Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.     

Freezability of equine semen after glass beads column separation

REASONS FOR PERFORMING STUDY: The success rate of artificial insemination following the freezing of stallion semen is limited; therefore, improving the stallion semen quality after the freezing and thawing process is a necessary objective. OBJECTIVES: To investigate the influence of glass bead column separation on the freezability of stallion semen. HYPOTHESIS: Glass beads in a column separator remove damaged and dead spermatozoa in the ejaculate during centrifugation. METHODS: In total, 50 ejaculates from 6 Lipizzaner stallions were studied. Each ejaculate was divided into 2 parts, one half processed following standard procedure and the second half used for the column separation procedure. After freezing, semen quality was evaluated using standard tests for motility, morphology and viability of semen. RESULTS: Motility and progressive motility of the column-separated (CS) semen were significantly higher (P < 0.001) before freezing and immediately, 24 and 48 h after thawing. A significant increase (P < 0.001) in the percentage of hypoosmotic positive spermatozoa was observed in CS samples. The percentage of total morphological changes in the separated samples before and after freezing was significantly lower (P < 0.001) compared with samples prepared using the standard procedure. A substantial decrease (P < 0.001) was found in the percentage of spermatozoa with damaged acrosomes. However, the percentage of spermatozoa with coiled tails was increased in the separated samples (P < 0.001). CONCLUSIONS: Column separation before freezing has a positive effect on the quality of thawed equine semen. POTENTIAL RELEVANCE: The quality of CS frozen/thawed samples indicates their potential use for increasing insemination success in mares.     

Effect of cooling rate and cryoprotectant on the cryosurvival of equine spermatozoa

Cryosurvival of cells is reduced if the cooling rate used is suboptimal. If cells cool too rapidly, intracellular water will freeze, causing intracellular ice crystals. However, if spermatozoa are cooled too slowly, excessive cellular dehydration occurs, causing irreversible damage to cellular compartments. In addition, cryoprotectants are added to the freezing diluent to protect cells from damage during cryopreservation. This study was conducted to determine the optimal cooling rate for stallion spermatozoa frozen in the presence of three different cryoprotectants. Spermatozoa were frozen in a skim milk, egg yolk diluent containing 4% glycerol, and ethylene glycol or dimethyl formamide at 10 different cooling rates ranging from 5°C/min to 50°C/min. The percentage of viable spermatozoa was higher for spermatozoa cooled at 10°C/min than at 50°C/min (P < .05). Spermatozoa frozen using glycerol as the cryoprotectant had higher percentages of motile and progressively motile spermatozoa compared with spermatozoa frozen using the other two cryoprotectants (P < .05). In conclusion, the cryosurvival of stallion spermatozoa is similar when cooling rates of 5°C/min to 45°C/min are used, and when 4% cryoprotectant is used, glycerol is a more effective cryoprotectant than ethylene glycol or dimethyl formamide.     

Effect of Sperm Cryopreservation on the European Eel Sperm Viability and Spermatozoa Morphology

The main objective of the present work was to study the effect of cryopreservation of European eel sperm both on the sperm viability and the spermatozoa head morphology. Spermatozoa morphology was evaluated with computer-assisted morphology analysis after collection in fresh samples, after adding the freezing medium containing dimethyl sulfoxide as cryoprotectant and, finally, after the cryopreservation process and thawing. Cell viability was assessed, in both fresh and thawed samples, by Hoechst 33258 staining. Computer-assisted sperm analysis (CASA) was used to determine the percentage of motile cells and to measure motility parameters in sperm samples. A significant decrease of head perimeter (12.56%) and area (17.90%) was detected from spermatozoa in fresh to thawed samples, indicating that cells do not recover the original size after the cryopreservation process. CASA was used to measure the percentage of motile cells (51.9%) and spermatozoa motility parameters such as curvilinear, straight line and angular path velocities, as well as beating cross frequency. This technique was employed in the fresh sperm samples but proteins present at the freezing medium (L-alpha-phosphatidylcholine) made impossible to use this last technique in thawed samples. When sperm viability was assessed by Hoechst staining, a significant decrease of approximately 15% (73.10 vs 58.26%) of alive spermatozoa was registered from fresh to thawed samples. The percentage of motile cells measured by CASA in fresh samples (51.9%) was lower than the percentage of alive cells determined by Hoechst stainning, suggesting the existence of different batches of spermatozoa in different stages of development, even during the eight to tenth weeks of treatment, when the highest sperm quality was found.     

