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1.
Two experiments were conducted to evaluate effects of 3 stains and 2 fixatives on morphologic features of bovine spermatozoa. In experiment 1, the morphologic features of acrosomes of raw and incubated, extended spermatozoa were evaluated after staining with Hancock's Blom's or Wells-Awa's stains or after fixation with buffered glutaraldehyde. Evaluations were done of stained smears by bright field microscopy and of fixed, unstained preparations, by differential interference contrast microscopy, using wet mounts. Raw semen samples from 1st ejaculates of 80 bulls were evaluated. The percentage of spermatozoa with intact acrosomes averaged 83.5% in unstained preparations fixed in glutaraldehyde, compared with averages of 68.1, 74.5, and 67.4% for smears stained with Hancock's, Blom's, or Wells-Awa's procedures (P less than 0.01). From these results, it appeared that procedures for preparing stained smears were detrimental to acrosomes. Although counts for other acrosomal abnormalities differed (P less than 0.01) in each treatment, patterns were inconsistent. With incubated, extended spermatozoa from 57 bulls, glutaraldehyde-fixed, unstained samples had more (55%) intact acrosomes (P less than 0.01) than did samples stained with Hancock's or Blom's procedures (24.0 and 34.7%, respectively, but the former were not significantly different from Wells-Awa-stained smears (49.3% intact acrosomes). In experiment 2, several morphologic characteristics of spermatozoa from 15 1st ejaculates of 7 bulls were evaluated after staining with Hancock's or Blom's stains or after fixation in buffered glutaraldehyde or buffered formal saline fixatives. Higher counts (P less than 0.01) of head abnormalities were found in wet, unstained fixed preparations (4.83, 4.47, 7.87, and 7.93% respectively, for Hancock's, Blom's, glutaraldehyde, and formol saline methods). There were more (P less than 0.05) separated heads on stained, dry smears (1.43, 1.23, 0.47, and 0.47%, respectively, for Hancock's, Blom's, glutaraldehyde, and formol saline procedures). Fixation with buffered glutaraldehyde resulted in higher counts (P less than 0.01) of proximal protoplasmic droplets (2.47, 1.03, 0.67, and 1.43%, respectively, for glutaraldehyde, Hancock's, Blom's, and formol saline procedures). Although not significant, the same trend was observed for distal protoplasmic droplets...  相似文献   

2.
Seven mature Japanese black bears were used as semen donors, and a total of 7 semen samples collected from the animals by the electroejaculation method were cryopreserved in liquid nitrogen. Egg yolk-TRIS-citrate-glucose extender was used, and the effects of different final concentrations of glycerol, at 4-12% (v/v), on frozen-thawed spermatozoa were examined. No significant difference was observed in percent motility or percent abnormal morphology of frozen-thawed spermatozoa among the different glycerol concentrations. Percent viability and percent intact acrosomes of spermatozoa cryopreserved with 4 and 6% glycerol were significantly higher than those with 10 and 12% glycerol. These results suggest that a suitable glycerol concentration for freezing Japanese black bear semen within the range tested would be 4-6%.  相似文献   

3.
The aim of this study was to evaluate the influence of Hoechst 33342 (H‐42) concentration and of the male donor on the efficiency of sex‐sorting procedure in canine spermatozoa. Semen samples from six dogs (three ejaculates/dog) were diluted to 100 × 106 sperm/ml, split into four aliquots, stained with increasing H‐42 concentrations (5, 7.5, 10 and 12.5 μl, respectively) and sorted by flow cytometry. The rates of non‐viable (FDA+), oriented (OS) and selected spermatozoa (SS), as well as the average sorting rates (SR, sorted spermatozoa/s), were used to determine the sorting efficiency. The effects of the sorting procedure on the quality of sorted spermatozoa were evaluated in terms of total motility (TM), percentage of viable spermatozoa (spermatozoa with membrane and acrosomal integrity) and percentage of spermatozoa with reacted/damaged acrosomes. X‐ and Y‐chromosome‐bearing sperm populations were identified in all of the samples stained with 7.5, 10 and 12.5 μl of H‐42, while these two populations were only identified in 77.5% of samples stained with 5 μl. The values of OS, SS and SR were influenced by the male donor (p < 0.01) but not by the H‐42 concentration used. The quality of sorted sperm samples immediately after sorting was similar to that of fresh samples, while centrifugation resulted in significant reduction (p < 0.05) in TM and in the percentage of viable spermatozoa and a significant increase (p < 0.01) in the percentage of spermatozoa with damage/reacted acrosomes. In conclusion, the sex‐sorting of canine spermatozoa by flow cytometry can be performed successfully using H‐42 concentrations between 7.5 and 12.5 μl. The efficiency of the sorting procedure varies based on the dog from which the sperm sample derives.  相似文献   

