutta), rainbow trout (S. gairdneri) and lake trout (Salvelinus namaycush) were examined using scanning and transmission microscopy. The zona radiata in all species examined consisted of an outer adhesive coating, a thin densely staining zona radiata externa with pore canal plugs and a thick, fibrous zona radiata interna with a fibrous network on the inner surface. There was a two-layer adhesive coating over the zona radiata externa in all species except pink salmon in which only one layer was observed. There were structural differences among species in the adhesive layer, zona radiata externa and plugs in the pore-canal openings.

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PubMed1987

Effects of low-molecular-weight fractions (LMWF) from milk, egg yolk, and seminal plasma on freezability of bovine spermatozoa.

The effects of the dialyzable fractions from bovine seminal plasma, egg yolk, and milk and of two buffer systems (TEST and sodium citrate) on post-thaw sperm motility were studied. Each basic salt solution was used in the experimental design. These solutions were used as extender systems in combination with egg yolk and glycerol. After collection, semen samples were extended (1:20), cooled to 5 degrees C in 1.5 hr, and frozen in 0.5-cc French straws after 3 hr of equilibration. Post-thaw samples were assayed for percentage of motile cells immediately after thawing and after 4 hr of incubation at room temperature (22 degrees C). Egg yolk (25%) provided the same protection as did the combination of colloidal material present in the skim milk-yolk extenders. The use of TEST as a buffer provided significantly higher (P less than 0.01) sperm post-thaw motility than milk salts or Na citrate. Sperm survival in extenders containing high concentrations of seminal plasma and/or egg yolk salts was significantly lower (P less than 0.01). Spermatozoa frozen in the presence of 6% glycerol resulted in sperm motility significantly (P less than 0.05) higher than that of spermatozoa frozen with 3% glycerol. However, no difference was observed between these two concentrations when TEST solution was used.

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PubMed1987

Factors affecting the removal of low-molecular-weight fractions (LMWF) from egg yolk and seminal plasma in extended semen by dialysis: effect on post-thaw sperm survival.

Three factors affecting dialysis of bovine semen were studied. These factors were (1) dialysis rates of egg yolk, seminal plasma, and glycerol, (2) temperature (37 degrees C, 5 degrees C, and while cooling from 37 to 5 degrees C), and (3) dialysis ratios between retentate and dialysate (1:1, 1:10, 1:20, 1:50, and 1:100). Ninety percent of the low-molecular-weight fraction (LMWF) from seminal plasma, egg yolk, and glycerol was removed from the retentate in a 2-hr period at 5 degrees C, and only slight changes were detected after the third hour of dialysis. Temperature affected dialysis and was faster at 37 degrees C. It was also found that a 1:20 dialysis ratio was sufficient to obtain 90% clearance of the LMWF. The effect of sperm dilution ratio, dialysis ratio, and exchange of the LMWF from egg yolk and/or seminal plasma for buffer systems was also studied. An improvement in post-thaw motility of spermatozoa (P less than 0.05) was obtained when the LMWF from both seminal plasma and egg yolk were replaced. A third experiment was conducted to study the effect of different combinations between the buffer systems, TEST and Na citrate, in the dialysate. The results indicated that a 1:1 combination of iso-osmotic solutions (320-325 mOsm/Kg, pH 7.0) between these two buffers, with 5% glycerol (v/v), yielded significant (P less than 0.05) sperm post-thaw motility as compared with the individual use of TEST-glycerol or Na citrate-glycerol. Dialyzed samples also yielded sperm post-thaw motility higher than that of the nondialyzed samples. Colloidal materials in the dialysate did not affect survival of spermatozoa.

Animals↗
PubMed1987

Dialysis of bovine semen and its effect on fresh and freeze-thawed spermatozoa.

