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

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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Selective sperm binding to pig oviductal epithelium in vitro

Reproduction ◽

10.1530/rep.0.1210889 ◽

2001 ◽

pp. 889-896 ◽

Cited By ~ 76

Author(s):

AM Petrunkina ◽

R Gehlhaar ◽

W Drommer ◽

D Waberski ◽

E Topfer-Petersen

Keyword(s):

Seminal Fluid ◽

Binding Capacity ◽

Sperm Binding ◽

Oviductal Epithelium ◽

Female Donor ◽

Sperm Reservoir ◽

The Individual ◽

Boar Spermatozoa ◽

Initial Binding

The sperm reservoir in the caudal isthmus of the oviduct of a number of species is created by binding of spermatozoa to oviductal epithelium. The sperm reservoir fulfills a number of functions such as control of sperm transport, maintenance of sperm viability and modulation of capacitation. The initial capacities of ejaculated and epididymal boar spermatozoa to bind to oviductal epithelium were investigated using a modified pig oviductal explant assay. The number of spermatozoa that bound to 0.01 mm(2) of explant surface was used as the parameter of binding capacity. Binding of spermatozoa to oviductal epithelial explants was dependent in a linear manner on the number of spermatozoa added (P < or = 0.05). No difference was found in initial sperm binding between isthmic and ampullar explants. There was no effect of the stage of the oestrous cycle or the reproductive status of the female donor. There was a significant effect (P < or = 0.05) of the individual boar on the binding index. The binding index correlated negatively with the percentage of spermatozoa with cytoplasmic droplets and the percentage of morphologically abnormal spermatozoa (P < or = 0.05). Epididymal spermatozoa showed significantly lower initial binding capability than did ejaculated spermatozoa from the same boars (P < or = 0.05); therefore, components of seminal plasma may play a role in the binding process. The individual differences revealed by this study and their relation to morphology and contact of spermatozoa with seminal fluid indicate a selective function of sperm-oviduct binding.

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18 EFFECTS OF SKIM MILK ON THE QUALITY AND FERTILITY OF BOAR SEMEN FOLLOWING LIQUID PRESERVATION AT 5°C AND 15°C

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab18 ◽

2011 ◽

Vol 23 (1) ◽

pp. 115 ◽

Cited By ~ 1

Author(s):

Z. Namula ◽

R. Kodama ◽

Y. Kaedei ◽

F. Tanihara ◽

V. L. Vien ◽

...

Keyword(s):

Sperm Motility ◽

Storage Temperature ◽

Semen Quality ◽

Sperm Concentration ◽

Membrane Integrity ◽

Skim Milk ◽

Control Group ◽

Boar Semen ◽

Boar Spermatozoa

Liquid preservation of semen can be an alternative to frozen–thawed semen for artificial insemination. The success of a selection of boar semen extenders has been studied over storage periods of 5 to 7 days. The objective of this study was to evaluate the effects of skim milk on the viability and in vitro fertility of boar spermatozoa preserved in Modena-based extenders at 5°C and 15°C for 2 weeks. A total of 7 ejaculates were collected from one boar. The sperm-rich fraction of each ejaculate was centrifuged and diluted in Modena extenders supplemented with 0 (control), 7.5, and 15 mg mL–1 of dry skim milk. The final sperm concentration was adjusted to 1 × 108 cells mL–1, and then the semen was stored at 5°C and 15°C for 2 weeks. In the first experiment, the motility, viability (live/dead fluorescence viability assay), plasma membrane integrity (hypoosmotic swelling test; HOST), and acrosome integrity (FITC-labelled peanut agglutinin staining) of semen stored for 2 weeks were assessed. In the second experiment, the fertilization of stored semen after 20 h of co-incubation with in vitro matured oocytes and their development were examined. Data were analysed using ANOVA. When the semen was stored at 5°C for 2 weeks, the mean total sperm motility of semen stored with 7.5 and 15 mg mL–1 of dry skim milk was significantly higher than that of semen in the control group (41.4% and 41.5% v. 17.4%; P < 0.05). However, the beneficial effects of skim milk on the sperm motility were not observed in the semen stored at 15°C. Moreover, there were no significant differences in the other parameters of semen quality among the groups in each storage temperature. Significantly higher penetration rates of semen stored with 7.5 and 15 mg mL–1 of dry skim milk were observed in the storage at 5°C (41.1% and 34.8% v. 19.8%; P < 0.05) but not at 15°C (38.9% and 26.0% v. 30.0%; P > 0.05) when compared with the control group. When the semen was stored at 5°C, the development rate to the blastocyst stage of oocytes fertilized with semen stored with 7.5 mg mL–1 of dry skim milk was significantly higher than that with control and 15 mg mL–1 of dry skim milk (15.4% v. 1.1% and 7.8%; P < 0.01). However, there were no significant differences in the development rates of oocytes fertilized with semen stored at 15°C among the groups (9.6–11.9%). In conclusion, our results indicate that the effect of skim milk on the viability and in vitro fertility of liquid-stored boar spermatozoa is dependent on the storage temperature. The addition of 7.5 mg mL–1 of dry skim milk may be effective for the improvement of viability and fertility of semen stored at 5°C but not at 15°C.

