Cooling of equine semen at 16ºC for 36h with the addition of cysteine in different concentrations

Abstract

Summary: Equine semen manipulation during the cooling process reduces sperm viability and fertility in consequence to, among others, membrane lipid peroxidation, because of the high content of polyunsaturated fatty acids, which makes cells highly susceptible to free radicals and reactive oxygen species. The objective ofthe present study was to evaluate the effect of in vitro addition of cysteine in four concentrations (0, 1, 1.5, and 2.5 mM) for cooling spermatozoa of 12 stallions at 16 ºC for 36 h. Evaluated variables were total motility, strength, viability and plasmatic and acrosomal membrane integrity at four different time points (0, 12, 24 and 36 h). With the exception of acrosomal integrity, it was verified a reduction in total motility, strength and plasmatic membrane integrity in all samples, during cooling. In the evaluations at 36 h of cold storage, total motility (mot) and viability (viab) were higher in groups treated with 1mM (mot:46,5 ± 6,1/viab:76,5 ± 6,9) and 1.5 mM (mot:46,0 ± 4,6/viab:76,9 ± 3,7) cysteine, respectively, compared to control (mot:35,5 ±18,4/viab:68,1±13,4) and 2.5mM (mot:39,7 ±12,4/viab:66 ±17,2) (P < 0.05). As for strength (str) and plasmatic membrane integrity (plasm), 1 mM cysteine (str:3.6 ± 0.5/plasm:57.2 ± 9.5) showed better results compared to control (str:3.2 ±1.1/plasm:54.1±11.8), 1.5 mM (str:3.5 ± 0.6/plasm:52.2 ±13.3) and 2.5 mM (str:3.2 ±1.1/plasm:55.8 ±12.5) (P < 0.05). Regarding acrosomal membrane integrity, in general, there was no loss of integrity (70.5 ±10.4; 69.4 ± 4.4; 68.0 ± 7.2 and 70.3 ± 0.5), control, 1 mM, 1.5 mM and 2.5 mM respectively. The concentration of 1mM cysteine was more efficient for the protection of sperm cells in the commercial system of passive cooling at 16 ºC for 36 h, with higher values for total motility, strength, viability and plasmatic membrane integrity.Keywords: antioxidant / cooled semen / genetic resources / spermatozoon / stallion Die Kühlung von Sperma über 36 Std. auf 16°C mit Zusatz von Cystein in verschiedenen KonzentrationenDie Manipulation von Pferdesamen während des Kühlprozesses reduziert deren Lebensfähigkeit und Fruchtbarkeit. Dies geschieht unter Anderem wegen der membranen Lipidperoxidation sowie des hohen Gehalts an mehrfach ungesättigten Fettsäuren, der die Zellen sehr anfällig für freie Radikale und reaktive Sauerstoff-Spezies macht. Das Ziel der vorliegenden Studie ist es, die Wirkung von künstlicher Zugabe von Cystein in vier Konzentrationen (0, 1, 1,5 und 2,5 mM) zu Kühlsperma von 12 Hengsten bei 16°C für 36 Stunden zu beurteilen. Ausgewertete Variablen waren Motilität, Lebensfähigkeit sowie plasmatische und akrosomale Membran-Integrität zu vier verschiedenen Zeitpunkten. Mit Ausnahme der akrosomalen Integrität wurde eine Reduktion der Motilität, der plasmatischen Membran-Integrität während der Abkühlung in allen Proben festgestellt. Die Bewertung nach 36 Stunden Kühlung ergab, dass die gesamte Motilität (Mot) und Lebensfähigkeit (Lbf) jeweils höher in der mit 1 mM Cystein behandelten Gruppe (Mot:46,5 ± 6,1/Lbf...

Comparison of extenders and storage temperature in chilling canine semen

“…Low levels of reactive oxygen species (ROS) results in a reduction-capacitation (redox)-regulated event (1) . However, spermatozoa are highly sensitive to oxidative damage and cooling of the semen induces plasma membrane damage because the membrane is susceptible to lipid peroxidation when exposed to ROS (1,2,3) . Sperm is particularly susceptible to oxidative stress and peroxidative attack by ROS.…”

“…Sperm is particularly susceptible to oxidative stress and peroxidative attack by ROS. It is, particularly, vulnerable to superoxide anion and hydrogen peroxide due to its limited ability to store antioxidant enzymes and its unsaturated fatty acid-rich membrane (3,4) . ROS is responsible for physiologically driving signal transduction processes associated with sperm capacitation.…”

“…To protect sperm cells from oxidative damage caused by higher concentrations of ROS and preserve the integrity and fertility potential of the spermatozoa, research concerning semen extenders is highly important. Chilling and cryopreservation protocols have a number of steps to reduce the temperature of the sperm, dehydrate the sperm, and induce intra-and extra-cellular freezing (3,5) . Dog spermatozoa are more resistant to cold shock than to bull, boar, and ram spermatozoa due to a low ratio of phospholipid-bound polyunsaturated/saturated fatty acids.…”

“…Additionally, egg yolk can interfere with semen quality assumptions, generating artefacts that obscure the visualisation of sperm, especially when using automated analyses (9) . Skimmed milk is also used as an extender for liquid or frozen semen from bulls and stallions (3) . The efficacy of skimmed milk is considered as good as an egg yolk based extender in preserving sperm (10) .…”