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1.
The aim of this research was to study the effect of hyaluronic acid on bovine cryopreserved spermatozoa compared with heparin as regards the variation of capacitation induction, cellular oxidative metabolism and intracellular signal induced by membrane‐associated adenylate cyclase to propose hyaluronic acid as a capacitation inductor. Heparin or hyaluronic acid and lysophosphatidylcholine were used to induce sperm capacitation and acrosome reaction, respectively. 2′,5′‐dideoxyadenosine was used as a membrane‐associated adenylate cyclase inhibitor. The highest percentages of capacitated spermatozoa and live spermatozoa with acrosome integrity were obtained by incubating sperm for 60 min using 1000 μg/ml hyaluronic acid. In these conditions, capacitation induced by hyaluronic acid was lower compared with heparin; nonetheless both glycosaminoglycans promote intracellular changes that allow true acrosome reaction in vitro induced by lysophosphatidylcholine in bovine spermatozoa. Oxygen consumption in heparin‐capacitated spermatozoa was significantly higher than in hyaluronic acid‐treated spermatozoa. With all treatments, mitochondrial coupling was observed when a specific uncoupler of the respiratory chain was added. The inhibition of membrane‐associated adenylate cyclase significantly blocked capacitation induction produced by hyaluronic acid, maintaining a basal sperm oxygen uptake in contrast to heparin effect in which both sperm parameters were inhibited, suggesting that the membrane‐associated adenylate cyclase activation is involved in the intracellular signal mechanisms induced by both capacitation inductors, but only regulates mitochondrial oxidative phosphorylation in heparin‐capacitated spermatozoa.  相似文献   

2.
We aimed to elucidate whether NO acts in in vitro sperm capacitation in bovine via cGMP/PKG1 pathway. For this, cryopreserved bovine sperm were capacitated in vitro with 20 µg/ml heparin (Control) plus treatments: 1 mM L‐arginine (L‐arg, NO precursor), 50 µM Rp‐8‐Bromo‐β‐phenyl‐1,N2‐ethenoguanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐Br‐cGMPS, selective inhibitor of the binding site for cGMP in PKG1), 1 mM 2‐Phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide (PTIO, NO scavenger), and the combinations of L‐arg + RP‐8‐Br‐cGMPS and L‐arg + PTIO. Sperm motility and vigour were determined by phase‐contrast microscopy, capacitation status by chlortetracycline staining, and the intracellular concentration of cGMP was measured by ELISA. Data were subjected to analysis of variance and means compared with SNK test at 5% probability. Motility and vigour were lower in sperm treated with PTIO when compared to Control and other treatments (p < .05). The L‐arg treatment showed the highest percentage of capacitated sperm when compared to the Control and other treatments (Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO) (69.8 ± 3.4%, 51.2 ± 3.0, 51.1 ± 2.1, 51.2 ± 3.0 and 45.5 ± 2.7, respectively) (p < .05). The capacitation ratio (%) was lower in treatments with Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO, respectively (p < .05). Lastly, cGMP concentration (pmol/ml) was lower in PTIO and L‐arg + PTIO (1.3 ± 0.3 and 1.6 ± 0.4) and was higher in Rp‐8‐Br‐cGMPS and L‐arg + Rp‐8‐Br‐cGMPS (3.7 ± 0.4 and 4.0 ± 0.5) treatments. We showed that during in vitro capacitation of cattle: (a) NO influences sperm motility and vigour; (b) NO is associated with cGMP synthesis through two independent pathways and (c) the cGMP/PKG1 pathway has a partial role in sperm capacitation and does not involve the L‐arg/NO.  相似文献   

3.
The magnitude of damage to buffalo spermatozoa during incubation with different levels of H2O2 was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris–egg yolk–citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5°C) and cryopreserved in 0.5‐ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen–thawed semen was separated by centrifugation (1500 g ; 15 min) and were washed with sperm TALP. The sperm cells were re‐suspended in incubation TALP at the rate of 108 sperm cells per millilitre and incubated with 0, 10, 25, and 50 μm H2O2 per ml at 37°C. Sperm motility, viability and intact acrosome percentages were assessed at 15‐min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H2O2. Among the different levels of H2O2, the 50‐μm H2O2‐incorporated group had significantly (p < 0.05) higher malonaldehyde (MDA) level than the other groups. In the 50‐μm H2O2‐incorporated group, the MDA levels in fresh, equilibrated and frozen–thawed semen after incubation for 60 min were 961.6 ± 12.7, 991.8 ± 10.3 and 1234.9 ± 9.6 nm per 109 spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H2O2 and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H2O2 was significantly (p < 0.05) higher in frozen–thawed than fresh and equilibrated spermatozoa.  相似文献   

