Effect of isolation protocols and cryoprotectants on freezing of stallion epididymal spermatozoa.

BACKGROUND: The recovery of spermatozoa from the cauda epididymis may be the only option to obtain genetic material from elite stallions that had undergone castration or sudden death due to colic or severe injury. OBJECTIVE: To evaluate two different protocols for retrieval of stallion epididymal spermatozoa and to evaluate different cryoprotectants on the freezability of the epididymal spermatozoa. MATERIALS AND METHODS: Six epididymides from three stallions were collected immediately after routine castration under general anesthesia. In the first experiment, each epididymis (of two testes) of the same stallion were processed using different methods for retrieval of the epididymal spermatozoa and were pooled and cryopreserved either using 5% glycerol or 5% dimethyl formamide (DMF) as cryoprotectant. The semen quality parameters viz., progressive motility, HOST, viability and acrosome integrity were evaluated at the fresh, pre-freeze and post-thaw stages. RESULTS: Retrograde method of flushing of epididymis yielded significantly (p < 0.05) higher concentration of the stallion sperm than that of the floating method. The qualitative semen parameters i.e., viability, plasma membrane integrity and acrosome integrity were found to be significantly restored using 5% DMF as cryoprotectant in comparison to when 5% glycerol was used. CONCLUSION: Retrograde flushing method of epididymis yielded significantly higher sperm concentration to that of the floating method, and 5% DMF as cryoprotectant provided acceptable freezability of stallion epididymal spermatozoa. DOI: 10.54680/fr23310110312.

Effects of extenders and cryoprotectants on cryopreservation of Thai red junglefowl (Gallus gallus gallus) spermatozoa.

Semen cryopreservation is crucial maintain genetic diversity of avian species. Little is known about suitable extenders for post-thaw survival of spermatozoa from Thai red junglefowl (Gallus gallus gallus). Therefore, the present study aimed to compare the suitability of the modified Thai red junglefowl extender (TRJFE) for cryopreservation of Thai red junglefowl spermatozoa with other extenders, including the Schramm extender, the red junglefowl extender (RFE), and the HS1 extender, in terms of sperm viability, motility, and fertility. First, the effects of adding 6% and 9% (v/v) N,N-dimethylacetamide, N,N-dimethylformamide (DMF), or N-methyl acetamide to these extenders on the post-thaw sperm quality of pooled ejaculates from 25 male fowls. The viability of thawed sperm was assessed by using nigrosin-eosin staining and sperm motility was determined by using computer-assisted sperm analysis. Fertility was assessed by inseminating 144 laying hens. The TRJFE +6% DMF combination significantly improved post-thaw viability (64.88 ± 0.51%) and motility (68.58 ± 1.13%) of Thai red junglefowl sperm, and the semen had the highest fertility (60.97 ± 0.72%). The findings suggest that TRJFE +6% DMF is a suitable freezing medium to conserve Thai red jungle fowl genetic resources.

Freezing chicken semen: Influence of base medium osmolality, cryoprotectants, cryoprotectant concentration, and cooling rate on post-thaw sperm survival.

A classical chicken semen diluent (Lake's 7.1 diluent) was modified to have lowered osmolalities (ranging from 290 to 410 mOsm/kg). The modified medium with physiological osmolality of 325 mOsm/kg allowed cold storage of fresh semen for several days with very little loss of membrane integrity and motility, while high osmolalities inhibited motility. This modified medium was then used as base for freezing medium to test effects of the type and concentration of cryoprotective agent (CPA), and the cooling rate (CR). A number of CPAs (methylformamide, methylacetamide, dimethylformamide (DMF), dimethylacetamide (DMA), diethylformamide, and propylene glycol) were first compared by freezing semen with 0.6 mol/l of the respective CPA at a cooling rate of 250 °C/min. Post-thaw motility and membrane integrity were highest with DMA and DMF. Finally, in more detailed factorial experiments, semen from individual cocks or pooled semen was frozen using CRs of 4, 50, 250, and 440 °C/min and DMA concentrations ([DMA]) of 0.4, 0.6, 1.0, and 1.5 mol/l. Straws from each semen sample x treatment combination were divided for semen assessment at three different research groups for sperm motility, membrane integrity, kinked tails, and DNA fragmentation, using microscopy, computer assisted motility analysis, and flow cytometry. There were clear effects of both CR and [DMA] and their interaction. CRs 50 and 250 °C/min gave best post-thaw sperm performance. Higher DMA concentrations gave better post-thaw membrane integrity, but concentrations above 1.0 mol/l can decrease sperm velocity or even inhibit sperm motility. Therefore [DMA] may best be 0.6-1.0 mol/l at a CR of 50-250 °C/min.

