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1.
For successful breeding programs, it is important to quantify the useful period of a male's reproductive life and it is often done simply by measurement of semen quality. This information is lacking for Japanese quail so we tested whether there is a decline in ejaculate quality and sperm kinematics with age, and whether the decline varies among strains. Nine males (n = 9) from each of 5 strains (A, B, C, D and E) were subjected to 4 semen collections (n = 16 per male) at 8, 16, 26 and 36 weeks of age. Ejaculate volume, sperm concentration and total sperm per ejaculate were measured, and sperm kinematics were analysed using a Sperm Class Analyser (SCA®). There was a significant effect of age for ejaculate volume, total sperm per ejaculate and per cent medium sperm. The effect of the interaction between age and strain was significant for percent progressive motile sperm, percent rapid sperm, velocity curvilinear, velocity straight line, velocity average path, linearity, straightness and beat cross frequency. Ejaculate volume peaked at Week 26 in all strains, while peak values for sperm concentration and total sperm per ejaculate were observed at Week 16 for most strains. There were declines in percent motile sperm, progressive motile sperm and rapid sperm, and in velocity curvilinear velocity, velocity straight line and velocity average path, by Week 16 for most strains. Linearity declined by Week 26 in some strains, and all strains showed a significant decline in beat cross frequency by that age. In conclusion, the ability of CASA to detect age-related changes in sperm kinematics makes it a valuable tool for identifying the best males and thus improving quail flock fertility. It is essential that breeders understand that age affects both sperm production and sperm kinematics, and that the changes vary with strain.  相似文献   

2.
European eel, Anguilla anguilla, is a target species for future captive breeding, yet best methodology to estimate sperm density for application in in vitro fertilization is not established. Thus, our objectives were to evaluate methods to estimate European eel sperm density including spermatocrit, computer‐assisted sperm analysis (CASA) and flow cytometry (FCM), using Neubauer Improved haemocytometer as benchmark. Initially, relationships between spermatocrit, haemocytometer counts and sperm motility were analysed, as well as the effect of sperm dilution on haemocytometer counts. Furthermore, accuracy and precision of spermatocrit, applying a range of G‐forces, were tested and the best G‐force used in method comparisons. We found no effect of dilution on haemocytometer sperm density estimates, whereas motility associated positively with haemocytometer counts, but not with spermatocrit. Results from all techniques, spermatocrit, CASA and FCM, showed significant positive correlations with haemocytometer counts. The best correlation between spermatocrit and haemocytometer counts was obtained at 6000 ×  g (r = 0.68). Of two CASA variants, one or three photographic fields (CASA‐1 and CASA‐2), CASA‐2 showed a very high accuracy to haemocytometer counts (r  =  0.93), but low precision (CV: CASA‐2 = 28.4%). FCM was tested with and without microfluorospheres (FCM‐1 and FCM‐2), and relationships to haemocytometer counts were highly accurate (FCM‐1: r  =  0.94; FCM‐2: r  =  0.88) and precise (CV: FCM‐1 = 2.5; FCM‐2 = 2.7%). Overall, CASA‐2 and FCM‐1 feature reliable methods for quantification of European eel sperm, but FCM‐1 has a clear advantage featuring highest precision and accuracy. Together, these results provide a useful basis for gamete management in fertilization protocols.  相似文献   

3.
Only a few studies have described hormonal treatments for induction of synchronicity and gamete collection in Nile tilapia (Oreochromis niloticus), both important for assortative matings in breeding programmes and essential for polyploidy technologies. In this study, we compared the effectiveness of carp pituitary extract (CPE), Nile tilapia pituitary extract (TPE), human chorionic gonadotropin (hCG) and gonadotropin‐releasing hormone (GnRH) protocols on the induction of spawning and egg production in Nile tilapia. Among the hormonal treatments analysed, only hCG was effective for producing viable gametes for in vitro fertilization. To verify the viability of this hormonal treatment, hCG was tested using different doses (1000, 2000, 3000, 4000 and 5000 IU/kg) in a large number of females (208 animals) from two Nile tilapia lines. The results indicated that hCG doses between 1000 and 5000 IU/kg could be used to induce final oocyte maturation in Nile tilapia with collection of stripped oocytes. This is the first study to report differential reproductive responses to hormonal treatment between tilapia lines: line 1 was more efficient at producing eggs and post‐hatching larvae after hCG induction than line 2. In conclusion, we demonstrated that the hCG protocol may be applied on a large scale to induce final oocyte maturation in Nile tilapia. The development of a protocol for in vitro fertilization in Nile tilapia may aid in breeding programmes and biotechnological assays for the development of genetically modified lines of Nile tilapia.  相似文献   

