Lactose-egg yolk diluent supplemented with N-acetyl-D-glucosamine affect acrosome morphology and motility of frozen-thawed boar sperm

These experiments were carried out to investigate the effect of N-acetyl-D-glucosamine, and to obtain additional information about the effect of orvus es paste (OEP) and egg yolk concentration in the freezing of boar sperm in the maxi-straw. The highest post-thaw acrosomes of normal apical ridge (NAR) and motility were obtained with 0.025 or 0.05% N-acetyl-D-glucosamine concentration in the first diluent. However, there were no effects of N-acetyl-D-glucosamine among the diluents with or without N-acetyl-D-glucosamine at the second dilution. The N-acetyl-D-glucosamine in the first and second diluents was added at room temperatures (20-23 degrees C) and 5 degrees C, respectively. It is suggested that the temperature of N-acetyl-D-glucosamine addition is important for the effect of boar sperm protection during freezing and thawing. When the 0.05% N-acetyl-D-glucosamine was supplemented in the first diluent, the optimum final OEP content was 0.5%. The optimum content of egg yolk in the diluent with 0.05% N-acetyl-D-glucosamine concentration was 20% and egg yolk was one of the main cryoprotective agents. In conclusion, we found out that the diluent with 0.025 or 0.05% soluble N-acetyl-D-glucosamine in the first diluent, 0.5% final orvus es paste concentration and 20% egg yolk concentration significantly enhanced NAR acrosomes and motility of boar sperm after freezing and thawing.     

Effect of egg yolk, cryoprotectant, and various sugars on semen cryopreservation in endangered Cuvier's gazelle (Gazella cuvieri)

Cryopreservation of spermatozoa from endangered species is a valuable tool for genetic management. Previous studies showed the feasibility of cryopreservation of spermatozoa from various endangered gazelles but have also revealed difficulties with available protocols for semen freezing in Cuvier's gazelle (Gazella cuvieri). Experiments were carried out to investigate the effect of (a) 5% or 20% egg yolk or 4% or 6% glycerol, and (b) addition of sugars (glucose, fructose, lactose and raffinose) on cryopreservation using a Tes-Tris-based diluent (TEST). A diluent containing 13.5% raffinose, 5% or 20% egg yolk, and 6% glycerol (REYG) was also evaluated. Semen was obtained by electroejaculation from 22 G. cuvieri males. Diluted samples were loaded into 0.25 ml straws, cooled to 5 °C over 1.5 h (−0.16 °C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapours for 10 min and plunged into liquid nitrogen. Subsamples were assessed for motility and acrosome integrity upon collection, after refrigeration–equilibration, after freezing and thawing, and 2 h after thawing. Use of TEST with 20% egg yolk or with 4% glycerol led to worse motility preservation, whereas TEST with 5% egg yolk and 6% glycerol led to better results. Addition of fructose, lactose or raffinose to TEST resulted in similar or worse preservation of motility than inclusion of glucose. On the other hand, use of a raffinose-based medium with 20% egg yolk and 6% glycerol (REYG) afforded better preservation of motility than use of TEST. With REYG, 20% egg yolk was better than 5% egg yolk for motility preservation. Differences were noted between males in their responses to cryopreservation when using different egg yolk or glycerol concentrations. Moreover, spermatozoa from most males exhibited better cryopreservation with REYG although some were better cryopreserved in TEST. The raffinose-based diluent thus represents an improvement over previous results but more work is needed to better characterize cryopreservation conditions for future routine banking of Cuvier's gazelle spermatozoa.     

