Sperm Motility Characteristics Research Articles

Changes in Motility Characteristics of Goat Spermatozoa During Glycerol-Equilibration and the Relevance to Cryopreservation

Artificial Insemination (AI) with frozen Boer goat semen could be a viable option for genetic upgradation of non-descript local goats. To maximize the utilization of Boer germplasm, minimizing the losses due to poor freezability and improving the quality of frozen semen are the areas to be addressed. A study was conducted to assess the deterioration in the quality of semen in terms of motility characteristics of spermatozoa during the processing of semen for cryopreservation using Computer Assisted Semen Analysis (CASA) technique. A highly significant decline in sperm motility, progressive motility, path velocity and progressive velocity was noticed during equilibration. Semen samples with acceptable freezability differed from samples not freezing well by not showing any significant decrease in velocity characteristics of the spermatozoa during glycerol­ equilibration. The decline was significant in semen samples that froze poorly. Comparing the motility characteristics of spermatozoa during glycerol-equilibration in the good and poor samples, the progressive velocity in poor samples was significantly lower than that of good samples at 3 and 4 h of equilibration. The curvilinear velocity of spermatozoa was also significantly less in poor samples than good samples after 4 h. These results indicate that veloci ty of sperm motion plays a vital role in determining the post-thaw quality of frozen semen. Cryopreservation significantly affected the quality of frozen semen by lowering all the motion characteristics post-thawing. This study also revealed that the duration of exposure of spermatozoa to the cryoprotectant during equilibration is critical in cryopreservation of Boer cross goat semen. A shorter duration of2 h of equilibration with glycerol at SoC may be beneficial for Boer goat semen cryopreservation.

76 COMPUTER-ASSISTED SPERM ANALYSIS OF FROZEN SPERM MOTILITY AFTER DIFFERENT TIMES OF EXPOSURE AT 15°C

One of the key factors for successful long-term cryopreservation in liquid nitrogen is maintaining the samples at –130°C or lower at all times to avoid cell damage (Barth 1991 Proc. 10th Ann. Conv. Am. Embr. Transf. Assoc., 20–26). Previous data indicated that exposure of the semen straw to ambient temperature for more than 15 s can raise the temperature above –130°C and reduce sperm motility, as determined by subjective evaluation (Berndtson et al. 1976 Proc. 6th NAAB Tech. Conf. Artif. Insem. Reprod., 51–60). The computer-assisted semen analysis (CASA) system provides an opportunity to assess multiple motility characteristics on a semen sample objectively and with high repeatability. An experiment was designed to evaluate the effect of exposing frozen semen in 0.5-mL straws to room temperature for 15, 30, 60, or 120 s on motility characteristics assessed by CASA system. Twenty-eight ejaculates from different bulls (19 Angus, 7 Hereford, 1 Brangus, 1 Shorthorn) were diluted using a chemically semi-defined media (Andromed, Minitüb, Tiefenbach, Germany) and frozen in an automatic freezer (Digicool, IMV, Paillette Crista, France). Five frozen straws per bull were used, one for each time of exposure and one as control (0 s = 0 time). Straws were exposed to room temperature (15°C ± 0.78) for different times and then placed back into liquid nitrogen. Semen thawing was conducted in a water bath at 37°C for 1 min. Motility characteristics were evaluated by the IVOS SpermAnalyzer (Hamilton Thorne Research, Beverly, MA, USA). Two chambers of 20-μm depth and 5 fields per chamber were analyzed (30 frames/0.5 s for each field). Seven motility parameters were evaluated: motile sperm (%), progressive sperm (%), VAP (path velocity, μm s–1), VCL (track speed, μm s–1), ALH (lateral amplitude, μm), BCF (beat frequency, Hz), and LIN (Linearity, %). The Kruskal–Wallis test was used to compare variables among groups, and results are shown in Table 1. There is a significant difference (P < 0.05) in the % of motile and progressive sperm when time of exposure was increased. There was a drastic and significant reduction in the percentage of motile and progressive sperm when exposure to 15°C was longer than 30 s. The live cells had similar motile characteristics: VAP, VCL, ALH, BCF, and LIN. In conclusion, sperm motility would be affected if straws are exposed for more than 30 s. More research should be done to test higher room temperatures, detect viability effects, and determine pregnancy rates after AI. Table 1. CASA of frozen sperm motility characteristics at different times of exposure at 15°C This research was supported by Centro Genetico Bovino Eolia S.A.

