C. Castaño

Influence of season on the freezability of free-range poultry semen.

The purpose of the present study was to examine the seasonal variation in freezing damage in free-range rooster sperm. Over a period of 1 year, heterospermic semen samples were collected weekly by massage from the roosters of 14 Spanish chicken breeds, all housed under natural photoperiod and climatic conditions. All samples were frozen in straws using DMA as a cryoprotectant, placing them first in nitrogen vapour and then plunging them into liquid nitrogen. No seasonal effects on fresh sperm quality were found. Neither did season affect the percentage of viable frozen-thawed spermatozoa nor the percentage with an intact acrosome. However, the collection season influenced (p < 0.05) most frozen-thawed sperm motility values. The percentage of immotile frozen-thawed spermatozoa was lower (p < 0.05) in spring-collected sperm than in summer- or autumn-collected samples. The percentage of spermatozoa showing progressive motility was higher in spring-collected sperm compared with winter-, summer- or autumn-collected samples (p < 0.05). The curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) values of spring-collected sperm were also higher (p < 0.05). In conclusion, spring would appear to be the best season for collecting and freezing the semen of free-range Mediterranean chicken breeds.

Seasonal variation in reproductive physiological status in the Iberian ibex (Capra pyrenaica) and its relationship with sperm freezability

The present work examines the relationship between seasonal changes in testicular function, accessory gland size, and horn growth in Iberian ibexes, as well as the relationship between these changes and the resistance of ibex spermatozoa to freezing-thawing. The size of the bulbourethral glands and seminal vesicles showed pronounced monthly variation (P < 0.001), which was correlated positively with the plasma testosterone concentration (P < 0.001) and scrotal circumference (P < 0.001). The size of the accessory sex glands peaked during the autumn. Overall, semen quality was markedly improved during autumn and winter. When horn growth was at a minimum during autumn and winter, semen quality and accessory gland size were all increased compared to in spring and summer. However, increased plasma testosterone levels in the autumn were strongly associated with reduced sperm freezability; thus, the cryosurvival of spermatozoa collected during the autumn was poorer than at other times of the year. In winter, however, when the plasma testosterone concentration fell to baseline, the negative effects of cryopreservation on the percentage of motile spermatozoa and on the integrity of the plasma membrane of frozen-thawed sperm cells were significantly less intense (P < 0.05). These findings show a clear relationship between the functional and morphological status of the different parts of the reproductive tract that optimises reproductive function during the breeding season in the ibex male. They also show that winter is the most suitable season for the collection and cryopreservation of ibex spermatozoa.

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa--but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P<0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P<0.001) and viability (P<0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.

Sperm–egg penetration assay assessment of the contraceptive effects of glycerol and egg yolk in rooster sperm diluents

Glycerol (GLY) and egg yolk (EY) are good cryoprotectants of avian and mammalian sperm, but in birds, they strongly inhibit the eventual fertilization of ova. Using the sperm penetration (SP-holes) assay and fertility trials, the present study investigates (1) the possible mechanism by which this contraceptive effect occurs in chickens and (2) the maximum concentrations of GLY and EY tolerated by fresh rooster sperm. Seventy Black-Barred Andaluza hens (five per treatment) were inseminated four times (twice per week) with 0.1 mL of fresh semen from roosters of the same breed diluted 1:1 (v:v) with Lake and Ravie medium containing different concentrations of GLY or EY. No adverse effects on acrosome integrity, sperm motility, or viability were seen with any concentration of GLY or EY. The number of SP-holes on perivitelline layer samples taken from above the germinal disc became progressively lower at GLY concentrations of 1.5% or greater (P > 0.05). No holes caused by sperms were seen in unfertilized eggs. The corresponding fertility results showed similar reductions when the GLY concentration was 1.5% or greater. No changes in the number of SP-holes were seen with increasing EY concentrations (0%-7.5%), nor were any differences in fertility observed, except for a reduction when 15% EY was used. The results therefore reveal that GLY affects the transit of sperms through the oviduct in their attempt to reach the infundibulum area, limiting their access to the ovum perivitelline layer. Egg yolk had no such effect, nor did it influence acrosome reaction capacity; its mechanism of contraceptive action therefore remains unknown. The maximum GLY and EY concentrations tolerated by the rooster sperm were 0.75% and 7.5%, respectively.

