Characterization of viability, mitochondrial activity, acrosomal integrity and capacitation status in boar sperm during in vitro storage at different ambient temperatures

2002 ◽  
Vol 14 (8) ◽  
pp. 509 ◽  
Author(s):  
Li-Jun Huo ◽  
Kui-Zhong Yue ◽  
Zeng-Ming Yang
Keyword(s):  
Low Fertility ◽  
Blue Staining ◽  
Mitochondrial Activity ◽  
Hoechst 33258 ◽  
In Vitro Storage ◽  
Coomassie Blue Staining ◽  
Boar Sperm ◽  
Boar Semen ◽  
Acrosome Integrity

Extended storage of unfrozen boar semen becomes an alternative because the use of frozen–thawed boar sperm results in low fertility. Sperm viability, mitochondrial activity, capacitation and acrosome integrity of freshly ejaculated boar semen stored in vitro for up to 48 h at 4°C, 15°C, 20°C and 39°C was characterized during the study. The viability of boar sperm was assessed by both Hoechst 33258 and SYBR-14/PI staining. Mitochondrial function was assessed by JC-1 staining. Capacitation status was determined by chlortetracycline (CTC)/Hoechst 33258 staining. The acrosome integrity was analysed with Coomassie blue staining. These data were derived from three ejaculates each from three crossbred boars. The viabilities assessed with SYBR-14/PI, Hoechst 33258 and JC-1 staining correlated highly (r > 0.980). In freshly ejaculated boar semen, 96 ± 1% of the sperm did not take up the Hoechst 33258, whereas 95 ± 2% were stained by SYBR-14 and 96 ± 2% of the sperm had mitochondria exhibiting positive JC-1 staining. Staining with CTC/Hoechst 33258 suggested that a high percentage of sperm became capacitated after 24 h storage at 15°C and 20°C. There were 62 ± 2% (15°C) and 89�±�2% (20°C) capacitated sperm by 48 h. Moreover, most of the capacitated sperm were acrosome intact. These results suggest that SYBR-14/PI, Hoechst 33258 or JC-1 staining can be used to effectively evaluate the quality of boar sperm during in vitro storage.

scholarly journals 79 EFFECT OF PRE-FREEZE ADDITION OF PLATELET-ACTIVATING FACTOR AND PLATELET-ACTIVATING FACTOR:ACETYLHYDROLASE ON THE POST-THAW INTEGRITY OF FROZEN - THAWED BOAR SPERM

2005 ◽  
Vol 17 (2) ◽  
pp. 189
Author(s):  
R. Bathgate ◽  
B.M. Eriksson ◽  
W.M.C. Maxwell ◽  
G. Evans
Keyword(s):  
Platelet Activating Factor ◽  
Egg Yolk ◽  
Large White ◽  
Boar Sperm ◽  
Thawing Process ◽  
Potential Benefits ◽  
Boar Semen ◽  
Acrosome Integrity ◽  
And Control