Effect of extender, centrifugation and removal of seminal plasma on cooled-preserved Amiata donkey spermatozoa

In the donkey species, the application of cooled semen artificial insemination could aid the survival of endangered breeds. Fifteen ejaculates collected from three Amiata donkeys were used to evaluate the effect of three extenders on spermatozoal motility characteristics after cooling and preservation for up to 72 h. Semen was diluted at a 1:4 semen:extender ratio in INRA96, INRA82 and INRA82 added of 2% centrifuged egg yolk (INRA82-Y) and motility was evaluated by the computerized analyzer CEROS 12.1 at hours 0, 24, 48 and 72. Total motility, and rapid spermatozoa after 24, 48 and 72 h of preservation were higher in INRA82-Y than in INRA96 or INRA82, as was progressive motility after 72 h. INRA82-Y was thus used in a second study, where the effects of centrifugation and of removal of seminal plasma on cooled donkey semen were evaluated on 12 ejaculates from four males. Rapid spermatozoa after 24 and 72 h, and total motility after 72 h were better preserved in the non-centrifuged samples than when seminal plasma was removed, the contrary was true for the proportion of spermatozoa keeping their progressive motility at hour 48. In conclusion, INRA82-Y kept sperm motility characteristics during cooled storage better than INRA82 or INRA96, and removal of seminal plasma during in vitro preservation did not seemed advantageous. Further studies are needed to better understand the changes in motility patterns of donkey spermatozoa caused by seminal plasma and semen extenders, and their relation to fertility.

Effects of Extracellular Adenosine 5′-Triphosphate on Human Sperm Motility

Extracellular adenosine 5'-triphosphate (ATP) previously has been shown to increase the fertilization percentage in human in vitro fertilization (IVF) performed for male factor infertility. The objective of this study is to determine the effects of extracellular adenosine 5'-triphosphate (ATPe) on human sperm function by examining its effects on end points of sperm capacitation. Sperm obtained from healthy volunteers with normal semen parameters, asthenozoospermic men, and cryopreserved samples were incubated in medium with or without 2.5 mM ATPe. The effects of ATPe on acrosomal exocytosis, protein tyrosine phosphorylation, and sperm motility parameters were quantified. Although ATPe did not affect acrosomal exocytosis or protein tyrosine phosphorylation in sperm from healthy donors, it significantly altered several motility parameters, with the largest effects manifested in increased curvilinear velocity and percentage hyperactivation. ATPe similarly affected sperm selected for poor motility and thawed cryopreserved sperm but to a lesser extent than its effects on sperm with normal motility. ATPe increased straight-line velocity and linearity of sperm obtained from asthenozoospermic men. Human sperm motility characteristics are altered by ATPe; this finding may explain its previously reported beneficial effect on human IVF. These results suggest that ATPe could constitute a new therapeutic modality in the treatment of male infertility.

Polymorphism of transferrin of carp seminal plasma: Relationship to blood transferrin and sperm motility characteristics

Transferrin (Tf) is a major protein of carp ( Cyprinus carpio) seminal plasma. Its relationship with milt quality is unknown. In this study, we sought to determine if Tf is polymorphic in carp seminal plasma and if this polymorphism is related to sperm motility characteristics. We screened males of purebred common carp line (Polish line R6) for Tf polymorphism in blood plasma. The majority of Tf genotypes represented only DD and DG variants. We then collected milt from preselected DD and DG genotypes and tested their sperm motility characteristics using computer-aided sperm analysis (CASA). Tf polymorphism in seminal plasma was found to be identical with that of blood. However, the relationships between Tf polymorphism and iron metabolic parameters were different for blood and semen. These data suggest different regulation of Tf in liver and testis. We found substantial differences in sperm motility characteristics between both genotypes. Spermatozoa of DG males were characterized by lower curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), higher linearity (LIN) and straightness (STR) of movement as compared to DD males. No differences were found in other sperm characteristics such as sperm concentration and percentage of sperm motility. Our results suggest that sperm motility parameters are related to Tf polymorphism and therefore this polymorphism may be related to sperm competitive ability.