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The in Vitro Effect of Taurine on Boar Spermatozoa Quality

Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis ◽

10.11118/actaun201866010131 ◽

2018 ◽

Vol 66 (1) ◽

pp. 131-137

Author(s):

Dušan Paál ◽

František Strejček ◽

Eva Tvrdá ◽

Grzegorz Formicki ◽

Sabine Klein ◽

...

Keyword(s):

Flow Cytometry ◽

Fluorescent Dyes ◽

Semen Quality ◽

Sperm Analysis ◽

Spermatozoa Motility ◽

Boar Semen ◽

Acrosome Integrity ◽

Boar Spermatozoa ◽

Vitro Effect

The aim of this in vitro study was to evaluate the effects of taurine (TAU) supplementation on boar spermatozoa motility, viability, acrosome integrity and morphology. Eighteen boar semen samples were diluted with the Androhep PlusTM extender containing no TAU (control) or supplemented with 1.5 mM, 7 mM, 12.5 mM TAU and cultured at 4 °C for 18 days. Sperm motility was evaluated using the computer-aided sperm analysis (CASA) system. Furthermore, the samples were fixed and assessed for the occurrence of morphological abnormalities using phase contrast microscopy. Fluorescent dyes SYBR – 14 and propidium iodide were used to determine the sperm viability. Acrosome integrity was examined using PNA – Alexa Fluor 647 and flow cytometry. A gradual decrease of the semen quality was detected in all experimental groups over the course of the study. CASA revealed no selective advantage of TAU supplementation on the spermatozoa motility (p > 0.05). TAU administration showed to be ineffective in preserving spermatozoa viability as well as acrosome integrity as measured by flow cytometry (p > 0.05). Under the conditions of this study, no significant positive effect of TAU was recorded following its administration to the Androhep PlusTM boar semen extender with respect to spermatozoa quality.

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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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Value of semen parameters, with special reference to TNF-α, in predicting the quality of boar semen after short-term storage

Acta Veterinaria Hungarica ◽

10.1556/avet.2013.009 ◽

2013 ◽

Vol 61 (2) ◽

pp. 209-219 ◽

Cited By ~ 5

Author(s):

Janko Mrkun ◽

Marjan Kosec ◽

Petra Zrimšek

Keyword(s):

Semen Quality ◽

Semen Parameters ◽

Short Term ◽

Progressive Motility ◽

Tnf Α ◽

Semen Storage ◽

Boar Semen ◽

Α Level ◽

Term Storage

The aim of this study was to address the question whether changes in boar semen quality after short-term storage could be predicted on the basis of standard semen parameters and TNF-α level determined on the day of semen collection under commercial conditions. Progressive motility showed the highest positive correlation with morphology on day 0 of collection, and progressive motility on day 3 (P < 0.05) showed a negative correlation with acrosome abnormalities (P < 0.05). According to the area under receiver operating characteristics (ROC) curves (AUCs), progressive motility could also be used in predicting semen quality after 3 days of storage (AUC > 0.5; P < 0.05). TNF-α in seminal plasma is the only parameter measured on day 0 to show a significant correlation with the percentage of viable spermatozoa after 3 days of semen storage (r = 0.495, P < 0.05). ROC analysis shows that TNF-α level is helpful in discriminating viability outcome after semen storage (AUC = 0.94, P < 0.001). We can predict with 92.35% certainty that fresh semen samples with more than 150 pg/ml of TNF-α in the seminal plasma will retain more than 85% of viable spermatozoa after 3 days of storage. Thus, TNF-α can contribute to predicting the quality of short-term stored semen.