Effect of lactose and glycerol on the motility, normal apical ridge, chromatin condensation and chromatin stability of frozen boar spermatozoa

The effect of lactose and glycerol concentration, as well as the equilibration time with glycerol was studied on motility, normal apical ridge (NAR), and chromatin state of boar spermatozoa after the freezing and thawing process. In the first experiment, samples were frozen in first and second extenders containing different concentrations of lactose (11, 12 and 14%). In the second experiment samples were frozen using second extenders with different concentrations of glycerol (4, 6, 8 and 10%) and were incubated at 5 degrees C for 0 and 30 min. Motility, motility after caffeine treatment, NAR, chromatin condensation and stability (susceptibility to de-condense after heparin treatment) were evaluated. The results indicated that freezing spermatozoa in extenders with increasing concentrations of lactose adversely affected motility but provided a protective effect on acrosomes. Increased lactose concentration induced higher chromatin condensation but maintained the same stability. Increasing the glycerol concentration in the second extender from 4-6 to 8% led to higher motility and NAR as well as lower chromatin condensation and stability. When 30 min equilibration time was allowed after dilution with the same extenders, spermatozoa showed higher NAR and lower chromatin condensation and stability. The longer equilibration time was detrimental for motility when freezing in the 8% glycerol extender but favourable when using the 4% glycerol extender. Compared to the 8% glycerol, spermatozoa frozen in the 10% glycerol extender showed similar motility and increased chromatin condensation and stability, as well as low values of NAR that did not improve by longer incubation time.     

Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa

Four experiments were conducted to investigate the effects of egg yolk during the freezing step of cryopreservation (namely, the process except for the cooling step), on the viability of goat spermatozoa. The effects of egg yolk on sperm motility and acrosome integrity during the freezing step were investigated in Experiment 1. Spermatozoa diluted with Tris-citric acid-glucose (TCG) solution containing 20% (v/v) egg yolk were cooled to 5 degrees C, washed, and then frozen in TCG with egg yolk (TCG-Y), TCG without egg yolk (TGG-NY), 0.370 M trehalose with egg yolk (TH-Y), or trehalose without egg yolk (TH-NY). All extenders contained glycerol. In frozen-thawed spermatozoa, the inclusion of egg yolk in the freezing extenders increased (P<0.05) percentages of motile sperm, progressively motile sperm, and the recovery rate (ratio of post-thaw to pre-freeze values), but decreased (P<0.05) acrosomal integrity. Moreover, extenders with trehalose had better (P<0.05) post-thaw sperm viability. In Experiment 2, the effects of egg yolk on acrosome status before and after freezing were studied. Egg yolk significantly decreased the proportion of intact acrosomes before freezing, leading to fewer (P<0.05) intact acrosomes post-thaw and lower (P<0.05) recovery rates for intact acrosomes. In Experiment 3, including sodium dodecyl sulfate (SDS) in a diluent containing egg yolk tended to preserve the acrosome compared with the egg yolk containing diluent free of SDS, however, spermatozoa had a lower (P<0.05) proportion of intact acrosomes than those in a yolk-free diluent. However, after cooling, spermatozoa were diluted with a glycerolated extender containing egg yolk. Therefore, the objective of Experiment 4 was to explore whether the egg yolk or glycerol was responsible for the reduced intact acrosome percentage. In this experiment, after cooling and washing the spermatozoa were diluted in TCG with glycerol and/or egg yolk. The combination of glycerol and egg yolk in the extender reduced (P<0.05) the proportion of intact acrosomes compared with egg yolk or glycerol alone. In conclusion, the inclusion of egg yolk significantly improved sperm motility, indicating its beneficial effects during the freezing step of cryopreservation; trehalose appeared to synergistically increase its cryoprotective effects. Furthermore, although neither glycerol nor egg yolk per se affected the proportion of intact acrosomes, the combination of the two significantly reduced the proportion of acrosome-intact spermatozoa.     

Semen preservation in Macaca fascicularis.

Semen was collected from adult male Macaca fascicularis using a rectal probe for electro-ejaculation. The effect on sperm motility of varying semen extender egg yolk concentration, pH, glycerol concentration, and equilibration times of sperm with glycerol was examined. No significant difference was observed between motilities at extender egg yolk concentrations of 10% to 40%. Progressive motility was significantly greater at pH 7.2 and 8.0 than at 5.8, 6.5, and 8.7 (p less than 0.05). Glycerol concentrations of 7% and 10% yielded optimum progressive motility after freezing. A 1-minute equilibration of semen in extender containing glycerol resulted in greater sperm motility after freezing than did equilibration for 25 or 45 minutes.     

Effects of egg yolk, glycerol and the freezing rate on the viability and acrosomal structures of frozen ram spermatozoa.