Improving the fertilizing ability of sex sorted boar spermatozoa

The sex sorting of spermatozoa by flow cytometry induces damage, since sperm cells are highly diluted, affecting their functionality and fertilizing ability. In this work it was investigated whether the concentration of sex sorted spermatozoa by the sedimentation method, rather than centrifugation, in combination with the presence of the seminal plasma protein PSP-I/PSP-II heterodimer may improve their fertilizing ability. Spermatozoa were sorted by flow cytometry and collected in BTS with 10% of seminal plasma (group C: control) or with 1.5 mg/mL of PSP-I/PSP-II heterodimer (group H). Collected spermatozoa from each medium were split into two aliquots. One aliquot of each group was centrifuged (800 × g/5 min) just after sorting and stored 16–18 h at 17 °C (groups Cc and Hc) at 6 × 10 6 sperm/mL. The second aliquot was directly stored at 17 °C for 16–18 °C (group Cs and Hs). After storage the supernatant was discarded and the sedimented pellet adjusted to 6 × 10 6 sperm/mL. Membrane integrity, acrosome status and motility characteristics of spermatozoa from all groups were assessed. Post-weaning pre-ovulatory sows were inseminated by laparoscopy into the oviduct with 0.3 × 10 6 sex sorted spermatozoa to assess their ability to penetrate oocytes in vivo. Putative zygotes were collected 18 h after insemination by washing the oviduct. Penetration and monospermic rates were evaluated. After 16–18 h of storage, centrifuged spermatozoa collected with 10% seminal plasma or 1.5 mg/mL PSP-I/PSP-II heterodimer after sex sorting showed lower ( p < 0.05) percentages of membrane integrity, motility and fertilization than sedimented spermatozoa. Overall, the presence of 10% seminal plasma or PSP-I/PSP-II heterodimer did not affect the results. However, a positive effect of PSP-I/PSP-II heterodimer ( p < 0.05) was observed in sedimented spermatozoa. Hence, our results indicate that the sedimentation method in the presence of PSP-I/PSP-II heterodimer improves the in vivo fertilizing ability of sex sorted boar spermatozoa.

Autologous Whole Ram Seminal Plasma and its Vesicle-free Fraction Improve Motility Characteristics and Membrane Status but not In Vivo Fertility of Frozen?Thawed Ram Spermatozoa

Motility characteristics (assessed subjectively and with computer-assisted semen analysis) and membrane status (after staining with chlortetracycline) of washed and non-washed frozen-thawed ram spermatozoa were evaluated after incubation in buffer and buffer containing autologous whole seminal plasma or one of its two fractions: the pellet of membrane vesicles obtained by ultracentrifugation (and used at three times normal protein concentration) or the vesicle-free supernatant fraction. Whole seminal plasma and supernatant, but not membrane vesicles, improved the motility characteristics of spermatozoa after 3 and 6 h of post-thaw incubation compared with the control buffer. Resuspension and incubation with whole seminal plasma, supernatant or membrane vesicles lowered the proportion of acrosome-reacted frozen-thawed spermatozoa compared with the control buffer. Unwashed frozen-thawed semen from three rams, incubated with autologous whole seminal plasma or its fractions and inseminated using cervical or intrauterine artificial insemination, had no effect on pregnancy rates of ewes in synchronized oestrus. However, fertility was higher after laparoscopic than cervical insemination (44.9 vs 12.3%, p < 0.001). In conclusion, resuspension and incubation of frozen-thawed ram spermatozoa in autologous whole seminal plasma or its vesicle-free supernatant fraction improved their motility characteristics and, with membrane vesicles, membrane status, but these benefits were not reflected in improved fertility after cervical or intrauterine insemination.

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.

The influence of homogenous zona pellucida on human spermatozoa hyperactivation, acrosome reaction and zona binding

