Polymyxin B enhances acrosomal exocytosis triggered by calcium and the calcium ionophore A23187 in ejaculated boar spermatozoa

Polymyxin B (PMB) is beneficial for boar semen storage since it neutralizes the endotoxin of bacteria. However, the direct effect of PMB on boar spermatozoa has been unknown. This study aimed to examine the effect of PMB on acrosomal exocytosis, an essential process for successful fertilization in boar spermatozoa. Ejaculated spermatozoa stored with BTS extender at 17°C were washed and incubated with 0–100 μM PMB for 20 min and then examined for % total motililty, vigor grade and viability. None of the parameters was significantly different between 0 and 50 μM PMB with a gradual decline at higher concentrations. Thus the effect on acrosomal exocytosis was investigated at 0–50 μM of PMB. Spermatozoa were preincubated with PMB for 10 min, incubated for stimulation of acrosomal exocytosis with Ca²⁺ and the calcium ionophore A23187 and then fixed with glutaraldehyde at 5, 10 and 15 min. Preincubation with PMB at 0.01–50 μM and 0.05–50 μM resulted in significant enhancement of acrosomal exocytosis at 10 min and 15 min of incubation, respectively. Preincubation with PMB followed by incubation without A23187 did not affect acrosomal exocytosis. These results suggest that PMB exerts effects on the acrosomal exocytosis triggered by Ca²⁺ and A23187 in boar spermatozoa.     

Seasonal changes in semen characteristics, composition of seminal plasma and frequency of acrosome reaction induced by calcium and calcium ionophore A23187 in Large White boars

This study attempted to explain the mechanisms regulating boar fertility by examining seasonal changes in semen characteristics, the composition of seminal plasma and responsiveness of sperm acrosomes to Ca(2+) and the Ca(2+) ionophore A23187 (Ca(2+)/A23187). Sperm-rich and sperm-poor fractions were separately collected from 3 mature fertile Large White boars once a month over a one-year period. During the period of study, ambient temperature and relative humidity were recorded for within the stall in which the boars were kept and the semen characteristics, composition of the seminal plasma of sperm-rich fractions, and occurrence of the acrosome reaction in response to Ca(2+) (3 mM)/A23187 (0.3 microM) were examined. The highest mean maximum and minimum ambient temperatures were recorded in August-September, whereas the lowest mean maximum and minimum ambient temperatures were recorded in December and January, respectively. There was a moderate peak in relative humidity from July to October. The lowest percentages of motile spermatozoa and of spermatozoa with intact acrosomes and highest percentage of spermatozoa with abnormal morphology and strongest agglutination were seen in August-September. The total protein and albumin concentrations were lowest in August-September. Testosterone levels increased gradually as day length decreased after the summer solstice (June) and peaked in October-November. The percentage of acrosome reactions in response to Ca(2+)/A23187 was highest with the quickest response in August-September, as shown by the shortest time required for 50% of relative acrosome reactions. The farrowing rates were lowest in these same 2 months. These results suggest that seasonal infertility in Large White boars may be due, at least in part, to a combination of low motility, abnormal morphology including acrosomal abnormality, and early occurrence of the acrosome reaction in response to stimulus, possibly resulting from a decrease in acrosomal stabilizing proteins in the seminal plasma during summer. These changes may be modulated by heat/humidity stress and/or photoperiod-regulated testosterone.     

In vitro induction of the acrosome reaction in ovine spermatozoa by calcium ionophore A23187

