Epidemiología de la ventilación mecánica en Argentina. Estudio observacional multicéntrico EpVAr / Epidemiology of Mechanical Ventilation in Argentina. The EpVAr Multicenter Observational Study

Objetivo Describir las prácticas relacionadas a ventilación mecánica (VM) en Argentina y explorar los factores asociados a la mortalidad en UCI en esta población. Diseño Se realizó un estudio observacional, prospectivo, multicéntrico. Ámbito Unidad de Cuidados Intensivos. Pacientes Incluimos pacientes mayores de 18 años ingresados en las UCI participantes que requirieron VM invasiva durante al menos 12 horas desde el ingreso a la institución de salud. Intervenciones Ninguna. Variables Todas las variables se clasificaron en tres categorías: variables relacionadas con factores demográficos y clínicos antes de la VM, factores relacionados con el primer día de VM, y factores relacionados con los eventos ocurridos durante la VM (complicaciones y destete de la VM). El destete de la ventilación mecánica y la mortalidad se clasificaron según WIND (Weaning according to a New Definition). Resultados El análisis primario incluyó a 950 pacientes. La principal indicación de VM fue insuficiencia respiratoria aguda (58% de los pacientes). El modo de ventilación inicial fue ventilación mandatoria continua con control de volumen en el 75% de los casos. La mortalidad en UCI y hospitalaria fue del 44,6% y 47,9%, respectivamente. Las variables identificadas como predictoras independientes de mortalidad en UCI fueron edad (OR 3,48 IC 95%, 1,22-11,66; p=0,028), fracaso en la implementación de ventilación mecánica no invasiva (VNI) antes de VM (OR 2,76, IC 95%, 1,02-7,10; p=0,038), diagnóstico de sepsis (OR 2,46, IC 95%, 1,09-5,47; p=0,027) y fracaso de la extubación (OR 4,50, IC 95%, 2,05-9,90; p<0,001). Conclusiones El presente estudio permitió describir las características y evolución clínica de los pacientes que recibieron ventilación mecánica en Argentina, encontrando como principal resultado que la mortalidad fue mayor a la reportada en estudios internacionales (AU)

Objetive To describe mechanical ventilation (MV) practices in Argentina, and to explore factors associated with ICU mortality in this population. Design A prospective, multicenter, observational study was carried out. Setting Intensive Care. Patients We enrolled patients above 18 years old admitted to any of the participating ICUs requiring invasive MV for at least 12hours since the admission to the healthcare institution, including MV initiation in emergency department, operating room or other hospitals. Interventions None. Variables All variables were classified into three categories: variables related to demographic and clinical factors before the MV, factors related to the first day on MV, and factors related to events happening during the MV (complications and weaning from MV). Mechanical ventilation weaning and mortality were classified according to WIND. Results The primary analysis included 950 patients. The main indication for MV was acute respiratory failure (58% of patients). Initial ventilation mode was volume control-continuous mandatory ventilation in 75% of cases. ICU and hospital mortality were 44.6% and 47.9% respectively. The variables identified as independent predictors of mortality in ICU were age (OR3.48 IC95% 1.22-11.66; p=0.028), failure to implement NIV before MV (OR 2.76, IC 95%, 1.02-7.10; p=0.038), diagnosis of sepsis (OR 2.46, IC 95%, 1.09-5.47; p=0.027) and extubation failure (OR 4.50, IC 95%, 2.05-9.90; p<0.001). Conclusions The present study allowed us to describe the characteristics and clinical course of the patients who received mechanical ventilation in Argentina, finding as the main result that mortality was higher than that reported in international studies (AU)

Epidemiology of mechanical ventilation in Argentina. The EpVAr multicenter observational study.

