Cat Epididymal Spermatozoa Research Articles

Mito-Tempo improves acrosome integrity of frozen-thawed epididymal spermatozoa in tomcats.

In tomcats, epididymal spermatozoa provide an additional source of male gametes available for cryopreservation. While this procedure is feasible, the survival rate and motility of epididymal cat spermatozoa are both low after thawing. Cryopreservation is known to induce oxidative stress in spermatozoa, with mitochondria and the plasma membrane being the two major generation sites, and an imbalanced presence of free radicals is a possible cause for this low survival rate. Different antioxidants have been tested before for their effect on cryopreserved cat spermatozoa quality, with varying results. Here, we used Mito-Tempo, which is a synthetic mitochondria-targeted antioxidant and a specific scavenger of the mitochondrial superoxide system. By supplementing Mito-Tempo with the freezing extender, we aimed to improve the sperm quality of frozen-thawed cat epididymal spermatozoa. Epididymal spermatozoa obtained from twelve tomcats were assessed for motility and concentration. Prior to freezing, samples were diluted in TRIS buffered extender with egg yolk and glycerol and divided into five aliquots supplemented with 0 (control), 0.5, 5, 50, and 1005M of Mito-Tempo. After thawing, sperm motility, concentration, morphology, plasma membrane integrity, acrosome integrity, and mitochondrial membrane potential were evaluated. A Friedman rank sum test with a Bonferroni post-hoc test was used to determine statistical in-between group differences in post-thaw semen parameters. The results indicated a slight improvement in acrosome integrity across all groups that were supplemented with Mito-Tempo, with the group that received 55M of Mito-Tempo showing the greatest improvement [(median of 67.99%, IQR of 5.55) compared to the control group (median of 65.33%, IQR of 7.75; P = 0.05)]. For all other sperm parameters, no significant differences (P > 0.05) were detected between different Mito-Tempo concentrations. These findings highlight the protective effect of Mito-Tempo on acrosome integrity and suggest that 55M is the most effective concentration for maintaining acrosome integrity. Since Mito-Tempo has shown a positive effect on multiple sperm parameters in other species, such as men, boars, roosters, rams, and bulls, we need to conclude that species-specificity may play a role here.

Effect of the addition of antifreeze protein type I on the quality of post-thawed domestic cat epididymal sperm

Cryopreservation of domestic cat semen is mainly performed as a model for the establishment of endangered wild feline protocols. The supplementation of antifreeze protein type I (AFP I) to cryopreservation medium has shown improvement in frozen-thawed sperm quality in other species, but its effect on cat semen has not yet been tested. This study aimed to assess the addition of AFP I to cryopreservation medium in domestic cats. Sperm was obtained from the cauda epididymis of orchiectomized cats; sperm was then pooled in Tris buffer and allocated into three treatments, according to AFP I final concentration: 0 (control), 0.1, and 0.5 µg/ml. Nine replicates were cryopreserved in a two-step protocol and subsequently thawed at 37°C for 30 s. There was no difference (P > 0.05) among the control, 0.1 and 0.5 µg/ml groups for parameters such as motility, vitality, functional membrane integrity, mature chromatin, normal morphology, and sperm binding to egg perivitelline membrane. In the 0.5 μg/ml group only, percentages of live sperm with intact acrosome and of sperm with most inactive mitochondria (DAB III) showed a significant reduction, along with a tendency (P = 0.053) to an increase in the percentage of sperm with most active mitochondria (DAB II). In conclusion, the supplementation of 0.1 and 0.5 µg/ml of AFP I did not promote consistent beneficial effects on the overall sperm cryotolerance in domestic cats.

Ultra-Rapid Freezing Preserves Morphofunctional Integrity and Fertilizing Ability of Epididymal Cat Spermatozoa.

