E.F. Graham

Some biochemical changes in spermatozoa due to freezing

Summary Several chemical analyses were conducted in an attempt to assess differences in extracellular medium before and after freezing semen of the bull, boar, and turkey. The objectives of the study were 1) to take advantage of some of the chemical components that are primarily associated with the cell, testing the leakage of these components into the extracellular medium, and 2) to demonstrate a few methods of how the analysis may be employed in selecting extenders, cryophylactic agents, and freezing rates that may improve the quality of spermatozoa after freezing. Data indicated that wide differences in some chemical constituents can be analyzed and taking advantage of these differences may be used as indicators of cell damage. Although limited data are reported, the analysis of GOT, LDH, and possibly acid phosphatase may be used as indicators of cell damage by their release into the extracellular medium. Other tests that are being investigated at the present time include lipids, protein, oxidases, and hyaluronidase. It is hoped that a sensitive test or a combination of tests may be developed to assess cellular damage due to freezing.

Some factors affecting loss of intracellular enzymes from spermatozoa.

Abstract Diluted semen from the bull, boar, and turkey was plunged into liquid nitrogen. After separation of the cells from the extracellular medium, the total releasable amounts of glutamic oxaloacetic transaminase (GOT), lactic dehydrogenase (LDH), cholinesterase, acid phosphatase, and alkaline phosphatase were determined. Turkey semen was low in enzyme content when compared to the bull and boar. Correlations between sperm cell concentration and enzyme concentration along with the amounts of enzyme in the extracellular medium after minimum and maximum damage indicated that GOT was present primarily in the cell. Therefore, of the five enzymes studied, GOT release into the extracellular medium was the best indicator of cell damage. Comparison of filtering semen through cellulose powder with discontinuous centrifugation to separate the cells from the extracellular medium was studied. Discontinuous gradient centrifugation resulted in significantly lower release of GOT from the cells in the boar and turkey than filtration. Thus discontinuous gradient centrifugation was the superior method for separating spermatozoa from the extracellular medium.

Studies on the absence of glycerol in unfrozen and frozen ram semen: Development of an extender for freezing: Effects of osmotic pressure, egg yolk levels, type of sugars, and the method of dilution☆

Abstract Four experiments were conducted to study the effects of: (1) osmolality (275 to 500 mOsm at 25-mOsm increments); (2) egg yolk levels (0 to 40% at 5% increases); (3) 10 sugars, 10% ( v v ); and (4) two different dilution methods (soon after collection at 37 °C or after cooling to 5 °C) on percentage of motility of spermatozoa before freezing and on frozen-thawed ram spermatozoa diluted in TEST [Tes ( N -tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) titrated with tris(hydroxymethyl)aminoethane to a pH of 7.0]. Glycerol was not used in any of the experiments. Before freezing, ram semen cooled to 5 °C, held for 3 h after collection, and then diluted with TEST buffer at 300–375 mOsm and 25–40% egg yolk ( v v ) had the best progressive motility. Overall, the presence of 10% of any sugar ( v v ) did not significantly affect the motility of spermatozoa before freezing. Maximum post-thaw motility was obtained when ram semen was diluted cold at 5 °C 3 h after collection in TEST buffer, pH 7.0, 375–400 mOsm, with 25–30% egg yolk ( v v ) and 10% maltose monohydrate ( v v ).

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.

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%).

Studies on the presence and absence of glycerol in unfrozen and frozen ram semen: Fertility trials and the effect of dilution methods on freezing ram semen in the absence of glycerol

Trials were conducted to study fertility of ram semen under various breeding conditions. In trial 1, 60 cycling ewes were randomly divided into five treatment groups of 12 ewes. Group 1 was bred naturally. Groups 2 through 5 were artificially inseminated with pooled, diluted semen: group 2 with fresh, diluted semen; group 3 with semen stored at 5 °C for 6 h; group 4 with semen containing 3% glycerol (vv) stored at 5 °C for 6 h; and group 5 with frozen-thawed glycerolated (3%) semen. Lambing rates were 83, 91, 83, 41, and 33%, respectively. Trial 2 studied the site of semen deposition and the effect of dialysis on frozen-thawed glycerolated ram semen fertility. Seventy-eight ewes were divided into three treatment groups. Two groups of 33 ewes each were inseminated in the cervix; group 1 with nondialyzed glycerolated thawed semen and group 2 with glycerolated thawed semen dialyzed (1:10) against TEST-yolk-glucose extender for 30 min at 5 °C. In group 3, 12 ewes received intrauterine inseminations of glycerolated frozen-thawed semen. Percentage lambing rates were 33, 48, and 67%, respectively. Dialysis of frozen-thawed semen did not improve lambing rate significantly (P > 0.05). However, intrauterine insemination of glycerolated frozen-thawed semen yielded a significantly higher lambing rate (P < 0.05). The effect of different dilution methods on survival of ram sperm frozen in the absence of glycerol is described. Ram semen diluted by the cold dilution method and frozen in the absence of glycerol was inseminated into 46 cycling ewes. A lambing rate of 52% was obtained.