OBJETIVE: To describe mechanical ventilation (MV) practices in Argentina, and to explore factors associated with ICU mortality in this population. DESIGN: A prospective, multicenter, observational study was carried out. SETTING: Intensive Care. PATIENTS: We enrolled patients above 18 years old admitted to any of the participating ICUs requiring invasive MV for at least 12 h since the admission to the healthcare institution, including MV initiation in emergency department, operating room or other hospitals. INTERVENTIONS: None. VARIABLES: All variables were classified into three categories: variables related to demographic and clinical factors before the MV, factors related to the first day on MV, and factors related to events happening during the MV (complications and weaning from MV). Mechanical ventilation weaning and mortality were classified according to WIND. RESULTS: The primary analysis included 950 patients. The main indication for MV was acute respiratory failure (58% of patients). Initial ventilation mode was volume control-continuous mandatory ventilation in 75% of cases. ICU and hospital mortality were 44.6% and 47.9% respectively. The variables identified as independent predictors of mortality in ICU were age (OR 3.48 IC 95% 1.22-11.66; p = 0.028), failure to implement NIV before MV (OR 2.76 IC 95% 1.02-7.10; p = 0.038), diagnosis of sepsis (OR 2.46 IC 95% 1.09-5.47; p = 0.027) and extubation failure (OR 4.50 IC 95% 2.05-9.90; p < 0.001). CONCLUSIONS: The present study allowed us to describe the characteristics and clinical course of the patients who received mechanical ventilation in Argentina, finding as the main result that mortality was higher than that reported in international studies.

First pregnancies in jennies with vitrified donkey semen using a new warming method.

Sperm vitrification has been recently developed, but fertility trials have not been performed yet in equine species. In this study, a new warming technique for vitrified donkey semen was developed and the uterine inflammatory response and fertility were compared to conventional freezing. In Experiment 1, sperm was vitrified in straws and warmed in 3 ml of extender or in a water bath at: 37 °C/30 s; 43 °C/10 s; and 60 °C/5 s. Sperm motility, plasma and acrosome membranes and DNA integrity were compared between treatments. In Experiment 2, jennies were inseminated twice (500 × 106 sperm) in the uterine body either with vitrified or frozen semen (2 cycles/jenny). Pregnancy rates and the uterine inflammatory response (polymorphonuclear neutrophil concentration; PMN) were evaluated after artificial insemination (AI). No differences between warming in extender/water bath were found and 43 °C/10 s was better than lower temperatures in terms of total (53.8 ±â€¯13.2%) and progressive sperm motility (41.4 ±â€¯11.4%). No differences in PMN concentration (×103 PMN/ml) were found between vitrified (276.8 ±â€¯171.6) or frozen (309.7 ±â€¯250.7) semen after AI. However, PMN decreased faster (P < 0.05) using vitrified semen. Pregnancy rates were greater for vitrified (22%) than frozen semen (10%) but not statistically different. In conclusion, donkey sperm vitrified in straws could be directly warmed in a water bath at 43 °C/10 s, reducing the uterine inflammatory response obtained after AI and promoting positive pregnancy outcomes. These findings confirm the possibility to use vitrified semen as an alternative for AI in jennies.

Antimicrobial activity of silver-carbon nanoparticles on the bacterial flora of bull semen.

The spermicidal effects of silver nanoparticles (AgNPs) hinder its application in the field of artificial insemination. In this study, silver-carbon NPs (Ag@C NPs) was synthesized and applied as an alternative antibiotic agent for bull semen extender. Ag@C NPs were characterized using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), atomic absorption flame spectroscopy, transmission electron microscope (TEM), and high-resolution TEM (HR-TEM). Data analysis revealed the successful synthesis of Ag@C NPs with a particle size of 1-5 nm (average particle size of 2.5 nm) embedded into carbon. The antimicrobial activity of Ag@C NPs was tested against bacteriospermia of fresh semen collected from five fertile bulls (three ejaculates/bull). Escherichia coli (E. Coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa) were isolated from fresh semen samples and identified by culture, staining, and conventional biochemical tests. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Ag@C NPs against bacteriospermia was determined at 5 and 37 °C. Ag@C NPs showed efficient antimicrobial activity (MIC: 3.125-12.5 µg/mL) against the tested strains and strong bactericidal effect on S. aureus, and P. aeruginosa (MBC: 3.125 µg/mL), with no detrimental effect (P ˃ 0.05) on the percentage of sperm motility (70.71 ± 4.82; 74.65 ± 4.46), plasma membrane integrity (68.39 ± 4.31; 72.38 ± 4.91), acrosome integrity (88.40 ± 13.21; 86.77 ± 14.23), and normal sperm morphology (86.85 ± 7.43; 87.82 ± 8.15) at concentrations of 15 and 30 µg/mL, respectively, after a cold storage of 48 h. However, Ag@C NPs showed a detrimental effect on sperm parameters in a dose dependent manner at concentrations ≥60 µg/mL. Ag@C NPs showed no adverse effect on the sperm's ultrastructure with limited sperm internalization at MIC. In conclusion, Ag@C NPs could be used as an alternative antibiotic agent for bull semen extender without a significant cytotoxic effect on the sperm during cold storage. However, further investigations for their effects on embryo production and female genitalia are still required.

