Fractionated Seminal Plasma of Boar Ejaculates Analyzed by LC–MS/MS: Its Effects on Post-Thaw Semen Quality

This study aimed to characterize the protein composition of fractionated seminal plasma (SP) by liquid chromatography mass spectrometry (LC–MS/MS) analysis and investigate its effects on survival of frozen-thaw (FT) boar spermatozoa following storage. Seminal plasma (SP) was fractionated by gel filtration chromatography to give two fractions, SP1 with more than 40 kDa (>40 kDa) and SP2 with less than 40 kDa (<40 kDa). SP1 and SP2 were subjected to LC–MS/MS and bioinformatics analysis. Following cryopreservation, FT boar semen (n = 7) was thawed in Beltsville Thawing Solution (BTS), BTS + SP1 or BTS + SP2, stored at different periods and subjected to post-thaw (PT) quality assessment. A total of 52 and 22 abundant proteins were detected in SP1 and SP2, respectively. FN1, ANGPTL1, and KIF15 proteins were more abundance in SP1, whereas a high abundance of spermadhesins (PSP-I and PSP-II) was detected in SP2. Proteins of the fractionated SP were involved in various biological processes, such as cell motility and signal transduction. The dominant pathway of SP1 proteins was the apelin signaling pathway (GNA13, MEF2D, SPHK2, and MEF2C), whereas a pathway related to lysosome (CTSH, CTSB, and NPC2) was mainly represented by SP2 proteins. In most of the boars, significantly higher motility characteristics, membrane integrity, and viability were observed in FT spermatozoa exposed to SP1 or SP2 compared with BTS. The results of our study confirm that a combination of several proteins from the fractionated SP exerted beneficial effects on the sperm membrane, resulting in improved quality characteristics following PT storage.

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Effect of Fractionated Seminal Plasma on Sperm Characteristics Following Cryopreservation of Boar Semen

Annals of Animal Science ◽

10.2478/aoas-2019-0016 ◽

2019 ◽

Vol 19 (3) ◽

pp. 695-712 ◽

Cited By ~ 1

Author(s):

Karolina Wasilewska-Sakowska ◽

Łukasz Zasiadczyk ◽

Leyland Fraser

Keyword(s):

Seminal Plasma ◽

Semen Quality ◽

Membrane Integrity ◽

Gel Filtration ◽

Sperm Characteristics ◽

Apoptosis Assay ◽

Sperm Membrane ◽

Boar Semen ◽

Acrosome Integrity ◽

Protein Components

AbstractThis study aimed to investigate the effect of fractionated seminal plasma (SP) on boar sperm characteristics following cryopreservation. Gel filtration chromatography yielded two fractions: SP1 with more than 40 kDa (>40 kDa) and SP2 with less than 40 kDa (<40 kDa). The fractionated SP (SP1 and SP2), whole seminal plasma (wSP) and Beltsville Thawing Solution (BTS) were used for the treatment of semen before freezing-thawing. Besides the analysis of sperm motility characteristics, plasma membrane integrity (PMI), acrosome integrity, and viability (Vybrant Apoptosis Assay) were analyzed in pre-freeze and post-thaw (PT) semen. Among the analyzed pre-freeze sperm parameters, rapid movement was markedly affected by boar and treatment. Furthermore, boar and treatment were significant sources of variations in PT semen quality. Treatment with wSP caused a marked reduction in PT semen quality compared with BTS, SP1 or SP2. Wide variations in PT acrosome integrity and viability were observed in spermatozoa treated with BTS and the fractionated SP, being significantly higher in the SP1- and SP2-treated samples. However, PT semen quality did not differ between semen samples treated with SP1 and SP2. Representative electrophoretic profiles of sperm proteins from each treatment showed quantitative and qualitative differences, indicating varying effects of the cryopreservation procedure on the sperm membrane integrity. The findings of this study indicated that the cryoprotective effects of the SP components varied among boars and that different components of the fractionated SP exerted varying effects on sperm functions following cryopreservation. It could be suggested that the variable protective protein components of either fractionated SP ameliorated alterations in the sperm membranes during cryopreservation, resulting in reduced susceptibility to cryo-damage.