The effect of the removal of the low-molecular-weight fraction (LMWF, less than 12,000-14,000 Da) from the seminal plasma present in extended semen by dialysis and by centrifugation (1,376g for 20 min at 5 degrees C) were compared with the current methods of freezing bovine semen. Significantly higher sperm post-thaw motility (P less than 0.05) was obtained in the dialyzed samples than with the other two methods. The appropriate time and temperature for dialysis of semen was also studied. Semen aliquots were dialyzed (1:50, retentate:dialysate) for 30 min, 1 or 2 hr at 5 degrees C, and during the cooling process from 37 to 5 degrees C over a 2-hr period. Superior sperm motility (P less than 0.05) in prefreeze and post-thawed samples was observed when semen was dialyzed for 1 or 2 hr during the cooling process as compared with that of semen dialyzed at 5 degrees C. A third experiment was conducted to establish the effect of the use of dialysis bags of different molecular weight cutoffs (MWCO) on sperm motility. Semen samples were dialyzed (1:50) during the cooling process in dialysis bags of 1,000, 3,500, 6,000-8,000, 12,000-14,000, 25,000, and 50,000 MWCO. No statistical differences (P greater than 0.05) in sperm post-thaw motility were found after evaluation of the number of cells that passed through the Sephadex filter and all the dialyzed values obtained were significantly (P less than 0.05) superior to the results obtained with no dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1986

The effect of dialysis of extended ram semen prior to freezing on post-thaw survival and fertility.

The effect of dialysis on extended ram semen prior to cryopreservation was studied. Techniques were developed to improve post-thaw recovery of dialyzed semen and a fertility trial was used to evaluate the viability of dialyzed and frozen semen. Dialysis prior to freezing was shown to increase post-thaw recovery of motile cells and percentage of cells passing through a Sephadex filter. Freezing semen in pellets on dry ice was superior to freezing in French straws. Pellets were thawed in an aluminum thaw block at 42 to 45 degrees C before insemination of progestagen-PMSG synchronized ewes. Double inseminations were made at 12-hr intervals. Natural service of synchronized ewes was also made at 12-hr intervals as a control. There was no significant difference (P greater than 0.05) in fertility between naturally serviced ewes (44.4%) and ewes inseminated with frozen semen (44.7%).

Animals↗
PubMed1986

The effects of nonpenetrating cryoprotectants added to TEST-yolk-glycerol extender on the post-thaw motility of ram spermatozoa.

The effects of adding five different concentrations of 17 polymeric compounds to TEST-yolk-glycerol extender on ram spermatozoa survival was studied. These were Aquacide (I, II, and III); dextran (0.8-1.6, 1.9, 15-20, 70, and 200-300 kDa); three types of Dri-Sweet; hydroxyethyl starch; methylcellulose, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, and Supercol 912. All the compounds tested except the Dri-Sweet compounds and hydroxyethyl starch significantly (P less than 0.05) decreased percentages of motile cells in unfrozen samples. The use of dextran (0.8-1.6 kDa; hydrolyzed dextran separated by ethanol) and Aquacide II significantly (P less than 0.05) increased post-thaw motility of spermatozoa frozen in pellets. Dextran (15-20 kDa), dextran (0.8-1.6 kDa), Aquacide II, and hydroxyethyl starch significantly (P less than 0.05) increased the percentages of post-thaw motility of ram spermatozoa frozen in the presence of glycerol and egg yolk.

Animals↗
PubMed1983

Effects of various degrees of supercooling and nucleation temperatures on fertility of frozen turkey spermatozoa.

The relative roles of the degree of supercooling and nucleation temperatures on turkey sperm cell survival and fertilizing capacity during a freeze-thaw cycle were investigated. Basically, the higher the degree of supercooling, which produced lower spontaneous nucleation temperatures during the freezing process, the worse the sperm cells survived, in the present study. When induced nucleation was applied, which eliminated the high degree of supercooling, an improvement in sperm cell survival was noted. During the fertility trial, it was shown that in treatment 3 (induced nucleation) the fertility was higher (P less than 0.05) than in treatment 2 (spontaneous nucleation). In general, the degree of supercooling prior to freezing is an important variable and should be considered very seriously during the overall freezing process.

Animals↗
PubMed1983

Alteration of seminal proteins during freeze-drying of bovine semen.