Higroma quístico y la importancia de su diagnóstico prenatal: a propósito de un caso / Cystic hygroma and the importance of the prenatal diagnosis: about a case

RESUMEN El higroma quístico es la malformación del sistema linfático que más frecuentemente se observa en el período prenatal y que se ubica principalmente en el cuello y/o la nuca. Su tasa de detección ha aumentado desde la implementación de la translucencia nucal fetal (TN) en el primer trimestre de embarazo, y su presencia se ha relacionado con anomalías congénitas, aneuploidías, pérdida del embarazo y trastornos en el desarrollo. El objetivo de la presentación de este caso es resaltar la importancia del diagnóstico antenatal del higroma quístico, con el fin de realizar una intervención precoz y evitar la muerte fetal. Se recibe para estudio anatomopatológico, feto de sexo indeterminado producto del primer embarazo de una madre de 19 años de edad sin previos controles prenatales, con presencia de una gran masa quística que se extiende desde el rostro hasta la nuca. Mediante el estudio histológico se confirma el diagnóstico de higroma quístico. Al carecer de análisis de cariotipo no fue posible establecer la preexistencia de alguna anomalía genética. El también conocido como linfangioma quístico, es un tumor vascular benigno cuyo diagnóstico antenatal mediante la ultrasonografía resulta fundamental en la evolución y pronóstico de la enfermedad. Desafortunadamente en nuestro caso, la falta de controles prenatales y la ausencia de estudios ultrasonográficos que permitieran conocer las características de este linfangioma, pudo impactar significativamente en el desenlace fatal.

ABSTRACT The cystic hygroma is the malformation of the lymphatic system that is most frequently observed in the prenatal period and is located mainly in the neck and/or the nape of the neck. Its detection rate has increased since the implementation of fetal nuchal translucency (NT) in the first trimester of pregnancy and its presence has been associated with congenital abnormalities, aneuploidies, pregnancy loss, and developmental disorders. The aim of this case is to highlight the importance of antenatal diagnosis of cystic hygroma in order to perform early intervention and avoid fetal death. It is received, for anatomopathological study, a fetus of undetermined sex product of the first pregnancy of a 19 year-old mother without previous prenatal controls, with the presence of a large cystic mass that extends from the face to the neck. The histological study confirms the diagnosis of cystic hygroma. As there was no karyotype analysis, it was not possible to establish the preexistence of any genetic abnormality. Also known as cystic lymphangioma, is a benign vascular tumor whose antenatal diagnosis by ultrasonography is essential in the evolution and prognosis of the disease. Unfortunately in our case, the lack of prenatal controls and the absence of ultrasonographic studies that would allow knowing the characteristics of this lymphangioma, could significantly impact in the fatal outcome.

Effect of season on individual stallion semen characteristics.

