Seminal plasma arising from the whole boar sperm-rich fraction increases the stability of sperm membrane after thawing1,2

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

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Porcine zona pellucida ZP3? glycoprotein mediates binding of the biotin-labeled Mr 55,000 family (ZP3) to boar sperm membrane vesicles

Molecular Reproduction and Development ◽

10.1002/mrd.1080360315 ◽

1993 ◽

Vol 36 (3) ◽

pp. 382-389 ◽

Cited By ~ 43

Author(s):

Edward C. Yurewicz ◽

Beverley A. Pack ◽

D. Randall Armant ◽

Anthony G. Sacco

Keyword(s):

Zona Pellucida ◽

Membrane Vesicles ◽

Boar Sperm ◽

Sperm Membrane

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Adición de proteínas del plasma seminal ovino durante la congelación del espermatozoide y efectos sobre su motilidad y viabilidad

Ciencia y Tecnología Agropecuaria ◽

10.21930/rcta.vol10_num1_art:128 ◽

2009 ◽

Vol 10 (1) ◽

pp. 51 ◽

Cited By ~ 2

Author(s):

Jaime Antonio Cardozo ◽

Patricia Grasa ◽

María Teresa Muiño B. ◽

José Álvaro Cebrián P.

Keyword(s):

Membrane Protein ◽

Sperm Motility ◽

Seminal Plasma ◽

Gel Electrophoresis ◽

Plasma Proteins ◽

Two Dimensional ◽

Plasma Séminal ◽

Sperm Membrane ◽

Seminal Plasma Proteins ◽

Ram Sperm

Este estudio se adelantó para evaluar el efecto de la adición de proteínas del plasma seminal de cordero en la criopreservación sobre la motilidad e integridad de la membrana espermática, y los cambios en el perfil electroforético de las proteínas de la membrana espermática inducidos por la criopreservación. Se usaron eyaculados de ocho corderos adultos de la raza rasa aragonesa, se les determinó su viabilidad y motilidad espermáticas y posteriormente se sometieron a un procedimiento de congelación. Las proteínas se separaron por el método de electroforesis en geles de acrilamida en dos dimensiones. Se obtuvo un mejoramiento significativo (p < 0,05) en la calidad del semen congelado, cuando se adicionaron proteínas del plasma seminal. El análisis bidimensional comparativo entre el semen fresco y el congelado evidenció la pérdida de 8 puntos de proteína en el espermatozoide descongelado. La concentración de un punto de proteína de membrana espermática, de bajo peso molecular (punto 2), fue más alta (p < 0,05) en el espermatozoide descongelado al que se adicionaron proteínas del plasma seminal. Se encontraron correlaciones entre algunos puntos de proteína y la motilidad y viabilidad espermáticas, lo cual sugiere que pueden jugar papeles importantes en el mantenimiento de la integridad y funcionalidad del espermatozoide. Se puede concluir que la adición de proteínas del plasma seminal en la congelación mejora la integridad del espermatozoide descongelado, y que la criopreservación del semen de cordero produce variaciones en la composición de las proteínas de membrana. Effect of seminal plasma proteins at freezing on ram sperm motility and viability The aim of the study was to evaluate the cryoprotective effect of seminal plasma proteins on ram sperm motility, membrane integrity and the changes in the profile of ram sperm membrane proteins induced by cryopreservation. Fresh ejaculates from 8 mature Rasa aragonesa rams were used. Sperm motility and cell viability was assessed. The freezing procedure was based on the method described by Fiser et al. (1987). Proteins extracted from fresh and frozen-thawed semen were subjected to the Two-dimensional polyacrilamide gel electrophoresis. A significant improvement in the quality of frozenthawed sperm was obtained after addition of seminal plasma proteins (p < 0.05). Comparative two-dimensional polyacrilamide gel electrophoresis analysis between fresh and frozen semen, either with or without seminal plasma proteins in the cryopreservation medium, revealed that eight protein spots were lost in frozen-thawed sperm. The concentration of one sperm membrane protein spot of low Mr (spot 2) was higher (p < 0.05) in proteinadded frozen sperm. Correlations found between certain protein spots sperm motility and viability suggests that these proteins could play important roles in the maintenance of sperm integrity and functionality. In conclusion, the addition of seminal plasma proteins to freezing extender improved frozen-thawed ram sperm integrity quality and cryopreservation of ram semen produced variations in the sperm membrane protein composition.

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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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Sperm Proteome after Interaction with Reproductive Fluids in Porcine: From the Ejaculation to the Fertilization Site

International Journal of Molecular Sciences ◽

10.3390/ijms21176060 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6060 ◽

Cited By ~ 1

Author(s):

Chiara Luongo ◽

Leopoldo González-Brusi ◽

Paula Cots-Rodríguez ◽

Mª José Izquierdo-Rico ◽

Manuel Avilés ◽

...

