Cryopreservation effects on canine sperm morphometric variables and ultrastructure: Comparison between vitrification and conventional freezing.

Semen cryopreservation is an increasingly demanded technique in canids, particularly in order to preserve and spread high genetic value material. Sperm vitrification may represent an interesting alternative to costly and time consuming conventional freezing. The objective of this study was to evaluate the effect of sperm vitrification on sperm morphometry and ultrastructure compared to conventional freezing. Pools of nine beagle dogs were both frozen and vitrified. Computerized morphological parameters (length, wide, area and perimeter) and sperm ultrastructure, using scanning and transmission microscopy, were analysed in both fresh and in thawed/warmed samples. There were no differences (p > 0.05) between post-thaw and fresh morphometric variables of the sperm heads. However, cluster analysis revealed that sperm-heads turned out to be smaller after thawing (p < 0.05) in two of the four subpopulations. Vitrification-warming process led to an overall increase in sperm-head size. Furthermore, the sperm head size increased after warming in two subpopulations (p < 0.05). In conclusion, the variations in the sperm head area depended on the cryopreservation procedure (conventional freezing or vitrification). Conventional freezing tended to decrease the head dimensions, at least in some subpopulations, and vitrification led to an overall increase in the sperm head size. Decondensation of chromatin and plasma membrane blebbing in the head region was observed by transmission electron microscopy in several vitrified sperm, which might explain the increase of head dimensions detected by CASA-Morph system.

Vitrification of dog spermatozoa: Effects of two cryoprotectants (sucrose or trehalose) and two warming procedures.

Sperm vitrification is a low cost and simple technique that does not require special equipment and may represent an attractive alternative to the costly and time consuming conventional dog spermatozoa cryopreservation techniques. The objective of this study was to evaluate different cryoprotectants and warming temperatures on the vitrification of dog spermatozoa. Pooled semen samples from 10 beagle dogs were vitrified with four extenders, based on Tris, citric acid and glucose, 20% egg yolk (TCG-20% EY) and different combinations of sucrose and/or trehalose: 250 mM sucrose; 250 mM trehalose; 125 mM sucrose + 125 mM trehalose; 250 mM sucrose + 250 mM trehalose. Samples were vitrified by dropping 50 µL of sperm suspension directly into liquid nitrogen. After vitrification, warming was done either fast (at 65 °C for 2-5 s) or slow (at 37 °C for one minute). Motility was assayed using a computer-aided sperm analysis (CASA) system; membrane integrity and acrosomal status were analyzed by fluorescence microscopy. For comparison, samples were also conventionally frozen in liquid nitrogen vapor using a TCG-20% egg yolk extender plus 5% glycerol. Frozen straws were thawed in a water bath at 37 °C for 30 s. Poorer motility results (P < 0.05) but similar viability were obtained when vitrification was performed, compared to conventional freezing (P > 0.05). When vitrification was used, cryoprotectants containing either 250 mM sucrose or 250 mM trehalose and warmed at 37 °C returned the best sperm quality variables.

Cryopreservation of testicular tissue from the dog (Canis familiaris) and wild boar (Sus scrofa) by slow freezing and vitrification: Differences in cryoresistance according to cell type.

Sperm cryopreservation is the most common procedure used to establish germplasm banks for endangered species - but sometimes sperm cells cannot be obtained. In such cases, freezing testicular tissue may be the only option. The testes contains germ cells at different stages of differentiation, including spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa, among which differences in cryoresistance might be expected. The present work compares the viability and DNA integrity of 'rounded' cells, and of elongated spermatids and spermatozoa, from the dog and wild boar, following the cryopreservation of testicular tissue by slow freezing or vitrification. Cell viability was analyzed by PI/SYBR14 staining, and DNA integrity via the TUNEL technique. For wild boar, no significant differences were seen between the two methods with respect to the percentage of viable cells, nor in the percentage of cells with DNA damage. In the dog, the percentage of viable rounded germ cells (65.0 ± 2.4%) was higher (P < 0.05) after vitrification than after slow freezing (45.1 ± 6.7%). No difference was found between the two methods in terms of the viability of elongated cells. For rounded cells, the percentage of intact DNA was greater (P < 0.05) after vitrification (90.5 ± 2.1%) than after slow freezing (42.6 ± 11.0%), while for elongated spermatids and spermatozoa it was higher (P < 0.05) after slow freezing (66.9 ± 6.1%) than after vitrification (50.7 ± 4.5%). Thus, the response to cryopreservation is cell type-, cryopreservation type-, and species-dependent. Vitrification would appear to be the most appropriate method for preserving dog testicular tissue given the associated high cell viability and low degree of DNA fragmentation, while in wild boar, either method might be used.

Vitrification of Iberian wolf (Canis lupus signatus) sperm: A possible alternative to conventional cryopreservation.

