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Highlights • Toluidine blue staining is effective in the evaluation of feline sperm. • Head dimensions of feline spermatozoa in the different epididymal portions. • Feline spermatozoa with larger head presented looser chromatin packaging. Abstract When using assisted reproductive technologies, there is seldom an evaluation of DNA integrity during sperm analysis, which is an important variable for proper embryo development. The toluidine blue staining technique allows the simultaneous evaluation of sperm chromatin and sperm head dimensions. The objectives of this study were to evaluate the applicability of the toluidine blue staining method for analyzing DNA abnormalities in epididymal sperm (from the caput, corpus, and cauda) of cats and to investigate the correlations among DNA condensation, morphology, and sperm head dimensions. The DNA alteration indexes were obtained using the toluidine blue and acridine orange techniques, and comparisons of these indexes indicated there was a 65.4% (r = 0.654; P < 0.001) correlation. The sperm from the cauda had greater chromatin stability (97.9%) than the sperm from the epididymal head (92.1%; P = 0.0023). There, however, was no difference in chromatin stability between sperm obtained from the corpus and cauda regions, indicating that these sperm were already mature. The sperm head dimension was correlated with chromatin decondensation, and the sperm head size decreased as the sperm were transported through the three epididymal regions (P < 0.0001). In addition, the percentage of sperm that were deficient in chromatin condensation decreased as the sperm were transported through the epididymal caput, corpus and cauda (26.4, 15.7, and 3.4%, respectively; P < 0.0001). Thus, the sperm head size predicts the quality of chromatin condensation in sperm cells. [ABSTRACT FROM AUTHOR]

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 60 min after incubation. Experiment II: spermatozoa were treated with 2 μM 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the efficacy of subcutaneous melatonin implants to temporarily and reversibly suppress spermatogenesis in male cats. Tomcats (n = 8) were housed in a conditioned room with alternating long and short 2-month photoperiod cycles to maintain sperm production and quality. Animals were randomly assigned to one of the two treatments. Four animals received a subcutaneous melatonin implant (MEL, 18 mg; Syntex, Argentina), whereas the other four received a subcutaneous placebo implant (PLA, 0 mg; Syntex). Semen samples were collected by electroejaculation every 14 days for 252 days. Sperm parameters were evaluated in all ejaculates, and data were analyzed by ANOVA. Melatonin-implanted cats significantly decreased their sperm quality in all the parameters studied compared with the control group (MEL vs. PLA; least squares means ± SEM; motility, 71.3 ± 3.4 vs. 82.1 ± 3.6; velocity, 3.4 ± 0.1 vs. 4.6 ± 0.1; total sperm count, 2.6 ± 2.2 vs. 19.4 ± 3.3; acrosome integrity, 48.7 ± 5.6 vs. 62.8 ± 5.6; plasma membrane integrity, 52.2 ± 4.7 vs. 72.9 ± 5.5; normal sperm morphology, 45.8 ± 3.3 vs. 63.7 ± 3.4; P < 0.05). Conversely, volume and serum testosterone concentrations were similar in both groups (volume, 0.15 ± 0.02; serum testosterone concentrations, 1.1 ± 0.1; CV 18.9%; P > 0.05). At 91 ± 7 days after implant insertion, sperm motility decreased 38.5%, velocity 26.5%, total sperm count 82%, acrosome integrity 22%, plasma membrane integrity 30%, and normal sperm morphology decreased 32% of preimplant values. This effect was present until 120 ± 15 days after implant insertion. After that, seminal parameters started to increase and reached preimplant values at about 140 ± 7 days after implant insertion. Nevertheless, treated animals conserved the capacity to produce semen during the treatment period. In conclusion, a single subcutaneous melatonin implant effectively and reversibly reduced sperm production and quality in male domestic cats for approximately 120 ± 15 days without clinically detectable adverse effects. [ABSTRACT FROM AUTHOR]

