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

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.

A drastic shift in the energetic landscape of toothed whale sperm cells

“…Three to five movies of 1.5 s were recorded at 60 frames/s using the software Pylon Viewer provided by Basler, capturing at least 100 moving spermatozoa. 64 The motility and sperm kinetics were analyzed using the free software ImageJ 52 with the plugin ISAS bmg following instructions for analyzing dolphin spermatozoa. 65 Motility was evaluated using a computer-assisted sperm analysis (CASA system; ISAS, Proiser R+D, Paterna, Spain) coupled with a Nikon Eclipse E400 phase-contrast microscope (Nikon, Tokyo, Japan) using a 10 x objective with negative phase.…”

Bull spermatozoa selected by thermotaxis exhibit high DNA integrity, specific head morphometry, and improve ICSI outcome

“…The medium used in this study for migration by thermotaxis was the FERT, supplemented with 5 mmol/L of caffeine instead of heparin to avoid sperm head-to-head agglutination. In these conditions, migration by thermotaxis was achieved in bull sperm, obtaining a net thermotaxis of 3.6% which is higher than that obtained for stallion (1.1%), mouse (0.5%) and human (0.8%) using the same sperm selection system [7,8]. The spermatozoa have to be capacitated to migrate by thermotaxis [6] and under capacitating conditions, we know that only about 10% of them are capacitated at a given point [40].…”

“…We applied here the system developed by Pérez-Cerezales et al [7] as it can recover mice and human spermatozoa with high quality and in the case of mice, improve ICSI outcomes. Consequently, in this study, we adapted the temperature of sperm selection for bulls with respect to mice, humans and horses [7,8] as the gradient established here went from 36 to 39 ℃ instead of 35 to 38 ℃. This change was made taking into consideration the normal core body temperature of a healthy cow, and the temperature of incubation in bovine IVF which is 38.5 ℃, therefore, this gradient would be the most suitable for bull sperm selection.…”

“…Different guidance mechanisms have been described in vitro which are chemotaxis [4], rheotaxis [5] and thermotaxis [6]. Sperm thermotaxis is defined as the ability of the spermatozoa to move in a temperature gradient from the lower to the higher [6], and it is present in different species such as rabbits, humans [6], mice [7], horses [8] and also in bull [9]. However, in bull, it has been evaluated the ability of the spermatozoa to sense the gradient but not the quality of the spermatozoa migrated [9].…”

Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen–Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status

“…We studied these sperm concentration and sperm total motility from 0 to 120 min because there are different protocols described for post-thawed sperm incubation times for research studies, such as sperm capacitation a process with high relevance in ARTs applied in equines. Those concentrations range between 10 × 10 6 sperm/mL to 200 × 10 6 sperm/mL [ 57 , 58 , 59 , 60 , 61 , 62 ] and times ranged between 10 and 120 min [ 58 , 63 , 64 ]. Sperm concentrations used were chosen based on the CASA system optimal-concentration working range (10–50 × 10 6 sperm/mL).…”