The relationship between concentration of calcium ionophore A23187 and incubation time upon the proportion of spermatozoa undergoing acrosome reaction (AR) in vitro was investigated in rams from a commercial artificial insemination (AI) program. Two ejaculates were collected by artificial vagina from each of nine rams of three breeds (Finn Dorset, Charolais and Suffolk) aged 8-36 months. Each ejaculate was diluted in a skimmed milk extender. Spermatozoa were thereafter incubated for 45 or 60 min in modified Tyrode's medium (TALP) which contained either zero, 0.1 or 1.0 microM/l A23187. After fixing in 10% formaldehyde, the number of spermatozoa that had undergone AR was determined by phase contrast microscopy. In pre-incubation samples, 21.3 +/- 3.3% of spermatozoa had undergone AR. Percentages of acrosome reacted spermatozoa were significantly (P < 0.001) increased after incubation with A23187. After incubation with 0.1 microM/l A23187 for 45 and 60 min there were 22.4 +/- 3.0% and 31.7 +/- 4.3% acrosome reacted spermatozoa, respectively. After incubation with 1.0 microM/l A23187 for 45 and 60 min there were 46.2 +/- 6.5% and 53.8 +/- 5.9% acrosome reacted spermatozoa, whilst corresponding numbers in control samples were 17.0 +/- 2.7% and 22.3 +/- 4.2%. There was also a significant (P < 0.001) effect of individual animals upon the responses to different concentrations of A23187. These findings indicate that (i) A23187 can be used to assess the AR of ovine spermatozoa in vitro and (ii) there are effects of individual animals upon the proportion of spermatozoa undergoing AR.     

Evaluation of glucose as a cryoprotectant for boar semen

Fertility parameters of boar spermatozoa were evaluated in vitro, after freeze-thawing the semen in three different extenders containing permeable and non-permeable cryoprotectants: A (111.0 mM Tris, 31.4 mM citric acid, 185.0 mM glucose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G); B (200 mM Tris; 70.8 mM citric acid, 55.5 mM glucose, 20 per cent egg yolk, three per cent glycerol and 100 iu/ml penicillin G); C (200 mM Tris, 70.8 mM citric acid, 55.5 mM fructose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G). The freeze-thawing techniques were the same for each extender. Eight ejaculates from four boars were obtained; the sperm-rich fraction of each ejaculate was extended in each of the three media at a final concentration of 400 x 106 sperm/ml, loaded into 0.5 ml straws and frozen at a rate of 30 degrees C/minute to -196 degrees C. The straws were thawed at 60 degrees C for eight seconds. Sperm motility, acrosomal integrity and in vitro sperm penetration through the zona pellucida of gilt oocytes matured in vitro were evaluated. The motility of unfrozen spermatozoa was 93.1 per cent compared with 60.7 per cent, 48.2 per cent and 35 per cent for sperm frozen in extenders A, B and C respectively; these values were all significantly different (P<0.05). There was no significant decline in sperm motility after incubation for 30 minutes in extender A, but there were significant decreases in sperm motility after 30 minutes of incubation in B and C. The percentage acrosomal integrities were 97.2 per cent for the control and 45.5 per cent, 30.3 per cent and 16.8 per cent for the frozen-thawed spermatozoa in extenders A, B and C respectively. The results of the in vitro penetration assay were 80.7 per cent when using control spermatozoa, and 42.2 per cent, 18.4 per cent and 3.3 per cent when using frozen-thawed spermatozoa in extenders A, B and C respectively     

Comparative evaluation of the effect of antioxidants in the conservation of ram semen

The aim of the present study was to develop a treatment supporting the membrane of ram spermatozoa. Semen of different ejaculates collected from breeding rams was mixed and samples of 10(9) sperm cells per ml and Tris-egg yolk extender were completed with the following antioxidants: alpha-tocopherol acetate (E), glutathione peroxidase (GP), Aromex (AR), resveratrol (R), resveratrol + vitamin E (RE), resveratrol + Aromex (RAR), resveratrol + GP (RGP). Peroxidation was evaluated by the analysis of malondialdehyde (MDA) during incubation for 30, 60 and 120 min at 37 degrees C as well as during a 24-h incubation at 5 degrees C. The success of preservation was checked in a 9-day-long period by observing the acrosomal defects and the motility of spermatozoa. Concentration of MDA was 4.06 nmol/10(9) spermatozoa in samples treated with 15 micrograms R while the control sample contained 69.79 nmol MDA per 10(9) spermatozoa after 24-h incubation. Following 30-, 60- and 120-min storage the concentration of MDA in control and R-treated samples was 25.89, 36.91, 49.57 and 3.69, 3.74, 3.74 nmol/10(9) spermatozoa, respectively. Moreover, a significantly higher proportion of motile sperm cells was observed in the treated than in the control samples. The frequency of acrosomal defects was lower in the treated groups than in the control. These results indicate that RAR treatment can improve the effects of ram semen preservation.     