Effect of staining and freezing media on sortability of stallion spermatozoa and their post-thaw viability after sex-sorting and cryopreservation

Sex‐sorted, frozen–thawed stallion spermatozoa remain out of reach of commercial horse breeders because of the low efficiency of the sex‐sorting process and unacceptable fertility rates after insemination. Two experiments were designed to test the effects of alternative staining and freezing media to improve the viability of sex‐sorted frozen–thawed stallion spermatozoa. Experiment 1 compared two freezing media, INRA 82^® and a modified lactose‐ethylenediaminetetraacetic acid (EDTA), for the cryopreservation of sex‐sorted stallion spermatozoa. No significant differences between the two freezing media could be identified, suggesting that both cryodiluents would be suitable for incorporation into a sex‐preselection protocol for stallion spermatozoa. Experiment 2 compared Kenney’s modified Tyrode’s (KMT) and Sperm TALP (Sp‐TALP) as the staining and incubation medium for stallion spermatozoa prior to sex‐sorting. A significant increase in the percentage of acrosome‐reacted spermatozoa occurred after staining and incubation in the clarified Sp‐TALP compared with KMT. As no improvements in sorting rates were achieved using Sp‐TALP, it was concluded that stallion sorting protocols could include KMT as the staining and incubation medium while either INRA 82^® or lactose‐EDTA could be employed as a cryodiluents.     

Effects of single layer centrifugation (SLC) on bull spermatozoa prior to freezing on post‐thaw semen characteristics

Single layer centrifugation (SLC) has been shown to select the most robust spermatozoa from the ejaculate in several species. Here the effects of SLC prior to freezing on various parameters of frozen‐thawed bovine sperm quality are reported. Semen from 8 bulls was layered on top of a species‐specific colloid, Bovicoll. After centrifugation for 20 min at 300 g, the resulting sperm pellet was resuspended in OPTIXcell^® (IMV Technologies, l′Aigle, France); the SLC‐selected sperm samples and uncentrifuged controls were frozen. On thawing, all sperm samples were analysed for membrane integrity, production of reactive oxygen species, mitochondrial membrane potential (MMP) and chromatin integrity. The SLC‐treated samples had a higher percentage of live, superoxide‐positive spermatozoa than uncentrifuged samples (27.9 ± 5.1% versus 21.7 ± 6.7%; p = .03). They had a higher proportion of spermatozoa with high mitochondrial membrane potential than uncentrifuged samples (55.9 ± 8.2% versus 40.5 ± 15.1%; p = .03) and also a lower proportion of spermatozoa with low mitochondrial membrane potential than non‐treated samples (42.0 ± 8.5% versus 55.9 ± 14.4%; p = .04). No significant effects of treatment were found for membrane integrity or chromatin integrity. The effect of bull was significant on the proportions of dead, superoxide‐positive spermatozoa and live, hydrogen peroxide‐negative spermatozoa, as well as on membrane integrity, but it was not significant for mitochondrial membrane potential or chromatin integrity. These results suggest that SLC selects the most metabolically active bull spermatozoa from the rest of the population in normal ejaculates; the pattern of reactive oxygen species production may be different in SLC‐selected spermatozoa compared to unselected samples.     

Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa

Slow-cooled stallion spermatozoa, with and without seminal plasma removed by centrifugation, were diluted in Kenney's extender (KE) containing nonfat dry skim milk with glucose and antibiotics or in KE supplemented by adding a modified high-potassium Tyrode's medium (KMT). Four ejaculates from each of four stallions were collected and divided factorially across these four treatments. Percentage of motile sperm, path velocity, and linearity immediately after treatment (0 h) and after storage at 4 degrees C for 24, 48, and 72 h were evaluated objectively by use of a HTM-2030 sperm motility analyzer. Stallions were a significant source of variation (P less than .01) throughout. After sperm had cooled, effects of stallion, extender, centrifugation, and their interactions were all found to be significant (P less than .01). The motility at 0, 24, 48, and 72 h for centrifuged KE was 74, 47, 39, and 24%; for uncentrifuged KE was 76, 56, 50, and 37%; for centrifuged KMT was 76, 75, 72, and 64%; and for uncentrifuged KMT was 80, 50, 26, and 13%, respectively. The extender x centrifugation interaction, after 24, 48, and 72 h of storage, accounted for half or more of the variation. Whereas centrifugation of semen extended in KE seemed to be harmful to sperm, motility of sperm extended in KMT after centrifugation was remarkably conserved for 72 h and was superior to all other treatments (P less than .05). This extender is promising for preserving liquid stallion semen when it must be transported before use in artificial insemination.     

