Feline Sperm Research Articles

In search of freezability predictors for feline spermatozoa – osmotic challenge tests and markers of sperm membrane structure

Semen cryopreservation is a crucial part of assisted reproductive techniques (ART) in animals, and recently it is gaining more and more attention among cat breeders. Even if fresh semen quality is good, sometimes spermatozoa do not survive freezing. The freezability prediction was widely studied in many species, but not in the domestic cat. The aim of this study was to verify the usefulness of osmotic challenge tests and membrane structure markers (Yo-Pro 1 and Merocyanine 540) for the prediction of the quality of post-thawed feline semen. Semen was collected by urethral catheterization from 22 male cats. After a basic evaluation of semen, 20×106 spermatozoa were cryopreserved; the rest were evaluated by flow cytometry for membrane integrity (SYBR-14/PI), acrosome status (lectin PNA/PI), mitochondrial potential (JC-1) and membrane stability (Yo-Pro 1/M540 staining). Hypo- and hyperosmotic challenge tests were also performed. The thawed samples were evaluated as fresh ones. The Pearson correlation between all parameters in fresh semen and all parameters in cryopreserved spermatozoa was assessed. Although some moderate correlations were found between the results of the osmotic tests and markers of sperm membrane stability (Yo-Pro 1 and Merocyanine 540) and post-thaw semen quality parameters, the predictive value of studied markers was rather weak – no cut-off values could be established and, based on regression models, they explained less than 40 % of variability in post-thaw quality. Our results confirm that cryodamage is a complex matter, in which many different factors play a role, affecting sperm motility and membrane integrity differently.

Fatty acid composition and biophysical characteristics of the cell membrane of feline spermatozoa

Sperm membrane composition and biophysical characteristics play a pivotal role in many physiological processes (i.e. sperm motility, capacitation, acrosome reaction and fusion with the oocyte) as well as in semen processing (e.g. cryopreservation). The aim of this study was to characterize the fatty acid content and biophysical characteristics (anisotropy, generalized polarization) of the cell membrane of domestic cat spermatozoa. Semen was collected from 34 adult male cats by urethral catheterization. After a basic semen evaluation, the fatty acid content of some of the samples (n = 11) was evaluated by gas chromatography. Samples from other individuals (n = 23) were subjected to biophysical analysis: membrane anisotropy (which is inversely proportional to membrane fluidity) and generalized polarization (describing lipid order); both measured by fluorimetry at three temperature points: 38 °C, 25 °C and 5 °C. Spermatozoa from some samples (n = 10) were cryopreserved in TRIS egg yolk-glycerol extender and underwent the same biophysical analysis after thawing. Most fatty acids in feline spermatozoa were saturated (69.76 ± 24.45%), whereas the polyunsaturated fatty acid (PUFA) content was relatively low (6.12 ± 5.80%). Lowering the temperature caused a significant decrease in membrane fluidity and an increase in generalized polarization in fresh spermatozoa, and these effects were even more pronounced following cryopreservation. Anisotropy at 38 °C in fresh samples showed strong positive correlations with viability and motility parameters after thawing. In summary, feline spermatozoa are characterized by a very low PUFA content and a low ratio of unsaturated:saturated fatty acids, which may contribute to low oxidative stress. Cryopreservation alters the structure of the sperm membrane, increasing the fluidity of the hydrophobic portion of the bilayer and the lipid order in the hydrophilic portion. Because lower membrane fluidity in fresh semen was linked with better viability and motility after cryopreservation, this parameter may be considered an important factor in determination of sperm cryoresistance.

Evaluation of a commercial proAKAP4 kit for the assessment of fresh and frozen-thawed feline spermatozoa.