Ubiquitin is a 8.5-kDa peptide that tags other proteins for proteasomal degradation. It has been proposed that ubiquitination might be responsible for the elimination of defective spermatozoa during transit through the epididymis in humans and cattle, but its exact biological function in seminal plasma has not yet been clarified. In the domestic cat ( Felis catus ), the percentage of immature, unviable, and abnormal spermatozoa decreases during the epididymal transit, indicating the existence of a mechanism that removes defective spermatozoa. Magnetic cell separation techniques, based on the use of magnetic beads coated with anti-ubiquitin antibodies, may allow the selective capture of ubiquitinated spermatozoa from semen, thus contributing to the identification of a potential correlation between semen quality and ubiquitination process. Moreover, the selective identification of all the ubiquitinated proteins in different epididymal regions could give a better understanding of the ubiquitin role in feline sperm maturation. The aims of this study were as follows: (1) to verify the possibility of separating ubiquitinated spermatozoa with magnetic ubiquitin beads and identify the morphological and acrosomal differences between whole sample and unbound gametes, (2) to characterize all the ubiquitinated proteins in spermatozoa retrieved in the three epididymal regions by a proteomic approach. The data indicated the presence of ubiquitinated proteins in cat epididymal semen. However, a correlation between abnormal and ubiquitinated spermatozoa has not been found, and ubiquitin cannot be considered as a biomarker of quality of epididymal feline spermatozoa. To the author's knowledge, this is the first identification of all the ubiquitinated proteins of cat spermatozoa collected from different epididymal regions. The proteomic pattern allows a further characterization of cat epididymal semen and represents a contribute to a better understanding of the ubiquitin role in feline sperm maturation. [ABSTRACT FROM AUTHOR]

Contents The aim of this study is to produce live kittens from oocytes fertilized by intracytoplasmic sperm injection ( ICSI) with frozen/thawed testicular spermatozoa. Spermatozoa were collected from thawed testicular tissue and subsequently injected into in vitro matured cat oocytes. At 24 h post- ICSI, presumptive zygotes/cleaved embryos were treated with 10 μ m forskolin for 24 h to reduce intracellular lipid content of embryos (delipidation). At 48 h after oocyte injection, cleaved embryos (2- to 8-cell stage) were frozen in 10% (v/v) ethylene glycol-based medium by a slow controlled rate method and stored in liquid nitrogen. To evaluate in vitro and in vivo developmental competence, frozen embryos were thawed and then cultured for 6 days (n = 155) or cultured for 2 h before transferred (n = 209) to hormonal (equine chorionic gonadotropin/h CG)-treated cat recipients. Cleavage frequency at day 2 after ICSI with frozen/thawed testicular spermatozoa was ~30%. The percentages of frozen/thawed embryos that developed to morula and blastocyst stage (on day 3 and day 6 of in vitro culture, respectively) were significantly lower than that of fresh ICSI embryos (22.6 vs 45.2% and 21.3 vs 38.7%, respectively; p < 0.05). However, no difference was found in the number of blastomeres between frozen/thawed (242.5 ± 43.1) and fresh (320.2 ± 28.1) blastocysts. Three of seven cat recipients were pregnant and one pregnant cat delivered two healthy kittens. This is the first report of the birth of kittens after the transfer of frozen-thawed embryos produced by ICSI with frozen/thawed testicular sperm. [ABSTRACT FROM AUTHOR]

Contents In dogs and cats, computer-assisted sperm analysis ( CASA) was originally described almost 20 years ago. Subsequently, numerous CASA systems were validated and used for various applications in dogs and to a lesser extent in cats. CASA systems offer an accurate, rapid, objective and simultaneous assessment of different semen parameters allowing the visualization of subtle changes in sperm characteristics, which cannot be identified by conventional semen analysis. The main problems of these computerized measuring devices are the relatively high investment costs and the need for standardization and validation before any practical use is possible. In comparison with automated motility and concentration assessment, automated morphometry and morphology assessment is more complex and time-consuming. Once validated, CASA systems can be routinely used in veterinary centres for assessment of fertility and for the improvement of sperm diluters, cooling and cryopreservation procedures in dogs and cats. Furthermore, information obtained by CASA systems could also be important when monitoring for example the effect of environmental stress on spermatozoa and for toxicity studies. In cats, CASA is less documented, and most studies describe the characteristics of epididymal sperm, which is frequently used for in vitro fertilization in cats. Implementation of the CASA technique in cat reproduction could be interesting to further optimize assisted reproductive techniques in domestic cats and endangered wild felids. [ABSTRACT FROM AUTHOR]

