Birchen and Blue Leonesa sperm cryopreservation: a new technique for evaluating the integrity of cockerel sperm membranes.

1. Birchen and Blue Leonesa are two endangered chicken breeds mainly raised in Curueño Valley in North Spain. The establishment of a germplasm bank to guarantee the preservation of these breeds is needed. However, cockerels from different breeder flocks can show variance in semen cryoresistance.2. The following work focused on the sperm characterisation and cryopreservation of Birchen and Blue Leonesa cockerels from four different breeders. A total of 30 semen pools were analysed. Besides conventional sperm analysis, including motility by computer-aided sperm analysis (CASA) and DNA fragmentation by TUNEL, the present study tested a double staining method (MitoTrackerTM Green FM/propidium iodide). This gave simultaneous assessment of plasma and acrosomal and mitochondrial membranes, which were previously validated by SYBR-14/PI, CASA, aniline blue and TUNEL.3. No significant differences were found among fresh semen variables between breeds and breeders. For post-thawed variables, significant differences (P < 0.05) were found between breeders in sperm viability (58.0 ± 1.90 breeder D vs. 35.2 ± 7.41 breeder A, 37.2 ± 4.09 breeder B and 22.3 ± 5.92 breeder C) and DNA fragmentation (62.4 ± 9.91 breeder C vs. 31.8 ± 7.08 breeder B and 24.5 ± 5.49 breeder D). The lowest DNA fragmentation values for semen from breeder D birds were coincident with higher integrity of the mitochondrial membrane.4. The results revealed higher sperm cryoresistance in the cockerels from one of the breeders, possibly due to differences in management system (e.g. diet, housing, control of stress elements and pathogens, reproduction practices or maintenance of genetic diversity). These differences may determine the sperm freezability, and thus the effectiveness of developing a germplasm bank.

Conventional slow freezing cryopreserves mouflon spermatozoa better than vitrification.

This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre-freezing equilibration times (2-3 hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500 mm). The highest quality (p < .01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3 hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60-85°C min-1 ), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100 mm) provided the highest (p < .05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified-warmed sperm variables were at their best when the spermatozoa was diluted in TCG-6% egg yolk + 100 mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p < .05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing.

Spermiotoxicity of commercial condoms made from polyurethane, polyisoprene and latex, using domestic ruminants as an experimental animal model.

The use of condoms could provide a means of collecting high-quality spermatozoa from different species under physiological ejaculation conditions. However, certain condom materials may affect sperm functionality. This study examined the spermiotoxicity of different commercial condom materials towards ram and goat spermatozoa. Sperm samples were diluted in Tyrode's medium and placed in contact with a piece of condom material (polyurethane, polyisoprene or latex) and incubated for 30 or 90 min. Contact time in the polyisoprene and latex treatments affected some sperm variables; no such effects were seen, however, in the polyurethane treatments. For ram spermatozoa in contact with polyisoprene, the percentage of dead spermatozoa with a damaged acrosome increased at 90 min, while for spermatozoa in contact with latex, the percentage of live spermatozoa with an intact acrosome decreased. For goat spermatozoa in contact with both polyisoprene and latex, the percentage of dead spermatozoa with a damaged acrosome increased at 90 min, while for spermatozoa in contact with polyisoprene, the percentage of live spermatozoa with an intact acrosome decreased. In conclusion, latex and polyisoprene contain components that affect sperm motility, plasma membrane integrity and acrosome function. Polyurethane does not seem to reduce the quality of semen.

Giant panda (Ailuropoda melanoleuca) sperm morphometry and function after repeated freezing and thawing.

This work examines the effects of subsequent cycles of freezing-thawing on giant panda (Ailuropoda melanoleuca) sperm morphometry and function, and assesses whether density-gradient centrifugation (DGC) can increase the number of freezing-thawing cycles this sperm can withstand. A sperm sample was collected by electroejaculation from a mature giant panda and subjected to five freezing-thawing cycles. Although repeated freezing-thawing negatively affected (P < 0.05) sperm motility and membrane integrity, in both nonselected and DCG-selected sperm samples, >60% of the sperm cells in both treatments showed acrosome integrity even after the fifth freezing cycle. In fresh semen, the sperm head length was 4.7 µm, the head width 3.6 µm, area 14.3 µm(2) and perimeter length 14.1 µm. The present results suggest that giant panda sperm trends to be resistant to repeated freezing-thawing, even without DGC selection.