ProAKAP4 is gaining increasing attention as a potential marker of semen quality in many species, but while there is a commercial kit for assessing proAKAP4 in the domestic cat, there are no publications about its use. The aim of this study was to evaluate the commercial proAKAP4 kit - Cat 4MID® Kit (SPQI - 4BioDx, Lille, France) for the assessment of feline semen. Semen was collected from 54 male cats by urethral catheterization. After a basic semen evaluation (subjective motility, CASA, viability, morphology), proAKAP4 levels in each sample were assessed using the Cat 4MID® Kit according to the manufacturer's protocol or with some modifications related to incubation time, sample storage conditions and number of spermatozoa used. Finally, the Spearman correlation of proAKAP4 concentration and sperm motility parameters was calculated. The most reliable results (acceptable intraassay coefficient of variation) were obtained with an optimized protocol of overnight incubation and isolation of proAKAP4 protein from 1 × 106 spermatozoa stored at -80°C. For fresh semen, there were no significant correlations between proAKAP4 concentration and sperm motility parameters, despite a strong correlation between motility parameters and sperm viability and morphology. A predominant effect of other sperm parameters and highly variable performance of lysis buffer question the usefulness of Cat 4MID® Kit for the assessment of feline semen. For frozen-thawed semen, there was a moderate, negative correlation between proAKAP4 concentration and two CASA parameters, VAP and VSL. As there were no correlations between proAKAP4 concentration in fresh semen and motility parameters in cryopreserved samples, proAKAP4 cannot be used as freezability marker in cats. More studies are needed to establish potential correlation with long-lasting motility.

Cushioned and high-speed centrifugation improve sperm recovery rate but affect the quality of fresh and cryopreserved feline spermatozoa

The development of endoscopic transcervical catheterization (ETC) in the queen increases the interest in handling fresh and cryopreserved feline semen. The ETC requires a small volume of the insemination dose with a high concentration, not easily reached with the actual frozen technique in this species. Centrifugation is widely used to concentrate spermatozoa for several purposes, but this process is detrimental to spermatozoa. This study verified the effects of conventional and cushioned centrifugation on fresh and cryopreserved feline spermatozoa. To this, semen was collected from 20 toms, grouped in seven pools and diluted. After dilution, the pools were divided into two aliquots, the first used for centrifugation on fresh semen, and the second, after freezing, on cryopreserved semen. Centrifugation regimens were: conventional at 500×g, conventional at 1000×g, and cushioned (iodixanol) at 1000×g. The sperm recovery rate was calculated for the three centrifugation regimens, and sperm kinematics, membrane and acrosome integrity, and plasma membrane stability on viable spermatozoa were assessed as endpoints.The data reported in this study showed that the centrifugation at 500×g resulted in negligible effects on both fresh and cryopreserved spermatozoa, but the lower recovery rate (62.4 ± 3.1 % and 60.2 ± 1.6 %, respectively) underlines the loss of a large proportion of spermatozoa, unfavourable in a species with small total sperm ejaculated. On the other hand, the centrifugation at 1000×g improved the recovery rate (86.9 ± 4.3 % and 89.8 ± 2.4 % in fresh and cryopreserved samples, respectively), but was more deleterious for feline spermatozoa, especially in cryopreserved samples (i.e. total motility of 40.7 ± 5.4 % compared with 57.2 ± 9.8 % in cryopreserved uncentrifuged samples, P < 0.05), resulting in artificial insemination doses of lower quality. The recovery rate in cushioned centrifugation appeared less efficient, likely due to the small volume of feline samples, which makes difficult the separation of sperm pellet and cushioned fluid. Interestingly, in cryopreserved samples centrifuged at 1000×g the number of viable spermatozoa with membrane destabilization (31.3 ± 3.2 %) was greater than uncentrifuged (4.1 ± 0.7 %, P < 0.05) and those centrifuged at 500×g (9.8 ± 1.3 %, P < 0.05), suggesting modifications induced by the cryopreservation amplifies centrifugation sublethal damage on feline spermatozoa. Cushioned centrifugation on cryopreserved samples showed kinematics similar to uncentrifuged samples, but higher viable spermatozoa with membrane destabilization (37.4 ± 3.4 % vs 4.1 ± 0.7 %; P < 0.05). In felines, g-force is crucial for sperm recovery rate during centrifugation, with better results at 1000×g; on the other hand, greater g-forces could have a significant impact on the quality of feline insemination dose, especially in cryopreserved samples.

