Freezability of Iberian ibex (Capra pyrenaica) spermatozoa according to the glycerolization temperature and plasma testosterone concentration.

Ibex spermatozoa can be successfully frozen using glycerolated media. However, no information is available regarding the most effective method of glycerol addition in this species. The aim of the present work was to evaluate the effect of the glycerolization temperature on the response to freezing-thawing of ibex spermatozoa collected by electroejaculation. The effect of the interaction glycerolization temperature x plasma testosterone concentration was also evaluated. The spermatozoa used in this work came from six adult ibexes maintained in captivity. Each ejaculate was divided into two aliquots in a Tris-egg yolk-based medium. One fraction was subjected to single step dilution with 5% glycerol at room temperature (23°C). The other fraction was diluted in two steps, first by dilution at room temperature with an extender identical to that described above but without glycerol, followed by the addition of glycerol after cooling to 5°C. The glycerolization temperature did not affect any sperm variable after thawing. Heterospecific artificial insemination involving domestic goats, revealed no differences in the fertilization rate for frozen-thawed spermatozoa diluted by the one or two step procedures (18.2% vs. 20.0%). The interaction glycerolization temperature x plasma testosterone concentration had no affect on the freezing-thawing of the sperm cells. The results revealed, however, that high plasma testosterone levels during the pre-rutting season may interfere with the freezing-thawing process, having a negative influence on sperm cryosurvival.

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.

Endogenous circannual cycles of ovarian activity and changes in prolactin and melatonin secretion in wild and domestic female sheep maintained under a long-day photoperiod.

The present study examines the ovulatory activity of wild and domesticated ewes subjected to either a constant photoperiod of long days (16L:8D) or natural changes in daily photoperiod for 16 mo. The aim was to determine whether an endogenous reproductive rhythm controls seasonal reproductive activity in these sheep, and how the photoperiod might affect this. The effects of long-day photoperiods on long-term changes in prolactin and melatonin secretion were also evaluated. The two species showed changes in reproductive activity under the constant photoperiod conditions, suggesting the existence of an endogenous rhythm of reproduction. This rhythm was differently expressed in the two types of ewe (P < 0.05), with the domestic animals exhibiting much greater sensitivity to the effects of long days. A circannual rhythm of plasma prolactin concentration was also seen in both species and under both photoperiod conditions, although in both species the amplitude was always lower in the long-day animals (P < 0.01). The duration of the nocturnal melatonin plasma concentrations reflected the duration of darkness in both species and treatments. The peak melatonin concentration did not differ between seasons either under natural or long-day photoperiods.

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.

Characteristics of the oestrous cycle of Iberian red deer (Cervus elaphus hispanicus) assessed by progesterone profiles.

This study examines the length of the oestrous cycle in 16 Iberian red deer females assessed by means of changes in progesterone concentrations, along with the changes in the profile of this hormone. Samples were collected three occasions per week from the week after calving (15 May to 15 June) up to May of the following year. The oestrous cycle lasted 19.57+/-0.29 days (range 10-27 d) calculated in 130 oestrous cycles examined. Progesterone titres did not rise above 0.5 ng/ml in the follicular phase, except in four samples. The maximum peak in progesterone concentration during the luteal phase remained above 1 ng/ml in most cases. Twenty-five percent of the individuals studied (4 out of 16) showed an oestrous cycle lasting shorter than the mean (15.2+/-0.30 days) before the start of the reproductive season, followed by a period of sexual inactivity. The standard progesterone profile in natural oestrous cycles rose from basal levels to those above 0.5 ng/ml four days after onset of oestrus, reached a peak of 1.71+/-0.07 ng/ml and then declined to less than 0.2 ng/ml after day 20. Following the rapid decline of progesterone after day 14, the concentration remained around the baseline level of 0.1 to 0.2 ng/ml during the immediate pre- and post-ovulatory phase of the cycle.