4.
Laboratory trials were conducted to study the effect of various concentrations of cloprostenol on the motility and morphological changes of the acrosomes of boar spermatozoa. As found, a cloprostenol concentration of 250 ng per ml of semen to 2500 ng per ml of diluted semen has no adverse effect on the motility of spermatozoa and on the morphological changes of their acrosomes. The concentration of 5000 ng of cloprostenol (in the Oestrophan Spofa product) per 1 ml of diluted semen negatively influences the motility of spermatozoa. An insemination dose of 100 ml of diluted sperm treated with 500 ng of cloprostenol was used for the artificial insemination of 152 sows; 166 sows of the same farm inseminated with untreated semen were used as controls. No gilts were included in the trial. Out of the 152 test sows, 113 conceived after the first insemination, i.e. 74.35%, and the average litter size was 10.04 piglets. In the control group, 125 sows delivered their litters, i.e. 75.30% of the total number, the average litter size being 9.96 piglets. Comparing the reproduction parameters of the experimental and control groups it can be said that the treatment of an insemination dose with 500 ng of cloprostenol immediately before insemination had no influence on the pregnancy rate and on the size of litters.  相似文献   

5.
The knobbed acrosome defect was found at levels of 25 to 100 percent of spermatozoa from 16 of 2054 beef bulls. The incidence of this defect appeared to be particularly high in the Charolais breed. Pedigree analysis of some of the affected Charolais bulls indicated there may be a genetic predisposition for this sperm defect. In eosin-nigrosin stained semen smears the most common form of the abnormality was a flattened or indented apex of the sperm head. A refractile bead at the apex of the sperm head was seen less commonly. Electron microscopy of the spermatozoa from one bull showed that the abnormality was similar to the knobbed sperm defect previously described in Friesian bulls. A breeding trial confirmed that bulls producing spermatozoa with a high incidence of knobbed acrosomes are infertile.  相似文献   

6.
Microencapsulation of bovine spermatozoa   总被引：1，自引：0，他引：1  
Two experiments were conducted to examine the efficacy of microencapsulation of bovine spermatozoa for use in artificial insemination. In Exp. 1, sperm were encapsulated at three different concentrations (45, 90 and 180 X 10(6) sperm/ml) in either .75- or 1.5-mm (diameter) microcapsules and incubated in vitro for 24 h at 37 C. Unencapsulated samples of each concentration served as controls. Capsule contents were evaluated for percentage of sperm motility and intact acrosomes at 2, 12 and 24 h of incubation. Capsule fragility was evaluated after 24 h incubation. Viability of spermatozoa was not influenced by sperm concentration or capsule size, and compared with controls, cellular injury after encapsulation was not apparent. Fragility of capsules was unaffected by capsule size; however, as the sperm concentration increased, integrity of the capsules decreased (P less than .05). In Exp. 2, using frozen-thawed semen, the effect of egg yolk content, presence of glycerol and viability of spermatozoa on the success of microencapsulation was measured. The extender was 2.9% sodium citrate with glycerol (7% v/v) and either 0, 5, 10 or 15% egg yolk (v/v). Uniformity of capsules in size and shape was evaluated subjectively. Capsule integrity and uniformity were unaffected by glycerol, sperm viability or egg yolk level up to 10% v/v; however, encapsulation of spermatozoa in 15%-yolk buffer increased the heterogeneity in capsule size and shape. Viability of encapsulated spermatozoa was maximal for extenders containing 10 or 15% yolk v/v. Reduced viability for the 5% yolk extender was due to pre-encapsulation injury associated with freezing. Microencapsulation procedures are compatible with sperm viability and can be adapted to an acceptable extender system used in artificial insemination.  相似文献   

