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1.
This study was carried out to assess the in vitro quality of canine semen frozen in an ultrafreezer at -152 degrees C and to evaluate the male-to-male variation of frozen semen in five male dogs of the Canarian Mastiff breed. Four ejaculates of each dog were processed individually (5% glycerol and 0.5% Equex) to reach a final concentration of 100 x 10(6) spermatozoa/ml. Then, two freezing techniques were tested to assess the seminal quality (sperm motility, live spermatozoa and abnormal sperm cell percentages) at 1, 30, 60, 120 and 360 days after freezing: (i) semen was frozen and stored in liquid nitrogen; (ii) semen was frozen and stored in the ultrafreezer at -152 degrees C. After freezing-thawing, both freezing protocols showed no significant differences in sperm motility and the percentages of live and abnormal spermatozoa. On the other hand, the microscopic characteristics of spermatozoa in fresh semen were practically similar among males; however, after the semen processing and freezing, significant differences were observed (p < 0.05) among males, especially as regards sperm motility. This inter-individual variability was detected in both freezing protocols, showing that the male-to-male variation in the seminal quality post-freezing was independent of the freezing technique used. The in vitro results obtained in the Canarian Mastiff breed confirmed that the use of ultra-freezers at -152 degrees C is a potential alternative to liquid nitrogen for storing canine semen for long periods of time.  相似文献   

2.
Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.  相似文献   

3.
A study was conducted to evaluate the relationship between boar sperm motility and membrane integrity following exposure to media with 150–1120 mOsm. Total sperm motility was defined as the percentage of spermatozoa that had any form of motility was subjectively assessed under a light microscope. Sperm cell damage was expressed as a loss of membrane integrity as measured by a combination of fluorescent stains, carboxyfluorescein diacetate (CFDA) and propidium iodide (PI), and Hoechst 33258 (H33258). There were no significant differences between sperm motility and membrane-intact spermatozoa, as measured by CFDA-PI and H33258, in media with 250 and 300 mOsm. In anisosmotic conditions, a higher amount of membrane-intact spermatozoa than motile spermatozoa was observed. In hypo-osmotic conditions (150 mOsm), a high proportion of spermatozoa had curled or coiled tails and most of them retained their entire membrane integrity, as detected by CFDA-PI. In media with 350–1120 mOsm, some spermatozoa accumulated PI in the head region and CFDA in the mid-piece. These spermatozoa fluoresced blue at the lower region of the head, as detected by H33258. The ATP content in spermatozoa exposed to hypo- and hyperosmotic conditions was markedly reduced. There was no recovery of sperm motility on returning the spermatozoa to isosmotic conditions after 10 min incubation in anisosmotic conditions, indicating that the spermatozoa suffered an almost complete and irreversible loss of motility. This irreversible loss of motility may be a consequence of reduced ATP production in spermatozoa subjected to anisosmotic conditions. The results of this study demonstrate that plasma membrane integrity assessment in combination with sperm motility, using a range of media varying in osmolality, can give valuable information about the status and function of different sperm membranes, which might be relevant for semen preservation.  相似文献   

