Comparison of different cryoprotectants for freezing donkey (Equus asinus) semen.

The aim of this study was to evaluate two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) in two concentrations (5 and 7 %) in vitro in donkey semen using a rapid freezing technique and the effect on pregnancy rates in mares. Twenty-four ejaculates from 8 jacks (n = 8; r = 3) were divided into 4 extenders: BotuSemen Gold with 5 % or 7 % MF and 5 % or 7 % DMF, all containing 11 % lactose, 20 % egg-yolk and 0.5 % Equex. Post-thaw evaluations included: sperm motility, membrane function and acrosome status. A linear mixed effect model was used to test the effect of different freezing media on semen parameters. No differences were observed between the 4 freezing media used, for any of the seminal parameters (P > 0.05). However, samples with 5 % DMF showed the highest percentages of sperm with acrosomes and functional membranes (DMF: 5 %: 53.67 ± 22.01; 7 %: 33.92 ± 23.4; MF: 5 %: 44.5 ± 20.46; 7 %: 38.75 ± 27.4) (Data: mean ± SD; P > 0.05). Hence, thirty mares were inseminated: 15 with 5 % DMF and 15 with 7 % DMF. The pregnancy rate was 46 % (7/15) and 0 % (0/15) using the extender with 5 % or 7 % DMF, respectively (P = 0.003). To conclude, the use of 5 % or 7 % of MF or DMF did not affect the in vitro parameters. Despite the lack of differences in vitro with the two DMF concentrations, in vivo results only showed pregnancies when using 5 % DMF. Thus, the results of this study demonstrate the importance of accompanying in vitro semen evaluations with studies that evaluate post-insemination pregnancy rates.

Diluent Containing Dimethylformamide Added With Sucrose Improves In Vitro Quality After Freezing/Thawing Stallion Sperm.

The aim of this study was to investigate the effects of sucrose on post-thawed equine semen quality. Semen samples (n = 24) were collected from six stallions. They were diluted (200 × 106 sperm/mL) in a freezing medium based on skimmed milk, egg yolk, dimethylformamide, and supplemented with sucrose at concentrations of 0 (Control), 25, 50, and 100 mM and in a commercial extender (BotuCrio). Subsequently, they were filled in straws (0.5 mL) and subjected to freezing and storage (-196°C). Immediately after thawing (37°C, 30 seconds), semen samples were evaluated for kinetics (CASA), plasma and acrosomal membrane integrity, and mitochondrial membrane potential (flow cytometry). The addition of 50 and 100mM sucrose to the freezing extender increased (P < .05) the parameters of TM, PM, VCL, VSL, and VAP, compared to the control group. The WOB parameter of the group supplemented with 100 mM sucrose was higher (P < .05) than the control group. Higher values ​​(P < .05) of ALH and BCF were observed in groups treated with sucrose (25, 50, and 100 mM), compared to BotuCrio. The semen frozen in the presence of 100 mM sucrose presented higher percentages (P < .05) of sperm with intact plasma and acrosomal membranes, and high mitochondrial membrane potential in relation to the other groups. It is concluded that the addition of sucrose to equine semen freezing extender increase motility (50 and 100 mM), plasma and acrosomal membrane integrity preserve, and high sperm mitochondrial membrane potential (100 mM) after thawing.

Dimethylformamide Preserves the Integrity of Cryopreserved Goat Semen in a Soybean Lecithin-Based Extender.