Influence of Post-Mortem Sperm Recovery Method and Extender on Unstored and Refrigerated Rooster Sperm Variables.

Many post-mortem sperm collection techniques have been described for mammalian species, but their use in birds is scarce. This paper compares the efficacy of two post-mortem sperm retrieval techniques - the flushing and float-out methods - in the collection of rooster sperm, in conjunction with the use of two extenders, i.e., L&R-84 medium and Lake 7.1 medium. To determine whether the protective effects of these extenders against refrigeration are different for post-mortem and ejaculated sperm, pooled ejaculated samples (procured via the massage technique) were also diluted in the above extenders. Post-mortem and ejaculated sperm variables were assessed immediately at room temperature (0 h), and after refrigeration at 5°C for 24 and 48 h. The flushing method retrieved more sperm than the float-out method (596.5 ± 75.4 million sperm vs 341.0 ± 87.6 million sperm; p < 0.05); indeed, the number retrieved by the former method was similar to that obtained by massage-induced ejaculation (630.3 ± 78.2 million sperm). For sperm collected by all methods, the L&R-84 medium provided an advantage in terms of sperm motility variables at 0 h. In the refrigerated sperm samples, however, the Lake 7.1 medium was associated with higher percentages of viable sperm, and had a greater protective effect (p < 0.05) with respect to most motility variables. In conclusion, the flushing method is recommended for collecting sperm from dead birds. If this sperm needs to be refrigerated at 5°C until analysis, Lake 7.1 medium is recommended as an extender.

Effect of the interaction between cryoprotectant concentration and cryopreservation method on frozen/thawed chicken sperm variables.

This work examines the effect of the interaction between different concentrations of two cryoprotectants - glycerol (GLY) and dimethylacetamide (DMA) - and two methods of cryopreservation - pellets produced by plunging into liquid nitrogen and gradual in-straw freezing - on frozen/thawed chicken sperm variables. Sperm was cryopreserved using: (i) 6% DMA, following the in-straw and the pellet methods (ii) 11% GLY, following the in-straw and the pellet methods; and (iii) 8% GLY in the in-straw method and 3% DMA in the pellet method (i.e. reduced cryoprotectant concentrations). When 6% DMA was used as the cryoprotectant, no differences were seen between the in-straw and pellet methods in terms of frozen/thawed sperm variables or fertility (10.8% and 12.8%, respectively). The viability and motility variables of the frozen/thawed sperm produced using the in-straw method with 11% GLY were higher (p < 0.05) than those recorded for the sperm preserved using the same cryoprotectant and concentration in the pellet method. However, fertility was extremely low in both groups (2.1% and 4.2% for the in-straw and pellet methods, respectively). Finally, the use of 8% GLY in the in-straw method returned higher sperm viability, intact acrosome and motility values than the use of 3% DMA in the pellet method (p < 0.01). No differences were seen, however, in the fertility results obtained (28.8% and 25.0%, respectively). These results suggest that cryoprotectant concentrations can be reduced and still provide acceptable fertility rates.

Influence of Staining Method on the Values of Avian Sperm Head Morphometric Variables.

Computer-assisted systems for the assessment of sperm morphometry (ASMA systems) have been used successfully with several mammalian species. Unfortunately, they have so far been of little use for assessing bird semen, a consequence of the filiform shape of avian spermatozoa. This study compares two staining techniques (Hemacolor(®) and aniline blue staining) for the morphometric analysis of rooster and red-legged partridge spermatozoa as part of a computer-assisted light microscopy method. For both species, Hemacolor(®) staining provided a significantly higher percentage of measurable cells (93.7 ± 11.7% in roosters and 71.9 ± 15.3% in red-legged partridges). Hemacolor(®) also showed greater repeatability (lower coefficients of variation) for length and area in roosters' sperm and for width in the case of red-legged partridge's sperm. In the roosters, the Hemacolor(®) technique returned significantly (p < 0.05) larger sperm head width and area values than did the aniline blue technique, while the latter resulted in greater sperm head length values (p < 0.05). In the red-legged partridge, no differences were seen in the results for sperm head width and area provided by the two techniques, but aniline blue staining was associated with longer length measurements. In conclusion, the morphometric values recorded differed depending on the staining method and species. However, the Hemacolor(®) technique might be deemed the more appropriate for computerized sperm assessment systems as it provides larger percentages of measureable cells and shows greater repeatability.