Contents Artificial insemination ( AI) is potentially invaluable as an adjunct to natural breeding for the conservation management of non-domestic felid populations. The efficacy of AI, however, must be substantially improved for applied use, especially when using frozen semen. Our recent advances in using laparoscopic oviductal AI ( LO- AI) with low sperm numbers and freezing of cat semen in a soy lecithin-based cryoprotectant medium suggest that combining these two approaches might improve pregnancy outcomes with frozen-thawed spermatozoa. In this study, our objectives were to (i) assess the effect of two gonadotropin dosages (100 vs 150 IU eCG) on ovarian response in domestic cats and (ii) compare the relative fertility of frozen-thawed and fresh semen in vivo following LO- AI. All 16 females ovulated after gonadotropin treatment and were inseminated with fresh semen from one male and frozen-thawed semen from a second male. There were no differences between gonadotropin dosages in CL number, pregnancy percentage or litter size. Half (8/16) of the females conceived, with seven females giving birth to a total of 36 offspring. Paternity analysis showed that more kittens resulted from LO-AI with fresh (28/36, 78%) than frozen-thawed (8/36, 22%) semen, possibly due to impaired motility and longevity of thawed sperm. These results demonstrated that viable offspring can be produced by AI using semen frozen in a soy lecithin-based medium. Insemination with greater numbers of frozen-thawed spermatozoa, combined with further refinement of cat sperm cryopreservation methods, may be necessary to optimize pregnancy success with LO- AI in domestic and nondomestic cats. [ABSTRACT FROM AUTHOR]

Abstract: This study examined the effects of cold storage on plasma membrane, DNA integrity, and fertilizing ability of domestic cat spermatozoa. Intact cat testes were stored at 4°C in Dulbecco''s phosphate buffered saline (DPBS) for 7 days. Membrane integrity (experiment 1) and DNA integrity (experiment 2) of extracted spermatozoa were assessed over time during storage. Testicular spermatozoa were also tested for their fertilizing ability via intracytoplasmic sperm injection (ICSI) in term of gamete activation and early embryonic development at 18h (experiment 3). The membrane integrity of testicular spermatozoa was well preserved in DPBS for 4 days compared to non-preserved control (Day 0) (P <0.05). The incidence of testicular sperm DNA fragmentation was <1% after 7 days of cold storage and was not significantly affected by the duration of cold storage (P >0.05). Finally, testicular spermatozoa could form pronuclei and sustain embryo development following ICSI regardless of the storage time (P >0.05). In conclusion, cat testicular spermatozoa can be preserved at 4°C for up to 7 days without severely compromising of plasma membrane and DNA integrity while retaining a normal fertilizing ability. [Copyright &y& Elsevier]

The impact of different storage conditions of epididymal spermatozoa (including prolonged storage, cryopreservation and freeze-drying) on their fertilisation capacity was tested using intracytoplasmic sperm injection (ICSI). This kind of information is urgently needed when applying assisted reproductive technology to endangered felids in zoos. In particular, the utilisation of epididymal spermatozoa of castrated or deceased felids often requires time-consuming transportation and is therefore susceptible to loss of gamete quality. Sperm cells were stored at 4°C for up to 72h followed by cryopreservation or freeze-drying. Thawed motile and immotile spermatozoa were used for ICSI and the embryo cleavage rate was assessed 36h after injection. A significant impact on the fertilisation rate of oocytes could only be detected when using immotile thawed or rehydrated spermatozoa. Cryopreservation or storage at 4°C showed no influence. The simulation of transport conditions using domestic cat spermatozoa revealed that in vitro production of felid embryos with gametes from euthanised individuals is possible if testes are stored cool and arrive at the laboratory within 72h. An essential prerequisite is the application of ICSI to achieve fertilisation even with single motile spermatozoa. Additional cryopreservation of spermatozoa after transportation is possible and will allow the establishment of a sperm bank for felids. [ABSTRACT FROM AUTHOR]

Abstract: The collection of semen from tomcats by urethral catheterization (CT) after medetomidine administration offers a novel and easy approach to obtain good quality sperm for in vitro fertilization. This study was designed to compare the sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in seventeen adult cats by urethral catheterization, after which the cat was orchiectomized. Motility, morphology, plasma membrane integrity, acrosomal status, and in vitro fertilizing capacity of both fresh CT and EP samples were evaluated. The results showed that both total and progressive motility, as well as the percentage of normal spermatozoa, were higher for EP sperm than for CT sperm (P < 0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P < 0.01), while CT sperm contained more spermatozoa with tail abnormalities (P < 0.01). Other morphological parameters, as well as plasma membrane integrity, did not differ (P > 0.05) between CT and EP sperm. Nevertheless, no difference (P > 0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the two different methods was found. In conclusion, semen collection by means of urethral catheterization after medetomidine administration yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Since this novel catheterization technique is repeatable, is easy to perform and facilitates semen preparation protocols, it may be preferable for routine IVF experiments with fresh spermatozoa. [Copyright &y& Elsevier]