4.
Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z‐VAD‐FMK 100 μM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO2 and evaluated motility and kinetic parameters by computer‐assisted semen analysis (CASA) and viability (propidium iodide), apoptotic‐like membrane changes (YO‐PRO‐1), acrosomal status (PNA‐FITC), intracellular calcium (FLUO‐3), membrane fluidity (M540) and ROS production (CM‐H2DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO‐PRO‐1+ and acrosome‐reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca2+ levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.  相似文献   

5.
The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well‐known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD‐ and NADP‐dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.  相似文献   

6.
Acrosin is an important proteolytic enzyme that is capable of hydrolysing the zona pellucida in bovine oocyte. Lysophosphatydic acid (LPA) derivated from lysophosphatidylcholine (LPC) is known to trigger the acrosome exocytosis. The present study was aimed at examining the acrosin activity variations in LPC‐induced acrosome exocytosis and its regulation by tyrosine kinase, protein kinase C (PKC) and voltage‐dependent calcium channels (VDCC) in spermatozoa previously capacitated with heparin or quercetin. The enzyme activities were spectrophotometrically measured using N‐α‐benzoyl‐DL‐arginine p‐nitroanilide as an acrosin‐specific substrate. The capacitation and acrosomal reaction were evaluated by chlorotetracycline assay, and the viability and acrosome integrity were evaluated by the trypan blue stain/differential interference contrast. It was observed that LPC induced acrosome exocytosis and increased the activity of acrosin in spermatozoa previously capacitated with heparin. In heparin/LPC‐treated samples, it was observed that the inhibition of tyrosine kinase and PKC blocked the acrosome exocytosis and the acrosin activity (p < 0.05). Under these conditions, in heparin‐capacitated spermatozoa, the LPC provokes an acrosin activity increase that is independent of calcium influx through VDCC Type L. In cryopreserved bovine spermatozoa, LPC might require modulation, mainly tyrosine kinase participation with respect to PKC activity to induce acrosome exocytosis and increase acrosin activity.  相似文献   

7.
The function of prion‐like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post‐thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim‐up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post‐swim‐up spermatozoa incubated as above was tested through heterologous fertilization of bovine in vitro matured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p ≤ 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p ≤ 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 ± 3.0%) than control (39.1 ± 2.2%) and 40 ng/ml rDpl (39.8 ± 2.7%). Preliminary data suggest that Doppel protein may enhance in vitro spermatozoa fertilizing ability.  相似文献   

8.
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9.
Extending the shelf life of chilled rabbit spermatozoa is vital for the expansion of the farmed rabbit industry. This study evaluated the relationship between sperm concentration and packaging on in vitro quality of chilled rabbit semen over 96 h. Semen was collected from adult bucks (n = 4) and pooled at 37°C following evaluation. Pooled ejaculates were diluted with a Tris‐based extender supplemented with 100 μm quercetin to a concentration of 15, 30 or 60 × 106 spermatozoa/ml, packaged into plastic tubes or 0.5‐ml straws and stored at 15°C. Sperm quality was assessed by computer‐assisted sperm Analysis [total motility (tMOT)] and flow cytometry [viability, acrosome integrity, H2O2 production, plasma membrane disorder, apoptosis and DNA fragmentation index (DFI)] at 0, 48, 72 and 96 h. From 48 h, concentrations of 30 and 60 × 106 spermatozoa/ml reported the highest tMOT, irrespective of storage vessel (p < 0.05). Storage in straws reduced oxidative stress and improved plasma membrane stability. The %DFI, mean DFI and SD‐DFI were increased in spermatozoa stored in tubes compared with straws (p < 0.05). Although the use of low sperm concentrations in artificial insemination doses would facilitate greater dispersion of genetically superior rabbit bucks, dilution to 15 × 106 spermatozoa/ml had a detrimental impact on motility. As such, chilled storage at 30 × 106 spermatozoa/ml may provide a suitable balance between motility and H2O2 production to best maintain overall sperm function and should be evaluated in a large‐scale AI trial.  相似文献   