This study investigated the cryoprotectant effects of dimethylformamide (DMF), ethylene glycol (EG), and dimethyl sulfoxide (DMSO) as substitutes for glycerol (GLY) in a soybean lecithin (SL)-based extender in the cryopreservation of buck sperm. In this study, the semen of three Saanen bucks was individually extended in SL supplemented with 5% GLY (control), DMF, EG, or DMSO. After this, the extended semen was cryopreserved and two straws from each group were thawed (37°C for 30 seconds), pooled, and analyzed for sperm motion parameters, plasma membrane integrity (PMI), acrosomal integrity (ACI), and high mitochondrial membrane potential (HMMP). Samples were analyzed after 15 minutes (T0) and after 2 hours of incubation at 37°C (T2). The results revealed higher values of motility (total and progressive) and sperm motion parameters for DMF than the other cryoprotectants (p < 0.0001). PMI and HMMP did not differ (p > 0.05) between GLY and DMF, but ACI was higher (p < 0.01) for DMF compared with GLY. Based on these results, DMF and GLY samples were used in heterologous in vitro fertilization assays by using bovine oocytes (n = 337) obtained from a slaughterhouse. No differences (p > 0.05) were observed between GLY and DMF for unfertilized (GLY: 38.8%; DMF: 25.33%), pronucleus (GLY: 25.68%; DMF: 27.92%), and cleavage rates (GLY: 35.52%; DMF: 46.75%). Based on these results, it is concluded that DMF preserves sperm motion characteristics and ACI better than GLY, EG, and DMSO, and it is the penetrating cryoprotectant of choice for the cryopreservation of buck sperm in SL extender.

Effect of Different Extenders on Sperm Motility and Vitality in Goose Semen Cryopreservation

This study aimed to investigate the usability of different diluents containing 6% Dimethylformamide (DMF) for cryo-preservation of the semen of geese (Anser cygnoides). The diluents of Glucose (G), Tris-Glucose (T), Lactated Ringer's-Glucose (LG), and Lactated Ringer's (L), all of which contained 6% DMF, were used as cryoprotectants. The researchers collected semen samples from four geese, twice a week over a four-week period, by means of abdominal massage; they then calculated how much sperm each goose ejaculated. Next, the semen samples were pooled and their spermatological parameters were determined. Their volume (4x (mL)), concentration (×108/mL), pH, motility (%), and vitality (%) rates were 0.31±0.01, 3.49±0.32, 7.13±1.06, 67.75±1.28, and 70.00±2.03, respectively. Then, these pooled semen samples were equally divided into four groups. Once they were frozen and thawed, the researchers discovered that the diluent L had the highest motility rate: 40.12% ± 1.35. The motility rates of the other diluents were as follows: LG (28.25%± 1.48), G (21.50% ± 1.41), and T (5.12% ± 0.83). Likewise, the vitality rates (%) of the diluents were as follows: L (41.93% ±1.87), LG (31.50%±1.88), G (29.43% ±1.45), and T (10.56%±1.34), respectively. Freezing and thawing appeared to lower each diluent's vitality and motility rates. However, for the Lactated Ringer's (L), this decrease was predictable. Therefore, Lactated Ringer's diluent containing 6% DMF can be used in cryo-preservation of goose semen.(AU)

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.

The Effect of N-N-Dimethylformamide on the Membrane Characteristics of Canine Spermatozoa After Cryopreservation, and its Relationship With Post-Thaw Motility.

Some studies have demonstrated that glycerol is superior to amides in preserving sperm motion characteristics of canine sperm; however, little is known about the effect of these cryoprotectants on the membrane characteristics of canine spermatozoa after freezing/thawing. In this study, the effects of using either N-N-dimethylformamide (DMF) or glycerol (GLY) on the integrity and function of the canine sperm, after cryopreservation were determined. We hypothesized that the use of a multiparametric approach for assessing the effect of DMF on the membranes of canine sperm would explain the lower values reported for post-thaw motility. Ejaculates from 12 dogs were collected, split into 2 groups, and frozen using a tris-fructose-citrate-egg yolk-based extender containing either 7% (v/v) GLY or 7% (v/v) DMF. Frozen straws (n = 120) were thawed and analyzed for subjectively-assessed sperm progressive motility, normal morphology, plasma membrane integrity, plasma membrane function (HOST+), acrosome membrane integrity, high mitochondrial membrane potential, and simultaneous assessment of sperm membrane integrity and function by a triple-staining fluorescent procedure. Overall, sperm motility and membrane intactness/function were higher when GLY was used as a cryoprotectant, as compared to DMF (P < .05). A model to explain the variation in progressive motility using the values obtained from the sperm integrity and function parameters was designed. The percent HOST+ sperm and high mitochondrial membrane potential sperm were mostly associated with the changes observed in the progressive motility (r2 = 0.84; P = .043) when either GLY or DMF were used as cryoprotectants. These results may explain the overall reduced sperm quality observed after cryopreservation, as a reflection of sublethal damage sustained by the sperm membranes.

