Effects of post-thaw incubation on motility, acrosomal integrity and in vitro fertilizing capacity of boar spermatozoa cryopreserved in 0.5 and 5ml straws

Aminoguanidine is a selective inhibitor of the inducible nitric oxide synthase (iNOS) and a scavenger of reactive oxygen species (ROS). Numerous studies have shown the antioxidant properties of aminoguanidine in several cell lines, but the in vitro effects of this compound on spermatozoa under oxidative stress are unknown. In this study, we tested the hypothesis that aminoguanidine may protect against the detrimental effects of oxidative stress in boar spermatozoa. For this purpose, sperm samples were incubated with a ROS generating system (Fe2+/ascorbate) with or without aminoguanidine supplementation (10, 1, and 0.1 mM). Our results show that aminoguanidine has powerful antioxidant capacity and protects boar spermatozoa against the deleterious effects of oxidative stress. After 2 h and 3.5 h of sperm incubation, the samples treated with aminoguanidine showed a significant increase in sperm velocity, plasma membrane and acrosome integrity together with a reduced lipid peroxidation in comparison with control samples (p < 0.001). Interestingly, except for the levels of malondialdehyde, the samples treated with 1 mM aminoguanidine did not differ or showed better performance than control samples without Fe2+/ascorbate. The results from this study provide new insights into the application of aminoguanidine as an in vitro therapeutic agent against the detrimental effects of oxidative stress in semen samples.

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Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda

Reproduction ◽

10.1530/rep.1.00034 ◽

2004 ◽

Vol 127 (5) ◽

pp. 547-556 ◽

Cited By ~ 26

Author(s):

R E Spindler ◽

Y Huang ◽

J G Howard ◽

P Wang ◽

H Zhang ◽

...

Keyword(s):

Sperm Motility ◽

Giant Panda ◽

Calcium Ionophore ◽

Sperm Cryopreservation ◽

Management Tools ◽

Giant Pandas ◽

Before And After ◽

Cryopreservation Protocol ◽

Acrosomal Integrity

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze–thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid (− 40 and − 100 °C/min) cryopreservation by incubation in HEPES-buffered Ham’s F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean±s.e.m. sperm motility post-thaw (56.1 ± 3.9% at 0 h) was less (P < 0.05) than pre-freeze (71.7 ± 6.0%), there was no difference (P > 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 ± 1.7% versus cryopreserved–thawed, 81.7 ± 4.7% at 0 h). Incidence of capacitation was greater (P < 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 ± 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 ± 1.1%). Frozen–thawed sperm preincubated without accelerators underwent capacitation (49.6 ± 1.1%) to a greater extent (P < 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 ± 1.4%) and without accelerators (9 h: 41.2 ± 1.1%) did not differ (P > 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitation in vitro with or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.

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“In vitro” capacitation and further progesterone‐induced acrosome exocytosis are linked to specific changes in the expression and location of threonine phosphorylation of boar spermatozoa

Reproduction in Domestic Animals ◽

10.1111/rda.13478 ◽

2019 ◽

Vol 54 (8) ◽

pp. 1085-1094

Author(s):

Laura Ramió‐Lluch ◽

Olga Blanco Prieto ◽

Alfredo Ramírez ◽

Josep M. Fernández‐Novell ◽

Alejandro Peña ◽

...

Keyword(s):

Boar Spermatozoa

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Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

Reproduction ◽

10.1530/rep-20-0123 ◽

2020 ◽

Vol 160 (5) ◽

pp. 725-735

Author(s):

Julieta Gabriela Hamze ◽

María Jiménez-Movilla ◽

Raquel Romar

Keyword(s):