Cryopreservation of epididymal sperm from ibexes (Capra pyrenaica) using short equilibration time with glycerol

Two experiments were conducted to study the effect of shortening the equilibration time with the cryoprotectant glycerol before freezing epididymal sperm recovered postmortem from Iberian ibex. In the first experiment, the standard equilibration time of 3 hours was compared with 2 hours, and subjective sperm motility and quality of movement were greater (P < 0.05) in the latter group. In the second experiment, reducing the equilibration time from 2 hours to 15 minutes did not affect sperm motility (evaluated subjectively and objectively), viability, acrosomal integrity, or membrane functional integrity. In conclusion, shortening the equilibration time can be used as a technique to simplify the cryopreservation process and this provides practical advantages under field conditions.

Sperm selection by Capripure(®) density-gradient centrifugation versus the dextran swim-up procedure in wild mountain ruminants.

This study compares the effectiveness of two methods of sperm selection - Capripure(®) density-gradient centrifugation (DGC) and dextran swim-up (DSU) - in semen samples from Iberian ibex (Capra pyrenaica) and European mouflon (Ovis musimon). During the increasing photoperiod, Capripure(®) DGC improved the percentage of sperm with progressive motility (P<0.05) in ibexes, and selected 60.6% of the initial total number of spermatozoa contained in the ejaculate samples. In mouflon, Capripure(®) DGC selection was unaffected by photoperiod, had no influence on any sperm variable, and selected 47.8% of the initial total number of mouflon spermatozoa in ejaculate samples. Photoperiod had no influence on the effectiveness of DSU in either ibexes or mouflons. In the ibexes, DSU reduced (P<0.05) the percentage of sperm cells with morphological abnormalities, but only selected 11.3% of the initial total number of spermatozoa in ejaculate samples. In the mouflons, DSU had no significant influence on any sperm variable, and selected 27.8% of the initial total number. Capripure(®) DGC improved ibex and mouflon sperm motility (P<0.05) following 30min and 2h of post-centrifugation, stress-inducing incubation, respectively. In both ibexes and mouflon, sperm cells showing non-progressive motility were found after only 20 h of post-centrifugation incubation following Capripure(®) DGC selection. In conclusion, Capripure(®) DGC would seem a useful method for selecting the best spermatozoa from both ibex and mouflon ejaculates.

Photoperiod and melatonin treatments for controlling sperm parameters, testicular and accessory sex glands size in male Iberian ibex: A model for captive mountain ruminants

This study examines whether photoperiod and/or melatonin treatments can improve sperm variables outside the breeding season in the Iberian ibex-a model species for wild mountain ruminants-thus helping in the collection of high quality sperm beyond the normal breeding season for depositing in genetic resource banks. Adult Iberian ibex males (n=17) were divided into four treatment groups: (1) controls under the natural photoperiod (control group; n=4), (2) treatment with melatonin implants on December 22nd, February 22nd and April 22nd (group WS-M; n=5), (3) treatment with short photoperiod cycles, i.e., 2 months of long days followed by melatonin implants (to emulate 2 months of short days) throughout the year (group PHPld+M; n=4), and (4) treatment with melatonin implants on June 22nd and August 22nd (group SS-M; n=4). The interaction treatment x season had a strong influence on testis size (P<0.05), the size of the seminal vesicles (P<0.001), the percentage of abnormal sperms (P<0.05), and percentage non-progressive (P<0.05) and progressive (P<0.001) sperm motility. In groups WS-M and PHPld+M, the normal springtime physiological reductions in testis size, non-progressive sperm motility and acrosome integrity were prevented. The values for the studied sperm variables were, however, reduced in the natural breeding season at the end of the experimental period in group PHPld+M, although not in group WS-M. The pattern of melatonin administration in group SS-M conferred no advantages on reproductive functionality. These results suggest that lengthening the short day period after the winter solstice (the WS-M treatment) extends reproductive activity in this species, allowing good quality sperm to be recovered for conservation purposes during the non-breeding season.

