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1.
The aim of this study was to develop a new method for morphometric assessment of the sperm head and acrosome in the ram. Ejaculates from 10 adult males were collected using an artificial vagina. For each ejaculate, 10 semen smears were prepared, air‐dried and divided (in pairs) into the following five treatment groups: (i) washed in distilled water and allowed to dry without further processing (DRY); (ii) fixed in 50% methanol (MET); (iii) fixed in 2% glutaraldehyde (GLUT); (iv) fixed and stained with Hemacolor® (HEM) and (v) fixed and stained with SpermBlue® (SB). The prepared slides were examined with a 40 × Relief Contrast® objective (RC) and processed with ISAS® commercial software. The use of RC optics increased the contrast between acrosome and sperm head, allowing capture and morphometric analysis by ISAS of sperm heads and the acrosome, even in non‐stained samples. MET and GLUT groups resulted in a lower number of acceptable, that is, correctly delineated, sperm heads than those in the SB, and SB and HEM groups, respectively (p < 0.05). The higher proportion of sperm discarded in MET and GLUT groups may be explained by a higher presence of artefacts. For the majority of the primary morphometric parameters of the sperm head and the acrosomal area, the relationship between treatments was the following: GLUT> HEM≥ MET≥ SB> DRY. When studying the proportion of the sperm head covered by the acrosome, the relation between treatments was: MET> DRY = GLUT = SB> HEM. It was concluded that the new method for sperm morphometric assessment allows the simultaneous assessment of sperm head and acrosome in the ram by the first time, even in unprocessed semen smears.  相似文献   

2.
South American camelid sperm characteristics are poorly known compared with those of other domestic animals. The long‐term duration of ejaculation makes difficult to gather all the seminal fluid, implying possible ejaculation portion losses. Thus, the aim of this research was to evaluate the characteristics of the morphology and morphometry of the spermatozoa change during ejaculation. The morphometric characterization was tested on nine specimens of the Lanuda breed, using a special artificial vagina. In five of the animals, a fractioning of the ejaculate was performed by taking samples every 5 min. for a total of 20 min. Air‐dried seminal smears were stained with Hemacolor and mounted permanently with Eukitt. Morphometric analysis was carried out with the morphometry module of the ISAS® CASA system. Almost 350 cells were analysed per sample, with a total number of 3207 spermatozoa. Mean values were given as follows: length: 5.51 μm; width: 3.38 μm; area: 17.75 μm2; perimeter: 14.8 μm; ellipticity: 0.24; elongation: 0.56; rugosity: 0.87; regularity: 1.07; and shape factor: 1.41. Different animals showed differences in their morphometric values. When we compared the values from different fractions, only two samples showed differences in morphometric parameter values and four samples showed differences in shape parameters. Multivariate analysis allowed the size classification of the cells into three classes and five classes of shapes. The distribution of classes among fractions showed no differences. Despite the individual morphometric differences observed in some fractions, the characteristics of the sperm head morphometry can be considered constant along the ejaculatory period in the llama.  相似文献   

3.
Viability assessment is an important part of semen analysis, and various live/dead staining protocols have been used in semen of avian species. Results of live/dead count differed between dyes, staining protocols and bird species, impeding comparability between studies and requiring species-specific comparisons of viability stains. In raptor semen, similar comparisons are absent. Thus, the aim of the present study was to compare eight conventional viability stains. Eosin blue 2% [EB], eosin blue 2% with the addition of 3% sodium citrate [EB2], eosin blue–nigrosin 5% [EBN5], eosin yellow–nigrosin 5% [EYN5], eosin yellow–nigrosin 10% [EYN10], eosin blue–aniline blue [EBA], eosin yellow–aniline blue [EYA] and bromophenol blue–nigrosin [BBN] were evaluated in comparison with the fluorescence stain SYBR® Green–propidium iodide [SYBR-PI] in spermatozoa of falcons. The comparison was performed using conventional light microscopy which is applicable in breeding centres, veterinary practices and field studies. Additionally, live/dead stains were correlated to motility values of the same samples to validate sperm viability. Light microscopy using EB and using SYBR-PI enabled an effective and clear differentiation between alive and dead spermatozoa of falcons. Motility values correlated significantly and strongly with EB only (r = .629; p < .001), but not with any other stain used in the study. Therefore, our results suggest EB as the most suitable stain for viability assessment in the semen of large falcons.  相似文献   

