FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Biology of reproduction2019; 101(5); 1056-1074; doi: 10.1093/biolre/ioz131

pH-dependent effects of procaine on equine gamete activation†.

Abstract: Procaine directly triggers pH-dependent cytokinesis in equine oocytes and induces hypermotility in stallion spermatozoa, an important event during capacitation. However, procaine-induced hyperactivated motility is abolished when sperm is washed to remove the procaine prior to sperm-oocyte co-incubation. To understand how procaine exerts its effects, the external Ca2+ and Na+ and weak base activity dependency of procaine-induced hyperactivation in stallion spermatozoa was assessed using computer-assisted sperm analysis. Percoll-washed stallion spermatozoa exposed to Ca2+-depleted (+2 mM EGTA) procaine-supplemented capacitating medium (CM) still demonstrated hyperactivated motility, whereas CM without NaCl or Na+ did not. Both procaine and NH4Cl, another weak base, were shown to trigger a cytoplasmic pH increase (BCECF-acetoxymethyl (AM)), which is primarily induced by a pH rise in acidic cell organelles (Lysosensor green dnd-189), accompanied by hypermotility in stallion sperm. As for procaine, 25 mM NH4Cl also induced oocyte cytokinesis. Interestingly, hyperactivated motility was reliably induced by 2.5-10 mM procaine, whereas a significant cytoplasmic cAMP increase and tail-associated protein tyrosine phosphorylation were only observed at 10 mM. Moreover, 25 mM NH4Cl did not support the latter capacitation characteristics. Additionally, cAMP levels were more than 10× higher in boar than stallion sperm incubated under similar capacitating conditions. Finally, stallion sperm preincubated with 10 mM procaine did not fertilize equine oocytes. In conclusion, 10 mM procaine causes a cytoplasmic and acidic sperm cell organelle pH rise that simultaneously induces hyperactivated motility, increased levels of cAMP and tail-associated protein tyrosine phosphorylation in stallion spermatozoa. However, procaine-induced hypermotility is independent of the cAMP/protein tyrosine phosphorylation pathway.
© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.
Get Full Text
Publication Date: 2019-08-03 PubMed ID: 31373616PubMed Central: PMC6877780DOI: 10.1093/biolre/ioz131Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research investigates the pH-dependent impact of the drug procaine on the activation of horse reproductive cells, revealing it can trigger important capacitative events in these cells, but can also hinder the fertilization process if administered incorrectly.

The role of procaine in reproductive cell activation

  • The study first observed that procaine, a local anesthetic, can influence equine reproduction.
  • Procaine was found to directly induce pH-dependent cytokinesis (cell division) in equine oocytes (egg cells) and hypermotility (increased movement) in stallion spermatozoa — an essential process during capacitation (preparation for fertilization).
  • However, they also found that this procaine-induced hyperactivity is halted if sperm is washed to remove procaine before being combined with the oocyte.

Investigating mechanism of procaine effect

  • To better understand how procaine brings about these effects, the researchers tested the dependency of procaine-induced hyperactivity on external calcium (Ca2+) and sodium ions (Na+) and weak base activity.
  • This was accomplished by using computer-assisted sperm analysis of stallion spermatozoa that had been washed with Percoll, a solution often used for sperm washing in in vitro fertilization.
  • The researchers found that procaine still induced hyperactivity under conditions of calcium depletion, but not when the medium was devoid of sodium.
  • Procaine and another weak base, NH4Cl, were shown to trigger an increase in the cytoplasmic pH, with an accompanying increase in sperm motility.

