Ceramide enhances acrosomal exocytosis triggered by calcium and the calcium ionophore A23187 in boar spermatozoa

Mammalian spermatozoa must undergo acrosomal exocytosis prior to penetration of the oocyte at fertilization. The mechanisms underlying acrosomal exocytosis have not yet been fully elucidated. This study explored the possible involvement of ceramide in exocytosis of the boar sperm acrosome. Ejaculated boar spermatozoa, stored with the Beltsville TS extender at 17 degrees C for up to 3 days, were washed and preincubated for 10 min with C2-ceramide, an analogue of endogenous ceramide, C2-dihydroceramide (C2-DH-ceramide), a negative control to C2-ceramide, or with (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP), an inhibitor of alkaline ceramidase, followed by incubation and stimulation with 3 mM Ca2+ and 0.3 microM A23187 (Ca2+/A23187) at 37 degrees C in air in a water bath. Spermatozoa fixed at specific intervals were examined, and the % of acrosomal exocytosis was monitored. Stimulation of spermatozoa with Ca2+/A23187 resulted in a time-dependent increase. There were no obvious changes at 5 min, but this was followed by a rapid increase at 10 min, reaching nearly a maximum level after 15 min or more of incubation. Preincubation with C2-ceramide or D-erythro-MAPP enhanced acrosomal exocytosis triggered by Ca2+/A23187 in a dose-dependent manner, whereas C2-DH-ceramide was without effect. These results suggest the possibility that ceramide may be involved in the mechanisms underlying acrosomal exocytosis.     

Morphological observations on frozen and thawed Muscovy spermatozoa

1. Fresh Muscovy drake spermatozoa were examined using a scanning electron microscope (SEM). The average lengths of the segments were: acrosome 1·8 μm, nucleus 10‐9 μm, midpiece 3·6 μm and flagellum (exclusive of midpiece) 71 μm.

2. Under the light microscope, the incidence of abnormal spermatozoa in Muscovy semen subjected to freezing and thawing (almost all with crooked necks) was about 5% higher than that in diluted unfrozen semen.

3. In thawed semen, various abnormalities of the acrosome were observed under the SEM. It seemed that the most radical change was the complete separation of the acrosome from the apical part of the nucleus.

4. The incidence of abnormal acrosomes was increased more than 20% by freezing and thawing.

5. These results suggest that low fertility in thawed semen may be related to increases in the proportion of spermatozoa with crooked necks and acrosomal damage.

     

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.     

Influence of Thawing Diluents on Vitality,Acrosome Morphology,Ultrastructure and Enzyme Release of Deep Frozen Roar Spermatozoa*

The present investigation was performed to study the effect of freezing and thawing on boar spermatozoa. Thirty-one ejaculates from four boars were investigated after thawing in three different thawing diluents (seminal plasma, OLEP, isotonic glucose solution).From each ejaculate one sample of 1 × 10⁹ spermatozoa was thawed in each of the thawing diluents. Each sample was examined in a thermoresistance test in which motility was stimulated with caffeine 30 min. and 3 hrs. after thawing. Furthermore, acrosome morphology and ASAT release from the spermatozoa were investigated for each sample. One ejaculate from the two most frequently used boars was examined by electron microscopy after thawing in each of the thawing diluents.Differences in the aspects studied appeared between isotonic glucose solution and the other two thawing diluents in the thermoresistance test, in the response to caffeine stimulation 3 hrs. after thawing and in the amount of ASAT released from the spermatozoa. The influence on the acrosome morphology varied between the thawing diluents, but the acrosomal alterations did not seem to be connected with the damage reflected by the thermoresistance test and by the measurement of extracellular ASAT activity.The ultrastructural investigation showed that all spermatozoa examined had some degree of ultrastructural alteration as compared with freshly ejaculated boar spermatozoa treated in the same way. This alteration could not be related to any of the thawing diluents.Of the various laboratory tests the thermoresistance test and the measurement of ASAT release are suggested to be sensitive indicators of sperm damage during freezing and thawing. These tests might be useful indicators of variations in sensitivity of spermatozoa to the freezing-thawing procedure.     

