Effects of Three Semen Extenders, Breeding Season Month and Freezing–Thawing Cycle on Spermatozoa Preservation of Portuguese Merino Sheep

Abstract

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.

Morphometric assessment of cryopreserved livestock bull spermatozoa in the tropics

“…Moreover, these parameters are linked to genetic factors, and the breed of the bull can influence results (Terán et al, 2021). Moreover, solutions in which sperm are suspended and processed can influence these variables and cause alterations in measurements depending on the semen extender used for the samples (Fernandes et al, 2021; Fernandez‐Novo et al, 2021). The evaluation of the semen extenders by breed can be useful for determining the adequate diluent and maximizing the fertilizing potential.…”

Effect of In Vitro Addition of Melatonin and Glutathione on Seminal Parameters of Rams in Diluted Semen and after Thawing

“…Semen extender was formulated in the EZN laboratory according to Fernandes et al [ 38 ] and consisted of egg yolk (15%), glycerol (6%), TRIS (2.1805 g), citric acid (1.1194 g), glucose (0.3 g), penicillin (0.05 g), and ultrapure water (Milli Q, 38 ml). We used egg yolks from fresh hen eggs, which were removed using an egg yolk separator and filter paper.…”

“…Following collection with an artificial vagina, ejaculate samples were placed in a 30 °C water bath and processed and evaluated according to Fernandes et al [ 38 ]. Only ejaculates with a volume >0.4 mL and >55% of individual motility (IM; 200× magnification at 38 °C) were processed.…”

Ram Semen Cryopreservation for Portuguese Native Breeds: Season and Breed Effects on Semen Quality Variation

“…For semen cryopreservation, a semen extender that were formulated in our Andrology laboratory placed at EZN and named EZN S-EXT [16] was used.…”

“…About one week after freezing, the straws were thawed in a water bath at 38 • C for 1 min. The content of each straw was diluted in 1 mL of saline solution and homogenized for 90 s. After 2 min, semen aliquots (10 µL) were used to evaluate motility, viability, and morphological defects as previously described by [16].…”