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
Postcopulatory sexual selection through sperm competition may be an important evolutionary force affecting many reproductive traits, including sperm morphometrics. Environmental factors such as ...pollutants, pesticides, and climate change may affect different sperm traits, and thus reproduction, in sensitive bird species. Many sperm-handling processes used in assisted reproductive techniques may also affect the size of sperm cells. The accurately measured dimensions of sperm cell structures （especially the head） can thus be used as indicators of environmental influences, in improving our understanding of reproductive and evolutionary strategies, and for optimizing assisted reproductive techniques （e.g., sperm cryopreservation） for use with birds. Computer-assisted sperm morphometry analysis （CASA-Morph） provides an accurate and reliable method for assessing sperm morphometry, reducing the problem of subjectivity associated with human visual assessment. Computerized systems have been standardized for use with semen from different mammalian species. Avian spermatozoa, however, are filiform, limiting their analysis with such systems, which were developed to examine the approximately spherical heads of mammalian sperm cells. To help overcome this, the standardization of staining techniques to be used in compute~-assessed light microscopical methods is a priority. The present review discusses these points and describes the sperm morphometric characteristics of several wild and domestic bird species.
Sperm cryopreservation is a complex process that needs to be adapted to wild and domestic avian species to ensure proper efficiency. Because of its accessibility, the peregrine falcon may be used as ...a good model for studying other raptor species. To find the most optimal cryopreservation protocol for peregrine falcon ejaculates, sperm parameters such as motility, viability, DNA fragmentation, acrosome integrity, and mitochondrial activity were analyzed under different conditions by varying the freezing method (slow freezing in straws vs. ultrarapid freezing in pellets), thawing conditions (37 °C for 30 s vs. 5 °C for 1 min), type of cryoprotectant (DMA vs. DMSO), and concentration of DMSO (4% vs. 8%). Results show that slow cryopreservation in straws yielded greater percentages (
< 0.05) of motile spermatozoa (22.5% ± 4.4% vs. 0.0% ± 4.1%), viable spermatozoa with intact acrosomes (84.6% ± 4.3% vs. 77.4% ± 4.3%), and spermatozoa with active mitochondria (41.0% ± 6.7% vs.12.8% ± 6.7%), compared with those obtained by the ultrarapid freezing in pellets. However, no differences were found between different thawing conditions. Moreover, all sperm motility parameters were greater (
< 0.05) when DMSO was used during freezing compared with DMA, although the use of 3% and 8% DMSO produced similar results. In conclusion, these results represent important progress in the study of falcon semen cryopreservation protocol, highlighting the crucial steps of the process and the most suitable conditions.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The American flamingo is a useful model for the development of successful semen cryopreservation procedures to be applied to threatened related species from the family Phoenicopteridae, and to permit ...genetic material banking. Current study sought to develop effective sperm cryopreservation protocols through examining the influences of two permeating cryoprotectants and the seminal plasma removal. During two consecutive years (April), semen samples were collected and frozen from American flamingos. In the first year, the effect of two permeating cryoprotectants, DMA (dimethylacetamide) (6%) or Me
SO (dimethylsulphoxide) (8%), on frozen-thawed sperm variables were compared in 21 males. No differences were seen between DMA and Me
SO for sperm motility, sperm viability, and DNA fragmentation after thawing. In the second year, the role of seminal plasma on sperm cryoresistance was investigated in 31 flamingos. Sperm samples were cryopreserved with and without seminal plasma, using Me
SO (8%) as a cryoprotectant. The results showed that samples with seminal plasma had higher values than samples without seminal plasma for the following sperm variables: Straight line velocity (22.40 µm/s vs. 16.64 µm/s), wobble (75.83% vs. 69.40%), (
< 0.05), linearity (62.73% vs. 52.01%) and straightness (82.38% vs. 73.79%) (
< 0.01); but acrosome integrity was lower (55.56% vs. 66.88%) (
< 0.05). The cryoresistance ratio (CR) was greater in samples frozen with seminal plasma than without seminal plasma for CR-progressive motility (138.72 vs. 54.59), CR-curvilinear velocity (105.98 vs. 89.32), CR-straight line velocity (152.77 vs. 112.58), CR-average path velocity (122.48 vs. 98.12), CR-wobble (111.75 vs. 102.04) (
< 0.05), CR-linearity (139.41 vs. 113.18), and CR-straightness (124.02 vs. 109.97) (
< 0.01). This research demonstrated that there were not differences between Me
SO and DMA to successful freezing sperm of flamingos; seminal plasma removal did not provide a benefit for sperm cryopreservation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Background
The Cantabrian capercaillie (Tetrao urogallus cantabricus) is critically endangered. This subspecies has the lowest genetic variability and it is in regression. It belongs to Phasianidae ...family; therefore, the domestic chicken (Gallus gallus domesticus) could be a good model for developing reproductive technologies for use in capercaillie populations with low availability of animals.
