Metabolite Profiling of Pig Seminal Plasma Identifies Potential Biomarkers for Sperm Resilience to Liquid Preservation

Metabolomic approaches allow the study of downstream gene expression events since metabolites are considered as the products of cell signaling pathways. For this reason, many studies in humans have already been conducted to determine the influence of the metabolites present in seminal plasma (SP) on sperm physiology, and to identify putative biomarkers. However, in livestock species, these relationships are yet to be uncovered. Thus, the present study aimed to explore: (i) if concentrations of metabolites in pig SP are related to sperm quality and functionality, and (ii) if they could predict the sperm resilience to liquid storage at 17°C. To this end, 28 ejaculates were individually collected and split into three aliquots: one was used for SP analysis through nuclear magnetic resonance (NMR) spectroscopy; another served for the evaluation of sperm concentration and morphology; and the last one was utilized to determine sperm functionality parameters using computer-assisted sperm analysis (CASA) and flow cytometry after 0 h and 72 h of liquid-storage at 17°C. NMR analysis allowed the identification and quantification of 23 metabolites present in pig SP which, except for fumarate, were not observed to follow a breed-dependent behavior. Moreover, specific relationships between metabolites and sperm variables were identified: (i) glutamate, methanol, trimethylamine N-oxide, carnitine, and isoleucine were seen to be related to some sperm quality and functionality parameters evaluated immediately after semen collection; (ii) leucine, hypotaurine, carnitine and isoleucine were found to be associated to the sperm ability to withstand liquid storage; and (iii) Bayesian multiple regression models allowed the identification of metabolite patterns for specific sperm parameters at both 0 h and 72 h. The identification of these relationships opens up the possibility of further investigating these metabolites as potential sperm functional biomarkers.

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201 EFFECTS OF REMOVING SEMINAL PLASMA DURING 8-h STORAGE BEFORE SEX-SORTING BOVINE SPERM

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab201 ◽

2012 ◽

Vol 24 (1) ◽

pp. 213 ◽

Cited By ~ 1

Author(s):

C. A. Burroughs ◽

K. M. Evans ◽

R. W. Lenz ◽

G. E. Seidel

Keyword(s):

Seminal Plasma ◽

Sperm Concentration ◽

Computer Assisted ◽

Sperm Analysis ◽

Long Term Storage ◽

Bovine Sperm ◽

Standard Procedures ◽

Term Storage ◽

Artificial Vagina

We evaluated sex-sorting parameters and post-thaw motility for sperm stored with or without seminal plasma for 8 h before sorting. One first ejaculate was collected from each of 6 bulls routinely collected via artificial vagina; ejaculates contained at least 70% motile and 75% morphologically normal sperm and sperm concentrations ranged from 0.75 to 2.21 × 109 sperm mL–1. Ejaculates were divided into 2 samples and centrifuged at 1000 × g for 15 min. Seminal plasma from 1 sample was replaced with TALP (pH 7.4) to a sperm concentration of 1.4 × 109 sperm mL–1. The seminal plasma/sperm admixture of the other sample (control) was suspended to initial ejaculate sperm concentration. Both samples were stored for 8 h at 16°C before being subjected to standard sex-sorting procedures. Sperm were analyzed and bulk sorted on a MoFlo SX (XY Inc., Navasota, TX, USA) flow cytometer/cell sorter for percentage of live-oriented cells, percentage of membrane-impaired sperm (cell membranes permeable to red food colouring, which were discarded during sorting) and resolution between X- and Y-bearing sperm populations (peak to valley ratio). Sorted sperm were frozen according to standard procedures and post-thaw motility was determined immediately after thawing using computer-assisted sperm analysis. Treatments were compared using a paired t-test. Control sperm stored with seminal plasma resulted in a higher percentage of live-oriented cells (55%) versus those stored without seminal plasma (51%; P = 0.02). The percentage of membrane-impaired sperm was lower for control sperm (19%) than that of samples without seminal plasma (28%; P < 0.001). Resolution was greater for sperm stored without seminal plasma (34%) than for control sperm (10%; P = 0.04). Post-thaw, both total and progressively motile sperm were higher for samples without seminal plasma (63 and 53%, respectively) compared with those of the control samples (52 and 45%, respectively; P < 0.04). In conclusion, sperm stored for 8 h without seminal plasma had greater resolution between X- and Y-bearing populations and higher post-thaw motility than control sperm. However, these samples had a higher percentage of membrane-impaired sperm that were removed during sorting. Long-term storage of sperm in their seminal plasma before sex-sorting appears to be detrimental to post-sorting, post-thaw sperm motility.