The study aimed to evaluate the changes in sperm motion characteristics and the occurrence of hyperactivation among sperm populations after exposure to human zona pellucida. Motile spermatozoa samples were used to evaluate the sperm-zona binding capacity, zona-induced acrosome reaction and changes in sperm motion characteristics. Sperm motion characteristic changes studied included straight line velocity, curvilinear velocity, amplitude of lateral head displacement, straightness and beat cross frequency. Recordings were performed on semen immediately after liquefaction, 3 h capacitation and after exposure to solubilised human zona pellucida. The semen samples were divided into morphology categories, namely six (16 +/- 1.4% normal forms, normal patterns), 31 (8 +/- 1.7% normal forms, G-pattern) and 27 (3 +/- 1.3% normal forms, P-pattern). The Hemizona Indices for the three morphology groups namely normal, G-patterns and P-patterns, were 77 +/- 6%, 61 +/- 5% and 41 +/- 5% respectively (P <or= 0.05). The zona-induced acrosome reaction for the three groups were 25.0 +/- 2% (normal group), 18.0 +/- 2% (G-patterns) and 14.1 +/- 0.6% (P-pattern) (P <or= 0.05). The number of hyperactivated sperm showed an increase in the ejaculates (1%), after capacitation (2%) and exposure to zona pellucida (5%) (P <or= 0.05). When divided into the three morphology groups, the number of spermatozoa with hyperactivated motility was more profound in men with normal morphology compared with G- and P-pattern men. Human zona pellucida is a strong inducer of hyperactivated motility among spermatozoa and the effect is more profound among sperm populations with normal morphology.

Effect of Body Weight, Age and Breeding History on Canine Sperm Quality Parameters Measured by the Hamilton‐Thorne Analyser

During the last decade, several computer assisted sperm analysis (CASA) systems have been validated for canine sperm quality assessment. Regarding the impressive possibilities of these systems, further research is required to determine which CASA measurements are of clinical importance in canine andrology. In the present study, the sperm quality parameters obtained by the Hamilton-Thorne Semen Analyser (Ceros 12.1; HTR) were correlated with the body weight and the age of the dogs. Moreover, the sperm quality parameters of dogs with a different breeding history were compared. The sperm-rich fraction was collected from 111 dogs of 50 different breeds, which were presented at our department. Immediately after collection, the concentration, the total sperm output (TSO) and 13 different sperm motility and velocity characteristics were measured by the HTR. The percentage of live spermatozoa and the spermatozoal morphology were examined on eosin/nigrosin stained smears. Based on their breeding history, the dogs were divided in three groups: 'fertile' (n = 60), 'subfertile' (n = 17) or 'not used for breeding' (n = 34). Significant (p < 0.05) correlations were established between the body weight of the dogs and the TSO (r = 0.245) and velocity curvilinear (VCL; r = -0.220), respectively. The age was negatively correlated with the percentage of normal spermatozoa (r = -0.203; p < 0.05). The correlations with all the other evaluated sperm parameters were low and not significant. Significant differences between the 'fertile' and the 'subfertile' group were found for all of the evaluated sperm quality parameters (except for BCF, LIN, STR and MEDIUM). In conclusion, dogs tend to produce ejaculates with a lower percentage of normal spermatozoa with increasing age and dogs with higher body weights produce ejaculates with a higher TSO and a lower VCL. Significantly poorer sperm characteristics were found for dogs with lower in vivo fertility results.

Computer automated motion analysis of crossbred bull spermatozoa and its relationship with in vitro fertility in zona-free hamster oocytes

The objective of this study was to determine the effective relationship between different motion characteristics of bull spermatozoa assessed by computer assisted semen analyzer (CASA) and in vitro fertilization percentage in zona-free hamster oocytes. A total of 64 frozen semen samples from 16 different crossbred bulls ( Bos taurus × Bos indicus) with four ejaculates from each bull were taken for analysis. Various motion characteristics of spermatozoa like progressive motility, path velocity, progressive velocity, beat cross frequency, straightness and linearity were recorded. Hypo-osmotic swelling test and sperm penetration bioassay were conducted to assess the membrane integrity and fertilization percentage of spermatozoa respectively. Significant positive correlation ( P < 0.01) was found between fertilization percentage and progressive motility ( r = 0.791) and between velocity parameters (VAP; r = 0.612 and VSL; r = 0.625) and fertilization percentage. Among different CASA variables, progressive motility alone contributed to 62.6% variation in the fertilization percentage. The velocity measurements (VAP and VSL) together with progressive motility and %HOS spermatozoa contributed to 66.1% of variation in fertilization percentage which was found to be significant ( P < 0.05).