4.
Three experiments evaluated the effects of dietary Se and vitamin E on the ultrastructure of spermatozoa, ATP concentration of spermatozoa, and the effects of adding sodium selenite to semen extenders on subsequent sperm motility. The experiment was a 2 x 2 arrangement of treatments in a randomized complete block design. A total of 10 mature boars were fed from weaning to 18 mo of age diets fortified with two levels of supplemental Se (0 or .5 ppm) or vitamin E (0 or 220 IU/kg diet). The nonfortified diets contained .06 ppm Se and 4.4 IU vitamin E/kg. In Exp. 1, the spermatozoa from all boars were examined by electron microscopy. Vitamin E had no effect on structural abnormalities in the spermatozoa. When the low-Se diet was fed the acrosome or nuclei of the spermatozoa was unaffected, but the mitochondria in the tail midpiece were more oval with wider gaps between organelles. The plasma membrane connection to the tail midpiece was not tightly bound as when boars were fed Se. Immature spermatozoa with cytoplasmic droplets were more numerous when boars were fed the low-Se diet, but the occurrence of midpiece abnormalities occurred in boars fed diets with or without Se or vitamin E. Our results suggest that Se may enhance spermatozoa maturation in the epididymis and may reduce the number of sperm with cytoplasmic droplets. In Exp. 2, the concentration of ATP in the spermatozoa was evaluated in the semen of all treatment boars. When the low-Se diet was fed, ATP concentration was lower (P < .01), whereas vitamin E had no effect on ATP concentration. Experiment 3 investigated the effect of diluting boar semen with a semen extender with sodium selenite added at 0, .3, .6, or .9 ppm Se. Three ejaculates from each boar were used to evaluate these effects on sperm motility to 48 h after dilution. Sperm motility declined (P < .01) when Se was added to the extender, and this decline was exacerbated as the concentration of added Se increased (P < .01). The added Se was demonstrated to be tightly adhered to the spermatozoa. Overall, these results suggest that low Se-diets fed to boars resulted in abnormal spermatozoal mitochondria, a lower ATP concentration in the spermatozoa, and a loose apposition of the plasma membrane to the helical coil of the tail midpiece, but no effect from inadequate vitamin E was demonstrated. Adding sodium selenite to the semen extender reduced sperm cell motility.  相似文献   

5.
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   

6.
Cyclodextrins improve post-thaw viability and motility of semen as well as mediate cholesterol efflux and subsequent acrosome reaction in spermatozoa from several species. The objectives of this study were: (a) to assess the effect of prefreeze addition of 60 mM hydroxypropyl-β-cyclodextrin (β-CD) on post-thaw viability and motility of jack and stallion semen cryopreserved in ethylene glycol-based freezing extenders containing 5% or 20% (v/v) egg yolk (LEY and HEY, respectively), and (b) to evaluate the ability of 1 μM calcium ionophore A23187 and/or 60 mM β-CD to induce acrosome reaction in thawed jack and stallion spermatozoa. Post-thaw motility of spermatozoa cryopreserved in HEY was higher (P < .05) for jack but lower (P < .05) for stallion spermatozoa when compared with LEY. Jack and stallion spermatozoa both exhibited higher (P < .05) motility when cryopreserved in 60 mM β-CD than without β-CD. Curvilinear velocity was faster (P < .05) for jack and stallion spermatozoa cryopreserved in LEY than in HEY. A treatment × time interaction affected (P < .05) the proportion of spermatozoa that underwent acrosome reaction. Post-thaw incubation of jack and stallion spermatozoa with β-CD for 90 minutes induced acrosome reaction in 85% and 22% of viable sperm cells, respectively; however, only 32% of jack and 8% of stallion spermatozoa incubated with calcium ionophore underwent acrosome reaction. This study is the first to evaluate the effect of β-CD (not loaded with cholesterol) on jack semen cryopreservation, and results reveal that β-CD may be a useful tool to enhance semen cryopreservation and to induce post-thaw acrosome reaction in jack spermatozoa.  相似文献   

7.
旨在探讨辅酶Q10对绒山羊精液冷冻保存效果的影响。利用添加不同浓度辅酶Q10(4、40、400?滋g/mL)的精液冷冻稀释液对绒山羊精液样本进行冷冻保存,待冷冻精液解冻后,采用流式细胞仪和计算机辅助精液分析系统(CASAS)分别检测不同精液样本的精子活率、质膜完整率、顶体完整率、DNA完整率、线粒体膜电位和细胞内ROS水平。结果表明,当冷冻稀释液中添加浓度为40μg/mL辅酶Q10时,经历冷冻—解冻过程的绒山羊精液样本的精子活率、质膜完整率、顶体完整率均显著高于对照组(P<0.05);在冷冻稀释液中添加浓度为40μg/mL或400μg/mL的辅酶Q10均能显著提高线粒体膜电位并降低细胞内ROS水平(P<0.05)。综上所述,在冷冻稀释液中添加40μg/mL的辅酶Q10能够显著提高绒山羊精子抗氧化能力和冷冻保存效果。  相似文献   

