Effect of cryopreservation protocol on postthaw characteristics of stallion sperm

Three ejaculates from each of eight stallions were subjected to cryopreservation in a milk/egg yolk-based freezing extender or an egg yolk-based freezing extender. Semen was exposed to a fast prefreeze cooling rate (FAST; semen immediately subjected to cryopreservation) or a slow prefreeze cooling rate (SLOW; semen pre-cooled at a controlled rate for 80 min prior to cryopreservation). Postthaw semen was diluted in initial freezing medium (FM) or INRA 96 (IMV Technologies, L'Aigle, France) prior to analysis of 10 experimental end points: total motility (MOT; %), progressive motility (PMOT; %), curvilinear velocity (VCL; μm/s), linearity (LIN; %), intact acrosomal and plasma membranes (AIMI; %), intact acrosomal membranes (AI; %), intact plasma membranes (MI; %), and DNA quality. Eight of 10 experimental endpoints (MOT, PMOT, average-path velocity [VAP], mean straight-line velocity [VSL], LIN AIMI, AI, and MI) were affected by extender type, with egg yolk-based extender yielding higher values than milk/egg yolk-based extender (P < 0.05). Exposure of extended semen to a slow prefreeze cooling period resulted in increased values for six of eight endpoints (MOT, PMOT, VCL, AIMI, AI, and MI), as compared with a fast prefreeze cooling period (P < 0.05). As a postthaw diluent, INRA 96 yielded higher mean values than FM for MOT, PMOT, VCL, average-path velocity, and mean straight-line velocity (P < 0.05). Treatment group FM yielded slightly higher values than INRA 96 for LIN and MI (P < 0.05). In conclusion, a slow prefreeze cooling rate was superior to a fast prefreeze cooling rate, regardless of freezing extender used, and INRA 96 served as a satisfactory postthaw diluent prior to semen analysis.     

Computerized analysis of the motility parameters of hamster spermatozoa during maturation

A computer-aided semen analysis system was used for the objective assessment of hamster spermatozoa during epididymal maturation. The caput epididymal spermatozoa were extremely sluggish, achieved very little progression, and the three velocity parameters, namely curvilinear velocity (VCL), progressive velocity (VSL), and path velocity (VAP), were low. These spermatozoa during progressive movement alternated between the linear shape and “U” shape or attained an “S” shape prior to changing to the “U”; shape. The corpus epididymal spermatozoa were faster, displayed greater VSL, VAP, and VCL compared to caput epididymal spermatozoa, and, during forward motility, attained “U,” “C,”; and (or) “?” shape as in the wriggling motility pattern. The proximal cauda epididymal spermatozoa were actively motile and VSL, VAP, and VCL in these spermatozoa were more than 10 times greater compared to the caput epididymal spermatozoa. The proximal cauda epididymal spermatozoa predominantly moved in circles and with time became slower and more circular in their trajectories and exhibited a reduction in LIN (linearity). The distal cauda epididymal spermatozoa were very similar to the proximal cauda epididymal spermatozoa with respect to their fast motility (VSL, VAP, and VCL are similar) and beat cross frequency (BCF), but showed larger values for STR (straightness) and LIN and moved along curved trajectories. The amplitude of lateral head displacement (ALH) was also considerably lower in the distal cauda epididymal spermatozoa compared to the proximal cauda epididymal spermatozoa. Thus, this study provides for the first time data related to seven motility parameters for caput and corpus epididymal spermatozoa of hamster. It also provides additional data with respect to VCL, LIN, BCF, and ALH for proximal and distal cauda epididymal spermatozoa of hamster. © 1994 Wiley-Liss, Inc.     

Effect of α-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes

Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of α-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of α-tocopherol (0, 100, 200, 400, 600 and 800 μM) using the straw-freezing procedure and stored at −196 °C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P < 0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 μM α-tocopherol supplemented frozen-thawed groups. In α-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 °C for 3 h, the expression in 200 and 800 μM α-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P < 0.01) lower in α-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, α-tocopherol, supplemented at 200 μM concentration in boar semen extender during cryopreservation had a positive effect on post-thawed sperm survivability.     