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Quercetin and Naringenin Provide Functional and Antioxidant Protection to Stored Boar Semen

Animals ◽

10.3390/ani10101930 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1930

Author(s):

Eva Tvrdá ◽

Mégane Debacker ◽

Michal Ďuračka ◽

Ján Kováč ◽

Ondřej Bučko

Keyword(s):

Mitochondrial Activity ◽

Ros Generation ◽

Dna Integrity ◽

Membrane Stabilization ◽

New Information ◽

Semen Storage ◽

Boar Semen ◽

Boar Spermatozoa ◽

The Impact ◽

Sample Dilution

In this study, we evaluated the impact of 5–50 μM quercetin (QUE) and naringenin (NAR) on extended boar spermatozoa in the BTS (Beltsville Thawing Solution) medium for 72 h. Spermatozoa motion, membrane, acrosome, and DNA integrity were investigated immediately after sample dilution (0 h) as well as after 24 h, 48 h, and 72 h of semen storage. Furthermore, reactive oxygen species (ROS) and superoxide production, as well as the extent of oxidative damage to the sperm proteins and lipids, were assessed to determine the potential of QUE and NAR to prevent a potential loss of sperm vitality due to oxidative stress development. Our results indicate that the most notable parameter influenced by QUE was the mitochondrial activity, which remained significantly higher throughout the experiment (p < 0.001 and p < 0.0001; 10 μM), and which correlated with the most prominent maintenance of sperm motility (p < 0.01, 48 h; p < 0.05, 72 h). A significant membrane stabilization (p < 0.01, 24 h and 48 h; p < 0.0001, 72 h) and prevention of lipid peroxidation (p < 0.05, 24 h and 48 h; p < 0.01, 72 h) was primarily observed following administration of 10 and 25 μM NAR; respectively. Administration of 10 μM QUE led to a significant decrease of superoxide (p < 0.0001, 48 h and 72 h) while the most notable decline of ROS generation was recorded in the case of 10 and 25 μM NAR (p < 0.001). This study may provide new information on the specific mechanisms of action involved in the favorable effects of natural biomolecules on spermatozoa.

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Cryopreservation of boar semen

Czech Journal of Animal Science ◽

10.17221/47/2020-cjas ◽

2020 ◽

Vol 65 (No. 4) ◽

pp. 115-123

Author(s):

Marija Jovičić ◽

Eva Chmelíková ◽

Markéta Sedmíková

Keyword(s):

Animal Species ◽

Semen Quality ◽

Egg Yolk ◽

High Rate ◽

Freezing And Thawing ◽

Long Term Storage ◽

Boar Semen ◽

Boar Spermatozoa ◽

Term Storage

Sperm cryopreservation is the best technology for long-term storage of the semen. However, the damage of boar spermatozoa by cryopreservation is more severe than in other animal species and a standardized freezing protocol for efficient cryopreservation has not been established yet. Semen quality and freezability vary greatly between breeds as well as between individual boars and even the season. Boar spermatozoa are sensitive to low temperatures; they sustain damage and a high rate of mortality and freezing/thawing the boar semen may strongly impair the sperm function and decrease the semen quality. The freezability of boar semen can be influenced by a cryopreservation procedure, and also by using various additives to freezing and thawing extenders such as antioxidants. In order to obtain acceptable results after thawing the boar semen, it is necessary to combine an optimal amount of additives (glycerol, egg yolk, sugars, antioxidants), cooling and warming velocities.

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Effects of feeding tuna oil on the lipid composition of pig spermatozoa and in vitro characteristics of semen

Reproduction ◽

10.1530/rep.0.1210315 ◽

2001 ◽

pp. 315-322 ◽

Cited By ~ 101

Author(s):

JA Rooke ◽

CC Shao ◽

BK Speake

Keyword(s):

Fatty Acids ◽

Fatty Acid Composition ◽

Fatty Acid ◽

Acid Composition ◽

Basal Diet ◽

Tuna Oil ◽

Total Fatty Acids ◽

Boar Semen ◽

Boar Spermatozoa

The aim of the present study was to characterize the effects of feeding tuna oil on the lipid and fatty acid composition of boar spermatozoa and to relate changes in composition to boar semen characteristics. Ten boars were paired by age and allocated to one of two diets (five boars per diet). The diets, which were offered for 6 weeks, consisted of a basal diet that was either unsupplemented or supplemented with 30 g tuna oil kg(-1) diet. Adding tuna oil to the diet increased the ether extract concentration of the diets fed from 65 to 92 g kg(-1) dry matter and supplied 10.5 g long chain polyunsaturated (n-3) fatty acids per 100 g total fatty acids. There were no changes in semen fatty acid composition after 3 weeks of feeding tuna oil. However, after 5 and 6 weeks, the proportions (g per 100 g total fatty acids) of 22:6(n-3) in sperm phospholipid fatty acids were increased from 34.5 to 42.9 g by feeding tuna oil and 22:5(n-6) decreased from 29.8 to 17.9 g. No changes were observed in other sperm lipids or seminal plasma phospholipids as a result of the diets fed. Feeding tuna oil increased the proportion of spermatozoa with progressive motility and with a normal acrosome score and reduced the proportion of spermatozoa with abnormal morphologies.