Bovine semen in TEST-yolk extender was frozen, freeze-dried to 50, 25, 12, 6, and less than 2% residual moisture, and stored at -196 degrees C. The freeze-dried semen was rehydrated, sampled for protein analysis, and used to inseminate cattle. Agar gel electrophoresis revealed no moisture was reduced to less than 6%. At that point, the percent of cationic proteins and neural proteins decreased with a concurrent increase in anionic migrating proteins. Immunodiffusion data with antisera against spermatozoa and seminal plasma revealed no difference in formation of precipitin lines if residual moisture was at least 6%. However, semen freeze-dried to 2% residual moisture was modified antigenically as certain precipitin lines were lost, new lines appeared, and concentration of other seminal antigens decreased. The absence of fertility with semen freeze-dried to 2% residual moisture is hypothesized to 2% residual moisture is hypothesized to be from alteration of the tertiary structure of certain essential seminal proteins.

Animals↗
PubMed1982

Development of extender and techniques for frozen turkey semen. 1. Development.

An extender for turkey semen with a frozen-thawed recovery of greater than or equal to 50% motile spermatozoa and a vigorous swirl was developed. The effect of glucose, a comparison of 10 different sugars in the extender, the use of sodium acetate and potassium acetate, the ratio of dimethylsulfoxide to ethylene glycol, the percent total cryoprotectant, equilibration time, and osmotic pressure were all tested. Replacement of approximately half of the extender with an iso-osmolar glucose solution yielded higher percentages of motile spermatozoa with both fresh and frozen-thawed semen samples. Replacement of glucose with other carbohydrates did not enhance recovery of frozen-thawed semen. The proportion of sodium and potassium acetates to TESNaK2 in the extender had no effect on motility. Approximately equal proportions of ethylene glycol to dimethylsulfoxide, with a final concentration of 11.2% after dilution, produced at or near optimal recovery of spermatozoal motility after freezing. Twenty to 90 min equilibration times before freezing yielded significantly higher recovery of motile sperm postthaw than shorter periods. A wide range of extender osmotic pressures were compatible with recovery of spermatozoal motility postthaw. The final extender consisted of 4.70 g sodium acetate, 3.39 g potassium acetate 9.23 g TES, .353 g sodium hydroxide, .492 g potassium hydroxide, 32.00 g glucose, H2O q.s. 1000 ml, 370 mOsm/kg, pH 7.2. Ethylene glycol and dimethylsulfoxide were added (1:1 ratio) for a final concentration of 11.2% cryoprotectant after dilution. Semen was held undiluted for 6 min, extended 1:4 at 22 C, and equilibrated for 30 min at 0 C before pellet freezing.

Animals↗
PubMed1982

Development of extender and techniques for frozen turkey semen. 2. Fertility trials.

The effects of cryoprotectant concentrations, redilution, centrifugation, and dialysis techniques on fertility of fresh and frozen-thawed turkey semen were tested. Fresh extended semen with final concentrations of 4.8, 5.6, 9.6, and 11.2% cryoprotectant (dimethylsulfoxide and ethylene glycol, 1:1 ratio) showed a significant (P less than .05) decrease in fertility when compared to semen without cryoprotectant. Fertility of fresh semen extended with 4.8% cryoprotectant when centrifuged or rediluted and centrifuged, was unaffected when compared to cryoprotectant treated semen. Fertility of frozen-thawed semen after redilution and centrifugation was low, but samples rediluted one part semen for two parts extender yielded significantly (P less than .05) higher fertility than semen rediluted 1:1. Fresh semen extended with 11.2% cryoprotectant showed a highly significant (P less than .01) decrease in fertility despite dialysis, while semen with 9.6% cryoprotectant showed no difference when compared to dialyzed semen without cryoprotectant. Fertility of frozen-thawed semen dialyzed against blood plasma (approximately pH 7.9) and blood plasma adjusted with TES2 to pH 7.4 or pH 7.2 was not significantly different. Fertility of frozen-thawed semen dialyzed for 30 min was significantly higher (P less than .01) than semen dialyzed for 15 min or 0 min. Frozen-thawed semen dialyzed with a semen to dialysate ratio of 1:15 and 1:24 supported significantly higher (P less than .01) fertility than at 1:50 ratio. The AGPT3 dialysate yielded significantly higher (P less than .01) fertility than blood plasma adjusted to pH 7.2 with TES.

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