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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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Metabolic activity of boar semen stored in different extenders supplemented with ostrich egg yolk lipoproteins

Journal of Veterinary Research ◽

10.1515/jvetres-2017-0016 ◽

2017 ◽

Vol 61 (1) ◽

pp. 127-133 ◽

Cited By ~ 3

Author(s):

Anna Dziekońska ◽

Marek Kinder ◽

Leyland Fraser ◽

Jerzy Strzeżek ◽

Władysław Kordan

Keyword(s):

Oxygen Consumption ◽

Metabolic Activity ◽

Storage Temperature ◽

Membrane Integrity ◽

Egg Yolk ◽

Atp Production ◽

Beneficial Effects ◽

Semen Storage ◽

Boar Semen ◽

Boar Spermatozoa

AbstractIntroduction:The aim of this study was to evaluate the effect of lipoprotein fraction isolated from ostrich egg yolk (LPFo) on the metabolic activity of boar spermatozoa following liquid semen storage in different extenders and temperatures.Material and Methods:Boar ejaculates were extended in Androhep, Beltsville thawing solution (BTS), and Martín-Rillo and Alias (MR-A) without (control) or with the addition of LPFo and stored for three days at either 5°C or 16°C. The analysed sperm parameters included total motility (TMOT), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), oxygen consumption, and adenosine triphosphate (ATP) production.Results:The sperm metabolic activity seemed to be higher in the LPFo-based extenders following storage for three days, irrespective of the storage temperature. Compared with the LPFo-free extenders, significantly higher (P < 0.05) sperm PMI and MMP were observed in BTS and MR-A extenders supplemented with LPFo during storage for three days at 5°C. Spermatozoa stored in the BTS-LPFo extender exhibited higher (P < 0.05) TMOT and oxygen consumption, whereas higher (P < 0.05) PMI was observed in spermatozoa stored in Androhep-LPFo and MR-A-LPFo for three days at 16°C. No significant differences (P > 0.05) in ATP content were observed between the LPFo-free and LPFo-based extenders during storage.Conclusions:Supplementation of LPFo to semen extenders had varying effects on the metabolic activity of boar spermatozoa stored at different temperatures. It can be suggested that the interactions of various components of the extenders and seminal plasma with LPFo exert beneficial effects on the sperm metabolic activity during liquid storage of boar semen.

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Use of Novel Methods to Assess Seasonal Differences in the Quality of Boar Semen Stored Up to 7 Days at 17°C

Annals of Animal Science ◽

10.2478/aoas-2019-0035 ◽

2019 ◽

Vol 19 (4) ◽

pp. 967-978

Author(s):

Barbara Szczęśniak-Fabiańczyk ◽

Michał Bochenek ◽

Piotr Gogol ◽

Monika Trzcińska ◽

Magdalena Bryła ◽

...

Keyword(s):

Semen Quality ◽

Membrane Integrity ◽

Winter Period ◽

Summer Period ◽

Sperm Membrane ◽

Boar Semen ◽

Structure Assessment ◽

Novel Methods ◽

Integrity Analysis

AbstractThe objective of the present study was to determine the seasonal changes in boar semen quality by the assessment of sperm membrane integrity, analysis of chromatin structure, assessment of oxidative stress and of apoptotic changes in spermatozoa. Semen from 16 boars (172 ejaculates) was investigated. The males were aged between 7 months and 7 years. Semen was extended with BTS diluent and stored at +17°C. During seven days of storage, the semen was subjected to standard evaluation and novel methods for semen assessment. In the autumn-winter period, the semen had higher evaluations than in the spring-summer period, but only sperm membrane integrity examination showed significantly lower (P≤0.01) percentage of moribund spermatozoa and the semen had a significantly (P≤0.05) lower (by 0.5%) percentage of sperm with damaged chromatin. Examination performed after 7-day storage showed significantly (P≤0.01) higher percentage of live spermatozoa and with high mitochondrial membrane potential for the autumn-winter period. The level of apoptotic cells was significantly (P≤0.01) lower for the autumn-winter period. Examination of sperm membrane integrity after 7 days of storage showed a lower (P≤0.05) percentage of moribund spermatozoa for the autumn-winter period. In our opinion, novel methods for sperm assessment may be used to monitor new parameters of sperm function.