Effects of protein phosphatase inhibitor calyculin a on the postacrosomal protein serine/threonine phosphorylation state and acrosome reaction in boar spermatozoa incubated with a cAMP analog

In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.     

Bestimmung der osmotischen Resistenz von Eberspermien und deren Beziehungen zur Konservierungsfähigkeit von Samenproben

Contents Determination of the osmotic resistance of boar spermatozoa and the relations to sperm freezing and long storage The osmotic behaviour of fresh boar spermatozoa (482 ejaculates from 34 A.I. boars) was examined in a standard medium but with different osmotic pressures ranging from 150 mosm/kg till 500 mosm/kg. The percentage of spermatozoa with normal acrosomal ridge (NAR) was determined after incubation of 0.2 ml fresh semen in 3 ml of the osmotic test solutions at 39° C for 15 and 120 min. In the non-isotonic media % NAR decreased and could be quantified depending to incubation time and to the different osmolalities; NAR decreased strongest at 150 or 500 mosm/kg and after 120 min incubation time. Differences in % NAR were large between ejaculates and between boars but low within boars and statistically significant correlations had been found to % NAR and motility in froren/thawed semen samples or stored samples. Still higher correlations were found by calculation of an osmotic resistance test-value (ORT-value) which is defined as follows: ORT = 1/2 % NAR in isotonic medium/(after 15 min) +% NAR in 150 or 500 mosm/kg/(after 15 or 120 min) ORT-values can be calculated for hypotonic (150 mosm/kg) as well as for hypertonic (500 mosm/kg) test solutions and for 15 and 120 minutes incubation time. The hypotonic ORT-value for 120 min. is the most accurate; the values varied from 34 till 83 between ejaculates and from 44 till 76 between boars with a low variability within boars. Grouping of boars by their ORT-values in “good”, “moderate” and “poor” corresponded to a similar grouping of these boars by their semen quality (% NAR and motility) in preserved semen samples. Also a grouping of single ejaculates by their ORT-values corresponded to a similar qualitative classification of these ejaculates. ORT seems to become a good and practicable assessment test for the selection of boars suitable dor deep freezing or long storage of their spermatozoa. On the other hand preservation techniques too can be examined by ORT. But more investigations with this test must be done especially its relation to the fertilization capacity of semen samples with different ORT-values.     

Platelet-activating factor in Iberian pig spermatozoa: receptor expression and role as enhancer of the calcium-induced acrosome reaction

Platelet-activating factor (PAF) is a phospholipid involved in reproductive physiology. PAF receptor is expressed in some mammalian spermatozoa species where it plays a role in these germ-cell-specific processes. The aim of this study is to identify PAF receptor in Iberian pig spermatozoa and to evaluate PAF's effects on motility, viability and acrosome reaction. Semen samples from Iberian boars were used. PAF receptor identification was performed by Western blotting. Spermatozoa motility was analysed by computer-assisted sperm analysis system, whereas spermatozoa viability and acrosome reaction were evaluated by flow cytometry. Different PAF concentrations added to non-capacitating medium during 60 min have no effect on any spermatozoa motility parameter measured. Acrosome reaction was rapid and potently induced by 1 μm calcium ionophore A23187 showing an effect at 60 min and maximum at 240 min. PAF added to a capacitating medium is not able to induce spermatozoa acrosome reaction at any time studied. However, PAF, in the presence of A23187, significantly accelerates and enhances the calcium-induced acrosome reaction in a concentration-dependent manner in Iberian boar spermatozoa. Exogenous PAF does not affect at all spermatozoa viability, whereas slightly exacerbated the A23187-induced loss in viability. This work demonstrates that PAF receptor is expressed in Iberian pig spermatozoa and that its stimulation by PAF regulates the calcium-induced acrosome reaction. This work contributes to further elucidate the physiological regulation of the most relevant spermatozoa functions for successful fertilization: acrosome reaction.     