Semen quality and fertility after heat stress in boars

Sperm morphology and the fertilizing capacity of ejaculated spermatozoa were examined in 6 Swedish Landrace boars before and after heat stress. The boars were exposed to 35° C during 100 h in a climatic room. Fertility was measured by insemination of gilts before and at various times after heat stress. Each gilt (n = 44) was inseminated with a total of 5×10⁹ spermatozoa diluted to 10O ml with EDTA-glucose diluent and fertilization was assessed by examining recovered ova 2 days after insemination.Changes in semen quality varied among the boars from a very weak response in 2 boars to pronounced semen alterations occurring 2–6 weeks after heat stress in the other boars. A close relationship was found between seminal changes and fertilization rates, all ejaculates which had high fertilization rates being of the same quality as the pre-exposure ejaculates. The ejaculates that had poor fertility were characterized by lowered sperm motility and increased numbers of spermatozoa with abnormal heads, proximal cytoplasmic droplets and nuclear pouch formations.     

Removal of Seminal Plasma Enhances Membrane Stability on Fresh and Cooled Stallion Spermatozoa

Fertility is reduced after semen cooling for a considerable number of stallions. The main hypotheses include alterations in plasma membrane following cooling and deleterious influence of seminal plasma. However, interindividual variability is controversial. We hypothesized that the removal of seminal plasma could enhance motility in some ‘poor cooler’ stallions, but could also affect, negatively or positively, membrane quality in some stallions. This study examined the effect of centrifugation, followed or not by removal of seminal plasma, on parameters indicating semen quality after 48 h at 4°C: motility, plasma membrane integrity as evaluated by hypo‐osmotic swelling test, acrosome integrity and response to a pharmacological induction of acrosome reaction using ionophore A23187. Sixty‐six ejaculates from 14 stallions were used, including stallions showing high or low sperm motility after cooled storage. Centrifugation without removal of seminal plasma did not affect sperm parameters. Removal of seminal plasma did not affect motility, but significantly stabilized sperm membranes, as demonstrated by a higher response to the osmotic challenge, and a reduced reactivity of the acrosome. Moreover, for the same semen sample, the response to an induction of acrosome reaction was significantly higher when the induction was performed in the presence of seminal plasma, compared with the induction in the absence of seminal plasma. This was observed both for fresh and cooled semen. When the induction of acrosome reaction with ionophore A23187 is used to evaluate sperm quality, care must therefore be taken to standardize the proportion of seminal plasma between samples. For the 10 stallions serving at least 25 mares, the only variable significantly correlated with fertility was motility. The influence of membrane stabilization regarding fertility requires further investigations.     

Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.     

A comparative study on the chemical composition of plasma from the cauda epididymidis, semen fractions, and whole semen in boars

