Previous experiments have established that filtration of bovine semen through a Sephadex ion-exchange column improves its quality before and after freezing. The present study was conducted to determine the post-thaw membrane integrity of bull spermatozoa separated with a Sephadex ion-exchange column and to determine the kind of protection to spermatozoa is provided by glycerol during freezing and thawing. Semen from Holstein bulls diluted in TEST-yolk extender (with and without glycerol) was filtered through a Sephadex ion-exchange column and frozen in liquid nitrogen (-196 degrees C). After thawing, there were more normal acrosomes in filtered spermatozoa than nonfiltered (P < 0.01). Post-thaw plasma membrane integrity and swelling ability in a hypoosmotic solution revealed that the filtered spermatozoa had a stronger (P < 0.005) plasma membranes than the nonfiltered. Filtered spermatozoa demonstrated higher zona-free hamster oocyte penetration than the nonfiltered (30.5 vs 11.5%; P < 0.0005). Spermatozoa extended in TEST-yolk without glycerol had the lowest (P < 0.001) normal acrosomes, intact plasma membranes and swelling ability. Plasma membrane over the post-acrosomal region of the head and post-midpiece region of the tail was more sensitive to damages caused by freezing and thawing than acrosomal and midpiece regions of spermatozoa. Glycerol in the extender provided significant (P < 0.05) protection to the sensitive regions of filtered and nonfiltered spermatozoa during freezing and thawing. Filtered plus glycerolated spermatozoa had the highest (P < 0.01) normal acrosomes, intact plasma membranes and swelling ability. In conclusion, the pre-freezing filtration of bovine semen harvested the spermatozoa possessing stronger plasma membranes which enabled them to endure freezing and thawing stresses. The addition of glycerol to the extender protected the post-acrosomal region of the head and post-midpiece region of the tail of spermatozoa from freezing and thawing shocks.

In this study, the role of sperm motility and acrosome integrity in filtration of bovine semen was investigated. In Experiment 1, the treatment of semen with formaldehyde, hyperosmotic buffer, heating and direct freezing immobilized the spermatozoa completely but their acrosomal status varied significantly (P < 0.01). The immotile spermatozoa, of any kind, did not pass through the Sephadex ion-exchange column at room temperature. In Experiment 2, semen samples possessing different percentages of immobilized spermatozoa (0, 50, 75 and 100%) were filtered through the Sephadex ion-exchange column. The immotile/dead spermatozoa were removed proportionately to their number in the semen by Sephadex ion-exchange column. The type and number of immotile spermatozoa in semen had no effect (P > 0.05) on the post-filtration recovery rate of motile spermatozoa. Filtered spermatozoa exhibited higher (P < 0.01) motility (> 90%), progressive motility (> 70%) and normal acrosomes (> 95%) than non-filtered spermatozoa. In conclusion, sperm motility seems to be more important than acrosome integrity for semen filtration, and the Sephadex ion-exchange column can remove the known quantities of different kinds dead/immotile spermatozoa.

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.

Semen from four Holstein bulls was diluted in TEST-yolk buffer (1:5 to 1:10) and then percolated through Sephadex ion-exchange filters at a rate of 1.5 ml min−1 using positive and negative pressures. Filters contained a fixed quantity of Sephadex (0.6 g), with a varied quantity of ion-exchanger, i.e. diethylaminoethane (DEAE) and carboxy-methyl (CM) celluloses (0, 0.125, 0.25 or 0.50 g). A filter containing only Sephadex gel was used as a control. Post-filtration quality of semen samples was ameliorated by all types of filter. The addition of ion-exchangers on a Sephadex column improved all the characteristics of spermatozoa involving motility and viability (P< 0.01), regardless of their quantity in the column (P>0.05). Post-filtration recovery rates of spermatozoa varied with different filters (P<0.01) and was highest with the control filter (82.5%). Motility (P<0.02) and progressive motility (P<0.02) were better in the filtrates obtained with negative pressure. A column composed of 0.6 g of Sephadex and 0.5 g of ion-exchangers with negative pressure was selected for use in further experiments. A Sephadex ion-exchange filter is effective in improving the quality of bovine semen.

Filter and semen related factors were studied to improve the post-filtration recovery rate of bovine spermatozoa using a Sephadex (0.6 g) ion-exchange (diethylaminoethane (DEAE) and carboxy-methyl (CM) celluloses, 0.25 g each) column. Mixing together the components of the column did not improve the recovery rate of spermatozoa compared with layering (P>0.05). A second post-filtration washing of the column with clean buffer improved the recovery rate by 8–9%, but the quality of spermatozoa was lower than after a first wash (P<0.01). Higher rates of filtration did not increase the number of spermatozoa recovered in the filtrates (P>0.05). The recovery rates of spermatozoa were 2.4%, 68.3% and 70.5% at 4°C, 22°C and 37°C (P<0.01) respectively. The quality of filtrate was significantly less at 37°C than at 22°C in terms of visual motility (P<0.025), progressive motility (P<0.0005), progressive velocity (P<0.025), linear index (P<0.0005), and percentages of live cells (P<0.0005) and normal acrosomes (P<0.025). Motility of spermatozoa seemed to be essential for successful filtration. An inverse relationship was found between the recovery rate and the quality of the semen or spermatozoa. It was concluded that a single wash of the column with extended semen at 22°C with a flow rate of 1.5 ml min−1 yielded the best semen quality with an optimum recovery rate of spermatozoa.