10.
This study was conducted to determine the effect of pre‐exposure of oocytes to Ricinus communis (RCA‐1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm–egg binding and fertilization. In vitro‐matured bovine oocytes were incubated (39°C, 5% CO2 in air) for 2 h in the following treatments: (i) 500 μl of fertilization medium (FM); (ii) 250 μl of FM with 0.25 ml of non‐luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 μl of FM with 250 μl of NLAUTF and 4 μl of RCA‐1 lectin; (iv) 250 μl of FM with 250 μl of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA‐1 lectin; (v) 500 μl of FM and RCA‐1 lectin. Following incubation, oocytes were washed, placed in FM with 2 μg heparin, and incubated with 1 × 105 frozen–thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate‐orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean ± SEM) when oocytes were incubated in treatment 3 (59.0 ± 5.5) than in treatments 2 (46.4 ± 5.6), 4 (18.1 ± 5.4), 5 (33.4 ± 5.6) or 1 (32.5 ± 5.6). More oocytes were fertilized when incubated in treatment 3 (91% ± 3.0) than in 2 (84% ± 3.0), 4 (40% ± 3.0), 5 (77% ± 3.0) or 1 (76% ± 3.0). As in previous studies, this study suggests that RCA‐1 lectin enhances binding of UTF‐derived OPN to bovine oocytes, resulting in increased sperm–egg binding and fertilization in vitro and a possible role in fertilization.  相似文献   

11.
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   

12.
In the spermatozoa of some species, the ubiquitin–proteasome system detects altered proteins and tags them for elimination by the proteasome. In some species' ejaculates, a high proportion of ubiquitinated spermatozoa (i.e. those having ubiquitin bound to the altered or damaged membrane proteins) has been related to infertility. The aim of this study was to assess whether the percentage of ubiquitinated spermatozoa relates to fertility of dairy bulls and whether ubiquitination increases during protein remodelling that occurs during in vitro spermatic capacitation. Thirty‐two frozen semen straws from four high‐fertility (ReproMax®) and four normal‐fertility (Normal) Holstein‐Friesian sires were evaluated. Ubiquitinated and capacitated spermatozoa were quantified by sperm ubiquitin tag immunoassay and chlortetracycline stain, respectively. Fertilizing capacity of sires was assessed by in vitro fertilization. No differences were found between Normal and ReproMax® sires with regard to the observed percentage of ubiquitinated spermatozoa (42.97 ± 3.69% and 49.68 ± 9.27%, respectively; p > 0.05). Additionally, no differences were found in the percentage of ubiquitinated spermatozoa as a consequence of spermatic capacitation in either Normal (42.97 ± 3.69% before capacitation vs 44.67 ± 7.5% after; p > 0.05) or ReproMax® sires (49.68 ± 9.27% before vs 45.05 ± 7.51% after; p > 0.05). The percentage of ubiquitinated spermatozoa in a thawed sperm samples did not correlate with its in vitro fertilizing capacity; thus, this assay does not prove useful to detect in vivo fertility differences between sires. Additionally, protein degradation occurring during remodelling of the spermatozoon plasma membrane during the capacitation process does not seem to involve the ubiquitin–proteasome system.  相似文献   