97 COMPARISON OF MOTILITY AND ACROSOMAL INTEGRITY OF ELECTRO-EJACULATED AND EPIDIDYMAL RAM SPERM AFTER EXPOSURE TO VARIOUS ANISOSMOTIC SOLUTIONS, CRYOPROTECTIVE AGENTS, AND TEMPERATURES

Effective ram sperm cryopreservation protocols, which would yield acceptable lambing rates following AI, are currently lacking. The objectives of the current studies were to compare the effects of various anisosmotic conditions, cryoprotective agents (CPA), and temperatures on the motility and acrosomal integrity of electro-ejaculated and epididymal ram sperm. Three experiments were conducted. In Experiment 1, electro-ejaculated and epididymal ram sperm were exposed to 75, 150, 225, 600, 900, and 1200 mOsm kg–1 sucrose solutions, held for 5 min, and then returned to isosmotic conditions. Motility characteristics of sperm during exposure to each anisosmotic solution and after return to isosmotic conditions were determined. In Experiment 2, electro-ejaculated and epididymal ram sperm were exposed to 1 m glycerol (Gly), dimethyl sulfoxide (DMSO), ethylene glycol (EG), or propylene glycol (PG) for 5 min and then returned to isosmotic conditions. Motility characteristics of sperm samples during exposure to each CPA solution and after return to isosmotic conditions were determined. In Experiment 3, effects of various temperatures on motility characteristics of electro-ejaculated and epididymal ram sperm were determined after exposure to three different sub-physiologic temperatures (4, 10, and 22�C) for 30 min and subsequently return to 37�C. A computer-assisted semen analysis system was used to determine sperm motility characteristics. Epifluorescence microscopy was used to determine sperm acrosomal integrity after fixing and staining with Alexa Fluor-488-PNA (Molecular Probes, Eugene, OR, USA). The data were analyzed by ANOVA by using SAS (SAS Institute, Inc., Cary, NC, USA). The motility of electro-ejaculated ram sperm was significantly more affected by anisosmotic stress than was that of epididymal ram sperm (P &lt; 0.05). While anisosmotic stress had no effect on acrosomal integrity of epididymal ram sperm, there was a significant reduction in acrosomal integrity for electro-ejaculated ram sperm after the addition and removal of a 75 mOsm sucrose solution. The abrupt addition and removal of 1 m Gly, DMSO, EG, or PG had no effect on the motility and acrosomal integrity of epididymal ram sperm (P &gt; 0.05). However, there was a slight decrease in acrosomal integrity for electro-ejaculated ram sperm after exposure to 1 m Gly, DMSO, or EG (P &gt; 0.05). Both epididymal and electro-ejaculated ram sperm exhibited temperature-dependent loss of motility and acrosomal integrity (P &lt; 0.05). However, electro-ejaculated ram sperm were more sensitive to chilling stress than were epididymal sperm (P &lt; 0.05). In conclusion, current data overall suggest that while epididymal ram sperm are extremely resilient to various cryobiologically relevant stress conditions, ejaculated ram sperm demonstrate greater sensitivity to such stressors. These current findings should be taken into account when handling and developing cryopreservation protocols for ejaculated and epididymal ram sperm.