The effects of freezing and thawing rates, sample geometry and dilution methods on the preservation of ram spermatozoa in the absence of glycerol were studied. Percentage of motile spermatozoa was recorded for unfrozen (5oC at 4 and 24 hr) and frozen-thawed (22oC at 0 and 4 hr) samples. The highest post-thaw motility was obtained with 0.25cc straws frozen at 73.8oC/min and thawed at 49oC/min in a 37oC water bath. Cold diluted semen yielded higher motility (P<0.05) for both unfrozen and frozen spermatozoa.

The bent neck and bent tail spermatozoa increased with the increase of the concentration of glycerol, whereas the bent tail spermatozoa increased when glycerol was diluted. Glycerol addition and dilution had a more detrimental effect on the chicken sperm morphology than freezing and thawing.

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 student's 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 manufacturer's 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.

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 ).

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 ( v v ) 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.

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%).

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.

Electroejaculated semen from Santa Gertrudis bulls was used to study the effect of centrifugation (600 x g for 5 min) or dialysis [molecular weight cutoff <14,000 Daltons (Da)] on post-thaw motility and on fertility in beef cattle. Analysis of post-thaw motility showed that the main effects (bulls and semen treatment) were significant (P<0.05). Dialysis significantly improved post-thaw motility in four of seven bulls. Initial volume of seminal plasma in the ejaculate was negatively correlated to post-thaw motility (r = -0.73). No significant improvement in post-thaw motility was observed for bulls with high volumes of ejaculate. In a fertility trial, calving rates of heifers synchronized with PGF(2)alpha and inseminated at 72 and 96 h after the second PGF(2)alpha injection with dialyzed or commercial semen were not statistically different (P>0.05; 54.4% vs 55.4%). These results show that dialysis could be used to improve post-thaw motility of electroejaculated bull semen without altering its fertilizing capacity. However, a high initial volume of seminal plasma seems to have a deleterious effect on sperm freezability that cannot be reversed by dialysis.

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.

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.

The effect of N-TRIS(hydroxymethyl)methyl-2-aminoetane sulfonic acid (TES); N,N BIS (2 hydroxvethyl)-2 aminoethane sulfonic acid (BES), N-2(hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES), morpholinopropane sulfonic acid (MOPS), and piperazine-N-N-BIS(2-ethane sulfonic acid (PIPES) solutions on dialyzed semen was studied. Each was titrated to pH 7.0 with TRIS-(hydroxymethyl)-amino methane (TRIS) solution and the osmotic pressure was adjusted to between 320 to 325 mOsm/kg. The new solutions were identified as TEST, BEST, HEPEST, MOPST and PIPEST, respectively. The solutions were used 1) alone, 2) in a composite with equal parts (V/V) of each solution and 3) in a 1:1 (V/V) combination with isosmotic trisodium citrate solution. Later, TRIS and sodium hydroxide (NaOH) were compared as titration bases for piperazine-N-N-BIS (2-ethane sulfonic acid) (PIPES) and N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES). Ejaculates were diluted 1:10 (V/V) in extenders containing buffer, 20% egg yolk and 5% glycerol (V/V). The samples were dialyzed (1:50) during cooling for a period of 2 h. Each sample was dialyzed against the same buffer system containing 5% glycerol without egg yolk and later it was frozen in pellets. The treatments were evaluated by observation of sperm motility in fresh and thawed semen samples. The latter were also analyzed by electronic count of cells that passed through the Sephadex column. Sperm survival was higher in PIPEST (PIPES titrated with TRIS) or the composite buffer, and the inclusion of 50% sodium citrate (Na citrate) improved significantly (P<0.05) sperm motility in fresh and frozen-thawed semen samples. There was no difference (P>0.05) between the titration bases. In the second experiment, sperm survival was superior in extenders containing PIPEST (P<0.05) than in those containing TEST independently of the inclusion of Na citrate.

The ultrastructure of spermatozoa from the domestic tom cat and the Siberian tiger was studied. Semen was collected from anesthetized tom cats and Siberian tigers by electroejaculation. Spermatozoa were fixed and processed for examination by transmission electron microscopy. The principle differences between the spermatozoa from the two species were the head shape, mitochondrial organization in the neck area and structure of the fibrous sheath. Tom cat spermatozoa had an elongated oval-shaped head, while tiger spermatozoa had a more rounded head shape. Circularly oriented mitochondria in the neck area, near the proximal centriole, were frequently observed in tiger cells but rarely observed in tom cat cells. The semicircular ribs of tom cat spermatozoa were larger than the ribs of tiger spermatozoa. Also, the dense fibers (Numbers 3 and 8) of the corresponding microtubule doublets were fused or connected to the longitudinal columns in tiger spermatozoa but showed only occasional attachment in tom cat spermatozoa. These differences could influence results when the tom cat is used as a model for studying tiger semen.

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.

The tolerance of fathead minnow Pimephales promelas embryos to cooling depended upon developmental stage; late blastulae and gastrulae were more tolerant of cooling to 0–5°C from 23°C than were early blastulae. Survival of embryos or their ability to continue development was reduced as temperature declined to 0°C and length of exposure to cold temperatures increased. Embryos survived better after cooling if they were maintained in full-strength fish Ringerˈs solution than if they were kept in 10% fish Ringerˈs solution or in 10% fish Ringerˈs solution with 0.2 M glucose. Embryo survival was greatest when gastrulae were held in 100% fish Ringerˈs at 10°C, a temperature at which development was slowed but not stopped.