Cryopreservation of llama semen using a combination of permeable cryoprotectants.

Semen cryopreservation is not available for massive use in South American Camelids (SACs) due to the lack of an efficient protocol and the low pregnancy rates obtained with artificial insemination (AI). The use of a single cryoprotectant (CP) is commonly used in SACs frozen semen. The objective of the study was to evaluate the combined cryoprotective capacity of two permeable CPs at different stages of the cryopreservation protocol in llama semen. Sixteen ejaculates from 4 llama males were analysed, and sperm quality was assayed in raw semen, at 5°C, after equilibration of samples with the CPs and when samples were thawed. The following CPs and combination were used: 6% glycerol (GL), 6% dimethylformamide (DMF) and the combination of both CPs: 3% GL and 3% DMF. A Kruskal-Wallis test and an experimental factorial design, considering one factor with four levels (raw semen, 6% GL, 6% DMF and GL/DMF), were used. Total sperm motility and live sperm with intact acrosomes remained unchanged after equilibration of samples (p > .05). A significant decrease in the percentage of functional membrane, motile and live sperm with intact acrosomes was observed when samples were thawed (GL, DMF and GL/DMF). Nevertheless, the cryopreservation protocols used preserved sperm DNA quality; thus, sperm chromatin condensation and DNA fragmentation were unaffected (p > .05) when GL, DMF and GL/DMF were used. To conclude, no superiority was found between the use of a single or a combination of permeable cryoprotectants to freeze llama semen.

Use of dimethylformamide to cryopreserve alpaca semen previously incubated with collagenase.

The objective of this study was to evaluate the effect of collagenase and two final dimethylformamide (DMF) concentrations (4% and 7%) on alpaca frozen-thawed sperm quality. A total of 25 ejaculates from 5 alpaca were obtained using electroejaculation. Each individual ejaculate was evaluated and then diluted 4:1 in a solution of 1 mg/ml collagenase in HEPES-TALP medium and incubated for 4 min at 37°C. Subsequently, samples were diluted in TRIS-fructose-citric acid-egg yolk and cooled to 5°C. Then, each sample was divided in two aliquots and DMF at final concentration of 4% or 7% was added, equilibrated for 1 hr at 5°C and frozen over liquid nitrogen vapours. A Kruskal-Wallis test was used to evaluate the sperm morphometry, and Completely Random Block designs were used to analyse sperm motility, viability, membrane function and acrosome status. After collagenase incubation, none of the samples showed thread formation, and sperm parameters were preserved. Non-progressive motile sperm were higher (p < .05) in equilibrated samples (4% DMF: 31.8 ± 8.3% and 7% DMF: 36.3 ± 11.8%) compared to raw (10.1 ± 4.3%) and frozen-thawed semen (4% DMF: 9.7 ± 1.8% and 7% DMF: 7.5 ± 3.2%). Sperm membrane function, membrane integrity and intact acrosomes were higher (p < .05) in raw semen (40.1 ± 12.2%, 94.6 ± 3.2% and 91.3 ± 8.1%) compared to frozen-thawed samples (4% DMF: 19.8 ± 4.7%, 53.2 ± 2.7%, 65.7 ± 8.7% and 7% DMF: 20.4 ± 4.5%, 54.1 ± 1.4%, 64.6 ± 9.1%). Length of the sperm head was lower in frozen-thawed samples, being statistically different with 4% DMF compared to pre-freezing samples. The ratio between acrosome and head areas was greater (p < .05) in frozen-thawed samples. Incubation of raw alpaca semen with collagenase decreased the thread formation without affecting sperm quality. Frozen of collagenase treated alpaca semen with 4% or 7% DMF did not preserve the sperm parameters in thawed samples.

