Metapopulation Patterns of Iberian Butterflies Revealed by Fuzzy Logic.

Metapopulation theory considers that the populations of many species are fragmented into patches connected by the migration of individuals through an interterritorial matrix. We applied fuzzy set theory and environmental favorability (F) functions to reveal the metapopulational structure of the 222 butterfly species in the Iberian Peninsula. We used the sets of contiguous grid cells with high favorability (F ≥ 0.8), to identify the favorable patches for each species. We superimposed the known occurrence data to reveal the occupied and empty favorable patches, as unoccupied patches are functional in a metapopulation dynamics analysis. We analyzed the connectivity between patches of each metapopulation by focusing on the territory of intermediate and low favorability for the species (F < 0.8). The friction that each cell opposes to the passage of individuals was computed as 1-F. We used the r.cost function of QGIS to calculate the cost of reaching each cell from a favorable patch. The inverse of the cost was computed as connectivity. Only 126 species can be considered to have a metapopulation structure. These metapopulation structures are part of the dark biodiversity of butterflies because their identification is not evident from the observation of the occurrence data but was revealed using favorability functions.

Influence of recovery method and microbial contamination on the response to freezing-thawing in ibex (Capra pyrenaica) epididymal spermatozoa.

The method of sperm recovery may influence the initial quality of sperm samples and their response to freezing-thawing. The aim of the present work was to compare two methods for collecting epididymal spermatozoa in order to improve the quality of recovered sperm and reduce possible contamination. Testes were obtained from 23 legally hunted, adult ibex males. The sperm mass of the right epididymis was collected by small longitudinal and transverse cuts made in the cauda epididymidis. The sperm mass of the left epididymis was collected by retrograde flushing from the vas deferens to the cauda epididymidis (using a cannula), employing a Tris, citric acid, glucose, egg yolk-based medium. The flushing method recovered more spermatozoa (P<0.001) than the cutting method. After freezing-thawing, greater acrosomes damage (P<0.001) and more morphological abnormalities (P<0.05) were seen among the sperm cells recovered by the cutting method than among those obtained by retrograde flushing. The method of sperm recovery did not, however, influence the microbial contamination rate. In frozen-thawed samples that were microbially contaminated, motility was significantly reduced (P<0.05) and membrane integrity tended to be poorer (P=0.06). In conclusion, retrograde flushing is recommended for ibex sperm collection since it would appear that microbial contamination is no more of a problem than that encountered with the cutting method, while a larger number of sperm cells more resistant to freezing-thawing can be obtained.

A comparison of the protective action of chicken and quail egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa.

Egg yolk-based diluents provide adequate cryoprotection for the sperm of several mammalian species. Traditionally, chicken egg yolk has been used as additive for the freeze preservation of spermatozoa because of its wide availability. Variations in the chemical composition of the egg yolk of different avian species appear to influence the protection afforded during cooling, freezing, and thawing. The aim of the present study was to assess the use of quail egg yolk as a novel additive for the epididymal spermatozoa of a threatened wild ruminant species-the Spanish ibex-and to compare its efficacy with chicken egg yolk. Epididymal spermatozoa were diluted using a Tris-citric acid-glucose medium (TCG) composed of 3.8% Tris (w v(-1)), 2.2% citric acid (w v(-1)), 0.6% glucose (w v(-1)), 5% glycerol (v v(-1)), and 6% egg yolk (v v(-1)). Sperm masses from the right epididymes were diluted with TCG-6% chicken egg yolk medium, while those from the left were diluted with TCG-6% quail egg yolk. The thawed spermatozoa preserved with TCG-6% quail egg yolk extender exhibited lower motility (P<0.001), membrane integrity (P<0.001), and viability (P<0.01) than those diluted with the TCG-6% chicken egg yolk extender. The fertility of spermatozoa frozen in TCG-6% chicken egg yolk tended to be higher than in those frozen with TCG-6% quail egg yolk (63.3% vs 36.4%, P=0.19). These results show that quail egg yolk offers no advantages over chicken egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa.

Recovery and cryopreservation of Spanish ibex epididymal spermatozoa.

A Tris-citric acid-glucose (TCG) diluent containing low concentrations (6%) of egg yolk, and a TCG extender containing lactose (without egg yolk), were compared for use in the cryopreservation of Spanish ibex (Capra pyrenaica) epididymal spermatozoa. To optimize the collection of epididymal spermatozoa, two spermatozoa recovery methods were tested: i) by using small cuts in the cauda epididymides and ii) by the application of air pressure (from a syringe) inside the vas deferens. The percentage of viable spermatozoa recovered was lower (P < 0.05) with the air pressure method. No significant differences were seen in the efficacy of the two diluents as determined by percentage viability of thawed sperm, membrane integrity (as determined by the hypo-osmotic swelling test), or acrosome integrity. The use of the TCG-lactose medium strongly reduced sperm motility (P < 0.001). The sperm samples that had been diluted with TCG-6% egg yolk extender showed a greater incidence (P < 0.05) of morphological abnormalities. TCG-lactose alone, does not well preserve motility when cryopreserving Spanish ibex epididymal spermatozoa.

Horn quality and postmortem sperm parameters in Spanish ibex (Capra pyrenaica hispanica).

