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Introduction and objectiveOsmotic changes during the process of freeze-thawing involve changes in the location of aquaporins (AQPs) in membrane domains of spermatozoa. Some AQPs, like aquaporin 3 (AQP3), are linked to sperm cryotolerance in the porcine species. Conspicuous individual variability exists between rams and their ejaculates, which may be classified as displaying good freezability (GFE) or poor freezability (PFE), depending on several endogenous and environmental factors. The present work aimed to examine whether differences in freezability could even involve changes in location and expression of AQP3 in ram spermatozoa.MethodsThirty ejaculates from 10 rams (three of each) were evaluated and subsequently classified as GFE (n = 13) or PFE (n = 17) through a principal component analysis (PCA) and k-means cluster analysis. Spermatozoa were examined for the presence, abundance and distribution of AQP3 by western blot and immunocytochemistry, employing a commercial rabbit polyclonal antibody (AQP3 - ab125219).Results and discussionAlthough AQP3 was found in the sperm acrosome, midpiece, principal and end piece of the tail in both fresh and after frozen-thawed samples, its highest immunolabeling was found in the mid- and principal piece. In the GFE group, the expression of AQP3 in the mid- and principal piece was greater (P < 0.05) in frozen-thawed samples than in fresh specimens while such differences were not detected in the PFE group. Sperm cryotolerance relates to changes in AQP3 expression and thus AQP3 could be used as a biomarker for cryotolerance.ConclusionA greater capacity of AQP3 localization in mid- and principal piece of the spermatozoa could be linked to an increase the osmo-adaptative capacity of ejaculates with better capacity to withstand freeze-thawing processes.

Spermatozoa collected from the cauda epididymis of wild ruminants are more cryoresistant than ejaculated spermatozoa. Changes in the membrane location of aquaporins (AQPs) follow the osmotic changes that occur during freeze-thawing, and might influence the cryosurvival of spermatozoa depending on their source. This work reports the location of AQP3, AQP7 and AQP10 in the cauda epididymal and post-ejaculation spermatozoa of three wild mountain ungulate species (Iberian ibex, mouflon, and chamois), as determined by Western blotting (WB) and immunocytochemistry (ICC) using commercial rabbit polyclonal primary antibodies. WB confirmed the presence of all three AQPs in the spermatozoa of all the studied species, while ICC showed AQP3 to be mainly located in the sperm acrosome, mid-piece, principal piece, and end piece, both in cauda epididymal and ejaculated cells. The percentage of ejaculated spermatozoa showing AQP3 in the principal piece was higher in the ibex than in the chamois (P

Introduction and objectiveCryopreservation of testicular tissues offers new possibilities to protect endangered species, genetically valuable individuals or even the fertility potential of prepubertal individuals who have died unexpectedly. However, the use of this technique still remains a challenge. In this study, slow freezing and vitrification of testicular tissue was investigated to find out which cryopreservation method could better preserve the viability and DNA integrity of testicular germ cells in diverse wild species.MethodsTestes were obtained post-mortem from 18 artiodactyls (wild boar, roe deer, dwarf goat, mhor gazelle, European mouflon, African forest buffalo, Malayan tapir, dorcas gazelle, Iberian ibex, gnu, red river hog), 5 primates (colobus monkey, capuchin monkey, mandrill), 8 carnivores (gray wolf, Persian leopard, binturong, European mink, American black bear, suricata), and 2 rodents (Patagonian mara). The testicles belonged to adult individuals and were cut into small pieces and cryopreserved by needle immersed vitrification or uncontrolled slow freezing using a passive cooling device. After warming or thawing, testicular tissues were enzymatically digested and two germ cell types were differentiated based on their morphology: rounded cells (spermatogonia, spermatocytes, and early spermatids) and elongated cells (elongated spermatids and spermatozoa). Cell viability was assessed by SYBR-14/propidium iodide while DNA fragmentation by TUNEL assay with fluorescence microscope.Results and discussionOur preliminary results revealed that our uncontrolled slow freezing method better preserved the viability and DNA integrity of elongated cells than vitrification. Such trend was observed in all species, being significant in artiodactyls, carnivores, and primates. Similarly, the viability and DNA integrity of rounded cells was also better maintained in primates by uncontrolled slow freezing, while in carnivores, vitrification by needle immersion showed better results in this type of cells. In artiodactyls and rodents both techniques preserved the viability of rounded cells in a similar manner, although the DNA integrity of these cells was greater after needle immersed vitrification in artiodactyls.ConclusionsIn conclusion, the effectiveness of each cryopreservation method is affected by the phylogenetic diversity between species and cell type.