4.
Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 106 sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm®. A single layer of 40% PureSperm® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.  相似文献   

5.
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   

6.
In this study, we developed an in vitro model for studying sperm–oviduct binding in the buffalo. Oviduct explants were prepared by overnight culture of epithelial cells in TCM‐199 medium under 5% CO2 at 38.5 °C. Cryopreserved spermatozoa from buffalo bulls (n = 4) were incubated with the oviduct explants, and the sperm–oviduct explants complex was stained with JC‐1. The effect of sperm concentration (2, 3 and 4 million), size of the oviduct explants (<0.2, 0.2–0.3, 0.3–0.4 and >0.4 mm2) and time of incubation (1 hr and 4 hr) on binding index (BI—number of sperm bound to unit area of explants) was studied. No significant difference was observed in the BI among <0.2, 0.2–0.3 and 0.3–0.4 mm2 size of explants; however, the BI decreased significantly (p < .05) when the size of explants exceeded 0.4 mm2. The BI decreased significantly (p < .05) when the sperm concentration was increased to 4 million, while the duration of incubation did not have any significant effect on the BI. The interaction of bulls with explants size, sperm concentration and incubation time was not significant. The developed assay has the potential to be used as an in vitro model for studying sperm–oviduct binding in the buffalo.  相似文献   

7.
Males of Muscovy duck (Cairina moschata) are mainly used for mule duck production via artificial insemination of females originated from wild mallard duck (Anas platyrhynchos); therefore, the quantity and quality of drake semen play a crucial role. The assessment results of male reaction to sexual stimulation by dummy female and basic semen characteristic (ejaculate volume, sperm concentration and morphology) of 12 individually kept Muscovy drakes carried out during the entire reproductive season are described. The male and period of the reproductive season effect on scored semen traits are documented. In total, 792 individual semen collections and evaluations were performed. The average of positive reaction in the entire reproductive season varied from 90.6% in December and April to 50.0% in July, while for individual males, it varied between 97.1% and 29.0%. Throughout the season, the ejaculate volumes ranged from 0.05 to 2.45 ml, sperm concentration from 0.15 × 109 ml−1 to 4.44 × 109 ml−1, total number of live spermatozoa from 68.0% to 100% and live normal (properly formed, with any deformations) from 51.0% to 99.0%. Our study indicates the necessity of male breeders pre-selection before the onset of the reproductive season, and the need to leave an appropriate number of males to ensure adequate amount of semen for female insemination, especially when using Muscovy drakes (Cairina moschata) for interspecies crossing with Anas platyrhynchos ducks.  相似文献   

8.
Breed and sire differences in sperm cryosurvival have been noted, with negative implications for sperm cryobanking and assisted reproduction programmes. This study hypothesized that these differences could be modified by using lower molecular weight cryoprotectants. Therefore, the effect of replacing glycerol (GLY) with ethylene glycol (EG) on differential cryosurvival of semen from two Sanga cattle breeds (Mashona vs. Tuli) was determined. Three to five ejaculates were collected from each of ten bulls (3-8 years) by electro-ejaculation, diluted in three Tris-egg yolk extenders (Triladyl®, 7% GLY-based and 7% EG-based) and evaluated for sperm motility, viability and morphology at three time periods (fresh – 0 hr, pre-freeze – 4 hr and post-thaw). Tuli bulls produced larger (11.8 ± 0.31 ml vs. 8.5 ± 0.38 ml) and more concentrated ejaculates of lower fresh semen quality. Breeds differed across time for motility and morphology, but not viability. Mashona bull semen had significantly higher motility and normal morphology values at each sampling time. Bulls classified as poor freezers had lower concentration (0.70 ± 0.09 × 109 sperm/ml vs. 1.37 ± 0.10 × 109 sperm/ml), sperm motility index (SMI, 35.0 ± 3.4 % vs. 67.8 ± 2.1 %) and viability (69.7 ± 1.1 % vs. 75.7 ± 1.0 %) compared to good freezers. Maintenance of semen quality by GLY and EG did not differ between breeds, poor and good freezers, or age groups. The interaction breed by extender across time did not reach statistical significance for all variables. The study revealed that bull and breed variation in sperm quality and cryosurvival is not modified by replacing GLY with EG, suggesting that cryostress tolerance of sperm may be under control of mechanisms other than differential response to GLY cytotoxicity.  相似文献   

9.
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   

10.
The objectives of this study were to evaluate if vitrified porcine spermatozoa are able to maintain their capacity to produce zygotes in vitro using intracytoplasmic sperm injection (ICSI) and to evaluate the zygote development in two in vitro atmospheric conditions: 5% CO2 and tri‐gas. A group of porcine oocytes maturated in vitro were injected with vitrified‐warmed sperm (treatment group) and another group, with sperm diluted and conserved at 17°C (control group). To evidence parthenogenetic activation, some oocytes were submitted to a Sham test. The injected oocytes were cultured in G1 medium at 38°C, 100% humidity and 5% CO2 or tri‐gas. No significant differences (p > .05) were observed in embryo development between the oocytes injected with vitrified‐warmed sperm (31.8%; 36/113), and those injected with semen diluted and conserved at 17°C (35.5%; 32/90), when cultured in 5% CO2 or under tri‐gas atmosphere (42.9%; 39/91 vs. 34.2%; 26/76, respectively). No significant differences (p > .05) were observed in the percentage of pronuclei (PN) obtained between 5% CO2 and tri‐gas, within each treatment either. Of the 52 oocytes submitted to the Sham test, only two presented a female PN (activation) indicating that the PN observed in the treatment group were a product of fertilization and not parthenogenetic activation. To conclude, porcine sperm vitrified using spheres, at a concentration of 5 × 106 spermatozoa/ml in TALP medium with 1% bovine serum albumin (BSA), conserve condensed and intact chromatin capable of producing early embryo development up to the pronuclear stage.  相似文献   

11.
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   

12.
Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN2) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.  相似文献   

13.
In this study, we investigated the effects of multiple collections of sperm on the endangered Persian sturgeon, Acipenser persicus, in terms of a number of sperm functional parameters (percentage of motile spermatozoa, total time period of motility and sperm concentration) as well as on the ionic composition, protein concentration and osmolality of seminal plasma. Semen samples were collected from 12 induced male fish in three experimental groups that had been injected intramuscularly with LHRH‐A2, at dosages of 5 μg/kg body weight, at a number of time regimes: at 12 h, 17 h and 24 h after spawning induction (1); at 24, 29 and 34 h after spawning induction (2); and at 36, 41 and 46 h after spawning induction (3). The percentage of motile spermatozoa and the period of sperm motility decreased significantly (p < 0.05) after the second and third collections. The concentration of spermatozoa decreased after the third collection, but this decline was not significant. No significant effect of multiple collections on protein concentration and ionic content (with exception of the Cl? ion) of seminal plasma was observed. In all experimental groups, a moderate impact of sequential collection on the osmolality (p < 0.05) of seminal plasma was observed. This study provides new data on the effects of multiple collections on spermatological characteristics in the Persian sturgeon. Our results confirm that sequential stripping after the third collections has a negative effect on a number of functional parameters associated with sperm.  相似文献   

14.
Laparoscopic artificial insemination technique (LAI) is described to overcome reduced fertility problems in sheep artificial insemination (AI) programmes with frozen semen. Later on, this technology was modified for endangered non-domestic cats to deposit low quality or reduced number of sperm cells hardly obtained by electro-ejaculation into the oviduct. This technique by passes the complex structure of cervix and efficiently transfers the sperm cells to the point of fertilization. In recent years, rabbits are becoming popular transgenic animal models producing various therapeutic and commercial products, as well as being experimental animals for disease models. The worldwide transportation of frozen semen and re-establishment of transgenic lines using AI technology has become a common practice. Therefore, this study was designed to describe a laparoscopic intrauterine insemination technique, which might assist in conceiving the animals with limited number of sperm cells. The female rabbits were laparoscopically (n = 22) or vaginally (n = 13) inseminated with frozen–thawed semen samples containing approximately 10 × 106 motile sperm. The laparoscopic insemination technique provided higher pregnancy rate (45.5%) than vaginal insemination technique (7.7%) (p < .05). In conclusion, the described laparoscopic AI might be a new alternative technique, thus enabling limited or low-quality frozen sperm samples to establish pregnancy in rabbits.  相似文献   