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Short-term liquid storage of ram semen in various extenders

South African Journal of Animal Science ◽

10.4314/sajas.v48i4.13 ◽

1970 ◽

Vol 48 (4) ◽

pp. 717-723

Author(s):

N. Maksimović ◽

A. Milovanović ◽

T. Barna ◽

V. Caro Petrović ◽

V. Pantelić ◽

...

Keyword(s):

Sperm Motility ◽

Sperm Morphology ◽

Sperm Quality ◽

Animal Husbandry ◽

Field Analysis ◽

Blue Staining ◽

Computer Assisted ◽

Short Term ◽

Sperm Analysis ◽

Liquid Storage

The aim of this study was to investigate the effects of three extenders on ram sperm quality after short-term liquid storage (24 hours’ holding time). The study included 20 crossbred rams (Pirot Pramenka x Wurttemberg x Ile de France), 12 months old. Animals were housed at the experimental sheep farm of the Institute for Animal Husbandry in Belgrade, Serbia. Semen was collected through electro ejaculation. The ejaculates were obtained from single services and routine field analysis of the semen was performed immediately after the collection. The semen was split and diluted with three extenders, namely Optidyl®, Andromed® and ultrahigh temperature processed (UHT) milk, in ratios of 1 : 50 or 1 : 100. The ejaculates were examined for sperm motility variables (sperm cell motility percentage, the progressive motility percentage, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), sperm linearity (LIN), straightness (STR), amplitude of lateral sperm head displacement (ALH), beat cross frequency (BCF) and circular tracks), and sperm morphology (live sperm percentage, percentage of normal sperm forms with intact acrosome, percentage of abnormal sperm forms and total damaged acrosome) by computer-assisted sperm analysis (CASA) and classic sperm cytology after supravital eosin/nigrosine/trypan blue staining, respectively. It was observed that the type of extender used in diluting ram semen is an important factor in the successful short-term liquid preservation (at 4 °C) of ram spermatozoa. In conclusion, this study showed that egg yolk (Optidyl) and soybean (Andromed)-based extenders gave better results of both sperm morphology and sperm motility parameters compared with UHT milk.Keywords: Diluents, morphology, motility, sperm

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Impact of Seminal Plasma Trace Elements on Human Sperm Motility Parameters

Romanian Journal of Internal Medicine ◽

10.1515/rjim-2017-0034 ◽

2018 ◽

Vol 56 (1) ◽

pp. 15-20 ◽

Cited By ~ 3

Author(s):

Mohammad Mostakhdem Hashemi ◽

Nasser Behnampour ◽

Mojgan Nejabat ◽

Afsaneh Tabandeh ◽

Behrouz Ghazi-Moghaddam ◽

...

Keyword(s):

Trace Elements ◽

Sperm Motility ◽

Seminal Plasma ◽

Sperm Quality ◽

Semen Analysis ◽

Sperm Concentration ◽

Negative Relationship ◽

Human Sperm ◽

Computer Assisted ◽

Human Seminal Plasma

Abstract Introduction. Human seminal plasma contains a variety of macro and trace elements including magnesium (Mg), copper (Cu), zinc (Zn), and iron (Fe) that have essential roles in normal functioning of semen and its quality. The imbalance of these elements has been reported in several pathologic and male infertility disorders. Therefore, this study aimed to determine the levels of these elements in seminal plasma samples, their relationships with each other and their impact on sperm motility. Methods. Overall, 192 males (96 normospermic and 96 asthenospermic males) were enrolled in the study. Semen samples were collected by masturbation and computer-assisted/aided semen analysis of sperm motility was performed. The samples were centrifuged and seminal levels of Mg, Cu, Zn and Fe were measured using atomic absorption spectroscopy. Results. The levels of Zn did not differ between the two groups, while the levels of Mg, Cu, and Fe were significantly higher in normospermic males. Fe showed a positive correlation with Mg and Cu in asthenospermic group. However, a negative relationship was found between Mg and Fe levels and between Mg and sperm concentration in the normospermic group. Fe levels were higher in the normospermic group compared to the asthenospermic group. Nevertheless, increased Fe levels caused a decrease in most of sperm motility fractions. Conclusion: Elements play major roles in male fertility and directly affect sperm quality. According to the results of this study, the levels of Zn do not affect the sperm quality and motility, while Fe, Cu and Mg are decreased in males with sperm motility problems. Nevertheless, Fe levels can adversely affect sperm motility in normospermic men.