8.
The phylogenetic order Squamata has many representatives that could benefit from the use of semen preservation as a tool for assisting conservation. To date, few studies have been made evaluating the potential for collecting and preserving semen from snakes. The objectives of this study were to characterize semen parameters of the corn snake (Elaphe guttata), including appearance, volume, concentration, sperm motility, and sperm morphology, and to determine the longevity of corn snake sperm motility stored at 4 degrees C. Single semen samples were collected from 22 adult corn snakes. The appearance of the corn snake semen was generally cloudy, and the color was white to tan. Corn snake spermatozoa initially exhibited a median motility of 92.5%. Corn snakes were found to produce small-volume ejaculates (median 0.01 ml). However, the overall concentration of the snake ejaculate was high (chi = 852 x 10(6) +/- 585 x 10(6) spermatozoa/ml). Morphologically, a mean of 75.7 +/- 9.3% of the sperm cells in an ejaculate were normal. Snake ejaculate with a white appearance had significantly higher sperm concentrations (chi = 1,859 x 10(6) +/- 1,008 x 106 sperm cells/ml; F = 15.74, P = 0.001) than tan ejaculates (chi = 601 x 10(6) +/- 439 x 106 sperm cells/ml). Sperm motility decreased significantly in samples that were stored at 4 degrees C for greater than 48 hr in a refrigerator or Equitainer I. This is the first study to characterize semen volume, appearance, and concentration; sperm motility; and sperm morphology in captive corn snakes. The information derived from this study can be used to develop a model for a collection, cooled storage, and shipping program for semen from endangered or threatened captive and wild snakes.  相似文献   

9.
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   

10.
The use of artificial insemination in cattle breeding has evolved to global extent, and insemination doses are often shipped via air transport which requires strict radiation‐based examinations. For the determination of effect of non‐ionizing radiation (NIR), to which are beings frequently exposed due to protection of airport or cultural event security, freshly ejaculated and cryopreserved bovine spermatozoa were used as experimental model. Following radiation with hand‐held metal detector in various exposition times (0, 10 s, 15, 30 and 60 min—groups FR, FR10, FR15, FR30 and FR60) the spermatozoa underwent motility and DNA fragmentation analyses. Study on cryoconserved semen treated with NIR was performed in time intervals 0, 10 s, 1 and 5 min (insemination doses radiated before cryoconservation—CB, CB10, CB1, CB5; samples radiated after freezing—CA, CA10, CA1 and CA5). Fresh semen and insemination doses radiated after cryoconservation showed significantly lower total and progressive motility. No effect on motility parameters was detected in semen extended with cryopreservative medium and radiated prior to freezing. Surprisingly, NIR showed a potential to stimulate spermatozoa velocity; however, the effect was modulated throughout the post‐thawing incubation. Based on the DNA fragmentation assay, sperm DNA stayed intact. Present study underlines the potential harm of NIR, which is frequently used in everyday life, with overall adverse impact on human and animal reproduction. Current study also points out on interesting short‐term spermatozoa stimulation induced by NIR.  相似文献   

