Effect of Exogenous Progesterone on Post-thaw Capacitation and Acrosome Reaction of Bovine Spermatozoa

The aim of this work was to study the effect of progesterone (P4) on capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa in vitro. Spermatozoa were incubated (0-180 min) in capacitation medium supplemented with 0, 0.1, 1.0 and 10.0 microg/ml of P4. At different time intervals aliquots were taken to determine sperm plasma membrane lipid destabilization, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment aimed to study the effects of P4, as potential inducer of AR in heparin-capacitated spermatozoa. The acrosomal status and viability of spermatozoa were evaluated under an epifluorescence microscope using Ethidium homodimer/peanut agglutinin fluorescein isothiocyanate staining method. Plasma membrane scrambling in spermatozoa was assessed by a flow cytometer, using merocyanine staining. The results show that P4 at the concentrations used had no negative effects on sperm viability. Progesterone significantly enhanced sperm capacitation (p < 0.001), but had no effect on plasma membrane lipid stability (p > 0.05) and did not significantly increase the AR of heparin-capacitated spermatozoa (p > 0.05). Progesterone displayed its effects in a dose-dependent manner with a maximum effect of 10 microg/ml P4 at 180 min of incubation. The results demonstrate that in cryopreserved bovine semen, P4 acts as capacitating, but not as an AR-inducing agent.     

Relationship between Sperm Response to Glycosaminoglycans in vitro and Non-return Rates of Swedish Dairy AI Bulls

In this study, the relations between fertility (56‐day non‐return rates, 56‐day NRR) after artificial insemination (AI) and bull sperm characteristics post‐thaw, after swim‐up and after co‐incubation with heparin (Hep) and hyaluronan (HA), respectively, were determined, attempting to determine if such a procedure could be of value to evaluate the potential fertilizing ability of frozen‐thawed AI bull spermatozoa. Spermatozoa from 20 semen batches derived from 20 Swedish Red and White AI bulls ranging widely in their field fertility after AI (55–79% 56‐day NRRs) were evaluated with regards to post‐thaw motility, membrane integrity, and migration through a simple swim‐up procedure. Sperm viability and capacitation status were evaluated by two different vital staining procedures and chlortetracycline hydrochloride staining. Sperm motility and membrane integrity post‐thaw (e.g. indicators of sperm viability) were significantly correlated (r = 0.53, p < 0.05 and r = 0.59, p < 0.01, respectively) with fertility. Heparin (5 µg/ml) significantly (p lt; 0.001) increased the frequencies of capacitation and acrosome‐reaction (AR) among swim‐up separated spermatozoa, whereas HA at a concentration of 50 ng/ml did not have any significant capacitating effect. The incidences of capacitated or AR‐spermatozoa following Hep‐treatment were not correlated with fertility. On the other hand, the percentage of viable spermatozoa was significantly (p < 0.001) lower in Hep‐treated samples than in control and HA‐treated samples and was significantly (r = 0.49, p < 0.05) correlated with fertility after AI (56‐day NRR). The results indicate that the percentage of viable spermatozoa after swim‐up separation and heparin‐exposure from a selected population of AI bulls were significantly and positively related to the AI fertility of the donors and thus could be used as a parameter to determine the fertilizing ability of frozen—thawed AI bull spermatozoa.     

Effect of incorporation of additives in tris-based egg yolk extender on buffalo (Bubalus bubalis) sperm tyrosine phosphorylation during cryopreservation