The use of frozen-thawed boar sperm is not widespread, owing to reduced fertility rates and high cost per dose (Eriksson et al. 2004 Proc. Aust. Assoc. Pig Vet., 61–69). Improvements in post-thaw sperm survival are required for commercialization. Platelet-activating factor (PAF) is a phospholipid involved in regulating sperm function. PAF:acetylhydrolase (PAF:AH) regulates PAF activity by conversion to its inactive isoform. Both occur naturally in boar semen (Kordan et al. 2003 Pol. J. Vet. Sci. 6, 55–60). Removal of PAF and PAF:AH along with seminal plasma during the cryopreservation process may inhibit the ability of sperm to withstand the freeze-thawing process. The aim of this study was to assess the effect of PAF and PAF:AH, added to boar semen pre-freeze, on the post-thaw motility and acrosome integrity of sperm. The sperm rich fraction was collected from a mature Large White × Landrace boar, diluted with Androhep (1:2, semen:Androhep; Minitube, Verona, WI, USA), cooled to 17°C over 2 h, and then centrifuged (10 min, 800g). The sperm pellet was resuspended in cooling extender (11% (w/v) lactose solution with 20% (v/v) egg yolk; control), cooling extender plus 100 ng/mL PAF (PAF), or cooling extender plus 0.4% (v/v) PAF:AH (Pafase; ICOS Corporation, Seattle, Washington, USA), and cooled to 5°C over 2.5 h. Sperm were further diluted with cooling extender plus 9% (v/v) glycerol and 1.5% (v/v) Equex STM (freezing extender), loaded into 0.5-mL straws, and frozen. Straws were thawed (20 s, 42°C) and the motility and acrosome integrity (FITC-PNA; Mortimer etal. 1990 Hum. Reprod. 5, 99–103) assessed at 0, 3, and 6 h post-thaw after incubation at 37°C. Data from three replicates were analyzed by ANOVA and a Tukey test applied where significant differences were found. Post-thaw motility (0 and 3 h) was higher for PAF (60.0 ± 0.0% and 25.0 ± 2.9%) than for control (41.7 ± 1.7% and 10.0 ± 2.9%; P < 0.05), but was similar for Pafase (41.7 ± 1.7% and 16.7 ± 1.7%; P > 0.05). By 6 h post-thaw, motility was similar for PAF (1.7 ± 1.7%), Pafase (6.7 ± 6.8%), and control (1.7 ± 1.7%, all respectively; P > 0.05). Acrosome integrity was higher at 0, 3 and 6 h post-thaw for Pafase (55.7 ± 3.2%, 45.7 ± 3.7% and 23.0 ± 3.1%) than for control (42.7 ± 1.5%, 25.7 ± 5.7% and 12.3 ± 2.7%) and PAF (33.0 ± 3.7%, 26.3 ± 2.2% and 11.7 ± 0.3%, all respectively; P < 0.05), but was similar between control and PAF (P > 0.05). Supplementation of cooling extender with 100 ng/mL PAF increased initial post-thaw motility, but this benefit was lost after 6 h post-thaw. Pafase in the cooling extender improved the proportion of intact acrosomes, even after 6 h post-thaw. In vitro studies investigating the interaction between Pafase-treated frozen-thawed sperm and oviducal epithelial cells would be of interest to further establish the potential benefits of pre-freeze addition of Pafase on the fertilizing potential of frozen-thawed boar sperm.

scholarly journals A comparative study of two methods to determine acrosome integrity of frozen-thawed boar sperm

2021 ◽  
Vol 52 (6) ◽  
Author(s):  
Janyaporn Rungruangsak ◽  
Junpen Suwimonteerabutr ◽  
Kakanang Buranaamnuay ◽  
Arun Chankrachang ◽  
Padet Tummaruk
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Blue Staining ◽  
Computer Assisted ◽  
Sperm Viability ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Boar Sperm ◽  
Acrosome Integrity ◽  
Sperm Acrosome

Coomassie blue staining has been reported as an effective and inexpensive method for evaluating the acrosome integrity of spermatozoa, though to date its use to evaluate cryopreserved boar sperm has not been reported. Moreover, there is no information concerning the agreement between Coomassie blue staining and fluorescein isothiocyanate conjugated peanut agglutinin and ethidium homodimer (FITC-PNA/EthD-1) methods for assessing sperm acrosome integrity for any species. The current study was performed to determine the efficacy and agreement between Coomassie blue and FITC-PNA/EthD-1 staining methods for evaluating the acrosome integrity of frozen-thawed boar sperm. A total of 25 semen samples were cryopreserved using lactose-egg yolk-based extender and loaded into 0.5 PVC-French straws. Sperm motility and motion characteristics were determined using a computer-assisted sperm analysis system. Sperm viability and plasma membrane integrity were evaluated using the SYBR-14/EthD-1 and hypo-osmotic swelling test, respectively. Acrosome integrity of frozen-thawed boar sperm was evaluated using both FITC-PNA/EthD-1 and Coomassie blue staining to assess the association between sperm acrosome integrity and agreement between these two methods. The average percent acrosome integrity of frozen-thawed boar sperm as determined by FITC-PNA/ EthD-1 and Coomassie blue staining was 48.8 ± 12.6% and 52.6 ± 13.6%, respectively (P&gt;0.05). Interestingly, Coomassie blue staining found a correlation between sperm viability and acrosome integrity (r=0.609, P=0.002), while FITC-PNA/EthD-1 staining did not (P&gt;0.05). However, the acrosome integrity of frozen-thawed boar sperm evaluated by FITC-PNA/ EthD-1 and Coomassie blue staining was significantly correlated (r=0.448, P=0.025, n=25). The Bland-Altman plot determined that this agreement was acceptable. In conclusion, the acrosome integrity of the frozen-thawed boar sperm assessed via Coomassie blue staining was significantly correlated with that obtained via the FITC-PNA/EthD-1 staining method, and the two methods showed good agreement. Moreover, the significant association between the acrosome integrity of frozen-thawed boar sperm determined by Coomassie blue staining with other sperm quality parameters indicates that this is an effective method for assessing the acrosome integrity of frozen-thawed sperm in pigs.