11.
The aim of the present study was to develop a treatment supporting the membrane of ram spermatozoa. Semen of different ejaculates collected from breeding rams was mixed and samples of 10(9) sperm cells per ml and Tris-egg yolk extender were completed with the following antioxidants: alpha-tocopherol acetate (E), glutathione peroxidase (GP), Aromex (AR), resveratrol (R), resveratrol + vitamin E (RE), resveratrol + Aromex (RAR), resveratrol + GP (RGP). Peroxidation was evaluated by the analysis of malondialdehyde (MDA) during incubation for 30, 60 and 120 min at 37 degrees C as well as during a 24-h incubation at 5 degrees C. The success of preservation was checked in a 9-day-long period by observing the acrosomal defects and the motility of spermatozoa. Concentration of MDA was 4.06 nmol/10(9) spermatozoa in samples treated with 15 micrograms R while the control sample contained 69.79 nmol MDA per 10(9) spermatozoa after 24-h incubation. Following 30-, 60- and 120-min storage the concentration of MDA in control and R-treated samples was 25.89, 36.91, 49.57 and 3.69, 3.74, 3.74 nmol/10(9) spermatozoa, respectively. Moreover, a significantly higher proportion of motile sperm cells was observed in the treated than in the control samples. The frequency of acrosomal defects was lower in the treated groups than in the control. These results indicate that RAR treatment can improve the effects of ram semen preservation.  相似文献   

12.
The Japanese black bear (Ursus thibetanus japonicus) is endangered for extinction in some areas of Japan, and semen collection and cryopreservation are an important means to preserve genetic resources. The aim of this study was to characterize and cryopreserve semen of free-ranging Japanese black bears. Semen was collected by electroejaculation procedure from 4 free-ranging Japanese black bears at the capture point in the field. Ejaculates containing motile sperm were recovered from all of the animals and ejaculate volume, total sperm count, % motility (percentage of motile spermatozoa), % viability (percentage of spermatozoa that excluded eosin) and % abnormal morphology (range (mean)) were 0.65-2.20 (1.51) ml, 99-1082 (490) x 10(6), 5-100 (31), 42-97 (66) and 20-87 (53), respectively. Three of the 4 ejaculates were diluted with an egg yolk-TRIS-citrate-glucose extender and cryopreserved in liquid nitrogen. Motile spermatozoa were observed after freezing and thawing in all cases. This study showed that electroejaculation would be a useful method for collecting semen from free-ranging Japanese black bears and that at least motile spermatozoa would be obtained by freezing the thus collected electroejaculates.  相似文献   

13.
Orchiopexy of the cryptorchid (CR) testis and castration of the scrotal testis were performed in three unilaterally CR beagles at six months of age. Induction rates for ejaculated sperm hyperactivation (HA) and the acrosome reaction (AR) in vitro in these orchiopexied dogs were compared with five those in normal beagles one year later. Canine spermatozoa were incubated for 9 hr at 38 degrees C under 5% CO2 in air in canine capacitation medium at a concentration of 30 x 10(6) sperm/ml. HA was observed using high-speed videomicrography. The AR spermatozoa were evaluated by the triple stain technique. As a result, there was no significant difference between 'the CR dogs after orchiopexy' (CDO) and the normal dogs (ND) with respect to sperm motility just after ejaculation. However, sperm motility of CDO decreased markedly during incubation. There was a significant difference in sperm motility between CDO (Mean +/- SD; 47 +/- 12%) and ND (80 +/- 9%) after three hours of incubation (p less than 0.01). No significant difference was observed between CDO and ND with respect to the HA rate of motile spermatozoa throughout the incubation period. The peak of HA rate was found in both CDO (58 +/- 5%) and ND (61 +/- 16%) after seven hours of incubation. The AR rate of spermatozoa in CDO was lower than that in ND after six hours of incubation. The AR rate of CDO (26 +/- 4%) was significantly lower than ND (46 +/- 5%) after eight hours of incubation (p less than 0.01). It is assumed that there might be relation between a rapid decrease of motility and low AR rate in spermatozoa of CDO during incubation.  相似文献   

14.
Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen‐thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post‐thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP‐αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post‐thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post‐thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time.  相似文献   

15.
A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 106 spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca2+, capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca2+ and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 106 spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 106 spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 106 spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca2+); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca2+, low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca2+, medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 106 spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing–thawing-associated damages in bubaline species.  相似文献   