The influence of egg yolk, glycerol and the freezing rate on the survival of ram spermatozoa and on the structure of their acrosomes after freezing was investigated. Egg yolk was shown to be beneficial not only during chilling but also during freezing; of the levels examined, 1-5% gave the greatest protection. Although the presence of glycerol in the diluent improved the survival of spermatozoa, increasing concentrations produced significant deterioration of the acrosomes. With closely controlled linear cooling rates, no overall difference was detected in the survival of spermatozoa frozen at rates between 6 and 24 degrees C per min. However, a significant interaction between freezing rate and the inclusion of glycerol in the diluent showed that glycerol was less important at the highest freezing rate. A sudden cooling phase near to the freezing point following the release of the latent heat of fusion was not detrimental to spermatozoa.     

Fertilizing potential of mouse spermatozoa cryopreserved in a medium containing whole eggs

Experiments were conducted to improve survival of mouse spermatozoa through the cryopreservation process. In the first experiment, percentages of motile spermatozoa and fertilizing capacities of spermatozoa were evaluated when mouse spermatozoa were cryopreserved using three previously reported cryopreservation media: (1) 18% raffinose in 3% skim milk; (2) Tes/Tris medium containing 25% egg yolk and 1.25% glycerol; and (3) PBS containing 18% raffinose and 1.75% glycerol, each at three different cooling rates (-3, -10, and -50 degrees C/min). Spermatozoa frozen in the skim milk/raffinose medium exhibited the highest percentage of motile spermatozoa (39%) when cells were frozen at -10 degrees C/min (P<0.05). The second experiment evaluated the effects of modifying the Tes/Tris/egg yolk medium, comparing different concentrations of egg yolk, BSA, and sodium dodecyl sulfate. Reducing egg yolk from 25% of the medium volume to 5%, increased percentages of motile spermatozoa after cryopreservation from 29 to 36% (P<0.05). Addition of 1% BSA and sodium dodecyl sulfate to medium containing 5% egg yolk further improved percentages of motile spermatozoa after freezing. In the final experiment, 20% whole egg was substituted for 5% egg yolk and 1% BSA used in previous experiments and resulted in percentages of motile spermatozoa (51%) equal to that of the skim milk-raffinose medium. However, fertility rates were higher (68%) than for spermatozoa frozen in the skim milk-raffinose medium (P < 0.05) and were comparable to the fertility rates of fresh spermatozoa (77%; P>0.05). In conclusion, freezing mouse spermatozoa in a medium containing 20% whole egg, 0.035% sodium dodecyl sulfate, and 1.25% glycerol using a cooling rate of -10 degrees C/min preserves the motility and fertilization capacity of mouse spermatozoa.     

Cryopreservation of epididymal sperm collected postmortem in the Tasmanian devil (Sarcophilus harrisii)

Effective sperm cryopreservation protocols are limited to a small number of marsupial species. In this study, postmortem gamete rescue (PMGR) epididymal sperm samples from Tasmanian devils (N = 34) euthanized due to the fatal Devil Facial Tumor Disease were used to develop long-term sperm storage techniques for the species. Cryoprotectant toxicity associated with equilibration of sperm samples in a TEST yolk diluent (TEST; 189 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 85 mM Trizma base [Tris], 11 mM glucose, 20% vol/vol egg yolk; pH 7.1, and 315.0 ± 5.0 mOsm/kg) with a final concentration of 0.06 M trehalose, or 4%, 10%, and 18% vol/vol of either glycerol or dimethyl sulfoxide (DMSO), was examined over 12 h at 15 °C. Trehalose supplementation resulted in an immediate decline (P < 0.05) of total motility. After 12 h, total motility was reduced (P < 0.05) in treatments containing 18% glycerol, and 10% and 18% dimethyl sulfoxide. The effects of final glycerol concentration (4% and 10%), glycerol equilibration duration (10 min 1 h, or 3 h) prefreeze, freezing rate and the addition of 0.10 M lactose or a combination of 0.10 M lactose and 0.11 M raffinose were assessed during three experiments on the cryopreservation of postmortem gamete rescue samples in TEST. In all experiments, motility and viability were reduced (P < 0.01 postthaw). Samples cryopreserved in TEST supplemented with lactose or lactose with raffinose using a fast freezing rate (−8 °C/min from 4 to −40 °C, then −65 °C/min until −165 °C) produced the highest (P < 0.05) postthaw motility (18.6 ± 5.5% and 16.9 ± 8.5%, respectively), which represented 35% to 48% retention of prefreeze motility. These results apparently were the best postthaw results of dasyurid sperm reported to date and will help lay the foundations for developing assisted reproductive technologies for marsupial species.     