Effect of glycerol, n, n-dimethylformamide and n-methyl-2-pyrrolidone on rabbit sperm stored at 4 °C and 16 °C

Artificial insemination with cooled semen is the most common practice in rabbit farms and any improvement on it helps to increase the efficiency and productivity of rabbit meat farms. Therefore, the aim of this study was to assess whether different cryoprotectant agents (CPA) as glycerol, N, N-Dimethylformamide (DMF) and N-Methyl-2-Pyrrolidone (NMP) can improve cooled rabbit sperm quality stored at 4ºC and 16ºC. Sperm samples were diluted with INRA 96® (Extender A), INRA 96® with 6% glycerol (Extender B) or 6% DMF (Extender C) or 6% NMP (Extender D) respectively and stored at 4ºC and 16ºC. Samples were then analysed at 4, 24, 48 and 72 hours after refrigeration by integrated sperm analysis system (ISAS®), eosin-nigrosin stain (vitality), hypo-osmotic swelling test (HOS test) and acrosome integrity test. Extender C showed higher percentage of motility, vitality and HOS test than extender B and D (p<0.05). Whereas sperm quality decreased over time (p<0.05), data showed that the addition of DMF kept the motility and sperm plasma membrane integrity after 24 hours of storage better than other diluents. These results suggest that the addition of DMF to INRA 96® exerts a protective effect on the membrane of spermatozoa improving seminal quality.(AU)

Effect of glycerol, n, n-dimethylformamide and n-methyl-2-pyrrolidone on rabbit sperm stored at 4 °C and 16 °C

Artificial insemination with cooled semen is the most common practice in rabbit farms and any improvement on it helps to increase the efficiency and productivity of rabbit meat farms. Therefore, the aim of this study was to assess whether different cryoprotectant agents (CPA) as glycerol, N, N-Dimethylformamide (DMF) and N-Methyl-2-Pyrrolidone (NMP) can improve cooled rabbit sperm quality stored at 4ºC and 16ºC. Sperm samples were diluted with INRA 96® (Extender A), INRA 96® with 6% glycerol (Extender B) or 6% DMF (Extender C) or 6% NMP (Extender D) respectively and stored at 4ºC and 16ºC. Samples were then analysed at 4, 24, 48 and 72 hours after refrigeration by integrated sperm analysis system (ISAS®), eosin-nigrosin stain (vitality), hypo-osmotic swelling test (HOS test) and acrosome integrity test. Extender C showed higher percentage of motility, vitality and HOS test than extender B and D (p<0.05). Whereas sperm quality decreased over time (p<0.05), data showed that the addition of DMF kept the motility and sperm plasma membrane integrity after 24 hours of storage better than other diluents. These results suggest that the addition of DMF to INRA 96® exerts a protective effect on the membrane of spermatozoa improving seminal quality.

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 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.

Comparison among different cryoprotectants for cryopreservation of epididymal sperm from agouti (Dasyprocta leporina).

We verify the effects of different cryoprotectants on the cryopreservation of agouti (Dasyprocta leporina) epididymal sperm. We used 16 pairs of testes-epididymis complexes of sexually mature animals. We immediately evaluated epididymal sperm obtained by retrograde flushing for concentration, motility, vigor, viability, osmotic response, and morphology. Samples were extended in a coconut water extender plus 20% egg yolk, containing glycerol, ethylene glycol, dimethylsulfoxide - DMSO, or dimethylformamide. Finally, samples were stored in 0.25 mL straws, frozen in liquid nitrogen, and thawed after one week, being reevaluated and assessed for membrane integrity using fluorescent probes. The higher values for postthawing sperm motility, vigor, and membrane integrity were achieved by the usage of glycerol, when compared to ethylene glycol and dimethylformamide (P < 0.05); however, no differences were found between glycerol and DMSO (P > 0.05). All cryoprotectants provided a similar effect on the preservation of sperm morphology, osmotic response, and viability (P > 0.05). Therefore, here onwards, there was testing of glycerol and DMSO at 3 and 6% concentrations using the same freezing-thawing protocol reported previously. As the main result, DMSO at 6% concentration provided a decrease in sperm parameters, as well as in the chromatin integrity and in the binding capability of sperm. In conclusion, glycerol 3 or 6% and DMSO 3% can be used as alternative cryoprotectants for agouti epididymal sperm cryopreservation.