16.
Spermatozoa are highly specialized cells, and energy metabolism plays an important role in modulating sperm viability and function. Rosiglitazone is an antidiabetic drug in the thiazolidinedione class that regulates metabolic flexibility and glucose uptake in various cell types, but its effects on boar sperm metabolism are unknown. In this study, we investigated the potential effect of rosiglitazone against time‐dependent deterioration of boar spermatozoa during liquid preservation at 17°C. Freshly ejaculated semen was diluted with Beltsville Thawing Solution (BTS) containing different concentrations of rosiglitazone, and the motility, membrane and acrosome integrity of sperm were detected. Besides, we measured glucose uptake capacity, l ‐lactate production level, mitochondrial membrane potential, adenosine triphosphate (ATP) content and mitochondrial reactive oxygen species (mROS) production of sperm after boar semen had been incubated with or without rosiglitazone, iodoacetate (glycolysis inhibitor) and rotenone (electron transport chain inhibitor) for 5 days. The addition of rosiglitazone significantly enhanced sperm quality and had a strong protective effect on the sperm membrane and acrosome integrity during storage. BTS containing 50 μM rosiglitazone maintained the total motility of liquid‐preserved sperm above 60% for 7 days. Rosiglitazone improved sperm quality by regulating energy metabolism manner of preserved sperm, protected the sperm mitochondrial membrane potential, enhanced sperm ATP production and in the meanwhile reduced mROS through enhancing glycolysis but not oxidative phosphorylation. The data suggested the practical feasibility of using rosiglitazone for improving boar spermatozoa quality during semen preservation.  相似文献   

17.
The aim of present study was to evaluate frozen canine semen with ACP-106 (Powder Coconut Water) using an in vitro sperm--oocyte interaction assay (SOIA). Ten ejaculates from five stud dogs were diluted in ACP-106 containing 20% egg yolk, submitted to cooling in a thermal box for 40 min and in a refrigerator for 30 min. After this period, a second dilution was performed using ACP-106 containing 20% egg yolk and 12% glycerol. Samples were thawed at 38 degrees C for 1 min. Post-thaw motility was evaluated by light microscopy and by using a computer aided semen analysis (CASA). Plasma membrane integrity and sperm morphology/acrosomal status were evaluated by fluorescent probes (C-FDA/PI) and Bengal Rose respectively. Moreover, frozen-thawed semen was analysed by a SOIA. Subjective post-thaw motility was 52.0 +/- 14.8% and it was significant higher than the total motility estimated by CASA (23.0 +/- 14.8%) because this system considered the egg yolk debris as immotile spermatozoa. Although normal sperm rate and acrosomal integrity evaluated by Bengal Rose stain was 89.6 +/- 3.1% and 94.3 +/- 3.1%, respectively, post-thaw percentage of intact plasma membrane was only 35.1 +/- 14.3%. Regarding SOIA, the percentage of interacted oocytes (bound, penetrated and bound and/or penetrated) was 75.3%. Using regression analysis, it was found significant relations between some CASA patterns and data for SOIA. In conclusion, the freezing-thawing procedure using ACP-106 was efficient for maintain the in vitro fertility potential of dog spermatozoa.  相似文献   