Examination of some processing methods for freezing boar semen.

Five experiments were conducted to examine the effect of processing methods and diluents on survival and morphology of boar spermatozoa after freezing. Post-thawing survival of spermatozoa was better for Beltsville-F3 (BF3) than for tris-fructose-EDTA freezing diluent when the seminal plasma and glycerol were removed prior to freezing (method A). Both freezing diluents yielded similar viability results when the spermatozoa were frozen in the presence of siminal plasma and glycerol (method B). Viability of spermatozoa after thawing was better when glycerol concentration in the prefreezing diluent (method A) or in the freezing medium (method B) was 2-5 and 5-0 rather than 7-5%. Cooling of diluted semen to 5 degrees C beyond 4 h decreased the post-thawing survival of spermatozoa. The proportion of spermatozoa with undamaged acrosomes after processing and thawing by different methods was indistinguishable and relatively low. When the semen was frozen at cell concentrations ranging from 0-25 to 2-0 X 10(9)/ml, the viability of spermatozoa declined with increasing concentration following freezing in BF3, and S-1 diluents. Viability results were very similar for all cell concentrations examined when tris-fructose-EDTA diluent was used, indicating the possibility of freezing boar semen in a concentrated state.     

Effects of egg yolk and glycerol levels in lactose-EDTA-egg yolk extender and of freezing rate on the motility of frozen-thawed stallion spermatozoa

Two experiments were designed to evaluate the effects of egg yolk and glycerol concentrations, freezing rate, and clarification of a lactose-EDTA-egg yolk extender on the post-thaw motility of stallion spermatozoa. In both experiments there was no influence of freezing rate (vapor vs controlled) on the percentage of progressively motile spermatozoa after thawing. Furthermore, no significant interaction among treatments was detected. In Experiment 1, clarified (centrifuged at 34,400 × g for 30 min) lactose-EDTA-egg yolk extenders containing 16 or 20% egg yolk and 3 or 4% glycerol were superior to those containing 12% egg yolk or 2% glycerol, based on the percentage of progressively motile stallion spermatozoa at 0, 30, 60, and 90 min after thawing. However, in Experiment 2, clarification of the lactose-EDTA-egg yolk extender was detrimental to the ability of the stallion spermatozoa to survive after thawing; 4% glycerol was superior to 2% glycerol. The best extender based on the percentage of progressively motile spermatozoa after thawing was nonclarified lactose-EDTA-egg yolk extender containing 20% egg yolk and 4% glycerol.     

Effects of the supplementation of trehalose extender containing egg yolk with sodium dodecyl sulfate on the freezability of goat spermatozoa

Two experiments were conducted to determine the effect of sodium dodecyl sulfate (SDS) added to a trehalose-egg yolk extender on the cryopreservation of goat spermatozoa. In Experiment 1, semen from four goats was frozen in trehalose extender (osmolality = 370, pH = 7) containing 4 and 20% (v/v) glycerol and egg yolk, respectively, and 0.035-0.2% SDS. After thawing, sperm motility and acrosome integrity were assessed using a computer-assisted sperm analysis (CASA) system and fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Both motility and progressive motility were improved (P < 0.05) by increasing the concentration of SDS in the trehalose-egg yolk extender, with the best results obtained with SDS at 0.1% (80.0 +/- 1.5% and 65.0 +/- 1.7%, respectively). There were no significant differences in path velocity when spermatozoa were frozen in a diluent containing 0.035, 0.05, 0.075, or 0.1% SDS, but path velocity decreased significantly with 0.2% SDS. The percentage of acrosome-intact sperm were highest (P < 0.05) when 0.05% (74.0 +/- 1.1) and 0.075% (70.0 +/- 1.2) SDS were used. In Experiment 2, the effect of diluent storage time (6, 24, or 48 h) before freezing on the cryoprotective effect of SDS was investigated. Prolonged storage of the diluent had slight cryoprotective effects when 0.2% SDS is used, while motility and the acrosome integrity of the cryopreserved spermatozoa improved slightly when the extender was stored for 48 h at 5 degrees C before use. In conclusion, goat sperm freezability was significantly improved when sperm were frozen in a trehalose-egg yolk extender containing an adequate concentration of SDS.     