Single-step solubilization of milk samples with N,N-dimethylformamide for inductively coupled plasma-mass spectrometry analysis and classification based on their elemental composition.

A single-step procedure for trace elements analysis of milk samples is presented. Solubilization with small amounts of dymethylformamide (DMF) was assayed prior to inductively coupled plasma mass spectrometry (ICPMS) detection with a high efficiency sample introduction system. All main instrumental conditions were optimized in order to readily introduce the samples without matrix elimination. In order to assess and mitigate matrix effects in the determination of As, Cd, Co, Cu, Eu, Ga, Gd, Ge, Mn, Mo, Nb, Nd, Ni, Pb, Pr, Rb, Sm, S, Sr, Ta, Tb, V, Zn, and Zr, matrix matching calibration with (103)Rh as internal standard (IS) was performed. The obtained limits of detection were between 0.68 (Tb) and 30 (Zn) µg L(-1). For accuracy verification, certified Skim milk powder reference material (BCR 063R) was employed. The developed method was applied to trace elements analysis of commercially available milks. Principal components analysis was used to correlate the content of trace metals with the kind of milk, obtaining a classification according to adults, baby or baby fortified milks. The outcomes highlight a simple and fast approach that could be trustworthy for routine analysis, quality control and traceability of milks.

A purely vibrational insight on the tetragonal distortion of [Cu(DMF)6](2+).

Raman spectra of N,N-dimethylformamide (DMF) solutions containing copper perchlorate at different compositions have been recorded. The spectral changes exhibited at the rmethyl region of DMF, in the presence of the Cu(II) salt, were reported for the first time. A correlation with the δOCN region is made. The quantitative Raman analysis performed at the rmethyl region allowed the determination of an average number of 6 DMF molecules per Cu(II) in the first solvation shell. This value as combined to the spectral variations observed in both regions of DMF allow us to propose the existence of axial bond lengths longer than equatorial ones in the octahedron. The spectra also reveal that the counter-ion is not capable of affecting the stability of the [Cu(DMF)6](2+) complex for the whole investigated concentration range.

Comparison of vitrification and conventional freezing for cryopreservation of caprine embryos.

The experiment aimed to compare conventional freezing and different vitrification protocols for cryopreservation of caprine embryos at morphological, ultrastructural, and functional levels. Caprine embryos produced in vivo were allocated randomly to three groups: (1) conventional freezing with ethylene glycol (EG); (2) dimethyl sulfoxide + EG (DMSO/EG) vitrification; and (3) dimethylformamide + EG (DMF/EG) vitrification. All groups were scored for cell viability (propidium iodide staining and ultrastructural levels) and re-expansion rate after thawing or warming. Embryos subjected to DMSO/EG vitrification showed higher cell viability (73.33%), compared with DMF/EG vitrification and conventional freezing group embryos (40.00 and 66.66%, respectively). The ultrastructural study revealed that vitrified embryos had greater preservation of cellular structure than embryos from conventional freezing with EG. DMSO/EG vitrification resulted in higher rates of re-expansion in vitro (47.36%) than DMF/EG vitrification (31.58%), and conventional freezing (25.00%). In conclusion, caprine embryos produced in vivo are better cryopreserved after vitrification than conventional freezing, therefore we conclude that DMSO/EG vitrification is the most effective protocol for cryopreservation.

Comparison of conventional freezing and vitrification with dimethylformamide and ethylene glycol for cryopreservation of ovine embryos.

