Short communication. Stallion sperm quality after combined ejaculate fractionation and colloidal centrifugation

Abstract

This study investigated the possible additive benefit of ejaculate fractionation and colloidal centrifugation on stallion sperm quality. Using an open-end artificial vagina, the sperm-rich fraction (FRAC-1) was separated from the rest of the ejaculate (FRAC-2) and a third sperm sample representing the combined ejaculate was reconstituted post-ejaculation (RAW). Each semen sample was processed for colloidal centrifugation. The percentage of abnormal spermatozoa was 17.8 ± 7.0% in RAW and 14.6 ± 9.5% in FRAC-1 but decreased to 11.4 ± 4.7% and 9.6 ± 6.9% respectively, after colloidal centrifugation. A sperm DNA fragmentation index of 10.9 ± 5.1% was observed in RAW and 7.5 ± 2.4% in FRAC-1 semen collected with the AV but this decreased to 7.8 ± 2.8% and 5.2 ± 2.3% after colloidal centrifugation. The rate of increase in sperm DNA fragmentation during the first 6 h of incubation at 37 ºC was 1.8 ± 0.9% per hour in RAW semen and 2.0 ± 2.0% per hour in FRAC-1 but this significantly decreased to 1.3 ± 1.4% and 0.9 ± 0.8% respectively after colloidal centrifugation. While stallion seminal characteristics can be improved using colloidal centrifugation, further enhancement is possible if the ejaculate is initially fractionated.Additional key words: Equus caballus; ejaculate fractionation; colloidal centrifugation; sperm DNA fragmentation; DNA longevity; SCD.Abbreviations used: FRAC-1 (first jet, sperm-rich fraction); FRAC-2 (rest of the ejaculate); RAW (combined ejaculate reconstituted post-ejaculation); ROS (reactive oxygen species); rSDF (rate of increase in sperm DNA fragmentation); SCD (sperm chromatin dispersion), SDF (sperm DNA fragmentation).

Equivalent seminal characteristics in human and stallion at first and second ejaculated fractions

“…The second fraction exhibits a low sperm count, and it is composed of secretions from the seminal vesicles, rich in reactive oxygen species with a negative effect on the seminal characteristics, since they contain chelating agents, alkaline pH and low zinc availability (Arver, 1982;Mortimer, 1994). This basic structuration of the two ejaculated fractions is known to be equivalent in both species (Crespo et al, 2015;Hebles et al, 2014;Kumar et al, 2011;Sieme, Katila, & Klug, 2004). Within this scenario, it is interesting to stress the apparent homogeneity found in the present prospective study, regarding the relative improvement achieved by ejaculate fractionation in both human and stallion, two distantly related species (with a presumably high seminal plasma proteome divergence) that also largely differ in the baseline seminal parameters considered as normal.…”

“…Sperm DNA damage was assessed by using the sperm chromatin dispersion test (SCD, Halosperm for human and Halomax for stallion; Halotech DNA, Madrid, Spain) as it was, respectively, used in both species (Crespo et al, 2015;Hebles et al, 2014). CON and PM were assessed with a CASA system-specific module (SCA, Microptic SL Barcelona, Spain), using fixed volume chamber slides (Leja, Holland) and by selecting default settings for human application.…”

“…CON and PM were assessed with a CASA system-specific module (SCA, Microptic SL Barcelona, Spain), using fixed volume chamber slides (Leja, Holland) and by selecting default settings for human application. Sperm DNA damage was assessed by using the sperm chromatin dispersion test (SCD, Halosperm for human and Halomax for stallion; Halotech DNA, Madrid, Spain) as it was, respectively, used in both species (Crespo et al, 2015;Hebles et al, 2014).…”

“…Thus, there are new technologies that can all be addressed for the best selection of the spermatozoa that shall be used for assisted reproductive techniques (ART). For example, the case of recurrent ejaculation, coupled or not with sperm selection (Carlsen, Petersen, Andersson, & Skakkebaek, 2004;Gosálvez, González-Martínez, López-Fernández, Fernández, & Sánchez-Martín, 2011;Levitas et al, 2005), or ejaculate fractionation with the first fraction being substantially better than the second (Crespo, Gosálvez, Johnston, & de la Torre, 2015;Hebles, Dorado, González-Martínez, & Sánchez-Martín, 2014;Kumar, Kalthur, Mascarenhas, Kumar, & Adiga, 2011;Valsa, Skandhan, Khan, Sumangala, & Gondalia, 2012), are logistically easy strategies to be implemented at the time of sperm collection. All these techniques aim to improve general sperm characteristics such as motility and morphology and reducing levels of DNA damage (Delbes, Herrero, Troeung, & Chan, 2013;Parmegiani et al, 2010).…”

“…Before applying these invasive technologies, other strategies, not requiring a direct intervention or treatment of the semen sample, and connected on the physiology of ejaculation, can be implemented to improve the whole quality of the sample that will be used for ART. For example, the case of recurrent ejaculation, coupled or not with sperm selection (Carlsen, Petersen, Andersson, & Skakkebaek, 2004;Gosálvez, González-Martínez, López-Fernández, Fernández, & Sánchez-Martín, 2011;Levitas et al, 2005), or ejaculate fractionation with the first fraction being substantially better than the second (Crespo, Gosálvez, Johnston, & de la Torre, 2015;Hebles, Dorado, González-Martínez, & Sánchez-Martín, 2014;Kumar, Kalthur, Mascarenhas, Kumar, & Adiga, 2011;Valsa, Skandhan, Khan, Sumangala, & Gondalia, 2012), are logistically easy strategies to be implemented at the time of sperm collection.…”