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.

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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.

Over the years, there has been much confusion in defining molecular markers of mesenchymal stem cells (MSCs) for other than human species due to a lack of species-specific antibodies. Therefore, the aim of our study was to define rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs) and to reflect upon the current identification of AF-MSCs by providing a summary of detected surface markers in different species. The expression of rAF-MSC surface markers was analyzed at the protein and mRNA level. Flow cytometry analyses showed that rAF-MSCs were positive for CD29 and CD44, low positive for CD90, but negative for CD73, CD105, and CD166. Interestingly, RT-PCR (reverse transcription-polymerase chain reaction) exposed a discprepancy between transcribed mRNA and protein expression, as the cells expressed mRNA of all MSC markers: CD29, CD44, CD73, CD90, CD105, and CD166. Our results also confirmed the mesenchymal nature of isolated cells by morphology, ultrastructure, and intracellular marker expression profile. In addition, the expression of few pluripotency markers was also detected. We also found that passaging did not affect apoptosis and viability. Similarly, changes in karyotype were not observed during passaging. In conclusion, the provided characteristics may be used as a comprehensive set of criteria to define and characterize rAF-MSCs, required for the identification of these cells in preclinical investigations. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1601-1613, 2017.

The objective of this study was to assess the effect of different diluent type and storage time on the quality of rooster insemination doses in terms of the spermatozoa motility and apoptosis (in vitro) and spermatozoa fertility potential (in vivo). The motility parameters were significantly (P<0.001) higher in the spermatozoa diluted in saline solution than those diluted in avian diluent at each storage time (0.5, 1 and 2 hours). The proportion of apoptotic spermatozoa was significantly (P < 0.05) higher in the semen samples diluted in the avian diluent in comparison to the semen samples diluted in the saline solution after 1 hour of storage. The proportion of dead spermatozoa was significantly (P < 0.05) higher in avian diluent in comparison to the semen samples diluted in the saline solution after 2 hours of storage. The fertility of spermatozoa diluted in the avian diluent significantly (P < 0.01) decreased 1 hour after the ejaculate collection. In conclusion, saline solution maintained better quality of rooster spermatozoa in terms of their motility values and the proportion of apoptotic and dead cells (in vitro) than the commercial avian diluent when stored at 4 °C for 2 hours. According to in vivo results, the saline solution seems to be suitable for semen dilution at low temperature, since similar fertility results were obtained over the whole storage time. Thus the commercial avian diluent could be replaced by the saline solution under certain conditions. Furthermore, the saline solution is cheaper and more accessible for practical use than the commercial avian diluent.