Breeders’ chilling, a valuable option to cryopreserve dog semen collected in field conditions

Introduction and aim: Cryopreservation of dog semen is the best option for breeders to store or ship the genetic material of valuable individuals, but this service is usually only available in some specialized centers. In field conditions, as in local breeding kennels, small clinical practices or during expositions, the unavailability of specific equipment and liquid nitrogen makes freezing procedures unfeasible and breeders ask to collect the semen in loco and ship it cooled to a cryopreservation facility. Although, before cryopreservation, prolonged semen chilling in laboratory setting gave different results (1–4), its effect on sperm characteristics when performed in field conditions has never been investigated. The aim of this study was to evaluate whether the cryopreservation of dog semen after 24 or 48 hr of storage in a chilling box results in a satisfying sperm quality. The ultimate goal is to suggest a valuable option to breeders for cryopreserving semen without moving animals. Materials and methods: Ejaculated spermatozoa from 9 healthy stud dogs (mixed breeds, 2–13 years old) were collected by digital manipulation: one aliquot was evaluated as fresh control (FRESH) and one immediately cryopreserved (CRYO T0) by Uppsala method (5), whereas other two aliquots were diluted (1:1) with chilling extender (TRIS buffer, antibiotics and 20% egg yolk) and kept in a Minitube® styrofoam transport box for 24 hr (CRYO T24) or 48 hr (CRYO T48), until cryopreservation. Briefly, maintaining spermatozoa (CRYO T24 and CRYO T48) at the chilling temperature, the samples were centrifuged, the surnatant was discarded, and spermatozoa were diluted (1 × 105 sp/mL) with freezing extender (TRIS buffer, 10% glycerol, 1% EquexSTM Paste, antibiotics and 20% egg yolk). At thawing,samples were analyzed for motility, morphology (Sperm Deformity Index, SDI: 6), membrane and acrosome integrity, and ability to interactwith oocytes by Zona pellucida Binding Assay (ZBA). To also check whether dog age influenced the results, data were analyzed by ANCOVA (covariate = age) followed by Tukey's test for multiple comparisons (significance level set at p < 0.05). Results: In frozen spermatozoa, morphology, membrane and acrosome integrity were not affected by different treatments (CRYO T0-T24- T48). Although a decrease in motility in CRYO T48 was observed, the ability to adhere to oocytes zona pellucida was maintained at the same extent in both fresh and cryopreserved spermatozoa. Dog age did not influence sperm qualities of fresh or frozen samples. Conclusions: Freezing of freshly collected semen is the optimal way to ensure a good sperm survival. However, present results showed that a cooled transport of 24 hr before freezing could be proposed to dog breeders to meet their demand for semen cryopreservation. References: 1) Hermansson et al., Theriogenology 2006;65:584–93. 2) Ponglowhapan et al., Theriogenology 2006;66:1633–6. 3) Santana et al., Reprod Domes Anim 2006;48:165–70. 4) Hidalgo et al., Vet Rec 2014;175:20. 5) Linde-Forsberg C. Proc. Soc. Theriogenology 2002;303–20.