In vitro sperm characterization and development of a sperm cryopreservation method using directional solidification in the killer whale (Orcinus orca)

Research was conducted to characterize seminal traits and to develop a sperm cryopreservation method using directional freezing (DF) for the killer whale (Orcinus orca). Experiments evaluated effects of: (i) freezing rate (SLOW, MED, FAST) by diluent (BF5F, Biladyl®, EYC) in 0.5 mL straws; and (ii) freezing method (straw or DF) by glycerol (3, 6, or 9% final concentration, v:v) on in vitro sperm quality. Fresh ejaculates (n = 161) were (mean ± SD) 7.8 ± 7.4 mL at 740 × 10⁶ sperm/mL with 92.2 ± 6.3% total motility (TM), 85.4 ± 6.9% progressive motility (PM), 89.6 ± 9.0% viability and 89.8 ± 9.2% acrosome integrity. Samples frozen using straws by the MED or SLOW method were improved (P < 0.05) over FAST across all diluents. At 3 h post thaw (PT), TM, PM, Rapid motility (RM), VAP, VCL, ALH and viability for 3% and 6% glycerol were improved (P < 0.05) over 9% glycerol. Directional freezing samples at 0 h and 3 h PT, at all glycerol concentrations, displayed higher (P < 0.001) TM, PM, RM, VAP, VSL, VCL and viability /intact acrosomes (PI/FITC-PNA) than straw. These data provided the first information on ejaculate characteristics and the development of a semen cryopreservation method using DF in the killer whale.     

Sperm quality of Colossoma macropomum after room‐temperature and cold storage

The objective of this study was to evaluate the effects of cold and room‐temperature storage on the quality of Colossoma macropomum sperm. The experiment was carried out in December (end of Spring), in Nova Mutum‐MT, Brazil, involving nine C. macropomum males (4 years old; 6.4 ± 1.5 kg average weight). The fish were selected and transferred to masonry tanks (4 m³) in a laboratory (water renewal rate: 10 L/s; average water temperature: 28°C). Subsequently, reproduction was induced using 2.5 mg of crude carp pituitary extract/kg and the semen was harvested 240 degree hours after hormonal induction. The following sperm characteristics were analyzed every 5 hr using Image J/casa software: total motility (MOT), curvilinear velocity (VCL), average path velocity (VAP), straight‐line velocity (VSL), straightness of sperm path (STR), wobble (WOB), progressive motility (PROG), beat cross frequency (BCF) and total number of spermatozoa (NSPZ). A fresh sample of semen from each animal was kept at room temperature (25.3 ± 1.2°C). For analysis of cooled semen, syringes were kept in cooling boxes at an average temperature of 16.9 ± 2.1°C. The reduction (p < 0.05) of MOT in semen kept at room temperature occurred at 10 hr (13.95%); in cooled semen, however, MOT declined at 15 hr (76.87%). At 15 hr, there was practically no MOT in the semen kept at room temperature (0.20%), whereas in the cooled semen this situation was observed only at 35 hr (2.91%) The MOT of cooled sperm was higher (p < 0.05) at all times (except zero time), compared with the semen maintained at room temperature. At 15 hr, the cooled spermatozoa showed higher (p < 0.05) VCL (142.18 μm/s) and BCF (29.72 Hz) than those maintained at room temperature (VCL: 51.18 μm/s; BCF: 19.57 Hz). After 15 hr, only the cooled sperm showed quality. In conclusion, semen cooling allows for extending the viability of C. macropomum spermatozoa from 5 to 10 hr without compromising their quality in most characteristics. At 15 and 25 hr of cooling, sperm viability is still observed, though with decreased quality.     

The effect of temperature and pH on the motility and viability of ostrich sperm

As the chemical environment of semen diluents can have a profound effect on sperm quality, we examined the effect of temperature and pH on the motility and viability of sperm in the ostrich. Semen was collected from four males, each male being replicated three times. Ejaculates were diluted and incubated for 10 min at 20°C and 40°C in four different buffers, temperature adjusted at pH 6, 7, 8 and 9 respectively. Average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), beat cross frequency (BCF) and amplitude of lateral displacement (ALH) were then recorded for each sample using CASA. The viability of sperm was assessed using nigrosin-eosin staining. Sperm incubated at 40°C had higher motility parameters, except for ALH. At 40°C, VAP, VSL and LIN increased with pH while VCL, BCF and ALH were higher for lower pHs. The viability of sperm was not affected by temperature but decreased at pH values>7. A pH in the neutral range appeared to yield higher quality sperm after in vitro storage at 20°C. However, the effect of different pH levels and temperatures on sperm longevity needs to be investigated further to develop viable ostrich specific diluents.     

