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1.
Astaxanthin is a member of the carotenoid family well known for its anti-cancer, anti-diabetic, anti-inflammatory and antioxidant nature. This study was designed to investigate the effects of astaxanthin supplementation of the extender (buffer 2) on post-thaw dog semen quality. Semen from four healthy dogs was collected by digital manipulation twice a week. The ejaculates were pooled, washed, divided into four equal aliquots, diluted with the extender supplemented with different concentrations of astaxanthin (0, 0.5, 1 and 2 µM) and cryopreserved. The results showed that 1 µM astaxanthin was the optimum concentration that led to significantly higher (p < .05) post-thaw motility, kinematic parameters and viability than the other groups. In comparison with the control group, sperm samples supplemented with 1 µM astaxanthin showed significantly higher (p < .05) sperm counts with intact membranes (55.7 ± 0.6% vs. 51.3 ± 0.9%), intact acrosome (58.4 ± 0.7% vs. 53.5 ± 0.6%), active mitochondria (54.9 ± 0.5% vs. 42.6 ± 0.6%) and normal chromatin (67.6 ± 0.9% vs. 61.7 ± 0.6%). Furthermore, astaxanthin-supplemented samples showed significantly lower expression levels (p < .05) of pro-apoptotic (BAX), oxidative induced DNA damage repair (OGG1), oxidative stress-related (ROMO1) genes and higher expression levels of anti-apoptotic (BCL2), and sperm acrosome-associated (SPACA3) genes compared to the control. Thus, supplementation of 1 µM astaxanthin in semen extender results in improved freeze-thaw sperm quality of the dog.  相似文献   

2.
The objective of this study was to compare different extenders for post‐thaw in vitro sperm function and in vivo fertility of buffalo semen. Accordingly, sperm of 30 ejaculates extended in egg yolk (TRIS with 20% egg yolk; EY), two soya lecithin‐based (SL‐1; AndroMed® and SL‐2; Bioxcell®) and a liposome‐based extender (LS; OptiXcell®) were tested. The post‐thaw semen was evaluated for computer‐assisted sperm analysis (CASA), sperm viability, membrane and acrosome integrity, DNA integrity and acrosome reaction and first service pregnancy rate (FSPR) in a fixed‐time artificial insemination programme. Total motility and VCL were the only CASA‐based parameters that exhibited significantly higher (p < .05) percentage in LS among these extenders. Post‐thaw percentage of acrosome integrity (55.9 ± 1.4, 58.1 ± 2.0, 55.8 ± 2.0, 56.6 ± 2.3) and DNA integrity (68.8 ± 2.0, 69.2 ± 2.3, 71.3 ± 2.1, 69.1 ± 2.1) did not differ (p > .05) in EY, SL‐1, SL‐2 and LS extender, respectively. However, a variable response in terms of efficacy of different extenders for sperm viability and plasma membrane integrity was observed. Assessment of inducibility of acrosome reaction showed significant differences between extenders (51.9 ± 2.1, 44.3 ± 2.4, 46.1 ± 2.3 and 58.1 ± 3.1%, respectively, for EY, SL‐1, SL‐2 and LS). Furthermore, field trials revealed significantly higher (p < .05) FSPR of LS‐extended semen as compared to that for EY, SL‐1 and SL‐2 extender (46.3%, 41.2%, 31.2% and 29.7%, respectively). It is concluded that the liposome‐based extender is more effective than egg yolk‐ and soya lecithin‐based extenders and may be used for cryopreservation of buffalo semen in the future.  相似文献   

3.
The study was designed to evaluate AndroMed® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 106 spermatozoa/ml) in tris‐citric egg yolk or AndroMed® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed® extender (45.5% vs. 49%). It is concluded that AndroMed® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.  相似文献   