7.
Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.  相似文献   

8.
To improve the results obtained with a reference cryopreservation extender (control extender: Triladyl® + 20% (v/v) egg yolk + 6.4% (v/v) glycerol) for freezing caprine semen, glutamine was added to 18 split ejaculates at concentrations of 0, 20, 40, 80 and 120 m m (experiment 1). In experiment 2, glutamine was added to 18 split ejaculates at concentrations of 20, 25, 30, 35 and 40 m m . In the third experiment, the egg yolk was replaced with the low-density lipoprotein (LDL) fraction of egg yolk. The quality of frozen then thawed spermatozoa in each extender was compared using computer-assisted semen analysis. In experiment 1, glutamine at concentrations of 20 m m and 40 m m significantly improved sperm motility compared with the control extender. However, at 120 m m , a significant decrease in motility and velocity was observed. In experiment 2, motility, curvilinear velocity and amplitude of lateral head displacement (ALH) were improved in glutamine at 25 m m compared with the control. In experiment 3, 8% LDL and 25 m m glutamine significantly improved sperm motility, straight line velocity and ALH. In the fourth experiment, the quality of the previously defined freezing extender (Triladyl® + 8% (v/v) LDL + 25 m m glutamine + 6.4% (v/v) glycerol) was tested by comparing acrosome, tail membrane, plasma membrane and DNA integrity in 18 split ejaculates of frozen then thawed spermatozoa with spermatozoa that had been frozen then thawed in the control extender, and with spermatozoa from fresh, unfrozen sperm. The percentage of spermatozoa with intact acrosomes and tail membranes was significantly higher with the newly defined extender than that observed with the control extender. There was no significant difference in the percentage of spermatozoa with intact DNA between the frozen and fresh semen.  相似文献   

9.
Four bulls that produced spermatozoa with a high percentage of abnormal acrosomes were individually placed in pens with females for 21 days. Frozen semen from 2 of the bulls was used for artificial insemination. One of the bulls was placed in a competitive mating situation with normal bulls at pasture. First service pregnancy rates were determined by transrectal ultrasonography 28 days after bull removal from breeding pens, or after the last artificial insemination. The results of competitive mating at pasture were determined from breeding observations, the phenotypic characteristics of calves sired, and blood typing for parentage. The results of these studies suggest that bulls that produce a high percentage of spermatozoa with indented acrosomes may have normal fertility when used in artificial insemination or in single sire mating; however, their fertility may be low when breeding competitively with bulls with normal spermiograms.  相似文献   

10.
The influence on sperm morphology of different methods for preparation of semen and of storage in a fixative solution was examined in 27 beef bulls subjected to a regular breeding health examination. Sperm head morphology under light microscopy did not differ between smears of fresh semen stained with carbol-fuchsin-eosin (Williams staining) or Nigrosin-Eosin. Nor was there any difference between samples stained immediately after collection and those stained after 1 month of storage at + 4 degrees C in buffered formal-saline solution. Formol-saline fixed spermatozoa examined in wet preparations under phase contrast microscopy had a higher prevalence of acrosome defects and cytoplasmic droplets than stained smears of fresh semen under light microscopy. One month of storage in formol-saline did not affect the prevalence of acrosome defects or cytoplasmic droplets. There was no influence of fixation method (wet or dry), staining, examination technique, or storage time on midpiece or sperm tail morphology. The affinity of spermatozoa to eosin at staining with Nigrosin-Eosin ("live and dead count") did not differ between fresh semen and spermatozoa that had been stored in formol-saline for 1 month. It is concluded that bull semen can be stored for at least 1 month at + 4 degrees C in buffered formal-saline without major changes in sperm morphology. Furthermore, examination of wet preparations of fixed spermatozoa under phase contrast microscope is likely to yield the most accurate results for morphological characteristics like acrosome morphology and cytoplasmic droplets.  相似文献   