Keyword(s):

Seminal Plasma ◽

Proteome Analysis ◽

Interacting Proteins ◽

Sperm Transport ◽

Two Fluids ◽

Boar Sperm ◽

Uterine Fluid ◽

Proteome Changes ◽

Oviductal Fluid

Ejaculated sperm are exposed to different environments before encountering the oocyte. However, how the sperm proteome changes during this transit remains unsolved. This study aimed to identify proteomic changes in boar sperm after incubation with male (seminal plasma, SP) and/or female (uterine fluid, UF; and oviductal fluid, OF) reproductive fluids. The following experimental groups were analyzed: (1) SP: sperm + 20% SP; (2) UF: sperm + 20% UF; (3) OF: sperm + 20% OF; (4) SP + UF: sperm + 20% SP + 20% UF; and (5) SP+OF: sperm + 20% SP + 20% OF. The proteome analysis, performed by HPLC-MS/MS, allowed the identification of 265 proteins. A total of 69 proteins were detected in the UF, SP, and SP + UF groups, and 102 proteins in the OF, SP, and SP + OF groups. Our results showed a higher number of proteins when sperm were incubated with only one fluid than when they were co-incubated with two fluids. Additionally, the number of sperm-interacting proteins from the UF group was lower than the OF group. In conclusion, the interaction of sperm with reproductive fluids alters its proteome. The description of sperm-interacting proteins in porcine species after co-incubation with male and/or female reproductive fluids may be useful to understand sperm transport, selection, capacitation, or fertilization phenomena.

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67 EFFECTS OF ANTIOXIDANTS LACTOFERRIN AND CATALASE ON STALLION FROZEN SEMEN

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab67 ◽

2015 ◽

Vol 27 (1) ◽

pp. 126

Author(s):

H. S. Martins ◽

M. F. Brito ◽

I. B. M. Sampaio ◽

R. Stahlberg ◽

M. R. Souza ◽

...

Keyword(s):

Sperm Motility ◽

Seminal Plasma ◽

Skim Milk ◽

Straight Line ◽

Frozen Semen ◽

Significant Difference ◽

Sperm Membrane ◽

Positive Effect ◽

Artificial Vagina ◽

Motility Parameters

During cryopreservation, the sperm were submitted to an increased generation of reactive oxygen species. Furthermore, because of the large portion of seminal plasma removal, there is a decrease of sperm antioxidant protection. Addition of antioxidants proteins found in seminal plasma, such as lactoferrin (Lf) and catalase (Cat), to the freezing semen extenders could protect the sperm during cryopreservation. Lactoferrin is a transferrin, which prevents the hydroxyl radicals generation, and Cat plays an antioxidant role. The aim of this study was to determine the effects of Lf and Cat supplementation to the INRA 82 freezing extender (Battelier et al. 1997) on sperm motility parameters and membrane functionality of stallion frozen semen. Semen from 6 stallions was collected with an artificial vagina, diluted with Kenney extender (1 : 1), and centrifuged (500 × g, 10 min). The supernatant was discarded, and sperm number per milliliter was calculated. Semen was resuspended with 3 extenders to 100 × 106 sperm mL–1. The treatments were distributed in (F1) control, INRA 82 freezing extender (Battelier et al. 1997), (F2) F1+ 500 μg mL–1 lactoferrin, and F3) F1 + 200 IU mL–1 catalase. Semen samples were packaged in 0.5-mL straws and cooled to 5°C (0.27°C min–1). For semen freezing, the straws were laid over the LN vapor for 20 min and plunged into the LN. The straws were thawed at 37°C for 30 s. Motility parameters of frozen semen were determined using a computer sperm cell analysis, and sperm membrane functionality was assessed by the hyposmotic swelling test (Lagares et al. 1998). The data were analysed using Friedman test using stallion as a block. A probability of P < 0.05 was considered significant. There was no significant difference between the percentage of total sperm motility (median, minimum-maximum value; F1: 29.9, 11.0–82.7; F2: 49.8, 7.7–55.2; F3: 39.8, 5.7–92) and progressive sperm motility (F1: 7.1, 3.2–23.3; F2: 13.4, 2.6-22.4; F3: 15.6, 1.1–29.6), and functional sperm membrane (F1: 26.7, 14.7–56.2; F2: 50.5, 15.7–61.7; F3: 46.6, 13.8–50.9) with regard to the treatment. However, the velocity parameters: velocity average path (F1: 29.3, 22.1–33.80; F2: 34.6, 24.8–44; F3: 35.7, 18.2–42.6), velocity curvilinear (F1: 36.9, 30.5–45.1; F2: 42.5, 34.7–51; F3: 44.6, 25.5–50.9), and velocity straight line (F1: 23.4, 17–3.60; F2: 28.9, 18.8–38.2; F3: 26.6, 13.6–37.2) in the treatment with Lf (F2) were higher compared with the control (F1; P < 0.05). These results corroborate with studies reporting the lack of positive effect on equine sperm motility when antioxidants were added to skim milk-based extenders. Although the addition of Lf or Cat to skim milk-based extenders did not improve the motility sperm characteristics and sperm membrane functionality, more studies about the positive effect of Lf on the velocity parameters are necessary. Lactoferrin could then play an important role on the oxidative metabolism, which provides energy to the sperm movement.Acknowledgments to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, for the financial support.