Characterization of red-legged partridge (Alectoris rufa) sperm: Seasonal changes and influence of genetic purity.

The general decline in wild Iberian populations of the red-legged partridge (Alectoris rufa) has been accompanied by an increase in game-farm facilities producing hybrids with chukar partridges (Alectoris chukar). Genetic introgression from chukar partridges is thought to modify male red-legged partridge reproductive indicators. The aim of the present study was to determine the effects of such genetic introgression on seasonal reproductive patterns by comparing the sperm and plasma testosterone concentrations of males from pure red-legged and hybrid red-legged/chukar populations. Semen was collected twice monthly over a 12-mo period using a massage technique. Both types of bird showed a clear seasonal pattern of spermatogenic activity. The proportion of males ejaculating sperm was higher (P<0.05) among the pure red-legged birds. The greatest sperm production was recorded in March to May among the pure birds and April to May among the hybrids. Reproductive activity in both groups decreased in June, to reach a minimum in August to December among the hybrids and in September to December among the pure birds. Spermatogenic activity resumed in January in both groups. The sperm concentration produced by the pure birds was smaller than that of the hybrids (P<0.001), but the percentage of motile sperm was higher in the pure birds (P<0.001). The sperm of the hybrids showed greater straight-line velocity (P<0.05), linearity (P<0.001), straightness (P<0.001), sperm wobble (P<0.05), and beat-cross frequency values (P<0.001). The length and area of the sperm head were smaller in the pure birds (P<0.05). The seasonal plasma testosterone concentration pattern followed a trend roughly parallel to the ejaculatory response. The present results suggest that genetic introgression influences the reproductive variables of the red-legged partridge.

Role of sperm velocity variables associated with poultry breed in 'last male precedence'.

It is well known that when a hen mates with multiple roosters, it is the sperm of the last male that usually fertilizes most of the eggs ('last male precedence'). Sperm quality varies between males within a breed, but also between breeds, and thus, sperm competitiveness after mating may depend on the breeds of the roosters involved. The aim of the present work was to identify differences in sperm competitiveness between breeds, especially with respect to motility. A multibreed mating model was used. Blue Andaluza (BA) and Black Castellana (BC) hens left for 21 days with BA and BC roosters, respectively, were then left with Black-barred Andaluza (Bb) roosters for another 21 days (experimental groups hBA-rBC-rBb and hBC-rBA-rBb). Bb roosters (as the second breed replacing the first) fertilized the majority of eggs in both the hBC-rBA-rBb and hBA-rBC-rBb groups. The percentage of offspring sired by BA roosters (8.0%) was higher (p < 0.05) than the percentage of chicks sired by BC roosters (2.1%). The fertility of the BC hens in the hBC-rBA-rBb group was higher (p < 0.01) than that of the BA hens in the hBA-rBC-rBb group. No difference in sperm concentration was seen between the breeds. Within the rapid sperm subpopulation (sperm velocity, >50 µm/s), Bb sperm showed a higher straight-line velocity (VSL) and average path velocity (VAP) (p < 0.05) than BC sperm. The VSL and VAP values for Bb and BA sperm were similar. In conclusion, the present results show that the sperm of the BA breed, traditionally regarded as of moderate fertility, compensates for this drawback via sperm movement characteristics that afford it an advantage in competition scenarios involving males of other breeds. The VSL and VAP of the rapid sperm subpopulation may play the most important role in securing last male precedence.

Review: Sperm cryopreservation in wild small ruminants: morphometric, endocrine and molecular basis of cryoresistance.