Preliminary comparisons of a unique freezing technology to traditional cryopreservation methodology of equine spermatozoa

With recent large-breed organization acceptance, the use of frozen semen is gaining more attention in the equine industry. However, cryopreserved stallion semen is commonly associated with poor quality and decreased pregnancy rates as compared with those produced during normal mating or with cooled semen techniques. Therefore our objective was to investigate a new unique freezing technique (UFT) with the intent of improving fertility outcomes. A series of experiments tested the UFT compared with traditional liquid nitrogen methodology in combination with influence of the extenders and stallions used. In Experiment 1, post-thaw motility results of UFT variations were compared with those from liquid nitrogen methods. The averaged post-thaw motility percentages of the 4 UFT treatments were similar when compared with the traditional liquid nitrogen control group (P = .845). In Experiment 2, 2 egg-yolk–based freezing extenders, Biladyl AB intended for bovine samples and Freezing Medium Test Yolk Buffer used for human samples, were compared. A significant difference in the average post-thaw motilities was found between Biladyl AB (17%) and Freezing Medium Test Yolk Buffer (25%) (P < .002). In the third experiment, we compared variability among stallions using the UFT with the intention of creating a more consistent outcome. Post-thaw motilities and percent of original motility returns among the 4 stallions were significantly different (P < .001). In a field trial using shipped semen from a regional stallion station, the UFT demonstrated very promising results.In conclusion, the UFT may potentially be used as an alterative freezing method to replace current liquid nitrogen methodology. However, further investigation is needed to refine techniques.     

Does single-layer centrifugation with Bovicoll improve sperm quality of frozen-thawed semen in Fleckvieh bulls?

The aim of the present study was to evaluate the effect of sperm selection by single-layer centrifugation (SLC) performed before freezing on sperm quality after thawing of Fleckvieh bull semen. Ejaculates from 22 bulls were collected by artificial vagina and divided into two aliquots. One aliquot (control sample) was diluted with Steridyl^® and frozen over nitrogen vapour in a Digitcool freezer (IMV Technologies). Sperm from the second aliquot (SLC sample) was selected using the SLC technique with Bovicoll colloid and then frozen over nitrogen vapour in a Digitcool freezer. After thawing, both samples (control and SLC) were evaluated by computer-aided sperm analysis (CASA; SCA 6.4 System; Microptic S.L) for sperm motility parameters. Integrity of the plasma membrane (viability), high mitochondrial membrane potential (HMMP) and acrosome integrity were assessed using a Guava® easyCyte flow cytometer (IMV Technologies). Morphological examination of spermatozoa was performed by Differential Interference Contrast microscopy (Leica DMi8). Morphological examination of live, immobilized spermatozoa was analysed under high magnification (≥6,600×). After thawing, the mean sperm viability of the control sample was 51.57%, compared to 40.37% for the SLC sample (p < .01). HMMP was higher (p < .01) in the control sample (40.37% versus 28.96%), and the mean of live spermatozoa with damaged acrosome was significantly higher (p < .03) in the SLC sample (1.63% versus 1.95%). The mean percentage of motile spermatozoa was 80.17% in the control sample, compared to 75.14% in the SLC sample (p < .0195), and rapid subpopulation reduced from 20.08% to 8.99% (p < .0001) after SLC. Percentage of hyperactivated sperm decreased from 12.23% to 4.28% (p < .0001) after SLC. Given the overall results, the sperm quality of thawed Fleckvieh bull semen was not improved when sperm were selected by SLC before freezing.     

Use of Glutamine and Low Density Lipoproteins Isolated from Egg Yolk to Improve Buck Semen Freezing