Effect of egg yolk concentration on cryopreserving Spanish ibex (Capra pyrenaica) epididymal spermatozoa

Tris-egg yolk based diluents provide adequate cryoprotection for the sperm of most wild species in which they have been tested. The objective of the current study was to evaluate various Tris-based diluents containing different concentrations of egg yolk, for the fertilizing ability of epididymal spermatozoa of the Spanish ibex (Capra pyrenaica) after freezing and thawing. For this purpose, we used heterologous in vivo fertilization by intrauterine insemination of domestic goats (Capra hircus). In Experiment 1, a Tris-citric acid-glucose (TCG) diluent containing 6% (v/v) egg yolk and a TCG extender containing 20% egg yolk were compared. In Experiment 2, a TCG-6% egg yolk extender was compared with Triladyl-20% egg yolk. Diluted samples were cooled slowly to 5 degrees C over 1 h and equilibrated at that temperature for 2 h. At that point, aliquots of samples were loaded into 0.25 ml straws, and frozen in nitrogen vapor for 10 min. The fertility of spermatozoa frozen in TCG-6% egg yolk was higher (P<0.05) than for those extended with TCG-20% egg yolk, and tended to be higher than for those frozen with Triladyl-20% egg yolk. From the results of this study, the use of Tris-based extenders containing low concentrations of egg yolk (6%) is recommended for cryopreserving Spanish ibex epididymal spermatozoa.     

The effect of zwitterion buffers on the freezability of Boer goat semen

Twenty semen samples with mass activity greater than +3 were collected from six healthy, mature Boer goat bucks. Each ejaculate was divided into four equal parts and extended at 37 degrees C in Tris, Test, Tes and Bes buffers containing egg yolk and glycerol. Semen was placed into medium size French strawsand after 2 hours of equilibration at 5 degrees C, frozen in the vapour phase and stored in liquid nitrogen for 7 days at -196 degrees C. Progressive motility, the number of live spermatozoa and glutamic oxaloacetic transaminase (GOT) release were studied after the initial extension, after equilibration and after 15 minutes and 7 days of freezing of semen. Semen samples when extended with Tris yolk glycerol showed significantly (P<0.01) higher progressive motility and live spermatozoa than when extended with the other zwitterion buffer-based extenders. The change of extenders did not influence the release of GOT at various stages of freezing of semen (P>0.05).     

Effect of egg yolk lipids on the freezing of goat semen

Jamunapari goat buck semen contained both phospholipase and lysophospholipase activities which remained active during dilution (Step I) with diluents containing egg yolk, cooling to 5 degrees C (Step II), glycerolization and equilibration (Step III) and freezing and thawing (Step IV). A quantitative estimate of the phosphatidyl choline and phosphatidyl ethanolamine before and after freezing revealed that the lipids in egg yolk added to dilute goat semen were not hydrolysed to lysophospholipids and free fatty acids. Seminal plasma was, therefore, not removed and goat semen was frozen in egg yolk citrate-glucose, egg yolk-tris and skim milk-egg yolk. Dilution of goat semen 20 times with the three extenders containing 7% glycerol and an equilibration time of 3 h yielded optimum results. A comparative evaluation of freezing in the three diluents based on the assessment of sperm motility, live sperm count and acrosomal damage showed egg yolk-tris to be best extender for the successful freezing of goat semen. Insemination trials conducted with frozen semen and the number of actual kiddings yielded a fertility rate of approximately 81% in our study.     