The relationship between procaine dosage, capacitation indicators, and fertilization

  • Fascinatingly, hyperactivity in sperm was reliably induced by 2.5-10 mM concentration of procaine, but there was a significant increase in two capacitation markers — high cytoplasmic cAMP levels and protein tyrosine phosphorylation of the sperm tail — only observed at a concentration of 10 mM.
  • While a high concentration of NH4Cl also induced oocyte cell division, the same capacitation characteristics observed with procaine were not seen.
  • Additionally, the study uncovered that cAMP levels in boar sperm were more than 10 times higher than in stallion sperm under similar capacitating conditions.
  • Finally, the research showed that equine oocytes were not fertilized by stallion sperm pre-treated with a 10 mM concentration of procaine, demonstrating that despite its stimulatory effects, procaine administration can impede subsequent fertilization.

Conclusion of the study

  • The comprehensive conclusion of this study is that procaine triggers a simultaneous increase in pH, hyperactivity, and capacitation characteristics in stallion spermatozoa, but these effects are largely independent of each other.
  • Meanwhile, the increased motility provoked by procaine is not dependent on the cAMP/protein tyrosine phosphorylation pathway familiar to capacitation, implying that procaine uses a different mechanism to incite hyperactivity.
  • Thus, while procaine does alter equine gamete activity, more investigation is required to ensure it can do so in a way that supports, rather than hinders, successful fertilization.

Cite This Article

APA
Leemans B, Stout TAE, Soom AV, Gadella BM. (2019). pH-dependent effects of procaine on equine gamete activation†. Biol Reprod, 101(5), 1056-1074. https://doi.org/10.1093/biolre/ioz131

Publication

Biology of reproduction
ISSN: 1529-7268
NlmUniqueID: 0207224
Country: United States
Language: English
Volume: 101
Issue: 5
Pages: 1056-1074

Researcher Affiliations

Leemans, Bart
  • Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
Stout, Tom A E
  • Departments of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
  • Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
Soom, Ann Van
  • Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
Gadella, Bart M
  • Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
  • Departments of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
  • Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

MeSH Terms

  • Animals
  • Calcium
  • Cytoplasm / chemistry
  • DNA
  • Embryonic Development
  • Fertilization in Vitro / veterinary
  • Horses / embryology
  • Horses / physiology
  • Hydrogen-Ion Concentration
  • Image Processing, Computer-Assisted / methods
  • Male
  • Oocytes
  • Organelles / chemistry
  • Procaine / pharmacology
  • Semen Analysis / veterinary
  • Sodium
  • Spermatozoa / drug effects