Objectives
In this study, we analyzed the response of capercaillie sperm to the freezing–thawing process for contributing to the development of a semen cryobank of Cantabrian capercaillie.
Methods
We used domestic chicken as the animal model in order to obtain the freezing protocol before applying on capercaillie. In the first experiment, two different extenders (EK and LR84) and different concentrations 4% and 6% dimethyl‐acetamide (DMA) v:v of cryoprotectants were evaluated using in‐straw freezing method in domestic chickens. A pilot study in capercaillie males, using the same conditions evaluated in chicken, was performed.
Results
In chicken, we found that the LR84‐4% DMA media provided the best results for freezing semen. In capercaillie study, LR84 extender seemed to be the most appropriate diluent and 4% was the better dose of DMA cryoprotectant agent. Further, based on previous studies carried out in rooster samples, we also tested the glycerol (8% v/v) as a cryoprotectant for capercaillie semen cryopreservation.
Conclusions
Our results suggest that sperm from both domestic and wild species had a similar response to freezing–thawing processes. Mediterranean chickens may be used as a suitable model for developing sperm freezing protocols that can be extrapolated to threatened capercaillie populations. In addition, LR84 media with glycerol was the most efficient extender to freeze capercaillie sperm native.
In this work, we obtained and described, for the first time, the ejaculate of Cantabrian capercaillie (Tetrao urogallus cantabricus). Also, we used a local Spanish breed (Gallus gallus) as the animal model in order to obtain the freezing protocol before applying on capercaillie. Then, we analyzed the response of capercaillie sperm to these freezing‐thawing process and our results suggest that sperm from both domestic and wild species have a similar response to freezing‐thawing processes.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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
The freeze-thawing process induces osmotic changes that may affect the membrane domain location of aquaporins’ (AQP) in spermatozoa. Recent studies suggest that changes in AQP3 localization allows ...better sperm osmo-adaptation, improving the cryoresistance. Ultra-rapid freezing is an alternative cryopreservation technique that requires less equipment than conventional freezing, and it is faster, simpler and can be used in the field. This study aimed to determine the influence of freezing-thawing rates (slow (control) vs. ultra-rapid) on AQP3 expression and location in the spermatozoa from small ruminants (sheep and goats) and its relationship with sperm cryo-damage. Spermatozoa were collected from 10 Merino rams and 10 Murciano-Granadina bucks. The presence and distribution of AQP3 were assessed by Western blotting and immunocytochemistry (ICC), employing a commercial rabbit polyclonal antibody. Sperm motility was CASA system-analyzed, and membrane and acrosome integrity assessed by fluorescence (PI/PNA-FITC). Western blotting did not detect a significant effect of freezing-thawing rate on the amount of AQP3 while ICC found freezing-thawing rate affecting AQP3 location (P < 0.05). In both species, the percentages of spermatozoa showing AQP3 in the post-acrosome region, mid-piece, and principal piece of the tail were greater in samples cryopreserved by slow freezing-thawing (control) than ultra-rapid freezing-thawing rates (P < 0.05). Spermatozoa cryopreserved using ultra-rapid freezing-thawing showed decrease motility, plasma membrane, and acrosome integrity (P < 0.05), which might be related, at least in part, to a lower expression of AQP3. In conclusion, the cooling rate modifies the location of AQP3 in spermatozoa of sheep and goat, which might be associated with sperm cryosurvival.