Contents The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70°C) and to investigate the effects of post-thaw dilution on the sperm quality and longevity of ejaculated cat spermatozoa. Six ejaculates of each of six male cats were collected using an electroejaculator (total 36 ejaculates). The semen was frozen in 0.25-ml straws using a Tris egg yolk extender containing Equex STM paste. Four straws prepared from each ejaculate were thawed at four different occasions; (i) at 37°C for 15 s, (ii) at 37°C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70°C for 6 s, (iv) at 70°C for 6 s and diluted 1 : 2 with Tris buffer (v/v). The percentages of motile spermatozoa, the scores of progressive motility, the percentages of spermatozoa with intact plasma membrane (using SYBR-14/EthD-1 stains) and intact acrosome (using fluorescein isothiocyanate conjugated peanut agglutinin/propidium iodide stains) were evaluated in fresh semen at 0, 2, 4 and 6 h after thawing. The thawing temperature had no effect on any sperm parameters throughout the incubation period (p > 0.05). The dilution after thawing improved sperm motility, progressive motility and acrosome integrity (p < 0.05). The thawing of cat spermatozoa and subsequently diluting with Tris buffer resulted in an immediate (at 0 h) overall (combined over temperature) percentage of motile sperm of 64.8 ± 10.7 (mean ± SD), a score of progressive motility of 4.0 ± 0.5, a percentage of spermatozoa with intact plasma membrane of 64.4 ± 12.1 and intact acrosome of 44.8 ± 20.2. In conclusion, frozen cat semen can be thawed either at 37 or 70°C and post-thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post-thaw incubation. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the sperm characteristics of four cloned male cats (Felis catus) to assess their reproductive potential. Fresh and frozen-thawed sperm were assessed for motility, viability, and morphology, and their functional competence was evaluated by in vitro fertilization (IVF) of domestic cat oocytes. All fresh semen characteristics varied among cats and collection times. Sperm concentration (× 106/mL) of Cat A (512±140, range 368 to 685) was significantly higher, whereas that of Cat C (335±92, range 274 to 469) was significantly lower than that of Cloned B (459±159, range 336 to 510) and control cats (680±452, range 360 to 479). After thawing, motility and progressive motility of sperm from Cat B were significantly lower than that of the other cloned and control cats. The curvilinear, straight line, and average path velocities of sperm from Cat B were significantly higher, whereas the straightness was lower, than that of the other cloned and control cats. Frozen sperm from Cats A, B, and C successfully fertilized oocytes (cleavage=74.4%, 71.4%, and 86.2%, respectively) and produced embryos that developed to the blastocyst stage after IVF/In vitro culture (IVC) (34.4%, 26.7%, and 48.0%) at frequencies similar to the cleavage rate (82.0%) and blastocyst rate (43.9%) obtained with sperm from the control male. In conclusion, seminal characteristics of cloned male cats did not differ markedly from those of our noncloned, control male cats. [Copyright &y& Elsevier]

Cryopreservation of gametes is an important tool in assisted reproduction programmes; long-term storage of oocytes or spermatozoa is necessary when in vitro fertilization (IVF) or artificial insemination is to be performed at a future date. Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of endangered populations. The objectives of this work were to: (1) examine sperm motility, viability, abnormality and acrosome integrity of frozen-thawed domestic cat epididymal spermatozoa; and (2) evaluate the same cryopreservation method on wild feline spermatozoa, needed to preserve their genetic resources. Epididymides were collected from 20 domestic cats during routine neutering procedure and from two wild felines at autopsy. The sperm samples, diluted with 4% glycerol/Tris/egg yolk, were loaded into 0.25 ml mini-straws, exposed to nitrogen vapour and stored in liquid nitrogen. After 4 weeks, samples were thawed and re-evaluated. The quality of each fresh and frozen-thawed sperm sample was tested by determining the motility (54.7±11.3% and 32±13.1% respectively for cat spermatozoa; 38.3±18.7% and 21.5±16.8% respectively for tiger spermatozoa), viability (74.3±8.6% and 45.2±9.4% respectively for cat spermatozoa; 42.4±14.5% and 33.5±12.9% respectively for wild felid spermatozoa), morphology and acrosomal status. The present study showed that feline epididymal spermatozoa can be frozen in egg-yolk extender with 4.0% glycerol in 0.25 ml straws. The procedure used in the present study for epididymal cat sperm cryopreservation may be applied to bank the genetic resources of wild felid species. [ABSTRACT FROM AUTHOR]

