Membrane-intact Spermatozoa Research Articles

Effects of addition of vitamin B_{12} to the extender on post-thaw motility, acrosome morphology, and plasma membrane integrity in bull semen

To investigate the effects of supplementation of vitamin B_{12} on bovine sperm post-thaw quality, vitamin B_{12} was added at the concentrations of 1.25, 2.50, 3.75, and 5.00 mg/mL to bovine semen cryoprotective medium. The results indicated that the motility and VSL, VCL, STR, and VAP values of sperm supplemented with 2.50 mg/mL vitamin B_{12} were significantly higher than those of other concentrations (P < 0.05). No significant difference was observed for LIN and ALH values and the percentage of grade a spermatozoa between the extenders containing 2.50 mg/mL and 3.75 mg/mL vitamin B_{12} (P > 0.05). The percentages of acrosome-intact and plasma membrane-intact spermatozoa were significantly improved (P < 0.05) by supplementing with 2.50 mg/mL vitamin B_{12}. With all parameters measured, the concentration of 2.50 mg/mL vitamin B_{12} showed the best results on the quality of bovine semen in freezing-thawing. In conclusion, the addition of 2.50 mg/mL vitamin B_{12} to the extender improved the bovine frozen semen quality.

Use of image analysis to assess the plasma membrane integrity of ram spermatozoa in different diluents

Sperm membrane integrity can be assessed by examining a large number of fluorochrome-stained sperm cells over a relative short period of time by flow cytometry or fluorimetry. However, many small laboratories lack a flow-cytometer or fluorimeter for sperm analysis. This study was designed to develop a new image analysis method to evaluate the membrane integrity of ram spermatozoa with the aid of open software, and was divided into three experiments. In the first experiment, the new computer-assisted method was validated by mixing fresh semen samples with different volumes of killed semen in order to know the proportions of damaged spermatozoa in the samples. In the second trial, the new method was compared with the traditional manual counting, and the effect of three extender media on the suitability of the new developed method was evaluated. In the third experiment, the method proposed was tested by comparing the use of milk-, citrate- or TRIS-based diluents for ram semen preservation at 15 degrees C. In all experiments, semen was assessed for plasma membrane integrity and for percentage of motile and progressive sperm by CASA. In the new computer-assisted method, two images of the sperm cells in a given microscopy field are captured and the number of total- and membrane-damaged cells counted. In the first trial, proportions of damaged sperm cells in each sample determined by the automated procedure agreed closely (r2=0.98, P<0.001) with the predicted theoretical values. In experiment 2, the results of membrane integrity obtained using the new method were highly correlated with those provided by the conventional manual counting after PI-CFDA double staining (r=0.99, P<0.001), and also correlated with sperm motility and progressive motility percentages. Viability was significantly higher after dilution with citrate-, than with Tris-based medium, but similar to PBS (70.32+/-3.93, 55.48+/-5.76 and 65.38+/-3.15, respectively), After 0, 24 and 48h of storage, significantly higher percentages of motile, progressive, and membrane-intact spermatozoa were recorded for the milk than for the Tris extenders. Our results validate the new computer-assisted method for assessing sperm membrane integrity in the sheep, and indicate that the milk extender is less damaging to the sperm of this species than citrate or Tris extenders.

Interrelationship between plasma membrane integrity, mitochondrial membrane potential and DNA

The objective of this flow cytometric study was to examine plasma membrane integrity, mitochondrial membrane potential (MMP) and the degree of DNA fragmentation of cryopreserved bovine sperm immediately (0 h) and 3 h after thawing and to compare the results with each other and with the fertility of bulls. Cryopreserved spermatozoa from 4 consecutive ejaculates of 20 bulls were examined. Percentages of plasma membrane intact sperm (PMI) and sperm showing a high MMP (HMMP), respectively, were determined by the SYBR14/PI- and the JC-1 assays. DNA fragmentation was analysed by the standard deviation of the DNA fragmentation index (SD-DFI) and the percentage of sperm with a high degree of DNA fragmentation (%DFI) by using SCSA(TM). The mean non-return rate on day 56 (NRR 56) ranged from 63.7% to 78.0% (mean +/- SD: 71.8% +/- 3.7%). Mean values for PMI and HMMP decreased from 37.4% +/- 6.8% to 31.2% +/- 6.1% and from 38.8% +/- 7.1% to 23.8% +/- 7.7% respectively. SD-DFI increased from 56.9% +/- 8.0% to 69.0% +/- 12.9% and %DFI from 6.4% +/- 2.5% to 12.4% +/- 5.8%. The correlation between PMI 0 h and HMMP 0 h (r = 0.95; p 0.05), nor was there a correlation (p > 0.05) between DFI 3 h and PMI 3 h; but %DFI 3 h and HMMP 3 h were significantly correlated (r = -0.31; p < 0.05). SD-DFI and %DFI 3 h were the only parameters related to NRR 56 (r = -0.58; p < 0.05). In conclusion, plasma membranes and mitochondria are similarly affected by the freezing and thawing process, but not during the incubation period after thawing.

