Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.     

Low-Dose Insemination in Pigs: Problems and Possibilities

Low-dose AI procedures are required by the pig industry to efficiently utilize emerging sperm technologies, such as cryopreservation and sex-sorting. Currently, several different procedures for inseminating with a low or very low number of spermatozoa have been described. Deep intrauterine insemination allows the deposition of the spermatozoa in the depth of the uterine horn, allowing a significant reduction in the number of spermatozoa inseminated with maintenance of optimal reproductive performance. Intra-oviductal laparoscopic insemination has been recently applied in pigs. This technique has proved to be applicable with diluted and sex-sorted spermatozoa. This review discusses several problems encountered during the development of deep intrauterine insemination and intra-oviductal laparoscopic insemination of pigs and provides potential solutions for the practical application of both the technologies.     

The Effects of Hoechst 33342 Staining and the Male Sample Donor on the Sorting Efficiency of Canine Spermatozoa

The aim of this study was to evaluate the influence of Hoechst 33342 (H‐42) concentration and of the male donor on the efficiency of sex‐sorting procedure in canine spermatozoa. Semen samples from six dogs (three ejaculates/dog) were diluted to 100 × 10⁶ sperm/ml, split into four aliquots, stained with increasing H‐42 concentrations (5, 7.5, 10 and 12.5 μl, respectively) and sorted by flow cytometry. The rates of non‐viable (FDA+), oriented (OS) and selected spermatozoa (SS), as well as the average sorting rates (SR, sorted spermatozoa/s), were used to determine the sorting efficiency. The effects of the sorting procedure on the quality of sorted spermatozoa were evaluated in terms of total motility (TM), percentage of viable spermatozoa (spermatozoa with membrane and acrosomal integrity) and percentage of spermatozoa with reacted/damaged acrosomes. X‐ and Y‐chromosome‐bearing sperm populations were identified in all of the samples stained with 7.5, 10 and 12.5 μl of H‐42, while these two populations were only identified in 77.5% of samples stained with 5 μl. The values of OS, SS and SR were influenced by the male donor (p < 0.01) but not by the H‐42 concentration used. The quality of sorted sperm samples immediately after sorting was similar to that of fresh samples, while centrifugation resulted in significant reduction (p < 0.05) in TM and in the percentage of viable spermatozoa and a significant increase (p < 0.01) in the percentage of spermatozoa with damage/reacted acrosomes. In conclusion, the sex‐sorting of canine spermatozoa by flow cytometry can be performed successfully using H‐42 concentrations between 7.5 and 12.5 μl. The efficiency of the sorting procedure varies based on the dog from which the sperm sample derives.     

Soil‐plant relationships,micronutrient contents,and cardenolide production in natural populations of Digitalis obscura

The production of secondary metabolites by plants growing in natural populations is conditioned by environmental factors. In the present study, we have investigated the relationships among soil properties, micronutrients in soils and plants, and cardenolide production from wild Digitalis obscura (Scrophulariaceae) populations. Young and mature leaves and soil samples were collected in ten different populations, corresponding to three Mediterranean bioclimatic belts (Thermo‐, Meso‐, and Supramediterranean belts). Soil (total and EDTA‐extractable) and leaf micronutrients (Fe, Mn, Zn, and Cu), and leaf cardenolide accumulation have been determined. Significant negative correlations were observed between Fe, Mn or Zn concentration in leaves and soil pH, as well as between Fe or Mn in leaves and carbonate content of soils. Only EDTA‐extractable Mn was significantly correlated with Mn content in the plants. With regard to cardenolide content in leaves, this parameter was negatively correlated with Zn_leaf in young leaves and with Mn_leaf in old leaves. Positively correlated, however, were Fe and cardenolide content in young leaves. The influence of environmental conditions and leaf micronutrient contents on cardenolide accumulation is discussed.     

Stay-green phenotype slows the carotenogenic process in Capsicum annuum (L.) fruits

Stay-green mutants have been very useful for elucidating the chlorophyll catabolism pathway in higher plants. In the present study the possible relationship between the retention/catabolism of chlorophylls and the carotenogenic process taking place in ripening Capsicum annuum (L.) fruits has been investigated. Phytylated, dephytylated and oxidized chlorophyll derivatives, and total and individual carotenoids were analyzed over the whole ripening period. In general terms, the biosynthesis of carotenoid pigments taking place during the ripening of C. annuum fruits is identical in both red and stay-green lines, so that the carotenogenic process is independent of the retention of chlorophylls. However, it has been found that the carotenogenesis is slowed in the stay-green lines. Therefore, although the catabolism of chlorophylls and biosynthesis of carotenoids seem to be separate processes, the fact that they are taking place in the chloroplast/chromoplast suggests that some kind of interaction between the two processes may occur at different levels. Plastids corresponding to the wild genotype (red color fruit phenotype) show high plastoglobuli density and thylakoids are almost absent, whereas in the case of stay-green phenotype, thylakoids and plastoglobuli coexist in the same plastid (chlorochromoplasts). The role of carotenoid pigments on the physiological mechanism for protecting the preserved thylakoid structures is discussed.     

Distinguishing between natural and anthropogenic sources for potentially toxic elements in urban soils of Talcahuano,Chile

Purpose

The objectives of this study were (1) to determine the concentrations and background concentrations of Ba, Co, Cr, Mn, and Ni in the urban soils of Talcahuano (Chile); (2) assess the level of contamination in the urban soils based on different pollution indexes; and (3) to identify natural or anthropogenic sources in order to obtain a spatial distribution of the pollutants.

Material and methods

A total of 420 samples were collected from the study area as follows: 140 topsoil samples (TS) (0–10 cm), 140 subsoil samples (SS) (10–20 cm), and 140 deep soil samples (DS) (150 cm). The soils were characterized, and the concentrations of Ba, Co, Cr, Mn, and Ni were analyzed by atomic absorption photospectrometry following aqua regia digestion. Correlations and principal component analysis combined with spatial analysis were implemented in order to distinguish the sources and their classification as geogenic or anthropogenic. Several simple and robust statistical methods were applied to datasets in order to explore their potential in the evaluation of a useful and robust background values. The degree of contamination along with the geoaccumulation index, enrichment factor, and contamination factor were also evaluated.

Results and discussion

The median concentrations obtained for various elements includes Ba 461 mg kg^?1, Co 82.7 mg kg^?1, Cr 134 mg kg^?1, Mn 311 mg kg^?1, and Ni 56.1 mg kg^?1. In general, the concentrations of Ba, Co, Cr, Mn, and Ni decrease with depth. Correlations and principal component analysis suggest that Cr, Mn, and Ni are contributed by external sources. The spatial distribution of Cr, Mn, and Ni in TS displays a spatial pattern extending along industrial environments and emission sources.

Conclusions

The estimated background values determined with the iterative 2σ-technique includes 536 mg kg^?1 for Ba, 95.9 mg kg^?1 for Co, 208 mg kg^?1 for Cr, 464 mg kg^?1 for Mn, and 90.5 mg kg^?1 for Ni. The geochemical index, enrichment factor, and the contamination factor register a moderate to considerable contamination in some soil samples.