A comparative study was carried out on the chemical composition of plasma from the cauda epididymidis, semen fractions, and whole semen of boars. A total of 22 boars were used in this study. The boars, which ranged in age from 8 to 14 months, were of Swedish Landrace and Swedish Yorkshire breed. All boars used presented a normal semen picture. A dummy sow and an artificial vagina were employed for semen collection. The semen was collected as whole semen and as semen fractions in 10 nil volumes. The contents of the cauda epididymidis was removed post mortem.The following parameters were investigated: sperm concentration, dry weight of spermatozoa and of seminal plasma, osmotic pressure, sodium, potassium, chloride, inorganic phosphorus, calcium, magnesium, total protein, GOT, GPT, and alkaline phosphatase in seminal plasma. Paper electrophoresis was carried out on seminal plasma. Tlxe results of the analysis are summarized in Tables 1–6.The sperm concentration was approximately 3.2 mill./mm3 in the cauda epididymidis, 1 mill./mm³ in the sperm-richest fraction (II) and 0.25 mill./mm3 in whole semen. The dry weight (expressed in per cent dry matter) of spermatozoa was highest in the cauda epididymidis (25.47 %), showing a tendency to decreasing in semen fractions I—IV and was lowest in whole semen (15.29 %). The per cent dry weight in plasma was higher in the cauda epididymidis (4.56 %) than in semen fraction I (2.20 %). In semen fractions I—IV the per cent dry weight rose from 2.20 (U to 4.51 % and reached the level of approximately 3.80 % in the sperm-free fractions V—VII. The osmotic pressure was significantly higher in the cauda epidi-dymal plasma than in the whole seminal plasma or the seminal plasma fractions. The same phenomenon was observed in a boar where the cauda epididymal content was collected in vivo from a patent established fistula. There appears to be a connection between the per cent dry weight of spermatozoa and the osmotic pressure, which means that the per cent dry weight of the cauda epididymal spermatozoa decreases when mixed with the accessory gland secretions, which have a lower osmotic pressure. The fall in per cent dry weights is thought to be caused by an intake of water.The amount of sodium, chloride and magnesium was higher in ejaculated seminal plasma than in cauda epididymal plasma. The reverse was true for inorganic phosphorus and potassium. Moreover the sperm-free fractions contained more sodium, chlorides and magnesium than the sperm-containing fractions, while the concentration of potassium and inorganic phosphorus was comparatively higher in the sperm-containing fractions. A connection is apparent between sperm concentration and the potassium, inorganic phosphorus and magnesium levels. Statistical analysis of the values of chloride and magnesium revealed significant differences between individual boars for most of the semen fractions.The concentration of plasma proteins in the cauda epididymidis was approximately the same as in whole semen and in the semen fractions except for fraction I, which contained a relatively low concentration. As regards total protein there were significant differences between individual boars in most of the semen fractions as well.The paper electrophoretic pattern of epididymal plasma was different from that of semen plasma. Thus there were three or four distinct components in the cauda epididymidis numbered 1, 2, 3, and 4, and three distinct components in whole seminal plasma numbered 3, 4, and 5, while the sperm-richest semen fractions contained four components (2, 3, 4, and 5) and the others three components, namely 3, 4, and 5.The level of GOT was high in the cautlu cpiflidymill contents (99.1 i. u./ml) compared with that for whole seminal plasma (99.1 i.u/ml). In semen fractions there was a clear positive correlation between the level of GOT and the sperm concentration. The GPT concentration wis as a whole low and. in contrast to GOT. somewhat higher in the sperm-free fractions than in the sperm-containing fractions. The concentration of alkaline phosphatase was very high in cauda epididymal plasma (31,463 i. u./ml) as well as in the sperm-rich fractions (e.g. 7,096 i. u./ml in fraction II). Preliminary investigation has moreover revealed a very low alkaline phosphatase concentration in seminal plasma of vasectomized boars, which condition suggests thai the main origin for alkaline phosphatase in boars is the testis and epididymis.     

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.     

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   

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

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

Assessment of viral presence in semen and reproductive function of frozen-thawed spermatozoa from Pallas' cats (Otocolobus manul) infected with feline herpesvirus.

Although herpesviruses are known to contaminate the semen of several mammalian species, the occurrence of feline herpesvirus type 1 (FHV-1) in semen of infected cats has not been reported. Our objectives in this study were to investigate the presence of FHV-1 DNA in seminal fluid and frozen-thawed spermatozoa from FHV-1 infected Pallas' cats (Otocolobus manul) and assess the functionality of their frozen-thawed spermatozoa in vitro. Over a 3-yr period, semen (n = 33 ejaculates) was collected periodically via electroejaculation from four Pallas' cats chronically infected with FHV-1. Spermic ejaculates were frozen by pelleting on dry ice and stored in liquid nitrogen. After thawing, sperm motility and acrosome status were assessed over time during in vitro culture. For vitro fertilization (IVF), viable domestic cat (Felis silvestris catus) oocytes were inseminated with frozen-thawed Pallas' cat spermatozoa and evaluated for embryo cleavage. For FHV-1 polymerase chain reaction (PCR) analysis, DNA was extracted from seminal fluid, frozen-thawed spermatozoa, inseminated oocytes, heterologous IVF embryos, and conjunctival biopsies and analyzed for presence of a 322-base pair region of the FHV-1 thymidine kinase gene. Immediately post-thaw, sperm motility and percentage of intact acrosomes were decreased (P < 0.05) compared to fresh samples, and declined further (P < 0.05) during culture. However, all frozen-thawed IVF samples were capable of fertilizing domestic cat oocytes (overall, 46.1 +/- 6.0% cleavage). PCR analysis did not identify FHV-1 DNA in any reproductive sample despite the repeated detection of FHV-1 DNA in conjunctival biopsies. These results suggest that semen collected from Pallas' cats infected with FHV-1 does not contain cell-associated or non-cell-associated virus and that frozen-thawed spermatozoa exhibit adequate function for potential genetic rescue with minimal risk of FHV-1 transmission.     