The cryoprotective effect of low-density lipoproteins in extenders on bull spermatozoa following freezing–thawing

Egg low-density lipoprotein (LDL) was added at concentrations of 7–10% to the extenders used to freeze bull semen and its effects on the motility, mitochondria activity, acrosome integrity, membrane integrity and DNA integrity of frozen–thawed sperm were assessed. Analysis of data showed that the motility and characteristics of spermatozoa movement were higher with LDL in the extender, as compared to the extender containing 20% egg yolk. The results indicated that 8% LDL supplementation provided the highest sperm motility (55.8%) and movement characteristics (VSL, straight linear velocity: 33.8 μm/s; VCL, curvilinear velocity: 50.2 μm/s; LIN, linearity index: 56.5%; STR, mean coefficient: 76.7%; VAP, average path velocity: 35.9 μm/s; WOB, wobble coefficient: 63.9%). A concentration of 10% LDL resulted in a significant decline in the VSL, LIN, VAP and WOB values (P < 0.05). Supplementation of LDL at 8% LDL resulted in significantly higher spermatozoa mitochondrial activity, acrosome integrity, membrane integrity and DNA integrity (P < 0.05). According to all measured parameters, the extender containing 8% LDL showed beneficial cryoprotective effects on frozen–thawed bull spermatozoa. In conclusion, our results indicated that the extender containing 8% LDL extracted from egg yolk could be used successfully in the cryopreservation of bull semen with an efficacy that would be greater than present extenders containing 20% egg yolk.     

A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Bird’s Nest on the quality of chilled Arabian stallion semen

The aim of this study was to evaluate the effects of adding different concentrations of edible bird’s nest (EBN) which is secreted by swiftlet birds (Aerodramus fuciphagus), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using computer assisted sperm analysis (CASA) and viability were assessed using Hoechst33342/PI stain. In both extenders, total motility (TM) and progressive motility (PM) were significantly higher at 0.12% and 0.24% compared to 0.24% + SP at 24 and 48 h. At 0.12%, E-Z mixin® treated semen had significantly higher TM and PM than EquiPlus^® at 24 and 48 h. At 0.12% and 0.24%, average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were significantly higher in E-Z mixin® treated semen compared to EquiPlus^® at 24 and 48 h. Comparisons between the two extender types at different concentrations of EBN showed no significant difference in lateral head amplitude (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF) and viability, irrespective of the storage time. The percentage of viable was significantly higher in E-Z mixin® than EquiPlus® at 0 and 48 h in control and 0.12%. Supplementation of the E-Z mixin^® extender with 0.12% and 0.24% EBN concentrations in the absence of SP provided better CASA parameters such as TM, PM, VAP, VSL, and VCL at 24 and 48 h storage time. In conclusion, the results of this study indicated that chilled semen from Arabian stallion that was extended using E-Z mixin® and supplemented with 0.12% and 0.24% EBN concentrations performed better and yielded superior results in sperm kinetic parameters and % viable compared to EquiPlus® at 24 and 48 h storage time.     

Semen characteristics, cryopreservation, and successful artificial insemination in the Blue rock pigeon (Columba livia)

The present study was undertaken in the Blue rock pigeon (Columba livia) to evaluate the annual semen characteristics, to identify a suitable extender for semen short-term storage, to determine a protocol for cryopreservation of semen and finally to check whether intracloacal insemination would lead to the birth of a chick. Semen characteristics such as semen volume, sperm concentration, sperm motility, and percentage of normal spermatozoa were maximum during the monsoon season. TALP was observed to be the most suitable semen extender and the sperm survived best at 37 degrees C at a dilution of 1:100 in TALP. Further, cryopreservation studies on pigeon semen indicated that 8% DMSO with or without egg yolk (20%) proved to be a better cryoprotectant compared to glycerol and polyethylene glycol. In addition, the slow freezing protocol was better than the fast-freezing protocol and about 40% of the cryopreserved spermatozoa were motile following thawing. Computer-aided semen analysis indicated that pigeon spermatozoa were extremely active immediately after dilution in TALP and exhibited linear trajectories persisting up to 9h. But, with time there was a time-dependent decrease in the velocity parameters (VAP, VSL, and VCL). Cryopreserved spermatozoa following thawing also exhibited linear trajectories but had reduced velocity as evident from the significant decrease in VAP, VSL, and VCL. Further, artificial inseminations using fresh semen resulted in 45% fertilization and birth of a live chick.     