References

This article includes 55 references
  1. Palmer E, Bezard J, Magistrini M, Duchamp G. In vitro fertilization in the horse. A retrospective study.. J Reprod Fertil Suppl 1991; 44:375–384.
    pubmed: 1795281
  2. Bézard J, Magistrini M, Battut I, Duchamp G, Palmer E. In vitro fertilization in the Mare.. Rec Med Vet 1992; 168:993–1003.
  3. Dell'Aquila ME, Cho YS, Minoia P, Traina V, Fusco S, Lacalandra GM, Maritato F. Intracytoplasmic sperm injection (ICSI) versus conventional IVF on abottoir-derived and in vitro-matured equine oocytes.. Theriogenology 1997; 47:1139–1156.
    pubmed: 16728064
  4. Dell'Aquila ME, Cho YS, Minoia P, Traina V, Lacalandra GM, Maritato F. Effects of follicular fluid supplementation of in-vitro maturation medium on the fertilization and development of equine oocytes after in-vitro fertilization or intracytoplasmic sperm injection.. Hum Reprod 1997; 12:2766–2772.
    pubmed: 9455850
  5. Roasa LM, Choi YH, Love CC, Romo S, Varner DD, Hinrichs K. Ejaculate and type of freezing extender affect rates of fertilization of horse oocytes in vitro.. Theriogenology 2007; 68:560–566.
    pubmed: 17614128
  6. Leemans B, Gadella BM, Stout TA, De Schauwer C, Nelis H, Hoogewijs M, Van Soom A. Why doesn't conventional IVF work in the horse? The equine oviduct as a microenvironment for capacitation/fertilization.. Reproduction 2016; 152:233–245.
    pubmed: 27651517
  7. Leemans B, Stout TAE, De Schauwer C, Heras S, Nelis H, Hoogewijs M, Van Soom A, Gadella BM. Reproduction 2019.Feb 1. pii: REP–18–0541.R1. doi: 10.1530/REP–18–0541. [Epub ahead of print] Review. 30721132.
    pubmed: 30721132doi: 10.1530/rep–18–0541google scholar: lookup
  8. McPartlin LA, Suarez SS, Czaya CA, Hinrichs K, Bedford-Guaus SJ. Hyperactivation of stallion sperm is required for successful in vitro fertilization of equine oocytes.. Biol Reprod 2009; 81:199–206.
    pubmed: 19208544
  9. Leemans B, Gadella BM, Stout TA, Nelis H, Hoogewijs M, Van Soom A. An alkaline follicular fluid fraction induces capacitation and limited release of oviduct epithelium-bound stallion sperm.. Reproduction 2015; 150:193–208.
    pubmed: 26242588
  10. Leemans B, Gadella BM, Stout TA, Heras S, Smits K, Ferrer-Buitrago M, Claes E, Heindryckx B, De Vos WH, Nelis H, Hoogewijs M, Van Soom A. Procaine induces cytokinesis in horse oocytes via a pH dependent mechanism.. Biol Reprod 2015; 93:23.
    pubmed: 26085521
  11. Loux SC, Crawford KR, Ing NH, Gonzalez-Fernandez L, Macias-Garcia B, Love CC, Varner DD, Velez IC, Choi YH, Hinrichs K. CatSper and the relationship of hyperactivated motility to intracellular calcium and pH kinetics in equine sperm.. Biol Reprod 2013; 89:123.
    pubmed: 24048572
  12. Ambruosi B, Accogli G, Douet C, Canepa S, Pascal G, Monget P, Moros Nicolas C, Holmskov U, Mollenhauer J, Robbe-Masselot C, Vidal O, Desantis S. Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species.. Reproduction 2013; 146:119–133.
    pubmed: 23722152
  13. Suarez SS. Control of hyperactivation in sperm.. Hum Reprod Update 2008; 14:647–657.
    pubmed: 18653675
  14. Lishko PV, Kirichok Y, Ren D, Navarro B, Chung JJ, Clapham DE. The control of male fertility by spermatozoan ion channels.. Annu Rev Physiol 2012; 74:453–475.
    pmc: PMC3914660pubmed: 22017176
  15. Ferrer-Buitrago M, Bonte D, De Sutter P, Leybaert L, Heindryckx B. Single Ca(2+) transients vs oscillatory Ca(2+) signaling for assisted oocyte activation: limitations and benefits.. Reproduction 2018; 155:105–119.
    pubmed: 29122969
  16. Mattioli M, Barboni B, Gioia L, Loi P. Cold-induced calcium elevation triggers DNA fragmentation in immature pig oocytes.. Mol Reprod Dev 2003; 65:289–297.
    pubmed: 12784251
  17. Petr J, Rajmon R, Lanska V, Sedmikova M, Jilek F. Nitric oxide-dependent activation of pig oocytes: role of calcium.. Mol Cell Endocrinol 2005; 242:16–22.