Vitrification and ultra-rapid freezing, which are more commonly used for oocytes and embryos, have recently been applied to spermatozoa in an attempt to make semen cryopreservation in field conditions easier compared to conventional freezing. It is well-known that in case of unexpected death of rare and wild animals, preserving epididymal spermatozoa from isolated testicles represents a great chance of salvaging male germplasm for future use in assisted reproductive technologies. The aim of this study was to evaluate the morphofunctional integrity of cat epididymal spermatozoa ultra-rapid frozen in pellets or straws with two different extenders [E1 (Tris buffer with 20% egg yolk and 0.25 M sucrose) or E2 (Ham's F10 with 1% bovine serum albumin and 0.4 M sucrose)] and to test whether spermatozoa preserved by the best combination were able to fertilize oocytes and produce embryos in vitro by intracytoplasmic sperm injection (ICSI) of in vitro matured cat oocytes. The results showed that E1 and E2 in straw or pellet were comparable (at warming, about 30% normal morphology, 45% intact membranes, and 20% intact acrosomes), except for post-warming motility that was better maintained along time by E1 pellet (21.7 ± 7.4% at warming and 3.6 ± 2.9% after 6 h). Such spermatozoa could fertilize conspecific oocytes and support embryonic development (cleavage 35.5%) as well as frozen control spermatozoa (cleavage 54.29%, p = 0.22). In conclusion, cat epididymal spermatozoa better maintained their morphofunctional features after ultra-rapid freezing with E1 and could successfully produce embryos in vitro after ICSI. This underscores their usefulness as cryobanked material for fertility and biodiversity preservation purposes.

Effects of green tea polyphenols and α-tocopherol on the quality of chilled cat spermatozoa and sperm IZUMO1 protein expression during long-term preservation

Sperm IZUMO1 protein was recently found to be a crucial mediator in the interaction and fusion with eggs, indicating an important role in assuring the favourable outcome from long-term preservation of chilled semen. The purpose of this study was to investigate whether supplementation of chilled semen extender with green tea polyphenols together with α-tocopherol would provide synergistic effects to prolong sperm survival and maintain IZUMO1 protein stability in cat spermatozoa. Sperm samples were collected from the cat epididymis before being diluted with semen extender containing various concentrations of α-tocopherol (0, 2.5, 5 and 7.5 µg/ml) and 0.75 mg/ml green tea polyphenols and cooled to 4 °C. One sample without antioxidants served as a control. Sperm characteristics and IZUMO1 protein expression were investigated before and after chilling at 3, 6, 9, 12 and 15 days. Using α-tocopherol at 5 µg/ml together with 0.75 mg/ml green tea polyphenols in the semen extender is the most suitable condition to retain the sperm characteristics up to nine days of preservation. Cat IZUMO1 proteins, 17 kDa, were identified at the equatorial segment of acrosome reacted sperm. Without antioxidant, cold storage can gradually degrade the IZUMO1 protein level. Sperm IZUMO1 protein was markedly conserved by supplementation of 5 µg/ml α-tocopherol together with 0.75 mg/ml green tea polyphenols up to 12 days in cold storage. These findings indicate that green tea polyphenols and α-tocopherol have protective effects on the preservation of sperm characteristics and IZUMO1 protein integrity of cat epididymal sperm during long-term chilling.

Brazilian green propolis doesn't have a beneficial effect on the cryopreservation of domestic cat epididymal spermatozoa

This study evaluated the effect of different concentrations of Brazilian green propolis on the cryopreservation of epididymal sperm from domestic cats. Spermatozoa were collected from cauda epididymis by slicing technique in a TRIS extender, preheated to 37ºC. Then, the sperm were evaluated and divided randomly between the control group (without adding propolis in the extender) and the treatment groups (adding different concentrations of propolis extract in the extender): P1 (0.1 mg/mL), P2 (0.3 mg/mL) e P3 (0.6 mg/mL). Subsequently, sperm were cryopreserved. The evaluated parameters were sperm kinetics by the Computer Assisted Semen Analysis, vigor, viability, membrane functionality, chromatin condensation and morphology. The parameters were measured before and after cryopreservation. Propolis didn’t show toxicity to the sperm in any concentrations, with no changes observed in the motility pattern. There was no significant influence of propolis on motility, vigor, percentage of viable spermatozoa, chromatin quality and other kinetic parameters in the cryopreserved samples. In sperm morphology, it was demonstrated a reduction in the percentage of normal cells in P1 and P2 groups in relation to the fresh sample, control and P3 group. In addition, an interesting effect from the propolis groups was observed on possible growth inhibition of microorganisms in the contaminated samples. Propolis didn’t provide superiority to the sperm parameters evaluated after thawing, except for the values of plasma membrane functionality, which were better. The propolis is an interesting component to be incorporated in the cryoprotectant medium due to its non-toxicity and the potential inhibition of microbial growth.

