Fertilizing Capacity of Frozen Epididymal Sperm Collected from Dogs

Abstract

The collection of epididymal sperm may be a valuable tool for canine reproduction especially since it can enable collection of cells after death of a valuable dog. The aim of the present study was to evaluate the viability of epididymal sperm after freeze-thawing. Epididymides were obtained from four adult dogs by elective orchiectomy. The caudal portion of the epididymides and part of the deferential ducts were squeezed by means of an anatomic clamp into a Petri dish containing either 0.9% saline solution (Group 1) or Ringer solution without lactate (Group 2). Samples were centrifuged at 800 x g for 10 min, the supernatant was removed and the pellet was diluted in one step with a Tris/citric acid/OEP (Orvus Es Paste) extender containing 7% glycerol and subjected to semen freezing. Oocytes were obtained from canine ovaries, after ovariohysterectomy. Only oocytes that were approximately 100 mum in diameter, with a dark ooplasm surrounded by three- or four-well formed cumulus cell layers were used for sperm testing. Frozen semen samples were thawed in a water bath at 70 degrees C for 8 s and analysed at room temperature for sperm motility and velocity. Oocytes were incubated with spermatozoa in humidified atmosphere containing 5% CO(2) at 38 degrees C for 18 h. Morphological and functional characteristics of spermatozoa were similar in both groups. However, the percentage of sperm cells bound to oocytes was significantly higher in Group 2 than in Group 1. This result suggests that the Ringer solution without lactate was a more suitable medium for collecting epididymal canine sperm than 0.9% saline.

Comparison of Two Different Extenders for Cryopreservation of Epididymal Dog Sperm

“…The results of motility sperm parameters obtained in this study, frozen with both extenders, were similar to those noted in the study by Martins et al. (). The effects of cold storage were not equal for both extenders; we observed a significant increase in the percentages of total abnormalities, altered acrosomes, which may be related to the 24 h of storage at 5°C of the testicles after surgery until the time for processing of semen as related in other study (Tittarelli et al.…”

“…Obtaining semen directly from the epididymal tails and vas deferens is a technique that has been frequently used for the purposes of assisted reproduction (Martins et al. ). The process of cryopreservation may cause irreversible damage to the sperm membrane interfering with its viability.…”

Influence of different anaesthetic protocols over the sperm quality on the fresh, chilled (4°C) and frozen‐thawed epididymal sperm samples in domestic dogs

“…However, the motility of frozen‐thawed epididymal spermatozoa showed lower values (ranging between 25% and 35%) to that observed in fresh and chilled semen, and no influence of the final concentration of glycerol over the sperm motility was detected. Mean values of frozen‐thawed sperm motility were comparable to that described in different studies where epididymal semen was frozen immediately after equilibration (Hewitt et al., ; Ponglowhapan & Chatdarong, ; Martins, Padilha, Souza, & Lopes, ) or was frozen after 2 days of cool storage at 4°C (Ponglowhapan et al., ). Therefore, the motility observed in epididymal sperm cells did not show different features to that described in ejaculated semen and apparently retained sufficient quality for use both fresh and after cooling with sufficient guarantees of success; in fact, epididymal dog spermatozoa are capable of fertilizing oocytes (Klinc et al., ) and their fertilizing ability remained even after freezing (Marks, Dupuis, Mickelsen, Memon, & Platz, ; Hori et al., ).…”

Canine epididymal spermatozoa: A hidden treasure with great potential

“…Sperm–oocyte in vitro interaction has already proved that canine epididymal spermatozoa have zona binding (Kawakami, Morita, Hori, & Tsutsui, ; Martins et al., ; Yu & Leibo, ) and oocyte penetration capabilities (Hewitt et al., ; Hishinuma & Sekine, , ), and it is noteworthy that sperm penetration test gave similar results with fresh and frozen spermatozoa (Hewitt et al., ).…”

“…Extenders such as Tris with egg yolk (Hewitt, Leahy, Sheldon, & England, ; Hori, Ichikawa, Kawakami, & Tsutsui, ; Hori, Matsuda, Kobayashi, Kawakami, & Tsutsui, ; Hori et al., , ; Korochkina, Johannisson, Goodla, Morrell, & Axner, ; Martins, Justino, Sant'Anna, Trautwein, & Souza, ; Martins, Padilha, Souza, & Lopes, ; Ponglowhapan et al., ; Varesi, Vernocchi, Morselli, & Luvoni, ), Tris with low‐density lipoproteins (Prapaiwan et al., ), or commercial extenders (Biladyl, Nöthling et al., ; Bovimix, Martins et al., ; powdered coconut water extender, ACP‐106c, Mota Filho et al., ) were tested with variable results of post‐thaw motility (20%–50%) and membrane/acrosome integrity (5%–60%). As for ejaculated spermatozoa, the beneficial effect of the supplementation with Equex STM Paste (sodium dodecyl (lauryl) sulphate), in the presence of egg yolk, was also demonstrated (Ponglowhapan & Chatdarong, ).…”