13.
Objective To characterize responses to different doses of propofol in horses pre‐medicated with xylazine. Animals Six adult horses (five females and one male). Methods Each horse was anaesthetized four times with either ketamine or propofol in random order at 1‐week intervals. Horses were pre‐medicated with xylazine (1.1 mg kg?1 IV over a minute), and 5 minutes later anaesthesia was induced with either ketamine (2.2 mg kg?1 IV) or propofol (1, 2 and 4 mg kg?1 IV; low, medium and high doses, respectively). Data were collected continuously (electrocardiogram) or after xylazine administration and at 5, 10 and 15 minutes after anaesthetic induction (arterial pressure, respiratory rate, pH, PaO2, PaCO2 and O2 saturation). Anaesthetic induction and recovery were qualitatively and quantitatively assessed. Results Differences in the quality of anaesthesia were observed; the low dose of propofol resulted in a poorer anaesthetic induction that was insufficient to allow intubation, whereas the high dose produced an excellent quality of induction, free of excitement. Recorded anaesthesia times were similar between propofol at 2 mg kg?1 and ketamine with prolonged and shorter recovery times after the high and low dose of propofol, respectively (p < 0.05; ketamine, 38 ± 7 minutes; propofol 1 mg kg?1, 29 ± 4 minutes; propofol 2 mg kg?1, 37 ± 5 minutes; propofol 4 mg kg?1, 50 ± 7 minutes). Times to regain sternal and standing position were longest with the highest dose of propofol (32 ± 5 and 39 ± 7 minutes, respectively). Both ketamine and propofol reversed bradycardia, sinoatrial, and atrioventricular blocks produced by xylazine. There were no significant alterations in blood pressure but respiratory rate, and PaO2 and O2 saturation were significantly decreased in all groups (p < 0.05). Conclusion The anaesthetic quality produced by the three propofol doses varied; the most desirable effects, which were comparable to those of ketamine, were produced by 2 mg kg?1 propofol.  相似文献   

14.
LaBranche, T. P., Ehrich, M. F., Eyre, P. Characterization of bovine neutrophil β2‐adrenergic receptor function. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2009.01143.x. This study compares bovine leukocyte β‐adrenergic receptor densities to that of the rat, demonstrates for the first time a functional β2‐adrenergic receptor signaling pathway in steer neutrophils, and investigates the effect of an inflammatory stimulus on that signaling pathway. The β1‐/β2‐adrenergic antagonist [3H]CGP‐12177 demonstrated that rat lymphocyte specific binding‐site density was highest, followed by steer and dairy cow lymphocytes, and lastly steer and dairy cow neutrophils. The β2‐adrenergic agonist terbutaline stimulated steer neutrophil adenosine 3,5‐cyclic monophosphate (cAMP) production, an effect increased by inclusion of ≥1 × 10?8 m phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C. Both terbutaline and the nonselective phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX) independently decreased steer neutrophil superoxide anion production in a concentration‐dependent manner, with 1 × 10?4 m IBMX enhancing both the potency and efficacy of the terbutaline effect (up to 74% reduction in superoxide anion production). Superoxide anion production was also reduced by the synthetic cAMP analog 8‐bromo‐cAMP, which increased the potency of the IBMX effect on superoxide anion production. Taken together, these data demonstrate the presence of a β2‐adrenergic receptor signaling pathway in bovine neutrophils much like that described in other animal species, as well as the potential for an inflammatory stimulus to alter its function.  相似文献   