Acrosome Reaction ◽

Cumulus Cell ◽

Cumulus Cells ◽

In Vitro Fertilisation ◽

Sperm Binding ◽

Fertilisation Rate ◽

Gamete Recognition ◽

Boar Spermatozoa

The role of specific zona pellucida (ZP) glycoproteins in gamete interaction has not yet been elucidated in many species. A recently developed 3D model based on magnetic sepharose beads (B) conjugated to recombinant ZP glycoproteins (BZP) and cumulus cells (CBZP) allows the study of isolated ZP proteins in gamete recognition studies. The objective of this work was to study the role of porcine ZP2, ZP3 and ZP4 proteins in sperm binding, cumulus cell adhesion and acrosome reaction triggering. ZP protein-bound beads were incubated with fresh ejaculated boar spermatozoa and isolated cumulus cells for 24 h. The number of sperm bound to the beads, the acrosomal shrouds (presence of acrosomal content) on the bead’s surface, and the acrosome integrity (by means of PNA-FITC lectin) in bound and unbound sperm were studied. Finally, in vitro matured porcine oocytes mixed with BZP2 were inseminated in vitro using fresh sperm and fertilisation results evaluated. Over 60% of beads had at least one sperm bound after 2 h of coincubation. ZP2-beads (BZP2) and cumulus-ZP2-bead complexes (CBZP2) reached the highest number of sperm per bead, whereas BZP3 and BZP4 models showed the highest number of unbound reacted sperm cells and acrosomal shrouds. Fertilisation efficiency and monospermy rate increased when oocytes were fertilised in the presence of BZP2. We, therefore, conclude that in pigs, it is mainly ZP2 that is involved in sperm-ZP binding whereas ZP3 and ZP4 induce acrosome reaction. Using magnetic sepharose ZP2-bound beads might be a valuable tool to improve the fertilisation rate in pigs.

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Effects of Seminal Plasma Components on Motility and Acrosomal Integrity of Meishan Boar Spermatozoa after Cooling Treatments

Nihon Chikusan Gakkaiho ◽

10.2508/chikusan.69.720 ◽

1998 ◽

Vol 69 (8) ◽

pp. 720-727

Author(s):

Hiroshi HARAYAMA ◽

Akira IMANO ◽

Masashi MIYAKE ◽

Seishiro KATO

Keyword(s):

Seminal Plasma ◽

Boar Spermatozoa ◽

Acrosomal Integrity

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Viable and morphologically normal boar spermatozoa alter the expression of heat-shock protein genes in oviductal epithelial cells during co-culture in vitro

Molecular Reproduction and Development ◽

10.1002/mrd.22350 ◽

2014 ◽

Vol 81 (9) ◽

pp. 805-819 ◽

Cited By ~ 21

Author(s):

Marc Yeste ◽

William V. Holt ◽

Sergi Bonet ◽

Joan E. Rodríguez-Gil ◽

Rhiannon E. Lloyd

Keyword(s):

Heat Shock ◽

Epithelial Cells ◽

Heat Shock Protein ◽

Culture In Vitro ◽

Heat Shock Protein Genes ◽

Boar Spermatozoa

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Binding of boar spermatozoa to oviductal epithelium in vitro in relation to sperm morphology and storage time

Reproduction ◽

10.1530/rep.1.00814 ◽

2006 ◽

Vol 131 (2) ◽

pp. 311-318 ◽

Cited By ~ 28

Author(s):

D Waberski ◽

F Magnus ◽

F Ardón ◽

A M Petrunkina ◽

K F Weitze ◽

...

Keyword(s):

Semen Quality ◽

Binding Capacity ◽

Storage Period ◽

Sperm Function ◽

Sperm Binding ◽

Oviductal Epithelium ◽

Semen Storage ◽

Boar Semen ◽

Boar Spermatozoa

In vitro short-term storage of boar semen for up to 72 h before insemination negatively affects fertility, but this often remains undetected during semen quality assessment. One important sperm function is the ability to form the functional sperm reservoir in the oviduct. In the present study, we used the modified oviductal explant assay to study sperm binding to oviductal epithelium in vitro in diluted boar semen stored for 24 or 72 h. First, we determined the kinetics of in vitro sperm binding to oviductal epithelium in relation to co-incubation time of sperm and oviductal tissue pieces. Then, we studied how the binding of sperm to oviductal epithelium was affected by in vitro semen storage and by differences among individual boars. Sperm binding after different incubation times was significantly higher when semen was stored 24 h than after 72-h storage (P < 0.05), and peaked at 30–90 min of incubation. Sperm binding differed between boars (n = 44), and was negatively correlated to the percentage of sperm with cytoplasmic droplets (R = −0.51, P < 0.001). There were no significant changes in motility, acrosome integrity and propidium iodide stainability during the 72-h storage period. However, sperm-binding indices were significantly lower after 72 h in vitro storage than after 24-h storage in sperm from boars with normal semen quality (P < 0.05); in contrast, the binding capacity of sperm from boars with higher percentages of morphologically altered sperm remained at a low level. The sperm-binding capacity of sperm from four of the five boars with known subfertility was lower than the mean binding index minus one standard deviation of the boar population studied here. It is concluded that changes in the plasma membrane associated with in vitro ageing reduce the ability of stored boar sperm to bind to the oviductal epithelium. This study shows the potential of sperm–oviduct binding as a tool to assess both male fertility and changes in sperm function associated with in vitro ageing.

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