162 FREEZING OF SEMEN FROM ENDANGERED ASTURIANA DE LA MONTAÑA BULLS IN ZWITTERONIC LIPIDS-BASED EXTENDERS

This experiment was designed to test the efficacy of 2 different preparation protocols of zwitteronic soyabean-origin lipids for the production of lipidsglycerol liposomes for use in bull semen cryopreservation. Lipids liposomes were prepared at 10% concentration in Tris buffer by 1. highpressure homogenization (Panda 2K, Parma, Italy) and then 8% glycerol were added, extender-1 (E-1); Lipids were homogenized together with glycerol, extender-2 (E-2). Bioxcell extender (E-3) was used as control. Semen was collected 3 times from 3 endangered Asturiana de la Monta�a bulls by the means of an artificial vagina. Ejaculates with at least 70% motility were processed further by a standard freezing protocol used in our AI station. Semen was diluted at 37�C with each of the 4 extenders to a concentration of 92 � 106 spermatozoa per mL, cooled to 4�C over 4 h, aspirated into 0.25-mL plastic straws (IMV Technologies, Aigle, France), frozen in a bio-freezer (IMV Technologies) in 3 steps from 4 to –140�C, and then plunged into liquid N2. Straws were thawed in a water bath at 37�C for 30 s. Sperm motility was analyzed microscopically immediately after collection, after dilution, and after 4, 24, 48, and 72 h of storage at 4�C. Post-thaw semen progressive motility was assessed microscopically, and sperm movement characteristics were analyzed by computer-assisted semen analysis (CASA) (SCA�, Microptic, Barcelona, Spain). Data were compared between extenders and bulls by 2-way ANOVA; percentages were transformed by arcsine transformation before ANOVA. Total and progressive sperm motility at 0 h after dilution ranged from 90 to 70% and was not different between extenders and bulls. There was no difference between bulls in total and progressive motility after 24 h of cold storage; however, both were significantly greater (P &lt; 0.05) for Control (62.4 � 14.7 and 41.4 � 14.9) and E-1 (70.1 � 12 and 33.8 � 7.0) extenders than for the E-2 extender (22.5 � 17 and 1.2 � 1.3). Average post-thaw sperm motility was not different between bulls for either extender, but motility for Bioxcell (Control, 48.1 � 14.6%) and E-1 extenders (43.2 � 13.0%) were significantly greater (P &lt; 0.05) than for E-2 extender (18.7 � 8.8%). There were no differences between bulls for all kinematic semen parameters: curvilinear (VCL), straight line (VSL), average path (VAP) velocities, linearity (LIN) and straightness (STR), evaluated by CASA before and after freezing; however, all were lower (P &lt; 0.05) for the E-2 extender and not different between Control and E-1 extenders. Sperm movements derived from heads (VCL) and linearity of sperm(LIN), both closely related to field fertility, were in the range of 90.9 � 2.1 and 63.0 � 5.5 for E-3 (Control) extender; 99.1 � 3.4 and 49.4 � 3.5 for E-1; and 21.8 � 2.2 and 29.9 � 4.0 for E-2. In summary, zwitteronic soyabean lipid liposomes are an effective egg yolk substitute for the cryopreservation of Asturiana de la Monta�a bull semen; however, the homogenization protocol of the lipids-glycerol mixture must be improved.

229 EFFECTS OF NONYLPHENOL ON MOTILITY, ACROSOMAL INTEGRITY, AND MITOCHONDRAL MEMBRANE POTENTIAL OF EPIDIDYMAL RAT SPERM

Nonylphenol (NP), a derivative of an estrogenic environmental endocrine disrupter called alkylphenol polyethoxylates (APEs), has been shown to have toxic, estrogenic, and carcinogenic effects on both aquatic and terrestrial organisms. NP is widely used in common cosmetic products and detergents. The primary objective these studies was to determine the effects of NP (dissolved in either ethanol or dimthyl sulfoxide (Me2SO)) on rat sperm viability characteristics such as motility, acrosomal integrity, and mitochondrial membrane potential. The epididymal rat sperm were suspended (50 � 106 spermatozoa mL–1) in sodium bicarbonate-buffered Tyrode's lactate solution containing 3 mg mL–1 bovine serum albumin (BSA) after removal of the cauda epididymis of sexually mature Sprague Dawley male rats (n = 5). Aliquots of 0.5 mL sperm suspension were added into 5-mL test tubes containing 1, 10, 100, 250, 500, and 1000 µg NP mL–1. As negative controls, no NP–no solvent, no NP+ethanol, and no NP+Me2SO were used. All sperm treatments were incubated for 1, 2, 3, and 4 h at 37�C with 5% CO2 in air. Computer-aided sperm analysis system (Hamilton Thorne, Beverly, MA, USA) was used to determine sperm motility characteristics. An epifluorescent microscopy method was used to determine sperm acrosomal status after staining with 20 µg mL–1 Alexa Fluor-488-PNA (Molecular Probes, Eugene, OR, USA) and a flow cytometric method was used to determine mitochondrial membrane potential after staining with 2 µg mL–1 JC-1 (Molecular Probes). The data were analyzed by ANOVA by using SAS (SAS Institute, Inc., Cary, NC, USA). The results showed that, as compared to negative controls (no NP–no solvent, no NP+ethanol), exposure of rat sperm to 250, 500, or 1000 µg NP mL–1 was highly detrimental to their motility (P &lt; 0.05). Total loss of sperm motility was observed after exposure to 500 µg NP mL–1. However, the time course did not seem to have a significant effect on sperm motility (P &gt; 0.05). Nonylphenol induced dose-dependent acrosomal reaction in rat sperm (P &lt; 0.05). The effects of NP on the mitochondrial membrane potential (MMP) of rat sperm were also significant (P &lt; 0.05). The percentage of rat sperm with high MMP declined drastically after exposure to 100 and 250 µg NP mL–1 as compared to negative controls (P &lt; 0.05). However, duration of exposure to NP did not have an effect on MMP of rat sperm (P &gt; 0.05). In conclusion, these data obtained from epididymal rat sperm provide important information about the mechanism of detrimental effects of NP on mammalian sperm, which must be seriously considered in the case of short- or long-term exposure to NP in humans as well as livestock species.