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INFLUENCE OF GLYCEROL CONCENTRATION AND FREEZING TO −79 C ON OXYGEN UPTAKE AND LIVABILITY OF BOAR AND BULL SPERMATOZOA

Canadian Journal of Animal Science ◽

10.4141/cjas72-007 ◽

1972 ◽

Vol 52 (1) ◽

pp. 65-72 ◽

Cited By ~ 2

Author(s):

L. M. SANFORD ◽

G. J. KING ◽

J. W. MACPHERSON

Keyword(s):

Oxygen Uptake ◽

Incubation Period ◽

Skim Milk ◽

Egg Yolk ◽

Freezing Process ◽

Glycerol Concentration ◽

Motile Cells ◽

Boar Semen ◽

Boar Spermatozoa

Boar and bull spermatozoa were diluted in a skim milk–egg yolk–glucose extender containing 0, 7.5, or 15% glycerol (v/v) and incubated aerobically for 6 hr at 37 C. Other partially diluted boar semen samples were cooled to 5 C. Glycerol was added to a final concentration of 0, 7.5, and 15%. Samples were frozen to −79 C, rewarmed, and incubated for 3 hr at 37 C. The presence of glycerol in the extender depressed (P < 0.01) the oxygen uptake by nonfrozen boar and bull spermatozoa during the 6-hr incubation period. The reduction of oxygen uptake by semen samples increased as the level of glycerol in the extender increased. There was a corresponding decrease (P < 0.01) in the number of motile cells at the conclusion of the incubation period. Glycerol appeared to have more of a detrimental effect on boar spermatozoan oxygen uptake. The rate of oxygen uptake by boar semen samples postfreezing was extremely depressed, suggesting that spermatozoa surviving the freezing process metabolize at a much lower rate than normal. Active progressive motility of most of the surviving boar spermatozoa ceased within 1–2 hr of incubation under the in vitro conditions of this experiment.

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Elimination of apoptotic boar spermatozoa using magnetic activated cell sorting

Acta Veterinaria Brno ◽

10.2754/avb201483010013 ◽

2014 ◽

Vol 83 (1) ◽

pp. 13-18 ◽

Cited By ~ 1

Author(s):

Janko Mrkun ◽

Tamara Dolenšek ◽

Tanja Knific ◽

Anja Pišlar ◽

Marjan Kosec ◽

...

Keyword(s):

Cell Sorting ◽

Semen Quality ◽

Annexin V ◽

Alexa Fluor ◽

Liquid Storage ◽

Boar Semen ◽

And Control ◽

Boar Spermatozoa ◽

First Time ◽

Magnetic Activated Cell Sorting

One of the features of apoptosis is the externalization of phosphatidylserine which could be used to remove apoptotic cells from semen preparations. Magnetic-activated cell sorting using annexin V-conjugated microbeads which bind to phosphatidylserine could be used to enhance semen quality. Twelve boar semen samples after 3 days of liquid storage at 16–17 °C were subjected to magnetic-activated cell sorting. Bound and unbound fractions and control samples were subjected to flow cytometry following the staining of spermatozoa with Annexin V conjugated with Alexa Fluor 488 and propidium iodide. Four subpopulations were obtained: live, early apoptotic live, late apoptotic, early necrotic dead and late necrotic dead. The frequency of early apoptotic and late necrotic spermatozoa was significantly higher (P< 0.05) in bound (14.1 ± 10.6% and 24.1 ± 10.2%, respectively) than in unbound fractions (3.4 ± 2.1% and 12.7 ± 3.1%) and control (3.5 ± 1.6% and 12.0 ± 5.0%). The lowest concentration of live spermatozoa was found in the bound fraction (10.6 ± 8.0 %), which differed significantly (P< 0.05) from the control. In unbound fractions there was a significantly higher concentration (P< 0.05) of morphologically normal spermatozoa (31.8 ± 12.6%) compared to bound ones (5.9 ± 7.3%). A significantly (P< 0.05) lower proportion of morphologically normal spermatozoa was observed in both fractions compared to control (67.2 ± 17.0%). Boar spermatozoa were separated by the above method for the first time, however, the results showed this method to be inappropriate for boar semen separation under the tested conditions.

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