15.
Propofol anaesthesia for surgery in late gestation pony mares   总被引：2，自引：0，他引：2  
Objective To characterize propofol anaesthesia in pregnant ponies. Animals Fourteen pony mares, at 256 ± 49 days gestation, undergoing abdominal surgery to implant fetal and maternal vascular catheters. Materials and methods Pre‐anaesthetic medication with intravenous (IV) acepromazine (20 µg kg?1), butorphanol (20 µg kg?1) and detomidine (10 µg kg?1) was given 30 minutes before induction of anaesthesia with detomidine (10 µg kg?1) and ketamine (2 mg kg?1) IV Maternal arterial blood pressure was recorded (facial artery) throughout anaesthesia. Arterial blood gas values and plasma concentrations of glucose, lactate, cortisol and propofol were measured at 20‐minute intervals. Anaesthesia was maintained with propofol infused initially at 200 µg kg?1 minute?1, and at 130–180 µg kg?1 minute?1 after 60 minutes, ventilation was controlled with oxygen and nitrous oxide to maintain PaCO2 between 5.0 and 6.0 kPa (37.6 and 45.1 mm Hg) and PaO2 between 13.3 and 20.0 kPa (100 and 150.4 mm Hg). During anaesthesia flunixin (1 mg kg?1), procaine penicillin (6 IU) and butorphanol 80 µg kg?1 were given. Lactated Ringer's solution was infused at 10 mL kg?1 hour?1. Simultaneous fetal and maternal blood samples were withdrawn at 85–95 minutes. Recovery from anaesthesia was assisted. Results Arterial blood gas values remained within intended limits. Plasma propofol levels stabilized after 20 minutes (range 3.5–9.1 µg kg?1); disposition estimates were clearance 6.13 ± 1.51 L minute?1 (mean ± SD) and volume of distribution 117.1 ± 38.9 L (mean ± SD). Plasma cortisol increased from 193 ± 43 nmol L?1 before anaesthesia to 421 ± 96 nmol L?1 60 minutes after anaesthesia. Surgical conditions were excellent. Fetal umbilical venous pH, PO2 and PCO2 were 7.35 ± 0.04, 6.5 ± 0.5 kPa (49 ± 4 mm Hg) and 6.9 ± 0.5 kPa (52 ± 4 mm Hg); fetal arterial pH, PO2 and PCO2 were 7.29 ± 0.06, 3.3 ± 0.8 kPa (25 ± 6 mm Hg) and 8.7 ± 0.9 kPa (65 ± 7 mm Hg), respectively. Recovery to standing occurred at 46 ± 17 minutes, and was generally smooth. Ponies regained normal behaviour patterns immediately. Conclusions and clinical relevance Propofol anaesthesia was smooth with satisfactory cardiovascular function in both mare and fetus; we believe this to be a suitable anaesthetic technique for pregnant ponies.  相似文献   

16.
A post‐breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex‐sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre‐ or post‐ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep? (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre‐ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 ± 189 × 106 leucocytes/uterine horn) or not (580 ± 153 × 106). Post‐ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 ± 198 × 106, AH+S: 162 ± 102 × 106). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 ± 6 × 106, SP+S: 73 ± 27 × 106) and after ovulation (SP: 60 ± 32 × 106, SP+S: 51 ± 33 × 106) did not differ significantly from controls using phosphate buffered saline (PBS) (pre‐ovulatory: 1 ± 1 × 106, post‐ovulatory: 11 ± 9 × 106). Quantitative in vitro transmigration assays with blood‐derived PMN proved that AH‐induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin‐8 (rhCXCL8) (AH: 14 ± 5% migration rate vs controls: 37 ± 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis‐inhibiting properties. SP at ≥0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.  相似文献   

17.
In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time‐dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)‐induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H‐89, PD9809 and GF‐109) and enhancer (dbcAMP, H2O2 and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F‐actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa.  相似文献   

18.
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   

19.
The objective of this study was to evaluate the quality of chilled dog semen processed with extenders containing various concentrations of N‐acetyl‐L‐cysteine (NAC). Ejaculates from five dogs were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF‐movement), viability, acrosomal integrity and by the hypo‐osmotic swelling test (HOST). In addition, superoxide anion (O2‐?) production, hydroxyl radicals (OH?) and total reactive oxygen species (tROS) were determined. The pool was divided into five aliquots, which were diluted to a final concentration of 66.66 × 106 spermatozoa/ml with Tris‐glucose‐egg yolk extender containing one of the following concentrations of NAC (0, 0.5, 1, 2.5 or 5 mm ). The semen aliquots were chilled and preserved at 4°C. Semen quality was evaluated after rewarming at 72 h. Sperm motility was significantly higher with the 0.5 mm concentration compared with the control group (p = 0.001). Rapid steady forward movement was higher with the 0.5 and 1 mm concentrations compared with the control and 5 mm group (p < 0.001). Viability and HOST percentages were not significantly altered. Compared with the control, the 5 mm concentration showed significantly reduced percentages of spermatozoa with normal acrosomes (p = 0.049). None of the ROS values at 72 h were significantly affected by the presence of NAC in semen extenders, although all NAC concentrations showed lower O2‐? and OH? values compared with the control. Only the concentrations of 1 and 5 mm inhibited the significant increase of tROS values after 72 h, compared with the fresh semen value. In conclusion, NAC supplementation of semen extenders is beneficial to semen motility of canine spermatozoa during chilling with the 0.5 mm concentration being the most effective, although no significant ROS inhibition was observed at 72 h.  相似文献   

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