Feline sperm head morphometry in relation to male pedigree and fertility

Computer-assisted sperm morphometry analysis is an advanced tool which allows to precise measure sperm head parameters like length, width, area, and perimeter. On the basis of these and calculated parameters, morphometric subpopulations of spermatozoa can be distinguished. In many species, the distribution of subpopulation within the ejaculate is related to male fertility. There is no information about such a relation for domestic cats; therefore, the aim of this study was to evaluate whether spermatozoa from non-pedigree and purebred domestic cats differ in morphometric parameters. The second aim was to check if there is a relationship between sperm morphometry and fertility. Urethral semen was collected from 27 tomcats, divided into three study groups: non-pedigree cats of unknown fertility, purebred infertile cats and purebred fertile cats. The morphometric assessment was performed by CASMA, followed by principal component analysis and clustering. The results revealed huge intra- and inter-individual variation in sperm head morphometric parameters and three sperm-head morphometric subpopulations were identified in feline semen. Neither mean values of morphometric parameters nor the distribution of spermatozoa between morphometric subpopulations differ between non-pedigree cats of unknown fertility and purebred infertile and fertile cats. We hypothesize that other factors, especially abnormalities of the midpiece and tail, and overall worse quality of the semen of infertile males could have masked the effect of subtle changes in the sperm head morphometry.

Application of the FISH Technique to Visualize Sex Chromosomes in Domestic Cat Spermatozoa.

Simple SummaryThe study was conducted with the aim of visualizing sex chromosomes in domestic cat sperm. Our research was carried out using fluorescence in situ hybridization (FISH) technique and the microscopic analysis showed the presence of X and Y chromosomes in domestic cat spermatozoa. The procedure used for sperm decondensation and FISH is adequate to visualize chromosomes in domestic cat sperm.Fluorescence in situ hybridization is a molecular cytogenetics technique that enables the visualization of chromosomes in cells via fluorescently labeled molecular probes specific to selected chromosomes. Despite difficulties in carrying out the FISH technique on sperm, related to the need for proper nuclear chromatin decondensation, this technique has already been used to visualize chromosomes in human, mouse, cattle, swine, horse, and dog spermatozoa. Until now, FISH has not been performed on domestic cat sperm; therefore, the aim of this study was to visualize sex chromosomes in domestic cat sperm. The results showed the presence of X and Y chromosomes in feline spermatozoa. The procedure used for sperm decondensation and fluorescence in situ hybridization was adequate to visualize chromosomes in domestic cat spermatozoa and, in the future, it may be used to determine the degree of chromosomal abnormalities in these gametes.

Epididymal tail solid-surface vitrification as an effective method for domestic cat sperm cryobanking.

This study aimed to describe the viability of domestic feline spermatozoa after epididymal tail vitrification. For this, 10 pairs of testis-epididymis complexes were used. The epididymal tails were vitrified using the solid-surface vitrification (SSV) method, in which two vitrification media containing ethylene glycol (EG) 40% or glycerol (GLY) 40% were tested. Vitrification with the presence of EG resulted in better results for all sperm motility parameters (motility, vigour and CASA) compared with GLY (P < 0.05). There were no statistical differences for sperm viability and acrosome integrity, plasma membrane integrity, or overall health of morphologically normal sperm before or after vitrification among experimental groups. In conclusion, epididymal tail vitrification appears to be a suitable method for long-term storage of cat sperm, especially if the procedure is performed with EG as the cryoprotectant.

Can amides be alternative cryoprotectors for the preservation of feline semen?