Cryopreservation Studies on Silver Carp (Hypophthalmichthys molitrix) Embryos.

BACKGROUND: Cryopreservation is an effective tool for the preservation of live biological materials. OBJECTIVE: This study examined the suitability of cryopreservation protocols and the effectiveness of ultrasound for silver carp embryos. MATERIALS AND METHODS: Embryos at three developmental stages were exposed to 10, 15, 20, and 25% of five cryoprotectants (CPAs), namely propylene glycol (PG), dimethylformamide (DFA), DMSO, MeOH, and ethylene glycol (EG) for 20 min. Embryos were exposed to twelve vitrification solutions (VSs) for 10 (five steps of 2 min), 15 (five steps of 3 min), 20 (five steps of 4 min) min. Embryos were also exposed to ultrasound in VSs prior to cooling for cryopreservation. RESULTS: Hatching rates decreased with increasing CPA concentrations while toxicity varied in the order of PG < DMSO < EG < MeOH < DFA. Tail elongation stage was more tolerant to CPA than 6-somites and morula stages. The survival of embryos exposed to ultrasound in VS was remarkably lower than in water. Embryos exposed to ultrasound in VSs under the best conditions did not response well after attempted vitrification. CONCLUSION: Ultrasound-mediated CPA impregnation could be effective but other innovative methods may be needed to attain successful cryopreservation.

Comparison of TNC and standard extender on post-thaw quality and in vivo fertility of Thai native chicken sperm.

Semen extender has a vital role in preservation of sperm cells properties in terms of sperm viability, motility, acrosome integrity, and mitochondrial membrane potential. The objective of the present study was to evaluate a new extender, known as Thai native chicken (TNC) extender compared to BHSV-based and modified Sasaki extenders for freezing chicken semen. Semen from Thai native roosters was collected, pooled, and randomly divided into three groups. Semen was frozen with a simple freezing method using nitrogen vapor and dimethylformamide. In the first experiment, post-thaw motion parameters, viability, acrosome integrity, mitochondrial function, and lipid peroxidation levels were analyzed using computer-assisted sperm analysis, propidium iodide, fluorescein isothiocyanate-conjugate peanut agglutinin, JC-1, and the thiobarbituric acid reaction. Results showed that the type of extender had no effect on the percentage of total motile and curvilinear velocity. The percentage of progressive motile, straight-line velocity, and average path velocity of post-thawed semen were significantly lower in TNC compared to the modified Sasaki extender. However, the percentages of post-thawed acrosome integrity and active mitochondria were significantly higher in TNC extender (P < 0.05). For the second experiment, semen was thawed by using each of extenders thereafter, was inseminated to 48-layer breeder hens to determine the fertility rate. Among the three extenders used, the highest fertility rate was found in TNC extender. In conclusion, TNC extender can be recommended as an appropriate and useful cryopreservation media for native chicken semen since it maintains the quality of rooster semen and fertility after freezing and thawing process.

Overexpression of ATG5 Gene Makes Granulocyte-Like HL-60 Susceptible to Release Reactive Oxygen Species.