Interrelationship Between Plasma Membrane Integrity, Mitochondrial Membrane Potential and DNA Fragmentation in Cryopreserved Bovine Spermatozoa

The objective of this flow cytometric study was to examine plasma membrane integrity, mitochondrial membrane potential (MMP) and the degree of DNA fragmentation of cryopreserved bovine sperm immediately (0 h) and 3 h after thawing and to compare the results with each other and with the fertility of bulls. Cryopreserved spermatozoa from 4 consecutive ejaculates of 20 bulls were examined. Percentages of plasma membrane intact sperm (PMI) and sperm showing a high MMP (HMMP), respectively, were determined by the SYBR14/PI- and the JC-1 assays. DNA fragmentation was analysed by the standard deviation of the DNA fragmentation index (SD-DFI) and the percentage of sperm with a high degree of DNA fragmentation (%DFI) by using SCSA(TM). The mean non-return rate on day 56 (NRR 56) ranged from 63.7% to 78.0% (mean +/- SD: 71.8% +/- 3.7%). Mean values for PMI and HMMP decreased from 37.4% +/- 6.8% to 31.2% +/- 6.1% and from 38.8% +/- 7.1% to 23.8% +/- 7.7% respectively. SD-DFI increased from 56.9% +/- 8.0% to 69.0% +/- 12.9% and %DFI from 6.4% +/- 2.5% to 12.4% +/- 5.8%. The correlation between PMI 0 h and HMMP 0 h (r = 0.95; p < 0.0001) was higher (p < 0.05) than that between PMI 3 h and HMMP 3 h (r = 0.88; p < 0.0001). %DFI 0 h was neither related to PMI 0 h nor to HMMP 0 h (p > 0.05), nor was there a correlation (p > 0.05) between DFI 3 h and PMI 3 h; but %DFI 3 h and HMMP 3 h were significantly correlated (r = -0.31; p < 0.05). SD-DFI and %DFI 3 h were the only parameters related to NRR 56 (r = -0.58; p < 0.05). In conclusion, plasma membranes and mitochondria are similarly affected by the freezing and thawing process, but not during the incubation period after thawing.

Effects of Equex STM Paste on the quality of frozen-thawed epididymal dog spermatozoa

This study was carried out to investigate the cryoprotective efficacy of Equex STM Paste on the quality of canine post-thaw epididymal spermatozoa. Following castration, spermatozoa were flushed from the cauda epididymides. Epididymal spermatozoa from 13 of 16 dogs with a sperm motility of >70% were frozen in an egg yolk-Tris extender, supplemented with Equex STM Paste (0.5%, v/v); the extender free of Equex STM Paste served as a control cryoprotective diluent. The quality of spermatozoa, judged by its motility, plasma membrane integrity and acrosome integrity, was evaluated on four occasions, immediately after collection, after equilibration and at 0 and 2h post-thaw. Reducing the temperature to 4 degrees C for 2h prior to freezing decreased sperm motility (P=0.001), but had no effects on membrane integrity or acrosome integrity. Immediately after thawing, the percentage of acrosome-intact spermatozoa significantly decreased in samples frozen without Equex STM Paste compared to freshly collected or Equex-treated samples. After incubation at 37 degrees C for 2h post-thaw, a greater percentage of motile spermatozoa (P=0.018) and spermatozoa with intact acrosomes (P=0.001) were observed in Equex-treated samples compared with the control. The percentage of membrane-intact spermatozoa did not differ significantly between Equex-treated and control samples at any time. Supplementation with Equex STM Paste in the semen extender was effective for freezing canine epididymal spermatozoa because it protected acrosome integrity against damage induced by cryopreservation and it prolonged post-thaw sperm motility during in vitro incubation at 37 degrees C.

274 LYSOLECITHIN TREATMENT OF ELAND, BONGO, AND BOVINE SPERMATOZOA AND CLEAVAGE OF BOVINE OOCYTES AFTER INTERSPECIES INTRACYTOPLASMIC SPERM INJECTION

Compared to success rates in human, intracytoplasmic sperm injection (ICSI) is inefficient in ungulate species. Although factors such as injection of membrane-intact sperm and toxic effects of acrosome contents are suspected causes, the reasons for the inefficiency are unclear. A recent report in mice demonstrated that ICSI using spermatozoa treated with a physiological detergent, lysolecithin, improved oocyte activation, cleavage, and offspring production after embryo transfer (Morozumi K et al. 2006 PNAS 109, 17 661–17 666). The objectives of the present study were to evaluate the effects of detergent treatment on motility and membrane integrity of frozen thawed eland, bongo and bovine spermatozoa and to examine sperm decondensation/embryonic cleavage following ICSI of in vitro-matured bovine oocytes. In experiment 1, sperm motility was observed on a warm microscope stage during exposure to 3 lecithin concentrations, 0.04, 0.02, and 0.01%, and the time at which 100% of the spermatozoa lost motility was recorded. In experiment 2, spermatozoa were exposed to 0.02% lecithin for 22 s, and the membrane integrity and acrosome status of spermatozoa were determined using a combined trypan blue-Giemsa staining (Nagy et al. 1999 Theriogenology 52, 1153–1159). In experiment 3, bovine oocytes were injected, using the piezo drill, with lecithin-treated (0.02%, immobilized) or untreated (piezo pulse immobilized) eland, bongo, or bovine spermatozoa and subsequently cultured for 2 days in CR1aa containing 3 mg mL–1 BSA. Each experiment was replicated at least 3 times. Lecithin induced time- and concentration-dependent loss of sperm motility. The average time to loss of motility in 100% of the spermatozoa at 0.04, 0.02, and 0.01% lecithin was 107, 222, and 344 s in bovine; 82, 135, and 179 s in eland; and 65, 115, and 158 in bongo, respectively. Data on membrane integrity (intact or nonintact) and acrosome status (reacted or nonreacted) of detergent-treated or control spermatozoa are shown in Table 1. Sperm head decondensation and embryonic cleavage were observed following homologous and interspecies (antelope into bovine) ICSI of lecithin-treated or control spermatozoa. In conclusion, lecithin treatment induced concentration and time-dependent loss of motility and was effective in damaging the sperm membrane and acrosome in eland, bongo, and domestic bulls. Eland and bongo spermatozoa underwent decondensation and activated bovine oocytes after interspecies ICSI. Table 1.