    pubmed: 15967570
  18. Yue C, White KL, Reed WA, Bunch TD. The existence of inositol 1,4,5-trisphosphate and ryanodine receptors in mature bovine oocytes.. Development 1995; 121:2645–2654.
    pubmed: 7545575
  19. Viets LN, Campbell KD, White KL. Pathways involved in RGD-mediated calcium transients in mature bovine oocytes.. Cloning Stem Cells 2001; 3:105–113.
    pubmed: 11945220
  20. Cahalan MD. Local anesthetic block of sodium channels in normal and pronase-treated squid giant axons.. Biophys J 1978; 23:285–311.
    pmc: PMC1473517pubmed: 687766
  21. Takei GL, Fujinoki M. Regulation of hamster sperm hyperactivation by extracellular Na.. Reproduction 2016; 151:589–603.
    pubmed: 26952096
  22. Gonzalez-Fernandez L, Macias-Garcia B, Loux SC, Varner DD, Hinrichs K. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.. Biol Reprod 2013; 88:138.
    pubmed: 23595906
  23. Gonzalez-Fernandez L, Macias-Garcia B, Velez IC, Varner DD, Hinrichs K. Calcium-calmodulin and pH regulate protein tyrosine phosphorylation in stallion sperm.. Reproduction 2012; 144:411–422.
    pubmed: 22843772
  24. Leemans B, Gadella BM, Sostaric E, Nelis H, Stout TA, Hoogewijs M, Van Soom A. Oviduct binding and elevated environmental ph induce protein tyrosine phosphorylation in stallion spermatozoa.. Biol Reprod 2014; 91:13.
    pubmed: 24829033
  25. Kirichok Y, Navarro B, Clapham DE. Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2+ channel.. Nature 2006; 439:737–740.
    pubmed: 16467839
  26. Fois G, Hobi N, Felder E, Ziegler A, Miklavc P, Walther P, Radermacher P, Haller T, Dietl P. A new role for an old drug: ambroxol triggers lysosomal exocytosis via pH-dependent Ca(2)(+) release from acidic Ca(2)(+) stores.. Cell Calcium 2015; 58:628–637.
    pubmed: 26560688
  27. Ostrowski PP, Fairn GD, Grinstein S, Johnson DE. Cresyl violet: a superior fluorescent lysosomal marker.. Traffic 2016; 17:1313–1321.
    pubmed: 27621028
  28. Chavez JC, De la JL, Jose O, Torres P, Nishigaki T, Trevino CL, Darszon A. Acrosomal alkalization triggers Ca(2+) release and acrosome reaction in mammalian spermatozoa.. J Cell Physiol 2018; 233:4735–4747.
    pubmed: 29135027
  29. Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin.. Biol Reprod 1988; 38:1171–1180.
    pubmed: 3408784
  30. Tremoleda JL, Stout TAE, Lagutina I, Lazzari G, Bevers MM, Colenbrander B, Galli C. Effects of in vitro production on horse embryo morphology, cytoskeletal characteristics, and blastocyst capsule formation.. Biol Reprod 2003; 69:1895–1906.
    pubmed: 12904313
  31. McPartlin LA, Littell J, Mark E, Nelson JL, Travis AJ, Bedford-Guaus SJ. A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis.. Theriogenology 2008; 69:639–650.
    pubmed: 18242679
  32. Flesch FM, Brouwers JF, Nievelstein PF, Verkleij AJ, van Golde LM, Colenbrander B, Gadella BM. Bicarbonate stimulated phospholipid scrambling induces cholesterol redistribution and enables cholesterol depletion in the sperm plasma membrane.. J Cell Sci 2001; 114:3543–3555.
    pubmed: 11682613
  33. Boerke A, Brouwers JF, Olkkonen VM, van de Lest CH, Sostaric E, Schoevers EJ, Helms JB, Gadella BM. Involvement of bicarbonate-induced radical signaling in oxysterol formation and sterol depletion of capacitating mammalian sperm during in vitro fertilization.. Biol Reprod 2013; 88:21.
    pubmed: 23115269
  34. Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G. Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.. Anim Reprod Sci 2007; 98:39–55.
    pubmed: 17101246
  35. Loomis PR, Graham JK. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols.. Anim Reprod Sci 2008; 105:119–128.
    pubmed: 18178040