Horses are long-day seasonal breeding animals, however, with modern stallion reproductive management it is important for collection of semen during periods that are not part of the traditional breeding season. This study was conducted to examine variation in the seminal characteristics of individual stallions in Avila, Spain during 1 year with a particular emphasis on sperm DNA fragmentation. Semen was collected twice per season from a total of 20 stallions. There was a marked seasonal effect on all seminal characteristics, with the greatest on progressive motility, % membrane integrity and least for SDF in the spring months; there was also an interaction effect with respect to individual stallion, indicating that some stallions did not fit this generalised pattern for semen quality. Sperm DNA fragmentation was assessed both immediately after semen collection (T0) and following incubation of extended semen for 24 h (T24) to broadly mimic changes in SDF that might occur in the female reproductive tract. While SDF evaluated at T0 was also generally less in spring, the proportion of stallions with the least SDF values in spring increased from 45% to 60% when assessed at T24, therefore, being consistent with the importance of dynamic SDF assessment in detecting DNA damage that was not detected at T0 or cryptic DNA damage. The results from this study indicate there is individual seasonal variation among stallions in all aspects of seminal characteristics; such variation needs to be considered when prioritising stallions that are to be used for breeding.

Fertilizing capacity of vitrified stallion sperm assessed utilizing heterologous IVF after different semen warming procedures.

The aim of this study was to evaluate the fertilizing capacity of frozen or vitrified stallion sperm after assessing different warming procedures. In Experiment 1, different warming procedures were compared after sperm vitrification: immersion in extender at 43 °C (C), or in a water bath at 37 °C/30 s (W37), 43 °C/10 s (W43) or 60 °C/5 s (W60). With the W60 treatment, there were greater values (P < 0.05) for VCL (83.93 ± 3.6 µm/s) and ALH (3.00 ± 0.2 µm) than freezing and with the C group, and greater values (P < 0.001) for PM (35.33 ± 2.5 %) than with the W43 treatment. In Experiment 2, the fertilizing capacity of vitrified and frozen sperm was assessed utilizing heterologous IVF procedures, using cattle oocytes. Vitrification resulted in greater values (P < 0.05) than freezing for the number of bound sperm (1.36 ± 0.3 and 0.69 ± 0.2, respectively). There were no differences between frozen or vitrified sperm in pronuclear formation (26 hours post-insemination - hpi; 14.08 ± 4.2 % and 22.78 ± 4.8 %, respectively) or cleavage rate (32.77 ± 4.3 % and 39.66 ± 4.6 %, respectively). In conclusion, vitrified stallion sperm warmed in a water bath at 60 ºC had the capacity to penetrate cattle oocytes, leading to pronuclear formation and hybrid embryo cleavage after heterologous IVF.

Factors Affecting Embryo Recovery Rate, Quality, and Diameter in Andalusian Donkey Jennies.

Embryo transfer and the vitrification of embryos could be used for the conservation and recovery of endangered donkey breeds. It is important to develop techniques that optimize recovery rates and the cryotolerance of donkey embryos. This study evaluates factors affecting the recovery rate, quality, and diameter of embryos obtained from donor jennies as a starting point for the use of vitrification and embryo transfer in the conservation of the Andalusian donkey. A total of 100 embryos were recovered out of 124 estrous cycles (80.6%). The donor jenny affected the rates of positive flushings (PFR; p = 0.040) and embryo recovery (ERR; p < 0.05) as well as embryo quality (p = 0.004). ERR was also affected by the number of flushings (p < 0.001), donor age (p < 0.05), successive cycle within donor (p < 0.001), and jacks (p < 0.05). Number of flushings (p < 0.001) and jack (p < 0.05) had a significant effect on PFR, whereas the day of flushing influenced the developmental stage (p < 0.001), embryo quality (p < 0.05), and diameter of embryos (p < 0.001). The number of flushings significantly influenced the diameter (p = 0.038) and embryo developmental stage (p = 0.001), whereas the developmental stage was statistically different between herds (p = 0.020). The factors influencing the success of this assisted reproductive technique were donor jenny, donor age, successive cycle within donor, day of flushing, number of flushings, and jack. The identification of these key points is crucial to achieve a higher efficiency of embryo transfer and vitrification processes, before considering their application in the conservation of endangered donkey breeds.

One-step warming does not affect the in vitro viability and cryosurvival of cryotop-vitrified donkey embryos.