The horns are secondary sexual characteristics used by males of many ungulate species for intra-sexual fights during the rut. Thus, the dominant males with most developed horns are naturally selected for reproduction. Several studies have suggested that the quality of the horn, in many wild ruminants, may be correlated with semen quality. The aim of the present study was to determine whether inter-individual differences in levels of horn asymmetry and horn size are related to differences in sperm quality in a wild population of Spanish ibex by the assay of epididymal spermatozoa collected postmortem. In order to test this hypothesis we collected morphometric horns data from a total of 59 mature males (9-15 years of age) that were legally hunted during rutting season. The testicles were recovered, and the collection of epididymal spermatozoa was done at different times after death (2-60 h). The percentage of motile spermatozoa, motility rate, plasma membrane integrity, sperm viability, sperm morphology, and acrosome integrity were evaluated. Our findings showed that viable epididymal spermatozoa may be retrieved from dead animals many hours after death. However, sperm parameters were affected by the elapsed time between the death of the animal and spermatozoa collection. The study revealed that the horn quality was firstly associated with sperm motility.

Effect of egg yolk concentration on cryopreserving Spanish ibex (Capra pyrenaica) epididymal spermatozoa.

Tris-egg yolk based diluents provide adequate cryoprotection for the sperm of most wild species in which they have been tested. The objective of the current study was to evaluate various Tris-based diluents containing different concentrations of egg yolk, for the fertilizing ability of epididymal spermatozoa of the Spanish ibex (Capra pyrenaica) after freezing and thawing. For this purpose, we used heterologous in vivo fertilization by intrauterine insemination of domestic goats (Capra hircus). In Experiment 1, a Tris-citric acid-glucose (TCG) diluent containing 6% (v/v) egg yolk and a TCG extender containing 20% egg yolk were compared. In Experiment 2, a TCG-6% egg yolk extender was compared with Triladyl-20% egg yolk. Diluted samples were cooled slowly to 5 degrees C over 1 h and equilibrated at that temperature for 2 h. At that point, aliquots of samples were loaded into 0.25 ml straws, and frozen in nitrogen vapor for 10 min. The fertility of spermatozoa frozen in TCG-6% egg yolk was higher (P<0.05) than for those extended with TCG-20% egg yolk, and tended to be higher than for those frozen with Triladyl-20% egg yolk. From the results of this study, the use of Tris-based extenders containing low concentrations of egg yolk (6%) is recommended for cryopreserving Spanish ibex epididymal spermatozoa.

Birth of live Spanish ibex (Capra pyrenaica hispanica) derived from artificial insemination with epididymal spermatozoa retrieved after death.

As a consequence of increasing limitations to maintaining genetic variability in endangered wildlife species, methods of assisted reproduction widely used in domestic animals are being applied to nondomestic species. However, practical efforts have met limited success to date. The Spanish ibex (Capra pyrenaica hispanica) is a wild caprine originating exclusively in the mountains of Spain. This study was designed to evaluate the fertilizing capability of cryopreserved Spanish ibex epididymal spermatozoa recovered postmortem. For this purpose, we have previously evaluated the effect of time elapsed between death and sperm recovery on spermatic parameters, and the fertilization ability of frozen-thawed spermatozoa using heterologous in vivo fertilization by intrauterine insemination in domestic goat (Capra hircus). The time of death significantly affected most sperm quality parameters (motility, viability and intact acrosomes). The fertility obtained by heterologous artificial insemination was 18.7%, and only goats inseminated with spermatozoa recovered within 8h after death became pregnant. Our findings showed that heterologous in vivo fertilization is a useful method to evaluate the fertilizing capacity of sperm samples in rare or wild species. Sperm samples, with verified fertilization ability in the previous trial, were used to inseminate a total of six ibex females. Inseminations resulted in one pregnancy. The study demonstrated for the first time the feasibility of applying artificial insemination in Spanish ibex.

Seasonal ovulatory activity and plasma prolactin concentrations in the Spanish ibex (Capra pyrenaica hispanica) maintained in captivity.

Seasonal changes in ovulatory activity, assessed by the measurement of plasma progesterone and plasma prolactin concentrations were monitored in 10 Spanish ibex females captured in the National Wildlife Reserve of Sierra Nevada (South Spain, 37 degrees N). Five of the 10 female ibex showed ovulatory activity with a mean (+/- s.e.m.) duration of the oestrous cycle of 19.4 +/- 1 days (range: 17-23 days). On average, the five cyclic females weighed 28 +/- 0.9 kg. Progesterone cycles occurred only in animals older than 4 years of age. Ovulatory activity extended from December to January. The duration of the breeding season was 43.2 +/- 7.7 days. Ibex females younger than 4 years of age had a body weight lower (P < 0.01 ) than that of adults and none of them displayed ovulations. Plasma concentrations of prolactin levels were significantly affected by season (P < 0.05), following a trend that was roughly parallel to daylength. The highest values occurred in the spring (119.7 +/- 21.4 ng x mL(-1)) and the summer (139.3 +/- 19.8 ng x mL(-1)), the lowest values in the autumn (26 +/- 7.4 ng x mL(-1)) and in the winter (19.7 +/- 3.2 ng x mL(-1)). Our results showed a very restricted breeding season, despite the fact that the Spanish ibex originates from and lives in a temperate latitude, revealing a remarkably good adaptation to the harsh climatic and nutritional conditions of their mountainous habitat.