  36. Hoogewijs M, Rijsselaere T, De Vliegher S, Vanhaesebrouck E, De Schauwer C, Govaere J, Thys M, Hoflack G, Van Soom A et al.. Influence of different centrifugation protocols on equine semen preservation.. Theriogenology 2010; 74:118–126.
    pubmed: 20207406
  37. Wertheimer EV, Salicioni AM, Liu W, Trevino CL, Chavez J, Hernandez-Gonzalez EO, Darszon A, Visconti PE. Chloride is essential for capacitation and for the capacitation-associated increase in tyrosine phosphorylation.. J Biol Chem 2008; 283:35539–35550.
    pmc: PMC2602906pubmed: 18957426
  38. Marquez B, Suarez SS. Bovine sperm hyperactivation is promoted by alkaline-stimulated Ca2+ influx.. Biol Reprod 2007; 76:660–665.
    pubmed: 17182893
  39. Szatkowski MS. The effect of extracellular weak acids and bases on the intracellular buffering power of snail neurones.. J Physiol 1989; 409:103–120.
    pmc: PMC1190434pubmed: 2555474
  40. Winkler MM, Grainger JL. Mechanism of action of NH4Cl and other weak bases in the activation of sea urchin eggs.. Nature 1978; 273:536–538.
    pubmed: 26876
  41. Wennemuth G, Carlson AE, Harper AJ, Babcock DF. Bicarbonate actions on flagellar and Ca2+-channel responses: initial events in sperm activation.. Development 2003; 130:1317–1326.
    pubmed: 12588848
  42. Gibb Z, Lambourne SR, Quadrelli J, Smith ND, Aitken RJ. L-carnitine and pyruvate are prosurvival factors during the storage of stallion spermatozoa at room temperature.. Biol Reprod 2015; 93:104.
    pubmed: 26316064
  43. Suarez SS, Varosi SM, Dai X. Intracellular calcium increases with hyperactivation in intact, moving hamster sperm and oscillates with the flagellar beat cycle.. Proc Natl Acad Sci USA 1993; 90:4660–4664.
    pmc: PMC46572pubmed: 8506314
  44. Ho HC, Suarez SS. An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca(2+) store is involved in regulating sperm hyperactivated motility.. Biol Reprod 2001; 65:1606–1615.
    pubmed: 11673282
  45. Marquez B, Ignotz G, Suarez SS. Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm.. Dev Biol 2007; 303:214–221.
    pmc: PMC1885980pubmed: 17174296
  46. Ho HC, Suarez SS. Characterization of the intracellular calcium store at the base of the sperm flagellum that regulates hyperactivated motility.. Biol Reprod 2003; 68:1590–1596.
    pubmed: 12606347
  47. Marquez B, Suarez SS. Different signaling pathways in bovine sperm regulate capacitation and hyperactivation.. Biol Reprod 2004; 70:1626–1633.
    pubmed: 14766720
  48. Loux SC, Macias-Garcia B, Gonzalez-Fernandez L, Canesin HD, Varner DD, Hinrichs K. Regulation of axonemal motility in demembranated equine sperm.. Biol Reprod 2014; 91:152.
    pubmed: 25339104
  49. Begg DA, Wong GK, Hoyle DH, Baltz JM. Stimulation of cortical actin polymerization in the sea urchin egg cortex by NH4Cl, procaine and urethane: elevation of cytoplasmic pH is not the common mechanism of action.. Cell Motil Cytoskeleton 1996; 35:210–224.
    pubmed: 8913642
  50. Westhues M, Fritsch R. Animal Anaesthesia.. Philadelphia: Lippincott; 1964.
  51. Johnson RG, Carlson NJ, Scarpa A. deltapH and catecholamine distribution in isolated chromaffin granules.. J Biol Chem 1978; 253:1512–1521.
    pubmed: 24053
  52. Ohkuma S, Poole B. Cytoplasmic vacuolation of mouse peritoneal macrophages and the uptake into lysosomes of weakly basic substances.. J Cell Biol 1981; 90:656–664.
    pmc: PMC2111913pubmed: 7287819
  53. Poole B, Ohkuma S. Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages.. J Cell Biol 1981; 90:665–669.
    pmc: PMC2111912pubmed: 6169733
  54. Zucker RS, Steinhardt RA, Winkler MM. Intracellular calcium release and the mechanisms of parthenogenetic activation of the sea urchin egg.. Dev Biol 1978; 65:285–295.
    pubmed: 355009
  55. Franks NP, Lieb WR. Molecular and cellular mechanisms of general anaesthesia.. Nature 1994; 367:607–614.
    pubmed: 7509043