The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O₃/mL. The semen samples were diluted (200 x 10⁶ spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p<0.05). There was no difference (p>0.05) between the control and treatment (6, 8, and 12 μg of O₃/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.     

Dynamics of motile-sperm subpopulation structure in boar ejaculates subjected to "in vitro" capacitation and further "in vitro" acrosome reaction

Incubation of diluted boar sperm from fresh ejaculates in a previously established "in vitro" capacitation medium induced a significant, time-dependent increase in several mean parameters of sperm motility, such as curvilinear velocity (VCL), linear velocity (VSL), mean velocity (VAP), linearity coefficient (LIN), straightness coefficient (STR) and wobble coefficient (WOB). Furthermore, motile boar-sperm semen samples were structured in four definite subpopulations. Subpopulation 1 showed the lowest values of VCL, VSL and VAP and also low values of linearity. Subpopulation 2 showed the second lowest values of VCL and VAP and higher values of LIN and STR. Subpopulation 3 was characterized by high values of velocity and low values of linearity. Finally, Subpopulation 4 was characterized by high values of velocity and linearity. "In vitro" capacitation and further acrosome reaction induced changes in the motility characteristics of each subpopulation as well as in their percentage distribution, Subpopulations 3 and 4 being those that showed the most significant changes. However, despite these changes, the observed, overall four-subpopulation structure was firmly maintained during the entire "in vitro" capacitation and acrosome-reaction process. Our results suggest that capacitation-induced motility changes are related to specific changes in the percentage of each motile-sperm subpopulation in the ejaculate without losing the overall, specific four-subpopulation structure. In this way, the maintenance of a four-subpopulation structure seems to be important in the control of the whole ejaculate physiology.     

Seasonal changes of semen quality and freezability in Franches-Montagnes stallions

The objective of this study was to investigate seasonal changes of semen quality parameters in Franches-Montagnes stallions and to compare the freezability of ejaculates collected in autumn and winter. Experiments were performed using 15 stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and evaluated every month during 1 year as well as cryopreserved in autumn and winter (September to February). In fresh semen the gel-free volume, concentration, motility and morphology (normal sperm, major defects, vacuoles and acrosome defects) were evaluated and in frozen-thawed semen the motility as well as the viability (SYBR-14/PI) were performed. To analyse seasonal differences four periods of 3 months each were defined as autumn (September, October, November), winter (December, January, February), spring (March, April, May) and summer (June, July, August). During the 1-year experiment all fresh semen quality parameters demonstrated a clear seasonal and individual pattern. The gel-free volume was significantly (P<0.05) higher in spring and summer compared to autumn and winter while sperm concentration was significantly (P<0.05) lower in spring than at any other time of the year. Total sperm number was significantly (P<0.05) higher and sperm motility significantly (P<0.05) lower in summer than in other seasons. Regarding sperm morphology, normal sperm was significantly (P<0.05) higher in autumn than in winter and summer and major defects were lowest (P<0.05) in autumn. In frozen-thawed semen motility was significantly (P<0.05) improved in the ejaculates collected in autumn compared to winter, while viability showed no obvious differences. Our results clearly demonstrate that individual and seasonal differences occurred in semen quality of Franches-Montagnes stallions. Ejaculates collected in autumn (September, October, November) demonstrated good quality, especially regarding sperm morphology, and were more suitable for cryopreservation because of better motility in frozen-thawed semen collected during autumn than in winter.     