'In vitro' capacitation and further 'in vitro' progesterone-induced acrosome exocytosis are linked to specific changes in the expression and acrosome location of protein phosphorylation in serine residues of boar spermatozoa

The aim of this study is to determine changes in the expression and location of protein serine phosphorylation (pSer) during 'in vitro' capacitation (IVC) and 'in vitro' acrosome exocytosis (IVAE) in boar spermatozoa. This was performed in both mono- and bi-dimensional analyses of protein expression through Western blot, as well as through immunocytochemistry. Furthermore, IVC was induced through incubation in an IVC medium, and afterwards, progesterone-induced IVAE was performed. The mono-dimensional Western blot analysis showed the presence of a predominant pSer band of approximately 70-75 kDa, which was accompanied by fainter bands, especially three with molecular weights of approximately 50, 35 and 32 kDa. Neither IVC nor IVAE significantly modified this pattern. Bi-dimensional analyses showed a more complex pattern, with at least five protein clusters. The attainment of IVC caused the disappearance of the proteins with the highest molecular weight concomitantly with the appearance of pSer proteins of 75-kDa/pI 9.5 and 80-kDa/pI 10. The induction of IVAE caused the appearance of new pSer proteins of a 75-kDa/pI 6.5-7.5 and 75-kDa/pI 10. Immunocytochemistry showed that the main pSer expression in boar expression before the attainment of IVC was located at the midpiece. The IVC induced the appearance of acrosomal pSer, which was greatly increased during IVAE. Our results indicate that the changes in serine protein phosphorylation associated with IVC and IVAE comprise not only the appearance of specific phosphorylated proteins, such as the pSer-75 kDa, but also changes in pI and displacements in the sperm location of phosphorylated proteins, like the specific acrosomal pSer signal induced during IVC.     

黄芪多糖对猪精液冷冻保存效果的影响

为了评价黄芪多糖（Astragalus polysaccharides）对外来公猪精液冷冻保存的影响情况,为猪精液冷冻稀释液配方的改良提供理论依据,试验采集美系长白、大约克与杜洛克3种公猪的精液,用添加不同浓度（0、0.01%、0.02%、0.03%、0.04%、0.05%、0.06%）黄芪多糖的冷冻稀释液稀释,0.25 mL塑料细管分装并冷冻,测定解冻后精子活力、畸形率及顶体完整率,并进行相同浓度3种公猪之间和同种公猪不同浓度之间的比较。结果表明,在相同黄芪多糖浓度下,仅杜洛克公猪冻后精子顶体完整率在不添加黄芪多糖时显著高于大约克公猪（P<0.05）,其余均无显著差异（P>0.05）;每种公猪的最佳黄芪多糖添加浓度均为0.04%,在该浓度下精液冻后质量均显著优于对照组（P<0.05）;各种公猪最佳黄芪多糖添加浓度下的精液冻后质量指标之间均无显著差异（P>0.05）。总之,稀释液中添加0.04%黄芪多糖在长白、大约克与杜洛克3种公猪的精液冷冻都可以取得较好的效果。     

Roles of Intracellular Cyclic AMP Signal Transduction in the Capacitation and Subsequent Hyperactivation of Mouse and Boar Spermatozoa

It is not until accomplishment of a variety of molecular changes during the transit through the female reproductive tract that mammalian spermatozoa are capable of exhibiting highly activated motility with asymmetric whiplash beating of the flagella (hyperactivation) and undergoing acrosomal exocytosis in the head (acrosome reaction). These molecular changes of the spermatozoa are collectively termed capacitation and promoted by bicarbonate, calcium and cholesterol acceptors. Such capacitation-promoting factors can stimulate intracellular cyclic AMP (cAMP) signal transduction in the spermatozoa. Meanwhile, hyperactivation and the acrosome reaction are essential to sperm fertilization with oocytes and are apparently triggered by a sufficient increase of intracellular Ca²⁺ in the sperm flagellum and head, respectively. Thus, it is necessary to investigate the relationship between cAMP signal transduction and calcium signaling cascades in the spermatozoa for the purpose of understanding the molecular basis of capacitation. In this review, I cover updated insights regarding intracellular cAMP signal transduction, the acrosome reaction and flagellar motility in mammalian spermatozoa and then account for possible roles of intracellular cAMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa.     