The objective of this study was to compare the effects of two warming protocols (three-step vs. one-step dilution) on embryo quality, post-warming embryo survival and embryo cell viability of donkey embryos vitrified by the Cryotop method. Twenty, Day 7-8, grade 1-2 donkey embryos were measured, morphologically evaluated and vitrified using the Cryotop technique. Embryos were then randomly warmed using two different warming procedures: (i) W3 (three-step dilution; n = 11): embryos were warmed in 1 M, 0.5 M and 0 M sucrose, and (ii) W1/0.5 (one-step dilution; n = 9): embryos were warmed directly in 0.5 M sucrose. After 3 and 24 h of warming, the embryos were measured and evaluated for their morphology, developmental stage and viability (Propidium Iodide-Hoechst 33,342 dyes). Although both treatments decreased embryo quality after warming (P < 0.05), no significant differences (P > 0.05) were observed between protocols in terms of post-warming embryo quality, diameter and embryo survival. Greater percentages of dead cells (P < 0.001) were observed when embryos were warmed directly in 0.5 M sucrose (one-step dilution) when compared to the three-step protocol. The percentage of ruptured embryos was 27.3% and 0% in W3 and W1/0.5 protocols (P = 0.0893), respectively. In conclusion, warming Cryotop-vitrified donkey embryos directly in 0.5 M sucrose had no negative effects on embryo quality and post-warming embryo survival. Moreover, one-step protocol may help to prevent rupture when donkey embryos warmed directly in 0.5 M sucrose. These results observed in vitro must be verified by embryo transfer.

Nano-depletion of acrosome-damaged donkey sperm by using lectin peanut agglutinin (PNA)-magnetic nanoparticles.

Lectin is considered as a suitable biomarker for nano-depletion of acrosome-damaged sperm. The aim of this study was to synthetize magnetic nanoparticles (MNPs) coated by peanut (Arachis hypogaea) agglutinin lectin (PNA) and investigate its beneficial effect in improving of sperm characteristics. MNPs were obtained by co-precipitation method, functionalized with chitosan and coated by PNA at a concentration of 0.04 mg/mL. Semen was frozen either with glycerol-based or sucrose-based extenders. Frozen-thawed straws from five donkeys (three ejaculates per donkey) were incubated with lectin-MNPs (2 mg/mL), and then exposed to an external magnet enabling the non-bound sperm to be collected as nanopurified sperm. Sperm were evaluated post-thawing (control) and after nanopurification for motility, plasma membrane integrity, acrosome integrity, morphology, DNA fragmentation and concentration. The statistical analyses were extended to investigate the correlation between the initial quality of the frozen-thawed semen samples and the effect of nanopurification after thawing. The obtained MNPs were biocompatible to the sperm and significantly improved the progressive motility (P < 0.05) for the glycerol nanopurified group (43.08 ± 3.52%) in comparison to control (33.70 ± 2.64%). Acrosome-damaged sperm were reduced (P < 0.05) in both nanopurified groups (19.92 ± 2.69 for G and 21.57 ± 2.77 for S) in comparison to control (36.07 ± 3.82 for G and 35.35 ± 3.88 for S). There were no significant changes in sperm morphology and membrane integrity after nanopurification. The average sperm recovery after nanopurification was 80.1%. Sperm quality index was significantly higher (P < 0.001) in nanopurified groups regardless of the initial quality of the frozen thawed semen samples. However, in the high sperm quality group, nanopurification significantly improved the progressive motility and membrane integrity besides the increasing of acrosome-intact sperm. Sperm nanopurification using lectin-magnetic nanoparticles can be considered as a suitable method to reduce the proportion of acrosome-damaged sperm and to increase the quality of frozen thawed donkey semen.

Seasonal variations in sperm DNA fragmentation and pregnancy rates obtained after artificial insemination with cooled-stored stallion sperm throughout the breeding season (spring and summer).