Physiological responses and characteristics of sperm collected after electroejaculation or transrectal ultrasound-guided massage of the accessory sex glands in anesthetized mouflons (Ovis musimon) and Iberian ibexes (Capra pyrenaica)

The objective was to characterize the stress response and the seminal parameters obtained with electroejaculation (EE) or transrectal ultrasound-guided massage of the accessory sex glands (TUMASG) in two captive but nondomestic ruminants, the mouflons and the Iberian ibex under general anesthesia. In mouflons, the physiological responses (heart and respiratory rate, rectal temperature, cortisol, creatine kinase, potassium and glucose concentrations) changed similarly with both procedures. The TUMASG procedure was faster than EE in mouflons (21.7 ± 1.4 vs. 12.4 ± 1.2 minutes, P < 0.01). In ibexes, respiratory rate, cortisol and creatine kinase concentration changes were greater with EE than with TUMASG (final respiratory rate: 62.7 ± 5.5 vs. 38.1 ± 5.6 breaths/min [P < 0.05]; final cortisol: 51.4 ± 5.1 vs. 25.3 ± 5.6 ng/mL [P < 0.001]; and final creatine kinase: 300.9 ± 99.9 vs. 87.1 ± 16.9 U/L [P < 0.001]). Electroejaculation provided better results in some sperm parameters (mouflons: sperm score: 3.4 ± 0.3 vs. 2.6 ± 0.2 [P < 0.01]; total number of sperm ejaculated: 982.4 ± 299 vs. 710.0 ± 542.2 [P < 0.05]; ibexes: sperm with progressive motility: 47.7 ± 6.2 vs. 20.5 ± 8.3 [P < 0.05]). The transrectal ultrasound-guided massage of the accessory sex glands appears to be an alternative technique to collect sperm from wild ruminants, reducing the need for electrical stimuli and thus decreasing the undesired responses of EE in the more sensitive species. On the other hand, better fresh sperm may be collected with EE. However, TUMASG provides practical advantages in animal welfare, firstly in these wild species more sensible to stress management and capture myopathy.

Descriptive analysis of sperm head morphometry in Iberian ibex (Capra pyrenaica): Optimum sampling procedure and staining methods using Sperm-Class Analyzer®

Sperm morphology has been identified as one characteristic which can be useful in prediction of fertility in a species. The development of computer automated sperm morphometry analysis allows for objective analysis of sperm head dimensions. The aim of the current study was to develop an optimum sampling procedure to characterize the Iberian ibex (Capra pyrenaica) sperm head morphometrically. Fresh semen from 11 males was collected using transrectal ultrasonic-guided massage of accessory sex glands and electroejaculation and prepared on slides for morphometric analysis to evaluate technical variation and standardize automated sperm morphometry analysis procedures by Sperm-Class Analyzer(®). Three staining methods (Diff-Quik(®), Hemacolor(®), Spermblue(®)), number of sperm cells necessary to sample and repeatability of the staining technique were assessed. There were significant differences in size of sperm head depending on stain used. Hemacolor(®) was stain most suitable for sperm head morphometry evaluation (length=8.42 μm; width=4.21 μm; area=29.37 μm(2); perimeter=21.93 μm; elongation=0.33; elipticity=2.01; regularity=0.95; rugosity=0.77). Morphometric values obtained from samples of 50, 100, 150, 175 and 200 sperm heads were compared. The most efficient method of analyzing sperm morphometry was to evaluate 100 sperm cells at 60× objective magnification. Thus, this study has allowed for description of optimal sample processing to determine morphometric parameters of sperm heads (size and shape) in Iberian ibex by Sperm-Class Analyzer(®) and provides a basis for future studies on the relationship with freezability and fertility in this species.

CHARACTERIZATION OF NATURAL EJACULATES AND SPERM CRYOPRESERVATION IN A GOLDEN EAGLE (AQUILA CHRYSAETUS)

This paper describes the sperm characteristics and response to cooling and freezing of naturally ejaculated semen from a captive, adult golden eagle (Aquila chrysaetus) trained to allow sperm recovery via cooperative copulation. A basic spermiogram was prepared, and sperm motility and morphometric variables recorded using a computer-aided system. For sperm storage, the effects of a polyvinylpyrrolidone-based extender were evaluated at 5°C. The same extender was also used in freezing procedures in which glycerol (11%) and dimethylacetamide (6%) were compared as cryoprotectants. The extender preserved sperm viability over storage periods of up to 6 days. Although sperm motility and percentage live sperm values were poorer for frozen-thawed (5.8-14.6% and 44-42%, respectively) than for fresh samples (46.7 and 74.6%, respectively), no differences were seen between the effects of the two cryoprotectants. These results could be of use when attempting to store the sperm of golden eagles and other raptors.