Sperm cryopreservation is a tool for the conservation of the genetic material of animals of genetic importance or for species preservation. In the case of domestic cats, this can be used to generate information about seminal harvest, evaluation and preservation, which is especially important due to its applicability to wild felids. This study evaluated seminal samples harvested by urethral catheterisation from 13 adult domestic cats. Samples were cryopreserved with experimental groups of extenders were defined by the penetrating cryoprotectant: 6% glycerol (GLY6%), 3% dimethylacetamide (DMA3%) and 3% dimethylformamide (DMF3%). The samples were thawed and evaluated by conventional microscopy and by computer-assisted sperm analysis (CASA). The structural and functional membrane integrity was assessed by supravital tests (EOS), hypoosmotic swelling tests (HOST) and flow cytometry (FC). There was a correlation (P < 0.05) between total motility and EOS (r = 0.54), HOST and FC (r = −0.62) and total motility and flow cytometry (r = 0.63), indicating that these are complementary parameters that increase the accuracy of the feline sperm quality evaluation post-thaw. The results regarding the structural and functional integrity of the sperm plasma membrane did not differ (P > 0.05) among groups. However, the DMA3% group had a lower (P < 0.05) percentage of morphological changes in the sperm tail compared to samples cryopreserved with GLY6% and DMF3%. Additionally, DMA3% provided lower values of immobile sperm post-thaw when compared to DMF3%. DMA is an interesting alternative to GLY and superior to DMF for the cryopreservation of feline semen at the studied concentrations.

Effect of refrigeration at -1°C on spermatozoa quality of domestic cats

ABSTRACT: The objective of this study was to evaluate the sperm quality obtained of domestic cats by electroejaculation and recovery of the tail of the epididymis after cooling at -1°C and 4°C for 24 and 48 hours. Twenty-nine adult cats (2 to 6kg) were used. Sperm collection was performed by electroejaculation (EEJ), and after 48 hours, the cats were orchiectomized, and sperm sample was obtained from the vas deferens and epididymis tail (EPD). The samples were diluted in ACP-117® extender, and the sperm characteristics were evaluated at three different moments: when still fresh, 24 and 48 hours after cooling. In order to compare the two refrigeration temperatures, the first stage was to analyze if there was a difference between the harvesting techniques. After this, two experiments were conducted: in the first, sperm sample from 14 cats were used and the cooling was performed at -1°C; and in the second, sample from 15 cats were used and the sperm were refrigerated at 4°C. Sperm kinetics were evaluated by computerized analysis (CASA) and concentration by Neubauer chamber, spermatic morphology was evaluated by modified Karras staining, and membrane integrity was evaluated by eosin nigrosine. The results obtained were analyzed in R software, version 3.2.5 using the Mann-Whitney test for variables with abnormal distributions, considering significance at the level of 5%. In ejaculate samples, higher values of total morphological defects were observed after 24 and 48 hours of refrigeration at 4°C (P&lt;0.022) compared to refrigeration at -1°C, using Friedman test. To quantify the decrease in sperm quality, parameter reductions were calculated among time points (F-24h/F-48h/24h-48h). In EPD samples, a greater reduction in sperm quality was detected after 24 hours of refrigeration at 4°C, both in motility and sperm kinetics and in the movement and velocity indices, compared to refrigeration at -1°C. Based on the results, it can be concluded that cooling of feline spermatozoa at -1°C for up to 48 hours was efficient in maintaining spermatic quality collected by EEJ and EPD, and it could be an alternative to spermatozoa cryopreservation in domestic felines.

Effect of supplementation of Aloe vera extracts in cold storage media and cryopreservation of domestic cat epididymal spermatozoa.