Citations

This article has been cited 8 times.
  1. Kirby J, Standfest M, Binkley J, Barnes C, Brown E, Cairncross T, Cartwright A, Dadisman D, Mowat C, Wilmot D, Houseman T, Murphy C, Engelsman C, Haller J, Jones D. The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification. J Appl Oral Sci 2024;32:e20240224.
    doi: 10.1590/1678-7757-2024-0224pubmed: 39356951google scholar: lookup
  2. Henning H, Luther AM, Höfner-Schmiing L, Waberski D. Compensability of an enhanced incidence of spermatozoa with cytoplasmic droplets in boar semen for use in artificial insemination: a single cell approach. Sci Rep 2022 Dec 17;12(1):21833.
    doi: 10.1038/s41598-022-26020-5pubmed: 36528611google scholar: lookup
  3. Leemans B, Bromfield EG, Stout TAE, Vos M, Van Der Ham H, Van Beek R, Van Soom A, Gadella BM, Henning H. Developing a reproducible protocol for culturing functional confluent monolayers of differentiated equine oviduct epithelial cells†. Biol Reprod 2022 Apr 26;106(4):710-729.
    doi: 10.1093/biolre/ioab243pubmed: 34962550google scholar: lookup
  4. Maitan PP, Bromfield EG, Hoogendijk R, Leung MR, Zeev-Ben-Mordehai T, van de Lest CH, Jansen JWA, Leemans B, Guimarães JD, Stout TAE, Gadella BM, Henning H. Bicarbonate-Stimulated Membrane Reorganization in Stallion Spermatozoa. Front Cell Dev Biol 2021;9:772254.
    doi: 10.3389/fcell.2021.772254pubmed: 34869370google scholar: lookup
  5. Gimeno BF, Bariani MV, Laiz-Quiroga L, Martínez-León E, Von-Meyeren M, Rey O, Mutto AÁ, Osycka-Salut CE. Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status. Animals (Basel) 2021 Jan 3;11(1).
    doi: 10.3390/ani11010074pubmed: 33401609google scholar: lookup
  6. Huang H, Liu Y, Gao Z, Wu X. Efficacy of procaine combined with ketamine and propofol in pediatric epidural anesthesia. Exp Ther Med 2020 Nov;20(5):23.
    doi: 10.3892/etm.2020.9151pubmed: 32934688google scholar: lookup
  7. Ruiz-Díaz S, Oseguera-López I, De La Cuesta-Díaz D, García-López B, Serres C, Sanchez-Calabuig MJ, Gutiérrez-Adán A, Perez-Cerezales S. The Presence of D-Penicillamine during the In Vitro Capacitation of Stallion Spermatozoa Prolongs Hyperactive-Like Motility and Allows for Sperm Selection by Thermotaxis. Animals (Basel) 2020 Aug 21;10(9).
    doi: 10.3390/ani10091467pubmed: 32825582google scholar: lookup
  8. Darszon A, Nishigaki T, López-González I, Visconti PE, Treviño CL. Differences and Similarities: The Richness of Comparative Sperm Physiology. Physiology (Bethesda) 2020 May 1;35(3):196-208.
    doi: 10.1152/physiol.00033.2019pubmed: 32293232google scholar: lookup
Subscribe to our newsletter
Join 99,727 horse owners like you who receive our email updates on horse health and nutrition.