不同甘油浓度与平衡时间对食蟹猴精液冷冻效果的影响

探讨了不同甘油浓度(3%、5%、7%、11%)和不同平衡时间(30、60、90、120min)对食蟹猴(Macaca fascicularis)精液冷冻效果的影响,以建立和优化食蟹猴精液冷冻的程序。参照TTE稀释液成分组成改良型TTE,冷冻前和解冻后均检测精子的活力、畸形率、质膜完整性、顶体完整率。结果显示,平衡时间为30min时精子的冷冻解冻后活力、复苏率均高于平衡时间90min和120min组,差异显著(P<0.05),比60min组稍好;甘油浓度为3%、5%组的精子冷冻解冻后活力及复苏率均高于甘油浓度11%组,差异显著(P<0.05),比7%组好;不同甘油浓度各组间以及不同平衡时间各组间畸形率、质膜完整性、顶体完整率差异不显著(P<0.05)。由此得出如下结论,在食蟹猴精液冷冻中,在改良TTE中加入3%~5%的甘油且平衡30min可以获得较好效果,精子冻后活率和复苏率达到45%和62%。     

Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing

Influence of different sugars supplemented to the extender on the motility, viability and intact acrosome rates of dog spermatozoa during dilution, equilibration and freezing was studied. The ejaculate was divided into 10 aliquots, which were diluted 1:3 with TRIS-citric acid extender containing 240 mMTRIS, 63 mM citric acid, 8% (v/v) glycerol, 20% (v/v) egg yolk and 70 mM sugar, which was either fructose, galactose, glucose, xylose (monosaccharide), lactose, trehalose, maltose, sucrose (disaccharide) or raffinose (trisaccharide). No sugar was added to the extender in the control group. Extended semen samples were cooled to 5 degrees C over 45 min, packaged in 0.25-mL straws, equilibrated for 2 h at 5 degrees C and frozen in liquid nitrogen vapor. Samples were thawed by placing straws into 37 degrees C water for 30 sec. Motility, viable sperm and intact acrosome rates decreased gradually in all groups after equilibration and consecutively freezing (P<0.001). The type of sugar significantly effected motility, viability and acrosomal integrity during equilibration and freezing (P<0.05). Galactose, lactose, trehalose, maltose and sucrose reduced damaged acrosome percentages in equilibrated samples (P<0.05). Sugar supplementation did not enhance motility and viability during equilibration. The disaccharides, except lactose, reduced post-thaw dead sperm and/or damaged acrosome percentages without promoting post-thaw motility (P<0.01), whereas monosaccharides, especially fructose and xylose, improved motility (P<0.05) along with viability and intact acrosome rates (P<0.05). Trehalose, xylose and fructose significantly increased total active sperm rates (motility x live sperm rate x normal acrosome rate) compared to other sugars (P<0.01) and control (P<0.0001) in frozen thawed samples. Therefore, sugar supplementation of the extender influenced post-equilibration and post-thaw sperm quality, and the type or locality of protective impact of the sugar on dog spermatozoa vary according to type of the sugar.     

Comparisons between three different extenders for canine intrauterine insemination with frozen-thawed spermatozoa

