Lead (Pb) poisoning via ingestion of shot pellets is a frequent cause of death in wild birds and also has a wide range of subclinical effects. Here we report on the sublethal effects Pb exposure has ...on the breeding performance of red-legged partridges (Alectoris rufa). We studied the effects of Pb exposure on sperm quality, reproductive success, egg properties, laying performance, antioxidant levels, and carotenoid-based coloration. Birds were exposed by oral gavage to one or three No. 6 Pb shot pellets (2.8 mm in diameter, mean mass ± SD: 109 ± 7.97 mg). We show that exposure to three pellets (330 mg) reduced the hatching rate of females and decreased the acrosome integrity and sperm motility of males. In addition, females exposed to 1 pellet (110 mg) produced heavier eggs and chicks, whereas males exposed to 1 pellet presented an increase in sperm vigor. Sperm viability, concentration, progressiveness or fecundation rate were not affected by Pb treatment. Pb exposure increased circulating antioxidant levels in males, whereas the percentage of carotenoid-pigmented eye-ring area decreased in exposed females. Several sperm parameters showed positive relationships with coloration and antioxidant levels, suggesting that males displaying redder ornaments may be more capable of protecting sperm from oxidative stress in the event of sublethal Pb exposure.
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IJS, KILJ, NUK, PNG, UL, UM
Transrectal ultrasonic‐guided massage of the accessory sex glands (TUMASG) is a technique that allows collecting semen requiring few electrical stimuli or even no pulse. A long‐acting analogue of ...oxytocin (carbetocin, 0.1 mg) was i.v. administered before TUMASG in 10 conscious bucks (Experiment 1) and 10 anaesthetized Iberian ibexes (Experiment 2) to shorten the time of semen collection, decrease the number of electrical stimuli and/or improve the semen quality. The ejaculated volume, concentration, quality parameters and kinetics variables of the sperm were determined in fresh semen. The time length of the procedures and the number of electric pulses applied were recorded. Furthermore, stress response indicators (number of vocalizations in Experiment 1; heart and respiratory rates, rectal temperature, cortisol levels, totals proteins and neutrophil‐to‐lymphocyte ratio in Experiment 2) were documented. In bucks, the administration of carbetocin tended to shorten the time needed for semen collection but no‐showed differences in the fresh seminal quality. In the Iberian ibexes, there were no significant differences between groups in the time length of procedures or in the number of animals that ejaculated. Carbetocin administration only reduced the respiratory rate, did it modify fresh semen characteristics in ibexes. In conclusion, the administration of carbetocin did not appear as a useful tool to improve welfare during semen collection with TUMASG or semen quality in conscious bucks and anaesthetized ibexes, having only slight advantages related to the procedure.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
This study examines the effect of L‐carnitine (LC) on chilled ram semen stored for up to 96 hr. Semen samples were collected, placed in a skimmed milk + 6% egg yolk extender, pooled, aliquoted and ...diluted with the same extender supplemented with different LC concentration: 0 (control), 1 mM (LC1), 2.5 mM (LC2.5), 5 mM (LC5), 7.5 mM (LC7.5) or10 mM (LC10). Sperm kinetics and membranes (plasma, acrosome and mitochondrial) were examined using the CASA system and triple fluorescence staining (PI/ PNA‐FITC/Mitotracker). The progressive motility was greater (p < .05) with LC7.5 treatment than the control sperm at 96 hr. The curvilinear velocity (p < .01) and the percentage of sperm with intact membranes (plasma/acrosome/mitochondria) (p < .01) were greater with all LC treatments than the control group at all times. Straight line velocity was greater (p < .01) with LC5 and LC7.5 treatments than the control group after 48 hr. The LC5 group also returned lower ALH values (p < .05) than these seen for the control groups after 48 hr. The fertilizing capacity of LC5 samples stored at 15°C for 2 hr (LC5‐15°C‐2h) and at 5°C for 24 hr (LC5‐5°C‐24h) was tested in three ewe groups via cervical fixed‐time artificial insemination. In two groups, the fertilizing capacity of the LC5‐5°C‐24h was reduced (p < .001). In the remaining group, however, no significant difference was seen between the LC5‐15°C‐2h and LC5‐5°C‐24h sperm in this respect (pregnancy rates 52.4% versus 42.8%; p > .05). Overall, the present results suggest that supplementing skimmed milk–egg yolk‐based extenders with LC has a positive effect on chilled sperm variables and fertilizing capacity.