Protective effect of taurine, glutathione and trehalose on the liquid storage of ram semen

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen during liquid storage is possibly one of the main causes for the decline in motility and fertility during storage—the other detrimental cause is low temperature on the destabilisation of sperm membrane structure. The aim of this study was to determine the effects of the addition of the anti-oxidants taurine and glutathione (GSH), and the membrane structure stabiliser, trehalose, on sperm viability during low temperature liquid storage. A total number of 36 ejaculates were collected using the artificial vagina from four Chios rams and nine replicates of the ejaculates were diluted with a Tris-based extender containing additives as the control. The sperm motility, percentage abnormal sperm, plasma membrane intact sperm and the hypo-osmotic swelling test (HOST) were determined during storage of semen at 5 °C for a period of 0, 6, 24 and 30 h of liquid storage, respectively. Trehalose at a level of 50 mM provided the best maintenance of motility at 6 and 30 h ( P < 0.05), and gave the highest percentage (69.0 ± 2.0% and 64.6 ± 1.8%, respectively) of viable sperm at 24 and 30 h ( P < 0.01). Trehalose treatment at a concentration of 50 mM also resulted in the highest percentage of membrane-intact sperm (53.7 ± 2.9%) after performing HOST at 30 h. The anti-oxidant treatments GSH 5–10 mM and taurine at 50 mM provided a significant improvement in sperm survival during the 6 h of liquid storage at 5 °C ( P < 0.05). In conclusion, many aspects of sperm protection, e.g. sperm motility, viability and membrane stabilisation of the sperm cells during relative low temperature storage, are the key factors determining the preservation of sperm function. Future efforts toward improving function of ram sperm kept in low temperature storage should concentrate on anti-oxidant additives. The results of this study provide a new approach to the preservation of sperm from rams of the Chios and related breeds, and so contribute to the improvement of these breeds for the world sheep industry.

258 THE EFFECT OF A PLANT PROTEIN COMPONENT OF MEDIA USED FOR BULL SPERM SEXING ON SPERM MEMBRANE STATUS

The aim of the work was to examine the effect of modified TALP medium (TALP/Pp, Animal Pharma B.V., Hengelo, The Netherlands)—used in the sperm sexing procedure—on bull sperm membrane status. The TALP was modified by replacement of bovine serum albumin (BSA) with a mixture of several plant proteins and soya lecithin (Pp). The Pp component was prepared using a high pressure homogenization process. The TALP/Pp had the same pH and osmotic pressure as the original TALP medium (TALP/BSA). The work was divided into 2 parts: (1) Nine ejaculates collected from 2 bulls (Holstein and Polish Red) were used. Immediately after collection, each ejaculate was split into 2 parts and diluted (1:2) with TALP/BSA or TALP/Pp. The sperm membrane status was examined after 3 days of storage at 15�C. (2) Fifteen ejaculates collected from 5 bulls (Holstein, Polish Red, and Simmental) were used. Each ejaculate was split into 2 parts: the first part was diluted with TALP/BSA, stained, incubated, and sexed according to the XY Inc. bull semen sexing procedure; the second part was diluted, stained, incubated, and collected after sexing into TALP/Pp with no egg yolk addition. In both groups no red food due was used to identify and exclude the dead spermatozoa from the sorted fractions. The sperm sexing procedure was performed with an SX MoFlo high-speed sorter at a speed of 3000–4000 cells/s. After collecting about 10 million spermatozoa, both fractions, X andY, were mixed, centrifuged at 700g for 15 min to concentrate the spermatozoa (20 million mL–1), and the sperm membranes examined. For sperm membrane examination, 'live/dead' samples were stained with SYBR-14/propidium iodide fluorochromes and analyzed by flow cytometry. The data from 20 000 spermatozoa were collected for each sample. The percentage of membrane-intact ('live') spermatozoa was taken for statistical analysis. The mean percentage of live spermatozoa stored for 3 days in TALP/BSA v. TALP/Pp was 25.7% (SD = 7.48) v. 28.58% (SD = 7.04), respectively (P &lt; 0.01). The mean percentage of live spermatozoa in samples of sexed semen was 33.57% (SD = 18.97) for TALP/BSA and 38.51% (SD = 20.22) for TALP/Pp (P &lt; 0.01). It can be concluded that Pp should be considered as a replacement for BSA in the TALP medium used for bull sperm sexing because (1) it results in significantly higher numbers of live spermatozoa after storage and/or sexing; (2) it eliminates a possible source of transmissible diseases (such as bovine spongiform encephalopathy); and (3) it decreases the total cost of the basic media used for the bull sperm sexing procedure.