Characteristics of sperm motility in boar semen diluted in different extenders and stored for seven days at 18 degrees C

Although numerous extenders exist for diluting boar semen, little research has been conducted comparing commercial extenders with regard to maintaining sperm motility during storage. The objective was to use a computer- assisted sperm analysis system to assess motility of boar spermatozoa diluted in Beltsville Thawing Solution, Merck-III, Androhep-lite, Sperm Aid, MR-A, Modena, X-Cell, VSP, and Vital. Ejaculates from boars (n=10) were collected and sub-samples were diluted (35x10(6) spermatozoa/ml) in the different extenders and stored for seven days at 18 degrees 90C. Extender by day interactions were detected (p<0.01) and on each day post collection, there were numerically small, but statistically significant differences in characteristics of sperm motility among extenders. For example, on day 7, the percentages of motile and progressively motile spermatozoa were highest (p<0.05) in X-Cell (90.7%) and Modena (63.9%), respectively. The average velocity measured over the actual point-to-point track followed by the sperm cell (VCL; 198.2 microm/s) and path velocity of the smoothed cell path (VAP; 106.4 microm/s) were highest (p<0.05) in Vital and Modena, respectively. Average velocity measured in a straight line from the beginning to the end of the track (VSL; 78.3 microm/s), average value of the ratio VSL/VAP (straightness; 73.2) and average value of the ratio VSL/VCL (linearity; 44.1) on day 7 were highest in Androhep-lite. In summary, changes in sperm motility during storage were affected by the extender utilized, but with the exception of Sperm Aid, all extenders maintained a high degree of sperm motility through 7 days of storage.     

Caffeic acid improves microscopic sperm parameters and antioxidant status of buffalo (Bubalus bubalis) bull semen following freeze-thawing process

This study investigates the effects of cryopreservation and supplementation of buffalo's semen with Caffeic acid. It studies the effects of different Caffeic acid concentrations on cryopreservation capacity of the buffalo and evaluates their influence on various sperm parameters like motility, viability, progressive motility, sperm plasma membrane integrity, and antioxidant status. Twenty-four semen samples were collected with an artificial vaginal from three adult water buffalos. The semen samples were evaluated and the qualified ejaculates were separated and were diluted in a Tris-based extender. The resulting samples were classified into 5 groups: No antioxidant (control), Control sham (NaOH), Caffeic acid 50 μM, Caffeic acid 100 μM, and Caffeic acid 200 μM. The semen samples encountered cryodamage and the quality was deteriorating during the cryopreservation (P < 0.05). The semen evaluation after thawing showed that the groups of samples receiving 100 μM Caffeic acid had higher viability, total motility, and lower abnormal sperm and better linearity (LIN), curvilinear velocity (VCL), straight-line velocity (VSL) and path velocity and higher intact plasma membrane (P < 0.05) compared to other groups. It is notable that adding 100 μM Caffeic acid to freezing extenders enhances the CAT, GPx, SOD, and GSH and also ameliorates total antioxidant capacity of spermatozoa after thawing. It is notable that the addition of 100 μM Caffeic acid decreases the amount of Malondialdehyde. These reactions lead us to conclude that 100 μM Caffeic acid enhances the semen quality of water buffalo after thawing.     

Zwitterionic buffers preserve ram semen quality more efficiently than TRIS during storage at 15 °C

This study was designed to evaluate the effect of various buffers on the storage of ram semen at 15 °C. Second ejaculates from six adult males were collected using an artificial vagina and diluted in either MOPS, TRIS, TES, HEPES, citrate, or phosphate-based extenders. Semen samples were stored at 15 °C and the sperm motility and viability (membrane integrity) variables assessed after 0, 24 and 48 h intervals. Significantly higher progressive sperm motility rates were recorded at 0 h of storage, and higher motile and progressive sperm motility at 24 and 48 h, when zwitterionic-based extenders (MOPS, TES and HEPES) were used, compared to citrate, TRIS, and phosphate-based extenders—with the last group showing a drastic reduction in sperm motility during storage. The zwitterionic groups were also superior to the other treatments in terms of sperm velocity (straight line velocity, VSL; curvilinear velocity, VCL; average path velocity, VAP) at 0 h of storage, although at 24 and 48 h the differences were minimal in the CITRATE group—regarding all velocity variables, and in the TRIS group, regarding the VCL parameter. Sperm diluted in the TRIS-based extender showed a marked increase in the proportion of circular sperm trajectories (lower sperm linearity, LIN, and straightness, STR), and a decrease in the VAP. The reduction in the vigour of the sperm in the TRIS extender (measured by VCL) was less pronounced than in the other groups. At the same time, VSL was reduced, as more sperm moved in circles, and the amplitude of lateral head displacement (ALH) was also dramatically increased. The CITRATE diluent recorded intermediate results—between that of TRIS and the other treatment groups, regarding the variables related to the quality of sperm movement at 0 h storage. However, following CITRATE dilution, semen underwent a clear improvement after a period of 24 and 48 h, so that differences with the zwitterionic groups were attenuated or disappeared. Similarly, the CITRATE group obtained similar or higher viable sperm values, compared to zwitterionic buffers during storage. The TRIS, and particularly the PHOSPHATE diluents, recorded poorer sperm viability after 24 and 48 h of storage. It was concluded that zwitterion-based buffers may be an acceptable alternative for inclusion in the composition of diluents for chilled ram semen storage. On the other hand, TRIS may be seen as having caused drastic modifications of certain sperm kinematic parameters during storage at 15 °C.     