11.
Semen samples collected postmortem from 142 yearling beef bulls (11-13 months old) of three different breeds (Charolais, Hereford and Simmental) were examined to evaluate the proportion of bulls with mature spermiograms. Before slaughter, testes and epididymides were clinically examined and scrotal circumferences were measured. Aliquots of the cauda epididymal contents taken postmortem were used for sperm morphology examination. Sperm head morphology was studied in dry smears stained with carbol-fuchsine. For each preparation, 500 spermatozoa were counted in each smear under light microscope (x 1000). The presence of proximal cytoplasmic droplets, abnormal acrosomes, detached heads and abnormalities of the midpiece and tail were recorded in wet preparations of formol-saline-fixed spermatozoa. For each preparation, 200 spermatozoa were counted in each preparation under a phase-contrast microscope (x 1000). The abnormalities were classified according to a classification system developed by Bane (1961). Morphological abnormalities were recorded as a percentage of the total number of counted spermatozoa. Criteria for a spermiogram to be considered mature included <15% abnormal heads and <15% proximal droplets. According to this definition approximately 48% (68 of 142) of the examined bulls were considered mature. The bulls in this study represent approximately one-fifth of the total amount of performance-tested beef bulls in Sweden during 5 years. Our results indicate that only less than half of the Swedish yearling beef bulls at the testing station appear to have a mature spermiogram at the time they are offered for breeding purposes.  相似文献   

12.
The appearance and incidence of sperm abnormalities was studied in 115 ejaculates, collected periodically over 1 year covering all seasons from five mature, healthy swamp buffalo (Bubalus bubalis) bulls reared under tropical conditions and serving as the current source of semen for artificial insemination (AI) in Thailand. Light microscopy of stained smears was used to investigate sperm head shape morphology, while unstained wet smears were used to examine other sperm abnormalities. The most commonly found morphological aberrations were pear-shaped spermatozoa, knobbed acrosomes, proximal cytoplasmic droplets, simple bent tails and coiled tails under the head, whose ultrastructure (scanning electron microscopy) corresponded to what has been found in other species of bovidae, including varieties of buffalo. The mean prevalence (as least squares mean +/- SEM) of sperm abnormalities was low (below 15%), corresponding to healthy spermiograms. The younger bulls (<10 years old, n = 3) had less abnormalities than the older ones (10.1 +/- 0.6% versus 14.1 +/- 0.8%, P < 0.001, n = 2), including abnormalities of sperm head shape (1.1 +/- 0.3% versus 3.6 +/- 0.3, P < 0.001), acrosome defects with knobbed acrosomes (1.1 +/- 0.2% versus 1.2 +/- 0.3%, P < 0.001), spermatozoa with proximal cytoplasmic droplets (2.7 +/- 0.1% versus 1.4 +/- 0.2%, P < 0.001), defective mid-pieces (0.2 +/- 0.1% versus 0.3 +/- 0.1%) and abnormal sperm tails (3.1 +/- 0.3% versus 5.7 +/- 0.4%, P < 0.001). The within-bull effect of the year solely affected the incidence of pear-shaped spermatozoa while the incidences of abnormal contour, variable size of sperm head shapes, abnormal mid-piece and simple bent tail among bulls were affected by ejaculate (week of collection). Interaction between age and ejaculate affected only the prevalence of spermatozoa with proximal cytoplasmic droplets. In conclusion, the types of defects encountered were similar to those found in other bovidae, with a very low prevalence over the year the AI sires were followed through.  相似文献   