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Sperm cryopreservation by ultra-rapid cooling based on dropping small volumes of sperm suspension directly into liquid nitrogen, has been successful in some wild ruminant species, including the ...Iberian ibex (Capra pyrenaica). In ultra-rapid cooling, the contents of these droplets are expected to enter a stable, glass-like state, but to the best of our knowledge no information exists regarding the presence or absence of ice formation in the extracellular milieu when using this technique. Different modifications to the extracellular milieu likely inflict different types of damage on the plasmalemma, the acrosome and mitochondrial membranes. The aims of the present work were: 1) to examine the physical state of the extracellular milieu after cryopreservation at slow and ultra-rapid cooling rates-and thus determine whether ultra-rapid cooling vitrifies the extracellular milieu; and 2) to compare, using conventional sperm analysis techniques and scanning and transmission electron microscopy, the damage to sperm caused by these two methods. Sperm samples were obtained by the transrectal ultrasound-guided massage method (TUMASG) from anesthetized Iberian ibexes, and cryopreserved using slow and ultra-rapid cooling techniques. Sperm motility (22.95 ± 3.22% vs 4.42 ± 0.86%), viability (25.64 ± 3.71% vs 12.8 ± 2.50%), acrosome integrity (41.45± 3.73% vs 27.00 ± 1.84%) and mitochondrial membrane integrity (16.52 ± 3.75% vs 4.00 ± 0.65%) were better after slow cooling (P<0.001) than after ultra-rapid technique. Cryo-scanning electron microscopy (Cryo-SEM) suggested that the vitrified state was not achieved by ultra-rapid cooling, and that the ice crystals formed were smaller and had more stretchmarks (P<0.001) than after slow cooling. Scanning electron microscopy revealed no differences in the types of damage caused by the examined techniques, although transmission electron microscopy showed the damage to the plasmalemma and mitochondrial membrane to be worse after ultra-rapid cooling. In conclusion ultra-rapid cooling provoked more membrane damage than slow cooling, perhaps due to the extracellular ice crystals formed.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In both captive wildlife and production animals is important to develop strategies for population control. Immunization against GnRH is an easy and inexpensive immunocastration method that reduces ...the concentration of testosterone and decreases sperm quality. However, its effectiveness depends on the species and repetition of the treatment. This study aimed to compare the effectiveness of a single treatment (initial immunization plus a booster with Improvac) vs repeated treatment (six doses of Improvac) to inhibit testicular function and maintain the contraceptive status during long periods in bucks. Three Dwarf bucks (Capra hircus) received two doses of Improvac, the first on Week 0, and the booster 4 weeks later (single immunization, group SI) while three Dwarf bucks received one dose of Improvac every 6 months during 3 consecutive years (repeated immunization, group RI). The other three Dwarf bucks remained untreated (control bucks, group CON). Bucks from RI had a greater decrease in scrotal circumference, testosterone concentration, male odor intensity, and sperm quality than SI bucks. However, there were no differences between SI and CON bucks in any of the variables studied. Overall, repeated treatment of Improvac decreased the testicular function of Dwarf bucks, although did not produce complete infertility. However, the repetition of the treatment produced more intensive negative effects, indicating that the strength of the effects of Improvac is rapidly lost in bucks.
Repeated treatment with Improvac (an anti‐GnRH vaccine) results in decreased sperm concentration and quality, among other reproductive changes in Dwarf bucks.
Highlights
The control of reproduction is a key point in zoos due to limitations in budgets and space.
Immunocontraceptive emerges as an alternative to surgical castration, as it reduces problems related to animal welfare and also may be reversible.