Reproductive technologies can help to protect wild ruminant species from becoming extinct. In addition, the decline in some wild game species has also raised interest in reproductive technologies to increase the number of animals that can be produced. Most biobanking efforts have focused on developing effective protocols for preserving sperm, oocytes, and embryos. Cryopreservation of sperm remains the least invasive method and the cheapest procedure for germplasm storage. Over the last few years, several reproductive biotechnologies have been developed beyond the conventional freezing of spermatozoa. These include ultra-rapid freezing techniques. Nevertheless, fertility results after artificial insemination using frozen-thawed spermatozoa are not always acceptable in wild small ruminants. Moreover, these technological efforts have met variable success related to the sample's origin (epididymal retrieved postmortem or ejaculated) and the season of sperm sample collection and storage. Epididymal sperm shows higher cryoresistance than ejaculated sperm. Changes in sperm proteome between epididymal and ejaculated sperm seem to contribute to this different cryotolerance. The role of endocrine status has been studied in some wild species to better understand the underlying mechanism of the annual variation in ruminant sperm cryoresistance. Seasonal changes in testosterone and prolactin are involved in sperm cryoresistance; sperm recovery and cryopreservation are recommended around the end of the rutting season, when good quality sperm samples can still be obtained, testosterone levels have already decreased, and prolactin concentrations remain low. The mechanisms of hormone action on sperm freezability are not well known. Still, it has been suggested that testosterone affects cell proliferation in the testis, during spermatogenesis, and membrane properties of sperm cells during their transit through the reproductive tract, which might influence their cryotolerance. Recent studies have revealed that the expression of aquaporins in the sperm cells of small wild ruminants could also be involved in the androgen-related seasonal variation of sperm cryoresistance. Along with epididymal and ejaculated spermatozoa, the cryopreservation of testicular tissue may provide a suitable source of male gametes, becoming an alternative for establishing germplasm banks when semen cannot be collected for whatever reason.

Cryopreservation of testicular tissue from the dog (Canis familiaris) and wild boar (Sus scrofa) by slow freezing and vitrification: Differences in cryoresistance according to cell type.

Sperm cryopreservation is the most common procedure used to establish germplasm banks for endangered species - but sometimes sperm cells cannot be obtained. In such cases, freezing testicular tissue may be the only option. The testes contains germ cells at different stages of differentiation, including spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa, among which differences in cryoresistance might be expected. The present work compares the viability and DNA integrity of 'rounded' cells, and of elongated spermatids and spermatozoa, from the dog and wild boar, following the cryopreservation of testicular tissue by slow freezing or vitrification. Cell viability was analyzed by PI/SYBR14 staining, and DNA integrity via the TUNEL technique. For wild boar, no significant differences were seen between the two methods with respect to the percentage of viable cells, nor in the percentage of cells with DNA damage. In the dog, the percentage of viable rounded germ cells (65.0 ± 2.4%) was higher (P < 0.05) after vitrification than after slow freezing (45.1 ± 6.7%). No difference was found between the two methods in terms of the viability of elongated cells. For rounded cells, the percentage of intact DNA was greater (P < 0.05) after vitrification (90.5 ± 2.1%) than after slow freezing (42.6 ± 11.0%), while for elongated spermatids and spermatozoa it was higher (P < 0.05) after slow freezing (66.9 ± 6.1%) than after vitrification (50.7 ± 4.5%). Thus, the response to cryopreservation is cell type-, cryopreservation type-, and species-dependent. Vitrification would appear to be the most appropriate method for preserving dog testicular tissue given the associated high cell viability and low degree of DNA fragmentation, while in wild boar, either method might be used.

The acidic probe LysoSensor is not useful for acrosome evaluation of cryopreserved ram spermatozoa.

To try new acrosomal probes for assessing ram spermatozoa, we compared the LysoSensor probe, which labels acidic organelles, with the frequently used peanut agglutinin acrosomal probe (PNA-PE; phycoerythrin as fluorescent moiety). The previous microscopic observations showed a lack of relationship of LysoSensor with acrosomal status. Semen was obtained from five rams and frozen in four pools. Each pool was analysed carrying out a triple staining propidium ioide/PNA-PE/LysoSensor Green DND-189 to test acrosome labelling, and a double staining SYBR-14/PI, to assess sperm viability. Stained samples were analysed by flow cytometry. All measurements were replicated. Data were processed using agreement and repeatability tests. LysoSensor labelling did not agree with PNA (mean of differences: 30.8%; coefficient of agreement: 22.6%), confirming microscopic observations. Nevertheless, when LysoSensor was compared with SYBR-14/PI, the agreement was high (mean of differences: -0.05%; coefficient of agreement: 5.07%). Repeatability of both methods was high and similar. LysoSensor did not seem to specifically stain the acrosome, but it may accumulate in the cytoplasm and label viable spermatozoa. Therefore, LysoSensor might not be used as an acrosomal probe in ram spermatozoa, but it could be used in other kind of studies, taking advantage of its pH sensitivity.

Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity.

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 degrees C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 microm Fe(2+)/1 mm ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 +/- 2.9%) and OXI (11.6 +/- 7.6%) (p < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI (p < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 +/- 0.8% OXI vs. 17.4 +/- 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa.