Phosphorylation of tyrosine residues on sperm protein is a known indicator of capacitation and a major intracellular signalling event. There is evidence that sperm cryopreservation promotes tyrosine phosphorylation and is associated with reduced fertility of spermatozoa. Under this study, cryoprotective role of different additives namely taurine, trehalose, catalase and 4-bromophenacyl bromide on buffalo sperm quality was evaluated. Buffalo semen was cryopreserved in tris-based egg yolk extender supplemented with additives like taurine (50 mm) or trehalose (100 mm) or 4-bromophenacyl bromide (200 μm) or catalase (100 U/ml) and used for assessment of levels of tyrosine phosphorylation in frozen-thawed spermatozoa. The results obtained were compared with the level of protein tyrosine phosphorylation of semen cryopreserved in tris-based egg yolk extender without additives. Proteins were extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random and analysed for tyrosine phospho-proteins using SDS-PAGE followed by immunoblotting. Monoclonal anti-phosphotyrosine antibody (Clone pT-154) was used as primary antibody followed by treatment with HRP-conjugated secondary antibody. Signals were detected on X-ray film using chemiluminescence. Nine proteins (p20, p30, p32, p38, p49, p56, p59, p72 and p86) were found to be tyrosine phosphorylated in cryopreserved spermatozoa. Supplementation of additives significantly (p<0.05) reduced the level of protein tyrosine phosphorylation in spermatozoa. Moreover, this study showed improved (p<0.05) post-thaw motility, viability and membrane integrity of spermatozoa on addition of these additives. The results obtained clearly indicate reduced level of capacitation like changes on supplementation of additives in terms of protein tyrosine phosphorylation.     

In vitro capacitation of stallion spermatozoa assessed by the lysophosphatidylcholine-induced acrosome reaction and the penetration rate into in vitro-matured, zona-free mare oocytes

The purpose of this study was to evaluate the ability of various chemicals to induce capacitation of stallion spermatozoa using 2 different assay systems. In Experiment 1, freshly ejaculated spermatozoa were treated for 0, 3 and 6 h with 10 μ g/ml heparin, 0.5 mM hypotaurine or 5 mM caffeine, or were incubated for 0, 3 and 6 h following 1 min exposure to 0.1 μ M ionophore A23187. The acrosome reaction (AR) in the capacitated spermatozoa was induced by 15 min challenge with 100 μ g/ml lysophosphatidylcholine (LPC). In the BO/BSA-control medium (Brackett and Oliphant medium with 0.3% BSA), mean percentage of AR spermatozoa at 0 h was 30%, and the AR rates increased to 40 and 48% after 3 and 6 h incubation, respectively. There was no significant further increase of the AR rates in the spermatozoa treated with heparin (50% at 6 h) and hypotaurine (58% at 6 h) when compared to the control. Caffeine had a beneficial effect on inducing sperm capacitation after 3 and 6 h incubation (AR rates; 61 and 66%, respectively, P<0.01). Immediately after ionophore A23187 treatment, the AR rate increased to 56%, and reached 68 and 67% after 3 and 6 h incubation, respectively (P<0.01). Spermatozoal motility at any time points did not differ between control and any chemical treatment groups, except one treatment (ionophore; 3 h group).In Experiment 2, frozen-thawed spermatozoa were treated with 4 different chemicals as described above. Aliquot of spermatozoa was added to a microdrop of BO/BSA medium in which 6 to 10 in vitro-matured, zona-free mare oocytes were placed, and the oocytes were fixed and stained 20 h after insemination. The penetration rate by BO/BSA-treated spermatozoa was 76%, which was comparable to the results with heparin (73%), hypotaurine (78%) and caffeine (58%). In contrast, treatment of spermatozoa with ionophore A23187 gave a significantly lower penetration rate (30%) than the control value. Surprisingly these two experiments had different conclusions in assessing capacitation of stallion spermatozoa.     

Assessment of intracellular Ca2+, cAMP and 1,2-diacylglycerol in cryopreserved buffalo (Bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender

In mammalian spermatozoa, intracellular calcium plays a major role in sperm functions like motility and capacitation. Cryopreservation-induced modifications to sperm membrane result in an influx of intracellular calcium affecting calcium-dependent intracellular signalling pathways. Intracellular calcium activates adenyl cyclase to produce cAMP that activates phospholipase A(2) (PLA(2) ) and phospholipase C (PLC) generating lysophosphatidyl choline, 1,2-diacylglycerol (DAG) and IP(3) , acting as intracellular secondary messengers required for sperm capacitation. Present study was designed to determine levels of intracellular calcium, cAMP and DAG in fresh and frozen-thawed buffalo spermatozoa cryopreserved in the presence and absence of taurine or trehalose. A total number of nine ejaculates from three randomly chosen buffalo bulls were cryopreserved in Tris-based egg yolk extender and thawed in warm water at 37°C. The cAMP was measured by enzyme immuno assay, and intracellular calcium was quantified using fluorescent dye FURA 2-AM. Total lipid was extracted from spermatozoa, and DAG was estimated using thin layer chromatography followed by spectrophotometric analysis. Intracellular calcium, cAMP and DAG levels in spermatozoa were significantly (p < 0.01) increased following cryopreservation as compared to fresh ejaculate. Addition of taurine or trehalose to the freezing medium significantly decreased (p < 0.01) the levels of intracellular calcium and cAMP in frozen-thawed spermatozoa. 1,2-diacylglycerol content was also decreased significantly (p < 0.01) in spermatozoa cryopreserved in presence of additives. Moreover, significant (p < 0.01) improvement in post-thaw motility, viability and membrane integrity of spermatozoa on addition of taurine or trehalose clearly indicated the reduced level of capacitation-like changes in buffalo spermatozoa.     