To improve the results obtained with a reference cryopreservation extender (control extender: Triladyl® + 20% (v/v) egg yolk + 6.4% (v/v) glycerol) for freezing caprine semen, glutamine was added to 18 split ejaculates at concentrations of 0, 20, 40, 80 and 120 m m (experiment 1). In experiment 2, glutamine was added to 18 split ejaculates at concentrations of 20, 25, 30, 35 and 40 m m . In the third experiment, the egg yolk was replaced with the low-density lipoprotein (LDL) fraction of egg yolk. The quality of frozen then thawed spermatozoa in each extender was compared using computer-assisted semen analysis. In experiment 1, glutamine at concentrations of 20 m m and 40 m m significantly improved sperm motility compared with the control extender. However, at 120 m m , a significant decrease in motility and velocity was observed. In experiment 2, motility, curvilinear velocity and amplitude of lateral head displacement (ALH) were improved in glutamine at 25 m m compared with the control. In experiment 3, 8% LDL and 25 m m glutamine significantly improved sperm motility, straight line velocity and ALH. In the fourth experiment, the quality of the previously defined freezing extender (Triladyl® + 8% (v/v) LDL + 25 m m glutamine + 6.4% (v/v) glycerol) was tested by comparing acrosome, tail membrane, plasma membrane and DNA integrity in 18 split ejaculates of frozen then thawed spermatozoa with spermatozoa that had been frozen then thawed in the control extender, and with spermatozoa from fresh, unfrozen sperm. The percentage of spermatozoa with intact acrosomes and tail membranes was significantly higher with the newly defined extender than that observed with the control extender. There was no significant difference in the percentage of spermatozoa with intact DNA between the frozen and fresh semen.     

牛皮肤成纤维细胞的体外培养与冻存

利用牛皮肤组织块直接培养法，得到牛皮肤细胞的原代培养物，再用酶消化法和反复贴壁法处理，能够纯化成纤维细胞。成纤维细胞的冻存是通过选用6种分别含有二甲基亚砜（DMSO）、甘油（GL）及乙二醇（EG）的保护液，以相同的冻前处理方法，对牛皮肤成纤维细胞进行缓慢冷冻，冰箱预冷平衡1－2h，逐步投入液氮（－196℃）中保存，再经37℃水浴解冻，Hanks液脱保护剂，以贴壁率评价冻存效果。结果表明，20％DMSO保护液对牛皮肤成纤维细胞表现出较好且稳定的冷冻保护效果，其平均贴壁率达87．9％。     

Comparison of spermatozoal movement and semen characteristics with fertility in stallions: 64 cases (1987-1988).

Information pertaining to evaluation of single ejaculates of semen and records for 2 consecutive breeding seasons were obtained. In all, data for 99 individual breeding seasons (n = 43 Standardbreds and 56 Thoroughbreds) were evaluated. Included in each semen evaluation was examination of semen characteristics and computer-aided analysis of spermatozoal movement characteristics. On the basis of the analysis of breeding records for 4,175 mares (7,017 estrous cycles), a per-estrous cycle fertility rate was calculated from data for 96 of the breeding seasons. Stallions with lower fertility than the mean overall season fertility had significantly (P less than 0.01) lower mean values for subjective appraisal of the percentage of motile and progressively motile spermatozoa and for percentage of morphologically normal spermatozoa. Lower mean values were obtained for computer-aided movement analysis of the percentage of motile and progressively motile spermatozoa, and for mean velocity of motile spermatozoa. Semen characteristics, including spermatozoal movement characteristics, and fertility were significantly (P less than 0.05) correlated for Thoroughbred and Standardbred stallions when analyzed individually and when data for both breeds were combined. Characteristics most highly correlated (P less than 0.01) with fertility data for both breeds combined were: subjective appraisal of the percentage of motile (r = 0.40) and progressively motile (r = 0.46) spermatozoa; percentage of morphologically normal spermatozoa (r = 0.36); and computer-aided analysis of percentage of motile spermatozoa (r = 0.34). However, on the basis of evaluation of a single ejaculate for each stallion, the variation in these characteristics only accounted for approximately 20% of the observed variation in fertility rate.     

Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa

Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen‐thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP‐7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid‐piece and principal piece domains in ejaculated spermatozoa. AQP‐9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP‐7, but not that of AQP‐9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP‐9 but relocated AQP‐7 towards the acrosome. AQP‐7, but not AQP‐9, appears as a relevant marker for non‐empirical studies of sperm handling.     