Effect of dietary selenium and vitamin E on the ultrastructure and ATP concentration of boar spermatozoa, and the efficacy of added sodium selenite in extended semen on sperm motility

Three experiments evaluated the effects of dietary Se and vitamin E on the ultrastructure of spermatozoa, ATP concentration of spermatozoa, and the effects of adding sodium selenite to semen extenders on subsequent sperm motility. The experiment was a 2 x 2 arrangement of treatments in a randomized complete block design. A total of 10 mature boars were fed from weaning to 18 mo of age diets fortified with two levels of supplemental Se (0 or .5 ppm) or vitamin E (0 or 220 IU/kg diet). The nonfortified diets contained .06 ppm Se and 4.4 IU vitamin E/kg. In Exp. 1, the spermatozoa from all boars were examined by electron microscopy. Vitamin E had no effect on structural abnormalities in the spermatozoa. When the low-Se diet was fed the acrosome or nuclei of the spermatozoa was unaffected, but the mitochondria in the tail midpiece were more oval with wider gaps between organelles. The plasma membrane connection to the tail midpiece was not tightly bound as when boars were fed Se. Immature spermatozoa with cytoplasmic droplets were more numerous when boars were fed the low-Se diet, but the occurrence of midpiece abnormalities occurred in boars fed diets with or without Se or vitamin E. Our results suggest that Se may enhance spermatozoa maturation in the epididymis and may reduce the number of sperm with cytoplasmic droplets. In Exp. 2, the concentration of ATP in the spermatozoa was evaluated in the semen of all treatment boars. When the low-Se diet was fed, ATP concentration was lower (P < .01), whereas vitamin E had no effect on ATP concentration. Experiment 3 investigated the effect of diluting boar semen with a semen extender with sodium selenite added at 0, .3, .6, or .9 ppm Se. Three ejaculates from each boar were used to evaluate these effects on sperm motility to 48 h after dilution. Sperm motility declined (P < .01) when Se was added to the extender, and this decline was exacerbated as the concentration of added Se increased (P < .01). The added Se was demonstrated to be tightly adhered to the spermatozoa. Overall, these results suggest that low Se-diets fed to boars resulted in abnormal spermatozoal mitochondria, a lower ATP concentration in the spermatozoa, and a loose apposition of the plasma membrane to the helical coil of the tail midpiece, but no effect from inadequate vitamin E was demonstrated. Adding sodium selenite to the semen extender reduced sperm cell motility.     

Studies of the factors causing abnormal acrosomes and crooked-necks in fowl spermatozoa during freezing and thawing

In order to investigate the factors causing crooked-necked spermatozoa (CNS) or those with abnormal acrosomes during freezing and thawing, fowl spermatozoa in NaCl or glucose solutions containing 92 ml glycerol/l were examined using a scanning electron microscope before and after freezing and thawing. The incidence of CNS in NaCl solution significantly increased after freezing and thawing, but not in glucose solution. The acrosomal damage caused by freezing and thawing was considerable in both solutions, and the incidence of damage in glucose solution was significantly higher than that in NaCl solution. In neither solution was there a significant difference between the incidence of acrosomal damage in CNS and in non-CNS. The ratios of incidences of abnormal acrosome after, versus before, freezing were higher in non-CNS than in CNS. It appears from these results that the factors during freezing and thawing which cause CNS may differ from those causing acrosomal damage.     

Effects of filtration of semen doses from subfertile boars through neuter Sephadex columns.

This study was designed to develop a method of improving the quality of sperm obtained from subfertile Piétrain boars. Seminal doses were filtered through neuter Sephadex columns (G-25 Medium, G-50 Fine, G-50 Medium and G-75, length 10 +/- 0.5 cm, flow rate 1 ml/20 s). Doses were prepared by pooling 10 ml semen samples collected from 58 asthenoteratospermic boars and diluted the sperm-cell rich fraction 1 : 6 in Betsville thawing solution extender. Sperm quality was determined before and after the filtering process. Sperm morphology and motility were assessed using the computer program SCA 2002 production, and sperm vitality was evaluated by fluorescence multistaining. ORT and HRT tests were used to determine the osmotic resistance of spermatozoa, and metabolic performance was assessed by measuring l-lactate production. Results indicate that the filtration process rendered increased proportions of mature spermatozoa and of viable spermatozoa with an intact acrosome, nucleus and mitochondrial sheath. Sperm filtration led to decreased percentages of spermatozoa with proximal and distal droplets and of agglutinated spermatozoa, along with slightly diminished ORT values. HRT scores and L-lactate production were unaffected. Our findings indicate that filtering through a Sephadex column improves the sperm morphology and vitality of seminal doses obtained from subfertile boars, but produces no functional changes in the spermatozoa. All four column types yielded similar results.     