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Macro- and microelements in serum and seminal plasma as biomarkers for bull sperm cryotolerance

Acta Veterinaria Scandinavica ◽

10.1186/s13028-021-00590-2 ◽

2021 ◽

Vol 63 (1) ◽

Author(s):

Maja Zakošek Pipan ◽

Petra Zrimšek ◽

Breda Jakovac Strajn ◽

Katarina Pavšič Vrtač ◽

Tanja Knific ◽

...

Keyword(s):

Seminal Plasma ◽

Semen Quality ◽

Sperm Quality ◽

Membrane Integrity ◽

Quality Characteristics ◽

Freezing And Thawing ◽

Additional Tool ◽

Bull Semen ◽

Bull Sperm ◽

Fresh Semen

ABSTRACT Background Wide variation in fertility rates is observed when using frozen bull semen, even when the bulls have met quality standards for semen production. Therefore, a simple and reliable test to assess the freezing potential of bull semen based on the analysis of fresh semen or blood would be of great value. Attention is now turning to assessment of seminal plasma components such as proteins and elements. In the present study, the concentrations of macro- and microelements in fresh bull semen plasma and in serum and their correlation with quality characteristics of fresh semen and with semen quality after freezing and thawing were determined. Ejaculates were collected from 30 mature bulls, and semen volume, concentration, sperm motility, morphology, tail membrane integrity, plasma membrane permeability and DNA fragmentation were determined on the day of collection and after freezing and thawing. The concentrations of macroelements (Na, Mg, K and Ca) and microelements (Cu, Fe, Zn and Se) were determined in the seminal plasma and serum. The semen samples were classified into satisfactory and unsatisfactory groups according to the fresh semen quality. Results Zinc and Se levels measured in serum were associated with almost all fresh and frozen-thawed semen quality characteristics, while Fe levels were associated only with acrosomal defects in fresh semen. Zinc and Fe levels in fresh seminal plasma were associated with various quality characteristics of fresh and frozen-thawed semen, while Se level in fresh seminal plasma was not associated with any of the semen quality characteristics. Conclusions Microelements were shown to be useful as biomarkers involved in the analysis of bull sperm quality and could be used as an additional tool to predict bull semen quality after freezing and thawing. Our results confirm that the analysis of Zn and Se levels in serum and Zn, Cu and Fe levels in fresh seminal plasma can provide information to discriminate between bull semen samples with spermatozoa with high or low cryotolerance.

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Spectrophotometric Analysis of the Resazurin Reduction Test as a Tool for Assessing Canine Semen Quality

Bulletin of the Veterinary Institute in Pulawy ◽

10.2478/bvip-2013-0049 ◽

2013 ◽

Vol 57 (2) ◽

pp. 281-285 ◽

Cited By ~ 1

Author(s):

Rafał Strzeżek ◽

Krystyna Filipowicz ◽

Marta Stańczak ◽

Władysław Kordan

Keyword(s):

Sperm Motility ◽

Seminal Plasma ◽

Semen Quality ◽

Membrane Integrity ◽

Spectrophotometric Analysis ◽

Normal Morphology ◽

Additional Tool ◽

Reduction Test ◽

Highly Correlated

Abstract The resazurin reduction test (RRT) was subjected to spectrophotometric analysis to evaluate the quality of canine semen. Twenty four samples of canine semen were analysed. The absorption peaks for resazurin and resorufin were determined at 615 and 580 nm, respectively. The RRT ratio (RRTsperm-the ratio for samples containing spermatozoa, RRTplasma-the ratio for samples containing seminal plasma) was calculated by dividing the absorbance at 580 nm by the absorbance at 615 nm. Spearman’s correlation test was used to determine the significance of correlations between the analysed sperm parameters and the results of the resazurin reduction assay. The RRT ratio was highly correlated with sperm motility (r=0.68, P<0.01), progressive sperm motility (r=0.61, P<0.01), the subpopulation of cells with rapid velocity (r=0.72, P<0.01), and the subpopulation of cells with medium velocity (r= -0.54, P<0.05). A negative correlation was observed between the reducing capacity of seminal plasma vs. sperm with plasma membrane integrity (r= -0.60, P<0.01) and sperm with normal morphology (r= -0.58, P<0.01). The RRT test can be used as an additional tool for evaluation of the quality of canine semen.