The aim of this work was to evaluate the efficiency of the cryoprotectants dimethylformamide and ethylene glycol for cryopreservation of ovine embryos using vitrification and conventional freezing. The recovered embryos were distributed randomly in three treatment groups: Gr. 1: conventional freezing (n = 44), Gr. 2: vitrification with ethylene glycol (n = 39) and Gr. 3: vitrification with dimethylformamide (n = 38). Quality of fresh embryos in control group as well as of frozen and vitrified embryos was examined by three methodologies: staining with propidium iodide and Hoechst 33258 and evaluation under fluorescent microscopy, evaluation of re-expansion and hatching rates after culture, and determination of apoptotic index with TUNEL technique. It was established that re-expansion rate in all treatment groups was similar. In the same time, hatching rates were higher in Gr. 1 (40.5%) and Gr. 2 (35.3%) in comparison with Gr. 3 (15.5%, p < 0.05). The number of dead cells in vitrified embryos of Gr. 2 and Gr. 3 was higher (42.6 ± 26.2 and 63.2 ± 34.65, respectively) in comparison with Gr. 1 (conventional freezing, 10.1 ± 8.5, p < 0.05). Embryos vitrified with dimethylformamide included the same quality of apoptotic cells that Gr. 1 (conventional freezing) and fresh embryos. In conclusion, the dimethylformamide and ethylene glycol used as cryoprotectant to vitrify ovine embryos, in the concentrations and exposition time tested in this work, were not as efficient as the conventional freezing for cryopreservation of ovine embryos Thus, the conventional freezing with ethylene glycol was the most efficient method to cryopreserve ovine embryos in comparison with vitrification.

Synthesis, spectroscopic properties and photodynamic activity of porphyrin-fullerene C60 dyads with application in the photodynamic inactivation of Staphylococcus aureus.

A covalently linked porphyrin-fullerene C60 dyad 5 was synthesized by 1,3-dipolar cycloaddition using 5-(4-formylphenyl)-10,15,20-tris[3-(N-ethylcarbazoyl)]porphyrin, N-methylglycine and fullerene C60. Methylation of 5 was used to obtain a cationic dyad 6. Spectroscopic properties were compared in toluene, N,N-dimethylformamide (DMF) and toluene/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water reverse micelles. Absorption spectra of the dyads were essentially a superposition of the spectra of the porphyrin and fullerene reference compounds, indicating a very weak interaction between the chromophores in the ground state. The fluorescence emission of the porphyrin moiety in the dyads was strongly quenched by the attached fullerene C60 unit. The singlet molecular oxygen, O2((1)Δg), productions (ΦΔ) were strongly dependent on the solvent polarity. Similar ΦΔ values were obtained for 5,10,15,20-tetrakis[3-(N-ethylcarbazoyl)]porphyrin (TCP) in both solvents. Also, dyad 5 showed a high O2((1)Δg) generation in toluene. However, O2((1)Δg) production mediated by 5 considerably diminished in the more polar solvent DMF. Also, a high photodynamic activity involving O2((1)Δg) was found for both dyads in a simple biomimetic system formed by AOT reverse micelles. The photoinactivation ability of these dyads was investigated in Staphylococcus aureus cell suspensions. Photosensitized inactivation of S. aureus by dyad 6 exhibits a 4.5 log decrease of cell survival (99.997% cell inactivation), when the cultures are treated with 5 µM photosensitizer and irradiated with visible light (350-800 nm) for 30 min. Under these conditions, a lower photocytotoxic effect was found for 5 (3.2 log decrease). Furthermore, photoinactivation induced by 6 was higher than those obtained with the separate moieties of the dyad. Therefore, molecular structures formed by porphyrin-fullerene C60 dyads represent interesting photosensitizers to inactivate S. aureus.

Spectroscopic and DFT study of solvent effects on the electronic absorption spectra of sulfamethoxazole in neat and binary solvent mixtures.