This study evaluated the effect of the extract of Aloe vera at concentrations of 10% and 20% on the cryopreservation of sperm from the epididymis of domestic cats. Epididymal spermatozoa were recovered using the flotation technique and used in the treatments: control (TRIS-egg yolk at 20%), T10% (TRIS plus 10% of A. vera extract), and T20% (TRIS plus 20% of A. vera extract). The spermatozoa were subjected to 4ºC for 60 minutes, followed by 20 minutes in nitrogen vapors, and stored in a cryogenic cylinder. The samples were thawed at 37°C for 30 seconds. The sperm motility decreased (P<0.05) after thawing in the three treatments. Only the spermatozoa in the control treatment maintained post-thawing vigor. The viability of spermatozoa decreased in the treatments with A. vera (P<0.05). According to the hypoosmotic test, all treatments maintained the sperm membrane functionality (P>0.05) during freezing; however, after thawing, it decreased (P<0.05) in the T10% and T20% treatments. The morphology and chromatin condensation of spermatozoa did not differ, regardless of the treatments and time of evaluation (P>0.05). The effect of the crude A. vera extract was not satisfactory on the cryopreservation of epididymal spermatozoa of domestic cats after thawing; although the motility of spermatozoa was similar to that found with the use of egg yolk, and it presented maintenance of the chromatin integrity. However, it is necessary to understand the action of the substances present in A. vera with the feline spermatozoa, well as the standardization and adjustment of physicochemical characteristics aiming at the future application of the vegetal extract.

Assisted reproduction for felid species conservation-Sperm competences at risk.

Cryobanking of gametes in combination with artificial insemination is an essential option to support conservation programmes for endangered and threatened species. About two-thirds of the felid species are classified as 'near threatened', 'vulnerable' or 'endangered' (www.cites.org), and mostly, epididymal sperm are collected from euthanized or castrated male felids and cryopreserved. However, epididymal compared with ejaculated and cryopreserved compared with fresh sperm have a limited potential to fertilize if vaginal non-surgical insemination is applied in feline species. Missing or highly diluted seminal fluid in epididymal and cryopreserved sperm, as well as a potential interference of extender ingredients with the natural interactive properties of sperm in the female genital tract is discussed as potential drawback which hampers a proper sperm transit and fertilization besides the limited longevity of cryopreserved feline sperm. Individual components in seminal fluid as well as cryoextenders may adversely alter sperm properties and have a different impact on fertility and preservation success. The identification and investigation of beneficial as well as detrimental components is a precondition to deduce options for improving the process of cryopreservation in felids, particularly, if only epididymal sperm are available.

Optimal concentration of Rho-associated coiled-coil kinase (ROCK) inhibitor improved sperm membrane functionality and fertilizing ability of cryopreserved-thawed feline sperm

Sperm cryopreservation induces irreversible loss of viability and fertilizing ability. This study aimed at examining the effects of Rho-associated, coiled-coil kinase (ROCK) inhibitor on quality of frozen-thawed feline sperm. Ejaculated semen from individual cats (n = 6) was examined for the expression of LIMK1 and LIMK2 mediated ROCK cascade. The effects of ROCK inhibitor during cooling and cryopreservation on sperm quality and fertilizing ability were also examined. Feline sperm were treated with different concentrations of ROCK inhibitor (10, 20 and 40 μM) during cooling at 4 °C and cryopreservation. Sperm cooled and conventionally cryopreserved without ROCK inhibitor (0 μM) served as a control group.The ROCK cascade was confirmed in feline sperm as they expressed mRNA of LIMK1 and LIMK2 genes. Cryopreservation significantly reduced sperm quality in terms of viability (91.63 ± 3.96 vs. 60.11 ± 8.93), progressive motility (91.67 ± 3.54 vs. 46.67 ± 8.66) and acrosome integrity (93.49 ± 3.64 vs. 63.81 ± 5.31) for fresh and frozen-thawed sperm, respectively (p < 0.05). The positive effects of ROCK inhibitor on sperm quality were pronounced at 1 and 3 h post-thaw. ROCK inhibitor at 10 μM significantly improved sperm motility and membrane functionality compared to those observed in a control group (0 μM) (p < 0.05). In vitro fertilization revealed that supplement ROCK inhibitor at 10 μM during cryopreservation significantly improved in vitro fertilizing ability of the frozen-thawed sperm (p < 0.05). However, it did not subsequently increase morula and blastocyst rates (p > 0.05). Increased concentrations of ROCK inhibitor to 20 and 40 μM did not further improve the quality of frozen-thawed sperm. In conclusion, an optimal concentration (10 μM) of the ROCK inhibitor added into cooling medium could improve post-thaw sperm quality.