Hyaluronic Acid as Capacitation Inductor: Metabolic Changes and Membrane‐Associated Adenylate Cyclase Regulation

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.     

CLC对冻融牛精子体外获能及抗氧化、抗凋亡能力的影响

本研究目的是评价CLC对牛冻融精子获能、抗氧化及抗凋亡能力的影响。试验分对照组和CLC处理组(0.5、1.0、1.5及2.0 mg/mL),检测冻融牛精子体外获能后的酪氨酸磷酸化水平以及冻融牛精子抗氧化基因和细胞凋亡基因的表达。结果:1)在0.5 mg/mL CLC处理组冷冻效果最好,与对照组和1.0 mg/mL CLC处理组相比无显著差异;2)冻融牛精子获能处理后,在1.0 mg/mL CLC处理组精子蛋白酪氨酸磷酸化水平最高,与对照组相比无显著差异;3)在0.5 mg/mL CLC处理组冻融牛精子抗氧化基因CAT、GPX和SOD的表达量与对照组相比显著升高(P<0.05);4)在0.5和1.0 mg/mL CLC处理组凋亡基因Caspase-3、Caspase-8和BAX的表达量与对照组相比显著降低(P<0.05)。结论:添加CLC可以改进冻融牛精子获能和抗氧化、抗凋亡能力。     

Effect of Progesterone and Oestradiol on Sperm–zona Binding and Acrosome Reaction in Bovine Spermatozoa after Thawing

This study was conducted assess spermatozoa binding capacity to the oocyte in the presence of 0.1 or 0.5 microg/ml progesterone (P4) or a combination of 0.5 microg/ml P4 with 0.1 microg/ml oestradiol (OE). The number of oocyte-bound spermatozoa in the presence of progesterone was significantly higher (p < 0.05 to p < 0.001) when compared with the control samples, that were incubated in the absence of P4. Spermatozoa binding to the zona pellucida (ZP) of the oocyte were concentration-dependent - significantly higher numbers of spermatozoa were bound in the presence of 0.5 microg/ml P4, when compared with that of 0.1 microg/ml P4. Oestradiol at 0.1 microg/ml concentration used impaired the effect of progesterone-mediated sperm-oocyte binding. The incidences of acrosome-reacted (AR) spermatozoa bound to the ZP - following 0, 60, 120 and 180 min. incubation in the presence and absence of 1 microg/ml progesterone was also assessed. Only at 180 min of incubation a higher (p < 0.001) incidence of the AR-spermatozoa was found in sperm-ZP complexes incubated in the presence or absence of progesterone, being 56.5 +/- 11.1 and 43.2 +/- 8.8 % respectively. In conclusion, progesterone enhances the sperm-ZP binding capacity. Progesterone affects the incidences of AR on zona-bound spermatozoa only after prolonged co-culture.     

Influence of different cellular concentrations of boar sperm suspensions on the induction of capacitation and acrosome reaction

We aimed to analyze the influence of different cellular concentrations of boar sperm suspensions on the induction of capacitation and acrosome reaction. When spermatozoa were incubated at 100 or 200 mill/ml, significant increases in protein tyrosine phosphorylation in the p32 protein were observed, compared to those at 50 mill/ml. In addition, sperm concentration-dependent increases were observed in plasma membrane lipid disorganization (50 mill/ml vs. 200 mill/ml), induction of the acrosome reaction (50 mill/ml vs. 100 mill/ml and 200 mill/ml), and sperm viability (50 mill/ml vs. 100 mill/ml and 200 mill/ml). Our data indicate that an increase in sperm concentration stimulates the induction of capacitation and acrosome reaction in boars.     