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Seminal Plasma Cytokines Are Predictive of the Outcome of Boar Sperm Preservation

Frontiers in Veterinary Science ◽

10.3389/fvets.2019.00436 ◽

2019 ◽

Vol 6 ◽

Cited By ~ 6

Author(s):

Isabel Barranco ◽

Lorena Padilla ◽

Cristina Pérez-Patiño ◽

Juan M. Vazquez ◽

Emilio A. Martínez ◽

...

Keyword(s):

Seminal Plasma ◽

Boar Sperm ◽

Plasma Cytokines

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Proteins and Heparin Binding Proteins of Spermatozoa and Seminal Plasma in Beetal Bucks: Purification, Characterization and Role in In vivo Fertility

Indian Journal of Animal Research ◽

10.18805/ijar.b-3974 ◽

2020 ◽

Author(s):

Ranjna S. Cheema ◽

Navjot S. Dhillon ◽

Sumit Singhal

Keyword(s):

Seminal Plasma ◽

Binding Proteins ◽

Proteome Analysis ◽

Fertility Rate ◽

Heparin Binding ◽

Sds Page ◽

Sperm Extract ◽

Sperm Membrane ◽

Special Relevance

Background: The proteome analysis of seminal plasma and spermatozoa is of special relevance in livestock. Heparin binding proteins (HBPs) found in the seminal plasma of several mammals are shown to bind to sperm membrane and affect a series of events that contribute to normal fertility, such as sperm capacitation, formation of the oviduct reservoir and binding to the oocyte. Profiles of HBPs from seminal plasma and sperm membranes have been associated with sperm fertility. Although, HBPs present in the SP are described in several species, but little is known about HBPs in buck. Methods: Seminal plasma (SP) and sperm extracts (SE) of 13 bucks were subjected to heparin-sepharose affinity chromatography. Sperm extract, seminal plasma and purified HBPs and Non-HBPs were fractionated by SDS-PAGE. Total 78 females (6 per buck) were mated with 13 bucks. Bucks were divided into two groups, G-I (high fertile, 83.3-100% FR) and G-II (low fertile, 50-66.7% FR). Relationship between HBPs and fertility rate was observed. Result: SDS – PAGE of SP and SE resulted in resolution of 22 (10-240 kDa) and 21 (10-270 kDa) bands, respectively. Based on fertility rate 15 and 13 kDa proteins were absent in SP of higher number of GI-compared to G-II bucks. Fourteen bands ranging from 10 – 180 kDa and 10 – 150 kDa were separated from SP-NHBP and SP-HBP. SP-HBPs of 75, 35, 30, 28, 25 and 13 kDa were present in higher (28.6%, 42.5%, 26.2%, 40.5%, 14.3% and 36.2%) number of high fertile than low fertile bucks. NHBP and HBP purified from SE resolved into 11 bands ranging from 10 – 135 kDa and 10 – 120 kDa, respectively. SE-HBP of 53 kDa, 50/45 kDa and 25 kDa were present in higher percentage of high fertile than low fertile bucks.

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80 CHOLESTEROL-LOADED METHYL-BETA-CYCLODEXTRIN IMPROVES BOAR SPERMATOZOA CRYOPROTECTANT ADDITION AND REMOVAL TOLERANCE

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab80 ◽

2009 ◽

Vol 21 (1) ◽

pp. 140

Author(s):

E. M. Walters ◽

J. D. Benson ◽

A. Rieke ◽

J. K. Graham ◽

J. K. Critser

Keyword(s):