Osmotic tolerance of feline epididymal spermatozoa

During the cryopreservation process, spermatozoa are exposed to hypertonic solutions contributed by the high concentration of cryoprotectant. During addition and removal of cryoprotectant the spermatozoa are subjected to a substantial osmotic stress. Spermatozoa of different species and different stages of maturation may have different susceptibility to osmotic stress depending on the biology of the cell membrane and this will affect their tolerance to the freezing-thawing stress. The aims of this study were to determine the osmotic tolerance limits for motility, membrane integrity and mitochondrial membrane potential of feline epididymal spermatozoa and to study the effect of osmotic stress on the feline spermatozoa of different epididymal regions. Epididymal spermatozoa from three regions (caput, corpus and cauda) were pre-exposed to various osmolalities (75, 300, 600, 900, 1200 mOsm) in a single step for 10min and returned to 300 mOsm afterward. Percentage of motile spermatozoa was measured subjectively and membrane integrity (SYBR-14 positive cells) was evaluated prior to and after exposure to different osmolalities. The mitochondrial membrane potential (JC1) of spermatozoa were evaluated using flow cytometer and compared between epididymal regions (caput, corpus and cauda). All the parameters were compared using a mixed procedure. The percentage of motile epididymal spermatozoa decreased significantly when spermatozoa were exposed to 75 mOsm and 600 mOsm. Epididymal spermatozoa showed signs of damage when pre-exposed to 900 and 1200 mOsm and returned to isotonic condition as significant reduction of membrane integrity and mitochondrial membrane potential were observed (P<0.05). The plasma membrane of spermatozoa from the cauda epididymal region showed higher susceptibility to osmotic stress than the other regions as demonstrated by a significant difference between regions after return to isotonicity from 900 mOsm (P>0.01) and a difference between caput and corpus after return from 1200 mOsm (P<0.05). The corpus and cauda epididymal spermatozoa had higher percentage of spermatozoa with high mitochondrial membrane potential than those from caput when exposed to 75, 300 and 600 mOsm (P<0.05). In conclusion, a single step exposure to hypertonic solution of greater than 600 mOsm prior to return to isotonic condition can cause severe damage to sperm membrane and mitochondrial membrane potential compared to non-returning (exposure to various osmolality but not returned to isotonic condition). Changes in osmolality impacted mostly on sperm motility. Spermatozoa from cauda epididymis were more susceptible to osmotic stress compared to those from corpus and caput indicating that the maturation changes in the sperm membrane during passage through the epididymis increase susceptibility to the osmotic changes that may occur during sperm cryopreservation.

In vitro fertilizing potential of urethral and epididymal spermatozoa collected from domestic cats (Felis catus).

The aim of this study was to provide a comparative analysis of in vitro fertilizing potential of frozen-thawed urethral and epididymal feline spermatozoa. Both types of semen were collected from 7 cats and cryopreserved in liquid nitrogen. To perform in vitro fertilization, both urethral and epididymal samples from the same individual were thawed and spermatozoa were co-incubated with in vitro matured cat oocytes. Obtained embryos were cultured in vitro for 7 days in a commercial medium. Cleavage rate, morula rate and blastocyst rate were calculated. Experiment was run in 10 replicates. The examined parameters showed no significant differences between urethral and epididymal spermatozoa (p>0.05). Cleavage rate and embryo's development were highly variable between replicates, even for the different sperm samples collected from one individual. There was no significant correlation between fertilizing capacity of two types of spermatozoa collected from the same male. In this study we confirmed that cryopreserved urethral spermatozoa have equally good fertilizing potential as epididymal ones, and both can be successfully used for in vitro fertilization in cats with the use of commercial medium.