Neutrophils represent the first line of defense against pathogens using various strategies, such as phagocytosis, production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. Recently, an autophagy-independent role of autophagy related (ATG) gene 5 in immune cells, including neutrophils, was emphasized. Our aim was to investigate the role of ATG5 protein in neutrophils' antimicrobial functions, proliferation and apoptosis. To this end, we used genetically modified human promyelocytic leukemia (HL-60) cells overexpressing ATG5, differentiated toward granulocyte-like cells with all-trans retinoic acid (ATRA) and dimethylformamide. The level of differentiation, phagocytosis, proliferation and apoptosis were determined by flow cytometry. ROS production and NETs release was assessed by fluorometry and fluorescent microscopy. ATG5 gene expression was evaluated by real-time PCR, whereas the protein level of ATG5 and LC3-II was determined by Western blot. We did not observe the induction of autophagy in differentiated HL-60 cells overexpressing ATG5. The increased expression of ATG5 affects the differentiation of HL-60 cells with ATRA, ROS production and phagocytosis. However, we did not detect changes in NETs release. Moreover, ATG5 protects differentiated HL-60 cells from apoptosis but does not cause changes in proliferation rate.

Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis.

The synergy obtained by the combination of cryoprotectants is a successful strategy that can be beneficial on the optimization of zebrafish sperm cryopreservation. Recently, a protocol was established for this species using an electric ultrafreezer (-150 °C) performing cooling rate (-66 °C/min) and storage within one step. The ultimate objective of sperm cryopreservation is to generate healthy offspring. Therefore, the objective of this study was to select the most adequate cryoprotectant combination, for the previously established protocol, that generate high quality offspring with normal skeletogenesis. Among the permeating cryoprotectant concentrations studied 12.5% and 15% of N,N-dimethylformamide (DMF) yielded high post-thaw sperm quality and hatching rates. For these two concentrations, the presence of bovine serum albumin (10 mg/mL), egg yolk (10%), glycine (30 mM) and bicine (50 mM) was evaluated for post-thaw sperm motility, viability, in vitro fertilization success and offspring skeletal development (30 days post fertilization). Higher concentration of permeating cryoprotectant (15%) decreased the incidence of deformed arches and severe skeletal malformations, which suggests higher capacity to protect the cell against cold stress and DNA damage. Extender containing 15% DMF with Ctrl, Bicine and egg yolk were the non-permeating cryoprotectants with higher post-thaw quality. The use of these compounds results in a reduction in vertebral fusions, compressions and severity of skeletal malformations in the offspring. Therefore, these extender compositions are beneficial for the quality of zebrafish offspring sired by cryopreserved sperm with -66 °C/min freezing rate. To the best of our knowledge, this is the first report on skeletal development of the offspring sired by cryopreserved sperm performed with different freezing media compositions in zebrafish.

Metabolic stress is a barrier to Epstein-Barr virus-mediated B-cell immortalization.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus that has been causally linked to the development of B-cell and epithelial malignancies. Early after infection, EBV induces a transient period of hyperproliferation that is suppressed by the activation of the DNA damage response and a G1/S-phase growth arrest. This growth arrest prevents long-term outgrowth of the majority of infected cells. We developed a method to isolate and characterize infected cells that arrest after this early burst of proliferation and integrated gene expression and metabolic profiling to gain a better understanding of the pathways that attenuate immortalization. We found that the arrested cells have a reduced level of mitochondrial respiration and a decrease in the expression of genes involved in the TCA cycle and oxidative phosphorylation. Indeed, the growth arrest in early infected cells could be rescued by supplementing the TCA cycle. Arrested cells were characterized by an increase in the expression of p53 pathway gene targets, including sestrins leading to activation of AMPK, a reduction in mTOR signaling, and, consequently, elevated autophagy that was important for cell survival. Autophagy was also critical to maintain early hyperproliferation during metabolic stress. Finally, in assessing the metabolic changes from early infection to long-term outgrowth, we found concomitant increases in glucose import and surface glucose transporter 1 (GLUT1) levels, leading to elevated glycolysis, oxidative phosphorylation, and suppression of basal autophagy. Our study demonstrates that oncogene-induced senescence triggered by a combination of metabolic and genotoxic stress acts as an intrinsic barrier to EBV-mediated transformation.

The influence of agents differentiating HL-60 cells toward granulocyte-like cells on their ability to release neutrophil extracellular traps.