Heterologous in vitro fertilization is a good procedure to assess the fertility of thawed ram spermatozoa.

A heterologous in vitro fertilization (IVF) test using calf oocytes with zona pellucida was employed to assess the fertility of thawed ram sperm samples. Six males with significant differences in fertility (P=0.003) were used. The males were classified as having high fertility (>or=42%) and low fertility (

Refrigerated storage of red deer epididymal spermatozoa in the epididymis, diluted and with vitamin C supplementation.

We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti-oxidant. Twenty-nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 x 10(6) sperm/ml in Tris-citrate-fructose with 20% egg yolk. Control group was stored as such, and Anti-oxidant group was supplemented with 0.8 mm vitamin C. The remaining epididymides and the diluted samples were stored at 5 degrees C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility [computer-assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.

Identification of sperm-head morphometric subpopulations in iberian red deer epididymal sperm samples.

Computer-assisted sperm morphometry analysis (CASMA) was used in this study to identify sperm morphometric subpopulations in Iberian red deer epididymal sperm samples. Epididymal sperm samples were collected from 37 mature stags and were divided. One portion was diluted in a Tris-citrate-egg yolk medium. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours using a conventional protocol. After thawing, sperm smears were prepared as described for extended samples. All slides were air-dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 145 sperm-heads were analyzed from each sample by means of the Sperm-Class Analyser, and the mean measurements recorded. Each sperm-head was measured for four primary sperm-head parameters, and five parameters of head shape. All sperm morphometric parameters evaluated were placed in a statistical database and a multivariate cluster analysis was performed. The clustering analyses, based on 10 867 individual spermatozoa, revealed the existence of three subpopulations (SP(1), SP(2), SP(3)) of spermatozoa with different morphometric characteristics (p < 0.001). The proportion of spermatozoa present in any of the three subpopulations remained constant (p > 0.05) through the cryopreservation process. Pre-freeze and post-thaw sperm quality was in vitro evaluated by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. In conclusion, our results show that applying the CASMA techniques and multivariate cluster analyses, it was possible to determine that three subtle subpopulations of spermatozoa with different morphometric characteristics coexist in red deer semen.

DNA status on thawed semen from fighting bull: a comparison between the SCD and the SCSA tests.

The assessment of sperm chromatin status is compulsory in a complete spermiogram. Here we applied the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test to assess the chromatin status of three fighting bulls. Cryopreserved semen (two straws/bull) were analysed by duplicate after thawing and after 6 h at 37 degrees C with and without oxidative stress (1 mm FE(2+)). Results (SCD: percentage of spermatozoa with halo; SCSA: SD-DFI, %DFI and HDS) were analysed for differences between bulls and treatments, sensitivity and specificity (receiver operating characteristic curves) and repeatability (repeatability coefficients as 2SD of duplicate differences).%DFI for the three bulls was below 2% at 0 h, indicating no risk for fertility according to previous reports. It increased slightly for two of the bulls after FE(2+) treatment (%DFI < 5%) and more pronouncedly for the other bull (C, %DFI approximately 10%), which merits further investigation. SCD rendered higher percentage of halos for bull C, but could not discriminate between samples with and without oxidizing treatment (AUC: 0.52). SCSA (%DFI) showed a high discriminating ability between treatments (AUC: 0.96). The repeatability coefficient was also higher for SCD (5.9) than for %DFI (1.8), indicating lower repeatability for SCD. Overall, %DFI might be the most useful parameter for assessing sperm chromatin on fighting bull. SCD might yield different information than SCSA, hence further research is warranted.

Semen cryopreservation in black-footed (Spheniscus demersus) and gentoo (Pygoscelis papua) penguins: Effects of thawing temperature on semen characteristics.