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.

Recovery and cryopreservation of epididymal sperm from domestic cat using powdered coconut water (ACP-117c) and TRIS extenders

Cryopreservation of cats epididymal spermatozoa allows the conservation of the genetic material and the study of the cryogenic effect applied to the gametes of other felines. However, this biotechnique still presents variable results, being necessary the investigation of alternative extenders. Powdered coconut water (ACP-117c) has been efficient in the sperm freezing of several species and in the cat sperm refrigeration. Therefore, we aimed to evaluate the effect of the freezing stages and the quality of the cats’ epididymal spermatozoa after thawing, using ACP-117c. Epididymides (n = 36) from 18 cats were processed using TRIS (n = 18) or ACP-117c (n = 18) for sperm recovery. The sperm were immediately evaluated. Then, this was cooled, glycerolized, frozen and thawed, and re-evaluated at each stage for sperm kinetics by Computer Assisted Semen Analysis, viability, functionality (HOST), mitochondrial activity (DAB) and morphology. There was a reduction in total motility and progressive motility after thawing in both groups, and TRIS was superior to ACP-117c. The curvilinear velocity reduced after thawing with ACP-117c. Viability decreased after glycerolization in TRIS. Although it also reduced after thawing in both groups, it was higher in TRIS. There was no change on HOST. Mitochondrial activity decreased during the cryopreservation steps for both extenders. Nevertheless, TRIS presented a higher percentage of spermatozoa from DAB class I and II after thawing. Morphology did not differ between extenders. Therefore, ACP-117c is an alternative for the recovery of cat epididymal spermatozoa; however, it is not efficient for freezing. Glycerolization and thawing are the most critical stages, regardless of the extender.

Morphological and morphometric characterization of domestic cat epididymal sperm.

Sperm morphometry is the tool that confers objectivity to the morphological evaluation by accurately measuring the dimensions of the gamete and its structures. Thus, the aim of the study was to perform a morphometric characterization of the domestic cat sperm. Therefore, sperm samples were collected from twenty pairs of epididymis in a TRIS extender at 37ºC. An aliquot of the sample was used to make a smear with Rose Bengal solution, and afterwards, the morphology and morphometry were analysed. In the morphology, were quantified the percentage of normal sperm cells, morphological changes of head, midpiece and tail. In morphometry, each normal sperm cell was measured for length, width, area and perimeter of head and midpiece, tail length and total length. The parameters ellipticity, elongation, regularity and rugosity were also determined. The percentage of normal sperm was 67.21%. Of the abnormalities, the curled/folded tail, followed by the curved midpiece, abnormal shaped head and detached head were the most quantified. The sperm head presented 5.56±0.01μm and 3.10±0.01μm of length and width, respectively. The head area was 16.94±0.05μm2 , while the perimeter was 16.16±0.03μm. In the derived parameters, the values were as follows: ellipticity of 1.81±0.00; elongation of 21.39±0.12; regularity of 0.81±0.00; and rugosity of 0.14±0.00. The midpiece presented length and width of 7.96±0.01μm and 0.76±0.01μm, respectively. The mean length of the sperm tail was 45.12±0.06μm, and the total cell size was 58.67±0.06μm. Thus, it was concluded that the cat sperm is an elongated cell, with high rugosity and regularity. The spermatic tail represents more than ¾ of the total length of the cell and the midpiece exceeds the length of the head.

Sperm cryopreservation in the Far-Eastern wildcat (Prionailurus bengalensis euptilurus).