In vitro capacitation of stallion spermatozoa assessed by the lysophosphatidylcholine-induced acrosome reaction and the penetration rate into in vitro-matured, zona-free mare oocytes

The purpose of this study was to evaluate the ability of various chemicals to induce capacitation of stallion spermatozoa using 2 different assay systems. In Experiment 1, freshly ejaculated spermatozoa were treated for 0, 3 and 6 h with 10 μ g/ml heparin, 0.5 mM hypotaurine or 5 mM caffeine, or were incubated for 0, 3 and 6 h following 1 min exposure to 0.1 μ M ionophore A23187. The acrosome reaction (AR) in the capacitated spermatozoa was induced by 15 min challenge with 100 μ g/ml lysophosphatidylcholine (LPC). In the BO/BSA-control medium (Brackett and Oliphant medium with 0.3% BSA), mean percentage of AR spermatozoa at 0 h was 30%, and the AR rates increased to 40 and 48% after 3 and 6 h incubation, respectively. There was no significant further increase of the AR rates in the spermatozoa treated with heparin (50% at 6 h) and hypotaurine (58% at 6 h) when compared to the control. Caffeine had a beneficial effect on inducing sperm capacitation after 3 and 6 h incubation (AR rates; 61 and 66%, respectively, P<0.01). Immediately after ionophore A23187 treatment, the AR rate increased to 56%, and reached 68 and 67% after 3 and 6 h incubation, respectively (P<0.01). Spermatozoal motility at any time points did not differ between control and any chemical treatment groups, except one treatment (ionophore; 3 h group).In Experiment 2, frozen-thawed spermatozoa were treated with 4 different chemicals as described above. Aliquot of spermatozoa was added to a microdrop of BO/BSA medium in which 6 to 10 in vitro-matured, zona-free mare oocytes were placed, and the oocytes were fixed and stained 20 h after insemination. The penetration rate by BO/BSA-treated spermatozoa was 76%, which was comparable to the results with heparin (73%), hypotaurine (78%) and caffeine (58%). In contrast, treatment of spermatozoa with ionophore A23187 gave a significantly lower penetration rate (30%) than the control value. Surprisingly these two experiments had different conclusions in assessing capacitation of stallion spermatozoa.     

Fluorescent Stain Method for the Simultaneous Determination of Mitochondrial Potential and Integrity of Plasma and Acrosomal Membranes in Boar Sperm

The purpose of this study was to validate a technique for simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa, using an association of fluorescent probes: Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and JC-1. Three ejaculates from each of four different boars, all showing motility >or=80% and abnormal morphology 相似文献   

Elucidation of the mechanism responsible for the temperature-dependent reversible inactivation of the motility of fowl spermatozoa

1. When washed fowl spermatozoa were held at 30 degrees C in a Ca++-free medium they retained internal Ca++, assimilated from the seminal plasma in vivo, which maintained their motility. 2. When this preparation was warmed to 40 degrees C, Ca++ was lost to the medium and the spermatozoa became immotile. 3. On subsequent cooling to 30 degrees C, the spermatozoa were capable of re-sequestering the Ca++, which restored their motility. 4. Removal of internal Ca++, with subsequent reduction of cellular activity, improved the survival of fowl spermatozoa held at 30 degrees C.     