15.
Although incorporating proteases into sperm medium is considered the most effective procedure to eliminate camel semen viscosity, it drastically affects viability, morpho-functional properties and, hence, fertilization potential of spermatozoa. The present work aimed at evaluating adequacy of employing magnetic nanoparticles-based sperm purification technique for eluting impaired and apoptotic camel spermatozoa from cryopreserved semen doses following protease-based semen liquefaction. Thirty cryopreserved semen doses (50 x 106 sperm/straw) representing the following liquefaction treatments: control (untreated), 0.1 mg/ml papain or 5 U/ml bromelain were used (n = 10 straws per treatment). Immediately after thawing (38°C for 40 s), sperm concentration of each straw within treatment was readjusted to 15 x 106 sperm/mL by dilution in PBS (37°C). Sperm physical and cytological properties were then assessed (non-purified semen). Thereafter, each specimen was subjected to lectin-functionalized DNA-defrag magnetic nanoparticles sperm purification, and the same sperm traits were re-evaluated after undergoing purification (purified semen). Sperm DNA fragmentation level within each group, prior to and after magnetic nano-purification, was also determined by fluorescent imaging. The results showed a dramatic improvement (p < .05) in post-thaw motility (%), viability (%), normal sperm (%), intact acrosome (%) and HOST-reacted (%) spermatozoa in protease-liquefied semen following sperm magnetic nano-purification. Additionally, the highest (p < .05) DNA fragmentation level was recorded in all cryopreserved semen groups prior to purification, whereas the lowest (p < .05) was observed in the protease-treated specimens after magnetic nano-purification. These results indicate that protease-based semen liquefaction prior to cryopreservation in conjunction with magnetic nano-purification post-thawing holds potential for reducing the proportion of damaged and dead spermatozoa, hence improving camel sperm fertilization competence.  相似文献   

16.
The aim of the present study was to evaluate the effect of sperm selection by single-layer centrifugation (SLC) performed before freezing on sperm quality after thawing of Fleckvieh bull semen. Ejaculates from 22 bulls were collected by artificial vagina and divided into two aliquots. One aliquot (control sample) was diluted with Steridyl® and frozen over nitrogen vapour in a Digitcool freezer (IMV Technologies). Sperm from the second aliquot (SLC sample) was selected using the SLC technique with Bovicoll colloid and then frozen over nitrogen vapour in a Digitcool freezer. After thawing, both samples (control and SLC) were evaluated by computer-aided sperm analysis (CASA; SCA 6.4 System; Microptic S.L) for sperm motility parameters. Integrity of the plasma membrane (viability), high mitochondrial membrane potential (HMMP) and acrosome integrity were assessed using a Guava® easyCyte flow cytometer (IMV Technologies). Morphological examination of spermatozoa was performed by Differential Interference Contrast microscopy (Leica DMi8). Morphological examination of live, immobilized spermatozoa was analysed under high magnification (≥6,600×). After thawing, the mean sperm viability of the control sample was 51.57%, compared to 40.37% for the SLC sample (p < .01). HMMP was higher (p < .01) in the control sample (40.37% versus 28.96%), and the mean of live spermatozoa with damaged acrosome was significantly higher (p < .03) in the SLC sample (1.63% versus 1.95%). The mean percentage of motile spermatozoa was 80.17% in the control sample, compared to 75.14% in the SLC sample (p < .0195), and rapid subpopulation reduced from 20.08% to 8.99% (p < .0001) after SLC. Percentage of hyperactivated sperm decreased from 12.23% to 4.28% (p < .0001) after SLC. Given the overall results, the sperm quality of thawed Fleckvieh bull semen was not improved when sperm were selected by SLC before freezing.  相似文献   