13.
Incubation of dog spermatozoa in a medium without glucose and in the presence of lactate and pyruvate (l-CCM) for 4 h at 38.5 degrees C in a 5% CO(2) atmosphere induced in vitro capacitation of these cells. This was verified after the combined specific capacitation-like changes in percentages of viability and altered acrosomes, motility characteristics, sperm location of reactivity against Pisum sativum, Arachis hypogaea and Helix pomatia lectins and the tyrosine phosphorylation pattern. Furthermore, a feasible acrosome reaction (AR) was induced when spermatozoa incubated in l-CCM for 4 h were further co-incubated for 1 h with canine oocytes. This was demonstrated by AR-like changes in percentages of viability, altered acrosomes, motility characteristics and sperm location of reactivity against P. sativum, A. hypogaea and H. pomatia lectins. All these results clearly indicate that in vitro capacitation, and subsequent AR, can be feasibly achieved without the presence of sugars. This ability can be related to the specific characteristics of energy-metabolism regulation reported in dog spermatozoa.  相似文献   

14.

Background

During epididymal transit, functional and structural modifications leading to full maturation enable male gametes to reach, recognize and fertilize the oocytes. In dogs, little is known on the modifications of spermatozoa during the passage in the epididymis. The aim of this study was to describe the motility, morphology and acrosomal patterns of canine spermatozoa retrieved from the epididymis caput, corpus and cauda.

Results

After the dilution required for the collection of epididymal content, sperm motility was significantly higher (P <0.0001) in the cauda compared to corpus and caput.Proportions of spermatozoa with normal morphology were significantly higher in corpus (P =0.02) and cauda (P <0.0001) compared to caput. Overall morphological abnormalities of the head and neck/midpiece were similar in the three different epididymal regions. A significantly increased prevalence of tail defects, mainly represented by single bent tails, was observed in the corpus compared to caput (P <0.0001) and cauda (P =0.006).Numbers of immature sperm with cytoplasmic droplets decreased from the proximal to the distal region of the epididymis. Particularly, proximal cytoplasmic droplets were more frequently found in spermatozoa collected from the caput epididymis than in the corpus (P <0.0001) and in the cauda (P <0.0001), whereas the occurrence of distal cytoplasmic droplets was higher in the corpus than in the caput (P =0.0003) and in the cauda (P <0.05).Significantly higher proportions of spermatozoa with intact acrosomes were retrieved from the cauda epididymis than from the caput (P =0.03) and the corpus (P =0.008). This difference was mainly due to a lower proportion of spermatozoa with abnormal acrosomes (mainly swollen acrosomes) rather than with absent acrosomes.

Conclusions

Canine spermatozoa undergo several modifications in the epididymis. The acquisition of progressive motility, migration of the cytoplasmic droplet and acrosomal reshaping lead to mature spermatozoa which are then stored in the cauda epididymis. From this site, spermatozoa can be retrieved and used in assisted reproductive techniques as a valuable tool for propagating genetic traits of high value individuals that dies accidentally or undergoes orchiectomy for medical purposes. Further investigations should be also focused on the potential use of spermatozoa recovered from other epididymal regions.  相似文献   

15.
Ejaculated boar spermatozoa subjected to a conventional freezing and thawing process, were ultra-rapidly fixed, freeze-substituted and examined by electron microscopy to monitor the presence of real or potential intracellular ice and the degree of cell protection attained with the different extenders used during the process. Numerous ice crystal marks representing the degree of hydration of the cells were located in the perinuclear space of those spermatozoa not in proper contact with the extender containing glycerol (i.e. prior to freezing). The spermatozoa which were in proper contact with the extenders presented a high degree of preservation of the acrosomes, plasma membranes as well as the nuclear envelopes. No ice marks were detected in acrosomes before thawing, indicating that the conventional assayed cryopreservation method provided a good protection against cryoinjury. The presence of acrosomal changes (internal vesiculization, hydration and swelling) in thawed samples however, raises serious questions about the thawing procedure employed.  相似文献   