57 TREHALOSE SUPPLEMENTED EXTENDER PRESERVE ACROSOMAL INTEGRITY IN POST-THAW BOAR SPERM

2013 ◽  
Vol 25 (1) ◽  
pp. 176
Author(s):  
R. Athurupana ◽  
H. Funahashi
Keyword(s):  
Egg Yolk ◽  
Low Fertility ◽  
Boar Sperm ◽  
Lower Fertility ◽  
Freezing Procedure ◽  
Boar Semen ◽  
Acrosome Integrity ◽  
Post Hoc ◽  
Fresh Semen ◽  
Acrosomal Integrity

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The use of glycerol for boar semen cryopreservation may be a reason of low fertility results. Trehalose is a nonreducing disaccharide known to stabilize proteins and biologic membranes during processes such as cryopreservation. The aim of this study was to evaluate the effects of trehalose on boar sperm cryosurvival in an egg-yolk-based extender. The semen samples collected from different individual Berkshires were diluted in egg-yolk-based freezing extender containing glycerol (final concentration 68.5 and 274 mM) or trehalose (50 and 100 mM). Then the samples were cryopreserved using the straw freezing procedure. Frozen sperms were thawed at 39°C in water. Post-thawed sperm were analyzed for motility (under microscope by a conventional method), viability, and acrosome integrity (under fluorescence microscope following LIVE/DEAD or CTC staining, respectively). Statistical analyses of results from 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post hoc test (significance; P < 0.05). The extender supplemented with 100 mM trehalose exhibited significantly higher acrosomal integrity (41.6%) compared with other extenders (P < 0.05, n = 5; Table 1). The trend of motility and viability was higher in 274 mM glycerol (28.0 and 52.9%) and 100 mM trehalose extenders (23.6 and 53.9%), but those were not significant. These results demonstrate that the presence of trehalose at 100 mM during cryopreservation improves the acrosome integrity of boar sperm, without any reduction in viability and motility, after thawing. Table 1.Effect of glycerol and trehalose on post-thaw boar sperm

scholarly journals Effects of Taurine on Sperm Characteristics during In vitro Storage of Boar Semen

2006 ◽  
Vol 19 (11) ◽  
pp. 1561-1565 ◽  
Author(s):  
H. Y. Jang ◽  
H. S. Kong ◽  
C. K. Park ◽  
J. D. Oh ◽  
S. G. Lee ◽  
...  
Keyword(s):  
In Vitro Storage ◽  
Sperm Characteristics ◽  
Boar Semen

Influence of progesterone on boar sperm capacitation

1995 ◽  
Vol 144 (1) ◽  
pp. 13-18 ◽  
Author(s):  
B Barboni ◽  
M Mattioli ◽  
E Seren
Keyword(s):  
Pisum Sativum ◽  
Direct Effect ◽  
Acrosome Reaction ◽  
Sperm Capacitation ◽  
Fertilizing Ability ◽  
Boar Sperm ◽  
The Status ◽  
Boar Semen ◽  
Fertilization System

Abstract This research investigates the effect of progesterone (P4) on boar sperm capacitation. Ejaculated spermatozoa were washed and incubated under capacitating conditions with or without P4. At different times of incubation samples of sperm were exposed to solubilized zonae pellucidae (ZP) and the degree of capacitation was evaluated by the incidence of zona-induced acrosome reaction (AR). The status of the acrosome was studied by using an FITCconjugated lectin (Pisum sativum agglutinin; FITC-PSA). The effect of P4 on the fertilizing ability of semen was then evaluated in an in vitro fertilization system by exposing in vitro matured oocytes to sperm preincubated for 2 or 4 h with or without P4, under capacitating conditions. PSA staining showed that P4 does not affect the incidence of spontaneous AR. By contrast, spermatozoa incubated with P4 showed a higher percentage of AR than controls after the exposure to solubilized ZP. This enhanced reactivity to ZP suggests a direct effect of P4 on sperm capacitation. The in vitro fertilization assay was consistent with these results demonstrating a higher fertilizing ability in sperm preincubated with P4 than in controls while the steroid was without effect when added only during the fertilization step. These results demonstrate that P4 improves the fertilizing ability of boar semen essentially by facilitating the process of capacitation. Journal of Endocrinology (1995) 144, 13–18

Analysis of proteins synthesizedin vitroby the erythrocytic stages ofPlasmodium knowlesi