Abstract: Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat (Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91±2.02, 48.21±1.47, and 43.03±1.32) and after thawing (51.81±3.02, 41.90±2.14, and 42.35±1.95) of sperm recovered from epididymides stored for 0, 48, or 72h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33±1.38% before and 52.50±1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22±1.25% normal sperm after recovery). When epididymides were stored for 72h, time of sperm equilibration with glycerol (30 vs. 120min) resulted in significant differences in both motility (SMI=39.17±2.76 and 45.00±2.65, respectively) and the percentage of intact acrosomes (45.76±4.91% and 60.67±3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24h of cool storage and when they are equilibrated with glycerol during 120min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids. [Copyright &y& Elsevier]

Abstract: Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll® centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR®14-PI) and DNA fragmentation (TUNEL). After addition of a Tris–glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4°C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P <0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation. [Copyright &y& Elsevier]

Abstract: The aims were to evaluate the susceptibility of feline ejaculated and epididymal spermatozoa to cold shock and to evaluate the effect of egg yolk in the preservation extender. Ejaculated and epididymal spermatozoa from eight males were subjected to a slow (0.5°C/min) or a fast (3°C/min) cooling rate with controls kept in room temperature. Ejaculated and epididymal spermatozoa from another eight males were cooled in a plain Tris buffer (Tris) or in Tris with 20% egg yolk (EYT) and evaluated for 96h. Subjective motility (MOT), plasma membrane integrity (PMI), and acrosome integrity (ACRI) were evaluated. Cooling did not induce sperm damage regarding PMI (P =0.6) or ACRI (P =0.19) and chilled spermatozoa had better overall MOT (P =0.046) than controls. EYT was better for MOT (P >0.05) from 48h of cold storage than Tris. EYT was also better for overall ACRI (P <0.0001) while Tris was better for overall PMI (P =0.0004). There were no interactions between time and treatment (P >0.05) for PMI or ACRI. Ejaculated spermatozoa had better overall MOT (P <0.05) and PMI (P <0.05) than epididymal spermatozoa, and higher ACRI in experiment 1 (P =0.0003) but not in experiment 2 (P =0.117). Source of spermatozoa did not affect the susceptibility to cooling or the effect of egg yolk as there were no interactions (P >0.05) between source of spermatozoa and treatment (cooling or control) or between time, source and extender (P >0.05). In conclusion cat spermatozoa were tolerant to cold shock and egg yolk was beneficial for preservation of MOT and ACRI but not PMI. [Copyright &y& Elsevier]

Contents There are limited data on feline sperm production. We exhausted epididymal spermatozoa (i.e. the number of ejaculated spermatozoa <5 × 106) by frequent semen collections using the artificial vagina method in five tomcats and determined the number of spermatozoa stored in the epididymis. We investigated the time (days) required for the number of epididymal spermatozoa to return to the pre-exhaustion level and determined the number of spermatozoa produced per day. After spermatozoa were exhausted by frequent semen collection, 6 or more days were required to return to the pre-exhaustion level. Based on the duration of resting (days) and total number of spermatozoa, the mean number of spermatozoa produced per day was 30 × 106. [ABSTRACT FROM AUTHOR]