Effects of epididymis cold storage on frozen-thawed epididymal sperm quality in tomcats (Felis catus)

The effect of cold storage of testes and epididymides at 4 °C for 12 h on the cryopreservation capacity of epididymal feline sperm was evaluated. Ten domestic cats were castrated, and testes and epididymides collected. Specimens were randomly assigned to two groups: in Group A, epididymal samples were immediately processed and frozen in 0.25-ml straws; in Group B, both testes and epididymides were maintained in saline at 4 °C for 12 h and sperm was then processed and frozen. Motility, morphology, acrosome status, sperm viability and DNA integrity were assessed in epididymal sperm samples before freezing (baseline), at thawing (0 h) and 6 h post-thawing (6 h). Although values were lower in Group B, no significant intergroup difference was observed for any of the parameters tested either at baseline or at 0 h. However, significantly higher values (P &amp;lt; 0.05) were observed in Group A at 6 h for total sperm motility (29.0 ± 2.4% vs 13.0 ± 4.3%), sperm viability (35.2 ± 5.4% vs 15.4 ± 1.4%) and normal morphology (47.6 ± 0.8% vs 40.0 ± 2.1%). It was observed that motility and acrosome status of epididymal sperm are the most sensitive parameters when both types of sperm samples (from fresh epididymis or from 12 h cold-stored epididymis) are frozen-thawed. When sperm quality was assessed 6 h after thawing, spermatozoa precooled in the epididymides showed significantly lower values for motility, viability and morphology than spermatozoa from fresh epididymal samples.

Retained fertilizing capability in cryopreserved feline spermatozoa.

Sperm cryopreservation offers a long-term preservation of male genetic materials for future assisted reproductive technologies. However, dramatic changes in temperature during freezing and thawing injure sperm cells. While motility is essential for AI and membrane integrity is crucial for in vitro fertilization (IVF), sperm DNA integrity is a common index of fertilizing capability required for AI, IVF and intracytoplasmic sperm injection (ICSI). In endangered felids died unexpectedly, attempts have been made to recover as many DNA intact spermatozoa as possible from epididymis and testis to increase the opportunity to produce offspring in future. Although sperm from caudal epididymis has shown retained fertilizing capability after freezing and thawing (27.3% conception rate after unilateral intrauterine insemination), sperm recovery from the corpus epididymis has been suggested as an alternative to increase the amount of preserved genetic materials. To improve epididymal sperm quality, pre-treatment with single-layer centrifugation resulted in selection of sperm cells with intact DNA while post-thaw treatment with extracellular ATP incubation promoted the blastocyst rate. Cold storage of domestic cat testis for 7days at 4°C demonstrated <1% of sperm cells with fragmented DNA. Moreover, isolated testicular sperm cells, stored for 7days at 4°C, produced after ICSI poorer percentages of cleavage, morula and blastocyst than the fresh control. In wild felids, a death-to-necropsy time of 2hr after a jungle cat (Felis chaus) aged 10years died during anaesthesia plus another necropsy-to-sperm recovery time of 25hr has been reported to yield the post-thawed testicular sperm with 22.2% intact DNA. In summary, the chromatin structure of feline ejaculated and epididymal sperm seems to be tolerated to cold storage and cryopreservation; thus, fertilizing capability is well protected. In contrast, the cat testicular sperm DNA is generally damaged through the cryopreservation.

Choice of staining technique affects the morphological assessment of epididymal feline sperm

Morphological examination of spermatozoa remains a valuable tool for determining the quality of feline sperm for cryopreservation and/or for use in artificial insemination. Numerous staining techniques have been employed for feline sperm assessment, and several variations have also been reported. Forty-eight domestic cats were castrated and epididymal sperm samples were obtained. Sperm morphology was assessed using two staining methods, an air-dried stain (Giemsa-Wright) and a wet stain (eosin-nigrosin), to determine their relative abilities for estimation of the proportion of normal and abnormal spermatozoa. The effects of centrifugation and the cooling process on sperm morphology were also analysed. The mean percentage of morphologically normal spermatozoa was significantly lower in eosin-nigrosin-stained samples than in Giemsa-Wright-stained preparations (46.5% vs 53.4%; P < 0.01), both examined by conventional light microscopy. Results concerning head, midpiece and tail sperm abnormalities suggest that these methods differ in their capacity to identify sperm anomalies in cat. Although no significant differences were observed for secondary abnormalities, cytoplasmic droplets were significantly lower in Giemsa-Wright-stained samples (P < 0.01). Centrifugation or refrigeration of sperm samples did not affect the epididymal sperm morphology in evaluated samples. This study highlights the necessity of minimising morphological assessment variations in order to eliminate subsequent errors and to improve the reproducibility of sperm morphology assessment in different laboratories.

