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The aim was to compare the effect of dimethylacetamide (DMA) and N-methylacetamide (NMA) concentrations on the quality and fertility of post-thaw chicken semen. Ejaculates were obtained from 30 Hi-Line White roosters and processed according to the following treatments: lake pre-freezing extender + 0.1 M trehalose (LPF-T) + 6% DMA (control treatment), LPF-T + 9% DMA, LPF-T + 6% NMA, and LPF-T + 9% NMA. Sperm quality (viability, motility, and kinetic traits) was assessed before and after cryopreservation. A total of 15 laying hens per treatment were inseminated to assess fertility and embryo viability. Sperm cryopreserved in presence of DMA had significantly better in vitro quality compared to NMA, showing the highest proportion of viable and progressive motile sperm recovered after thawing. Furthermore, proportion of progressive motile sperm and the VCL, LIN, ALH, and WOB mean values were significantly improved in semen samples frozen/thawed with 6% compared to 9% cryoprotectant concentration. However, the best cryoprotective action on sperm quality played by DMA and the lowest cryoprotectant concentration did not translate into a concomitant advantage in in vivo semen fertility that showed no differences between cryoprotectant and cryoprotectant concentration treatments. Finally, the cryoprotectant DMA and NMA showed an opposite effect on embryo viability in comparison with the effect played on in vitro semen quality, being NMA more efficient than DMA on preserving viable embryos. The present results suggest the urgency to further decrease the cryoprotectant concentration in poultry semen freezing procedures and to assess the specific toxic effect of cryoprotectant on sperm integrity, fertility, and embryo development., (© 2019 Poultry Science Association Inc.)

This study was designed to optimize the semen freezing protocol of the native Mediterranean brown trout inhabiting the Molise rivers through two experiments: an in vitro analysis of the effects of two basic extenders combined with three cryoprotectants on post-thaw semen quality; and an in vivo test to assess the fertilization and hatching rate. Semen was diluted at a ratio of 1:3 in a freezing medium composed of a glucose extender (A) or mineral extender (B). Each basic component contained 10% dimethylsulfoxide, dimethylacetamide or methanol. The post-semen quality was evaluated considering motility, duration of motility, viability and DNA integrity. The basic extender and cryoprotectant were shown to have significant effects on these variables, and the best results were obtained using extender A or B combined with dimethylsulfoxide (P < 0.05). These freezing protocols were selected for fertilization trials in vivo. Fertilization and hatching rates were significantly higher in fresh semen. No significant differences were observed in frozen semen using extender A or B, although higher percentages of eyed eggs and hatching rates were recorded using extender A. According to our in vitro and in vivo results, the glucose-based extender and dimethylsulfoxide emerged as the best combination for an effective cryopreservation protocol for semen of this trout.

There is need for standardization of freezing-thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post-thaw motility and to analyse combined effect of the best permeating cryoprotectant (P-CPA) with one of four non-permeating cryoprotectants (N-CPA) on post-thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N-methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N-CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing-thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen-thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post-thaw motility. Moreover, ficoll addition to EG-based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N-CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank., (© 2017 Blackwell Verlag GmbH.)

The aim of this study was to assess the combined effect of dimethylacetamide (DMA) and two non-permeating cryoprotective agents, trehalose and sucrose, on the quality of post-thaw chicken semen. Adult Lohmann roosters (n = 27) were used. Semen was processed according to the following treatments: Lake pre-freezing extender +6% DMA (LPF, control treatment), LPF + 0.1 M trehalose (LPF-T treatment), LPF + 0.1 M sucrose (LPF-S treatment) and LPF + 0.1 M trehalose + 0.1 M sucrose (LPF-TS treatment). Semen was loaded into straws and frozen in nitrogen vapour. Sperm quality (viability, mobility and kinetic parameters) was assessed immediately after thawing (T0) and at 5 (T5), 10 (T10) and 15 min (T15) thereafter. The different cryodiluent combinations significantly affected the kinetic parameters. The presence of trehalose, alone or with sucrose, combined with DMA improved the quality of motion in cryopreserved sperm in comparison to DMA alone (LPF) and DMA with sucrose (LPF-S). In particular, the highest values in linearity (LIN) and wobble (WOB) were measured in the treatment LPF-T. The treatments significantly affected the recovery rate of progressive motile sperm that presented the best value soon after thawing in the LPF-T treatment; moreover, the presence of trehalose, alone (LPF-T) or with sucrose (LPF-TS), significantly improved the recovery rate of progressive motile sperm also at T5 and T10 compared to LPF and LPF-S. The present results show a positive synergic action of DMA and trehalose on motile function of thawed chicken sperm., (Copyright © 2016 Elsevier Inc. All rights reserved.)