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.     

Fine surface structure of bovine acrosome-intact and reacted spermatozoa observed by atomic force microscopy

The atomic force microscope (AFM) provides nanometer resolution, topographic data of the natural surface structure of materials. We studied the topology of the surface structure of bovine sperm heads during the acrosome reaction by AFM. In addition, we numerically analyzed the areas of the median sagittal plane of the sperm heads. Bovine frozen-thawed spermatozoa were washed, capacitated by heparin, and incubated with lysophosphatidylcholine (LPC) to induce the acrosome reaction, smeared on a cover glass, air-dried, and observed with AFM using the dynamic force (tapping) mode. AFM analysis of spermatozoa showed the clear surface structure of acrosomes, equatorial segments, postacrosomal regions and necks. Although AFM images of spermatozoa capacitated by heparin had complete acrosomes, most spermatozoa treated with LPC had no acrosomal caps as shown by AFM. These observations coincided with those obtained by light microscopy after staining with naphthol yellow S and erythrosin B. Furthermore, numerical analysis of AFM images indicated that areas of the median sagittal plane of the anterior portions of acrosome-reacted sperm heads (2679 +/- 616 pixels) were approximately 40% less than those of intact heads (4535 +/- 174 pixels, P<0.05). These results indicate that AFM can usefully observe and numerically analyze the fine surface structures of bovine spermatozoa.     

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。     

Effect of washing on motility and acrosome morphology of frozen-thawed goat spermatozoa

Semen from 4 bucks was collected using an artificial vagina and was pooled and divided into 6 aliquots. Three aliquots were washed twice, 15 minutes each time, with Ringer's solution, and the fluid was removed by centrifugation at 950 X g between washes. All 6 aliquots (3 washed and 3 unwashed) were extended with skim milk-glycerol, lactose-egg yolk-glycerol, or tris (hydroxymethyl) aminomethane-citric acid-egg yolk-glycerol and were frozen in straws to -196 C. The semen was then thawed and kept at 37 C for 8 hours. Percentage of sperm motility was estimated, and the percentage of normal acrosomes (NA) was determined at 0, 2, 4, 6, and 8 hours after thawing. The experiment was repeated 7 times. The data indicated a significant positive effect (P = 0.0009) of washing on motility, but no effect (P = 0.5347) of extender. There was also a significantly higher percentage of NA in washed semen (P less than 0.0001). Sperm extended in tris aminomethane-citric acid-egg yolk-glycerol had more NA than those extended in lactose-egg yolk-glycerol. Sperm motility and acrosome morphology were depressed also in the presence of seminal plasma for the milk extender, which did not contain egg yolk. Removal of seminal plasma from goat semen was beneficial in preserving the integrity of the spermatozoa after freezing, regardless of the extender used.     

Effect of season and social environment on testis size and semen quality of the adult Landrace boar

Eight adult Landrace boars were housed for 12 mo in one of two social environments. Socially nonrestricted boars were penned adjacent to and allowed minimal physical contact with ovariectomized gilts hormonally induced into estrus every 2 wk. Socially restricted boars were penned behind solid walls to eliminate visual and physical contact with other pigs. All animals were subjected to natural changes in daylength. Semen was collected weekly; gel-free volume, gel weight, sperm concentration and number per ejaculate, sperm motility (percent and type) and semen pH were determined. Total protein, citric acid contents and alkaline phosphatase activity were measured in seminal plasma. Testis length and width and various body temperature measurements were recorded monthly. Except for percent motile sperm and alkaline phosphatase activity, all semen characteristics varied (P less than .05) with month. The pattern of seasonal change in semen volume was modified by social environment (group X month, P less than .05). Sperm numbers were highest in winter and lowest in spring and summer. Ejaculate protein and citric acid contents were highest in fall and winter; decreases in spring were associated with moderate ambient temperatures and increases in daylength (r = -.80, P less than .05). Testicular length for socially nonrestricted boars was maximum in November through January and minimum in April through July, and did not vary as extensively for socially restricted boars. Scrotal temperature was elevated during periods of high ambient temperature, but not to values detrimental to spermatogenic function.(ABSTRACT TRUNCATED AT 250 WORDS)