18.
Twenty ejaculates from five dairy AI‐bulls were used to compare, in a split‐sample experiment, the fertility [56 day‐non‐return‐rate (NRR) from more than 14000 AI) and sperm viability post‐thaw of semen diluted with an egg yolk‐ (Triladyl®) or soybean‐based (Biociphos‐Plus®) commercial extender. The in vitro evaluations were divided in two experiments. Experiment 1 (n = 20) included post‐thaw evaluations of motility (subjective and computerized), membrane integrity (CalceinAM/EthD‐1, SYBR‐14/PI, and osmotic resistance test; ORT), and capacitation status (CTC/EthD‐1). Experiment 2 (n = 10) included evaluations of the capacitation‐(CTC/EthD‐1) and acrosome status (FITC‐PSA/EthD‐1) during incubation with/without a challenge with solubilized zona pellucida proteins (SZP). No significant difference in the fertility (69.1 ± 0.8 versus 69.2 ± 0.8) results was found between the two extenders. In experiment 1, the computerized motility evaluations post‐thaw (CASA) showed higher values for Biociphos‐Plus® processed semen for the velocity patterns and lateral sperm head displacement. After 6 h at room temperature (20–22°C) all the CASA motility patterns were significantly higher for Biociphos‐Plus®. The proportion of spermatozoa with intact membranes assessed by CalceinAM was significantly higher in Biociphos‐Plus® (p < 0.001) compared to Triladyl®, but such difference was not seen when using SYBR‐14 or the ORT‐assay. When using the CTC/EthD‐1 assay, a lower proportion of acrosome reacted (AR) spermatozoa post‐thaw (p < 0.01) was found in Biociphos‐Plus® processed semen, as well as a tendency (p < 0.07) for a higher number of uncapacitated spermatozoa. In experiment 2, the proportion of uncapacitated spermatozoa was significantly higher for Biociphos‐Plus® when semen was incubated (38°C and 5% CO2) without SZP at both 0 (p < 0.001) and 30 min (p < 0.05). Concomitantly, Triladyl® showed a higher percentage of capacitated spermatozoa at 0 (p < 0.01), 30 (p < 0.05) and 120 min (p < 0.05). A higher (p < 0.05) incidence of AR‐spermatozoa was seen in Triladyl® at the beginning of the incubation with SZP. No significant difference between extenders was detected for the acrosome status by the FITC‐PSA‐assay. Incubation with SZP induced acrosome reaction of capacitated spermatozoa in both extenders, which was detected by CTC and FITC‐PSA assays. In conclusion, fertility was not affected by Biociphos‐Plus® when 15 × 106 of spermatozoa per AI dose were inseminated. The finding that higher frequencies of spermatozoa seemed more membrane stable post‐thaw, when frozen in Biociphos‐Plus®, might indicate that this extender better protects the sperm viability compared with Triladyl®.  相似文献   

19.
Deep-frozen semen from 28 bulls belonging to 6 different A.I. centres was studied after thawing and the ATP content in the spermatozoa was assayed using a bioluminescence technique. The sperm motility was subjectively estimated under a phase contrast microscope and the sperm concentration of each ejaculate was calculated in a haemocytometer. The overall mean ATP content was 16.6 nmoles ATP/spermatozoa x 10(8). There was a significant variation in ATP content between A.I. centres. Significant differences between bulls in ATP content were found as well as a significant correlation between ATP concentration and the number of motile spermatozoa. This may indicate that ATP assessment may be useful as an additional, objective laboratory test.  相似文献   

20.
The effect of Mycoplasma bovis (Donetta strain) on the ability of bull spermatozoa to interact with zona pellucida-free hamster oocytes was studied in an in vitro assay. Ejaculates of semen from a fertile Holstein bull were used fresh on the day of collection (unextended semen) as well as diluted with egg yolk-citrate and used the following day (extended semen). The addition of M. bovis to both unextended and extended semen at a mycoplasma to sperm cell ratio of 10:1 significantly reduced sperm penetration rates and the mean number of sperm per penetrated egg. Similarly, the ability of spermatozoa to form pronuclei and the activation of penetrated oocytes were adversely affected by M. bovis. No apparent effect on sperm motility was detected. When M. bovis was added to the oocytes, there was a marked reduction in the sperm penetration rates and fertilization processes suggesting that the organism affects certain oocyte function(s). The results indicate that the presence of M. bovis in semen or in the female reproductive tract may affect fertilization. Moreover, the in vitro assay with hamster oocytes was found to be useful for demonstrating the effects of contaminating microbial agents on bovine fertilization processes.  相似文献   

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