Effect of technical settings on canine semen motility parameters measured by the Hamilton-Thorne analyzer

Computerized measuring devices are needed to assess canine semen quality objectively both for research and practical purposes. As internal image settings may influence the results considerably, the effect of different technical settings and semen processing on the parameters assessed by the Hamilton-Thorne Ceros 12.1 semen analyzer (HTR Ceros 12.1) was investigated. The frame rate (15, 30 or 60 frames/s) significantly (P<0.05) influenced most of the measured motility characteristics in experiment 1 while no differences in the motility parameters were found using a different sampling duration (0.5 or 1 s, i.e. 30 or 60 frames scanned) in experiment 2. In experiment 3, an increase in sperm velocity (VAP, VSL, VCL), in linearity and in the percentage of motile and rapidly moving spermatozoa was observed with increasing sperm concentrations (25 x 10(6), 50 x 10(6) or 100 x 10(6) ml(-1)). In experiment 4, a clear effect of the diluent used was visible with higher velocity parameters (VAP, VSL, VCL) and higher percentages of motile, progressive and rapid spermatozoa for semen samples diluted in Hepes-TALP or prostatic fluid in comparison with physiological saline or egg-yolk-Tris extender. In experiment 5, significant (P<0.01) and high correlations were found between the conventional dog semen analysis methods and HTR Ceros 12.1 measurements (n=97 semen samples) for the sperm concentration (r=0.91), the motility (r=0.74) and the progressive motility (r=0.84). In experiment 6, the ejaculates from 21 proven, fertile dogs were compared with the ejaculates of a population (N: 11) of young beagles (1.5 years) but no significant differences in HTR Ceros 12.1 measurements were found between the two groups. Based on our results, diluting dog semen samples to 50 x 10(6) ml(-1) with physiological saline solution and scanning 30 frames at a frame rate of 60 frames/s (i.e. a scanning time of 0.5 s), are the set-up parameters proposed to obtain objective and standardized canine semen motility results using the HTR Ceros 12.1.     

Effect of single layer centrifugation using Androcoll-E-Large on the sperm quality parameters of cooled-stored donkey semen doses

The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Significant differences (P<0.05) were found between SLC-selected and unselected semen samples for all parameters assessed, obtaining better results after SLC. In the second experiment, semen samples were classified in two groups according to their sperm progressive motility (PM) before SLC. Then, the increments obtained in semen quality parameters after SLC were compared between groups. No significant differences were found between groups, indicating that SLC improved the sperm quality parameters of entire set of semen samples processed with independence to their original PM. In conclusion, SLC with Androcoll-E-Large can be used in donkeys, increasing the sperm quality of cooled-stored donkey semen doses after 24 h of cool storage.     