Low levels of apoptotic-like changes in fresh and cryopreserved feline spermatozoa collected from the urethra and epididymis

The aim of this study was to examine apoptotic markers in fresh and frozen-thawed feline spermatozoa collected via urethral catheterization and epididymal slicing. Caspase activation, DNA fragmentation, and phosphatidylserine externalization were evaluated using flow cytometry in sperm cells from both sources before and after cryopreservation. The study revealed no differences between urethral and epididymal spermatozoa, both in fresh and frozen-thawed samples. The level of apoptotic changes in sperm cells in fresh feline semen was low: 0.8 ± 0.8% of live urethral and 0.4 ± 0.4% of live epididymal spermatozoa showed active caspases; 1.6 ± 0.9% and 2.1 ± 1.9%, respectively, showed DNA fragmentation; and 0.3 ± 0.2% and 1.0 ± 1.3%, respectively, showed phosphatidylserine externalization. In both types of sperm cells, cryopreservation did not induce a significant increase in caspase activation (urethral: from 3.9 ± 3.2% to 7.5 ± 5.0%; epididymal: from 4.7 ± 2.9% to 11.7 ± 8.5%). In urethral spermatozoa, phosphatidylserine externalization in live cells was significantly (P < 0.05) increased after thawing (from 0.3 ± 0.2% to 2.7 ± 2.5%). This increase was not noted for epididymal spermatozoa (from 1.0 ± 1.3% to 1.7 ± 1.3%). No significant changes in DNA fragmentation were observed (2.1 ± 0.8% and 1.7 ± 1.0%). In conclusion, both urethral and epididymal feline spermatozoa showed equally low levels of apoptotic-like changes. Hence, apoptotic alterations seem to play only a minor role, if any, in urethral and epididymal feline spermatozoa. The deterioration of sperm quality after freezing and thawing is more likely connected with direct damage to the cells than to activation of apoptotic processes.

The ability of feline spermatozoa in different epididymal regions to undergo capacitation and acrosome reaction

The sperm maturation process that occurs in the epididymis is a necessary process for spermatozoa to acquire motility and the ability to undergo capacitation, which is an important key for fertilization. The aim of this study was to evaluate the ability of feline spermatozoa from different regions of the epididymis to undergo capacitation and acrosome reaction. Experiment I: epididymal spermatozoa from caput, corpus and cauda regions were placed in phosphate buffered saline (control medium) and in vitro fertilization medium (capacitating conditions). Sperm motility, motility patterns, plasma membrane integrity and tyrosine phosphorylation were evaluated at time 0 and 60min after incubation. Experiment II: spermatozoa were treated with 2μM of calcium ionophore (A23187) to induce the acrosome reaction and acrosome reaction was evaluated. The results showed a significant effect of region with a higher percentage of tyrosine phosphorylation in spermatozoa from the cauda than in the caput or corpus regions (P=0.0061; P=0.0088). Spermatozoa from corpus and cauda showed higher values in the majority of the measured motility parameters than spermatozoa from the caput (P<0.0001). Spermatozoa from all epididymal regions can undergo the acrosome reaction in vitro in response to induction by calcium ionophore with no difference between regions (P>0.05). Spermatozoa from all epididymal regions were able to undergo capacitation. Higher percentage of tyrosine phosphorylation in spermatozoa from the cauda reflect that they more easily underwent capacitation compared to spermatozoa from caput and corpus which required more time of incubation for capacitation. In conclusion feline epididymal spermatozoa from all regions can undergo capacitation and acrosome reaction in vitro and do not require incubation under capacitating conditions.

DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa

Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies.