Nanotechnology-based approach for safer enrichment of semen with best spermatozoa
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摘要
Background: Advances in nanotechnology have permitted molecular-based targeting of cells through safe and biocompatible magnetic nanoparticles(MNP). Their use to detect and remove damaged spermatozoa from semen doses could be of great interest. Here, MNP were synthesized and tested for their ability to target apoptotic(annexin V) and acrosome-reacted(lectin) boar spermatozoa, for high-throughout retrieval in a magnetic field(nanoselection). The potential impacts of nanoselection on sperm functions and performance of offspring sired by sperm subjected to nanoselection were determined. Fresh harvested and extended boar semen was mixed with various amounts(0, 87.5, and 175 μg) of MNP-conjugates(Annexin V-MNP or Lectin-MNP) and incubated(10 to15 min) for 37 °C in Exp. 1. In Exp. 2, extended semen was mixed with optimal concentrations of MNP-conjugates and incubated(0, 30, 90, or 120 min). In Exp. 3, the synergistic effects of both MNP-conjugates(87.5 μg– 30 min)on spermatozoa was evaluated, followed by sperm fertility assessments through pregnancy of inseminated gilts and performance of neonatal offspring. Sperm motion, viability, and morphology characteristics were evaluated in all experiments.Results: Transmission electron microscopy, atomic force microscopy, and hyperspectral imaging techniques were used to confirm attachment of MNP-conjugates to damaged spermatozoa. The motility of nanoselected spermatozoa was improved(P < 0.05). The viability of boar sperm, as assessed by the abundance of reactive oxygen species and the integrity of the acrosome, plasma membrane, and mitochondrial membrane was not different between nanoselected and control spermatozoa. The fertility of gilts inseminated with control or nanoselected spermatozoa, as well as growth and health of their offspring were not different between(P > 0.05).Conclusions: The findings revealed the benefit of magnetic nanoselection for high-throughput targeting of damaged sperm, for removal and rapid and effortless enrichment of semen doses with highly motile, viable,and fertile spermatozoa. Therefore, magnetic nanoselection for removal of abnormal spermatozoa from semen is a promising tool for improving fertility of males, particularly during periods, such as heat stress during the summer months.
Background: Advances in nanotechnology have permitted molecular-based targeting of cells through safe and biocompatible magnetic nanoparticles(MNP). Their use to detect and remove damaged spermatozoa from semen doses could be of great interest. Here, MNP were synthesized and tested for their ability to target apoptotic(annexin V) and acrosome-reacted(lectin) boar spermatozoa, for high-throughout retrieval in a magnetic field(nanoselection). The potential impacts of nanoselection on sperm functions and performance of offspring sired by sperm subjected to nanoselection were determined. Fresh harvested and extended boar semen was mixed with various amounts(0, 87.5, and 175 μg) of MNP-conjugates(Annexin V-MNP or Lectin-MNP) and incubated(10 to15 min) for 37 °C in Exp. 1. In Exp. 2, extended semen was mixed with optimal concentrations of MNP-conjugates and incubated(0, 30, 90, or 120 min). In Exp. 3, the synergistic effects of both MNP-conjugates(87.5 μg – 30 min)on spermatozoa was evaluated, followed by sperm fertility assessments through pregnancy of inseminated gilts and performance of neonatal offspring. Sperm motion, viability, and morphology characteristics were evaluated in all experiments.Results: Transmission electron microscopy, atomic force microscopy, and hyperspectral imaging techniques were used to confirm attachment of MNP-conjugates to damaged spermatozoa. The motility of nanoselected spermatozoa was improved(P < 0.05). The viability of boar sperm, as assessed by the abundance of reactive oxygen species and the integrity of the acrosome, plasma membrane, and mitochondrial membrane was not different between nanoselected and control spermatozoa. The fertility of gilts inseminated with control or nanoselected spermatozoa, as well as growth and health of their offspring were not different between(P > 0.05).Conclusions: The findings revealed the benefit of magnetic nanoselection for high-throughput targeting of damaged sperm, for removal and rapid and effortless enrichment of semen doses with highly motile, viable,and fertile spermatozoa. Therefore, magnetic nanoselection for removal of abnormal spermatozoa from semen is a promising tool for improving fertility of males, particularly during periods, such as heat stress during the summer months.
Background: Advances in nanotechnology have permitted molecular-based targeting of cells through safe and biocompatible magnetic nanoparticles(MNP). Their use to detect and remove damaged spermatozoa from semen doses could be of great interest. Here, MNP were synthesized and tested for their ability to target apoptotic(annexin V) and acrosome-reacted(lectin) boar spermatozoa, for high-throughout retrieval in a magnetic field(nanoselection). The potential impacts of nanoselection on sperm functions and performance of offspring sired by sperm subjected to nanoselection were determined. Fresh harvested and extended boar semen was mixed with various amounts(0, 87.5, and 175 μg) of MNP-conjugates(Annexin V-MNP or Lectin-MNP) and incubated(10 to15 min) for 37 °C in Exp. 1. In Exp. 2, extended semen was mixed with optimal concentrations of MNP-conjugates and incubated(0, 30, 90, or 120 min). In Exp. 3, the synergistic effects of both MNP-conjugates(87.5 μg – 30 min)on spermatozoa was evaluated, followed by sperm fertility assessments through pregnancy of inseminated gilts and performance of neonatal offspring. Sperm motion, viability, and morphology characteristics were evaluated in all experiments.Results: Transmission electron microscopy, atomic force microscopy, and hyperspectral imaging techniques were used to confirm attachment of MNP-conjugates to damaged spermatozoa. The motility of nanoselected spermatozoa was improved(P < 0.05). The viability of boar sperm, as assessed by the abundance of reactive oxygen species and the integrity of the acrosome, plasma membrane, and mitochondrial membrane was not different between nanoselected and control spermatozoa. The fertility of gilts inseminated with control or nanoselected spermatozoa, as well as growth and health of their offspring were not different between(P > 0.05).Conclusions: The findings revealed the benefit of magnetic nanoselection for high-throughput targeting of damaged sperm, for removal and rapid and effortless enrichment of semen doses with highly motile, viable,and fertile spermatozoa. Therefore, magnetic nanoselection for removal of abnormal spermatozoa from semen is a promising tool for improving fertility of males, particularly during periods, such as heat stress during the summer months.
引文
1.Bungum M,Humaidan P,Axmon A,Spano M,Bungum L,Erenpreiss J,et al.Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome.Hum Reprod.2007;22(1):174-9.
2.Lopes S,Sun J-G,Jurisicova A,Meriano J,Casper RF.Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection.Fertil Steril.1998;69(3):4.
3.Broekhuijse M,Gaustad A,Bolarin Guillén A,Knol E.Efficient boar semen production and genetic contribution:the impact of low-dose artificial insemination on fertility.Reprod Domest Anim.2015;50(S2):103-9.
4.Foote RH.Fertility estimation:a review of past experience and future prospects.Anim Reprod Sci.2003;75(1-2):119-39.
5.Shannon P,Curson B.Toxic Effect and action of dead sperm on diluted bovine semen.J Dairy Sci.1972;55(5):614-20.
6.Sharma RK,Agarwal A.Role of reactive oxygen species in male infertility.Urology.1996;48(6):835-50.
7.Feugang JM.Novel agents for sperm purification,sorting,and Imaging.Mol Reprod Dev.2017;9999:1-10.
8.Said TM,Land JA.Effects of advanced selection methods on sperm quality and ART outcome:a systematic review.Hum Reprod Update.2011;17(6):719-33.
9.Jain S,Park SB,Pillai SR,Ryan PL,Willard ST,Feugang JM.Applications of Fluorescent Quantum Dots for Reproductive Medicine and Disease Detection.In:Unraveling the Safety Profile of Nanoscale Particles and Materials-From Biomedical to Environmental Applications.Edited by Gomes AC,Sarria MP.Rijeka:InTech;2018:Ch.06.
10.Pankhurst QA,Connolly J,Jones SK,Dobson J.Applications of magnetic nanoparticles in biomedicine.J Phys D Appl Phys.2003;36(13):R167.
11.Sutovsky P,Kennedy CE.Biomarker-based nanotechnology for the improvement of reproductive performance in beef and dairy cattle.Ind Biotechnol.2013;9(1):6.
12.Rath D,Tiedemann D,Gamrad L,Johnson L,Klein S,Kues W,et al.Sex-sorted boar sperm-an update on related production methods.Reprod Domest Anim.2015;50(S2):56-60.
13.Barkalina N,Jones C,Coward K.Nanomedicine and mammalian sperm:lessons from the porcine model.Theriogenology.2016;85(1):74-82.
14.Feugang JM,Youngblood RC,Greene JM,Willard ST,Ryan PL.Selfilluminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes.J Nanobiotechnol.2015;13(1):38.
15.Sutovsky P,Lovercamp K.Molecular markers of sperm quality.Reproduction in Domestic Ruminants VII;2011.p.247.
16.Hozaien MM,Elqusi KM,Hassanen EM,Hussin AA,Alkhader HA,El Tanbouly SM,et al.A comparison of reproductive outcome using different sperm selection techniques;density gradient,testicular sperm,PICSI,and MACS for ICSI patients with abnormal DNA fragmentation index.Fertil Steril.2018;110(4):e19-20.
17.Mortimer D.Sperm preparation methods.J Androl.2000;21(3):357-66.
18.Kaneko S,Oshio S,Kobanawa K,Kobayashi T,Mohri H,Iizuka R.Purification of human sperm by a discontinuous Percoll density gradient with an innercolumn.Biol Reprod.1986;35(4):1059-63.
19.Ericsson RJ,Langevin CN,Nishino M.Isolation of fractions rich in human Ysperm.Nature.1973;246(5433):421-4.
20.Gil M,Sar-Shalom V,Melendez Sivira Y,Carreras R,Checa MA.Sperm selection using magnetic activated cell sorting(MACS)in assisted reproduction:a systematic review and meta-analysis.J Assist Reprod Genet.2013;30(4):479-85.
21.Valcarce DG,Herraez MP,Chereguini O,Rodriguez C,Robles V.Selection of nonapoptotic sperm by magnetic-activated cell sorting in Senegalese sole(Solea senegalensis).Theriogenology.2016;86(5):1195-202.
22.Miltenyi S,Müller W,Weichel W,Radbruch A.High gradient magnetic cell separation with MACS.Cytometry.1990;11(2):231-8.
23.Morrell J.Colloids:applications in sperm preparation for assisted reproduction.In:Advances in Colloid Science.InTech;2016.
24.Morrell JM,Rodriguez-Martinez H.Colloid Centrifugation of Semen:Applications in Assisted Reproduction.American Journal of Analytical Chemistry.2016;7(08):597.
25.Morrell JM,Rodriguez-Martinez H.Colloid centrifugation of semen:applications in assisted reproduction.Am J Anal Chem.2016;7(08):597.
26.Morrell J,Lagerqvist A,Humblot P,Johannisson A.Effect of Single Layer Centrifugation on reactive oxygen species and sperm mitochondrial membrane potential in cooled stallion semen.Reproduction,Fertility and Development.2017,29(5):1039-45.
27.Martinez-Alborcia MJ,Morrell JM,Gil MA,Barranco I,Maside C,Alkmin DV,et al.Suitability and effectiveness of single layer centrifugation using Androcoll-P in the cryopreservation protocol for boar spermatozoa.Anim Reprod Sci.2013;140(3):173-9.
28.Sutovsky P.New approaches to boar semen evaluation,Processing and Improvement.Reprod Domest Anim.2015;50(Suppl 2):11-9.
29.Feugang J,Liao S,Crenshaw M,Clemente H,Willard S,Ryan P.Lectinfunctionalized magnetic iron oxide nanoparticles for reproductive improvement.J Fertil.2015;3(145):17-9.
30.Durfey CL,Burnett DD,Liao SF,Steadman CS,Crenshaw MA,Clemente HJ,et al.Nanotechnology-based selection of boar spermatozoa:growth development and health assessments of produced offspring.Livest Sci.2017;205(Supplement C):137-42.
31.Odhiambo JF,DeJarnette JM,Geary TW,Kennedy CE,Suarez SS,Sutovsky M,et al.Increased conception rates in beef cattle inseminated with nanopurified bull semen.Biol Reprod.2014;91(4):97.
32.Lee WY,Lee R,Kim HC,Lee KH,Cui XS,Kim NH,et al.Pig spermatozoa defect in acrosome formation caused poor motion parameters and fertilization failure through artificial insemination and in vitro fertilization.Asian Australas J Anim Sci.2014;27(10):1417.
33.Ni?ański W,Partyka A,Prochowska S.Evaluation of spermatozoal function-useful tools or just science.Reprod Domest Anim.2016;51(S1):37-45.
34.Knox RV.Semen processing,Extending&storage for artificial insemination in swine.Swine Reproductive Extension Specialist Department of Animal Sciences,University of Il inois,USA.2011.
35.Vasquez ES,Feugang JM,Willard ST,Ryan PL,Walters KB.Bioluminescent magnetic nanoparticles as potential imaging agents for mammalian spermatozoa.J Nanobiotechnol.2016;14(1):20.
36.Feugang JM,Youngblood RC,Greene JM,Fahad AS,Monroe WA,Willard ST,et al.Application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa.J Nanobiotechnol.2012;10(1):45.
37.Feugang JM,Rodriguez-Munoz JC,Dillard DS,Crenshaw MA,Willard ST,Ryan PL.Beneficial effects of relaxin on motility characteristics of stored boar spermatozoa.Reprod Biol Endocrinol.2015;13(1):24.
38.Martinez-Alborcia MJ,Morrell JM,Parrilla I,Barranco I,Vazquez JM,Martinez EA,et al.Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior freezing.Theriogenology.2012;78(5):1117-25.
39.Lassalle B,Testart J.Lectins binding on human sperm surface increase membrane permeability and stimulate acrosomal exocytosis.Mol Hum Reprod.1996;2(9):651-8.
40.Sutovsky P.Proteomic analysis of mammalian gametes and sperm-oocyte interactions.Control of Pig Reproduction VIII.2010;66:103.
41.Wu W,Wu Z,Yu T,Jiang C,Kim W-S.Recent progress on magnetic iron oxide nanoparticles:synthesis,surface functional strategies and biomedical applications.Sci Technol Adv Mater.2015;16(2):023501.
42.Bepari RA,Bharali P,Das BK.Controlled synthesis ofα-andγ-Fe 2 O 3nanoparticles via thermolysis of PVA gels and studies onα-Fe 2 O 3catalyzed styrene epoxidation.J Saudi Chem Soc.2014.
43.Falchi L,Khalil WA,Hassan M,Marei WFA.Perspectives of nanotechnology in male fertility and sperm function.Int J Vet Sci Med.2018;6(2):265-9.
44.Vermes I,Haanen C,Steffens-Nakken H.Reutellingsperger C.a novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V.J Immunol Methods.1995;184(1):39-51.
45.Nicolson GL,Usui N,Yanagimachi R,Yanagimachi H.Lectin-binding sites on the plasma membranes of rabbit spermatozoa:changes in surface receptors during epididymal maturation and after ejaculation.J Cel Biol.1977;74(3):950-62.
46.Schwarz MA,Koehler JK.Alterations in lectin binding to Guinea pig spermatozoa accompanying in vitro capacitation and the acrosome reaction.Biol Reprod.1979;21(5):1295-307.
47.Caldeira DF,Paulini F,Silva RC,Azevedo RBd,Lucci CM.In vitro exposure of bull sperm cells to DMSA-coated maghemite nanoparticles does not affect cell functionality or structure.Int J Hyperth 2017(just-accepted):1-26.
48.Singh N,Jenkins GS,Asadi R,Doak S.Potential toxicity of superparamagnetic iron oxide nanoparticles(SPION).Nano Rev.2010;1(1):5358.
49.Gupta AK,Wells S.Surface-modified superparamagnetic nanoparticles for drug delivery:preparation,characterization,and cytotoxicity studies.IEEE Trans Nanobioscience.2004;3(1):66-73.
50.Knox RV.Artificial insemination in pigs today.Theriogenology.2016;85(1):83-93.
51.Knox R,Levis D,Safranski T,Singleton W.An update on north American boar stud practices.Theriogenology.2008;70(8):1202-8.
52.Katz DF,Diel L,Overstreet JW.Differences in the movement of morphologically normal and abnormal human seminal spermatozoa.Biol Reprod.1982;26(4):566-70.
53.Katz DF,Drobnis EZ,Overstreet JW.Factors regulating mammalian sperm MigrationThrough the female ReproductiveTract and oocyte vestments.GameteRes.1989;22:26.
54.Liu DY,Clarke GN,Baker HW.Relationship between sperm motility assessed with the Hamilton-thorn motility analyzer and fertilization rates in vitro.J Androl.1991;12(4):231-9.
55.Kawaguchi T,Kawachi M,Morikawa M,Kazuta H,Shibata K,Ishida M,et al.Key parameters of sperm motion in relation to male fertility.J Toxicol Sci.2004;29(3):217-31.
56.Yanagimachi R.Fertility of mammalian spermatozoa:its development and relativity.Zygote.1994;2(4):371-2.
57.Hernandez M,Roca J,Calvete JJ,Sanz L,Muino-Blanco T,Cebrian-Perez JA,et al.Cryosurvival and in vitro fertilizing capacity Postthaw is improved when boar spermatozoa are frozen in the presence of seminal plasma from good freezer boars.J Androl.2007;28(5):689-97.
58.Yeste M.Recent advances in boar sperm cryopreservation:state of the art and current perspectives.Reprod Domest Anim.2015;50:71-9.
2.Lopes S,Sun J-G,Jurisicova A,Meriano J,Casper RF.Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection.Fertil Steril.1998;69(3):4.
3.Broekhuijse M,Gaustad A,Bolarin Guillén A,Knol E.Efficient boar semen production and genetic contribution:the impact of low-dose artificial insemination on fertility.Reprod Domest Anim.2015;50(S2):103-9.
4.Foote RH.Fertility estimation:a review of past experience and future prospects.Anim Reprod Sci.2003;75(1-2):119-39.
5.Shannon P,Curson B.Toxic Effect and action of dead sperm on diluted bovine semen.J Dairy Sci.1972;55(5):614-20.
6.Sharma RK,Agarwal A.Role of reactive oxygen species in male infertility.Urology.1996;48(6):835-50.
7.Feugang JM.Novel agents for sperm purification,sorting,and Imaging.Mol Reprod Dev.2017;9999:1-10.
8.Said TM,Land JA.Effects of advanced selection methods on sperm quality and ART outcome:a systematic review.Hum Reprod Update.2011;17(6):719-33.
9.Jain S,Park SB,Pillai SR,Ryan PL,Willard ST,Feugang JM.Applications of Fluorescent Quantum Dots for Reproductive Medicine and Disease Detection.In:Unraveling the Safety Profile of Nanoscale Particles and Materials-From Biomedical to Environmental Applications.Edited by Gomes AC,Sarria MP.Rijeka:InTech;2018:Ch.06.
10.Pankhurst QA,Connolly J,Jones SK,Dobson J.Applications of magnetic nanoparticles in biomedicine.J Phys D Appl Phys.2003;36(13):R167.
11.Sutovsky P,Kennedy CE.Biomarker-based nanotechnology for the improvement of reproductive performance in beef and dairy cattle.Ind Biotechnol.2013;9(1):6.
12.Rath D,Tiedemann D,Gamrad L,Johnson L,Klein S,Kues W,et al.Sex-sorted boar sperm-an update on related production methods.Reprod Domest Anim.2015;50(S2):56-60.
13.Barkalina N,Jones C,Coward K.Nanomedicine and mammalian sperm:lessons from the porcine model.Theriogenology.2016;85(1):74-82.
14.Feugang JM,Youngblood RC,Greene JM,Willard ST,Ryan PL.Selfilluminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes.J Nanobiotechnol.2015;13(1):38.
15.Sutovsky P,Lovercamp K.Molecular markers of sperm quality.Reproduction in Domestic Ruminants VII;2011.p.247.
16.Hozaien MM,Elqusi KM,Hassanen EM,Hussin AA,Alkhader HA,El Tanbouly SM,et al.A comparison of reproductive outcome using different sperm selection techniques;density gradient,testicular sperm,PICSI,and MACS for ICSI patients with abnormal DNA fragmentation index.Fertil Steril.2018;110(4):e19-20.
17.Mortimer D.Sperm preparation methods.J Androl.2000;21(3):357-66.
18.Kaneko S,Oshio S,Kobanawa K,Kobayashi T,Mohri H,Iizuka R.Purification of human sperm by a discontinuous Percoll density gradient with an innercolumn.Biol Reprod.1986;35(4):1059-63.
19.Ericsson RJ,Langevin CN,Nishino M.Isolation of fractions rich in human Ysperm.Nature.1973;246(5433):421-4.
20.Gil M,Sar-Shalom V,Melendez Sivira Y,Carreras R,Checa MA.Sperm selection using magnetic activated cell sorting(MACS)in assisted reproduction:a systematic review and meta-analysis.J Assist Reprod Genet.2013;30(4):479-85.
21.Valcarce DG,Herraez MP,Chereguini O,Rodriguez C,Robles V.Selection of nonapoptotic sperm by magnetic-activated cell sorting in Senegalese sole(Solea senegalensis).Theriogenology.2016;86(5):1195-202.
22.Miltenyi S,Müller W,Weichel W,Radbruch A.High gradient magnetic cell separation with MACS.Cytometry.1990;11(2):231-8.
23.Morrell J.Colloids:applications in sperm preparation for assisted reproduction.In:Advances in Colloid Science.InTech;2016.
24.Morrell JM,Rodriguez-Martinez H.Colloid Centrifugation of Semen:Applications in Assisted Reproduction.American Journal of Analytical Chemistry.2016;7(08):597.
25.Morrell JM,Rodriguez-Martinez H.Colloid centrifugation of semen:applications in assisted reproduction.Am J Anal Chem.2016;7(08):597.
26.Morrell J,Lagerqvist A,Humblot P,Johannisson A.Effect of Single Layer Centrifugation on reactive oxygen species and sperm mitochondrial membrane potential in cooled stallion semen.Reproduction,Fertility and Development.2017,29(5):1039-45.
27.Martinez-Alborcia MJ,Morrell JM,Gil MA,Barranco I,Maside C,Alkmin DV,et al.Suitability and effectiveness of single layer centrifugation using Androcoll-P in the cryopreservation protocol for boar spermatozoa.Anim Reprod Sci.2013;140(3):173-9.
28.Sutovsky P.New approaches to boar semen evaluation,Processing and Improvement.Reprod Domest Anim.2015;50(Suppl 2):11-9.
29.Feugang J,Liao S,Crenshaw M,Clemente H,Willard S,Ryan P.Lectinfunctionalized magnetic iron oxide nanoparticles for reproductive improvement.J Fertil.2015;3(145):17-9.
30.Durfey CL,Burnett DD,Liao SF,Steadman CS,Crenshaw MA,Clemente HJ,et al.Nanotechnology-based selection of boar spermatozoa:growth development and health assessments of produced offspring.Livest Sci.2017;205(Supplement C):137-42.
31.Odhiambo JF,DeJarnette JM,Geary TW,Kennedy CE,Suarez SS,Sutovsky M,et al.Increased conception rates in beef cattle inseminated with nanopurified bull semen.Biol Reprod.2014;91(4):97.
32.Lee WY,Lee R,Kim HC,Lee KH,Cui XS,Kim NH,et al.Pig spermatozoa defect in acrosome formation caused poor motion parameters and fertilization failure through artificial insemination and in vitro fertilization.Asian Australas J Anim Sci.2014;27(10):1417.
33.Ni?ański W,Partyka A,Prochowska S.Evaluation of spermatozoal function-useful tools or just science.Reprod Domest Anim.2016;51(S1):37-45.
34.Knox RV.Semen processing,Extending&storage for artificial insemination in swine.Swine Reproductive Extension Specialist Department of Animal Sciences,University of Il inois,USA.2011.
35.Vasquez ES,Feugang JM,Willard ST,Ryan PL,Walters KB.Bioluminescent magnetic nanoparticles as potential imaging agents for mammalian spermatozoa.J Nanobiotechnol.2016;14(1):20.
36.Feugang JM,Youngblood RC,Greene JM,Fahad AS,Monroe WA,Willard ST,et al.Application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa.J Nanobiotechnol.2012;10(1):45.
37.Feugang JM,Rodriguez-Munoz JC,Dillard DS,Crenshaw MA,Willard ST,Ryan PL.Beneficial effects of relaxin on motility characteristics of stored boar spermatozoa.Reprod Biol Endocrinol.2015;13(1):24.
38.Martinez-Alborcia MJ,Morrell JM,Parrilla I,Barranco I,Vazquez JM,Martinez EA,et al.Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior freezing.Theriogenology.2012;78(5):1117-25.
39.Lassalle B,Testart J.Lectins binding on human sperm surface increase membrane permeability and stimulate acrosomal exocytosis.Mol Hum Reprod.1996;2(9):651-8.
40.Sutovsky P.Proteomic analysis of mammalian gametes and sperm-oocyte interactions.Control of Pig Reproduction VIII.2010;66:103.
41.Wu W,Wu Z,Yu T,Jiang C,Kim W-S.Recent progress on magnetic iron oxide nanoparticles:synthesis,surface functional strategies and biomedical applications.Sci Technol Adv Mater.2015;16(2):023501.
42.Bepari RA,Bharali P,Das BK.Controlled synthesis ofα-andγ-Fe 2 O 3nanoparticles via thermolysis of PVA gels and studies onα-Fe 2 O 3catalyzed styrene epoxidation.J Saudi Chem Soc.2014.
43.Falchi L,Khalil WA,Hassan M,Marei WFA.Perspectives of nanotechnology in male fertility and sperm function.Int J Vet Sci Med.2018;6(2):265-9.
44.Vermes I,Haanen C,Steffens-Nakken H.Reutellingsperger C.a novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V.J Immunol Methods.1995;184(1):39-51.
45.Nicolson GL,Usui N,Yanagimachi R,Yanagimachi H.Lectin-binding sites on the plasma membranes of rabbit spermatozoa:changes in surface receptors during epididymal maturation and after ejaculation.J Cel Biol.1977;74(3):950-62.
46.Schwarz MA,Koehler JK.Alterations in lectin binding to Guinea pig spermatozoa accompanying in vitro capacitation and the acrosome reaction.Biol Reprod.1979;21(5):1295-307.
47.Caldeira DF,Paulini F,Silva RC,Azevedo RBd,Lucci CM.In vitro exposure of bull sperm cells to DMSA-coated maghemite nanoparticles does not affect cell functionality or structure.Int J Hyperth 2017(just-accepted):1-26.
48.Singh N,Jenkins GS,Asadi R,Doak S.Potential toxicity of superparamagnetic iron oxide nanoparticles(SPION).Nano Rev.2010;1(1):5358.
49.Gupta AK,Wells S.Surface-modified superparamagnetic nanoparticles for drug delivery:preparation,characterization,and cytotoxicity studies.IEEE Trans Nanobioscience.2004;3(1):66-73.
50.Knox RV.Artificial insemination in pigs today.Theriogenology.2016;85(1):83-93.
51.Knox R,Levis D,Safranski T,Singleton W.An update on north American boar stud practices.Theriogenology.2008;70(8):1202-8.
52.Katz DF,Diel L,Overstreet JW.Differences in the movement of morphologically normal and abnormal human seminal spermatozoa.Biol Reprod.1982;26(4):566-70.
53.Katz DF,Drobnis EZ,Overstreet JW.Factors regulating mammalian sperm MigrationThrough the female ReproductiveTract and oocyte vestments.GameteRes.1989;22:26.
54.Liu DY,Clarke GN,Baker HW.Relationship between sperm motility assessed with the Hamilton-thorn motility analyzer and fertilization rates in vitro.J Androl.1991;12(4):231-9.
55.Kawaguchi T,Kawachi M,Morikawa M,Kazuta H,Shibata K,Ishida M,et al.Key parameters of sperm motion in relation to male fertility.J Toxicol Sci.2004;29(3):217-31.
56.Yanagimachi R.Fertility of mammalian spermatozoa:its development and relativity.Zygote.1994;2(4):371-2.
57.Hernandez M,Roca J,Calvete JJ,Sanz L,Muino-Blanco T,Cebrian-Perez JA,et al.Cryosurvival and in vitro fertilizing capacity Postthaw is improved when boar spermatozoa are frozen in the presence of seminal plasma from good freezer boars.J Androl.2007;28(5):689-97.
58.Yeste M.Recent advances in boar sperm cryopreservation:state of the art and current perspectives.Reprod Domest Anim.2015;50:71-9.