•This is the first study identifying AQP3 in spermatozoa of goat.•Cooling rate modifies the location of AQP3 in spermatozoa of sheep and goat.•Changes in the location of AQP3 appears to be related to sperm cryosurvival.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
This work examined the influence of induced changes in prolactin (PRL) secretion on sperm cryoresistance of ibex and the mouflon.
PRL secretion was modified in a first experiment by the use of ...bromocriptine (BCR, dopamine agonist) during the non-breeding season, and in a second experiment by the use of sulpiride (SLP, dopamine D
-receptor antagonist) during the rutting season. Slow and ultra-rapid freezing protocols were used to cryopreserve sperm samples.
BCR decreased blood plasma PRL concentrations, whereas SLP increased them. Cryoresistance ratios (CRs) for curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP) in BCR-treated mouflons were lower than in controls using slow-freezing (
< 0.05), while CRs of motility and morphologically normal sperm of BCR-treated mouflons were greater than controls with ultra-rapid freezing (
< 0.05). BCR increased the head sperm dimensions in ibexes (
< 0.001); conversely, BCR decreased the head dimensions in mouflons (
< 0.001). CR-motility, CR-amplitude of lateral head displacement (ALH), CR-viability, and CR-acrosome integrity in SLP-treated mouflons were lower than in controls with slow-freezing (
< 0.01); CR-viability and CR-acrosome were lower than controls with ultra-rapid freezing (
< 0.05). In ibexes, CR-ALH was lower for SLP-treated (
< 0.05). SLP treatment increased head dimensions in ibexes (
< 0.001) but did not affect the sperm head of mouflons.
Our findings show that high levels of blood plasma PRL negatively affect the cryoresistance of ibex and mouflon sperm.
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
Cryopreservation of testicular tissues offers new possibilities to protect endangered species, genetically valuable individuals or even the fertility potential of prepubertal individuals who have ...died unexpectedly. However, the use of this technique still remains a challenge. In this study, slow freezing and vitrification of testicular tissue was investigated to find out which cryopreservation method could better preserve the viability and DNA integrity of testicular germ cells in diverse wild species.
Testes were obtained post-mortem from 18 artiodactyls (wild boar, roe deer, dwarf goat, mhor gazelle, European mouflon, African forest buffalo, Malayan tapir, dorcas gazelle, Iberian ibex, gnu, red river hog), 5 primates (colobus monkey, capuchin monkey, mandrill), 8 carnivores (gray wolf, Persian leopard, binturong, European mink, American black bear, suricata), and 2 rodents (Patagonian mara). The testicles belonged to adult individuals and were cut into small pieces and cryopreserved by needle immersed vitrification or uncontrolled slow freezing using a passive cooling device. After warming or thawing, testicular tissues were enzymatically digested and two germ cell types were differentiated based on their morphology: rounded cells (spermatogonia, spermatocytes, and early spermatids) and elongated cells (elongated spermatids and spermatozoa). Cell viability was assessed by SYBR-14/propidium iodide while DNA fragmentation by TUNEL assay with fluorescence microscope.
Our preliminary results revealed that our uncontrolled slow freezing method better preserved the viability and DNA integrity of elongated cells than vitrification. Such trend was observed in all species, being significant in artiodactyls, carnivores, and primates. Similarly, the viability and DNA integrity of rounded cells was also better maintained in primates by uncontrolled slow freezing, while in carnivores, vitrification by needle immersion showed better results in this type of cells. In artiodactyls and rodents both techniques preserved the viability of rounded cells in a similar manner, although the DNA integrity of these cells was greater after needle immersed vitrification in artiodactyls.
In conclusion, the effectiveness of each cryopreservation method is affected by the phylogenetic diversity between species and cell type.
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