Sperm vitrification is a simple, inexpensive method that allows the cryopreservation of sperm in the field and for endangered species is a useful alternative to conventional freezing. The study, therefore, is focused on the suitability of vitrification for cryopreserving Iberian wolf sperm and utilizing plasma testosterone concentration as a marker for procedure efficacy. Sperm and blood samples were collected from 17 wolves. There were 14 samples suitable for cryopreservation (12 ejaculated and two epididymal). Immediately after collection, these samples were proportioned into two aliquots for conventional freezing using a Tris-citric acid-glucose based extender (TCG) or vitrification utilizing an animal protein free extender (HTF®). Vitrification occurred by directly plunging a sperm suspension into liquid nitrogen. Sperm were assessed for motility, membrane integrity, acrosomal status and DNA integrity before and after cryopreservation. With both techniques, there were similar post-thaw/warming results (P > 0.05) with respect to progressive motility, kinetic variables VCL, VSL, VAP and BCF, DNA fragmentation, sperm membrane functionality and morphological abnormalities. Total motile sperm, progression ratios LIN, STR, and WOB, the ALH, sperm viability and sperm with intact membrane and acrosome were greater (P < 0.05) in the conventional frozen-thawed sperm than vitrified-warmed sperm. Plasma testosterone concentrations varied from 0.0 ng/mL to 7.7 ng/mL. For epididymal sperm, sperm motility and viability following thawing were greater in vitrified-warmed samples than conventionally-frozen samples; however, small sample numbers precluded statistical analysis. When considered together, these results indicate vitrification may be a possible alternative for wolf sperm cryopreservation.

Using n-alkanes to estimate diet composition of herbivores: a novel mathematical approach.

N-alkanes are long-chain saturated hydrocarbons occurring in plant cuticles that can be used as chemical markers for estimating the diet composition of herbivores. An important constraint of using n-alkanes to estimate diet composition with currently employed mathematical procedures is that the number of markers must be equal or larger than the number of diet components. This is a considerable limitation when dealing with free-ranging herbivores feeding on complex plant communities. We present a novel approach for the estimation of diet composition using n-alkanes which applies equally to cases where the number of markers is lower, equal or greater than the number of plant species in the diet. The model uses linear programming to estimate the minimum and maximum proportions of each plant in the diet, and avoids the need for grouping species in order to reduce the number of estimated dietary components. We illustrate the model with two data sets of n-alkane content of plants and faeces obtained from a sheep grazing experiment conducted in Australia and a red deer study in Portugal. Our results are consistent with previous studies on those data sets and provide additional information on the proportions of individual species in the diet. Results show that sheep included in the diet high proportions of white clover (from 0.25 to 0.37), and relatively high proportions of grasses (e.g. brome from 0.14 to 0.26) but tended to avoid Lotus spp. (always less than 0.04 of the diet). For red deer we found high proportions of legumes (e.g. Trifolium angustifolium and Vicia sativa reaching maximum proportions of 0.42 and 0.30 of the diet, respectively) with grasses being less important and Cistus ladanifer, a browse, also having relevance (from 0.21 to 0.42 of the diet).

Relación entre apolipoproteínas A-1 y B-100 e infarto al miocardio / Relation between apolipoproteins A-1 and B-100 e infarction myocardial

La enfermedad ateroesclerótica (EA) es la primera causa de muerte en Venezuela, determinándose marcadores más específicos para EA, en 47 sobrevivientes al infarto de miocardio y 40 controles (sexo masculino, 30-60 años). Indicadores: apolipoproteínas (Apo) A-1 y B-100, Colesterol total (Ct), HDLc, LDLc, Vldlc, triglicéridos y sus relaciones. Diferencias estadísticamente significativas: Apo A-1, Ct/HDLc y LDLc/HDLc (p<0.0005); B-100 y LDLc (p<0.05); HDLc (p<0.025); Apo A-1/Apo B-100, LDLc/Apo A-1 y Ct/Apo A-1 (p>0.0001). Apo A-1 [CD=68 por ciento; Vp(+)=71], Apo A-1/Apo B-100 [CD=75 por ciento, Vp(+)=75 por ciento], LDLc/Apo A-1 [CD=66 por ciento, Vp(+)=71 por ciento] y Ct/Apo A-1 [CD=70 por ciento, Vp(+)=71 por ciento] constituyeron marcadores de riesgo más especifícos para EA que el perfil lipídico y que las relaciones utilizadas actualmente [Ct/HDLc: CD=66 por ciento, Vp(+)=67 por ciento, LDLc/HDLc: CD=65 por ciento, Vp(+)=67 por ciento]

Relación entre apolipoproteínas (APO A1 y APO B100) y el perfil lipídico tradicional como marcadores de riesgo cardiovascular en mujeres / Relation apolipoproteins (APO A1 and APO B100) and the profile lipid traditional with markers of risk cardiovascular in women

La enfermedad arterial coronaria, ha sido la principal causa de muerte en hombres de edad media y mujeres de mayor edad. Se determinó el valor predictivo de la apolipoproteínas (APO A1, APO B100) para comparar su capacidad diagnóstica con el perfil lipídico tradicional. Las apolipoproteínas se analizaron nefelométricamente y el perfil lipídico tradicional por métodos colorimétricos y la fórmula de fridewald. Los triglicéridos (Tg) resultaron ser mejores marcadores (p<0.025), presentaron valor predictivo positivo 82 por ciento, capacidad diagnóstica 87 por ciento, especificidad 80 por ciento, sensibilidad 93 por ciento. La APO B100 no resultó ser un buen marcador de riesgo. La correlación entre Tg y LDLc con APO B100 fue significativa (p<0.05). Los resultados obtenidos sugieren que los Tg constituyen un mejor marcador para determinar riesgo cardiovascular en mujeres