4.
A limiting factor in canine artificial insemination (AI) is the low number of insemination doses obtained per ejaculate. In this study, semen was collected from dogs (n = 28) either once and frozen directly after collection or the same dogs were submitted to a dual semen collection with a 1-hr interval and the two ejaculates were combined for cryopreservation. We hypothesized that combining two ejaculates increases semen doses per cryopreservation process without negative effects on semen characteristics. Total sperm count was lower in semen from a single semen collection in comparison with the combination of the first and second ejaculate of a dual semen collection (p < .001). The percentage of motile and membrane-intact spermatozoa determined by computer-assisted sperm analysis (CASA) in raw semen did not differ between single and combined dual ejaculates and was reduced (p < .001) by cryopreservation to the same extent in single (motility 73.7 ± 1.8%, membrane integrity 65.6 ± 2.2%) and combined dual ejaculates (motility 72.7 ± 2.3%, membrane integrity 64.6 ± 2.5%). The percentage of spermatozoa with morphological defects increased after cryopreservation (p < .001) but was similar in single and combined dual ejaculates. The CASA sperm velocity parameters decreased with cryopreservation (p < .001) but did not differ between single and combined dual ejaculates. The number of insemination doses increased from 2.7 ± 0.4 for single to 4.7 ± 0.8 for combined dual ejaculates (p < .01), based on 100 million motile spermatozoa per frozen-thawed semen dose. In conclusion, combining two ejaculates collected at short interval for one cryopreservation process increases the number of AI doses without compromising semen quality.  相似文献   

5.
The current study was designed to investigate the effect of idebenone (Id), an antioxidant on ram semen quality. Semen samples were collected, pooled and diluted in a Tris‐based extender supplemented with 0, 1, 2, 4 and 8 µM idebenone. Computer‐assisted sperm analysis was used to evaluate spermatozoa kinematics. Sperm viability and membrane functionality were assessed respectively, by eosin‐nigrosin staining and HOS test. Biochemical assays were carried out to measure different metabolites in spermatozoa and medium at 0, 24, 48 and 72 hr. Total and forward progressive motility were greater in 1, 2 and 4 µM idebenone treated groups compared to control at 24, 48 and 72 hr time points (p < 0.05). Semen supplementation with Id significantly increased viability and functionality of spermatozoa membrane during storage (p < 0.05). Lower amounts of lipid hydroperoxides in medium and spermatozoa were observed in Id‐treated groups compared to control one at 24 and 48 hr of storage (p < 0.05). Medium and spermatozoa amounts of malondialdehyde and nitric oxide were less in Id 4 µM group compared to the control at 72 hr (p < 0.05). Total antioxidant capacity values and superoxide dismutase activity of spermatozoa and medium were greater in 2 and 4 µM idebenone treated groups in comparison with the control at 72 hr (p < 0.05). Results of the current study indicated that ram semen supplementation with Id at 4 µM level improved quality by ameliorating nitrosative and peroxidative stress, hence could be considered as an antioxidant additive during storage at 4°C.  相似文献   

6.
Centrifugation of boar semen through one layer of 40% colloid (Porcicoll) was previously shown to separate spermatozoa from bacteria without having a detrimental effect on sperm quality. However, some spermatozoa were lost. The purpose of the present study was to determine whether 20% or 30% Porcicoll could be used to recover most of the spermatozoa without impacting on sperm quality. Insemination doses (n = 10) from a commercial boar station were sent to the laboratory at the Swedish University of Agricultural Sciences and processed by Single Layer Centrifugation with 20% and 30% Porcicoll approximately 7 hr after semen collection. The resulting sperm samples and controls were evaluated for sperm quality immediately and again after storage at 16–18°C for 4 and 7 days. Sperm recovery was 94 ± 18% and 87 ± 15% for 20% and 30% Porcicoll, respectively (p > .05). Sperm mitochondrial membrane potential and chromatin integrity were unaffected (p > .05). The proportion of live spermatozoa producing superoxide (9 ± 8%, 7 ± 6% and 3 ± 1%; p < .05), and the proportion of spermatozoa with high stainability DNA (0.68 ± 19%, 0.61 ± 0.22% and 0.96 ± 0.23%; p < .05- <0.01), were marginally increased whereas membrane integrity, although high, was lower in the centrifuged samples than in the controls (82 ± 8%, 83 ± 5% versus 92 ± 4%; p < .05). In conclusion, centrifugation through 20% or 30% Porcicoll enables most spermatozoa to be recovered, without having a major effect on sperm quality. These results are encouraging for further studies involving microbiological investigation of the processed samples, and scaling-up to process larger volumes of boar ejaculates.  相似文献   