The prion-like protein Doppel enhances ovine spermatozoa fertilizing ability

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.     

Acrosomal and viability status of bovine spermatozoa evaluated by two staining methods

Artificial insemination with frozen-thawed spermatozoa is commonly used in cattle breeding. A simple and fast procedure is needed for routine evaluation of the acrosomal status of frozen-thawed bovine sperm. Therefore, the purpose of this study was to test two staining procedures used to determine the viability and integrity of acrosome of frozen-thawed bovine spermatozoa. Double staining and Hoechst/FITC-Pisum sativum agglutinin (FITC-PSA) labelling were tested for evaluating the viability and acrosome reaction induced by calcium ionophore of bull spermatozoa. In our experiments no significant differences were detected in the frequency of acrosome-reacted sperm either by double staining (37.98%) or by FITC-PSA labelling (39.33%). The viability of sperm stained by the double staining method was 67.17%, and a higher portion of viable sperm (82.67%) was observed by staining with the Hoechst procedure (P < 0.01). On the basis of the results obtained it is concluded that both methods can be used for detecting the acrosome reaction of frozen-thawed bovine spermatozoa.     

Effect of Progesterone and Ionomycin on Domestic Cat Sperm Motility Patterns and Acrosome Reaction

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P₄). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25°C, were incubated for 30 min at 38°C in 5% CO₂ and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P₄ was added (10 μg/ml) to the P1 group. Groups P2 and P3 were supplemented with P₄ (10 and 20 μg/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 μ m , respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P₄ treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P₄ groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 μ m seems to be more suitable to trigger AR in domestic cat sperm.     

Hyperactivation and acrosome reaction in vitro in spermatozoa ejaculated by cryptorchid dogs after orchiopexy.

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.     

Methyl‐β‐Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida‐Binding Ability of Frozen/Thawed Bovine Spermatozoa

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein‐free media. However, the additive capacitating effect of methyl‐β‐cyclodextrin (MβCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen–thawed bovine spermatozoa in a BSA‐containing medium supplemented with MβCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MβCD in a dose‐dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MβCD. However, pre‐incubation of spermatozoa in MβCD‐supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)‐binding ability (p < 0.05). Thus, we conclude that MβCD supplementation is able to enhance the capacitation status of frozen–thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.     

Differential proteins associated with plasma membrane in X- and/or Y-chromosome bearing spermatozoa in indicus cattle

The differential proteins associated with plasma membrane of spermatozoa are less known, identification of which shall help overcome limitations of currently used methods of sperm sexing, considered as a high priority for livestock sector of many countries. This study has reported plasma membrane proteomics of unsorted spermatozoa and differential expression of plasma membrane-associated proteins between X- and Y-chromosome bearing spermatozoa of indicus cattle (Bos indicus). Isolation of plasma membrane fraction using percoll gradient, relatively a rapid method, from bovine spermatozoa has been reported to enrich isolation of plasma membrane proteins. Significant enrichment for plasma membrane-associated proteins was observed in plasma membrane fraction (p < .05) as compared to the total cell lysate using LC-MS/MS. Furthermore, these experiments were conducted in flow cytometry sorted, sexed-semen samples. Thirteen proteins were identified as differentially abundant between X- and Y-sorted spermatozoa. Among these, two proteins were downregulated in Y-sorted spermatozoa compared to the X-sorted spermatozoa (p < .05), while four and seven proteins could be noted in X- and Y-sorted spermatozoa, respectively. Proteins that are presumed to support sperm capacitation and sperm migration velocity were found to be abundant in Y-sorted spermatozoa while those associated with structural molecule activity were identified as abundant in X-sorted spermatozoa in the present study. Our study provides better insight into the plasma membrane proteomics of spermatozoa of indicus cattle and furnishes data that might aid in design and development of alternate and open technology for sex-sorting of semen.     