The aim of this study was to assess seasonal variations during different periods of the breeding season (spring and summer) on stallion sperm DNA fragmentation and in vivo fertility associated with cooled-stored semen samples. Ejaculates were collected from eleven stallions and assessed for sperm motility (assessed by computer-assisted sperm analysis) and plasma membrane integrity (evaluated under fluorescence microscopy). Sperm DNA fragmentation (evaluated by the Sperm Chromatin Dispersion test) was assessed in cooled-stored semen at 5 °C for up to 24 h. Artificial insemination was performed throughout the breeding season. Mares were inseminated with cooled-stored semen (up to 24 h) every other day until ovulation. Pregnancy rates per cycle were determined detecting the embryonic vesicle by ultrasonography fifteen days after ovulation. Values (mean ± SD) for progressive sperm motility were significantly higher (P < 0.05) in spring (53.57 ± 9.97%) in comparison to summer (41.37 ± 10.81%). No significant differences in plasma membrane integrity were found between seasons (P > 0.05). Sperm DNA fragmentation was significantly lower (P < 0.01) in spring in comparison to summer after 0h (4.81 ± 1.87% vs. 8.77 ± 5.78%), 6h (9.00 ± 3.19% vs. 18.73 ± 8.22%) and 24h (14.6 ± 4.13% vs. 30.14 ± 9.85%) of cooled-storage. Pregnancy rates per cycle were also significantly higher (P < 0.01) in spring (50%) in comparison to summer (37%). There was a moderate negative relationship between positive pregnancies and sperm with fragmented DNA (r = - 0.619; P < 0.001). Semen samples associated with moderate fertility levels (Pregnancy rate < 50%) showed a higher percentage of sperm with fragmented DNA compared to samples obtaining higher fertility levels. In conclusion, seasonal variations were found during the breeding season, obtaining lower sperm DNA fragmentation and higher pregnancy rates in spring. Additionally, samples with the highest proportion of sperm with fragmented DNA showed the lowest fertility levels throughout the breeding season.

The cryoprotective effect of Ficoll 70 on the post-warming survival and quality of Cryotop-vitrified donkey embryos.

Many domestic donkey breeds are at risk of extinction, there is a critical urgency for genome resource banking. In the present study, we examined whether the use of Ficoll 70 added to the vitrification medium containing ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose improves the cryotolerance of donkey in vivo derived embryos. Day 7-8, grade 1-2 donkey embryos were measured and morphologically evaluated and then vitrified-warmed using the Cryotop technique. Before vitrification, embryos were randomly distributed into two groups: (i) VS1 (n = 14): vitrified using 15% EG + 15% DMSO + 0.5 M sucrose; and (ii) VS2 (n = 10): vitrified in the same medium supplemented also with 18% of Ficoll 70. After 24 h of warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). Post-warming survival was numerically higher but not significantly different (P > 0.05) when embryos were vitrified in VS2 (70%) compared to VS1 (57.1%). Embryo rupture was only observed in the VS1 group (21.4%, 3/14). Higher embryo diameter was observed in all groups after 24 h culture (P < 0.05). No significant differences (P > 0.05) were observed among treatments in terms of percentages of cell death. These results demonstrate that the addition of Ficoll 70 to the vitrification medium is not a pre-requisite for successful vitrification of donkey embryos. However, its addition seems to enhance some of the post-warming embryo quality characteristics. Since no statistically significant evidence was found, further studies should be conducted in order to confirm our findings.

Cryopreservation of Andalusian donkey (Equus asinus) spermatozoa: Use of alternative energy sources in the freezing extender affects post-thaw sperm motility patterns but not DNA stability.

The aim of this study was to compare the effect of three sugars and Equex paste in a freezing extender for donkey sperm cryopreservation. Ejaculates (n = 18) were collected from six Andalusian donkeys of proven fertility were pooled (two ejaculates per pool) and cryopreserved using a freezing extender containing three different sugars (glucose, fructose and sorbitol), with or without the addition of Equex paste. Sperm quality was assessed before and after freezing-thawing for motility, morphology, plasma membrane integrity, acrosome integrity and DNA integrity. The use of sorbitol in the freezing extender improved total and progressive sperm motility (P < 0.05) and amplitude of lateral head displacement (P < 0.01), but it reduced the values for other sperm motility variables compared with glucose (P < 0.001). The use of fructose resulted in a reduction in values for most CASA variables (P < 0.05), whereas addition of Equex paste did not have any beneficial effect on values for these variables (P > 0.05). Glucose was more effective in maintaining sperm morphology (P < 0.05), while there was no beneficial effect with the addition of Equex paste (P > 0.05). Supplementation of fructose and Equex paste in the freezing extender decreased plasma membrane integrity (P < 0.05) as compared with glucose, but there were no differences between treatments for acrosome and DNA integrity (P > 0.05), even after 24 h of incubation. The use of different sugar sources in the extender could affect the in vitro post-thaw quality of cryopreserved donkey spermatozoa, with sorbitol being an interesting alternative for improving the sperm quality. Results of the present study indicate the use of Equex paste could negatively affect post-thaw outcomes for sperm viability in this species.