7.
Microbial environment is one of the important factors that affect the quality of preserved semen. Iodine methionine (IM), participating in the production and activation of metabolic enzymes, is a new type of amino acid chelate. To date, there has been no report to evaluate the effects of IM on boar semen preservation at 17°C. This study was designed to investigate the effects of IM on boar sperm quality and reproductive performance during liquid storage at 17°C and its antibacterial effect. Semen samples collected from six Yorkshire boars were diluted with basic liquid containing different concentrations of IM (0, 20, 40, 80, 160 and 320 μM). Subsequently, sperm motility, plasma membrane integrity and acrosome integrity were determined. After 6 days of preservation, the difference in microbial composition between control group and 80 μM IM group was compared using 16S rDNA sequencing, and the effects of IM on reproductive performance were also compared and analysed between the two groups. The results demonstrated that 20, 40 and 80 μM IM improved boar sperm motility, plasma membrane integrity and acrosome integrity. 80 μM IM was the optimum concentration. Conversely, 160 and 320 μM IM resulted in deleterious consequences to boar sperm quality compared to the control group and other treatment groups (p < .05). After 6 days of preservation, sperm motility, plasma membrane integrity and acrosome integrity were 56.0%, 51.8% and 59.4%, respectively. There was no significant difference in non‐return rate between the two groups (p > .05). But the litter size of 80 μM IM group was significantly higher than that of control group (p < .05). 80 μM IM inhibited proliferation of the phylum Proteobacteria and the genus Staphylococcus as well as Pseudomonas (p < .05). Further studies are required to understand the antibacterial mechanism of IM in liquid‐preserved boar semen.  相似文献   

8.
The objective of this study was to evaluate the effect of collagenase and two final dimethylformamide (DMF) concentrations (4% and 7%) on alpaca frozen-thawed sperm quality. A total of 25 ejaculates from 5 alpaca were obtained using electroejaculation. Each individual ejaculate was evaluated and then diluted 4:1 in a solution of 1 mg/ml collagenase in HEPES-TALP medium and incubated for 4 min at 37°C. Subsequently, samples were diluted in TRIS-fructose-citric acid-egg yolk and cooled to 5°C. Then, each sample was divided in two aliquots and DMF at final concentration of 4% or 7% was added, equilibrated for 1 hr at 5°C and frozen over liquid nitrogen vapours. A Kruskal–Wallis test was used to evaluate the sperm morphometry, and Completely Random Block designs were used to analyse sperm motility, viability, membrane function and acrosome status. After collagenase incubation, none of the samples showed thread formation, and sperm parameters were preserved. Non-progressive motile sperm were higher (p < .05) in equilibrated samples (4% DMF: 31.8 ± 8.3% and 7% DMF: 36.3 ± 11.8%) compared to raw (10.1 ± 4.3%) and frozen-thawed semen (4% DMF: 9.7 ± 1.8% and 7% DMF: 7.5 ± 3.2%). Sperm membrane function, membrane integrity and intact acrosomes were higher (p < .05) in raw semen (40.1 ± 12.2%, 94.6 ± 3.2% and 91.3 ± 8.1%) compared to frozen-thawed samples (4% DMF: 19.8 ± 4.7%, 53.2 ± 2.7%, 65.7 ± 8.7% and 7% DMF: 20.4 ± 4.5%, 54.1 ± 1.4%, 64.6 ± 9.1%). Length of the sperm head was lower in frozen-thawed samples, being statistically different with 4% DMF compared to pre-freezing samples. The ratio between acrosome and head areas was greater (p < .05) in frozen-thawed samples. Incubation of raw alpaca semen with collagenase decreased the thread formation without affecting sperm quality. Frozen of collagenase treated alpaca semen with 4% or 7% DMF did not preserve the sperm parameters in thawed samples.  相似文献   

9.
The dilution effect and effect of restoring seminal plasma (SP) proportion in diluted semen were determined in chilled Asian elephant sperm. Semen was collected from eight males, and samples with ≥30% motile sperm were used in the study. Tris‐glucose‐egg yolk extender (TE) was used for cooled storage at 4°C for 48 hr. In experiment 1 (n = 18), semen was diluted to 1:1, 1:3, 1:7 and 1:15 with TE (volume per volume). There were no significant changes in sperm viability and sperm with normal acrosome integrity among dilutions, but sperm motility and motility velocities were greater (p < .05) in the 1:1 dilution than those of the 1:7 and 1:15 dilutions at 48 hr of storage. In experiment 2, supplemented SP was derived from elephants and stallions. In experiment 2.1, diluted semen (1:7 dilution) was restored with SP to obtain a 1:2 proportion (n = 8). Sperm motility, viability and sperm with normal acrosome integrity were similar among treatments, but motility velocities were greater (p < .05) with stallion SP at 48 hr of storage. In experiment 2.2, diluted semen (1:15 dilution) was restored with SP to obtain a 1:3 proportion (n = 10). Sperm viability and sperm with normal acrosome integrity were similar among treatments at 48 hr of storage. However, sperm motility and motility velocities were greater (p < .05) with stallion SP than those of others. In conclusion, elephant sperm motility was affected by a dilution effect and restoration of SP proportion with stallion SP, but not with elephant SP, could improve motility in chilled highly diluted sperm.  相似文献   