Assessment of Sperm Apoptosis in Cryopreserved Bull Semen After Swim-up Treatment: A Flow Cytometric Study

The techniques used to prepare bovine spermatozoa for in vitro fertilization, to enhance the percentage of motile sperm cells include the swim-up (SU) method, among others. The objective of the present study was to evaluate the phosphatidylserine (PS) translocation and plasma membrane integrity as the indicator of apoptosis and necrosis in post-thaw bull sperm after SU treatment using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay. A flow cytometric method was employed to measure apoptosis levels on frozen-thawed bull spermatozoa. The assay detects PS translocation across the plasma membrane using a fluorescein-labelled annexin-V and PI. By using the annexin V/PI assay four different subpopulations of sperm were observed: (i) a population of apoptotic sperm, labelled with annexin V-FITC but not with PI; (ii) a population of early necrotic spermatozoa, sperm labelled with annexin-FITC and PI; (iii) a population of necrotic sperm, labelled with PI but not with annexin-FITC; and (iv) a population of fully viable sperm cells, sperm not labelled with annexin V-FITC and without PI. Results clearly indicated that SU technique itself could have an adverse effect on the spermatozoa membrane stability. It has also been found, significant differences between bulls in the levels of apoptotic sperm, after SU treatment.     

In vitro Capacitation and Acrosome Reaction of Dog Spermatozoa can be Feasibly Attained in a Defined Medium Without Glucose

Incubation of dog spermatozoa in a medium without glucose and in the presence of lactate and pyruvate (l-CCM) for 4 h at 38.5 degrees C in a 5% CO(2) atmosphere induced in vitro capacitation of these cells. This was verified after the combined specific capacitation-like changes in percentages of viability and altered acrosomes, motility characteristics, sperm location of reactivity against Pisum sativum, Arachis hypogaea and Helix pomatia lectins and the tyrosine phosphorylation pattern. Furthermore, a feasible acrosome reaction (AR) was induced when spermatozoa incubated in l-CCM for 4 h were further co-incubated for 1 h with canine oocytes. This was demonstrated by AR-like changes in percentages of viability, altered acrosomes, motility characteristics and sperm location of reactivity against P. sativum, A. hypogaea and H. pomatia lectins. All these results clearly indicate that in vitro capacitation, and subsequent AR, can be feasibly achieved without the presence of sugars. This ability can be related to the specific characteristics of energy-metabolism regulation reported in dog spermatozoa.     

Western Blot Analysis of Proacrosin/Acrosin in Frozen Dog Sperm During In Vitro Capacitation

Acrosin is an acrosomal protease synthesized as an inactive precursor, proacrosin, which is processed via autoproteolysis into active forms alpha- and beta-acrosin. In this paper, a comparative study on the immunoreactivity of acrosin during in vitro capacitation of frozen and fresh (control) canine sperm using Western blot analysis is reported. Semen samples were obtained by digital stimulation and ejaculates processed as fresh and frozen samples and then capacitated for 0, 30, 60 and 90 min. At each time period, samples were analyzed with monoclonal antibody C5F10 by Western blot. The antibody specifically recognized, in fresh and frozen/thawed spermatozoa, a 40-, 32- and 27-kDa bands corresponding to proacrosin, alpha- and beta-acrosin, respectively, during capacitation. Western immunoblots showed that the beta-acrosin reactivity in fresh sperm was directly proportional to the time of capacitation, whereas a decreased reactivity of active form of acrosin was observed with frozen-thawed sperm (p   < 0.05). These results suggest that proacrosin is activated to beta-acrosin earlier in frozen/thawed dog spermatozoa than in fresh dog spermatozoa.     

牛精子体外获能前后超微结构的研究

应用透射电镜技术,对牛精子体外获能前后超微结构观察的结果表明,牛精子的超微结构相似于其他哺乳动物,但顶体较大;获能后精子发生了顶体反应.在顶体反应中,囊泡化有3种形式:(1)顶体外膜相互融合,形成囊泡;(2)顶体外膜内陷形成囊泡化;(3)顶体外膜和质膜融合形成囊泡.此外,对精子的结构特点和囊泡形成过程进行了讨论.     

Effect of Ram Age on Structural and Functional Competence of Frozen–Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen–thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1–2 years (young) or 4–5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of ≥ 2.5 × 10⁹ sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell^® medium and frozen in 0.25 ml straws. The end points of post‐thawing semen evaluation were computer‐assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per‐cell analysis of lipid peroxidation using C11‐BODIPY^581/591, sperm‐hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen‐synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non‐capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ?0.63 to ?0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.