Ejaculates from 7 dogs were obtained on the same day and were pooled. This pooled semen was separated into 3 equal fractions and processed simultaneously, the only difference being in the extender used for freezing. The extenders were laiciphos (containing laiciphos, egg yolk, distilled water and glycerol- Group 1); Tes/Tris (containing Tes/Tris, egg yolk, distilled water and glycerol- Group 2); and biociphos (containing biociphos with glycerol in it, egg yolk and distilled water- Group 3). Spermatozoa were conditioned in 0.5ml French straws and presented normal characteristics before freezing and after thawing. The sperm concentration of the pooled was 683 x 10(6) sperm/ml; sperm motility was above 95%, the percentage of live spermatozoa was above 95% and was of good quality and mobility. Characteristics of the spermatozoa after thawing were the same for spermatozoa frozen with laiciphos and Tes/Tris. Mean sperm concentration was 201.5 +/- 4.95 x 10(6) sperm/ml, sperm motility was 65%, the percentage of live spermatozoa was 80% and the quality of motility.was good. Spermtozoa frozen with biociphos had the following post-thaw characteristics: sperm concentration was 201 x 10(6) sperm/ml, sperm motility was 50%, the percentage of live spermatozoa was 78% and the quality of mobility was medium. Abnormalities were less than 15% for all spermatozoa after thawing. Intrauterine artificial inseminations were performed by laparoscopic intrauterine insemination twice at Days 3 and 5 after the estimated LH peak in 15 normally cyclic Beagle bitches (5 per group) presenting normal hormonal profiles. There were no differences between groups. The females were inseminated with 1.0 ml of spermoatozoa (concentration of 200 x 10(6) sperm/ml) diluted with 1.0 ml of extender. A 60% pregnancy rate was obtained in bitches inseminated with frozen-thawed spermatozoa extended with laiciphos or Tes/Tris and 100% in bitches inseminated with spermatozoa extended with biociphos. Females inseminated with laiciphos, Tes/Tris and biociphos had a mean litter size of 5 +/- 2.6, 3 +/- 1 and 3.4 +/- 1.3 pups, respectively. This study demonstrated that post-thaw assessment of sperm characteristics is not the best technique for evaluating sperm fertility after freezing or for assessing different semen extenders.     

Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing-thawing

Whole ejaculate or sperm-rich fraction, collected from four sexually mature boars, was frozen in an extender containing lactose-hen egg yolk with glycerol (lactose-HEY-G) or extender containing lactose, lyophilized lipoprotein fractions isolated from ostrich egg yolk and glycerol (lactose-LPFo-G), and Orvus Es Paste, respectively. The sperm samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Sperm DNA integrity was assessed using a modified neutral comet assay. Sperm characteristics such as motility, plasma membrane integrity (SYBR-14/PI), mitochondrial function (rhodamine 123) and acrosome integrity were monitored. Freezing-thawing caused a significant increase (P<0.05) in sperm DNA fragmentation, irrespective of the procedures of ejaculate collection and extender type. Sperm DNA fragmentation was significantly lower (P<0.05) in the whole ejaculate compared with the sperm-rich fraction, indicating that spermatozoa maintained in the whole seminal plasma prior to its removal for freezing-thawing procedure were less vulnerable to cryo-induced DNA fragmentation. Furthermore, spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender exhibited lower (P<0.05) DNA fragmentation than those frozen in the absence of cryoprotective substances. The levels of sperm DNA damage, as expressed by comet tail length and tail moment values, were significantly higher (P<0.05) in sperm samples frozen in the absence of cryoprotective substances. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. It can be suggested that evaluation of DNA integrity, coupled with different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, may aid in determining the quality of frozen-thawed boar semen.     

Effect of cryoprotectants on certain seminal attributes and on the fertility of buck spermatozoa

Semen samples were obtained from 12 bucks (3 Beetal, 3 Black Bengal and 6 Beetal x Black Bengal) and 10 different extenders were constituted with varying concentrations of glycerol, DMSO, glycerol + DMSO and glycerol + lactose as the sperm cryoprotective agents. After the collection of semen samples, they were assessed for quality, diluted in different extenders after removal of seminal plasma, packaged in ministraws and frozen after equilibration (5'C) for 5 h. The samples were evaluated immediately after equilibration and again 24 h after freezing for progressive motility, percentage of live spermatozoa and acrosome, head and tail abnormalities. Both motility and the percentage of live spermatozoa were most affected by extenders containing only DMSO and these values improved in glycerol + DMSO extenders as the concentration of glycerol was increased while DMSO was decreased. However, these values were significantly higher in extenders containing glycerol + lactose as the cryoprotective agents, and were found to increase with increased concentration of lactose, being highest in TYGL (180). Acrosomal and tail abnormalities tended to increase between post equilibration and post thawing stage, and were higher in extenders containing the higher levels of DMSO. Significantly (P < 0.01) lower percentages of abnormalities were recorded in the glycerol + lactose extenders. The fertility results showed nonsignificant effect of extenders on the conception rate of does.     

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.