Studies on neutrophil extracellular traps (NETs) are challenging as neutrophils live shortly and easily become activated. Thus, availability of a cell line model closely resembling the functions of peripheral blood neutrophils would be advantageous. Our purpose was to find a compound that most effectively differentiates human promyelocytic leukemia (HL-60) cells toward granulocyte-like cells able to release NETs. HL-60 cells were differentiated with all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and stimulated with phorbol 12-myristate 13-acetate (PMA) or calcium ionophore A23187 (CI). Cell differentiation, phagocytosis and calcium influx were analyzed by flow cytometry. Reactive oxygen species production and NETs release were measured fluorometrically and analyzed microscopically. LC3-II accumulation and histone 3 citrullination were analyzed by western blot. ATRA most effectively differentiated HL-60 cells toward granulocyte-like cells. ATRA-dHL-60 cells released NETs only upon PMA stimulation, DMSO-dHL-60 cells only post CI stimulation, while DMF-dHL-60 cells formed NETs in response to both stimuli. Oxidative burst was induced in ATRA-, DMSO- and DMF-dHL-60 cells post PMA stimulation and only in DMF-dHL-60 cells post CI stimulation. Increased histone 3 citrullination was observed in stimulated DMSO- and DMF-, but not in ATRA-dHL-60 cells. The calcium influx was diminished in ATRA-dHL-60 cells. Significant increase in autophagosomes formation was observed only in PMA-stimulated DMF-dHL-60 cells. Phagocytic index was higher in ATRA-dHL-60 cells than in control, DMSO- and DMF-dHL-60 cells. We conclude that ATRA, DMSO and DMF differentiate HL-60 in different mechanisms. DMF is the best stimulus for HL-60 cell differentiation for NETs studies.

Sulfated vizantin induces formation of macrophage extracellular traps.

Vizantin is an insoluble adjuvant that activates macrophages and lymphocytes. Recently, 2,2',3,3',4,4'-hexasulfated-vizantin (sulfated vizantin), which enables solubilization of vizantin, was developed by the present team. Sulfated vizantin was found to enhance bactericidal activity against multi-drug resistant Pseudomonas aeruginosa in RAW264.7 cells. In addition, spread of P. aeruginosa was inhibited in RAW264.7 cells treated with sulfated vizantin. When only sulfated vizantin and P. aeruginosa were incubated, sulfated vizantin did not affect growth of P. aeruginosa. Formation of DNA-based extracellular traps (ETs), a novel defense mechanism in several types of innate immune cells, helps to eliminate pathogens. In the present study, ET-forming macrophages constituted the majority of immune cells. Sulfated vizantin induced ET formation in RAW264.7 cells, whereas a Ca-chelating reagent, EDTA, and T-type calcium channel blocker, tetrandrine, inhibited ET formation and attenuated inhibition of spread of P. aeruginosa in sulfated vizantin-treated cells. Thus, sulfated vizantin induces ET formation in phagocytic cells in a Ca-dependent manner, thus preventing spread of P. aeruginosa. Hence, sulfated vizantin may be useful in the management of infectious diseases.

Dimethylformamide is a novel nitrilase inducer in Rhodococcus rhodochrous.

Nitrilases are of commercial interest in the selective synthesis of carboxylic acids from nitriles. Nitrilase induction was achieved here in three bacterial strains through the incorporation of a previously unrecognised and inexpensive nitrilase inducer, dimethylformamide (DMF), during cultivation of two Rhodococcus rhodochrous strains (ATCC BAA-870 and PPPPB BD-1780), as well as a closely related organism (Pimelobacter simplex PPPPB BD-1781). Benzonitrile, a known nitrilase inducer, was ineffective in these strains. Biocatalytic product profiling, enzyme inhibition studies and protein sequencing were performed to distinguish the nitrilase activity from that of sequential nitrile hydratase-amidase activity. The expressed enzyme, a 40-kDa protein with high sequence similarity to nitrilase protein Uniprot Q-03217, hydrolyzed 3-cyanopyridine to produce nicotinic acid exclusively in strains BD-1780 and BD-1781. These strains were capable of synthesising both the vitamin nicotinic acid as well as ß-amino acids, a compound class of pharmaceutical interest. The induced nitrilase demonstrated high enantioselectivity (> 99%) in the hydrolysis of 3-amino-3-phenylpropanenitrile to the corresponding carboxylic acid.