Boar variability affects sperm metabolism activity in liquid stored semen at 5 degrees C

Metabolic activity of boar spermatozoa, liquid stored for three days at 5 degrees C, was measured using bioluminescence for ATP content, fluorescent assay (JC fluorochrome) of mitochondrial activity and oxygen consumption. Sperm motility and plasma membrane integrity (PMI) were simultaneously analyzed. Apart from the statistically significant effect (P < 0.001) of semen storage time, the importance of the individual source of the ejaculate for the analyzed parameters of metabolic efficiency of spermatozoa was shown. This phenomenon was manifested in the interaction of the individual source of the ejaculate with spermatozoa motility, integrity of their membranes and metabolic activity with the passing time of semen preservation. Recorded results indicate that the individual factor may have a significant influence on the technological usefulness of boar spermatozoa for liquid storage. Quality analyses conducted on boar semen stored at 5 degrees C may be used for pre-selection of boars producing sperm with an enhanced tolerance to cold shock.     

Hyperactivation and acrosome reaction in vitro in spermatozoa ejaculated by cryptorchid dogs after orchiopexy.

Orchiopexy of the cryptorchid (CR) testis and castration of the scrotal testis were performed in three unilaterally CR beagles at six months of age. Induction rates for ejaculated sperm hyperactivation (HA) and the acrosome reaction (AR) in vitro in these orchiopexied dogs were compared with five those in normal beagles one year later. Canine spermatozoa were incubated for 9 hr at 38 degrees C under 5% CO2 in air in canine capacitation medium at a concentration of 30 x 10(6) sperm/ml. HA was observed using high-speed videomicrography. The AR spermatozoa were evaluated by the triple stain technique. As a result, there was no significant difference between 'the CR dogs after orchiopexy' (CDO) and the normal dogs (ND) with respect to sperm motility just after ejaculation. However, sperm motility of CDO decreased markedly during incubation. There was a significant difference in sperm motility between CDO (Mean +/- SD; 47 +/- 12%) and ND (80 +/- 9%) after three hours of incubation (p less than 0.01). No significant difference was observed between CDO and ND with respect to the HA rate of motile spermatozoa throughout the incubation period. The peak of HA rate was found in both CDO (58 +/- 5%) and ND (61 +/- 16%) after seven hours of incubation. The AR rate of spermatozoa in CDO was lower than that in ND after six hours of incubation. The AR rate of CDO (26 +/- 4%) was significantly lower than ND (46 +/- 5%) after eight hours of incubation (p less than 0.01). It is assumed that there might be relation between a rapid decrease of motility and low AR rate in spermatozoa of CDO during incubation.     

Comparative Effects of Autologous and Homologous Seminal Plasma on the Viability of Largely Extended Boar Spermatozoa

Sperm handling, associated to artificial reproduction technologies (ART) such as in vitro fertilization (IVF) or the use of flow cytometry for cell analysis or sorting imposes volumetric extension of the sperm suspension and decreases sperm viability, presumably because of the removal of seminal plasma (SP) components. This study evaluated whether a 10% v/v of autologous SP (retrieved from the same donor boar) or homologous SP (e.g. from any of the four fertile boars included, other than the one providing the spermatozoa) would differently affect the viability of boar spermatozoa subjected to large extension in a simple saline medium [phosphate-buffered saline and 0.1% ethylenediaminetetraacetic acid (EDTA), PBSm] to a concentration of 0.3 x 10(6) spermatozoa/ml and incubated for 2 h at 30 degrees C. Sperm viability was monitored as membrane integrity [using the fluorophore carboxyfluorescein diacetate (C-FDA) and propidium iodide (PI)], mitochondrial function (using the fluorophore R-123) and motility characteristics [using Computer Assisted Sperm Analysis (CASA)]. Substraction of the SP and extension followed by incubation in PBSm significantly (p < 0.05) decreased sperm viability, which could be restored by addition of autologous SP. Furthermore, exposure of the extended spermatozoa to homologous SP (from any other individual boar) significantly (p < 0.05) varied with the source of the sire; some boars exerting beneficial effects (even surpassing the effects of the autologous SP; p < 0.05) while at least one boar negatively (p < 0.05) influencing the viability of the incubated spermatozoa. It is concluded that SP should be present when incubating highly extended spermatozoa. As a result of the obvious differences among boars, it would be advantageous to examine the ability of SP to maintain sperm viability prior to the use of SP pools during sperm handling in vitro.     