4.
The aim of the study was to evaluate semen quality in the two most popular colour morphs of the Arctic fox Alopex lagopus L., blue and white, based on ejaculate parameters, acrosin activity and analysis of sperm morphology. The research material consisted of ejaculates collected once by manual stimulation from 20 one‐year‐old male Arctic foxes (10 individuals of the blue morph and 10 of the white morph). Ejaculates were evaluated in terms of volume, sperm concentration, total number of spermatozoa and the percentage of spermatozoa with major and minor defects. The study revealed that male blue Arctic foxes produce ejaculates with much higher concentration (148.75 × 106/ml) and total number of spermatozoa (98.16 × 106) compared to white Arctic foxes (42.88 × 106/ml and 35.2 × 106 respectively). The level of acrosin activity from white foxes seemed to be higher compared to blue foxes but the difference was not statistically confirmed. Semen from Arctic foxes is characterized by high inter‐individual variability in sperm morphology. The frequency of morphological changes in sperm from Arctic foxes does not significantly depend on ejaculate volume, sperm concentration or the total number of spermatozoa in the ejaculate, but is associated with acrosin activity.  相似文献   

5.
The importance of standardizing the procedures of sample and slide preparation for computer‐assisted morphologic analysis has been emphasized in human and veterinary andrology. The purpose of this study was to optimize slide preparation (dilution grade and sperm washing), staining procedures and analysis conditions (colour of light source and objective magnification) for the morphometric analysis of bull spermatozoa using the Hamilton Thorne morphology analyzer integrated visual optical system (IVOS). For experiment 1, one ejaculate was collected from one bull and diluted to 200 000–300 000 spermatozoa/μl. Slides were prepared and stained using seven different procedures: rapid Papanicolaou (PAP), rapid Papanicolaou with prolonged staining times (PAP+), Diff‐Quik (DIF), haematoxylin (HEM), Farelly (FAR), Spermac (SPER) and the modified GZIN (MGZIN) staining. All slides were analysed using a Hamilton Thorne Morphology Analyser IVOS equipped alternatively with a red, green or blue light source, and a 40× or 100× oil immersion objective. Recognition and digitization errors as well as morphometric parameters were determined. The IVOS was unable to detect DIF‐stained spermatozoa. The GZIN and the SPER staining as well as the blue light source led to unsatisfactory results. Among the staining methods examined, the FAR, HEM, PAP+, and PAP staining, preferably in combination with the green light source, and the 40× objective yielded optimal results concerning sperm recognition and digitization. The 100× objective did not allow reliable analysis of the sperm heads because of a frequently appearing digitization error. For experiment 2, three ejaculates were collected from each of three bulls and diluted to five dilution grades (100 000–500 000 spermatozoa/μl). An aliquot of each dilution grade was washed additionally. The percentage of correctly digitized sperm heads decreased with increasing spermatozoal concentration. However, the evaluation speed increased. The range of 200 000–300 000 spermatozoa/μl appeared to be a reasonable compromise for both criteria. Sperm washing failed to further improve the analysis results. Sperm head dimensions were influenced significantly by all variations of the methods in both experiments. In conclusion, using the proposed methods, the IVOS allows precise and reliable morphometric analyses of bull spermatozoa. The consistent application of these procedures may lead to an inter‐laboratory standardization and to further establishment of generally accepted morphometric criteria used in human andrology (e.g. World Health Organisation or strict criteria).  相似文献   

6.
Development of new semen cryopreservation techniques improving sperm survival and ensuring availability of viable spermatozoa for a prolonged time‐period after AI is promising tools to reduce sensitivity of timing of AI and enhance overall fertility. The SpermVital® technology utilizes immobilization of bull spermatozoa in a solid network of alginate gel prior to freezing, which will provide a gradual release of spermatozoa after AI. The objective of this study was to compare post‐thaw sperm quality and in vitro sperm survival over time of Norwegian Red bull semen processed by the SpermVital® (SV) technology, the first commercialized production line of SpermVital® (C) and by conventional procedure applying Biladyl® extender (B). Post‐thaw sperm motility was not significantly different between SV, C and B semen (p > .05). However, sperm viability and acrosome intactness were higher for SV than C and B semen (p < .05). Small differences in DNA quality were observed (p < .05). Sperm viability after storage in uterus ex vivo was higher for SV than for C semen (p < .05). Furthermore, sperm survival in vitro over time at physiological temperature was significantly higher for SV semen than C semen as well as B semen during the incubation period of 48 hr (p < .05). In conclusion, the SpermVital® technology is improved and is more efficient in conserving post‐thaw sperm quality and results in higher sperm viability over time in vitro for SV than for C and B semen.  相似文献   

