Semen cryopreservation for the creation of a Spanish poultry breeds cryobank: Optimization of freezing rate and equilibration time

Abstract

A sperm cryopreservation protocol requiring dimethylacetamide (DMA, 6%) as a cryoprotectant was optimized via assays involving different prefreezing equilibration times (1, 10, 30, 60, and 120 min at 5°C) and different freezing rates achieved by the following: 1) using nitrogen vapor to reduce the temperature from 5°C to -85°C at 10°C/min (slow freezing rate); 2) using a biological freezer unit in a 2-step method to reduce the temperature from 5°C to -35°C at 7°C/min and then from -35°C to -140°C at 60°C/min (medium freezing rate); or 3) using a biological freezer unit in a 1-step freezing method to reduce the temperature from 5°C to -180°C at 60°C/min (rapid freezing rate). Heterospermic semen samples from chicken breeds raised as part of a Spanish genetic resource conservation program were used in all assays. The 1-min equilibration treatment was associated with a lower percentage of viable thawed spermatozoa than the 30-min treatment (P < 0.05). The remaining sperm variables studied were not affected by equilibration time. The medium-rate 2-step freezing method was associated with a higher percentage of motile spermatozoa after thawing and with greater acrosome integrity (P < 0.05) than the slow nitrogen vapor or rapid 1-step methods. Thawed sperm movement quality and plasma membrane integrity (as assessed by the hypoosmotic swelling test) were better (P < 0.05) in samples frozen by the medium-rate 2-step freezing method than in those subjected to the slow nitrogen vapor method. Fertility was not influenced by freezing method, although that achieved with the medium rate 2-step freezing method showed a trend toward being greater than that achieved with the rapid 1-step method (P = 0.07). Together, the present results suggest that slow cooling rates are not recommendable when using dimethylacetamide. The 2-step freezing method may be useful in the establishment of a germplasm bank for Spanish chicken breeds.

Recent advances in bird sperm morphometric analysis and its role in male gamete characterization and reproduction technologies

“…However, given the space limitations faced by zoos and animal parks, long-term germplasm storage would appear to offer a means of preserving genetic diversity while minimizing space requirements. A number of national programs (e.g., in France, the USA, The Netherlands, and Spain) are currently trying to cryopreserve chicken spermatozoa 1516. Although the conservation of primordial germ cells (PGCs) would open up new possibilities, a number of technical limitations have prevented this being feasible.…”

Methods for Cryopreservation of Guinea Fowl Sperm

“…It is well known that for safe gene conservation, both in vivo and in vitro gene banks are necessary. Presently, indigenous poultry genetic materials are stored in vitro in only four national gene banks (France, Netherlands, North-America and Spain) [4], [5], [6]. In addition to in vivo management, i n vitro conservation is a strategic tool to secure genetic diversity, considering the risk of epidemic diseases [7].…”

Optimization of Sperm Cryopreservation Protocol for Peregrine Falcon (Falco peregrinus)

“…Semen cryopreservation is a complex process with a number of unanswered questions, and this is particularly true in bird species [15]. There is a need to adapt the cryopreservation protocols for domestic species to wild species to improve the efficacy of the freezing process and to allow for its application in germplasm banks, reintroduction projects, and the breeding of domesticated falcons.…”

“…These inconsistent results might be explained by species-specific differences in the membrane composition and seminal plasma content. In domestic bird species, DMA is often recommended for sperm cryopreservation in pellets, and it yields better results when applied to protocols with rapid cooling rates [15,20,23]. In contrast, DMSO is better suited to slow freezing methods [24].…”