17.
The present investigation was carried out to study the effect of various levels of dissolved oxygen (DO) on reactive oxygen species (ROS) and cryocapacitation‐like changes in bull sperm. Egg yolk–Tris–glycerol (EYTG) extender was split into four subextenders; viz., Extender I (control; no flushing with liquid nitrogen (LN2)), Extender II, Extender III and Extender IV were flushed with LN2 for 40, 16 and 8 min, respectively. The DO levels were standardized to 11.7, 2, 4 and 8 ppm, respectively, in control (Extender I), Extender II, Extender III and Extender IV. Ejaculates with mass motility of ≥ 3+ were divided into group I (diluted with Extender I), group II (diluted with Extender II), group III (diluted with Extender III) and group IV (diluted with Extender IV) up to 80 × 106 sperm/ml. Extended semen samples were packed in French mini straws (0.25 ml), equilibrated and cryopreserved. Semen samples were evaluated at prefreeze and post‐thaw stage for various parameters (DO, progressive motility (PM), viability (VIB), acrosomal integrity (AI), hypo‐osmotic swelling (HOS) test, ROS, cholesterol (C) and phospholipid (P). The percentage of PM, VIB, AI, HOS test, cholesterol (C) and phospholipid (P) levels, and capacitated sperm were significantly (p < 0.05) higher in groups III and IV as compared to groups I and II. However, the acrosome‐reacted sperm (%; pattern AR) were significantly (p < 0.05) decreased in group III as compared to all other groups. Besides the proportion of sperm displaying tyrosine‐phosphorylated pattern, EA (fluorescence at both equatorial and anterior acrosomal regions, i.e. high capacitation level) was significantly (p < 0.05) reduced in group III compared to all other groups. In conclusion, varying DO levels in the extender significantly affect sperm quality, ROS production and capacitation‐like changes in bulls.  相似文献   

18.
Acipenser sinensis and Acipenser dabryanus are critically endangered species, so germplasm conservation via cryopreservation of sperm is necessary. Disaccharides can act as membrane‐impermeable cryoprotectants, and enolase3 (ENO3) and plasma membrane Ca2+ ATPase isoform (PMCA2) are proteins associated with sperm quality. We considered seven characteristics of sperm quality in cultured brood stock from A. sinensis and A. dabryanus. We tested use of sucrose or trehalose alone and in combination at different concentrations for cryopreservation of A. dabryanus sperm. A low concentration of sucrose plus trehalose (S15T15) was optimal. Mixing of the extender with sucrose, lactose, or trehalose alone or with pairwise mixtures revealed that a mixture of lactose and trehalose (L15T15) gave the best results for both A. sinensis and A. dabryanus. Enolase3 and PMCA2 expression levels were measured in cryopreserved A. sinensis sperm via Western blotting. Relative ENO3 and PMCA2 expression levels were examined, and the relationship between disaccharide composition, sperm quality and protein expression was explored in A. sinensis. The results showed that relative ENO3 and PMCA2 expression levels were the highest at L15T15 in cryopreserved A. sinensis sperm. There were significant positive correlations between ENO3 expression and percentage membrane integrity, and between PMCA2 expression and sperm motility parameters (percentage of motile sperm, curvilinear velocity, straight‐line velocity and average path velocity; p < .05) in cryopreserved A. sinensis sperm. Our results indicate the optimal disaccharide combination and concentrations for cryopreservation of A. sinensis and A. dabryanus sperm and suggest that ENO3 and PMCA2 expression levels could serve as a valuable indicator of sperm quality in A. sinensis.  相似文献   

19.
Seminal plasma (SP) is known to induce motility and capacitation in spermatozoa curtailing their lifespan when preserved. Hence, this study was conducted to examine the effects of removal of SP from sperm surface prior to liquid preservation either by high dilution (1/15) or by washing and the poststorage treatment with SP (15% and 25%, v/v) on the quality attributes of liquid‐preserved ram semen. Over the period of storage, the rapid motility (66.0% and 71.1% vs. 58.3%), straightness (87.1% and 82.1% vs. 79.4%), average path velocity (152.3 and 152.0 µm/s vs. 133.3 µm/s) and the straight‐line velocity (131.3 and 127.8 µm/s vs. 108.5 µm/s) were significantly (p < 0.05) higher in both the high‐dilution and wash groups as compared to the control (1/3 dilution). The functional membrane integrity (82.3% vs. 77.2%) and noncapacitated sperm count (65.0% vs. 58.7%) were also significantly (p < 0.05) higher in the high‐dilution and wash groups, respectively, as compared to the control. The poststorage treatment of sperm with SP significantly (p < 0.05) increased the functional membrane integrity (70.1% vs. 53.8%) and most of the motility attributes as compared to the control (without SP). In conclusion, both the removal of SP prior to liquid preservation and poststorage treatment with SP significantly improved the quality attributes of ram spermatozoa.  相似文献   

20.
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   

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