7.
This study aimed to compare the ability of sperm chromatin structure assay (SCSA®) and Sperm‐Ovis‐Halomax® to detect DNA fragmentation in frozen‐thawed ram spermatozoa incubated under capacitating conditions in synthetic oviductal fluid (SOF) supplemented with oestrous sheep serum (SOF‐ESS) at multiple time points (0–240 min). Incubation in SOF‐ESS had no significant effects on SCSA® parameters while the percentage of spermatozoa with fragmented DNA measured by Sperm‐Ovis‐Halomax® increased after 180 min of incubation. In addition, no correlation or agreement was found between the techniques, suggesting that SCSA® and Sperm‐Ovis‐Halomax® may quantify different types of DNA damage in ram spermatozoa under these experimental conditions.  相似文献   

8.
Although computer‐assisted systems for sperm morphometry and morphological analysis are important tools in the study of male fertility, their use in extensive systems in alpacas is limited by factors such as the expense of equipment and the high altitudes of the Andean region. The objectives of this study were to evaluate alpaca sperm head morphometry using a nonautomated digital method and determine the frequency of sperm abnormalities based on strict criteria for sperm morphology in fertile male alpacas. Ejaculates (n = 15) from seven alpacas were collected, and sperm smears stained with modified Papanicolaou were processed. For morphometric analysis, 3,000 sperm (200 cells/sample) images were captured at 400× magnification and Quick Photo MICRO 3.0 software was used for manual measurement of basic (sperm head length, width, perimeter and area) and derived variables (ellipticity, shape factor, elongation and regularity). For morphology assessment, smears were observed at 1000× magnification according to WHO and strict criteria. Average morphometric parameters were length 5.48 μm, width 2.99 μm, perimeter 13.62 μm, area 12.43 μm2, ellipticity 1.86, shape factor 1.20, elongation 0.29 and regularity 1.05. Significant between‐individual and within‐individual differences were found in morphometric parameters. Based on morphometric study, sperm heads were classified as elliptical or normal (49%), long (18%), short (2%), pyriform (12%), round (9%), large (6%) and small (4%). Morphological analysis found no additional sperm head defects in 49% of normal sperm obtained by morphometry, although a 4% incidence of neck/mid‐piece defects and a 16% incidence of principal‐piece defects were found. We conclude that sperm head morphometry assessment in fertile alpacas using a nonautomated digital method is feasible, and that defects in sperm heads constitute the main morphological alteration (>50% of the sperm population), based on WHO and strict criteria.  相似文献   

9.
Cryopreservation of epididymal spermatozoa is often performed after shipping the excised testis–epididymis complexes, under refrigeration, to a specialized laboratory. However, epididymal spermatozoa can be collected immediately after excision of the epididymis and sent extended and refrigerated to a laboratory for cryopreservation. In this experiment, we evaluated the effect of both methods of cold storage bovine epididymal spermatozoa as well as of two different extenders on spermatozoa characteristics after freeze–thawing. For that, spermatozoa collected from the caudae epididymis of 19 bulls were extended and cryopreserved in either AndroMed® or a Tris–egg yolk (TEY)‐based extender. Cryopreservation of sperm cells was performed immediately after castration (Group A, n = 9) or after cold storage for 24 h diluted in the two extenders and (Group B, n = 9) and also after cold storage for 24 h within the whole epididymis (Group C, n = 10). Sperm subjective progressive motility (light microscopy), plasma membrane integrity (hypoosmotic swelling test) and sperm viability (eosin–nigrosin) were evaluated. In vitro fertilization and culture (IVF) was performed to assess the blastocyst rate. No differences (p > 0.05) were observed on post‐thaw sperm parameters between samples from Group A, B and C. TEY extended samples presented a higher (p < 0.01) percentage of progressive motile and live sperm, than those extended in AndroMed®. Blastocyst rate after IVF differed only (p < 0.05) between the reference group (IVF performed with frozen semen with known in vitro fertility) and Group A extended in AndroMed®. We conclude that when cryopreservation facilities are distant from the collection site, bovine epididymal sperm can be shipped chilled overnight either within the epididymal tail or after dilution without deleterious effect on post‐thaw sperm quality. TEY extender was more suitable for cold storage and freezing bovine epididymal sperm, than the commercial extender AndroMed®.  相似文献   