[Effects of N, N-dimethylformamide on hepatic antioxidant capacity and liver PPARs mRNA levels in rats].

OBJECTIVE: To investigate the effects of N, N-dimethylformamide(DMF)exposure on liver anti-oxidative capacity and peroxisome proliferator activated receptor(PPAR)αand PPARγin rats. METHODS: A total of 30 male SD rats were randomly divided into 6 groups and orally administered with DMF 150 mg/kg body weight. Blood and liver tissues were collected on day 0(before DMF exposure), 1, 3, 7, 14 and 28 after DMF exposure. Blood were collected for blood routine examination and liver tissues for H&E staining. Glutathione peroxidase(GSH-Px)and catalase(CAT)were detected by kits, the mRNA levels of PPARαand PPARγwere detected by real-time PCR, and proinflammatory cytokines[interleukin-1 beta(IL-1ß), interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)]were detected by ELISA kits. RESULTS: Compared with day 0(control group), white blood cell(WBC)level in blood was significantly increased after 1 day exposure[(8. 30±0. 61)×10~9/L vs. (12. 64±1. 02)×10~9/L, P<0. 05]. As exposure time increases, WBC levels were increasing. In addition, DMF causes serious liver damage, including cell swelling, lymphocyte infiltration, and punctuate necrosis. GSH-Px was significantly increased on the first day after DMF exposure[(1006. 00±168. 60)U vs. (1437. 00±321. 00)U, P<0. 05]. However, after DMF exposure, the CAT levels increased significantly on day 28[(35. 17±4. 90)U/mg vs. (51. 80±10. 32)U/mg, P<0. 05]. Compared with day 0, the mRNA levels of PPARαand PPARγwere significantly increased on day 7 after DMF exposure(1. 35±1. 30 vs. 35. 70±10. 88, 1. 04±0. 33 vs. 191. 10±44. 70, P<0. 01). IL-1ß, IL-6 and TNF-αdecreased after DMF exposure, in which IL-1ß significantly decreased on day 28 after DMF exposure when compared with day 0[(34. 75±5. 94)pg/mL vs. (25. 52±1. 65)pg/mL, P<0. 05]. IL-6 decreased continuously after DMF exposure and the lowest value on day 14 after DMF exposure[(139. 10±23. 10)pg/mL vs. (97. 86±4. 15)pg/mL, P<0. 01], and TNF-αalso continuously decreased after DMF exposure, and decreased on the bottom value on day 28 after DMF exposure[(295. 40±29. 31)pg/mL vs. (217. 10±7. 43)pg/mL, P<0. 01]. CONCLUSION: A large amount ROS was produced during DMF metabolized by CYP2E1, which can cause oxidative stress and lead to inflammation. Therefore, it negative feedback up-regulates the transcription levels of PPARs and inhibits the proinflammatory cytokines secretions so as to reduce the inflammatory reaction.

Full-term potential of goat in vitro produced embryos after different cryopreservation methods.

Cryopreservation of preimplantation embryos represents a major challenge due to their shape and relatively large cells. Embryo source and cryopreservation method are key factors to cryotolerance efficiency and few reports have investigated more promising protocols for goat embryos. The study was aimed to compare different cryopreservation methods for goat in vitro produced (IVP) embryos. Goat blastocysts were subjected to conventional freezing (CF), Dimethyl sulfoxide vitrification (DMSO-V) and Dimethylformamide vitrification (DMF-V). Cryopreserved blastocysts were assessed for re-expansion, cell viability and in vivo development rates. Blastocyst re-expansion after cryopreservation was similar between groups, but cell viability was lower for DMF-V (32%) than CF (68%) and DMSO-V (60%). Pregnancy and delivery rates were similar for CF (60% and 50%) and DMSO-V (50% and 45%) and higher then DMF-V (20% and 15%), respectively. Finally, kidding rates were also indistinguishable for CF (40%) and DMSO-V (35%), but higher then DMF-V (12.5%). In conclusion, conventional freezing and vitrification using DMSO have similar efficiencies for cryopreservation of goat IVP embryos and cryoprotectant for vitrification affects its outcome.