Contents The aim of this study was to assess whether refractoriness to long photoperiod ( LP) could be reversed by subjecting tomcats to a period of short days. Our hypothesis was that photoperiod changes can avoid refractoriness and restore sperm quality and production to that before refractoriness. Tomcats (n = 6) were housed in a conditioned room with LP (12 L: 12 D) for 45 days of acclimation and then maintained under LP for 18 month. Then, tomcats were changed to a period of decreasing light at a rate of 8 min/day for 1 month. Tomcats stayed for 1 month with short photoperiod ( SP; 8 L: 16 D) and then were switched back to a period of increasing light at a rate of 8 min/day for 1 month. The experiment was completed after tomcats remained in LP for 2 months. Toms were anaesthetized and semen samples were collected by electroejaculation every 2 weeks. Sperm parameters were evaluated in all ejaculates, and data were analysed by anova. Motility, velocity, volume, sperm concentration, total sperm count, viability, acrosome integrity, plasma membrane integrity and sperm morphology were higher during LP compared with a refractory LP (p < 0.01). Likewise, velocity, viability, acrosome integrity, plasma membrane integrity and sperm morphology were higher in a LP compared with a SP (p < 0.05). On the other hand, motility, volume, concentration and total sperm count were similar between LP and SP (p > 0.20).Whereas motility, velocity, viability, acrosome integrity and plasma membrane integrity were similar in a refractory LP compared with SP (p > 0.05), volume, sperm concentration, total sperm count and sperm morphology were lower in a refractory LP compared with SP (p < 0.05). In conclusion, refractoriness and reduced sperm production and quality induced by a prolonged LP of 18 month can be restored after placing tomcats to a SP. [ABSTRACT FROM AUTHOR]

Development of a chemically defined, plant-based cryopreservation media would reduce extender variability and the potential for transmission of zoonotic pathogens compared with traditional egg-yolk-based extenders. The objective of this study was to compare effects of egg yolk- and soy lecithin-based cryopreservation media and the temperature of glycerol addition on sperm parameters following freezing and thawing of domestic cat spermatozoa. Fresh semen was collected by manual stimulation on 3 separate occasions from 4 adult male cats. Each ejaculate was washed to remove seminal plasma, divided into 4 equal aliquots, and extended at room temperature in one of the following treatments: 1) TEST-egg yolk (Irvine Scientific Inc., Santa Ana, CA, USA) medium with 4% glycerol (EYG); 2) TEST-egg yolk, with 4% glycerol added after cooling to 5°C (EY); 3) TES-Tris buffer with soy-lecithin (1%) and 4% glycerol (SLG); and 4) TES-Tris buffer with 1% soy-lecithin, and 4% glycerol added after cooling to 5°C (SL). Sperm progressive motility (%) and rate of progressive movement (scale of 0–5) were evaluated at 0, 1, 3, 6, and 24h post-thaw. Sperm capacitation (chlortetracycline staining), acrosome integrity (FITC-PNA staining), and DNA integrity (acridine orange staining) were assessed at 15min post-thaw. Data were exponentially transformed to achieve normal distribution and then subjected to GLM analysis to determine effects of media and temperature of glycerol addition on sperm traits. At 0 and 1h post-thaw, acrosome integrity, DNA integrity and % sperm motility did not differ (P>0.05) among treatments. However, % sperm motility was greater in the soy-based media compared to egg yolk-based media at 3, 6, and 24h post-thaw (Table 1; P<0.05). A higher percentage of uncapacitated spermatozoa were present in soy-based compared to egg-yolk based cryopreservation media (63.9±9.3 v.51.2±11.5, respectively; P<0.05), regardless of temperature of glycerol addition. Finally, addition of glycerol at 5°C resulted in higher % sperm motility compared to room temperature at 6 and 24h post-thaw in both medium types (Table 1; P<0.05). Our results suggest that use of a chemically defined, soy-based medium improves long-term motility and capacitation status of frozen–thawed domestic cat spermatozoa compared with cryopreservation in a traditional egg yolk-based extender. Table 1.Motile spermatazoa and motility score at 3, 6, and 24h [ABSTRACT FROM AUTHOR]