87 OSMOTIC SENSITIVITY OF OKAPI SPERMATOZOA AND DEVELOPMENT OF CRYOPRESERVATION PROTOCOLS USING CRYOMICROSCOPY

Okapi would benefit from artificial insemination with frozen-thawed sperm in cases where aggression prevents mating or where individuals are geographically disparate. Effective sperm cryopreservation is a prerequisite to this goal. Ejaculates (n = 20) were collected from 7 anesthetized adult male okapi housed individually, or with a female for breeding, throughout the year by electroejaculation, and semen and sperm parameters were assessed. Semen aliquots were centrifuged; resuspended in 500 µL of PBS with the osmolarity adjusted to 35, 75, 150, 600, 1200, and 2400 mOsm; and incubated for 30 min before returning to isosmotic conditions. Semen was extended in TEST containing 1%, 2%, or 4% glycerol with or without 0.5% Equex (Minitube, Verona, WI, USA); 5-µL aliquots were cooled in a Latin square design on a Linkam BCS 196 cryomicrostage (Linkam Scientific, Tadworth, Surrey, UK) at 20�C min–1 to –6� –12�C at which point ice crystal formation was induced (seeded), and cooled further to –70�C before warming at 50�C min–1 to 35�C (okapi body temperature). To investigate cooling effects only, raw ejaculate was cooled to –6�C without seeding and warmed to 35�C. Percent sperm motility and plasma membrane integrity (PMI) were recorded before and after treatments. Differences were examined using one-way repeated measures analysis of variance. No differences in motility, total sperm numbers, or percent normal morphology were observed throughout the year (P &gt; 0.05), although the power of the test was low so that negative findings should be interpreted cautiously. Mean semen volume was 1.3 � 0.19 mL, sperm motility was 29 � 3.2%, with a PMI of 39 � 6.8%; 48 � 2.8% were morphologically normal. High proportions of non-motile, plasma membrane-damaged cells were noted in every ejaculate, and whiplash motility, possibly indicating spontaneous capacitation, was observed in several ejaculates 1 h after collection. Motility was dramatically reduced on either side of isosmotic conditions and was more sensitive to osmotic pressure than was plasma membrane integrity. Cooling of raw ejaculate to sub-zero temperatures without freezing did not result in any loss of motility or PMI, indicating cold tolerence. Superior results were obtained when sperm were frozen-thawed in TEST containing 4% glycerol with 0.5% Equex. Findings indicate that okapi semen collected by electroejaculation routinely contain high numbers of non-motile and plasma membrane-damaged spermatozoa, apparently unrelated to season or the length of time since the male was housed with a breeding female. Okapi spermatozoa are remarkably intolerant of departures from isosmotic conditions, indicating a lack of ability to regulate or withstand volume excursions during osmotic stress events; however, cooling to sub-zero temperatures in the absence of cryoprotectant did not reduce percent sperm motility or PMI, indicating resistance to cold shock. Increasing and maintaining proportions of motile, membrane-intact spermatozoa prior to and during cryopreservation will be critical for development of freezing protocols for this species.

Influence of bacteria and gentamicin on cooled-stored stallion spermatozoa

This study investigated effects of bacteria from the genital tract of horses and the effect of gentamicin in semen extender on spermatozoal function in cooled-stored stallion semen. Semen was collected from healthy stallions and processed with a milk-based extender with or without gentamicin (1 g/l). Pseudomonas ( Ps.) aeruginosa, Staphylococcus ( St.) aureus, Streptococcus ( Sc.) equi subsp. equi ( Sc. equi), Sc. equi subsp. zooepidemicus ( Sc. zooepidemicus), Sc. dysgalactiae subsp. equisimilis ( Sc. equisimilis) or culture medium alone (control) were added. Immediately after addition of bacteria and after storage at 5 °C for 24, 48 and 72 h, motility, velocity and membrane integrity of diluted semen were determined with a CASA system. After 24 h, semen with Ps. aeruginosa and Sc. equisimilis showed significantly lower motility and velocity compared to all other groups; after 72 h these differences still existed for Ps. aeruginosa ( p < 0.05). The percentage of membrane-intact spermatozoa was significantly lower after 24 h of storage in spermatozoa incubated with Sc. equisimilis and after 72 h with Sc. equisimilis and Ps. aeruginosa. Addition of gentamicin to extender resulted in decreased motility and velocity in semen without addition of bacteria and did not improve motility parameters in semen with bacteria added. In conclusion, certain bacteria may have detrimental effects on semen quality during cooled-storage. These effects are not reduced by addition of gentamicin. Gentamicin can negatively affect spermatozoal function in extended semen during cooled-storage and therefore, optimal concentrations have to be tested for the respective extender medium.

