Abstract
Simple SummarySpermatozoa (SPZ) viability, morphology and the kinetics of fresh and thawing Portuguese Merino Semen were evaluated at different breeding season months using an owner egg yolk-based semen extender (S-EXT) and two commercial lecithin-based semen extenders. The main significant differences between S-EXT were observed for thawed semen. The viability (30% vs. 15% of alive SPZ; p < 0.001), total motility (74% vs. 34%; p < 0.001), total progressive motility (18% vs. 5%; p < 0.001), straight line velocity (37 vs. 22 μm/s; p < 0.001) and beat cross frequency (13 vs. 6 Hz; p < 0.05) were higher in the owner egg yolk than in egg yolk-free-based S-EXT. Significant interactions between S-EXT, semen processing and/or months of semen collection were observed on several of the 22 evaluated SPZ parameters and should be elucidated in further studies. The egg yolk-based S-EXT was more consistent across the months in cryopreserving SPZ than commercial egg yolk- free-based S-EXT and still seems, to the present day, the most appropriate S-EXT to be used in (Merino) sheep.This study aimed to evaluate and compare the effect of three semen extenders (S-EXT) on 22 spermatozoa (SPZ) parameters (subjective and computer-assisted sperm analysis evaluations), before and after semen cryopreservation throughout different months of the breeding season in the Portuguese Merino breed. According to the multivariable model, the SPZ viability (alive %), kinetics subjective individual motility, total motility, total progressive motility and its subpopulations, and beat cross frequency) were higher in the egg yolk-based S-EXT improved by Estação Zootécnica National (Portugal) than in Ovixcell® or Andromed® extenders. All the differences were only observed in thawed semen, except for total motility and total progressive motility, in which Ovixcell® also showed the poorest results on fresh semen. An interaction effect between S-EXT and semen processing was observed on 72.3% (17/22) of the evaluated parameters, evidencing a variable cryoprotective action between S-EXT. The SPZ viability was poorer in the onset of the breeding season (end of April/early May) than in the previous middle breeding season (November/early December), suggesting the influence of a short anoestrous season on ejaculate quality, even though the volume and SPZ concentration of the ejaculates remained stable throughout the experiment. Additionally, S-EXT x semen processing x month interaction effect on 59.1% (13/22) of the evaluated parameters evidenced the importance of SPZ time collection in a natural environment to cryopreserve ram’s semen. We concluded that, overall, the egg yolk-based S-EXT provided a greater value to the cryopreservation of Merino rams´ semen. Nevertheless, the causes of the interaction effect between S-EXT, semen processing and/or month on several SPZ parameters should be addressed, including SPZ molecular research in new studies, in order to improve egg yolk-based as well as in egg yolk-free-based S-EXT.
Highlights
IntroductionCryopreservation of spermatozoa (SPZ) has several advantages apart from the longterm conservation, such as a cost reduction (e.g., decreasing the number of rams in the flock), easy storage, the prevention of genetic drift and the promotion of genetic resource exchange and transportation all over the world [1,2,3,4,5]. cryopreservation has many positive aspects, the cooling process involves fast structural changes in SPZ, which can promote different cell damages such as the reduction in the membrane fluidity or the alteration of lipid and protein organization [6,7,8].In view of the above, semen is usually diluted before cryopreservation using freezing semen extenders (S-EXT), which can be formulated using different components such asTris, fructose or glucose, citric acid, glycerol, antibiotics, and egg yolk (EY) [3,4]
All SPZ parameters were affected by semen processing: 40.1% (9/22) by semen extenders (S-EXT)
EZN S-EXT was more than double both soybean lecithin-based S-EXT (OVIX—13.7%; ANDR—15.1%), and was similar to the results reported by Savvulidi et al [54]
Summary
Cryopreservation of spermatozoa (SPZ) has several advantages apart from the longterm conservation, such as a cost reduction (e.g., decreasing the number of rams in the flock), easy storage, the prevention of genetic drift and the promotion of genetic resource exchange and transportation all over the world [1,2,3,4,5]. cryopreservation has many positive aspects, the cooling process involves fast structural changes in SPZ, which can promote different cell damages such as the reduction in the membrane fluidity or the alteration of lipid and protein organization [6,7,8].In view of the above, semen is usually diluted before cryopreservation using freezing semen extenders (S-EXT), which can be formulated using different components such asTris, fructose or glucose, citric acid, glycerol, antibiotics, and egg yolk (EY) [3,4]. There are two main limitations, namely the difficulty for the SPZ to cross the ewe cervix and reach the uterus for fertilization [14,15]; and the complex anatomy of the cervix which impedes vaginally depositing semen directly into the uterus [16]. Several studies have been carried out to overcome this difficulty, optimizing methods to cross the cervical barrier [17,18,19] or improving the sperm [9,18,20] In this sense, many studies [21,22,23,24,25] have been published evaluating different S-EXT and improving its effectiveness in order to reach an acceptable fertility in commercial flocks.
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