The solvatochromic behavior of sulfamethoxazole (SMX) was investigated using UV-vis spectroscopy and DFT methods in neat and binary solvent mixtures. The spectral shifts of this solute were correlated with the Kamlet and Taft parameters (α, ß and π(*)). Multiple lineal regression analysis indicates that both specific hydrogen-bond interaction and non specific dipolar interaction play an important role in the position of the absorption maxima in neat solvents. The simulated absorption spectra using TD-DFT methods were in good agreement with the experimental ones. Binary mixtures consist of cyclohexane (Cy)-ethanol (EtOH), acetonitrile (ACN)-dimethylsulfoxide (DMSO), ACN-dimethylformamide (DMF), and aqueous mixtures containing as co-solvents DMSO, ACN, EtOH and MeOH. Index of preferential solvation was calculated as a function of solvent composition and non-ideal characteristics are observed in all binary mixtures. In ACN-DMSO and ACN-DMF mixtures, the results show that the solvents with higher polarity and hydrogen bond donor ability interact preferentially with the solute. In binary mixtures containing water, the SMX molecules are solvated by the organic co-solvent (DMSO or EtOH) over the whole composition range. Synergistic effect is observed in the case of ACN-H2O and MeOH-H2O, indicating that at certain concentrations solvents interact to form association complexes, which should be more polar than the individual solvents of the mixture.

Avaliação da eficácia de crioprotetores permeantes e não permeantes no descongelamento rápido e lento do sêmen canino / Evaluating the effectiveness of permeating and non-permeating cryoprotectants in fast and slow defrosting of canine semen

The aim of this study was to evaluate the association between cryoprotectants little studied in Brazil such as dimethylformamide and trehalose amid thinner, using protocols of fast and slow defrosting. Three adult Labrador Retrievier male, healthy dogs, weekly submitted to one semen collection during five-weeks period, were used. The base diluent medium used in this study was tris-citrate added with 3% of dimethylformamide + 3% glycerol (D1), 3%  dimethylformamide and trehalose (D2) and 4% glycerol (D3). At defrosting, half of the semen samples from each diluent medium was defrosted by rapid method in water-bath at 75 C for seven minutes, followed by a new immersion at 37 C for 1 minute. The other half of the samples was defrosted by slow method, in water-bath at 37 C for 1 minute. The semen was evaluated for sperm progressive motility and vigor, besides membrane integrity. For this, the semen samples were submitted to either hyposmotic and membrane integrity tests of the plasmatic membrane and acrosome  (fluorescence). The results indicated that the use of glycerol as cryoprotector in TRIS diluter provides greater efficacy in cryopreserving spermatozoa of the canine species, when compared to dimethylformamide associated with trehalose or glycerol.(AU)

O objetivo do trabalho foi avaliar o efeito da associação entre crioprotetores ainda pouco estudados no Brasil como a dimetilformamida e a trealose em meio diluidor, por meio de protocolos de descongelamento rápido e lento. Foram utilizados três cães machos, adultos, sadios da raça Labrador do Retrievier, que foram submetidos a uma coleta de sêmen semanal durante o período de cinco semanas. Os três meios diluentes utilizados neste estudo foram: (D1)-tris-citrato, acrescido de 3% de dimetilformamida + 3% de glicerol, (D2)-3% de dimetilformamida e de trealose e (D3)-4% de glicerol. No descongelamento, metade das amostras de cada meio diluente foi descongelada pelo método rápido, em banho-maria a 75 ºC por sete segundos, seguido de nova imersão a 37 ºC por 1 minuto. A outra metade foi descongelada pelo método lento, em banho-maria à 37 ºC por 1 minuto. O sêmen foi avaliado quanto à motilidade progressiva, vigor espermáticos e integridade de membrana. Pra isso, as amostras foram submetidas aos testes hipo-osmótico e de integridade da membrana plasmática e do acrossoma (fluorescência). Os resultados indicam que o uso do glicerol como crioprotetor em diluidor TRIS proporciona maior eficácia na criopreservação dos espermatozoides da espécie canina, quando comparados com a dimetilformamida associada ao glicerol ou trealose.(AU)