16.
The aim of this study was to determine the best concentration of low-density lipoproteins (LDL) in a semen extender to improve the percentage of motile spermatozoa in equine sperm after freezing and thawing in comparison with standard extenders. Ten extenders were compared: 1 with 2% egg yolk (EY), 8 with different concentrations of LDL (0.25%, 0.50%, 0.75%, 1%, 2%, 3%, 4%, and 5%), and INRA 96; all of the extenders contained 2.5% glycerol. Fourteen ejaculates were collected from four different stallions. The first dilution was made with equal parts at +37°C, centrifuged (600 × g/10 min), and resuspended in the corresponding extenders to obtain a final concentration of 100 × 106 spermatozoa/ml. The resulting mixture was cooled to 4°C over 1 hour, packed into four 0.5-ml straws, and left for a further 30 minutes at +4°C. Finally, the straws were frozen in nitrogen vapors 4 cm over liquid nitrogen for 10 minutes before being immersed in liquid nitrogen at −196°C and stored. Two straws per extender and per ejaculate were thawed in a water bath at +37°C for 30 seconds. The contents of each straw were recovered into a cryotube and placed in a water bath at +37°C for 10 minutes before being examined with an image analyzer. The best post-thaw motility results were obtained with the extenders made with 0.5%, 2%, and 3% LDL and with the control extender made with egg yolk; no significant difference was observed between these extenders. The last two straws were thawed to perform four sperm function tests. The hypo-osmotic test was used to assess the integrity of the plasma membrane; the 2% and 3% LDL treatments were the most suitable and were comparable to that with whole egg yolk for protecting stallion sperm during cryopreservation (32.3%, 32.4%, and 31.3%, respectively). The Pisum sativum agglutinin-fluorescein isothiocyanate test was used to verify the integrity of the acrosomes; the best results were obtained with the 0.5%, 0.75%, and 3% LDL and INRA96 extenders; no significant differences were observed among the 85.8%, 85.0%, 84.7%, and 84.8% extenders. The acridine orange test was used to assess DNA integrity; there were no significant differences among the various extenders: the DNA was preserved in 98% of the spermatozoa. Finally, spermatozoal morphology was examined using Spermac stain; 78% of the spermatozoa did not present any anomalies in the 0.25% and 2% LDL extenders. In conclusion, the 2% LDL extender gave the best post-thaw percentage of motile spermatozoa. The results of the sperm function test were also superior for this extender.  相似文献   

17.
In a study of the absorptive activities of the excurrent ducts of the testis of birds, six adult cocks of the Ovambo breed of domestic fowl were deeply anaesthetised and intravascularly perfused with buffered 3 per cent glutaraldehyde. Epididymal tissue was prepared conventionally for electron microscopy. In favourable sections in three of these birds avid ingestion of spermatozoa was revealed in the non-ciliated (Type I) cells of the epithelial lining of the initial part of the proximal efferent duct, at and a little beyond its junction with the rete testis. No obvious defects were detected in luminal spermatozoa. The testicular excurrent ducts were neither distended nor obstructed, and mononuclear cell mobilisation was absent in both the ductal and periductal tissue. The significance of this observation with regard to the specific segment of the efferent duct system involved and how certain spermatozoa are identified for elimination from the duct lumen, must await precise studies.  相似文献   

18.
Tail and mid-piece morphology of ram spermatozoa were compared using wet preparations of semen diluted in buffered formal saline at temperatures of 10 degrees C and 65 degrees C. The temperature of the diluent did not affect the occurrence of abnormalities. Tail and mid-piece morphology were also examined in semen smears stained by a modified Williams' stain using glass slides at temperatures of 10 degrees C, 38 degrees C and 65 degrees C. The occurrence of the abnormalities was not affected by the slide temperature. The occurrence of tail and mid-piece abnormalities was compared using the 2 methods of preparation. Only the occurrence of distal cytoplasmic droplets was affected by the method of preparation. In the stained smear preparations, most of the distal cytoplasmic droplets were lost. However, only a few of the proximal droplets were lost when this method was used.  相似文献   

19.
Evaluation of glucose as a cryoprotectant for boar semen   总被引：2，自引：0，他引：2  
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  相似文献   

20.
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.  相似文献   

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