Porcine sperm vitrification II: Spheres method.

Owing to current problems in boar sperm cryopreservation, this study proposes to evaluate vitrification in spheres as an alternative cryopreservation procedure, comparing the use or not of permeable cryoprotectants and two warming methods. Extended (n = 3; r = 4) and raw (n = 5; r = 2) porcine spermatozoa were diluted in media, in the absence or presence of either 4% dimethylformamide or 4% glycerol, to a final concentration of 5 × 106  spermatozoa/ml and vitrified using the spheres method. Two warming procedures were evaluated: a rapid method (30 s at 37°C) and an ultrarapid method (7 s at 75°C, followed by 30 s at 37°C). Percentages of total motility (phase contrast), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), sperm viability (6-carboxyfluorescein diacetate and propidium iodide stain), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated in the samples before and after vitrification. Results, analysed using Friedman's test, suggest that rapid warming of raw porcine spermatozoa vitrified without permeable cryoprotectants may preserve DNA condensation and integrity better than the other processing methods studied in this work. Hence, porcine sperm vitrification using spheres could be used to produce embryos with ICSI to further validate this method.

The effect of some cryoprotectants on dromedary camel frozen-thawed semen.

The cryopreserved camel semen is often associated with poor quality and fertility. This study aimed to improve the dromedary frozen semen quality by comparing the efficiency of four cryoprotectant agents (CPAs) on sperm freezability. Semen samples were collected from seven male Maghrabi camels, diluted with Shotor diluent supplemented with glycerol (Sh-G), dimethyl formamide (DMF, Sh-DF), dimethyl sulfoxide (DMSO, Sh-DS) or ethylene glycol (EG, Sh-EG), all at 6% final concentration, and the samples were subjected to cryopreservation. The results revealed the superiority of Sh-DF over Sh-G and Sh-DS in terms of post-thaw motility (55.83 ± 2.20 vs. 47.50 ± 4.33 and 45.00 ± 2.89%, respectively), sperm membrane (49.00 ± 0.58, 39.33 ± 3.33 and 42.67 ± 1.45%, respectively) and acrosomal integrities (53.00 ± 0.58, 57.33 ± 0.88 and 52.33 ± 1.45%, respectively). Sh-EG group showed the lowest post-thaw motility, plasma membrane and acrosome integrities (12.50 ± 1.44, 22.67 ± 1.45 and 30.67 ± 1.45, respectively). In conclusion, the protocols of dromedary camel semen cryopreservation could be enhanced using 6% DMF as a cryoprotectant agent.

[Study on apoptosis mechanism of H9c2 cardiomyocytes induced by N, N-dimethylformamide].