10.
DNA fragmentation of frozen‐thawed feline epididymal sperm from corpus and cauda regions was evaluated by three different techniques. The DNA fragmentation index (DFI) was compared between techniques: the sperm chromatin structural assay (SCSA®), acridine orange staining techniques (AOT) and the sperm chromatin dispersion (SCD). There were significant differences in DFI among the techniques (p < 0.05) with no correlations. Only DFI values obtained from SCD revealed a significantly higher DFI in corpus compared with cauda spermatozoa (p < 0.05). The discrepancy between techniques might be due to the sensitivity of each technique, differences in severity of DNA damaged that can be detected. The difference in DFI between epididymal regions from SCD technique might indicate different maturational stages of spermatozoa, with less chromatin condensation of spermatozoa in corpus compared with cauda epididymis.  相似文献   

11.
In the spermatozoa of some species, the ubiquitin–proteasome system detects altered proteins and tags them for elimination by the proteasome. In some species' ejaculates, a high proportion of ubiquitinated spermatozoa (i.e. those having ubiquitin bound to the altered or damaged membrane proteins) has been related to infertility. The aim of this study was to assess whether the percentage of ubiquitinated spermatozoa relates to fertility of dairy bulls and whether ubiquitination increases during protein remodelling that occurs during in vitro spermatic capacitation. Thirty‐two frozen semen straws from four high‐fertility (ReproMax®) and four normal‐fertility (Normal) Holstein‐Friesian sires were evaluated. Ubiquitinated and capacitated spermatozoa were quantified by sperm ubiquitin tag immunoassay and chlortetracycline stain, respectively. Fertilizing capacity of sires was assessed by in vitro fertilization. No differences were found between Normal and ReproMax® sires with regard to the observed percentage of ubiquitinated spermatozoa (42.97 ± 3.69% and 49.68 ± 9.27%, respectively; p > 0.05). Additionally, no differences were found in the percentage of ubiquitinated spermatozoa as a consequence of spermatic capacitation in either Normal (42.97 ± 3.69% before capacitation vs 44.67 ± 7.5% after; p > 0.05) or ReproMax® sires (49.68 ± 9.27% before vs 45.05 ± 7.51% after; p > 0.05). The percentage of ubiquitinated spermatozoa in a thawed sperm samples did not correlate with its in vitro fertilizing capacity; thus, this assay does not prove useful to detect in vivo fertility differences between sires. Additionally, protein degradation occurring during remodelling of the spermatozoon plasma membrane during the capacitation process does not seem to involve the ubiquitin–proteasome system.  相似文献   

12.
Routine semen evaluation includes volume, motility, vital staining for live‐dead ratio and pathomorphology including Spermac® staining for evaluation of the acrosome. In recent years, depending on the species, also the hypoosmotic swelling (HOS) test has been applied routinely for evaluation of semen quality. In this respect, a significant correlation between the ability of spermatozoa to swell in HOS test and the fertilizing ability has been reported. Also for evaluation of dog semen, reference has been made to the HOS test; however, its correlation to conventional semen parameters so far is discussed controversially. In the present study, the results of 400 semen examinations from stud dogs presented at our clinic were evaluated for their correlations between conventional semen parameters (motility, live/dead ratio, pathomorphology), conventional semen parameters and age, Spermac® staining and HOS test, respectively. We found a significant correlation of age and sperm concentration (p < 0.01), total sperm count (p < 0.0001), percentage of progressively motile sperm (p < 0.01) and live spermatozoa (p = 0.012). Furthermore, several correlations between conventional semen parameters were identified. Percentage of sperm with normal acrosome identified by Spermac ® staining correlated significantly with live spermatozoa (p < 0.0001) and percentage of progressively motile sperm (p < 0.01). A significant correlation was proven between curled tails in HOS test and age (p < 0.001), motility (p < 0.0001), live sperm (p < 0.0001), acrosomal status (p < 0.05), pathomorphology (p < 0.0001) and sperm concentration (p = 0.011). These results indicate that Spermac® staining and the HOS test are useful in improving canine semen analysis.  相似文献   