鸡胚19期原始生殖细胞慢速冷冻和玻璃化冷冻保存的研究

本研究对发育至第19期的鸡胚性腺原始生殖细胞(primordial germ cells，PGCs)分离提纯后，在DMEM中添加冷冻保护剂DMSO（二甲基亚砜）、EG（乙二醇）、蔗糖、PVP（聚乙烯吡咯烷酮）分别组成6种慢速冷冻保护液和6种玻璃化冷冻保护液，进行超低温冷冻保存。复苏后苔盼蓝染色测定细胞存活率，体外接种培养、传代。结果：①慢速冷冻保存中，PGCs在慢速冷冻液V（10％EG+10% FBS+0.1 mol/L蔗糖）条件下复苏后存活率最高（92.20%），且与慢速冷冻液I（10％ DMSO+10% FBS）存活率之间差异显著（P<0.05）。②玻璃化冷冻保存中，PGCs在玻璃化冷冻液I（10% DMSO+10% EG+20% FBS+10% PVP）条件下复苏后存活率最高（84.15%），且与其余5种玻璃化冷冻液下复苏后存活率之间差异均极显著（P<0.01）。③培养传至第3代的慢速冷冻复苏后PGCs细胞和培养传至第2代的玻璃化冷冻复苏后PGCs细胞，PAS染色、AKP染色呈阳性并保持完整的二倍体核型。     

Cryopreservation of Stallion Spermatozoa with INRA96 and Glycerol

The aim of the present study was to improve success of cryopreservation of stallion spermatozoa. Semen from eleven stallions was collected and frozen in INRA 96 with two different concentrations of glycerol (3.5% and 6.0%) and compared with a control freezing process. The mean post-thaw motility for the eleven stallions of 57.93% (3.5% glycerol) and 66.50% (6.0% glycerol), which was statistically higher (P < 0.05) when compared with the mean post-thaw motility (39.7%) for semen in a control egg-yolk extender (Equipro^® CryoGuard™ Complete, Minitube). The Equipro^® CryoGuard™ Complete is a commercial semen freezing protocol that has been one of the standard processes used in our laboratory for freezing equine spermatozoa. INRA 96 with 6% added glycerol was used in the fertility trial as it provided the highest spermatozoa survival. To evaluate fertility of the frozen semen, eight mares were bred over two cycles with both fresh and frozen semen. The pregnancy rate of mares bred with frozen semen (55.6%) was not statistically different (P > 0.05) from the pregnancy rate of mares bred with fresh semen (55.6%). INRA 96 with 6.0% glycerol improved the survivability of stallion spermatozoa through the cryopreservation process, and subsequent fertility was not different (P > 0.05) from fresh, extended semen.     

A Scanning Electron Microscopy Study of Frozen / Thawed Dog Sperm During In Vitro Gamete Interaction

The aim of this work was to study the effects of cryopreservation on the binding and penetration of dog spermatozoa to the zona pellucida (ZP) by scanning electron microscopy (SEM). The sperm-rich fraction of six ejaculates from five dogs was divided into two aliquots and washed by centrifugation. One aliquot was processed as fresh control sample and the other aliquot frozen in Tris-fructose extender. Gamete interaction was assessed using in vitro matured bitch oocytes, which were co-incubated for up to 3 h. At hourly intervals after the start of co-incubation, in vitro fertilized (IVF) oocytes were processed by SEM. The results were analysed statistically using the anova test. Differences in binding and penetration of the spermatozoa to the ZP occurred; a lower proportion of oocytes with spermatozoa bound to ZP was observed using frozen sperm (p < 0.05) than with fresh sperm (61%, 57% and 53% vs 42%, 40% and 44% at 1, 2 and 3 h, respectively). The percentage of ZP penetration by fresh sperm was directly proportional to the time of co-incubation (9%, 25% and 34%; p < 0.05); in contrast, no differences were observed in the penetration rate with frozen-thawed sperm (21%, 17% and 21%). More acrosome reacted sperm were observed in frozen sperm than in fresh sperm on the surface of the ZP. The differences in the percentage of binding and penetration between fresh and frozen sperm during the co-culture could indicate that the time course of penetration is faster in frozen-thawed dog spermatozoa than in fresh sperm, but that fresh spermatozoa can penetrate more oocytes over a given period of time, which may be related to their reacted or non-reacted initial status.     

Acrosomal ultrastructure of stallion spermatozoa cryopreserved with ethylene glycol using two packaging systems

