The Presence of D-Penicillamine during the In Vitro Capacitation of Stallion Spermatozoa Prolongs Hyperactive-Like Motility and Allows for Sperm Selection by Thermotaxis.

Sara Ruiz-Díaz,Serafin Perez-Cerezales,Ivan Oseguera-López,Alfonso Gutiérrez-Adán,Maria José Sanchez-Calabuig,David De La Cuesta-Díaz,Belén García-López,Consuelo Serres

doi:10.3390/ani10091467

Abstract

Assisted reproductive technologies (ARTs) in the horse still yield suboptimal results in terms of pregnancy rates. One of the reasons for this is the lack of optimal conditions for the sperm capacitation in vitro. This study assesses the use of synthetic human tubal fluid (HTF) supplemented with D-penicillamine (HTF + PEN) for the in vitro capacitation of frozen/thawed stallion spermatozoa by examining capacitation-related events over 180 min of incubation. Besides these events, we explored the in vitro capacity of the spermatozoa to migrate by thermotaxis and give rise to a population of high-quality spermatozoa. We found that HTF induced higher levels of hyperactive-like motility and protein tyrosine phosphorylation (PTP) compared to the use of a medium commonly used in this species (Whitten's). Also, HTF + PEN was able to maintain this hyperactive-like motility, otherwise lost in the absence of PEN, for 180 min, and also allowed for sperm selection by thermotaxis in vitro. Remarkably, the selected fraction was enriched in spermatozoa showing PTP along the whole flagellum and lower levels of DNA fragmentation when compared to the unselected fraction (38% ± 11% vs 4.4% ± 1.1% and 4.2% ± 0.4% vs 11% ± 2% respectively, t-test p < 0.003, n = 6). This procedure of in vitro capacitation of frozen/thawed stallion spermatozoa in HTF + PEN followed by in vitro sperm selection by thermotaxis represents a promising sperm preparation strategy for in vitro fertilization and intracytoplasmic sperm injection in this species.

Highlights

Despite nearly two decades of efforts, in vitro fertilization (IVF) in the horse remains unavailable, and intracytoplasmic sperm injection (ICSI) in this species still yields suboptimal results
The objectives of this study were: (i) to assess the effect of penicillamine supplementation on capacitation-related events during incubation under capacitating conditions in frozen/thawed stallion spermatozoa, (ii) to examine the capacity of these spermatozoa to migrate by thermotaxis, and (iii) to determine DNA integrity and tyrosine phosphorylation in the spermatozoa selected by thermotaxis
We examined the effect of incubating frozen/thawed stallion spermatozoa processed by density gradient centrifugation (DGC) in Whitten’s medium (WHI) and synthetic human tubal fluid (HTF), two media commonly used for the capacitation of mammalian spermatozoa

Summary

Introduction

Despite nearly two decades of efforts, in vitro fertilization (IVF) in the horse remains unavailable, and intracytoplasmic sperm injection (ICSI) in this species still yields suboptimal results. One of the main causes of these limitations are suboptimal in vitro conditions for sperm capacitation, preventing successful IVF [1] and low ICSI outcomes [2]. Reports exist of capacitation-related events that occur in equine spermatozoa under different incubation conditions employing the media. Protein tyrosine phosphorylation (PTP) in the sperm flagellum has been related to the hyperactive motility of sperm, and both these factors are considered hallmarks of mammalian sperm capacitation [10,11]. While PTP in the equine spermatozoon flagellum is elevated under different capacitating conditions in vitro, so far all attempts to establish its relationship with hyperactive motility have had limited success [12]. We propose here that it could be the suboptimal in vitro conditions for stallion spermatozoa that prevent a true or complete hyperactivation response

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