The Far-Eastern wildcat (Prionailurus bengalensis euptilurus) is a rare and poorly investigated nondomestic felid species. An attempt of freezing and cryopreserving Far-Eastern wildcat spermatozoa in CaniPlus Freeze (CPF) medium is reported. Sperm was collected by electroejaculation from five adult Far-Eastern wildcat captive-born males. Epididymal spermatozoa from five adult randomly bred domestic cat males were used as a reference. The viability of frozen-thawed spermatozoa evaluated by double staining with SYBR Green I and PI followed by the subsequent confocal laser scanning microscopy (CLSM) was 38.2%±3.0% for the domestic cat and 38.0%±10.2% for the Far-Eastern wildcat. The motility of frozen-thawed spermatozoa was 30.8%±9.8% for the domestic cat and 33.7%±15.1% for the Far-Eastern wildcat. Sperm morphology was assessed by light microscopy. The total percentage of normal spermatozoa after freezing and thawing was 51.9±5.9 for the domestic cat and 55.0%±6.4% for the Far-Eastern wildcat. Defects of flagella were the most frequently observed abnormalities in both species (32.2%±4.8% and 30.8%±4.4% of all reported anomalies for the domestic cat and Far-Eastern wildcat, respectively). Domestic cat epididymal and Far-Eastern ejaculatory spermatozoa fertilized in vitro-matured oocytes of the domestic cat (30.0%±5.5% and 35.5%±15.0%, respectively). Taken together, these results suggest that the freezing of Far-Eastern wildcat spermatozoa with CPF medium is a suitable method for Felidae cryopreservation.

Cryopreservation and storage of cat epididymal sperm using ‒75 °C freezer vs liquid nitrogen

The quality of cat epididymal sperm cryopreserved and stored by four methods was assessed. Epididymal sperm were suspended in Tris-glucose-citrate egg yolk extender, loaded in 0.25 mL straws and then cryopreserved. The samples in a standard protocol (LN) were cryopreserved and stored in liquid nitrogen (LN2). The sperm straws in the LN-Fr-LN group were cryopreserved in LN2 and stored in a −75 °C freezer; the straws were returned to LN2 prior to thawing. The loaded straws in the Fr group were transferred directly from 4 °C to the freezer and maintained in the freezer until thawing. The Fr-LN samples were cryopreserved and stored in the freezer and were introduced into LN2 before thawing. The sperm thawing was conducted on days 30, 60, 90 and 120 of cryopreservation. The sperm motility, viability, membrane integrity and acrosome integrity were evaluated at 15 and 180 min after thawing. The quality of post-thaw sperm in all three modified protocols was comparable (P > 0.05) and did not differ from that in the standard protocol except the membrane integrity of the 60 days stored samples evaluated at 15 min after thawing, which was significantly higher for the LN-Fr-LN than the Fr-LN groups (P = 0.04). The length of cryopreservation time had no effect (P > 0.05) on the sperm parameters assessed at 15 min after thawing. The sperm motility was significantly greater (P = 0.01 to P = 0.02) for the 15 min than the 180 min incubation. In conclusion, cat epididymal sperm could alternatively be cryopreserved and/or stored by using the −75 °C freezer for 120 days. To use, the cryopreserved sperm in the freezer could be thawed immediately or after being transferred to LN2. This was useful for the application of the −75 °C cryopreserved sperm in remote areas.

Characteristics and fertility of domestic cat epididymal spermatozoa cryopreserved with two different freezing media