Sperm Motility ◽

Membrane Integrity ◽

Tolerance Limits ◽

Permeability Characteristics ◽

Flow Cytometry Data ◽

Osmotic Tolerance ◽

Motile Cells ◽

Boar Sperm ◽

Chilling Sensitivity ◽

Sperm Membrane

Difficulties associated with the cryopreservation of boar sperm include their sensitivities to osmotic stresses and chilling sensitivity. We investigated the effects of cholesterol-loaded cyclodextrin (CLC) on boar sperm motility and membrane integrity following exposure to various osmolalities. Samples were collected using the gloved hand method from crossbred boars, and ejaculates having greater than 75% motility were extended 1:3 with Androhep (Minitube Inc., Verona, WI) for this study. Samples were centrifuged at 700g for 5 min, and the resulting pellets were resuspended to 1.2 × 108 cells mL–1 in Androhep. Samples were then treated with 0, 1.5, or 3.0 mg of CLC/1.2 × 108 cells mL–1 for 10 min at room temperature. In experiment 1, samples were aliquoted into 1.5-mL centrifuge tubes, centrifuged at 700g for 5 min and the sperm exposed to Dulbecco’s PBS at different osmolalities for 5 min before being returned to 300 mOsm by adding Dulbecco’s PBS solutions at differing osmolalities. After returning the sperm to isosmotic conditions, sperm motility was analyzed. In experiment 2, samples were treated as in experiment 1 and following exposure to the various osmolalities, sperm were stained with Alexa 488-PNA and propidium iodide to determine sperm membrane integrity. Ten thousand sperm per treatment were analyzed by flow cytometry. Data were analyzed by standard ANOVA. The CLC-treated sperm (normalized means ± SEM; 33 ± 16, 80 ± 8, 86 ± 5, 100, 64 ± 4, 7 ± 3, 0 ± 0, respectively) exhibited greater percentages of motile cells following hypo-isosmotic exposure than control sperm (4 ± 1.6, 33 ± 9.6, 84 ± 7.1, 100, 37 ± 5.5, 3 ± 1.6, 0 ± 0, respectively), and there was a tendency for CLC-treated sperm (P = 0.0225) to maintain motility following hyper-isosmotic exposure. In addition, CLC-treated sperm (87 ± 4, 93 ± 1, 95 ± 1, 93 ± 2, 88 ± 4, 83 ± 3, 41 ± 9, respectively; P < 0.05) maintained greater percentages of membrane integrity following treatment with anisosmotic solutions compared with controls (29 ± 8, 63 ± 10, 81 ± 7, 92 ± 3, 73 ± 8, 44 ± 5, 21 ± 9, respectively). Using a combination of these osmotic tolerance data with previously published boar sperm membrane permeability characteristics, we mathematically modeled the number of steps needed for the addition or removal of cryoprotectants. Computer simulations indicate that an abrupt addition of 1 m glycerol will cause boar sperm to exceed their osmotic tolerance limits unless they are treated with 3 mg of CLC. Moreover, the addition of 1 m EG causes boar sperm to exceed all osmotic tolerance limits and therefore, the addition and removal of EG requires multiple-step protocols. However, the addition and removal of 1 m DMSO maintains volume excursions well within the osmotic tolerance limits with the addition of cholesterol (1.5 and 3 mg). Empirical data for addition of CPA have shown similar results as seen with the computer simulation. These data support the hypothesis that adding cholesterol to porcine sperm broadens their osmotic tolerance limits and potentially provide a mechanism to increase post-thaw survival of porcine sperm.

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Seminal Plasma Osteopontin: A Marker for Potential Fertility in Dogs

Indian Journal of Animal Research ◽

10.18805/ijar.b-4131 ◽

2020 ◽

Author(s):

S.N. Abedin ◽

V. Leela ◽

P. Devendran ◽

G. Suganya ◽

S. Rangasamy ◽

...

Keyword(s):

Seminal Plasma ◽

Matrix Protein ◽

Reproductive Tract ◽

Linear Regression Analysis ◽

Domestic Animals ◽

Extracellular Matrix Protein ◽

Group I ◽

Sperm Membrane ◽

Males And Females ◽

Breeding Soundness

Background: Osteopontin (OPN) is an extracellular matrix protein with multifunctions and has been identified in the reproductive tract of both males and females. OPN from the seminal plasma (SP) has been closely associated with male fertility in several domestic animals. Different isoforms of OPN has been identified in the SP and sperm membrane in dogs but with unknown functions. The current study investigated the comparison of key canine sperm microscopic parameters and SP markers between dogs exhibiting different fertility potential.Methods: In this investigation during 2018-2019, 21 dogs coming for breeding soundness examination were selected and divided into fertile (Group I) and hypofertile (Group II). Evaluation included assessment of sperm microscopic characteristics and estimation of OPN, Malondialdehyde (MDA), Catalase (CAT), Glutathione peroxidase (GPx) and Superoxide dismutase (SOD) concentration in the SP.Result: The OPN level showed positive correlation with all the sperm evaluated characteristics (p less than 0.01). Linear regression analysis depicted a significant (p less than 0.001) influence of OPN on the evaluated sperm characteristics. These findings suggest that OPN in the SP along with the microscopic characteristics of the spermatozoa can be used as a potential fertility marker in discriminating fertile from the hypofertile ones.

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