10.
Addition of Glutathione to an Extender for Frozen Equine Semen   总被引：1，自引：0，他引：1  
The manipulation of equine semen during cryopreservation reduces sperm viability and fertility because of, among other factors, membrane lipid peroxidation that makes cells highly susceptible to free radicals and reactive oxygen species (ROS). The oxidative effect caused by the generation of ROS can be reduced by the addition of antioxidants to the seminal plasma or to the extenders used for freezing. The current study was performed to test the in vitro effect of exogenous glutathione added in five different concentrations (control, 2.5 mM, 5.0 mM, 7.5 mM, and 10 mM [treatments 1-5, respectively]) to the extender for 12 stallions. Analyzed parameters were sperm motility, viability, and acrosome and plasmatic membrane integrity. Total motility was higher in treatments 1 and 2 (P < .05); viability, progressive motility, and plasmatic membrane integrity were higher in treatment 2 (P < .001). As for acrosome membrane integrity, treatment 3 showed the best results (P < .05). The addition of 2.5 mM glutathione to the freezing extender preserves total motility and increases sperm viability, progressive motility, and plasmatic membrane integrity. Concentrations above 2.5 mM were deleterious to spermatozoa.  相似文献   

11.
The aim of this experiment was to evaluate the effects of adding ascorbic acid 2‐glucoside (AA2G), a water‐soluble antioxidant and stable derivative of ascorbate, to the semen extender and compare it to the addition of vitamin C (Vit. C) and the fat‐soluble antioxidant α‐tocopherol (α‐Toh), both individually and in combination, on the seminal variables of equine sperm submitted to cooling for 72 h. We used two ejaculates from 10 stallions and evaluated them for motility, membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation. In the analysis of lipid peroxidation, the control group showed 2506.2 ± 796.4 ng malondialdehyde/108 sperm, which was higher (P < 0.05) than the groups treated with antioxidants. The average value of motility in the AA2G group was 68.4 ± 18.1%, which was higher (P < 0.05) than that observed in the control group (62.1 ± 16.2%). The variables membrane integrity, chromatin fragmentation and mitochondrial activity did not show significant difference (P > 0.05) between treatments. It was concluded that the antioxidants protected sperm cells from lipid peroxidation and that AA2G was effective during the cooling process of equine semen at 5°C for72 h, providing increased levels of total motility.  相似文献   

12.
During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome‐reacted sperm were evaluated with a computer‐assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 μM/μg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.  相似文献   

13.
Metformin is clinically used to treat diabetes. Given its role‐impacting metabolism, metformin has been also added to semen cryopreservation media showing specie‐dependent effects. We aimed to investigate metformin effects in both fresh (38.5°C for 2, 24 hr) and refrigerated (17°C for 10 days) boar spermatozoa. Metformin (2 hr) does not affect fresh sperm viability, membrane lipid organization nor acrosome integrity. However, metformin (24 hr) blocks sperm ΔΨm and significantly reduces % motile spermatozoa (65%), % progressive spermatozoa (50%), % rapid (100%), velocities VCL (69%), VSL (86%), VAP (78%) and motility coefficients. Metformin‐including extender does not modify sperm viability, membrane lipid organization or acrosome integrity. Furthermore, it significantly reduces high ΔΨ‐population spermatozoa at refrigeration day 4. Metformin also significantly reduces sperm motility during refrigeration. Summarizing, metformin inhibits both boar sperm ΔΨ and motility in any sperm condition studied: fresh and refrigerated. These findings dissuade metformin as an additive to improve boar sperm quality.  相似文献   

14.
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   

15.
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   

16.
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   

17.
Glycerol‐based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post‐thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol‐based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio®). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.  相似文献   