13.
The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well‐known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD‐ and NADP‐dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.  相似文献   

14.
The semen movement and sperm head size patterns of boar ejaculates were analysed using computer-assisted semen analysis (CASA)-Mot and -Morph systems. The aim of the present study was to compare morphometric and kinematics variables from boars and to determine the relationship with sow fertility variables related to litter size. The females were from maternal crossing schemes such as the continuous 3-generation cross between York (Y), Landrace (L), and Pietrain (P) hybrid sows and Pietrain boars. Semen samples were collected from 11 sexually mature boars from two sire lines. Samples were analysed using the ISAS®v1 system to evaluate eight kinematic variables of sperm velocity, progressiveness and undulations. Four morphometric parameters of sperm head size (length, width, area and perimeter) were analysed. Bayesian analysis revealed relevant differences in four kinematic variables (VSL, LIN, STR and WOB) between sire lines, with a probability of relevance (PR) of 0.79–0.91, and Pietrain boars were associated with higher progressive motility compared with Duroc x Pietrain boars. Moreover, there were relevant differences in all morphometric variables (PR = 0.82–0.85) between sire lines. The dam line Y-L-50 (½ Y × ½ L) had higher total born per litter and piglets born alive, and YLP-75 (1/8 Y × 1/8 L × 3/4 P) was associated with higher values of litter weight at birth (highest posterior density region at 95% = 9.92, 16.41 kg). There are relevant differences in kinematic variables between the assessed sire lines and the differences in morphometric and litter size variables were also relevant. The York-Landrace hybrid sows had higher total born per litter and piglets born alive, and there were relevant differences when compared with YLP-50 (¼ York × ¼ Landrace × ½ Pietrain). Differences in kinematic and morphometric variables between sire and dam lines related to fertility need to be further studied.  相似文献   

15.
The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post‐thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post‐thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll®, Puresperm® and Bovipure?, and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post‐thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure? yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll® and Puresperm® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll® selected less live and more apoptotic spermatozoa than Puresperm® and Bovipure? for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.  相似文献   

16.
This study was conducted to investigate the predictive capacity of fertility and litter size of sperm head morphometric measurements when the ejaculates fulfilled the minimum requirements commonly used in artificial insemination (AI). Semen samples from 11 rabbits (77 ejaculates) were evaluated for sperm motility, abnormal spermatozoa and sperm head morphometry using computer automated sperm analysis system. Morphometric dimensions for length, width, area and perimeter were analysed. Only ejaculates with more than 70% of motility rate and <15% of abnormal sperm were used for AI. A total of 1031 individual AI were performed in commercial rabbitries. Our results showed significant differences among animals for all sperm head measurements. The mean values for fertility and litter size obtained were 68.4 ± 0.01% and 9.3 ± 0.1% respectively. To assess the predictive value of morphometric dimensions in fertility, a logistic regression analysis was applied. Moreover, multiple linear regression analyses were used to examine the relationship between litter size and sperm head morphometric parameters. Logistic regression analysis rendered a significant model between fertility and area and perimeter, explaining the 0.65% variation. Multiple linear regression analysis rendered a significant model between litter size and width, area and perimeter that explained the 1.3% variation. By conclusion, the sperm head morphometric parameters assay showed low potential to predict fertility and litter size when the ejaculates fulfilled the minimum requirements commonly used in AI (motility and abnormal spermatozoa) in rabbit.  相似文献   