The study represents a comparison of cryopreservation of domestic cat epididymal spermatozoa with two commercially available freezing media: CaniPlus Freeze (CPF) and SpermFreeze (SF). The viability of nonfrozen spermatozoa evaluated by the VitalScreen test was 68.7 ± 3.0%. These figures were lower for the frozen-thawed spermatozoa: 51.2 ± 6.3% for CPF group and 54.4 ± 3.1% for SF group. The motility of nonfrozen spermatozoa was 57.2 ± 4.5%. These figures were reduced in both frozen-thawed groups; however, there was no significant difference in these parameters between CPF (30.8 ± 7.1%) and SF (27.4 ± 8.1%) groups. The percentage of nonprogressively moving motile spermatozoa after freezing-thawing was decreased in both frozen-thawed groups (23.5 ± 5.9 and 12.0 ± 2.4 for CPF and SF frozen correspondingly) as compared with nonfrozen controls (42.1 ± 4.1%). Morphology of spermatozoa was assessed by light microscopy. The mean percentages of normal spermatozoa were 28.5 ± 4.1% for nonfrozen group, 26.0 ± 2.3% for CPF frozen group, and 23.9 ± 1.9% for SF frozen group. The most frequent anomalies in all the three groups were flagella and combined defects. In vitro fertilization (IVF) of domestic cat oocytes with nonfrozen and frozen-thawed spermatozoa produced developing embryos. The percentage of in-vitro-derived embryos was 43.6% after using nonfrozen spermatozoa. Frozen-thawed spermatozoa developed at a similar rate (44.0%) after using SF. However, the rate of embryo development was lower (20.1%) when CPF was used. The in-vitro-derived embryos in the nonfrozen group consisted of 46.9 ± 2.5 cells after 5-day culturing. After cryopreservation with SF and CPF the cell numbers per embryo were 39.9 ± 2.7 and 31.8 ± 3.4 correspondingly. In CPF group these numbers were lower than in nonfrozen controls. Cryopreservation of spermatozoa with either of two freezing media led to a decrease in post-thaw viability and motility of spermatozoa but did not affect the rate or spectrum of their morphological anomalies. The use of CPF, but not SF led to a decrease of sperm fertilizing abilities.

Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72hours).

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post-mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer-assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3'3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7±6.3%) and total defects (53.4±6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6±2.2%) occurred after 72hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48hr of cooling time.

Structural integrity and developmental potential of spermatozoa following microwave-assisted drying in the domestic cat model

Characterizing the resilience of mammalian cells to non-physiological conditions is necessary to develop preservation and long-term storage strategies at low or ambient temperatures. Using the domestic cat model, the objective of the study was to characterize structural integrity (morphology and DNA damage) as well as functional properties (sperm aster formation and embryo formation after sperm injection) of spermatozoa after microwave-assisted drying to a moisture content compatible with storage in a glassy state at supra-zero temperatures. In Experiment 1, cat epididymal spermatozoa were porated with hemolysin and dried (using a commercial microwave oven set to 20% power) in the presence of trehalose for up to 50 min in a low humidity environment (11%) before measuring moisture content and sample temperature. In Experiment 2, morphology and DNA integrity were evaluated in sperm dried for up to 30 min (using the same method as above) versus fresh spermatozoa. In Experiment 3, the functionality of sperm dried for 30 min versus fresh sperm cells was evaluated after injection into oocytes based on sperm aster formation (5 h post-injection) and embryo development in vitro over 7 days. Moisture contents compatible with dry state storage were reached after 30 min of microwave-assisted drying. After rehydration, sperm morphology was not affected and the percentages of cells with damaged DNA (∼6.5%) was similar to the fresh controls. Sperm aster diameters appeared to be generally smaller for dried-rehydrated cells compared to the fresh controls. This observation was consistent with a lower proportion of blastocyst formation after injection with dried spermatozoa (6.5%) compared to fresh spermatozoa (15%). However, the blastocyst quality based on the total blastomere number was not affected by the sperm treatment. This is the first and encouraging report in any species so far demonstrating that spermatozoa can be dried using microwaves without causing irreversible damage to the cellular structure and function.

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.

Effect of extracellular adenosine 5′-triphosphate on cryopreserved epididymal cat sperm intracellular ATP concentration, sperm quality, and in vitro fertilizing ability