Anti‐oxidative Status and Semen Quality during Cooled Storage in Stallions

Activity of the anti-oxidative enzymes glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT), content of thiobarbituric acid reactive substances (TBARS) and SH-groups were determined in native stallion semen (n = 8 stallions). Semen was then diluted in Kenney extender, EquiPro((R)) extender either with or without addition of N-acetyl cysteine or phosphate-buffered saline (PBS) and stored for 72 h at 5 degrees C. Correlations between initial activity of enzymes and development of semen motility and membrane integrity were calculated. Activities of GSH-Px, SOD and CAT immediately after semen collections were 10.0 +/- 0.6 picokatals, 0.40 +/- 0.03 SOD units and 0.70 +/- 0.05 nanokatals/10(6) spermatozoa respectively. TBARS content was 0.06 +/- 0.01 nmol and SH-group content 1.7 +/- 0.5 mmol/10(6) spermatozoa. The loss of motile spermatozoa during storage did not differ between extenders. N-acetyl cysteine had no effect on semen motility and membrane integrity. The loss in membrane-intact spermatozoa was highest (P < 0.05) in semen diluted in PBS. Motility and membrane integrity after addition of extender were positively correlated with GSH-Px and CAT, indicating that anti-oxidative mechanisms contribute to the initial high percentage of motile and membrane-intact spermatozoa. However, in these samples the decrease in semen quality was most pronounced. No correlations existed between initial activity of anti-oxidative enzymes, peroxidation products and semen quality during storage. This indicates that once extender has been added, peroxidative damage to sperm membranes is not the predominant cause of losses in semen quality.

Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 °C

A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen was diluted with PBS or defined milk protein extender and was either stored directly or 90% of seminal plasma was removed by centrifugation and replaced by defined milk protein extender before storage. In both experiments, eight stallions were available for semen collections. Motility, velocity and membrane integrity of spermatozoa were determined by CASA immediately after semen processing and after 24, 48 and 72 h of storage at 5 °C. Total motility after 24 h of storage was lowest in semen diluted with PBS ( p < 0.05 versus all extenders). At 48 and 72 h, motility of spermatozoa in defined milk protein extender was significantly ( p < 0.05) higher than in PBS or skim milk extender. Velocity of spermatozoa after storage was highest in defined milk protein extender. Membrane integrity after storage was significantly ( p < 0.05) lower in semen diluted with PBS than in semen diluted with both extenders. Addition of NAC was without effect on the examined parameters. Centrifugation further increased the percentage of motile and membrane-intact spermatozoa in the defined milk protein extender ( p < 0.05). Velocity of spermatozoa in this extender was not negatively affected by centrifugation.

113 EFFECT OF SPERM COATING ON THE QUALITY OF BOVINE FROZEN - THAWED SPERMATOZOA

The substantial decrease of sperm quality after cryopreservation remains an important issue in the artificial insemination industry. Sperm coating with Triladyl® (Minitübe, Tiefenbach, Germany) during ejaculation can preserve sperm characteristics and oocyte penetrating capacity of fresh bovine spermatozoa stored in egg yolk diluent for up to 6 days (De Pauw et al. 2003 Theriogenology 59, 1109–1122). Since collecting semen in a tube containing egg yolk-Tris extender (sperm coating) limits the period of contact between spermatozoa and seminal plasma, the present experiment was conducted to assess if this slightly adjusted method of sperm collection could also have a significant effect on bovine sperm quality after cryopreservation. Semen of five young Holstein Friesian bulls was collected by means of an artificial vagina connected to an empty tube (Group 1; five ejaculates per bull) or a tube containing 4 mL of an egg yolk-Tris extender (Groups 2 and 3; each five ejaculates per bull). The semen samples of Group 1 were conventionally diluted in straws (60 × 106 sperm/mL), frozen, and stored in liquid nitrogen. The samples of Group 3 were centrifuged, and after removing diluent and seminal plasma, the sperm pellet was conventionally diluted and processed. The samples of Group 2 were processed without removal of the supernatant. After thawing each ejaculate was analyzed for average path velocity (VAP), beat cross frequency (BCF), and progressive motility (PROG) using CASA (Minitübe, Tiefenbach, Germany). Furthermore, the membrane integrity of each sample was evaluated using fluorescent SYBR®–14/PI staining (BD Biosciences, Erembodegem, Belgium). All parameters were compared among the three groups of sperm using univariate analysis of variance (SPSS 12.0; SPSS, Inc., Chicago, IL, USA). No significant differences could be observed among the three groups for all of the evaluated sperm characteristics (Table 1). A significant effect of the bull could be determined for all analyzed parameters (P ≤ 0.02), except for the percentage of moribund cells. Nevertheless, the group-bull interaction was never statistically significant. Coating bovine sperm with an egg yolk-Tris extender during ejaculation cannot prevent the substantial deterioration of the spermatozoa that occurs during freezing and thawing since this method of sperm collection does not significantly influence the motility parameters or the membrane integrity after thawing. Table 1. VAP, BCF, PROG, and percentage of membrane-intact, dead, and moribund spermatozoa for the three groups of sperm This research was supported by IWT (no. IWT/020727).

Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival

Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sperm were maintained after thawing when 1.5 mg CLC was added to sperm from stallions whose sperm do not survive freezing well, compared to control sperm from those same stallions (67% vs. 50%; P < 0.05). Addition of CLCs increased the percentages of membrane intact sperm surviving cryopreservation compared to untreated sperm for all stallions ( P < 0.05). The amount of cholesterol that incorporated into the membranes of the sperm cells increased in a polynomial fashion ( R 2 = 0.9978) and incorporated into all sperm membranes. In addition, there was a significant loss of cholesterol from sperm membranes after cryopreservation; however, addition of CLCs to sperm prior to cryopreservation maintained higher cholesterol levels in the sperm after freezing and thawing than untreated sperm ( P < 0.05). Addition of CLCs also resulted in more sperm binding to the zona pellucida of bovine oocytes after cryopreservation than control sperm (48 vs. 15; P < 0.05). In conclusion, CLCs improved the percentage of post-thaw viability in equine sperm as well as increased the number of sperm that bind to zona pellucida. Addition of CLCs to stallion sperm prior to cryopreservation is a simple procedure that increases the cryosurvival of cells.

Cryopreservation of boar semen: equilibrium freezing in the cryomicroscope and in straws

Supercooling causes very abrupt temperature and osmotic changes and can thus lead to freezing damage. Supercooling can be prevented by seeding, using a sample volume and geometry that allows rapid spreading of the ice throughout the sample. In a split-sample comparison of such samples on the cooling stage of a cryomicroscope and seeded at −5 and −15 °C, respectively, the percentages of membrane-intact sperm and sperm with acrosomes with a ‘normal apical ridge’ (NAR) were 72.5 ± 3.8 and 75.8 ± 2.0 versus 46.3 ± 4.8 and 36.0 ± 3.7 (means ± S.E.M., n = 4). In ejaculates of 15 unselected AI boars, after seeding at −5 °C, the post-thaw % live and % NAR were 66.3 ± 10.4 and 74.8 ± 7.5, respectively. Our present research is aimed at translating these findings to freezing in straws and at a high sperm concentration. We have designed a novel type of freezing apparatus for controlled-rate freezing of straws, in which supercooling can be effectively prevented in the entire straw. In a split-sample comparison of semen frozen in straws at a sperm concentration of 1.5 × 10 9 cells/ml with nine ejaculates from eight unselected AI boars, we found 54.8 ± 1.9% versus 40.7 ± 1.7% (means ± S.E.M.) membrane-intact sperm for the new apparatus and a conventional freezing apparatus, respectively. With bull semen (eight ejaculates from six bulls), we obtained 67.3 ± 3.0% versus 59.3 ± 2.9% (means ± S.E.M.) membrane-intact sperm for the new apparatus and conventional freezing, respectively. Additionally, the temperature curve after ice nucleation is of great importance. We have developed a model that allows us to predict that optimal cryopreservation requires a non-linear cooling curve in which the cooling rate varies as a function of subzero temperature.

258ULTRASTRUCTURAL STUDY OF STALLION SPERM FOLLOWING THAWING, CAPACITATING AND IVF PROCEDURES: EFFECT OF THE PRESENCE OF CUMULUS-ENCLOSED OOCYTES

The use of IVF in horses has a limited efficiency, reflecting low oocyte developmental competence and inadequate sperm capacitation procedures. In a preliminary study, using carboxyfluorescein diacetate/propidium iodide staining, we determined that the freezing-thawing procedure left only 56.6±3.4 % of the sperm cells with an intact membrane. The following incubation in TALP-IVF induced membrane damage at high rates with only 9.58±1.8 % of them intact after 18h. However, the presence of at least four cumulus-enclosed oocytes (CEO) in the medium significantly increased the number of membrane-intact spermatozoa at the end of incubation (53.87±1.99%). This indicated that the sperm thawing and capacitating procedures can damage the cell membrane but the presence of four or more CEO in TALP-IVF could prevent further damages. The aim of the study was to investigate in detail the membrane damages and to analyze the differences induced by the presence of CEO. Spermatozoa were thawed in water at 37°C, and centrifuged for 30 minutes at 600g in a 45–90% Percoll gradient made with modified Tyrode’s medium. The sperm pellet was washed once in the same medium and diluted to a final concentration of 1×106 spermatozoa/ml TALP supplemented with 0.6% (w/v) BSA fatty acid free and 12μgmL−1 heparin (TALP-IVF). Sperm cells were incubated with 0 or 4 in vitro-matured CEO. Sperm cells were examined after thawing, 0, 2 and 18h from the beginning of incubation in TALP-IVF. Each experiment was replicated at least 3 times. Both scanning and transmission electron microscopy were performed on sperm samples fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.2, using standard procedures. Specimens for scanning electron microscopy were examined under a field emission gun JEOL JSM 6301 microscope. For transmission electron microscopy the samples were examined with a JEOL JEM 100 SX. A minimum of 25 cells were analyzed for each group. Immediately after thawing, damaged spermatozoa showed, on the surface of their heads, small vesicles correlated to a progressive process of vacuolisation and degeneration of membrane integrity. The same lesions were visible at all the successive time points taken into account. Moreover, a loss of the acrosome integrity with acrosomal swelling and a decrease of content homogeneity were observed particularly in the spermatozoa cultured for 18 h without CEO. When CEO were present in the IVF medium lesions were visible in a lower percentage of spermatozoa but the type of lesions did not differ from those observed in their absence. These observations confirmed our previous data and gave more details on the lesions that occur during the IVF procedures in the horse. Supported by MURST COFIN grant n. 2001078849.