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Paternal effect on embryo quality in diabetic mice is related to poor sperm quality and associated with decreased glucose transporter expression

Reproduction ◽

10.1530/rep-08-0167 ◽

2008 ◽

Vol 136 (3) ◽

pp. 313-322 ◽

Cited By ~ 60

Author(s):

Sung Tae Kim ◽

Kelle H Moley

Keyword(s):

Glucose Transporter ◽

Sperm Quality ◽

Sperm Concentration ◽

Diabetic Mouse ◽

Blastocyst Stage ◽

Computer Assisted ◽

Diabetic Mice ◽

Sperm Analysis ◽

Fertilization Capacity

The objective of this study was to determine whether sperm quality, fertilization capacity, and subsequent embryo development are altered in diabetic male mice and whether differences in facilitative glucose transporter (GLUT; now known as solute carrier family 2, SLC2A) expression in the testis and sperm exist. Using two type 1 diabetic mouse models, SLC2A expression in the testis and sperm was determined by western immunoblotting and immunofluorescence staining. To address sperm quality and fertilization capacity, computer-assisted sperm analysis andin vitrofertilization were performed. SLC2A1, SLC2A3, and SLC2A5 did not change in expression in the testes or sperm between diabetic and non-diabetic mice. SLC2A8 and SLC2A9b were less expressed in the testes of both diabetic models versus controls. SLC2A9a was not expressed in the Akita testis or sperm when compared with strain-matched controls. 3β-hydroxysteroid dehydrogenase (HSD3B) expression was significantly decreased in the Leydig cells from the diabetic mice. Sperm concentration and motility were significantly lower in both the diabetics when compared with the control. These parameters normalized in Akita diabetic males treated with insulin. In addition, fertilization rates were significantly lower in the Akita group (17.9%) and the streptozotocin (STZ)-injected male group (43.6%) when compared with the normal group (88.8%). Interestingly, of the fertilized zygotes, embryo developmental rates to the blastocyst stage were lower in both diabetic models (7.1% Akita and 50.0% STZ) when compared with controls (71.7%). Male diabetes may cause male subfertility by altering steroidogenesis, sperm motility, and SLC2A expression. This is the first study to link a paternal metabolic abnormality to a sperm effect on cell division and subsequent embryonic development.

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Expanding the Limits of Computer-Assisted Sperm Analysis through the Development of Open Software

Biology ◽

10.3390/biology9080207 ◽

2020 ◽

Vol 9 (8) ◽

pp. 207

Author(s):

Jesús Yániz ◽

Carlos Alquézar-Baeta ◽

Jorge Yagüe-Martínez ◽

Jesús Alastruey-Benedé ◽

Inmaculada Palacín ◽

...

Keyword(s):