Use of computer-assisted sperm analysis (CASA) to evaluate the quality of cryopreserved sperm in red seabream ( Pagrus major)

In the present study, the quality of post-thaw sperm of red seabream Pagrus major frozen with 6–24% DMSO was investigated. The motility, average path velocity and fertilizing capacity of fresh and their corresponding post-thaw sperm were examined for evaluation of the post-thaw sperm motion characteristics and its association with fertilizing capacity. An analysis of sperm motility before and after cryopreservation has been performed using computer-assisted sperm analysis (CASA). For post-thaw sperm frozen with 12–21% DMSO, the percentages of motile sperm were not significantly ( P > 0.05) changed 10 s after activation. Moreover, the main motility pattern and swimming velocity of the motile post-thaw sperm were not significantly ( P > 0.05) changed and the progressive linear motion was still the dominant pattern. However, the total motility of post-thaw sperm (72.3 ± 6.3%) 30 s after activation was ( P < 0.05) lower than the corresponding fresh sperm (82.7 ± 7.2%). Additionally, the fertilizing capacity of post-thaw sperm was investigated with a standardized sperm to egg ratio 500:1. There is a linear regression relationship between the percentage of motile post-thaw sperm and fertilizing capability. These data demonstrate that 12–21% DMSO can provide good protection to the sperm during the freezing–thawing process.

Effects of liquid storage on amidase activity, DNA fragmentation and motility of turkey spermatozoa

Short-term liquid storage of turkey semen is of great interest in the management of turkey reproduction due to the extensive use of artificial insemination. This study examined changes in DNA fragmentation (using a comet assay), sperm motility characteristics (using computer-aided sperm analysis), and amidase activity (using a colorimetric assay) of turkey sperm stored for 24 and 48 h. In addition we found that turkey spermatozoa contain besides acrosin, additional two serine proteinases of molecular weight of 34 and 42 kDa. We found that, after 48 h of liquid storage, decreases in sperm motility characteristics and increases in amidase activity and DNA fragmentation occurred. An increase of amidase activity was found after 24 h. Decreases in sperm motility and increase in DNA fragmentation were found after 48 h of storage. These data suggest that a decrease in turkey sperm quality during short-term storage is related to disturbances to the acrosome, presumably related to premature activation of acrosomal serine proteinases, and to a lesser extent a decrease in sperm motility characteristics and damage of sperm DNA.

Sperm motion characteristics of Garole × Malpura sheep evolved in a semi-arid tropical environment through introgression of FecB gene

The arid and semi-arid tropical climates of India are endowed with vast diversity of non-prolific sheep breeds. The Garole × Malpura sheep has been evolved in a semi-arid tropical environment by introgression of FecB gene via artificial insemination of Malpura ewes using diluted semen of prolific microsheep Garole and subsequently multiplied by inter se mating among Garole × Malpura halfbreds. The aim of the present study was to identify FecB mutation in sexually mature Garole × Malpura rams by forced RFLP-PCR of BMPR-1B gene and evaluate: (i) semen production and sperm motion characteristics of GM rams and (ii) influence of age and FecB genotype on their semen attributes. Semen was collected during autumn season from 12 donor rams by artificial vagina on 8 occasions at weekly interval. The overall means of traits which did not differed significantly with age or FecB genotyping were volume (0.72 ml), mass motility (4.44), sperm concentration (2721.56 × 10 6 ml −1), curvilinear velocity (134.51 μm/s), motility (81.3%), amplitude of lateral head displacement (6.24 μm), beat frequency (44.43 Hz), sperm head elongation (48.9%) and sperm head area (10.01 μm 2). The FecB genotyping had a significant effect ( P < 0.05) on percent linearity and rapid motile sperms, which did not vary significantly with age. Although sperm concentration was higher in FecB BB and FecB B+, compared to FecB ++ genotypes but the effect was non-significant. The age and FecB genotyping had significant effects ( P < 0.05) on straightness, average path velocity, straight-line velocity and percentage of medium or slow motile sperms. It is concluded that Garole × Malpura rams with introgressed FecB gene are capable of producing good quality semen in a semi-arid tropical climate.