202FERTILIZING ABILITY OF EPIDIDYMAL CAT SPERMATOZOA AFTER CRYOPRESERVATION OR STORAGE AT 4°C.

The possibility of harvesting cat epididymal spermatozoa from excised testis represents a potentially important tool for preserving valuable genetic material from males that die unexpectedly. The purpose of this study was to evaluate plasma membrane integrity, acrosomal status and in vitro fertilizing capability of epididymal cat sperm after short- and long-term storage at 4°C and after cryopreservation. Spermatozoa were collected by flushing the cauda epididymis and the vasa deferentia removed from adult cats undergoing orchiectomy. Spermatozoa were split into three aliquots: A-fresh, B-frozen, and C-cooled. The A portion was immediately tested on the day of collection (control), the B portion was frozen in pellets in Test Yolk Buffer (TYB) with 8% glycerol, while the C portion was extended in TYB and stored at 4°C for 1 day, 2 days or 7 days. Cat oocytes recovered from minced ovaries were matured for 24h in TCM 199+1UI/mL hCG+0.5UI/mL FSH+0.3% BSA at 38.5°C in 5% CO2. In vitro fertilization (IVF) of in vitro-matured (IVM) oocytes was performed using spermatozoa from portions A, B or C in modified Tyrode’s solution supplemented with 0.6% BSA at 38.5°C in 5% in air. Before IVF, aliquots of sperm samples from the three portions were stained simultaneously with fluorescein isothiocyanate-labelled Pisum Sativum agglutinin (FITC-PSA) and propidium iodide to evaluate the percentage of plasma membrane-intact spermatozoa with acrosomes present. At least 200 spermatozoa were counted in duplicate for each sample. After 24h of incubation, to evaluate fertilization rate, the oocytes were stained with aceto-lacmoid. Oocytes with two pronuclei, presumably male and female, were classified as fertilized. The percentages of spermatozoa maintaining plasma membrane integrity and intact acrosomes after 1 day or 2 days of storage at 4°C were 75% and 65%, respectively, and were similar to that of fresh epididymal spermatozoa (78%). However, the percentages of spermatozoa with intact plasma membranes that had intact acrosomes after 7 days of storage at 4°C (52%) or after cryopreservation (48%) were significantly lower (P&amp;lt;0.01, ANOVA) than those of samples stored for 1 or 2 days at 4°C. As shown in the Table, fertilization rates of oocytes inseminated with fresh spermatozoa and spermatozoa stored for 1 or 2 days were similar. In contrast, the fertilization rates of oocytes inseminated with spermatozoa that had been cryopreserved or stored for 7 days were significantly lower than those obtained with spermatozoa used immediately after collection or storage for 1 day. From our results, we suggest that storage of epididymal spermatozoa at 4°C may be a potentially useful method for temporary storage of spermatozoa from endangered cats, and may allow more efficient use and long-distance transport of genetically important germplasm. This work was supported by MIUR (ex 40%). Table 1 In vitro fertilization of in vitro-matured cat oocytes with fresh, stored at 4°C and cryopreserved epididymal cat spermatozoa

Relationship between thirty post-thaw spermatozoal characteristics and the field fertility of 11 high-use Australian dairy AI sires

This study determined the relationship between two measures of field fertility of 11 high-use Australian artificial insemination (AI) dairy bulls and thirty standard laboratory assessments of spermatozoal post-thaw viability. The two measures of field fertility used, conception rates (cCR) and non-return rates (cNRR), were both corrected for all major non-bull variables. Sperm viability assessments were conducted on semen collected within the same season as that used to derive the field fertility estimates. These assessments measured sperm concentration, motility, morphology and membrane integrity at thawing, after 2 h incubation and after the swim-up sperm selection procedure. Derivations of these measures and in vitro embryo fertilizing and developmental capacity were also determined. The Genstat Statistical Package [Genstat 5 Release 4.2 Reference Manual, VSN International, Oxford, 2000] was used to conduct an analysis of variance on the viability parameters across semen straws and bulls, and to calculate the strength of correlation between each semen parameter, cNRR and c CR in a correlation matrix. Step forward multiple regression identified the combination of semen parameters that were most highly correlated with c CR and with cNRR. The sperm parameters identified as being most predictive of c CR were the percentage of morphologically normal sperm immediately post-thaw (zeroNorm), the number of morphologically normal sperm after the swim-up procedure (nSuNorm), and the rate of zygote cleavage in vitro (Clv); the predictive equation formed by these parameters accounted for 70% of variance. The predictive equation produced for cNRR contained the variables zeroNorm, the proportion of membrane intact sperm after 2 h incubation at 37 °C (twoMem) and Clv and accounted for 76.5% of the variation. ZeroNorm was found to be consistent across straws and semen batches within-bull and the sperm parameter with the strongest individual predictive capacity for both cCR ( P=0.1) and cNRR ( P=0.001). Post-thaw sperm parameters can be used to predict field fertility of Australian dairy sires; the calculated predictive equations are particularly useful for identifying and monitoring bulls of very high and very low potential fertility within a group.