Intracellular adenosine 5′-triphosphate (ATP) is essential for supporting sperm function in the fertilization process. During cryopreservation, damage of sperm mitochondrial membrane usually leads to compromised production of intracellular ATP. Recently, extracellular ATP (ATPe) was introduced as a potent activator of sperm motility and fertilizing ability. This study aimed to evaluate (1) levels of intracellular ATP in frozen–thawed epididymal cat sperm after incubation with ATPe and (2) effects of ATPe on epididymal cat sperm parameters after freezing and thawing. Eighteen male cats were included. For each replicate, epididymal sperm from two cats were pooled to one sample (N = 9). Each pooled sample was cryopreserved with the Tris–egg yolk extender into three straws. After thawing, the first and second straws were incubated with 0-, 1.0-, or 2.5-mM ATPe for 10 minutes and evaluated for sperm quality at 10 minutes, 1, 3, and 6 hours after thawing and fertilizing ability. The third straw was evaluated for intracellular ATP concentration in control and with 2.5-mM ATPe treatment. Higher concentration of intracellular sperm ATP was observed in the samples treated with 2.5-mM ATPe compared to the controls (0.339 ± 0.06 μg/2 × 106 sperm vs. 0.002 ± 0.003 μg/2 × 106 sperm, P ≤ 0.05). In addition, incubation with 2.5-mM ATPe for 10 minutes promoted sperm motility (56.7 ± 5.0 vs. 53.3 ± 4.4%, P ≤ 0.05) and progressive motility (3.1 ± 0.2 vs. 2.8 ± 0.4, P ≤ 0.05), mitochondrial membrane potential (36.4 ± 5.5 vs. 28.7 ± 4.8%, P ≤ 0.05), and blastocyst rate (36.1 ± 7.0 and 28.8 ± 7.4%, P ≤ 0.05) compared with the controls. In contrast, ATPe remarkably interfered acrosome integrity after 6 hours of postthawed incubation. In sum, the present finding that optimal incubation time of postthaw epididymal cat sperm under proper ATPe condition might constitute a rationale for the studies on other endangered wild felids regarding sperm quality and embryo development.

Determination of appropriate cryopreservation protocols for epididymal cat spermatozoa.

Effects of Equex and glycerol additions and sample dilution step on frozen-thawed epididymal cat spermatozoa were investigated. The epididymal sperm pellets were resuspended in extenders using one- (groups III and IV) or two- (groups I, II, V and VI) step dilution. For one-step dilution, the pellets were resuspended in plain egg yolk-Tris medium (EYT)+5% glycerol with (IV)/without (III) 0.5% Equex and cooled (4(°) C, 1h). For two-step dilution, the pellets were resuspended in EYT (I and V) and in EYT+3% glycerol (II and VI), cooled and further diluted with EYT+10% glycerol with (I)/without (V) 1% Equex and with EYT+7% glycerol with (II)/without (VI) 1% Equex. Immediately after freeze-thawing, no differences (p>0.05) were found in the motility, viability and membrane integrity (HOST) among the groups except the lowest HOST in IV (p=0.005 to p=0.04). The acrosome integrity (FITC) in group I was comparable to that in group II (p>0.05) and was higher than the rest (p<0.001 to p=0.02). At 2h after thawing, the motility, viability and HOST were comparable among the groups (p>0.05) except the lower percentages of viability in III (p=0.008 to p=0.3) and of HOST in IV (p=0.005 to p=0.2). Two-step dilutions with Equex (I, II) were more beneficial for the FITC at 2h than without Equex (V) (p=0.005 and p=0.02) and than one-step dilutions (III, IV) (p<0.001 to p=0.02). In conclusion, epididymal cat sperm quality after freeze-thawing could be improved when Equex was added and two-step dilution was performed during freezing. The extenders prepared for the first step of dilution could be with (3%) or without (0%) glycerol.

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.

L-carnitine Supplemented Extender Improves Cryopreserved-thawed Cat Epididymal Sperm Motility.

Cryopreservation of epididymal sperm is an effective technique to preserve genetic materials of domestic cats and wild felids when they unexpectedly die. However, this technique inevitably causes detrimental changes of cryopreserved-thawed spermatozoa, for example, by physical damage and excessive oxidative stress. L-carnitine is an antioxidant that has been used to improve sperm motility in humans and domestic animals. This study aimed to investigate the effects of L-carnitine on cat epididymal sperm quality following cryopreservation and thawing. After routine castration, cauda epididymides were collected from 60 cat testes. The epididymal spermatozoa from 3 cauda epididymides were pooled as 1 replicate. Spermatozoa samples (16 replicates) were examined for spermatozoa quality and then randomly divided into 4 groups: 0 mM L-carnitine (control), 12.5 mM, 25 mM and 50 mM L-carnitine. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, plasma membrane integrity, DNA integrity and acrosome integrity were evaluated. The 25 mM L-carnitine significantly improved sperm motility compared with a control group (p<0.05), although this was not significantly different among other concentrations. In conclusion, supplementation of 25 mM L-carnitine in freezing extender improves cauda epididymal spermatozoa motility. The effects of L-carnitine on the levels of oxidative stress during freezing and thawing remains to be examined.