Objective: To observe the change levels of nuclear factor-kappa B (NF-κB) p65 protein in cytoplasm and nuclear, phosphorylation of inhibitor of kappa B (p-IκB) protein and cytochrome C (Cyt-c) , cleaved cysteinyl aspartate specific proteinase-3 (Cleaved caspase-3) , B-cell lymphoma/leukemia-2 (Bcl-2) in cytoplasm in the process of N, N-dimethylformamide (DMF) -induced apoptosis in H9c2 cardiomyocytes, and explore the tentative mechanism of apoptosis. Methods: H9c2 cardiomyocytes were exposed to 200 mmol/L DMF. Western blotting was used to detect the protein expression levels of p65 in cytoplasm and nuclear, p-IκB after exposure for 0, 2, 4, 6, 8, 12 h, and the protein expression levels of Cyt-c, Cleaved caspase-3, Bcl-2 in cytoplasm after exposure for 0, 2, 4, 8, 12, 24 h. Immunofluorescencecytochemistry (IFC) was used to observe the location of Cyt-c after 200 mmol/L DMF exposure for different times. Results: The levels of p65 in cytoplasm and nuclear and p-IκB among groups were statistically significant (F were 7.79, 33.11, 90.25, respectively, all P<0.01) . Compared with the control group, the levels of p65 in cytoplasm of 2, 4, 6 h group were significantly decreased (all P<0.01) ; the levels of p65 in nuclear of 2, 4, 6, 8 h were significantly increased (all P<0.01) ; the levels of p-IκB of 2, 4, 6 h group were significantly increased (all P<0.01) . The levels of Cyt-c, Cleaved caspase-3 and Bcl-2 among groups were statistically significant (F were 51.42, 503.68, 73.37, respectively, all P<0.01) . Compared with the control group, the levels of Cyt-c of 8, 12 h group were significantly increased (both P<0.01) ; the levels of Cleaved caspase-3 of 2, 4, 8, 12, 24 h were significantly increased (all P<0.01) ; the levels of Bcl-2 of 2, 4, 8, 12, 24 h group were significantly decreased (all P<0.01) . IFC showed that Cyt-c was released from the mitochondria to the cytoplasm gradually as the extension of the exposure time. Conclusion: NF-κB signaling pathway and mitochondrial pathway are involved in the mechanism of DMF-induced apoptosis in H9c2 cardiomyocytes.

The role of amides in seminal cryopreservation of wild silverside, Odontesthes bonariensis.

Amides were tested as internal cryoprotectants for the preservation of wild silverside (Odontesthes bonariensis) sperm. The semen was diluted in modified Mounib's medium and cryopreserved by adding 2, 5, 8 or 11% of dimethyl acetamide (DMA), dimethyl formamide (DMF) or methyl formamide (MF). Dimethyl sulfoxide (DMSO) at a concentration of 10% diluted in modified Mounib's medium was used as a control. The rate motility (17.7 ± 1.9%) and time motility (143.2 ± 9.7 s) (P < 0.05) of the sperm were higher with 2% DMF when compared with the other treatments. Despite the better motility results obtained with 2% DMF, the solution was not able to maintain cellular structure integrity of the cryopreserved sperm. The 10% DMSO and 8% MF treatment allowed for completeness of the plasma membrane (34.8% and 29%), functional mitochondria (19.8% and 16.2%) and plasma membrane fluidity (39.4% and 46.4%); furthermore, rate motility (11.8% and 10%) and time motility (81.4 s and 71.8 s) of the sperm were found to be at suitable levels when compared with 2% DMF. Thus, our evaluation suggests that 10% DMSO and 8% MF provide better cryopreservation of O. bonariensis sperm cells.

Cryopreservation of boar sperm comparing different cryoprotectants associated in media based on powdered coconut water, lactose and trehalose.

In swine spermatozoa, the damage caused by cryopreservation is more severe than other species, provoking reduced potential for fertilization. Adjustments in the freezing extender composition may be an important alternative to increase its efficiency. The objective of this study was to test the efficiency of different cryoprotectant solutions during cryopreservation of swine semen with a controlled cooling curve. Three cryoprotectant solutions (5% dimethylformamide, 3% glycerol and the combination of these two cryoprotectants) were used in association with three base media (powdered coconut water, lactose and trehalose), constituting nine different treatments. The semen was frozen using a controlled-rate freezer (TK-3000). After thawing, semen was evaluated for total sperm motility, vigor, morphology, plasma membrane integrity and acrosome integrity. Cryopreservation with the controlled curve using an automated system showed satisfactory results, guaranteeing practicality and repeatability for the process of freezing swine sperm. With this curve, the solutions of lactose, trehalose and powdered coconut water associated with glycerol, as well as the solution of coconut water containing dimethylformamide, presented higher quality of sperm compared to the other solutions. Powdered coconut water associated with dimethylformamide appears as a new solution for swine sperm cryopreservation. The freezing controlled curve used in this study allowed standardization of the cryopreservation technique.