18.
Comparative effect of slow and rapid freezing on sperm functional attributes and oxidative stress parameters of goat spermatozoa cryopreserved with tiger nut milk (TNM) extender was examined in this study. Pooled semen samples obtained from West African Dwarf (WAD) goat bucks were diluted with Tris‐based extenders containing different levels of TNM (0, 5, 10, 15 and 20 ml/100 ml extender). The diluted semen samples were subjected to slow and rapid freezing for a period of 7 days and thereafter evaluated for sperm functional attributes (percentage motility, acrosome integrity, membrane integrity, abnormality and livability) and oxidative stress (malondialdehyde [MDA] concentration and acrosin activity) parameters. Results showed that higher (p < 0.05) motility, livability, membrane and acrosome integrities in semen cryopreserved with slow freezing compared to rapid freezing. These parameters (motility, livability and membrane integrity) were higher (p < 0.05) in semen cryopreserved with 15% TNM in both slow and rapid freezing protocols. The results revealed that semen cryopreserved in slow freezing had lower (p < 0.05) abnormality compared to rapid freezing. Acrosin activity was higher in slow freezing compared to rapid freezing. Acrosin activity was higher at 15% TNM in both slow and rapid freezing. Lower (p < 0.05) MDA concentration was observed in semen cryopreserved using slow freezing compared to rapid freezing. The findings revealed improved post‐thaw sperm functional attributes and oxidative stress parameters of WAD goat spermatozoa cryopreserved with 15% TNM using slow freezing.  相似文献   

19.
Glycerol is used as a bovine semen osmotic cryoprotectant that greatly improves the quality of frozen and thawed bovine sperm. However, high glycerol concentrations can have a toxic effect on frozen and thawed bovine sperm. Therefore, this experiment investigated the effect of replacing a portion of the glycerol in a cryoprotectant solution with crocin on the sperm apoptosis, protamine deficiency and membrane lipid oxidation of frozen and thawed Yanbian yellow cattle sperm. The experiment included a control group (6% glycerol) and four treatment groups: I (3% glycerol), II (3% glycerol +0.5 mM crocin), III (3% glycerol + 1 mM crocin) and IV (3% glycerol + 2 mM crocin). Computer assisted semen analysis was used to detect sperm motility, Hoechst 33,342, propidium iodide, and JC-1 staining were used to analyse sperm viability and mitochondrial membrane potential, chromomycin A3 staining was used to detect protamine deficiency and DNA damage, flow cytometry was used for sperm membrane lipid disorder detection and analysis, and real-time quantitative RT-qPCR was used to detect the mRNA expression levels of protamine-related genes (PRM2, PRM3), sperm acrosome-associated genes (SPACA3), oxidative stress-related genes (ROMO1) and apoptosis-related genes (BCL2, BAX). Compared to the control group, replacing a portion of glycerol with 1 mM crocin significantly improved sperm motility, plasma membrane integrity, membrane lipid disorders (p < .05) and viability, mitochondrial membrane potential, protamine deficiency (p < .01). The expression level of PRM2, PRM3, SPACA3 and BCL2 significantly increased (p < .05), while the expression levels of ROMO1 and BAX significantly decreased (p < .05). Accordingly, the BCL2/BAX ratio significantly increased (p < .05). In summary, the substitution of a portion of glycerol with crocin in cryoprotective solution improved the quality of Yanbian yellow cattle sperm after freezing and thawing.  相似文献   

20.
Cryopreservation damages permeability of sperm mitochondrial membranes, with formation of a mitochondrial permeability transition pore (mPTP). Mitochondria are both a primary synthesis site and principle target for melatonin, which can directly inhibit mPTP formation. The objective was to determine effects of melatonin on mPTP opening of frozen-thawed ram sperm and elucidate underlying pathways by antagonist and agonists of melatonin receptors (MTs), and antagonists of PI3K and GSK 3β treatments; furthermore, plasma membrane integrity, mitochondrial membrane potential (ΔΨm), mitochondrial cytochrome c (Cyt c) release and fertilization were analysed to assess the effect of mPTP status mediated by melatonin on quality of frozen-thawed sperm. Fresh ram semen was diluted in glucose-egg yolk buffer with 0 or 10–7 M melatonin (frozen and frozen + melatonin groups, respectively) and slow-frozen. In frozen-thawed sperm, melatonin added at initiation of 4°C equilibration was most effective for inhibiting mPTP opening, decreasing peptidyl-prolyl-cis/trans isomerase activity of cyclophilin D and increasing plasma membrane integrity, ΔΨm, mitochondrial Cyt c concentration and fertilizing ability (p < .05). In a mechanistic study, the melatonin receptor (MT)1 antagonist eliminated inhibition of melatonin on mPTP opening, whereas MT1 agonist had opposite effects (p < .05). Neither MT2 antagonist nor agonist had significant effect, but PI3K and/or GSK 3β antagonist decreased inhibition of MT1 agonist on mPTP opening (p < .05). In conclusion, melatonin improved sperm cryopreservation, perhaps by acting on MT1 via the PI3K-Akt-GSK 3β pathway to inhibit mPTP opening.  相似文献   

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