Fluorometric assessments of viability and mitochondrial status of boar spermatozoa following liquid storage

A study was conducted to assess viability and mitochondrial status of boar spermatozoa stored at 5 degrees C and 16 degrees C. Gel-free ejaculates, collected from 3 mature boars, were extended in a standard diluent (K3) supplemented with a low-density lipoprotein fraction (LDF) isolated from egg yolk, and stored for 96 h at 5 degrees C and 16 degrees C. Motility analysis was conducted after semen dilution (D0) and on D1-D4 of storage. A double staining method, rhodamine 123 (R123) and propidium iodide (PI), was used to assess sperm viability and mitochondrial status. Sperm viability was also assessed using Hoechst 33,258 (H33258) stain. In fresh semen samples, the percentage of motility was significantly correlated with the percentage of viable spermatozoa with functional mitochondria (R123-PI), viable spermatozoa determined by H33258 staining and ATP content (r = 0.88, p < or = 0.01; r = 0.69, p < or = 0.05; r = 0.77, p < or = 0.01, respectively). The ATP content was also positively correlated with the percentage of viable spermatozoa with functional mitochondria (r = 0.76, p < or = 0.01). Sperm cells progressively lost motility, viability and mitochondrial capacity when stored in the supportive media at 5 degrees C and 16 degrees C. Motility estimates were lower (p < or = 0.05) than the percentage of viable spermatozoa with functional mitochondria during storage in K3 and LDF-based diluents on D4 and D3-D4, respectively. Deterioration in motility and membrane integrity was less marked in spermatozoa stored in LDF-based diluents. Spermatozoa doubly-stained with R123-PI appeared to possess some functional mitochondria, particularly in LDF-based diluent semen. Estimates of sperm viability, as determined by R123-PI staining, were equivalent (p > or = 0.05) to estimates made using H33258 staining. A decrease in mitochondrial activity, as measured by R123 uptake, was accompanied by lower ATP content in spermatozoa stored in K3 and LDF-based diluents after 48 h and 72 h of storage, respectively. Fluorometric measurements of viability and mitochondrial status of boar spermatozoa during liquid storage seem to provide reliable information about the sperm functional membranes.     

利用CTC染色进行猪精子不同体外获能方法的比较

为了探讨不同方法对猪精子体外获能的影响,本试验以猪的新鲜精液为研究对象,分别经肝素上游法、钙离子载体诱导法和咖啡因诱导法进行精子不同获能时间的处理,并利用金霉素荧光染色法（chlortetracycline,CTC）对其获能效果进行检验。结果表明,采用肝素上游法孵育40 min处理后,精子的获能比例显著高于其他各处理组（P<0.05）,说明肝素上游法可以较好地诱发猪精子在体外获能。     

Variations in the Proportion of Glycolytic/Non-glycolytic Energy Substrates Modulate Sperm Membrane Integrity and Function in Diluted Boar Samples Stored at 15–17°C

In this work the role of energy substrates in the maintenance of boar-sperm survival during storage at 15-17 degrees C was tested. For this purpose, boar spermatozoa were stored at 15-17 degrees C in several defined media with separate combinations of a monosaccharide, glucose and a non-monosaccharide, either citrate or lactate, energy substrates. Our results indicate that the medium containing the highest concentration of glucose together with low lactate levels was the most suitable to maintain sperm quality for 168 h at 15-17 degrees C. This was confirmed after observation of the results of the percentages of viability and altered acrosomes, the osmotic resistance test, the hyperosmotic resistance test and the rhythm of L-lactate production. The survival ability of boar sperm was greater in this experimental medium than in the standard Beltsville Thawing Solution extender, which contains only glucose as an energy substrate, although at a concentration far higher than that of all the tested experimental media. Our results indicate that the exact composition, more than the pure quantity of energy substrates, is a very important modulatory factor which affects survival ability of boar sperm in refrigeration. Thus, the exact combination of several energy substrates would have to be taken into account when optimizing the design of commercial extenders to store boar spermatozoa at 15-17 degrees C.