Our purpose was to compare in vitrosurvivability and functionality of cat epididymal spermatozoa cryopreserved in TEST egg-yolk buffered extender (TYB) with that obtained by use of clear Tris-citrate and HEPES-buffered extenders containing BSA. Testes were transported to the lab in HEPES saline; epididymides were dissected in HEPES-199 medium (HE-199) and repeatedly sliced. The sperm suspension was filtered (40 μm), layered onto a density gradient column (Isolater, Irving Scientific, Santa Ana, CA), and centrifuged at 600gfor 20 min. Aliquots of the sperm pellet were extended in TYB, Human Sperm Preservation Medium (HSPM), or Tris-citrate + 10% BSA (TCBSA). After cooling to 4°C, samples were diluted 1:1 with extender + 12% glycerol in 4 steps as modified from Gao DY et al.1995 Hum. Reprod. 10, 1109–1122. Then, samples were loaded into 0.25-mL straws, sealed, and frozen on a dry ice block (–80°C) for 20 min before storage in LN2. Straws were thawed by exposure to air (22°C) for 5 s and immersion in a 60°C water bath for 5 s. Samples were diluted by addition of HE-199 in 7 steps as modified from Gao DY et al.1995 Hum. Reprod. 10, 1109–1122, centrifuged at 200gfor 10 min and pellets resuspended in HE-199. Motility (MOT, phase contrast, 37°C), membrane integrity (MI, SYBR 14–PI), and acrosomal status (AS, FITC–PNA) were evaluated at 0 h, after gradual cooling to 4°C, and after freezing at 0 h and 3 h post-thaw (37°C). Cumulus oocyte complexes (COC) were placed in modified TCM-199 and cultured for 24 h in 5% O2, 5% CO2, and 90% N2at 38°C (IVM). For IVF, COC were co-incubated with spermatozoa frozen in either TYB or HSPM in droplets (1 million sperm mL-1) of IVF medium under 5% CO2in air at 38°C. After 18 h, oocytes were rinsed and cultured using a 3-step system (Pope CE et al.2006 Theriogenology 66, 59–71) until blastocyst development was evaluated (Day 8). There were no treatment differences at any time/temperature point for the 3 sperm parameters evaluated (one-way ANOVA; P> 0.05). As shown in Table 1, sperm motility in TCBSA and HSPM decreased by 20% after cooling to 4°C and another 20% after freezing, whereas motility in TYB was maintained after cooling and decreased <30% after freezing. Membrane integrity and acrosomal status values were 12 to 15% greater at collection, at 4°C and at 0 h post-thaw, and 25% greater at 3 h post-thaw than were the motility values. Cleavage frequency and blastocyst development rate of 203 IVM oocytes after IVF using sperm frozen in TYB and HSPM was 36 v. 33% and 50 v. 44%, respectively. In summary, we have shown that cat epididymal spermatozoa can be frozen successfully in cryoprotectant solutions that do not contain egg yolk.Table 1.Motility, membrane integrity and acrosomal status of cat epididymal sperm after cryo-storage [ABSTRACT FROM AUTHOR]

Cryopreservation of testicular tissue is beneficial for valuable animals that die unexpectedly or when elective castration is required. Until recently, knowledge regarding cryopreservation of testicular tissue/sperm in the domestic cat has been limited. The objective of this study was to determine the effects of freezing techniques and cryoprotectants on quality of testicular sperm. In Experiment 1, each testis was cut into 10 equal small pieces (~2×3×5mm) and cryopreserved in freezing medium containing with 5% (v/v) glycerol using conventional (10min in liquid nitrogen vapors) or controlled-rate freezing techniques. In Experiment 2, testicular tissues were conventionally frozen with different types of 5% (v/v) cryoprotectants [glycerol (Gly), dimethylsulphoxide (DMSO), 1,2-propanediol (PrOH), or ethylene glycol (EG)]. Non-cryopreserved testicular sperm was used as a control. After thawing, testicular sperm were extracted and examined for viability and DNA integrity using non-membrane-permeable DNA staining (Ethidium homodimer-1) and TUNEL assay, respectively. Viability of testicular sperm cryopreserved by controlled-rate cryopreservation (45.9±3.7) was significantly lower than non-frozen control (60.3±0.9) and conventional freezing technique (55.0±2.7). Gly (58.2±2.6) and EG (53.3±2.3) yielded a similar viability compared with non-frozen control (P>0.05), whereas DMSO and PrOH demonstrated an inferior cryoprotectant for feline testicular sperm (% viability for DMSO and PrOH: 46.3±3.3 and 44.3±2.9, respectively). In both experiments, DNA integrity of frozen–thawed testicular sperm did not significantly differ from the control group. In conclusion, cat testicular tissue can be frozen as small pieces using both conventional technique and controlled rate freezing. However, freezing technique and type of cryoprotectant markedly affect the post-thawed quality of testicular sperm. Further study requires examination of the optimal cooling rate during cryopreservation and also the fertilizability of frozen–thawed testicular sperm. This study was financially supported by CHE-TRF Senior Research Scholars RTA-5080010. [ABSTRACT FROM AUTHOR]