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Assessment of quality in specific fractions of Large White Yorkshire boar semen

Journal of Veterinary and Animal Sciences ◽

10.51966/jvas.2021.52.2.155-160 ◽

2021 ◽

Vol 52 (2) ◽

Author(s):

K. G. Ambily ◽

Malati Naik ◽

Hiron M. Harshan ◽

C. Jayakumar ◽

M. P. Unnikrishnan ◽

...

Keyword(s):

Cold Shock ◽

Sperm Concentration ◽

Membrane Integrity ◽

Membrane Cholesterol ◽

Large White ◽

Progressive Motility ◽

Sperm Membrane ◽

Boar Semen ◽

Quality Assessments

Boar semen is voluminous and ejaculated as jets or fractions of pre-sperm, sperm rich (SRF) and post-sperm rich fractions. Recent studies have reported more resilient characteristics of sperm in initial portions of SRF towards cold shock and cryopreservation. The present study was conducted to assess the quality of specific fractions of SRF, namely, first 10mL of SRF (F1) and rest of SRF (F2) in Large white Yorkshire (LWY) boar semen. Ejaculates were collected using gloved-hand technique and were subjected to quality assessments of volume, pH, sperm progressive motility, concentration, plasma membrane integrity, abnormality, acrosome integrity and sperm membrane cholesterol. Upon statistical analysis, significant differences were noticed in volume, pH, sperm concentration and sperm membrane cholesterol between fractions of the ejaculate.

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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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Relationship between semen quality and seminal plasma cholesterol, triacylglycerols and proteins in the domestic cat

Journal of Feline Medicine and Surgery ◽

10.1177/1098612x19889072 ◽

2019 ◽

Vol 22 (10) ◽

pp. 882-889 ◽

Cited By ~ 1

Author(s):

María F García ◽

Romina Nuñez Favre ◽

María C Stornelli ◽

Ramiro Rearte ◽

María C García Mitacek ◽

...

Keyword(s):

Seminal Plasma ◽

Sperm Morphology ◽

Semen Quality ◽

Sperm Count ◽

Sodium Dodecyl ◽

Sperm Concentration ◽

Membrane Integrity ◽

Domestic Cat ◽

Sds Page ◽

The Relationship

Objectives The current study aimed to evaluate the relationship between specific seminal plasma components – cholesterol (CHOL), triacylglycerols (TAG) and total protein (PROT) concentrations – and semen quality in cats. A further aim was to determine the relationship between specific seminal protein bands and semen quality. Methods Thirteen toms, 2–5 years of age, were included. Semen collection was performed by electroejaculation every 4 weeks. Fifty-eight ejaculates were assessed for motility, velocity, volume, sperm concentration, total sperm count, viability, acrosome integrity, plasma membrane integrity and sperm morphology. Samples were divided into two groups: good semen quality (GSQ) and poor semen quality (PSQ). After evaluation, seminal plasma was separated from the sperm by centrifugation and stored at −20°C. CHOL, TAG and PROT concentrations were then assessed and seminal plasma protein profile was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results Seminal plasma CHOL and TAG concentrations, motility, velocity, sperm concentration, total sperm count and sperm morphology were significantly higher in GSQ cats compared with PSQ cats ( P <0.01). Moreover, seminal plasma SDS-PAGE analysis showed an identifiable extra band exclusively in the GSQ group. Conclusions and relevance Data obtained in this study showed that seminal plasma CHOL and TAG concentrations and specific protein bands could be used to improve semen evaluation in toms. In this sense, the 14 kDa protein band could be a valuable marker for semen quality in the cat and should be further investigated. However, more studies are necessary to determine its relationship with fertility.

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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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