Finding an Effective Freezing Protocol for Turkey Semen: Benefits of Ficoll as Non-Permeant Cryoprotectant and 1:4 as Dilution Rate

Abstract

The present study aimed to find an effective cryopreservation protocol for turkey semen through the combined use of dimethylsulfoxide (DMSO) and three non-permeant cryoprotectants (NP-CPAs), sucrose, trehalose, and Ficoll 70. In addition, the action of two dilution rates (1:2 and 1:4) were also investigated. Semen was processed according to two final dilution rates and the following treatments: Tselutin extender (TE)/DMSO (control), TE/DMSO + sucrose or trehalose 50, 100, 200, or 400 mM, and TE/DMSO + Ficoll 0.5, 0.75, 1, or 1.5 mM. In total 26 different combinations treatments were achieved. The diluted semen was filled up into straws and frozen on liquid nitrogen vapor. The post-thawing sperm quality was assessed by analyzing motility, membrane integrity, osmotic resistance, and DNA integrity. The results obtained revealed a significant effect of NP-CPA concentration on total and progressive motility, on most of the kinetic parameters, on membrane integrity and DNA integrity, while the post-thaw quality was less affected by dilution rate. The highest post-thaw quality for all sperm quality parameters assessed except curvilinear velocity (VCL) and DNA integrity were found in semen frozen with 1 mM Ficoll/1:4 (p < 0.05). Our findings provide an important contribution for the identification of a reference procedure for turkey semen cryopreservation, in order to create the first national avian semen cryobank.

Validation of the Turkey Semen Cryopreservation by Evaluating the Effect of Two Diluents and the Inseminating Doses

“…In the present study, in order to obtain a freezing procedure with a fertilization rate as similar as possible to that of fresh semen, we investigated the effect of the two extenders (Tselutin and Lake) on in vitro sperm cryosurvival and on the fertilizing ability by testing three sperm concentration doses. It is to be highlighted that for the first time, we evaluated the fertilizing ability by testing three sperm concentration doses using the optimized freezing protocol [24]. Examining the results obtained in vitro it emerged that the Lake extender preserved the post-thaw progressive sperm motility and related kinetic parameters better (p < 0.05).…”

“…Subsequently, each pool was divided into two equal aliquots, diluted with Tselutin or Lake extender (Table 1), in order to obtain a sperm concentration of 6 × 10 9 spermatozoa/mL, which was cooled at 4 • C for 25 min. Thereafter, the pre-extended semen was further diluted (1:1, v/v) with the freezing medium composed of Lake or Tselutin extender, both containing 20% of dimethylsulfoxide (DMSO) as P-CPA and 1 mM of Ficoll 70 as NP-CPA [24] to reach a final sperm concentration of 3 × 10 9 spermatozoa/mL. The diluted semen was loaded into 0.25 mL straws through the aid of a manual micro-aspirator (IMV-Technologies, Piacenza, Italy) and then equilibrated at 4 • C for 20 min.…”

“…The diluted semen was loaded into 0.25 mL straws through the aid of a manual micro-aspirator (IMV-Technologies, Piacenza, Italy) and then equilibrated at 4 • C for 20 min. Straws were frozen and thawed in accordance to our previous studies [7,24].…”

“…In the last few years, we have identified in vitro as an effective freezing protocol which is based on the use of dimethylsulfoxide (DMSO) as a permeant cryoprotectant (P-CPA) combined with Ficoll as a non-permeant cryoprotectant (NP-CPA) [7,24], with a final sperm concentration of 3 × 10 9 sperm/mL. However, despite our efforts and encouraging results obtained so far, we still aim to obtain a freezing procedure with a fertilization rate as similar as possible to that of fresh semen.…”

“…In the last few years, we have identified in vitro as an effective freezing protocol which is based on the use of dimethylsulfoxide (DMSO) as a permeant cryoprotectant (P-CPA) combined with Ficoll as a non-permeant cryoprotectant (NP-CPA) [ 7 , 24 ], with a final sperm concentration of 3 × 10 9 sperm/mL.…”