Impact of Ultra‐Rapid Freezing on the Motility, Morphology, Viability and Acrosome Integrity of Epididymal Cat Sperm Diluted in Sucrose‐Based Extenders

The objective of the present study was to investigate the influence of different sucrose-based extenders on the motility, morphology, viability and acrosomal integrity of epididymal cat spermatozoa cryopreserved by ultra-rapid freezing method. Nine cats were castrated, and collected semen was diluted 1:1 with Dulbecco`s phosphate-buffered saline-BSA1%-based extender supplemented with different sucrose concentrations (0, 0.25, 0.4 and 0.6m). After ultra-rapid freezing, samples were thawed and sperm motility, morphology, viability and acrosome status were assessed. At thawing, the number of progressively motile (p<0.01) and morphologically normal (p<0.01) sperm was higher in the sucrose-supplemented groups than in the sucrose-free group. Viability of spermatozoa cryopreserved without sucrose was significantly reduced. In extender supplemented with 0.4m sucrose, spermatozoa viability showed higher values (57.0±4.7; p<0.01). No significant differences were detected among groups for sperm acrosome integrity. Results support that cat sperm survive after ultra-rapid freezing using sucrose as a cryoprotectant, and the best results were achieved when 0.4m of sucrose was used. This is the first report on sperm ultra-rapid freezing of cat sperm and further studies on extenders, sperm management or cryovials should be carried out to improve sperm cryosurvival.

Sperm‐TALP: An Alternative Extender for Retrieving and Diluting Epididymal Sperm in the Domestic Cat

Effects of sperm-TALP (TALP) on the quality of fresh-extended and frozen-thawed epididymal cat sperm were evaluated. The epididymides suspended in Tris-glucose-citrate solution (Tris), a conventional medium, and TALP were cut into small pieces to recover epididymal sperm. In experiment 1, the sperm pellets remained after centrifugation were re-suspended (1:2, v/v) in Tris and TALP. The sperm quality in all four groups, that is, sperm retrieved with Tris (I and II) or TALP (III and IV) and diluted with Tris (I and III) or TALP (II and IV) was assessed. The sperm motility at the 0-h incubation in TALP-TALP was superior to that of the rest (p<0.001 to p=0.04). At the 2-h incubation, the motility in Tris/TALP-TALP was greater than that in Tris/TALP-Tris (p≤0.001). In experiment 2, after centrifugation, the sperm pellets were added with freezing extenders and frozen. The thawed sperm previously retrieved from the epididymides with Tris and TALP were allotted so as not to further diluted (Tris/TALP-O) and to further diluted (1:1, v/v) with Tris (Tris/TALP-Tris) and TALP (Tris/TALP-TALP) and were evaluated the quality. At both incubation times, the motility of frozen-thawed sperm recovered with TALP (TALP-O/Tris/TALP) was comparable with or significantly higher than that in the Tris groups (Tris-O/Tris/TALP; p=0.003 to p>0.05). The motility and viability of thawed sperm in Tris-Tris were significantly decreased during the 2-h incubation (p=0.007 for the motility and p=0.01 for the viability). In both experiments, neither type of diluent (Tris vs TALP) nor incubation time (0 vs 2h) significantly affected the sperm membrane integrity under hypo-osmotic condition (p>0.05). According to beneficial effects on the quality of fresh-extended and frozen-thawed sperm demonstrated, sperm-TALP could be used as an alternative medium for recovering sperm from the epididymides and for diluting epididymal sperm in the domestic cat.