1. This study was designed to identify a suitable protocol for freezing turkey semen in straws exposed to nitrogen vapour by examining the effects of dimethylacetamide (DMA) or dimethylsulfoxide (DMSO) as cryoprotectant (CPA), CPA concentration, freezing rate and thawing rate on in vitro post-thaw semen quality. 2. Pooled semen samples were diluted 1:1 (v:v) with a freezing extender composed of Tselutin diluent containing DMA or DMSO to give final concentrations of 8% or 18% DMA and 4% or 10% DMSO. The semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen (LN2) surface (1, 5 and 10 cm) for 10 min. Semen samples were thawed at 4°C for 5 min or at 50°C for 10 s. After thawing, sperm motility, viability and osmotic tolerance were determined. 3. Cryosurvival of turkey sperm was affected by DMSO concentration. Freezing rate affected the motility of sperm cryopreserved using both CPAs, while thawing rates showed an effect on the motility of sperm cryopreserved using DMA and on the viability of sperm cryopreserved using DMSO. Significant interactions between freezing rate × thawing rate on sperm viability in the DMA protocol were found. 4. The most effective freezing protocol was the use of 18% DMA or 10% DMSO with freezing 10 cm above the LN2 surface and a thawing temperature of 50°C. An efficient protocol for turkey semen would improve prospects for sperm cryobanks and the commercial use of frozen turkey semen.

The aim of this study was to evaluate the effect of the addition of Ficoll 70 into the cryopreservation medium containing sucrose and dimethyl sulfoxide (DMSO) on rabbit spermatozoa characteristics following freezing/thawing. This large molecular weight polymer elevates the viscosity of medium and, therefore, could better protect spermatozoa during the freezing process. Only ejaculates of good initial motility (>80%) were used in the experiments. Heterospermic pools were diluted in a freezing medium composed of commercial diluent, 16% dimethyl sulphoxide (DMSO) and 2% sucrose (control) or in the same medium enriched with 4% Ficoll 70 (Ficoll) and frozen in liquid nitrogen vapours for 10 min before being plunged in liquid nitrogen. The quality of fresh and frozen/thawed spermatozoa samples was evaluated in vitro using the Computer Assisted Semen Analysis (CASA) system, fluorescent probes (peanut agglutinin (PNA)-Alexa Fluor®; annexin V-FLOUS) and by electron microscopy. Better cryoprotective effect was observed when Ficoll 70 was added, compared with the semen cryopreserved with sucrose and DMSO only. The higher values (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing and at 30 min following incubation at 37°C were obtained in the Ficoll group. Moreover, the higher number (P < 0.05) of acrosome intact sperm was found in the Ficoll compared with the control group. Furthermore, no significant differences in kindling rates and number of pups born between frozen/thawed and fresh semen group were found. In conclusion, our study showed that the addition of Ficoll 70 might improve several characteristics of rabbit spermatozoa measured in vitro following freezing/thawing.