Parasitology ◽  
1980 ◽  
Vol 81 (1) ◽  
pp. 177-198 ◽  
Author(s):  
A. A. McColm ◽  
P. G. Shakespeare ◽  
P. I. Trigg
Keyword(s):  
Developmental Stages ◽  
Plasmodium Knowlesi ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electrophoretic Analysis ◽  
The Other ◽  
Blue Staining ◽  
Coomassie Blue Staining

SUMMARYStudies were performed to identify specific parasite proteins synthesized withinPlasmodium knowlesi-infected rhesus erythrocytes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of whole parasites freed from the host erythrocyte by immune lysis, of membranous and cytoplasmic parasite fractions, and of isolated merozoites, detected several parasite-specific components after Coomassie Blue staining of the separated proteins. However, significant contamination with host erythrocyte material generally occurred, particularly in the whole parasite and parasite membrane preparations. Improved identification of plasmodial proteins was subsequently afforded by a radioisotope labelling technique in which parasitized erythrocytes were cultivatedin vitrowith [3H] isoleucine prior to electrophoretic analysis. Of 11 principal labelled peaks ranging in molecular weight from approximately 17000 to 145000 which were detected upon electrophoresis of whole parasites harvested from culture, all were observed in the cytoplasmic fraction while at least 5 were also associated with the membranous cell fraction. Analysis of different developmental stages of the intra-erythrocytic parasite revealed no significant stage-specific qualitative variations in the electrophoretic profiles. Quantitatively, however, the middle to late trophozoites incorporated more [3H] isoleucine into protein than the other intra-erythrocytic stages. Analysis of merozoites purified from labelled schizonts showed a protein pattern similar to the other stages. This confirmed that host components did not contribute to the labelling pattern and that none of the labelled proteins were specific to the residual cytoplasm remaining after merozoite formation.

Effect of staining and sorting on boar sperm membrane integrity, mitochondrial activity and in vitro blastocyst development

2005 ◽  
Vol 64 (1) ◽  
pp. 191-201 ◽  
Author(s):  
M. Spinaci ◽  
M. De Ambrogi ◽  
S. Volpe ◽  
G. Galeati ◽  
C. Tamanini ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Mitochondrial Activity ◽  
Blastocyst Development ◽  
Boar Sperm ◽  
Sperm Membrane

scholarly journals Molecular Mechanisms Involved in the Impairment of Boar Sperm Motility by Peroxynitrite-Induced Nitrosative Stress

2020 ◽  
Vol 21 (4) ◽  
pp. 1208 ◽  
Author(s):  
Rebeca Serrano ◽  
Nicolás Garrido ◽  
Jose A. Céspedes ◽  
Lauro González-Fernández ◽  
Luis J. García-Marín ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Molecular Mechanisms ◽  
Nitrosative Stress ◽  
Mitochondrial Activity ◽  
Dependent Protein Kinase ◽  
Plasma Membrane Fluidity ◽  
Boar Sperm ◽  
Dependent Protein ◽  
Boar Spermatozoa

Excessive levels of reactive nitrogen species (RNS) produce nitrosative stress. Among RNS is peroxynitrite, a highly reactive free radical generated when nitric oxide reacts with superoxide anion. Peroxynitrite effects have been mainly studied in somatic cells, and in spermatozoa the majority of studies are focused in humans. The aim of this study is to investigate the in vitro peroxynitrite effect on boar spermatozoa functions and the molecular mechanisms involved. Spermatozoa were exposed to the donor 3-morpholinosydnonimine (SIN-1) in non-capacitating or capacitating medium, motility was evaluated by CASA, functional parameters by flow cytometry and sperm protein phosphorylation by Western blotting. SIN-1 treatment, that significantly increases peroxynitrite levels in boar spermatozoa, potentiates the capacitating-stimulated phosphorylation of cAMP-dependent protein kinase 1 (PKA) substrates and GSK-3α. SIN-1 induced peroxynitrite does not decrease sperm viability, but significantly reduces sperm motility, progressive motility, velocities and motility coefficients. Concomitantly, peroxynitrite does not affect mitochondrial membrane potential, plasma membrane fluidity, or A23187-induced acrosome reaction. However, peroxynitrite significantly increases sperm lipid peroxidation in both media. In conclusion, peroxynitrite compromises boar sperm motility without affecting mitochondrial activity. Although peroxynitrite potentiates the phosphorylation of pathways leading to sperm motility, it also causes oxidative stress that might explain, at least partially, the motility impairment.

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