The present experiments aimed to examine the substitution of glycerol (G) by ethylene glycol (E) as a cryoprotective agent for stallion spermatozoa. Two different ethylene glycol concentrations (5% and 10%) and also the association of glycerol (2%) and ethylene glycol (3%) (E/G) were studied (Experiment 1). In Experiment 2, two packing systems (0.5 x 4.0 ml) were evaluated using both cryoprotectors. In both experiments, the sperm membrane integrity after freezing was evaluated using transmission electron microscopy. The mean post-thaw motility was 34.25, 36.5, 29.25 and 34.75% for G5%, E5%, E10% and E/G, respectively. It was observed that the percentage of motile spermatozoa was significantly smaller (P<0.05) when semen was processed with E10%. A decrease in the acrosome integrity was observed in frozen thawed spermatozoa from all treated groups. It was observed that 28.0, 22.5, 25.5 and 22.5% of the sperm cells had a normal acrosome following freezing with G5%, E5%, E10% and E/G, respectively. Undulation of the outer acrosomal membrane, acrosomal swelling and loss of acrosomal content density and homogeneity were the most evident ultrastructural alterations observed. In Experiment 2, the post-thaw motility was higher (P<0.05) for sperm frozen in 0.5 ml straws than in 4.0 ml straws, regardless of the cryoprotector used. The ultrastructural evaluation showed 26.7 and 16.0% of intact acrosomes for sperm frozen in 0.5 ml and 4.0 ml straws, respectively. We concluded that ethylene glycol has similar cryoprotective properties to glycerol and that utilisation of 0.5 ml straws improved the ability of horse sperm cells to withstand damage after the cryopreservation process.     

Influence of the Freezing Technique (Nitrogen Liquid vs Ultrafreezer of −152°C) and Male-to-Male Variation Over the Semen Quality in Canarian Mastiff Breed Dogs

This study was carried out to assess the in vitro quality of canine semen frozen in an ultrafreezer at -152 degrees C and to evaluate the male-to-male variation of frozen semen in five male dogs of the Canarian Mastiff breed. Four ejaculates of each dog were processed individually (5% glycerol and 0.5% Equex) to reach a final concentration of 100 x 10(6) spermatozoa/ml. Then, two freezing techniques were tested to assess the seminal quality (sperm motility, live spermatozoa and abnormal sperm cell percentages) at 1, 30, 60, 120 and 360 days after freezing: (i) semen was frozen and stored in liquid nitrogen; (ii) semen was frozen and stored in the ultrafreezer at -152 degrees C. After freezing-thawing, both freezing protocols showed no significant differences in sperm motility and the percentages of live and abnormal spermatozoa. On the other hand, the microscopic characteristics of spermatozoa in fresh semen were practically similar among males; however, after the semen processing and freezing, significant differences were observed (p < 0.05) among males, especially as regards sperm motility. This inter-individual variability was detected in both freezing protocols, showing that the male-to-male variation in the seminal quality post-freezing was independent of the freezing technique used. The in vitro results obtained in the Canarian Mastiff breed confirmed that the use of ultra-freezers at -152 degrees C is a potential alternative to liquid nitrogen for storing canine semen for long periods of time.     

Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa

The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sperm treatment groups. Semen was collected from 2 stallions with an artificial vagina and processed for each treatment. Treatment 1 (n = 10 mare cycles) consisted of fresh, nonsorted spermatozoa and Treatment 2 (n = 16 mare cycles) of fresh, flow sorted spermatozoa. Spermatozoa to be sorted were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Treatment 3 (n = 16 mare cycles) consisted of frozen/thawed nonsorted spermatozoa (frozen at 33.5 x 106 sperm/ml in 0.25 ml straws) and Treatment 4 (n = 15 mare cycles) of flow sorted frozen/thawed spermatozoa (frozen at 64.4 x 10(6) sperm/ml). Concentrations of sperm in both cryopreserved treatments were adjusted, based on predetermined average post-thaw motilities, so that each insemination contained approximately 5 x 10(6) motile spermatozoa. Hysteroscopic insemination of 5 x 10(6) motile spermatozoa in a volume of 230 microd was used for all treatments. Pregnancy was determined ultrasonographically 16 days postovulation. No differences were found (P>0.1) in the pregnancy rates for mares inseminated with fresh nonsorted (4/10 = 40.0%), fresh flow sorted (6/16 = 37.5%), frozen/thawed nonsorted (6/16 = 37.5%) and flow sorted frozen/thawed spermatozoa (2/15 = 133%). Pregnancy rates tended (P = 0.12) to be lower following insemination of frozen/thawed flow sorted spermatozoa. Further studies are needed with a larger number of mares to determine if fertility of flow sorted frozen/thawed spermatozoa can be improved.     

Morphology and Chromatin Integrity of Stallion Spermatozoa Prepared by Density Gradient and Single Layer Centrifugation Through Silica Colloids

The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p < 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p < 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p < 0.001), and mid-piece defects from 5.3% to 1.4% (p < 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p < 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.