Effects of surrounding fluid on motility of hyperactivated bovine sperm

Mammalian spermatozoa in organisms with internal fertilization are required to swim in the cervical and oviductal mucus, whose rheological properties differ substantially from those of water. Moreover, on the way to the oviduct, a change in sperm motility called hyperactivation may occur. In the present study, we focused on the motion characteristics of hyperactivated bovine sperm and investigated the effect of the surrounding fluid on motility. We prepared two kinds of polyacrylamide with high-viscosity non-Newtonian fluid properties, similar to the actual cervical and oviductal mucus. Using semen from Japanese cattle, we evaluated curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). Additionally, we estimated linearity (LIN), straightness (STR), and wobble (WOB) as sperm motility parameters for several surrounding fluids. We successfully induced hyperactivation of bovine sperm in high-viscosity non-Newtonian fluid. Hyperactivation resulted in an increase in VCL and a decrease in VSL. In the high-viscosity non-Newtonian fluid, the hyperactivated sperm moved in a zig-zag pattern with regularity, different from the movement observed in a diluted solution. The increase in WOB in the non-Newtonian fluid suggests that hyperactivated sperm efficiently progress along the groove that exists on the oviductal mucus wall. These results improve our understanding of the motility of bovine sperm when they undergo hyperactivation in the actual cervical and oviductal mucus.     

Computerized analysis of motility, motility patterns and motility parameters of spermatozoa of carp following short-term storage of semen

A computer-aided semen analysis system was used to assess the % motile cells following storage of carp semen in 11 different buffers at 2, 5 or 22° C. BWW and TLP were the most suitable storage buffers because carp semen stored at 5° C in these buffers following activation showed no significant decrease in % motile spermatozoa up to 24 h. But, in most of the other buffers (Fish Ringer, Cytomix, Cortland, FRT, Mannitol, FPS, NAS and TSM) the motility potential was lost by 2 h. Storage was best at pH 6–9 and at 5° C. Carp spermatozoa exhibit three distinct motility patterns, namely 'linear', 'circular' and 'haphazard', the proportion of spermatozoa with a particular motility pattern depending on storage buffer and time. All spermatozoa with a linear trajectory had high VSL, STR and LIN; those moving in circles had low VSL, STR, LIN and BCF and those with a haphazard trajectory were distinct in that they had the highest ALH and their VSL, STR, LIN and BCF were higher than the circular moving spermatozoa and lower than the spermatozoa exhibiting linear trajectory. The study also demonstrates a pronounced time-dependent decrease in VCL, VAP, VSL and ALH of carp spermatozoa following activation with water or low osmolality solutions. This study provides for the first time data related to seven motility parameters of carp spermatozoa and demonstrates how these parameter values could be used to evaluate quality of carp milt following storage in different buffers. It confirms that carp spermatozoa exhibit linear or circular trajectories and provides evidence for a third type of trajectory described as haphazard. All three motility patterns could be discriminated objectively on the seven motility parameters.     

Semen characteristics and their ability to predict sperm cryopreservation potential of Atlantic cod, Gadus morhua L

There is a lack of biomarkers or indices that can be used to predict the quality of fish semen samples following the freezing and thawing cycle. In the present study, a series of semen indices were tested to assess if they could accurately forecast the cryopreservation potential of Atlantic cod (Gadus morhua) semen. Fresh and frozen-thawed sperm activity variables were compared, and relationships between frozen-thawed sperm activity and fertilization success were examined. In comparison with fresh sperm, activity variables of frozen-thawed spermatozoa were reduced. Of the 18 males examined, mean (± SEM) spermatocrit of fresh sperm was 40.72 ± 4.23%, osmolality of the seminal plasma 366.32 ± 4.95 mOsmol/kg, pH 8.32 ± 0.04, protein concentration 1.05 ± 0.08 mg/mL, anti-trypsin activity 153.83 ± 19.25 U/L, and total antioxidant capacity 0.15 ± 0.03 μmol Trolox equivalents/mL. Frozen-thawed fertilization success was highly variable among males with values ranging from 18.5 to 90.2%. Regressions yielded significant positive relationships between frozen-thawed motility, velocity, track crossing frequency, and subsequent fertilization success. Sequential multiple regressions explained up to 95% of the variation in frozen-thawed sperm activity. Spermatocrit and pH of fresh semen were negatively related, whereas osmolality and antioxidant capacity were positively related to frozen-thawed motility and velocity. Each of these indices can be measured within minutes of collecting a fresh sample of semen and are thus early indicators of the capacity of semen samples to withstand cryopreservation. These results have many benefits for conservation of wild stocks, aquaculture production, and for understanding semen biology and cryobiology of fishes.