This study was designed to identify the most effective non-permeable cryoprotectant (CPA) for the cryopreservation of rabbit semen by comparing the effects of different concentrations of low-density lipoproteins (LDL) on post-thaw sperm quality with those of whole egg yolk or sucrose. In a second experiment, the performance of the non-permeable CPAs identified as most effective was assessed in vivo by determining reproductive performances. Pooled semen samples were diluted to a ratio of 1:1 (v:v) in freezing extender (Tris-citrate-glucose and 16% dimethylsulfoxide as permeable CPA) containing as non-permeable CPAs 6, 8, 10 or 15% LDL from egg yolk, 0.1M sucrose, or 15% egg yolk. The semen was loaded in 0.25mL straws and frozen in liquid nitrogen vapor. After thawing, we determined sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Our results clearly revealed a significant effect of LDL concentration on semen quality. Also, at an optimal concentration of 10%, motility and acrosome integrity were improved over the values recorded for egg yolk (P<0.05). Based on the in vitro data, 3 groups of does (n=30 each) were inseminated with fresh semen or semen frozen using sucrose or 10% LDL. Sucrose led to a significantly higher conception rate than LDL and reproductive performance was similar to that observed for fresh semen. Our findings indicate the markedly better performance of sucrose in vivo as a non-permeable CPA for the cryopreservation of rabbit semen., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This study was designed to improve current freezing protocols for rabbit sperm by examining: (1) the toxicity of different permeable cryoprotectants (CPAs) used for standard vapor freezing (conventional freezing); (2) the feasibility of ultrarapid nonequilibrium freezing (vitrification) of sperm in the absence of permeating CPAs; and (3), the addition of bovine serum albumin (BSA), alone or with sucrose or trehalose as osmoprotectants. First, we evaluated the effects on sperm motility of the incubation time (5 to 60 minutes) with different final concentrations (5% to 20%) of glycerol, N-N-dimethylacetamide, dimethylsulfoxide (DMSO), ethylene glycol, propylene glycol, and methanol. N-N-dimethylacetamide (5%) and DMSO (5% and 10%) showed the least toxic effects; the use of 10% DMSO producing the best postthaw sperm motility and membrane integrity results (P < 0.05) after conventional freezing. For vitrification, semen was diluted in the absence of permeable CPAs and frozen by dropping semen directly in liquid nitrogen. However, this led to the low or null cryosurvival of sperm postvitrification (0.16 ± 0.4%, 1.8 ± 1.6%, and 94.5 ± 1.4% of motile, membrane-, and DNA-intact sperm cells, respectively). To assess the effects of albumin and osmoprotectants on sperm cryosurvival, sperm was conventionally frozen with 10% DMSO or vitrified in the absence of permeable CPAs without or with 0.5% BSA alone or combined with sucrose or trehalose (range, 0-0.25 M). In the conventional freezing procedure, the addition of BSA alone failed to improve sperm cryosurvival, however, in the presence of BSA plus either sucrose or trehalose, the postthaw motility (using 0.1 M sucrose or trehalose) and DNA integrity (using all additive concentrations) of sperm were significantly better (P < 0.05) than control. Higher numbers of motile and membrane-intact cells were observed when semen was vitrified with BSA alone or with BSA and sucrose (0.1 and 0.25 M) or BSA and trehalose (0.25 M) and a best recovery of DNA-intact sperm was recorded for BSA plus sucrose compared with semen vitrified without osmoprotectants (P < 0.05). Finally, the cryodiluent combinations BSA/sucrose and BSA/trehalose were compared in an insemination trial. Rabbit does were inseminated with fresh semen (N = 56), semen conventionally cryopreserved in the BSA-based cryodiluents containing 0.1 M sucrose or trehalose (N = 56 per group), or semen vitrified in the presence of 0.25 M sucrose or trehalose (N = 8 per group). Fertility rates and live born kids were similar for semen cryopreserved with BSA/sucrose (77% and 7.6) compared with fresh semen (84% and 8.1) and significantly higher than the figures recorded for the conventionally frozen semen in the BSA/trehalose group (52% and 6.1; P ≤ 0.05). In contrast, only one doe inseminated with semen vitrified in the presence of BSA/sucrose became pregnant, though no kids were delivered. The conclusions to be drawn from our study are: (1) incubation times and concentration toxicities established for the main permeable CPAs used for conventional freezing of rabbit sperm indicated that DMSO 10% was the least damaging; (2) CPA-free vitrification of rabbit semen led to a low or null sperm cryosurvival; and (3) enriching the freezing medium with BSA plus adequate amounts of sucrose or trehalose can improve the cryosurvival of rabbit sperm after conventional freezing or vitrification. In our working conditions, BSA/sucrose was more effective than BSA/trehalose at preserving the in vivo fertilization capacity of rabbit sperm cryopreserved using the standard procedure., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This study was designed to identify a suitable freezing protocol for rabbit semen by comparing the effects of different concentrations and equilibration times of dimethylacetamide (DMA) and dimethylsulfoxide (DMSO) on the postthaw quality of the semen. After establishing the best protocols for each cryoprotectant, their efficacy was compared by examining the in vivo fertilizing capacity of the semen samples. Pooled semen samples diluted in freezing medium containing 4%, 6%, or 8% DMA or DMSO (all combined with 1% sucrose as a nonpermeating cryoprotectant) were loaded in straws and equilibrated for 5, 15, or 45 min before freezing in liquid nitrogen vapor. The variables assessed after thawing were sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Marked effects on these variables were shown by the cryoprotectant concentration and equilibration time, with best results obtained using DMA 6% or DMSO 8% and equilibration times of 45 min. These freezing protocols were selected to compare the two cryoprotectants in an insemination trial. Three groups of 114 rabbit does (28 nulliparous and 86 multiparous in each group) were inseminated with fresh semen or with semen frozen using the optimized DMA or DMSO protocols. Fertility rates and numbers of kids born were similar, respectively for the DMSO-frozen (79.8% and 7.7 ± 0.3 young per kindling) and fresh semen (81.6% and 8.6 ± 0.3) yet higher (P ≤ 0.05) than the rates returned using the DMA-frozen semen (47.4% and 6.7 ± 0.4). Moreover, the numbers of rabbits born alive when DMSO was used in the freezing protocol, despite being lower than those recorded using fresh semen, were higher than when DMA was used as the cryoprotectant (P < 0.05). The physiological status of the does (nulliparous or multiparous) had no influence on the fertility and prolificacy results. Our findings indicate that the cryosurvival of rabbit sperm frozen using DMSO or DMA as the cryoprotectant is highly influenced by the concentration of cryoprotectant used and the time the semen is exposed to the agent before freezing. According to our in vivo fertility and prolificacy data, DMSO emerged as more effective than DMA for the cryopreservation of rabbit sperm., (Copyright © 2012 Elsevier Inc. All rights reserved.)