Open Source ◽

Sperm Quality ◽

Sperm Concentration ◽

Control Group ◽

Computer Assisted ◽

Sperm Analysis ◽

Potential Applications ◽

Computer Assisted Sperm Analysis ◽

High Level ◽

Almost All

Computer assisted sperm analysis (CASA) systems can reduce errors occurring in manual analysis. However, commercial CASA systems are frequently not applicable at the forefront of challenging research endeavors. The development of open source software may offer important solutions for researchers working in related areas. Here, we present an example of this, with the development of three new modules for the OpenCASA software (hosted at Github). The first is the Chemotactic Sperm Accumulation Module, a powerful tool for studying sperm chemotactic behavior, analyzing the sperm accumulation in the direct vicinity of the stimuli. This module was validated by comparing fish sperm accumulation, with or without the influence of an attractant. The analysis clearly indicated cell accumulation in the treatment group, while the distribution of sperm was random in the control group. The second is the Sperm Functionality Module, based on the ability to recognize five sperm subpopulations according to their fluorescence patterns associated with the plasma membrane and acrosomal status. The last module is the Sperm Concentration Module, which expands the utilities of OpenCASA. These last two modules were validated, using bull sperm, by comparing them with visual counting by an observer. A high level of correlation was achieved in almost all the data, and a good agreement between both methods was obtained. With these newly developed modules, OpenCASA is consolidated as a powerful free and open-source tool that allows different aspects of sperm quality to be evaluated, with many potential applications for researchers.

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Sperm Quality Assessment in Honey Bee Drones

Biology ◽

10.3390/biology9070174 ◽

2020 ◽

Vol 9 (7) ◽

pp. 174

Author(s):

Jesús L. Yániz ◽

Miguel A. Silvestre ◽

Pilar Santolaria

Keyword(s):

Honey Bee ◽

Multiple Testing ◽

Semen Quality ◽

Sperm Quality ◽

Sperm Concentration ◽

Membrane Integrity ◽

Domestic Animal ◽

Computer Assisted ◽

Sperm Analysis ◽

Sperm Volume

The quality of honey bee drone semen is relevant in different contexts, ranging from colony productivity to pathology, toxicology and biodiversity preservation. Despite its importance, considerably less knowledge is available on this subject for the honey bee when compared to other domestic animal species. A proper assessment of sperm quality requires a multiple testing approach which discriminates between the different aspects of sperm integrity and functionality. Most studies on drone semen quality have only assessed a few parameters, such as sperm volume, sperm concentration and/or sperm plasma membrane integrity. Although more recent studies have focused on a broader variety of aspects of semen quality, some techniques currently used in vertebrates, such as computer-assisted sperm analysis (CASA) or multiparametric sperm quality testing, still remain to be developed in the honey bee. This may be attributed to the particular sperm morphology and physiology in this species, requiring the development of technologies specifically adapted to it. This article reviews the present knowledge of sperm quality in honey bee drones, highlighting its peculiarities and proposing future lines of research.

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Post-Thaw Sperm Quality and Functionality in the Autochthonous Pig Breed Gochu Asturcelta

Animals ◽

10.3390/ani11071885 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1885

Author(s):

José Néstor Caamaño ◽

Carolina Tamargo ◽

Inmaculada Parrilla ◽

Felipe Martínez-Pastor ◽

Lorena Padilla ◽

...

Keyword(s):

Genetic Resource ◽

Sperm Quality ◽

Genetic Material ◽

Mitochondrial Activity ◽

Computer Assisted ◽

Kinematic Parameters ◽

Linear Mixed Effects ◽

Sperm Analysis ◽

Computer Assisted Sperm Analysis ◽

Analysis System

Genetic resource banks (GRB) preserve the genetic material of endangered, valuable individuals or genetically relevant breeds. Semen cryopreservation is a crucial technique to reach these goals. Thus, we aimed to assess the sperm parameters of semen doses from the native pig breed Gochu Asturcelta stored at the GRB of Principado de Asturias (GRB-PA, Gijón, Spain), focusing on intrinsic and extrinsic (boar, season) factors. Two straws per boar (n = 18, 8–71 months of age) were thawed, pooled, and assessed after 30 and 150 min at 37 °C by CASA (computer-assisted sperm analysis system; motility and kinematic parameters) and flow cytometry (viability, acrosomal status, mitochondrial activity, apoptosis, reactive oxygen species, and chromatin status). The effects of age, incubation, and season on post-thawing quality were determined using linear mixed-effects models. Parameters were on the range for commercial boar breeds, with chromatin status (SCSA: fragmentation and immaturity) being excellent. Incubation decreased sperm quality and functionality. The boar age did not have a significant effect (p > 0.05), but the between-boar variability was significant (p < 0.001). The season significantly affected many parameters (motility, kinematics, viability, acrosomal status, mitochondrial activity), especially after 150 min of incubation. In general, samples collected in spring and summer showed higher quality post-thawing, the lowest in winter. In conclusion, the sperm doses from the Gochu Asturcelta breed stored at the GRB-PA showed excellent chromatin status and acceptable characteristics after thawing. Therefore, boar and seasonal variability in this autochthonous breed could be relevant for cryobank management.