17.
Single layer centrifugation (SLC) has been shown to select the most robust spermatozoa from the ejaculate in several species. Here the effects of SLC prior to freezing on various parameters of frozen‐thawed bovine sperm quality are reported. Semen from 8 bulls was layered on top of a species‐specific colloid, Bovicoll. After centrifugation for 20 min at 300 g, the resulting sperm pellet was resuspended in OPTIXcell® (IMV Technologies, l′Aigle, France); the SLC‐selected sperm samples and uncentrifuged controls were frozen. On thawing, all sperm samples were analysed for membrane integrity, production of reactive oxygen species, mitochondrial membrane potential (MMP) and chromatin integrity. The SLC‐treated samples had a higher percentage of live, superoxide‐positive spermatozoa than uncentrifuged samples (27.9 ± 5.1% versus 21.7 ± 6.7%; p = .03). They had a higher proportion of spermatozoa with high mitochondrial membrane potential than uncentrifuged samples (55.9 ± 8.2% versus 40.5 ± 15.1%; p = .03) and also a lower proportion of spermatozoa with low mitochondrial membrane potential than non‐treated samples (42.0 ± 8.5% versus 55.9 ± 14.4%; p = .04). No significant effects of treatment were found for membrane integrity or chromatin integrity. The effect of bull was significant on the proportions of dead, superoxide‐positive spermatozoa and live, hydrogen peroxide‐negative spermatozoa, as well as on membrane integrity, but it was not significant for mitochondrial membrane potential or chromatin integrity. These results suggest that SLC selects the most metabolically active bull spermatozoa from the rest of the population in normal ejaculates; the pattern of reactive oxygen species production may be different in SLC‐selected spermatozoa compared to unselected samples.  相似文献   

18.
The aim of this study was to evaluate home‐made and commercial extenders for the cryopreservation of Rusa deer semen. After collection by electroejaculation, six ejaculates were diluted and frozen in TES‐based, Tris‐based and Triladyl® extenders. Subjective motility, viability, morphology, acrosome integrity and membrane functionality were assessed post‐thawing and after 1‐hr incubation at 37°C (Thermal stress test). Total and progressive motility, and kinematic parameters were also assessed through CASA system. Post‐thawing sperm progressive motility (PM), velocity according to the straight path (VSL) and linearity (LIN) showed significant differences, and higher values were detected for spermatozoa diluted with Triladyl® and TES (p < 0.05) as compared with Tris (PM of Triladyl® 14.7% vs. 3.2% TES and 2.5% Tris; VSL 56 for Triladyl®, 59.2 for TES and 41.7 for Tris; LIN 45.6 for Triladyl®, 52 for TES and 36.5 for Tris). Triladyl® and TES extender led to better post‐thawing sperm parameters, but these preliminary results need to be verified through artificial insemination trials.  相似文献   

19.
Sperm plasma membrane is an essential structure of sperm resistance to freezing. Signs of cryodamage can be visible on the sperm plasma membrane. The aim of our study was to evaluate the appearance of plasma membrane and acrosome in fresh and frozen‐thawed chicken sperm using electron and fluorescence microscopy. Semen was collected from 12 sexually mature roosters of Ross PM3 heavy line, diluted with Kobidil+ extender with 16% of ethylene glycol (KEG; control) or with KEG in combination with one of following non‐permeating cryoprotectants: trehalose (KEG‐TRE) or glycine (KEG‐GLY). Fluorescence staining was used for detection of the membrane integrity, apoptotic changes and viability (Annexin V, Yo‐PRO‐1, PI, respectively). Ultrathin sections (70 nm) from samples were prepared to examine sperm head ultrastructure. Freezing process significantly worsened the status of the sperm plasma membranes. In all frozen groups, only about a quarter of the evaluated sperm were graded as class I quality. In the KEG and KEG‐GLY groups, about half of sperm had severe plasma membrane damages (III class). In sperm with extensively damaged membranes (III class), the acrosome–sperm head junction was mostly disturbed. The use of trehalose was more beneficial (p < 0.05) for sperm plasma membrane than the use of glycine. In contrast, a decrease (p < 0.05) in the apoptotic sperm ratio (Yo‐PRO‐1) was noted in the KEG‐GLY group when compared to other treatments. In conclusion, we identified different plasma membrane and acrosome damages in cryopreserved chicken sperm. The loss of acrosomes can contribute to diminishing of fertilization ability of cryopreserved chicken sperm.  相似文献   

20.
The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb® filtration (53.6 ± 22.3%). GWS or Leucosorb® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 106 pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.  相似文献   

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