Immunization against GnRH‐induced gonadal suppression, but treatment should be repeated over time and does not induce complete infertility.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
In most goat breeds, testosterone serum concentration and semen quality decrease during the nonbreeding season. However, bucks reproductive activity may be stimulated with the administration of ...equine chorionic gonadotropin (eCG). Therefore, the aim of this study was to determine whether the repeated administration of eCG stimulates the reproductive status of bucks during the nonbreeding season. The study was performed with 19 bucks that were assigned to a group that was treated with eCG (GeCG) and an untreated control group (GCon). The GeCG bucks received an initial dose of 800 IU of eCG (Day 0), followed by four doses of 500 IU administered every 5 days beginning on Day 5. Serum testosterone and anti‐eCG antibody concentrations, testicular and seminal traits were determined until Day 60. Testosterone concentration (from Day 3 to 21: p < 0.0001), anti‐eCG titre (from Day 12 to 44: p ≤ 0.01), percentage of motile spermatozoa (Day 6: p = 0.006 and 14: p = 0.001) and of spermatozoa with progressive motility (Day 6: p = 0.01 and 14: p = 0.002) and the percentage of spermatozoa with functional membrane (Day 6: p = 0.02 and 22: p = 0.008) were higher in GeCG than in GCon bucks. Also in frozen‐thawed samples, the percentage of motile spermatozoa tended to be higher in GeCG than that of GCon bucks (p = 0.07). In conclusion, the administration of eCG during the nonbreeding season stimulated the secretion of testosterone and improved fresh and possibly frozen‐thawed semen quality. However, it also resulted in an increase in anti‐eCG antibody titre.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were ...to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat ('good freezer') and Black-Red Andaluza ('bad freezer') showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Chicken semen cryopreservation is a tool for programs of genetic diversity management and endangered breeds conservation. Due to physiological features, the fertility rates of cryopreserved poultry ...sperm are lower than mammal species. Thus, improvement of the semen cryopreservation methods is required. A first study was performed by a 2 × 2 factorial design consisting of 2 methods of adding the cryoprotectant Direct or Diluted (mixed with extender medium) and 2 cryoprotectants (glycerol and dimethylacetamide). Then sperm quality indicators were evaluated after freezing. A second study with a 2 × 2 design was conducted to evaluate the effectiveness of bovine serum albumin (BSA) on the optimization of 2 different extenders (Lake and Animal Sciences Group ASG). Viability and motility variables were evaluated before and after freezing. There was no significant difference in sperm viability and motility variables between Direct or Diluted methods. Supplementation of extenders with BSA improved most of the sperm motility variables in both extenders before and after freezing. Progressive sperm, non-progressive sperm before freezing, and all post-thaw sperm motility parameters, except amplitude of lateral head displacement and beat-cross frequency, were increased in BSA-supplemented extenders (P < 0.05), and BSA improved sperm viability in ASG extender after thawing (P < 0.05). After thawing, the interaction between extender and BSA (P < 0.05), eliminated the differences between the 2 BSA-supplemented media in curvilinear velocity, straight-line velocity, average path velocity, and amplitude of lateral head displacement which were higher in non-supplemented ASG extender than nonsupplemented Lake medium. In conclusion, the direct or diluted methods of adding glycerol or dimethylacetamide, did not significantly affect the post-thaw sperm characteristics. BSA positively affected most of the post-thaw sperm motility indicators regardless of the type of extender and resulted in significantly higher post-thaw sperm viability in ASG medium.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background
Recent studies have noted that the circulating testosterone concentration may affect the ability of spermatozoa to survive cryopreservation. However, few attempts to confirm such a ...relationship have been made. Wild ruminant species have very marked seasonal changes in their reproductive function and strong annual changes in their plasma testosterone concentration.
Objectives
The present work examines the influence of induced changes in testosterone secretion on sperm variables following conventional slow freezing and ultra‐rapid freezing, using the Iberian ibex as an experimental model.
Materials and Methods
In a first experiment, testosterone levels were reduced in the middle of the rutting season (December) using the antiandrogen cyproterone acetate (CA). In a second experiment, testosterone levels were increased at the end of the rutting season (January) via the use of the androgen testosterone propionate (TP).
Results
During December, the testosterone concentration was found to be higher in the blood and seminal plasma of untreated males than in those of CA‐treated males (p < 0.001 and p < 0.05, respectively). Compared with controls, the TP‐treated animals had higher blood plasma testosterone concentrations but lower seminal plasma testosterone concentrations during January (p < 0.01 and p < 0.001, respectively). The seminal vesicles of the TP‐treated males were larger than those of untreated males (p < 0.05). When CA was administered, sperm viability improved compared with controls (p < 0.05), irrespective of the freezing protocol followed. For the ultra‐rapid freezing procedure, the cryoresistance ratio for motility decreased when TP was administered (p < 0.05). The values for fresh sperm morphometric variables decreased during the 50 days after the end of CA treatment (p < 0.001) and increased over the same time after the end of TP treatment (p < 0.001).
Discussion and Conclusion
The circulating testosterone concentration appears to influence sperm cryoresistance. This may explain the seasonal changes seen in sperm freezability in some species, independent of fresh sperm quality.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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