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Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer

Zygote ◽

10.1017/s096719941600037x ◽

2016 ◽

Vol 25 (1) ◽

pp. 85-97 ◽

Cited By ~ 7

Author(s):

María Elena Arias ◽

Esther Sánchez-Villalba ◽

Andrea Delgado ◽

Ricardo Felmer

Keyword(s):

Gene Transfer ◽

Sperm Quality ◽

Computer Assisted ◽

Exogenous Dna ◽

Sperm Analysis ◽

Functional Parameters ◽

Transgenic Embryos ◽

Fertilization Capacity ◽

Motility Parameters

SummarySperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) orin vitrofertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

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Absence of seminal plasma from sperm-rich fraction decreases boar sperm quality characteristics during the course of liquid storage

Animal Reproduction Science ◽

10.1016/j.anireprosci.2018.08.029 ◽

2018 ◽

Vol 198 ◽

pp. 20-26 ◽

Cited By ~ 3

Author(s):

D.F. Leal ◽

M.A. Torres ◽

G.M. Ravagnani ◽

S.M.M.K. Martins ◽

F.V. Meirelles ◽

...

Keyword(s):

Seminal Plasma ◽

Sperm Quality ◽

Quality Characteristics ◽

Liquid Storage ◽

Boar Sperm

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Induced hyperactivity in boar spermatozoa and its evaluation by computer-assisted sperm analysis

Reproduction ◽

10.1530/rep.1.00153 ◽

2004 ◽

Vol 128 (2) ◽

pp. 171-179 ◽

Cited By ~ 56

Author(s):

Harald Schmidt ◽

Günter Kamp

Keyword(s):

Seminal Plasma ◽

Roc Curve ◽

Curve Analysis ◽

Computer Assisted ◽

Ionophore A23187 ◽

Threshold Values ◽

Roc Curve Analysis ◽

Sperm Analysis ◽

Computer Assisted Sperm Analysis ◽

Boar Spermatozoa

Hyperactivity, a form of sperm motility characterized by vigorous flagellar movements, has been proposed as essential for fertilization in mammals. The objective of the present study was to establish a method for inducing hyperactivityin vitroin boar spermatozoa and to define threshold values to differentiate between hyperactive and non-hyperactive spermatozoa by computer-assisted sperm analysis (CASA) as a prerequisite for analyzing the energy metabolism during hyperactivity. In TALP-HEPES medium, non-frozen boar spermatozoa were stimulated to hyperactivity by 50 μmol l−1Ca2+within 15 min at 37 °C if 5 μmol l−1of the Ca2+ionophore A23187 was present. If 25% seminal plasma was present, boar spermatozoa required higher Ca2+concentrations (about 700 μmol l−1) for hyperactivity. Under both conditions, immobilization and head-to-head agglutination were low so that hyperactive spermatozoa could be analyzed for at least 40 min. The transition from normal to hyperactive movement was characterized by an increase in flagellar beat angle from 49° ± 12° to 200° ± 36° (n= 32) and a decrease in flagellar curvature ratio from 0.89 ± 0.04 to 0.47 ± 0.11 (n= 32). For quantification of hyperactive boar sperm, kinematic parameters of hyperactive and non-hyperactive spermatozoa were measured by CASA and statistically evaluated (receiver operating characteristic (ROC) curve analysis). The threshold values of the following four parameters were well suited for differentiating between hyperactive and non-hyperactive boar spermatozoa (ROC curve analysis: >50% specificity at 100% sensitivity). Hyperactive boar spermatozoa showed mean lateral head displacement >3.5 μm, curvilinear velocity >97 μm s−1, linearity <32% and wobble <71%. According to this multiparametric definition, induction of hyperactivity increased significantly (P< 0.0001) the fraction of hyperactive spermatozoa in semen samples from 5.1 ± 4.3% (n= 13) to 48.3 ± 6.6% (n= 7) in the absence and to 44.2 ± 7.6% (n= 10) in the presence of 25% seminal plasma, while the overall percentage of motile spermatozoa did not change significantly.

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