In this study, there was an examination of the effect on the characteristics of cryopreserved black-footed (Spheniscus demersus) and gentoo (Pygoscelis papua) penguin semen, of thawing at 37 and 5 °C. For two consecutive years, semen was collected and frozen during the April-June period from six gentoo penguins, and during the October-November period from 13 black-footed penguins. After thawing, sperm motility variables were examined by computer-assisted sperm analysis. Propidium iodide and SYBR-14 were used as fluorochromes for the examination of membrane integrity. For the gentoo penguins, no differences were detected in the values of frozen-thawed semen characteristics after thawing at 37 or 5 °C. For the black-footed penguins, however, thawing at 5 °C resulted in greater values (P < 0.05) for straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), and wobble (WOB) as compared with thawing at 37 °C. After thawing at 37 ºC, there were greater values with gentoo penguin sperm for percentage motile sperm, progressive motility, curvilinear velocity (VCL), VSL VAP, LIN, STR, WOB and beat-cross frequency (BCF; P < 0.05) than that for black-footed penguin sperm. After thawing at 5 ºC, there were no differences in values for any variables between the two species. In conclusion, thawing temperature affects semen characteristics in a species-specific manner. The present data strongly suggest that cryopreservation procedures should be adapted for use with each penguin species. Cryopreserved black-footed penguin semen should be thawed after cryopreservation at 5 ºC, while that of gentoo penguins can be thawed at either 5 or 37 ºC.

Effectiveness of ultra-rapid cryopreservation of sperm from endangered species, examined by morphometric means.

This study compares the effectiveness of the ultra-rapid and conventional freezing of sperm from captive bovids, giraffids, cervids, ursids, a cercopithecid, a delphinid and a phascolarctid. The relationship between sperm head dimensions and cryosurvival was also examined. Compared to conventional freezing, the ultra-rapid freezing of epididymal sperm from the dama gazelle, giraffe and brown bear returned higher cryoresistance ratios (CR, the ratio, in percentage, between the value of the variable after thawing/value before thawing) for sperm viability and motility. In the remaining species, the conventional freezing of epididymal sperm returned better CR values. The conventional freezing method also returned better CR values for ejaculated samples from all species. The head dimensions of both fresh epididymal and ejaculated sperm differed widely among species: for epididymal sperm, dolphin sperm heads were the smallest (7.189 ±â€¯0.049 µm2) and dama gazelle sperm heads the largest (43.746 ±â€¯0.291 µm2), while for ejaculated sperm, giant panda sperm heads were the smallest (15.926 ±â€¯0.150 µm2) and mouflon sperm heads the largest (38.258 ±â€¯0.104 µm2). However, no significant correlations were detected between the CR for motility, viability, membrane functional integrity or acrosome integrity and the sperm head area, either for epididymal or ejaculated sperm. In conclusion, ultra-rapid freezing is especially recommended for the cryopreservation of dama gazelle, giraffe and brown bear epididymal sperm. Sperm head dimensions appear not to be useful predictors of how well sperm might survive freezing.

Mitochondrial activity and forward scatter vary in necrotic, apoptotic and membrane-intact spermatozoan subpopulations.

In the present study, we have related mitochondrial membrane potential (DeltaPsim) and forward scatter (FSC) to apoptotic-related changes in spermatozoa. Thawed red deer spermatozoa were incubated in synthetic oviductal fluid medium (37 degrees C, 5% CO2), with or without antioxidant (100 microm Trolox). At 0, 3, 6 and 9 h, aliquots were assessed for motility and were stained with a combination of Hoechst 33342, propidium ioide (PI), YO-PRO-1 and Mitotracker Deep Red for flow cytometry. The proportion of spermatozoa YO-PRO-1+ and PI+ (indicating a damaged plasmalemma; DEAD) increased, whereas that of YO-PRO-1- and PI- (INTACT) spermatozoa decreased. The proportion of YO-PRO-1+ and PI- spermatozoa (altered plasmalemma; APOPTOTIC) did not change. Both DEAD and APOPTOTIC spermatozoa had low DeltaPsim. Most high-DeltaPsim spermatozoa were INTACT, and their proportion decreased with time. The FSC signal also differed between different groups of spermatozoa, in the order APOPTOTIC > DEAD > INTACT/low DeltaPsim > INTACT/high DeltaPsim; however, the actual meaning of this difference is not clear. APOPTOTIC spermatozoa seemed motile at 0 h, but lost motility with time. Trolox only slightly improved the percentage of INTACT spermatozoa (P < 0.05). The population of APOPTOTIC spermatozoa in the present study may be dying cells, possibly with activated cell death pathways (loss of DeltaPsim). We propose that the sequence of spermatozoon death here would be: (1) loss of DeltaPsim; (2) membrane changes (YO-PRO-1+ and PI-); and (3) membrane damage (PI+). INTACT spermatozoa with low DeltaPsim or altered FSC may be compromised cells. The present study is the first that directly relates membrane integrity, apoptotic markers and mitochondrial status in spermatozoa. The results of the present study may help us understand the mechanisms leading to loss of spermatozoon viability after thawing.