P-843: Lipid peroxidation, Superoxide dismutase, catalase levels and sperm motility: Protective role of Vitamin C and resveratrol prior to sperm cryopreservation

It is estimated that the sperm motility decreases by 25% to 75% after cryopreservation. The reasons for the decline observed in sperm motion characteristics after cryopreservation is not clear, but seems to be related to factors such as sperm membrane stress induced during the freeze-thaw process. Vitamin C and resveratrol (a red wine antioxidant) are considered important antioxidants that may prevent the oxidative stress during the freeze-thaw process. Also, Superoxide dismutase (SOD) and catalase are important antioxidant enzymes that can quench excess free radicals such as: superoxide anion and hydrogen peroxide respectively.

Ram sperm motility after intermittent scrotal insulation evaluated by manual and computer-assisted methods

To study whether additional measurements of motility characteristics of spermatozoa by computer assisted semen analysis (CASA) were more sensitive indicators of reduced semen quality than estimates of percentages of motile, rapid or progressive cells. Intermittent scrotal insulation was applied to 6 rams for 16 h per day for 21 days or to 2 of these for 12 h per day for 28 days in the following year. Semen was collected and evaluated by CASA immediately and either frozen or stored at 30 degrees Celsius or 5 degrees Celsius before re-evaluation. Intermittent scrotal insulation caused falls in the percentage of motile, progressive and rapid sperm, as did freezing-thawing and storage at 30 degrees Celsius or 5 degrees Celsius. Motility characteristics (amplitude of lateral head displacement, mean path velocity, mean progressive velocity and curvilinear velocity), as determined by CASA fell only when the percentage of motile sperm was already reduced. Freezing and thawing or liquid storage of the semen from insulated rams caused a greater fall in the percentage of motile and rapid sperm than control semen, but only affected the motility characteristics when the percentage of motile sperm was already reduced. Intermittent scrotal insulation affected not only the motility of the freshly collected sperm, but also their ability to withstand the additional stress of storage. The additional data on motility characteristics obtained by CASA appeared to be no more a sensitive indicator than the percentage of motile cells of reductions in semen quality.

The Cryoprotective Effect on Frozen-thawed Boar Semen of Egg Yolk Low Density Lipoproteins

In order to protect the spermatozoa against cold shock, hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders for domestic animals. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). The effects of LDL on sperm quality of bull and northern pike (Esox lucius) after freezing-thawing have been reported, but no study has been made to evaluate the effect of LDL on boar sperm motility and other characteristics. The experiment was carried out to investigate the effect of LDL on the freezing of boar sperm in 0.25 ml straws. The aim was to evaluate the quality of boar spermatozoa cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Tris-citric acid-glucose (TCG) and Tris-citric acid-fructose (TCF), two basic freezing extenders containing egg yolk. Similarly, acrosome and plasma membrane integrity were also evaluated and compared to semen cryopreserved with TCG and TCF. Analysis of sperm quality after freeze-thaw showed that the motility, acrosome and plasma membrane integrity were improved with LDL in the extender, as compared to the TCG and TCF. The highest post-thaw integrity of acrosome and plasma membrane and motility were obtained with 9% LDL (w/v). Consequently, the optimum LDL concentration in the extender was 9%. It is also suggested that the concentration of LDL addition is important for the effect on boar sperm protection during freezing and thawing. The percentage of motile spermatozoa was significantly higher after freezing in 9% LDL than in TCG and TCF 54.4% versus 30.4% and 30.1% (p<0.05), respectively. The integrity of acrosome and plasma membrane were also significantly higher at 70.3% and 50.5% respectively with semen frozen in 9% LDL extender compared to TCG at 37.8% and 30.3% and TCF at 36.4% and 29.9%, respectively (p<0.05),. In conclusion, we propose that extender containing LDL extracted from hen egg yolk could be used as a cryoprotective media with a better efficiency than TCG and TCF. LDL improved boar semen quality, allowing better spermatozoa motility, acrosome and plasma membrane integrity after the freeze-thaw process. Furthermore, we found out that the extender with 9% LDL concentration significantly enhanced motility, acrosome and plasma membrane integrity of boar sperm after freezing and thawing. (Asian-Aust. J. Anim. Sci. 2006.