Effect of filtration on post-thaw quality of bull semen

Semen from 4 Holstein bulls was diluted in 4 different extenders, filtered with Sephadex ion-exchange column, and frozen in liquid nitrogen. Sperm motility, progressive motility, path velocity, progressive velocity and the percentage of normal acrosomes of filtered and nonfiltered semen were recorded before and after freezing. Semen characteristics were significantly influenced by extender, filtration and freezing. Before and after freezing, motility measurements and the percentage of normal acrosomes were higher (P < 0.001) in filtered than in nonfiltered spermatozoa. Post-thaw recovery rate of motile spermatozoa was higher in filtered semen than nonfiltered (68 vs 39%, P < 0.0001). The reduction in motility, progressive motility and the percentage of normal acrosomes during freezing and thawing processes were significantly lower (P < 0.0001) in filtered semen (34, 34 and 4%, respectively) than nonfiltered (59, 54 and 15%, respectively). Post-thaw viability of spermatozoa was significantly affected by extender, filtration and time (P < 0.0001). Immediate (0 h) post-thaw motility of nonfiltered semen (29%) was similar to 4-h post-thaw motility of filtered semen (25%; P > 0.05). In conclusion, bull spermatozoa recovered by Sephadex ion-exchange filtration showed better post-thaw viability.

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)

Development of a buffer system for dialysis of bovine spermatozoa before freezing. III. Effect of different inorganic and organic salts on fresh and frozen-thawed semen

Three experiments were conducted to study the effect of inorganic and organic acids on survival of dialyzed bovine spermatozoa. Ejaculates were pooled, extended (1:10), dialyzed (1:50) for 2 h during cooling, and 1 h later they were frozen in pellets and stored in liquid nitrogen. The pellets were thawed in aluminum block depressions (preheated at 45 degrees C) and transferred to a test tube at room temperature as the last ice melted. Sperm motility was recorded in all samples before freezing and after thawing. The number of spermatozoa that passed through the Sephadex column was analyzed in all the postthaw samples. No statistical difference (P>0.05) was found between the use of potassium (KOH) or sodium hydroxide (NaOH) as titration bases. However, solutions containing calcium (Ca++) or magnesium (Mg++) provided significantly less (P<0.05) protection to the cells during freezing and thawing. No significant difference (P>0.05) was found in sperm survival of the postthaw samples when Ca++ or Mg++ were present. Inorganic salts of phosphates, carbonates or chloride provided significantly less protection to the cells than the control extenders with Na citrate (P<0.05). Results of the second experiment indicated that citrate, tartrate and oxalate salts provided superior (P<0.05) protection to the cells than salts of succinate, acetate or formate. It was concluded that an appropriate solution for use as a dialysate of extended bovine spermatozoa may be formulated as 30% (V/V) isosmotic Na salt of Piperazine-N-N-BIS (2-ethane sulfonic acid) (PIPES) plus 30% (V/V) isosmotic glucose plus 5% (V/V) glycerol plus 35% (V/V) of isosmotic solutions of Na or K citrate or tartrate, or a (1:1) combination of them.

Efficacy of the Hamilton Thorn Motility Analyzer (HTM-2030) for the evaluation of bovine semen.

Semen samples from four Holstein-Friesian bulls were evaluated by the Hamilton Thorn Motility Analyzer (HTM-2030) for sperm concentration, motility and other motion parameters. In the first trial, the extender preparation (P<0.005) and the program settings (P<0.001) of the motility analyzer significantly effected the accuracy of sperm concentration estimates. The students t-test revealed that setting the variables on the HTM-2030 Analyzer according to the dimensions and brightness of bull spermatozoa and the background of the extender was better than using the settings for bull semen as recommended in the manufacturers manual. In the second trial, different quantities of dead cells were added to semen samples to evaluate the accuracy of the HTM-2030 Analyzer for the estimation of percentage of motile cells and other motion characteristics. All motion parameter estimations except mean path velocity were similar for the settings studied. Addition of dead spermatozoa had a significant effect (P<0.0001) on all parameters of sperm movement. High correlation coefficients between the percentage of dead cells added and the decline in sperm motility verified the accuracy of this system. Use of the HTM-2030 system yielded simple, rapid and objective analysis of the studied spermatozoal parameters.