Vitrification of stallion sperm using 0.25 ml straws: Effect of volume, concentration and carbohydrates (sucrose/trehalose/raffinose).

Sperm vitrification is a rapid freezing method in which carbohydrates are used as cryoprotectants. The aim of this study was to determine the optimal volume, concentration and type of carbohydrates for stallion sperm vitrification using 0.25 ml straws in comparison to conventional freezing. Ejaculates (n = 54) were collected from six stallions. For vitrification, straws were filled with different volumes (30, 70, 100 µl), sperm concentrations (50, 100, 200 × 106 sperm/ml) and extenders containing sucrose (20, 100, 200 mM), trehalose (50, 100, 200 mM) and raffinose (50, 100, 200 mM) and plunged into LN2. Conventional freezing was performed in 0.5 ml straws frozen in LN2 vapors. Sperm motility, plasma and acrosome membrane integrities and DNA fragmentation were compared among treatments. The use of straws filled with 100 µl at 100 × 106 sperm/ml with the extender containing 100 mM trehalose resulted in greater values for sperm quality than the other concentrations, volumes and carbohydrates. With vitrification, there were greater values (mean ± SEM; P < 0.05) than freezing for progressive motility (48.2 ± 2.3 compared with 37.3 ± 2.2%), plasma membrane integrity (82.8 ± 1.5 compared with 74.1 ± 1.9%), and intact acrosomes (50.2 ± 1.2 compared with 43.1 ± 1.4%); and less DNA fragmentation (6.4 ± 0.7 compared with 8.2 ± 0.3%). In conclusion, stallion sperm can be vitrified in 0.25 ml straws filled with 100 µl of sperm at 100 x 106 sperm/ml using an extender with 100 mM of trehalose, obtaining better sperm quality after warming than conventional freezing.

Optimization of donkey sperm vitrification: Effect of sucrose, sperm concentration, volume and package (0.25 and 0.5 mL straws).

The aim of this study was to assess the effect of different factors affecting vitrification success of donkey sperm: extender, sperm concentration, volume and storage vessel type. In Experiment 1, sucrose supplementations at 0.25 and 0.1 M were compared using two base extenders (containing or not egg-yolk); in Experiment 2, three sperm concentrations were assessed: 100, 200 or 300 million sperm/mL; and in Experiment 3, three different sperm volumes (100, 160 and 200 µL) and two different storage vessels (0.25 and 0.5 mL straws) were assessed. Sperm motility variables (CASA), plasma membrane and acrosome (evaluated under fluorescence microscopy) and sperm DNA integrity (flow cytometry) were evaluated after warming with comparisons of protocols. There was a greater total (55.7 ± 16.4%) and progressive (44.0 ± 11.5%) motility using the extender with egg-yolk and 0.1 M sucrose. There were no effects of sperm concentrations on vitrification results (P > 0.05). The 0.25 mL covered straw showed higher values than the 0.5 mL straw for total (50.0 ± 17.3% vs 2.0 ± 6.7%) and progressive (40.5 ± 14.9% vs 0.9 ± 1.5%) motility, plasma membrane (43.9 ± 14.4% vs 14.0 ± 16.4%) and acrosome integrity (51.5 ± 13.6% vs 28.0 ± 14.7%), respectively. In conclusion, values for donkey sperm quality variables after vitrification were greater using an extender containing egg-yolk and 0.1 M sucrose, at 300 million sperm/mL in 0.25 mL straws with outer covers.