Effect of semen collection practices on sperm characteristics before and after storage and on fertility of stallions

This study analyzed effects of different methods and intervals of semen collection on the quantity and quality of fresh, cool-stored, and frozen–thawed sperm and fertility of AI stallions. In Experiment 1, ejaculates were obtained from six stallions (72 ejaculates per stallion) using fractionated versus non-fractionated semen collection techniques. Initial sperm quality of the first three jets of the ejaculate was not different from that of total ejaculates. Centrifugation of sperm-rich fractions before freezing improved post-thaw motility and sperm membrane integrity when compared to non-centrifuged sperm-rich fractions or non-fractionated centrifuged ejaculates ( P<0.05). In Experiment 2, semen from four stallions (60–70 ejaculates per stallion) was collected either once daily or two times 1 h apart every 48 h. The first ejaculates of double collections had significantly higher sperm concentrations, percentages of progressively motile sperm (PMS) after storage for 24 h at 5 °C and lower percentages of midpiece alterations than single daily ejaculates. Semen collected once daily showed significantly lower values of live sperm after freezing and thawing than the first ejaculate of two ejaculates collected 1 h apart every 48 h. In Experiment 3, semen was collected from 36 stallions (≥12 ejaculates per stallion) during the non-breeding season and the time to ejaculation and the number of mounts was recorded. When time to ejaculation and the number of mounts increased, volume and total sperm count (TSC) also increased ( P<0.05), whereas a decrease was observed in sperm concentration, percentage of PMS after storage for 24 h at 5 °C, percentage of membrane-intact sperm in fresh semen ( P<0.05) as well as motility and percentage of membrane-intact sperm of frozen–thawed sperm ( P<0.05). In Experiment 4, AI data of 71 stallions were retrospectively analyzed for the effect of number of mounts per ejaculation and frequency, time interval of semen collections on pregnancy, and foaling rates (FRs) of mares. Semen volume increased, but sperm concentration and percentage of PMS after 24-h cool-storage decreased with increasing number of mounts on the phantom ( P<0.05). A statistically significant inter-relationship was demonstrated between frequency and interval of semen collection and FR. Mares inseminated with stallions from which semen was collected frequently (≥1 on an average per day) showed significantly higher FRs than mares inseminated with semen from stallions with a daily collection frequency of 0.5–1 or <0.5. FR of mares inseminated with stallions having 0.5–1 days between semen collections was significantly better than FR of mares that were inseminated with stallions having semen collection intervals of 1–1.5 days or >2.5 days.

Comparison of methods to evaluate the plasmalemma of bovine sperm and their relationship with in vitro fertilization rate

The objectives of this study were to compare different methods of evaluating sperm plasmalemma and to determine their relationship with in vitro fertilization rate. A single batch of frozen semen from each of eight beef bulls was used for assessment of sperm viability and for in vitro fertilization. Conventional viability tests included sperm morphology, motility, acrosome integrity, and abnormal DNA condensation. Methods for evaluation of the sperm plasmalemma included eosin/nigrosin (EN) and trypan-blue (TB) vital stains, propidium iodide (PI) in combination with carboxyfluorescein diacetate (CFDA) or SYBR-14 (SYBR) fluorescent vital stains, and the hypoosmotic swelling test (HOST). A total of 133–150 oocytes were fertilized in vitro with sperm from each bull and cleavage rates were determined. There were high correlations between the results obtained with vital stains and good to excellent interclass correlation coefficients of agreement, indicating that these stains provide measures of the same sperm attribute, i.e. plasmalemma integrity. However, the proportions of membrane-intact sperm identified by EN or TB stains were greater ( P<0.0001) than identified by CFDA/PI or SYBR/PI fluorescent stains. The results obtained with the HOST had moderate correlations but poor agreement with the results of the vital stains. The proportion of viable sperm identified by the HOST was lower ( P<0.05) than the proportion identified by vital stains, indicating that response to the HOST did not depend only on the integrity of the plasmalemma. Although there were significant differences in fertilization rates and sperm viability among bulls, there was no sharp distinction for the results of sperm viability tests from bulls producing different in vitro fertilization rates. Proportions of normal, motile, acrosome-intact, and HOST-responsive sperm were identified as significant predictors of in vitro fertilizing potential; each of these endpoints explained 12–18% of the variation when evaluated separately (linear regression) and 48% when evaluated collectively (stepwise regression). In conclusion, EN and TB stains overestimated the proportion of plasmalemma-intact sperm compared to PI-based fluorescent stains. Vital stains evaluated the morphological integrity of the plasmalemma, whereas the HOST assessed plasmalemma function. In that regard, the HOST was the only plasmalemma evaluation method that significantly contributed to conventional sperm quality tests in predicting in vitro fertilization rate, indicating that the test could be incorporated to the routine of semen analysis.